diff --git "a/deduped/dedup_0059.jsonl" "b/deduped/dedup_0059.jsonl"
new file mode 100644--- /dev/null
+++ "b/deduped/dedup_0059.jsonl"
@@ -0,0 +1,824 @@
+{"text": "The identification of disease-associated genes using single nucleotide polymorphisms (SNPs) has been increasingly reported. In particular, the Affymetrix Mapping 10 K SNP microarray platform uses one PCR primer to amplify the DNA samples and determine the genotype of more than 10,000 SNPs in the human genome. This provides the opportunity for large scale, rapid and cost-effective genotyping assays for linkage analysis. However, the analysis of such datasets is nontrivial because of the large number of markers, and visualizing the linkage scores in the context of genome maps remains less automated using the current linkage analysis software packages. For example, the haplotyping results are commonly represented in the text format.Here we report the development of a novel software tool called CompareLinkage for automated formatting of the Affymetrix Mapping 10 K genotype data into the \"Linkage\" format and the subsequent analysis with multi-point linkage software programs such as Merlin and Allegro. The new software has the ability to visualize the results for all these programs in dChip in the context of genome annotations and cytoband information. In addition we implemented a variant of the Lander-Green algorithm in the dChipLinkage module of dChip software (V1.3) to perform parametric linkage analysis and haplotyping of SNP array data. These functions are integrated with the existing modules of dChip to visualize SNP genotype data together with LOD score curves. We have analyzed three families with recessive and dominant diseases using the new software programs and the comparison results are presented and discussed.The CompareLinkage and dChipLinkage software packages are freely available. They provide the visualization tools for high-density oligonucleotide SNP array data, as well as the automated functions for formatting SNP array data for the linkage analysis programs Merlin and Allegro and calling these programs for linkage analysis. The results can be visualized in dChip in the context of genes and cytobands. In addition, a variant of the Lander-Green algorithm is provided that allows parametric linkage analysis and haplotyping. The oligonucleotide Mapping 10 K arrays -4 and thHere we report the development of a new software tool called CompareLinkage that can be used for automated conversion of Mapping 10 K genotype data into the \"Linkage\" format for linkage analysis in Merlin, GeneHunter and Allegro . In addiTo analyze large pedigrees rapidly and to compare the linkage analysis results of different software packages, we developed a software tool called CompareLinkage to automate the following processes: (1) Converting of Affymetrix Mapping 10 K genotype data, pedigree files and marker information into the \"Linkage\" format , and detA graphical user interface (GUI) for Windows was also implemented in Java. In this GUI users are allowed to set their own working directory and the location of the Perl interpreter through the \"Setting\" menu. CompareLinkage's functions of converting file formats and getting dChip input files are incorporated through the \"Convert\" and \"GetCurve\" menu Figure . Since cThe Affymetrix Mapping 10 K array CEL files and genotype TXT files can be imported into dChip and visualized along cytobands and genes as previously reported ,11. The v involves the summation over all the possible real genotypes :We implemented a variant of the Lander-Green ,6,16 algi represents the ith of all the possible founder allele configurations and is independent of v. P is 1 since an inheritance vector and founder allele configuration uniquely determines the real genotypes, and P involves comparing the real genotype and observed genotype for all the individuals and multiplying the probability by the error rate of 0.01  for each disagreement and 0.99 for each agreement. We also use the matrix-vector multiplication algorithm and bit reduction due to founder phase symmetry described in [where Fribed in , and theribed in ,17 to spWe use the forward-backward computation in the Lander-Green algorithm to obtain the marginal probability distribution of inheritance vector at each SNP marker position given the data of all the markers on a chromosome. In addition the most likely inheritance vector at each marker given the genotype data of all the markers on this chromosome is calculated . ConditiCompareLinkage can format Affymetrix Mapping 10 K SNP genotype output files and genotype files into the \"Linkage\" format and convert genome information and pedigree files into the formats suitable for Merlin (Version 0.10.2), GeneHunter (Version 2.1) and Allegro (Version 1.2). CompareLinkage removes all non-informative markers and calls the PedCheck software  to detecFigures To do parametric linkage analysis in dChipLinkage, a pedigree file is needed Figure . The fil1. Open dChip.Analysis menu and the Get External Data function to read in the genotype file in the text format  with LOD scores of greater than 2.3 Figure  and the After the linkage computation is finished, the inferred haplotype information can be visualized. In the haplotype view Figure  and 16, We have developed the CompareLinkage software for easy comparison and analysis of genotype datasets with common multi-point linkage analysis software programs. It provides functions such as automated data formatting and the calling of linkage analysis software programs to facilitate comparative linkage analysis. The results can be visualized in a chromosome window in the context of genes, cytobands and SNPs in dChip's user friendly graphical interface. The linkage scores of other linkage software packages can be saved into the dChip score file format through CompareLinkage and viewed in the dChip chromosome viewer. This provides the interface to view other computed statistics such as linkage disequilibrium scores along the chromosomes. We have also implemented a variant of the Lander-Green algorithm as the dChipLinkage module for parametric linkage analysis of small pedigrees. It can analyze all chromosomes for families with up to 18 bits within one hour on a PC with one gigabyte memory. This is useful for recessive and consanguineous families whose bits are often small.The comparison analysis of three Mapping 10 K array data sets show similar results in regions with significant LOD scores across all the four software packages. The regions with concordant LOD/NPL scores should provide more confidence in the candidate disease loci. However, there are clear differences in isolated regions. This emphasizes the challenge of a comparative analysis using different linkage algorithm implementations. We hypothesize that the differences between the software programs in peak locations are attributable to:1. The specific algorithm implementation in each program.2. The difference between parametric \u2013 and non-parametric analysis.3. The existence of undetected genotype errors in the data sets which could falsely deflate LOD scores ,22. dChiIn light of the discordance between the results from common linkage software packages and from dChipLinkage, we will validate dChipLinkage implementation using additional datasets and the CompareLinkage software.In summary, the CompareLinkage and dChipLinkage software automate the comparative linkage analysis and visualization using multiple software packages. With these tools users will be able to increase their confidence in candidate regions and can use the visualization tools to explore the disease associated genome regions.Project name: The CompareLinkage software and the dChipLinkage software moduleProject home page: Operating system(s): Windows (dChipLinkge); Windows , Unix (CompareLinkage command line version)Programming language: Visual C++ 6.0 (dChipLinkge); Perl and Java (CompareLinkage software)Other requirements: NoneLicense: None.Any restrictions to use by non-academics: No restrictionsCR, CL and WHW conceived of the study, and participated in its design and coordination. NM and RJHS generated the 5026.10 family data, and MP generated the CR and ER family data. IL implemented the CompareLinkage software and performed the comparative analysis using multiple linkage analysis software packages. JC implemented its graphical user interface (GUI). CL implemented the dChipLinkage module. KH participated in the design and analysis of the study. IL, CR and CL drafted the manuscript. All authors read and approved the final manuscript."}
+{"text": "Unlike a pedigree initiated with two inbred lines, a full-sib family derived from two outbred parents frequently has many different segregation types of markers whose linkage phases are not known prior to linkage analysis.We formulate a general model of simultaneously estimating linkage, parental diplotype and gene order through multi-point analysis in a full-sib family. Our model is based on a multinomial mixture model taking into account different diplotypes and gene orders, weighted by their corresponding occurring probabilities. The EM algorithm is implemented to provide the maximum likelihood estimates of the linkage, parental diplotype and gene order over any type of markers.Through simulation studies, this model is found to be more computationally efficient compared with existing models for linkage mapping. We discuss the extension of the model and its implications for genome mapping in outcrossing species. The construction of genetic linkage maps based on molecular markers has become a routine tool for comparative studies of genome structure and organization and the identification of loci affecting complex traits in different organisms . Statist2 or backcross, always has two alleles and a consistent segregation ratio across different markers. Second, linkage phases among different markers are not known a priori for outbred parents and, therefore, an algorithm should be developed to characterize a most likely linkage phase for linkage analysis.Genetic analyses and statistical methods in outcrossing species are far more complicated than in species that can be selfed to produce inbred lines. There are two reasons for this. First, the number of marker alleles and the segregation pattern of marker genotypes may vary from locus to locus in outcrossing species, whereas an inbred line-initiated segregating population, such as an FTo overcome these problems of linkage analysis in outcrossoing species, Grattapaglia and Sederoff  proposedIn this article, we construct a unifying likelihood analysis to simultaneously estimate linkage, linkage phases and gene order for a group of markers that display all possible segregation patterns in a full-sib family derived from two outbred parents  is generated due to the combination of two-marker haplotypes [11] and [22], whereas diplotype [12] [21] (denoted by ) is generated due to the combination of two-marker haplotypes [12] and [21]. If the probability of forming diplotype [11] [22] is p, then the probability of forming diplotype [12] [21] is 1 - p. The genotype of parent Q and its possible diplotypes [33] [44] and [34] [43] can be defined analogously; the formation probabilities of the two diplotypes are q and 1 - q, respectively.In general, the genotypes of the two markers for the two parents can be observed in a molecular experiment, but the allelic arrangement of the two markers in the two homologous chromosomes of each parent  is not known. In the current genetic literatuire, a linear arrangement of nonalleles from different markers on the same chromosomal region is called the , 1 and , with a probability of pq, p(1 - q), (1 - p)q and (1 - p) (1 - q), respectively. The estimation of the recombination fraction in the full-sib family should be based on a correct diplotype combination  [22] \u00d7 [33] [44]), [11] [22] \u00d7 [34] [43], [12] [21] \u00d7 [33] [44] and [12] [21] \u00d7 [34] [43], expressed as 11, 1bination . The foufor [11] [22] \u00d7 [33] [44],for [11] [22] \u00d7 [34] [43],for [12] [21] \u00d7 [33] [44] and and , respectively.for [12] [21] \u00d7 [34] [43]. Note that these matrices are expressed in terms of the combinations of the progeny genotypes for two markers n = (nj1j2)4 \u00d7 4 denote the matrix for the observations of progeny where j1,j2 = 1 for 13, 2 for 14, 3 for 23, or 4 for 34 for the progeny genotypes at these two markers. Under each parental diplotype combination, nj1j2 follows a multinomial distribution. The likelihoods for the four diplotype combinations are expressed asLet N1 = n11 + n22 + n33 + n44, N2 = n14 + n23 + n32 + n41, N3 = n12 + n21 + n34 + n43, and N4 = n13 + n31 + n24 + n42. It can be seen that the maximum likeihood estimate (MLE) of r  under the first diplotype combination is equal to one minus  under the fourth combination, and the same relation holds between the second and third diplotype combinations. Although there are identical plug-in likelihood values between the first and fourth combinatins as well as between the second and third combinations, one can still choose an appropriate  from these two pairs because one of them leads to  greater than 0.5. Traditional approaches for estimating the linkage and parental diplotypes are to estimate the recombination fractions and likelihood values under each of the four combinations and choose one legitimate estimate of r with a higher likelihood.where In this study, we incorporate the four parental diplotype combinations into the observed data likelihood, expressed as\u0398 =  is an unknown parameter vector, which can be estimated by differentiating the likelihood with respect to each unknown parameter, setting the derivatives equal to zero and solving the likelihood equations. This estimation procedure can be implemented with the EM algorithm  [222], [112] [221], [121] [212] and [122] [211]. Relative to the fixed allelic arrangement 1|2| of the first marker on the two homologous chromosomes 1 and 2, the probabilities of allelic arragments 1|2| and 2|1| are denoted as p1 and 1 - p1 for the second marker and as p2 and 1 - p2 for the third marker, respectively. Assuming that allelic arrangements are independent between the second and third marker, the probabilities of these four three-marker diplotypes can be described by p1p2, p1(1 - p2), (1 - p1)p2 and (1 - p1) (1 - p2), respectively. The four diplotypes of parent Q can also be constructed, whose probabilities are defined as q1q2, q1(1 - q2), (1 - q1)q2 and (1 - q1) (1 - q2) respectively. Thus, there are 4 \u00d7 4 = 16 possible diplotype combinations (whose probabilities are the product of the corresponding diplotype probabilities) when parents P and Q are crossed.Consider three markers in a linkage group that have three possible orders r12 denote the recombination fraction between markers  and , with r23 and r13defined similarly. These recombination fractions are associated with the probabilities with which a crossover occurs between markers  and  and between markers  and . The event that a crossover or no crossover occurs in each interval is denoted by D11 and D00, respectively, whereas the events that a crossover occurs only in the first interval or in the second interval is denoted by D10 and D01, respectively. The probabilities of these events are denoted by d00, d01, d10and d11, respectively, whose sum equals 1. According to the definition of recombination fraction as the probability of a crossover between a pair of loci, we have r12 = d10 + d11, r23 = d01 + d11 and r13 = d01 + d10. These relationships have been used by Haldane  [oo] \u00d7 [aa] [oo] (cis \u00d7 cis) and (2) [ao] [oa] \u00d7 [ao] [oa] (trans \u00d7 trans). The MLE of the linkage under combination (2), in which two dominant alleles are in a repulsion phase, is not as precise as that under combination (1), in which two dominant non-alleles are in a coupling phase  [oo] \u00d7 [aa] [oo], one can easily select cis \u00d7 cis as the best estimation of phase combination because it corresponds to a larger likelihood and a smaller   is biased since it is far less than the value of 0.20 hypothesized. Our model gives the same estimation precision of the linkage for the data derived from [ao] [oa] \u00d7 [ao] [oa] as obtained when the analysis is based on a correct diplotype combination (Table  =  = 0 ).For our data set simulated from [r  Table . Our modns Table . In this of r 0.093 is biaaaa] [ooo] \u00d7 [aaa] [ooo] and (2) [aao] [ooa] \u00d7 [aao] [ooa]. The traditional approach is to calculate the likelihood values under three possible gene orders and choose one of a maximum likelihood to estimate the linkage. Under combination (1), a most likely gene order can be well determined and, therefore, the recombination fractions between the three markers well estimated, because the likelihood value of the correct order is always larger than those of incorrect orders   [aV] \u00d7 [AV] [av] or [AV] [av] \u00d7 [Av] [aV]. The other two recombination fractions and the corresponding parental displotypes are estimated as , [Vx] [vX] \u00d7 [VX] [vx] or [VX] [vx] \u00d7 [Vx] [vX] and , [AX] [ax] \u00d7 [AX] [ax], respectively. From the two-point analysis, one of the two parents have dominant alleles from markers  and  are repulsed with the dominant alleles from marker .We use an example from published literature  to demon. The MLEs of the recombination fractions are ,  and . The parental diplotype combination is [XAV] [xav] \u00d7 [XAv] [xaV] or [XAv] [xaV] \u00d7 [XAV] [xav]. The three-point analysis for these three markers by Ridout et al. [ was not considered.Our subsequent three-point analysis combines parental diplotypes and gene orders to estimate the linkage between these three dominant markers. The estimated gene order is t et al.  led to tSeveral statistical methods and software packages have been developed for linkage analysis and map construction in experimental crosses and well-structured pedigrees -6, but tIn this article we present a unifying model for simultaneously estimating the linkage, parental diplotype and gene order in a full-sib family derived from two outbred parents. As demonstrated by simulation studies, our model is robust to different parameter space. Compared to the traditional approaches that calculate the likelihood values separately under all possible linkage phases or orders ,10,18, oThird, and most importantly, our unifying approach can significantly improve the estimation precision of the linkage for dominant markers whose alleles are in repulsion phase. Previous analyses have indicated that the estimate of the linkage between dominant markers in a repulsion phase is biased and imprecise, especially when the linkage is not strong and when sample size is small . There aOur approach will be broadly useful in genetic mapping of outcrossing species. In practice, a two-point analysis can first be performed to obtain the pairwise estimates of the recombination fractions and using this pairwise information markers are grouped based on the criteria of a maximum recombination fraction and minimum likelihood ratio test statistic . The parIn any case, our two- and three-point analysis has built a key stepping stone for map construction through two approaches. One is the least-squares method, as originally developed by Stam , that caOur maximum likelihood-based approach is implemented with the EM algorithm. We also incorporate the Gibbs sampler  into theQL derived the genetic and statistical models and wrote computer programs. YHC participated in the derivations of models and statistical analyses. RLW conceived of ideas and algorithms, and wrote the draft. All authors read and approved the final manuscript."}
+{"text": "Autism is a neurobehavioral spectrum of phenotypes characterized by deficits in the development of language and social relationships and patterns of repetitive, rigid and compulsive behaviors. Twin and family studies point to a significant genetic etiology, and several groups have performed genomic linkage screens to identify susceptibility loci.We performed a genome-wide linkage screen in 158 combined Tufts, Vanderbilt and AGRE (Autism Genetics Research Exchange) multiplex autism families using parametric and nonparametric methods with a categorical autism diagnosis to identify loci of main effect. Hypothesizing interdependence of genetic risk factors prompted us to perform exploratory studies applying the Ordered-Subset Analysis (OSA) approach using LOD scores as the trait covariate for ranking families. We employed OSA to test for interlocus correlations between loci with LOD scores \u22651.5, and empirically determined significance of linkage in optimal OSA subsets using permutation testing. Exploring phenotypic correlates as the basis for linkage increases involved comparison of mean scores for quantitative trait-based subsets of autism between optimal subsets and the remaining families.positive correlations of linkage between the 19p locus and 17q, between 19p and 6q, and between 7q and 5p. While potential phenotypic correlates for these findings were not identified for the chromosome 7/5 combination, differences indicating more rapid achievement of \"developmental milestones\" was apparent in the chromosome 19 OSA-defined subsets for 17q and 6q. OSA was used to test the hypothesis that 19p linkage involved more rapid achievement of these milestones and it revealed significantly increased LOD* scores at 19p13.A genome-wide screen for autism loci identified the best evidence for linkage to 17q11.2 and 19p13, with maximum multipoint heterogeneity LOD scores of 2.9 and 2.6, respectively. Suggestive linkage (LOD scores \u22651.5) at other loci included 3p, 6q, 7q, 12p, and 16p. OSA revealed Our results further support 19p13 as harboring an autism susceptibility locus, confirm other linkage findings at 17q11.2, and demonstrate the need to analyze more discreet trait-based subsets of complex phenotypes to improve ability to detect genetic effects. Autism OMIM # 209850) is a neurobehavioral disorder involving deficits in language and social abilities and patterns of repetitive behaviors, restricted interests and resistance to change. The most recent estimate of population prevalence for the broader autism spectrum indicates a rate of 34/10,000 (~1/300) 09850 is -8, and tGenome-wide screens of multiplex autism families for susceptibility loci -22 have We report a second generation 10-cM microsatellite-based genomic screen of multiplex autism families. The dataset for this screen includes 71 families recruited by the Tufts/New England Medical Center, a well-characterized set of 85 families from the Autism Genetics Resource Exchange (AGRE), and 2 families from Vanderbilt University. Several sites of suggestive linkage are identified, although none meet criteria for genome-wide significance. The loci with greatest support for linkage were 17q11.2 and 19p13; the latter site demonstrated significantly increased allele-sharing when the Ordered-Subset Analysis (OSA) algorithm was employed using a quantitative trait-based autism phenotypic subset related to specific \"developmental milestones\" as a covariate to rank families.The demographics for the 158 family dataset comprising the studies in this report are shown in Table DNA from Tufts and Vanderbilt samples was obtained from peripheral blood or immortalized lymphoblastoid cell lines using the PureGene Kit (Gentra Systems). While a minority of families from the Tufts/NEMC cohort had been genotyped previously , both neGenotype data for each chromosome underwent thorough error detection and genotype confirmation. Initially, data were tested for Mendelian inconsistencies using PEDCHECK  and RELPAllele frequencies were estimated using genotype data from all unrelated individuals in the combined dataset, consisting of more than 300 chromosomes. Allele frequencies were compared with available data from other Caucasian populations, and no significant differences were observed (data not shown). The LAPIS program of the PEDIGENE system  was usedSpairs scoring function as recommended by McPeek [Linkage was analyzed using both model-dependent and model-independent methods. For autosomes, two-point and multipoint heterogeneity LOD (HLOD) scores were calculated under both dominant and recessive models using Allegro . Diseasey McPeek . NPL scoy McPeek  and FASTy McPeek  to calcuth percentile of the resulting distribution.The nonparametric genome-wide significance threshold ,49 for lOSA  identifi(1) To walk unaided; (2) to sit unaided on flat surface; (3) age of first single words; (4) age of first phrase; (5\u20136) acquisition of bladder control: daytime, night; (7) acquisition of bowel control.\"To explore potential genetic interaction or other genetic correlations between sites of main effect (i.e. suggestive linkage), OSA was applied using family-specific LOD scores as the covariate trait. Families were ranked according to the family-specific LOD score at peak sites demonstrating LOD scores \u22651.5. Allele-sharing analysis was performed for the other six chromosomes using the OSA algorithm. For instances of empirically significant increases in evidence for linkage, we explored the nature of the genetic correlation to ask whether it reflected clinical correlations in the respective subsets. We employed ADI-based factor subsets, identified by principal components analyses of ADI/ADI-R items, to represent putative phenotypic subsets in autism ,56. The Analysis of the \"developmental milestones\" factor as a potential phenotypic subset related to the autism linkage correlations was performed by applying the OSA algorithm. We used \"developmental milestones\" family means, normalized via SAS and Box-Cox transformation procedures, as an ascending ranking covariate. LOD* scores were calculated according to the OSA algorithm, and the resulting increase in linkage achieved with the OSA-determined family subset was analyzed through permutation testing.Approval for these studies was granted by the respective Institutional Review Boards at Tufts University School of Medicine/New England Medical Center and Vanderbilt University Medical Center. Additionally, all studies were performed with informed consent provided by the families participating in the research.Seven chromosomes revealed one or more regions of linkage with a model-dependent or model-independent LOD score \u22651.5 Figure . Data prSLC6A4) locus, long considered to be an attractive functional candidate gene for autism and other neuropsychiatric conditions. Figure For 17q11.2, peak linkage was observed at 53 cM, corresponding to marker D17S1294  and 6 (30 families) for the \"developmental milestones\" cluster. The families in the optimal OSA subset have lower scores and therefore are more rapidly achieving developmental milestones. A similar procedure for the chromosome 7-based subset revealed no obvious differences in any of the factors (data not shown).To directly test the hypothesis that chromosome 19 linkage was related to reduced affection for the \"developmental milestones\" factor, we performed an OSA analysis in which families were ranked in ascending order based on mean values for the milestones factor score. Figure We have presented evidence in support of autism susceptibility loci on chromosomes 17q and 19p. Our results suggest that the 19p locus is related to a phenotypic profile involving a more rapid achievement of particular \"developmental milestones\". Features indexed in this ADI-based factor are: (1) ability to walk unaided; (2) ability to sit unaided on a flat surface; (3) age of first single words; (4) age of first phrase; (5\u20136) acquisition of bladder control: daytime and night; and (7) acquisition of bowel control. Analyses leading to this conclusion also showed positive genetic correlations between optimal OSA-defined subsets contributing to linkage at 19p13 and increases in linkage at loci on 17q21 and 6q23. A similar positive genetic correlation was shown for chromosomes 7q and 5p, however this observation lacks evidence of an underlying phenotypic relationship based on available ADI variable clusters. While the increase in linkage at 17q21 was not empirically significant, the differences in \"milestone\" score means between the optimal chromosome 19 subsets seen for both chromosomes 17 (52 families) and 6q (30 families) were significant. These exploratory data led to the significant finding of increased linkage in the single direct test of our hypothesis concerning the phenotypic correlation related to chromosome 19 linkage.Despite the significance of the final results on 19, we remain cautious in the interpretation of the overall results. As with a number of other genomic screens in autism, no single main effect locus achieved genome-wide significance. Support for a number of these loci, particularly at 17q11.2 and 19p13 comes from similar suggestive linkage in other genomic screens for autism. Although not all screens detect these loci (not an uncommon finding in linkage studies for complex genetic disorders), the evidence is strong regarding an effect at 19p, within 10 cM of our peak: (1) Shao et al reported an MMLS = 1.21 and an MLOD = 1.38 ; (2) theSLC6A4 [SLC6A4 gene on chromosome 17q11.2. Our own more preliminary analysis of linkage in this region with a highly overlapping dataset to that in the current study, revealed very similar results [nd-stage screen [Similarly, several groups have reported evidence for linkage at 17q11. The recently published AGRE follow-up genomic screen identified an MLS of 2.83 near SLC6A4 . A genomSLC6A4 . An IMGSSLC6A4  reported results . Given oe screen , howeverITGB3) locus, which was identified recently from a genome-wide quantitative trait locus (QTL) association screen for platelet serotonin levels [The 17q21 locus is worth further consideration. Our data support the premise that the adjacent linkage peaks represent distinct loci and are not an artifact of primary linkage at 17q11.2. The evidence for linkage at 17q21, while weaker than that at 17q11.2 only 16 cM centromeric, specifically showed an, albeit nonsignificant, interlocus correlation with 19p13 linkage. Linkage at 17q11.2 in this subset of families actually decreases slightly. Of particular interest is the fact that the distal region harbors the integrin \u03b23 (n levels . We see The other \"suggestive\" (LOD \u2265 1.5) loci reported here have also been detected in other genome-wide scans for autism loci. A broad region of 7q has been detected in most screens ,25,27,28that linkage to 19p13 was related to a more rapid achievement for specific milestones. We tested this hypothesis with a single analysis revealing an empirically significant increase in linkage at 19p13. Our results of autism linkage and its increase using an ascending milestone score covariate in OSA, taken in the context of replicated observations of suggestive linkage by other groups, strengthens support for the presence of an autism gene at this site. In the end, ultimate interpretation will rely upon replication of these phenomena with independent samples to confirm these observations.The application of OSA to detect putative interlocus correlations between the 19p13 and 17q21, 19p13 and 6q23, and between 7q35 and 5p are limited to some degree in significance by their highly exploratory and hypothesis-generating nature. Given the number of comparisons between loci, and the number of comparisons between optimal subset pairs (on 19p or 7q) for the traits means, the potential for type I error is increased. Therefore our interpretation must be cast alongside appropriate caveats. Nevertheless, the multiple exploratory comparisons generated a hypothesis: Finally, our results highlight the utility of using trait-based subsets of autism to identify putative susceptibility loci for this complex disorder. We and others have hypothesized a likely increased specificity of individual risk genes and corresponding alleles for traits or subphenotypes comprising the broader autism spectrum. Therefore methods such as OSA with power to identify more homogeneous samples and QTL (quantitative trait locus) linkage and association analyses should provide greater sensitivity in the discovery of disease genes in the context of locus and clinical heterogeneity. Additionally, OSA or other forms of conditional linkage analyses, have the ability to uncover potential interactions between loci, an important concept since the inherent interdependence of proteins in common pathways or networks acting during development and normal neuronal function could be easily imagined to act genetically in concert with one another.We report evidence to support linkage of autism to 17p11.2 and 19p13. Exploratory analyses to test for correlations between suggestively-linked loci, using the OSA method revealed positive correlations of linkage (i.e. in overlapping families) between 7q and 5p, 19p and 6q, and possibly 19p and 17q22, distal to peak linkage at 17q11.2. Comparing mean scores for ADI-derived factor traits from families above and below the OSA-defined split maximizing linkage, suggested a positive correlation between 19p13 linkage and a more rapid achievement of \"developmental milestones\" as measured by items in this cluster of ADI variables. We tested this hypothesis by applying OSA with descending \"developmental milestone\" scores as the ranking covariate, and detected an empirically-significant increase in linkage to 19p13. These findings further support evidence for an autism susceptibility locus in 19p13 and underscore the utility in applying trait subsets in complex disorders to identify genetic risk factors.The authors declare that they have no competing interests.JLM and LMO were responsible for conducting the genome-wide linkage analyses; JLM conducted all OSA analyses, was largely responsible for coordinating and executing the bulk of the reported studies, was key to drafting and editing the manuscript text, and in preparing the figures. CL and JLH provided input into the design and interpretation of statistical results, and provided guidance for conducting the genome-wide simulations required to determine the significance threshold. JLH established the Core-based infrastructure in the Center for Human Genetics Research that facilitated this work and provided very helpful input into the development of the manuscript. SEF developed, with her group, the ADI-based clusters so crucial to examining phenotypic subsets in this paper, which she helped to edit. SEF was responsible for overseeing recruitment and the phenotypic assessment of families from her research group, then at Tufts/NEMC. GC is the clinical coordinator at the Vanderbilt site and oversees ascertainment, recruitment and detailed phenotypic assessment of affected individuals. KG is the Vanderbilt data coordinator and is responsible for management and oversight of family information, pedigree data, and status of DNA, blood or cell line samples for family members. She is directly responsible for preparation of Table The pre-publication history for this paper can be accessed here:"}
+{"text": "Plasmodium chabaudi chabaudi can be considered as a rodent model of human malaria parasites in the genetic analysis of important characters such as drug resistance and immunity. Despite the availability of some genome sequence data, an extensive genetic linkage map is needed for mapping the genes involved in certain traits.P. c. chabaudi was determined in 28 independent recombinant progeny clones. These, AFLP markers and 42 previously mapped Restriction Fragment Length Polymorphism (RFLP) markers  were organized into linkage groups using Map Manager software.The inheritance of 672 Amplified Fragment Length Polymorphism (AFLP) markers from two parental clones (AS and AJ) of Plasmodium falciparum614 AFLP markers formed linkage groups assigned to 10 of 14 chromosomes, and 12 other linkage groups not assigned to known chromosomes. The genetic length of the genome was estimated to be about 1676 centiMorgans (cM). The mean map unit size was estimated to be 13.7 kb/cM. This was slightly less then previous estimates for the human malaria parasite, P. c. chabaudi genetic linkage map presented here is the most extensive and highly resolved so far available for this species. It can be used in conjunction with the genome databases of P. c chabaudi, P. falciparum and Plasmodium yoelii to identify genes underlying important phenotypes such as drug resistance and strain-specific immunity.The  Plasmodium chabaudi chabaudi is a malaria parasite of murine rodents. It has been widely used as a model to study various aspects of parasite biology and disease which are difficult to investigate using human malaria parasites. For instance, P. c. chabaudi is being used to study the genetic basis of drug resistance  ATP. PCR products were run on acrylamide gels and AFLP bands visualised on autoradiography films. Polymorphic bands between the two parental strains were used as markers for the genetic linkage map.The AFLP method was carried out according to the original protocol  with sliet al . BrieflyFor every marker, the parental alleles identified in each of the progeny clones were entered in an Excel spreadsheet. The absence of a band in one parent was treated as the presence of the other parental allele at that locus. Data were then prepared for analysis with the Map Manager QTX software  accordinPrior to linkage analysis, markers were tested for random assortment using a chi-square test, to exclude markers segregating in a non-random fashion from the initial analysis with Map Manager, and thus to avoid spurious linkage inferences between such markers. Because of the large number of tests for non -random assortment performed (n = 672), some markers showing apparent non-random assortment may have been falsely excluded from our analysis; i.e. some valid markers may indeed show non-random assortment and should be included. A Bonferroni correction was therefore applied to the chi-square test to decrease the stringency of the statistical test. The value of the Bonferroni correction represents the number of 'independent' comparisons and here was arbitrarily set at 24. This value was chosen as representing the likely number of chromosomal fragments at meiosis, and is supported by data presented in this paper. Markers within these fragments are not independently inherited. The chosen value (24) is a compromise between 672  and 14 . Markers not following random assortment in the initial test were thus divided into two groups, i.e. those segregating in a non-random fashion before and after Bonferroni correction, and those segregating in a non-random fashion before but not after the Bonferroni correction. The markers in the latter group were added separately after linkage groups had been determined (see below).Linkage groups using AFLP and RFLP markers were formed with an initial p-value of 0.0001 using the \"Make Linkage Groups\" command in Map Manager. p-values in Map Manager indicate the probability of a Type 1 error; that is, the probability of a false positive linkage. Following formation of linkage groups, the p-value was raised to 0.001. Linkage at p < 0.001 is considered significant. Using the command \"Distribute\", linkage groups were brought together. Then, other previously unlinked markers were allocated to these new linkage groups, again using the \"Distribute\" command. Markers with non-random assortment after statistical analysis without Bonferroni correction were added next, and those still segregating in a non-random fashion after Bonferroni correction were added last. The \"Ripple\" function was then used to position markers in an order which maximizes the total LOD (logarithmic odds) score for linkage. The software also estimated the optimum order and genetic distance between markers in centiMorgans (cM) by using the \"Kosambi\" function in the software. It was then possible to calculate a map unit size .P. c. chabaudi chromosomes [The presence of 42 previously characterised RFLP markers which had been physically mapped onto omosomes  served aP. c. chabaudi AJ and either clone AS (3CQ) or AS (30CQ). The majority of the AFLP markers showed independent assortment in the 28 progeny clones, as illustrated previously [The inheritance of 672 AFLP markers was determined in 28 progeny clones derived from two crosses between eviously . Howevereviously . EstimatP. c. chabaudi chromosomes 1 and 5\u201313, by virtue of their linkage to RFLP markers previously assigned to specific chromosomes by physical mapping [The 672 AFLP markers formed a total of 22 linkage groups with a final p-value of 0.001. 58 AFLP markers, 21 of which segregated in a non-random fashion, could not be allocated to any linkage groups. Physical mapping of these markers would be required to assign them to specific chromosomes. Alternatively, unassigned linkage groups or unallocated markers might map to the small mitochondrial or apicoplast genomes, although these combined represent only 0.2% of the genome. Several RFLP markers could not be allocated to linkage groups by the Map Manager software, probably due to the small numbers of clones analysed for these markers. These markers were added to assigned linkage groups according to their chromosomal assignment, as previously determined by physical mapping [assigned markers because of factors such as a high rate of recombination between two linkage groups (one linked to the RFLP anchor) or because of an intervening region with a low density of AFLP markers. Both factors, or a combination of the two, may prevent two physically linked groups from being identified as genetically linked. Conversely an apparent unusually high frequency of AFLP markers (as in chromosome 7) may arise from strong linkage disequilibrium between loci on two different chromosomes. Some markers located on one chromosome may thus appear to be genetically linked to markers on another. This could arise where one locus exerts a strong constraint on another unlinked locus. For instance, the AJ allele of an enzyme in a metabolic pathway may only function with the presence of the product of the AJ allele encoding another enzyme in the same pathway. This constraint might be structural or functional. The same may be true of AS alleles of the same enzymes. In this case, the genes encoding these enzymes, and markers strongly linked to them, may appear in the same genetic linkage group.With the exception of chromosomes 2, 3, 4 and 14 (discussed above), chromosomes 9 and 10 showed the lowest density per Mb of AFLP markers. Chromosome 7 showed the highest density. This may simply reflect natural variation in the frequency of AFLP polymorphisms on particular chromosomes. However, for chromosomes with a low apparent density of AFLP markers such as chromosomes 9 and 10, it is likely that some of the markers in unassigned linkage groups would physically map to these chromosomes. These unassigned groups may not show genetic linkage with (groups of) AFLP markers were initially ordered on the linkage groups as described in Materials and Methods. After inspection of the predicted marker order, occasional manual adjustments were made to correct markers which appeared to be inappropriately positioned.Because Map Manager failed to allocate some RFLP markers to an assigned linkage group, these were positioned manually. The final distribution of the markers on the 10 linkage groups assigned to chromosomes is shown in Fig. If markers and recombination events were both uniformly distributed across the genome, then we would expect the number of predicted recombination events  in each chromosome to correlate with its physical size. The predicted total number of recombination events occuring in each linkage group is shown in the P. c. chabaudi genome consists of about 20 Mb [Plasmodium falciparum [Assuming that the ut 20 Mb  the valulciparum .P. falciparum, and were interpreted as being due to non-reciprocal conversion events [A significant number of double crossover events around a single AFLP marker were observed in many linkage groups. Any such occurrences were re-evaluated on the original X-ray film to detect any possible errors or ambiguous bands. Many distinct double crossover events were observed. The same phenomenon was also commonly observed in n events .The apparent genetic length of each linkage group in cM was calculated by Map Manager based on the number of recombination events . When adran, 40 cM from its nearest AFLP marker. At its other end, an RFLP marker, Ag 3027, lies about 35 cM from its nearest AFLP marker. Chromosome 10 has RFLP marker cDNA121 about 70 cM from its nearest AFLP marker and RFLP marker, 5S rRNA, a further 70 cM distant. These large gaps may be artefacts which arise for two reasons. Firstly, some unreliability in the typing of clones using RFLPs was previously noticed [ran, Ag3027, cDNA121 and 5S rRNA were typed for only 9, 17, 9 and 16 clones, respectively. The characterisation of inheritance of RFLP markers in all of the 28 progeny clones, and the correction of possible mistakes may reduce the estimated genetic length. This would lead to an increase in map unit size. Nevertheless, it is notable that the value reported above (13.7 kb/cM) is close to estimates made for P. falciparum , althou30 kb/cM .Of the 400 AFLP markers placed on chromosomes, 37 AS-AJ pairs  shared the same selective bases at both primer ends and showed complementary segregation in the cross-progeny clones. Most of these markers also differed in size by only a few base pairs. They are likely to be alleles at the same loci. This was confirmed by sequencing two such pairs, namely ASTA01AC and AJTA01AC (chromosome 5), and ASTT02CA and AJTT02CA (chromosome 13) (sequence data not shown). This suggests that a significant proportion of the polymorphisms observed between AS and AJ may be due to small insertions or deletions. In fact, it was observed that small indels tend to occur in introns or intergenic regions (data not shown).A few AFLP markers which were originally identified between clones AJ and AS  were notOngoing work on linkage between chloroquine resistance and markers on chromosome 11  suggesteP. falciparum [P. c. chabaudi [Eimeria tenella [Toxoplasma gondii [Theileria parva [Cryptosporidium parvum [P. falciparum exceeds its resolutionGenetic or physical linkage maps have been determined and reported for a number of apicomplexan parasites, including lciparum , P. c. cchabaudi , Eimeria tenella , Toxoplaa gondii , Theileria parva  and Crypm parvum . The mapOf the AFLP markers analysed, most were assigned to 10 of the 14 chromosomes, while some were placed on 12 unassigned linkage groups, which probably include groups located on chromosomes 2, 3, 4 and 14. The remaining AFLP markers could not be allocated to any linkage group. Unallocated AFLP markers and unassigned linkage groups may arise in a number of possible ways, discussed in the Results section above, including mistakes or considerable gaps in the recorded inheritance pattern of RFLP markers and, to a lesser extent, AFLP markers. Other factors include variations in the density of AFLP markers or polymorphism in general, areas of the genome where the rate of recombination is particularly high, linkage disequilibrium between loci on different chromosomes and non-random representation of clones in our sample. The numbers of markers allocated, the approximate genetic lengths and the number of recombination events were estimated for each of the linkage groups. The genetic length of the entire genome was estimated to be 1684 cM and the overall size of map unit 13.7 kb/cM. The genetic length and number of recombination events were expected to increase with the size of chromosomes. This was generally found to be the case, although a number of factors may influence these data. These factors include the failure to assign some linkage groups, incomplete or incorrect inheritance data, particularly for RFLPs, variation in the frequency of AFLP markers across the genome, variation in the recombination rate across the genome, and incorrect assignation of some linkage groups due to linkage disequilibrium.The presence and frequency of small indel mutations was confirmed. These markers could prove suitable for rapid typing of clones by size polymorphism and quantitative analysis by Real Time Quantitative PCR.P. chabaudi was originally conceived as an essential step towards the identification of loci linked to genes encoding important phenotypes, such as drug resistance. Indeed, the identification of a locus underlying chloroquine resistance in P. chabaudi within approximately 250 kb of chromosome 11 [P. c. chabaudi  [P. falciparum (complete) [P. falciparum genome. Because of the extensive gene synteny between P. chabaudi  and P. falciparum [P. falciparum are likely to be closely linked in P. chabaudi too.The generation of a complete AFLP genetic linkage map for osome 11  relied uosome 11  which moosome 11 . A genetpartial)  and P. fomplete) , and seqlciparum ,9,10, maP. falciparum genome in the studies discussed above [P. c. chabaudi, Plsmodium yoelii and P. falciparum genomes [P. falciparum genome , will allow us to assign unallocated AFLP markers and unassigned linkage groups to particular chromosomes on the assumption that gene synteny is conserved.The correspondence between the genetic linkage map reported here and the mapping of AFLP markers to the ed above ,5,26 has genomes ,10. The AM characterized the AFLP markers in the cross progeny, generated the genetic linkage map and drafted the article, PH helped in the generation of the linkage map, analysed genetic data from it and drafted the article, RF helped in the characterization of the AFLP markers in the cross progeny, PC and DW provided the recombinant clones and revised the article, RC designed and coordinated the study, revised the article and gave final approval. All authors read and approved the final manuscript.The file (.XLS) contains the following data for markers assigned to chromosomes and, where possible, for markers in unassigned linkage groups and the overall genome: \u2013 physical size of each chromosome (Mb), the number of markers , the number of AFLP markers per Mb, the number of recombination events predicted for the 28 clones, the frequency of recombination events per Mb and per AFLP marker, the predicted genetic length of all linkage groups, and the estimated size of map unit (kb/cM).Click here for fileThis file is the original PPT files from which figure 1 was derived.Figures 1-3 contain the linkage map for the chromosomes 1 and 5-13.Click here for fileThis file is the original PPT files from which figure 2 was derived.Figures 1-3 contain the linkage map for the chromosomes 1 and 5-13.Click here for fileThis file is the original PPT files from which figure 3 was derived.Figures 1-3 contain the linkage map for the chromosomes 1 and 5-13.Click here for fileThis file is the original PPT files from which figure 4 was derived. Figure 4contains various unassigned linkage groups.Click here for file"}
+{"text": "Zlr statistic as calculated in the program ALLEGRO; a conditioning approach in which the per-family contribution on one chromosome was weighted according to evidence for linkage on the other chromosome; and a novel multimarker regression approach. The multipoint and multimarker approaches were generally successful in localizing known susceptibility loci on chromosomes 1 and 3, and were found to give broadly similar results. No advantage was found with the per-family conditioning approach. The effect on power and type I error of different choices of weighting scheme (to account for different numbers of affected siblings) in the multimarker approach was examined.The purposes of this study were 1) to examine the performance of a new multimarker regression approach for model-free linkage analysis in comparison to a conventional multipoint approach, and 2) to determine the whether a conditioning strategy would improve the performance of the conventional multipoint method when applied to data from two interacting loci. Linkage analysis of the Kofendrerd Personality Disorder phenotype to chromosomes 1 and 3 was performed in three populations for all 100 replicates of the Genetic Analysis Workshop 14 simulated data. Three approaches were used: a conventional multipoint analysis using the  Zlr statistic ; 2) the Suarez and Hodge scheme ; 4) a scheme where each pair contributes equally regardless of the number of affected sibs in the family. These weighting schemes were incorporated into the multimarker analysis via use of importance weights in the statistical analysis package STATA.An issue not investigated in the previously proposed approach  was the e scheme , in whicactor of /3 and 3 . The Karangar study provides reasonable evidence for the disease locus on chromosome 3  but little evidence on chromosome 1 , while the Aipotu study provides good evidence for the disease locus on chromosome 3  and some evidence for the disease locus on chromosome 1 . The Zlr scores from the conditional weighted analyses are lower than those from the unweighted analysis, indicating no advantage from using conditioning weights.Figure z-score results from the multimarker approach are given in Table z-score on chromosome 4 is close to 0 with variance close to 1 and approximate normality  for all four weighting schemes. The positions of the maximum Zlr from the multipoint approach and the maximum z-score from the multimarker approach are shown in Figure The Overall, the multimarker and multipoint approaches appear to provide quite similar results, particularly when using the Hodge or Suarez and Hodge weighting schemes. Slightly greater power for the multimarker approach is obtained using the 'Equal pairs' weighting scheme, which is consistent with the results of Sham et al. . The genZlr scores from the conditional weighted analyses are lower than those from the unweighted analysis, indicating no improvement in power from using conditioning weights, and no power to detect an interaction. The exact form of the proposed interaction is not specified in the \"answers\" and could potentially correspond to a number of different underlying scenarios [The cenarios . Only thThe multipoint and multimarker approaches were generally successful in localizing known susceptibility loci on chromosomes 1 and 3, and were found to give broadly similar results. No advantage was found with a per-family conditioning approach. For the multimarker approach, greatest power and acceptable type I error was seen with the 'Equal pairs' weighting scheme.ASP: Affected sib pairGEE: Generalized estimating equationKPD: Kofendrerd Personality DisorderIBD: Identity by descentMJB developed and applied the multimarker regression approach. EW applied the per-family weighted multipoint approach and generated the figures. HJC directed the project and drafted the final manuscript."}
+{"text": "To compare different strategies for linkage analyses of longitudinal quantitative trait measures, we applied the \"revisited\" Haseman-Elston (RHE) regression model  to cross-sectional, summary, and repeated measurements of systolic blood pressure (SBP) values in replicate 34, randomly selected from the Genetic Analysis Workshop 13 simulated data. RHE linkage scans were performed without knowledge of the generating model using the following phenotypes derived from untreated SBP measurements: the first, the last, the mean, the ratio of the change between the first and last over time, and the estimated linear regression slope coefficient. Estimates of allele sharing in sibling pairs were obtained from the complete genotype data of Cohorts 1 and 2, but linkage analyses were restricted to the five visits of Cohort 2 siblings. Evidence for linkage was suggestive (p < 0.001) at markers neighboring SBP genes Gb35, Gs10, and Gs12, but weaker signals (p < 0.01) were obtained at markers mapping close to Gb34 and Gs11. Linkage to baseline genes Gb34 and Gb35 was best detected using the first SBP measurement, whereas linkage to slope genes Gs10-12 was best detected using the last or mean SBP value. At markers on chromosomes 13 and 21 displaying strongest linkage signals, marginal RHE-type models including repeated SBP measures were fit to test for overall and time-dependent genetic effects. These analyses assumed independent sib pairs and employed generalized estimating equations (GEE) with a first-order autoregressive working correlation structure to adjust for serial correlation present among repeated observations from the same sibling pair. Many complex quantitative traits (QT) such as blood pressure exhibit temporal variation and age-dependent penetrances, and as is the case with the Framingham Heart Study, are studied longitudinally. Statistical methods for QT linkage analyses include variance components (VC) analyses ,2, tradiPredecessors of VC analyses, traditional HE models  regress In this paper, we apply the RHE model to Genetic Analysis Workshop 13 (GAW13) simulated data to compare different strategies for conducting linkage analyses within the framework of a longitudinal study. The dependent variable in the RHE model is the cross product of sib-pair trait measures corrected for the sample mean. We also examine an RHE-type marginal model, similar to that of Ziegler et al. , that emReplicate 34 was randomly selected from among the 100 simulated data sets available to GAW13, and single-point IBD sharing was estimated using the complete genotype data of Cohorts 1 and 2 via SAGE/GENIBD software . Blind tp < 0.001) and were located close to SBP susceptibility genes. At these loci, the IBD-sharing estimates for Cohort 2 sib pairs were extracted from SAGE/GENIBD output, and using SAS software we calculated five different phenotypes: the mean-adjusted cross product of the FirstSBP, LastSBP, MeanSBP, \u0394SBP, and RegSBP sib-pair values. RHE-type models that regressed these sib-pair cross products on IBD sharing were fit with SAS/GENMOD. Assuming no residual familial correlation among multiple sib pairs from the same pedigree, the RHE-type models were extended via GEE [Based on the results of our genome scans and knowing the generating model, a marker on chromosome 13 and four markers on chromosome 21 were selected for longitudinal analyses. All these markers showed suggestive linkage evidence  correlation structure. Specific to each visit, we considered the average age of the sib pair at the time of measurement adjusted for the overall population mean across measurements (SibAge). To test whether age had an influence on genetic variability, we included an interaction term between SibAge and IBD allele sharing. Similarly, we considered the visit number (1 to 5) and its interaction with IBD allele sharing to assess time-dependent effects.p < 0.0001) was detected using MeanSBP at the two neighboring markers located on chromosome 21 within the interval between Gs12 and Gs10 (Table p < 0.001) at the other two markers, GATA129D11 and GATA70B08, on chromosome 21 flanking Gs12 and Gs10, respectively (Table p < 0.01) was seen at marker GATA88H02 on chromosome 15, mapping close to Gs11  at marker ATA26D07 on chromosome 13 close to Gb35 and weak (p < 0.01) at marker GATA6E05 on chromosome 5 close to Gb34 , and thus this approach remains an important tool for QT linkage analyses. A more stringent testing level of p < 0.001 for MeanSBP was sufficient to eliminate the three apparent false positive results at the price of two false negatives . Analyses using early phenotypes may be more powerful to detect baseline genes whose contribution to trait variance decreases over time. However, analyses using single phenotype measures are unable to distinguish between genes with a constant effect and those with a variable age effect. As seen in these data, when the gene effect increases with age, a longitudinal model may be preferable because it provides comparable results to mean summary values, but facilitates a test for interaction between age/time and gene effects.The RHE scan for cross-sectional and summary SBP phenotypes identified five of six SBP susceptibility genes (using a criterion of"}
+{"text": "Genetic maps based on single-nucleotide polymorphisms (SNP) are increasingly being used as an alternative to microsatellite maps. This study compares linkage results for both types of maps for a neurophysiology phenotype and for an alcohol dependence phenotype. Our analysis used two SNP maps on the Illumina and Affymetrix platforms. We also considered the effect of high linkage disequilibrium (LD) in regions near the linkage peaks by analysing a \"sparse\" SNP map obtained by dropping some markers in high LD with other markers in those regions.The neurophysiology phenotype at the main linkage peak near 130 MB gave LOD scores of 2.76, 2.53, 3.22, and 2.68 for the microsatellite, Affymetrix, Illumina, and Illumina-sparse maps, respectively. The alcohol dependence phenotype at the main linkage peak near 101 MB gave LOD scores of 3.09, 3.69, 4.08, and 4.11 for the microsatellite, Affymetrix, Illumina, and Illumina-sparse maps, respectively.The linkage results were stronger overall for SNPs than for microsatellites for both phenotypes. However, LOD scores may be artificially elevated in regions of high LD. Our analysis indicates that appropriately thinning a SNP map in regions of high LD should give more accurate LOD scores. These results suggest that SNPs can be an efficient substitute for microsatellites for linkage analysis of both quantitative and qualitative phenotypes. Microsatellites are commonly used as markers for linkage analysis. More recently, single-nucleotide polymorphisms (SNPs) have been increasingly used as genotyping markers due in part to lower cost and ease of use. This study compares SNP marker linkage results for qualitative and quantitative phenotypes to the corresponding microsatellite results. For this study we focused on chromosome 7, an area shown to have linkage and association for both alcohol dependence, a qualitative trait, and a neurophysiologic measure, a quantitative trait -4. For tThe GAW14 population data consisted of phenotypic and genotypic information for 1,614 individuals in 143 pedigrees. Because of a significant difference in the allele frequencies for African Americans and Caucasians in this population, our analyses were restricted to the larger Caucasian subpopulation of 1,214 individuals in 112 pedigrees .n-1 sib pair method with the proband being matched to all other affected siblings. This strategy was chosen because the proband is generally more severely affected and more likely represents a \"true\" case.For the alcohol dependence phenotype an individual was defined as affected by DSM-III-R alcohol dependence and Feighner definite alcoholism. For this phenotype, an affected sibling pair method was used using GENEHUNTER, where only individuals who are affected are analyzed, though unaffected individuals can contribute information regarding IBD sharing. We used the We used a new version of GENEHUNTER , which hp = 2 \u00d7 10-14).The neurophysiology phenotype was the target case frontal theta band , denotedMultipoint IBD matrices were computed at 1-cM intervals using LOKI and output into SOLAR format. Two-point identity-by-descent (IBD) values were computed using SOLAR.It was found by Hinrichs et al.  that a sAlthough genetic maps were provided and were used for IBD computations and linkage analyses, chromosome lengths for microsatellites, Affymetrix SNPs, and Illumina SNPs were different  and therefore markers were adjusted to their physical locations for the purpose of plotting and ease of comparison. All findings were then placed on a common map defined by the physical map obtained from the NCBI database (Build 34.3).In the present analyses, linkage results were stronger overall for SNPs than for microsatellites. We found higher LOD scores and more narrowly defined linkage peaks with SNPs for both the quantitative and qualitative phenotypes Figures  and 2. FIn order to interpret our linkage results, we compared the dense and sparse Illumina maps near the two linkage peaks. There were 19 markers within 5 MB of the peak near 130 MB and 7 of these were dropped for the sparse map. There were 17 markers within 5 MB of the peak near 101 MB and 2 of these were dropped for the sparse map.We also performed a two-point analysis for the neurophysiology phenotype at the linkage peak near 130 MB. The Illumina SNPs produced a LOD score of 1.65 and the microsatellite markers gave a LOD score of 2.32.These analyses provide a comparison of the use of microsatellite and SNP markers in linkage analysis for quantitative and qualitative phenotypes. For both phenotypes linkage results were stronger overall for SNPs than for microsatellites. Does this represent a true or a false increase in the evidence of linkage? The SNP maps have higher information content than microsatellite maps , and thiAs one expects, the two-point analysis of the neurophysiology phenotype gave lower linkage signals than the multipoint analysis with a much greater difference evident with SNPs than with microsatellites. Such differences are often exaggerated in regions of high LD. In addition, it is known that single-point analysis with SNPs has substantially lower information than with microsatellites. In this case we believe that the differing linkage results are more due to reduced information in the SNPs than to LD in the region.There was also some difference in LOD scores between the Affymetrix and Illumina SNPs, with the Illumina SNPs giving higher scores at the main linkage peaks in these analyses. These differences may be due to the differing number of SNPs typed and their distribution on the chromosome. Though the overall information content for the Illumina and Affymetrix maps were similar, by chance it appears that the information content is lower in the Affymetrix map at these linkage peaks.Our results indicate that SNPs can perform as well as or better than microsatellites for linkage analysis. In conjunction with the results of , it seemGAW14: Genetic Analysis Workshop 14IBD: Identity-by-descentLD: Linkage disequilibriumSNP: Single-nucleotide polymorphismGD, SB, and ALH formatted the data files and analyzed the data. GD, LJB and ALH drafted the manuscript. JSKK provided analyses identifying Caucasian subgroups. GD, SB, ALH, LJB, CHJ, and BKS participated in the design and coordination of the study. All authors read and approved the final manuscript."}
+{"text": "We conclude that parametric methods, using even over-simplified models of complex phenotypes, may complement nonparametric methods and decrease false positives.Many investigators of complexly inherited familial traits bypass classical segregation analysis to perform model-free genome-wide linkage scans. Because model-based or parametric linkage analysis may be the most powerful means to localize genes when a model can be approximated, model-free statistics may result in a loss of power to detect linkage. We performed limited segregation analyses on the electrophysiological measurements that have been collected for the Collaborative Study on the Genetics of Alcoholism. The resulting models are used in whole-genome scans. Four genomic regions provided a model-based LOD > 2 and only 3 of these were detected ( Although model-based or parametric linkage analysis on extended families is generally considered the most powerful means to localize genes when a model can be approximated, the requirement for reasonable model parameter values is often perceived to be unattainable for complex traits. As a result, the potential advantages of the method are frequently passed over in favor of \"model-free\", nonparametric statistics that may be less powerful ,2. The r2, and age3, and the remaining variables were adjusted for sex, age, and age2; all effects were highly significant. Additionally, the regression residuals for each of the 143 families were re-centered on zero to remove any family-specific effects on the mean.We evaluated 13 quantitative traits representing neurological function in the COGA dataset (143 families). These included one EEG phenotype, ecb21, and 3 sets of 4 related ERP phenotypes , ttth1, tth2, tth3, and ttth4; ttdt1, ttd2, ttd3, and ttdt4; and ntt1, ntt2, ntt3, and ntt4. Linear regression was used to adjust for the effects of age and sex. The variable ecb21 was adjusted for sex, age, ageComplex segregation analyses (CSA), using PAP v. 5, was used to estimate co-dominant (no overdominance) and dominant mixed models for each adjusted trait ,9. MultiUsing the more parsimonious CSA model for each trait, we performed a two-marker genomic scan using LINKMAP ,11. Thusp < 0.05 (data not shown). Using the Mendelian dominant genetic model for each trait, the parametric 2-marker genome scans detected 4 regions with a LOD score of at least 2.0 for 3 of the 13 traits  the dominant model was found to be more parsimonious  have been estimated. However, the models available in current linkage software are overly simple for complex traits and the utility of model-based methods under these limitations is unclear. An incorrect model may lead to loss of power in the presence of true linkage as well as an overestimation of recombination . Severalp < 0.05 but did not detect the chromosome 4 region at this probability level. Two of the regions identified with LOD > 2 using parametric linkage appear to have been previously detected in published analyses of these data, while two others were not. Both previously detected regions were found by the program, SOLAR, which calculates a likelihood ratio derived LOD score by comparing a model in which the additive genetic variance at a specified map position is compared to one in which this component is set to zero. The chromosome 3 region (max LOD = 2.01 for ttdtla), near D3S2406-GATA128C02-D3S2459, is the same location in which Porjesz et al. [Our findings indicate that model-based linkage of complex traits may add information not furnished by nonparametric analyses. Our two-marker parametric linkage results suggest four regions with LOD > 2 for three traits. The multipoint nonparametric analysis detected three of these regions with a p-value of 0.0005. Some support for linkage on this chromosome is suggested by the MERLIN results in that, of the 4 regions analyzed by multipoint linkage, chromosome 12 gave the highest KC-LOD, 0.9, and the smallest p(KC), 0.02.On chromosome 4 we obtained a LOD of 2.08 for ecb21 with D4S1558-D4S2361 whereas Williams et al.  obtainedAlthough, in this instance, our procedure resulted in the identification of a possibly 'real' linkage that was missed by standard nonparametric analysis, the use of nonparametric linkage methods shouldn't be viewed as inferior nor should our CSA be viewed as sufficient. The nonparametric linkage tests found candidate loci that our model-based procedure did not and our CSA was limited to a single major gene and forced convergence within a restricted sample space. Complex traits with multiple genetic and environmental effects will often result in no reasonable model. We found linkages for only four of thirteen modeled traits. Failure to detect linkage may have been due to unaccounted sources of familial correlation  or to modeling a single major gene when one did not exist. Also, when linkage is found using an overly-simple model, sensitivity testing to evaluate parameter values, e.g., marker allele frequencies and QTL penetrance/quantitative effect, can assess potential misspecification. However, overall, we recommend obtaining maximum likelihood genetic models from CSA whenever possible; by definition no 'truer' trait models can be obtained, given the single major gene restraint conditions under which we modeled.Our results indicate that model-based linkage procedures using simple models from CSA may detect candidate loci for complex traits that are not revealed by commonly used model-free techniques. Parametric methods that allow more complex modeling, such as MCMC methods, are being implemented ,18,19. HCOGA: Collaborative Study on the Genetics of AlcoholismCSA: Complex segregation analysisEEG: ElectroencephalogramERP: Event-related potentialGAW: Genetic Analysis WorkshopHLOD: Heterogeneity LODIBD: Identity by descentKC-LOD: Kong-Cox LODMCMC: Markov chain Monte CarloQTL: Quantitative trait locusMDB formulated the study question, did the analyses, and wrote the manuscript; ELG and GPJ provided critical input on the analyses and manuscript."}
+{"text": "To find specific genes predisposing to heavy alcohol consumption , we studied 330 families collected by the Framingham Heart Study made available to participants in the Genetic Analysis Workshop 13 (GAW13).Parametric and nonparametric methods of linkage analysis were used. No significant evidence of linkage was found; however, weak signals were identified in several chromosomal regions, including 1p22, 4q12, 4q25, and 11q24, which are in the vicinity of those reported in other similar studies.Our study did not reveal significant evidence of linkage to heavy alcohol use; however, we found weak confirmation of studies carried out in other populations. While heavy drinking is not necessarily indicative of alcohol abuse or alcoholism, individuals who binge drink are at a higher risk for alcohol-related disorders than others. \"Heavy\" (or \"hazardous\") drinking is a serious public health condition that has been defined in different ways. The Centers for Disease Control and Prevention describes it as \"more than 14 drinks per week for men and more than 7 drinks per week for women\". Using this definition, there are 8.7 percent of males and 6.7 percent of females who are heavy drinkers among current drinkers in the United States . This peThat alcoholism has a genetic component has been known for at least three decades and part of the Genetic Analysis Workshop 11 (GAW11) was dedicated to this phenotype . Previoun = 193; offspring cohort n = 578), whereas subjects who consistently reported no consumption of alcohol at any time in the year previous to all examinations were \"unaffected\" for the heavy drinking phenotype . The remaining family members, subjects who reported alcohol use in the year previous to at least one examination but who never consumed on average > 24 grams/day (men) or > 12 grams/day (women), were excluded from the analysis .The Framingham Heart Study data set provided for the GAW13 included genotypes and longitudinal data for 330 families collected during 1948\u20131998  and between 1971\u20131999 (offspring cohort). \"Heavy drinking\" was defined as self-reported consumption of an average of more than 24 grams of alcohol per day during the year before the examination among men and an average of more than 12 grams per day among women. Data were available from 11 (out of 21) examinations in the original cohort and from all five examinations in the offspring cohort. Subjects who reported heavy drinking during the year previous to any one examination were classified as affected  analysis was performed using the S (pairs) option of GENEHUNTER-Plus, and maximized nonparametric LOD scores (\"K&C LOD scores\") were calculated under an exponential model with \u03b4 constrained between 0 and 2 .p-values were obtained. While this sib-pair linkage method was originally explained for a continuous trait, it is also applicable to binary traits [Finally, two nonparametric affected sib-pair analyses were performed. Maximum-likelihood estimates of the proportions of sib pairs sharing 0, 1, or 2 alleles identical by descent (IBD) at marker loci were estimated with the routine SIB-MLS of the software GAS (v. 2.0) . This noy traits .There were 86 families with one heavy drinker, 73 with two, 59 with three, 38 with four, and 35 with five or more. One family had 16 heavy drinking members (26526). In all, four markers showed some evidence for linkage in parametric two-point LOD score analysis , our analyses failed to provide significant evidence of linkage to heavy alcohol use in the Framingham Heart Study data set. However, if we accept the suggestion of Rao and Gu [p = 0.0023 and LOD score of 1.75), we identified several areas of potential interest, including 1p22, 11q24, and 12q. Our finding on chromosome 1 overlaps with that of Reich et al. [Assuming the recommendations of Lander and Kruglyak  that we o and Gu  to relaxh et al. ,20. We fSeveral reasons might explain our failure to find significant evidence of linkage to heavy alcohol use and the seemingly different results using alternative analytic methods. First, the phenotype we chose was extreme and yielded a low sample size, particularly of informative affected sib pairs. Second, individuals were classified as \"affected\" if they consumed, on average, high amounts of alcohol in the year previous to any examination. This might have been an inadequate choice, since it probably included a substantial number of heavy \"social\" drinkers, for whom a genetic susceptibility is unlikely. If the sample size had allowed it, we would have used the definition of the U.S. Substance Abuse and Mental Health Services Administration (see Background), which requires at least five episodes of heavy drinking in a 30-day period. Finally, the data on alcohol exposure was self-reported. We are unaware of reliability studies for alcohol consumption in this population. If misclassification bias exists, it might have reduced the statistical power and decreased the likelihood of finding significant results.Our study did not reveal significant evidence of linkage to heavy alcohol use; however, we found weak confirmation of studies carried-out in other populations."}
+{"text": "P300 amplitude is an electrophysiological quantitative trait that is correlated with both alcoholism and smoking status. Using the Collaborative Study on the Genetics of Alcoholism data, we performed model-free linkage analysis to investigate the relationship between alcoholism, P300 amplitude, and habitual smoking. We also analyzed the effect of parent-of-origin on alcoholism, and utilized both microsatellites (MS) markers and single-nucleotide polymorphisms (SNPs). We found significant evidence of linkage for alcoholism to chromosome 10; inclusion of P300 amplitude as a covariate provided additional evidence of linkage to chromosome 12. This same region on chromosome 12 showed some evidence for a parent-of-origin effect. We found evidence of linkage for the P300 phenotype to chromosome 7 in non-smokers, and to chromosome 17 in alcoholics. The effects of alcoholism and habitual smoking on P300 amplitude appear to have separate genetic determinants. Overall, there were few differences between MS and SNP genome scans. The use of covariates and parent-of-origin effects allowed detection of linkage not seen otherwise. A receptor alleles [A reduced amplitude of the P300 event-related potential is a heritable phenotype that correlates with alcoholism and other psychiatric disorders. Quantitative trait linkage analysis of P300 amplitude in the Collaborative Study on the Genetics of Alcoholism (COGA) subjects identified linkage to five regions on chromosomes 2, 5, 6, 13, and 17 . It is uThis study analyzed the COGA dataset released for the Genetic Analysis Workshop 14. Subject ascertainment and data collection have been previously described . GenotypAs a measure of P300 amplitude, we used the COGA variable ttth4, which is measured at the posterior electrode Pz. P300 amplitude is usually recorded most reliably, shows its maximum amplitude, and is least affected by artifact in the posterior electrodes. P300 amplitude at the Pz electrode is correlated with amplitudes at other locations. The P300 amplitude variable was adjusted for sex and age using linear regression prior to linkage analysis. To evaluate the effect of smoking status on P300 amplitude, we stratified individuals into smoking and non-smoking subgroups using the COGA variable habitual smoker. To evaluate the effect of alcoholism on P300 amplitude, we stratified individuals into alcoholism and non-alcoholism subgroups, using the ALDX2-derived binary variable described above.1m and \u03b21p, are estimated, as measures of the recurrence risk ratio, \u03bb1, where \u03bb1m = exp(\u03b21m) is the recurrence risk ratio for a sib pair that shares exactly one allele IBD, the maternal allele. Testing for a parent-of-origin effect involves testing whether \u03b21m = \u03b21p. SIBPAL performs linear regression-based modeling of sib-pair traits as a function of marker allele IBD sharing. For quantitative traits, sib-pair regression-based linkage analysis was performed in SIBPAL using the weighted combination of squared trait difference and squared mean-corrected trait sum, further adjusted for the non-independence of sib pairs and the non-independence of squared trait sums and differences. SIBPAL models allow for binary and continuous traits, as well as inclusion of covariates and epistatic interactions. IBD sharing probabilities were estimated using GENIBD in S.A.G.E [p = 2.2 \u00d7 10-5 and p = 7.4 \u00d7 10-4 as thresholds for significant and suggestive linkage, respectively, as proposed by Lander and Kruglyak [p-values, for the regions with small nominal p-values from the asymptotic distribution. Under this test, we permuted the allele sharing  among the pairs (both within sibships and across sibships of the same size).Two methods of model-free linkage analyses were performed using the SIBPAL and LODPAL programs in S.A.G.E. . LODPAL  S.A.G.E . We chosKruglyak . We usedp = 2.0 \u00d7 10-5). No evidence for linkage was found using the MS genome scan. Adding P300 as a covariate, suggestive evidence for linkage was seen to 12q  or any other region. Restricting the analyses to only those relative pairs that had both MS and SNP genome scans had no significant effect on the results above.The dataset included 1,253 White, non-Hispanic individuals in 116 pedigrees, comprising 633 men and 620 women. MS marker data was available for 1,062 individuals, SNP data for 1,044 individuals. Model-free ARP linkage analysis was performed on chromosomes 1 through 22 using LODPAL. Using the binary trait for alcohol dependence, there was significant evidence for linkage to chromosome 10q using the SNP genome scan  and SNP  genome scans. Parameter estimates indicate a maternal effect, or increased maternal transmission at this locus .Interestingly, linkage analysis under a parent-of-origin model showed some evidence for linkage to this same region of 12q  analysis was performed using SIBPAL. Evidence for linkage was found on chromosome 7 using both MS markers , long implicated to have a role in alcohol dependence. Interestingly, linkage analysis with a parent-of-origin model also detects linkage to this same region on chromosome 12, suggesting the presence of an imprinted gene in this region influencing susceptibility to alcoholism.In this study the use of covariate-based linkage analysis and the inclusion of parent-of-origin effects identified possible linkage signals that would not have been detected otherwise, suggesting an important role of such strategies in the dissection of genetic heterogeneity in complex disease.ARP: Affected relative pairCOGA: Collaborative Study on the Genetics of AlcoholismIBD: Identity-by-descentMS: MicrosatelliteQTL: Quantitative trait linkageSNP: Single-nucleotide polymorphismJFB helped design the study, performed affected relative-pair and parent-of-origin linkage analyses, and drafted the manuscript. SREQ performed sib-pair linkage analyses and drafted the manuscript. ARP performed IBD calculations. KABG designed and coordinated the study and helped draft the manuscript. All authors read and approved the final manuscript."}
+{"text": "Elevated blood pressure in middle age is a major risk factor for subsequent cardiovascular complications. An important longitudinal characteristic of blood pressure is the \"tracking phenomenon\". Tracking is defined as the persistence of the rank of a person's blood pressure level in a group over a long period of time. In this analysis, we used the Framingham data to investigate whether there are some genes responsible for this phenomenon.Both two-point and multipoint linkage analyses were applied to family members with complete data only and to all family data with missing values imputed by a Gaussian model. The results of two-point linkage analysis indicated that two loci for linkage with the intercept were on chromosomes 10 and 13, and two loci for linkage with both slope and intercept were on chromosomes 1 and 3. Multipoint linkage analysis indicated only one region, 200\u2013240 cM on chromosome 1, to be linked with both intercept and slope. For the intercept of SBP, the highest LOD (4.43) was found at 214 cM when missing data were imputed, and the highest LOD (2.81) was at 231 cM for the complete case data. For the slope of SBP, the highest multipoint LODs were 3.63 at 227 cM and 2.02 at 234 cM for the complete case data and imputation data, respectively.One or more genes in the range of 200\u2013240 cM on chromosome 1 may be related to the tracking phenomenon of SBP. Elevated blood pressure in middle age is a major risk factor for subsequent cardiovascular complications, such as congestive heart failure, left ventricular hypertrophy, stroke, renal dysfunction, and renal failure. An important longitudinal characteristic of blood pressure is the \"tracking phenomenon\". Here, tracking is defined as the persistence of the rank of a person's blood pressure level in a group over a long period of time. A large number of longitudinal studies have provided evidence of \"tracking\" of systolic blood pressure (SBP) . The traSeveral genome-wide scan studies in which cross-sectional SBP was used as the phenotype have been reported in the literature. The Framingham Heart Study, which began in 1948 and characterized SBP in a two-generation cohort, provides a unique opportunity to detect risk factors of SBP via longitudinal traits. In this study, a two-stage approach was used to detect genes related to the tracking of SBP. First, two phenotypes, intercept and slope, were derived using a mixed model to represent the initial value and the rate of increase in SBP. Second, both two-point and multipoint linkage analyses were conducted using these phenotypes.A mixed model was fitted to both the original and offspring cohorts as follows:Yi = Xi \u03b2 + Zibi + \u03b5i (1)+ \u03b5i(2)bi ~ N i (1)~ N  \u00a0\u00a0\u00a0 (1)\u03b5i (2)~ N \u03b5b1,...,bN,\u03b5(1)1,...,\u03b5N(1), \u03b5(2)1,...,\u03b5N(2) independent,Yi is the vector of ni SBP measurements  for the ith individual, each element of this vector being one observation in a time sequence for the individual i:Xi and Zi are matrices of covariates; \u03b2 is a parameter vector representing fixed effects; bi is a parameter vector representing random effects; \u03b5i (1)is an extra component of measurement error, and \u03b5i (2)is a component of error due to serial correlations.where In this analysis, all individuals having at least three SBP measurements were selected from the 330 families in the Framingham data. There were 15,509 observations from the original cohort and 6155 observations from the offspring cohort. Because individuals in the different cohorts might be different in many respects, the model was fitted to the original cohort and offspring cohorts, respectively.Assuming that each individual profile could be approximated by a linear function over age with subject-specific intercept as well as slope, our final model is:ij = \u03b21M + \u03b22F + \u03b23BMI + (\u03b24M + \u03b25F + \u03b26BMI) age + b0i + b1i age + \u03b5(1)i + \u03b5(2)i, \u00a0\u00a0\u00a0 (2)YYij is the jth observation of individual i, M is a binary variable for male, F is a binary variable for female, bi 0is the intercept, and bi 1is the slope. Thus bi 0and bi 1were defined as intermediate quantitative phenotypes for individual i. PROC MIXED in SAS\u00ae was used to fit this model assuming an unstructured covariance D and a Gaussian serial correlation H.where Because of the various treatments and different drug responses in patients, we decided not to use any blood pressure values recorded after the subject started to receive treatment. In this analysis, observations after the start of hypertension treatment were coded as missing.\u00ae. The process was conducted separately for the original and the offspring cohorts.The mixed model was fitted to both the complete case data, i.e., only individuals without missing values, and the data that included both complete and imputed cases. Because of missing values, 8988 observations in the original cohort and 41 observations in the offspring cohort were excluded in the complete case data. Imputation was based on the Gaussian model for quantitative variables, which was fitted by the EM algorithm  implemenMendelian inconsistencies and the relationships were checked using the S.A.G.E. programs MARKERINFO and RELTEST. Heritability of the intercept and slope were estimated using SOLAR.Two-point and multipoint linkage analyses were conducted using a model-free approach. The SIBPAL program in S.A.G.E. was used for two-point linkage analysis. This program modeled the mean corrected cross product trait data from full sib pairs as a function of marker allele sharing identical by descent (IBD) . The mulIn both cohorts, the mean SBP increases with age for both males and females Figure . The meaTo verify the tracking of SBP in the Framingham population, Pearson correlation coefficients among the different age groups were evaluated. All the correlation coefficients were statistically significant Table .p < 0.0001) and 0.406  for the intercept and slope, respectively.The heritability of SBP was 0.321 and chromosome 13 (p < 0.001). The most interesting result was that linkage with both the slope and intercept was detected on chromosome 1 and on chromosome 3   or hypertension (a dichotomous trait). Because blood pressure changes with time, a trait which can include longitudinal information is better suited to linkage analysis than a trait that only incorporates cross-sectional information.In order to derive this kind of intermediate phenotype for SBP, we noticed that the \"tracking phenomenon\" is a longitudinal characteristic of SBP. There is general agreement that tracking exists among different populations. In our analysis, all Pearson correlation coefficients were statistically significant among the different age groups, suggesting tracking also exists in our study population.This phenomenon involves the initial value as well as the change of SBP over time, so both the intercept and slope in the random effect components of a linear mixed model were used. Because the mixed model was subject-specific, it was able to characterize individual behavior through the best linear unbiased predictor. In addition, this model made use of the prior information that phenotypes are correlated between time points, so it was considered more efficient than a standard regression model.We hypothesized that genes play a role in the \"tracking phenomenon\". To test this hypothesis, the heritability of the intercept and slope were first estimated. The heritability of each trait was statistically significant, suggesting further linkage analysis would be worthwhile. To avoid false-positive results, linkage analyses were conducted based on both the Haseman-Elston model and the variance component model.One challenge in the analysis was how to deal with the data when subjects had received treatment. The adjustment for complex diseases like hypertension is difficult, and can even be misleading, with bias that cannot be estimated. In this analysis, SBP values after receiving treatment were considered as missing, although this approach takes the risk of losing some information.Another challenge arose from the large number of missing values. Two methods, complete case analysis and model-based imputation analysis, were used. Complete case analysis was simple but, in theory, less efficient than the imputation analysis, which assumed a distribution for all the data together, both the missing and observed data. From the result of our multipoint linkage analysis, the imputation appears to be more efficient. However, its efficiency was not shown in the two-point linkage analysis. This implies that imputation is not always efficient in linkage analysis. An inappropriate imputation model may result in more bias than the complete case analysis. In our analysis, the results from complete case data and imputation data were consistent, implying that our imputation model did not introduce large extra bias.Our analysis was actually a two-stage approach, in which the pedigree structure was not considered at the first stage when a mixed model was fitted. An approach that simultaneously fits the mixed model and the linkage model ought to be more efficient. To fulfill this goal, further extension of the mixed model and the imputation model are needed for longitudinal pedigree data .In conclusion, our results provide evidence for our hypothesis that there might be gene(s), specifically in the region of 200\u2013240 cM on chromosome 1, related to the \"tracking phenomenon\" of SBP. If there is only a single causative gene in this region, then it has a pleiotropic effect on both the intercept and slope of SBP. The correlation between the intercept and slope might make contribution to the consistency, but this kind of consistency on this region was not found on other chromosomes, implying that it was unlikely to be the only cause. This result provides a focus for further study, including fine mapping based on linkage disequilibrium and the evaluation of functional consequences of genes in this region.Our results also suggested there are two other loci linked only with the intercept of SBP, suggesting that hypertension is heterogeneous and that genes for either the intercept or the slope also play roles in some cases of hypertension."}
+{"text": "Our Markov chain Monte Carlo (MCMC) methods were used in linkage analyses of the Framingham Heart Study data using all available pedigrees. Our goal was to detect and map loci associated with covariate-adjusted traits log triglyceride (lnTG) and high-density lipoprotein cholesterol (HDL) using multipoint LOD score analysis, Bayesian oligogenic linkage analysis and identity-by-descent (IBD) scoring methods. Each method used all marker data for all markers on a chromosome. Bayesian linkage analysis detected a linkage signal on chromosome 7 for lnTG and HDL, corroborating previously published results. However, these results were not replicated in a classical linkage analysis of the data or by using IBD scoring methods.We conclude that Bayesian linkage analysis provides a powerful paradigm for mapping trait loci but interpretation of the Bayesian linkage signals is subjective. In the absence of a LOD score method accommodating genetically complex traits and linkage heterogeneity, validation of these signals remains elusive. The aim of our analyses was to detect and localize trait loci associated with quantitative traits logtriglyceride (lnTG) and high-density lipoprotein cholesterol (HDL) in the Framingham Heart Study data. Based primarily on Shearman et al. , we focuWe first did routine analysis and investigation, removing uninformative individuals. Based on data availability, we decided for later analyses to use time point 11 for Cohort 1 and time point 1 for Cohort 2. For a few covariates unavailable at time point 11 for Cohort 1, the information was taken from time point 10 or 12. The program ECLIPSE was used to investigate pedigree uncertainties or errors on the basis of all available marker data. We also estimated sex-averaged and sex-specific recombination frequencies via an expectation maximization (EM) algorithm based on the MCMC program lm_auto in the MORGAN package (URL information below).We analyzed quantitative traits using the MORGAN EM program PolyEM for fitting multivariate polygenic models. We used the phenotypic traits and covariates shown in Table Two approaches were used to obtain models for HDLAA to be used in linkage analysis. Loki , the BayOur linkage studies focused primarily on chromosome 7. Linkage signals have been reported on chromosome 7 for lnTG, HDL, and log(HDL/TG) ,2, whichWe used Loki to analyze several quantitative traits based on HDL and lnTG. The binary HDLA and HDLAA traits were subjected to IBD scoring linkage detection methods based on lm_auto . We usedAn ECLIPSE analysis of putative sib trios identified individual 590513 in pedigree 27096 as being an unlikely member of the stated sibship. Whereas true sib trios give log-likelihood differences in the range 40 to 80 relative to half-sib alternatives, trios including this individual gave values close to 0. This pedigree has substantial missing marker data, suggesting there may have been Mendelian errors. The data set that was left after removing 35 individuals with no data, 593 unobserved founder couples each with only one offspring, and the individual 590513, consisted of 3470 individuals in 362 pedigree components, ranging in size from 1 to 74. Two pedigrees contained loops due to sibship exchanges, and several had more than one founder couple. Our segregation and joint linkage/segregation analyses used the reduced data set of 3470 potentially informative individuals. Genome sharing, map re-estimation, and LOD score methods used the subset of 3444 individuals in non-singleton pedigrees.Re-estimated maximum likelihood recombination frequencies were obtained for chromosome 7. The genetic distance from marker 7_1 to marker 7_22 increased from 191 cM (given) to 205 cM (estimated). Overall, there were few large differences between the given and estimated sex-averaged and sex-specific maps. Marker intervals 7_13\u201314, 7_18\u201319 and 7_19\u201320 showed the highest relative increase in sex-averaged recombination rates: 37%, 45%, and 58%, respectively.The results of two polygenic segregation analyses are shown in Table Bayesian analyses using Loki detected one main signal on chromosome 7 around marker 7_21. Figure The strength of Loki signals was sensitive to the prior distribution assumed for QTL effects. It was also sensitive to changes in the marker genetic map: the MCMC EM estimated map resulted in reductions in the peak IR, relative to the supplied map, of 11.6% and 17.3% for HDLA and lnTGA, respectively. As shown in Figure Using the IBD S-pairs scoring statistic of , lm_autoUsing penetrances based on commingling and Loki analyses, LOD scores for the \"very-high\" binary and ordinal HDLA and HDLAA traits were computed using lm_bayes Figure . AlthougIn combination, our segregation and linkage analysis results suggest both oligogenic inheritance of HDLA and lnTGA, and a negative genetic correlation, which may be the result of loci affecting these traits at chromosome 7-qter. The chromosome 7 signals were consistently stronger for HDLA than for HDLAA, the latter trait being adjusted for lnTG. For genetically correlated traits, adjustment may weaken the signal, whereas the ratio-trait of Shearman et al.  will reiThe weak signals of the model-free analyses of binary traits may be due to low power, and the problems of the multipoint LOD score analyses due to model sensitivity. In the presence of oligogenic inheritance, Loki can detect weak signals, imputing linked QTL only in certain families and modeling other heritable variation with unlinked QTL. However, interpretation of the strength of the signal provided by Loki remains an open question. Thus, in the absence of a LOD score method accommodating genetically complex traits and linkage heterogeneity, validation of these signals remains elusive."}
+{"text": "Previous genome scan linkage analyses of the disease Kofendrerd Personality Disorder (KPD) with microsatellites led to detect some regions on chromosomes 1, 3, 5, and 9 that were identical for the three populations AI, KA, and DA but with large differences in significance levels. These differences in results may be explained by the different diagnosis definitions depending on the presence/absence of 12 traits that were used in the 3 populations AI, KA, and DA. Heterogeneity of linkage was thus investigated here according to the absence/presence of each of the 12 traits in the 3 populations. For this purpose, two methods, the triangle test statistic and the predivided sample test were applied to search for genetic heterogeneity. Three regions with a strong heterogeneity of linkage were detected: the region on chromosome 1 according to the presence/absence of the traits a and b, the region on chromosome 3 for the trait b, and the region on chromosome 9 for the traits k and l. These 3 regions were the same as those detected by linkage analyses. No novel region was detected by the heterogeneity tests. Concerning chromosome 1, linkage analyses showed a much stronger evidence of linkage for traits a and b and for a combination of these traits than for KPD. Moreover, there was no indication of linkage to any of the other traits used to define the diagnosis of KPD. A genetic factor located on the chromosome 1 may have been detected here which would be involved specifically in traits a and b or in a combination of these traits. Different diagnosis definitions depending on the presence/absence of 12 traits were used in 3 populations . These diagnosis definitions were not precisely known in each population. Linkage analyses of the Kofendrerd Personality Disorder (KPD) with microsatellites . These aWe had no knowledge of the \"answers\" at the time we did the analyses. Three of the 4 regions detected by previous linkage analyses of KPD were also detected by the heterogeneity tests: the regions on chromosomes 1, 3, and 9 according to the presence/absence of the traits a and b, the trait b, and the trait a, respectively. No novel regions were detected by these tests. For the region on chromosome 1, linkage analyses showed a much stronger evidence of linkage for the traits a and b and for a combination of these two traits than for KPD.2, z1 and z0, respectively) given the observed marker genotypes. If a susceptibility gene is linked to the marker, the respective proportions z2, z1, and z0 are constrained within a triangle by: 2z0 \u2264 z1 \u2264 0.5, referred to as the triangle constraints . When likelihoods are estimated in the different groups without constraints, the PST follows a \u03c72 distribution with 2 df [c1), L(zc2) and L(zc) are the likelihoods of the parameter vector, Zc, estimated in respectively the concordant sib pairs for phenotype A, the concordant pairs for A' and the whole sample. Here, because the triangle constraints were applied to estimate the likelihoods and the parameter vector Z, the PST would not exactly follow a \u03c72 with 2-df distribution. We showed by bootstrap analyses that the PST distribution was close to a \u03c72 with 1-df distribution.The PST tests whether IBD distributions in different groups of sib pairs are similar. Let us consider two sub-phenotypes, A and A'. Under the null hypothesis of genetic homogeneity , the Zith 2 df  (number p \u2264 0.005 in the first set of families were followed up in the second set. Results were considered replicated if the same genomic regions was significantly detected (p \u2264 0.005) in the second sample for the same trait, the same population, and using the same method (TTS or PST). A genomic region was defined as including the marker with a maximum score in the first dataset and two adjacent markers at every side. For regions, traits, and populations with which genetic heterogeneity were detected, linkage analyses were conducted with the MLS method in the whole sample of subjects . All tests  were applied using all possible independent pairs in sibships  pairs per sibships with n affected sibs). Analyses with these 3 tests were conducted using multipoint information on the microsatellites.We conducted analyses to search for linkage heterogeneity using the information provided by the entire set of microsatellites covering the 10 chromosomes and 5 replicates of family samples (rep 1 to 5) for each of the 3 populations . These replicates were pooled for the analyses and led to a sample of 500 families for each population. This represents a reasonable size to apply tests of heterogeneity . The PST and TTS tests were applied on affected sib pairs to search for heterogeneity of linkage with KPD according to each trait consecutively. More precisely, the PST was applied on affected sib pairs concordant for the presence or the absence of the trait and the TTS on affected sib pairs discordant for the trait, i.e., one sib had the trait, the other did not. Only 7 traits  were considered because among affected subjects, some of the traits  were always present and some  were always confounded with c. To account for problems of multiple tests increasing type I error, we repeated genetic heterogeneity analyses for replications in a second set of 500 families (5 pooled replicates: rep 6 to 10). Only the populations, genomic regions, and traits that were found significant at a p \u2264 0.005) simultaneously in the two sets of data for the same trait in the same population using the PST test (see Table p \u2264 0.005) but not with the same methods and in the same populations for the two data sets: with TTS for the first set in DA and with PST in AI for the second set.Three chromosomal regions led to a genetic heterogeneity  in 60 affected sib-pairs with the trait a vs.  in the 496 affected sib pairs without this trait. The IBD distributions were equal to  and , respectively, in the 358 and 148 affected sib pairs with and without trait b in the same population and set of data. In the absence of trait b, there was thus no longer IBD distortion from the IBD distribution expected under no linkage. Because traits a and b appeared to be important for the detection of linkage, we conducted separate and combined linkage analyses of these two traits in the whole sample of subjects (affected or not). We then considered the following phenotypes for linkage analyses: presence of trait a, presence of trait b, presence of both traits a and b, presence of trait a and/or of trait b and KPD. We conducted linkage analyses of these phenotypes in the first and second sets of data of the population AI (population in which the strongest heterogeneity was detected for traits a and b). Results are presented in Table p-value \u2264 0.005 according to the presence/absence of the trait a: with the TTS in DA in the first set, and with the PST in AI for the second set.In agreement with our results, the underlying model included a locus on chromosome 1 involved in both traits a and b, in affected and unaffected subjects. Concerning the linked region on chromosome 3, we found a significant heterogeneity according to the presence/absence of trait b in affected subjects. The underlying model indeed assumed a locus located in this region, involved in trait b and a combination of this trait with other traits, in affected subjects. For the linked region on chromosome 9, we found heterogeneity according to the presence/absence of traits k and l, which indeed depend on a locus located in this region according to the underlying model. The region in chromosome 5, which included a locus involved in trait a and in trait b combined with other traits, was not detected here with our criteria. However, heterogeneity of linkage was suggested with a Our analyses with the heterogeneity tests have permitted the detection of 3 of the 4 genetic factors, the 3 located on chromosomes 1, 3, and 9 and moreover to determine which of the 12 traits depend on these genetic factors. For example, we have detected here a genetic factor on chromosome 1 in the region of markers 23\u201325 that is not specifically involved in KPD disease, but which is involved in only some traits used to define the disease: traits a and b. Moreover, the much stronger detection of linkage between traits a and b with the chromosome 1 region than the one detected with KPD illustrates well the importance of the diagnosis definition in linkage analysis for the detection of linkage.IBD: Identical by descentKPD: Kofendrerd Personality DisorderMLS: Maximum Likelihood scoreNPL: Nonparametric linkagePST: Predivided sample testTTS: Triangle test statisticMHD conceived the design of the study, participated in the analysis and the interpretation of results, drafted the manuscript. EG carried out the heterogeneity test analyses and participated in the interpretation of results and the draft of the paper. MCB and CB participated in the analysis."}
+{"text": "Genome-wide association will soon be available to use as an adjunct to traditional linkage analysis. We studied alcoholism in 119 families collected by the Collaborative Study on the Genetics of Alcoholism and made available in Genetic Analysis Workshop 14, using genome-wide linkage and association analyses.Genome-wide linkage analysis was first performed using microsatellite markers and a region with the strongest linkage evidence was further analyzed using single-nucleotide polymorphisms (SNPs). Family based genome-wide association test was also conducted using the SNPs.Nonparametric linkage analysis revealed weak linkage evidence on chromosome 7, and association analysis identified SNP tsc0515272 on chromosome 3 as significantly associated with alcoholism.Linkage analysis may require large sample sizes and high quality genotyping and marker maps to adequately improve power, while association analysis could hold more promise in efforts to identify variants responsible for complex traits. Alcoholism is a complex trait affected jointly by genetic components and environmental factors. Linkage and association are often used to search for the responsible genetic variants of a complex trait. It is believed that association analysis has more power than linkage analysis in the genetic dissection of complex traits such as alcoholism, providing that strong linkage disequilibrium is present between a testing marker and the disease locus . BecauseThe COGA dataset included 1,294 White individuals in 119 families. These individuals were enrolled for a linkage and association study. We selected ALDX1 as the phenotype. ALDX1 has five categories: 0: no information; 1: pure unaffected; 2: never drank; 3: unaffected with some symptoms; 5: affected. Fourteen individuals are classified in group 2 (never drank). In our analysis, we then defined 5 as affected, 1 and 2 as unaffected, and the remaining as unknown. The analysis results of coding 2 as unknown were essentially the same as that of coding 2 as unaffected. Our data then consisted of 528 affected individuals, among them, 487 offspring. The data also included 315 microsatellite markers evenly spaced across the genome with average marker distance of about 10 cM. There are also 10,081 single-nucleotide polymorphisms (SNP) across genome genotyped using GeneChip Mapping 10 K Array marker set of Affymetrix Inc.ALL statistic [Both single- and multipoint genome-wide nonparametric linkage (NPL) analyses were performed and the Statistic  was usedtatistic . We usedtatistic . We thenWe next performed family-based association testing (FBAT) by applying the program FBAT using the SNP . The metALL statistic suggested by Sengul et al. [p = 0.00027). We also observed five additional markers on chromosome 7 with LOD scores \u2265 1.0. The linkage information for a single marker was lower than multiple markers. We then conducted multipoint linkage analysis and the results were generally consistent with the single-point analyses (Table p = 0.002), 13 cM away from marker D7S820. Although the linkage information was improved in multipoint analysis, the observed LOD scores were sometimes lower than the single-point analyses. This is perhaps due to the fact that multipoint linkage analysis is sensitive to genotyping errors and map misspecification [We first performed single-point NPL analysis  using SAl et al. . The LODl et al. . Table 1p-values of the test statistic on each SNP were calculated based on the empirical variance, as described in Lake et al. [p < 0.01). These SNPs were excluded from further analyses. We observed 670, 167, and 19 SNPs with p-value less than 0.05, 0.01, 0.001, significantly exceeding 457, 91, and 9 SNPs expected under the null hypothesis of no association or linkage, suggesting true association and linkage between SNP and alcoholism. Table p-values less than 0.001. Interestingly, only two associated SNPs (tsc0668988 and tsc1177811 on chromosome 1) were close to the region where weak linkage evidence was observed. SNP tsc0515272 on chromosome 3 showed the most significant association and linkage evidence to alcoholism  and was close to genome-wide significance . For the further comparison with the linkage result on chromosome 7, we also listed the 5 SNPs with nominal p-values less than 0.01 in the association analysis. All the 5 SNPs were located at least 28 cM away from the linkage peak.We then performed genome-wide association using FBAT on the SNP data. The e et al. . The proWe conducted genome-wide linkage and association analyses using microsatellite markers and SNPs on the data provided by GAW14. Both single- and multipoint NPL analyses showed suggested linkage evidence on chromosome 7. We could not replicate the linkage evidence on chromosome 3 (LOD = 0.37 at 71 cM) that was reported by Foroud et al. . HoweverIn contrast, association analysis may hold great promise in the genetic dissection of complex traits. In this study, we observed genome-wide significant evidence of SNP tsc0515272 associated with alcoholism after Bonferroni correction for multiple comparisons. Such a correction is usually conservative because of existence of linkage disequilibrium between SNPs located close to one another. Interestingly, we did not observed consistent results between linkage and association analyses. For example, we did not observed significant association evidence for SNPs under the linkage peak on chromosome 7. A possible reason is that the SNP genotypes in this study are still not able to capture all of the genetic variation in this region. Theoretical studies suggest that 250,000\u2013800,000 SNPs are required for a genome-wide association study ,11. SomeCOGA: Collaborative Study on the Genetics of AlcoholismFBAT: Family-based association testGAW14: Genetic Analysis Workshop 14NPL: Nonparametric linkageSNP: Single-nucleotide polymorphism"}
+{"text": "Study design strategies are of critical importance in the search for genes underlying complex diseases. Two important design choices in planning gene mapping studies are the analytic strategy to be used, which will have an impact on the type of data to be collected, and the choice of genetic markers. In the present paper, we used the simulated behavioral trait data provided in the Genetic Analysis Workshop14 to: 1) investigate the usefulness of incorporating unaffected sibs in model-free linkage analysis and, 2) compare linkage results of genome scans using a 7-cM microsatellite map with a 3-cM single nucleotide polymorphisms map. To achieve these aims, we used the maximum-likelihood-binomial method with two different coding approaches. We defined the unaffected sibs as those totally free of phenotypes correlated to the disease. Without prior knowledge of the answers, we were able to correctly localize 2 out of 5 loci (LOD > 3) in a sample of 200 families that included the unaffected sibs but only one locus when based on an affected-only strategy, using either microsatellite or SNPs genome scan. LOD scores were considerably higher using the analytic strategy which incorporated the unaffected sibs. In conclusion, including unaffected sibs in model-free linkage analysis of complex binary traits is helpful, at least when complete parental data are available, whereas there are no striking advantages in using single nucleotide polymorphisms over microsatellite map at marker densities used in the current study. The maximum-likelihood-binomial (MLB) approach is one such method, and unlike other sib-pair approaches, it analyzes sibships of arbitrary size as a whole. In the context of a proposed strategy to account for covariates in the MLB approach, Alca\u00efs and Abel have shown an increase in the power to detect a susceptibility locus when making use of the information carried by unaffected sibs . The valTo date, genome scans of complex traits have been performed using a set of 300\u2013400 microsatellite markers (MS) evenly spaced (~10 cM) across the genome. More recently, single nucleotide polymorphisms (SNPs) have emerged as attractive alternative tools to conduct genome scans of complex traits, mainly motivated by their more rapid and highly automated genotyping as compared to MS. The merit of SNPs in the context of a genome-wide scan of complex traits is relatively undocumented. One recent study comparing MS with SNPs whole-genome scans concluded in favor of SNPs, mainly because of the more refined position of loci .The purpose of our study is twofold: first, to investigate the effects of an alternative sibship design, which uses both affected and unaffected sibs, and to compare it with the frequently used affected-only design; and second, to compare genome-wide scan linkage results obtained from MS with SNPs.We used the simulated behavioral trait data generated in the context of the Genetic Analysis Workshop 14 (GAW14). The analyses were performed without any knowledge of the answers. For the present paper, the data from the first two replicates of the simulated population \"Aipotu\" were used (REP001 and REP002). In order to increase the power to detect linkage while maintaining a realistic sample size, the two replicates were combined for these analyses. The combined replicate data included 200 nuclear families. Details on the distribution of the families by number of sibs according to affection status are presented in Table The affected sibs were those with the diagnosis of the simulated behavioral trait, Kofendrerd Personality Disorder (KPD). The unaffected sibs were those not only without KPD, but also free of the 12 phenotypes associated with KPD. There was no difference in affection status according to sex or age and therefore, no covariate adjustment for these variables was done.2 distributions with 0 and 1 degree of freedom; the statistic is usually expressed as a LOD score, which has exactly the same distribution as a classical model-based LOD score estimating the recombination fraction parameter [Model-free two-point and multipoint genome scan linkage analyses were performed using the MLB method , as implarameter ,7. The lFigure The multipoint MLB-binary analysis identified one region with evidence of linkage as defined by a LOD score > 3.00, both in the MS and the SNPs scan. The peak signal is on chromosome 3 at D3S127 with a LOD score of 3.73 in the MS scan, and 3.40 at C03R0278 with the SNPs scan. Suggestive evidence of linkage is reported on chromosome 5, with a peak multipoint MLB-binary LOD score of 2.60 at D5S172 (MS) and of 2.39 at D05R0380 (SNPs). No MLB-binary LOD score above 3.00 was identified in the two-point analyses (data not shown).Using both affected and strictly unaffected individuals  leads to a marked increase in the LOD score for the signals on chromosome 3 and chromosome 5. The multipoint MLB-categorical LOD score is 6.37 at D3S217, the same marker as in the MLB-binary analysis, and 4.76 at C03R0280, a marker in the region identified in the MLB-binary analysis. The signal on chromosome 5 now provides evidence for linkage with peak MLB-categorical LOD scores of 3.67 at D5S172 (MS) and 3.14 at C05R0380 (SNPs). The increase is marked enough to be picked up as evidence for linkage even in the two-point analyses (data not shown). The peak two-point MLB-categorical LOD scores were 4.61 and 3.46 for D3S127 and D5S172, respectively. None of the two-point LOD scores for the SNPs markers were over 3.00. The higher LOD scores observed for the MS scans are likely due to the higher information content for MS vs. SNPs Figure . For exap-value = 0.74). Therefore, the LOD scores for these two markers are more likely independent. In a second step, we investigated putative locus heterogeneity. For this, we identified the families not contributing positively to the LOD score at D3S127, i.e., the marker providing the highest LOD score in the genome scan analysis. There were 106 such families. We then conducted two additional MS genome scans, one that included the affected-only and another that included the affected-unaffected, of the \"unlinked\" families. In the multipoint MLB-binary analysis, only one LOD score above 2 (LOD 2.43 at D5S203), provided some suggestive evidence for linkage. No other LOD scores, MLB-binary or -categorical, were above 2.00.To further investigate the two loci localized, we first aimed at identifying evidence for putative epistasis affecting the major loci identified. For this, we computed the correlation between the LOD scores at markers D3S127 and D5S172 in the 200 nuclear families. The correlation is 0.02 and non-significant , simulated chromosome 3 and 5 loci, with both MS and SNPs genome scans. Using a looser criterion, that is a LOD score > 1, we identified additional loci (chromosome 1 and 9 loci) with no false positives for the MS scan, but at the cost of three false positives for the SNPs scan (data not shown). The locus on chromosome 10 was missed by both MS and SNPs scans.GAW: Genetic Analysis WorkshopKPD: Kofendrerd Personality DisorderMLB: Maximum-likelihood binomialMS: MicrosatellitesSNP: Single-nucleotide polymorphism"}
+{"text": "This Genetic Analysis Workshop 13 contribution presents a linkage analysis of hypertension in the Framingham data based on the posterior probability of linkage, or PPL. We dichotomized the phenotype, coding individuals who had been treated for hypertension at any time, as well as those with repeated high blood pressure measurements, as affected. Here we use a new variation on the multipoint PPL that incorporates integration over the genetic model. PPLs were computed for chromosomes 1 through 5, 11, 14, and 17 and remained below the 2% assumed prior probability of linkage for 73% of the locations examined. The maximum PPL of 4.5% was obtained on chromosome 1 at 178 cM. Although this is more than twice the assumed prior probability of linkage, it is well below a level at which we would recommend committing substantial additional resources to molecular follow-up. While the PPL analysis of this data remains inconclusive, Bayesian methodology gives us a clear mechanism for using the information gained here in further studies. The posterior probability of linkage, or PPL, has several advantages over other parametric methods . First, Here we exploit a fourth feature of the PPL, which allows us to integrate over the parameters of the trait model, resulting in a statistic that is model-free in the sense that it does not fix trait parameters at specific values. Thus, we are able to allow for heterogeneity, reduced penetrance, and phenocopies. Additionally, using integration in this way allows us to avoid the inflationary effects on the likelihood of maximizing over multiple parameters. This makes the model-integrated PPL an ideal tool for the analysis of complex diseases such as hypertension.This also represents the first analysis using a model-integrated version of the PPL based on multipoint likelihoods. The multipoint PPL gives us an indication of whether or not there is a disease gene close to each position on the chromosome , in contWhen analyzing the Framingham Heart Study data set, two issues needed attention. First, it was necessary to establish a dichotomous phenotype definition that reliably captured the information from the multiple measurements. Our phenotype definition combined the \"treated for hypertension\" variable and the \"high blood pressure\" variable. An individual treated for hypertension at any time point was called affected. Of those not treated for hypertension, individuals having high blood pressure at four or more time points were coded as affected; the majority (57%) of people who had four or more high blood pressure readings were also treated for hypertension at some point. Of the people who had three or fewer high blood pressure readings, those who had fewer than eight recorded measurements were coded as unknown. Those with eight or more measurements were coded as unaffected. Individuals with no history of treatment and no blood pressure measurements were coded as unknown. This definition was intended to minimize the number of misclassified individuals by coding those without a clear propensity for high or normal blood pressure as unknown. was unable to use 60 due to their structure. Therefore, we split these pedigrees into their constituent nuclear families using MEGA2 ; \u03b1 is the admixture parameter [g is a vector of parameters that describe the genetic model . The HLOD is the multipoint heterogeneity (admixture) LOD score [t, \u03b1 and g. The prior distributions for g and \u03b1 are represented by h(g) and h(\u03b1).where arameter ; and g iOD score  as a funh(|t - t0|) on the disease gene location is used to summarize the probability that a risk locus is close to each position. This prior is constructed to mirror the one used in the two-point case, and places positive probability at all points within 44 cM, with 95% of the probability concentrated within \u00b1 5 cM [HLOD) is computed at each position to approximate the integration of the likelihood surface over the genetic model. A 2% prior probability was assumed [The moving-window prior n \u00b1 5 cM . Multipon \u00b1 5 cM , varying assumed .73% of the PPLs were below the prior probability of linkage. The maximum PPL of 4.5% was observed on chromosome 1 at 178 cM. The next highest PPL of 4.2% was achieved on chromosome 3 at 135 cM. The PPL was below 4% on the remaining chromosomes. The graphs of the PPLs for chromosomes 1 through 5, 11, 14, and 17 are presented in Figure At this point we have no compelling evidence for a hypertension-predisposing gene on any of the chromosomes we examined. Our largest PPLs of 4.5% and 4.2% were observed on chromosomes 1 and 3. A PPL of 4% indicates a location that is twice as likely to be linked to the trait being studied than one chosen at random. However, this is well below the level at which we would suggest committing substantial additional resources to fine mapping or molecular work.Overall, PPLs obtained do not differ much from the assumed prior probability of linkage. This indicates that we have little evidence for or against linkage across the genome. The PPL has the property that as the amount of genetic information increases the PPL converges to 1 under linkage and 0 under no linkage . One exp"}
+{"text": "Genome-wide scan data from a community-based sample was used to identify the genetic factors that affect body mass index (BMI). BMI was defined as weight (kg) over the square of height (m), where weight and height were obtained from the first measurement available between the ages of 40 and 50 years.p < 0.05) of loci to BMI at 23 markers. Multi-point sib-pair regression analysis provided nominal evidence for linkage to BMI at 42 loci on 12 chromosomes. Empirical p-values showed results consistent with the multi-point results; all but three of the loci identified by multi-point analysis were also significant.Significant familial correlations were observed in mother:father (spouse) relative pairs and in all relative pairs examined except parent:daughter pairs. Single-point sib-pair regression analysis provided nominal evidence for linkage (The largest regions of nominally significant linkage were found on chromosomes 2, 3, and 11. The most significant evidence for linkage was obtained with markers D2S1788, D2S1356, D2S1352, D3S1744, and D11S912 from multi-point sib-pair single-trait regression analysis. Our results are in agreement with some of the recently published reports on BMI using various data sets including the Framingham Heart Study data. Obesity is a major risk factor for morbidity and mortality from many chronic diseases including CVD [Data from the ongoing NHLBI-supported Framingham Heart Study (FHS) of factors that contribute to cardiovascular disease (CVD) was made available to the Genetic Analysis Workshop 13 (GAW13). The FHS has collected physical exam and lifestyle information from a community-based sample that has identified major CVD risk factors, such as high blood pressure, cholesterol, smoking, and obesity ding CVD . Most stding CVD . To idending CVD -6, Pima ding CVD ,8, and Ading CVD ,9. In thp = 0.03) in a meta-analysis of studies performed on individuals with Caucasian-, African-, and Mexican-American ethnic ancestry [Recently two groups reported results of genome-wide linkage scans for BMI. Wu et al. found strong evidence for the presence of a quantitative trait locus (QTL) for BMI at 3q27  in an attempt to minimize the positive correlation of age on BMI.Subjects for this analysis were participants in the ongoing NHLBI-supported Framingham Heart Study (FHS), which included 5209 subjects in Cohort 1 who were recruited in 1948 and 5124 children of the original subjects and spouses of these children in Cohort 2 who were recruited in 1971. Selected data on these subjects were made available to GAW13. The available data included family histories on all FHS participants, phenotypic information including height and weight data, as well as results of genetic analysis of 401 polymorphic markers on chromosomes 1 to 22 in the largest families.2 and 26.54 kg/m2, respectively, differed significantly . We tested for significance of sex, cohort, and calendar year on BMI values across the entire data set of 2252 individuals. Sex was significant in all models while cohort was significant when included in models without calendar year. Calendar year was never significant (results not shown). Therefore sex and cohort were used as covariates in all linkage analyses. Our assumption was that sibs would be highly correlated for age at BMI and calendar year of BMI. We tested this assumption; this will be discussed later (see Discussion). Individuals without height and weight measurements between ages 40 to 50 years were removed from all analyses.The data investigated for phenotypic assessment and familial correlation in this analysis were obtained from Cohort 1 and Cohort 2 and included the BMI calculated from the height and weight information provided by the FHS. The first recorded height and weight measurement between the ages of 40 and 50 years for each subject was used for the analysis. BMI was calculated by dividing weight in kilograms by the square of height in meters. The average BMI at ages 40 to 50 for individuals in Cohorts 1 and 2, 25.70 kg/mp-values for all the markers were calculated using the SIBPAL program from S.A.G.E. 4.2. The covariates sex and cohort were included in the regression models.GAW13 phenotypic and genotypic data were imported into a Microsoft Access database and exported in appropriate files for familial correlation and linkage analysis in S.A.G.E.  4.2 . FamiliaThe total number of individuals in the total FHS sample supplied to GAW13 is 4692, where 1702 have genotype information and 2252 have weight and height information. The total number of participants with BMI \u2265 30  is 248. Two hundred and forty-five individuals among the 1702 that were genotyped lacked data for BMI between the specified age range and were removed from all analyses. Eight individuals had BMI values that were 4 standard deviations above the mean (mean = 25.98). These eight outliers, all female with mean BMI of 47.5, were removed from all analyses.Familial correlation analysis was done using S.A.G.E. 4.2 (FCOR). Table p < 0.05) for linkage to BMI was found at 23 markers on 12 chromosomes  on chromosome 19q.A genome-wide linkage analysis was performed using the GENIBD and SIBPAL programs of S.A.G.E 4.2. Through single-point linkage analysis, nominal evidence   = 0.0003) on chromosome 11q  was plotted for all markers for chromosomes 2, 3, and 11 that showed the strongest evidence for linkage  was also plotted for all markers on chromosome 6 for comparison with the findings of Atwood et al. [To further evaluate the linkage results, -Log Figures . -Log10 with age at BMI as a covariate. We found that including age at BMI as a covariate did not affect the linkage results (data not shown).At least three other studies have suggested linkage of BMI to marker D3S2427 and/or other markers within that region of chromosome 3. Marker D3S2427, located on 3q27, was found to be strongly linked to BMI in the study by Wu et al. . In the Several groups have also reported evidence of linkage of BMI to markers on chromosome 2. Deng et al. found significant linkage of BMI with marker D2S347 on 2q14 at map location 131.51 cM . ComuzziOur results supporting nominal evidence of linkage to markers D11S4464, D11S912, and D11S2359 on chromosome 11 (123\u2013147.77 cM) are in agreement with reports by several groups. Hanson et al. reported strong evidence of linkage of BMI to markers D11S4464 and D11S912 at 136.6\u2013143.9 cM on chromosome 11q in Pima Indians [Our results also suggest possible linkage of BMI to markers D6S503 and D6S1027 on chromosome 6 (184.51\u2013187.23 cM) Table . Duggirap-values for all markers using the Framingham Heart Study data.We report evidence for nominally significant linkage of BMI to three regions on chromosomes 2, 3, and 11 by performing multi-point sib-pair single-trait regression analysis and calculating empirical"}
+{"text": "We present a method for using slopes and intercepts from a linear regression of a quantitative trait as outcomes in segregation and linkage analyses. We apply the method to the analysis of longitudinal systolic blood pressure (SBP) data from the Framingham Heart Study. A first-stage linear model was fit to each subject's SBP measurements to estimate both their slope over time and an intercept, the latter scaled to represent the mean SBP at the average observed age (53.7 years). The subject-specific intercepts and slopes were then analyzed using segregation and linkage analysis. We describe a method for using the standard errors of the first-stage intercepts and slopes as weights in the genetic analyses. For the intercepts, we found significant evidence of a Mendelian gene in segregation analysis and suggestive linkage results (with LOD scores \u2265 1.5) for specific markers on chromosomes 1, 3, 5, 9, 10, and 17. For the slopes, however, the data did not support a Mendelian model, and thus no formal linkage analyses were conducted. In the conventional epidemiology literature, much has been written about utilizing data in which measurement of quantitative traits are periodically taken from each subject over time -3. HowevIn this paper, we also adopt a two-stage modeling approach. In our first stage, we fit a linear regression of SBP on age to obtain subject-specific intercepts and slopes. This first-stage model includes adjustment for any time-varying covariates of interest, such as calendar year, body mass index, and hypertension treatment. Also estimated in this first stage are the subject-specific standard errors of the corresponding intercepts and slopes. The second stage analysis consists of a segregation analysis of the subject-specific intercepts from the first stage model, and a separate segregation analysis of the slopes. We claim that the standard errors from the first stage should be used in weighting the contribution of each subject in the segregation analysis, and we describe how this can be accomplished. Based on the results of the segregation analyses, we conducted a genome screen using parametric linkage analysis applied to all pedigrees in the Framingham Heart Study. We demonstrate increased LOD scores using the weighed analysis, compared with the analogous approach that does not use weights.ij denote the SBP of the ith subject at the jth study visit, Tij be the corresponding age of the subject at the visit, and let Xij denote a matrix of time-dependent covariates. We propose using a first-stage model of the formLet YYij = ai + bi (Tij - ) + X\u03b3'ij + eij, \u00a0\u00a0\u00a0 (1) is the overall mean age in the sample and eij are residuals, assumed to be independent and normally distributed with mean 0 and variance \u03c92. The goal of this first-stage model is to estimate the subject-specific intercepts (ai) and slopes on age (bi), and their corresponding standard errors. We denote these standard errors by sai for intercepts and sbi for slopes. Note that the intercepts have the interpretation as the predicted mean Y at age  when all X values are zero. We center any continuous X variables on their corresponding sample means to increase interpretability of the intercepts.where ai and slopes bi as continuous phenotype data in a genetic analysis. We first perform segregation analysis to determine the evidence for a Mendelian gene and to estimate the associated model parameters. For analysis of the intercepts, the penetrance model used in the segregation analysis has the formThe second-stage model utilizes the first-stage intercepts ai = \u03b1 + \u03b2Gi + Xi \u03b7'+ ei, \u00a0\u00a0\u00a0 (2)Gi is a covariate based on an unobserved major gene gi, and Xi is a matrix of time-independent covariates. An analogous model was used for the slopes. The residual ei is assumed to be normally distributed with mean 0 and variance (\u03c32 + sai2), where sai2 is the square of the first-stage standard error of the intercept, and \u03c32 is the between-subject residual variance to be estimated. Note that this variance expression has the effect of weighting each subject's contribution to the genetic analysis based on the precision (standard error) of their intercept estimate. We thus denote the use of this variance for ei as a 'weighted' analysis. Generally speaking, these first-stage standard errors will be smallest for those with many measurements, and with measurements at ages that span the overall average age . One could also perform an 'unweighted' analysis by assuming that the variance of ei was simply \u03c32, which would treat the intercepts for all subjects as equally informative.where To estimate the parameters of the above model, we maximized the likelihoodF indexes family, gF is a vector of unobserved major genotypes, and YF and XF are the trait and covariate data for family F. The parameters \u03a9 = {\u03b1, \u03b2, \u03b7, \u03c3 } are the parameters of the penetrance model, qA is the population frequency of the variant allele 'A', and \u03c4 = {\u03c4AA, \u03c4Aa, \u03c4aa} are the probabilities that a parent with the subscripted genotype transmits an 'A' to their offspring. Computation of the above likelihood requires use of the peeling algorithm [AA, \u03c4Aa, and \u03c4aa were treated as free parameters to be estimated. This general model was compared to the Mendelian models, in which \u03c4AA, \u03c4Aa, and \u03c4aa were constrained to their theoretical values of 1.0, 0.5, and 0.0, respectively. Likelihood ratio tests (LRTs) were used to compare the general model to the Mendelian models, and also to the no-major-gene model. We also computed Akaike's Information Criteria (AIC) for each model as -2(log-likelihood at the maximum likelihood estimator (MLE)) + 2(number of model parameters estimated). A lower AIC indicates a more parsimonious model.where the lgorithm ,6. We coYij in equation (1) is ln(SBPij). Only observations with age in the range 30 to 80 were utilized, to further linearize the relationship between ln(SBP) and age. The average age was  = 53.7. Time-dependent covariates defining Xij in equation (1) included body mass index (BMI), calendar year (CY), CY2, hypertension treatment (HRX), CY \u00d7 HRX, CY \u00d7 male, CY \u00d7 cohort, CY \u00d7 BMI, CY \u00d7 age, male \u00d7 age, and BMI \u00d7 HRX. The continuous variables BMI and CY were centered on their respective sample means, while HRX and male were indicators of treatment status and male sex, respectively. The CY2 term was included to account for observed nonlinearity between SBP and CY. The intercepts from the first-stage model have interpretation as the subject-specific mean ln(SBP) adjusted to a female, untreated person of average age (53.7 years) and BMI (26.3 kg/m2) in calendar year 1969.5. PROC MIXED in SAS, Release 8.2 , was used to fit the first-stage model and obtain person-specific intercepts and slopes, and their respective standard errors.The GAW13 data set of the Framingham Heart Study included a total of 4692 subjects, of which 1213 subjects provided longitudinal observation data from the first cohort, and 1672 subjects from the offspring cohort. The outcome variable of interest in this paper was systolic blood pressure (SBP). A natural log transform was used to linearize the SBP relationship with age; thus ai values) and 2787 person-specific slopes  were obtained from the first-stage analysis. These estimates were used as trait data in the second-stage segregation and linkage analyses. Covariates Xi in equation (2) included male sex and cohort, the latter an indicator of membership in Cohort 2. We fit the segregation and linkage models using a version of the Genetic Analysis Package , modified by one of the authors (WJG) to utilize sai2 (and sbi2) in a weighted analysis. As will be demonstrated below, a Mendelian model was supported for the intercepts, but not for the slopes. We therefore focused our linkage analysis only on the intercepts. We fixed the segregation-model parameters to their MLEs from the weighted analysis, and performed two-point LOD-score linkage analysis to estimate the recombination fraction (\u03b8) between g and each of 399 markers. Allele frequencies at each marker locus were fixed to the values provided with the data. For comparison, we also performed an unweighted linkage analysis, in which a segregation analysis was re-run without standard error weights, and these MLEs then used in linkage analysis.A total of 2883 person-specific intercepts . On the other hand, the remaining Mendelian models and the no-major-gene model could be rejected (p < 0.001 for each). The codominant model also provided the lowest AIC, again indicating that this model provided the best fit to the data. The estimated allele frequency from this model was qA = 0.31, translating into 48% of subjects with g = aa, 42% with g = Aa, and 10% with g = AA. Compared with subjects with g = aa, SBP was estimated to be 12% higher (exp(0.115)) for subjects with g = Aa, and 33% higher (exp (0.283)) for subjects with g = AA. Segregation analysis of the slopes, on the other hand, did not support evidence of Mendelian transmission (Table p < 0.001), and provided the lowest AIC. The estimate of the transmission parameter \u03c4AA was 0.0, far from its Mendelian expectation of 1.0.Segregation analysis of the intercepts supported a Mendelian codominant model, with strong evidence of a genetic effect Table . SpecifiGiven the findings in segregation analysis, linkage analysis was conducted only on the intercepts. The parameters of the segregation model were fixed to the values shown for the Mendelian codominant model in Table g = Aa or AA) that leads to some elevation in average SBP relative to genetically normal (g = aa) individuals. Subsequent analysis revealed modest evidence of linkage on chromosomes 1 (202 and 212 cM), 5 (40 cM), 9 (32 cM), 10 (125 cM), and 17 (100 cM). Levy et al. [Our segregation analysis indicates that SBP, specifically average SBP at age 53, has a significant genetic basis. We estimated that approximately half of the population carries a genotype (2 cM), 5 0 cM, 9 . We chose to include HRX as a time-dependent covariate in our first-stage model. However, since the decision to treat is based on SBP, this approach may lead to invalid estimates of the HRX effect, and may ultimately affect our genetic inferences as well. Levy et al.  propose We adopted a parametric modelling approach in our genetic analysis. An advantage of this approach is that it utilizes all available data in each pedigree. A disadvantage, however, is that the model form was likely misspecified, particularly if SBP is determined by several genes with differing allele frequencies and effects on the trait. As an alternative, one could replace our second-stage parametric model with a weighted nonparametric linkage approach, for example using a variance components (VC)  or HasemIn conclusion, we have proposed a two-stage modelling approach to the genetic analysis of longitudinal data for a quantitative trait. Additional work is necessary to evaluate the method, including simulation studies and comparisons to other two-stage and joint-analysis approaches."}
+{"text": "P \u2266 0.0001) were identified. We found that the general patterns for nonparametric linkage (NPL) scores from SNP and microsatellite genome scans are fairly consistent; however, the peaks of the NPL scores are mostly higher in the SNP-based scan than those using microsatellite markers, which might be located at different regions. Furthermore, SNPs identified from linkage screens were not so strongly associated with alcoholism  as those identified from association genomic screening .We conducted genome-wide linkage scans using both microsatellite and single-nucleotide polymorphism (SNP) markers. Regions showing the strongest evidence of linkage to alcoholism susceptibility genes were identified. Haplotype analyses using a sliding-window approach for SNPs in these regions were performed. In addition, we performed a genome-wide association scan using SNP data. SNPs in these regions with evidence of association ( Genome-wide linkage scans are typically conducted to narrow down regions prior to association fine mapping. However, Risch and Merikangas  claim thA total of 143 pedigrees (or 364 nuclear families) comprising 1,614 subjects  were analyzed. There were 328 microsatellite markers and 11,120 Affymetrix SNP markers available for analysis. To test for Hardy-Weinberg equilibrium (HWE), one subject from each pedigree was randomly sampled and a chi-square goodness-of-fit test was performed using PROC ALLELE procedure in SAS/GENETICS package. Four hundred and thirty-one SNPs and 69 microsatellites were excluded as a result of departure from HWE. To avoid potential bias caused by rare alleles, 192 SNPs with minor allele frequencies less than 0.02 were further excluded. In addition, to reduce the impact of LD on our linkage results, we computed the pairwise LD measure |D'| sequentially using FBAT computing package .Genome-wide microsatelite or SNP linkage screens were conducted using GENEHUNTER 2.1 ; linkageThe average information content from 7,328 SNPs after excluding 3,169 SNPs was almost identical to the original 11,120 SNP markers. The peak NPL scores on individual chromosomes dropped slightly on most chromosomal regions compared to those using all the markers. For example, the peak NPL scores dropped from 3.81 to 3.72 on chromosome 2, from 2.86 to 2.59 on chromosome 4, from 3.76 to 3.08 on chromosome 10, and from 2.94 to 2.44 on chromosomes 11, respectively  on chromosome 2 and the 591st\u2013600th SNPs (tsc0549932...tsc0517919) on chromosome 10 with NPL scores greater than 3.0 (p < 0.0017) were selected for haplotype analysis at sliding-window sizes from 1 to 6 (results not shown). The haplotypes with an overall significance level less than a nominal level of 0.05 were constructed by the SNP of tsc1278942 (p = 0.024), and the interval of five SNPs  (p = 0.04) on chromosome 2. None of the haplotypes on chromosome 10 with an overall significance level less than 0.05 were observed. The most significant single haplotype was found to be \"1 1 1 1 2 1\" constructed by six SNPs  on chromosome 2, with a p-value of 0.0044. On the contrary, 15 markers across the genome have significance levels less than a nominal level of 0.0001 when testing for the null hypothesis of no association and no linkage , the significance level of 0.05 with a conservative Bonferroni correction for 10,187 SNPs used in the association analysis. Nevertheless, none of these SNPs were located in the regions showing evidence of linkage. These results indicated that the genetic effects from the alcoholism causal variants might be too weak to be identified through linkage analysis, yet can be detected by genomic association studies.The 325Our analyses illustrated that the typical gene-mapping procedure to identify target regions through a genome-wide linkage scan using markers at a density of 1 marker/~10 cM prior to a fine-scale mapping on the targeted regions, could possibly fail to identify disease loci due to either limited major gene effects, misplacement of markers, or insufficient information content of microsatellite markers. The initial genome-wide scan turns out to be extremely critical to select regions harboring disease genes for further fine-mapping analysis in the typical process. The availability of SNP markers provides substantially greater information content than the microsatellites, thus linkage signals missed by microsatellites could be picked up by SNPs. However, the presence of LD among the SNPs, the inability to detect Mendelian errors, and the inability to accurately validate genetic maps have complicated linkage studies using SNPs. Association studies, on the other hand, have greater power to detect genes of modest effect [HWE: Hardy-Weinberg equilibriumLD: Linkage disequilibriumNPL: Nonparametric linkageSNP: Single-nucleotide polymorphismYFC made contributions to the study design, statistical analysis, interpretation, and draft of the manuscript. SYL participated in the design of the study and performed the data analysis. YYT conceived of the study and helped to draft the manuscript. All authors read and approved the final manuscript."}
+{"text": "Problem 1 of the Genetic Analysis Workshop 13(GAW13) contains longitudinal data of cardiovascular measurements from 330 pedigrees. The longitudinal data complicates the phenotype definition because multiple measurements are taken on each individual. To address this complication, we propose an approach that uses generalized estimating equations to obtain residuals for each time point for each person. The mean residual is then taken as the new phenotype with which to use in a variance components linkage analysis. We compare our phenotype definition approach to an approach that first reduces the multiple measurements to a single measurement and then models these summary statistics as regression terms in a variance components analysis. For each approach, multipoint linkage analysis was performed using the residuals and the SOLAR computer program. Our results show little difference between the methods based on the LOD scores. The phenotype definition is an important component of gene mapping. In gene mapping studies, each individual is assigned a phenotypic value for a particular trait of interest. This phenotype is then tested for cosegregation with certain markers. If a susceptibility locus exists, then individuals with similar trait values should have higher-than-expected allele sharing at this locus.The data from the Framingham Heart Study complicates the phenotype definition in that up to 21 measurements over a 40-year time span were taken from each person (Cohort 1) or five measurements taken over a 20-year time span (Cohort 2). Moreover, these multiple measurements from a person are correlated with each other. Thus, it is unclear as how to define a phenotype given the correlated repeated measures. One approach would be to model each time point in a regression analysis and then combine the parameter estimates . In theiWe hypothesize that our approach will provide more linkage information because all of the data, rather than summary statistics, are used to estimate the parameters in the regression model. The approach of Levy et al. does use all of the data, but by averaging the variables first, information about variability is lost. We test this hypothesis by comparing the genome-wide linkage results using our phenotype-definition approach with the genome-wide linkage results using the phenotype-definition approach of Levy et al.Problem I data consists of 2885 individuals from 330 pedigrees. Of the 2885 individuals, 1213 were from Cohort 1. Cohort 1 individuals were followed every 2 years for a total of 21 measurements. Cohort 2 individuals are offspring of Cohort 1 individuals, and they were followed every 4 years for a total of five measurements. At each follow-up, extensive amount of medical information was obtained, including SBP, age, height, weight, and high-blood pressure treatment information., for the ith individual. We then used linear regression to regress  on ( - ) and ( - ), where  is the mean age of an individual,  is the mean BMI of an individual,  is the sample mean age, and  is the sample mean BMI. The residuals from this regression analysis are then used as the quantitative phenotype for the linkage analysis.We are interested in finding the genes that increase the risk for cardiovascular (CV) disease. We used SBP as a surrogate to CV. We first analyzed the longitudinal data following the methods of Levy et al., denoted as Method 1. Specifically, we calculated the mean SBP, ith time point. Residuals were then obtained for each time point.We also analyzed the longitudinal data with an alternative approach (Method 2). We first found the residuals for each time point. We then calculate the average residual over all time points for each person to use as the phenotype in the linkage analysis. To calculate the residuals, we used generalized estimating equations (GEE) . This isFor both approaches, we analyzed the phenotype data from males and females and from Cohorts 1 and 2 data separately, resulting in four longitudinal analyses. This was done to allow for different rates of change for age and BMI for each of the male/female and Cohort 1/Cohort 2 combinations. The residuals obtained from each of these four analyses were combined into one set of residuals, which was then used in the linkage analysis. The correlation between the two phenotypes used in the linkage analysis is 0.97.Multipoint linkage analysis of the residuals were completed using a variance-component approach, which tests for linkage by testing whether the variance component associated with a particular chromosomal location is significantly greater than zero. The analyses were performed using SOLAR . Since w) between the two phenotypes. Moreover, neither of these methods reported LOD scores above 3. The largest multipoint LOD score that was observed was on chromosome 5, with a LOD score of 2.35 (Method 1) and a LOD score of 1.73 (Method 2) at 32 cM. These results were close to the result found by Levy et al.; they reported a multipoint LOD score of 1.9 at 23 cM.Results of the linkage analyses from the two different approaches are presented in Figure Levy et al. found significant evidence for linkage (LOD score = 4.7) at 67 cM on chromosome 17. Although we did not achieve that level of significance with either approach , we do see a peak at that location.We present an alternative approach to deriving a phenotype from longitudinal data based on the GEE methodology, which accounts for the repeated measures from each observation. We hypothesize that our approach would provide more linkage information than that of Levy et al. because our approach uses all of the data to estimate the parameter estimates in the regression model. The approach of Levy et al. averages the longitudinal data first, thereby reducing the variability of the data, and then models the summary statistics in a regression analysis. What we observed, however, was that the approaches provided essentially the same amount of genetic information based on how similar the LOD scores are across the genome and how correlated the two phenotypes were. The two regions that had LOD scores of about 1.5 or higher occurred on chromosomes 5 and 17. At both regions, Method 1 had a LOD score that was about 0.55 units greater than that of Method 2. For the other regions, the results were similar or inconclusive, such as that found on chromosome 22. Since the maximum LOD score was about 1 on chromosome 22, no conclusions can be made about the difference between the methods in this region. This is because of the strong potential of false positives with such a low LOD score. The overall similarity between the methods indicates that little or no loss of information occurs by reducing the multiple measurements from each person to a single measurement before adjusting for other covariates.a priori. With simulated data, empirical type I error rates and power can then be determined for both methods, but a simulation study such as this requires at least 1000 replicates to appropriately test at the 5% significance level; the simulated data from GAW had only 100 replicates. We suspect that the conclusions drawn from the use of the simulated data (with 100 replicates) would not have been much more accurate than what we observed from the used of the real data. Another limitation of our study is that our approach (Method 2) did not account for familial relationships in the analysis. An assumption of GEE is independence among subjects, and a violation of this assumption may bias parameter estimates. We do observe a difference in parameter estimates between Cohorts 1 and II. This difference could be due to the fact that Cohort 2 consists of related subject, due to the fact that more data are available in Cohort 1, or it even could be due to ascertainment differences between the cohorts. However, based on Figure A limitation of our study is that we use the real data to compare the two statistical methods. A more accurate comparison should be made with the use of simulated data, in which the true gene locations are known We note that our results from Method 1 differed from the published results of Levy et al. Even though our Method 1 was similar to that of Levy et al., our analysis method was not as extensive as theirs, e.g., Levy et al. accounted for treatment effects of hypertension, and they had inclusion criteria specifying which subjects to include in the analysis. In contrast, we included all subjects who had both phenotype and genotype information. Moreover, the Levy et al. analysis had two additional pedigrees. Thus, we expected the two results to differ. However, we emphasize that the primary goal here was to compare two analytical approaches rather than replicate the findings of Levy et al. In summary, since the two approaches provided similar results, we conclude that Method 1 is the more parsimonious approach to use because it requires fewer assumptions in the data analysis."}
+{"text": "We explored the power and consistency to detect linkage and association with meta-analysis and pooled data analysis using Genetic Analysis Workshop 14 simulated data. The first 10 replicates from Aipotu population were used. Significant linkage and association was found at all 4 regions containing the major loci for Kofendrerd Personality Disorder (KPD) using both combined analyses although no significant linkage and association was found at all these regions in a single replicate. The linkage results from both analyses are consistent in terms of the significance level of linkage test and the estimate of locus location. After correction for multiple-testing, significant associations were detected for the same 8 single-nucleotide polymorphisms (SNP) in both analyses. There were another 2 SNPs for which significant associations with KPD were found only by pooled data analysis. Our study showed that, under homogeneous condition, the results from meta-analysis and pooled data analysis are similar in both linkage and association studies and the loss of power is limited using meta-analysis. Thus, meta-analysis can provide an overall evaluation of linkage and association when the original raw data is not available for combining. Identifying the susceptibility genes for human complex traits, such as obesity, diabetes, and hypertension, represents a challenging task for human geneticists. Due to the moderate effect of each gene on the trait, it is difficult to acquire enough power to detect all disease susceptibility genes in one study with a moderate sample size. One potential solution to this challenge is to combine the primary studies to increase the power to identify the genes with small effects. When the raw data is available, pooling raw data should be the most powerful method to combine studies. However, in practice, raw data is usually hard to obtain and the alternative is to pool the results  of the primary studies instead.p-values or LOD scores. The advantage of such methods is that it does not require a common effect size, only that every study tests the same null hypothesis. Thus, these methods are quite general and flexible to use. The very first method for combining results from different studies to obtain consensus was developed by R. A. Fisher in 1925 [p-values is remarkably general and easy to use. Wise et al. [p-value or LOD score ranks. In this method, each chromosome is separated into independent bins with equal spacing, and the linkage evidence for each bin is ranked in each study. To combine the linkage evidence from different studies, an average rank is calculated for each bin across the studies.Although meta-analysis has been widely used in clinical trials and epidemiological studies, it is a relatively new approach in linkage studies. Methods for meta-analysis of linkage analysis can be roughly divided into two categories. In the first category, individual effect size and its variance are available for each study and can be combined using a fixed-effects or random-effects model to obtain an overall effect. Li and Rao  proposed in 1925 . Fisher'The main aim for this analysis is to identify the underlying genetic factors responsible for Kofendrerd Personality Disorder (KPD) using pooled data analysis and meta-analysis and to further compare the power to detect linkage and association with these two methods.The analysis was performed in the Aipotu population. The diagnosis for KPD in this population is based on all 3 different criteria, which is the most heterogeneous compared with the other 3 populations and represents the most realistic case. Replicates 1\u201310 were selected for the analysis, which represents a reasonable sample size for the meta-analysis of genome scans in reality.p-values was used in our analysis considering that 1) in practice, the designs and applied statistical methods are usually different in linkage analyses and it is difficult to obtain a common effect size for combining; 2) we can combine significance level at every marker location using Fisher's method, but only at every bin (~30 cM) using GSMA.Fisher's method of combining Zlr [Zlr asymptotically follows a normal distribution. We then combined the results of the 10 primary genome scans using Fisher's method of combining p-values [n independent tests are made of the same hypothesis, then we can calculate a combined p-value for all n tests by , where pi is the significance level for study i. The combined p-value is asymptotically distributed as a chi-square distribution with 2xn degrees of freedom. Under the null hypothesis of no linkage, 4.6 \u00d7 LOD follows a chi-square distribution with 1 degree of freedom. In practice, the test is one-sided because it is only declared significant when  < 1/2, where  is the estimated recombination rate. The combined p-value was transformed to LOD scores by calculating the quantile of the chi-square distribution.Linkage analysis with microsatellite markers was performed using a nonparametric allele-sharing method  implemenZlr  as the tp-values . This me2 [ in each replicate as well as in pooled data. The p-values were corrected for multiple testing for the number of single-nucleotide polymorphisms (SNPs) tested for association. To account for the non-independence between these SNPs, a spectral decomposition method was used to obtain the effective number of independent SNPs [. SNPs with allele frequency less than 5% were excluded from the analysis.Hardy-Weinberg equilibrium (HWE) was tested in founders using software package ARLEQUIN . Pair-wi2  using ldent SNPs . This meTo further narrow down the regions containing the genetic variants for KPD, we applied family association test implemented in software package FBAT (version 1.5.1)  for all Four regions were found showing significant evidence for linkage (LOD > 3.6 ) in the p = 0.0001) after adjustment for multiple testing in pooled data. LD was calculated around the linkage regions on chromosomes 1, 3, 5, and 9. The LD measurements from individual replicate and pooled data are similar. We defined strong LD as D' > 0.90 or r2 > 0.30 [2 = 0.024). Strong LD was found around B03T3063-B03T3065 region on chromosome 3, B05T4141-B054143 region on chromosome 5, and B09T8337-B09T8339 region on chromosome 9.Fine mapping of KPD by linkage analysis was further performed at these regions by analyzing additional SNP markers. The results from meta-analysis and pooled data analysis are presented in Table 2 > 0.30 . No strop < 0.05) in either meta-analysis or pooled data analysis are presented in Table p = 0.0004) and B09T8341 (p < 0.0001).The FBAT results for markers with nominal significance  was detected in both analyses for 8 out of 10 SNPs showing significant association in either analysis (Table We concentrated on single-SNP analysis in our association studies. The results of association analyses are quite consistent in both combined analyses. Significant association (is Table . Two SNPp-values is that the primary studies are independent from each other. Bias could be caused by including studies with overlapping samples in the meta-analysis. This could be avoided by a careful check of authorship of the publications and detailed information about these studies. However, this may not be possible to do under some circumstances. If a serious overlap is found, an easy solution to prevent such bias is to leave some studies out. However, we may waste useful information by doing this. New statistical methods will be needed to incorporate such dependency in the meta-analysis.An important assumption of meta-analysis using Fisher's method of combining The 10 replicates we chose for this analysis differ only in sampling and no heterogeneity exists among them. Our results show that meta-analysis has a similar power to detect linkage and association compared with pooled data analysis under homogeneous condition. In reality, different studies could differ in study design, marker set, statistical analysis and etc., further investigation need to be done to evaluate the effect of these factors on meta-analysis.Our analysis showed that, under homogeneous condition, the results from meta-analysis with Fisher's method and pooled data analysis are similar and the loss of power to detect linkage and association is limited for meta-analysis. Thus, meta-analysis can provide an overall evaluation of linkage and association when the original raw data is not available under this condition. More studies need to be done to investigate the power of meta-analysis when heterogeneity exists among primary studies.GSMA: Genome-scan meta-analysisHE: Haseman-ElstonHWE: Hardy-Weinberg equilibriumIBD: Identical by descentKPD: Kofendrerd Personality DisorderLD: Linkage disequilibriumSNP: Single-nucleotide polymorphism"}
+{"text": "Currently most pastoral farmers rely on anthelmintic drenches to control gastrointestinal parasitic nematodes in sheep. Resistance to anthelmintics is rapidly increasing in nematode populations such that on some farms none of the drench families are now completely effective. It is well established that host resistance to nematode infection is a moderately heritable trait. This study was undertaken to identify regions of the genome, quantitative trait loci (QTL) that contain genes affecting resistance to parasitic nematodes.Rams obtained from crossing nematode parasite resistant and susceptible selection lines were used to derive five large half-sib families comprising between 348 and 101 offspring per sire. Total offspring comprised 940 lambs. Extensive measurements for a range of parasite burden and immune function traits in all offspring allowed each lamb in each pedigree to be ranked for relative resistance to nematode parasites.Trichostrongylus spp. adults in the abomasum and small intestine at the end of the second parasite challenge. Two further QTL for associated immune function traits of total serum IgE and T. colubiformis specific serum IgG, at the end of the second parasite challenge, were identified on chromosome 23.Initially the 22 most resistant and 22 most susceptible progeny from each pedigree were used in a genome scan that used 203 microsatellite markers spread across all sheep autosomes. This study identified 9 chromosomes with regions showing sufficient linkage to warrant the genotyping of all offspring. After genotyping all offspring with markers covering Chromosomes 1, 3, 4, 5, 8, 12, 13, 22 and 23, the telomeric end of chromosome 8 was identified as having a significant QTL for parasite resistance as measured by the number of Despite parasite resistance being a moderately heritable trait, this large study was able to identify only a single significant QTL associated with it. The QTL concerned adult parasite burdens at the end of the second parasite challenge when the lambs were approximately 6 months old. Our failure to discover more QTL suggests that most of the genes controlling this trait are of relatively small effect. The large number of suggestive QTL discovered (more than one per family per trait than would be expected by chance) also supports this conclusion. Haemonchus contortus, Teladorsagia circumcincta and Trichostrongylus colubriformis [H. contortus is now making small ruminant production, in some areas of the tropics, unsustainable [Nematode parasites are the major animal health constraint in sheep production on pasture. Chemical control using anthelmintic drenches has been a reliable means of nematode control for the last 40 years but increasingly nematodes are becoming resistant to anthelmintics. A recent survey in New Zealand  has showriformis . Multipltainable -5.As a result of this failure of anthelmintic drenches, a major research effort has been underway for the past 15 years to examine alternatives to chemical control. The use of nematode trapping fungi , diets hA particularly popular nematode control method has been to select sheep which are genetically resistant to parasitic nematodes. This has the advantage of providing a long-term solution which involves challenging and phenotyping only a small proportion of the industry flocks, as the trait can be easily disseminated through the sale of resistant rams . In New Haemonchus infection than the South African Dorper breed [A wide range of studies in many different sheep breeds has shown that resistance to nematode parasites is a moderately heritable trait with most heritability estimates about 0.3 -19. Certer breed . French er breed .The major method of measuring parasite burden is by counting parasite eggs in sheep faeces, the so called faecal egg count (FEC), which is an unattractive and labour intensive technique. Furthermore the parasite challenge required can affect lamb growth and other important traits such as faecal soiling around the anus (dags). These constraints have limited the uptake of parasite resistance selection by the sheep breeding industry. A proposed solution to this dilemma has been to try to identify the genes responsible for the heritable trait and offer a more attractive DNA test to help identify resistant sheep .T. circumcincta. Markers located in the sheep gamma interferon gene on chromosome 3q have also shown significant associations with parasite resistance in Romney selection lines and wild populations of Soay sheep on the island of St Kilda off the west coast of Scotland. [In order to identify suitable DNA markers a number of association studies have been undertaken with markers from the MHC region on sheep chromosome 20 -26. Givecotland. ,28Trichostronglus colubriformis parasites no genome wide significant QTL were identified [H. contortus on 2 separate occasions at 6 months and 13 months of age. Four half-sib pedigrees were involved and a number of QTL were identified, but unfortunately their chromosomal location was not disclosed [H. contortus larvae 12 weeks apart followed by measurement of faecal egg counts and blood packed cell volume (PCV) were used to generate and measure the parasite burden. As with the previous study, two QTL were identified but their chromosomal location was not disclosed [More global searches for DNA markers involving genome scans for QTL have also been undertaken. This is the fifth such study to be reported for parasite resistance in sheep. In all cases half-sib experimental pedigrees derived from a sire known to be segregating for parasite resistance were used, with the sires derived from crossing either divergent selection lines or crossing resistant and susceptible breeds of sheep. In the first study which used Merino sheep derived from crossing divergent selection lines and an artificial challenge with entified , althougisclosed . The thiisclosed .Nematodirus FEC and on chromosome 3 and 20 associated with Strongyle FEC.The fourth study recently published by Davies et al  was the This study reports in full our linkage analysis involving 5 half-sib pedigrees in which sires derived from crossing resistant and susceptible lines of Romney sheep were out-crossed to Coopworth ewes and a total of 18 different measurements for parasite burden and associated traits were used to define the phenotype in the offspring.Decreases in the nematode parasite burden, as measured by FEC and internal worm numbers , were the major phenotypes used to measure parasite burden and hence resistance. Given the very high cost of developing the large pedigrees used in this experiment we also measured the humoral immune response, both IgG and IgE, to the parasite challenge, the development of dags and lamb growth rate in the hope that these associated phenotypes may also provide some insight into the genetic basis of parasite resistance. We did however concentrate on parasite burden as the primary trait of interest so that in choosing the most extreme animals for this trait for genotyping we were reducing our chances of finding QTL for the other traits examined.Results from the initial genome scan using the 44 most extreme offspring in the five pedigrees and the FEC1 and FEC2 traits, indicated the possibility of QTL for parasite resistance on chromosomes 1q, 3, 4, 5, 8, 12, 13, 20 and 23. Subsequently all progeny were genotyped with the markers used on these eight chromosomes. Given the very large number of phenotype and genotype measurements taken the results of all the analyses undertaken are presented in additional chromosome figures with results from each of the 26 chromosomes presented in a series of 21 graphs. [Additional files A total of six QTL with genome wide significance were identified and are summarised in Table Trichostrongylus spp adults and late stage larvae in the abomasum while the other is for the same nematode genus in the small intestine. The fact that two separate measures of parasite burden both show QTL in the same region of the genome strengthens the evidence for a QTL in this region. These two measures were moderately correlated .The 2 most interesting QTL are those found in family 124 near the telomere on Chromosome 8. The two phenotypes are complementary in that one is a measure of Trichostrongylus spp adults in the small intestine on chromosome 11 and the family 124 QTL for Trichostrongylus spp adults in the abomasum on chromosome 2 are both on chromosomes where only the most extreme animals were genotyped. In our initial genome scan we obtained similar results on the 8 chromosomes intensively genotyped and with the exception of Chromosome 8 the QTL we identified were not confirmed by the additional genotyping. There is a good chance that the chromosome 11 and 2 QTL would not be confirmed by further genotyping.The across family QTL for T. colubriformis L3 larvae. These two phenotypes have a correlation of 0.25 (P < 0.001). The QTL peaks, although approximately 30 cM apart on this small chromosome, do overlap so we are unable to determine whether the same or different genes are responsible.The two QTL in family 154 on Chromosome 23 are both concerned with the immune response to parasite infection measuring either total serum IgE or serum IgGspecific for the As expected with a genome scan involving a large number of measured traits, many suggestive QTL were also identified. We identified 21 of these across families combined (c.f. 26 expected if there are no true QTL) and 123 suggestive QTL within families (90 expected). All the suggestive QTL are indicated by an appropriately coloured asterisk above their most favoured position on the chromosome. The six significant QTL are indicated by a double asterisk.There is good evidence from a large range of studies in a variety of sheep populations, including the breeds used in this study , that paT. colubriformis IgG1 and total IgE in serum) involved in the immune response to parasite infection. Given that these two QTL are in regions without even suggestive parasite resistance QTL, it is unlikely that the genes affecting these traits directly affect parasite resistance.A further two QTL were identified for associated traits   means thTrichostrongylus spp. resistance in those regions where we had chosen to genotype all the animals. The QTL concerned adult parasite burdens at the end of the second parasite challenge when lambs are approximately 6 months old. Our failure to discover more QTL suggests that most of the genes controlling this trait are of relatively small effect hence we conclude that discovery strategies using linkage analysis should be replaced by strategies using linkage disequilibrium in unrelated populations.Despite being a moderately heritable trait this large study was only able to identify a single significant QTL for p divergence between lines . Rams used as the sires of five large half-sib families (Table p. Studies in these lines have shown FEC to be highly correlated (r = 0.85 \u2013 0.91) with Trichostrongyle adult worm burden in the intestine [The Wallaceville divergent FEC selection lines of Romneys commenced in 1979 . Selectintestine . The lamChallenge 1 \u2013 after weaning, lambs were drenched with 9 ml of ivermectin  then grazed on pasture known to be contaminated with parasite larvae. A subset of the lambs were monitored weekly by faecal sampling until the average strongyle egg count approached 1500 eggs per gram of faecal material (epg), then all lambs were measured. Three separate faecal samples were taken over 5 days and results (in epg) averaged. Faecal egg counts were determined using a modified McMaster method [Challenge 2 \u2013 After all the Challenge 1 faecal samples had been taken, lambs were again drenched  then returned to contaminated pasture and monitored. When the parasite burden again reached approximately 1500 epg 3 more faecal samples were taken and measured as described for the first challenge. Mean strongyle and Nematodirus infection levels achieved were 1091 epg (FEC1) and 70 epg (NEM1) respectively for the first challenge and 1462 epg (FEC2) and 36 epg (NEM2) respectively for the second challenge.Two natural field challenges of infective nematode larvae  were givr method  in whichAfter the completion of the second field challenge, and the collection of faecal samples, lambs were slaughtered. From each lamb, the abomasum and first 5 m of small intestine were collected with the ends sealed to retain the contents for analysis. Laboratory analysis of the contents was designed to count the number of large L4, L5 larvae and adult worms using the method of Robertson and Elliott . Direct 2T. colubriformis L3 total IgG antibodies [tibodies  were meatibodies  in the sLiveweight measurements were taken at birth, weaning, and after each challenge. Dag scores were recorded at weaning, and after each challenge. Dagginess was measured by visual examination of the hindquarters of each lamb and a score given from 0 to 5 depending on the degree of faecal contamination of the skin and wool surrounding the anus, with 0 = no contamination and 5 = very heavily soiled hindquarters .All DNA markers used were microsatellites. The 5 sires were screened to determine whether each of the markers was heterozygous and if heterozygous the genotypes of the progeny determined. Initially only the 22 most resistant and 22 most susceptible progeny within each family were genotyped. These extremes were chosen on the basis of LFEC1, LFEC2, SNEM1 and SNEM2 after adjusting for fixed effects. The adjusted trait values were combined into a single trait for ranking. This trait was the first principal component (the linear combination that explains the most variation), i.e. 0.19 * adjusted LFEC1 + 0.47 * adjusted LFEC2 + 0.49 * adjusted SNEM1 + 0.71 * adjusted SNEM2. If some suggestion of linkage was identified on a particular chromosome after this initial screen all progeny were genotyped for all informative markers for this chromosome. The method of genotyping used radioactively labelled primers and 12% polyacrylamide sequencing gels to resolve the microsatellite allele sizes as described by Crawford et al. . A compln microsatellite called Oar CDT2 and uses the primers 5'-CGTGCACAGAGATTGCATTC-3' and 5'-CACAGTATCAGTCATCTCTAGCTCC-3'. The amplification conditions were as previously described[With the exception of one marker developed as part of this project to improve the informativeness of Sheep chromosome 23 all information about the markers including, primer sequence, product sizes, and best Mg++ concentration can be found at the following web address, . The mardescribede(x+c), where x is the count and c is the unit count. Total IgE was also log transformed. Nematodirus counts were further transformed by scaling each cohort to the average standard deviation and modelling the standard deviation of cohorts as linearly related to their means.Raw cohort means and standard deviations of the traits are shown in Additional Table 2.  CorrelatGenotype data was checked as follows. Sire-offspring pairs were compared for consistency with Mendelian inheritance and sire families for equal segregation of sire alleles. Linkage maps were calculated using CRI-MAP  and ordeThe information content  was calcThe QTL detection method used was least squares interval mapping, using the Haley-Knott regression methods . Fixed eThe marker positions used were those estimated for male meioses, from  except tThe suggestive and significant permutation thresholds for all traits and pedigree combinations are provided in Additional Figure 1 (All families combined)  and AddiAMC: experimental design, genotype analysis and manuscript preparation; KAP: oversight of genotyping, marker development; KGD: experimental design and statistical analysis; CDT: genotyping, marker development; PAW: genotyping; MRT: genotyping, marker development; SAB: Selection line development and provision of F1 rams; AEB: Statistical analysis; GJG: Oversight of phenotype measurement; RSG: measurement of serum IgG; RW: animal husbandry and phenotype measurement; RJS: measurement of serum total IgE; KK: oversight of animal husbandry; JCM: conception of the experiment and overall management of project.Additonal figure 1. Significance thresholds for each trait across all families as determined by permutation.Click here for fileAdditonal figure 2. Significance thresholds for each trait/trait combination as determined by permutation.Click here for fileAdditional table 1. List of all DNA markers used in the linkage analysis.Click here for fileAdditional table 2. Raw means and standard deviations by cohort for traits analysed.Click here for fileAdditional table 3. Raw correlations between groups of traits.Click here for fileChr 1. Haley Knott linkage analysis of sheep chromosome 1.Click here for fileChr 2. Haley Knott linkage analysis of sheep chromosome 2.Click here for fileChr 3. Haley Knott linkage analysis of sheep chromosome 3.Click here for fileChr 4. Haley Knott linkage analysis of sheep chromosome 4.Click here for fileChr 5. Haley Knott linkage analysis of sheep chromosome 5.Click here for fileChr 6. Haley Knott linkage analysis of sheep chromosome 6.Click here for fileChr 7. Haley Knott linkage analysis of sheep chromosome 7.Click here for fileChr 8. Haley Knott linkage analysis of sheep chromosome 8.Click here for fileChr 9. Haley Knott linkage analysis of sheep chromosome 9.Click here for fileChr 10. Haley Knott linkage analysis of sheep chromosome 10.Click here for fileChr 11. Haley Knott linkage analysis of sheep chromosome 11.Click here for fileChr 12. Haley Knott linkage analysis of sheep chromosome 12.Click here for fileChr 13. Haley Knott linkage analysis of sheep chromosome 13.Click here for fileChr 14. Haley Knott linkage analysis of sheep chromosome 14.Click here for fileChr 15. Haley Knott linkage analysis of sheep chromosome 15.Click here for fileChr 16. Haley Knott linkage analysis of sheep chromosome 16.Click here for fileChr 17. Haley Knott linkage analysis of sheep chromosome 17.Click here for fileChr 18. Haley Knott linkage analysis of sheep chromosome 18.Click here for fileChr 19. Haley Knott linkage analysis of sheep chromosome 19.Click here for fileChr 20. Haley Knott linkage analysis of sheep chromosome 20.Click here for fileChr 21. Haley Knott linkage analysis of sheep chromosome 21.Click here for fileChr 22. Haley Knott linkage analysis of sheep chromosome 22.Click here for fileChr 23. Haley Knott linkage analysis of sheep chromosome 23.Click here for fileChr 24. Haley Knott linkage analysis of sheep chromosome 24.Click here for fileChr 25. Haley Knott linkage analysis of sheep chromosome 25.Click here for fileChr 26. Haley Knott linkage analysis of sheep chromosome 26.Click here for file"}
+{"text": "Genome wide linkage scans have often been successful in the identification of genetic regions containing susceptibility genes for a disease. Meta analysis is used to synthesize information and can even deliver evidence for findings missed by original studies. If researchers are not contributing their data, extracting valid information from publications is technically challenging, but worth the effort. We propose an approach to include data extracted from published figures of genome wide linkage scans. The validity of the extraction was examined on the basis of those 25 markers, for which sufficient information was reported. Monte Carlo simulations were used to take into account the uncertainty in marker position and in linkage test statistic. For the final meta analysis we compared the Genome Search Meta Analysis method (GSMA) and the Corrected p-value Meta analysis Method (CPMM). An application to Parkinson's disease is given. Because we had to use secondary data a meta analysis based on original summary values would be desirable.Data uncertainty by replicated extraction of marker position is shown to be much smaller than 30 cM, a distance up to which a maximum LOD score may usually be found away from the true locus. The main findings are not impaired by data uncertainty.Applying the proposed method a novel linked region for Parkinson's disease was identified on chromosome 14 (p = 0.036). Comparing the two meta analysis methods we found in this analysis more regions of interest being identified by GSMA, whereas CPMM provides stronger evidence for linkage. For further validation of the extraction method comparisons with raw data would be required. Genome wide linkage scans have often been successful in the identification of genes for monogenic diseases. However, the chance of success decreases by the multiplicity of genetic and environmental determinants involved in the aetiology of a complex disease. The contribution of each disease gene to overall risk is presumed to be small, and thus large sample sizes are required to detect the effect . An ad hAs the raw genotype data might not always be available to the public, more flexible approaches are required to carry out the meta analysis. In this context Allison and Heo used the Fisher method of combining the p-values across candidate regions in a study of obesity . ProvincIf researchers are reluctant to contribute even summary measures like test statistics for linkage (LS), rest assured one may introduce some bias into a meta analysis, similar to publication bias. Additionally, the power of the meta analysis will be decreased. Even if the meta analysis contains an amount of uncertain summary data, the results will provide a higher level of validity than by simply viewing the individual findings. Therefore they are highly valuable in deciding how to proceed next, e.g. which regions to pursue in further studies. That said, one should consider such approaches as preliminary, and the necessity to discuss the impact of data uncertainty onto the findings still remains.In this study we propose a way to reconstruct test statistics for linkage (LS) and corresponding marker positions as the key summary measure of genome wide scans from condensed materials such as figures in published papers. Furthermore we carry out the meta analysis of all published genome wide scans of susceptibility to PD taking into account the uncertainty of the summary measures by using the GSMA and CPMM.In this investigation we demonstrate with using PD as an example, how a meta analysis of genome wide linkage searches can be carried out to data with uncertainty when using data extraction. The influence of data uncertainty on the results is discussed and differences between methods are shown.By applying inclusion criteria, the meta analysis is based on all published investigations for genome wide linkage to PD as the phenotype of interest: Scottetal. , PankratWith the methodology proposed above, we were able to extract the same number of markers (n = 344) as originally investigated by Scottetal. . From thPositions and corresponding LSs of a total of 25 markers were provided in the original papers. The extracted LSs were almost similar to those reported . For two markers the extracted mean positions deviated from those reported by ~12 cM and ~16 cM . HoweveUsing our method of extraction, markers yielding higher LS are unambiguously identified in a figure. Thus, we may assume that missed markers are exclusively those with a low value of the LS. Moreover, the estimated standard error of LS ranges from approx. 0 to 0.07 LS-units. The largest deviation was 0.29 LS-units. On average LS of a marker was extracted within a range of 0.06 LS-units. This precision is satisfactory for the coarse grid of genome wide scans.In order to take into account the uncertainty in marker positions, the estimated standard errors of extracted positions for one marker ranges from approx. 0 to 4.15 cM. The largest span between two single extractions is 28 cM. Position uncertainty was exceptionally high on chromosome X, which was printed with an open ended axis in two figures. Hence, locating markers on this chromosome must be regarded as problematic.We found evidence for linkage on chromosomes 1 (p = 0.0074) and X (p = 0.0015). In a leave-one-(study-)out cross-validation analysis we did not find any significant linkage. This shows the heterogeneity of the scans, because both findings are primarily caused by the results of one single included genome scan each. The results obtained by CPMM did not reach the level of genome wide significance as suggested by the Lander and Kruglyak criteria . Howeverth 30 cM bin on chromosome 9 (pSR = 0.0145). Furthermore, for the 6th 30 cM bin on chromosome5 (pSR = 0.0363), for the 5th 30 cM bin on chromosome 14 (pSR = 0.0363) and for the 4th 30 cM bin on chromosome 1 (pSR = 0.0492) locally significant signals were achieved. The individual ranks for these regions ranged from 51 to 118. Neither for heterogeneity nor for homogeneity between studies evidence was given for any bin (phet from 0.1550 to 0.3320). Adjacent to the significant 6th bin, the 4th (pSR = 0.0874) and 5th bin (pSR = 0.0940) of chromosome 9 showed some trend towards linkage. Similarly, adjacent to the significant 6th bin of chromosome 5, the 5th bin (pSR = 0.0940) showed some trend towards linkage. The summed-rank-statistics of each 30 cM bin are shown in figure The most significant results by the summed-rank-statistic (SR) could be achieved for the 6pSR = 0.045. Furthth bin of chromosome 9 got slightly more significant for the weighted-summed rank-statistic (SRweight) (pSR-weight = 0.0120), the p-value for the 4th bin of chromosome 1 ascended above 5% (pSR-weight = 0.0546). In addition, for the 1st bin of chromosome 17 the weighted GSMA-analysis provided a locally significant finding .The results of the weighted and the unweighted GSMA-analysis were comparable. While the finding of the 6rd60 cM bin of chromosome 5 (corresponding to the 5th and 6th 30 cM bin) achieved nominal significance by pSR = 0.0186 and pSR-weight = 0.01438. The 3rd60 cM bin of chromosome 9 (corresponding to the 5th and 6th 30 cM bin) achieved a nominal significance of pSR = 0.0353 and pSR-weight = 0.0238.The p-values of the findings using 30 cM bins of chromosomes 5 and 9 changed slightly when using 60 cM bins, but they did not fall below 0.00847 (suggestive genome wide evidence). The 3All these findings remained significant when accounting for data uncertainty by simulation and achieved p-values less than 0.05 in all 333 replications table . No furtWe applied data extraction combined with assessing data uncertainty to carry out the meta analysis of genome wide scans of linkage to PD from all published investigations. If known studies without accessible data are not considered, a bias might be introduced in meta analysis so that this problem is reduced by using as much information as possible from published figures. To examine the validity of the extraction method a comparison with all summary measures from the considered genome wide scans would naturally be desirable. Such precise information about LS and the corresponding marker position was reported for only 25 markers in the papers considered. Please note that these markers are those relaying the most outstanding information about linkage. For these markers, we found the precision of the extraction, both for LS and position, to be satisfactory for GSMA where information is pooled within bins of 30 cM size. For the remaining markers, the use of extracted LSs and positions is based on two assumptions: Firstly, missed markers are exclusively those with a low LS. Secondly, a potentially greater uncertainty at markers with lower LS does not have any decisive influence on the results of the meta analysis. This is reasonable, since only the highest LS in each bin is used for GSMA. Furthermore and since none of the bins with exclusively low LS was even suggestively significant in none of the MC-replications, these assumptions may be met. However, a further validation of the extraction method is required. An adequate estimation of sensitivity and specificity of the findings when applying the extraction method can only be achieved by comparing with findings from a pooled analysis of all raw data.Both meta analysis methods considered here are robust with respect to design, as they can deal with differences in structure and number of families between studies, quantitative and qualitative phenotype definition, genetic markers analysed and methods of statistical analysis. In addition, no assumptions on the mode of inheritance or genetic heterogeneity are necessary for the valid application of these two methods. The distribution and interpretation of the linkage test statistics does depend on the statistical method applied. This is no problem for GSMA, since test statistics are ranked within the single scans. The key information used by CPMM is based on p-values, which may be converted from test statistics for linkage by a known relation. But CPMM requires the raw data to produce reliable results. That was one of the reasons for developing GSMA .To our knowledge no extensive comparison between these methods has been published yet. Thus the relative power of these methods is not yet clear. While with GSMA one searches for evidence for linkage across studies in pre-specified genomic segments (termed as bins), CPMM identifies regions of clustered markers with LS-values indicating towards linkage and assessing significance using p-values corrected for the size of the region. In the presence of uncertainty in marker position it remains unclear which of these approaches remains more powerful or robust. Please note, that it could be problematic to combine the lowest p-values from genome scans particularly for smaller scans, because of a severe bias towards linkage . Giving Data uncertainty in linkage statistics and marker positions does not deteriorate the strength of the main findings. Since markers are allocated into bins for GSMA, uncertainty in position is reduced to uncertainty of allocation. This allocation is ambiguous only for a small proportion of markers, of which only a small proportion is important for the ranking of bins. Consequently, one might expect less variability in GSMA results due to uncertainty in position. The direct comparison of extracted values of markers to reported values, if available, shows the robustness of the whole approach. The only notable differences appeared from the deviation of original reported marker position to those given by the Marshfield map. In summary, the extraction process led to tolerable uncertainty in both position and test statistic for linkage.In meta analysis it is important to consider departures from homogeneity between the included studies. For CPMM, the cross-validation as a test of heterogeneity addresses whether the overall results are primarily affected by one single scan. The test of heterogeneous ranks for a bin might lack power when the number of scan is low. So it does not come as a surprise that we were unable to find evidence of either homogeneity or heterogeneity for any of the major findings.GSMA appears to be robust towards imprecise data extracted from papers reporting genome wide scans. Setting the analysis into a Monte-Carlo framework and comparing results to those of different meta-analytical approaches is a possible way of investigating the sensitivity to uncertainty. However, GSMA and CPMM lead only in parts to concurrent results, applying both methods to our data collection. GSMA came up with more regions of interest, whereas CPMM provided stronger evidence for linkage by lower p-values. Lewis et al.  applied Finally, our approach is limited by the use of uncertain secondary data instead of original summary statistics. Hence, a meta analysis based on all real summary values to verify these preliminary results would be desirable both to further validate our approach and to give further support to the results regarding PD.GSMA yields weak evidence for linkage to PD for 30 cM bins on chromosomes 1, 5, 9 and 14. While evidence for linkage on chromosome 1 was also provided by CPMM, the findings for chromosomes 5 and 9 remain stable when enlarging the size of the bin to 60 cM or weighting studies according to their number of affected cases included. Additional evidence for linkage was also obtained on chromosome X by CPMM, not detected by GSMA.We are unable to find a genome wide significant or genome wide suggestive evidence of linkage in our meta analysis based on a total of 1384 affected individuals in 862 families.The conspicuous 30 cM bin on chromosome 1 (87\u2013116 cM) overlaps with the PARK10 region designated by Hicks et al. . This fiThe finding on chromosome 5 (132\u2013198 cM) was yet suspected before  by viewiThe finding on chromosome 9 112\u2013169 cM) was highlighted by DeStefanoet al. 2\u2013169 cM ,12.The linkage signal on chromosome 14 110 \u2013 138 cM) arises from the combination of weak signals (LS between 0.62 and 1.6) located within a 9 cM distance of three single genome scans  the density function of the normal distribution. \u0394 denotes the average marker spacing in Morgan. \u03bd (x) denotes the discreteness correction for the distance between markers; for x <2 we have v (x) \u2248 exp (-0.583x).This corrected p-value for such a region is, C because observed p-values less significant than 0.045 (LOD-scores of ~0.89) result in p* > 1. Applying CPMM, we proceeded as follows: On each chromosome the most significant marker, defined by the maximum LS, was identified across all scans. A region \u00b1 30 cM around this marker was considered a linkage region if p* < 0.01. Hence, all LS of the remaining scans within a linkage region were converted to p-values by using Holman's triangle[This equation differs from that used by Badner and Gershon and give triangle as imple triangle. For the triangle. These pFor each region the multiple scan probability According to the criteria for genome scans by Lander and Kruglyak we consiweight [Briefly, the GSMA ,8 methodSRweight .For the analysis we did not consider the X chromosome. The X chromosome was drawn on an open end scale in some of the figures. Hence the position of the extracted markers could only be determined rather imprecisely,15.We considered an approximate bin size of 30 cM as recommended by Wiseetal. In totaSR gives the probability of an arbitrary bin to achieve the observed SR or a higher value. SR analysis assesses the significance of each bin independently. Applying Bonferroni correction for the number of bins, significant genome wide evidence for linkage of 5%, as defined by Lander and Kruglyak [SR<0.00042 for 118 30 cM bins . Suggestive evidence  is given for a pSR < 0.00847.For each bin we calculated p-values of three kinds of tests. First, pKruglyak , will behet gives the probability of heterogeneous ranks across studies for a bin, conditional on the observed rank sum. Therefore we used ij is the rank of j-th bin in the i-th study and het indicates consistent evidence for linkage across studies, while a large phet indicates heterogeneity between the considered searches.Secondly, pIoannidis,31, wherWe assigned top ranks to known bins and the mean of the remaining ranks to empty bins to overcA Monte Carlo (MC) simulation approach was usedThe original studies forming the basis of this meta analysis were all carried out in accordance with the Declaration of Helsinki.We carried out a literature search in MEDLINE for MESH-headings Genetics, Parkinson's disease and genome scan (or screening), restricted from 1998 to 2004 and sourced references of neurological and genetic journals. In total we were able to identify seven genome wide linkage scans of Parkinson's disease . Three fThe following criteria for the inclusion of genome wide scans in the meta analysis were defined to ensure the quality of the individual studies and the data to be extracted:1. Patients are included by status of Parkinson's disease and not being selected e.g. by family history or therapy response.2. Statistical results are available in figures or tables for whole chromosomes, at least for the major findings.3. The statistical analysis is carried out by using established genetic epidemiological methods.4. The analysis concentrates exclusively on the susceptibility to PD, not e.g. to the age of onset. Thus the two genome scans based on other phenotypes are excluded .The study characteristics of the five identified and included genome wide scans on susceptibility to PD are given in table GSMA Genome Search Meta Analysis methodCPMM Corrected p-value Meta analysis MethodLS linkage statisticSR summed-rank statisticPD Parkinson's diseaseAR participated in the design of the project, carried out the data extraction and performed the meta analysis.MS participated in the design of the project and carried out the performed the meta analysis.BMM, ThG and HB participated in the design of the project and helped to draft the manuscript.All authors read and approved the final manuscript."}
+{"text": "There has been a lack of consistency in detecting chromosomal loci that are linked to obesity-related traits. This may be due, in part, to the phenotype definition. Many studies use a one-time, single measurement as a phenotype while one's weight often fluctuates considerably throughout adulthood. Longitudinal data from the Framingham Heart Study were used to derive alternative phenotypes that may lead to more consistent findings. Body mass index (BMI), a measurement for obesity, is known to increase with age and then plateau or decline slightly; the decline phase may represent a threshold or survivor effect. We propose to use the weight gain phase of BMI to derive phenotypes useful for linkage analysis of obesity. Two phenotypes considered in the present study are the average of and the slope of the BMI measurements in the gain phase (gain mean and gain slope). For comparison, we also considered the average of all BMI measurements available . Linkage analysis using the gain mean phenotype exhibited two markers with LOD scores greater than 3, with the largest score of 3.52 on chromosome 4 at ATA2A03. In contrast, no LOD scores greater than 3 were observed when overall mean was used. The gain slope produced weak evidence for linkage on chromosome 4 with a multipoint LOD score of 1.77 at GATA8A05. Our analysis shows how omitting the decline phase of BMI in the definition of obesity phenotypes can result in evidence for linkage which might have been otherwise overlooked. Since height and weight are collected in many studies, the convenience and cost-effectiveness of using BMI leads to its use as a quantitative trait in linkage studies of obesity. Unfortunately, finding the chromosomal locations of genes responsible for controlling BMI has proven difficult [Body mass index (BMI = weight (kg)/heightifficult . The curThe phenotypic definition of obesity is often a result of the study design, leaving little choice for more robust alternatives. Study designs for quantitative trait locus (QTL) mapping of BMI-related phenotypes vary substantially, from longitudinal -4 to crogain mean and gain slope) and evaluate them in a genome-wide linkage analysis.In a technical report by  from the Ontario Health Study (OHS), analysis of cross-sectional survey data provided significant evidence of a nonlinear association between BMI and age, characterized by an increasing phase to approximately age 55 followed by a slight decline as subjects aged. The decline phase may reflect both a survivor effect as well as a true threshold effect. The existence of such a nonlinear relationship between BMI and age implies that, for studies of obesity-related traits, any phenotype that incorporate BMI measurements across the whole age range (gain and decline phases), whether they be cross-sectional phenotypes (a mean or single measurement) or longitudinal (a slope), would be biased by the decline phase. As an alternative, we derive two obesity phenotypes based on BMI measurements in the gain phase only  a cross-sectional phenotype defined as the average of the BMI measurements in the gain phase, gain mean, measuring the tendency to be heavier-set, and 2) a longitudinal phenotype, the slope in the gain phase, gain slope, measuring the rate of weight gain.Within individuals, on average, BMI increased from age 18 until age 53 and then began to decline or stabilize. Depending on the age range of the individual during data collection, different components of this profile might be seen. For this reason and to address issues of nonlinearity, each individual's BMI by age profile was summarized by two phases: a gain slope for an individual was calculated as the coefficient for age from a simple linear model, regressing BMI on age, with the data restricted to that observed in the individual's gain phase. Thus, both the gain mean and the gain slope utilized the longitudinal nature of the data to obtain more reliable measures of cross-sectional and longitudinal BMI phenotypes for obesity-related traits.The gain mean and gain slope, it was required that they have at least three BMI measurements in their gain phase. This choice was arbitrary and made by the authors in an attempt to reduce the noise in the measurements while still ensuring sufficient subjects were included in the analysis. If a height measurement was missing at a particular visit for an individual whose data would otherwise be complete at that visit, height was imputed from other visits. Specifically, the measurement from the closest gain phase visit, with height information available, was used.For individuals to have a measurement for gain mean and gain slope were evaluated and then QTL mapping using SOLAR 1.6.7 [overall mean, a BMI phenotype defined as the average of all the BMI measurements including the decline phase of the individuals. Approximate multipoint linkage analyses [gain slope and gain mean phenotypes were also conducted. All genetic models used for linkage analysis included gender, cohort  and the cohort-by-gender interaction. The two-point LOD scores from the linkage analyses of gain mean and overall mean were compared to evaluate the utility of omitting the decline phase when defining a BMI phenotype.A natural log transformation was used to achieve approximate normality for the phenotypic distributions. The heritability and significance of covariate information in the additive genetic model for the age at maximum BMI, the gain slope, the gain mean, and the overall mean. The majority of subjects in the study were defined to have both gain phase phenotypes (N = 2226 of 2878 for whom BMI data was available in 330 pedigrees); 375 individuals had only a decline phase, with their maximum BMI measurement at their first observation; 277 individuals had only one BMI measurement before their age at maximum BMI. The gain slope appeared quite variable across individuals. The distributions of gain mean and overall mean were quite similar. The variability of the age at maximum BMI was large .Table gain slope, gain mean, and overall mean equal to 0.11 (0.04), 0.49 (0.04), and 0.49 (0.03), respectively.All the phenotypic models, accounting for the covariates, displayed significant heritability, with the heritability (standard error) for the gain mean or overall mean are listed in Table gain mean was larger than that for overall mean. There were two markers for gain mean with LOD scores greater than 3, the largest being 3.52 located on chromosome 4 at 93 cM. The largest two-point LOD score observed for overall mean was 2.49 on chromosome 9 at 92 cM. The LOD score at this marker for gain mean was 3.14.All markers exhibiting two-point LOD scores greater than 2 for gain slope was weak, with only four markers across the genome exhibiting LOD scores greater than 1, the largest being 1.29 on chromosome 4 at 158 cM. However, three of these markers were in the same general region on chromosome 4 . Table gain slope and the lack of evidence at locations in any other region or on any other chromosome, approximate multipoint analysis was also performed. The multipoint analysis produced larger LOD scores on chromosome 4 with the largest LOD score being 1.77 at 174 cM. The multipoint results for the gain slope on chromosome 4 are illustrated in Figure The two-point evidence of linkage for the gain mean phenotype on chromosome 4 and the information content across this chromosome. The largest multipoint result for the gain mean was also on chromosome 4 with a LOD score of 2.64 at 105 cM. The multipoint results for the gain mean phenotype on chromosome 9, the other chromosome that provided a two-point LOD score for the gain mean that was greater than 3, are illustrated in Figure gain mean phenotype were smaller than those observed in the two-point analysis. The information content across chromosomes 4 and 9 was low .Figure gain slope, gain mean and overall mean, were shown to be significantly heritable, with the phenotypes based on mean values exhibiting much larger heritability estimates than the gain slope. The most apparent gain from considering the nonlinear relationship in the definition of the BMI phenotype can be seen in the comparison of LOD scores for gain mean and overall mean. Namely, it appears that linkage analysis based on the gain mean phenotype provided us with possible chromosomal locations influencing an individual's tendency to be heavier-set, while the analysis using the overall mean phenotype (including both gain and decline phases) did not produce strong linkage evidence at these potential locations. The gain mean phenotype provided two regions with two-point LOD scores greater than 3 with no such regions for the overall mean phenotype. The proximity of the two elevated LODs for the adjacent markers on chromosome 9 provided additional evidence that this location is worthy of future study. Moreover, the use of the gain mean phenotype detected chromosomal locations that have already been implicated in previous studies using more direct measures of the obesity phenotype.The D9S257 at 92 cM) reported in the Qu\u00e9bec Family Study to influence abdominal subcutaneous fat.The Qu\u00e9bec Family Study , a genom157xh6 at 131 cM).A study of Pima Indians , who havgain slope. No chromosomal locations linked to a slope phenotype were reported in the Human Obesity Map [D4S2417), which has been suggested to contain a potential candidate gene.Only weak linkage signals were observed in our study for the sity Map . Howeversity Map , from thsity Map  . Additionally, the data available on some individuals did not consist of data in both the gain and decline phases. For example, if data were available for an individual only in their gain phase then their age at maximum BMI may have been underestimated. However, when choosing a BMI phenotype in a cross-sectional study, with the intent to omit decline phase measurements, the possibility of a large standard deviation in the age at maximum BMI should be considered.In cross-sectional study designs one might incorporate the results of this study by choosing to restrict BMI phenotypes on the basis of age; selecting the measurement for the BMI phenotype to be at ages less than the lower confidence limit for the average gain slope may provide a means to distinguish genetic components controlling the rate of weight gain for an individual by omitting the decline phase in the definition of this phenotype. The analysis using the gain slope phenotype did suggest a potential region for future study, although the evidence, as measured by the LOD score, was weak. Inclusion of the decline phase in the definition of this phenotype might have led some, who have attempted to use slope phenotypes in past studies, to overlook this potential region.It is unknown how prevalent the use of a slope phenotype is for studying obesity-related traits, because there has been little discussion of these phenotypes in the published literature and no slope phenotypes exhibiting linkage were reported in the Human Obesity Map . The gaigain mean phenotype exhibited higher LOD scores than the overall mean phenotype, some at loci that have already been implicated in a study using a more direct measure of obesity [gain slope phenotype exhibited some weak evidence for linkage while no such evidence for other obesity slope phenotypes have been previously reported [BMI is clearly an accessible surrogate measure for obesity and is available as a by-product of many studies with an alternative focus. Exploiting the nonlinearity between BMI and age to omit the decline phase of individuals in the definition of the phenotype proved fruitful in the current study; the  obesity . Furtherreported . The des"}
+{"text": "Often, multiple measures of a trait are available in a genetic linkage analysis. We compare Monte Carlo Markov chain analysis of two very different measures of hypertension in the simulated Genetic Analysis Workshop 13 data to examine how choice of measure affects the results. The measures selected were age-of-onset of hypertension and systolic blood pressure at first visit.In combined segregation and linkage analysis of the complete pedigrees using the first replicate of the simulated data with missing values, we found that the age-of-onset analysis was better at identifying \"slope\" genes, while the systolic blood pressure analysis was better at identifying \"baseline\" genes.Analysis of different trait measures may identify different trait-related genes. When linkage analysis is conducted on multiple trait measures, a linkage signal found for only one measure can represent a true trait locus. In studies such as the Framingham Heart Study (e.g. ) or the Here, we compared two different simulated measures of hypertension in analyses with Monte Carlo Markov chain (MCMC) oligogenic combined segregation and linkage analysis, as implemented in the program Loki . These mFrom the first replicate of the simulated GAW13 data, we selected two traits that were related, but had slightly different genetic characteristics. Both simulated traits we selected, AOH and SBP at the first visit, are related to hypertension. However, since the generating model divided the trait loci into those that affected \"baseline\" value and those that affected \"slope,\" we expected that SBP would localize \"baseline\" genes, while AOH would localize \"slope\" genes. For SBP we used the SBP value from the first examination in both cohorts. We used the simulated data with missing values. The data set did not contain age-of-onset data as a separate value, but it did contain an indicator of hypertension diagnosis at each visit. We used the age at the earliest visit with a hypertension diagnosis as AOH. Since \"baseline\" was age 20 at the generating model and the first visit occurred at different ages for different subjects, our SBP measure was not a pure measure of the \"baseline.\" Similarly, since AOH was determined by the crossing of a threshold, AOH is not a pure measure of \"slope.\" Such \"impure\" measures may, however, better reflect the measures found in studies of real data.To estimate the number, effects, and location of loci contributing to AOH and SBP, we applied the MCMC segregation and linkage analysis methods described in Heath  and Daw X is the incidence matrix for covariate effects, \u03b2 is the vector of covariate effects, Qi is the incidence matrix for the effects of QTL i, \u03b1i is the vector of effects for QTL i, e is the normally distributed residual effect, and k is the number of QTL currently estimated (k \u2265 0). The MCMC process samples \u03bc, \u03b2, \u03b1i, i, and e as well as parameters such as unobserved marker genotypes. All these parameters are sampled from the space of model values consistent with the data observed. Values are sampled proportional to their posterior probability. After the number of sampling iterations is sufficiently large, the sampled values provide an estimate of the posterior probability distribution over the space of possible parameter configurations. We chose a set of covariates that had an effect in the generating model. For comparability, nearly the same covariates were used for both traits. For SBP, the covariates included cigarettes smoked per day (CPD), sex, and age, as reported at the first visit. For AOH, the effects of CPD and sex were estimated as covariates. AOH covariates are a subset of those for SBP, with only age not used in the case of AOH. The censored trait model described in [where \u03bc is the \"reference\" trait value, ribed in  was usedWe conducted a complete genome scan for both traits. We first carried out analyses on both models on all 22 chromosomes using 50,000 iterations, while saving every fifth iteration. On chromosomes with evidence for linkage, we followed up with longer runs. Additionally, for each trait we conducted a longer multi-chromosome analysis including all chromosomes with an L score > 5. These longer runs were 200,000 to 500,000 iterations in length. All analyses were conducted with the sex-averaged Haldane map provided with the simulated data.To evaluate evidence for linkage, we considered Bayes factors estimated over 1-cM wide bins along the chromosomes. A Bayes factor is simply the posterior probability divided by the prior probability. In the absence of any data, a Bayesian analysis should have posterior probability equal to the prior probability. Thus, a Bayes factor of 1 indicates that the data contains no information for or against linkage. A Bayes factor < 1 indicates evidence against linkage, while a Bayes factor > 1 indicates evidence for linkage. We refer to these Bayes factors for linkage calculated in 1-cM intervals as \"L-scores.\" We used the Haldane map provided with the simulated data.In both analyses, there were three regions with L-scores that stood out with values > 50, while the next largest scores were < 25. All three regions that were identified in this way contained simulated trait loci (see Table Exactly where to draw a threshold for follow-up is not clear. In addition to the three locations with L-scores > 50, there were several regions with L-scores > 10 and < 50. For completeness, we list all regions with L-scores > 10. For AOH, these L-scores were: ~21 at ~80 cM on chromosome 5, ~22 at ~95 cM on chromosome 7, ~15 at 45 cM on chromosome 14, and ~11 at ~50 cM on chromosome 19. For SBP, these L-scores were: ~14 at ~220 cM on chromosome 3 and ~11 at ~95 cM on chromosome 16. Since no trait loci were placed on even-numbered chromosomes, the two signals on even numbered chromosomes represent weak false-positives. It is more difficult to say anything conclusive about the signals on the odd-numbered chromosomes because of the complexity of the generating model: all of the odd numbered chromosomes do, in fact, contain loci that contribute to the simulated hypertension trait. Since more signals in this range of 10 to ~20 are on odd numbered chromosomes, it seems likely that at least some of these are true weak positive signals. The largest effect height gene, Gb1, is at ~80 cM on chromosome 5, while a total of nine different genes are on chromosome 7, making these signals for AOH likely true weak positives and both of these are over 20. Any threshold value may depend on the investigators tolerance of false-positives, but while the three strong signals (Table We find that analysis of AOH is better at localizing slope genes, while analysis of SBP is better at identifying baseline genes. We noted with interest at GAW 13 that some other groups reported better localization of slope genes with methods that examine value at one time-point rather than \"slope.\" We believe we were able to identify Gs10 in our SBP analysis because most subjects were over the baseline age of 20 at the first visit and the slopes were generally positive, causing the variation in SBP to increase with age. Some alternate generating models were suggested at the GAW 13 meeting under which we would expect our SBP analysis to fail to localize slope loci. Exactly what cut-off should be used for declaring significance with L-scores remains an open question. Our results suggest that analysis of different trait measures can identify different trait loci. Thus, if one has multiple measures, conducts linkage analyses on all of them, and only focuses on those linkage signals replicated in analyses of several measures, one may miss some trait loci."}
+{"text": "MC1R) is known to be the main regulator of the switch between the two coat colour pigments: eumelanin (black pigment) and phaeomelanin (red pigment). Some breeds, such as Charolais and Simmental, exhibit a lightening of the original pigment over the whole body. The dilution mutation in Charolais (Dc) is responsible for the white coat colour of this breed. Using an F2-Backcross Charolais \u00d7 Holstein population which includes animals with both pigment backgrounds, we present a linkage mapping study of the Charolais dilution locus.In cattle, the gene coding for the melanocortin receptor 1 (SILV gene was examined as the strongest positional and functional candidate gene. A previously reported non-synonymous mutation in exon 1 of this gene, SILV c.64A>G, was associated with the coat colour dilution phenotype in this resource population. Although some discrepancies were identified between this mutation and the dilution phenotype, no convincing recombination events were found between the SILV c.64A>G mutation and the Dc locus. Further analysis identified a region on chromosome 28 influencing the variation in pigment intensity for a given coat colour category.A Charolais \u00d7 Holstein crossbred population was investigated for genetic effects on coat colour dilution. Three different traits representing the dilution of the phaeomelanin, eumelanin, and non-pigment-specific dilution were defined. Highly significant genome-wide associations were detected on chromosome 5 for the three traits analysed in the marker interval [ETH10-DIK5248]. The SILV c.64A>G as the causative mutation for the Charolais dilution phenotype, although other genetic effects may influence the coat colour variation in the population studied. A region on chromosome 28 influences the intensity of pigment within coat colour categories, and therefore may include a modifier of the Dc locus. A candidate gene for this effect, LYST, was identified.The present study has identified a region on bovine chromosome 5 that harbours the major locus responsible for the dilution of the eumelanin and phaeomelanin seen in Charolais crossbred cattle. In this study, no convincing evidence was found to exclude  Tyrp1, Tyrp2), the biology of melanocytes and melanosomes  and migration and survival of melanocytes during development   has been found exclusively in the Charolais breed, and suggested as possibly being the causative mutation for the coat colour dilution characteristic of this breed . In addition to the SILV c.64A>G mutation in exon 1, a previously unreported substitution in exon 2 was identified that affects the second residue of codon 36 (c.107G>T), causing an amino acid change from serine to leucine. A T>C substitution in intron 2 (c.187+56T>C) was also identified. The other polymorphisms observed have been previously reported: (i) a silent mutation in exon 6 affecting the third residue of the codon 374 (c.1122C>A) [EF363685], leading to an alanine for glutamic acid substitution. Apart from the c.64A>G mutation, none of these allelic variants were associated with the dilution phenotype of the 16 individuals analyzed.The coding region of the bovine tron 2 c.7+56T>C wDc and the Extension loci are mainly responsible for the variation in coat colour observed. As multiple alleles at the Extension (MC1R) locus were segregating in this population, the effect of the Dc locus on both types of backgrounds was confirmed by the observation of a complete or partial dilution affecting individuals with ED-, E+e or ee MC1R genotype. The pale colour observed in individuals with E+e MC1R genotype demonstrates the dilution of pigments produced by both Agouti-responsive (E+-) and non-responsive (ee) melanocytes. This consistency of the effect across MC1R genotypes was also supported by the results of the genome scan, in which the same region on chromosome 5 showed linkage with the three dilution-related traits analyzed. In addition, the additive effects estimated for Quantitative-Black and Quantitative-Red had similar size. The dominance effect was very small relative to the additive effect; therefore a single copy of the Dc allele originating from the Charolais is sufficient to dilute either eumelanin or phaeomelanin. Heterozygous individuals, Dc/dc+, are generally of intermediate phenotype (light-grey or light-red) and two copies of the Dc allele are required to produce a complete dilution of the original pigment (white phenotype). These results are consistent with the inheritance of the Charolais dilution locus described in the literature , and the complete DNA sequence of the gene, [GenBank: NC_007303], based on the bovine genome sequence assembly (Build 3.1) [The coding region of the bovine ild 3.1) . A pair ild 3.1) .Dc/Dc; Holstein genotype: dc+/dc+) using \u03c72-tests assuming that heterozygotes at the Charolais dilution locus showed intermediate coat colour [SILV c.64A>G mutation in the three genetic groups, against the hypothesis of fixation of alternative alleles in the founder lines. The effects of experimental variables on coat colour was also investigated using residual maximum-likelihood analysis , the observed proportions of individuals included in each of the classes were compared with those calculated under the hypothesis of fixation of alternative alleles in the founder lines (Charolais genotype: t colour . The disis (REML ) implemeis  as a fixed effect. To avoid the mix of colour backgrounds in the analysis of the two pigment-specific traits, Quantitative-Black and Quantitative-Red, the White animals included in the analysis were selected according their MC1R genotype . To test for a direct relationship of the SILV c.64A>G mutation [SILV c.64A>G genotype as a fixed effect in the regression model. The trait Grey-Intensity was later analyzed, using the SILV c.64A>G genotype as fixed effect .The primary regression analysis used the linkage map obtained from microsatellite data and assuming the founder lines to be fixed for alternative alleles at the Dc locus . It was Dc locus  for the mutation  on the lPermutation testing  was used to obtain the 5% and 1% chromosome-wide and genome-wide thresholds . The 95%BG-G carried out part of the genotyping experiments, performed the statistical analyses, performed error-checking on phenotype and genotype data, and drafted the manuscript. PW participated in the design and coordination of the study, compiled the phenotype data and helped to draft the manuscript. JLW conceived of the study, participated in its design and coordination, selected the initial marker panel and helped to draft the manuscript. All authors read and approved the final manuscript.SILV c.64A>G mutation is also presented (*).Linkage map details.Marker positions (cM Kosambi) are shown for the sex-average maps built for the Charolais \u00d7 Holstein population considered in this study. The average information content (IC) for each linkage group is also indicated. For chromosome 5, the map including the Click here for filePrimers used for sequencing analysis.Click here for file"}
+{"text": "The merle phenotype includes a lack of eumelanic pigmentation and developmental defects, hearing impairments and microphthalmia. It is similar to that observed in microphthalmia mouse mutants.Coat colours in canines have many natural phenotypic variants. Some of the genes and alleles involved also cause genetic developmental defects, which are also observed in humans and mice. We studied the genetic bases of the merle phenotype in a five-generation pedigree, comprising 96 sampled Australian shepherd dogs. Genetic linkage analysis allowed us to identify a locus for the merle phenotype, spanning 5.5 megabases, at the centromeric tip of canine chromosome 10 (CFA10). This locus was supported by a Lod score of 15.65 at a recombination fraction \u03b8 = 0. Linkage analysis in three other breeds revealed that the same region is linked to the merle phenotype. This region, which is orthologous to human chromosome 12 (HSA12 q13-q14), belongs to a conserved ordered segment in the human and mouse genome and comprises several genes potentially involved in pigmentation and development.Taking advantage of the dog as a powerful genetic model and using recently available genomic resources, we investigated the segregation of the merle coat colour in dogs to be at the centromeric end of CFA10. Genetic studies on other breeds segregating the merle phenotype should allow the locus to be defined more accurately with the aim of identifying the gene. This work shows the power of the canine system to search for the genetic bases of mammalian pigmentation and developmental pathways.This study has identified the locus for the  Melanocytes manufacture two types of melanin: the black/brown photo-protective eumelanin pigment, and the red-yellow cytotoxic phaeomelanin pigment. Several paracrine factors secreted primarily by surrounding keratinocytes are involved in the melanogenic pathway by stimulating the switch between phaeomelanin and eumelanin . In thisufacture ,3.melanocortine 1 receptor gene (MC1R),  [tyrosinase-related protein 1 (TYRP1) [melanophillin [TYRP1 in several dog breeds including the Australian Shepherd dog [Coat colour is highly polymorphic in dogs. In 1957, Little described, after observing the possible phenotypes, more than 20 loci affecting coat colours ,5. Untilnsion E) -8, variansion E) , variant (TYRP1)  and variophillin . Three mherd dog . Genomicherd dog ,14, a raherd dog , and a wherd dog . Altogetmerle phenotype because of its involvement in coat colour and developmental impairments. The merle phenotype is a dominant trait, with heterozygous dogs presenting a coat colour in which eumelanic regions are incompletely and irregularly diluted, leaving intensely pigmented patches. Merle is found throughout the body except on the pheomelanic regions of the black and tan coat colour  was responsible for the merle phenotype in different breeds [merle coat colour may be due to a transposable element, after the observation of two germinal reversions out of 66 merle offspring of a homozygous merle female [Kit Ligand, KITLG, was excluded as a candidate gene for the merle phenotype in dogs [When analysing the genetic basis of the t breeds . It was e female . Recentl in dogs  and the merle phenotype in dogs by considering well-described pigment disorders in mice. Mutations in the gene of the Mitf pathway cause specific coat colour phenotypes, some of which are similar to the merle phenotype in dogs. These include dilution of the coat colour in patches and complete or mild microphthalmia ; a Beauce shepherd family (five dogs) and a Border collie family (13 dogs).A pedigree comprising 96 Australian shepherd dogs (43 brown and 53 black dogs) was collected. This pedigree, called the \"complete pedigree\", included 42 TYRP1 gene in the complete pedigree. As the TYRP1 gene was previously associated with the brown coat colour in dogs [TYRP1. These markers, FH2319 and REN105I03, are 1.18 and 5.17 Mb from TYRP1, respectively  with markers flanking SOX10, with recombination fractions, \u03b8, ranging from 0.08 to 0.14   Table .merle phenotype. We obtained increased Lod scores for markers CFA10.1 to CFA10.8, with maximum Lod scores for CFA10.7 and CFA10.8 . The CFA10.9 marker (telomeric to CFA10.8) is unlinked to the phenotype  and the CFA10.9 marker (located at 8.5 Mb) defining the telomeric limit of the critical interval  and the CFA10.9 marker (8.5 Mb), with the highest Lod score of 19.87.We focused on three candidate genes belonging to the coat colour pathway: merle genes. This locus has many candidate genes, with at least a dozen being potential metabolic candidates as they, or their paralogs, belong to the pigmentation pathway. These include proteins involved in neural crest development (such as ERBB3), melanosome motility and transfer to surrounding keratinocytes .The corresponding orthologous human region is HSA12q13-q14 (position from 54.36 Mb to 60.94 Mb) and mouse region is MMU10D3 (position from 122.8 Mb to 128.7 Mb). These orthologous dog, human and mouse regions correspond to a unique conserved ordered segment, which has the same orientation in dogs and humans but is inverted between dogs and mice. In the dog region, 99 genes are predicted and 48 are known (Broad1), in the human region, 134 genes are predicted and 98 are known (NCBI 35), and in the mouse region, 112 genes are predicted and 95 are known (NCBI M34) . These gMITF gene itself has been excluded, the merle mutation should affect a gene interacting directly with the MITF gene in the pigmentation pathway. Alternatively, a more complex mechanism could explain the incomplete penetrance of eye defects observed in homozygous merle dogs. Although hearing loss may be due to an extreme white phenotype, including the absence of melanocytes in the cochlea, as in other white canine breeds [Although the e breeds , less ismerle phenotype occurs in several breeds and is commonly encountered in mongrel dogs. Breeds segregating merle are from the collie lineage : Shetland sheepdog, Border collie, collie, Australian shepherd dog, etc., and from other unrelated breeds belonging to different FCI groups and different clusters, as defined by Parker et al. [merle phenotype is most probably very old, with the merle coat colour being reported in old books [merle dogs have been selected and reproduced [The r et al. , such asld books ,31, fromproduced .merle phenotype is the same in all breeds and mongrels segregating this phenotype. A unique locus has been suggested as responsible for the merle coat colour [merle locus in dachshund, Beauce shepherd and Border collie families is consistent, at least in these breeds, with there being a unique locus for the merle coat colour. If all merle dogs share a common ancestor chromosome, all breeds segregating merle could be used to refine the locus. The sharing of the merle locus by several breeds and also by mongrels may be due either to a common ancestor chromosome region being transmitted throughout canine evolution and/or to backcrosses that introduced a merle haplotype in several breeds at different times.It is not yet known whether the genetic cause of the t colour . In the MITF, PAX3 and SOX 10 candidates genes in the merle phenotype. However, we identified the merle locus at the centromeric end of CFA10 in pedigrees of Australian shepherd dogs, dachshund, Beauce shepherd dogs and Border collies segregating the merle phenotype. This locus spans 5.5 Mb and is linked to the merle coat colour with a maximum Lod score of 19.87 and a recombination fraction of 0. We are currently analysing this locus in several breeds segregating merle, with a high density of single nucleotide polymorphic markers (SNP). This should help in identifying the merle gene. As well as benefiting breeding practices and canine veterinary medicine, identifying the merle gene will also help in understanding the genetic bases of mammalian pigmentation and developmental pathways.Using genetic linkage analysis, we excluded the involvement of the No dogs were housed for research purposes, and all dogs were privately owned pets.Blood samples and the accompanying pedigree and coat colour data (with pictures when possible) were collected by DVM veterinarians. All data were entered into a database. Genomic DNA was extracted from 5 ml of blood collected on EDTA, using the nucleon BACC 3 kit . For low concentration samples, the extracted DNA was \"whole genome amplified\" using the genomiphi kit (Amersham Biosciences).Pedigrees were constructed using the Cyrillic software (Cyrillic2.1) , which aMicrosatellite markers were selected from RH map data ,34 or frMicrosatellite markers were labelled using a two-step-PCR fluorescent labelling procedure . The firmerle phenotype using M-LINK software through the GLUE web interface [Haplotypes were constructed using the Cyrillic software. Two-point linkage analysis was carried out between each marker and the nterface  and Multnterface . We usedBH collected samples, constructed the pedigrees and performed genotyping experiments; BH also interpreted all dataand actively participated in writing the manuscript. SC helped with the genotyping experiments and interpretation of the dataand participated in writing the manuscript. CH carried out the statistical analyses and data interpretation, and critically revised the manuscript. SD helped with the genotyping experiments. TV extracted DNA from blood samples and commented critically on the work and manuscript. TD carried out the synteny analyses. BD contributed with knowledge on canines coat colours and critically revised the manuscript. FG provided intellectual input and critically revised the manuscript. MDG helped conceive and design the work and helped in the writing of the manuscript. CA conceived and designed the work and drafted the manuscript. All authors read and approved the final manuscript.Characteristics of the markers used in the genetic linkage studies. a CFA : Canis familiaris chromosome.b Starred markers were selected from the CanFam 1.0 canine sequence draft, c marker corresponding to marker FH2537 from Guyon et al. [d number of alleles as determined from the sub-pedigree. E Markers flanking SOX10 gene.n et al. , d numbeClick here for filemerle phenotype in the Australian Shepherd dog pedigrees on CFA10Scheme of two-point linkage analysis of the . Two-point linkage analysis of the merle phenotype in the Australian shepherd dog sub-pedigree (in black) and complete pedigree (in brown) (Lod scores at theta = 0) is shown on the right. An ordered list of genotyped markers (right) and genes (left) and their position in Mb are indicated in the middle. An ideogram of the canine chromosome 10 is shown on the left with the corresponding human chromosomal conserved segments. NB: genomic sequence systematically starts at an arbitrary coordinate of 3 Mb to include the non-sequenced centromeric region.Click here for file"}
+{"text": "GABRB1 at 51.4 cM and marker FABP2 at 116.8 cM .Multivariate linkage analysis using several correlated traits may provide greater statistical power to detect susceptibility genes in loci whose effects are too small to be detected in univariate analysis. In this analysis, we apply a new approach and perform a linkage analysis of several electrophysiological phenotypes of the Collaborative Study on the Genetics of Alcoholism data of the Genetic Analysis Workshop 14. Our approach is based on a variance-component model to map candidate genes using repeated or longitudinal measurements. It can take into account covariate effects and time-dependent genetic effects in general pedigree data. We compare our results with the ones obtained by SOLAR using single measurement data. Our multivariate linkage analysis found linkage evidence on two regions on chromosome 4: around marker  The Collaborative Study on the Genetics of Alcoholism (COGA) is a large, multisite genetic study to identify susceptibility genes for alcohol dependence and related phenotypes. COGA data include information from the visual oddball experiment and the eyes closed resting electroencephalogram (EEG) dataset. The four fields beginning with ttth contain data extracted from the target case of the visual oddball experiment for four electrode placements. The extracted measures correspond to the late time window, which is set at 300 to 700 ms following stimulus presentation , and the theta band power (3 to 7 Hz). The ttth1 measures have yielded a strong linkage signal on chromosome 7 ,3. The dMultivariate linkage analysis using several correlated traits may provide greater statistical power to detect susceptibility genes in loci whose effects are too small to be detected in univariate analysis. In this report, we analyzed the COGA data using an extension of the variance components models for repeated measurements, and considered simultaneously several of the electrophysiological phenotypes.y =  be a vector of m multivariate trait values for n members of the pedigree. The ith family member has m trait values observed at the age of ti, i = 1,...,n. Consider the model, for i = 1,...,n and j = 1,...,m,We assume independence between pedigrees, and consider one pedigree to describe our model. Let yij(ti) = f + s(ti)\u03b3i1 + \u03b3i2 + eij(ti), \u00a0\u00a0\u00a0 (1)f is a function of the fixed covariate effects Xi and time ti, s(ti) a simple parametric function to accommodate time variant genetic effects, \u03b3i1 the random effect for a major gene, \u03b3i2 the random effect for the cumulative effect of the residual genes, and eij(ti) the measurement error. We assume that \u03b3i1, \u03b3i2, and eij are independent, although eij(ti), j = 1,...,m, has a within-subject correlation structure. It follows:where yij(ti),ylk(tl)) = s(ti)s(tl)cov + cov + \u03b4i = l),cov is the covariance function for eij(ti) and elk(tl) and \u03b4i = l)  at the locus of interest, \u03c6 and \u03c4 are respectively the expected kinship coefficient and the expected probability of sharing 2 alleles IBD over the residual components of the genome,  and  are the additive and dominant genetic variances at the locus of interest, respectively, and  and  are the total additive and dominant genetic variances over the residual components of the genome, respectively. The \u03c0il, kil2,, \u03c6il and \u03c4il can be obtained using the SOLAR software program [\u03c72 distributions [where otterman  for the  program . Becauseibutions .s(t): constant and linear functions of the age. For the environmental covariance function \u03c3jk, we assumed the same environmental variance for every phenotype  and we considered either the same environmental covariance between any two phenotypes or all different environmental covariances between any two phenotypes.The dataset includes a total of 143 nuclear and multigenerational families with 1,614 individuals. We chose those genotyped individuals with no missing electrophysiological phenotypes and ages. This yields 140 families with a total of 819 individuals. We focus our analysis on ecb21 and ttth1 and ttth2 electrophysiological phenotypes that result in a total of 2,457 measurements for the 819 individuals. We noted that from a scatter plot of each electrophysiological phenotype versus the age at which the data were collected, there was a roughly quadratic trend of the phenotype over age. Therefore, in Equation (1), we incorporated age at which the electrophysiological data were collected and its square as covariates. We also incorporated sex as a covariate and included some dummy variables as covariates to allow different intercepts for the individual phenotypes. We also performed the analysis with or without smoking status as a covariate. We considered two forms of D7S1804 at 156.4 and D7S509 at 163.7 on chromosome 7 both have LOD scores around 3.5 and p-values reaching 0.00003, D7S1796 at 120 cM has a LOD score of 2.4 and a p-value of 0.0003, and D7S794 at 177.9 cM has a LOD score of 2.1 and a p-value of 0.0009. Consistent with existing analyses, individual ttth1 phenotypes produced strong linkage signals on chromosome 7. The results from SOLAR also showed that, for ecb21 data, marker D4S2382 at 43.3 cM on chromosome 4 has a p-value of 0.006 (LOD score = 1.4), GABRB1 at 51.4 cM on chromosome 4 has a p-value of 0.006 (LOD score = 1.4), and FABP2 at 116.8 cM on chromosome 4 has a p-value of 0.002 (LOD score = 1.7).We first used the SOLAR software program  to analyp-values. In the figure, curve a shows the linkage analysis results from SOLAR for ecb21 data, curve b shows the linkage analysis results from SOLAR for ttth1 data, and curve c shows the linkage analysis results from SOLAR for ttth2 data. Curve d shows the linkage analysis results from our model considering s(t) as a constant function for using both ecb21 and ttth1 data. Curve e shows the combined linkage analysis results for using ecb21, ttth1, and ttth2 from our model considering s(t) as a constant function and assuming the same environmental covariance between any two phenotypes. Curve f shows the combined linkage analysis results for using ecb21, ttth1, and ttth2 from our model considering s(t) as a constant function and assuming different environmental covariance between any two phenotypes. Curve g shows the combined linkage analysis results for using ecb21, ttth1, and ttth2 from our model considering s(t) as a linear function and assuming different environmental covariance between any two phenotypes. Because we do not have theoretical proofs for the asymptotic distributions of the test statistics, we computed the power from the simulated critical values instead of the asymptotic values. From curves a, b, and c, the univariate linkage analysis exhibits the largest value of -log, 6.10, (LOD score = 1.7) on curve a for ecb21 at marker FABP2 at 116.8 cM.Inclusion of smoking status as a covariate did not lead to a notable change in the test statistics. Thus, we present only the results without adjusting for smoking. Figure p-value) of 9.08 (LOD score = 2.9) at marker FABP2 at 116.8 cM on chromosome 4. And, at the same marker, curve e has a value of 7.83 (LOD score = 2.4), curve f has a value of 9.27 (LOD score = 3.0) and curve g has a value of 9.65 (LOD score = 3.9). Our multivariate linkage analysis led to peaks around these two regions, marker GABRB1 at 51.4 cM and marker FABP2 at 116.8 cM, while the evidence of linkage around the two regions has been enhanced. The marker GABRB1 at 51.4 cM on chromosome 4 has already been identified before to be associated with alcoholism [s(t) as one constant parameter different for each phenotype deserves further investigation. Our multivariate linkage analysis did not find any significant evidence of linkage on chromosome 7. Therefore the results are not reported here.As we can see from Figure GABRB1 at 51.4 cM on chromosome 4, but also some markers that were not suggested before such as marker FABP2 at 116.8 cM on chromosome 4. It is also important to note that the power can also be compromised by a multivariate analysis if only one of the phenotypes contains strong linkage. For example, for chromosome 7, univariate linkage analysis for ttth1 phenotypes revealed very strong linkage signals around 156.4 cM on chromosome 7 with p-values reaching 0.00003, but our multivariate linkage analysis considering ttth1 and ecb21 together or considering ttth1, ttth2, and ecb21 together did not find any significant region on chromosome 7 with linkage, which may be due to the noise introduced by the phenotypes that do not have linkage signals on the chromosome. In other words, a multivariate analysis is most effective when linkage evidence to the individual phenotypes is not strong.In this analysis, we conducted a simultaneous linkage analysis of multivariate phenotypes. We identified some candidate markers that were identified before using some single phenotypes such as marker COGA: Collaborative Study on the Genetics of AlcoholismEEG: ElectroencephalogramIBD: Identity by descentLD: Linkage disequilibriumQTL: Quantitative trait lociSNP: Single-nucleotide polymorphismHZ conceived of the study, participated in the design of the study, drafted the manuscript, revised it critically for intellectual content, and gave final approval of the version to be published. XZ participated in the design of the study, performed the statistical analysis, and drafted the manuscript. YY participated in the manuscript preparation, particularly in the initial data processing. All authors read and approved the final manuscript."}
+{"text": "Specifically, both scans detected confirmed evidence for linkage  to chromosome 1 in the AI families, chromosomes 1 and 3 in the DA families, and chromosomes 3, 5, and 9 in the KA families. An additional confirmed linkage to chromosome 5 in the AI families was detected only by the MS scan. We also observed slightly wider 1-LOD intervals for more of the SNP peaks than for the MS peaks, which is likely due to lower information contents for the SNPs. Subsequent fine-mapping association analysis further identified 2 to 3 markers significantly associated with disease status in each population; B03T3056, B03T3058, and B05T4139 in the AI population, B03T3056 and B03T3058 in the KA population, and B03T3056, B03T3057, and B03T3058 in the DA population. Among the four markers, three were chosen based on results obtained from the two scans, but one was solely from the SNP scan. In summary, our finding suggests that the SNP-based genome scan has the potential to be as powerful as the traditional MS-based scan and offers good identification of peak location for further fine-mapped association analysis.Several simulation studies have suggested that a high-density single-nucleotide polymorphisms (SNPs) marker set may be as useful as a traditional microsatellites (MS) marker set in performing whole-genome linkage analysis. However, very few studies have directly tested the SNPs-based genome-wide scan. In the present study, we compared the linkage results from the SNPs-based scan with a map density of 3-cM spacing with those from the MS scan using a 10-cM marker set among 300 nuclear families each from the Aipotu (AI), Danacaa (DA), and Karangar (KA) populations from the simulated Genetic Analysis Workshop 14 Problem 2 data. We found that information contents obtained from the SNPs scan were somewhat lower than those from the MS scan. However, the linkage results obtained from the two scans showed a high degree of similarity. Both scans identified a similar number of chromosomal regions attaining nominal significance ( Relative to microsatellites (MS), single-nucleotide polymorphisms (SNPs) are more abundantly and uniformly distributed along the human genome, and they are more reliably typed and require a smaller DNA sample . Using STo learn whether a SNP marker set could be as useful as the standard MS marker set for the linkage-based genome scan, we compared both MS- and SNPs-based genome-wide scans among nuclear families in the three populations using the simulated Genetic Analysis Workshop 14 (GAW14) Problem 2 data. Subsequent fine-mapped analyses were performed in regions showing `confirmed' evidence for linkage.We pooled together the last 3 replicates (98\u2013100), which provided us with 300 nuclear families each from the Aipotu (AI), Danacaa (DA), and Karangar (KA) populations. All analyses were performed without knowledge of the answers.2 < 0.7). We used a multipoint nonparametric linkage (NPL) scoring method as implemented in the program GENEHUNTER [Hardy-Weinberg (HW) tests were first carried out for the 917 SNPs using founder genotypes in each population. Seventeen, 16, and 20 SNPs in the AI, DA, and KA populations were not in HW equilibrium and were dropped from further analysis. Two genome-wide scans with 416 MS (10-cM intermarker spacing) and ~900 SNPs (3-cM intermarker spacing) were then performed among families from the three populations. All SNP pairs were not in strong linkage disequilibrium  + ln(Lcontrols) - ln(Lcombined))|, where L is the estimate of maximum likelihood for haplotype frequency. This has asymptotically a \u03c72 distribution with  degrees of freedom under the null hypothesis. Test for transmission of a two-marker haplotype from parents to an affected offspring employed a score test implemented in the TRANSMIT program [r2 statistic on the basis of founder genotypes with an EM algorithm implemented in the GOLD-LDMAX program [Subsequent fine-mapped association analysis was carried out by comparing the distribution of single-marker alleles and two-marker haplotypes between affected offspring from the three populations and unrelated controls. The frequencies of single marker alleles and two-marker haplotypes within each population were estimated by an expectation maximization (EM) algorithm implemented in the TRANSMIT program . Test fo program . The deg program . Multipl program .p < 0.05) at two or more adjacent markers. Specifically, both scans detected 8, 5, and 8 chromosomal regions in the AI, DA, and KA families, respectively (Table p = 2 \u00d7 10-5) (Table We observed that information contents (ICs) were lower in the SNP scan relative to those in the MS; the average ICs across the genome for the SNPs was ~10% lower  than that for the MS (93% for the AI and KA families and 92% for DA families). However, there was good concordance of results between the MS and SNP scans; both scans identified a similar number of chromosomal regions attaining nominal significance for linkage , on chromosome 6 from the AI families (22 cM between the peaks-D06S0229 and C06R0510), and on chromosome 7 from the DA families (60 cM between the peaks-D07S0272 and C07R00623), suggesting that the two scans detected different signals in these chromosomal regions. Additionally, most of the MS peaks covered slightly narrower 1-LOD intervals relative to the SNP peaks  in the two scans in any of the three populations. This resulted in 3 , 4 , and 6  candidate markers from the AI, DA, and KA families, respectively. Accordingly, we acquired 6 20-marker data from packets 28, 29, 153, 207, 208, and 417, with genotypes for the three populations and one control sample. Each packet covers an average spacing of 5 cM. Packets 29, 153, 208, and 417 covered the linkage signals detected by the MS scan, and packets 28, 153, 207, and 417, by the SNP scan.We repeated linkage analysis on chromosomes 1, 3, 5, and 9 with addition of the acquired markers. An increase in the ICs and NPL scores of the original peaks were observed, a clear confirmation of the original evidence for linkage  from packet 153 were significant in the DA population and 2 markers (B03T3056 and B03T3058) were significant in the AI and KA populations. Table We extended two-marker haplotype association on the two markers, B03T3056 and B03T3058, which showed significant single-marker association in the three populations. Using a likelihood ratio test, we further observed significant difference in haplotype distribution between the affected offspring and controls for the marker pair (Table In the present study, we observed a high degree of correspondence of results from the MS and SNP genome scans. Although lower ICs were present for the SNPs relative to those for the MS, both the MS and SNP scans detected similar number of signals attaining nominal significance. Specifically, the two scans detected confirmed evidence for linkage to chromosome 1 in the AI families, chromosomes 1 and 3 in the DA families, and chromosomes 3, 5, and 9 in the KA families. However, one confirmed linkage to chromosome 5 in the AI families was detected only by the MS scan. The peak locations obtained from the two scans were mostly close. Moreover, we observed somewhat wider 1-LOD peak intervals for most of the SNPs relative to those for the MS, likely due to the lower ICs for the SNPs. Subsequent fine-mapped linkage analysis confirmed the initial linkage results. We also observed significant associations of the 4 markers with affection status, all of which could be acquired from the SNP scan. One haplotype from two of these markers was shown to be over-transmitted from parents to offspring in the three populations.Two studies comparing a high-density SNP scan with a traditional MS scan have also shown a remarkable similarity of results from the two scans ,10. ThesIn summary, our findings suggest that the SNP-based genome-scan has the potential to be as powerful as the traditional MS-based scan. In the present study, an average intermarker spacing of 3 cM offers comparable linkage results from the nuclear families to those based on the MS scan with an average spacing of 10 cM. In addition, fine-mapped association results further confirmed the utility of SNPs with good identification of peak locations. With the availability of a dense map, accurate map position, and low-cost high-throughput genotyping, we anticipate that SNPs may soon become useful in conducting both linkage and association studies.EM: Expectation maximizationFDR: False-discovery rateHW: Hardy-WeinbergIC: Information contentLD: Linkage disequilibriumMS: MicrosatellitesNPL: Nonparametric linkageSNP: Single-nucleotide polymorphismBoth authors contributed to conception, analysis, and interpretation of data. JL drafted the article. Both authors read and approved the version for publication."}
+{"text": "Alcohol dependence is a serious public health problem. We studied data from families participating in the Collaborative Study on the Genetics of Alcoholism (COGA) and made available to participants in the Genetic Analysis Workshop 14 (GAW14) in order to search for genes predisposing to alcohol dependence. Using factor analysis, we identified four factors  related to the electroencephalogram traits. We conducted variance components linkage analysis with each of the factors. Our results using the Affymetrix single-nucleotide polymorphism dataset showed significant evidence for a novel linkage of F3  to chromosome 18 (LOD = 3.45). This finding was confirmed by analyses of the microsatellite data (LOD = 2.73) and Illumina SNP data (LOD = 3.30). We also demonstrated that, in a sample like the COGA data, a dense single-nucleotide polymorphism map provides better linkage signals than low-resolution microsatellite map with quantitative traits. Alcoholism is a complex disorder involving multiple genes likely interacting with one another and environmental factors. Quantitative endophenotypes, such as electroencephalogram (EEG) measurements, have been suggested as better indices of alcoholism susceptibility than the customary dichotomous affection status ,2. EGG dWe conducted a principal components analysis using the 12 EEG measures . EEG measures from the Visual Oddball experiment were represented as four letters followed by a number . The four letters denote different experiment conditions: ttth_ contain extracted measures from the target case correspond to the 'late' time window, which is set at 300 to 700 ms following stimulus presentation , and the theta band power (3 to 7 Hz). ttdt_ contain extracted measures which the delta band power is 1 to 2.5 Hz with other conditions same as ttth_. The fields labeled ntth_ contain extracted measures from the non-target case correspond to the 'early' time window, which is set at 100 to 300 ms following stimulus presentation, and the theta band power (3 to 7 Hz). The number following the four letters denotes the locations of the 4 electrode placements: 1 \u2013 FP1 , 2 \u2013 FZ , 3 \u2013 CZ , 4 \u2013 PZ .This was followed by a common factor analysis in order to identify the underlying dimensions measured by the EEG data. We examined each of the phenotypes for normality before including it in the analysis. In the common factor model, each new phenotype is expressed as a linear combination of the original variables. The relationship of factors to the EEG phenotypes is reflected by factor loadings. The contribution of each factor to the set of variables is evaluated by eigenvalues. Based upon the distribution of the eigenvalues and the composition of the factors, we retained four factors. This solution accounted for 88% of the total variance. We used an oblique rotation of the factor solution. Factor scores were obtained using PROC FACTOR implemented in SAS . We treated each of the four factor scores as a new derived quantitative trait.Quantitative data usually provide more statistical power than a binary affection status. However, using the quantitative traits alone may still not be powerful enough to identify disease susceptibility genes for complex traits. Kruglyak predicted that using single-nucleotide polymorphism (SNPs) with a heterozygosity of 0.50 and approximately two to three times the density of the current microsatellite marker sets would achieve a similar result in linkage analysis as a genome scan with microsatellite markers . Recentlr2 were calculated for all SNPs by HAPLOVIEW (v3.0) [Because linkage analysis algorithms assume linkage equilibrium between all markers, strong LD between SNPs may exaggerate the significance level of linkage and thus generate false positive results . So we kW (v3.0) . HaplotyW (v3.0) .We performed variance components analysis for each factor by using SOLAR (v2.13) . In vari2q + 2\u03a6\u03c32g + I\u03c32e,\u03a9 = \u03a0\u03c3qij, which is the expected proportion of genes two individuals share as identical by descent (IBD) at specific chromosomal location, \u03a6 is the kinship matrix, I is the identity matrix, \u03c32q is the variance component corresponding to the additive genetic effects from the major locus, \u03c32g is the variance component corresponding to the polygenic effects, and \u03c32e is the variance component corresponding to the environmental effects. The variance components analysis tested the null hypothesis that the additive genetic variance caused by the major quantitative trait locus (QTL) for a given trait equals zero . The hypothesis testing was conducted by comparing the maximum likelihood of a restricted model in which \u03c32q was constrained to zero with a more general model in which \u03c32q was estimated, using the likelihood ratio test. Twice the difference of the natural logarithm likelihoods of the two models yields a test statistic that is asymptotically distributed as a 50/50 mixture of a \u03c72 and a point mass of zero. The log10 of the likelihood ratio between the two models yields a LOD score that is equivalent to the classical LOD score of linkage analysis [h2) for each factor were estimated using SOLAR.where \u03a9 is the covariance matrix for a pedigree, \u03a0 is a matrix with elements \u03c0analysis . The IBDp < 0.05). Similar patterns were seen in ttdt3 and ttdt4.EEG measures and loadings on each of the four factors F1, F2, F3, F4) obtained from factor analysis are shown in Table , F3, F4 p < 0.001). We found significant evidence of linkage for F3 to chromosome 18 (LOD = 3.45 at 58 cM) in the Affymetrix SNP dataset. We had similar findings in the microsatellite (LOD = 2.73 at 61 cM) and Illumina SNP dataset (LOD = 3.30 at 56 cM) , F2 (32.1 \u00b1 5.9), F3 (30.7 \u00b1 6.2), and F4 (30.8 \u00b1 6.7) was all significant , representing the midline measures , was significantly different between affection status groups as defined by both ALDX1 and ALDX2.2+-dependent release of neurotransmitters and neuropeptides from the presynaptic nerve terminal. SYT4 expression was only detected in the brain, and was highest in the hippocampus [We found a novel genetic locus with significant evidence of linkage to F3  on chromosome 18, indicating this region (18q12.1-12.3) may harbor a gene that confers liability for alcohol dependence. A search of genome databases revealed a potential candidate gene SYT4 located in the genetic locus on 18q12.3 where we found significant linkage. Synaptotagmin-4, encoded by SYT4, may play an important role in the Capocampus . An animpocampus . Based oBy using the SNPs in the genome-wide linkage analysis we observed a higher LOD score than using the microsatellite markers. The peak of linkage was also sharper for the SNPs with a smaller confidence interval than for the microsatellite markers.In this study, our results from both SNPs and microsatellites suggest that there is a strong linkage of F3, which mostly consists of ttdt2, ttdt3 and ttdt4, to chromosome 18. We demonstrated that, in a sample like the COGA data, a dense SNP map with a quantitative trait could provide better linkage signals than low-resolution microsatellite scan for linkage analysis, and would also help define the peak of linkage more precisely.ANOVA: Analysis of varianceCOGA: Collaborative Study on the Genetics of AlcoholismEEG: ElectroencephalogramGAW: Genetic Analysis WorkshopIBD: Identical by descentLD: Linkage disequilibriumQTL: Quantitative trait locusSNP: Single-nucleotide polymorphismYY participated in the design of the study, performed the statistical analysis and drafted the manuscript. YM, QM, JF, and LAF participated in its design and coordination. MAW participated in the design of the study and helped to draft the manuscript. All authors read and approved the final manuscript."}
+{"text": "Plasma triglyceride and high density lipoprotein cholesterol levels are inversely correlated and both are genetically related. Two correlated traits may be influenced both by shared and unshared genes. The power to detect unshared trait-specific genes may be increased by incorporating correlated traits as covariates. The power to localize the shared genes may be improved by bivariate analysis. Univariate genome scans were carried out on triglyceride (high density lipoprotein cholesterol) with and without using high density lipoprotein cholesterol (triglyceride) as a covariate, and bivariate linkage analysis on triglyceride and high density lipoprotein cholesterol using the 330 Framingham pedigrees of the Genetic Analysis Workshop 13 data. The results of five genome scans were compared to determine the chromosomal regions which may harbor the genes influencing variation specific to triglycerides, specific to high density lipoprotein cholesterol, or the covariation of both triglyceride and high density lipoprotein cholesterol.The results of our five genome scans identified some chromosomal regions with possible quantitative trait loci (QTL) that may specifically influence one trait, such as the regions on chromosome 1 (at 1 cM near marker 280we5), on high density lipoprotein cholesterol, or control the covariation of both traits, such as the regions on chromosome 7 (at 169 cM near marker GATA30D09), chromosome 12 (at 3 cM near marker GATA4H03), chromosome 20 (at 49 cM near marker GATA29F06), chromosome 2 (at 146 cM near marker GATA8H05), and chromosome 6 (at 148 cM near marker GATA184A08) on triglyceride and high density lipoprotein cholesterol. The one on chromosome 6 had a LOD score of 3.1 with the bivariate linkage analysis.There is strong evidence for a QTL on chromosome 6 near marker GATA184A08 appearing to influence the variation of high density lipoprotein cholesterol and triglycerides in the Framingham population. Genetic components play important roles in determining serum lipid levels including plasma triglyceride (TG) and high density lipoprotein cholesterol (HDL-C) levels. Studies demonstrate that both TG and HDL-C concentrations are significantly related to cardiovascular diseases ,2. ThereThe real Framingham data , problem 1) were used in this study. The Framingham Heart Study began in 1948 with the recruitment of 5209 residents, 2336 men and 2873 women aged 28\u201362 years, from Framingham, Massachusetts. The subjects have undergone biennial examinations since the study began. In 1971, the Framingham Offspring Study was initiated, in part, to evaluate the genetic components of cardiovascular disease etiology. In total, there were 5124 subjects aged 5\u201370 years recruited. The offspring subjects have been examined every 4 years (except in the first two examinations with 8 years intervening). Our study population included the 330 largest, extended Framingham families with a total of 1702 genotyped individuals.TG, HDL-C, and the other phenotypes used in these analyses were measured at the original cohort examinations 10\u201312  and first offspring cohort examination. Both cohorts were measured almost at the same time, in the early 1970s. The measurement of these phenotypes has been detailed elsewhere .Variance-component univariate linkage analysis implemented in SOLAR (1.7.4)  was usedp < 0.01).The total number of individuals measured for TG, HDL-C as well as all covariates used for the heritability estimates and linkage analyses  was 1999. The correlation between logTG and logHDL-C was -0.4 , and the LOD scores for bivariate analyses were all decreased as well. No new chromosomal region was identified with a LOD score reaching 1.5 by scan 2.Our scan 1 was similar to a genome scan of logTG in the Framingham Heart Study . The twoScans 3 and 4 were of logHDL-C with and without logTG as a covariate. On scan 3, two chromosome regions were identified with maximum LOD scores equal to or greater than 1.5 . When an additional covariate, logTG, was included, the peak LOD score on chromosome 2 decreased from 2.6 to 1.7, and the peak LOD score on chromosome 6 decreased slightly from 2.9 to 2.6. This may indicate that these two regions harbor shared genes controlling the covariation for both traits, and that the shared gene on chromosome 2 may play a greater role in controlling the variation of HDL-C than that of TG, because this region was not identified by the scans of logTG. In the bivariate linkage analyses, the LOD score on the chromosome 6 region at 148 cM increased (LOD = 3.1) (Figure The region on chromosome 6 with highest multipoint LOD scores in the last three genome scans is near marker D6S1009-GATA184A08. Interestingly, the same region was identified by a genome scan of TG as well as BMI, a TG and HDL-C related trait, in nondiabetic Mexican Americans .In conclusion, the results of our five genome scans identified chromosomal regions in which QTL may reside and that might influence one of the traits, such as HDL-C, or control the covariation of both traits, TG and HDL-C. There is strong evidence for a QTL on chromosome 6 near marker GATA184A08 appearing to influence the covariation of HDL-C and TG in the Framingham population."}
+{"text": "The number of chromosome arms which were covered by these maps remained unknown.The development of large genomic resources has become a prerequisite to elucidate the wide-scale evolution of genomes and the molecular basis of complex traits. Linkage maps represent a first level of integration and utilization of such resources and the primary framework for molecular analyses of quantitative traits. Previously published linkage maps have already outlined the main peculiarities of the rainbow trout meiosis and a correspondance between linkage groups and chromosome arms has been recently established using fluorescent in situ hybridization results. The present map is therefore assumed to cover the 52 chromosome arms which constitute the rainbow trout karyotype. Our data confirm the occurrence of a high interference level in this species. Homeologous regions were identified in eleven linkage groups, reflecting the tetraploid nature of the salmonid genome. The data supported the assumption that gene orders are conserved between duplicated groups and that each group is located on a single chromosome arm. Overall, a high congruence with already published rainbow trout linkage maps was found for both gene syntenies and orders.We report an updated linkage map based on segregation analysis of more than nine hundred microsatellite markers in two doubled haploid gynogenetic lines. These markers segregated into 31 linkage groups spanning an approximate total map length of 2750 cM. Centromeres were mapped for all the linkage groups using meiogenetic lines. For each of the 31 linkage groups, the meta or acrocentric structure infered from centromere mapping was identical with those recently found with fluorescent This new map is likely to cover the whole set of chromosome arms and should provide a useful framework to integrate existing or forthcoming rainbow trout linkage maps and other genomic resources. Since very large numbers of EST containing microsatellite sequences are available in databases, it becomes feasible to construct high-density linkage maps localizing known genes. This will facilitate comparative mapping and, eventually, identification of candidate genes in QTL studies. The development of genomic resources is a key step towards further investigation of wide-scale genome evolution, identification and study of gene or gene networks involved in complex characters. Such efforts have naturally focussed on genetic model systems and species of applied biomedical or agronomical importance. In the case of rainbow trout, genetic resources have been primarily developed in order to study the gene networks controlling the phenotype of traits that affect breeding performances or fisheries sustainability and to identify mutations responsible for the genetic variation of these traits. However, the rainbow trout is also a suitable model to investigate more academic scientific questions (reviewed in ), such aThe construction of linkage maps is a primary step towards the description and understanding of the structure and evolution of whole genomes. In rainbow trout, several linkage maps based on segregations at AFLP and microsatellite loci have been already published -11 and sThese linkage maps have allowed to ascertain several important features of the meiosis and genome structure in the rainbow trout. Briefly, female maps are three to four times longer than male ones on the average, while telomeric regions show higher average recombination rate in male than in female . SeveralThe current consensus linkage map consists of 31 major linkage group ,9. SinceIn this article, we report a new rainbow trout map. The main objective was to cover all the chromosomes arms. This information is of importance in rainbow trout since, due to Robertsonian polymorphism, the number of chromosomes and, therefore, of independent linkage groups can vary among families while the number of chromosomes arms are the constant basic characteristic of the rainbow trout karyotype. The construction of the map was based on segregation analysis of nearly one thousand microsatellite markers in two doubled haploid lines, which are currently used in our laboratory. A particular effort was made to map the centromere of each linkage group using meiogynogenetic progeny in order to determine the acrocentric or metacentric structure of each linkage group.Finally, since approximately one third of the mapped microsatellites originated from ESTs sequences, we attempted to recover conserved syntenies between rainbow trout and zebrafish by comparing the map localisation of homologous sequences in the two species.Out of 1321 microsatellite primer pairs tested, 168 did not give any amplification at two annealing temperatures (58 and 52\u00b0C) and 366 were monomorphic in the test sample and 796 were informative in one family at least. Three hundred and eighty-nine microsatellite primer pairs were designed from EST sequences. Eighty primer pairs originated from USDA EST sequences, of which eight have been already mapped )Four linkage groups in INRA1  and one in INRA2 (RT7) were composed of two unlinked fragment. This could reflect a Robertsonian polymorphism or, alternatively, an insufficient local coverage of the linkage group in one family. In the case of a Robertsonian polymorphism, the two unlinked fragments would reflect the independence of the two acrocentric arms. When considering two independent acrocentric chromosomes as a single metacentric one, 1/8 of the progeny is expected to exhibit triple crossovers under complete interference: one crossover on each acrocentric group and one recombinant due to the co-segregation of centromeres from each grandparent. Since we did not find any convincing case of triple crossover at these linkage groups, Robertsonian polymorphism is unlikely to explain the occurrence of unlinked fragments in one of the family. Note also that only two triple crossovers were found instead of 15  when RT2A and RT2B are grouped, suggesting that these two fragments could be linked in our DH families as in ,23Assignment of each linkage group to a specific chromosome will allA total number of 214 microsatellite loci were common to the female-specific maps published by  and thisOne hundred and forteen microsatellite loci covering all the linkage groups were common to the map of  and oursOverall, both syntenies and gene order appear to be conserved among the different linkage maps.The localisation of type I marker on this map has provided some evidences of conservation of syntenies between zebrafish and rainbow trout, though it is not the rule. Of particular interest are the alignment of zebrafish linkage groups 3 and 19 on the linkage map of rainbow trout and the complex architecture of the syntenies found. Even if the comparison of the zebrafish and rainbow trout genome is still very preliminary and based on a crude assessment of sequence homologies, these findings suggest that chromosome rearrangements have been frequent between zebrafish and rainbow trout at least over long map distances, but that cases of conserved syntenies can be found.Extensive gene duplication has been reported early in salmonids and has been a crucial argument in favour of the hypothesis of tetraploidy in salmonids. Our data confirm several homeology between linkage groups already reported or hypothesized, namely RT2\u2013RT9, RT2\u2013RT29, RT3\u2013RT25, RT7\u2013RT15, RT10\u2013RT18, RT12\u2013RT16, RT14\u2013RT20 and RT27\u2013RT31  lines . The source population was an experimental INRA rainbow trout strain. A grand-parental population of all homozygous doubled haploids was first established, using the mitotic gynogenesis . BrieflyCentromeres were mapped using a diploid meiogenetic family . To produce this meiogenetic family, eggs from a single female were fertilized with UV irradiated sperm and diploidy was restored by retention of the second polar body .All the individuals  were fin clipped and fin clips were stored in 95% ethanol for subsequent analyses.-3), then manually validated. New microsatellite markers were named in accordance with the convention outlined by [A total number of 1321 primer pairs were tested. Name, references, primer sequences of markers not submitted to GenBank are given see . Five hulined by . For alllined by . PCR teslined by .++ concentration was fixed to 2 mM.Genomic DNA samples were prepared from ethanol preserved fin tissues following simplified phenol extraction and ethanol precipitation procedures . The PCRTwo sequences, BX087958 and BX087759, were mapped by SNP-based genotyping using sequencing and PCR-RFLP procedures. Fragments of 550 and 650 base pairs respectively were amplified by PCR. Amplifications  were carried out in 20 \u03bcl reactions containing 40 ng of genomic DNA template, 1X reaction buffer (Promega), 0.4 \u03bcM of each primer, 250 \u03bcM of each dNTP, 2 mM MgCl2, 0.6 units Taq polymerase (Promega). SNP polymorphism in females was detected by sequencing. PCR products were purified by treatment with 2 units of exonuclease 1 and one unit of shrimp alkaline phosphatase. SNP polymorphism in the mothers of DH progeny was detected by sequencing using the ABI Big Dye terminator chemistry on an ABI 310 sequencer. Progeny were genotyped by PCR-RFLP methods: BX087958 and BX087579 PCR products were cut with BsrB I and Taq I respectively following Biolabs protocol and fragments were separated in 2% agarose.Genotypes were converted into a backcross format with linkage phase deduced from the genotypes of the grandparents, each offspring being equivalent to one gamete. Since linkage phase was known, the two data files obtained for each female were merged and analysed as a single data set. Linkage groups, gene orders and map distances were obtained with CARTHAGENE . LinkageThough it was not expected in female meiosis, the occurrence of pseudo-linkage was checked using a genotype file with inverted linkage phase.et al. [et al. [The linkage group numbers followed the current nomenclature ,9. CorreGraphic representations of linkage groups were generated with MAPCHART version 2.1 . The perCentromeres were mapped through genotyping of meiogenetic progeny for several loci per linkage group. For each linkage group, loci were ordered manually in such a way that the number of recombination events was minimum. This condition is expected under the hypothesis of complete interference. The most likely interval in which the centromere lies was deduced from the gene-centromere distances , avoiding, as far as possible, double recombinants per arm (hypothesis of nearly complete interference). Details of this half-tetrad analysis can be found in .-5 were kept. The output file displayed the BLAST results and the location of the rainbow trout genes on the chromosomes of the zebrafish for each query sequence showing significant homology.The EST sequences of rainbow trout were masked from known repeats and vector sequences and compared to all the transcript sequences of the zebrafish using the Iccare Web server  with theRG conceived the project, supervised data analyses and interpretations and wrote the manuscript. SMR contributed to genotyping, collection of segregation data, data treatments and map construction. KTC produced the EST-based microsatellites from the EST-SIGENAE database, contributed to genotyping, segregation data collection and analyses. SMU contributed to the EST-SIGENAE database screening, genotyping, segregation data analyses. CG contributed to data analysis, performed comparative mapping with zebrafish and tentative annotation of rainbow trout sequences and contributed to manuscript drafting. FK contributed to EST-SYGENAE database screening and genotyping. EQ contributed to project conception and manuscript drafting and produced the mapping DH families. All authors read and approved the final manuscript.Correspondence of the map reported in this study with already published linkage maps and with chromosome identified by FISH. Number of markers and crossovers per linkage group.Click here for fileEST accession number or reference for microsatellite loci unavailable in GENBANK.Click here for fileFunctional Annotation of microsatellites markers associated to ESTs. Tentative annotations from Oncorhynchus mykiss UniGene Build  or from Click here for file"}
+{"text": "Using the simulated data set from Genetic Analysis Workshop 13, we explored the advantages of using longitudinal data in genetic analyses. The weighted average of the longitudinal data for each of seven quantitative phenotypes were computed and analyzed. Genome screen results were then compared for these longitudinal phenotypes and the results obtained using two cross-sectional designs: data collected near a single age (45 years) and data collected at a single time point. Significant linkage was obtained for nine regions (LOD scores ranging from 5.5 to 34.6) for six of the phenotypes. Using cross-sectional data, LOD scores were slightly lower for the same chromosomal regions, with two regions becoming nonsignificant and one additional region being identified. The magnitude of the LOD score was highly correlated with the heritability of each phenotype as well as the proportion of phenotypic variance due to that locus. There were no false-positive linkage results using the longitudinal data and three false-positive findings using the cross-sectional data. The three false positive results appear to be due to the kurtosis in the trait distribution, even after removing extreme outliers. Our analyses demonstrated that the use of simple longitudinal phenotypes was a powerful means to detect genes of major to moderate effect on trait variability. In only one instance was the power and heritability of the trait increased by using data from one examination. Power to detect linkage can be improved by identifying the most heritable phenotype, ensuring normality of the trait distribution and maximizing the information utilized through novel longitudinal designs for genetic analysis. Studies designed to identify genes contributing to complex disease have been ongoing for many years, utilizing assorted study designs and analysis methods with varied success. The Genetic Analysis Workshop 13 (GAW13) simulated data set provides an opportunity to explore the advantages of longitudinal as compared with cross-sectional study designs. For each univariate phenotype, we compared linkage results generated from a weighted average of the longitudinal data with results from two different cross sectional designs: data gathered at a single examination and data obtained from one examination collected during the age range (mid-40s) when the phenotypes were most heritable.All analyses were performed using the simulated data set without knowledge of the underlying model, including number of genes or their location. Analyses were performed with complete genotypic and phenotypic data from Replicate 1. The true model was obtained only at GAW13, and was used in this manuscript to identify true and false positives.The simulated data set included a number of cardiovascular phenotypes, blood pressure, lipid, and glucose measurements, which were collected longitudinally in the study subjects. Unfortunately, data were gathered at different intervals in the first and second cohort. In addition, the subjects in the two cohorts participated in the study at variable ages. To extract maximal informativeness for linkage studies, we developed phenotypes that were both heritable and capitalized on the longitudinal phenotypic information.A univariate phenotype was calculated for each of the measures: body mass index (BMI), total cholesterol (CHOL), fasting glucose (GLUC), high-density lipoprotein (HDL), height (HEIGHT), systolic blood pressure (SBP), and triglycerides (TG). Longitudinal data was provided for these traits at different time points and with differing amounts of time between consecutive measurements. The area under the curve obtained by joining consecutive measurements over the time scale capitalized on the longitudinal nature of the data, yet accounted for the non-uniform amount of time between consecutive measurements . This arData points were also selected to simulate a cross-sectional study design . Exam 12 was selected for Cohort 1, since it was the only patient visit where all phenotypes were collected . Exam 1 was selected for Cohort 2. Another cross-sectional design selected data points to simulate a study design that collected people in their mid-40s (AGE = 45). The patient visit closest to age 45, and within 5 years, was selected for each individual for each phenotype. The age of 45 was chosen because data were available for the largest number of individuals  and the phenotypes tended to be highly heritable at that age (0.71\u20130.81 versus 0.56\u20130.85). For these cross-sectional study designs, the number of individuals with available data was smaller than that for the longitudinal design. So, to make a valid comparison between the two methods, an additional set of longitudinal phenotypes were calculated for each of the cross-sectional designs that utilized the same individuals.  , and so p < 0.10). Traditionally, a nonparametric lod score of 3.6 has been used as a genome-wide significance level. However, given our use of seven phenotypes, we adjusted our significance level based on a Bonferroni correction. A LOD score of 3.6 corresponds to a p-value of 2.33 \u00d7 10-5. After correcting for seven tests, the new alpha would be 3.33 \u00d7 10-6, and so all chromosomal locations with LOD scores greater than 4.4 were identified and prioritized for further evaluation.Heritability estimates were calculated for each of the phenotypes using SOLAR. Multipoint linkage analysis was performed for all seven phenotypes  for each of the three study designs  using the pedigree-based variance component method implemented in the program SOLAR with simultaneous correction for [average] age in study, body mass index, cigarettes per day, alcohol consumption (drinks) and gender when they were significant covariates . The highest level of linkage evidence (LOD = 34.6) was found for the fasting glucose phenotype, directly at the Gs3 gene on chromosome 5. The HDL phenotype linked within 4 cM of Gb12 on chromosome 9 (LOD = 9.6) and between Gb19 and Gb20 on chromosome 17 (LOD = 7.4). Height linked to both Gb1 on chromosome 5 (LOD = 16.8) and Gb2 on chromosome 7 (LOD = 6.7). SBP linked to within 3 cM of Gs11 on chromosome 15 (LOD = 6.4) and to within 7 cM of Gs10 on chromosome 21 (LOD = 33.8). The longitudinal data for triglycerides did not yield a LOD score above 4.4. But more importantly, there were no false-positive linkage results at this level of significance.For the longitudinal data (AUC), nine chromosomal regions produced LOD scores greater than 4.4. Linkage (LOD = 28.7) was identified between BMI and chromosome 13, 6 cM from gene Gb14 at the q-terminus of chromosome 1. The two chromosomal regions that were no longer detected were the region on chromosome 15 for SBP and the linkage to chromosome 11 for cholesterol.Analyses performed using the cross-sectional study designs yielded consistently lower LOD scores in comparison with the longitudinal measures  in the full longitudinal sample were still detected in the smaller longitudinal samples.p = 0.02). Even with maximal information from the longitudinal study design and ideal conditions such as complete genotype and phenotype data, two genes contributing a large percentage of the variance to HDL and SBP  were not identified. Also, despite this large data set with complete information, we were unable to detect genes with smaller effects. No baseline gene accounting for less than 15% of the trait variance was detected in our analyses. However, the models we utilized also did not identify any false-positive linkage findings.Several conclusions can be made from these results. First, it is not surprising that the highest LOD scores were obtained near genes accounting for the greatest percentage of the trait variance. The baseline genes identified by the six highest LOD scores (4.4 \u2264 LOD \u2264 28.7) were among the genes accounting for the greatest trait variance (between 15% and 40%). The correlation between these LOD scores and the proportion of phenotypic variance each explained was 0.88 . Removal of the extreme phenotypic outliers reduced the number of false-positive results dramatically (data not shown). By removing these values, the kurtosis of the distribution was reduced from 37.5 to 2.2 and from 21.5 to 1.7 for GLUC and TG, respectively. But while this improved the sensitivity and specificity immensely, GLUC and TG still have the highest levels of kurtosis and are the only measurements with false-positive linkage results. From these data, it appears that it is more important to remove extreme values for the sake of specificity than it is to retain them for power.Third, when looking at the same phenotype measured cross-sectionally or longitudinally, the heritability of the trait tended to correlate with the magnitude of the linkage signal. This is of particular importance when examining TG levels. The heritability, and hence LOD scores, increased and reached our level of statistical significance only when the subset of subjects with an age near 45 were included in the analysis. By maximizing the heritability of the trait prior to genetic analysis, the power to detect genetic effects was increased.Our analyses demonstrated that the use of simple longitudinal phenotypes, AUC, was a powerful means to detect genes of major to moderate effect on trait variability. In a few instances, where the heritability of the trait was increased at a specific age, examining cross-sectional data at a uniform age (TG) provided higher LOD scores and linkage to genes that were not detected using other phenotypic modelling. We conclude that it is important to carefully examine phenotypic traits so as to maximize the ability to detect genes for complex traits. A variety of strategies may be appropriate depending on the situation and the underlying biology. In some instances, a multivariate phenotype that accurately models the underlying biology will provide the most power. However, in most instances, the underlying biology is unknown and assumptions must be made so as to develop phenotypes for genetic analyses. Other strategies to define the phenotypic trait accurately and improve power include identifying the most heritable phenotype, ensuring normality of the trait distribution, and maximizing the information utilized through novel longitudinal designs for genetic analysis."}
+{"text": "We explored the evidence for a quantitative trait locus (QTL)-specific genotype \u00d7 alcoholism interaction for an evoked electroencephalogram theta band oscillation (ERP) phenotype on a region of chromosome 7 in participants of the US Collaborative Study on the Genetics of Alcoholism. Among 901 participants with both genotype and phenotype data available, we performed variance component linkage analysis (SOLAR version 2.1.2) in the full sample and stratified by DSM-III-R and Feighner-definite alcoholism categories. The heritability of the ERP phenotype after adjusting for age and sex effects in the combined sample and in the alcoholism classification sub-groups ranged from 40% to 66%. Linkage on chromosome 7 was identified at 158 cM (LOD = 3.8) in the full sample and at 108 in the non-alcoholic subgroup (LOD = 3.1). Further, we detected QTL-specific genotype \u00d7 alcoholism interaction at these loci. This work demonstrates the importance of considering the complexity of common complex traits in our search for genes that predispose to alcoholism. Neurophysiological features extracted from electroencephalogram (EEG) data, such as event related potentials (ERPs), provide a non-invasive endophenotype to study cognitive functioning in humans . ERPs arThe US Collaborative Study on the Genetics of Alcoholism (COGA) began in 1989 in order to elucidate genetic mechanisms that influence susceptibility to alcohol abuse and related phenotypes [Details of the EEG phenotypes have been described elsewhere ,9. Briefn = 901). The \"affected\"  sample comprised those individuals who displayed at least one symptom in three out of four possible categories of symptoms (n = 457). The \"unaffected\"  sample comprised individuals who used alcohol but did not report any symptoms of alcohol dependence (n = 148). There is no overlap between affected and unaffected classifications. Individuals who failed to be classified as affected or unaffected (n = 296) were considered unknown because clinically no definite diagnosis could be made. These individuals are likely a heterogeneous group, which would impair our ability to detect gene \u00d7 environment interactions.Alcoholism was diagnosed using the Diagnostic and Statistical Manual of Mental Disorders, Third Edition, Revised (DSM-III-R) criteria for alcohol dependence and the Feighner et al.  criteria2, and age2 by sex interaction on the phenotype trait mean. Because data were adjusted for covariates, heritability reported will be the proportion of additive genetic variance over the phenotypic variance after adjustment for covariates. Additionally, a correction for ascertainment was made by conditioning the likelihood of each pedigree on the phenotypic values of its probands [t-distribution function was implemented to account for the slightly kurtotic distribution of the phenotype [Univariate quantitative genetic analysis was performed to partition the phenotypic variance of the ERP into its additive genetic and environmental variance components using maximum likelihood variance decomposition methods  implemenprobands . A genomprobands . The t-dhenotype . Marker To examine the effect of alcoholism on the phenotype of interest, we used four methods: inclusion of alcoholism as a covariate , test for additive G \u00d7 A interaction, linkage analysis by subgroup, and test for G \u00d7 A at the linkage peak.To test for evidence of G \u00d7 A, we extended the expected genetic covariance to have different effects based on environment:e1,Ge2) = 2 \u03c6 \u03c1G \u03c3ge1 \u03c3ge2,COV is the genetic correlation between the trait in the two environments, and \u03c3ge1 and \u03c3ge2 are the genetic standard deviations in the two environments (affected and unaffected) [o: \u03c1G = 1.0) and the genetic variances in the two groups should be equal (Ho: \u03c3ge1 = \u03c3ge2). Conversely, if there is G \u00d7 A interaction, the genetic correlation between the groups will be significantly less than one  < 1.0) and/or the genetic variances will not be equal between the groups (HA: \u03c3ge1 \u2260 \u03c3ge2). Rejection of either null hypothesis is evidence of an additive G \u00d7 A. To formally test these hypotheses, we used the likelihood ratio test to compare restricted models in which \u03c1G is constrained to one or the genetic standard deviations are constrained to be equal. When comparing models with standard deviations constrained to be equal, interpretation of significant differences were based on the assumption of an asymptotic  distribution for the likelihood test statistic. However, for the model that restricted the genetic correlation to one, the genetic correlation was constrained to the upper boundary of the parameter space (\u03c1G = 1.0), thus the test statistic is as a 1/2:1/2 mixture of a  distribution and a point mass at zero [where \u03c6 is the coefficient of kinship between the two individuals, \u03c1ffected) -17. In t at zero .To test for evidence of a QTL-specific G \u00d7 A, two additional parameters were added, the marker specific standard deviations for the affecteds and unaffecteds . Therefoe1,Ge2) = 2 \u03c6 \u03c1G \u03c3g e1 \u03c3ge2 +\u03c0q\u03c3qe1 \u03c3qe2,COV and 158 cM (p = 0.012), suggesting a stronger genetic effect in the unaffected group.Although no additive G \u00d7 A was detected, significant QTL-specific G \u00d7 A was detected when comparing affected participants to unaffected participants for both loci. Although the subset linkage analysis by alcoholism suggests that there would be a stronger effect in alcoholics as that group had the greatest linkage signal, the marker specific standard deviations for the unaffected group were larger than the affected group at locus 108 , both the linkage data from alcoholism subsets and formal tests of QTL-specific G \u00d7 A support differential genetic effects in alcoholic and non-alcoholic individuals.p-terminal end. It is unclear which factors are influencing this shift; possibilities include low information content of the markers between these two linkage peaks or two QTLs. However, an oligogenic linkage analysis did not support the presence of a second QTL on chromosome 7 (data not shown).The heritability of ERP phenotype in the full sample and in the alcoholism classification sub-groups ranged from 40% to 66%, but these differences were not statistically different because the standard errors of the estimates overlapped. When subsetting based on alcoholism, we found that alcoholic and non-alcoholic participants displayed a peak LOD score 50 cM toward the Significant QTL-specific G \u00d7 A interaction was detected when comparing affected participants to unaffected participants at 108 and 158 cM. When estimating QTL specific G \u00d7 A, a total of 605 individuals entered the analysis, representing 1,108 relative pairs, including 404 relative pairs discordant for alcoholism, 640 relative pairs concordant for alcoholism and 64 relative pairs concordant for non-alcoholism status. When compared to alcoholics, non-alcoholics displayed larger marker-specific standard deviations at 108 and 158 cM. This may seem counterintuitive because the non-alcoholics displayed lower evidence of linkage (LODs \u2264 1.0). However, given the small number of relative pairs concordant for unaffected status, the power to detect linkage in this group was low. Indeed, simulation analysis suggests that the LOD was underestimated by 15% while for affected, the LOD was overestimated by 25%. However, by formally modeling a genotype \u00d7 environment interaction, we are able to include the 404 pairs of relatives discordant for alcoholism and thus improve our ability to detect genetic effects.We also explored the effect of alcoholism classification, by examining the effect of inclusion of the individuals who were not clearly affected or unaffected (data not shown). We found that including these ambiguous individuals eliminated our ability to detect G \u00d7 A. There are several possible reasons for this such as genetic or environmental heterogeneity of the ambiguous group. Additionally, we examined only the White sample to remove race/ethnicity effects, and found that the G \u00d7 A interaction at 108 and 158 cM persisted.p-ter, suggesting a second QTL or poor localization of the trait. This work demonstrates the importance of considering the interaction of genes and environment in the etiology of common complex traits. In future studies, researchers must consider the impact of genetic and environmental heterogeneity and formally test for these effects to improve our ability to find and localize genes involved in complex traits such as alcoholism.In summary, using the COGA data, we attempted to disentangle the effects of alcoholism on an EEG theta band oscillation phenotype that has been linked to a region of chromosome 7. Although we failed to detect evidence for an additive G \u00d7 A, we detected evidence for a different magnitude of effect in alcoholics versus non-alcoholics at the QTL level. Further, the subsetting of this data shifted the linkage peak 50 cM COGA: Collaborative Study on the Genetics of AlcoholismEEG: ElectroencephalogramERP: Event-related potentialG \u00d7 A: Genotype \u00d7 alcoholismGAW14: Genetic Analysis Workshop 14QTL: Quantitative trait lociLJM and KEN performed statistical analyses and interpreted results. JTW and CLA assisted in the interpretation of the results. All authors read and approved the final manuscript."}
+{"text": "We describe a method for mapping quantitative trait loci that allows for locus heterogeneity. A genome-wide linkage analysis of blood pressure was performed using sib-pair data from the Framingham Heart Study. Evidence of linkage was found on four markers  at a significance level of 0.01. Two of them (GATA14E09 and 049xd2) seem to overlap with linkage signals reported previously, while the other two are not linked to any known signals. High blood pressure (BP) is an important risk factor for cardiovascular disease and is a leading cause of mortality in industrialized countries . Being aWe propose a model that allows for locus heterogeneity for quantitative trait loci (QTLs). This model generalizes the locus heterogeneity model for qualitative traits ,3 to theThe Framingham Heart Study consists of two cohorts with a total of 10,333 subjects. The first cohort includes 5209 individuals who were recruited initially in 1948 when the study began. The second cohort includes 5124 subjects who are offspring, or spouses of offspring, of the initial participants. Longitudinal data were recorded over follow-up years in both cohorts. In the total 330 pedigrees provided by the Genetic Analysis Workshop 13 (GAW13), 1702 subjects were genotyped at at least one marker and 2885 subjects were phenotyped for BP at least once.To remove the effect of hypertension treatment, we focused only on those subjects who never received any antihypertensive medication. There are 1909 such subjects. Their characteristics are summarized in Table A total of 400 markers were used in the genome scan. Not all sib pairs were genotyped at every marker. So the number of sib pairs used in the analysis varied from marker to marker, roughly from 400 to 1500.p-value < 0.0001). To reduce the impact of the non-normality of the data on the performance of the proposed statistic, the BP measurements were transformed based on the normal copula model ,l , the likAt significance level 0.01, four significant markers are identified by statistic HET-LRT. Among the four markers, GATA14E09 on chromosome 8 is very close to 8q21.11, a region that showed linkage to BP [We introduce a heterogeneity model for mapping QTLs using sib-pair data. A genome scan was performed to map QTLs affecting BP variation on a population-based data using this new method. At significance level 0.01, evidence of linkage is found in four marker regions on chromosomes 5, 8, and 16. Two of the markers seem to overlap with linkage signals in previous studies, while the other two are not linked to any previous findings.Of the three statistics used in the genome scan, H-E provides the most linkage signals, followed by HOM-LRT, and then by HET-LRT. At the four markers at which HET-LRT is significant (and at many nonsignificant markers that are not shown), the statistic value of HET-LRT is the same as that of HOM-LRT. Finer grid size may change the values of HOM-LRT and HET-LRT, but the changes are expected to be small. Experiments on the four significant markers with grid size 0.001 strongly support this claim. In these experiments, HET-LRT is still the same as HOM-LRT for all four markers.The proposed statistic HET-LRT is intended for a population sample. Its performance under selected samples is unknown. Although the normal copula model can be used on selected samples to recover normality, its effectiveness has yet to be investigated. It is worthwhile to point out that our analysis has excluded individuals on antihypertensive medication. Therefore, careful attention should be paid when generalizing our results to general population. For a recent review on the issues dealing with antihypertensive treatments, see Palmer .We have been using the posterior probability of linkage (PPL) to assess linkage signals across heterogeneous data sets -14. SincThe proposed model is for sib-pair data only. It is expected that the use of nonindependent sib-pairs, like what we did in this analysis, does not affect the type I error rate asymptotically. It is of interest to see how this model can be generalized to general pedigrees and how the generalized model performs."}
+{"text": "Genome-wide linkage analysis using microsatellite markers has been successful in the identification of numerous Mendelian and complex disease loci. The recent availability of high-density single-nucleotide polymorphism (SNP) maps provides a potentially more powerful option. Using the simulated and Collaborative Study on the Genetics of Alcoholism (COGA) datasets from the Genetics Analysis Workshop 14 (GAW14), we examined how altering the density of SNP marker sets impacted the overall information content, the power to detect trait loci, and the number of false positive results. For the simulated data we used SNP maps with density of 0.3 cM, 1 cM, 2 cM, and 3 cM. For the COGA data we combined the marker sets from Illumina and Affymetrix to create a map with average density of 0.25 cM and then, using a sub-sample of these markers, created maps with density of 0.3 cM, 0.6 cM, 1 cM, 2 cM, and 3 cM. For each marker set, multipoint linkage analysis using MERLIN was performed for both dominant and recessive traits derived from marker loci. Our results showed that information content increased with increased map density. For the homogeneous, completely penetrant traits we created, there was only a modest difference in ability to detect trait loci. Additionally, as map density increased there was only a slight increase in the number of false positive results when there was linkage disequilibrium (LD) between markers. The presence of LD between markers may have led to an increased number of false positive regions but no clear relationship between regions of high LD and locations of false positive linkage signals was observed. Genome-wide linkage analysis using microsatellite markers has been successful in the identification of numerous Mendelian and complex disease loci. Recently available high-density single-nucleotide polymorphism (SNP) maps theoretically provide greater information content (IC), which should help to both identify and narrow linkage regions. This is supported by a few published reports comparing genome-wide linkage analysis using microsatellites to studies of the same dataset using dense SNP maps ,2. Yet qAnalyses were performed (separately for each population and replicate) using all replicates of the Aiputo, Danaca, and Karanga populations. The full marker sets for both the MS (7.5 cM) and SNP (3 cM) maps were used. Additional fine mapping markers were purchased for chromosomes 8 and 9 (packets 400\u2013406 and 416\u2013419) to increase the density of the SNPs (0.3 cM). We had knowledge of the answers.Dominant or recessive traits were created using these marker loci: B08T8044, B08T8045, B08T8050, and B08T8051. Affection status for a dominant trait was defined as individuals with \u2265 1 copy of allele 1 at the marker and for a recessive trait as individuals with 2 copies of allele 1.Using a perl script, we created an interpolated genetic map that used MS markers from the deCode map and SNPs from both Illumina and Affymetrix. For each SNP, 2 MS markers from the deCode map were identified that flanked the SNP using the physical positions of these markers obtained from sequence build 34. From the physical and genetic position of the 2 flanking microsatellites and assuming a linear interpolation between the markers, the genetic position of the SNP was determined. Any MS or SNP without a physical position was removed. If SNP markers mapped to the same genetic location, the SNP with the largest physical location was kept.The following markers  were used to create a dominant and/or a recessive trait: rs0041510 , tsc2832191 , tsc0061481 . To avoid errors due to differences in allele frequencies between ethnic groups, analysis was limited to the white/non-Hispanic families, which comprised the largest ethnic subgroup.Using a perl script, we selected a subset of the SNP markers to create maps that were less dense. Our goal was to select markers with desired inter-marker distances. To avoid tight clusters of markers, we moved at least the desired distance minus 10% of that distance before another marker was selected. If there were multiple markers within \u00b1 10% of the desired distance, the marker with the major allele frequency (MAF) closest to 0.5 was selected. For example, for the 0.3-cM map, markers were forced to be at least 0.27 cM apart, and if there were multiple markers located between 0.27 cM and 0.33 cM from the last marker, the marker with the MAF closest to 0.5 was selected.p-values for Whittemore and Halpern's NPLAll [p-value thresholds  and 2 Lander-Krugylak [p-value less than the threshold within a 20 cM region (10 cM in either direction) of the trait loci. To assess the frequency of false positive results, we counted the number of regions where a p-value less than the above-mentioned cut-off occurred on chromosomes not containing the trait loci. In order to ensure that adjacent makers with p-values below the given level were not counted as multiple false positive results, a region with a p-value greater than or equal to 0.2 was required to occur between two false positive regions.We used the analysis program MERLIN for all linkage analyses . Allele s NPLAll  statistiKrugylak  genome-wTable There was a modest increase in power with increasing SNP map density in the simulated data Table . Power wp-value below a given level in a region unlinked to the trait loci) for the simulated data is in Tables p-value levels there were only a few false positive results, and no false positives were observed for any of the traits at genome-wide significant p-values (0.000049) [The number of false positive linkages (.000049) .Overall, IC was higher for the dense SNP maps as compared with the less dense SNP and MS maps. In the simulated data, there was a modest increase in power with increasing SNP map density. However in the COGA data, no consistent trends were observed in our ability to detect trait loci with increasing map density. There was variation in the LOD scores across maps, with more dense maps sometimes yielding lower LOD scores. This could be due to errors in map order and supports the need for precise genetic maps when using dense SNP maps for linkage. Unsurprisingly, power was dependent on disease prevalence for these homogeneous, completely penetrant traits.In the simulated data, in which there was no significant LD between markers, the number of false positives did not increase with increasing map density. In the COGA data, more false positives were observed for the densest map set, 0.25 cM, in which there was significant intermarker LD. Huang et al.  reportedCOGA: Collaborative Study of the Genetics of AlcoholismGAW14: Genetic Analysis Workshop 14IC: Information contentLD: Linkage disequilibriumMAF: Major allele frequencyMS: MicrosatelliteSNP: Single-nucleotide polymorphism"}
+{"text": "Using over 100 replicates, we found that if founders were not genotyped, multipoint IBD estimates and \u03b4 parameters were modestly inflated and NPLall and KC-LOD scores were biased upwards in the region with LD and no gene; rather than centering on the null, the mean NPLall and KC-LOD scores were 0.51 \u00b1 0.91 and 0.19 \u00b1 0.38, respectively. Reduction of LD by dropping markers reduced this upward bias. These trends were not seen in the non-LD region with no gene. In regions with genes (with and without LD), a slight loss in power with dropping markers was suggested. These results indicate that LD should be considered in dense scans; removal of markers in LD may reduce false-positive results although information may also be lost. Methods to address LD in a high-throughput manner are needed for efficient, robust genomic scans with dense SNPs.Current genome-wide linkage-mapping single-nucleotide polymorphism (SNP) panels with densities of 0.3 cM are likely to have increased intermarker linkage disequilibrium (LD) compared to 5-cM microsatellite panels. The resulting difference in haplotype frequencies versus that predicted may affect multipoint linkage analysis with ungenotyped founders; a common haplotype may be assumed to be rare, leading to inflation of identical-by-descent (IBD) allele-sharing estimates and evidence for linkage. Using data simulated for the Genetic Analysis Workshop 14, we assessed bias in allele-sharing measures and nonparametric linkage (NPL Gene-mapping endeavors currently assess linkage of up to 11,555 single-nucleotide polymorphisms (SNPs) distributed throughout the genome  iThe Aipotu population of 100 nuclear families simulated for GAW14 was used because of its relatively high prevalence of the phenotypes studied. One hundred replicates were separately analyzed. Analyses were performed with and without founder genotypes. Two dichotomous traits were analyzed: Trait H, due to Gene D2 in a region with LD, and Trait B, due to Gene D1 in a region without LD. Both traits were monogenic, dominant, and had no phenocopies. Penetrance and prevalence were 20% and 7.4% for Trait H and 30% and 2.1% for Trait B. All analyses were performed with full knowledge of the simulated genetic models [Four chromosomal regions were analyzed Figure . A regioTwo regions without simulated intermarker LD were analyzed Figure . These rr2 values based on the estimation maximization of founder haplotype frequencies in the second Aipotu replicate [LDMAX  and GOLDeplicate . One megall scores and Kong and Cox (KC)-LOD scores were calculated for each replicate using MERLIN v. 0.10.2 [all scores are normalizations of scores based on observed phenotypes and the binary inheritance vector at each location [\u03b4, the free parameter in a one-parameter allele-sharing model; under the null, \u03b4 equals 0, and, under the alternative, \u03b4 is greater than 0 [\u03b8 was converted to centimorgans using the Kosambi map function.Multipoint NPL. 0.10.2  which im. 0.10.2 . Both stlocation ,10. KC-Lr than 0 . \u03b8 was cWe compared regions with and without LD, and we compared regions with LD before and after LD reduction. We performed analyses under a variety of conditions: 1) whether allele frequencies were estimated from all individuals or from founders and 2) whether linkage statistics were calculated at five evenly spaced intervals between markers or at 0.2-cM intervals.n = 100), the mean probability of sharing 0, 1, and 2 alleles IBD across markers and across relative pairs was determined, and the mean value of  and the mean NPLall and KC-LOD scores  across markers pairs was determined. These statistics (prob(0), prob(1), and prob(2), , NPLall and p-value, KC-LOD and p-value) were then summarized across all replicates.For each replicate  and Trait B (chromosome 4 and 1). On average, each replicate contained 229 sibling pairs affected with Trait H and 119 sibling pairs affected with Trait B.LD was assessed among founders in the four regions. As expected, intermarker LD was observed on chromosomes 2 and 3 Figure . On chroThere was a modest increase in estimated allele-sharing in the region with LD and without a gene on chromosome 2 when founders were ungenotyped; prob(2) increased slightly from 0.336 \u00b1 0.468 with founders to 0.342 \u00b1 0.471 without founders. The non-gene region without simulated LD on chromosome 4 did not show any increase in allele-sharing with ungenotyped founders. Reduction of LD in the region with simulated LD reduced the upward bias in IBD allele-sharing (prob(2) = 0.340 \u00b1 0.469), suggesting that the bias may be due to LD.\u03b4 parameters are provided in Table  was elevated when a gene was present and centered on null otherwise. However, when founders were not genotyped, inflation in  was seen in the chromosome 2 region with LD and no gene (mean  = 0.06 \u00b1 0.10). This was not seen in the in chromosome 4 region with no LD and no gene (mean  = 0.00 \u00b1 0.11). Reduction of LD brought  slightly closer to null on chromosome 2 (mean  = 0.04 \u00b1 0.10), consistent with LD being the reason for the observed upward bias.Estimated When founders were genotyped and all markers were used, results were as expected based on simulation parameters Table . After Lall and KC-LOD scores was observed in the region with no gene but with LD on chromosome 2 . No differences in results were seen in the region without LD and without a gene (chromosome 4) when markers were removed. In the regions with genes, again, a reduction in power with dropping of markers was observed.With ungenotyped founders, an upward bias in NPLp-value distributions for regions without genes (simulated null distributions) also suggested an upward bias in the presence of LD. On chromosome 2 with simulated LD, the fifth percentile p-values for NPLall and KC-LOD scores were 0.06 and 0.06, respectively. When founders were not genotyped, these values decreased to 0.02 and 0.01, respectively, suggesting an increase in type I error. When LD was reduced, these values became 0.03 and 0.02, respectively. This trend was not seen on chromosome 4 without simulated LD.Comparison of the Results were similar when calculated on a grid, rather than evenly spaced between markers, and when allele frequencies were estimated from the dataset, rather than founders.all and KC-LOD scores via reducing overestimation of IBD when founders are not genotyped. In studies of late-onset diseases, pedigree founders are often not available and marker allele frequencies are required. It has been shown that, for two-point analysis, errors in marker allele frequencies may lead to false-positive results when a common marker is assumed to be rare [Our results suggest that reduction of intermarker LD may reduce false-positive rates  of NPL be rare . Becauseall and KC-LOD scores across regions and did not consider width of linkage peaks. We considered only nuclear families, but expect results to be similar with allele-sharing methods in extended pedigrees. We did not consider traditional LOD scores although these may be susceptible to inflated type I error rates as well [\u03b8 [This analysis has several limitations. Only 100 replicates were examined, and analyses were performed under a limited configuration of parameters. We examined effects of LD on mean NPL as well . We also well [\u03b8 .Issues arise in attempting to account for LD in linkage analysis using the methods described here. First, choice of an LD coefficient and its cut-off or other test for its significance will affect regions to be addressed. Although we removed |D'| greater than 0.73, this could be varied to optimize the balance between bias and informativeness. Second, specific markers to drop in an LD region must be selected. We dropped markers such that shorter map gaps were created; an alternative is to choose based on IC, as proved useful in a recent empirical report .Dropping markers in LD in the current analysis appeared to reduce power in areas with true linkage. This is an important loss, because, in reality one can not differentiate true and false positives. Software allowing for estimation and/or fixing of haplotype-frequencies in LOD score linkage analysis without dropping markers was developed for early restriction fragment length polymorphism studies (described in ). HoweveAs linkage analyses are conducted on dense SNP genome scans, one issue to weigh will be increased intermarker LD over microsatellite genome scans. Genome-wide analysis of LD should be performed preliminarily so that LD can be accounted for and bias away from the null can be minimized. Simple methods to account for LD, such as marker-dropping, or more sophisticated analytical approaches may improve validity of these types of linkage studies.GAW14: Genetic Analysis Workshop 14IBD: Identical by descentIC: Information contentKC-LOD: Kong and Cox LODLD: Linkage disequilibriumNPL: Nonparametric linkageSNP: Single nucleotide polymorphismELG designed the study, performed analyses, and wrote the manuscript. MDB provided critical input on analyses and manuscript. GPJ guided analyses and edited the manuscript."}
+{"text": "A genome-wide linkage analysis was conducted on systolic blood pressure using a score statistic. The randomly selected Replicate 34 of the simulated data was used. The score statistic was applied to the sibships derived from the general pedigrees. An add-on R program to GENEHUNTER was developed for this analysis and is freely available. Recently, score statistics have been proposed for mapping quantitative trait loci (QTLs) using sibship data or general pedigree data -4. TheseThe statistic to be used is the one in Wang and Huang  that assyk and yl be the phenotypic values of individuals k and l, respectively, in a pedigree. It is assumed that there is no locus-specific dominance effect. Then conditional on the number of alleles shared identical by descent (IBD) by k and l at a putative locus t, we have [Let  we have kl is the kinship coefficient between individual k and individual l,  is the additive variance due to locus t, and 1k} {\u03c4=is the indicator function which equals 1 when \u03c4 = k and equals 0 otherwise. The first term, Cov, is completely determined by the segregation parameters, such as means, the total additive variance, the total dominance variance, the variance of environmental effect, etc. These parameters can be estimated beforehand using phenotypic data only. The remaining term depends only on the pedigree structure through \u03a6kl and the linkage parameter . The hypotheses of interest arewhere \u03a6ni be the number of individuals in pedigree i and  be a vector of phenotypic values in pedigree i whose mean is denoted by \u03bci. The mean \u03bci may depend on other covariates in a linear or nonlinear fashion [\u03a3i0 = ), , andUnder the null hypothesis, the segregation parameters are independent of the linkage parameter , a prope fashion . Define  and  are the vectors of the probabilities that IBD = 1 and 2, respectively, for all the relative pairs in pedigree i. The score statistic, denoted by S, is defined as S = b2/Var(b) if b > 0; S = 0 otherwise, where b = \u03a3i (\u03c0i - E(\u03c0i))t(vi - E(vi)) and, for sibship data, Var(b) = Var(\u03c0)\u03a3i\u03a3k<lVar(wikwil). See Wang and Huang [b and Var(b). The score statistic S is asymptotically distributed as 0.5 \u03c702 + 0.5 \u03c712 [S is equivalent to the statistic in [where nd Huang  for more 0.5 \u03c712 . For sibS defined above. Systolic blood pressure (SBP) was used as the phenotype. To generate a single value of SBP from its repeated measurements for an individual, SBP was regressed over body mass index (BMI) and hypertensive treatment for each individual using the repeated measurements. The intercept of this individual-wise regression was used as the phenotype of the corresponding individual. We did not adjust the SBP further across different individuals using other covariates such as age, gender, or antihypertensive treatment that are believed to influence SBP, as pointed out by two anonymous reviewers. The power of our analysis may thus be hampered. Both Cohort 1 and Cohort 2 data were used.Replicate 34 of the simulated data was randomly chosen and a genome scan was done using the statistic S described in this report and the associated p-value computed based on the asymptotic distribution 0.5 \u03c702 + 0.5 \u03c712, which is p-value = 0.5Pr(\u03c712 \u2265 c), where c is the value of the score statistic S. This R program dissects a pedigree into nuclear families that are treated as if they were biologically unrelated. The sibship data from all nuclear families were used for the analysis.The genome scan was facilitated by an R  program.wikwil are also empirically estimated. See Wang [One reason for us to consider sibships instead of general pedigrees is that GENEHUNTER outputs IBD sharing probabilities for common relative pairs (including sib-pairs) only. For some pedigrees that are beyond GENEHUNTER's capacity, there is no output on IBD sharing probabilities for any relative pairs in such pedigrees. Such pedigrees are therefore not included in our analysis. In the R program, the mean of the phenotype is empirically estimated by its sample mean for all sibs in all sibships (so it is the same for each sib). The mean and variance of See Wang  for deta or . The source code is commented. It is expected that its usage be clear from the comments and this report. No separate documentation is provided.This R program used for the analysis can be freely downloaded from either p-value at each marker location is graphed in Figure The score statistic is calculated throughout the genome for the adjusted SBP. The p-value is smaller than, but very close to 0.01: one is marker c4g10 (106.13 cM) on chromosome 4 and the other one is marker c16g11 (109.60 cM) on chromosome 16.There is no strong linkage signal from our analysis. There are two markers whose We applied a score statistic to map QTLs that influence SBP using a randomly selected replicate. This score statistic is asymptotically equivalent to the likelihood ratio statistic and is straightforward to calculate. An R program was developed to facilitate the calculation of the score statistic using output on IBD sharing probabilities from GENEHUNTER. Although the score statistic is applicable to general pedigrees, we considered only sibships partly due to the fact that the IBD sharing probabilities from GENEHUNTER are available for common relative pairs (including sib pairs) only.p-values were less than 0.01 (Figure No significant linkage signal was found. There were two markers whose As mentioned earlier, the R program developed for this analysis handles sibships only. General pedigrees are dissected into sibships first. We are considering expanding its capacity to be able to handle nuclear families so the phenotypic information on the parents can be incorporated into the analysis. Because the calculation of IBD sharing probabilities for an arbitrary relative pair remains difficult, even using the MCMC technique, implementing the score statistic for large pedigrees does not seem to be simple at this moment.The size of the output file on IBD sharing probabilities from GENEHUNTER, which depends on the number of individuals in pedigrees and the number of scanned locations, may present a potential problem to the R program. In the genome scan in this report, the size of the output files is in the range of 6~34 MB. We experienced no problems with the R program in handling these files when running it on a Linux cluster with 512 MB memory on each node. We also tested it on a PC with 256 MB memory and experienced no problems. If the size of the output file from GENEHUNTER is too large for the R program to run, the output file can be split into smaller parts."}
+{"text": "Using the longitudinal Framingham Heart Study data on blood pressure, we analyzed the reproducibility of linkage measures from serial cross-sectional surveys of a defined population by performing genome-wide model-free linkage analyses to systolic blood pressure (SBP) and history of hypertension (HTN) measured at five separate time points.The heritability of SBP was relatively stable over time, ranging from 11.6 to 23.5% (coefficient of variation = 25.7%). However, the variability in linkage results was much greater. The average correlation in LOD scores at any pair of time points was 0.46 for HTN (NPL All LOD) and 0.17 for SBP (Variance Components LOD). No evidence of reproducible linkage results was found, with a mean \u03ba of 0.02 for linkage to HTN and -0.03 for SBP linkage. At loci with potential evidence for linkage (LOD > 1.0 at one or more time points), the correlation was even lower. The coefficient of variation at loci with potential evidence of linkage was 126% for HTN and 135% for SBP. None of 15 chromosomal regions for HTN and only one of 28 regions for SBP with potential evidence for linkage had a LOD > 1.0 at more than two of the five time points.These data suggest that, although heritability estimates at different time points are relatively robust, the reproducibility of linkage results in serial cross-sectional samples of a geographically defined population at successive time points is poor. This may explain in part the difficulty encountered in replicating linkage studies of complex phenotypes. The inability to replicate linkage findings has been a significant limitation in the advancement of understanding the genetics of complex human diseases . This prAlthough the study of complex disease genetics has thus far almost exclusively relied on the use of phenotypic data obtained at a single time point by a cross-sectional study, it is reasonable to hypothesize that longitudinally collected data may provide more insight into genetic susceptibilities for disease. Methodologies for the analysis of phenotypic data collected at multiple time points remain under development. An understanding of the reproducibility of linkage results using phenotypic data measured at different time points in serial cross-sectional surveys of a defined population will be important both to our understanding of the potential importance of sampling variation between studies and in developing robust methods for analyzing longitudinal data. The longitudinal Framingham Heart Study data made available through GAW13 allows these questions to be directly addressed. In this paper, we have explored the stability of linkage results to both a dichotomous and a quantitative trait in serial cross-sectional samples of a population-based sample in order to better understand the dependence of analytic results on sampling error and the particular time at which a sample is phenotypically assessed.The original (parent) cohort had phenotypic data regarding blood pressure collected at 21 time points spaced 2 years apart over 40 years while the offspring cohort was studied over 20 years at five time points every 4 years apart except for 8 years between the first two time points. To generate populations with maximal power, the parent and offspring cohorts were combined for this analysis. For each time point at which there were offspring data, phenotypic data from the closest date in the parent data set were used. This resulted in combining the 1971, 1979, 1983, 1987, and 1991 offspring data with the 1972, 1978, 1982, 1986, and 1988 parent data. The traits of interest were a diagnosis of hypertension (HTN) and systolic blood pressure (SBP). HTN was defined as systolic blood pressure \u2265 140 mm Hg, diastolic blood pressure \u2265 90 mm Hg, or use of medical therapy for hypertension. For those who were receiving antihypertensive medications, the SBP was assumed to be 10 mm Hg greater than the measured SBP as data suggest that this is the average reduction seen with medical therapy ,6.We used the Whittemore and Halpern NPL (nonparametric linkage) all statistic to test for allele sharing among all hypertensive individuals in a pedigree . The KonTo test for allele sharing using SBP as the quantitative trait of interest, a variance components model was constructed for each of the five time points. The total phenotypic variance  was based on a conventional covariate structure appropriate to the extended families present in the Framingham cohort. The model specified was:2Total = \u03c32A + \u03c32CS + \u03c32C + \u03c32E.\u03c32A represents additive genetic effects, \u03c32CS the effects of common sibling environment, \u03c32C the effects of a common family environment, and \u03c32E the residual variance . The narrow sense heritability (h2N) was calculated as \u03c32A / \u03c32Total. Age, gender, alcohol use (g/day), smoking (cigarettes/day), height, weight, and fasting glucose were included in the model as possible fixed effects. Linkage to the locus of interest was tested by comparing the likelihood of a model where the variance due to the locus of interest was constrained to zero versus an unrestricted model.In this model, \u03c3The linkage analyses of HTN were undertaken using the program MERLIN ; variancThe coefficient of variation (CV) of the heritability over the five time points was calculated to assess the reproducibility of this measure of additive genetic effect. To assess the reproducibility of linkage findings, Spearman correlations of LOD scores between each pair of time points were calculated over the entire genome as well as over the subset of loci where evidence for potential linkage was observed. In addition, the CV was calculated at each locus over the five time points to describe the variability in the LOD scores for linkage with each of the two traits. Mean and SD of the CVs at those loci where evidence for potential linkage was found are reported. The evidence for linkage was dichotomized at a threshold of LOD = 1.0 and Cohen's kappa statistic  was calcThe characteristics of the population are shown in Table As is shown in Table The results of the NPL analysis of HTN for the five time points are displayed in Figure The results of the variance components linkage analysis for SBP are plotted in Figure Our findings suggest that although heritability measurements are relatively stable over time, the finding of linkage to either a quantitative or dichotomous trait using cross-sectional data is highly dependent on the particular time point at which phenotypic data is collected. Evidence for linkage was markedly more labile when potential linkage (LOD > 1.0) was present on at least one time point. This may in part be due to regression to the mean but the poor reproducibility overall suggests other mechanisms are in play.Part of the explanation for the high degree of variability in linkage evidence may be a loss of power as the number of phenotyped individuals fell with time. However, the loss was not dramatic and in addition, over half of the instances of LOD > 1.0 for both traits occurred in the last two time points. Alternatively, the lack of reproducibility may reflect the importance of genes whose effects are highly dependent on age or on gene \u00d7 environment interactions. The disparate results of the linkage analyses at time points 3 and 4, where the average age is only 2 years apart, the sample sizes are within 3%, and the distribution of SBP and HTN are similar, counter this argument.Although there is variability in individual SBP and HTN assessments in this cohort, the correlations in these phenotype measures was much stronger than that found in linkage findings. Importantly, even analyses of highly variable phenotypic measures over time would not necessarily imply poor reproducibility in linkage results so long as the familial correlations in the measures remain relatively constant (as was suggested by the relative stability of the heritability estimates for SBP over time).The changing composition of the population over the five time points may have also contributed to the variation in linkage findings. One would expect the correlation to be greater if the identical population was studied at each time point. We deliberately did not restrict our analysis to pedigrees for which there were data at all time points both because this was not the primary hypothesis we wished to test and because we would have substantially limited our power to detect linkage. We also believe our approach more accurately reflects the data that would actually be available to researchers studying longitudinal populations. Assuming that the population studied at each time point is a random sampling of the general population of the town of Framingham, the linkage results for each cross-sectional study should represent an unbiased estimate of the true evidence for linkage in the entire population.A high degree of lability in complex phenotypic measures over time together with simple sampling error may be the most likely explanations for our findings. Stochastic processes may have a larger influence on complex phenotypes, and hence on the results of linkage studies, than has been previously appreciated. The implications of this are disturbing, given than most genetic linkage and association studies have been based on cross-sectional surveys. It is possible that phenotypic variability and random error may be restricted in highly ascertained samples, reducing this problem. However, in the absence of empirical evidence of this, given the high caliber of the Framingham Heart Study in terms of both design and quality of phenotyping, it is difficult to argue that other studies would be less subject to this phenomenon. Our results highlight the difficulty of replicating linkage results for complex phenotypes and make the need for large longitudinal studies of such phenotypes clear.These findings may help to explain the difficulties that have been experienced in replicating linkage results from cross-sectional genome scans . Our fin"}
+{"text": "Most methods for testing association in the presence of linkage, using family-based studies, have been developed for continuous traits. FBAT (family-based association tests) is one of few methods appropriate for discrete outcomes. In this article we describe a new test of association in the presence of linkage for binary traits. We use a gamma random effects model in which association and linkage are modelled as fixed effects and random effects, respectively. We have compared the gamma random effects model to an FBAT and a generalized estimating equation-based alternative, using two regions in the Genetic Analysis Workshop 14 simulated data. One of these regions contained haplotypes associated with disease, and the other did not. Testing association in a region with confirmed linkage may increase the rate of false positives in family-based studies. In a linked region one expects similarity between related individuals. If unaccounted for, this similarity may be mistaken for association. Different remedies have been suggested, ranging from using a robust variance estimator  for the We consider a random effects model for binary events, which is similar in spirit to the multivariate survival model in Zhong and Li , which mYi1, Yi2, ..., ) be the binary trait vector for family i and let j denote offspring . We allow for different family sizes Ji. We use \u03b8mj and \u03b8pj to denote the effect of the transmitted alleles to offspring j, with mj = 1, 2 the maternal alleles and pj = 3, 4 the paternal alleles, respectively. Conditional on the transmitted alleles, we write the probability of the trait for offspring j in family i as P. We consider a model with a log(-log) link of the formLet )) = log(\u03b8mj + \u03b8pj) + Xj\u03b2,log, isThe matrix .R (version 1.9.1) [\u03b2.We used the statistical software \u03c0i. We return to the choice of weights in the discussion.We have so far not described how to deal with incompletely observed inheritance vectors. In the context of testing association in the presence of linkage, Zhong and Li  suggest We compare the GRE with FBAT (version 1.5.1)  and a ge-o to find the optimal weight. We then applied the optimal weight to the phenotype score and used FBAT option -e to test our data. The function gee (in package gee) in R (version 1.9.1) was used for the GEE analysis. The gee package can be found at the R web page [We used FBAT option For details concerning how the simulation was performed see the GAW14 Data Description .All analyses were performed with knowledge of the data simulation process. We chose to analyze the data with respect to trait A. Trait A is known to be associated with haplotypes in the Region D3, while markers in the D2 region are known to not be associated with trait A. For the purpose of our comparison we therefore chose to \"purchase\" markers in the D3 region (B05T4135\u2013B05T4142) as well as markers from the D2 region (B03T3048\u2013B03T3067). Our aim was to use regions D2 and D3 to gain some insight into the performance of the different methods. More specifically, we were not expecting a signal in region D2, but were hoping for one in region D3.The Aipotu population (one of four simulated populations) only consists of nuclear families, although these are of different sizes. For simplicity, we chose to concentrate on the Aipotu population and to only include families of maximum size six .We merged 10 (out of 100) replicates in order to get a sample with reasonable power. This provided us with a total of 481 independent nuclear families. There was no missing data and we did not simulate any.We selected the markers described above and analyzed each marker separately in a set of single-point analyses. The method we have described can, however, be extended to multiple markers and a multipoint analysis.p-value of around 0.01  were used, compared with the FBAT procedure  indicated marker B03T3056 had borderline significance with a p-value around 0.01. However, taking the multiple testing into account, this p-value does not reach statistical significance. The results from all markers in the region are showed in Figure p-values than any other method.In the simulated data, region D2 harbored no locus associated with trait A. All three methods  gave a signal for association with marker B03T3056 with a In region D3, association with trait A was simulated at the haplotype level. We still chose to perform single-point analyses with each marker in turn. The GEE and the GRE turn out to be slightly better in detecting significant markers than FBAT.The GRE model presented here seems to work well, compared to both GEE and FBAT. It would be useful to perform simulation studies to assess validity and power of the three procedures under different genetic models. The GRE model requires more computational time, stemming from the fact that in spite of the closed form in (3) it is time consuming to evaluate and to maximize the likelihood.A problem with the GRE model is how to handle the missing information on transmission. In our single-point algorithm we propose using a weighted sum  over all compatible inheritance vectors, given parental and offspring genotypes. Following Zhong and Li  we compuFBAT: Family based association testsGAW14: Genetic Analysis Workshop 14GEE: Generalized estimating equationGRE: Gammar random effectsVCM: Variance components model"}
+{"text": "We consider a new Bayesian approach for heterogeneity that can take into account categorical covariates, if available. We use the Genetic Analysis Workshop 14 simulated data to first compare the Bayesian approach with the heterogeneity LOD, when no covariate information is used. We find that the former is more powerful, while the two approaches have comparable false-positive rates. We then include informative covariates in the Bayesian approach and find that it tends to give more precise interval estimates of the disease gene location than when covariates are not included. We had knowledge of the simulation models at the time we performed the analyses. One of the major difficulties in mapping genes that influence complex traits is locus heterogeneity. A widely used statistic for dealing with locus heterogeneity in linkage analysis is the Heterogeneity LOD (HLOD) score . RecentlIn the current study, we first used the simulated data of the AI population to compare the power and false-positive rates between the Bayesian approach and the HLOD. Then we investigated the performance of a new extension of the Bayesian approach that incorporates informative categorical covariates. We did this by applying the approach to the all-population-combined data using the population indicator as a covariate.k families in the sample. Let \u03b1j be the probability that the jth family is of the linked type, j = 1, ..., k, and let d be the position of the disease gene on the chromosome. Here \u03b1 =  is a set of nuisance parameters while d is the main parameter of interest. The likelihood of  is expressed in terms of mixture distributions, similar to that described in [N distances (locations) on the chromosome, labelled as 1, ..., N, at which the LOD scores are calculated. Let Id denote the index of distance d. Then Id \u2208 {1, ...., N}. The prior distribution for d consists of two components: \u03c0d < \u221e and \u03c0d = \u221e for linkage and no linkage, respectively, on the chromosome of interest. Further, for d < \u221e, there is a probability distribution of d at the N distances denoted by \u03c0d (Id). Let the prior distribution of \u03b1j  be \u03c0j(\u03b1j).This approach is described and investigated in detail by Biswas and Lin . Briefly\u03b1j values and d. The values of d < \u221e (linked subspace) and d = \u221e (unlinked subspace) lead to two different models with a different number of parameters. The linked subspace (L) consists of k+1 parameters  while the unlinked subspace (U) has no parameters, since d = \u221e renders the \u03b1 parameters meaningless. To allow moves between these two subspaces, we use the reversible jump Markov chain Monte Carlo sampler [The goal is to obtain the posterior distributions of  sampler .B iterations and then for another T iterations for estimating the posterior distributions. From the estimated posterior distribution of d, we obtain the posterior probability of linkage, , which is the proportion of times the chain is in the L subspace. If  is greater than a certain threshold, po, we take it as a signal for linkage. In that case, an estimator of the location of the disease gene under linkage is the mean, , of the estimated posterior distribution of d when the chain is in the L subspace. An interval estimate is obtained by computing a Bayesian credible set (CS) for d under linkage.The Markov chain is run initially for a burn-in period of \u03c0j(\u03b1j) is U, j = 1, ...,k, \u03c0d< \u221e = 1/10 (the probability that a randomly chosen chromosome of the simulated data will harbor the disease gene), \u03c0d = \u221e = 9/10, and \u03c0d (Id) = 1/N, Id = 1, ..., N. We let B = 10,000, T = 300,000, following Biswas and Lin [\u03c0d< \u221e and threshold, po, were, respectively, 1/22 and 0.5 (Bayes rule for the 0\u20131 loss function), which in turn corresponded to a Bayes factor of 21. Because we are using a different \u03c0d< \u221e, the Bayes factor of 21 is obtained when po = 0.7 and so we use this threshold in our analyses.We use the following prior distributions:  and Lin . In thei\u03b1 parameters have prespecified hyper-priors. We describe one implementation of this idea as follows: suppose there are G groups of families in the sample corresponding to G categories of covariate(s). These G categories may be G levels of one covariate or G combinations of levels of two or more covariates. Suppose the ith group has ni families, i = 1, ..., G. Denote the \u03b1 parameter of the jth family in the ith group by \u03b1ij, j = 1, ..., ni, i = 1, ..., G. Assume that the prior distribution of \u03b1ij, \u03c0ij(\u03b1ij), is Beta with hyper-parameter ai following Binomial. Here, ti, for 0 <ti \u2264 1, is a tuning parameter needed only for computational purposes (explained below). The value of \u03c6i is prespecified according to the covariate value in the ith category. If the ith group is likely to have more linked families, it is assigned a higher value of \u03c6i. The ti values may be set to 1. However, large ni values lead to large parameters for Beta distributions, which make them highly concentrated in a narrow range. This can dramatically increase the computation time for updating the \u03b1ij values for some families when using rejection sampling. We use ti = 0.1 for all i.One way of incorporating categorical covariates is using a hierarchical model in which the We used the following genetic model, where D denotes the disease allele: P(D) = 0.2, and penetrances for genotypes dd, Dd, and DD are 0.05, 0.5, and 0.7, respectively. This is a kind of incomplete penetrance model that one might use as an approximation to the true but unknown complex model. The data used are microsatellite markers and affection statuses. We used GENEHUNTER version 2.1_r5 beta  to compupo, to 0.73  as a covariate. These populations differ in the diagnostic schemes used, which are based on three broad categories of symptoms: communally-shared emotions (COM), behavioral (BEH), and anxiety-related (ANX). In AI and NYC, a person is considered to be affected by Kofendrerd Personality Disorder if he/she has any of the three types of symptoms. In KA, the criterion is the presence of COM or ANX, while in DA only BEH matters. NYC consists of extended pedigrees in contrast to the other three populations that comprise nuclear families only. Furthermore, NYC differs from the others in its ascertainment scheme. The varying diagnostic and ascertainment schemes can be expected to give rise to some heterogeneity across populations. Specifically, if there is a locus that influences COM and/or ANX but not BEH, then DA is not expected to contain information about this locus while KA is the most homogeneous and hence most informative for detecting the locus. On the other hand, DA is most informative for a locus influencing BEH. Also, although both AI and NYC are phenotypically heterogeneous, NYC families may contain more linkage information because the families ascertained are extended pedigrees with at least four affected members.i\u03c6) for : 1)  and 2) . We analyzed the first 25 replicates with both sets of covariate settings and without covariate. The results for the first 5 replicates only are shown in Table i \u03c6parameters that gave a higher posterior probability of linkage .In our first analysis, we found linkage signals on chromosomes 1, 3, 5, and 9 in numerous replicates. Although we used population AI only, all three types of symptoms  are present in the AI families, thus we decided to focus on these chromosomes only. Following the above reasoning concerning the informativeness of populations for different types of loci, we considered two possible sets of binomial parameters  can be used to form informative covariates that refine the linkage analysis by yielding narrower CSs and even uncovering signals for linkage that might otherwise be missed. However, we note that even when no covariate is included, the linkage signals in most of the replicates with all populations combined are so strong  influences BEH strongly and either ANX or COM moderately, with no effect on the other type of symptom. The genes on chromosomes 5 (D3) and 9 (D4) influence COM and ANX only while the one on chromosome 3 D2) affects all symptoms, with a slightly stronger effect on BEH. Our results are consistent with this simulation model, since of the two sets of  affects The approximate locations of D1, D2, D3, and D4 are, respectively, 169, 299 (location of the last SNP), 5, and 6 cM on their respective chromosomes based on the microsatellite map. Compared with the CSs in Table ANX: Anxiety-relatedBEH: BehavioralCOM: communally-shared emotionsCS: Credible setHLOD: Heterogeneity LODSB and SL developed the methodology. SB carried out the applications and drafted the manuscript. DAB provided insightful comments for the presentation of results. SL and DAB were involved in the critical revision of the manuscript. All authors read and approved the final manuscript."}
+{"text": "For the identification of susceptibility loci in complex diseases the choice of the target phenotype is very important. We compared results of genome-wide searches for linkage or for association related to three phenotypes for alcohol use disorder. These are a behavioral score BQ, based on a 12-item questionnaire about drinking behavior and the subject's report of drinking-related health problems, and ERP pattern and ERP magnitude, both derived from the eyes closed resting ERP measures to quantify brain activity. Overall, we were able to identify 11 candidate regions for linkage. Only two regions were found to be related to both BQ and one of the ERP phenotypes. The genome-wide search for association using single-nucleotide polymorphisms did not yield interesting leads. For the identification of susceptibility loci in complex diseases, genome-wide searches are a first step. Study design issues such as sample structure and marker choice play a role. However, of fundamental importance is a precise, homogeneous, and insightful phenotype definition. The right hunch might be the key to a particular disease pathway. The Genetic Analysis Workshop 14 (GAW14) family data on alcohol use disorders  provide the possibility to compose new phenotypes for alcoholism. These are either based on 12 questions regarding drinking behavior and the subject's report of drinking-related health problems or on eyes closed resting event-related potential (ERP) measures to quantify brain activity, possibly defining a phenotype closer to a biological pathway.To localize susceptibility regions for alcoholism we first separately construct one phenotype for drinking behavior and two for ERP measures, pattern (P) and magnitude (M), taking clustering within families into account. Genetic information is not used in this step. Previous studies of ERP focused on the amplitudes of measurements. However, the pattern of measures can be more important than the magnitude . For ERP we considered both the pattern of an individual's ERP measurements and the magnitude (amplitude). Secondly, we performed a genome-wide linkage search with microsatellite markers and an association search with single-nucleotide polymorphisms (SNPs). Then we compared the results yielded by the different phenotypes and searches.i of the score for \"affected\" and \"pure unaffected\" sibs of family i was calculated. Under the null hypothesis of no discrimination by the score the distribution of \u0394i must be located around E(\u0394) = 0. We performed a sign rank test and calculated a standardized effect size (\u03b4 = /SD) for each phenotype considered. As disease status we used the DSM-IV classification of alcohol dependence (in GAW14 coded as ALDX2) [Behavioral questions (BQ) and ERP measures are multidimensional. We employed a reduction to a single score each while conserving as much information in the whole set as possible. For inheritance patterns the pedigree structure must be considered. Before utilizing these scores in subsequent linkage analysis, we evaluated if a score discriminates affected and unaffected siblings within a sibship. Therefore the mean difference \u0394Prior to score definition, we analyzed the impact of covariates on pure ERP measures, independent of wave band, time window, and skull location, with a generalized additive model (GAM) . We conslnBQ).To construct a univariate score for behavior questions we applied a logistic regression model to discriminate best between \"affected\" and \"unaffected with some symptoms\" conditioning on whole families. \"Pure unaffected\" were not considered, because they did not show positive answers on any behavior item. Item 5 \"Spent most time for drinking\" was dichotomized to \"<1 month\" and \"\u22651 month\". All 12 behavior items and age were included as explanatory variables. Gender did not show a significant impact. All family data were used. Scores for \"pure unaffected\" were also calculated according to the estimated coefficients, which is equivalent to the age effect only. To reach approximate normality as needed for further analysis the score was transformed by addition of a constant and logarithmic transformation  of the Affymetrix and Illumina chips. Each SNP was tested for association using the FBAT program  with thensidered .\u00ae 8.6.All calculations and data handling were done with R or SASp < 0.0001) and non-linearly by age (p < 0.0001). Age is estimated as u-shaped with a minimum around 45 years. The largest effect by ethnicity (p < 0.0001) showed the contrast of Black non-Hispanic (lowest mean) compared with American Indian (in the mean 0.62 higher), but other populations also differed from each other. Almost half (49.6%) of the variability of the adjusted, nonstandardized ERPs is explained by the first principal component. The score coefficients .Visual inspection of the phenotype density functions Figure  shows th2 of 0.527 (max possible = 0.602). Score coefficients are given in Table BQ score an effect size of \u03b4 = 2.13 could be achieved. This may be considered equivalent to a group assignment being ~85% correct  and used later.The applied logistic model yielded a pseudo-RlnBQ five loci achieved a LOD > 1 , with the highest LOD score on chromosome 10 at 114 cM between D10S670 and D10S544 (max LOD = 1.4). For ERP-P three loci achieved a LOD > 1 , with the highest LOD score on chromosome 16 at 130 cM between D16S750 and D16S539 (max LOD = 1.2). For ERP-M four loci achieved a LOD > 1 , with the highest LOD score on chromosome 2 at 243 cM between D2S434 and D2S1323 (max LOD = 1.6). In second passes for all three phenotypes, conditional on the most significant QTL, no further evidence for linkage (LOD > 1) was found. The scans for the three phenotypes result in linkage signals at different locations across the genome, except for those on chromosomes 2 for lnBQ and ERP-M and 15 for lnBQ and ERP-P . Within these regions 0.0% to 7.11% are significantly (at the 5% level) associated with lnBQ, ERP-P, or ERP-M, respectively. Figure We achieved for ERP-M and ERP-P, from ERP measures. The poor ability to discriminate between affected and unaffected individuals may result from a small number of informative discordant sibships (n = 49) in the sample, weak classification of affection status by DSM-IV, or the properties of ERP-M and ERP-P as phenotypes, which were chosen specifically to be different from each other. We could identify 11 candidate regions for linkage on 10 chromosomes using all three phenotypes. Only one region (chromosome 2: 212\u2013273 cM) was found twice, for lnBQ and ERP-M. The region on chromosome 15 (117 cM to end) was identified clearly by lnBQ and showed a maximal LOD of 0.95 related to ERP-P. The three phenotypes differ in their information, so that heterogeneous findings are expected. ERP-M and ERP-P, different scores calculated on the same set of measurements, can be seen as overall brain activity (ERP-M) and within-brain activity contrast (ERP-P). Here the usability of these scores cannot be discussed regarding biophysiology, but only regarding results.For the identification of susceptibility loci in complex diseases, the choice of the target phenotype, the marker set, and the analysis strategy are important issues. For alcoholism one might expect an applicable impact of the social surrounding. As biophysiological measures we derived two different phenotypes, lnBQ have been reported before. Analyzing the COGA dataset, Reich et al. [Some linkage peaks are replications from previous studies. For example, 3 of the 4 linkage regions identified for h et al.  reportedh et al.  found lih et al. ,13.p-value \u2264 0.05) within these regions were checked to determine if they were on a gene potentially relevant for alcoholism. On chromosome 10 we found a SNP lying on SORCS3 that showed a significant association with lnBQ . SORCS3 potentially encodes for a neurotensin receptor.Genome-wide association tests of SNPs (lnBQ. We found HTR7 encoding serotonin receptor 7 and COMTD1 in the chromosome 10 area for lnBQ. The gene of \u03bc-opiod receptor (OPRM1) is located in the chromosome 6 area for ERP-M. Finally the gene SLC6A4 (serotonin transporter) is located about 30 cM away from the LOD peak on chromosome 17 for ERP-P. It should be noted that here we did not account for sex differences in linkage maps. Furthermore, we noticed differences between SNP and microsatellite marker maps up to 40 cM. Both can generate some false location comparisons , linkage analysis , association analysis , candidate genes (K Korb), and drafting the article . All authors gave their final approval of this manuscript."}
+{"text": "In this paper we apply two novel quantitative trait linkage statistics based on the posterior probability of linkage (PPL) to chromosome 4 from the GAW 14 COGA dataset. Our approaches are advantageous since they use the full likelihood, use full phenotypic information, do not assume normality at the population level or require population/sample parameter estimates; and like other forms of the PPL, they are specifically tailored to accumulate linkage evidence, either for or against linkage, across multiple sets of heterogeneous data.The first statistic uses all quantitative trait (QT) information from the pedigree ; we applied the QT-PPL to the trait ecb21 . The second statistic allows simultaneous incorporation of dichotomous trait data into the QT analysis via a threshold model (QTT-PPL); we applied the QTT-PPL to combined data on ecb21 and ALDX1. We obtained a QT-PPL of 96% at GABRB1 and a QT-PPL of 18% at FABP2 while the QTT-PPL was 4% and 2% at the same two loci, respectively. By comparison, the variance-components (VC) method, as implemented in SOLAR, yielded multipoint VC LOD scores of 2.05 and 2.21 at GABRB1 and FABP2, respectively; no other VC LODs were greater than 2.The QTT-PPL was only 4% at GABARB1, which might suggest that the underlying ecb21 gene does not also cause ALDX1, although features of the data complicate interpretation of this result. We have developed two new methods for quantitative trait (QT) linkage analysis based on the posterior probability of linkage (PPL) framework , which dThese methods were applied to chromosome 4 of the Collaborative Study on the Genetics of Alcoholism (COGA) data using the quantitative ecb21 phenotype, and the dichotomous phenotype ALDX1. ecb21 was chosen because it had yielded a variance components (VC) LOD score of 5.01 near GABRB1 in an analysis using the full set of COGA families [Analysis was performed on all 143 COGA families; average family size was 11.3 (range 5 to 32) and average generations was 2.8 (range 2 to 5). Two pedigrees contained loops and are therefore complex. The two phenotypes considered were resting electroencephalogram (EEG) beta2 spectral/spatial component (ecb21) and the categorical diagnosis of alcoholism (ALDX1). ALDX1 contained two additional categories beyond affected and unaffected which were recoded to unknown for the purpose of analysis. No other changes to phenotypes were made.Analysis was conducted on all chromosome 4 markers provided by COGA. Allele frequencies were required all of the analyses presented; we used the values provided with the data, which were estimated by the maximum likelihood method. Map positions were taken as given in the associated map file.VC analysis was conducted with SOLAR . AnalysiThe PPL is defined as the integral over [0...1/2) of the posterior density of the recombination fraction \u03b8, computed with the prior probability of linkage set to 2% , and a cL is the prior probability of linkage, G is the genotypic data, X is the trait data, g is the prior distribution function for the given parameter, and t is the vector ot trait parameters . We include \u03b1, the admixture parameter in the QT-PPL to better approximate a multilocus likelihood from the single-locus likelihood.where \u03c0Here we have used LIPED  to compuP(xi|gi) required by the likelihood, where xi is ith person's phenotypic value and gi is their corresponding latent trait genotype. All other subjects are assigned their quantitative (ecb21) trait values, with these same factors calculated using the density f(x) = P, for the t-distribution as before, and where j indexes the specific trait genotypic distribution. Here we use the t-distribution instead of the normal density for computational reasons involving the difficulty of estimating probabilities in the extreme tails of the normal distribution. From our experiences in developing this method, this substitution is not expected to have substantive effects on the reported results (data not shown).The threshold quantitative trait PPL (QTT-PPL) assumes that all individuals who are affected  are below some unknown threshold for the underlying quantitative trait . For affected individuals, the cumulative t-distribution (30 df) is used to generate the factors The QTT-PPL can be applied when a clinical diagnosis is available for some subjects for whom quantitative measures are not available, yet a relationship between the affection status and the quantitative trait is postulated. But it can also be used to investigate the underlying relationship between the QT and the clinical phenotype by contrasting results from categorical, QT, and QTT analyses because the latter assumes both of the former are related by a common trait locus.n = 192) more phenotypic information than the categorical analysis, while the QTT-PPL used 30% (n = 490) more phenotypic information. These large changes in the number of phenotypes, as well as who in the pedigree was phenotyped, might alone account for the large difference in the threshold QT PPL compared with the categorical and QT analyses. Alternatively, the low QTT-PPL might be indicating a lack of an underlying biological relationship between ecb21 and alcoholism as defined using ALDX1.Two regions are identified for possible follow-up genotyping. The QT-PPL was 96% and 18% at GABRB1 (51 cM) and FABP2 (116 cM), respectively. Categorical PPL analysis gave rise to lower PPLs, 26% and 2%, at these same loci. Multipoint VC analysis in these regions yielded VC LOD scores of 2.05  and 2.21 . Figure This paper indicates strong evidence for linkage of ecb21 to the GABRB1 region of chromosome 4. This result confirms a previous genome scan using this phenotype in an extended set of the COGA families, which yielded a VC LOD of 5.01 in this same region [However, there has been no equivalent indication of linkage to GABRB1 with a categorical alcoholism phenotype in the literature, while our results indicate a 26% of linkage to alcoholism. When we applied a unified threshold analysis of the categorical and QT phenotypes, implicitly assuming a relationship mediated by the QT, the PPL was only 4%, which is larger than the prior probability of 2%, but not appreciably so. Because the threshold analysis used the largest amount of phenotypic information of all the PPL analyses, we may conclude that it represents a solution closest to the correct assessment of the data; either the relationship of ecb21 phenotype to alcoholism is weak (perhaps non-existent) in this dataset or the relationship of ecb21 to alcoholism departs substantially from the assumed model of the QTT-PPL. The former conclusion is supported by the lack of a categorical linkage of GABRB1 to the alcoholism diagnosis in the literature.prima facie, it appears that the QT-PPL provided more compelling evidence for linkage than VC analysis of the GAW data. Because all PPL values are on the probability scale , a probability of 96% is a very strong indication that a gene for ecb21 is near GABRB1 even after considering 3 separate analyses of these same data. We are in the process of systematically examining the properties of the QT-PPL and threshold QT-PPL under a variety of single- and multi-locus QTL models, as well as implementing multipoint versions of both statistics. The result of such systematic evaluations will aid in interpretation of the PPL compared to other commonly used linkage methods.While issues of scale preclude a direct comparison between VC- and PPL-based methods, COGA: Collaborative Studies on the Genetics of AlcoholismEEG: ElectroencephalogramGAW: Genetic Analysis WorkshopIBD: Identity by descentPPL: Posterior probability of linkageQT: Quantitative traitQT-PPL: Quantitative trait posterior probability of linkageQTT-PPL: Threshold quantitative trait posterior probability of linkageVC: Variance componentsBoth authors were fully involved in all aspects of this collaborative research endeavor. Both authors read and approved the final manuscript."}
+{"text": "The repeated measures in the Framingham Heart Study in the Genetic Analysis Workshop 13 data set allow us to test for consistency of linkage results within a study across time. We compared regression-based linkage to variance components linkage across time for six quantitative traits in the real data.The variance components approach found 11 significant linkages, the regression-based approach found 4. There was only one region that overlapped. Consistency between exams generally decreased as the time interval between exams increased. The regression-based approach showed higher consistency in linkage results across exams.The low consistency between exams and between methods may help explain the lack of replication between studies in this field. The general lack of replication of genome scan results across data sets is an ongoing concern in statistical genetics . ConsistA new regression-based method of linkage analysis that works on arbitrary pedigrees has recently been published  and impl2, and cohort status were included as covariates in all genome scans. In order to test the robustness of regression-based linkage, we did not transform any of the variables to achieve normality (except triglycerides). To maintain as much consistency across data sets as possible, we did not correct for hypertension, diabetes, or any medication.The traits we analyzed were body mass index (BMI), total cholesterol (CHOL), glucose (GLU), high-density lipoprotein cholesterol (HDL), height (HGT), systolic blood pressure (SBP), and log-transformed triglycerides (LGTG). The effects of sex, age, ageThe 330 families were trimmed using the PEDSYS PEDTRIM program. This program removes individuals from a pedigree who do not contribute any phenotypic or genotypic information. Using the criterion of at least one marker on chromosome 22, PEDTRIM reduced the family set from 4692 individuals in 330 families to 2604 individuals in 334 families (trimming caused four families to each be split into two).The complexity of a family is measured by the formula 2N-F, where N is the number of nonfounders and F is the number of founders. Our prior experience with GENEHUNTER indicates that a practical upper limit for the complexity of a family is 21. After trimming, there were still nine families with complexity greater than 21 (range 23\u201346). These nine families were cut into 19 separate pedigrees, resulting in 2588 individuals in 344 families. All analyses described in this report used these trimmed and cut family structures.We constructed four data sets (denoted A-D) by filling out the families with data corresponding to the first four Cohort 2 exams and include individuals from Cohort 1 exams that overlapped in time with the four Cohort 2 exams, thus yielding four data sets, each with paired contemporaneous data for Cohorts 1 and 2. These four \"cross-sectional\" data sets represent four time points spanning approximately 16 years. For data set A, the variables were taken from Cohort 2 Exam 1 and Cohort 1 Exam 12, depending on the cohort of the individual. Cohort 2 Exam 1 and Cohort 1 Exam 12 overlap in time (1970\u20131971) and are thus the natural choice to achieve a cross-sectional data set of these families. Similarly, for data sets B through D, we combined Cohort 2 Exam 2 and Cohort 1 Exam 16 (1978\u20131979), Cohort 2 Exam 3 and Cohort 1 Exam 18 (1982\u20131983), and Cohort 2 Exam 4 and Cohort 1 Exam 20 (1986\u20131987), respectively. There were several exceptions to this scheme. There were no Cohort 1 Exams 16 or 18 for cholesterol and HDL, so data set C was not constructed for either trait and data set B used data from Cohort 1 Exam 15 rather than 16. Similarly, height was not available from Cohort 1 Exam 12, so both height and BMI data sets A were constructed using data from Cohort 1 Exam 10.We performed VC linkage analysis on all 23 traits using GENEHUNTER and MERLIN. Both programs computed multipoint identity-by-descent (IBD) probabilities at each marker and at four equally spaced loci between each pair of markers. We also performed regression-based linkage analysis [MERLIN computed IBD probabilities much faster than GENEHUNTER (approximately 4.5 times faster on our Pentium 4 computer). A full genome scan  on MERLIN took approximately 4.5 hours for VC linkage and 41 hours for regression-based linkage. In addition, MERLIN was limited to markers with less than 32 alleles (on our 32-bit system). One marker had 39 alleles; it was downcoded to 30 alleles.2), and heritability in this table were used as input to the regression-based linkage analyses. Heritability was computed by MERLIN as the ratio of additive polygenic variance to the total variance.Table p < 0.005) for VC and regression. For every LOD score greater than 1.44, the LOD score at the same point using the other method is included for comparison. For each trait, the LOD scores are ordered by chromosome, centimorgan location, and data set, in that order. There were 15 distinct regions for which one or the other method found a LOD score greater than 3.0 in at least one exam. VC analysis found 11 regions, regression found 4 regions, and there was one region in which both methods found significant linkage (chromosome 7 for cholesterol). Of the 11 significant regions from VC, 8 had some supporting evidence for linkage (LOD score > 1.0) from regression. Of the 4 significant regions from regression, 2 showed supporting evidence from VC. Perhaps surprisingly, of the 5 significant regions for which the other method did not provide some support, 4 were for height. The last line of Table Table p-values were highly significant (p < 0.0001).VC LOD scores from GENEHUNTER were compared to VC results from MERLIN. For all traits examined the correlation between LOD scores from the two programs was at least 0.993. For 18 of the 23 traits the correlation was 0.9999. All The glucose A data set, which had the highest kurtosis, was a special problem for regression-based linkage. The program gave no LOD score at all between 180 and 190 cM on chromosome 4, and it gave a LOD score of 12.5 at 1 cM on chromosome 3. In both cases, it gave heritabilities greater than 1. For these reasons, we exclude the glucose A data set from further consideration. In addition, results from MERLIN for weight were obviously incorrect , so we do not report any results for weight. The reasons for this anomaly are currently unknown to us.Table Table Table Two obvious differences between the two methods are the number of significant linkage regions and the improved consistency in LOD scores between data sets provided by the regression-based approach as opposed to the variance components approach. VC analysis found almost three times as many significant regions as regression-based linkage. This may be due to the well known inflation of LOD scores due to non-normality, and indeed two of the significant VC linkages were detected in glucose, which has high kurtosis. However, the other eight significant VC linkages are in traits with low kurtosis  is less than half the next lowest (0.51 for SBP). Height is unique in this study in at least two ways, much higher heritability than the other traits and the assortative mating for height in humans. However, it is not clear why either of the two methods should be sensitive to these unique aspects of height. In addition, kurtosis for height is negligible and the highest LOD score from both methods have some support from previously reported genome scans , thus t"}
+{"text": "Although many years of genetic epidemiological studies have demonstrated that genetics plays a significant role in determining smoking behavior, little information is available on genomic loci or genes affecting nicotine dependence. Several susceptibility chromosomal regions for nicotine dependence have been reported, but few have received independent confirmation. To identify susceptibility loci for nicotine dependence, 313 extended pedigrees selected from the Framingham Heart Study population were analyzed by both the GENEHUNTER and S.A.G.E. programs.After performing linkage analyses on the 313 extended Framingham Heart Study families, the EM Haseman-Elston method implemented in GENEHUNTER provided evidence for significant linkage of smoking rate to chromosome 11 and suggestive linkage to chromosomes 9, 14, and 17. Multipoint sib-pair regression analysis using the SIBPAL program of S.A.G.E. on 1389 sib pairs that were split from the 313 extended families identified suggestive linkage of smoking rate to chromosomes 4, 7, and 17. Of these identified positive regions for nicotine dependence, loci on chromosomes 7, 11, and 17 were identified by both GENEHUNTER and S.A.G.E. programs.Our genome-wide scan results on the Framingham Heart Study data provide evidence for significant linkage of smoking rate to chromosome 11 and suggestive linkage to chromosomes 4, 7, 9, 14, and 17. These findings suggest that some of these regions may harbor susceptibility loci for nicotine dependence, and warrant further investigation in this and other populations. Over the last several decades, a number of twin studies throughout the world have yielded results consistent with the overall conclusion that both genetic and environmental factors contribute to the risk of becoming a long-term smoker . AfteAlthough the twin studies suggest moderate genetic influences on nicotine dependence, little information is provided about the chromosomal locations harboring susceptibility loci/genes for nicotine dependence. Linkage-based genome-wide scans for smoking behavior have been reported by Straub et al.  on the CGenerally speaking, there are two approaches available to address this problem of identifying susceptibility loci for nicotine dependence and other complex disorders. The first approach is to repeat and extend these genome-wide linkage analyses in different populations; the second is to use higher marker densities for association genome scanning studies. Based on the availability of information on smoking phenotype in the Framingham Heart Study population, we adopted the first genome-wide scan approach to identify susceptibility loci for nicotine dependence in the present study.Data from the Framingham Heart Study along with clinical exam information from 1948 through 1988 for the original cohort and from 1971 through 1991 for the offspring cohort were provided through Genetics Analysis Workshop 13 (GAW13). On the basis of number of smokers present at each exam, the consistency of the clinical data and interviewing time between the two cohorts, and the potential environmental effect on smoking phenotype included in the Framingham Heart Study data, Exam 12 from 1970 for the original cohort and Exam 1 from 1971 for the offspring cohort were selected and used in this study.From the 330 extended families of the Framingham Heart Study, 313 were chosen in which there was at least one smoker present in the data from 1970\u20131971. Table Two linkage analysis programs (SIBPAL in S.A.G.E. v. 4.2 and GENEHUNTER v. 2.1) were used in the study. For the EM (expectation maximization) Haseman-Elston quantitative trait locus (QTL) regression method implemented in GENEHUNTER, we analyzed both log-transformed and categorized SR data sets. A detail description on the method can be found in Kruglyak et al.  and KrugTo maximally utilize the phenotypic information from the Framingham Heart Study data, we searched clinical records regarding smoking status of each subject from 1948 to 1988 for the original cohort, and from 1971 to 1989 for the offspring cohort. It appears that data from 1970 for the original cohort and from 1971 for the offspring cohort were more complete and contained significantly more smokers relative to other time points  for both cohorts. As shown in Table p = 0.000018) and the categorized SR data sets (p = 0.000001), based on the threshold suggested by Nyholt [p < 0.0017; [p < 0.01 . Furthe01 Table . To conf01 Table .In this study, by using the EM Haseman-Elston QTL regression method implemented in GENEHUNTER and the SIBPAL program of S.A.G.E., we obtained evidence for significant linkage of smoking rate to chromosome 11 and suggestive linkage to chromosomes 4, 7, 9, 14, and 17. Additionally, our results suggest that the genomic regions mapped on chromosomes 1, 6, 12, 15, 20, and 21 are of potential interest to harbor susceptibility genes for nicotine dependence at a significance level of 0.01. Of these loci, three loci on chromosomes 7, 11, and 17 were identified by both linkage analysis methods.p = 0.0063; see Table p-values for most regions reported in our study than other GAW13 analyses evaluating smoking as the phenotype may due to how the data measuring smoking phenotype were selected from the Framingham Heart Study data. As indicated earlier, we only used the smoking information obtained from the exam conducted during 1970\u20131971, instead of using the maximum number of cigarettes smoked per day across multiple exams over many years. Epidemiological studies have indicated that there has been a steady and dramatic decline of 40% in the prevalence of cigarette smoking by people 18 years or older in the US from 1965 to 1990 [p-values obtained in this study.Although our mapping results provided weak evidence for linkage of smoking rate to chromosome 20  from those who were never exposed to nicotine. As documented earlier, the transformed smoking phenotype still deviated slightly from a normal distribution; however, we do not feel that such remaining kurtosis would have a large effect on the linkage results reported herein, because only model-free methods were used in the analysis and they tend to be more robust to the presence of non-normality in the data. Also, the participants in the Framingham Heart Study are predominantly Caucasian Americans. Accordingly, it is of interest to know whether we can repeat these findings in other ethnic populations.The 313 extended Framingham Heart Study families were analyzed to identify susceptibility loci for smoking rate by the Haseman-Elston regression methods implemented in GENEHUNTER and the SIBPAL program of S.A.G.E. Our genome-wide scan results provided evidence for significant linkage of smoking rate to chromosome 11 and suggestive linkage to chromosomes 4, 7, 9, 14, and 17. Additionally, we found several regions located on chromosomes 1, 6, 12, 15, 20, and 21 are potentially of interest with a significance level of <0.01. Interestingly, the genomic regions on chromosomes 7, 11, and 17 were identified by both the linkage methods. To our knowledge, most of the susceptibility regions for smoking rate identified in this study have not been reported previously and thus replication of these findings is an important next step."}
+{"text": "Studies have shown that genetic and environmental factors and their interactions affect several alcoholism phenotypes. Genotype \u00d7 alcoholism (G\u00d7A) interaction refers to the environmental  influences on the autosomal genes contributing to variation in an alcoholism-related quantitative phenotype. The purpose of this study was to examine the effects of G\u00d7A interaction on the detection of linkage for alcoholism-related phenotypes.We used phenotypic and genotypic data from the Collaborative Study on the Genetics of Alcoholism relating to 1,388 subjects as part of Genetic Analysis Workshop 14 problem 1. We analyzed the MXDRNK phenotype to detect G\u00d7A interaction using SOLAR. Upon detecting significant interaction, we conducted variance-component linkage analyses using microsatellite marker data. For maximum number of drinks per a 24 hour period, the highest LODs were observed on chromosomes 1, 4, and 13 without G\u00d7A interaction. Interaction analysis yielded four regions on chromosomes 1, 4, 13, and 15. On chromosome 4, a maximum LOD of 1.5 at the same location as the initial analysis was obtained after incorporating G\u00d7A interaction effects. However, after correcting for extra parameters, the LOD score was reduced to a corrected LOD of 1.1, which is similar to the LOD observed in the non-interaction analysis. Thus, we see little differences in LOD scores, while some linkage regions showed large differences in the magnitudes of estimated quantitative trait loci heritabilities between the alcoholic and non-alcoholic groups. These potential hints of differences in genetic effect may influence future analyses of variants under these linkage peaks. Family, twin, and adoption studies have indicated that genetic and environmental factors and their interactions contribute to the development of alcoholism -3. SeverG) is less than 1 between exposure groups. In an interaction model, assuming the probability of an individual having a specific polygenotype is independent of environment, the expected additive genetic covariance between a pair of alcoholics is COV = 2\u03a6 \u03c32Galc or the covariance between a pair of non-alcoholics would be COV = 2\u03a6 \u03c32Gnalc, i.e., 2\u03a6 times the appropriate genetic variance.In this study, the Genetic Analysis Workshop 14 (GAW14) COGA data (Problem 1) consisting of 1,388 family members, have been analyzed. Prior to the analysis we recoded the affection status based on the definition of alcoholism according to COGA as well as DSM-IV criteria in two ways: diagnoses 1 and 2 correspond to COGA and DSM-IV and that a includes individuals with some symptoms as unaffected (diagnoses COGA-Aldxla and DSM-IV-Aldx2a), whereas b considers them unknowns (diagnoses COGA-Aldx1b and DSM-IV-Aldx2b). In the analysis of the GAW14 COGA data, we used a maximum likelihood variance components approach for the study of G\u00d7E interaction using related individuals in different environments . To miniThe covariance between an alcoholic individual and a nonalcoholic one is modeled as: = 2\u03a6 \u03c3Galc \u03c3Gnalc \u03c1G,COVG is the additive genetic correlation between the expression of the phenotype in the two environments, and \u03c3Galc and \u03c3Gnalc are the additive genetic standard deviations of alcoholics and nonalcoholics, respectively. We first screened for the presence of G\u00d7A interaction in several quantitative phenotypes, including MXDRNK, using a quantitative genetic method. After detecting significant G\u00d7A interaction for MXDRNK phenotype, we performed variance-component linkage analyses with a customized model to include diagnosis-specific quantitative trait loci (QTL) effects and using microsatellite marker multipoint identity by descent (MIBD) matrices estimated using LOKI. The customized variance component linkage model may be defined aswhere alc and nalc denote alcoholics and non-alcoholics, respectively, \u03a6 is the coefficient of kinship between the two individuals, \u03c1 = \u03a0 \u03c3Qalc \u03c3Qnalc + 2\u03a6 \u03c3Galc \u03c3Gnalc \u03c1G.COVc) scores assume that \u03c3Qalc \u03c3Qnalc are independent under the null, producing a test statistic distribution of \u00bc\u03c722, \u00bd\u03c721, \u00bc point mass at 0. This assumption may be overly conservative. These analytical techniques were implemented using the computer program SOLAR [This model has an additional QTL variance as compared with the standard linkage model. Corrected LOD (LODam SOLAR .G < 1), suggesting different genetic effects in the two environments. It is necessary to replicate this finding in the entire COGA sample to make sure that it is not a false positive. But if we correct for multiple testing, i.e., by multiplying p-values by 28 (7 traits \u00d7 2 diagnoses \u00d7 2 parameters tested), MXDRNK is still significant (p < 0.028).Results of G\u00d7A interaction analyses for several quantitative phenotypes according to two diagnostic criteria with a modified coding for affection status are shown in Table 2 \u00b1 SE, LNMXDRNK = 0.18 \u00b1 0.05, p < 0.0001) after adjusting for age, sex, and smoking influences. For MXDRNK, chromosomal regions with LODs > 1 obtained in both linkage analyses (non-G\u00d7A and with G\u00d7A) are presented in Table c) = 0.9 at 238 cM) near the marker D1S2141, which is located 44 cM centromeric to a region obtained initially in the analysis without G\u00d7A interaction. The chromosomal region near marker D4S1651 on chromosome 4 showed improved evidence for linkage  to the MXDRNK phenotype incorporating G\u00d7A interaction (Aldx1b diagnosis) near marker D4S1651 (126 cM). On chromosome 13, the initial analysis yielded a max LOD of 2.24 at 64 cM near marker D13S800, while the interaction analysis showed a reduced LOD of 1.21 (LODc = 0.8) at 59 cM in almost the same region between markers D13S318 and D13S800. Also, an additional region with a max LOD of 2.04 (LODc = 1.6) at 100 cM was obtained near marker D15S205 in the G\u00d7A interaction analysis.Descriptive statistics for the MXDRNK phenotype based on diagnostic criteria and affection status are reported in Table Environment may influence the variation in the expression of genes influencing a variety of phenotypes including alcohol-related phenotypes. Genotype \u00d7 alcoholism interaction was explored for a variety of quantitative phenotypes in the COGA dataset but was detected only for the MXDRNKs phenotype. Results of G\u00d7A analyses were consistent across the COGA and DSM-IV alcoholism diagnoses, but were affected by the categorization of individuals who were unaffected with some symptoms. Results were generally stronger when these individuals were categorized as unaffected, rather than unknown. However, this result may be strongly influenced by sample size considerations as the addition of the \"unaffected with some symptoms\" more than doubled the size of the nonalcoholic group.c = 1.6, 100 cM), which is only 25 cM away from a linkage with factor 2 [Our analysis also shows that accounting for G\u00d7E interaction may increase the linkage signal. For MXDRNK, interaction analysis failed to show evidence at the implicated region on chromosome 1 in the non-G\u00d7A analysis but yielded a slightly increased linkage signal at a different location (238 cM), which corresponds to the previously reported linkage with factor 2, a factor analysis-derived trait defined by harm avoidance, novelty seeking, and age of onset of drinking . On chrofactor 2  and QTL In conclusion, genotype \u00d7 alcoholism interaction analysis yielded interesting results. Drinking behavior appears to be influenced by environment-specific genes in both alcoholics and non-alcoholics. The implicated regions on chromosomes 1, 4, and 15 are consistent with previously reported linkage findings. These results indicate that further analyses may benefit from considering the possibility of differing genetic effects in alcoholics and non-alcoholics, for example by stratifying analysis on alcoholism diagnosis.COGA: Collaborative Study on the Genetics of AlcoholismERP: Event-related evoked potentialsG\u00d7A: Genotype \u00d7 alcoholismG\u00d7E: Genotype \u00d7 environmentGAW14: Genetic Analysis Workshop 14MIBD: Multipoint identity by descentMXDRNK: Maximum number of drinks per a 24-hour periodQTL: Quantitative trait lociRA contributed to the study design, performed the genetic analyses, interpreted the results, and prepared the manuscript. LA has contributed the study design, methodology, and interpretation of results. TDD provided MIBDs. DMW ran initial linkage screen based on which traits were selected for this study. RD and CPJ participated in the interpretation of results."}
+{"text": "The purpose of these analyses was to determine if incorporating or adjusting for covariates in genetic analyses helped or hindered in genetic analyses, specifically heritability and linkage analyses. To study this question, two types of covariate models were used in the simulated Genetic Analysis Workshop 14 dataset in which the true gene locations are known. All four populations of one replicate were combined for the analyses. The first model included typical covariates of sex and cohort (population) and the second included the typical covariates and also those related endophenotypes that are thought to be associated with the trait . A final best fit model produced in the heritability analyses was used for linkage. Linkage for disease genes D1, D3, and D4 were localized using models with and without the covariates. The use of inclusion of covariates did not appear to have any consistent advantage or disadvantage for the different phenotypes in regards to gene localization or false positive rate. The analyses of complex traits can be complex. Often the phenotype itself is not defined or well measured, and voluminous information is collected that relates to the trait. While this increased phenotyping can be helpful in analyses, there still remains a question as to the best methods for incorporating additional information into the genetic analyses: \"How do we analyze all the data together?\"The Genetic Analysis Workshop 14 (GAW14) dataset was simulated to reflect realistic issues in study design and data collection. Data were gathered from several different research groups using different ascertainment schemes and different affection criteria. The data therefore are heterogeneous, reflecting reality. The purpose of these analyses was to determine if the inclusion of typical covariates (sex and population or cohort) and endophenotype traits  improved the genetic analyses of Kofendrerd Personality Disorder (KPD) and the 12 endophenotypes.p < 0.05) were included in the final model.To imitate a realistic situation, only one replicate (23) of the simulated GAW14 dataset was used. All families from all four populations were included, even though each population had different ascertainment schemes. The affection status for KPD and all phenotypes  were studied. Each phenotype was analyzed with 3 types of variance component models: 1) without any covariates included in the model, 2) with sex and population , 3) with significant variables of sex, population, and the other 12 endophenotype traits. Each covariate was tested independently for significance, and only significant covariates (m2), unmeasured genetic effects (2\u03c6\u03c3g2), and other effects (I\u03c3e2). \u03a9 = \u03a0\u03c3m2 + 2\u03c6\u03c3g2 + I\u03c3e2, where \u03c3m2 is the additive genetic variance due to the major locus, and \u03a0 is a matrix of elements that provide the probability that individuals i and j are identical-by-descent (IBD) at a trait locus that is linked to a genetic marker locus. \u03a0 is a function of the estimated IBD matrix of the genetic marker itself and a matrix of the correlations between the proportion of genes IBD at the marker and at the trait. \u03c3g2 is the genetic variance due to residual additive genetic factors, \u03c6 is the kinship matrix, \u03c3e2 is the variance due to individual-specific environmental effects, and I is an identity matrix. The dichotomous variables were analyzed modeling the discrete affection status trait as a threshold model [2 variable and a point mass at zero. Two point linkage analyses were performed using all of the genome scan markers. LOD scores > 3.3 were considered significant for linkage from the genome scans [Heritability and linkage analyses were performed using variance component analyses or random effects models -4 as impld model , whereasme scans .The inclusion of the endophenotypes as covariates into the variance component models influenced some of the heritability estimates and linkage analyses.p < 0.000001). It appears that there may be a significant genetic component to KPD that is not explained by the associated phenotypes, however the heritability parameter is at a boundary that may indicate instability.Not all of the phenotypic traits were heritable Table . KPD wasFive of the phenotypes appear to have a significant genetic component either with or without covariates: phenotypes A, B, D, K, and L. Interestingly, phenotypes C and G appeared heritable when analyzed without any covariates, but the heritability is no longer significant when the associated phenotypes are included in the model.Surprisingly, population was not as much of a confounder as expected; it was only significant for phenotype D. Population was associated with KPD when only population and sex were included in the model. Sex was significant for KPD, phenotype I, and phenotype L.The inclusion of covariates in the linkage analyses did not produce consistent results among the phenotype variables as to the localization of genes Tables  and 3. DForty loci were significant for KPD with the inclusion of covariates, however none of them was the closest to the disease gene. These markers with LODs are listed, along with the LODs near the true disease locations in centimorgans: D01S0011 (3.68), D01S0016 (4.15), D01S0017 (5.07), D01S0018 (3.61), D01S0023 *D1* (2.80), D01S0034 (4.24), D02S0043 *D6* (3.25), D02S0057 (4.62), D02S0075 (3.36), D02S0076 (4.50), D02S0080 (3.94), D02S0081 (3.78), D02S0082 (3.42), D03S0126 (4.06), D04S0128 *D2* (0.00), D04S0145 (3.83), D04S0159 (4.40), D04S0160 (3.79), D04S0171 (4.12), D05S0172 *D3* (1.54), D05S0174 (3.84), D05S0183 (3.49), D05S0211 (3.37), D05S0213 (3.38), D06S0222 (3.86), D06S0229 (4.11), D06S0232 (3.50), D06S0244 (3.92), D06S0246 (4.83), D06S0248 (3.95), D07S0264 (3.78), D07S0271 (3.61), D07S0274 (4.69), D07S0276 (3.82), D07S0289 (4.06), D08S0304 (3.77), D08S0329 (4.32), D09S347 *D4* (1.19), D09S0350 (3.71), D09S0356 (3.83), D09S0376 (3.53), D09S0381 (4.41), D09S0388 (4.23), D10S0400 *D5* (0.47), D10S0413 (4.06), and D10S0414 (3.46). These false positives could be due to violations of assumptions about the distribution of the trait; specifically the assumption that there is a threshold and an underlying distribution. The simulation parameters show that the assumption is violated as unaffecteds and affecteds have different genetic liabilities. This could explain why heritability is unstable and goes to a boundary when adjusted for covariates.Disease genes D1, D3, and D4 for phenotypes A, B, and K were localized with or without the inclusion of covariates. Genes were only localized for Phenotypes C, D, G, and L with models that did not include the covariates.Heritability was no longer significant for phenotype C or G when covariates were in the model. The use of endophenotypes for covariates may have regressed out the major gene effects. Therefore, any genes revealed in the analyses without the covariates should reveal shared effects. Phenotypes C and G showed linkage to D3 and D4, genes identified in analyses of other phenotypes.Several false positives were found in the search for disease genes, including a false-positive region quite distal to any disease genes. The inclusion of covariates did not consistently alter the false-positive rates.Simulated disease genes D1, D3, and D4 were localized by variance component linkage analyses to the endophenotypes of the simulated KPD trait. Adjusting models for covariates and the other endophenotypes did not consistently alter the findings.GAW14: Genetics Analysis Workshop 14IBD: Identical by descentKPD: Kofendrerd Personality Disorder"}
+{"text": "We investigate the power of heterogeneity LOD test to detect linkage when a trait is determined by several major genes using Genetic Analysis Workshop 13 simulated data. We consider three traits, two of which are disease-causing traits: 1) the rate of change in body mass index (BMI); and 2) the maximum BMI; and 3) the disease itself (hypertension). Of interest is the power of \"HLOD2\", the maximum heterogeneity LOD obtained upon maximizing over the two genetic models.Using a trait phenotype Obesity Slope, we observe that the power to detect the two markers closest to the two genes  at the 0.05 level using HLOD2 is 13% and 10%. The power of HLOD2 for Max BMI phenotype is 12% and 9%. The corresponding values for the Hypertension phenotype are 8% and 6%.The power to detect linkage to the slope genes is quite low. But the power using disease-related traits as a phenotype is greater than the power using the disease (hypertension) phenotype. One of the issues in analysis of complex diseases is the power of considering an disease related trait as a phenotype than the disease itself . AnotherIn this work, we want to investigate the power of HLOD maximized over two genetic models (dominant and recessive) to detect linkage with the Obesity Slope phenotype and also with the hypertension phenotype, i.e., the disease itself. We also consider another trait, the individual's maximum observed BMI (Max BMI), which is related to both obesity and hypertension.ij) = \u03b1j + \u03b2jlog10(Xij), for i = 1,2,..., tj.The first phenotype considered is Obesity Slope. For each individual, we regressed BMI values on log of age values at each time where the BMI was recorded, using the following model: E. We defined individuals as \"High\" if Max BMI \u2265 m90 and \"Normal\" if Max BMI < m90, where m90 denotes the 90th percentile of the Max BMI values observed in each replicate being analyzed. The third phenotype is \"Hypertension.\" Individuals defined as \"affected\" are those with diastolic BP \u2265 90 and/or systolic BP \u2265 140, and \"Normal\" are those having diastolic BP < 90 and systolic BP < 140.The second disease-related phenotype considered here is the maximum of jSingle-point linkage analysis was done for the two markers closest to the two genes S1 and S2, and for five unlinked markers. One marker linked to S1 is 11g6 and the other, linked to S2, is 7g7. We also considered five unlinked markers on chromosomes 2, 4, 6, 8, and 10 , on which none of the disease genes were located.We used the linkage test specified on Abreu et al. . We obtaN = 500. Then we obtained power values for the three phenotypes by computing the percentage of replicates with higher HLOD2 (or LOD2) values than the critical value in all 100 replicates. Highest power is 13% for Obesity Slope phenotype at the 11g6 marker. Power values for three phenotypes are presented in the Table With the results of analysis of five unlinked markers using the Obesity Slope phenotype, the empirical critical values for a 0.05 level test are 0.947 for HLOD2 and 0.730 for LOD2 from the null distribution of size 2 = 19.5). On the other hand, there is no association in this sample between being in the top 10% for the Slope Phenotype and having hypertension (\u03c72 = 2.66).The Obesity Slope phenotype resulted in the highest power. The second highest is the Max BMI Figure . At a ceThere are several possible explanations for the low power to detect linkage observed here. The most likely one is that we lose power by dichotomizing the values rather than analyzing these traits as quantitative phenotypes using model-free methods. Second, even though we are focusing on the slope itself, which is the direct product of the gene, we do have error in our estimate of an individuals' slope due to the combination of random noises in the simulations models.The overall power values for HLOD2 analysis are quiet low in the case of the analysis of dichotomized BMI slope values and dichotomized BMI values. However, they are higher than those obtained using the hypertension phenotype."}
+{"text": "NRXN1 at 2p21 and a few others in the inter-gene regions. The relative magnitude of risks to the identified risky/protective haplotypes was elucidated.A thorough genetic mapping study was performed to identify predisposing genes for alcoholism dependence using the Collaborative Study on the Genetics of Alcoholism (COGA) data. The procedure comprised whole-genome linkage and confirmation analyses, single locus and haplotype fine mapping analyses, and gene \u00d7 environment haplotype regression. Stratified analysis was considered to reduce the ethnic heterogeneity and simultaneously family-based and case-control study designs were applied to detect potential genetic signals. By using different methods and markers, we found high linkage signals at D1S225 (253.7 cM), D1S547 (279.2 cM), D2S1356 (64.6 cM), and D7S2846 (56.8 cM) with nonparametric linkage scores of 3.92, 4.10, 4.44, and 3.55, respectively. We also conducted haplotype and odds ratio analyses, where the response was the dichotomous status of alcohol dependence, explanatory variables were the inferred individual haplotypes and the three statistically significant covariates were age, gender, and max drink (the maximum number of drinks consumed in a 24-hr period). The final model identified important AD-related haplotypes within a candidate region of  Alcohol dependence (AD) is a complex disorder accompanying familial aggregation and etiological heterogeneity. The development of AD involves genetic and environmental components as well as gene \u00d7 gene and gene \u00d7 environmental interactions. Due to these factors, results from different studies often diverge .Owing to the advancement of biotechnology, enormous numbers of short tandem repeat polymorphisms (STRPs) and single-nucleotide polymorphisms are available to help the process of gene mapping. In this report, STRP and SNP markers were integrated and a five-stage procedure was designed to identify the putative AD loci and to elucidate the genotype-phenotype-covariate relationship. Different methodologies  were considered for statistical analyses, different populations  for heterogeneity issues, different types of markers (STRPs and SNPs) for linkage mapping, different densities of SNPs (Illumina and Affymetrix) for association study, and different data structures (family data and case-control data) for study design to yield reliable conclusions.From the COGA ascertainment criteria, the numbers of total patients, pure unaffected individuals, and others were 643 (39.84%), 285 (17.66%), and 686 (42.50%), respectively. The category \"others\" was considered as \"unknown\" throughout our analyses. On average, 60% of parents' genotypes were available.In total, 315 STRPs, 4,720 Illumina SNPs, and 11,120 Affymetrix SNPs on the 22 autosomal chromosomes with average spacing of 11.53 cM, 0.75 cM, and 0.32 cM were considered. The genetic map was provided by the Genetic Analysis Workshop 14 (GAW14) working group.Ethnic heterogeneity was considered by stratifying the studied families as pure \"non-Black\" and \"non-White\" families, i.e., families where none of the members were from the Black population and vice versa. The non-Black population contained 1,300 individuals from 119 families and non-White families contained 247 individuals from 19 families. Other families were not included in this report. In addition to family data, founders from each family were selected for case-control data that contained 505 individuals with 52 affected (cases), 127 unaffected (controls) and 326 individuals with other phenotypes.To explore the phenotype \u00d7 genotype relationship and locate the AD predisposing genes, we carried out a five-stage procedure. The first stage was designed to search the potential candidate regions by considering a genome-wide linkage analysis using the STRP markers. GENEHUNTER  and SIMWThe second stage used denser SNP markers to confirm linkage results obtained in the first stage. On the basis of the NPL scores from the first stage, a candidate region was defined to be a segment in which all NPL scores exceeded 1 and the maximal NPL score exceeded 3. In the candidate regions, SIMWALK2 [In the third stage, association analyses were conducted using SNPs to further narrow the candidate region. Transmission disequilibrium tests were performed by using PDT  and FBATIn the fourth stage, anchor markers were selected on the basis of results from the third stage. HAPLOVIEW  was usedIn the fifth stage, the relationships between genotype, phenotype, and covariates underlying the complex alcoholism etiology was further explored. The individual haplotypes were inferred based on results obtained from SIMWALK2  for famiA genome-wide multipoint linkage analysis for the 22 pairs of autosomal chromosomes based on the 315-STRP markers using GENEHUNTER [To reduce false-positives due to population heterogeneity, stratified analyses by selecting non-Black and non-White subpopulations from the whole population was conducted. Whole-genome linkage mapping with STRP markers was applied to these two subpopulations and yielded rather different results compared with the whole population. The results are shown in Figure In the second stage, we conducted SNPs linkage analysis to confirm the STRP linkage results of chromosomes 1, 2, and 7 found in the first stage. The three candidate regions determined by the mentioned criteria were D1S518-D1S547, D2S320-D2S436, and D7S1790-D7S665. In these three candidate regions, the Linkage III Panel of SNPs of Illumina consists of 38, 151, and 103 SNPs and the inter-marker distances are 0.99, 0.53, and 0.74 cM in average. The GeneChip Mapping 10 K Array marker set of SNPs of Affymetrix consists of 113, 344, and 238 SNPs and the average distances between markers are 0.47, 0.23, and 0.30 cM. The results confirm the previous linkage results and find significant Illumina and Affymetrix SNPs with NPL scores > 3 on chromosome 2 as shown in Figure p-value < 0.01) without correcting multiple tests are shown in Table p-values are transformed by taking -log10.In the third stage, further fine mapping was pursued to narrow down the candidate regions using association tests. Based on family-based transmission disequilibrium tests . On chromosome 7, haplotype 111 from SNPs tsc0018713, tsc0018712, and tsc0593964 is a risk haplotype with an OR of 2.13 and corresponding 95% CI .In the fifth stage, haplotype regression analyses considering three significant covariates  were conducted, which were selected in preliminary analysis. Results of adjusted odds ratio are summarized in Table NRXN1 related to polymorphic cell surface proteins was identified, as well as two strongly protective haplotypes in inter-gene regions. On chromosome 7, one moderately risky haplotype in an inter-gene region was identified. These results should be useful to biologists for the advanced study of functional cloning.In summary, some potential candidate regions on chromosomes 1, 2, and 7 linked with AD susceptibility loci were found. These findings are consistent with previous reports ,11. MoreThe linkage scans based on three different marker sets were compared. The curves of NPL scores based on two SNP sets are quite similar; however, the SNP scans and STRP scan show somewhat inconsistent results on different chromosomes. On chromosome 2, SNP linkage scan confirms STRP scan and yields more and higher linkage signals in the same region. In other candidate regions, SNP scans fail to identify any important SNPs, probably due to their lower information content. We also compared the results from three association tests and found many different significant SNPs based on family-based and case control association tests. The differences were probably due to the different samples used in the analyses and information extracted from transmission and linkage disequilibrium tests.Our five-stage gene mapping procedure is elaborate though incomplete. Other analytical strategies, such as quantitative trait analysis, will provide complementary information to further dissect the etiology of AD.AD: Alcohol dependenceCOGA: Collaborative Study on the Genetics of AlcoholismGAW: Genetic Analysis WorkshopNPL: Nonparametric linkageSNP: Single-nucleotide polymorphismSTRP: Short tandem repeat polymorphismH-CY conceived the statistical analysis scheme, coordinated the project and drafted the manuscript. CSJF contributed to the discussion and preparation of the final manuscript. Other members carried out the data management, statistical analysis and technique assistance. All authors have approved the final manuscript."}
+{"text": "We present a meta-analysis procedure for genome-wide linkage studies (MAGS). The MAGS procedure combines genome-wide linkage results across studies with possibly distinct marker maps. We applied the MAGS procedure to the simulated data from the Genetic Analysis Workshop 14 in order to investigate power to detect linkage to disease genes and power to detect linkage to disease modifier genes while controlling for type I error. We analyzed all 100 replicates of the four simulated studies for chromosomes 1 (disease gene), 2 (modifier gene), 3 (disease gene), 4 (no disease gene), 5 (disease gene), and 10 (modifier gene) with knowledge of the simulated disease gene locations. We found that the procedure correctly identified the disease loci on chromosomes 1, 3, and 5 and did not erroneously identify a linkage signal on chromosome 4. The MAGS procedure provided little to no evidence of linkage to the disease modifier genes on chromosomes 2 and 10. Kofendred Personality Disorder (KPD), as simulated for Genetic Analysis Workshop 14 (GAW14), is a psychiatric syndrome characterized by an overwhelming concern with the meaning of personal inner emotions while regarding the emotions of others. Like other complex personality disorders, KPD has numerous behavioral and biological characteristics. Additionally, KPD, like other complex diseases, is believed to be linked to many genes. The possibility of finding the majority of these genes from one independent study is small. Instead, pooling data across independent studies  or pooling linkage results across independent studies  may be the best means to identify these numerous genes with small effects.p-values, LOD scores, or effect sizes.In a mega-analysis, combining raw data from several studies allows the investigator to increase sample size. A mega-analysis can lead to an increase in power to detect linkage and reduce the level of type I error. Combining raw data would be an ideal approach, but data are not always readily available or freely shared. In a meta-analysis, the investigator can still combine information from several studies to obtain a consensus for linkage. The information typically found in the literature can range from published p-value) at each marker. Combining results from significance tests can be limited [Caveats to mega- and meta-analyses involve among-study heterogeneity, which can include differing marker maps, informativeness, sample sizes, sampling plans, and linkage tests. Methods have been proposed to handle such problems. The genome-scan meta-analysis (GSMA) method proposed by Wise et al.  accommod limited -4 where  limited  develope limited  developeIn this paper, we present an updated meta-analysis method for assessing linkage to a quantitative trait locus (QTL) that generalizes the meta-analytic procedure first proposed by Etzel and Guerra  such thap-values).The MAGS method that we developed is based on procedures proposed by Loesgen et al.  and Etzek studies. Each study k has mk number of markers. It is not assumed that the studies have the same number of markers, mi \u2260 mk, i \u2260 j nor it is assumed that the studies have the same marker maps. For a specified chromosome, let Mst denote the tth marker from study s, for s = 1, ..., k and t = 1, ..., mk. Define {Lq,q = 1, ..., l} as the set of analysis points such that the Lq are equally spaced across the chromosome. For each set of Mst on a chromosome,The MAGS method is based on a weighted average of transformed normal variates that are obtained through the reported linkage summary statistics. Suppose that we wish to complete a meta-analysis on Mst to a p-value, pst, for example1. Transform the summary statistic for each marker Xst = 4.6* HLODst and obtain a p-value for each chi-square variate [a. HLOD to Chi-square variate:  variate : p-value: pst = Pr(Z <NPLst). This Z is not necessarily a standard normal random variable. Rather, Z is a normal variable with mean 0 and standard deviation of \u03c32. The calculation of \u03c32 is difficult and may be influenced by incomplete information. However, we have found that in practice (data not shown) that this situation does not erroneously affect the meta-analysis.b. Transform NPL to a p-value to a normal variate: Zst = \u03a6-1 (Pst)2. Transform the resulting Lq, calculate the weighted normal variate:3. For each analysis point wstq is the weight given to marker Mst and Zst is the normal variate for marker Mst. The indicator function  is defined as 1 if marker Mst is within a set distance D cM from analysis point Lq and 0 otherwise. The weight wstq for marker Mst can be a function of study sample size, information content at that marker, and/or distance  between marker Mst and analysis point Lq, say .where p-value for each meta-analytic variate: 4. Calculate the p-values from step 4 can then be compared to a set level to determine areas with combined evidence for linkage. NOTE: If all studies use the same marker map, then the combined set of markers can replace the analysis points Lq and step 3 simplifies to The p-values using step 1b. The subsequent normal variates were weighted by wst = Istns, where ns is defined as the total number of individuals used in the linkage analysis of study s and Ist is the information content (given as output from GENEHUNTER2) for marker t in study s. For AI, n1 = 683, for DA, n2 = 700, for KA n3 = 694, and for study NY, n4 = 943. Since the four studies had identical marker maps, we used the simplified version of step 3 to calculate the weighted MAGS estimate  for each marker location t.The provided genome-wide microsatellite marker maps were identical for all four simulated data sets. We used GENEHUNTER2 ,9) to asD = 10 cM. We chose D = 10 cM because a polymorphism at a marker linked to an analysis point can provide information about the polymorphism at the putative QTL. If an analysis point had only one marker within a 10-cM radius, then no meta-analysis was conducted. For studies DA and KA, we transformed the NPL scores obtained from GENEHUNTER to p-values using step 1b; for study NY, we transformed the MaxLOD scores obtained from LINKAGE using step 1a; for study AI, we transformed the t-value obtained from SAGE in a similar fashion to the NPL score in step 1b. The subsequent normal variates were weighted by wstq = (1 - 2\u03b8stq)2 ns, where ns is defined as the total number of individuals used in the linkage analysis of study s. Note that the weights in this application do not include information content because the measure was not available from all analysis packages . For study AI in which we used Sibpal, n1 = 483 possible siblings were included in the analyses. For the studies in which we used GENEHUNTER2 (DA and KA), n2 = 700 and n3 = 694 individuals, respectively, were included in the analyses. For study NY, n4 = 943 individuals were included in the analyses.The provided genome-wide SNP marker maps were also identical for all 4 simulated data sets. In order to fully evaluate the MAGS method using studies with differing marker maps and different analysis packages, we created unique genome-wide SNP marker maps for each study for chromosomes 1, 2, 3, and 10. The unique marker maps were created by simply randomly assigning the available markers on each chromosome to one of the four studies. We then used a different linkage test within each study to assess linkage to KPD one chromosome at a time: the Sibpal procedure from SAGE ,9 for st for the microsatellite analysis and  for the SNP analysis) exceeded a set critical value at varying alpha levels  [We calculated the frequency (across the 100 replicates) that the resulting MAGS values ( \u00d7 10-5)  to evalu-5.Figure -5. Additionally, this gene was not identified in any of our single-study replicates. Meta-analysis of chromosome 4, which did not contain any disease genes, did not detect any erroneous linkage signals for alpha levels less than 1%.For the disease modifier gene D6 (located approximately at 15 cM on chromosome 2) that affects the penetrance of phenotype 2, the MAGS method was not able to distinguish its signal from that of background genetic noise. Furthermore, D6 was not identified in any of our analyses of the individual studies. Likewise for the modifier gene D5 that is located on chromosome 10 (approximately 67 cM). The location of gene D5 was only identified in 10 of the 100 MAGS replicates when the alpha level was set at 1% and in none of the replicates when the alpha level was set at 2.2 \u00d7 10-5. Difficulty in identifying D2 was attributable to the sparseness of SNPs in the modified marker maps in the region at the very end of chromosome 3 surrounding D2. As with the microsatellite results, the modifier genes, D5 and D6, were not clearly identified.In the analyses using the modified SNP marker maps (data not shown), D1 was identified in over 80% of the replicates while D2 was only identified in 20% of the replicates at an alpha level 2.2 \u00d7 10Meta-analysis provides a means of combining information about linkage from smaller independent studies to identify genetic linkage to a complex trait while adjusting for among-study heterogeneity . Because multiple genes are believed to be involved in a complex disease, many with modest effects, the probability of identifying them from single studies and replicating the results is low. This meta-analysis procedure correctly identified the three major genes we analyzed with high power even under quite restrictive conditions. Furthermore, this method did not erroneously identify linkage where no linkage was simulated when alpha levels of 0.1% or lower were used. In fact, based on the results for chromosome 4 (where no linkage was simulated) depicted in Figure The MAGS method performed better for the microsatellite marker maps than for the modified SNP marker maps. The microsatellite maps had higher marker density than the modified SNP maps with possibly different information content per marker. Also, when we analyzed the modified SNP maps, we did not use the same test for linkage in each study and we did not include information content in the MAGS calculation. The linkage tests that we used  vary in the type of pedigree structure and data that is used to test for linkage and hence vary in power to detect linkage which therefore affected the SNP meta-analysis. However, any meta-analytic procedure that is conducted on studies using varying linkage tests (with varying levels power to detect linkage) will be affected by these among-study differences. Meta-analysis provides a way to obtain consensus for linkage to a disease and is clearly an important step in the localization of genes involved in complex diseases.GAW14: Genetic Analysis Workshop 14KPD: Kofendred Personality DisorderMAGS: Meta-analysis procedure for genome-wide linkage studiesNPL: Nonparametric linkageQTL: Quantitative trait locusSNP: Single-nucleotide polymorphismGSMA: Genome scan meta-analysisCJE conceived of the application of this methodology as well as developed the meta-analytic method to the simulated data provided by GAW14. ML conducted the analyses of the simulated data with the assistance of CJE and TJC. All authors contributed to the writing and approval of the final manuscript."}
+{"text": "Both theoretical and applied studies have proven that the utility of single nucleotide polymorphism (SNP) markers in linkage analysis is more powerful and cost-effective than current microsatellite marker assays. Here we performed a whole-genome scan on 115 White, non-Hispanic families segregating for alcohol dependence, using one 10.3-cM microsatellite marker set and two SNP data sets . Two definitions of alcohol dependence (ALDX1 and ALDX2) were used. Our multipoint nonparametric linkage analysis found alcoholism was nominal linked to 12 genomic regions. The linkage peaks obtained by using the microsatellite marker set and the two SNP sets had a high degree of correspondence in general, but the microsatellite marker set was insufficient to detect some nominal linkage peaks. The presence of linkage disequilibrium between markers did not significantly affect the results. Across the entire genome, SNP datasets had a much higher average linkage information content  than did microsatellite marker set (0.57). The linkage peaks obtained through two SNP datasets were very similar with some minor differences. We conclude that genome-wide linkage analysis by using approximately 5,000 SNP markers evenly distributed across the human genome is sufficient and might be more powerful than current 10-cM microsatellite marker assays. In traditional linkage analysis for identifying genomic regions related to disease phenotypes, a whole-genome scan is usually performed using a set of 300\u2013400 microsatellite markers evenly spaced across the genome. To maximize the chances of detecting linkage, the optimal amount of inheritance information is critical. This can be increased by genotyping more families and adding additional markers. With the rapid discovery of SNPs across the genome and the development of large-scale, high-throughput SNP genotyping approaches, high-density SNP assays throughout the genome may be a more rapid, powerful, and cost-effective tool than microsatellite marker assays in linkage analysis . RecentlThe COGA dataset provided to participants in GAW14 was analyzed in this study. Only families with ethnicity self-reported as White, non-Hispanic were kept for analysis. Two diagnostic criteria for alcoholism were used in our analyses. For the first criterion, a diagnosis of alcoholism required positive diagnosis by the DSM-III-R criteria  and defiSNP genetic map positions were interpolated on the deCODE genetic map  through Spairs scoring function [We performed multipoint nonparametric linkage analysis using an affected-only allele-sharing method, which was implemented in the ALLEGRO (version 1.2c) software . We emplfunction , which pfunction  to generfunction .We used 115 White, non-Hispanic families in our analysis. The total number of individuals was 1,245, of which 1,009 were genotyped. Linkage information content for two SNP datasets was very similar except that the less-dense Illumina set had lower linkage information content on the X chromosome due to its poor coverage , we found 12 genomic regions with nominally significant LOD scores  and X (Xp22) in the SNP assays were two examples of signals not detected in the microsatellite analyses. The good concordance between the two SNP marker sets (Affymetrix and Illumina) in both linkage information content and linkage findings suggests that >5,000 SNPs may be excessive for samples with structures similar to the COGA data, and a SNP scan with ~5,000 markers distributed evenly across the human genome is sufficiently dense and powerful in whole-genome linkage analysis. Also, with current technology SNP genotyping is more rapid, requires fewer samples, and is more accurate than microsatellite marker genotyping. High-density SNP marker sets also offer a better localization of linkage peaks, which may save work for fine mapping in regions showing linkage . Since bOur analysis found nominal linkage for alcoholism to 12 genomic regions under both definitions for alcohol dependence (ALDX1 and ALDX2). The results for the two phenotype definitions are somewhat different. It is not clear which criterion is best for identifying genetic susceptibility loci for alcoholism. However, if one genomic region is associated with alcoholism, there should be similar statistical evidence under both criteria. Our finding on chromosome 2 overlaps with that of Reich et al. , who repWe conclude that a high-density SNP scan may offer a more rapid, cost-effective and powerful tool in genome-wide linkage analysis compared to traditional 10-cM microsatellite marker scans. However, further investigation is warranted to explore the effects of genetic map and computational issues on the utility of high density SNP assays in linkage analysis.COGA: Collaborative Study on the Genetics of AlcoholismGAW14: Genetic Analysis Workshop 14LD: Linkage disequilibriumSNP: Single-nucleotide polymorphismQM reconstructed the genetic map, carried out statistical analysis and drafted the manuscript. YY participated in genetic map reconstruction. YM and JF managed the data. LAF supported this study and helped to draft the manuscript. MAW conceived of the study, and participated in its design and helped to draft the manuscript. All authors read and approved the final manuscript."}
+{"text": "We used a maximum-likelihood based multipoint linkage approach implemented in SOLAR to examine simultaneously linkage for three electrophysiological endophenotypes from the Collaborative Study of the Genetics of Alcoholism: TTTH1, TTTH2, and TTTH3. These endophenotypes have been identified as markers of alcohol dependence susceptibility. Data were from 905 individuals in 143 families. Measured covariates considered included sex, age at electrophysiology data collection, habitual smoking status, and the maximum number of drinks consumed in a 24-hour period. Comparisons were made among genome-wide univariate, bivariate, and trivariate linkage analyses using genotypes based on microsatellite markers supplied by the Center for Inherited Disease Research, and genotypes based on single-nucleotide polymorphism markers provided by Illumina. All LODs were corrected to a standard equivalent to 1 degree of freedom. Using the trivariate approach and the microsatellite-based genotypes, we estimated a maximum multipoint linkage signal of LOD = 2.66 on chromosome 7q at 157 cM. Analyses using the Illumina SNP genotypes produced similar results, yielding a maximum multipoint LOD of 2.95 on 7q at 174 cM. These regions of interest correspond to those identified in the univariate and bivariate linkage screens. Our results suggest that trivariate multipoint linkage analyses have utility in the further characterization of chromosomal regions potentially containing genes influencing the phenotypes being examined. Based on a comparison of the number of LOD scores achieving statistical significance, our results suggest that the microsatellite- and Illumina SNP-based genotypes have similar utility for detecting genomic regions of interest. Multipoint variance component linkage analyses are a commonly used tool of statistical geneticists attempting to locate genes influencing complex phenotypes. More recently, bivariate multipoint linkage methods have been developed to examine linkage for two traits of interest simultaneously . Here, wSeveral electrophysiological endophenotypes correlated with alcohol dependence have been examined in the Collaborative Study of the Genetics of Alcoholism (COGA). Observed and hypothesized physiological relationships among the endophenotypes suggest pleiotropy may have a role in endophenotype variation. Quantitative trait loci (QTLs) influencing multiple endophenotypes may be detectible using multivariate linkage screens. Thus, the primary objectives of this study were 1) to introduce trivariate linkage screens for quantitative traits, as implemented in SOLAR , 2) to aDetails about participant selection and other aspects of COGA can be found in Begleiter et al. . The COGg < 0.10), and were not significant (p > 0.05).Endophenotype values \u00b1 4 SD from the mean were blanked prior to analysis. After removing outliers, TTTH1 data were available for 901 individuals, TTTH2 data for 902 individuals, and TTTH3 data for 903 individuals. Measured covariates considered included sex, age at electrophysiology data collection (ERPAGE), habitual smoking status (SMOKER), and maximum number of drinks consumed in a 24-hour period (MXDRNK). Whereas the inclusion in models of covariates genetically correlated with traits of interest can reduce analytical power, genetic correlations between MXDRNK and SMOKER and the three endophenotypes examined were minimal  matrices were generated using LOKI . All LODd et al.  as 2.77 The assumptions of linkage equilibrium that underlie multipoint linkage analysis may be violated if genetic markers are densely spaced, thus increasing the likelihood of linkage disequilibrium (LD) between markers. LD between markers is a particular concern with SNP-based maps, which often have higher marker density than occurs in traditional microsatellite-based maps. A specific analysis of the potential impact of LD between the Illumina SNPs on the results of our trivariate linkage analyses was beyond the scope of this paper. However, in another paper  we exami2, or the proportion of the residual phenotypic variance explained by additive genetic effects), and the covariates meeting the threshold for model inclusion. For TTTH1, a genome-wide linkage scan using the microsatellite genotypes yielded a maximum multipoint LOD of 3.41 at 157 cM on chromosome 7q. Approximately 23 cM qter of this, a locus at 180 cM also has a LOD > 2 (LOD = 2.37). The strongest linkage signal (LOD = 1.80) for TTTH2 was in approximately the same location as that of TTTH1, at 160 cM on chromosome 7q. For TTTH3, the maximum multipoint LOD (2.42) was at 43 cM on chromosome 3p. No other LODs over 2 were estimated for TTTH1, TTTH2, or TTTH3.Table Results of linkage analyses conducted using the Illumina SNP genotypes were similar. For TTTH1, we estimated a maximum multipoint LOD of 3.38 at 139 cM on chromosome 7q. A second strong linkage signal (LOD = 3.06) occurred on this same chromosome, at 154 cM. For TTTH2, the strongest linkage signal (LOD = 2.01) was on chromosome 3p at 49 cM. The maximum multipoint LOD score for TTTH3 was 3.67, on chromosome 7q at 174 cM. The next highest LOD in the initial linkage pass for TTTH3 was 2.00, located on chromosome 3p at 53 cM. No other LOD scores over 2 were estimated for any of the three endophenotypes in the univariate linkage analyses using the Illumina SNP-based genotypes.g \u2265 0.71) and moderate to strong environmental (0.74 \u2265 \u03c1e \u2265 0.41) correlations occurred between the endophenotypes (Table p < 0.0001). When TTTH1 and TTTH2 were considered simultaneously, a genome-wide multipoint linkage screen using the microsatellite-based genotypes yielded one LOD > 2, a linkage signal with LOD = 3.49 at 158 cM on chromosome 7q. Using the Illumina SNP-based genotypes, we estimated a maximum multipoint LOD of 2.55 for TTTH1 and TTTH2 on chromosome 7q, at 140 cM. A weak linkage signal (LOD = 2.35) also occurred at 87 cM on chromosome 2p.Strong genetic  and 155 cM (LOD = 2.17).The strongest linkage signal (LOD = 2.15) estimated for TTTH2 and TTTH3 using the microsatellite-based genotypes occurred at 40 cM on chromosome 3p. The next strongest signal (LOD = 1.73) for these endophenotypes occurred at 183 cM on chromosome 7q. By contrast, using the Illumina SNP-based genotypes we estimated a maximum multipoint LOD score of 3.58 on chromosome 7q at 175 cM. No additional LODs over 2 occurred.Using the microsatellite-based genotypes and a genome-wide linkage screen that considered TTTH1, TTTH2, and TTTH3 simultaneously, we estimated a maximum multipoint LOD of 2.66 at 157 cM on chromosome 7q. Results of genome-wide linkage analyses utilizing the Illumina SNP-based genotypes were similar, yielding a maximum multipoint LOD of 2.95 for these endophenotypes at 174 cM on chromosome 7q. Additionally, a LOD of 2.39 occurred on chromosome 2p at 79 cM. No other LODs over 2 were estimated in either the microsatellite- or the Illumina SNP-based linkage screens. The results of the microsatellite- and SNP-based linkage scans of chromosome 7 are compared in Figure p > 0.05), we were able to detect regions of interest corresponding to those identified in the univariate and bivariate linkage screens. Based on a comparison of the number LOD scores achieving statistical significance, our results also suggest that the microsatellite- and Illumina SNP-based genotypes have similar utility for detecting genomic regions of interest.We have introduced a trivariate multipoint linkage screen, a novel approach investigators can use in SOLAR to examine regions of interest for genes having potential pleiotropic influence on three quantitative traits. Here, we used this approach to examine three electrophysiological traits correlated with susceptibility to alcohol dependence in the COGA families, TTTH1, TTTH2, and TTTH3. Whereas the strongest linkage signals estimated in the trivariate multipoint linkage analyses did not achieve statistical significance (genome-wide Our results suggest that the trivariate approach has utility in the further characterization of chromosomal regions potentially containing genes influencing the phenotypes being examined. However, additional analyses using other datasets, including simulation studies, are required to evaluate the power of this approach relative to other methods. Similar to bivariate linkage screens , trivariCIDR: Center for Inherited Disease ResearchCOGA: Collaborative Study of the Genetics of AlcoholismERP: Event-related potentialERPAGE: Age at Electrophysiology data collectionGAW14: Genetic Analysis Workshop 14LD: Linkage disequilibriumMIBD: Multipoint identity by descentMXDRNK: Maximum number of drinks consumed in a 24-hour periodQTL: Quanitative trait lociSMOKER: Habitual smoking statusSNP: Single-nucleotide polymorphismDMW performed the data analysis and drafted the manuscript. TDD generated the MIBD matrices used in the analyses. CPP provided the computer programs used to implement the analyses. MCM contributed to data analysis. JB developed the multivariate modeling methodology. LA was responsible for study design and implementation, and helped to draft the manuscript. All authors read and approved the final manuscript."}
+{"text": "We compare two new software packages for linkage analysis, LODPAL and GENEFINDER. Both allow for covariate adjustment. Replicates 1 to 3 of Genetic Analysis Workshop 13 simulated data sets were used for the analyses. We described the results of searching for evidence of loci contributing to a simulated quantitative trait related to systolic blood pressure (SBP). Individuals with SBP greater than 130 mm Hg were defined as affected individuals, and all others as unaffected. Total cholesterol was treated as a covariate.Using LODPAL, the power of detecting one of the three major genes related to SBP is 44.4% when a LOD score of 1 is used as the cut-off point. The power of GENEFINDER is lower than that of LODPAL. It is 22.2%.Based on the limited comparison, LODPAL provided the more reasonable power to detect linkage compared to GENEFINDER. After adjusting for the total cholesterol covariate, the current version of both programs appeared to give a high number of false positives. There has been great interest in developing linkage analysis methods that allow for the adjustment of covariates, because this type of analysis can potentially allow us greater power to detect genetic effects after adjusting traits for the possible effect of covariates. The object of this study is to compare the performance of two genetic model-free linkage analysis software packages: LODPAL and GENEFINDER. Below we present some brief theoretical background on the two statistical methods implemented in LODPAL and GENEFINDER.1 and \u03bb2, where \u03bb1 is the relative risk for a pair of relatives that shares exactly one allele IBD and \u03bb2 is the relative risk for a pair of relatives that shares two alleles IBD. Olson's original method requires two additional parameters for each covariate, while the new method needs only one parameter by using the relationship \u03bb2 = 3.634 \u00d7 \u03bb1 - 2.634. This idea of parameter reduction was based on the work described by Whittmore and Tu [LODPAL is an affected-relative-pair analysis method using a conditional-logistic model that allows covariates to adjust the relative risks associated with sharing alleles identity by descent (IBD) . GoddardLiang et al.  developeS(t) | \u03a6) = 1 + 2(E(S(\u03c4) | \u03a6) - 1)E 2 \u00d7 C,= 1 + (1-2\u03b8S(t) is the number of alleles shared IBD at an arbitrary locus t in the chromosomal region, \u03a6 is the event of affected siblings, \u03b8 is the recombination fraction between locus t and the unobserved trait locus \u03c4, and C is defined as (E(S(\u03c4) | \u03a6) - 1), which is the effect of the unobserved trait locus as characterized by the excessive IBD sharing due to the linkage to an unobserved trait locus. Using the generalized estimating equation (GEE) procedure, we can estimate the parameters of interest, \u03c4 and C, and their confidence intervals, directly. An interesting feature of this approach is that one can test the null hypothesis of no linkage to this region by testing C = 0, which follows a \u03c72 distribution with 1 df. Furthermore, this GEE approach has been extended to incorporate the linkage evidence from unlinked regions [where  regions  and to i regions . When inS(t) | x \u2208 l,\u03a6) = 1 + 2(E(S(\u03c4) | x \u2208 l, \u03a6) - 1)E2 \u00d7 Cl,= 1 +  is the value of this covariate, and Cl is defined as (E(S(\u03c4) | \u03a6) - 1) for the pairs with a covariate coded as 0, 1, and 2, respectively. Similarly, we can estimate \u03c4, Cl, and their confidence intervals. One can test the null hypothesis of no linkage to this region by testing C0 = C1 = C2 = 0, which follows a \u03c72 distribution with 3 df.where The details of the simulation data set of the Genetic Analysis Workshop 13 (GAW13) were described elsewhere . We wereWe identified 330 families with a total of 4692 individuals. The data contained the following relative pairs: parent-offspring (5840), sib-sib (2798), grandparent-child (6220), avuncular (2175), half-sib (77) and cousin (1747). For LODPAL, 175 relative pairs from Replicate 1, 177 relative pairs from Replicate 2, and 199 relative pairs from Replicate 3 with informative allele-sharing information were included in the analyses. The covariate \"total cholesterol\" was treated as a continuous variable.l = 0, 1, and 2, respectively.Since the current version of GENEFINDER only allows for affected sibling pairs, the information from pedigrees with affected relative pairs other than siblings will be discarded. For Replicate 1, 109 affected sibling pairs from 50 families were used; for Replicate 2, 107 affected sibling pairs from 60 families were used; and for Replicate 3, 112 affected sibling pairs from 65 families were used. Since GENEFINDER can only handle discrete covariates, we dichotomized the total cholesterol based on a cut point at 200 mg/dl. Therefore, individuals with total cholesterol greater than 200 mg/dl were considered to have high cholesterol. Based on this classification, there were 48 sibling pairs both with low cholesterol (LL), 44 pairs with only one low cholesterol (HL), and 17 pairs both with high cholesterol (HH) for Replicate 1. For Replicate 2, the numbers of sibling pairs used for analysis were 37, 40, and 30, respectively. For Replicate 3, the numbers were 53, 41, and 18, respectively. In our analysis, covariates for these three scenarios were coded as We performed a series of multipoint LODPAL analyses on Replicates 1 to 3 of the GAW13 data using SAGE version 4.3 . We alsop-value = 0.0013) in Replicate 1, two true loci on chromosome 5  and 13  in Replicate 2, and one true locus on chromosome 5  in Replicate 3 . After adjusting for covariate, it was detected on chromosome 5 in Replicate 1  and chromosome 7 in Replicate 2 . However, the estimates of the locations of trait loci were not close to the true ones on chromosomes 5 (at 176.08 cM) and 7 (at 47.49 cM). Relatively, GENEFINDER located a trait locus closer to the true location of quantitative trait locus B35 (at 85.16 cM) on chromosome 13 in Replicates 1 and 2. Further, the false-positive rates are much higher after adjusting for the covariate when compared to those without adjusting for the covariate ; see Table For the GENEFINDER analysis, without adjusting for covariate, the significance of linkage evidence was detected on chromosome 7 in Replicate 2 only . However, it is important to estimate empirical Although our comparison of LODPAL and GENEFINDER is limited, there are still some points worth mentioning. First, the addition of covariates increased the possibility of getting a false-positive result in our analyses. Because total cholesterol is a real risk factor for SBP, the models without adjustment are less powerful when compared with those with adjustment. Second, the appropriate selection of a cut point for total cholesterol for GENEFINDER may have had an impact on our power to detect linkage. An advantage of using LODPAL is that one does not have to dichotomize covariates, because it naturally models continuous covariates. Third, the cut point for SBP is 130 mm Hg, which is lower than the normal cut-off of 140 mm Hg. This may partly explain why we observed lower power, but higher false positive rates. Fourth, unlike LODPAL, GENEFINDER is designed to be used after previous evidence of linkage has been found  because 2 distribution with 1 df. Additional covariates gives an LRS with a distribution that is a 50:50 mixture of a \u03c72 with k df and a \u03c72 with (k+1) df. Also note that GENEFINDER is sensitive to the initial values of the estimates. Therefore, exploring different initial values to ensure that the solution reaches a global maximum is highly recommended because it can help reach the convergent criteria.Furthermore, we stress the advantages and disadvantages of these two methods. While both methods are model-free and allow for the adjustment of covariates, LODPAL also allows for continuous covariates not just categorical ones. GENEFINDER, on the other hand, only allows for categorical covariates, which presents a limitation. However, GENEFINDER can be used to test the location of an unobserved trait locus and also provide its 95% confidence interval. The other advantage is that GENEFINDER, a multipoint approach, can also be used to test the null hypothesis of no linkage using a test with 1 df without adjusting for a covariate and with 3 df after adjusting for a covariate. In LODPAL, as indicated by Goddard et al. , the disFinally, we would like to note that using the programs we evaluated required dichotomization of a quantitative trait and therefore may cause a noticeable amount of power loss. On the other hand, dichotomizing a trait such as blood pressure is not an uncommon practice in real studies. Therefore, it is of interest to examine the power to detect linkage under a situation where the design is less than optimal. Since we only performed analyses on three replicates , it is difficult to give solid guidelines to future users. However, we would like to caution the users with respect to the large number of false positives produced by both software packages."}
+{"text": "The central issue for Genetic Analysis Workshop 14 (GAW14) is the question, which is the better strategy for linkage analysis, the use of single-nucleotide polymorphisms (SNPs) or microsatellite markers? To answer this question we analyzed the simulated data using Duffy's SIB-PAIR program, which can incorporate parental genotypes, and our identity-by-state \u2013 identity-by-descent (IBS-IBD) transformation method of affected sib-pair linkage analysis which uses the matrix transformation between IBS and IBD. The advantages of our method are as follows: the assumption of Hardy-Weinberg equilibrium is not necessary; the parental genotype information maybe all unknown; both IBS and its related IBD transformation can be used in the linkage analysis; the determinant of the IBS-IBD transformation matrix provides a quantitative measure of the quality of the marker in linkage analysis. With the originally distributed simulated data, we found that 1) for microsatellite markers there are virtually no differences in types I and II error rates when parental genotypes were or were not used; 2) on average, a microsatellite marker has more power than a SNP marker does in linkage detection; 3) if parental genotype information is used, SNP markers show lower type I error rates than microsatellite markers; and 4) if parental genotypes are not available, SNP markers show considerable variation in type I error rates for different methods. A key issue in nonparametric linkage analysis is the accuracy in the estimation of the relative pair identity-by-descent (IBD) distributions. The Genetic Analysis Workshop 14 (GAW14) simulated data provide an opportunity to evaluate new or existing methods for linkage analysis since the \"answers\" were known to the designers of the simulated data. We applied two types of methods to find the locations of linkage and determine the power and type I errors for single-nucleotide polymorphism (SNP) and microsatellite markers according to whether or not parental genotypes are available. The first method is the affected pedigree member (APM) method implemented in Duffy's SIB-PAIR program . The secWe applied two types of methods to determine the performance (power and type I errors) for SNP and microsatellite markers with different data assumptions based on the availability of parental genotypes. The first method is the APM method implemented in Duffy's SIB-PAIR program, which was fully documented in . The secPij (M) = Pji(M) = 1/2 of the sum of frequencies for the genotypes ai/aj and aj/ai in the parental generation with ai being the alleles over the marker. Then in a full sib pair population without gender differences, the IBS and IBD probabilities are related byAssume that 1) parental mating is random; 2) in the parental population, for any genotype the two possible phase known genotypes have the same probability; 3) for each mating type that produces a sib-pair with IBD = 0, the two possible sib pairs have an equal probability to come; if the IBD = 1, the shared IBD allele has an equal probability to come from each one of the two parents. Let T = [Tij] with Tij = p(IBS = i|IBD = j) 0 \u2264 j \u2264 i \u2264 2 is given bywhere the transformation matrix T11 = Het(M)T21 = Hom(M)Hom(M) and Hom(M2) are the sums of all diagonal elements for the matrix [Pij(M)] and [Pij(M)]2, respectively, Het(M) and Het(M2) are the sums of all off-diagonal elements for the matrix [Pij(M)] and [Pij(M)]2, respectively, and Pi(M) is the frequency for the ith allele ai, . The above formula reduces into Lange's [Pij(M) = Pi(M)Pj(M). Our formula can transform the IBD distribution to that of IBS by the transformation matrix T or vice-versa through the inverse transformation matrix T-1. With the estimates for IBD or the IBS probabilities, the statistics for nonparametric linkage analysis can be calculated and tested in the usual manner. and HomM are the We performed all analyses without knowledge of the \"answers.\" We still do not have the \"answers,\" except those results appearing in the meeting abstracts.Figure To see the type I error rate, we listed the median of the numbers of significant replicates over all the markers, which could be viewed as an average type I error rate and graphically could be interpreted as the \"noise level.\" Table Based on our single-point linkage analysis, we observed the following results with respect to the comparison of SNP vs. Microsatellite markers (one SNP marker vs. one Microsatellite marker around the same location) and the effects of the parental genotype information in the comparison on the two types of markers.1) On average, a microsatellite marker showed higher rates of significant replications than a SNP marker over the linkage locations.2) Some SNP markers provided almost equally strong linkage evidence, for example, SNP C01R0052 in the Danacaa sample  For the microsatellites, our affected sib-pair only methods showed a modest \"noise\" level (3%\u20136%) while the affected pedigree method (Duffy's APM) has a stable \"noise\" level of 3%. Both methods have almost the same \"power.\" Since our method just used one affected sib-pair , it seems therefore that parental genotyping may not be very critical in linkage analysis for microssatellite markers.4) For SNP data, our affected sib-pair only IBS method has a \"noise\" level (4%\u20136%), IBS-IBD method has a high \"noise\" level (15%\u201321%) while affected pedigree method (Duffy's APM) has a stable \"noise\" level of 0%. The high \"noise\" level for IBS-IBD method reflects the fact that the IBS-IBD matrix for a SNP marker is close to singular. Thus, we conclude that for SNP data with parental genotypes, the false positive rate is very low in linkage analysis, and without parental genotype information the false positive rate can be relatively high.The different sites vary with respect to the power to detect linkage. Since the linkage evidence over a marker for a disease is inversely proportional to the number of markers which interact in determining the phenotype, our results may reflect some characteristics of the four population groups. For example, the relatively weak linkage over the four locations in the Aipotu group  for microsatellite markers there are virtually no differences in type I or type II error rates whether one uses or excludes parental genotypes. 2) On average, a microsatellite marker provides more power than a SNP marker does in linkage analysis. 3) If parental genotype information is used, SNPs show lower type I error rates than that of microsatellite markers. 4) If parental genotypes are not available, SNPs show variable type I error rates over different methods.In summary, other things being equal in the simulated sample analyzed, microsatellites are better than SNPs, although if parents are typed, SNPs can have slightly better type I error rates.APM: Affected pedigree memberGAW: Genetic Analysis WorkshopIBD: Identity-by-descentIBS: Identity-by-stateKPD: Kofendrerd Personality DisorderSNP: Single-nucleotide polymorphismQY developed the methodology, implemented the computer program, participated in the simulations and drafted the manuscript. VA participated in the simulations and GEB participated in analysis and coordination and helped to draft the manuscript."}
+{"text": "ADH3 is located, may influence the risk of alcoholism, variations of electroencephalogram, and the 4 ttdts of the event-related potential measures.Genetic components significantly contribute to the susceptibilities of alcoholism and its endophenotypes, such as event-related potential measures and electroencephalogram. An endophenotype is a correlated trait which identifies individuals at risk. Correlated traits could be influenced by shared genes. This study is intended to identify chromosome regions that may harbor common genetic loci contributing to alcoholism, event related potential measures and electroencephalogram. All 143 Collaborative Study on the Genetics of Alcoholism families with 1,614 individuals provided by the Genetic Analysis Workshop 14 were used for the analysis with aldx1 as an alcoholism diagnosis. We carried out factor and principal component analyses on the 12 event-related potentials, then bivariate genome scans on aldx1 and electroencephalogram (ecb21), as well as alcoholism and the principal component scores of the event-related potential measures. A univariate genome scan was also carried out on each trait. Factor and principal component analysis on the event-related potential measures showed that the 4 ttths and 4 ntths belong to one cluster (cluster 1), while the 4 ttdts belonged to another (cluster 2). From each cluster, one principal component was extracted and saved as pc1 (for cluster 1) and pc2 (for cluster 2). The results of genome scans revealed only one chromosome region, chromosome 4 q at about 100 cM, identified by several univariate genome scans including aldx1, ecb21, and pc2, and the evidence of linkage increased significantly in the bivariate genome scans of aldx1 and ecb21 and aldx1 and pc2. Our study suggests that the same quantitative trait locus on the chromosome 4 q region, where  In order to further identify and study genetic loci contributing to alcoholism susceptibility, investigators have recently focus on genome scans on alcoholic endophenotypes, such as event-related potential (ERP) and electroencephalogram (EEG). An endophenotype is a correlated trait with a relatively high heritability. It segregates with the illness in the affected relatives and identifies individuals at risk.Two correlated traits could be influenced by shared genes. The power to localize the shared genes could be improved by bivariate linkage analysis . HoweverThis study is intended to identify common genetic loci contributing to alcoholism and its endophenotypes, EEG and ERPs, through a bivariate genome scan on alcoholism and EEG (ecb21) as well as alcoholism and the factors of the 12 ERPs. Before bivariate genome scans, we carried out univariate genome scans on each trait.We used all 143 COGA families with 1,614 individuals provided by Genetic Analysis Workshop 14 (GAW14) for the analyses. \"aldx1\" was used as the alcoholism diagnosis.The ERP data are extracted from the case of the visual oddball experiment for 4 electrode placements, 1 for the far frontal left side channel, 2 for the frontal midline channel, 3 for the central midline channel, and 4 the for parietal midline channel. For the 4 ttths (ttth1 to ttth4) and 4 ttdts (ttdt1 to ttdt4), the data are extracted from the target case. The extracted measures correspond to the 'late' time window, which is set at 300\u2013700 ms following stimulus presentation , and the theta band power (3\u20137 Hz) for the 4 ttths, and the delta band power (1\u20132.5 Hz) for the 4 ttdts. The 4 ntths (ntth1 to ntth4) contain data extracted from the non-target case. The extracted measures correspond to the 'early' time window, which is set at 100 to 300 ms following the stimulus presentation, and the theta band power (3\u20137 Hz).Y = \u03bc + \u039bf + u, where Y denoted the 12 \u00d7 1 vector of the ERP variables, \u039b = {\u03bbij} denoted a 12 \u00d7 2 loading matrix, f = ' denoted the 2 \u00d7 1 vector of common factors, and u denoted the 12 \u00d7 1 vector of unique factors. This model assumed that there were 2 unobserved latent factors, f1 and f2, which explained most of the variations of the ERP measurements [Preliminary examination of the correlation matrix of the 12 ERP variables suggested that these variables could be roughly divided into two clusters, where there were high intra-cluster correlations and low inter-cluster correlations. To verify the fitness of a two-factor model for the ERP variables, we fitted the model urements , and obturements  and compVariance component linkage analysis implemented in SOLAR (v. 2.1.2)  was usedThe loading matrix computed from the 2-factor model on ERP showed that a) the loading estimates of the 4 ttth ERP variables and the 4 ntth ERP variables were all >0.7 for factor 1 and nearly zero (<0.001) for factor 2; b) the loading estimates of the 4 ttdt ERP variables were  for factor 1 and  for factor 2. These results suggested that the 4 ttth ERP and the 4 ntth ERP variables contributed almost exclusively to factor 1, while the 4 ttdt ERP variables contributed almost exclusively to factor 2. These 2 factors explained approximately 63% of the total variation of the ERP measurements. Our principal component analysis showed that pc1, the principal component score computed from the 8 ERP variables in cluster 1, accounted for approximately 80% of the total variation in cluster 1. Similarly, pc2, the principal component score for the 4 ERP variables in cluster 2, accounted for approximately 92% of the total variation in cluster 2. This approach effectively reduced the original 12 correlated ERP variables to two summary scores, pc1 and pc2.The heritability of aldx1, ecb21, pc1, and pc2 was 0.25 \u00b1 0.07, 0.30 \u00b1 0.05, 0.38 \u00b1 0.06, and 0.31 \u00b1 0.06, respectively, after adjusting for covariates age at interview and sex.Table Figure Our study suggests that the variations of the 4 ttths and the 4 ntths may be mostly controlled by a major factor while the 4 ttdts by another. Both factors have strong genetic components with relatively high heritabilities.There is one and only one chromosome region, chromosome 4 q about 100 cM, that is identified by several univariate genome scans, aldx1, ecb21 and pc2, and the evidence of linkage was significantly increased in the two bivariate genome scans: aldx1 and ecb21, aldx1 and pc2.ADH3 [Previous studies have reported significant linkage between alcoholism and this chromosome 4 region near the class I alcohol dehydrogenase locus ADH3 ,7. LinkaADH3 .ADH3 is located, may influence the risk of alcoholism, variations of EEG, and the 4 ttdts. The other 8 ERPs may be controlled by other genetic loci.Through a combination of statistical dimension-reduction techniques  and bivariate genome scans, our study further suggests that the same quantitative trait locus on the chromosome 4 region, where We also carried out bivariate genome scans on ecb21 and pc1 and ecb21 and pc2 (data not shown). The joint consideration of the traits did not increase the evidence of linkage of those traits to this chromosome 4 area.It would be interesting to see the results of a simultaneous trivariate genome scan on aldx1, ecb21, and pc2. However, the software we used (SOLAR) does not allow us to carry out this kind of analysis.ADH3 was located, may influence the risk of alcoholism, variations of EEG, and 4 ttdts of the 12 ERPs.Through factor and principal component analyses on the12 ERP variables, followed by univariate and bivariate genome scans on alcoholism, EEG, and principal component scores of the 12 ERPs, our study suggested that the same quantitative trait locus on the chromosome 4 q region, where COGA: Collaborative Study on the Genetics of AlcoholismEEG: ElectroencephalogramERP: Event-related potentialGAW14: Genetic Analysis Workshop 14J-PL carried out study design, data analysis and drafted the manuscript. CW participated in study design, data analysis, and helped to draft the manuscript."}
+{"text": "Complex diseases are generally thought to be under the influence of multiple, and possibly interacting, genes. Many association methods have been developed to identify susceptibility genes assuming a single-gene disease model, referred to as single-locus methods. Multilocus methods consider joint effects of multiple genes and environmental factors. One commonly used method for family-based association analysis is implemented in FBAT. The multifactor-dimensionality reduction method (MDR) is a multilocus method, which identifies multiple genetic loci associated with the occurrence of complex disease. Many studies of late onset complex diseases employ a discordant sib pairs design. We compared the FBAT and MDR in their ability to detect susceptibility loci using a discordant sib-pair dataset generated from the simulated data made available to participants in the Genetic Analysis Workshop 14. Using FBAT, we were able to identify the effect of one susceptibility locus. However, the finding was not statistically significant. We were not able to detect any of the interactions using this method. This is probably because the FBAT test is designed to find loci with major effects, not interactions. Using MDR, the best result we obtained identified two interactions. However, neither of these reached a level of statistical significance. This is mainly due to the heterogeneity of the disease trait and noise in the data. It is commonly believed that complex diseases are caused not by single genes acting alone, but by multiple genes interacting with one another. Due to the large number of single-nucleotide polymorphisms (SNPs) available in a genome-wide scan, the computational burden of testing each locus for main effects and all possible pair-wise, 3-way, and even higher-order interactions is overwhelming. One approach is to first identify a smaller number of candidate SNPs, using linkage analysis or a candidate gene approach. With a refined list, a more thorough statistical analysis can be performed. At this second stage, a univariate test is commonly used, which we refer to as single-locus method. Family-based association tests (FBAT) are used for pedigree data  and a chThere are several multilocus approaches that consider interactions of multiple genes and environmental factors in identifying susceptibility loci for complex diseases -5. The mWe chose to use to the simulated data provided to participants in the Genetic Analysis Workshop 14 (GAW14). To avoid analysis bias, we did the analysis without knowing the real answers prior to the GAW14 conference. Because we used simulated data with a fictitious trait, there were no a priori candidate genes to consider, so we used a positional approach to identify candidate regions. First, we performed linkage analysis using microsatellite markers with GENEHUNTER-PLUS. Next, we identified candidate regions near linkage peaks, and selected candidate SNPs in these regions. Finally, we performed association analyses on the candidate SNPs, using both FBAT and MDR.We used the simulated data from the country of Aipotu. The Aipotu families were selected when at least two offspring were present who had P1, P2, or P3. We chose disease status for Kofendrerd Personality Disorder (KPD) as the phenotype of interest. In order to get sufficient sample size for MDR analysis, we combined five replicates (REP001-005) of microsatellite marker and SNP data, with 500 nuclear families. We first performed genome-wide linkage analysis using microsatellite markers to identify candidate regions, then we selected SNPs in the candidate regions for the follow-up association analysis. To simulate discordant sib-pair design, we randomly selected 410 discordant sib pairs , with one discordant sib pair from each family. This dataset was then analysed using FBAT and MDR.We performed multipoint linkage analysis on microsatellite markers using GENEHUNTER-PLUS. This approach is based upon 1-parameter allele sharing model , which aAll 29 SNPs were tested for Hardy-Weinberg Equilibrium (HWE) to check the quality of the data. SNPs not in HWE were removed from the analysis.The FBAT program implements a series of family-based association tests . When tev-fold cross-validation ; 2) select a set of n candidate genetic and/or discrete environmental factors from all factors; 3) represent the n factors and their multifactor classes (m genotypes/locus means nm classes) in n-dimensional space; 4) estimate the ratio (R) of the number of affected sibs (A) to the number of unaffected sibs (U) within each multifactor class. Each multifactor class in n-dimensional space is labelled either as \"high-risk,\" if R \u2265 T (some threshold), or as \"low-risk,\" if R <T, thereby reduces the n-dimensional model to a uni-dimensional model. For balanced designs, the threshold is usually set to 1:1. 5) All possible combinations of n factors are evaluated sequentially for their ability to classify affected and unaffected individuals in the training data. The best n-factor model is selected. 6) The independent test data from the v-fold cross-validation is used to estimate the prediction error of the best model selected in Step 5. Steps 1\u20136 are repeated v times with the data split into v different training and testing sets.MDR  is a modp-values of MDR results were obtained by running 100 permutation tests.We analyzed the SNP dataset using MDR as described above, with an affected sibs-to-unaffected sibs threshold ratio of 1 and 10-fold cross-validation. We conducted exhaustive search of all possible 1- to 7-locus interactions. Due to the fact that the number of possible combinations is exponential to the number of loci tested for interaction, it is computationally overwhelming to do an exhaustive search of all higher order interactions. Cross-validation consistency (CVC) was used as the statistic to select the best model . Empiricp = 3.73 \u00d7 10-9), chromosome 3 , chromosome 5  and chromosome 9 . We used the 1-LOD score rule to get the 90% confidence regions as candidate regions. Because there was no uniform map for microsatellite markers and SNPs, we assumed two extreme scenarios in which either left ends of the two maps align, or the right ends of the two maps align, and then defined the combination of the candidate regions in both situations as the final candidate regions. We then selected the 29 SNPs , so all of them are included in the analyses. Based on real answers, four susceptibility loci (D1 to D4) were within the candidate regions; two susceptibility loci (D5 and D6) on chromosomes 10 and 2 were not in the candidate regions, hence were not included in the follow-up association analysis. D1, D2, D3, and D4 were within the candidate regions, but do not appear in the map. They are in the extra SNP packets, which we did not order at the time of our analysis. D1, D2, D3, and D4 cause disease via four 2-locus interactions . The closest SNPs to the four susceptibility loci in our map  were referred to as D1 to D4 for our purposes, unless otherwise noted. Using FBAT, we identified D4 (p = 0.043). However, after correcting for multiple tests, it is not significant  correction on chromosome 9). Using MDR, we did not identify any of 1- to 7-locus models as significantly different from the null hypothesis of no association. According to cross-validation consistency, the MDR 6-locus model was the best model (p = 0.84) including interaction of D1\u2013D2 ; each interaction caused disease alone. It has been shown by simulation that MDR has very limited power in the presence of heterogeneity , rangingMDR has been shown to be useful in the identification of gene \u00d7 gene interactions in real data from case-control studies ,6. In orCPM: combinatorial partitioning methodCVC: Cross-validation consistencyFBAT: Family-based association testFDR: False discovery rateGAW14: Genetic Analysis Workshop 14HWE: Hardy-Weinberg EquilibriumKPD: Kofendrerd personality disorderLD: Linkage disequilibriumMDR: Multifactor-dimensionality reductionSNP: Single-nucleotide polymorphismYM participated in the design of the study, performed the statistical analysis and drafted the manuscript. QM, YY, and JF participated in its design and coordination. LAF participated in the design of the study. MAW participated in the design of the study and helped to draft the manuscript. All authors read and approved the final manuscript."}
+{"text": "HD gene; however, several studies have indicated that other genetic factors also contribute to the variability in HD age at onset. To identify modifier genes, we recently reported a whole-genome scan in a sample of 629 affected sibling pairs from 295 pedigrees, in which six genomic regions provided suggestive evidence for quantitative trait loci (QTL), modifying age at onset in HD.Age at onset of Huntington's disease (HD) is correlated with the size of the abnormal CAG repeat expansion in the In order to test the replication of this finding, eighteen microsatellite markers, three from each of the six genomic regions, were genotyped in 102 newly recruited sibling pairs from 69 pedigrees, and data were analyzed, using a multipoint linkage variance component method, in the follow-up sample and the combined sample of 352 pedigrees with 753 sibling pairs.p = 0.002) increased to genome-wide significance for linkage in the combined sample , while suggestive evidence for linkage was observed at 18q22, in both the follow-up sample  and the combined sample . Epistatic analysis indicated that there is no interaction between 6q23-24 and other loci.Suggestive evidence for linkage at 6q23-24 in the follow-up sample (LOD = 1.87, In this replication study, linkage for modifier of age at onset in HD was confirmed at 6q23-24. Evidence for linkage was also found at 18q22. The demonstration of statistically significant linkage to a potential modifier locus opens the path to location cloning of a gene capable of altering HD pathogenesis, which could provide a validated target for therapeutic development in the human patient. HD gene, supporting the existence of genes capable of modifying HD pathogenesis. Although Wexler et al. [Huntington's disease (HD MIM 143100]) is a progressive neurodegenerative disorder with an age at neurological onset commonly in midlife. The major clinical features of HD include involuntary choreiform movements, psychiatric symptoms, and cognitive dysfunction . Random effect models (Proc MIXED in SAS) were used in these models to account for familial clustering. Each model was used to determine the expected age at onset for a given expanded CAG repeat, and the residual was computed as the difference between the observed and expected age at onset. Residuals were standardized to a mean of zero and a standard deviation of 1. Both models show similar modest negative skewness. The skewness of the residual generated by Model One is -0.42 and the kurtosis is 1.82. Corresponding values for Model Two were -0.49 and 1.11, respectively.Age at onset, defined as the onset of motor impairment, was reported for all affected participants ,10. Casepression . The quapression  and prevpression , adjusteResidual onset ages for all analyses were computed using the 836 combined sample plus 234 locally studied HD patients recruited from the New England HD Research Center and 303 brain specimens from the McLean Brain Tissue Resource Center, for a total sample of 1373. While the local and brain samples are not included in this linkage analysis, they provide an additional randomly ascertained samples to more accurately model the relationship between age at onset and CAG repeat sizes. Finally, because our future studies will involve SNP association studies in all of three samples , we sought to define the repeat adjusted age at onset uniformly across all three samples.The heritability analyses were conducted using maximum likelihood procedures as implemented in the SOLAR program .HD CAG repeat sizes were determined by polymerase chain reaction using an assay that does not include the adjacent proline (CCG) repeat. Cases with 36 or more repeats were designated HD mutation carriers.All the newly recruited HD samples were genotyped by three microsatellite markers at each of the six regions with maximum multipoint LOD scores greater than or equal to 1.5 in the original genome scan . The marVariance component linkage analysis to repeat adjusted age at onset was performed using MERLIN . We perfThe oligogenic linkage analysis and epistatic interaction analysis were performed by the SOLAR program . MERLIN h2 \u00b1 SE) for expanded repeat adjusted age at onset (Model One) are 0.72 \u00b1 0.09 for the Original Sample, 0.74 \u00b1 0.20 for the Follow-up Sample and 0.74 \u00b1 0.08 for the Combined Sample.Three sample sets, newly recruited (Follow-up Sample), original  and combined (Combined Sample) were used in this study. The Original, Follow-up and Combined samples are described in Table p = 0.002, Model One; or LOD = 2.27, p = 0.0006, Model Two). One additional region provided modest confirmation for linkage, 18q22 . However, no evidence for linkage was seen at 2q33 , 4p16 , 5q31-32 , and 6p22 .Multipoint linkage analyses were completed using both Model One, adjusting for expanded HD repeats only, and Model Two, adjusting for expanded HD repeats, normal repeats and their interactions. Multipoint LOD scores obtained in these three sample sets for the six chromosome regions  are presented in Table p = 0.00001) and suggestive linkage at 4p16  and 18q22 . The 2q33  and 6p22.3  regions achieved a LOD score greater than 1.0. However, the LOD score at 5q31-32 dropped to 0.98 (p = 0.02). The Model Two analysis, adjusting for the expanded repeat, normal repeats and their interaction, generated higher LOD scores than Model One at 6q23-24 in all the three tested sample sets: the Original Sample , Follow-up Samples  and Combined Sample . All of the other analyses using Model Two produced LOD scores very similar to those of Model One. Epistatic analysis indicated that there is no interaction between 6q23-24 and five other loci.Model One analysis in the Combined Sample yielded significant linkage at 6q23-24  and suggestive support for linkage was observed at 18q22 (p = 0.03). No evidence for linkage was observed in the Follow-up Sample at 2q33 (p = 0.2), 4p16 (p = 0.5), 5q31-32 (p = 0.2) and 6p22.3 (p = 0.4). The lack of confirmation for the latter four loci may indicate that these regions do not contain genes that modify the age at neurologic onset for HD or that this relatively small Follow-up study did not adequately sample families which carry modifier genes from these regions.The purpose of this study was to replicate our original linkage findings for repeat adjusted age at onset in HD  in an ex linkage . In thisp = 0.00001). The Model Two analysis, adjusting for the expanded repeat, normal repeats and their interaction, generated higher LOD score than Model One at 6q23-24. The LOD scores of the Original Sample , Follow-up Samples  and Combined Sample  are highly significant using this second model. All of the other analyses using Model Two produced LOD scores very similar to those of Model One. We emphasize results from Model One because beta coefficients from Model Two show greater variability compared with those computed in the original scan [In the Combined Sample, the Model One analysis, adjusting only for the size of the expanded repeat, yielded significant linkage at 6q23-24 . We reported a LOD score of 2.28 at 6q23-24 in our original genome scan [p = 0.00003) in the current study using the same model (Model Two). The difference is due to a modification of the method used to define the repeat adjusted age at onset. In the original genome scan only the 754 individuals were used to model the relationship of repeat size age at onset. In the present study, we used a sample of 1373 individuals, derived from the 836 in the combined sample, plus 234 locally studied HD affected persons and a sample of 303 brain specimens. While the local and brain samples do not represent sib-pairs that could be included in this linkage analysis, they provide additional randomly ascertained samples to more accurately model the relationship between age at onset and CAG repeat sizes. They also provide an increased sample size for subsequent fine-mapping association studies to assess candidate modifiers. The increased sample size provided a more accurate assessment of the relationship between repeat size and age at onset, yielding a residual that more accurately adjusts for the effect of repeat size on age at onset in HD.The results of the Combined Sample are generally similar to those of the original genome scan . The Combined Sample provides suggestive evidence for linkage at this locus . Suggestive linkage is still observed at 4p16  in the Combined Sample, although, this locus was not confirmed in the Follow-up study and the LOD score is lower than that of the original scan . The decreased LOD score at 4p16 may be a consequence of genetic heterogeneity, possibly reflecting the diverse ethnic background of the sample [In addition to the 6q23-24 peak, the Follow-up study supports evidence for linkage at 18q22  and metabotropic glutamate receptor 1 . A recent study reported that SGK levels are increased in brains of HD patients; SGK phosphorylates huntingtin at serine 421, protecting striatal neurons against toxicity caused by a polyQ-huntingtin amino-terminal fragment [SGK is a plausible candidate gene. GRM1 is located within 2 Mb of the peak marker  and is highly expressed in the cerebellum [GRM1 is to protect neurons from apoptotic death [GRIK2 is associated with early onset [GRIK2  is about 30 Mb proximal to the 1-LOD interval on the confirmed 6q23-24 (133 \u2013 153 Mb). However, the variance in onset age explained by GRIK2 is small and one would not expect that it would be detected by linkage. The 7-Mb 1-LOD unit support interval (50 \u2013 57 Mb) at 18q22 contains 36 known genes, according to the Ensembl database (v27) [NEDD4L , which encodes a neural precursor cell expressed, developmentally down-regulated 4-like gene. NEDD4L is an ubiquitin ligase and contains WW domains. Yeast two-hybrid studies found that huntingtin binds to a group of genes with WW domains [NEDD4L is that it may mediate degradation of the product of SGK, the above mentioned candidate gene located at 6q23 [The epistatic analysis indicated that there is no interaction between 6q23-24 with five other loci. Therefore, we assumed that the potential modifiers are not members in a common pathway. The 1-LOD unit support interval (133 \u2013 153 Mb) at 6q23-24 contains 128 known and predicted genes (Ensembl v27) . Two genfragment . SGK is rebellum . One proly onset ,23. GRIK at 6q23 .In conclusion, this replication study confirms evidence for linkage in the 6q23-24 region observed in our original genome scan. Although the other regions, particularly 18q22, may also contain genes that modify age at onset in HD, the 6q23-24 shows evidence for harboring one or more genetic modifiers that exceeds the level required for genome-wide statistical significance (LOD >3.6) . In our HD: Huntington's diseaseHD MAPS: Huntington's Disease Modifiers of Age at Onset in Pairs of SiblingsQTL: Quantitative Trait LociLOD: logarithm of the oddsThe author(s) declare that they have no competing interests.JL participated in the design of the study, performed the statistical analysis and drafted the manuscript. RP was responsible for the coordinating the study groups and sample collection. MRH, SCW, AD, PJM, MN, CAR, RLM, AR, FS, LF, EG, CAG, OS, MLK, RJT, EM, AN, MF, JSP, RJ, TA, AL, MHS, JJC, SMH, JBP, MBH, SLP, AZ, RKA, AJL, AD, KM, and PMC had ascertained the clinical status of the patients and provided the patient samples. JSM, TG, and MH were responsible for the genotyping. GX and LD participated in the data analysis. LAC, VCW, JFG and MEM participated in the study design, data generation, data analysis and manuscript preparation. RHM was responsible for study conception, design, and oversight and finalized the data analysis as well as manuscript preparation. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:"}
+{"text": "The Framingham Heart Study is a very successful longitudinal research for cardiovascular diseases. The completion of a 10-cM genome scan in Framingham families provided an opportunity to evaluate linkage using longitudinal data. Several descriptive traits based on simulated longitudinal data from the Genetic Analysis Workshop 13 (GAW13) were generated, and linkage analyses were performed for these traits. We compared the power of detecting linkage for baseline and slope genes in the simulated data of GAW13 using these traits. We found that using longitudinal traits based on multiple follow-ups may not be more powerful than using cross-sectional traits for genetic linkage analysis. In the past 50 years, the Framingham Heart Study has been a very successful longitudinal research study of cardiovascular diseases. Over the years, many of the major cardiovascular disease risk factors, i.e., high blood pressure, high blood cholesterol, smoking, obesity, diabetes, and physical inactivity, have been identified through careful monitoring of the Framingham Heart Study population. A large amount of valuable information on the effects of related factors, such as blood triglyceride and HDL cholesterol levels, age, gender, and psychosocial issues, has been collected. In the mid-1990s, a genome scan was conducted for 330 pedigrees selected from the Framingham Heart Study. This raised a series of interesting questions such as: how to use the longitudinal phenotypic data in linkage analysis? Would longitudinal data provide more power for demonstrating linkage?Longitudinal phenotypic data contain information not only on trait values at a specific time point, but also on the progression of a trait over time. In addition to identifying genes responsible for cross-sectional trait values, longitudinal data also provide the possibility of identifying genes related to the progression of a trait across time. The progression of a complex trait may depend on many environmental factors and gene \u00d7 environment interactions. Therefore, identifying genes related to the progression of a complex trait may help us study the environmental factors and the gene \u00d7 environment interactions involved. So far, three types of descriptive traits have been used for linkage analysis of longitudinal data: single visit value , within-The simulated data of GAW13 were generated based on the real data scenario of the Framingham Heart Study. The family structure was composed of 330 pedigrees selected for the genome scan of the Framingham Heart Study. The pedigrees consisted of 4692 subjects, of whom 2885 had participated in the Framingham Heart Study. Among the 2885 participants, there were 3041 parent-offspring pairs, 2796 sib-pairs, 2107 avuncular pairs, 183 grandparent-grandchild pairs, and 1595 first cousin pairs. The same family structure was used for simulating all 100 replicates. For each of the 100 replicates, a total of 399 microsatellite markers on the 22 autosomal chromosomes were simulated using the allele frequencies of the markers from the Framingham Heart Study data. Each replicate contained longitudinal data for two cohorts, with data collection on each cohort starting about 30 years apart. The first cohort was examined 21 times at 2-year intervals, while the second cohort was examined 5 times with an 8-year interval between the first two exams and 4-year intervals between subsequent exams. Both completed data and data with missing values were provided for analysis. For simplicity, we used complete genotype and phenotype data for our analysis.GAW13 simulated data provided phenotypic data on age, sex, height, weight, cholesterol, blood pressure, glucose, and various other traits. We focused our analysis on cholesterol and its related covariates, such as sex, age, body mass index (BMI), and triglycerides (TG). There were 21 visits for the first cohort, and only five visits for the second cohort. In order to take advantage of the longitudinal nature of the phenotypic data, we constructed several descriptive traits for linkage analysis. To make the variance of these descriptive traits as comparable as possible for both cohorts, visit 1, 5, 7, 9, and 11 were selected from the first cohort, corresponding to the time intervals of the visits in the second cohort, to generate the descriptive traits. Furthermore, only subjects with cholesterol data at all five visits were used in the analysis for both cohorts. The following traits were generated for linkage analysis.CHOL1: the total cholesterol level at the first visit. It contains genetics effects mainly from the baseline genes.1) CHANGE: the change of the total cholesterol level over 20 years. For Cohort 1, it was the change of the cholesterol level from visit 1 to visit 11; for Cohort 2, it was the change of the cholesterol level from visit 1 to visit 5. It contains the genetic effects mainly from the slope genes.2) MEAN: the within-subject mean of the total cholesterol level across the five visits. It contains the genetic effects from both the baseline and the slope genes.3) SLOPE: the within-subject slope of the total cholesterol level of each individual for the five visits regressed on age. It contains the genetic effects mainly from the slope genes.4) Among the four descriptive traits we considered here, CHOL1 was based on the data at the first visit only, CHANGE was generated based on the data from two out of five visits, and MEAN and SLOPE were generated based on data from all five visits.Two-point sib-pair linkage analysis was conducted using SIBPAL in S.A.G.E. 3.1 . Sex, agMultipoint sib-pair linkage analysis was conducted with MAPMAKER/SIBS in GENEHUNTER2.1_r3 Beta program package ,8. The rAccording to the answer key distributed by GAW13, Gb30-Gb33 and Gs7-Gs9 were the seven genes influencing cholesterol levels directly. Among the seven genes, Gb30-Gb33 were baseline genes and Gs7-Gs9 were slope genes. Both two-point and multipoint sib-pair linkage analyses were carried out for the four descriptive traits on the chromosomes containing the baseline and the slope genes for total cholesterol level, that is, chromosomes 1, 3, 7, 11, 13, 15, and 21. In order to evaluate the false-positive rate, we also analyzed chromosome 2, which does not have any trait locus.p-value for each marker locus. For each trait locus, we took the smallest p-value out of the four markers around the trait locus, which was equivalent to a 30-cM range, as the significance level for that trait locus. We counted the number of times the smallest p-value among the four markers was less than 0.0125  out of the 100 replicates as the power of detecting linkage. Table Two-point linkage analysis using SIBPAL produced a A multipoint LOD score was calculated for each locus by using MAPMAKER/SIBS. For each trait locus, again, we considered four markers around it. We counted the number of times that the largest LOD score among the four markers was greater than 1.67  as evidence for suggestive linkage. Table From Table According to data description of the simulated data of GAW13, the contributions of the four baseline genes (Gb30-Gb33) to the baseline variance of total cholesterol level were 0.20, 0.15, 0.1, and 0.05, respectively. In our linkage analysis results, Gb30 was detected with the highest power while Gb33 was detected with the lowest power. This demonstrated that genes with higher contribution to the trait variance could be detected with higher power. Among the three slope genes, Gs9 contributed to the slope of females only and its contribution to the variance of the slope is very low (~0.03). Here, we actually did not have any power to detect this gene using either two-point or multipoint linkage analyses. For the other two slope genes, the power for detecting Gs7 was higher than that for Gs8, which was also consistent with their contributions to the variance of the slope (0.36 for Gs7 and 0.08 for Gs8).For the two traits containing the genetic effects from baseline genes, CHOL1 and MEAN, the total cholesterol level at the first visit (CHOL1) had a similar or sometimes even higher power to identify baseline genes compared to MEAN. Thus CHOL1 is an acceptable trait for detecting linkage for baseline genes, especially when genetic effects are relatively large. Two longitudinal genetic studies ,10 also CHANGE, MEAN, and SLOPE were the three traits containing genetic effects from slope genes. For a slope gene with large effect, e.g., Gs7, SLOPE is the most powerful trait and MEAN is the least powerful trait from both two-point and multipoint analyses. However, improvement in power from using SLOPE to using CHANGE was limited  while SLOPE used much more data than CHANGE (five visits vs. two visits). For a slope gene with relatively small effect, e.g., Gs8, power was low for both analytic methods.In summary, in comparison with descriptive traits generated from multiple longitudinal data points (such as MEAN and SLOPE), CHOL1 had a similar or even higher power for detecting baseline genes as MEAN, and CHANGE had a similar power for detecting slope genes as SLOPE. The possible explanation for this is that genetic effects are relatively stable in an individual's lifetime, at least in this simulated situation. However, MEAN and SLOPE used the data of all five visits while CHOL1 and CHANGE required data of one or two visits only. Therefore, conducting a longitudinal analysis with multiple follow-ups may not be an effective way to identify susceptibility genes responsible for either baseline or change over time.In the simulated data of GAW13, environmental factors did not play an important role in total cholesterol levels. However, in reality, environmental exposures, medications, and gene \u00d7 environment interactions may play an important role in determining an individual's cholesterol levels, as well as many other complex traits such as DBP and SBP. Under such circumstances, the usefulness of a longitudinal study design with multiple visits should be further explored."}
+{"text": "There is currently a great interest in using single-nucleotide polymorphisms (SNPs) in genetic linkage and association studies because of the abundance of SNPs as well as the availability of high-throughput genotyping technologies. In this study, we compared the performance of whole-genome scans using SNPs with microsatellites on 143 pedigrees from the Collaborative Studies on Genetics of Alcoholism provided by Genetic Analysis Workhsop 14. A total of 315 microsatellites and 10,081 SNPs from Affymetrix on 22 autosomal chromosomes were used in our analyses. We found that the results from the two scans had good overall concordance. One region on chromosome 2 and two regions on chromosome 7 showed significant linkage signals  for alcoholism from both the SNP and microsatellite scans. The different results observed between the two scans may be explained by the difference observed in information content between the SNPs and the microsatellites. There is currently great interest in using SNPs in genetic linkage and association studies because of the abundance of SNPs as well as the availability of high-throughput genotyping technologies. Kruglyak  predicteCOGA data provided by GAW14 include 143 pedigrees with 1,614 individuals genotyped with both microsatellites and SNPs. In addition, the genetic maps for both the microsatellites and the SNPs were provided. We used the nonparametric linkage analysis implemented in MERLIN  was applp \u2264 0.025 was excluded [To avoid potential bias caused by possible genotyping errors on linkage signals, the error-checking algorithm implemented in MERLIN was applied. This algorithm identifies unlikely genotypes based on the inferred double recombination events, when erroneous genotypes can imply excessive and unlikely recombination events between tightly linked markers . We usedThe major advantage of using high density SNPs versus microsatellites is the increased information content (IC). IC was calculated using MERLIN to compare the microsatellites and the SNPs in order to investigate factors contributing to the differences between the two scans. The microsatellites were spaced an average of 13 cM apart, whereas the SNPs were spaced an average of 0.35 cM apart. To assess the effect of the reduced IC on the SNP scan, a 3,360-SNP map with an average spacing of 1.0 cM was randomly extracted from the full set of SNPs as a subset for a separate scan.The results from the whole-genome scans using the microsatellites and the SNPs had good overall concordance. Six regions showed some evidence of increased allele sharing, with a NPL cutoff value of 2 for either the SNP scan, the microsatellite scan, or both. The results were summarized in Table The mean IC for each individual chromosome for the full SNP set, SNP subset, and microsatellites across 22 autosomal chromosomes when the erroneous genotypes were either excluded or included were summarized in Table We have compared the genome-wide linkage analyses based on the microsatellites and the SNPs. We used the software MERLIN to conduct nonparametric linkage analysis to map regions associated with alcoholism on 22 autosomal chromosomes. The results from the two scans had good concordance in general, although more significant signals were obtained using the SNPs versus the microsatellites. Both scans suggested strong linkage evidence on chromosomes 2 and 7, where the two scans agreed especially well. The microsatellite scan had a peak at the marker D7S820 at 107.5 cM with an NPL score of 2.56 on chromosome 7, and the SNP scan had a peak at the marker tsc0046246 at 100.9 cM with an NPL score of 2.81. For chromosome 2, the microsatellite scan had a peak at the marker D2S1329 at 4.9 cM with an NPL score of 2.13, and the SNP scan had a peak at the marker tsc0056805 at 243.6 cM with an NPL score of 2.80. The differing results observed in the two scans were likely explained by the difference between the IC in the microsatellites and the SNPs. In fact, the higher IC is one major advantage of the high-density SNPs compared with the conventional microsatellite sets. The IC across the genome for the SNPs was uniformly higher than that for the microsatellites.As expected, the analysis based on the SNP subset showed decreased IC and reduced linkage signals compared with the SNP full set, which suggested that the difference in IC might be one key factor that contributed to the observed difference in the two scans. This was consistent with the conclusion from John et al. , who exaFinally, we noted that the SNP subset scan was able to detect some regions detected by the SNP full set scan, and the SNP subset had an average IC of 0.910 compared to the average IC of 0.950 for the full SNP set. With the NPL cutoff of 2, the SNP subset scan resulted in some loss of significance of several regions on chromosomes 2, 7, and 12.We have identified two regions that showed some evidence of linkage with alcoholism on chromosome 2 and chromosome 7 from both the microsatellite and the SNP scans. For these regions, we had stronger linkage signals using the SNPs than those using the microsatellites. Although results from the two scans had good overall concordance, three regions of significant linkages were detected in the SNP scan but not in the microsatellite scan. Lastly, the difference in IC between the SNPs and the microsatellites might explain the different results observed in the two scans.COGA: Collaborative Study on the Genetics of AlcoholismGAW: Genetic Analysis WorkshopIC: Information contentLD: Linkage disequilibriumSNP: Single-nucleotide polymorphismSW participated in the design of the study, performed the analysis, and drafted the manuscript. SH, NL, LC, and CO participated in the design and the discussion of the study. HZ conceived the study and helped to draft the manuscript. All authors read and approved the final manuscript."}
+{"text": "There are no well accepted criteria for the diagnosis of the metabolic syndrome. However, the metabolic syndrome is identified clinically by the presence of three or more of these five variables: larger waist circumference, higher triglyceride levels, lower HDL-cholesterol concentrations, hypertension, and impaired fasting glucose. We use sets of two or three variables, which are available in the Framingham Heart Study data set, to localize genes responsible for this syndrome using multivariate quantitative linkage analysis. This analysis demonstrates the applicability of using multivariate linkage analysis and how its use increases the power to detect linkage when genes are involved in the same disease mechanism. It has been shown that for correlated traits, multivariate approaches for genetic linkage analyses can increase the power and precision to identify genetic effects -4. When Our approach is based on the assumption that it is easier to detect a quantitative trait locus (QTL) involved in the metabolic syndrome using multivariate linkage analysis. Our aim is to show that using combinations of traits related to the metabolic syndrome, and then using them in multivariate linkage analysis software, gives reliable results for linkage to genes associated with this syndrome.There are no well accepted criteria for the diagnosis of the metabolic syndrome. However, the metabolic syndrome is identified by the presence of three or more of the variables listed in Table i Y= ' be a vector of m multivariate trait values for ki members of the ith family. Let N be the total number of families, \u03b2 a vector of dimension mp of the regression coefficients for the p covariates , Xi = ImXki x m an mki \u00d7 mp known matrix of covariate values for the ith family, where  is the Kronecker product, and i Va VC matrix of dimension mki\u00d7 mki. Then, the variance-covariance matrix of the traits is i = AVi + BGi + CZiI, where i Gis the ki \u00d7 ki matrix of the coefficients of relationship for the family i; i Zan ki \u00d7 ki matrix of estimated proportion of alleles identical by decent (IBD) for pairs of related individuals for the ith pedigree; i Iis the ki \u00d7 ki identity matrix; and A, B, and C, are, respectively, polygenic, major-gene, and environment variance-covariance matrices each of dimension m \u00d7 m. A more detailed description of these models was presented elsewhere [The multivariate variance-components (MVC) approach is an extension of the univariate approach described by Amos . For mullsewhere .0. The distribution of the multivariate test is a mixture of \u03c72 values [02, 3/8 \u03c712, 3/8 \u03c732 and 1/8 \u03c762. One-eighth of the time all the VCs are estimated to be positive with all the covariances different from 0 yielding 6 degrees of freedom. Three-eighths of the time, one of the VCs is estimated to be zero with two covariances fixed to zero (yielding 3 degrees of freedom). Another three-eighths of the time two VCs are fixed to zero with all covariances equal to zero yielding 1 degree of freedom. Finally, one-eighth of the time all the variances are fixed to zero resulting in a degenerate distribution of point mass at zero.To test for genetic linkage, we also construct a likelihood ratio test. Under the null hypothesis, the major gene parameter(s) are restricted to equal 2 values . For triFor the multivariate linkage analysis, we use the following four traits: triglycerides, HDL-cholesterol, systolic blood pressure (SBP), and fasting glucose. Since these variables, except for triglycerides, were measured at several time points, we applied a similar regression approach described in Levy et al.  for thesWe do observe small to moderate positive genetic correlations between SBP and triglycerides (0.187), SBP and fasting glucose 0.296), and triglycerides and fasting glucose (0.361); we also observe a strong negative correlation between HDL-cholesterol and triglycerides (-0.664), and small to moderate negative correlations between HDL-cholesterol and SBP (-0.048), and HDL-cholesterol and fasting glucose (-0.249). Table 96, and t 5.37, position 136 cM, P = 5.4 \u00d7 10-5); HDL, fasting glucose, and triglycerides ; SBP, HDL, and fasting glucose ; SBP, HDL, and triglycerides . The univariate maximum LOD scores for SBP, triglycerides, fasting glucose, and HDL were, respectively, 1.5 (34 cM), 1.75 (74 cM), 3.3 (136 cM), and 1.2 (38 cM). On chromosome 5, the following combination produced evidence for linkage: SBP, fasting glucose, and triglycerides ; HDL, fasting glucose, and triglycerides ; SBP, HDL, and triglycerides ; SBP, HDL, and fasting glucose . The univariate maximum LOD scores for SBP, triglycerides, fasting glucose, and HDL were, respectively, 2.21 (34 cM), 1.97 (0 cM), 1.53 (160 cM), and 0.16 (160 cM). On chromosome 6, the following combination produced evidence for linkage: SBP, fasting glucose, and triglycerides ; HDL, fasting glucose, and triglycerides ; SBP, HDL, and triglycerides . The univariate maximum LOD scores for SBP, triglycerides, fasting glucose, and HDL were, respectively, 0.12 (2 cM), 5.52 (152 cM), 0.64 (44 cM), and 0.25 (182 cM). On chromosome 17, the following combination produced evidence for linkage: SBP, fasting glucose, and triglycerides ; SBP, HDL, and triglycerides . The univariate maximum LOD scores for SBP, triglycerides, fasting glucose, and HDL were, respectively, 1.35 (66 cM), 1.76 (6 cM), 0 (-), and 0.22 (126 cM).Figure  3.14, position 12 cM, results not shown). Furthermore, evidence for linkage was also found on chromosomes 2, 5, and 6. We also showed that the pair-wise combinations with evidence for linkage are the ones that have either small to moderate genetic correlation or negative genetic correlation. In summary, the use of multivariate quantitative trait loci linkage analysis can increase the power to detect a QTL. However, this procedure is computationally intensive, i.e., the CPU time increases exponentially as the number of traits increases additively.The MVC approach appears to perform well in the identification of regions linked to genes associated with traits related to the metabolic syndrome, mainly on regions where the QTL effects were negatively correlated and there was a positively correlated polygenic effect as shown by Amos et al.  and Evan"}
+{"text": "Pedigree, demographic, square-root transformed maximum alcohol (SRMAXAPD) and maximum cigarette (MAXCPD) consumption, and genome-wide scan data from the Framingham Heart Study (FHS) were used to investigate genetic factors that may affect alcohol and cigarette consumption in this population-based sample.A significant sister:sister correlation greater than spouse correlation was observed for MAXCPD only. Single-point sib-pair regression analysis provided nominal evidence for linkage of loci to both SRMAXAPD and MAXCPD consumption traits, with more significant evidence of linkage to SRMAXAPD than to MAXCPD. One genomic region, chr9q21.11, exhibits significant multi-point sib-pair regression to SRMAXAPD.SRMAXAPD exhibits greater evidence for genetic linkage than does MAXCPD in the FHS sample. Four regions of the genome exhibiting nominal evidence for linkage to SRMAXAPD in the FHS sample correspond to regions of the genome previously identified as linked to alcoholism or related traits in the family data set ascertained on individuals affected with alcohol dependence known as COGA. Data from the ongoing NHLBI-supported Framingham Heart Study (FHS) on cardiovascular disease (CVD) was made available to Genetic Analysis Workshop 13 (GAW13). Two behaviors of general medical and psychiatric interest collected from this community-based sample were included in the data, i.e., alcohol consumption and cigarette consumption. Increased cigarette consumption in the FHS sample is associated with the development of CVD , but incth century, due to health concerns and restrictions placed on this behavior [The consumption of these two substances varies significantly based on both sex and age and there has been a long-term decline in the consumption of cigarettes in the US in the latter half of the 20behavior . The conbehavior . The genbehavior . MeasureN = 346) were included. Individual outlier trait values > +4 standard deviations were changed to missing for analysis (without outliers); the individuals whose values were converted were predominantly male for all three traits but were mostly (\u226580%) from Cohort 1 for APD traits  and from Cohort 2 for MAXCPD (N = 10). Removal of outliers brings SRMAXAPD and MAXCPD traits much closer to normality  measure and 2881 with a maximum cigarette consumption (MAXCPD) measure. Descriptive statistics of the MAXAPD and MAXCPD traits and a square root transformation of MAXAPD, SRMAXAPD, are reported in Table Familial correlations of relative and parental pairs (sex-specific and non-sex-specific) without extreme positive outliers for MAXCPD and SRMAXAPD are reported in Table p-value < 0.01  of marker loci that were nominally linked (p < 0.01) with the maximum alcohol consumption traits, SRMAXAPD. A broad region on chromosome 9 exhibited the most significant evidence for linkage, with the maximum linkage evidence at ~66 cM (Table N = 9) with evidence for nominally significant (p < 0.05) linkage and no loci at p < 0.021. The low number of loci exhibiting nominal evidence for linkage to MAXCPD suggests that the MAXCPD trait, as investigated in this linkage analysis, lacks power to detect the influence of genetic susceptibility factors on maximum cigarette consumption.In the genome-wide search for linkage evidence to maximum alcohol consumption, we found a number N = 7 of markp-values for the significance of linkage analysis results were not substantially different from asymptotic p-values for either trait, suggesting that assumptions of the SIBPAL regression model apply to the phenotypic and genotypic data analyzed in this study. Only the regions of maximum linkage to SRMAXAPD on chromosome 9 and the single-point linkage analysis result to SRMAXAPD on chromosome 17 at 89 cM provided statistical evidence for linkage at p < 0.0007, a level considered \"significant\" by Lander and Kruglyak [Empirical Kruglyak . MultiplLinkage studies of alcohol- and cigarette-related traits have identified regions of the genome with more than nominal evidence for linkage . RegionsWe observed several marker loci with nominal evidence for linkage to the square-root transformed maximum alcohol consumption traits, SRMAXAPD, in the Framingham Heart Study sample. Some of the regions of the genome have been previously linked to alcoholism or related traits in Caucasian samples based on different ascertainment criteria.The traits of interest were defined as the maximum reported number of grams of alcohol per day (MAXAPD), and the maximum reported number of cigarettes smoked per day (MAXCPD). Exams 1, 2, 4, and 7 from FHS Cohort 1 and exams 1, 2, 3, and 4 from FHS Cohort 2 were chosen to assess MAXAPD and MAXCPD to utilize multiple exams at the earliest age possible to obtain measurements. Covariates of interest included cohort, age of maximum consumption measure, and sex.GAW13 FHS data were imported into a Microsoft Access database and exported in appropriate files for descriptive statistical analysis in SPSS Advanced Statistics or Microsoft Excel and familial correlation and linkage analysis in S.A.G.E. . Familiap-values were obtained for single-point linkage analysis of MAXCPD and SRMAXAPD to evaluate possible deviation from asymptotic p-values. Multi-point linkage analysis was performed using IBD distributions at multiple markers for MAXCPD and SRMAXAPD on those chromosomes showing nominal evidence  for linkage at two or more consecutive loci.SIBPAL from S.A.G.E. 4.2 was used to model the sib-pair covariance of traits reported as a function of marker allele identity-by-descent (IBD) sharing. Our analyses used estimated IBD information from the GENIBD procedure described above to perform single-point linkage analysis in which the mean corrected cross product of the trait was regressed onto the IBD information one trait at a time. The single-point linkage analysis was carried out separately for traits MAXAPD, SRMAXAPD, and MAXCPD, treated as continuous variables. The covariates sex, age of trait report, and cohort were included in the regression models. SRMAXAPD single-point linkage analysis was only performed without outliers. Empirical AB nominated the traits, performed the descriptive and FCOR analyses, and wrote the manuscript, XY performed the linkage analysis of the alcohol consumption traits, YB performed the linkage analysis of the cigarette consumption trait, MB performed the GAW13 FHS databasing, selection, reduction, transformation, and export, and AG and LG provided analysis direction and recommendations at each phase of the analysis."}
+{"text": "The objective of this study is to evaluate the efficacy of a model-free linkage statistics for finding evidence of linkage using two different maps and to illustrate how the comparison of results from several populations might provide insight into the underlying genetic etiology of the disease of interest. The results obtained in terms of detection of the risk loci and threshold for declaring linkage and power are very similar for a dense SNP map and a sparser microsatellite map. The populations differed in terms of family ascertainment and diagnosis criteria, leading to different power to detect the individual underlying disease loci. Our results for the individual replicates are consistent with the disease model used in the simulation. The Genetic Analysis Workshop 14 (GAW14) simulated problem provided family data ascertained in four different populations. All members of the family were typed both with a relatively sparse map of microsatellites, and a denser map of single-nucleotide polymorphisms (SNPs). The objective of this study is to evaluate the efficacy of a model-free linkage statistics for finding evidence of linkage in the different populations using the two different maps. We also show how the comparison of several diagnostic criteria can provide clues to the underlying genetic model. This study was performed blind to the genetic model used to simulate the data provided.The disease under study, Kofendrerd Personality Disorder (KPD), results in an unknown combination of 12 sub-phenotypes. Families with this disorder were ascertained in four populations, with a different scheme. In 3 populations (Aipotu (AI), Danacaa (DA) and Karangar (KA)), ascertainment was based on the presence of at least two affected sibs in nuclear families, while in the last, New York (NY), large pedigrees including more than 4 affecteds were recruited. The populations differed also in the distribution of the sub-phenotypes. All family members were typed for markers on their 10-chromosome genome, without genotyping error. Two marker sets were available: 416 microsatellites spaced every ~7 cM, and a denser 917-SNP map, with ~3.5 cM inter-marker distances.To identify the number and location of the susceptibility loci involved in the simulated disorder, a pooled linkage analysis of all 100 replicates in a given population was performed with the nonparametric linkage (NPL) statistics  for the 0 (5 chromosomes \u00d7 3 populations \u00d7 100 replicates). Because of its ascertainment scheme, and computing limitations, the NY population was ignored in this step.After the pooled analysis, some chromosomes appeared not to harbor any susceptibility loci. Five chromosomes (see \"Results\") represented the null hypothesis of no linkage, whatever the population, giving a total of 1,500 replicates simulated under HThe value of the maxNPL that was exceeded in 0.5% of these 1,500 replicates, was then determined. It corresponds to the threshold for declaring linkage at the 5% genome-wide level, after a Bonferroni correction for 10 independent chromosomes.This was calculated as the number of replicates in which the value of the NPL at the putative disease locus exceeded the 5% genome-wide threshold value.Four, and possibly 5, linkage regions had evidence of linkage by the pooled analysis, as shown in Table Apart from the region on chromosome10, whatever the diagnosis criteria, one can conclude there is a susceptibility factor on chromosome 1, 3, 5, and 9. For these chromosomes, in all populations, the peak occurred at the same marker or the one immediately adjacent.In addition, the different results obtained in the populations AI, DA, and KA, which only differ by the definition of the affection status, show that the genotype-phenotype relationships vary widely across populations. This is well illustrated for chromosome 9, where the NPL ranges from 8.6 in DA to 37.9 in KA.In population AI and NY, the diagnosis criteria seem to be the same, as indicated evidenced by the similar distribution of sub-phenotypes among cases, but the ascertainment criteria and family structures differ. The NPL values are greater for the AI nuclear families than for the NY extended pedigrees for chromosome 1, 5, and 9, whereas they are similar for chromosome 3. This result is interesting in view of the debate \"sampling large extended pedigrees vs. smaller familial structures\". Here, we show that for the simulated model, two nuclear families with two affected sibs are more informative than one three-generation pedigree with four affected members.For chromosome 10, the signal is very weak because this NPL value was obtained for 10,000 families in AI, DA, and KA and 5,000 families for NY. So this could well represent a factor with an effect difficult to detect by linkage analysis or a factor observed only in a subgroup of affecteds.Similar observations were obtained in a pooled analysis using the denser SNP map. It was not possible to align the two maps, because no indication was given about merging the two maps. However, the peaks were located about the same distance from the first marker of each map.For the 5 chromosomes , the highest NPL obtained on the pooled data for the microsatellite map was 2.65. As explained in the \"Methods\" section, these 5 chromosomes were considered to carry no risk factor and were thus presumably simulated under the null hypothesis of no linkage. It is thus possible to establish the 5% genome-wide threshold from the distribution of the NPL scores observed in the individual replicates of the three populations AI, DA, and KA. This threshold was found to be NPL = 3.3 and 3.2, using the microsatellite and SNP maps, respectively.The power to detect linkage in the 5 regions found by the pooled analysis is given by the number of replicates for which the statistic value is over the 0.5% threshold, as shown in Table The detection of the different risk factors varies according to the diagnosis strategy and the chromosome, giving clues on the genetic basis of KPD. Let A denote the anxiety-related symptoms, B, the behavioral, and C the \"communally shared emotions\" sub-phenotypes. From the indication given to all participants prior to the analysis, individuals in AI are declared affected if they have A or B or C symptoms, while in DA, B is prominent. In KA, only those individuals with either A or C, whatever their B symptoms, are classified as affected, while those with prominent B symptoms are not.The chromosome 1 risk factor is very well detected in DA, and not in the other populations, suggesting that it is involved only in the determination of behavior B. On the other hand, the risk factors on chromosomes 5 and 9 do not seem to play a role in determining B (lack of evidence in DA), but are probably involved in the determination of A and C.Chromosome 3 is detectable in all populations, with varying intensities. It is probably involved whatever the diagnosis criteria. However, in the AI population, this locus is detected in 41% of the replicates, but the NPL values range from 1.38 to 5.71. This observation is true even when the power is high, such as in the DA population where the values range between 1.66 and 6.13. This risk locus illustrates the difficulty of replicating an earlier linkage finding, as shown by Clerget-Darpoux et al. [Finally, the chromosome 10 risk factor is never detected with sample sizes of 100 families. As we have seen in the pooled analysis, it is a factor difficult to detect by linkage analysis. Note that it was detected in DA and KA by association analysis .The disease model used in the simulation was given during GAW14. Four disease loci and two modifier genes were simulated, and their position on the SNP map was given. Neither D5 nor D6, which act as modifier genes involved in the phenotype P2 that regroups most of the traits defined as behavioral related traits B, are expected to be detectable by linkage analysis, even with the large sample size of the pooled analysis. In fact, disease locus D6 was not detected at all. The other loci were all detected at their exact location on the SNP map, with the exception of D2 on chromosome 3 in DA (maxNPL found at the adjacent SNP) and D5 on chromosome 10 in AI (maxNPL located 16 cM more centromeric). The value of these two maxNPL are given italics in Table Analysis of the individual replicates gave results consistent with the disease model used in the simulation. In population DA, individuals are declared affected when they have phenotype P1, determined by the two loci D1 and D2 on chromosome 1 and 3, respectively, with a highly penetrant dominant mode of inheritance. These two loci are therefore easily detected in this population.Locus D2, on chromosome 3, underlies all the phenotypes. This explains why it is very well detected in all 3 populations, whatever the ascertainment criteria. In contrast, locus D3 on chromosome 5, and locus D4 on chromosome 9 determine phenotype P2 and/or P3. This explains the high level of detection in KA and AI, but not in DA. Note that D4, which acts in a recessive manner with a high penetrance, displays more evidence of linkage than D3.The answers also provided some explanation of the difference in magnitude of the maxNPL in the pooled analysis of the AI and NY replicates. The ascertainment criteria were not only different; but showed greater heterogeneity in NY. In the NY study, the 4 affected individuals could each have different phenotypes, determined by different combination of the disease loci, thus lowering the resemblance between affecteds and the expected value of the linkage statistic.In this simulated problem, the results obtained in terms of detection of the risk loci, threshold and power were very similar for the microsatellite and SNP map. A sparser map, with very polymorphic markers, brings as much information on the IBD sharing than a denser, less polymorphic marker map, at a smaller genotyping cost. Whether this is true in all cases remains to be explored. However, this point should be kept in mind before embarking on a genome scan using SNPs.The power to detect linkage varies according to the population diagnosis criteria and to the disease locus.GAW14: Genetic Analysis Workshop 14IBD: Identical by descentKPD: Kofendrerd Personality DisorderNPL: Nonparametric linkageSNP: Single-nucleotide polymorphismM-CB performed the analyses and drafted the manuscript. CB and A-LL provided the file formatting programs. FC-D and M-CB designed the study. All authors read and approved the final manuscript."}
+{"text": "At the same location, our analysis showed a LOD score of 2.2. This decrease in the LOD score is the result of a drastic reduction (one-third) of the available GAW14 phenotypic data. We performed SNP and haplotype association analyses with the same phenotypic data under the linkage peak region on chromosome 4. Seven Affymetrix and two Illumina SNPs showed significant associations with ecb21 phenotype. A haplotype, a combination of SNPs TSC0044171 and TSC0551006 , showed a significant association with ecb21 (p = 0.015) and a relatively high frequency in the sample studied. Our results affirmed that the GABA region has potential of harboring genes that contribute quantitatively to the beta oscillation of the brain rhythms. The inclusion of the remaining 614 subjects, which in the GAW14 had missing data for the ecb21, can improve the strength of the associations as they have already shown that they contribute quite important information in the linkage analysis.This study, part of the Genetic Analysis Workshop 14 (GAW14), explored real Collaborative Study on the Genetics of Alcoholism data for linkage and association mapping between genetic polymorphisms (microsatellite and single-nucleotide polymorphisms (SNPs)) and beta (16.5\u201320 Hz) oscillations of the brain rhythms (ecb21). The ecb21 phenotype underwent the statistical adjustments for the age of participants, and for attaining a normal distribution. A total of 1,000 subjects' available phenotypes were included in linkage analysis with microsatellite markers. Linkage analysis was performed only for chromosome 4 where a quantitative trait locus with 5.01 LOD score had been previously reported. Previous findings related this location with the  In thisor genes  be reproIn the GAW14 data, which made available data from the Collaborative Study on the Genetics of Alcoholism (COGA), information on 1,614 subjects was made accessible, but 614 of the subjects had missing values for ecb21 (brain rhythm beta electric oscillations). In the Porjesz et al.  study, tq-value software of Storey in R language [q-value of SNPs tests provides the proportion of false positives occurring when that particular SNP association is declared significant. The phenotype ecb21 was adjusted for age (age at the time of the clinic visit).Identity-by-descent (IBD) coefficients were estimated on nuclear families with the MAPMAKER/SIBS program . Linkagelanguage ,9. The qPorjesz et al.  had repoWe restricted the association tests to the region of the linkage signal (39 cM = dist = 70 cM) in order to decrease the possibilities of having false-positive associations Figure . EmployiA) receptor, which is a multisubunit chloride channel that mediates the fastest inhibitory synaptic transmission in the central nervous system. It is located at 4p12, in the physical chromosomal interval 46.6\u201347.5 Mb, and at 51.4 cM based on GAW14 genetic map. GABAA is contained in a cluster consisting of genes encoding alpha 4, alpha 2, and gamma 1 subunits of the GABAA receptor. Modifications of this gene were implicated in the pathogenetics of schizophrenia [The heritability of the trait studied was high (68 \u00b1 6%). We found evidence of linkage for ecb21 at the published location. The LOD score we observed using the available data was lower than the previous published value. There were two possible peaks, with the main one located within 57\u201358 cM , showed a significant association with ecb21 (p = 0.015) and a relatively high frequency in the sample studied. The inclusion of the remaining 614 subjects, that in the GAW14 have missing data for the ecb21, can improve the strength of the associations as they have shown already that they contribute quite important information in the linkage analysis , none of them hit the location 46.6\u201347.5 Mb, but some of them were very close to the GABAOD score ).The previously reported linkage peak of 5.01 for ecb21 located on chromosome 4, was reproduced by this analysis, but attenuated to a 2.2 LOD score as a result of unavailable phenotypes for 614 subjects. With the reduced sample of 1,000 subjects, 9 SNPs, 7 of Affymetrix and 2 of Illumina, showed nominally significant associations with ecb21. A common haplotype of 2 SNPs states showed a significant association with ecb21. The presence of these findings around the GABA cluster supports the formerly proposed relation among this cluster, brain oscillations, and alcoholism. The inclusion of the other 614 subjects in the analysis may strengthen the association signals, and provide better evidence on the GABA cluster role, as it has been shown that these extra subjects carry important information in the linkage analysis.COGA: Collaborative Study on the Genetics of AlcoholismEEG: ElectroencephalogramFDR: False discovery rateA: Gamma-aminobutyric acidGABAGAW14: Genetic Analysis Workshop 14IBD: Identity by descentQTL: Quantitative trait lociSNP: Single-nucleotide polymorphism"}
+{"text": "Analysis of the White population from the COGA data with the reordered map for alcohol dependence led to a significant change of the linkage signal (p = 0.0365 decreased to p = 0.0039) on chromosome 1 between marker D1S1592 and D1S1598. Our results suggest that reordering fine-mapping markers in candidate regions when the genetic map is uncertain can be a critical step when considering a dense map.We developed a new marker-reordering algorithm to find the best order of fine-mapping markers for multipoint linkage analysis. The algorithm searches for the best order of fine-mapping markers such that the sum of the squared differences in identity-by-descent distribution between neighboring markers is minimized. To test this algorithm, we examined its effect on the evidence for linkage in the simulated and the Collaborative Studies on Genetics of Alcoholism (COGA) data. We found enhanced evidence for linkage with the reordered map at the true location in the simulated data ( Errors in map order may originate from the use of general genetic maps, e.g., Marshfield, that are based on a limited number of meioses and can lead to incorrect marker order and poor estimates of interpolated recombination fractions . MoreoveThe subject of map order in linkage analysis has been investigated and several statistical approaches have been suggested: 1) include order errors as nuisance parameters through the use of profile likelihoods , 2) use N = 700 in 100 pedigrees with 1,214 sib pairs) and REP085  with the highest and lowest LOD scores, 5.97 and 1.86, respectively. Our goal was to determine whether we could improve the evidence for linkage either or both of the replicates after reordering the markers.We screened all the groups from the simulated data with a binary trait (b) to obtain datasets representative of moderate to strong linkage. We selected the Danacaa population from the simulated data; the sample was ascertained as nuclear families. To identify replicates that provided the most information, we searched all the replicates in the Danacaa population using LODPAL (S.A.G.E. ) with onN = 1,219 in 115 pedigrees with 1,374 sib pairs). Prior to linkage analysis, we split two loops in the selected pedigrees. Two different measures of alcohol dependence were averaged to obtain the final measure of alcohol dependence, a semi-quantitative trait, and we adjusted for pack years and the interaction between sex and age at interview. We calculated values at age 80 and added them to the residuals from the final regression model. To obtain an appropriate Box-Cox transformation parameter, we used the SEGREG program in S.A.G.E. [For the COGA data, we restricted the analysis to individuals of White descent , we combined the SNP map with the microsatellite map using the Kosambi map function. For the COGA data the distances are given as Kosambi centimorgans for both SNPs and microsatellites, and for these data we simply placed the markers at the appropriate locations, interleaving SNPs with microsatellites.Initially, we selected microsatellite markers with an average intermarker distance of 7 cM as the framework map to identify candidate regions for further pursuit. At locations where we obtained evidence for linkage, we increased the density of markers using SNPs to cover an average spacing of 3 cM both in the simulated and in the COGA data (Affymetrix).In the simulated data, with prior knowledge of the true location, we selected the candidate region between markers D01S0021 and D01S0026, adding 11 SNP markers (C01R0047\u2013C01R0057). We also selected regions with no evidence for linkage between marker D01S0016 and D01S0019, adding 7 SNP markers (C01R0035\u2013C01R0041). For the COGA data, we selected the candidate region between marker D1S548 (0 cM) and D1S1631 (135.76 cM), based on our preliminary analysis and a previously published result [f0, f1, and f2 at one locus and , , and  at the other, our criterion to find the best order was to minimize the sum over all sibs and all pairs of neighboring loci, .We used, for two neighboring marker loci, the estimated IBD distribution for each sibpair, i.e., the probabilities that the sib pair shares 0, 1, or 2 alleles IBD. Denoting these probabilities Our reordering algorithm comprises three basic steps that are applied iteratively: initializing a list of markers as framework markers, multipoint IBD calculation, and reordering. First, all MS markers were placed in the list of framework markers. Second, for a fine-mapping marker between two framework markers, the sum of \u0394 over all sibs was calculated between each pair of these three markers  and the order of the three markers chosen that minimizes these sums. Third, the list of framework markers was updated to include the new fine mapping marker. The algorithm sequentially inserts fine-mapping markers among the framework markers until there are no more fine-mapping markers left. The GENIBD program and SIBPAL in S.A.G.E.  were usep-value for linkage at the true location in REP001 was 1.16 \u00d7 10-9  did not show any noticeable change compared with using microsatellites only . Using the reordered map  compared to the initial estimate . Table Increasing the map density by supplementing with additional SNP markers Figure  in the ip Figure , the higPrevious analysis of the COGA data  also shoFor the simulated data, we simply combined the microsatellite and SNP maps with the Kosambi map function, which introduced uncertainty in the local map order between microsatellite and SNP markers. Hence, the small local shifts modified the multipoint IBD distribution sufficiently to be detected as changes in the linkage signal. Moreover, the COGA data showed there are considerable map uncertainties present in the initial map, and the average IBD calculation greatly affected by the local changes of marker orders.In our study, we only considered marker order as the important parameter in construction of a genetic linkage map. However, a recent study  showed tWe investigated the impact of map order on the IBD distribution, and developed a maker-reordering algorithm to optimize the linkage evidence. In both the simulated and the COGA data, we found an improvement in the linkage signal with the reordered map using our algorithm. We believe that a more generalized approach including additional parameters (e.g. linkage disequilibrium and marker informativity) incorporated with our algorithm will help to construct a more accurate genetic map, and consequently improve the process of multipoint linkage analysis.COGA: Collaborative Studies on Genetics of AlcoholismGAW: Genetic Analysis WorkshopIBD: Identity by descentSNP: Single-nucleotide polymorphismGJ performed the overall statistical analysis and interpreted the data. YS prepared and analyzed the data. SKI designed the study, interpreted the results, and helped to draft the manuscript. RCE developed the algorithm, reviewed results, and helped to draft the manuscript."}
+{"text": "This region harbors OPRM1, a candidate gene for substance dependence.A simple multipoint procedure to test for parent-of-origin effects in samples of affected siblings is discussed. The procedure consists of artificially changing all full sibs to half-sibs, with distinct mothers or fathers depending on the parental origin to be evaluated, then analyzing these families with commonly used statistics and software. The procedure leads to tests for linkage through mothers or fathers and also leads to a test for imprinting effects in the presence of linkage. Moreover, simulations illustrate that in regions unlinked to susceptibility genes this multipoint procedure does not have an inflated type I error if a sex-averaged genetic map is used, even when large differences exist between male-specific and female-specific maps. In regions linked with susceptibility genes, the test of imprinting is biased under the null hypothesis if differences exist between sex-specific maps, irrespective of the map used in the analysis. The procedure is applied to the Collaborative Study on the Genetics of Alcoholism dataset from the Genetic Analysis Workshop 14. Results indicate that brothers categorized as affected according to the DMS-III-R and Feighner classification show evidence of linkage through fathers to the 6q25 region ( This procedure was recently used in an probands . The proprobands  and Karaprobands . Recentlprobands .In this paper I investigate some properties of the proposed procedure and apply it to the Collaborative Study on the Genetics of Alcoholism (COGA) dataset.Spairs statistic of Whittemore and Halpern [Zlr, that is robust against the incompleteness of the marker data, following the method of Kong and Cox [ be the value of the statistic when applied to nuclear families where the sibs have been artificially recoded as half-sibs with distinct mothers. Then the  statistic evaluates the excess sharing of alleles from fathers, and is a test of linkage through fathers (more precisely it is a test of Mendelian transmission from fathers to affected sibs). Similarly, let  be the value of the statistic when the distinct parents are fathers, which evaluates the sharing of alleles from mothers and is a test of linkage through mothers. I wrote a program, NUCULAR, that splits extended pedigrees into nuclear families with the option of recoding all sibs as half-sibs, with distinct mothers or fathers, who nevertheless maintain their original genetic material.The nonparametric  Halpern  is used  and Cox . It is a. This statistic has an approximately normal distribution. The mean of the distribution is 0 and the variance is 1 if the region under study is unlinked to any susceptibility gene (due to independent segregation from both parents), but the variance may be unequal to 1 if there is linkage, because the variance of  and  may be different than 1 and the statistics are likely to be correlated. Consequently, if the region is not linked to any susceptibility genes, the test of imprinting effects has the correct size (but has no power); if the region is linked to a susceptibility gene, the test has power to detect imprinting effects but the true type I error may be different from the nominal one. A solution, if the analysis is carried out using a sex-averaged map, could be a permutation procedure consisting of randomly permuting the genotypes of the parents. This procedure preserves linkage while creating replicates under the null hypothesis of no imprinting effects. It will not correct for the bias introduced if there are large differences in gender-specific genetic maps: the expected values of  and  may then be different under linkage, due to the reduced power of sparser maps to detect linkage [A test of imprinting effects in the presence of linkage can be constructed by evaluating  linkage .ALLEGRO v1.2 ,7 was usI applied the procedure to the COGA microsatellite dataset, using the DMS-III-R and Feighner classification to define the set of affected individuals. Only one nuclear family was extracted per pedigree, the one with the most affected individuals; this is to allow for a valid permutation procedure as described above. This study was approved by the McGill Institutional Review Board. statistic shows a bias when there is linkage and when differences exist between the maps, in that its expectation differs significantly from 0. Note that the bias is not eliminated if the analysis is carried out under the true sex-specific maps. Even if in our case the estimated true type I errors are not significantly different from the nominal ones, the inflation of the type I error is bound to become more important as the sample size increases.The type I error of the proposed methods was evaluated in samples of 250 nuclear families with 2 affected sibs by simulating 4 markers located 5 cM  from one another, each having 4 equally frequent alleles (heterozygosity of 0.75), under the rules of Mendelian inheritance. When creating replicates under linkage, an affection status was modelled based on penetrances of 0.18, 0.48, and 0.78 for carriers of 0, 1, or 2 copies of a susceptibility allele found in 20% frequency at a locus located halfway between the second and third marker. In all cases the tests were evaluated halfway between the second and third marker. The same procedure was repeated for sex-specific maps, where the markers were separated by 9 cM in females and 1 cM in males. Results from 10,000 replicates are shown in Table ij, where allele i is received from the mother, are given in Table  or  rather than Zlr on the full sibs, unless the imprinting effect is small. Power was also evaluated by simulating the sex-specific maps described above , and the results are essentially the same. Statistics similar to  and  can be obtained with ALLEGRO v1.2 [For illustration, the power of the tests was computed under two genetic models using the same simulation design as above. The disease locus has two alleles, labelled 1 and 2, with allele 2 conferring susceptibility, found in 20% frequency in the population. Penetrances for the ordered genotype GRO v1.2 ,7, which = 3.36; p = 0.00038), as well as evidence of imprinting . The significance of the latter result was confirmed by use of the permutation procedure described in Methods; out of 5,000 random replicates that preserve linkage, 1.6% gave an absolute value of  greater than or equal to 2.36 . Approximately 5 cM away from GATA165G02 lies OPRM1 (opioid receptor \u03bc-1), a candidate gene for substance dependence [Table pendence ,11.a priori, because without linkage the test has no power. Given prior evidence, the imprinting tests are moreover not bound to small genome-wide significance levels. This is a significant advantage because the power to detect imprinting, seemingly bounded from above by the power to detect linkage, can thus also be quite low for small genome-wide significance levels. The presence of a candidate gene in the 6q25.2 region provides additional strength to the significance of the test of imprinting.Interpretation of tests of imprinting is best if linkage with a susceptibility gene is suspected Spairs was used as the core statistic, other statistics such as Sall [Sad [Although  as Sall  or Sad [all [Sad  (a statiThe statistical properties of a simple procedure to test for the presence of parent-of-origin effects were evaluated. Simulations illustrated that the test of imprinting in the presence of linkage is biased under the null when differences exist between male and female genetic maps, irrespective of the map used in the analysis. We found evidence that the 6q25 region may harbor an imprinted susceptibility gene for alcoholism in males.COGA: Collaborative Study on the Genetics of Alcoholism"}
+{"text": "We compared linkage analysis results for an alcoholism trait, ALDX1 (DSM-III-R and Feigner criteria) using a nonparametric linkage analysis method, which takes into account allele sharing among several affected persons, for both microsatellite and single-nucleotide polymorphism (SNP) markers (Affymetrix and Illumina) in the Collaborative Study on the Genetics of Alcoholism (COGA) dataset provided to participants at the Genetic Analysis Workshop 14 (GAW14). The two sets of linkage results from the dense Affymetrix SNP markers and less densely spaced Illumina SNP markers are very similar. The linkage analysis results from microsatellite and SNP markers are generally similar, but the match is not perfect. Strong linkage peaks were found on chromosome 7 in three sets of linkage analyses using both SNP and microsatellite marker data. We also observed that for SNP markers, using the given genetic map and using the map by converting 1 megabase pair (1 Mb) to 1 centimorgan (cM), did not change the linkage results. We recommend the use of the 1 Mb-to-1 cM converted map in a first round of linkage analysis with SNP markers in which map integration is an issue. Using single-nucleotide polymorphisms (SNP) for linkage analysis is a relatively new strategy. With only two alleles, the homozygosity rate for SNP markers is high. Homozygous parents are not informative for linkage. SNP-based linkage analyses using a single SNP may not be as successful as linkage analyses using microsatellite markers. However, SNP-based linkage analyses may work better when several neighboring markers are combined together . These SNP combinations can be considered as one composite marker with more alleles . HoweverEven though on a single marker basis SNP markers are not as informative as microsatellite markers, technological advantages have pushed SNP-based linkage analysis forward and SNPs can now be used both for linkage and association studies. This advance is possible because SNPs are relatively inexpensive, have high-throughput production, and dense coverage of the genome. The Affymetrix 10 k chip ,4, for eComparisons between linkage analyses using SNPs and microsatellite markers have recently been reported in the literature . This woThe 143 families  were included in the pedigree data, though only roughly 1,300\u20131,400 people are genotyped with microsatellite markers, Affymetrix SNP, or Illumina SNP markers (the exact number of genotyped samples is given in the papers describing the data provided to GAW participants). The numbers of microsatellite markers, Illumina SNP markers, and Affymetrix SNP markers are 315 , 4,596 , and 10,805 . We used the same set of individuals for the microsatellite marker, Illumina SNP, and Affymetrix SNP genome scans.The phenotype we used was ALDX1, where affectation includes both DSM-III-R alcohol dependence and Feighner alcohol dependence. We coded an individual as affected if he or she was an alcoholic (ALDX1 = 5), as unaffected if they never drank or if they were unaffected with some symptoms , and as unknown if there was no information about their symptoms (ALDX1 = 0).MERLIN  is curreWe ran MERLIN twice on the microsatellite markers: once for single-point analysis and again for multipoint analyses. MERLIN was run twice for the Illumina SNP data also. The first time we used a genetic map that was converted from physical (base pair) position information (1 Mb is converted to 1 cM); and the second time we used the genetic map position provided by GAW14, but manually added an arbitrary small distance when two or more SNPs had the same position in centimorgans. MERLIN was run three times for the Affymetrix SNP data: 1) using the genetic map with the 1 Mb-to-1 cM conversion, 2) using the genetic map given by the GAW14, and 3) using the genetic map given by Affymetrix . We investigated the 1 Mb-to-1 cM map conversion because there may be other cases in which the genetic map information is not available. We were interested to see whether this simple method was successful with the COGA data.MERLIN (version 0.10.2) was run with 24 bits (a measure of the pedigree complexity), on a computer with 8 gigabytes of memory. Even with this upper memory limit, 9 pedigrees  were skipped by MERLIN. We used the same program, and the same individuals in the sample, for all analyses. As a result, the same exact set of 134 families  was analyzed in each case. Even with extended computer hardware and a moderately restricted sample, for an average size chromosome, the computing time was in excess of 2 to 3 hours for each SNP dataset. The total computing time for results included in this paper was in the range of 300 hours.10 curve with the p-value from the NPLall test is very similar to Figures 10 roughly equal to the maximum value of LOD plus 1 .Figures Figure Figure There are three sets of KC-LOD scores shown in Figure To compare linkage analyses from different types of markers, we plot the 3 sets of KC-LOD results for chromosome 7 in Figure This study addresses two questions: first, are the results consistent when comparing microsatellite marker and SNP-based linkage analyses? Second, for SNP-based linkage analysis, can the 1 Mb-to-1 cM genetic map be used when only the physical location is provided for the SNP markers instead of genetic map distance? The answer to the first question seems to be that the two linkage results match on a crude level, but do not match on a finer level. For all three sets of marker data, the highest linkage peaks appear in chromosome 7. This indicates that on the genome-wide level, microsatellite and SNP markers do lead to consistent results. On the other hand, for intermediate linkage peaks , three sets of marker data do not match. For example, a peak on chromosome 12 for Illumina and Affymetrix SNP data does not appear in the microsatellite data.One strategy to explain the discrepancy between the two sets of markers is to examine the information content. Evans et al.  simulateThe answer to the second question seems to be that we can indeed use the simple 1 Mb-to-1 cM rule to generate a genetic map for linkage analysis. Using the 1 Mb-to-1 cM conversion rule is equivalent to the assumption that the recombination rate was homogeneous along a chromosome, which is known to be unrealistic. The reason that this simplistic map is still able to reproduce the linkage result based on a more detailed genetic map is perhaps that the inter-marker distances are relatively short. If we consider Affymetrix's 10 k chip for example, as 10,000 SNP covers the whole human genome with 3000 Mb, the inter-marker distance is on average 300 kb, or roughly 0.3 cM. Even with a large variation in local recombination rate, the range of the absolute value of the inter-SNP distance is small. Our findings showed that the cM/Mb = 1 rule may prove to be a useful tool in situations when the genetic marker map is missing or incomplete.In conclusion, we have carried out genome-wide NPL multipoint analyses seven times using both microsatellite and Illumina or Affymetrix SNP markers, on the same pedigree data with identical affection status definition. The linkage signals obtained from the Illumina SNP data and Affymetrix SNP data are more similar than those obtained from the microsatellite markers. However, all three datasets point to chromosome 7 as having the strongest linkage signal. We also compare linkage analyses with SNP markers using the given genetic map and using the map derived from the simple rule of 1 Mb-to-1 cM. We showed that the linkage signals obtained from these two genetic maps are essentially the same.COGA: Collaborative Study on the Genetics of AlcoholismGAW14: Genetic Analysis Workshop 14KC-LOD: Kong-Cox LODNPL: Nonparametric linkageSNP: Single-nucleotide polymorphismsAU conducted the MERLIN runs on the microsatellite data, and WL conducted the MERLIN runs on SNP markers. Both authors read and approved the final manuscript."}
+{"text": "We analyzed the Genetic Analysis Workshop 13 (GAW13) simulated data to contrast and compare different methods for the genetic linkage analysis of hypertension and change in blood pressure over time. We also examined methods for incorporating covariates into the linkage analysis. We used methods for quantitative trait loci (QTL) linkage analysis with and without covariates and affected sib-pair (ASP) analysis of hypertension followed by ordered subset analysis (OSA), using variables associated with change in blood pressure over time.Four of the five baseline genes and one of the three slope genes were not detected by any method using conventional criteria. OSA detected baseline gene b35 on chromosome 13 when using the slope in blood pressure to adjust for change over time. Slope gene s10 was detected by the ASP analysis and slope gene s11 was detected by QTL linkage analysis as well as by OSA analysis. Analysis of null chromosomes, i.e., chromosomes without genes, did not reveal significant increases in type I error. However, there were a number of genes indirectly related to blood pressure detected by a variety of methods.We noted that there is no obvious first choice of analysis software for analyzing a complicated model, such as the one underlying the GAW13 simulated data. Inclusion of covariates and longitudinal data can improve localization of genes for complex traits but it is not always clear how best to do this. It remains a worthwhile task to apply several different approaches since one method is not always the best. In our analysis of the Genetic Analysis Workshop 13 (GAW13) simulated data for Replicate 1 with no missing data we had two goals: 1) use the longitudinal information to improve our linkage analysis and 2) incorporate covariates in our linkage analysis. At present, there are no standard tools for linkage analysis of longitudinal data. In addition, the incorporation of covariates in linkage analysis is a very active area of current methodological development. As a result there was no \"industry standard\" analysis for either of our goals. Consequently, we explored several approaches to detect the simulated genes. We opened the answers at the beginning and used the answers extensively to evaluate our approach.We investigated different approaches for handling data from repeated measurements, in this case multiple phenotype values collected at different time points. We used subject-specific slope values to represent change in systolic blood pressure (SBP) over time and considered the effects of adjusting slopes for potentially important covariates before the linkage analysis. We used these slopes directly as traits in quantitative analysis, and as covariates in linkage analysis of the hypertension as a binary affection status variable. We chose change over time in SBP as the quantitative trait for SOLAR multipoint linkage analysis, and used the presence of hypertension (HBP) to define a binary affection status for affected sib-pair (ASP) multipoint linkage analysis with SIBLINK . A recenA key issue in our investigation involved determining which covariates to use and at what point in our analyses to adjust for them. We were interested in examining how inclusion of all covariates in a general model and subsequent covariate elimination using a variance-component analysis method, SOLAR, compared with analysis with one covariate at a time using a method like OSA, in conjunction with ASP linkage analysis. A related question was whether covariates should be included at a family or individual level. With OSA, the mean covariate values are taken over all time points for each individual and then averaged at the family level. We examined how this approach might differ from the inclusion of individual means for all covariates in a mixed model using a variance-components approach.We used only one replicate in our analysis. Our initial goals were implementation of the analysis plan and a limited consideration of whether our analysis methods could detect any of the genes involved in change in blood pressure over time. An analysis of all replicates and further simulation studies are required to begin to make statements about the general performance of covariate-adjusted linkage methods.We used the GAW13 simulated complete data set for Replicate 1 consisting of 2860 genotyped individuals in 330 families. The data set was examined using three genetic analysis programs: SIBLINK [Subject-specific slopes reflecting individual change over time of SBP were computed with SAS-PROC MIXED  using thVariance components analysis (SOLAR) was used to perform multipoint linkage analysis using the normalized individual slope values for SBP  as the quantitative trait values. All families were included in this analysis. A common constant was added to make all estimated slope values positive, then the data were log-transformed and outliers were eliminated to approximate a normal trait distribution. Heritability was estimated from the best-fitting polygenic model, starting with a model that included age  and average values  of cholesterol, glucose, HDL, triglycerides, height, weight, and body mass index (BMI). Once this model was generated, multipoint linkage analysis using a variance components approach was carried out using SOLAR.p-value based on randomly permuting the order in which families were added.SIBLINK v. 3.0 was used to perform multipoint ASP linkage analysis using HBP as indicated in the data set as a dichotomous disease trait. There were 171 families with at least one ASP included in our SIBLINK analysis, for a total of 575 ASPs with hypertension. SIBLINK was used to calculate family-specific multipoint LOD scores across each chromosome, based on estimated identity by descent (IBD) status among affected sibling pairs. The OSA program then utilized the multipoint LOD scores and covariate values from each family, attempting to identify homogeneous subsets of families presenting increased evidence for linkage. In theory, examining the data from a homogeneous subset of linked families yields a more accurate estimate of disease gene location because the location estimate is not influenced by unlinked families. The OSA program takes as input, any multipoint set of additive, family-specific linkage scores, such as LOD scores from SIBLINK or nonparametric LOD scores from GENEHUNTER Plus (Kong and Cox). The OSA program ranked families by mean values of the covariates: the calculated slope values for SBP and additional covariates age , cholesterol, glucose, HDL, triglycerides, height, weight, and BMI. Family-specific means were calculated i) for all affected individuals and ii) for all family members regardless of affection status . Using one covariate at a time, family-specific multipoint LOD scores were added in the covariate-based rank order and the maximum LOD score for any subset of families was determined for that covariate. The significance of the increase in the subset-based LOD score over the global LOD score from all families was assessed with an empirical The linkage results for slope variables compared with the known locations of simulated SBP genes for chromosomes 5, 7, 13, 15, and 21 are shown in Table p-value < 0.05), we did not observe any linkage signals within 20 cM of any baseline SBP genes on chromosomes 5, 7, or 13 with ASP combined with ordered subsets analyses (SIBLINK/OSA) using the mean of normalized slope values for all family members or using variance component analysis on the calculated normalized SBP slope values. OSA was able to identify gene s11 on chromosome 15 . Not surprisingly, since s10 exerts a relatively strong effect on the variance of SBP over time, our methods were most successful at identifying slope gene s10 on chromosome 21 . Interestingly, non-normalized slope values used in SOLAR tended to have higher heritability estimates than normalized slopes , although the increase in the heritabilities did not automatically lead to larger LOD scores. The analysis of non-normalized adjusted slope (H2 = 44.5%) found s11 on chromosome 15 with LOD = 1.59 and s10 on chromosome 21 with LOD = 6.80. We analyzed Replicate 7 of the simulated data and confirmed that the non-normalized values do appear to give stronger results than the normalized values in that replicate as well.Using standard criteria for identifying regions of interest . In addition, ordered subset analysis detected b3  can lead to successful localization if the QTL is bi-allelic, has a relatively strong effect on the mean of the distribution, and if the genotype-specific means are close to an additive model, which is assumed in ASP analysis using SIBLINK. Incidentally, this was also the situation where variance component analysis using SOLAR performed best (chromosome 21).p-values for the increase in the OSA LOD scores in this region are below 0.05. We also analyzed simulated Replicate 7 to see if any of the patterns observed in Replicate 1 were observed in another replicate. Qualitatively the localization results were similar in that other genes were detected, however, we did not observe the same genes as those in Replicate 1.OSA appears to be useful in localization of complex trait genes, but it can also produce peaks for genes indirectly related to the trait of interest, particularly when there are multiple genes on the same chromosome that are correlated with the analyzed trait in a complex way. It is possible that our detection of the height genes is due to high heritability of height or strong correlations with other variables. The answers reveal that height is indirectly related to SBP through its direct contributions to other variables. When concentrating on a single maximum for the gene location, we did not detect any baseline genes for SBP with our approach. However, when there is more than one gene on a chromosome, localization of any single gene may be more difficult. For example, on chromosome 13, a high LOD score of 3.2 obtained from OSA occurs around 118 cM (near the height gene b3 at 120 cM), and 33 cM away from the SBP baseline gene b35 at 85 cM. The There is considerable methodological work to be done to provide insight into how proximity of other genes may influence detection and localization of any individual gene. We were pleasantly surprised that we were able to obtain results for any of the baseline genes, since our analysis was weighted toward detecting slope genes. The OSA results for chromosome 13 Figure  suggest We are able to conclude from our analyses that inclusion of covariates and longitudinal data can improve localization of genes for complex traits but it is not always clear how best to do this. The information used by each method is slightly different and thus it is not at all surprising that the different methods pick up different genes in this complex system. Thus we conclude that it remains a worthwhile task to apply several different approaches since one method is not always the best."}
+{"text": "Studies of model-based linkage analysis show that trait or marker model misspecification leads to decreasing power or increasing Type I error rate. An increase in Type I error rate is seen when marker related parameters  are misspecified and ascertainment is through the trait, but lod-score methods are expected to be robust when ascertainment is random (as is often the case in linkage studies of quantitative traits). In previous studies, the power of lod-score linkage analysis using the \"correct\" generating model for the trait was found to increase when the marker allele frequencies were misspecified and parental data were missing. An investigation of Type I error rates, conducted in the absence of parental genotype data and with misspecification of marker allele frequencies, showed that an inflation in Type I error rate was the cause of at least part of this apparent increased power. To investigate whether the observed inflation in Type I error rate in model-based LOD score linkage was due to sampling variation, the trait model was estimated from each sample using REGCHUNT, an automated segregation analysis program used to fit models by maximum likelihood using many different sets of initial parameter estimates.The Type I error rates observed using the trait models generated by REGCHUNT were usually closer to the nominal levels than those obtained when assuming the generating trait model.This suggests that the observed inflation of Type I error upon misspecification of marker allele frequencies is at least partially due to sampling variation. Thus, with missing parental genotype data, lod-score linkage is not as robust to misspecification of marker allele frequencies as has been commonly thought. In model-based linkage analysis, one assumes that the causative mechanisms of both the trait and marker phenotypes are known without error, including the number of loci involved, the mode of inheritance and allele frequencies. With the advancement of genetic research in complex traits, we are dealing more with uncertain mode of inheritance and unknown parameters  which ought to be known to apply model-dependent linkage analysis appropriately. Several studies have been reported in the literature about the implications of misspecifying the underlying genetic model of the trait in linkage analysis. Clerget-Darpoux et al.  quantifiIn previous studies, ,8 the poComputer simulations generating a quantitative trait and marker data in nuclear families were performed using G.A.S.P. V3.3 . The traLod-score tests of linkage between the trait and both linked and unlinked marker loci were performed using LODLINK  on each The same simulated data were reanalyzed using LODLINK as described above except that instead of assuming the \"true\" simulation model, parameters estimated by segregation analysis of the trait data were assumed for the trait model. The segregation analysis was performed on each replicate sample using REGCHUNT , an autoFor both methods of analysis, power was determined as the proportion of 10,000 samples in which analysis of a marker linked to the trait produced maximum LOD scores equal to or exceeding thresholds corresponding to nominal significance levels of 0.01, 0.001 and 0.0001. Type I error rate was determined in a similar manner based on analysis of the unlinked marker, using nominal significance levels of 0.01 and 0.001, since 10,000 replications are not adequate to accurately estimate Type I error at the 0.0001 level. Note that commonly used lod-score thresholds of 1.0, 2.0 and 3.0 correspond to p-values of approximately 0.0159, 0.0012, and 0.0001, respectively ,14.The power of lod-score linkage analysis of a linked marker (recombination fraction 0.01), using the \"correct\" generating model for the trait, is presented in Figure Where the power using the generating trait model showed an extreme inflation (or deflation) due to the misspecification of marker allele frequencies in the analysis, inflated (or deflated) power to detect linkage in the same situation was also observed using the estimated trait model from REGCHUNT. However, the extent of inflation (or deflation) was less than when using the generating trait model, except in some cases when inflation was limited by the upper bound of 100%. For significance levels 0.01 and 0.001, the power was always lower, compared to analyses assuming the true (generating) trait model (data not shown). However, the reverse was frequently true for significance level 0.0001. For example, when \"true\" marker allele frequencies of 0.001, 0.24975, 0.24975, 0.24975, and 0.24975 were misspecified as 0.1, 0.225, 0.225, 0.225, and 0.225, respectively, the power increased from 53% to 62%. Results are presented in Figure As the power of lod-score linkage analysis using the \"correct\" generating model for the trait was found often to increase substantially when the marker allele frequencies were misspecified, nominal type I error rates were investigated. Observed Type I error rates based on linkage tests at the unlinked marker are presented in Figure When the most common allele(s) were misspecified as even more common in the analysis, the observed Type I error rate tended to decrease slightly, unless the misspecification was relatively large.As shown in Figure When performing lod-score linkage analysis of a randomly sampled quantitative trait with missing parental marker information, the power of lod-score linkage analysis often appears to increase as the marker allele frequencies are misspecified even when the \"correct\" generating model for the trait is used in the analysis. The effect of misspecification on power is more pronounced when the nominal significance level is more significant. The apparent increase in power appears to be due at least in part to an increase in Type I error rates. A severe increase in the Type I error rate was observed for lod-score linkage analysis in a sample size of 300 independent sibpairs without parental data when marker allele frequencies were misspecified in the analysis by underestimating the most common marker allele. The inflation may be caused by sampling variation resulting in some extreme samples for which the \"true\" generating model is not a good fit. It is also possible that this inflation in Type I error rate is observed because these sample sizes were not large enough to display asymptotic properties. Different proportions of available parental information have a significant effect on the power of lod-score linkage analysis depending upon the sample sizes . OmissioIn an attempt to investigate the effect of misspecification of marker allele frequencies on trait heritability, similar analysis was done on a simulated trait with 50% heritability due to an additive major locus with two equifrequent alleles and the other one due completely to a random environmental effect. LODLINK was used to test for linkage in each of 10,000 replicates of 300 families with sibship size two. When the trait was completely environmentally determined, the Type I error rate of the lod-score linkage analysis was found to be robust to misspecification of marker allele frequencies, even if the allele frequencies were severely misspecified (\u00b1 0.8 of true frequencies). However, for the trait with heritability of 50%, the Type I error rate was sometimes as much as 21 times the nominal significance level of 0.001 when the allele frequency of a common allele in a five-allele polymorphic marker was misspecified in the analysis as rare (data not shown). In general, the Type I error rates for the trait with a heritability of 90% were the highest among the three traits studied. Thus, when the parental genotype data are missing, the model-based lod-score test of linkage is robust with respect to Type I error for environmentally determined traits but not for the traits which are largely genetically determined. The results for the trait with heritability of 50% were similar to that of the trait heritability of 90% but less dramatic [In an attempt to resolve the problem of inflated Type I error rates, the model was estimated from each sample using REGCHUNT followed by linkage analysis with LODLINK. The data obtained show that the observed power and Type I error rates of model-based LOD score linkage are often inflated when marker allele frequencies are misspecified, even when using this analytic strategy. However, the Type I error rates observed in this case are usually closer to the nominal levels than those obtained when assuming the generating trait model. This suggests that the observed inflation of Type I error upon misspecification of marker allele frequencies is at least partially due to sampling variation. However, sampling variation is inadequate to completely explain the pattern of inflation of Type I error rates due to marker allele misspecification. The observed Type I error rates are possibly due to not having asymptotic sample sizes. The effect of sibship size is expected to be small relative to the effect of locus heterozygosity on the ability to infer missing parental genotypes from sibship data. If there are two alleles in a marker, no sibship size can determine with certainty the parental mating type. On the other hand, parental mating type can often be determined with certainty  in a sibship of size two if there are four or more alleles. From our findings, we have observed that, for two-allele markers, sib-pair sample sizes of 100 and 300 with sibships of size five  always produce a better power than sib-pair sizes of 100 and 300 with sibships of size two  when \"correct\" allele frequencies are used in the analysis. However, as we increased the number of marker alleles to five, the power using 300 sib-pairs with sibships of size five  is not always better than the power achieved using 300 sib-pairs with a sibship size of two . However, when only 100 sibpairs are available for study, sibships of size five  yield better power than sibships of size two  even with a five-allele marker, since power is not optimal for a sample size of only 100 sib-pairs, so the extra power that is obtained by being able to use the multiple sibs in a large sibship to impute the missing parental genotype is important. In general, an increase in the marker heterozygosity leads to more complete inference of parental genotypes . The powIt is a common practice in simulation studies to use the generating model as the \"best\" model. The data from the present study suggest that the estimated model generally performs better, with respect to Type I error, than the generating model. Therefore, we need to use caution in interpreting simulation study results obtained using the generating model. These results also suggest that in order to avoid spurious inferences of linkage when performing linkage analysis of randomly ascertained quantitative traits with missing parental data, a very large sample size is needed. Moreover, both the trait model parameters and marker allele frequencies should be estimated from the sample data for randomly ascertained quantitative traits to attempt to reduce the false positive rates.A thorough investigation was previously conducted on power and Type I error using the model-free Haseman-Elston (H-E) sibpair linkage method ,16. PoweThe estimates of marker allele frequencies are irrelevant to linkage analysis when marker genotypes are known for all family members. However, if parental genotypes are not available, accurate estimates of allele frequencies for highly polymorphic DNA marker loci with high heterozygosities are often not available in the underlying study population. If the genotypes at a marker locus are not known and cannot be imputed for a family member, then inaccurate estimates of marker allele frequencies may lead to false positive evidence for linkage if model-based linkage analysis is used. However, if a quantitative trait is well characterized and marker allele frequencies are accurately estimated the type I error rate for the model-based linkage method is not substantially greater than the nominal rate.In conclusion, the model-based lod-score method of linkage analysis is not robust to misspecification of marker allele frequencies when the families are randomly ascertained and parental marker data are missing, especially when the true trait model is assumed, rather than one estimated from the data. There is some indication, however, that a slight overestimate of the most common marker allele would be safer to use than an underestimate.DMM participated in the design of the simulation studies, conducted the simulations, interpreted the results and drafted the paper. AJMS participated in the programming, interpretation and helped to draft the paper. LDA gave his expert comments on REGCHUNT. AFW provided his simulation software and helped in the design of the studies. JEBW participated in the design of the simulation studies, interpretation and provided useful suggestions. All authors read and approved the final manuscript."}
+{"text": "High triglycerides (TG) and low high-density lipoprotein cholesterol (HDL-C) jointly increase coronary disease risk. We performed linkage analysis for TG/HDL-C ratio in the Framingham Heart Study data as a quantitative trait, using methods implemented in LINKAGE, GENEHUNTER (GH), MCLINK, and SOLAR. Results were compared to each other and to those from a previous evaluation using SOLAR for TG/HDL-C ratio on this sample. We also investigated linked pedigrees in each region using by-pedigree analysis.Fourteen regions with at least suggestive linkage evidence were identified, including some that may increase and some that may decrease coronary risk. Ten of the 14 regions were identified by more than one analysis, and several of these regions were not previously detected. The best regions identified for each method were on chromosomes 2 , 5 , 7 , and 22 . By-pedigree multi-point LOD values in MCLINK showed linked pedigrees for all five regions, ranging from 3 linked pedigrees (chromosome 5) to 14 linked pedigrees (chromosome 7), and suggested localizations of between 9 cM and 27 cM in size.Reasonable concordance was found across analysis methods. No single method identified all regions, either by full sample LOD or with by-pedigree analysis. Concordance across methods appeared better at the pedigree level, with many regions showing by-pedigree support in MCLINK when no evidence was observed in the full sample. Thus, investigating by-pedigree linkage evidence may provide a useful tool for evaluating linkage regions. Obesity, diabetes, and hypertension are closely associated with low levels of high-density lipoprotein cholesterol (HDL-C) and elevated levels of triglycerides (TG), and are recognized as jointly increasing coronary risk . These fIn an evaluation of a genetic component of the metabolic syndrome, Shearman et al.  reportedIn contrast, parametric linkage analysis requires specification of the underlying model of genetic inheritance. These models are usually unknown and must be estimated. Commingling analysis can be used to estimate genetic parameters from the phenotypic data. Although parametric analysis requires model specification, it can provide statistical power beyond that of model-free analyses . Three lLINKAGE calculates exact two-point IBD probabilities for use in two-point linkage analysis, and can be used for pedigrees of arbitrary size. Two-point analysis is less sensitive to misspecification of the model parameters than multi-point, since these are absorbed into the maximization over the recombination fraction, \u03b8. For a detailed description see G\u00f6ring and Terwilliger . Two-poiParametric analysis in GENEHUNTER (GH) also is an exact likelihood method. Multi-point LOD analysis that is less sensitive to inaccurate allele frequencies and is superior at determining IBD probabilities can be calculated; however, multi-point LOD statistics are constrained for \u03b8 = 0 and thus can be prone to false-negative results (loss of power) if the model parameters are misspecified. Further, the pedigree size capacity for GH is limited and with large pedigrees can require extensive trimming, which can lead to loss in power due to the elimination of important genealogical and segregational information.MCLINK is a Markov chain Monte Carlo (MCMC) method that uses blocked Gibbs sampling to estimate multi-point IBD probabilities on extended pedigree structures. In addition, MCLINK supports a robust multi-point theta-LOD (TLOD) statistic ,10. The Thus, while multi-point analysis methods have become popular, exact multi-point methods cannot evaluate large, extended pedigrees as exact two-point methods can. Estimation methods may circumvent these issues, but may have their own weaknesses. The major objective of this study was to compare the linkage analysis results of LINKAGE, GH, and SOLAR to a potentially more robust parametric method, MCLINK, that incorporates all available pedigree information into the linkage analysis.The data for this study consisted of real FHS data for both the original and offspring cohorts, as provided in Problem 1 of the Genetic Analysis Workshop 13 (GAW13) data set. As noted by Shearman et al. [Prior to linkage analysis, the TG/HDL-C ratio was adjusted by regression for age, body mass index (BMI), systolic blood pressure (SBP), and glucose level because of the definition of the metabolic syndrome in the ATP III guidelines , with BMWe used commingling analysis to determine genetic parameters assuming normal distributions under a major gene model. Commingling analysis revealed significant evidence for two populations with mean TG/HDL-C residuals of 1.26 and -0.08, with common standard deviation 0.56. Gene frequencies of 0.01 and 0.1 were used for the dominant and recessive models. Four parametric models  were analyzed as we were interested in locating susceptibility genes for both low (protective) and high (risk) TG/HDL-C ratios. All LOD and TLOD scores were maximized over the proportion of families linked using heterogeneity LODs (\u03b1).p-value of 0.05. The region size was estimated using the 1-LOD drop from the peak of the summed pedigree LODs (sumLOD) for linked pedigrees only, and also from the observed recombinants. We used the pedigree multi-point graphs to estimate positions of recombinants, and used a drop of 0.5 LOD units to indicate the existence of a recombinant.We used by-pedigree classical multi-point LOD statistics (with \u03b8 = 0) available from MCLINK to assess if a pedigree was linked to a region. Each region was defined as the 20 cM surrounding the peak full sample LOD. A pedigree was considered linked to that region if a pedigree LOD > 0.588 was observed, which corresponds to a nominal General characteristics of the study population are given in Table For the parametric analyses, regions were identified under both the high and low TG/HDL-C ratio models. Analyses evaluating potential loci predisposing to a high TG/HDL-C ratio identified five potential regions on chromosomes 1, 3, 5, and 7 Table . The higParametric analyses to locate predisposition loci for low TG/HDL-C ratio, which could indicate a genetically protective effect, identified seven regions on chromosomes 2, 8, 10, 13, 17, 19, and 22 , 5 , 7 , and 22  using multi-point by-pedigree support as provided by MCLINK. Despite using only MCLINK to identify linked pedigrees, at least three linked pedigrees (LOD > 0.588) were found for each region. For each of the regions analyzed, no conflicting recombinants were evident. Table Several loci were found by multiple analysis methods, including a region on chromosome 17 identified by all methods. This is reassuring, although a large range of LODs was often seen for the same locus. Across all 14 regions identified, only four loci were identified using only a single analysis method , six regions were identified with two methods, three regions were identified with three methods, and one region on chromosome 17 at 126\u2013129 cM was located by all four methods, although only GH identified the region with at least suggestive evidence. This region on chromosome 17 was also the best region observed by Klos et al. [In the analyses here, no single method identified all the regions. MCLINK performed the best in this regard with LODs > 1.0 in 10 of the total 14 regions, and 3 of the 4 regions missed were those identified by only a single method.Shearman et al. , using SIn general, better agreement was found in this study between the parametric methods than with the results from SOLAR, and in particular between LINKAGE and MCLINK. This may be due to the similarity in pedigree structures analyzed. In addition, MCLINK agreed better with GH than it did LINKAGE. The better agreement of GH with MCLINK may be due to the multi-point nature of both analyses. Further, MCLINK obtained better agreement with SOLAR than the other two parametric methods. However, our model-free SOLAR results were more concordant with those of Shearman et al.  than witEven if a much less stringent LOD threshold were imposed on the analyses of these data, no single method used in this study would have identified all 14 regions here. MCLINK identified only moderate-size peaks compared across parametric methods, but located evidence for linkage (LOD > 1.0) for 10 of the total 14 regions, and had the best concordance with each of the other methods, LINKAGE located 8 out of 14 regions, GH 7 out of the total 14, and SOLAR only 4. Of the 7 locations it found, GH found them all with LOD > 1.5. The lower number located by GH may be partly due to loss of pedigree structure.The best four regions identified by each method were followed up with by-pedigree analysis using MCLINK multi-point LODs. Despite the lack of highly confirmatory full sample scores, the by-pedigree support identified at each locus was good (at least three pedigrees linked to each region). This may indicate better consistency of LOD findings at the pedigree level.Other evaluations of linkage for TG and HDL-C phenotypes have been published, in addition to those of Shearman et al.  and KlosThe generally poor concordance between this study's findings and the results of studies other than Shearman et al.  may be dThis study has identified loci linked to TG/HDL-C ratio that may relate to the metabolic syndrome or insulin resistance, including some that may increase coronary risk and some that decrease risk. Further, it indicates that multi-point and two-point analyses, based on exact inheritance probabilities  and estimation methods  IBD, and parametric  and model-free (SOLAR) analyses can all contribute to the identification of loci for complex quantitative traits. Although we attempted no formal comparison using simulations that would enable definitive conclusions about the comparison of methods, these results do provide a valuable practical comparison of approaches. We found reasonable consistency across multiple methods, and suggest that consistency of findings, in addition to LOD peak size, could be used in identifying potential regions. Our results indicate that the MCMC method, MCLINK, behaves as one might expect for a method that has parallels with both two-point full pedigree analyses and limited pedigree structure multi-point analyses, with reasonable correspondence with both methods. Our results further suggest the utility of inspecting by-pedigree support for regions, both to increase confidence and to increase region definition and narrowing."}
+{"text": "Complex disease mapping usually involves a combination of linkage and association techniques. Linkage analysis can scan the entire genome in a few hundred tests. Association tests may involve an even greater number of tests. However, association tests can localize the susceptibility genes more accurately. Using a recently developed combined linkage and association strategy, we analyzed a subset of the Collaborative Study on the Genetics of Alcoholism (COGA) data for the Genetic Analysis Workshop 14 (GAW14). In this analysis, we first employed linkage analysis based on frailty models that take into account age of onset information to establish which regions along the chromosome are likely to harbor disease susceptibility genes for alcohol dependence. Second, we used an association analysis by exploiting linkage disequilibrium to narrow down the peak regions. We also compare the methods with mean identity-by-descent tests and transmission/disequilibrium tests that do not use age of onset information. The Collaborative Study on the Genetics of Alcoholism (COGA) is a large, multi-site genetic study to identify susceptibility genes for alcohol dependence and related phenotypes . The COGAge of onset data are often collected in studies designed to map a complex disease. If age at onset is genetically mediated, it may carry useful linkage information. Genetic analysis that incorporates variable age of onset may improve the ability to map genes for complex diseases. In this report, we analyzed the COGA data using genetic methods based on additive genetic gamma frailty models to account for age of onset or covariate information ,4.n sibs. Let Tj be the random variable of age at disease onset for the jth sib. Let  be the observed data where tj is the observed age at onset if \u03b4j = 1, and age at censoring if \u03b4j = 0. Consider a marker locus d in the test chromosomal region. We assume that the hazard function of developing disease for the jth individuals at age tj is modelled by the proportional hazards model with random effect Zj,Consider a sibship with j(tj|Zj) = \u03bb0(tj)exp(Xj\u03b2)Zj, for j = 1, 2, ..., n,\u03bb0(t) is the unspecified baseline hazard function, and Xj is a vector of observed covariates for the jth sib and \u03b2 is a vector of regression parameters associated with the covariates. Zj is the unobserved genetic frailty. The genetic frailty is defined as the followingwhere \u03bbVd =  is the inheritance vector [d, vj - 1 2= 1 or 2, and vj 2= 3 or 4 to indicate the origins of the inherited alleles for j = 1, 2, ..., n. Ud1 and Ud2 represent the genetic frailties due to part of the genome on the two chromosomes of the father at locus d. Ud3 and Ud4 represent the same, though for the mother. The random frailty term, Up, takes into account possible genetic contributions to shared familial effects. Gamma distributions were used to model the frailties and retrospective likelihood ratio tests were constructed for linkage analysis [where e vector  of a sibThe dataset includes a total of 143 nuclear and multigenerational families with 1,614 individuals. There are two kinds of diagnostic definitions of alcohol dependence, labelled in the data set as ALDX1 and ALDX2. ALDX1 alcohol-dependent subjects were defined as those individuals who met both the DSM-III-R  criteria for alcohol dependence and the Feighner criteria for alcoholism. ALDX2 alcohol-dependent subjects were defined as those who met the DSM-IV criteria. Both ALDX1 and ALDX2 alcohol dependence phenotypes are coded in four levels: pure unaffected, never drank, unaffected with some symptoms, and affected. We combined the first three codings as unaffected. We extracted genotyped affected sib pairs with their parents from each pedigree to ensure independence between nuclear families. This yielded 142 affected sib pairs with their parents for a total of 568 individuals for the ALDX1 phenotype for alcoholism. For the ALDX2 phenotype, this yielded 117 affected sib pairs with their parents for a total of 468 individuals. We utilized MAPMAKER/SIBS linkage program [p-value < 0.01) to some candidate genes is established, we further applied the association method [For each of the two disease classifications, ALDX1 and ALDX2, we first performed linkage analysis over the whole genome, excluding the sex chromosome, using the methods of Li and Zhong , adjustin method  as well n method  to singlp-value of 0.006), 161 cM of chromosome 6 , and 157 cM of chromosome 12 . Evidence of linkage to ALDX2 was found in the regions around 128 cM of chromosome 7 , 14 cM of chromosome 8 , 169 cM of chromosome 6 , 5 cM of chromosome 2 , and 148 cM of chromosome 2 .A preliminary multipoint genome scan using mean IBD tests (figures not shown here) indicated some evidence of linkage to ALDX1 in the regions around 145 cM of chromosome 7  data was found between rs273954 and rs727714 on chromosome 7; for ALDX2, evidence was found for rs12142 and rs951149 on chromosome 4, and rs1705748, rs1705772, rs1843910, rs1846629, and rs1820545 on chromosome 12.For the association tests in the presence of linkage, we present here only the SNPs within a 10-cM vicinity of the two linkage peaks with smallest p-values reported here are not corrected for multiple comparisons.Although the markers identified under the two criteria, ALDX1 and ALDX2, are not entirely the same, several common regions were identified. The similarity of the regions supports some common genetic bases for ALDX1 and ALDX2, whereas the differences in the identified regions underscore the importance of using different phenotypes. As studied in ,4, the mCOGA: Collaborative Study on the Genetics of AlcoholismGAW: Genetic Analysis WorkshopIBD: Identical by descentSNP: Single-nucleotide polymorphismXZ conceived of the study, carried out the genetic studies, performed the statistical analysis and drafted the manuscript. HZ drafted the manuscript, revised it critically for important intellectual content, and gave final approval of the version to be published. All authors read and approved the final manuscript."}
+{"text": "In addition, the anecdotal observation that The applied pseudo-trait (APT) method was applied to the Affymetrix and Illumina SNPs in the Collaborative Study on the Genetics of Alcoholism dataset to determine appropriate critical values for regression of offspring on mid-parent (ROMP) and Haseman-Elston association and linkage analyses, investigating the occurrence of type I errors in different chromosomal locations, and the extent to which the critical values obtained depend on the type of pseudo-trait used.p-values does not seem to depend on chromosomal position for ROMP association analysis methods, but does in some cases for Haseman-Elston linkage analysis. Results vary with different pseudo-traits.On average, the 5 percentile critical values obtained for this study were less than the expected 0.05. The distribution of  The vast majority of markers in a genomic screen for linkage or association are not linked or associated with the trait being analyzed. This observation led to the applied pseudo-trait (APT) method ,2, a metThis method can also be used to investigate so-called \"spurious\" peaks that occur at the ends of chromosomes. A number of anecdotal accounts by our group and others suggest that values near the telomeres tend to be inflated . This observation has been noted in both two-point and multipoint linkage analysis. In this study we used APT to determine the distribution of the type I errors across chromosomes.Three objectives were addressed: 1) to determine the appropriate critical values for the Affymetrix and Illumina single-nucleotide polymorphism (SNP) datasets through the use of the APT method for linkage analysis (with the revised Haseman-Elston method) and association analysis (with variations of the regression of offspring on mid-parent (ROMP) method); 2) to determine the validity of the observation that the type I error rate may vary with respect to chromosomal location; 3) to determine to what extent the critical values generated is dependent on the type of pseudo-trait used.p arms of chromosomes 13\u201315 and 21\u201322, leaving 39 pseudo-trait markers. For each of these markers, an allele-count pseudo-trait was defined as the number of occurrences of allele 2 at that marker. Another type of pseudo-trait was created by adding a random quantity drawn from a standard normal distribution to the allele count. In addition, two randomly generated pseudo-traits were also created: one from a standard normal distribution and one from a uniform distribution. A total of 80 pseudo-traits were considered.Several different pseudo-traits were considered for use with the Collaborative Study on the Genetics on Alcoholism (COGA) dataset. For each chromosomal arm, a SNP was randomly chosen from a region excluding centromeric   and teloFor every combination of pseudo-trait and SNP marker, linkage analysis was performed using the revised Haseman-Elston method with single-point identity-by-descent (IBD) sharing probabilities and using the mean-corrected cross-product as the dependent variable, as implemented in S.A.G.E. 4.5 . The tesp-Values resulting from all SNP tests were determined for each pseudo-trait. For pseudo-traits based on a SNP marker, tests with syntenic markers were excluded, so that null hypothesis conditions were maintained. For each chromosome, the set of p-values obtained was considered as a single unit and also broken down by location on the chromosome . Each chromosome end (p term and q term) was defined as 10% (or 25%) of the chromosome, according to physical map distance. Alternative definitions of chromosomal ends using a fixed distance of 3 MB (Affymetrix) and 7 MB (Affymetrix and Illumina) were also investigated. For some chromosome arms, too few SNPs  were available in the terminal segment, and those segments were omitted from the partitioned segment summaries. For each segment and for the whole genome, descriptive statistics were obtained using SAS version 8.2 [p term v. middle and q term v. middle).To investigate the possible variation in type I error rate between telomeric and non-telomeric regions, means of each segment were computed separately. In addition, traditional analysis of variance and Kolmogorov-Smirnov two-sample tests were performed separately to compare each end with the middle segment . However, comparisons for Haseman-Elston linkage analysis did show significant results for some pseudo-traits. For example, using the decile segment definition, 41 of 80 pseudo-traits showed significant (at the 0.01 level) differences between the q term segment means and the middle segment means, according to both analysis of variance and Kolmogorov-Smirnov tests. P-values for different pseudo-traits ranged from 0\u20130.8, with a mean of 0.1.Analysis of variance and Kolmogorov-Smirnov tests  comparing Fifth percentile values for different types of pseudo-traits are summarized in Table Similar results were observed for the Illumina dataset.p-value distribution was, on average, less than 1 standard deviation below 0.05 for three of the ROMP methods , the fifth percentiles for the ROMP/ROOP method using all the sibs and the revised Haseman-Elston method were less than expected. This suggests that these methods may be liberal when used with nominal p-values. Alternatively, one could use a critical value derived with the APT method, but the range of empiric critical values obtained in this fashion is large.The determination of critical values with the APT method resulted in a large range of values. Overall the fifth percentile critical values obtained were less than the expected 0.05 level. Although the fifth percentile of the whole genome p-values across segments, there appeared to be little difference for the ROMP methods for tests of association. However, significant differences between the q term segment and the middle segment were seen for some pseudo-traits with the revised Haseman-Elston linkage analyses. This seems to corroborate the anecdotal reports for linkage analyses.With respect to the differences between means of Finally, the type of pseudo-trait chosen may in some cases have an effect on the resulting null distribution. However, there may be a large variation among critical values generated using different pseudo-traits of any given type. Additional studies and simulations will be required to investigate the statistical properties of the estimate of the critical value.APT: Applied pseudo-traitCOGA: Collaborative Study on the Genetics of AlcoholismROMP: Regression of offspring on mid-parentROOP: Regression on one parentSNP: Single-nucleotide polymorphismAll authors read and approved the final manuscript. Contributions: analysis , theory (GJP and AFW), overall concept (AFW), first draft, and project supervisor (GJP)."}
+{"text": "We used a random coefficient regression (RCR) model to estimate growth parameters for the time series of observed serum glucose levels in the Replicate 1 of the Genetic Analysis Workshop 13 simulated data. For comparison, a two time-point interval was also selected and the slope between these two observations was calculated. This process yielded four phenotypes: the RCR growth phenotype, a two time-point slope phenotype, and Time 1 and Time 2 serum glucose level phenotypes. These four phenotypes were used for linkage analyses on simulated chromosomes 5, 7, 9, and 21, those chromosomes that contained loci affecting the growth course for serum glucose levels. The linkage analysis of the RCR-derived phenotype showed overwhelming evidence for linkage at one locus (LOD 65.78 on chromosome 5), while showing elevated but nonsignificant LOD scores for two other loci , and no evidence of linkage for the final locus. The two time-point slope phenotype showed evidence for linkage at one locus (LOD 4.16 on chromosome 5) but no evidence for linkage at any of the other loci. A parallel cross-sectional approach, using as input phenotypes the endpoints of the two-point slope phenotype, gave strong linkage results for the major locus on chromosome 5  while showing elevated but nonsignificant linkage results on chromosome 7  and no evidence for linkage at the two remaining loci. The RCR growth parameter showed more power to detect linkage to the major locus than either the cross-sectional or two-point slope approach, but the cross-sectional approach gave a higher maximal LOD score for one of the minor loci. Longitudinal studies are often designed to investigate the progression of a trait over time by taking repeated measurements in each study participant. For many traits, there are assumed to be genetic influences not only upon baseline values, but also on the course of the trait over time. If individual growth curves share the same basic model with parameters varying by individual, e.g., if a trait varies linearly over time with the slope and intercept varying between individuals, then we might estimate growth parameters from longitudinal data using a random coefficient regression (RCR) model ,2.This analysis used an RCR model to estimate growth curve parameters for the glucose trait in Replicate 1 of the Genetic Analysis Workshop 13 (GAW13) simulated data with answers known at the time of analysis. The RCR model was implemented in SAS using the PROC MIXED procedure. The resulting parameters were then used as phenotypes for a variance-components based linkage analysis method implemented in the computer software package SEGPATH [The RCR model is a two-stage model which admits both individual-level and population-level effects. It is known to be robust against data that are not missing completely at random .n time points in m individuals, yielding m time series of n measurements each: yij . Note that some yij may be missing. Let us denote by yj the time series of observations for the jth participant. Population level effects, i.e., covariates which are assumed to affect the trait y in the same manner for all subjects, are modeled by C, an n \u00d7 p matrix of regressors. The jth participant's values for these p covariates are given by a p \u00d7 1 data vector, \u03bej .Let y be a trait measured at q \u00d7 1 data vectors, \u03b6j , consisting of the subjects observed values for the q individual level covariates, and n \u00d7 q matrices of regressors, Bj. Thus, there are different matrices of regressors (Bj) for each individual. An example of an individual level effect would be a linear dependence of a trait on age with slope and intercept values that vary from individual to individual. The conditional expected value of the time series is then a sum of the population level and individual level effects:Individual-level effects, that is the effects of covariates which may impact each subject differently, are modeled by a family of E = C \u03bej + Bj \u03b6j.V = Var, depends on the individual only through the number of, and time between, observations. We assume further that observations, while being correlated within individuals, are independent between individuals and that the distribution of the conditional time series is multivariate normal.We make a homoscedasticity assumption in that we assume that the conditional variance of the time series, Bj which are variable, are drawn from a joint multivariate normal distribution. Individual level covariance structures tested included linear, quadratic, and exponential models of age dependence. We shall hereafter refer to the estimates of the regression coefficient(s) in the individual level effects that correspond to age dependence as the growth or slope parameter(s).For the GAW13 simulated data, many possible RCR models were tested on the time series of observations of the serum glucose phenotype. The RCR modeling framework assumes that the individual level parameters, i.e., those coefficients of the individual level regressor matrices Population-level covariance structures tested included those with and without body mass index (BMI) and sex effects. Note that these models, while differing in the number of covariates or particular structure assigned to the individual and/or population levels of variance effects, all take the same basic form within the framework of the RCR model paradigm. Models were selected based upon their fit as quantified by the Akaike Information Criterion (AIC). The modeling and fitting was performed in SAS using the PROC MIXED procedure.For comparison, a two time-point subset of the sample was selected by taking the first and third observations in the first cohort  and the first and second observations in the second cohort . The slope between these observations was then used as an alternative growth parameter phenotype. In addition, the glucose levels at each of the two time points in this subset were separately analyzed as phenotypes. This cross-sectional approach serves as a contrast with the linkage analysis using the growth parameter(s).All four phenotypes  from the RCR model) were used as input phenotypes for a linkage analysis in SEGPATH. SEGPATH performs variance-components linkage analysis on complex, extended pedigrees ,4.Bj and \u03b6j were selected to be of the form:The RCR model selected was an exponential growth model (with one parameter) at the individual level. In particular the y) for a subject at age t (after regressing out population level effects) would be assumed to be given by:so that the expected value of the log of the glucose level (denoted by E (y) = Bj \u03b6j = \u03b1jt + \u03b2j.j and \u03b2j are assumed to follow a bivariate normal distribution.Furthermore, \u03b1j was then used as a phenotype for linkage analysis. This analysis was performed on chromosomes 5, 7, 9, and 21, the chromosomes which contained the QTLs that affected the growth curve for glucose levels.At the population level, following our rule to include or exclude covariates based upon the AIC of examined models, sex was included as a covariate while BMI was excluded. The individual growth parameter \u03b1One region was found to be linked significantly to the RCR model growth parameter phenotype. The peak was on chromosome 5 at 9 cM with a LOD score of 65.78 Figure . This phIn contrast, the two-point slope phenotype gave only one significant region of linkage on chromosome 5 LOD 4.16 at 9 cM). The two single-point phenotypes gave significant evidence for linkage to chromosome 5  and elevated LOD scores on chromosome 7  Table 6 at 9 cMThe model selected by PROC MIXED in SAS closely resembled the simulated disease model. The linear and quadratic models were correctly rejected in favor of an exponential growth model, and sex was correctly included as a population-level fixed effect. However, despite influencing the trait on a population level, BMI was not included as a population-level covariate based upon the AIC scores for the models, which included it. The model selection process did not appear to be affected by the presence of missing phenotypic data or by the difference in sampling procedures between the first and second cohorts.Of the four chromosomal regions linked to the growth course of serum glucose levels that were modeled, only one was discovered unambiguously. However, the one true signal discovered was discovered with an overwhelming LOD score (LOD 65.78). Two of the remaining three regions (on chromosomes 7 and 9) showed elevated but nonsignificant LOD scores, while one (on chromosome 21) showed no evidence for linkage to the RCR growth curve parameter.Evidence for linkage was present in the two time-point slope phenotype only in chromosome 5. The LOD score, while respectable (LOD 4.16), did not come close to matching the LOD score for the RCR growth parameter. The two-point slope phenotype showed no evidence for linkage at any of the other chromosomal regions simulated to affect the growth course of glucose levels. The LOD scores in those regions were well below the LOD scores found using the RCR growth parameter.The RCR growth parameter thus proved much more powerful at discovering the location of loci affecting the growth curve for serum glucose levels than the two time-point slope phenotype. Not only did the RCR model virtually recover the model used to simulate the glucose level time series, but it had the advantage over the two time-point slope phenotype of incorporating information from all of the recorded observations. However, the two single time-point phenotypes proved capable not only of detecting linkage to the major gene on chromosome 5, but also gave higher LOD scores than the RCR growth parameter for the minor locus on chromosome 7.The relatively high LOD scores provided by the two single-point phenotypes indicates, especially vis-\u00e0-vis the two-point slope phenotype, the utility of a cross-sectional approach to the detection of genes affecting growth courses, at least in cases such as this simulation, where a difference in means is easily distinguishable. In this simulation, genes affecting the growth of glucose levels caused large divergences beginning at birth. Had these genes been coupled with low initial glucose levels, for example, in order that their effects not been so easily detected, a cross-sectional approach might not have been so fruitful. In this case, we may be seeing an effect in which the differing means between groups of individuals with different growth course affecting genotypes at a given age are more easily seen and are less affected by noise and error than differing slopes for those groups, at least until a large number of measurements are available to estimate the growth course."}
+{"text": "There is growing evidence that a map of dense single-nucleotide polymorphisms (SNPs) can outperform a map of sparse microsatellites for linkage analysis. There is also argument as to whether a clustered SNP map can outperform an evenly spaced SNP map. Using Genetic Analysis Workshop 14 simulated data, we compared for linkage analysis microsatellites, SNPs, and composite markers derived from SNPs. We encoded the composite markers in a two-step approach, in which the maximum identity length contrast method was employed to allow for recombination between loci. A SNP map 2.3 times as dense as a microsatellite map (~2.9 cM compared to ~6.7 cM apart) provided slightly less information content (~0.83 compared to ~0.89). Most inheritance information could be extracted when the SNPs were spaced < 1 cM apart. Comparing the linkage results on using SNPs or composite markers derived from them based on both 3 cM and 0.3 cM resolution maps, we showed that the inter-SNP distance should be kept small (< 1 cM), and that for multipoint linkage analysis the original markers and the derived composite markers had similar power; but for single point linkage analysis the resulting composite markers lead to more power. Considering all factors, such as information content, flexibility of analysis method, map errors, and genotyping errors, a map of clustered SNPs can be an efficient design for a genome-wide linkage scan. Traditionally, genome-wide linkage scans employ low-density maps of microsatellite markers, or short tandem repeat polymorphisms (STRPs), spaced at intervals of ~10 cM across the genome. Although single-nucleotide polymorphisms (SNPs) are less informative than STRPs, they are distributed densely and uniformly throughout the genome, which can make up for their lack of informativeness. Moreover, SNP genotyping is easily automated, cost-effective, and low in error rate -5, as weBecause the power of a linkage study increases with the markers' information content (IC), comparison between SNP and STRP maps for linkage has mostly been focused on IC. When SNPs are uniformly distributed along the genome, multipoint analysis of dense SNPs can provide linkage IC comparable to that of less dense STRPs. To obtain equivalent IC, the ratio of the number of SNPs to STRPs has been estimated to be 1.7\u20132.5 . When thThe Genetic Analysis Workshop 14 (GAW14) simulated data mimic a genome scan of a behavioral disorder with a genome scan map of STRPs ~7.5 cM apart, a genome scan map of SNPs ~3 cM apart, and a fine map of SNPs ~0.3 cM apart. Thus, we have an opportunity to compare STRPs and SNPs in genome-wide linkage analysis. There are two specific aims in this paper: 1) to compare the IC provided by STRPs, evenly spaced SNPs, and composite markers derived from tightly linked SNPs; and 2) to investigate the influence of inter-SNP distance on linkage analysis.Replicate 33 of the 100 Karangar nuclear pedigrees was randomly chosen from the GAW14 simulated data. We analyzed chromosomes 1, 3, 5, and 9, at which the simulated disease susceptibility loci lie. In addition to the STRP map and the 3-cM SNP map, we also \"purchased\" 2 packages of 0.3-cM SNPs that spanned the regions covering the disease susceptibility loci on each chromosome. Specifically, packages 028, 029 (38 SNPs), packages 153, 154 (26 SNPs), packages 207, 208 (38 SNPs), and packages 417, 418 (38 SNPs) were purchased for chromosome 1, 3, 5, and 9, respectively.S(i) denote the score of identity length at locus i. If the two alleles at the ith SNP are different, S(i) = 0; if they are identical in state (IIS), we repeat the comparison process for the next SNP on each side, and this is repeated to determine S(i). After the S(i) values were calculated at each SNP between any pair of founder and non-founder haplotypes, every 3 (or 5) SNPs were grouped into a cluster as one composite marker and a mean score  was calculated for each cluster. The largest mean score was then used to assign haplotypes. Suppose, for example, that for a particular trio at a given cluster, P1 and P2 denote the father's two haplotypes, M1 and M2 the mother's two haplotypes, and O1 and O2 the child's two haplotypes. If the largest mean score was for the P1 - O1 pair, then the child inherited the haplotype P1 and the corresponding composite marker allele; the other haplotype inherited was then M1 or M2 depending on which pair (M1 - O2 versus M1 - O2) had the larger score. If the largest scores for P1 - O1 and M1 - O1 were equal, then O1 was randomly assigned to be from either parent. The map position of a composite marker was labelled as being in the middle of the cluster of SNPs.For a cluster of tightly linked SNPs, haplotypes are analogous to the alleles of a STRP marker, and thus the whole cluster forms a composite marker. A recombination within a cluster can lead to Mendelian inconsistency of genotypes. To avoid this type of inconsistency, and to study the influence of inter-SNP distance on linkage analysis, we encoded the composite markers in a two-step approach. First, we generated the most likely haplotype for every family member based on the SNP data and the given recombination fraction between consecutive pairs of SNPs using the software MERLIN  and encoThe multipoint IC, measuring the fraction of inheritance information extracted by the map relative to that extracted by an infinitely dense polymorphic map , is baseTable 10  by Haseman-Elston regression. Here we only report the results for chromosomes 5 and 9, because there was no signal reaching nominal significance  for chromosome 1 or 3 in this replicate. For chromosome 5, at the simulated disease susceptibility locus (~3.2 cM) only multipoint and single point analyses using 3-SNP markers from the 3-cM map detected linkage signals with p-values less than 5 \u00d7 10-2 . Both multipoint and single-point analyses using 1-, 3-, and 5-SNP markers from the 3-cM and 0.3 cM maps generated false linkage signals at other locations. For chromosome 9, at the simulated disease susceptibility locus (~3.5 cM) multipoint analyses using 1- and 3-SNP markers from the 3-cM map detected linkage signals with p-values of 2 \u00d7 10-5 and 3 \u00d7 10-2, respectively; single-point analyses detected linkage signals at the same position with p-values of 1 \u00d7 10-2 and 4 \u00d7 10-2, respectively. Analyses using 5-SNP markers did not detect linkage signals with p-values less than 5 \u00d7 10-2. When employing the 0.3-cM map, each analysis detect the designed linkage with p-value less than 1 \u00d7 10-5. When using the 3-cM map, the single point analysis had weak power to detect linkage because of the low informativeness of a single SNP; composite markers could not make any improvement \u2013 they even resulted in loss of signal on chromosome 9 by multipoint analysis. When using the 0.3-cM map, both composite markers and single SNPs gained power, and gave quite similar results with multipoint analysis. When employing the single-point approach, the composite markers produced higher and smoother signals than did the single SNPs.Figure The relationship between the IC of SNP and STRP maps is not simple . To achiIC varies as a function of SNP density. The denser the map, the more IC can be extracted. In this study of nuclear families with parental genotypes known, the 3-cM map gave an IC of 0.83 and the 0.3-cM map gave an IC of 0.98. Together with the observations of Evans and Cardon  that incThe recombination between loci in a cluster is usually ignored, given tight linkage. Wilson and Sorant  simulateA clustered map structure can be more useful than a uniform SNP map for linkage analysis from practical consideration . The cluGAW: Genetic Analysis WorkshopIBD: Identical by descentIC: Information contentIIS: Identical in stateLD: Linkage disequilibriumMILC: Maximum identity length contrastMPIC: Multilocus polymorphic information contentSNP: Single-nucleotide polymorphismSTRPs: Short tandem repeat polymorphismsCX, FRS, and RCE conceived the study, and participated in its design and coordination. CX, FRS, and GX carried out programming and analyzed chromosomes 1 and 3. QL analyzed chromosome 5, and TW analyzed chromosome 9. All authors read and approved the final manuscript."}
+{"text": "The feasibility of effectively analyzing high-density single nucleotide polymorphism (SNP) maps in whole genome scans of complex traits is not known. The purpose of this study was to compare variance components linkage results using different density marker maps in data from the Collaborative Study on the Genetics of Alcoholism (COGA). Marker maps having an average spacing of 10 cM (microsatellite), 0.78 cM (SNP1), and 0.31 cM (SNP2) were used to identify quantitative trait loci (QTLs) affecting maximum number of alcoholic drinks consumed in a 24-hour period .Heritability of lnmaxalc was estimated to be 15%. Multipoint variance components linkage analysis revealed similar linkage patterns among the three marker panels, with the SNP maps consistently yielding higher LOD scores. Robust LOD scores > 1.0 were observed on chromosomes 1 and 13 for all three marker maps. Additional LODs > 1.0 were observed on chromosome 4 with both SNP maps and on chromosomes 18 and 21 with the SNP2 map. Peak LOD scores for lnmaxalc were observed on chromosome 1, although none reached genome-wide statistical significance. Quantile-quantile plots revealed that the multipoint distribution of SNP results appeared to fit the asymptotic null distribution better than the twopoint results.In conclusion, variance-components linkage analysis using high-density SNP maps provided higher LOD scores compared with the standard microsatellite map, similar to studies using nonparametric linkage methods. Widespread application of SNP maps will depend on further improvements in the computational methods implemented in current software packages. Alcoholism is a complex trait influenced by both genetic and nongenetic factors. Previous linkage studies have identified several chromosomal regions harboring potential genes for alcoholism , althougTraditionally, studies involving genome-wide linkage scans have used microsatellite markers spaced evenly across the genome at ~10-cM intervals. An alternative and increasingly popular strategy is to use a high-density map of single nucleotide polymorphisms (SNPs). SNPs have several advantages over their microsatellite counterparts. They are found far more abundantly in the genome and are easier to genotype. Howerver, because of the lower heterozygozity on a per marker basis, a larger number of SNPs is necessary to achieve an information content similar to that of microsatellites .One way of increasing power for linkage studies is to use more closely spaced markers, which is now possible with the advent of high-throughput technology for large scale SNP genotyping. Simulation studies have indicated that SNPs can offer equal or superior power to detect linkage compared with low-density microsatellite maps . RecentlData for the Genetic Analysis Workshop GAW14) was obtained from the Collaborative Study on the Genetics of Alcoholism (COGA) 4 was obt. The traHeritability estimates and evidence for linkage were obtained using the variance components approach implemented in SOLAR version 2.1.2 . This meln 10) \u00d7 LOD is expected to be distributed as a 1/2:1/2 mixture of a  variable and a point mass at 0 [We examined the two-point and multipoint genome-wide LOD score distributions of each marker set against the asymptotic null distribution using quantile-quantile (Q-Q) plots. Under the null hypothesis of no linkage, the likelihood ratio test statistic that is given by  on chromosome 4q21.3, near marker D4S2407 . We wereGenome-wide two-point results from the three marker maps appeared to be comparable. However, the observed SNP-based LODs were lower than expected under the asymptotic null, which is probably due to the decreased information content of SNPs on an individual basis. For the multipoint results, the densest SNP map appeared to fit better, likely reflecting the increased information content provided by the larger number of markers or finer spacing. The asymptotic null distribution assumes independence across the genome, but LOD scores are correlated along a chromosome, even under the null hypothesis of no linkage. Our Q-Q plot comparisons may be affected by correlation among LOD scores. However, we would expect such correlation to cause greater deviations from the asymptotic null for the multipoint results rather than the two-point results as we observed.P-values corresponding to a given LOD score may also be higher for the SNP-based analyses. To fully compare the power of marker panels, extensive simulations should be carried out under various genetic models, map densities and sample sizes.Peak variance-components LOD scores were consistently higher for the SNP-based linkage analyses, similar to recent reports using nonparametric linkage methods -5. HowevDirect comparisons between SNP-based and microsatellite-based results in this analysis were hindered by several factors. First, we used the genetic maps as provided to us, which were not aligned among the three marker sets. Further, the presence of linkage disequilibrium among SNPs can lead to inflated LOD scores . HoweverMoving to denser SNP maps, however, comes at the expense of increasing computational time. We performed analyses in SOLAR using the RAM drive rather than the hard drive, which decreased the computation time by ~50%. Each analysis of the simulated chromosome 1 data took approximately 23 minutes for the microsatellite map (38 markers), 3\u20135 hours for the SNP1 map (381 markers), and 15\u201319 hours for the SNP2 map (864 markers). At the time of our analysis, SOLAR was unable to handle more than 500 markers per chromosome, so we had to break the SNP2 data into two to three sections for the larger chromosomes. This complicated the analysis as we had to \"bridge\" across sections to minimize boundary effects when estimating multipoint IBDs. The latest release of SOLAR 2.1.4 can handle up to 2000 markers, but the computation time may still be quite substantial.In conclusion, variance-components linkage analysis using high-density SNP maps provided higher LOD scores compared with the standard microsatellite map. These results demonstrate that using dense SNP maps in linkage analysis is feasible and may increase power. However, the computational challenges are not trivial and will only increase as denser SNP sets become available. More widespread application of SNPs in linkage analysis will depend on further improvements to current statistical methods and associated software packages.COGA: Collaborative Study on the Genetics of AlcoholismGAW: Genetic Analysis WorkshopIBD: Identity by descentQ-Q: Quantile-quantileQTL: Quantitative trait locusSNP: Single-nucleotide polymorphismAll authors participated in the study design and read and approved the final manuscript. HK performed statistical analyses and drafted the manuscript. CMH performed statistical analyses and provided manuscript support. SAM and KLE guided the statistical analyses."}
+{"text": "The average r2 between adjacent markers across the genetic map was 0.099 \u00b1 0.003 in the Illumina III panel and 0.17 \u00b1 0.003 in the Affymetrix 10 k array. In order to determine the effect of LD between marker loci in a nonparametric multipoint linkage analysis, markers in strong LD with another marker (r2 > 0.40) were removed  and the linkage analysis results were compared to the results using the entire marker sets. In all analyses using the ALDX1 phenotype, 8 linkage regions on 5 chromosomes  were detected (peak markers p < 0.01), and the Illumina panel detected an additional region on chromosome 6. Analysis of the same pedigree set and ALDX1 phenotype using short tandem repeat markers (STRs) resulted in 3 linkage regions on 3 chromosomes (peak markers p < 0.01). These results suggest that in this pedigree set, LD between loci with spacing similar to the SNP panels tested may not significantly affect the overall detection of linkage regions in a genome scan. Moreover, since the data quality and information content are greatly improved in the SNP panels over STR genotyping methods, new linkage regions may be identified due to higher information content and data quality in a dense SNP linkage panel.Genotype data from the Illumina Linkage III SNP panel ( For many years short tandem repeat (STR) or microsatellite markers have been used as the standard genetic markers for linkage mapping -5. STRs . Recent reports have shown SNP linkage panels have higher information content and data quality than STR marker panels [In contrast, single-nucleotide polymorphism (SNP) assays are more amenable to multiplexing and are easier to automate, which increases accuracy and reliability. Therefore, more complete linkage studies with thousands of markers, and studies with large numbers of DNA samples are more feasible using SNPs. In addition, there is an abundance of SNPs: currently over 8 million human SNPs are in public databases, many of which have been validated r panels -9. Howevn = 1,074). The next largest self-reported ethnic group was black, non-Hispanic (n = 191).One hundred forty-three pedigrees from the COGA study comprised 1,614 individuals  segregating alcoholism were used in all analyses. The 143 pedigrees had 256 sibships. Of these 256 sibships, 123 (48%) had both parents, 75 (29%) had 1 parent, and 58 (23%) had no parents available for SNP genotyping. The majority of the individuals had a self-reported ethnicity of White, non-Hispanic , low error rate (0.005%), and low rate of Mendelian inconsistencies (0.09%). From a genetic map generated from 28 CEPH pedigrees [9.95%, lor2, was determined for each pair-wise combination. In particular, LD between each adjacent marker on the genetic map used for linkage analysis was investigated. Loci in strong LD with another marker (r2 > 0.40) were removed and the linkage analysis was repeated using the subset of markers where all loci have no or weak LD (r2 < 0.40).Inter-marker LD was determined from between all SNP loci in the two SNP panels, respectively, in 244 unrelated individuals. LD strength was derived from estimated haplotypes using the expectation-maximization algorithm of Slatkin and Excoffier  as implep-values < 0.01.Linkage analysis of both SNP panels and the COGA STR panel were analyzed using the ALDX1 phenotype. Genotype data were analyzed using multipoint nonparametric methods as implemented in GENEHUNTER v2.1  on all pr2) between each adjacent marker pair on the genetic map was evaluated in both SNP panels. For the Illumina SNP panel, the average r2 between each adjacent marker pair on the genetic map was 0.099 \u00b1 0.003 (SD = 0.23). As shown in Figure r2 < 0.05. Figure r2 > 0.4 by chromosome. The X chromosome had the highest proportion of marker pairs with r2 > 0.4 . Overall, 471/4,697 marker pairs (10%) had r2 > 0.4. One marker from each of the 471 marker pairs with r2 > 0.40 was randomly removed. In the case where there were multiple adjacent SNPs with r2 > 0.40, the minimum numbers of loci were removed so that the remaining adjacent loci had r2 < 0.40. A subset of 4,149 loci was used for subsequent linkage analyses where all loci had weak or no LD. For the Affymetrix SNP panel, the average LD between loci was higher (as expected) because the SNPs are more closely spaced. The average r2 between each adjacent marker pair on the genetic map was 0.17 \u00b1 0.003 (SD = 0.30), and less than 65% of all marker pairs had an r2 < 0.05 . The same nine regions on six chromosomes were identified in this analysis. These results are also shown in Table p = 0.004), though less significant than the analysis with all loci (p = 0.0006). Even though there were moderate differences between the two analyses on these two chromosomes, the same regions were identified in both analyses with p-values \u2264 0.01. The six chromosomes with p-values \u2264 0.01 are shown in Figure p-values < 0.01 as shown in Table p-values  are shown in Figure Genotype data were analyzed using the ALDX1 phenotype and the NPL multipoint analysis methods as implemented in the program GENEHUNTER v2.1 . For the01 Table . The fivIC was also compared across all marker sets as shown in Table p < 0.01. The resulting peak loci and linkage intervals are summarized in Table Finally, STR data were analyzed using the ALDX1 phenotype. Two of the 9 linkage peaks detected using the high-density SNP map were detected with STRs at r2 < 0.05. Using the Affymetrix mapping array, a higher proportion of marker pairs had high LD genome-wide, and 65% of all marker pairs had an r2 < 0.05.An early study suggested that if LD exists between a trait and marker and if LD is not taken into account in a linkage analysis, the resulting LOD score would be reduced . Howeverp < 0.01, with modest changes in p-values and NPL scores. The NPL scores and p-values did not consistently become more or less significant with the removal of loci in LD. In addition, there was high concordance in terms of significance and location of linkage peaks between the two SNP mapping panels. The main difference in findings between the two panels was that the Affymetrix mapping array did not detect the linkage peak at p < 0.01 on chromosome 6. This result could have occurred for many reasons. The IC is lower in this region (IC = 0.81 at peak marker). In addition, because the peak marker does not map uniquely in the build 34 genome assembly, the genetic map position may not be accurate since the genetic map position is linked to physical map position through linear interpolation in this SNP panel. Finally, this result could be a false-positive result in the Illumina mapping panel.Overall, in both SNP panels there was very little difference between results using the entire marker set and the reduced marker set without loci in strong LD. Using both SNP panels, the same regions were detected in both analyses at p-values < 0.01. Two of these regions  overlapped with the SNP linkage results and the third region (7p14) did not overlap although it was between two of the SNP linkage peaks . In addition, the IC using the STR data was approximately 3\u201321% lower at the linkage peaks compared with the high density SNP data.The analysis of STR markers in the same pedigree set resulted in only 3 linkage peaks with corresponding The results on this study suggest that LD between loci in a linkage analysis does not significantly affect the overall detection of linkage regions in a genome scan. However, this result is dependent on the number of genotyped founders or unaffected siblings in the pedigrees because the potential bias in LOD score due to underlying LD between SNP loci in a linkage analysis is largest in pedigrees in which genotypes are not available for founding individuals. Therefore, one strategy to determine the effect that LD has on a linkage analysis in other pedigree collections might be to re-run a linkage analysis using a subset of loci with weak or no LD in the pedigree collection being studied. Therefore, one can determine if a linkage result is being inflated by underlying LD between SNP loci in the linkage peak. Despite the potential bias of a resulting LOD score, the high-density SNP panels provide an order of magnitude higher data quality compared to STR genotyping methods and also provide higher IC. In this study, several new linkage regions may have been identified that were not detected using a 10-cM STR marker panel due to higher IC and data quality in the dense SNP linkage panels.COGA: Collaborative Study of the Genetic of AcoholismIC: Information ContentLD: Linkage disequilibriumNPL: Nonparametric linkageSTR: Short tandem repeat markerSNP: Single-nucleotide polymorphism"}
+{"text": "The metabolic syndrome is characterized by the clustering of several traits, including obesity, hypertension, decreased levels of HDL cholesterol, and increased levels of glucose and triglycerides. Because these traits cluster, there are likely common genetic factors involved.We used a multivariate structural equation model (SEM) approach to scan the genome for loci involved in the metabolic syndrome. We found moderate evidence for linkage on chromosomes 2, 3, 11, 13, and 15, and these loci appear to have different relative effects on the component traits of the metabolic syndrome.Our results suggest that the metabolic syndrome components, diabetes, obesity, and hypertension, are under the pleiotropic control of several loci. Metabolic syndrome (MSX) is characterized by the aggregation of several risk factors, including obesity, impaired glucose tolerance, elevated triglyceride levels and blood pressure, and low HDL cholesterol . These fMethods that account for shared environmental influence on the components of MSX are necessary if the genetic variance is not to be overestimated. Structural equation models (SEM) comprise a valuable method for partitioning the variance into its genetic and shared environmental components. A previous SEM analysis has suggested that the components of MSX are pleiotropically influenced by common genetic factors . Thus, a2. Data from the final time point were used because we hypothesized that the study subjects would best demonstrate any progression to MSX by this time. Both GLUC and TG were log-transformed to reduce skewness. Because the distribution of GLUC was leptokurtotic after log transformation, we further transformed this variable using a generalized modulus power transformation [We analyzed data from the offspring cohort of the Framingham Heart Study, provided for Problem 1 of Genetic Analysis Workshop 13. The analysis was performed on all individuals with complete genotypic and phenotypic data for the fifth time point; this sample consisted of 1097 individuals from 381 pedigrees for a total of 1220 sibpairs. The proportion of males and females was roughly equal, and the mean age was 51.33 (\u00b1 10.01) years. We modeled MSX using the measurements taken at the fifth time point for systolic blood pressure (SBP), fasting plasma glucose (GLUC), triglycerides (TG), HDL cholesterol (HDL), and body mass index (BMI), which was calculated as weight(kg)/(height(m))Five adjusted phenotypes were used in multivariate linkage analyses that simultaneously incorporate the phenotypic and genetic marker information into a single SEM, as proposed by Eaves and colleagues  and implThe methodology is fully described in Eaves et al. . Brieflywhere AA' and EE' are the covariance matrices due to additive genetic and within-family environmental effects, respectively. The information contained about the QTL contained in cross-trait covariances is used to estimate the effects of a putative QTL. The resulting sib-pair covariance matrix isk is the expected proportion of pairs in a random sample sharing k alleles IBD , and AA' and EE' are residual additive genetic and environmental effects, after accounting for QQ', the contribution of the QTL effects at the genetic location in question. The SEM including QTL effects is illustrated in Figure where k is the number of alleles shared identical by descent (IBD) at the location s, fD 1/4, 1/, 1/4, anp-values and associated QTL contributions for each phenotype are provided , and the uncoupling protein 2 (OMIM 601693) [IRS-2, a diabetes candidate gene, has been mapped to chromosome 13 (102 cM) [We observed moderate linkage on chromosomes 2, 3, 4, 11, 13, and 15, although the relative effect of these putative QTL on each MSX phenotype appears to differ. Because the literature related to linkage signals for MSX components is vast, we have summarized reports within a 30cM window of our linkage signals . Though  601693) . An adip 601693) . Insulin 601693) , BMI [19 601693)  have bee 601693) . Finally(102 cM) .Given the multifactorial nature of MSX, the method of linkage analysis most suitable is one that incorporates all available trait information. Here we use SEM with multivariate data to increase power, account for shared sibling environment within a sib-pair linkage analysis, and depict the combined effects of the components of MSX simultaneously. This approach is uniquely capable of estimating the contribution of each phenotype to the QTL, which can illustrate facets of MSX biology. Another advantage of our approach is the direct use of quantitative traits. Currently, there are two different clinical definitions of MSX , which rThere are, however, limitations to these analyses. Although there were data available on blood pressure medication, there were no data on diabetes medication. Since both were not available, we did not adjust our quantitative variables for these covariates. Because of missing genotype and/or phenotype data, approximately 700 sibpairs were excluded from this analysis. Though our sample size was still substantial, it is unknown how these sibpairs might have increased power. There is an upward bias in the estimation of the QTL contributions. The sum of the squared QTL contributions to GLUC for all distinct linked regions is greater than 1, which is not possible if these values are interpreted to be QTL-specific heritabilities. Also, when partitioning genetic and shared-environmental components of variance, gene\u00d7environment interaction was unaccounted for, and allowing for such interaction would only exacerbate the discrepancy. Finally, in the SEM, all sibpairs were treated as though they were independent. It is unclear how to handle larger sibships within the SEM; however, the average sibship size is less than three, so this should not have a great impact on the analysis.Another point worth revisiting is our approach to non-normal data. The SEM assumes multivariate normality and, with increasing sample size, departure from this assumption will affect the likelihood ratio test adversely. In our analysis, the GLUC variable was transformed using a generalized modulus power transformation  to removA number of loci appear to be linked to MSX, most notably regions on chromosomes 2, 3, 11, 13, and 15. Diabetes, obesity, and hypertension were most influenced by these loci. These regions appear to influence the components of MSX in different ways, and they warrant further analysis, both in reference to the metabolic syndrome itself and to other disorders associated with insulin-resistance."}
+{"text": "Multivariate phenotypes underlie complex traits. Thus, instead of using the end-point trait, it may be statistically more powerful to use a multivariate phenotype correlated to the end-point trait for detecting linkage. In this study, we develop a reverse regression method to analyze linkage of Kofendrerd Personality Disorder affection status in the New York population of the Genetic Analysis Workshop 14 (GAW14) simulated dataset. When we used the multivariate phenotype, we obtained significant evidence of linkage near four of the six putative loci in at least 25% of the replicates. On the other hand, the linkage analysis based on Kofendrerd Personality Disorder status as a phenotype produced significant findings only near two of the loci and in a smaller proportion of replicates. A complex trait is usually a function of a multivariate phenotype comprising correlated quantitative variables. Since end-point traits are usually binary in nature (affected/unaffected) and hence contain minimal information on variation within trait genotypes, it may be statistically more powerful to use a correlated multivariate phenotype for identifying genes for the complex trait. Mapping a multivariate phenotype traditionally uses some function of quantitative values of sib-pairs or other sets of relatives as a response variable and marker identity-by-descent (IBD) scores as explanatory variables -3. In thFor our analysis, we considered data on the KPD status (affected or unaffected), twelve associated binary phenotypes, and genome-wide information separately on 416 microsatellite marker loci and 917 single nucleotide polymorphisms (SNPs) with average intermarker distances of 7.5 cM and 3 cM, respectively, distributed over 10 autosomal chromosomes for the New York population. Our method utilizes phenotype and marker data on 50 independent sibships of sizes varying from 2 to 9 and their parental genotypes for IBD computations. We analyzed data on all 100 available replicates.yijk denote the phenotypic value of the ith trait for the jth sib in the kth sibship, i = 1, 2, 3, 4, 5; j = 1, 2, ..., nk; k = 1, 2, ..., 50. The twelve phenotypes relate to personality traits and therefore may be associated with the end-point trait, the affectation status of KPD. Thus, instead of using the KPD status as a phenotype for linkage analysis, it may be statistically more powerful to use a multivariate phenotype comprising some of these personality traits, which are highly correlated to the disease status. In order to select a subset of the twelve traits, which may be used as a surrogate for the end-point trait, we performed a logistic regression of the KPD disease status on the twelve binary phenotypes. To ensure the independence of our observations, the regression was based on the 100 parents of the 50 sibships.Suppose The logistic model used was:zjk is the affectation status of KPD of the jth parent of the kth sibship; \u03b4 = 0 or 1 according to whether an individual is affected with KPD or not and xijk is the phenotypic value of the ith trait of the jth parent of the kth sibship. The test for association between the ith  personality trait with KPD is equivalent to testing ai = 0 versus ai \u2260 0. We used a level of 0.005 for testing each ai in the 12 tests. We obtained five of the phenotypes to be significantly correlated to the end-point trait (details are provided in the \"Results\" section). Thus, the multivariate phenotype we used for our linkage analysis comprises five binary personality traits.where Sham et al.  proposed is the estimated marker IBD score of the first and jth sibs of the kth sibship, j = 1, 2, ..., nk; k = 1, 2, ..., 50; ejk values are random environmental errors assumed to have mean 0 and equal variances. We note here that an advantage of using IBD scores instead of the squared sib-pair trait differences as the response variable is that in a sibship of size nk, the marker IBD scores \u03c0k,2k1, \u03c0k,3k1, ..., \u03c0k1,nk are independent, but the squared differences in trait values for these sib-pairs are not independent. Thus, for a sibship of size nk, we have nk - 1 independent. We wish to point out here that our method is not related to parity . While analyzing data, we suggest that the sib assigned \"1\" be chosen at random from the sibship. When we computed multipoint IBD scores, the conversion of recombination distances to physical distances (in cM) on chromosomes was based on the Haldane map function [where function .H0 : \u03b21 = \u03b22 = \u03b23 = \u03b24 = \u03b25 = 0 versus H1: \u03b21 < 0 U \u03b22 < 0 U \u03b23 < 0 U \u03b24 < 0 U \u03b25 < 0. In other words, under no linkage between the two loci, the estimated marker IBD score will not be correlated to the squared difference in sib-pair trait values. On the other hand, if the two loci are linked, the estimated marker IBD score will not be correlated to the squared difference in sib-pair values for at least one of the correlated traits [We define our test for linkage between the locus controlling KPD and the marker locus to be equivalent to testing The test statistic used isp-values for the observed value of the test statistic. We generate marker IBD scores at random using the marginal distribution of IBD scores  for normally distributed errors. Under the assumption of normality, the test statistic is distributed asymptotically as a mixture of chi-square distributions. It is very unlikely in practice for the errors to be distributed as normal. Thus, instead of making any assumptions on the distribution of the errors, we use Monte Carlo simulations to obtain the empirical d Elston  and markBecause our aim is to show that using the multivariate phenotype vector for the linkage scan is statistically more powerful than using the end-point trait (KPD status), we also perform the reverse regression analysis using only the KPD status. The regression procedure is identical to the one described above with the test for linkage based on only one parameter, i.e., the regression coefficient associated with the KPD status variable.Based on the logistic regression, five phenotypes: fear/discomfort with strangers, dislike of jokes told face to face, obsession with entertainers, humor impairment, and uncommunicative, contentless speech patterns were found to be significantly correlated to KPD status. As mentioned earlier, we performed two linkage analyses: one based on a multivariate phenotype vector comprising these five traits and the other based on only the KPD status as a phenotype. We used the statistical package MERLIN 0.10.2  for multWhen we used the multivariate phenotype, the linkage analyses based on the 416 microsatellite markers yielded significant peaks on 4 chromosomes: D01S0023 on chromosome 1, D03S0127 on chromosome 3, D05S0173 on chromosome 5, and D09S0347 on chromosome 9. The linkage analyses using the 917 SNP markers yielded significant peaks around the same regions as the peaks corresponding to the microsatellite markers: C01R0051 on chromosome 1, C03R0281 on chromosome 3, C05R0381 on chromosome 5, and C09R0763 on chromosome 9. When we used the end-point KPD status as our phenotype, we obtained significant peaks only at D03S0127 and C03R0281 on chromosome 3; and D05S0173 and C05R0381 on chromosome 5 for microsatellite markers and SNPs, respectively. It is clear from the tables that not only did the multivariate phenotype approach produce significant linkage findings at more locations, but also the proportions of replicates in which we obtained the significant findings for both microsatellite markers and SNPs were much lower when only the KPD status was used.Based on the multivariate phenotype, we have been able to detect linkage in at least 25% of the replicates for both microsatellite markers and SNP markers on four chromosomes  very close to the putative trait loci. The proportion of replicates in which we obtained significant linkage findings for the SNPs appears to be marginally lower than that for the microsatellite markers. This can be explained by the fact that since the SNPs are less polymorphic compared with microsatellite markers, the information content at the same marker density is higher with microsatellite markers, leading to more efficient estimation of marker IBD scores. Moreover, we used the same level of significance in our tests of linkage for both microsatellite as well as SNP markers. Since the SNPs are at a much higher density, at the same level of single-marker significance, the genome-wide significance level based on SNPs is higher than that for the microsatellite markers.Our proposed reverse regression method was able to detect linkage near four of the six putative loci controlling KPD in multiple replicates. We found that our linkage analyses based on the multivariate phenotype comprising five binary traits correlated with KPD was more powerful than those based on only the affectation status of KPD as the phenotype. Thus, using a multivariate phenotype vector comprising traits correlated with the end-point trait may be a prudent strategy for linkage mapping of a complex trait.While it is important to compare the power of our method with those of existing methodologies, the structure of the dataset did not permit a valid statistical comparison with most existing methods. The variance components methods like those implemented in MERLIN, GENEHUNTER, SEGPATH, and ACT assume multivariate normality of trait values within pedigrees and are designed for quantitative traits. However, all the personality traits in the dataset were binary in nature and assumption of normality for these traits would not be proper. The package SOLAR has an option of using a threshold model for binary traits , but likp-values in the linkage scan after accounting for the p-values in the first stage. Extensive simulations to examine this issue are being conducted.The overall level of significance would most likely be a function of the level of significance used in the first stage of our analysis in which we are selecting a subset of phenotypes that are significantly associated with the end-point trait. The nature of dependence of the two stages is quite complex and it is difficult to obtain exact adjustments of the GAW14: Genetic Analysis Workshop 14IBD: Identity by descentKPD: Kofendrerd Personality DisorderLRT: Likelihood ratio testSNP: Single-nucleotide polymorphismSG proposed and worked on the methodology. SB optimized the linkage statistics and wrote the computer codes. GB and SP managed the data and implemented the software packages/computer programs for IBD computations, logistic regression, and empirical power computations. PPM coordinated the analysis and participated in writing the manuscript."}
+{"text": "Using the dataset provided for Genetic Analysis Workshop 14 by the Collaborative Study on the Genetics of Alcoholism, we performed genome-wide linkage analysis of age at onset of alcoholism to compare the utility of microsatellites and single-nucleotide polymorphisms (SNPs) in genetic linkage study.A multipoint nonparametric variance component linkage analysis method was applied to the survival distribution function obtained from semiparametric proportional hazards model of the age at onset phenotype of alcoholism. Three separate linkage analyses were carried out using 315 microsatellites, 2,467 and 9,467 SNPs, spanning the 22 autosomal chromosomes.p < 0.001). We observed weak correlation, both in trend and strength, of genome-wide linkage signals between microsatellites and SNPs. Results from SNPs revealed more and stronger linkage signals across the genome compared with those from microsatellites. The only suggestive evidence of linkage from microsatellites was on chromosome 1 (LOD of 1.43). Differences in map densities between the two sets of SNPs used in this study did not appear to confer an advantage in terms of strength of linkage signals.Heritability of age at onset was estimated to be approximately 12% (Our study provided support for better performance of dense SNP maps compared with the sparse mirosatellite maps currently available for linkage analysis of quantitative traits. This better performance could be attributable to precise definition and high map resolutions achievable with dense SNP maps, thus resulting in increased power to detect possible loci affecting given trait or disease. Several reports have been published on the use of evenly spaced microsatellites, usually less than 500, for genome-wide linkage analysis of traits or diseases with reasonable heritability. Although this has lead to great successes in mapping of Mendelian disorders, comparable successes still remain to be achieved in complex disease mapping . It is nThe COGA dataset provided for GAW14 contained 1,614 family members in 143 families with different sizes. Each family was ascertained from an alcoholic proband in a treatment program. Self-reported ethnicity in this study population included American Indian, Asian, Pacific Islander, Black Hispanic and non-Hispanic, and White Hispanic and non-Hispanic. From the list of alcohol-related phenotypes available in the dataset, we chose the age at onset for alcohol dependence (ALDX1). ALDX1 was defined as lifetime diagnosis  criteriaBecause age at onset is a truncated quantitative trait \u2013 censored for those individuals for whom age at onset was not observed, we used semiparametric proportional hazards model as implemented in SAS  softwareThere were 315 microsatellites (11.53 cM average spacing), 4,596 Illumina SNPs (1.41 cM average spacing), and 10,801 Affymetrix SNPs (0.36 cM average spacing) provided across the autosomal chromosomes. In cases were there were multiple SNPs per locus, we carried out thinning by random sampling of one SNP per locus and then discarded others. This process thus resulted in 2,467 and 9,467 effective SNP loci for Illumina and Affymetrix, respectively, without affecting the original marker spacing. We then proceeded to use SOLAR [P < 0.001). The random environmental effects thus contributed 88.2% of the total variation for this trait. The distributions of the highest multipoint LOD scores (logarithm of the odds to the base 10) and their corresponding chromosome positions for each of the genetic marker sets are presented in Table Prior to the multipoint linkage analysis, we fitted a polygenic model and estimated the heritability of age at onset to be 11.8% \u00b1 3.9% (In this study we compared the linkage signals obtained through the use of limited number of microsatellites and two sets of relatively dense SNP maps in nonparametric multipoint linkage of age at onset of alcoholism across the 22 autosomal chromosomes. Although there was neither significant nor suggestive evidence of linkage  for age As seen in Figure This study provided support for better performance of dense SNP map compared with microsatellites for linkage analysis, even for traits with low heritability such as age at onset of alcoholism. This better performance is attributable to high map resolution achievable with dense SNP maps. Also, the more abundant genome-wide distribution of SNPs compared with microsatellites, and the availability of advanced genotyping technology to process rapidly and extract needed data from the samples place SNPs as very promising tools for linkage analysis.COGA: Collaborative Study on the Genetics of AlcoholismGAW: Genetic Analysis WorkshopIBD: Identity by descentSNP: Single nucleotide polymorphismBOT conceived of the study, performed all statistical analyses and wrote the manuscript. YL participated in the design, statistical analyses, and preparation of manuscript. SS and MT participated in the design, and preparation of manuscript. All authors read and approved the final manuscript."}
+{"text": "Using the Genetic Analysis Workshop 14 simulated datasets we carried out nonparametric linkage analyses and applied a log-linear method for analysis of case-parent-triad data with stratification on parental mating type. We proposed and applied a random effect modelling approach to explore the impact of population heterogeneity on tests of association between genetic markers and disease status. The estimated genetic effect may appear to be strongly significant in one population but nonsignificant in another population, leading to confusion about interpretation. However, when results are interpreted in the light of a random effects model, both studies may be making similar statements about a genetic effect that varies depending on environment and background. It has proven to be very difficult to validate linkage and association findings for complex diseases. Part of the reason for the inconsistency may be due to real differences in effect sizes across populations and studies. Methods that explicitly model potential heterogeneity across populations are useful in clarifying reasons behind this variability as well as in estimating model parameters accurately. The objective of our study is to use the Genetic Analysis Workshop 14 (GAW14) simulated datasets to explore the impact of population heterogeneity on tests of association between genetic markers and disease status, and to use random effects models to account for this heterogeneity. We were also interested in estimating gene \u00d7 covariate interactions and heterogeneity associated with these effects. We adopted a three-stage analytical approach without knowledge of the generating model: 1) linkage analysis to find \"interesting\" regions, 2) association analysis for selected markers in separate populations, and 3) use of random effects models to combine the association test results across populations and to examine heterogeneity.We conducted a combined linkage and association analysis using GAW14 simulated datasets. The simulated data consists of phenotypic as well as genotypic information from four populations: Aipotu, Danacaa, Karangar, and New York City for the study of Kofendred personality disorder (KPD). Twelve disease symptom traits were also included. Microsatellite and single-nucleotide polymorphism (SNP) markers were available for 10 chromosomes.We first carried out a nonparametric linkage (NPL) analysis  within eWe used a log-linear model for estimating association that was designed for the analysis of case-parent-trios ,3, and eBased on the linkage analysis results, we selected a small number of interesting regions for association mapping using \"purchased\" fine-mapping markers. From each population, we selected 300 independent case-parent trios. One affected child was randomly selected from each pedigree, with his/her parents. For populations Aipotu, Danacaa, and Karangar, replicates 1, 11, and 87 were used. For New York, where replicates contained only 50 pedigrees, trios were also selected from replicates 2, 3, and 4. Association analysis was carried out for selected markers with covariates based on gender and the anxiety-related sub-phenotype.\u03bc = E(Y|b) = h(\u03b7) with \u03b7 = X \u03b2 + Zb, where h is the logarithmic link function, X is a design matrix corresponding to the fixed-effect parameters \u03b2, and Z is a design matrix associated with the random-effect parameters b. Detailed notations and formulation of the basic Poisson model of Weinberg are given elsewhere [A generalized linear mixed modelling (GLMM) framework was used to explore variability across populations and to combine risk estimates . This frlsewhere ,3. The rlsewhere .All analyses were conducted without knowledge of the generating model.The NPL analyses using data from replicate 1 showed that, of the 10 chromosomes, only chromosomes 1, 3, 5, and 9 contained statistically significant regions, in at least one of the four populations (Table Fine mapping packages were purchased for chromosome 3 and 5 near the linkage peaks. We chose these regions in order to contrast linkage findings that appeared consistent across populations (chromosome 3) with a region demonstrating variability in the strength of the linkage evidence across populations (chromosome 5); we hoped that the generating models for this chromosome 5 region might vary across populations. We obtained packages 152 and 153 for chromosome 3 and packages 207\u2013211 for chromosome 5.We did not find significant allelic association for markers on chromosome 5, and therefore no results are reported. For chromosome 3, Table p-value for marker B03T3057 for the gene \u00d7 anxiety interaction was less striking but still significant  while the p-value for the main effect of testing association with the variant allele increased to p = 0.0170 . The random-effect variance components are not significantly different from zero.SNPs B03T3057 and B03T3056 are used to illustrate the results from the mixed model where we assumed that the allelic and interaction risk estimates, under an additive genetic model, were random across populations (a GLMM framework). After combining across populations, the p = 0.0791); however, the main effect remained significant . In this model the random effect variance components are significantly different from zero.For the adjacent marker B03T3056, the gene \u00d7 anxiety interaction effect appeared non-significant . Therefore, a trade-off becomes necessary between full-immunity to population stratification and reliable estimates. All models fitted here used only one set of intercept parameters (6 intercepts). These models will give unbiased estimates of the genetic associations if either the covariate frequency (gender or anxiety) does not vary across any hidden population substructure, or the covariate is not associated with the genetic effect. In the GAW simulation, there was no hidden population substructure within each of the four stated populations, and therefore the more parsimonious models were appropriate.Validation of association studies is a continuing problem, and part of the difficulty is attached to inadequate power in the various studies, as well as, of course, genetic model heterogeneity in different populations or samples. By estimating genetic risk parameters using the Weinberg model, we can see whether the estimates of genetic risk are similar in different populations, rather than just comparing parameters. The Poisson GLMM approach we applied here allows exploring differences across populations (variability in risk estimates) as well as combining estimates meta-analytically. The estimated genetic effect may appear to be strongly significant in one population but non-significant in another population, leading to confusion about interpretation. However, when results are interpreted in the light of a random effects model, both studies may be making similar statements about a genetic effect that varies depending on environment and background.The estimates of association from the GLMM were smaller than from the separate Poisson models. This may be due to inflation in the estimates of the association parameters from the individual populations due to small sample bias, or to parameter shrinkage associated with the incorporation of extra sources of variability. Further investigation of this effect is warranted.Our approach of combining results across populations is an implementation of a meta-analysis strategy to understand and summarize results across independent studies. Meta-analysis can also be used to identify factors that may explain heterogeneity. Such an approach may prove useful in genetic studies where results vary across populations. A further extension to our mixed model approach may be developed in a fully Bayesian framework.We identified several regions showing evidence for linkage and association in the GAW14 simulated data. We also proposed a strategy for examining heterogeneity of association test results by using models that can include covariates, and implemented mixed models to allow for genetic effects to vary across populations. Although there is still a long way to go to dissect the genetics of complex diseases, data integration approaches such as our multi-level modelling framework might help elucidate genetic and environmental contributions to the risk of diseases.GAW: Genetic Analysis WorkshopGLMM: Generalized linear mixed modelKDP: Kofendred personality disorderNPL: Nonparametric linkageSNP: Single-nucleotide polymorphismJY carried out the linkage and log-linear association analyses. JB wrote the manuscript and carried out the mixed model analyses, using ideas developed by himself together with CMTG. CMTG assisted in editing the manuscript."}
+{"text": "Recently, alcohol-related traits have been shown to have a genetic component. Here, we study the association of specific genetic measures in one of the three sets of electrophysiological measures in families with alcoholism distributed as part of the Genetic Analysis Workshop 14 data, the NTTH  phenotypes: ntth1, ntth2, ntth3, and ntth4. We focused on the analysis of the 786 Affymetrix markers on chromosome 4. Our desire was to find at least a partial answer to the question of whether ntth1, ntth2, ntth3, and ntth4 are separately or jointly genetically controlled, so we studied the principal components that explain most of the covariation of the four quantitative traits. The first principal component, which explains 70% of the covariation, showed association but not genetic linkage to two markers: tsc0272102 and tsc0560854. On the other hand, ntth1 appeared to be the trait driving the variation in the second principal component, which showed association and genetic linkage at markers in four regions: tsc0045058, tsc1213381, tsc0055068, and tsc0051777 at map distances 53.26, 85.42, 89.31, and 172.86, respectively. These results show that the partial answer to our starting question for this brief analysis is that the NTTH phenotypes are not jointly genetically controlled. The component ntth1 displays marked genetic linkage. Recently, evidence has been found to relate alcoholism to genetic factors -4. The CThere are four NTTH phenotypes: ntth1, ntth2, ntth3, and ntth4. For the four correlated traits a natural question is, are they mediated by the same set of genes, or, is each separately genetically controlled? Here we attempt to find a partial answer to these questions.In view of our desire to determine whether ntth1, ntth2, ntth3, and ntth4 are separately or jointly genetically controlled, we shall study the principal components that explain most of the covariation of the four quantitative traits. We shall then analyze the promising components separately.For the association and linkage analyses, we used our Genetic Epidemiology Models (GEMs) package, which has routines for the regressive models for quantitative traits ,6. TypicThe proportion of the total variance explained by the first, second, third and fourth components are: 0.698, 0.195, 0.084, and 0.024, respectively.The factor loadings for the first and second principal components are listed in Table 0.338ntth1 + 0.515ntth2 + 0.569ntth3 + 0.545ntth4.Instead of analyzing the second principal component, which, explains 20% of the covariation, we analyzed ntth1, which is the driving factor. The third and fourth principal components account for little covariation so they were not analyzed. However, we also performed univariate linkage analysis on ntth2, ntth3, and ntth4 for comparison.The results of the association study for the first principal component are summarized in Figure -8 level. This is a far stronger result than those using the principal component combinations. Although Figures The association analysis results for ntth1 alone are displayed in Figure Table The linkage results for ntth1 alone are presented in Table The linkage analysis results for ntth2, ntth3, and ntth4 are presented in Table Our analysis of the four NTTH phenotypes, although brief, is revealing. The first principal component, which explains 70% of the covariation, showed association but not linkage to two markers: tsc0272102 and tsc0560854. On the other hand, ntth1, which was the trait driving the variation in the second principal component, showed association and linkage at markers in four regions: tsc0045058, tsc1213381, tsc0055068, and tsc0051777 at map distances 53.26, 85.42, 89.31, and 172.86 respectively.COGA: Collaborative Study on the Genetics of AlcoholismGAW14: Genetic Analysis Workshop 14SNP: Single-nucleotide polymorphismAY worked on the methodology, participated in the computations, and drafted the manuscript. VA implemented the computer program and participated in the computations. JPH participated in the analysis. RET participated in the analysis and interpretation of results. GEB helped to decide on the methodology, participated in the analysis, and assisted in writing the manuscript."}
+{"text": "Alcohol dependence is a typical example of a complex trait that is governed by several genes and for which the mode of inheritance is unknown. We analyzed the microsatellite markers and the Affymetrix single-nucleotide polymorphisms (SNPs) for a subset of the Collaborative Study on the Genetics of Alcoholism family sample, 93 pedigrees of Caucasian ancestry comprising 919 persons, 390 of whom are affected according to DSM III-R and Feighner criteria. In particular, we performed parametric single-marker linkage analysis using MLINK of the LINKAGE package (for the microsatellite data), as well as multipoint MOD-score analysis with GENEHUNTER-MODSCORE (for the microsatellite and SNP data). By use of two liability classes, different penetrances were assigned to males and females. In order to investigate parent-of-origin effects, we calculated MOD scores under trait models with and without imprinting. In addition, for the microsatellite data, the MOD-score analysis was performed with sex-averaged as well as sex-specific maps. The highest linkage peaks were obtained on chromosomes 1, 2, 7, 10, 12, 13, 15, and 21. There was evidence for paternal imprinting at the loci on chromosomes 2, 10, 12, 13, 15, and 21. A tendency to maternal imprinting was observed at two loci on chromosome 7. Our findings underscore the fact that an adequate modeling of the genotype-phenotype relation is crucial for the genetic mapping of a complex trait. Alcohol dependence occurs in many populations; it represents a complex trait with clear familial aggregation. It is more common in males than in females, and, in addition to social and psychological gender differences, genetic factors are supposed to act in a sex-specific way. In addition, genomic imprinting, which is also called parent-of-origin-effect, is assumed to play a role.Here, we focus on the microsatellite and the Affymetrix single-nucleotide polymorphism (SNP) markers typed for the Collaborative Study on the Genetics of Alcoholism (COGA) family sample . A set oBecause the prevalence of alcoholism is higher in males than in females, it can be expected that, on average, the penetrances of a particular susceptibility locus are higher in males as well. Hence, we used separate liability classes for males and females. In order to investigate the role of parent-of-origin effects, we calculated MOD scores under models with and without imprinting. In addition, for the microsatellite data, we performed linkage analyses by using sex-averaged as well as sex-specific genetic maps.We focused on the 93 Caucasian pedigrees of the COGA dataset (including families with at most three founders without information on ethnicity). This subset consists of 468 males (50.9%) and 451 females (49.1%). We looked at the phenotype ALDX1, which is based on DSM-III-R [We performed parametric exploratory linkage analysis with 315 microsatellite markers on the 22 autosomes. Marker allele frequencies were obtained by maximum likelihood estimation using MENDEL . Here, sWhen analyzing the microsatellite data, we selected the 'modcalc single' option under which a separate maximization over trait model parameters is performed for each genetic position of the putative trait locus. The penetrances for both sexes were varied. We used the sex-averaged as well as the sex-specific COGA marker map provided by Stassen . After rWe also used the 11,145 autosomal SNP markers of the Affymetrix GeneChip Human Mapping 10 K Array for a multipoint MOD-score analysis. Starting with the raw  data, the comprehensive quality control and data conversion was managed by ALOHOMORA . We applSingle-marker analysis using LINKAGE yielded suggestive evidence of linkage for two genetic regions. A LOD score of 2.51 was obtained for chromosome 7 at marker D7S1790 (19 cM), and a LOD score of 2.02 for chromosome 10 at marker D10S670 (135 cM). All other two-point LOD scores were below 2 (data not shown).p obtained by a MOD-score analysis has the largest variance of all trait-model parameters. Furthermore, in some cases, the estimated disease allele frequency will be markedly higher than the true value. This is due to the fact that specifying a higher disease allele frequency can compensate for a general model misspecification and hence lead to robustness in a multipoint analysis [A genome-wide plot of the multipoint results obtained with GENEHUNTER-MODSCORE for the microsatellite data is shown in Figure analysis .With MOD-score analysis under imprinting models, the most prominent linkage peak was obtained for chromosome 1 at 140 cM. When using the sex-averaged map, the MOD score reached 5.29; it further increased to 5.93 with the sex-specific map. The best-fitting penetrances point to a dominant model with nearly complete penetrance in females and a recessive model with strongly reduced penetrance in males. A MOD score of 4.11 (sex-averaged map) and 4.34 (sex-specific map) was obtained for chromosome 2 at 136 cM, with a recessive model in males and a paternal-imprinting model in females. On chromosome 7, at 118 cM, the MOD score was 3.30 for the sex-averaged map; it dropped to 2.46 (at 122 cM) when using the sex-specific map. Two peaks were seen on chromosome 10 with the sex-averaged map, a MOD of 3.27 at 34 cM and a MOD of 3.43 at 61 cM. With the sex-specific map, the first peak drops to 2.75, whereas the second peak increases to 3.73. The best-fitting model at the second peak is additive with complete homozygous-mutant penetrance for males and indicates complete paternal imprinting in females, albeit with a strongly reduced penetrance of 0.16. MOD scores of 3.85 (sex-averaged map) and 3.68 (sex-specific map) were found on chromosome 12 at 172 cM, with the trait model pointing to paternal imprinting. On chromosome 15 at 127 cM, the MOD score reached 3.45 with the sex-averaged map and 3.67 with the sex-specific map; there was evidence for complete paternal imprinting at this locus, with complete penetrance in females but almost no effect in males. Finally, on chromosome 21, a MOD score of 3.76 was obtained for the sex-averaged map at 43 cM and a MOD of 3.86 for the sex-specific map at 38 cM, with the best-fitting model pointing to paternal imprinting.In order to conclude whether imprinting is present at a certain locus or not, it is possible to look at the difference between the MOD scores obtained under four-penetrance trait models and under standard trait models with three penetrances. This strategy has been proposed in the context of a linkage study of house dust mite allergy . Here, wFigure Alcoholism is most likely governed by a considerable number of genetic factors, and so the contribution of a single gene is small. In addition, it is known that environmental factors play an important role as well. As with almost any complex trait, the mode of inheritance is unknown for alcohol dependence. Therefore, we took the approach of parametric exploratory linkage analysis. In particular, we performed single-marker LOD score analysis using MLINK under seven different trait models, and multipoint MOD-score analysis using GENEHUNTER-MODSCORE. The highest linkage signals were seen on chromosomes 1, 2, 7, 10, 12, 13, 15, and 21. The loci on chromosomes 2, 10, 12, 13, 15, and 21 yielded evidence for paternal imprinting. A tendency to maternal imprinting was observed at two loci on chromosome 7. For the microsatellites, several linkage peaks decreased with the sex-specific map, while others increased; the latter was the case for the loci on chromosomes 10, 15, 21, and most prominently chromosome 1. Daw et al. [Most of the linkage peaks shown in Table p-Values of MOD scores can be obtained by performing simulations for the studied dataset under the null hypothesis of no linkage. However, because p-values should be calculated especially for high MOD scores, many replicates need to be analyzed. With the COGA family sample, a substantial amount of computation time was already required for the MOD-score analysis of the original dataset; thus, analyzing many replicates for each of the loci identified here would not be feasible. Instead, we relied on criteria given by Weeks et al. [A MOD-score analysis represents one of the most comprehensive ways to analyze linkage data; we believe this procedure is particularly well suited for the genetic dissection of a complex trait. On the other hand, a MOD-score analysis is clearly exploratory, and so it is difficult to control the type I error. s et al.  and Hodgs et al. . They haCOGA: Collaborative Study on the Genetics of AlcoholismGAW14: Genetic Analysis Workshop 14SNP: Single-nucleotide polymorphismKS is the author of the GENEHUNTER-MODSCORE program; he drafted the manuscript and coordinated the realization of the study. RF performed the MOD-score analyses for the microsatellites and curated for the database. FR performed the SNP analyses including quality checks. CW did the analyses with LINKAGE and the selection of the dataset. JD implemented the use of sex-specific maps into GENEHUNTER-MODSCORE. AF contributed allele frequency estimates and selected the genetic maps used. MPB decided on the design of the study and contributed to the discussion of methodology and results. TFW is the group leader and coordinated the collaboration of the study; he decided on the analytical approaches and contributed to the discussion of methodology and results. All authors read and approved the final manuscript."}
+{"text": "Currently, a commonly used strategy for mapping complex quantitative traits is to use a genome-wide linkage analysis to narrow suspected genes to regions on a scale of centiMorgans (cM), followed by an association analysis to fine map the genetic variation in regions showing linkage. Two important questions arise in the design and the resulting inference at the association stage of this sequential procedure: (1) how should we design an efficient association study given the information provided by the previous linkage study? and (2) can an association in a linkage region explain, in part, the detected linkage signal?We derive a quantitative linkage score (QLS) based on Haseman-Elston regression (Haseman and Elston 1972) and make use of this score to address both questions. In designing an association study, the selection of a subsample from the linkage study sample can be guided by the linkage information summarized in the QLS. When heterogeneity exists, we show that selection based on the QLS can increase the proportion of sample individuals from the subpopulation affected by a disease allele and therefore greatly improves the power of the association study. For the resulting inference, we frame as a hypothesis test the question of whether a linkage signal in a region can be in part explained by a marker allele. A simple one sided paired t-statistic is defined by comparing the two sets of QLSs obtained with/without modeling a marker association: a significant difference indicates that the marker can at least partly account for the detected linkage. We also show that this statistic can be used to detect a spurious association.All our results suggest that a careful examination of QLSs should be helpful for understanding the results of both association and linkage studies. Identifying genes underlying complex quantitative traits, which are often heterogeneous and multifactorial, is still a great challenge in genetic epidemiology studies. Currently, a commonly used strategy for mapping complex traits is to use a genome-wide linkage analysis to narrow suspected genes to regions on a scale of centiMorgans (cM), followed by an association analysis to fine map the genetic variation in regions showing linkage. At the association stage of this sequential process, we are often interested in two questions: 1) how should we design a powerful and efficient association study given the information provided by the previous linkage study? and (2) can an association in a linkage region explain, in part, the detected linkage signal? Although these questions that arise respectively at the design and inference stages are two quite different aspects of an association study, they are related because both questions essentially rely on the interdependence of linkage and association. Here, we derive a quantitative linkage score (QLS) from Haseman-Elston linkage regression . -16. IBDiPL score  and the PL score .We consider selecting a set of unrelated individuals from sibships previously used for a QTL linkage analysis. In the case of a complex quantitative trait where heterogeneity exists, the goal of an association study is to detect a variant with maximum power. We emphasize that such a study would not be a classic epidemiologlcal study done to determine the attributable risk, for which subjects should be drawn randomly from a population. Rather, the study we discuss here is done for gene finding and therefore the selection of the sample should be done to provide maximum power rather than to represent the whole population.q1 and q2 respectively (q1 + q2 = 1), where the gene variant has an effect in only one subpopulation (P1). To examine the usefulness of the QLS in selecting a sample for an association study, we theoretically compare the proportions of individuals affected by a disease allele selected from a homogenous subpopulation (P1) in two selected samples: one sample is obtained by randomly selecting sibships (proportion qr) and the other is obtained by selecting sibships with QLS>0 (proportion qqls). To simplify the theoretical derivation, we assume known IBD sharing and sibships of size 2 (independent sibpairs).Suppose that a population consists of two subpopulations (P1 and P2) with proportions Tk = T, where the superscript T denotes transpose and the subscripts 1 and 2 indicate two sibs in a sibship. With the assumption of normal individual effects eik, T ~ N, whereLet Tk as Zk, so that the correlation matrix of Zk isTo further simplify the presentation, we standardize \u03c1k = 0, 0.5qr = q1 andwhere \u03c1IBD = 1 is the correlation between two sibs of a pair with proportion 1 IBD sharing. (see Appendix 1). It is obvious that qqls is always \u2265 qr. From this inequality, we can also see that the difference between qqls and qr depends on (1) the proportion of P1: when q1 = 0.5, the difference is maximum; and (2) the variance explained by the QTL: the difference is an increasing function of \u03c1IBD = 1, i.e. qqls and qr is presented in Figure where k is given byTo answer the question of whether a linkage signal in a region can be in part explained by a marker allele used in an association study, we compare the QLS on incorporating and not incorporating this marker into the trait model (equation 1), which we call the first  level regression, to distinguish it from the second (or family) level regression (equation 2). We frame this problem as a hypothesis test. When a marker is included in the model at the individual level, the variance-covariance matrix of sibship xik is a genotype code for the marker and b is its effect on the trait, which may arise from a \"true\" association (the marker is the QTL itself or is in linkage disequilibrium with the QTL), or from a \"spurious\" association (e.g. due to population stratification). Based on the above equation, we can obtain the corresponding QLS with the marker included in the above regression model, which is given bywhere b and k\u03bc, respectively. In the following presentation, we denote the QLS obtained with and without modeling an association marker n be the total number of sibpairs. The statistic is then defined bywhere t distribution with degrees of freedom n - 1. The one sided p-value is given by P(tn - 1 > T).and under the null hypothesis follows a b is expected to be small and therefore the statistic is likely to be close to zero. However, when there is an association between the marker and the quantitative trait in a statistical sense, but it is not related to the detected linkage (for example it is due to the well-known bias from population stratification), we may not expect the allelic effect b to be small. In this scenario, we may look upon the marker as a covariate representing to some extent population stratification, and therefore modeling this marker would reduce the residual variance of the trait similarity measure coming from population stratification, and hence strengthen the linkage signal. So we can expect the statistic T to be more likely to be negative, and our test statistic would maintain the type I error rate in a conservative fashion in the case of population stratification. Our simulation results agree with this line of reasoning (see results). In this sense, a small lower sided p-value, i.e. P(tn - 1 <T), indicates a spurious association, which is also seen in the simulations.It is useful to examine this statistic under various situations. When the marker modeled is not associated with the phenotype, the allelic effect b and the sibship mean are k \u03bcknown and so can be specified correctly; it can then be easily shown that for sibpair  in family k, For simplicity, assume the allelic effect \u03c72 distribution with one degree of freedom for an additive model.Because in practice the number of alleles shared IBD is generally not known with certainty, owing to partially informative markers and missing parental genotypes, we also performed computer simulations to examine the usefulness of the QLS in sample selection for an association study by comparing, in various situations, the statistics from random samples of unrelated individuals and from samples based on the rank order of the QLS. The statistic used to make the comparison is the score statistic proposed by Schaid et al. , which fIn our simulations, we generate 1000 sibships of size 2 from different subpopulations. A total of 6 markers, evenly space at a 2 cM density in a 10 cM range and each with 4 equally frequent alleles, are used for the linkage analysis. A QTL with 2 equally frequent alleles is located midway between marker 3 and marker 4. We assume Hardy-Weinberg equilibrium at each marker, linkage equilibrium among the markers and a Haldane no-interference map function. Trait values are constructed as the sum of a major-gene effect generated by the QTL, normal random individual effects, polygenic effects and common environmental effects. We calculate the probabilities of the number of alleles shared IBD using the program GENIBD in the S.A.G.E. package , removin\u03c72 is shown in Figure We first compare random selection and the QLS selection with different sample sizes for the association study. We assume the population consists of two subpopulations, in equal proportions, from which 1,000 sibpairs have been used for the linkage analysis. In subpopulation 1, 20% of the total variance is explained by the QTL, 30% by the polygenic and common environmental effects and the rest by a random individual effect. In subpopulation 2, there is no QTL effect but the same other effects are simulated. We separately sample 50, 100, 300, 500 and 800 unrelated individuals from the 1000 sibpairs by the two selection approaches and compare their score statistics. In QLS selection, we first select sibships with largest QLS and then randomly select one sib from each of these pairs, while in random selection the sibpair is selected randomly. The average \u03c72 values obtained based on any of the QLSs are greater than those from random selection and that they have small differences between them  are explained by the QTL in just one subpopulation. In this simulation, linkage signals are quite small and even cannot be detected. We sampled 100 unrelated individuals for an association study. The results show that the sampling based on the QLS still improves the power of an association study, even in the case that the power to detect linkage is negligible  and subpopulation 2 (0%). We compared the statistics of a commonly used joint linkage/association method (awbw) for a random sample and QLS based sample of families . The resTo assess the properties of our tests to determine whether an association is responsible in part for the linkage of a complex quantitative trait, we carried out a limited simulation study. We examined the type I error rate of the proposed test under two scenarios: 1) no trait-marker association and 2) trait-marker association due to population stratification. Under no trait-marker association, we simulated 10,000 replicate data sets of 500 sibpairs or 500 sibships . Trait values were constructed as the sum of a major-gene effect generated by the QTL that explains 10% of the variance, and various proportions of random individual, polygenic and common environmental effects. An association marker with two equally frequent alleles was simulated to be in complete linkage equilibrium with the QTL and a fully informative linkage marker  was also simulated at the same location. For the case of linkage but no trait-marker association, the results show that the type I error rate of the proposed statistic is generally good for a complex quantitative trait for both sibpair data and sibship data  traits. We illustrated the usefulness of this score to answer two different questions posed by an association study: (1) how to select samples at the design stage when heterogeneity exists; and (2) how to test at the inference stage whether an observed association can explain in part a previous linkage signal. In this paper, we are not necessarily advocating a two-stage approach to analyze family data on which we have information on both linkage markers and association markers. For such data a joint linkage and association framework could be of more interest than a two-stage analysis approach. Recent work on this kind of joint analysis has included work on both regression-based methods  and variThe idea of selecting families with linkage evidence for further genotyping in a follow-up association study is not new and has been successfully implemented in practice. In the context of quantitative traits, the proposed score can conveniently be used to summarize quantitative linkage information from a sibpair (or sibship). We have shown that in a heterogeneous population, which is expected to commonly occur for a complex trait, selecting a sample of unrelated persons based on the order of the QLS magnitude results in a more homogeneous sample for an association study than does a random sample, and therefore can improve power for a given sample size. Other approaches to identifying sibpairs with linkage are available, for example using a regression diagnostic . CarefulAnother use of the QLS investigated in this paper is to test whether association can account in part for a detected linkage. To address this question, we simply compare two sets of QLSs, before and after incorporating an association marker into the individual level regression model. Essentially, the proposed test evaluates the interaction of the allele effect of an associated marker and IBD sharing. In this sense it may be likened to other methods, for example the regression model proposed by Cardon , though In conclusion, as proved by our simulations, the QLS is useful for the design of, and resulting inference from, an association study following a linkage study. We suggest that careful examination of the QLS should be helpful for understanding the results of both association and linkage studies.TW contributed to the conception of the study, performed the simulation analysis, and wrote the manuscript. RCE contributed to the conception of the study and the writing of the manuscript.Appendix 1 the derivation of qlsqk comprising sib 1 and sib 2, Zk follows the distribution f , which we assume to be a bivariate normal distribution. With the assumption that a random sample of full sibpairs is used for the linkage analysis, the proportions of pairs for which the number of alleles shared IBD is 0, 1 and 2 are \u03c00 = 1/4, \u03c01 = 1/2 and \u03c02 = 1/4, respectively. Let P1 and P2 refer to subpopulation 1 and 2 and let their proportions be denoted q1 and q2. We then haveFor sibpair Thus,q1 and \u03c1. We note that \u03c1 depends on the size of the effect and allelic frequencies of the QTL. On the other hand,which is an increasing function of Pr = Pr(QlS > 0|P2)Pr(P2) = so that ThusAppendix 2 - E(Ub) (- Ua)yik = k \u03bc+ xikb + eik, we assume the eik are identically and independently distributed with mean 0. Suppose k \u03bcand b are known. Let the subscripts 1 and 2 indicate the two sibs of a sibpair in family k. ThenUnder the trait model"}
+{"text": "The design of appropriate strategies to analyze and interpret linkage results for complex human diseases constitutes a challenge. Parameters such as power, definition of phenotype, and replicability have to be taken into account in order to reach meaningful conclusions. Incorporating data on repeated phenotypic measures may increase the power to detect linkage but requires sophisticated analysis methods. Using the simulated Genetic Analysis Workshop 13 data set, we have estimated a variety of systolic blood pressure (SBP) phenotypic measures and examined their performance with respect to consistency among replicates and to true and false positive linkage signals.The whole-genome scan conducted on a dichotomous hypertension phenotype indicated the involvement of few true loci with nominal significance and gave rise to a high rate of false positives. Analysis of a cross-sectional quantitative SBP measure performed better, although genome-wide significance was again not reached. Additional phenotypic measures were derived from the longitudinal data using random effects modelling for censored data with varying levels of covariate adjustment. These models provided evidence for significant linkage to most genes influencing SBP and produced few false positive results. Overall, replicability of results was poor for loci, representing weak effects.Longitudinally derived phenotypes performed better than cross-sectional measures in linkage analyses. Bearing in mind the sample design and size of these data, linkage results that fail to replicate should not be dismissed; instead, different lines of evidence derived from complementary analysis methods should be combined to prioritize follow up. Lander and Kruglyak  proposedPhenotype definition is a critical factor that affects the ability to detect trait loci. The simulated data from Genetic Analysis Workshop 13 (GAW13) provide enough clinical and environmental information collected over time to generate several potential phenotypes aimed at detecting loci influencing systolic blood pressure (SBP). Incorporating data on repeated phenotypic measures may increase the power to detect linkage and will be the only way to detect genes that influence variation in traits over time. Obtaining longitudinal data is far more resource-intensive than collecting cross-sectional data. It is, therefore, important to know whether using longitudinal phenotypes does have more power than cross-sectional phenotypes. The GAW13 simulated data therefore provide an excellent opportunity to compare the ability of cross-sectional with longitudinal phenotypic measures to detect linkage to known loci.Our aim was to explore the benefits in terms of power, scientific insight, and replicability of linkage analysis findings of a model-based approach to phenotype characterization. Several different phenotypic models of SBP from the GAW13 simulated data were considered, including a dichotomous hypertension phenotype, a cross-sectional measure of SBP and longitudinal measures of 'trait' SBP, adjusted for various sets of covariates. Evidence for linkage for each phenotypic measure was examined. Irrespective of the phenotype studied, it is still considered important to replicate evidence of linkage in an independent data set. Therefore, we analyzed three replicates to examine the consistency of results. All analyses were done blind to the simulation conditions, which were provided at the workshop.Replicates 4, 10, and 21 were used in all analysis. Families with more than 20 individuals were excluded from the data set to reduce computational time. Two hundred and seventy seven pedigrees were analyzed with a mean of 11 members (minimum 7 and maximum 20 members). Of the 3155 subjects in the pedigrees, 33% had genotype data available.Six phenotype models describing SBP were defined; hypertension, a cross-sectional measure of SBP, and four models derived from longitudinal data, Models 1\u20134. Absence or presence of hypertension was used as a qualitative phenotype; individuals were defined as affected if they had a diagnosis of hypertension at any examination. Eighty-five families, containing enough affected members with genotype data, contributed to linkage analysis in each of the three replicates.Taking the SBP value at the first examination for each subject generated a cross-sectional measure of systolic SBP. No one in replicates 4, 10, or 21 was recorded as receiving antihypertensive treatment at the first time interval; therefore, no adjustment for treatment was necessary.Longitudinal SBP data were analyzed with a subject-specific approach . All syyit made on individual i at time t the model was specified as yit = \u03b2Xit + ui + eit, where Xit is a vector of covariates (including a constant) that may vary over time, \u03b2 is a vector of regression coefficients, ui is a N subject-specific random effect, eit are N disturbance terms with corr = 0 for all t and corr = 0 for s \u2260 t. For censored observations Pr[Y >yit] = \u03a6 ((\u03b2Xit + ui - yij) / \u03c3e,1), where \u03a6 is the standard normal cumulative density function.For an untreated SBP measurement }) were extracted for use as adjusted longterm SBP phenotypes for input to SOLAR [The empirical Bayes' estimates of the individual random effects  were tested for evidence of association with the hypertension phenotype. The PDT method [Microsatellite markers under areas of increased allele sharing (T method  was usedNo NPL scores achieving genome-wide significance were detected using hypertension as a trait. All replicates produced linkage peaks with a NPL score > 1. The most significant results were on chromosomes 15 (109 cM), 18 28\u201370 cM) and 21 (23\u201350 cM) for replicates 4, 10, and 21, respectively. The former two loci did not contain true trait loci. There was very little consistency between replicates, with only chromosome 21 showing some evidence of linkage in more than one replicate. Table 8\u201370 cM aAll of the phenotypic measures of SBP were highly heritable (>70%) and are listed in Table There was little overall consistency in loci detected between replicates as shown in Table p < 0.05) using the sum and/or average PDT result. Of these, two mapped to true disease loci near Gb35 and Gs12.To provide further evidence of linkage for potential linkage regions, microsatellite markers mapping to weak regions of linkage on chromosomes 2, 5, 10, 11, 13, 15, 16, 20, and 21 were tested for evidence of association to hypertension using the PDT method. Seven markers showed evidence of preferential transmission .The categorical hypertension phenotype showed the least evidence of linkage to any of the true loci. Two of the most significant regions of linkage identified using hypertension as a qualitative trait  only showed any evidence for linkage in a single replicate and were not consistent with any other measure of SBP. These loci are in fact false positive results. The relatively poor performance of the hypertension phenotype may, however, be related to the general population sample design that yielded a rather small effective sample size (85 families per replicate). To overcome the loss of power inherent to such a study design, the PDT method was employed to provide complementary evidence toward the involvement of the implicated chromosomal regions. The quantitative cross-sectional measure derived from SBP at first exam detected three genes at suggestive evidence of linkage, one with a LOD score 1.8, and produced three false positive results on chromosomes 5, 8, and 12. None of the LOD scores reached genome-wide significance and there was little consistency between replicates. The longitudinally derived phenotypes were superior to the cross-sectional measure both in terms of the number of loci detected and the magnitude of the evidence for linkage. These phenotypes were able to detect five loci influencing SBP at suggestive evidence of linkage, four of these at a genome-wide significance level. In general, LOD scores also improved with adjustment for covariates, although this was not always the case, suggesting that using all the models provided the most information about linkage at any given locus.Because this analysis was performed blind to the answers, it was not possible at the time to determine which linkage peaks corresponded to true trait loci. This made the initial interpretation of the results difficult because many of the LOD scores were modest and there were inconsistencies in results between replicates. Deciding which loci are true or false is a difficult problem for researchers when faced with real whole-genome scan results. We therefore prioritized loci more likely to be true using the following criteria: evidence of linkage in more than one replicate, evidence of linkage with more than one phenotype, and evidence of linkage and association with a nearby microsatellite marker. Having followed this process, we concluded that we would suggest that chromosome 21 harbors at least one locus influencing BP and we would predict that the regions on chromosomes 5, 11, and 13 would also be worthy of further investigation. Interestingly, the chromosome 21 loci, detected through the use of each of the phenotypic measures, cumulatively accounted for 54% of the variance. We would therefore have followed up all the true loci except Gb7, which we did not detect, and Gs11, because it produced evidence for linkage in just a single replicate. We would also have followed up one false positive result (chromosome 11) and not followed up the one on chromosome 12.a priori scientific case for them to be less convincing. It is, therefore, perhaps surprising that we were able to detect the three slope genes influencing SBP with such high LOD scores. Two of these slope genes mapped to chromosome 21; Gb37 also mapped to the same locus as Gs12 and explained 40% of the variance of diastolic blood pressure. Because diastolic blood pressure directly affects SBP, it is not clear exactly what the linkage is picking up on chromosome 21.The aim of using the standardized residuals from the longitudinal BP models was to increase power to detect linkage to SBP. We used a random intercept residual and did not explore using random slope residuals that might have detected genes for late onset high SBP. While such residuals are easily estimable within our approach, we considered the This analysis of the GAW13 simulated data has demonstrated that longitudinally derived phenotypes have more power to detect baseline and slope genes than cross-sectional measures of the same trait, thus suggesting that they are likely to be essential for many genetic studies. The successful identification of all but one true trait loci using GLLAMM indicates that it is an effective method for deriving powerful longitudinal phenotypes. Furthermore, the limited overlap observed among replicates suggests that unreplicated linkage results should not be lightheartedly dismissed, especially when they may represent weak effects, as is the case in complex diseases. Complementary evidence derived from diverse data analyses should, therefore, drive the prioritization of linkage peaks for follow-up, in order to maximize use of the available resources to detect real disease genes."}
+{"text": "A standard multivariate principal components (PCs) method was utilized to identify clusters of variables that may be controlled by a common gene or genes (pleiotropy). Heritability estimates were obtained and linkage analyses performed on six individual traits , high and low density lipoproteins, triglycerides (TG), body mass index (BMI), and systolic blood pressure (SBP)) and on each PC to compare our ability to identify major gene effects. Using the simulated data from Genetic Analysis Workshop 13 (Cohort 1 and 2 data for year 11), the quantitative traits were first adjusted for age, sex, and smoking (cigarettes per day). Adjusted variables were standardized and PCs calculated followed by orthogonal transformation (varimax rotation). Rotated PCs were then subjected to heritability and quantitative multipoint linkage analysis. The first three PCs explained 73% of the total phenotypic variance. Heritability estimates were above 0.60 for all three PCs. We performed linkage analyses on the PCs as well as the individual traits. The majority of pleiotropic and trait-specific genes were not identified. Standard PCs analysis methods did not facilitate the identification of pleiotropic genes affecting the six traits examined in the simulated data set. In addition, genes contributing 20% of the variance in traits with over 0.60 heritability estimates could not be identified in this simulated data set using traditional quantitative trait linkage analyses. Lack of identification of pleiotropic and trait-specific genes in some cases may reflect their low contribution to the traits/PCs examined or more importantly, characteristics of the sample group analyzed, and not simply a failure of the PC approach itself. Principal component analyses often provide valuable information that allows data reduction and reveals relationships between variables that were not previously suspected. As we begin to better understand the scope of gene effects, we find that single genes often contribute to multiple phenotypes (pleiotropy). Therefore, when mapping genes for complex disorders, it can be helpful to identify groups of variables or phenotypes  that may be controlled by a single gene. Arya et al.  demonstrn = 27) were excluded in order to obtain valid low density lipoprotein (LDL) calculations. In addition, several (n = 8 > 4 SD) observations were excluded because they were judged to be highly influential in the PC analysis.All analyses were performed on the simulated data without missing observations. Replicate data set 57 was randomly selected and analysis was limited to the year 11 time point. Year 11 was selected because this was the first year in which Cohorts 1 and 2 both had data collected. Observations with triglyceride values greater than 400  were log-transformed in order to better conform to a normal distribution. Each QT was then regressed on sex, age, and cigarettes per day using linear regression modeling, and residuals were obtained. The residuals for each QT were then standardized. PCs were calculated from the correlation matrix of the standardized residuals corresponding to the six QTs using standard methods, in which all individuals are assumed to be independent. PC analysis was performed using PROC FACTOR in the SAS statistical software package , with PC extraction and varimax rotation : total cholesterol (Chol), triglycerides (TG), high density lipoprotein (HDL), LDL, systolic blood pressure (SBP), and body mass index (BMI). LDL was calculated using the Friedewald's equation :  . GenotypWe did not consult the GAW13 simulated data set answers prior to either the interpretation of the PCs or performing linkage analysis. Verification of genes modeled in the simulated data set at baseline  were considered verified if linkage analysis identified a marker with a peak LOD score (LOD > 1.0) within 20 cM of the gender-averaged chromosomal location for a simulated trait gene. While there is little consensus regarding the most appropriate LOD score threshold for complex disease, similar to other studies of complex disease reporting LODs less than 2.0, we considered LOD scores greater than 1.0 as suggestive evidence of linkage ,6.At year 11 we had complete data on 989 individuals (316 families) from Cohort 1, mean age 59.9 years, and 1511 individuals (330 families) in Cohort 2, mean age 53.4. Variable means for the QTs and confounders were comparable between cohorts, except for SBP, TGs, and cigarettes per day, where mean SBP and TG were higher in Cohort 1 than 2  and mean cigarettes per day were lower in Cohort 1 than 2 . After adjustment for age, sex, and cigarettes per day, cohort was a statistically significant predictor of only one of the QTs: SBP. The additional adjustment for cohort produced results (PCs and linkage) that were similar to those reported and did not change any of our conclusions.p < 0.0001), ranging from 0.60 for LDL to 0.79 for BMI . Two of the three LODs in this range were false-positive results according to our criteria, while the third LOD identified a minor gene (b10) contributing 1% of trait variation for height  were adjusted prior to PC analysis, consistent with the strategy used by Moser et al., although concerns about the effect of these adjustments on PC and heritability estimates arose [before and after PC analysis [data not shown] and found no significant differences in PCs, loading, or heritability. Overall, the PC analysis, in particular PC2, reflected the pleiotropic genes (HDL and TG) modeled in the simulated data.While our analysis was somewhat limited in terms of the number of variables available in the Heritability estimates were statistically significant for each of the three major PCs, as were those for the traits evaluated individually. Each PC heritability estimate was consistent in magnitude with the trait heritabilities comprising the PC. PC2, which reflected the simulated model best with respect to shared gene effects, had a heritability estimate slightly higher than the two individual variables (HDL and TG) in the PC and closer to that for BMI alone. This higher heritability estimate for PC2 may reflect the accuracy with which PC identifies/groups variables with common genetic influence or it may reflect the significant influence of BMI on this PC.Several factors that may have contributed to limited power in both our individual trait and PC linkage analyses include sample size and composition (single replicate), pedigree structure, and the number and size of genetic effects. One of the challenges facing linkage mapping for complex disease traits is adequate sample size. Risch and Merikangas state that the power of linkage for complex disease is limited to the detection of only the strongest loci unless thousands of small families are utilized . In thisStudies have shown the PC approach may improve the power to identify genes with pleiotropic effects involved in complex disease ,9,10. WhIn summary, PC analysis has been demonstrated in reported studies of complex disease to localize regions of the human genome likely to contain pleiotropic genes , but may"}
+{"text": "FLJ40852 gene , and SNP rs1859646 is located in the TAS2R5 gene (a taste receptor). The other four SNPs are not located in any known or annotated genes. We found the high density SNP scan to be superior to microsatellites because it is effective in downstream fine mapping due to a better defined linkage region. Our study proves the utility of high density SNP in genome-wide mapping studies.We performed linkage and linkage disequilibrium (LD) mapping analyses to compare the power between microsatellite and single nucleotide polymorphism (SNP) markers. Chromosome-wide analyses were performed for a quantitative electrophysiological phenotype, ttth1, on chromosome 7. Multipoint analysis of microsatellite markers using the variance component (VC) method showed the highest LOD score of 4.20 at 162 cM, near D7S509 (163.7 cM). Two-point analysis of SNPs using the VC method yielded the highest LOD score of 3.98 in the Illumina SNP data and 3.45 in the Affymetrix SNP data around 152\u2013153 cM. In family-based single SNP and SNP haplotype LD analysis, we identified seven SNPs associated with ttth1. We searched for any potential candidate genes in the location of the seven SNPs. The SNPs rs1476640 and rs768055 are located in the  Current strategy for complex disease gene mapping usually includes three stages. A genome-wide scan using microsatellite markers is performed to identify interesting chromosomal regions harboring the susceptibility loci. Then fine mapping is used as a follow-up to confirm and narrow the interesting regions. Finally, single nucleotide polymorphism (SNPs) are used to further saturate the regions and discover the candidate genes.Genetic Analysis Workshop 14 (GAW14) provided data from the Collaborative Study on the Genetics of Alcoholism (COGA), including genome-wide microsatellite markers, genome-wide SNPs and several alcoholism-related phenotypes. This data allowed us to compare the power to detect susceptibility loci between SNPs and microsatellite markers in the context of genome-wide linkage and linkage disequilibrium (LD) analyses. We particularly chose a quantitative electrophysiological phenotype, ttth1 , as our phenotype of interest because a strong linkage signal was previously detected on chromosome 7 [The COGA data set provided to GAW14 includes 1,350 members with genotype and phenotype information in 143 families. We used the quantitative data of ttth1, microsatellite markers, and two SNP panels (Illumina and Affymetrix panels) on chromosome 7 for our linkage and LD mapping analyses. First, a total of 31 microsatellite markers, at average inter-marker distance of 6.23 cM on chromosome 7, were used for chromosome-wide scan to identify the interesting regions for ttth1. Two-point and multipoint analyses of microsatellite markers were conducted using the variance component (VC) method implemented in the SOLAR [For microsatellite markers, two-point analysis showed the highest LOD score at D7S509 163.7 cM): unadjusted LOD = 2.70 and age- and sex-adjusted LOD = 3.83. The highest age- and sex-adjusted multipoint LOD score was 4.20 at 162 cM, with a 1-LOD support interval between 150 and 168 cM  in the Illumina SNP panel  haplotypes ranged from 0.002 to 0.007. The significant haplotype blocks were located at 150.2 cM , 153.036 cM (tsc0058416 and tsc0058418), and 153.912 cM (tsc0590614 and tsc0590615). Comparing the results of the single SNP analysis with SNP haplotype analyses, suggested that the risk-bearing haplotype can be primarily ascribed to one SNP. However, the flanking SNP in the haplotype also contributed additional information leading to a more significant p-value. We further searched the SNP database using CHIP Bioinformatics Tools  to find related information of the three closely linked haplotype blocks. The search showed: rs1476640 and rs768055 are located in the introns of the FLJ40852 gene, which is a hypothetical protein with unknown gene function, and rs1859646 is located in the intron of the TAS2R5 gene, which is a taste receptor. The biological relevance of the three SNPs  were unknown. The other four SNPs  are not located in any known or annotated genes.Our family-based LD analysis focused on the 1-LOD support interval. A total of 40 Affymetrix SNPs and 24 Illumina SNPs were in this interval. Single SNP LD analyses showed significant associations on three Illumina SNPs and two Affymetrix SNPs  by either linkage- or family-based LD analysis. Our joint SNP linkage and LD mapping pinpointed a critical region between 150 and 154 cM, which is much smaller than the 1-LOD region by microsatellite markers. Using two different SNP panels, we found that the highest LOD scores and their locations are very close. Using family-based single SNP and SNP haplotype LD analyses, we further identified seven SNPs associated with phenotype ttth1. Our results indicated that the haplotype analysis may be more power than single SNP LD mapping in this dataset. Among them, three SNPs  are located within two potential genes, Although MERLIN has the advantage of faster speed than SOLAR in analyzing SNP data, it cannot effectively handle large pedigrees when analyzing microsatellite markers. In this study, we had to increase the default 24 bits to 40 bits while using MERLIN for SNP analysis. In this way, we analyzed all families with MERLIN, but the bit increase is not unrestricted and it may be a problem for even larger pedigree sizes. While we obtained identical results from SOLAR and MERLIN, MERLIN provided results in several hours, while SOLAR required several days.Three recent studies comparing SNP and microsatellite analysis reported findings similar to ours: high-density SNPs defined a better critical region than microsatellite markers -8. John This study found that SNP panels provide sufficient meiotic information for linkage analysis. The high-density SNP genome scan is more effective for fine mapping and LD mapping due to a better definition of the linkage region. Multipoint analysis of microsatellite markers showed strong linkage evidence within a 1-LOD support interval from 150 to 168 cM on chromosome 7. Two-point analyses of SNPs showed the highest LOD scores of 3.98 and 3.45 around 153 cM for Illumina and Affymetrix SNP data, respectively. We identified seven SNPs associated with ttth1 in the candidate region harboring potential susceptibility loci using family-based single SNP and SNP haplotype LD analysis.COGA: Collaborative Study of the Genetics of AlcoholismGAW14: Genetic Analysis Workshop 14LD: Linkage disequilibriumRA: Rheumatoid arthritisSNP: Single-nucleotide polymorphismVC: Variance componentH-FL performed linkage analyses and summarized the results. RC was responsible for data management, family-based LD analyses and manuscript preparation. S-HHJ designed and oversaw the project and prepared the manuscript. All authors read and approved the final manuscript."}
+{"text": "Using data provided by the Collaborative Study on the Genetics of Alcoholism we studied the genetics of a quantitative trait: the maximum number of drinks consumed in a 24-hour period. A two-stage method was used. First, linkage analysis was performed, followed by association analysis in regions where linkage was detected. Additionally, the extent of linkage disequilibrium among single-nucleotide polymorphisms (SNP) associated with the phenotype was assessed. Linkage to chromosomes 2 and 7 was detected, and follow-up association analysis found multiple trait-associated SNPs in the chromosome 7 linkage region. Chromosome 4, which has been implicated in previous studies of the maximum drinks phenotype, did not pass our threshold for linkage evidence in stage 1, but secondary analyses of this chromosome indicated modest evidence for both linkage and association. The evidence suggests that chromosome 7 may harbor an additional locus influencing the maximum drinks consumption phenotype. The data provided by the Collaborative Study on the Genetics of Alcoholism (COGA) for Genetic Analysis Workshop 14 (GAW14) includes the \"maximum number of drinks consumed in a 24-hour period.\" This phenotype is closely related to alcoholism diagnosis, and a previous genome screen of this phenotype in COGA resulted in evidence for linkage to chromosome 4 in the vicinity of the alcohol dehydrogenase (ADH) gene cluster [In defining the quantitative trait, we set the phenotype of individuals who report a maximum drinks value of zero to be unknown, since these unexposed individuals have undetermined response to alcohol. We reduced skewness by taking the logarithm of the maximum number of drinks. A linear regression was used to correct for sex, and the final trait value was defined as the residual from the regression.Our primary analysis consisted of two stages, applied to the cleaned SNP data. In the first stage we tested for genetic linkage using the SNP marker data provided by Illumina. Nonparametric two-point quantitative trait linkage analysis was performed using the \"--qtl\" option of the program MERLIN . The metTo supplement our primary two-stage analysis, we carried out some related additional analyses. These are described below.For comparison with the stage 1 SNP linkage screen, linkage analysis of the autosomal microsatellite data was performed using the same two-point nonparametric analysis as above. We also repeated this microsatellite analysis using a 2-cM multipoint grid because LD among these markers was not a significant concern.For chromosome 4, prior linkage evidence for the maximum drinks phenotype has been reported , making D' = D/|D|max, where D = p11 - p1 p2, p11 is the frequency for the haplotype containing allele 1 at both markers, pi is the frequency of allele 1 at the ith marker of the pair, and |D|max is the maximum possible (absolute) value of D given the marker allele frequencies.LD between markers should be considered when using SNP data to search for disease or trait association. Thus, SNPs exhibiting trait association in our primary analysis that were also in close proximity to one another were tested to see if they were in LD with each other. Pair-wise LD coefficients were computed using the program LDMAX, which is part of the GOLD computer package ; resultsOut of a sample of 1,614 individuals, the maximum number of drinks was recorded for 1,388 (86%) individuals. The mean, median, and mode were 17.8, 12, and 24, respectively, with a standard deviation of 17.3. Due to computer memory constraints, 9 out of 143 families (6%), consisting of 243/1,614 (15%) individuals, were automatically dropped by MERLIN from the linkage analysis in stage 1, and this reduction in sample is a limitation of our analyses. The average maximum drinks value in the dropped families was slightly lower than the overall average (by 0.16 of a standard deviation); this difference does not appear to have a systematic cause and is not expected to affect the interpretability of the results.We used 4,710 markers from the Illumina SNP set. The mean spacing between markers was 0.77 cM on the genetic map and 621 kb on the physical map, and the mean minor allele frequency (MAF) was 0.39. We used 11,120 markers from the Affymetrix SNP set. The mean spacing was 0.32 cM , and the mean MAF was 0.27.In our primary analysis, only chromosomes 2 and 7 were found to have LOD scores over 1.8 at stage 1. Tables D' for the significantly associated SNPs on chromosomes 2 and 7  and chromosome 15 at D15S230  and D15S642 . For the multipoint analysis, the only LOD score over 1.0 was 1.49, which occurred on chromosome 15 at 142 cM. Thus, in a direct comparison of the two-point screens using SNPs versus microsatellites, it is curious that higher LOD scores were detected with the SNPs than with the microsatellites, even though the SNPs are individually less informative. It may be the density of the SNP map that was beneficial and allowed the detection of linkage evidence that was missed by the microsatellite screen. To examine this hypothesis, we compared the locations of the trait-linked SNP markers on chromosomes 2 and 7 to the locations of the microsatellite markers using integrated map information from the National Center for Biotechnology Information (NCBI) [Note also that in the microsatellite screens, the two-point analysis in many instances found higher LOD scores than the multipoint analysis. For example, the two-point analysis found weak evidence on chromosome 7 with a LOD score over 1 at 116.6 cM , while multipoint LOD scores did not reach 1 on chromosome 7 (the maximum LOD was 0.89 at 140 cM).Our two-stage genome-wide analysis implicates chromosomes 2 and 7 as potentially harboring loci influencing the maximum drinks consumption phenotype. Follow-up association analysis supports chromosome 7 more strongly than chromosome 2, and suggests that further fine-mapping efforts on chromosome 7 may be constructive. Interestingly, this region of chromosome 7q contains several TAS2R bitter taste receptor genes . The musChromosome 4, which has been implicated in a previous linkage study of maximum drinks, did not pass our threshold for linkage evidence in the first stage of our primary analysis, but additional analyses of this chromosome indicated modest evidence for both linkage and association. In Saccone et al.  linkage LD between markers is a concern when using multipoint linkage analysis, and our results show there is strong LD among SNPs on chromosome 7 which were found to be associated with maximum drinks. As an additional analysis we studied global LD structure using an algorithm we have previously described  to produCOGA: Collaborative Study on the Genetics of AlcoholismGAW14: Genetic Analysis Workshop 14LD: Linkage disequilibriumMAF: Minor allele frequencyNCBI: National Center for Biotechnology InformationQPDT: Quantitative pedigree disequilibrium testSNP: Single-nucleotide polymorphismSFS performed all statistical analyses and is the author of the program used to find LD blocks. All authors assisted in the design of the study and interpretation of results. In particular, NLS and JPR assisted in the design of the LD block program. SFS and NLS drafted the manuscript with input from all authors, and all authors read and approved the final manuscript."}
+{"text": "MLH1 germ line mutation.The apparent dominant model of colorectal cancer (CRC) inheritance in several large families, without mutations in known CRC susceptibility genes, suggests the presence of so far unidentified genes with strong or moderate effect on the development of CRC. Linkage analysis could lead to identification of susceptibility genes in such families. In comparison to classical linkage analysis with multi-allelic markers, single nucleotide polymorphism (SNP) arrays have increased information content and can be processed with higher throughput. Therefore, SNP arrays can be excellent tools for linkage analysis. However, the vast number of SNPs on the SNP arrays, combined with large informative pedigrees (e.g. >35\u201340 bits), presents us with a computational complexity that is challenging for existing statistical packages or even exceeds their capacity. We therefore setup a procedure for linkage analysis in large pedigrees and validated the method by genotyping using SNP arrays of a colorectal cancer family with a known Quality control of the genotype data was performed in Alohomora, Mega2 and SimWalk2, with removal of uninformative SNPs, Mendelian inconsistencies and Mendelian consistent errors, respectively. Linkage disequilibrium was measured by SNPLINK and Merlin. Parametric linkage analysis using two flanking markers was performed using MENDEL. For multipoint parametric linkage analysis and haplotype analysis, SimWalk2 was used.MLH1-region, a LOD score of 1.9 was found by parametric linkage analysis using two flanking markers. On chromosome 11 a small region with LOD 1.1 was also detected. Upon linkage disequilibrium removal, multipoint linkage analysis yielded a LOD score of 2.1 in the MLH1 region, whereas the LOD score dropped to negative values in the region on chromosome 11. Subsequent haplotype analysis in the MLH1 region perfectly matched the mutation status of the family members.On chromosome 3, in the We developed a workflow for linkage analysis in large families using high-density SNP arrays and validated this workflow in a family with colorectal cancer. Linkage disequilibrium has to be removed when using SNP arrays, because it can falsely inflate the LOD score. Haplotype analysis is adequate and can predict the carrier status of the family members. Already in 1913, familial aggregation of CRC was described by Warthin [l cancer . Clinical cancer  was instl cancer ,6. This er [MSH2  and MLH1H2 [MLH1 , respectS2 [MSH6 ,11 and r6 [MutYH  were ide6 [MutYH . Further6 [MutYH . This in6 [MutYH -20.Families with a clustering of colorectal cancer but without germ line mutations in CRC genes have been under surveillance in Leiden since the 1980s. Due to the long period of follow-up, with three to four affected generations, these Dutch HNPCC-like families have become informative for linkage analysis.Traditionally, linkage analysis is performed with multi-allelic microsatellite markers. Recently, however, the more advanced single nucleotide polymorphism (SNP) arrays were brought into use for linkage analysis. It was shown that the information content of a dense SNP map is significantly and uniformly higher than that of a genome wide microsatellite marker map . SeveralStudying large families with thousands of SNPs results in a computational complex analysis that is challenging for existing statistical packages and that may even exceed their capacity. Current linkage analysis programs can handle either large pedigrees or large numbers of markers, depending on the underlying algorithm. In order to perform linkage analysis in large pedigrees using SNP arrays, we explored the possibilities of currently available linkage analysis software. Most currently available programs are based on the Lander-Green or the Elston-Stewart algorithm or both. The computation time of the former algorithm increases exponentially with the number of bits  in a pedigree, whereas the latter scales exponentially with the number of markers. To perform multipoint linkage analysis in a large family with SNP arrays in one run would probably take several months computation time, if at all possible.Several programs are suitable for linkage analysis with bi-allelic markers. Genehunter and Merlin can handle a relative large numbers of markers, however the analysis is restricted to pedigrees of up to ~30-bits ,26. BothMLH1 germ line mutation on chromosome 3.MENDEL  is a proMLH1 gene  was studied. Nine family members are affected with colorectal cancer. Another two family members are affected with polyps and three cases with skin cancer (non-specified) and one case with endometrium cancer (non-specified) are seen as well. Peripheral blood lymphocytes were collected from the family members. DNA was extracted using standard procedures. A total of thirteen family members were genotyped on Affymetrix GeneChip Human Mapping 10K 2.0 SNP arrays. The arrays were processed according to the instructions of the manufacturer. The mean SNP call rate was 96.3% (89.0%-98.5%).A large colorectal cancer family Figure  with a rThe study was approved by the Medical Ethical Committee of the LUMC (protocol P01-019).We processed the data according to the following workflow: 1) First, the genotype data were generated by GeneChip DNA Analysis Software (GDAS) from Affymetrix. 2) These genotype data were combined with the pedigree and the marker information in Alohomora. 3) In this program the uninformative SNPs were removed as well. 4) To be able to perform linkage analysis in the desired program, the output files (in Merlin-format) of Alohomora were by Mega2 converted to the proper format. 5) Mega2 also removed the Mendelian inconsistent errors. 6) The files were then ready to perform parametric linkage analysis using 2 flanking markers in MENDEL; affected-only analysis as well as parametric linkage analysis using liability classes was performed. 7) Based on the second analysis, regions of interest were defined that were further tested for Mendelian consistent errors and 8) possible linkage disequilibrium was removed in SNPLINK. 9) Multipoint parametric linkage analysis using the liability classes was then performed in Simwalk2 for the ROIs and 10) finally, the haplotypes were inferred in Simwalk2.Genotype data of the individual family members were generated using GeneChip DNA Analysis Software (GDAS) from Affymetrix. In the Alohomora program  the pediMendelian consistent errors were identified by mistyping analysis. Since this analysis is computationally complex and therefore time consuming (2 1/4 hours for 35 SNPs), only the regions of interest were analyzed for Mendelian consistent errors. All chromosomal regions with LOD scores exceeding 1 and lacking negative LOD scores were defined as regions of interest (ROI). SimWalk2  was used2, as a measure of linkage disequilibrium (LD) between adjacent SNPs, was estimated using SNPLINK [2, we split the large family into nuclear families. In addition to the family under study, genotypes from 12 Dutch nuclear families from other studies (unpublished results) were used to calculate LD. The program SNPLINK provides a list of SNPs to be removed. We used as cut off value for LD removal an r2 \u2265 0.4. The information content was computed before and after removal of the SNPs using Merlin.In the ROI the pair-wise correlation coefficient r SNPLINK  and Merl SNPLINK . Since wMLH1 family, we performed a simulation study using Simlink [To determine the power to detect linkage in the  Simlink  under th Simlink .In MENDEL , an affeMLH1 in this family.Haplotype analysis was performed in the ROI, using SimWalk2. All SNPs in the region of interest (~18) were included in this analysis (computation time: 1 1/3 hours for 18 SNPs). The results of the haplotyping were visualized in HaploPainter . The hapLinkage analysis using bi-allelic genotype data from SNP arrays and large families is a computational challenge using commonly used, freely available analysis software. For the different steps of the linkage analysis; e.g. data formatting, detection of Mendelian inconsistencies, mistyping analysis, LD removal and single to multipoint linkage analysis, we have chosen the following programs that can handle large pedigrees and many SNPs where required; Alohomora , Mega2 , MENDEL In advance of the linkage analysis we performed a simulation study to calculate the power using Simlink. The mean LOD score in 1000 simulations in this family was 2.0.The Alohomora program  was usedSince errors in genotyping can easily mask linkage, the data were checked for different types of errors. First, we have estimated the genotyping error rate in five duplicate experiments. The mean genotyping error rate between the duplicates was only 0.0051.Mega2 was then used for several data validation checks, including errors in the pedigree data or Mendelian inconsistent errors. Mega2 was used since it supports 28 different programs, including the programs MENDEL and SimWalk2, which we have used for linkage analysis and haplotype analysis. The genotypes of 18 SNPs (0.21%) were removed from analysis, because of Mendelian inconsistencies. However, with bi-allelic markers not all errors appear as Mendelian inconsistent errors . The datMLH1 gene on chromosome 3 .Current linkage analysis programs assume LD between markers and a disease locus and importantly, linkage equilibrium between markers. The presence of linkage disequilibrium between two markers can falsely inflate the LOD score and missing genotypes can increase this effect. Therefore, the rMLH1-mutation carriers share the same haplotype, as well as the affected obligate carriers. Therefore, this haplotype perfectly co-segregates with the clinical phenotype of the family members  declare that they have no competing interests.AM performed SNP arrays and mutation analysis, statistical analyses and drafted the manuscript. SJC and QH assisted in the statistical analysis, and QH performed the LD analysis. HMVDK performed SNP arrays. CMJT provided DNA samples and mutation status. HFAV was responsible for family recruitment and surveillance. PD participated in study design. JJHD supervised the statistical analysis, JTW, HM and TVW designed and coordinated the study, TVW helped to draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:"}
+{"text": "Eucalyptus have been localized on pedigree-specific RAPD or AFLP maps seriously limiting the value of such QTL mapping efforts for molecular breeding. The availability of a genus-wide genetic map with transferable microsatellite markers has become a must for the effective advancement of genomic undertakings. This report describes the development of a novel set of 230 EMBRA microsatellites, the construction of the first comprehensive microsatellite-based consensus linkage map for Eucalyptus and the consolidation of existing linkage information for other microsatellites and candidate genes mapped in other species of the genus.Eucalypts are the most widely planted hardwood trees in the world occupying globally more than 18 million hectares as an important source of carbon neutral renewable energy and raw material for pulp, paper and solid wood. Quantitative Trait Loci (QTLs) in Eucalyptus, involves 234 mapped EMBRA loci on 11 linkage groups, an observed length of 1,568 cM and a mean distance between markers of 8.4 cM. A compilation of all microsatellite linkage information published in Eucalyptus allowed us to establish the homology among linkage groups between this consensus map and other maps published for E. globulus. Comparative mapping analyses also resulted in the linkage group assignment of other 41 microsatellites derived from other Eucalyptus species as well as candidate genes and QTLs for wood and flowering traits published in the literature. This report significantly increases the availability of microsatellite markers and mapping information for species of Eucalyptus and corroborates the high conservation of microsatellite flanking sequences and locus ordering between species of the genus.The consensus map covers ~90% of the recombining genome of Eucalyptus comparative genomics, opening stimulating perspectives for evolutionary studies and molecular breeding applications. The generalized use of an increasingly larger set of interspecific transferable markers and consensus mapping information, will allow faster and more detailed investigations of QTL synteny among species, validation of expression-QTL across variable genetic backgrounds and positioning of a growing number of candidate genes co-localized with QTLs, to be tested in association mapping experiments.This work represents an important step forward for  E. globulus is the premier species for temperate zones plantations in Portugal, Spain, Chile and Australia, elite hybrid clones involving E. grandis and E. urophylla are extensively used by the pulp and paper industry in tropical regions of Brazil, South Africa, India and Congo because of its wood quality, rapid growth, canker disease resistance and high volumetric yield [Eucalypts are the most widely planted hardwood trees in the world occupying globally more than 18 million hectares [ic yield .Pinus taeda [Populus [Genetic mapping became accessible to several forest tree species in the beginning of the 90's based on the combination of the speedy and inexpensive generation of dominant RAPD and AFLP markers and the pseudo-testcross strategy in two-generation pedigrees ,4 or theus taeda  and Popu[Populus . HoweverEucalyptus, including twelve from E. globulus named with the prefix EMCRC [E. nitens, eight from E. sieberi, 26 from E. globulus and 13 from E. leucoxyon named respectively with prefixes En, Es, Eg and El [E. grandis and E. urophylla named EMBRA [E. globulus [Symphyomyrtus varies between 78 and 100% depending on the section to which they belong. It still remains around 50 to 60% for species of different subgenera such as Idiogenes and Monocalyptus and goes down to 25% for the related genus Corymbia [Symphyomyrtus is very high not only in terms of microsatellite flanking sequence conservation, but also marker order along linkage maps [Eucalyptus still lacks a more comprehensive genetic map built exclusively with microsatellite markers.One hundred thirty seven autosomal microsatellite markers have been published to date for species of ix EMCRC , eight fg and El -27 and 7ed EMBRA ,15. Receglobulus . MicrosaCorymbia . Microsaage maps . Althougage maps , and 40 age maps , the genEucalyptus [Several QTL mapping reports have demonstrated the existence of major effect QTLs for a number of silviculturally and industrially relevant traits in calyptus -39. Rececalyptus . In a sucalyptus .Eucalyptus has grown and become increasingly sophisticated, the large majority of the mapped QTLs have been localized on RAPD or AFLP maps so that it is essentially impossible to compare positions of QTLs for the same or correlated traits, seriously limiting the long term value of such QTL mapping efforts for genomics and breeding applications. Exceptions are QTL studies where transferable markers such as a few microsatellites [Eucalyptus where breeders worldwide take advantage of the interspecific genetic variation for wood properties and disease resistance through hybridization, the availability of a robust, genus-wide genetic map with highly transferable microsatellite markers has become a must for the effective advancement of genomic undertakings including QTL validation across pedigrees, co-localization of QTL and candidate genes for guiding association mapping experiments, positional cloning of QTLs and eventually marker assisted selection.Although the number of QTL detection reports in tellites ,38 or catellites ,38 were Eucalyptus including a total 234 mapped loci making it, to our knowledge, the most complete genetic map of Eucalyptus and of a forest tree to date based exclusively on interspecific transferable microsatellites. Besides the linkage map, a comprehensive set of 230 novel microsatellite markers are reported and a subset of 35, selected as anchor loci, were characterized for mapping information content. Finally, based on a set of shared microsatellites with other Eucalyptus mapping studies, a further set of 41 microsatellites, candidate genes and QTLs for wood and flowering traits are assigned to the consensus map making it the first consolidated source of existing linkage information for species of Eucalyptus.This work reports on the construction of a consensus genetic linkage map covering all 11 linkage groups of E. grandis enriched libraries for poly-(AG) and poly-(AC) repeats, a total of 450 primer pairs complementary to microsatellite flanking sequences were designed. Seventy of them were previously mapped, characterized and had their primer pairs sequences published [From ten ublished ,15. In tublished ,15 incluE. grandis) informative and 32 only male (E. urophylla) informative. A total of 128 (55%) were fully informative, i.e. segregated from both parents with a total of three or four different alleles. No markers were observed segregating 1:2:1, i.e. equally heterozygous in both parents. However only 122 out of the 128 fully informative markers could be placed on the consensus map. Six markers, although segregating from both parents, could not be positioned on the consensus map. These markers, indicated in the map with an underline , E. grandis would still display 194 heterozygous markers, i.e. 17% more than E. urophylla. This difference in observed heterozygosity is most likely due to the variable inbreeding status of the particular parents and not a species characteristic.The 237 segregating markers used in the construction of the consensus map . This extra group is a set of three markers that mapped at the end of linkage group 4 in E. urophylla at a LOD threshold lower than 3.0. These three markers most likely belong to this group but will require more markers to bridge them consistently to group 4. Seven markers although informative, remained unlinked on the map built only with microsatellites. These markers were: EMBRA94, EMBRA96, EMBRA103, EMBRA163, EMBRA178, EMBRA190, from the novel set of 230 markers reported in this study, and EMBRA62 previously mapped to the RAPD marker framework of linkage group 11 of E. urophylla in Brondani et al. [E. urophylla  mapped when compared to E. urophylla (160). The consensus map had an observed length of 1,567.7 cM and a mean inter-marker distance of 8.4 cM. A total of 19 map intervals with a genetic distance greater than 20 cM were observed, scattered throughout almost all linkage groups, except groups 1 and 4, but only 5 out of the 19 intervals were larger than 30 cM, indicating a relatively homogeneous map coverage obtained when using exclusively microsatellites markers. Clustering of markers was observed on both parental maps and consequently on the consensus map, particularly on groups 2, 5 and 7, although no formal test for clustering was carried out. Interestingly, however, clustering of markers had also been observed on these same linkage groups when built with RAPD markers using this same set of progeny [The female map with 202 markers covered an observed length of 1,814.5 cM with a mean distance between adjacent markers of 10.7 cM calculated as the arithmetic mean of the map distances between adjacent markers in each linkage group and not just simply by dividing the total map length by the number of markers. For the est) of the consensus map was 1,683 cM while the observed length (Gobs) was 1,567.7  was estimated as Cexp = 88.6%.The estimated genome length  deviated from the expected 1:1:1:1 or 1:1 segregation ratio at alpha \u2264 0.05 but only one (EMBRA81) remained significant after applying a Bonferroni correction. All these markers but EMBRA81 on group 6 could be confidently placed onto the consensus map. Sixteen distorted markers clustered mainly into two linkage groups (group 2 and 8) and the remaining 14 were scattered across eight linkage groups , conservation of locus order was observed between the two parental maps, with 82% of the markers mapping with the same linear order along the individual linkage groups. This conserved linear ordering can be easily visualized by the dotted lines connecting the parental maps with the consensus map  kept the same order in the consensus map. In E. urophylla, out of the 152 markers, 140 (92%) kept the same order as in the Eucalyptus consensus map. Out of the 122 markers mapped on all three maps, thus comparable, 85% of them kept the same order. No evidence of rearrangement of chromosomal block between the two species was found. As expected, the size of the consensus map had an intermediate size  between the female  and male  parental maps.The 122 fully informative markers mapped, allowed a robust identification of homologous pairs of linkage groups of the E. urophylla and 10.66 \u00b1 2.59 for E. grandis, with 50% of the alleles shared between them and the other 50% appearing exclusively in one or the other species most likely due to sampling effect although suggesting that important differences in microsatellite allele frequencies do exist between these two species. These frequency differences increase the probability of detecting markers segregating in a fully informative fashion, thus more informative for QTL mapping. The highest diversity was observed for marker EMBRA201 \u2013 originally a 17 dinucleotide repeat long \u2013 displaying 22 alleles in 64 chromosomes sampled. The total number of alleles detected in the two species combined ranged from 8 to 22, with the maximum allele size of 320 bp and minimum of 75 bp. The average observed heterozygosity of the 35 loci was around 66%, while the expected heterozigosity was higher, around 85%. These results are in agreement with a small level of selfing and/or related matings in natural populations of Eucalyptus.Genetic diversity for 35 markers selected as anchor loci due to their transferability and ease of interpretation was determined to guide future mapping experiments . A similEucalyptus, it was possible to establish the homology among the linkage groups of this consensus map and those of other linkage maps published , seven from E. nitens (En) and four from E. sieberi (Es). Furthermore 18 candidate genes for wood fiber and floral traits, previously mapped by Thamarus et al. [Based on the EMBRA markers mapped in other linkage studies in ed Table . This ans et al.  could alEucalyptus. Although some RFLP markers were mapped in E. nitens [E. globulus [Eucalyptus genetic research. Furthermore, the linkage map presented, involving 234 mapped loci, spans an estimated ~90% of the recombining genome of Eucalyptus, making it the most comprehensive genetic linkage map of a forest tree to date based exclusively on microsatellite markers.Prior to this work, important advances were made in the construction of genetic maps of species of . nitens  and E. gglobulus , the mosglobulus ,42-45. Wglobulus , totals Eucalyptus pedigrees, a similar proportion of informative markers could be obtained when genotyping tropical eucalypt progenies within the section Latoangulatae of subgens Symphyomyrtus. In fact Missiaggia et al. [Eucalyptus map in a cross of E. grandis \u00d7 E. urophylla hybrid parents when mapping QTL for early flowering. Moving microsatellites to other commercially important species such as E. globulus (section Maidenaria) or E. camaldulensis (section Exsertaria) the issue becomes one of transferability first and then information content. While in an earlier study, based on 100 microsatellites, we indicated a transferability of 78% from E. grandis to E. dunni (section Maidenaria) [E. globulus [E. camaldulensis [E. globulus and E. tereticornis [Symphyomyrtus and across section, particularly Maidenaria where E. globulus and E. nitens belong, should remain around 80%. Once robustly transferred, it is likely that polymorphism should be detected at a rate similar to the one in this study, i.e. 70 to 80%. This entire set of 300 EMBRA microsatellite markers in addition to the 67 published by other groups should therefore allow positioning 200 or more informative markers on any segregating family involving any of the most planted species of Eucalyptus.Consolidating all the development and screening data since our initial studies , from 45a et al.  were abldenaria) , estimatglobulus ,44, E. cdulensis  and a slticornis . These sEucalyptus providing important anchor loci and candidate genes for positional cloning efforts as well as association mapping experiments. These markers, however, will demand high throughput typing techniques based on single nucleotide polymorphism assays to be able to be widely used across pedigrees. Other sources of microsatellites will also be important to sample regions of the genome that have not been contemplated so far. For example, 93 operational microsatellites were derived from a sample sequencing study of 3 megabases of shotgun DNA of Eucalyptus grandis [Eucalyptus genome; (2) microsatellites into transcribed regions, specifically in untranslated regions such as 5'-UTR, should be evolutionarily older than those in noncoding regions and thus are expected to be more polymorphic as reported in a survey of some major monocots and dicots species [Besides microsatellites, other sources of genetic markers such as EST, gene-based and SNP have been ,17 and w grandis . EST der grandis  we have  species ; (3) genEucalyptus, 33% and 36% of fully informative RFLP markers were detected respectively [et al. [E. globulus pedigree and also did not detect any marker segregating 1:2:1. These results taken together, and now based on a larger set of mapped markers from different sources, indicate that around 60% of a screened set of Eucalyptus microsatellites should segregate in a fully informative fashion. Furthermore the fact that the pedigree used in this study is interspecific, should not significantly increase the proportion of fully informative markers due to the fact that E. grandis and E. urophylla although separate species, belong to the same section (Latoangulatae).Fully informative markers that allow integration of the parental maps have been always considered key elements for a more detailed examination of interaction among alleles at QTL in forest trees . The pseectively ,17. We oectively , a propoectively . In thisE. urophylla but amplified both alleles in E. grandis . In thiis Table . The oveE. grandis map (202) than on the E. urophylla map (160). In principle this should be due to a higher level heterozigosity in the E. grandis parent tree. However, previous survey of randomly distributed sequence polymorphism with RAPD markers in these same two parents did not show significant difference in the number of segregating markers with 272 heterozygous markers from E. grandis and 286 from E. urophylla assayed with the same set of arbitrary sequence primers [E. grandis library. Fourteen microsatellites did not amplify in E. urophylla  and comAll linked microsatellites mapped consistently on the same linkage groups in the two parental maps and 82% of the markers mapped with the same order along the homologous parental linkage groups with most order changes concentrated on linkage groups 1, 8, 9 and 10  were identified but only one would remain significantly distorted after applying a stringent Bonferroni correction. All distorted markers but one were mapped on the consensus map and more than half clustered mainly into two linkage groups (group 2 and 8) with the others scattered across eight linkage groups  to evencalyptus , and SNPcalyptus , combineEucalyptus have varied between 919 cM and 1551 cM although most estimates to date have remained around 1300 to 1500 cM , based Eucalyptus species.Using EMBRA markers that were mapped in other independent studies, it was possible to assign other 41 microsatellites to this consensus map at the linkage group level. The definition of the exact order of these 41 microsatelites relative to the other markers on the linkage groups will require genotyping them on this same set of progeny individuals. However the consolidation of linkage data carried out in this study demonstrates the power that a more comprehensive map of microsatellites provides for expanding the opportunities of comparative mapping across Eucalyptus species to facilitate the continued addition of new markers and genes. While the numbering proposed here now makes a first step toward this direction, the establishment of a correct numbering system for the chromosomes and hence for the linkage groups, should derive from cytogenetic studies using previously screened BAC with specific microsatellites as in situ hybridization probes.Linkage group numbering adopted for this map follows the one originally established for RAPD marker maps. This was an arbitrary numbering that was nevertheless kept to allow integration of microsatellites on the existing maps. Other reports where RFLP, AFLP, EST, other microsatellites and candidate genes were mapped have used different numbering. There is clearly a need to unify linkage group numbering for Eucalyptus by Marques et al. [et al. [Eef1 was recently mapped by Missiaggia et al. [et al. [Eucalyptus functional equivalent of the Arabidopsis Apetala1 gene was mapped. As the ectopic expression of the EAP1 in Arabidopsis driven by the 35S promoter caused plants to flower earlier [Eef1 QTL. In a similar way, the linkage group assignment of the CCR gene at the tip of linkage group 10 of Thamarus et al. [Eucalyptus nitens and E. globulus [Eucalyptus species. In an analogous way, once microsatellites are mapped close to the EgMYB2 gene, recently shown to co-localize with a QTL for lignin content [This consolidation of microsatellite linkage mapping data will also expand the prospects of making comparative analysis of putative QTL synteny such as that carried out in s et al.  for vege [et al.  for wooda et al.  on linka [et al.  where EA earlier  this coms et al.  indicateglobulus . This co content , it willEucalyptus genome. Based on a set of shared microsatellites with other Eucalyptus mapping studies, a set of other 41 microsatellites, candidate genes and QTLs for wood and flowering traits were assigned to the consensus map making it the first consolidated source of existing linkage information for species of Eucalyptus. This report significantly increases the availability of microsatellite markers for species of Eucalyptus, corroborating the high conservation of microsatellite flanking sequences and locus ordering across species of the genus. This work represents an important step forward for Eucalyptus comparative genomics, opening stimulating perspectives for evolutionary studies and molecular breeding applications in species of the genus. The availability of microsatellites for Eucalyptus should undergo a quick expansion in the next few years based on the large EST and genomic sequence databases available. The generalized use of increasingly larger sets of interspecific transferable markers and consensus mapping information will allow faster and more detailed investigations of QTL synteny among species, validation of expression-QTL across variable genetic backgrounds and positioning of a growing number of candidate genes to be tested in association mapping experiments.This report describes the development of a novel set of 230 EMBRA microsatellites, the construction of the first consensus linkage map with 234 mapped loci, covering ~90% of the E. grandis (clone G44) and male E. urophylla (clone U28) [Symphyomyrtus. For the characterization of the mapping information content, 32 individual trees of Eucalyptus, 16 from E. grandis and 16 from E. urophylla, were randomly chosen from a germplasm collection composed of trees grown from seeds collected in natural populations. Genomic DNA from leaves stored at -20\u00b0C was extracted as described earlier [Genetic mapping of the new set of microsatellite markers was performed on a mapping population of 92 F1 individuals derived from a cross between a female s clone G4 and malEucalyptus Microsatellite from Brazil) adopted earlier [The development, PCR amplification, detection, inheritance and segregation analysis of the microsatellites were carried out as described previously . In this earlier , followeE. grandis and later adopted for microsatellite mapping [Separate linkage maps for each parent tree were constructed based exclusively on microsatellites that were in heterozygous state and thus segregating in the expected 1:1 ratio. Seventy microsatellites published previously (EMBRA1 through EMBRA70) ,15 were  mapping .A combined data set with the gametic classes of all 92 F1 progeny was created to construct an integrated linkage map. As a result of using outbred parents, two distinct types of segregation were found: less informative 1:1 segregating loci, with two different alleles segregating from only one of the two parents (pseudo-testcross), and a fully informative 1:1:1:1 segregating locus with both parents heterozygous and three or four different alleles segregating from the two parents. A chi-square test was performed  to test for deviation of genotypic classes from the expected Mendelian inheritance ratios. The integrated linkage analysis, including estimation of recombination fraction and locus order was performed using the windows-based package OUTMAP  specificest) of the consensus map was estimated with the commonly used Hulbert estimate [obs) was then estimated as the ratio between the total observed genome length (Gobs) to the estimated genome length (Gest). A theoretical expected genome coverage (Cexp) for the consensus map was also estimated using the equation Cexp = 1-e-XN/1.25Gest [est) is the estimated genome length.The estimated genome length  and expected (Hexp) heterozygosity for each species and combined.Allelic diversity and estimates of observed and expected heterozygosity of thirty-five selected microsatellites were obtained as described previously  after geE. grandis \u00d7 E. urophylla [Eucalyptus species [A compilation of all microsatellite linkage information available in the literature to date was carried out to establish homology between the linkage groups of this consensus map and other maps published for rophylla  and othe species ,30,44. ERPVB carried out most of the experimental work, including microsatellite development, genotyping and data analysis, parental map construction and drafting of the first version of the manuscript. ERW was responsible for constructing the first version of the consensus map. CB contributed to the development of microsatellites. DG was responsible for project conception, data analysis, compilation of existing linkage information from the literature and writing the final version of the manuscript.E. grandis G44 \u00d7 E. urophylla U28 mapping population. .Consolidated list of 300 EMBRA microsatellite markers. Summary of all the information for the 230 new microsatellite markers reported in this study (EMBRA71 through EMBRA395) plus 70 published earlier (EMBRA1 through EMBRA70) ,26 incluClick here for fileEucalyptus sp. .Genetic characterization of anchor microsatellite markers. Summary information on the genetic diversity of 35 microsatellites selected as anchor loci as evaluated on a panel of 32 unrelated individual trees of Click here for file"}
+{"text": "Family studies are often conducted in a cross-sectional manner without long-term follow-up data. The relative contribution of a gene to a specific trait could change over the lifetime. The Framingham Heart Study offers a unique opportunity to investigate potential gene \u00d7 time interaction. We performed linkage analysis on the body mass index (BMI) measured in 1970, 1978, and 1986 for this project.We analyzed the data in two different ways: three genome-wide linkage analyses on each exam, and one genome-wide linkage analysis on the mean of the three measurements. Variance-component linkage analyses were performed by the SOLAR program. Genome-wide scans show consistent evidence of linkage of quantitative trait loci (QTLs) on chromosomes 3, 6, 9, and 16 in three measurements with a maximum multipoint LOD score > 2.2. However, only chromosome 9 has a LOD score = 2.14 when the mean values were analyzed. More interestingly, we found potential gene \u00d7 environment interactions: increasing LOD scores with age on chromosomes 3, 9, and 16 and decreasing LOD scores on chromosome 6 in the three exams.The results indicate two points: 1) it is possible that a gene (or genes) influencing BMI is (are) up- or down-regulated as people aged due to aging process or changes in lifestyle, environments, or genetic epistasis; 2) using mean values from longitudinal data may reduce the power to detect linkage and may have no power to detect gene \u00d7 time, and/or gene \u00d7 gene interactions. An advantage of the Framingham Heart Study is its repeated measurement of several cardiovascular risk factors over a long period of time. The Framingham Heart Study offers a unique opportunity to investigate the value of follow-up family studies. Quantitative trait data from repeated measurements in follow-up studies often fluctuate due to changes in lifestyle, age-related covariates, gene \u00d7 gene interaction, and measurement errors. Although there are statistical tests, such as the generalized estimating equations (GEE) model  to analyIn this study 330 pedigrees from the Framingham Heart Study from Genetic Analysis Workshop 13 data were used. While most pedigrees consist of 4 to 10 subjects in two generations, there are also several large pedigrees (up to 29 participants) and a few pedigrees that include three generations. The pedigrees consist of 4692 subjects, of whom 2885 have phenotype data. Cleaned genotyping data with 401 molecular markers are provided for chromosomes 1 through 22 for 1702 of the 4692 subjects.2). All data including BMI, high-density lipoprotein-cholesterol , age, sex, cigarette smoking, and alcohol consumption were first explored to see their distributions and outliers. The BMI data were available from years 1970, 1978, (1976 for the Framingham Heart Study Cohort 1 and 1978 for Cohort 2), and 1986. If the data were missing in year 1970, the information was supplied from the data in 1968. The mean BMI was calculated from three time points . We took the mean of three exams for each individual . Covariates, age, sex, smoking, alcohol, HDL-C, and interactions between HDL-C and age, smoking, and sex were included in the linkage analyses. Mean HDL-C and mean age were calculated in same way. Due to many nonsmokers and non-alcohol drinkers, the smoking and alcohol data were highly skewed. Therefore, these two variables were recoded as categorical variables. Smoking information was equally divided into four categories, and alcohol consumption into five categories. Four different genome-wide linkage analyses were performed for the three individual measurement and the mean values. The program SOLAR version 1.7.4 [We restricted our focus to BMI  in BMI. First we performed the genome scan with outliers, and then analyses without outliers were carried out for four candidate chromosomal regions. Table We considered \"evidence\" of linkage if a LOD score was \u2265 2.2 in one or more of the four analyses . The results of the genome scan with outliers showed that chromosomes 3, 6, 9, and 16 have evidence of quantitative trait loci (QTLs) affecting BMI  has been invoked to signify potential linkages, though at a reduced genome-wide significance  . In thisAlthough analyzing the mean values of quantitative traits from longitudinal data offers the advantages of simplicity of analytic procedure and overall genetic effect over several years, it may lose power and lose the chance to detect interactions as we show in this study. However, summarizing the overall genetic effect from longitudinal family data is not easy. Several studies  have useDetected genetic effects on obesity-related traits are likely to be modified by several factors including 1) actual genetic changes over time ; 2) secular changes over time that are not genetic; and 3) random variation . Our linkage analysis results from BMI in the four chromosomal regions is an example of a mix of the three situations. The consistent suggestion of linkage in three measurements from the Framingham Heart Study and replicated findings from other independent data ,6,8 and In summary, our studies suggest that elaborate analyses of longitudinal data may provide more insight and improve statistical power. The evidence of QTLs on chromosomes 3, 6, 9, and 16 are potentially interesting and the trend of LOD scores on these chromosomes are likely to be due to gene \u00d7 environment (time) and/or gene \u00d7 gene interaction."}
+{"text": "For linkage analysis in affected sibling pairs, we propose a regression model to incorporate information from a disease-associated single-nucleotide polymorphism located under the linkage peak. This model can be used to study if the associated single-nucleotide polymorphism marker partly explains the original linkage peak. Two sources of information are used for performing this task, namely the genotypes of the parents and the genotypes of the siblings. We applied the methods to three significantly disease-associated single-nucleotide polymorphisms and five microsatellite markers at the end of chromosome 3 of replicate 1 of Aipotu population. Two out of five of the microsatellite markers showed a LOD score higher than 3. The question to be answered was whether one of the single-nucleotide polymorphisms partly explains these high LOD scores. We did not have the answers when we analyzed the data. When a region of interest is identified by a linkage study, one may proceed by typing single-nucleotide polymorphisms (SNPs) in the region and test whether these SNPs are associated with the outcome. If a SNP is significantly associated with the outcome, the question arises whether the identified SNP partly explains the original linkage peak. For quantitative traits observed in randomly selected siblings, Beekman et al.  proposedSpairs at a microsatellite marker [Spairs [Li et al.  proposede marker . For an  [Spairs  indicateAnother source of information is the SNP genotypes of the parents. When both parents are homozygous for the SNP, the SNP genotypes of the affected siblings are fixed (nonrandom). In the extreme situation of one causal SNP or a SNP in complete linkage disequilibrium (LD) with the causal SNP, the IBD status at the microsatellite marker is not informative for transmission from parents homozygous for the SNP to affected offspring at the SNP locus. Hence the IBD probabilities of affected offspring of these parents are the probabilities under the null hypothesis. On the other hand, for affected siblings with heterozygous parents, the IBD status at the microsatellite marker is informative for transmission of the risk allele to the affected offspring. The risk allele will be most likely transmitted to the affected offspring and hence the allele at a linked microsatellite marker with the same grandparental origin will likely be transmitted. Based on this argument, Dupuis and Van Eerdewegh  proposedAs an alternative to the approaches of Dupuis and Van Eerdewegh  and Li ej,Olson  showed t\u03b1i the prior probabilities that a sibling pair shares i alleles IBD and fij the IBD status at the marker locus for sibling pair j. If the IBD status cannot be derived with certainty, fij are the posterior weights. The parameter \u03b20 is set to zero to avoid nonidentifiability. Depending on the underlying genetic model, constraints may be set on \u03b21 and \u03b22. Here, we use an additive model, i.e., . This parameterization corresponds to the following relationship for the IBD sharing in the affected sibling pair: z0 = 0.25, z1 = 0.5, and z2 = 0.5 - 0.25. By using the parameterization proposed by Olson [x can be easily be added to LR (1):with x: z0(x) = 0.25, z1 = 0.5 and z2(x) = 0.5 - 0.25. Note that LR (2) is an overall test of linkage, i.e., it tests the null hypothesis of \u03b21 = \u03b4 = 0 versus the alternative. The difference between LR (2) and LR (1) can be used to test the model with the covariate x versus the model without x, i.e., the null hypothesis of \u03b4 = 0.Under the additive model the IBD sharing in the affected sibling pair depends on the covariate x is this indicator function minus its mean \u03bc in the sample. Note that \u03bc is the frequency of sibling pairs with parents homozygous for the SNP. If \u03b4 is zero the IBD sharing at the microsatellite is similar in offspring with homozygous parents to offspring with at least one heterozygous parent and the SNP does not explain the linkage peak at all. For \u03b4 < 0, the sharing of marker alleles IBD is higher in siblings with at least one parent heterozygous for the SNP compared with offspring of parents homozygous for the SNP. If \u03b4 is significantly smaller than zero, it can be concluded that the SNP partly explains the linkage peak.To verify if an associated SNP explains partly the linkage peak, we can incorporate the indicator function as a covariate. The indicator function is defined as one if both parents are homozygous for the SNP and zero otherwise. Thus the centralized x. Thus x is the number of risk alleles, which varies from 0 to 4, minus its mean in the sample of affected sib pairs. By doing so, we assume an additive model for the SNP [\u03b4 is zero the SNP does not explain the linkage peak at all. For \u03b4 > 0 the IBD sharing is higher in siblings who carry a high number of risk alleles and for \u03b4 < 0 the IBD sharing is higher in siblings who carry a low number of risk alleles. The models were fitted using the package SAGE 4.5 [P-values smaller than 0.05 were considered to be statistically significant.If the genotypes of the parents are not available, the genotypes of the siblings can be used. We can study if the sharing of marker alleles IBD depends on the siblings' genotypes. We propose to use the centralized number of carried 'risk' alleles by the sibling pair as covariate  the SNP , i.e., aSAGE 4.5 . P-valueWe applied the methods to the SNPs B03T3056, B03T3057, and B03T3066 and the five microsatellite markers at the end of chromosome 3 using replicate 1 of population Aipotu. This region was identified by performing linkage analysis of the micro-satellites using the affected sibling pairs from several replicates . In rep\u03b4 at marker D3S0127 was  = 0.56 (standard error of 0.89).To determine whether any of these SNPs partly explains the original linkage peak, we included the indicator function of both parental genotypes being homozygous as covariate in the model. The results are given in Table P = 0.02,  = 0.36). The LOD score at D3S0124 increased only by 0.64. Including the other SNPs in the model increased the LOD scores only slightly.Second, we considered the genotypes of the siblings as covariates. In line with the approach papers of Hsu et al.  and Putt = 0.34) and the estimate for the parental genotype was smaller but still positive ( = 0.21).Finally we added the covariate based on the parental B03T3056 genotypes to the model in addition to sibling's B03T3056 genotypes. For the microsatellite marker D3S0127, the LOD score increased by 0.14. The corresponding estimate for the sibling's genotype was similar to the first estimate (P = 0.02). From this analysis we conclude that only B03T3056 significantly explained a small part of the linkage signal. Other unknown genetic factors, probably in LD with B03T3056, are likely to be present in this region.In the original affected sibling pair linkage study, microsatellite markers D3S0124 and D3S0127 showed LOD scores above 3. In association analyses ,10, SNP Including the parental B03T3056 genotypes in the linkage analysis of marker D3S0127 increased the LOD score only with 0.25. Siblings of parents homozygous for the SNP even showed higher IBD sharing than siblings with at least one heterozygous parent. This result is somewhat unexpected and not in line with the significant result when using the sibling's genotypes of this SNP as covariate in the linkage analysis of marker D3S0127. To disentangle association signals using SNP genotypes of the siblings and of the parents, extended modelling will likely be needed. More research in this area will be fruitful in understanding the phenomenon observed in this data analysis.For situations in which the SNP genotypes of the parents are indeed significant, the question arises whether residual linkage exists, i.e., whether the SNP explains all genetic variation in this region. When the SNP is the only causal factor in the region or in complete LD with the causal factor, the IBD sharing for offspring of parents homozygous for the SNP should be similar to the probabilities under the null hypothesis of no linkage. A statistic could be formulated to test this null hypothesis. However, our analysis of parental genotypes does not support this hypothesis and therefore we did not perform such an analysis for residual linkage in these data.This paper is a first attempt to combine the information available in genotypes of the parents, the siblings, and the IBD status at a microsatellite marker to better understand the role of a significantly disease-associated SNP. After knowing the answers, the conclusion that B03T3056 only partly explained the linkage peak and that other unknown factors are present in this region was correct. In this sense the proposed method appeared to work well. However, more research will be needed to study the statistical properties and assumptions of the method.We conclude that SNP B03T3056 only partly explains the original linkage peak. Other unknown genetic factors are probably present in this region. The models of Olson [GAW: Genetic Analysis WorkshopIBD: Identity by descentLD: Linkage disequilibriumLR: Likelihood ratioSNP: Single-nucleotide polymorphismJJH-D performed the analyses and wrote the manuscript. JJH-D, H-WU, JJPL carried out the preliminary linkage analyses. All authors participated in the development of the methods and interpretation of the results of the analysis. All authors read and approved the final manuscript."}
+{"text": "Linkage analysis based on identity-by-descent allele-sharing can be used to identify a chromosomal region harboring a quantitative trait locus (QTL), but lacks the resolution required for gene identification. Consequently, linkage disequilibrium (association) analysis is often employed for fine-mapping. Variance-components based combined linkage and association analysis for quantitative traits in sib pairs, in which association is modeled as a mean effect and linkage is modeled in the covariance structure has been extended to general pedigrees . The QTDT approach accommodates data not only from parents and siblings, but also from all available relatives. QTDT is also robust to population stratification. However, when population stratification is absent, it is possible to utilize even more information, namely the additional information contained in the founder genotypes. In this paper, we introduce a simple modification of the allelic transmission scoring method used in the QTDT that results in a more powerful test of linkage disequilibrium, but is only applicable in the absence of population stratification. This test, the quantitative trait linkage disequilibrium (QTLD) test, has been incorporated into a new procedure in the statistical genetics computer package SOLAR. We apply this procedure in a linkage/association analysis of an electrophysiological measurement previously shown to be related to alcoholism. We also demonstrate by simulation the increase in power obtained with the QTLD test, relative to the QTDT, when a true association exists between a marker and a QTL. The resulting fixed effect model for the mean phenotype isLinkage analysis based on identity-by-descent (IBD) allele-sharing can be used to identify a chromosomal region harboring a quantitative trait locus (QTL), but lacks the resolution required for gene identification. Consequently, linkage disequilibrium (association) analysis is often employed for fine-mapping. Fulker et al.  proposedbb + \u03b2ww.E [y] = \u03bc + \u03b2b, a robust test of association is obtained by comparing a model in which both \u03b2b and \u03b2w are freely estimated with a model in which \u03b2w is fixed at 0. Moreover, the presence of stratification can be inferred when the estimates of \u03b2b and \u03b2w differ significantly.Because population structure will affect only the parameter \u03b2b) is defined for each non-founder, and deviations (w) from this expectation are used to score allelic transmissions. For founders, b is set to the observed genotype and w is set to 0. As in the sib-pairs analysis described above, the model parameters associated with the between-family and within-family components of the genotype scores, \u03b2b and \u03b2w, are used to test for population stratification and linkage disequilibrium. Siegmund et al. [Abecasis et al.  extendedd et al.  have proThe QTDT approach accommodates data not only from parents and siblings, but also from all available relatives. This test is also robust to population stratification. However, when population stratification is absent, it is possible to utilize even more information, namely the additional information contained in the founder genotypes. In this paper, we introduce a simple modification of the allelic transmission scoring scheme of Abecasis et al. that results in a more powerful test of linkage disequilibrium, but is only applicable in the absence of population stratification. This test, the quantitative trait linkage disequilibrium (QTLD) test, has been incorporated into a new procedure in the statistical genetics computer package SOLAR . Like thb' and w', where we have added primes to distinguish the QTLD components from those defined in the QTDT method. We modify the QTDT allelic transmission scoring in a simple way: founder genotypes are included in the within-family component rather than in the between-family component. That is, for founders, w' is set to the observed genotype and b' is set to 0. With this change in the scoring algorithm, our decomposition of the genotype scores no longer results in between- and within-family components, although we retain the notation for consistency.In the QTLD test, we model association as a fixed effect on the trait mean and we decompose the genotype scores into two components, The QTLD test procedure in SOLAR involves maximizing the likelihood of six genetic models:b = 0, \u03b2w = 0 the association parameters are both fixed to 0Model 1: \u03b2 both parameters are estimatedModel 2:  the parameters are held equalModel 3:  the within-family parameter is fixed to 0Model 4:  both parameters are estimatedModel 5:  the regression parameter on w' is fixed to 0Model 6: b and w components are defined according to the QTDT scheme, while in Models 5 and 6, the QTLD-defined components, b' and w', are used. All models are derived from a previously maximized model which includes the trait of interest, any relevant covariates, and a linkage component based on IBD allele-sharing estimated from a microsatellite genome scan.In Models 1 through 4, the The following four tests are performed as part of the QTLD procedure:b and \u03b2w, are estimated is compared to the likelihood of a model in which they are constrained to be equal, as would be the case in the absence of population stratification.1) Test for Stratification (Model 2 vs. Model 3) \u2013 The likelihood of a model in which the association parameters, \u03b22) Measured Genotype (Model 3 vs. Model 1) \u2013 This standard fixed effects regression  on the m3) QTDT (Model 2 vs. Model 4) \u2013 This test is valid whether or not the stratification test indicates that population stratification is present.absence of population stratification.4) QTLD (Model 6 vs. Model 5) \u2013 The QTLD test is only applicable in the 2 with one degree of freedom.All test statistics are distributed as a \u03c7The data are from families participating in the Collaborative Study on the Genetics of Alcoholism (COGA) . We restWe chose the TTTH1 electrophysiological endophenotype  because the information supplied to Genetic Analysis Workshop participants regarding the COGA data indicated that microsatellite markers on chromosome 7 are linked to a QTL affecting normal variation in this phenotype. Linkage analyses were run at every 1-cM interval on chromosome 7 using multipoint estimates of IBD allele-sharing derived from the microsatellite marker data by the program LOKI ,9. Age a2 test statistic for each of the association tests  given a true allelic association between a marker and a QTL. We used SOLAR to simulate a biallelic QTL responsible for up to 5% of the phenotypic variance for a quantitative trait having a total heritability of 30%. The family structure and pattern of missing data in the COGA dataset were assumed. We also simulated a fully informative marker completely linked to the QTL, which we used to calculate the IBD allele-sharing at the QTL location. Treating the QTL genotypes as if they had been observed for a marker in complete LD with the trait locus, we then ran the QTLD procedure, with a linkage component based on the IBDs included in the model, to obtain the \u03c72 statistic for each of the tests. The test statistics were averaged over 100 replicates of the simulation for various values of the marker-specific heritability .To demonstrate the increase in power that can be attained with the QTLD test, we conducted simulations to derive the expected \u03c7p < 0.0001). The covariates age and max drinks accounted for approximately 15% of the phenotypic variance. A maximum LOD score of 4.24 was achieved at a point 156 cM from pter  of the total number of SNPs might have been expected to generate a significant test statistic purely by chance. Nevertheless, these SNPs were omitted from the QTLD portion of the test procedure.2 statistic of 14.69 (p = 0.000127) would be required for significant evidence of association. By this criterion, none of the tests, including the measured genotype test, showed significant evidence of association for any of the SNPs in the region of linkage. The Illumina SNP rs1896887, at location 129.5 cM, yielded the highest \u03c72 value (11.296) for the QTDT, followed by the Affymetrix SNPs tsc0049494 (\u03c72 = 11.179) and tsc0022400 (\u03c72 = 10.507), both at a location 131.8 cM. The highest \u03c72 value (10.0598) for the QTLD was observed for the Affymetrix SNP tsc0510163, at location 143.6 cM.Assuming 395 independent tests were performed, a 1 df \u03c7As shown in Figure We have introduced the QTLD test, a novel approach for detecting association due to linkage disequilibrium in the absence of population stratification. We have confirmed in our simulations that the QTLD test provides a significant increase, relative to the QTDT, in power to detect an allelic association. Investigators can now use SOLAR to conduct a combined linkage and association analysis, using pedigrees of arbitrary size and complexity, that includes a test for population stratification along with several tests of association: measured genotype, QTDT, and, where appropriate, the QTLD test. Our application of this procedure to the COGA data for TTTH1 identified several SNPs under the chromosome 7 linkage peak which exhibit suggestive levels of association, although none were statistically significant after correction for multiple testing.COGA: Collaborative Study on the Genetics of AlcoholismIBD: Identity by descentQTL: Quantitative trait locusQTDT: Quantitative transmission disequilibrium testQTLD: Quantitative trait linkage disequilibriumSNP: Single-nucleotide polymorphismJB, LMH, and TDD developed the QTLD methodology. LMH, with the assistance of DKR and TDD, performed the analysis of the COGA data and drafted the manuscript. TDD conducted the power simulations. MCM provided a critical review of the methodology. All authors have read and approved this manuscript."}
+{"text": "Most linkage programs assume linkage equilibrium among multiple linked markers. This assumption may lead to bias for tightly linked markers where strong linkage disequilibrium (LD) exists. We used simulated data from Genetic Analysis Workshop 14 to examine the possible effect of LD on multipoint linkage analysis. Single-nucleotide polymorphism packets from a non-disease-related region that was generated with LD were used for both model-free and parametric linkage analyses. Results showed that high LD among markers can induce false-positive evidence of linkage for affected sib-pair analysis when parental data are missing. Bias can be eliminated with parental data and can be reduced when additional markers not in LD are included in the analyses. Most multipoint linkage programs assume linkage equilibrium among the markers being studied. This assumption is appropriate for the study of sparsely spaced markers with inter-marker distances exceeding a few centimorgans, because linkage equilibrium is expected over these intervals for almost all populations. However, with recent advances in high-throughput genotyping technology, much denser markers are available and linkage disequilibrium (LD) may exist among the markers. Applying linkage analyses that assume linkage equilibrium to dense markers may lead to bias. It is well known that misspecification of allele frequencies can cause inflation of LOD scores for both model-free  and modeRecently, Huang et al.  demonstrpairs [In order to examine the possible effect of LD on linkage analysis, we decided to study the markers from a dense marker dataset, because the inter-marker distances are smaller and the simulated LD was higher. Single-nucleotide polymorphism (SNP) packets from the non-disease related regions that were generated with LD were bought and used for the analyses. The inter-marker distance was 0.29 cM on average among these markers (20 SNPs per packet). Pedigree samples from the Aipotu population of simulated GAW14 data were used for the analyses. There were 100 nuclear families in the replicate sample and at least two sibs were affected with Kofendrerd Personality Disorder (KPD) in each family. We treated parents from each family as unrelated individuals and used them to estimate haplotype frequencies and LD. Haplotype frequencies were estimated by using the expectation maximization algorithm  and pairpairs . For thepairs , thus alr2 > 0.38. The pair-wise LD as measured by D' and r2 for this packet is shown in Table Although all the SNP packets that we examined were from regions that were generated with LD, LD was not strong in most of the regions and did not have an obvious effect on linkage analysis. However, strong LD existed between three markers in SNP packet 121: B03T2407, B03T2408, and C03R0221 with pair-wise D' > 0.95 and Single-point linkage analysis did not show any evidence of linkage both for the three markers in strong LD alone and for the whole marker set Fig. . HoweverFor multipoint linkage analysis of affected sib-pair data, for which parental phase information is inferred from the sib pairs, usual methods of linkage analysis assume linkage equilibrium between multiple linked markers and assigns equal probabilities to all possible phases. This assumption can cause overestimation of multipoint identity by decent (IBD) sharing and induces false positives for both model-free and parametric linkage analysis, as showed by Huang et al. . This stOur results indicate that LD among tightly linked marker should be examined, especially in the fine-mapping stage where strong LD is likely to exist between the markers. Markers that are in strong LD should not be used together for linkage analysis in order to avoid possible false positives. An alternative approach is to modify current linkage programs to allow for LD so that all marker information can be used in the search for a disease-related region.GAW14: Genetic Analysis Workshop 14IBD: Identity by descentKPD: Kofendrerd Personality DisorderLD: Linkage disequilibriumSNP: Single-nucleotide polymorphismQH did the analysis and prepared the manuscript. MS assisted in the development of data for this project and performed analysis of the LD patterns of simulated data, and also presented the results at the Genetic Analysis Workshop. SS provided guidance in concept development. CIA directed the project and revised the manuscript."}
+{"text": "The information content of a continuous variable exceeds that of its categorical counterpart. The parameterization of a model may diminish the benefit of using a continuous variable. We explored the use of continuous versus discrete environment in variance components based analyses examining gene \u00d7 environment interaction in the electrophysiological phenotypes from the Collaborative Study on the Genetics of Alcoholism.The parameterization using the continuous environment produced a greater number of significant gene \u00d7 environment interactions and lower AICs (Akaike's information criterion). In these cases, the genetic variance increased with increasing cigarette pack-years, the continuous environment of interest. This did not, however, result in enhanced LOD scores when linkage analyses incorporated the gene \u00d7 continuous environment interaction.Alternative parameterizations may better represent the functional relationship between the continuous environment and the genetic variance. Generally, there is more information when a risk factor is represented by a continuous variable than a categorical variable. The resulting gain of analytical power justifies the increased effort required to collect and use data in more refined detail, i.e., packs of cigarettes per day versus smoking status. One exception to this may be the unusual circumstance in which levels of exposure below the lowest unit of measurement are sufficient to generate the outcome of interest. Another instance may be when the parameterization or constraints of a data analytical tool offer no benefit from the use of a continuous variable. The VARCOMP procedure in SAS is an example of the latter case because it allows only class variables, i.e., variables that are not continuous .In this article, we examined the 12 continuous traits concerning event-related potentials (ERPs) and the continuous resting potential in the Genetic Analysis Workshop 14 (GAW14) dataset with regard to gene \u00d7 environment (G \u00d7 E) interaction, with the environmental exposure of interest being cigarette smoking. We considered the dichotomous indicator of habitual smoking (SMOKER), the continuous cigarette pack-years (CIGSPKY), and smoking status as a dichotomization of cigarette pack-years.We used a variance components model with one parameterization that allowed for separate discrete environment-specific genetic and environmental standard deviations and a second parameterization that modeled the genetic standard deviation as a function of the continuous environment. The first aim of these analyses was to determine whether there is G \u00d7 E interaction. The second aim was, given G \u00d7 E interaction, to determine whether incorporation of the dichotomous or the continuous variable affected our ability to detect linkage in variance components based linkage analyses. Finally, given linkage, we examined whether this incorporation provided additional information about the underlying quantitative trait loci (QTL).We obtained data from the Collaborative Study on the Genetics of Alcoholism (COGA) provided for the GAW14. Begleiter et al. have previously described the recruitment of the study participants . Bierut xg) and nonsmokers (\u03c3yg) and whether the genetic correlation (\u03c1g) between smokers and nonsmokers differed from 1. When the genes in both smokers and nonsmokers that influence the trait comprise identical sets, \u03c1g = 1, whereas when the genes in smokers and nonsmokers that influence the trait comprise completely different, nonoverlapping sets of genes, \u03c1g = 0. In this description, smoker is general for either SMOKER or SMK_STATUS. Towne et al. \u00a0\u00a0\u00a0(1)\u03c3g = exp [-\u03bb|CIGPKYRSi - CIGPKYRSj|]\u00a0\u00a0\u00a0(2)\u03c1g) decreases linearly with increasing disparity in CIGPKYRS, such that individuals with the same CIGPKYRS have \u03c1g = 1 and individuals with increasing differences in CIGPKYRS have decreasing ln(\u03c1g) with slope -\u03bb. Almasy et al. [Under this parameterization, the natural logarithm of the genetic correlation  was not statistically different from 1. When SMK_STATUS was the discrete environment, however, there was evidence that \u03c1g for TTTH3 and for TTTH4 differed statistically from 1 , i.e., the sets of genes in smokers and nonsmokers that influence the trait were not identical.We found evidence of a genotype-by-smoking interaction only for TTTH1, using either SMOKER or SMK_STATUS as the discrete environment. Table Upon performing a linkage analysis without incorporating the G \u00d7 discrete E interaction we found a maximum LOD score of 3.4116 at chromosome 7, 157\u2013158 cM. Upon incorporating the G \u00d7 discrete E interaction, for which SMOKER was the discrete environment of interest, we found a maximum LOD of 3.6190 at the same location. Table qx = 0.338 and \u03c3qy = 0.335, respectively. The difference in residual polygenic effect among smokers and nonsmokers, \u03c3gx = 0.271 and \u03c3gy = 0.550, respectively, appears intriguing, but remains statistically insignificant (p = 0.36). For the locus at chromosome 6, 96 cM, there is a statistical difference (p = 0.0139) between the genetic variation due to the locus among smokers and that among nonsmokers.The genetic SD due to the locus at chromosome 7, 157\u2013158 cM among smokers was not significantly different from that of nonsmokers, \u03c3We found evidence of G \u00d7 continuous E interaction for NTTH1, NTTH4, and TTTH4, but not for TTTH1. In each case, \u03b2 was positive, indicating that the genetic variance increased with increasing cigarette pack-years. Table These analyses suggest that the parameterization using the continuous environment seems to be a better choice as more results of G \u00d7 E investigations were significant for the continuous environment and the resulting AIC were lower. Whether this parameterization conveys greater power, however, is unknown. Further, as indicated by the linkage analyses, implementation of this parameterization may be sensitive to the particular functional relationship of the environment to the genetic variance. In particular, alternative parameterizations, such as described by Diego et al. , may proAIC: Akaike's information criterionCOGA: Collaborative Study on the Genetics of AlcoholismGAW14: Genetics Analysis Workshop 14ERP: Event-related potentialsG \u00d7 E: Gene \u00d7 environmentQTL: Quantitative trait lociKRV performed statistical analyses and wrote the manuscript. DMW performed the linkage analyses that did not incorporate interaction and assisted in editing the manuscript. AB provided programming assistance and statistical analyses. TDD provided the IBD matrices for the linkage analyses. THE assisted with interpretation of results. LA conceived the study and provided direction, in addition to editing the manuscript. All authors read and approved the final manuscript."}
+{"text": "We analyzed 143 pedigrees (364 nuclear families) in the Collaborative Study on the Genetics of Alcoholism (COGA) data provided to the participants in the Genetic Analysis Workshop 14 (GAW14) with the goal of comparing results obtained from genome linkage analysis using microsatellite and with results obtained using SNP markers for two measures of alcoholism . First, we constructed haplotype blocks by using the entire set of single-nucleotide polymorphisms (SNP) in chromosomes 1, 4, and 7. These chromosomes have shown linkage signals for MAXDRINK or EEG-TTTH1 in previous reports. Second, we randomly selected one, two, three, four, and five SNPs from each block  to conduct linkage analysis using variance component approach. Finally, results of all SNP analyses were compared with those obtained using microsatellite markers.The LOD scores obtained from SNPs were slightly higher but the curves were not radically different from those obtained from microsatellite analyses. The peaks of linkage regions from SNP sets were slightly shifted to the left when compared to those from microsatellite markers. The reduced sets of SNPs provide signals in the same linkage regions but with a smaller LOD score suggesting a significant impact of the decrease in information content on linkage results. The widths of 1 LOD support interval of linkage regions from SNP sets were smaller when compared to those of microsatellite markers. However, two linkage regions obtained from the microsatellite linkage analysis on chromosome 7 for LOG of TTTH1 were not detected in the SNP based analyses.The linkage results from SNPs showed narrower linkage regions and slightly higher LOD scores when compared to those of microsatellite markers. The different builds of the genetic maps used in microsatellite and SNPs markers or/and errors in genotyping may account for the microsatellite linkage signals on chromosome 7 that were not identified using SNPs. Also, unresolved map issues between SNPs and microsatellite markers may be partly responsible for the shifted linkage peaks when comparing the two types of markers. The identification of chromosomal segments showing association or linkage is only the first step toward discovery of genetic factors underlying susceptibility to disease. The typical genome-wide linkage analysis based on microsatellites with an average density of 10 cM results in large genomic regions for fine-mapping. In this regard, there is considerable interest in developing maps based on genomic markers that will lead to higher resolution linkage results with the hope of reducing future cost and time to conduct fine-mapping. With the availability of several million new SNPs in the public database and new technologies for large-scale, high throughput SNP genotyping at affordable costs, there is growing interests in using SNPs to create high resolution linkage maps. In this paper we evaluate strategies to systematically compare genome-wide linkage results from microsatellite and SNPs using different density maps.The dataset for the Collaborative Study on the Genetics of Alcoholism (COGA) was provided as problem 1 for GAW14. The dataset included 1,350 individuals in 143 pedigrees, 318 microsatellite genotypes for a 10 cM genome map, 4,763 SNP loci from Illumina, 11,555 SNP loci from Affymetrix and phenotypic information. We used MAXDRINK and TTTH1 as phenotypes and the panel of 4,763 Illumina SNPs. MAXDRINK is defined as maximum number of drinks in a 24-hour period [For each chromosome, we constructed haplotypes using GENEHUNTER2 (GH2) . Linkagep < 0.01), the location of the signals were for the most part similar GAW: Genetic Analysis Workshop 14SNP: Single-nucleotide polymorphismsSTRP: Short tandem repeat polymorphism"}
+{"text": "Several such methods use covariates to discriminate between linked and unlinked pedigrees with respect to a certain disease locus. Here we apply several such methods including two mixture models, ordered subset analysis, and a conditional logistic model to genome scan data on the DSM-IV alcohol dependence phenotype on the Collaborative Studies on Genetics of Alcoholism families, and compare the results to traditional nonparametric linkage analysis. In general, there was little agreement among the various covariate-based linkage statistics. Linkage signals with empirical  Etiological heterogeneity is inevitable when large sets of pedigree data are analyzed for complex diseases, where the susceptibility loci may vary from one pedigree to another. Such heterogeneity, if unrecognized, tends to reduce the power to detect linkage. Covariate-based methods attempt to adjust for heterogeneity by using covariate data to discriminate between pedigrees with different disease etiologies; however, since these methods are relatively new, few studies have applied them to real datasets -3. The mIn this study, we applied four covariate-based methods to the COGA families from the Genetic Analysis Workship 14 dataset. Our aim was to identify new genes responsible for alcoholism, as well as to study whether previously detected regions of linkage were also detected using these new methods. The methods included the pre-cluster and covariate-identity by descent (cov-IBD) models of Devlin et al. , orderedOne class of models assumes that a proportion of the pedigrees are linked to the disease gene, while the remaining pedigrees are affected due to some other reason. Membership in the linked group is predicted using one or more covariates assumed to be related to the disease. The pre-cluster, cov-IBD, and OSA models fall into this category. Regression-based models that condition on the covariate values are a second category of heterogeneity-based methods, Olson's method being an example.Pre-cluster and cov-IBD by Devlin et al. are mixture models that analyze affected sib-pair (ASP) data . Each ASa priori specification of the linked and unlinked subsets is not required; however, it ignores the magnitude of the covariate values, considering only the rank.OSA determines the ordered subset of the pedigrees that provides maximal evidence for linkage . Each peOlson's method uses a conditional-logistic representation of an affected relative pair (ARP) likelihood ratio that includes the effects of covariates as additional parameters in a test for linkage . This moThe DSM-IV alcohol dependence phenotype (ALDX2)  was recomclust .XG1 and G2, based on the Euclidian distance of their centres from the origin, G1 representing the nearer cluster. Because OSA allows for only one covariate per pedigree, we assigned Xped values as pedigree-level covariates prior to running OSA. We ran LODPAL on affected sib pairs using both the sum and the difference of each pair's covariate values, reporting the best score. The multiple-testing issue arising in this case was taken care of by the empirical p-value calculation.The two clusters were designated as all statistic. MEGA2 [We used all 143 multiplex pedigrees and the 315 microsatellite markers located on chromosomes 1 through 22. Our analysis was not appropriate for X-linked data. Due to software limitations, the seven largest pedigrees were broken into smaller components or trimmed of uninformative individuals, resulting in 156 pedigrees overall. Multipoint IBD probabilities were obtained using MERLIN version 0.10.2 [c. MEGA2  was usedG1 as the linked cluster, and with G2 as the linked cluster. Similarly, for OSA, we used both orderings of the Xped values: L2H, ordered small to large so that linked pedigrees have smaller covariate values; H2L, ordered large to small, so linked pedigrees have larger covariate values. Marker positions are reported by using Haldane map function. The chromosomal locations of the genes that were not included in the COGA marker map were obtained from the Marshfield web site and converted to Haldane map distances.For pre-cluster, we computed two likelihoods: with A small region on chromosome 7 spanning 27\u201361 cM was selected for determining the empirical significance of LOD scores obtained from the various covariate methods. We simulated 1,000 replicates of the genotype data using SIMULATE  under thThe NPL analysis produced a single peak with LOD score 2.68 at D10S544 on chromosome 10. We have not reported cov-IBD results because these were not significantly different from the pre-cluster results. Table th percentiles of the empirical null distribution of LOD scores for pre-cluster range between 1.17 and 1.34 for the four covariates; 99th percentiles for OSA are between 1.86 and 2.09; LODPAL's 99th percentile range from 1.99 to 2.24.Except for one region on chromosome 21 . The lack of agreement among the results may be also be due to the sensitivity of the covariate-based methods to the relationship between the covariate and trait under study.Our covariate selection was rather heuristic, based on evidence from clustering, rather than biological reasons. Ideal candidates for covariate statistics would be risk factors with a gene \u00d7 environment interaction effect and identifying such factors requires prior biological knowledge. A purely environmental risk factor would act as a confounder, reducing the power of the mixture model because it cannot cluster families into linked and unlinked groups. However, it is a challenging issue to determine which of the above classes a covariate falls into, and this bears further investigation within a systematic framework. We would also expect that the choice of the function for creating pedigree-level covariates from individual values would have an effect on the analysis. Indeed, when we used the mean value of the affecteds instead of our XTsai  observedARP: Affected relative pairASP: Affected sib pairCOGA: Collaborative Study on the Genetics of Alcoholismcov-IBD: Covariate identity by descentIBD: Identity by descentOSA: Ordered subset analysisBHR, NM, and H-JT contributed equally to the data processing, analysis, and the writing of the manuscript. DEW contributed to the design of the study and writing of the manuscript."}
+{"text": "Only one genome scan to date has attempted to make use of the longitudinal data available in the Framingham Heart Study, and this attempt yielded evidence of linkage to a gene for mean systolic blood pressure. We show how the additional information available in these longitudinal data can be utilized to examine linkages for not only mean systolic blood pressure (SBP), but also for its trend with age and its variability. Prior to linkage analysis, individuals treated for hypertension were adjusted to account for right-censoring of SBP. Regressions on age were fitted to obtain orthogonal measures of slope, curvature, and residual variance of SBP that were then used as dependent variables in the model-free linkage program SIBPAL. We included mean age, gender, and cohort as covariates in the analysis. To improve power, sibling pairs were weighted for informativity using weights derived from both the marker and trait. The most significant results from our analyses were found on chromosomes 12, 15, and 17 for mean SBP, and chromosome 20 for both SBP slope and curvature. Hypertension is a complex disorder that involves both environmental and genetic components . Unfortutij be the SBP measured at age xij, on the ith individual, j = 1, 2, ..., ni. To adjust for the effects of SBP due to treatment for hypertension, we chose to employ a simple method that imputes an \"untreated\" SBP by taking the maximum of: 1) SBP measured at time points when the individual was being treated to lower blood pressure plus a constant ), 2) 140 mm Hg, the clinical threshold for the diagnosis of hypertension for the Framingham study, and 3) the last SBP measurement before treatment commenced.Selection criteria, study design, and data collection methods for the Framingham Heart Study have been detailed previously ,4. The syij asDefine tik is the most recent untreated measure of SBP for individual i at some time-point k <j. Unlike other methods, we do not attempt to infer information about the tail of the uncensored SBP distribution from those individuals who, in spite of being clinically hypertensive, do not receive treatment for their condition. We judged that there was insufficient information on such individuals in the available Framingham data set to attempt that style of imputation.where ni values of yij into four new variables: the mean (bi1), the slope (bi2), the curvature (bi3), and log residual variance (log si2), by constructing orthogonal polynomials for unequally spaced variables [zi be an ni \u00d7 3 matrix,For each individual, we then transformed these ariables . Let zi ith individual are the elements ofThe first three new variables for the yi is the ni \u00d7 1 vector of SBP values for the ith individual, and the fourth new variable is the logarithm of the residual variance of the model,where Multipoint allele sharing identical by descent (IBD) was estimated at approximately 2-cM intervals using the GENIBD computer program from the S.A.G.E. package . After rya and yb for the pair of sibs a and b) on the mean proportion of alleles shared IBD and covariates for cohort membership, gender, mean age, and a conditional mean effect:Linkage analysis was then performed on approximately 1077 sibling pairs (varying by genetic marker informativity) by regressing the weighted squared sums and differences of each of the new variables , and aa and ab are the mean ages of a and b over all exams. Including the sum of the trait values as a covariate allows for the simultaneous estimation of the mean trait value conditional on marker allele sharing and all covariates, which in turn increases the power to detect linkage.where ferences -9,  is tD, within a simplex determined by the probability that a sibling pair shares i alleles IBD fi, whereThe above model was fitted using generalized least squares to account for the trait correlations among pairs of sibling pairs that occur in sibships of size greater than two ,8. Additbi1, bi2, and bi3 were calculated based on the inverse sum of the variance of each parameter estimate and the residual variance,Trait-based weights for  were calculated based on a first order approximation of the inverse of the variance of ,Trait-based weights for , where  is an estimate of \u03b22, and  is the corresponding estimated variance of .A combined weight that included information on both trait and marker informativity was obtained by taking the geometric mean of the marker and trait weights. We tested for the presence of significant linkage using usual right-tailed t-test statistics p-values lower than 10-4 and 10-5, respectively. Using these criteria, the most significant results for mean SBP were found on chromosome 12 near marker GATA47F05 (p = 0.00000029), on chromosome 15 between markers GATA22F01 and GATA27A03 (p = 0.00000021), and on chromosome 17 near marker GATA28D11 (p = 0.0000048), and on chromosome 20 near marker GATA47F05 for both slope and curvature of SBP , as shown in Table All results presented below include the covariates mentioned above in their models. However, the significance of these covariates will not be discussed because it was not the design of the analysis to obtain interpretable parameter estimates, but rather to test for linkage. Criteria for suggestive and significant evidence for linkage were chosen as markers having Although it was one of our goals, the analysis performed for the mean SBP is not directly comparable to the linkage scan performed by Levy et al. . A fundap-value was decreased by at least an order of magnitude when including marker, trait, or both weights. Though we were not able to conduct an analysis directly comparable to that performed by Levy et al. [Weighting our regression models by marker and trait informativity increased the power to detect linkage Figure . In sevey et al. , we pres"}
+{"text": "Three variants of the confidence set inference (CSI) procedure were proposed and applied to both the simulated and the Collaborative Study on the Genetics of Alcoholism (COGA) data. For each of the two applications, we first performed a preliminary genome scan study based on the microsatellite markers using the GENEHUNTER+ software to identify regions that potentially harbor disease loci. For each such region, we estimated the sibling identity-by-descent sharing probability distribution at the putative disease locus. Based on these estimated probabilities, the CSI procedures were employed to further localize the disease loci using the single-nucleotide polymorphism markers, leading to confidence intervals/regions for their locations. For our analysis with the simulated data, we had knowledge of the simulating models at the time we performed the analysis. A frequently used strategy in linkage analysis is to first screen the entire genome using microsatellite (MS) markers, and then to follow up on preliminary linkage regions using densely saturated (often single-nucleotide polymorphisms (SNP)) markers. Although many statistical methods are available for each step of this two-stage approach, only a limited number of them  are ablThe confidence set inference (CSI) procedure  can be um:This is a two-point approach that tests the following hypotheses for each marker 0m : \u03b8m \u2264 \u03b80 vs. Ham: \u03b8m > \u03b80,H\u03b8m denotes the true, but unknown, recombination fraction between the disease locus and marker m, and \u03b80 is a predetermined recombination fraction. Note that the above null and alternative hypotheses are the reversals of those in traditional linkage analysis. It is actually this formulation that allows us to construct the confidence region for the location of the trait locus [where it locus ,3. For tit locus  because it locus . Recentlit locus  suggesteL be the set of markers for which the corresponding null hypotheses are not rejected at level \u03b1. Then the probability that L includes at least one marker located within \u03b80 from the disease locus is at least (1 \u2013 \u03b1). From this set of markers, we can deduce the following confidence region for the disease locus:Let \u03c4m is the map position of marker m, while d (\u03b80) is the genetic distance that corresponds to recombination fraction \u03b80. In practice, \u03b80 is usually chosen to correspond to half of the maximum distance between any two adjacent markers in the region to be investigated.where \u03c4 in the preliminary linkage region, we test the following hypotheses:For an arbitrary location Hm 0: \u03c4 = \u03c4* vs. Ham : \u03c4 \u2260 \u03c4*\u03c4* is the true location of the disease locus in the region, and m is the marker closest to \u03c4. The following is our strategy for carrying out these tests. First, we consider a finite number of loci  in the region. For each of these loci, we test whether the IBD sharing at the nearest marker locus m is within the margin of error from what is expected if the disease locus is indeed at \u03c4 (the null hypothesis). We then iteratively refine the discretization strategy so that the locations for which the null hypotheses are not rejected constitute a (union of) \"continuous\" chromosomal segment(s).where m, information from all markers, not just marker m itself, are used.This is the multipoint extension of CSI-v1. The hypotheses and the discretization/ search strategies are the same as before. However, when calculating the observed sharing statistic at the nearest marker locus This is another multipoint extension with the same hypotheses as in CSI-v1 and CSI-v2, but the observed sharing statistic is calculated at the hypothesized locus itself given all the observed marker data.\u03c4 be the putative disease locus (at which the maximum score occurs) in a linkage region, and zk, k = 0, 1, 2, be the probabilities that an ASP shares k alleles IBD at \u03c4. Then, the likelihood of zk isIn Papachristou and Lin , the IBDn is the number of ASPs in the study. Note that the likelihood is parameterized in terms of z1 and z2 only, since z0 is completely determined by the other two. For more details on the computation of the above likelihood the reader is referred to Kruglyak et al. [zk values that maximize the above likelihood  are taken as estimates of the IBD probabilities.where k et al. . The zk For all four simulated populations, we extracted all possible families with at least two affected children. For families with three or more affected children all possible pairs were formed and were treated as if they were independent nuclear families. This method yielded an average of 150, 170, 150, and 180 ASPs for the AI, DA, KA, and NYC populations, respectively. For the application to the Collaborative Study on the Genetic Analysis of Alcoholism (COGA) data, ASPs were also extracted from the extended pedigrees, yielding a sample of 551 pairs. The ALDX1 diagnostic criterion was used, and only those who were confirmed to be affected were used in our study.After linkage regions are identified using the method of Kong and Cox (KAC)  as impleFirst, the KAC scores were calculated using the MS markers throughout the whole genome. Only chromosomes 1, 3, 5, and 9 yielded significant results using the threshold of 3.09 (corresponding to a pointwise significant level of 0.001) for more than one-third of the replicates in any of the populations. Therefore, we decided to focus on those 4 chromosomes for obtaining confidence regions using CSI and its variants based on the 3-cM-density SNPs.From the clinical ascertainment scheme, DA would be most informative for a locus influencing the behavioral symptoms. It turns out that, in the simulating model, locus D1 (on chromosome 1) plays an important role in the trait relating to these symptoms. Indeed, the KAC results reveal that only population DA has a majority of the replicates  showing linkage at the pointwise significance level of 0.001. Among the other three populations, only AI has more than one-third of the replicates (45) showing significant results. Similarly, as expected from the ascertainment schemes, all four populations contain information about the disease gene on chromosome 3, and thus they all have more than one-third of the replicates  with significant results. For the genes on chromosomes 5 and 9, on the other hand, only AI and KA contain information about their locations. Again, the results are consistent with the simulating model in that KA is more informative about these two loci than AI . Surprisingly, more than two-thirds of the replicates from NYC are not informative for these loci.Table Whole-genome screening using KAC based on the MS markers resulted in 6 chromosomal regions  with the maximum KAC scores exceeding the cutoff of 2.33 (a pointwise significant level of 0.01). As with the simulated data, our analysis scheme, after preliminary linkages are established, is to use the SNPs data to further narrow down the linkage regions. Specifically, the four CSI procedures, all with 95% coverage probability, were used to construct confidence regions for the disease gene locations. However, chromosomes 1 and 15 are not included in the CSI analysis as there are no SNPs in the preliminary linkage regions (toward the ends) on these two chromosomes. Figure The purpose of this contribution is two-fold. First, we want to demonstrate that, unlike most of other linkage methods, confidence regions with pre-specified coverage probabilities can be obtained by the CSI procedures. This is especially useful following preliminary linkage analysis. Specifically, after linkage is established, dense SNP markers can be genotyped in the linkage regions so that the CSI procedures can then be applied, perhaps as an intermediate mapping method before fine mapping association studies commence. Second, through the analyses of both the simulated and the COGA data, we show that the CSI procedure  can be fFor the COGA dataset, we are able to place the disease loci on three of the chromosomes to narrow confidence regions using CSI-v3 (ranging from 1.5 cM to 12.3 cM in length), which may have potential implications in studying the genetics of alcoholism. For the simulated data, however, the confidence regions are still quite large (around 30 cM for most of them) with the two multipoint variants. We speculate that this is mainly due to the limited informativeness of the still quite sparse 3-cM-density SNP markers. We believe that, with a much denser SNP map (say one with 0.25\u20130.5 cM inter-marker separation), further narrowing can be achieved. Moreover, we also plan to explore other methods for estimating relative risks (or IBD probability distributions at disease loci) to examine their effects on the results from the CSI procedures.ASP: Affected sib-pairCOGA: Collaborative Study on the Genetics of AlcoholismCSI: Confidence set inferenceEM: Expectation maximizationIBD: Identity-by-descentKAC: Kong and CoxMS: MicrosatelliteSNP: Single-nucleotide polymorphismBoth authors contributed equally to the conceptual development of the project. CP performed the analysis. SL drafted the manuscript. Both authors read and approved the final manuscript."}
+{"text": "We evaluate a method for the incorporation of covariates into linkage analysis using the Genetic Analysis Workshop 14 simulated data. Focusing on a randomly chosen replicate (42) we investigated the effect of the 12 subclinical phenotypes, sex, population, and parent-of-origin on the linkage signal from a model-free linkage analysis of Kofendrerd Personality Disorder.We detected a linkage peak on chromosome 1, at about 175 cM, which varied depending upon individuals' status for subclinical phenotype b. A linkage peak on chromosome 3 (310 cM) was found not to depend upon subclinical phenotype status. Further peaks were found on chromosomes 5 (12 cM), 9 (4 cM), and 10 (95 cM), depending on the status of subclinical phenotypes a, k, and c/d/g, respectively.Retrospective comparison of our results with the simulation model showed correct identification of disease loci D1-5 on chromosomes 1, 3, 5, 9 and 10, respectively. We chose to analyze all four populations of replicate 42 from the simulated data set. All analyses were performed without knowledge of the simulation model. The aim of the analysis was to utilize the information on the subclinical phenotypes of Kofendrerd Personality Disorder (KPD), sex, population, and parent-of-origin in a linkage analysis. Including covariates in the analysis allowed us to investigate models, such as locus heterogeneity, that give rise to different subclinical phenotypes within KPD. We present the results of our analyses and a retrospective comparison with the simulation model.Zlr test statistic from ALLEGRO [We began by screening the genome for linkage to KPD. We performed separate scans of the microsatellites and the single-nucleotide polymorphism (SNP) data using the  ALLEGRO , with thThe multipoint likelihood of the marker data of an affected relative pair at any point in the genome is given byj is the (unknown) probability that an affected relative pair share j alleles identically by descent (IBD), and fij, are the prior and posterior  probabilities that pair i shares j alleles IBD [pFS be the probability that a pair of affected full siblings share a given parental allele IBD. Following the suggestion of Rice [0 = (1 - PFS)2, z1 = 2 pFS (1 - P FS), and . Similar formulae apply for double-first-cousin pairs.where zeles IBD ,3. Theseeles IBD  and ALLEeles IBD , respect of Rice ,6, in thR, can only share 0 or 1 allele IBD. For these, z0 = 1 - PR, z1 = PR, and z2 = 0 (where PR is the IBD probability for affected relative pairs of type R).Other types of relative pair, PR in a logistic regression framework including a 3-level factor \u03b2 with levels corresponding to the status of the pair with respect to the covariate . That is,The effect of a binary covariate on the IBD sharing probabilities may be investigated by modelling OR is a fixed offset, ensuring that PR takes the correct value for a relative pair of type R in the absence of linkage . Under the null hypothesis of no covariate effect, \u03b1 is a measure of the divergence of IBD from the null in the sample as a whole. The subscript k indexes the status of the particular relative pair with respect to the covariate. Multiple pairs from the same pedigree were analysed as if they were independent, with parameters \u03b1 and \u03b2 in common. To ensure identifiability of the parameters, \u03b2-/- was set to zero (making \u03b1 a measure of IBD divergence from the null in -/- pairs). The degree of IBD sharing for the discordant (-/+) pairs was constrained to be less than or equal to the maximum IBD in the concordant pairs, to ensure that the model makes sense biologically. Each of the subclinical phenotypes (a - l) was modelled in this way, as was sex . Population membership was modelled as a four-level factor, with one level for each population (the first was set to zero). The total number of affected relative pairs in each category is shown in Table where Locus \u00d7 locus interactions between the peaks were investigated by including the estimated IBD sharing value for each pair at one location on a different chromosome  as a quantitative covariate in the logistic regression for IBD at the peak of interest . This isfi1 and , of sharing 1 allele IBD into components reflecting whether the paternal or maternal allele was shared. The IBD probabilities for affected pairs were expressed in terms of IBD probabilities for the paternal (ppat) and maternal (pmat) alleles , with the test statistic for parent-of-origin effect given by a likelihood-ratio test of ppat = pmat.Finally, parent-of-origin effect was modelled in affected sibling pairs only by splitting the prior and posterior probabilities, x, to give , and to both \u03b1 and \u03b2, giving . The ratio of the maximum likelihoods on the chromosome, with and without the covariate of interest, gives a LOD score, which was used as the test statisticTo test for effects of categorical or quantitative covariates, the likelihood was maximized with respect to \u03b1 alone at each position n replicates are generated in this manner, of which r give a test statistic greater than that in the actual data, the chromosome-wide p-value is estimated by (r + 0.5)/(n + 0.5).We allowed the location of the maximum likelihood to change when the covariate was added. This reflects the fact that linkage peaks from standard analyses are often some distance from the true disease locus . Incorpo. For example, an increase in LOD score of 2 to 3 is more significant than from 0 to 1 because the former is likely to occur by chance (in the absence of covariate effects) only in a linkage peak region, whereas the latter could occur anywhere on the chromosome. An estimate of genome-wide significance for a given chromosome, allowing for multiple testing, involves a joint Bonferroni-type adjustment for the relative length of the chromosome and the number of covariate tests conducted.For the test statistic chosen for this analysis, it was not possible to obtain a genome-wide significance level for covariate effects because this depends not only on the increase in LOD score given by the covariate, but also on the linkage evidence present without allowing for the covariate, i.e., based on The subclinical phenotypes c, d, and g were indistinguishable in the affected individuals and e, f, and h were all present in the affected individuals and hence provided no useful information for analysis. Therefore, we have carried out 10 covariate analyses on each chromosome . The interaction analyses were carried out between identified peak regions and hence were treated separately.Zlr = 4.97 at 177 cM), 3 (max Zlr = 5.58 at 310 cM), 5 (max Zlr = 5.11 at 12 cM) and 9 (max Zlr = 6.04 at 4 cM). On chromosome 1 the peak was narrower with the 3-cM SNP map than with the microsatellite map, but this effect was not seen for the other peaks.We found genome-wide significant linkage peaks on chromosomes 1 . The linkage evidence appeared to come entirely from the +/+ pairs . A similar effect was found on chromosome 5 with the subclinical phenotype a , with the linkage coming from the -/- pairs , and chromosome 10 (at 95 cM) with the subclinical phenotype c . On chromosome 9, the LOD increased from 7.65 to 18.13 with subclinical phenotype k , with increased sharing in both the -/- and +/+ pairs . No genome-wide significant effect of subclinical phenotype was observed on chromosome 3. No significant results were obtained for the analyses considering differences in IBD owing to sex, population, parent-of-origin, or interactions between the four identified linkage peaks. For each analysis, the maximum LOD score is presented in Table The linkage signal on chromosome 1 was found to increase substantially when the subclinical phenotype b was fitted as a covariate in the relative pair covariate linkage analysis, a LOD of 7.07 being increased to 14.29 (chromosome-wide p = 0.016), but this was not significant at the genome-wide level.Retrospective comparison of our results with the simulation model showed correct identification of disease loci D1-5 on chromosomes 1, 3, 5, 9, and 10, respectively. D1 influences phenotypes P1 and P3, which both have subclinical phenotype b, confirmed by the increased sharing we observed in the b+/+ affected pairs. D2 influences all three phenotypes, P1-3, with one or two of the subclinical phenotype b and c in a somewhat complicated manner. D2 also influences subclinical phenotype k. We observed increased IBD in the k+/+ pairs . This is because the penetrances of the low-risk genotype combinations were set to zero, giving a multiplicative model for interactions. Under such models, IBD sharing at one locus is independent of that at the other . The D1\u2013From analyzing the data blind to the simulation model, there appear to be five susceptibility genes for KPD, located on chromosomes 1, 3, 5, 9, and 10. Those on chromosomes 5 and 10 appear to influence disease only in the absence of subclinical phenotypes a and c/d/g respectively. The locus on chromosome 1 influences disease only in individuals with subclinical phenotype b, whereas that on chromosome 9 appears to have two variants, one giving rise to the presence of subclinical phenotype k in affected individuals, the other to its absence. No subclinical phenotype was found to have a significant genome-wide effect on the linkage of KPD to chromosome 3, although k reached chromosome-wide significance. Even with knowledge of the simulation model, it was difficult to detect the locus-locus interactions, suggesting that affected relative pairs give little power for such analyses.IBD: Identical by descentKPD: Kofendrerd Personality DisorderSNP: Single-nucleotide polymorphismAll authors contributed to the statistical analysis and interpretation of the data, and to the drafting of this article."}
+{"text": "Complex diseases are often reported along with disease-related traits (DRT). Sometimes investigators consider both disease and DRT phenotypes separately and sometimes they consider individuals as affected if they have either the disease or the DRT, or both. We propose instead to consider the joint distribution of the disease and the DRT and do a linkage analysis assuming a pleiotropic model. We evaluated our results through analysis of the simulated datasets provided by Genetic Analysis Workshop 14. We first conducted univariate linkage analysis of the simulated disease, Kofendrerd Personality Disorder and one of its simulated associated traits, phenotype b (fear/discomfort with strangers). Subsequently, we considered the bivariate phenotype, which combined the information on Kofendrerd Personality Disorder and fear/discomfort with strangers. We developed a program to perform bivariate linkage analysis using an extension to the Elston-Stewart peeling method of likelihood calculation. Using this program we considered the microsatellites within 30 cM of the gene pleiotropic for this simulated disease and DRT. Based on 100 simulations of 300 families we observed excellent power to detect linkage within 10 cM of the disease locus using the DRT and the bivariate trait. Due to the complexity of the transmission of complex diseases, more researchers are paying attention to disease-related traits (DRT), or endophenotypes. We define a DRT as an abnormality that 1) appears more frequently in cases  than in the population, and 2) has a higher frequency in unaffected siblings of cases than in the population. There can be different explanations of the relationship between the disease and the DRT. One explanation is that the traits are determined by a pleiotropic gene, a gene that controls more than one trait. For instance, a single gene mutation may cause an enzyme deficiency, which in turn may affect more than one tissue in one individual [The DRT, phenotype b, fear/discomfort with strangers (FDS), appears at a greater frequency in Kofendrerd Personality Disorder (KPD) affected individuals than in the general population. Additionally, the trait FDS appears more frequently in unaffected siblings of KPD individuals than in the general population. Moreover, according to Greenberg , both trThe study was conducted on nuclear pedigree datasets collected from Aipotu, Karangar, and Danacaa, respectively, and the combination of these three datasets. The simulated data from each city contained 100 nuclear families averaging about 7 members per family. There was no missing phenotype or marker data. Each dataset was simulated 100 times .In order to demonstrate that FDS was a DRT we investigated its distribution in offspring of these nuclear families. For our sample of probands, we used all offspring in these families who had KPD. All offspring who did not have KPD were considered unaffected siblings of probands. Upon doing this we noted that in Karangar 47% of the probands had FDS, 3% of the unaffected siblings of KPD probands had FDS; the rate of FDS in Karangar was 2% [We noted first that the two binary traits could be considered as a single trait with 4 phenotypes, which we defined as follows: 1) KPD positive and FDS positive, KPD+ FDS+; 2) KPD positive and FDS negative, KPD+ FDS- ; 3) KPD negative and FDS positive, KPD- FDS+; 4) KPD negative and FDS negative, KPD- FDS-.B, with entries gu(x). Here gu(x) denotes the penetrance of the phenotype x for the uth genotype, for u = 1, 2, 3 and x = 1, 2, 3, 4, where . A nuclear family dataset is composed of information on family size, the phenotype of interest, and the marker genotype for each individual. We used disease locus allele frequencies, and marker allele frequencies provided by Genetic Analysis Workshop 14 (GAW14) [There are then 12 penetrance values in penetrance matrix \u03c8Conditional on the genetic parameters and the joint distribution of the 4 phenotypes and marker genotypes in family members, we calculated the LOD score for the bivariate trait for a given nuclear family as follows:pstu denotes the probability that an offspring has genotype u at the disease/DRT locus given that the parents disease/DRT genotypes are s and t; gu(xi) denotes the probability of having trait phenotype x  given that the disease/DRT genotype is u for the ith offspring in the nuclear family; n denotes the number of offspring; pfm (pmm) denotes the probability that the father's marker (mother's marker) genotype is fm (mm); pt (ps) denotes the probability that the father (mother) has disease/DRT genotype s (t).Here, as in Elston and Stewart , pstu deThe algorithm described above was implemented in a C++ program, GAWBI ,5, whichWe computed the bivariate LOD score for the combined city samples of 300 nuclear families and the average of these bivariate LOD scores over 100 replicates (bivariate ELOD). This value was then compared to the average LOD score obtained on considering the disease status alone (ELOD-disease) and the trait status alone (ELOD-DRT) using the usual univariate LOD score method and using the marginal penetrance values of KPD and FDS assumed in the bivariate analysis. We then calculated the frequency of the bivariate LOD \u2265 3.0 out of 100 replicates and referred to this value as the bivariate power. Power was similarly obtained for disease and DRT.We used the gene frequencies given by GAW14  as the vThe markers in the region close to locus D1 contained in the microsatellite files were investigated. The number of alleles at each marker varied from 4 to 9.The ELOD results for the linkage analysis of the samples of 300 families are presented in Figure In Figure In Figures In all ELOD and power figures, the highest values are observed at marker position D01S0023, which is the closest marker to D1 (the major susceptibility gene).The results observed for the linkage analysis of FDS/KPD appear at first to be counterintuitive. We would expect that the bivariate approach would result in greater power than consideration of a single trait. This is indeed what we observed in comparing the two approaches under a wide range of generating models . HoweverWe did have a rough estimate of the generating model and perhaps did make a better than average guess than one could do in reality. However Ji  investigOur results indicate that the power of the bivariate analysis appears to be more sensitive to the accuracy of the penetrance values assumed than the analysis of the DRT alone. Ji  did noteThese findings indicate the need to consider several analysis model parameter values with a correction for the number of parameter values considered as is done in LOD score analysis of a single binary phenotype. We also need to realize that there are going to be some situations in which analysis of the trait alone might be as powerful as analysis of the bivariate trait.j, j = 1, 2, 3, or 4. In the parameter input file, the penetrances of each liability class are set to equal P(j|genotype), where genotypes are disease/DRT genotypes. A simple dataset was tested using both LINKAGE and GAWBI, and identical results were obtained.The bivariate analysis discussed in this study was done using software (GAWBI) developed by Ji and Yoo ,5; howevDRT: Disease-related traitsFDS: Fear/discomfort with strangersGAW14: Genetic Analysis Workshop 14KPD: Kofendrerd Personality DisorderNRM and FJ conceived the study, participated in its design and coordination, and drafted the manuscript. NRM presented this work. FJ developed GAWBI, the bivariate LOD score analysis program for this study. FJ and DL carried out all the genetic analyses and statistical analyses."}
+{"text": "Cigarette smoking behavior may have a genetic basis. We assessed evidence for quantitative trait loci (QTLs) affecting the maximum number of cigarettes smoked per day, a trait meant to quantify this behavior, using data collected over 40 years as part of the Framingham Heart Study's original and offspring cohorts.Heritability was estimated to be approximately 21% using variance components (VC) methods (SOLAR), while oligogenic linkage and segregation analysis based on Bayesian Markov chain Monte Carlo (MCMC) methods (LOKI) estimated a mean of two large QTLs contributing approximately 28% and 20%, respectively, to the trait's variance. Genome-wide parametric (FASTLINK) and VC linkage analyses (SOLAR) revealed several LOD scores greater than 1.0, with peak LOD scores using both methods on chromosomes 2, 17, and 20; multi-point MCMC methods followed up on these chromosomes. The most robust linkage results were for a QTL between 65 and 84 cM on chromosome 20 with signals from multiple sex- and age-adjusted analyses including two-point LOD scores of 1.30 (parametric) and 1.07  at 70.51 cM, a multi-point LOD score of 1.50  at 84 cM, and an intensity ratio of 12.0 (MCMC) at 65 cM.Familial aggregation of the maximum number of cigarettes smoked per day was consistent with a genetic component to this behavior, and oligogenic segregation analyses using MCMC suggested two important QTLs. Linkage signals on chromosome 20 between 65 and 84 cM were seen using multiple analytical methods. No linkage result, however, met genome-wide statistical significance criteria, and the true relationship between these regions and smoking behavior remains unclear. Many behaviors, such as smoking, offer a variety of possible phenotypes that may have differing genetic components. Genomic scans for current smoking status ,2, pack-Data from the Framingham Heart Study and the Framingham Offspring Study were analyzed as part of Genetic Analysis Workshop 13 (GAW13) and are described elsewhere ,5.Self-reported number of cigarettes smoked per day was available from at least one exam for 2883 participants. The quantitative trait maximum number of cigarettes per day represented the largest number of cigarettes smoked per day reported by each participant at any point throughout the study; this value was equal to zero for individuals who reported smoking no cigarettes at each exam. Skewness and kurtosis for maximum number of cigarettes per day were 1.0 and 3.7, respectively . A variety of transformations were used on the trait; however, because none markedly reduced skewness or kurtosis, analyses were performed on untransformed values. Covariates for some analyses included sex and the age and year at which the maximum number of cigarettes were smoked. These age and year variables were the first age and year at which the person reported smoking that quantity (including zero for non-smokers) if it was reported more than once.Intraclass correlation coefficients among pairs of relatives were calculated using FCOR, a component of S.A.G.E. 4.2  with pedGenome-wide two-point parametric LOD score analyses for maximum number of cigarettes per day were performed with FASTLINK -11 usingGenome-wide two-point VC linkage analysis was performed using SOLAR 1.7.3 [In order to assess possible cohort effects, additional two-point parametric LOD and VC analyses considered year of smoking maximum number of cigarettes as a covariate.p-values, the results were used to provide a count of the number of times a particular genomic position was accepted as the position of a QTL during an update of the model (a \"hit\"). Using the intensity ratio (IR), these estimated numbers of hits were then compared with the number that would be expected by chance, given the specified prior distributions. In map interval i, the IRi was calculated as follow: IRi = hi/ei, where hi was the observed number of hits in map interval i and ei, the expected number of hits in map interval i. The expected number of hits ei being (E(n)/L) \u00d7 bi \u00d7 I. E(n) was the expected number of QTLs in an iteration in the analysis, obtained from the prior distribution, L was the total map length, bi was the bin width, and I was the number of iterations. IRs were computed using a 2-cM bin and a total map length of 3000 cM.Chromosomes yielding LOD scores > 1.0 in both parametric and VC sex- and age-adjusted analyses were followed up with multi-point oligogenic joint linkage and segregation analyses using Bayesian MCMC methods implemented in LOKI 2.2  with sexOf 330 pedigrees , smoking history was available for 2883 individuals, including 1743 (37%) ever-smokers (653 genotyped) and 1140 (24%) never-smokers (402 genotyped). Among smokers, on average, the amount of smoking when individuals smoked their most was 24.2 cigarettes per day  occurring at an average age of 41.7 years  in 1967 (range 1948\u20131991).2 = 0.18 \u00b1 0.03, 2796 pairs); the correlation estimate did not vary after adjustment for age and sex (r2 = 0.18 \u00b1 0.03) or age, sex, and year (r2 = 0.16 \u00b1 0.03). Unadjusted correlation estimates were lower for parent-child pairs  and spouse pairs . Heritability of this trait was estimated to be 0.21 (SE = 0.03) using the VC approach (p < 0.001), and did not vary with adjustments for sex, age, or year . Allowing for a t-distribution did not change VC modeling results . Using MCMC oligogenic segregation analysis, the components of variance for maximum number of cigarettes smoked per day were as follows: residual variance 41%, age 3%, sex 5%, and total genetic variance 51%. Two large QTLs were estimated for the trait, with individual contributions of approximately 28% and 20% of the total variance, respectively, which explained approximately 55% and 39% of the genetic variance. MCMC analysis estimated the largest QTL to be overdominant.Maximum number of cigarettes smoked per day was correlated in sibling pairs (rp < 0.001). There were two LOD scores greater than 1.0 in unadjusted analyses; one on chromosome 2 at 70.32 cM (LOD = 1.98) and the other on chromosome 17 at 66.85 cM (LOD = 1.52) . Adjustment for sex and age reduced the significance of the signal on chromosome 1  number of cigarettes per day in the Framingham Heart Study's original and offspring cohorts was assessed in segregation and linkage analyses. Correlations between sibling pairs were consistent with the existence of a genetic component, as were variance components estimates of heritability (0.21\u20130.23); in addition, MCMC oligogenic segregation analysis estimated that 48% of the total phenotypic variance could be attributable to approximately two large QTLs.Genome-wide linkage analyses using parametric and VC methods, both unadjusted and adjusted for sex and age, revealed several regions with LOD scores greater than 1.0. Chromosomes 2, 17, and 20 harbored peak LOD scores using both methods (though only in similar positions on chromosome 20). Follow-up of these chromosomes using multi-point MCMC analysis were consistent with the existence of QTLs on chromosomes 2 and 20. The most robust linkage result in our analyses was between 65 and 84 cM on chromosome 20 in the 20q13.1 region; results from sex- and age-adjusted analyses included peak two-point LOD scores of 1.30  and 1.07  at 70.51 cM (marker GATA47F05), a peak multi-point LOD score of 1.50  at 84 cM, and an IR of 12.0 (MCMC) at approximately 65 cM. The trait distribution's non-normality may have affected accuracy of QTL detection, however, and no linkage result in this analysis was statistically significant.We chose to examine the genetics underlying a propensity to smoke large quantities of cigarettes at any age, a narrowly defined \"extreme\" quantitative phenotype that we hoped may have provided improved power -3. NonetTo our knowledge, no previous studies of any component of smoking behavior have indicated linkage with markers on chromosome 20 -3, with In conclusion, these results, though not definitive, may lend support to other evidence for a genetic influence on smoking behavior and may provide some clues as to where specific QTLs might lie. Additional research is needed to further understand the complex relationship of genes, the environment, and smoking behavior. Given that the best analysis method for a complex trait is often unknown, it is of particular interest whether the use of multiple methods with a conclusion based on the consistency of the findings would improve the reliability of linkage analyses."}
+{"text": "Cardiovascular disease-related traits, such as body mass index (BMI), systolic blood pressure (SBP), triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL), and glucose levels (GLUC), have moderate to high correlations with each other. We hypothesized that there might be some common factors underlying the correlations of these traits, and attempted to identify these factors and their genetic structures. Cross-sectional measurements from the 330 extended Framingham Heart Study families were used in this study. Principal component factor analysis was applied to obtain the factors that were then analyzed using variance components linkage analysis.With the above six traits three factors were generated: BMI-SBP-GLUC, HDL-TG, and TC-TG. The heritabilities for these factors were 32%, 45%, and 49%, respectively. Comparing the linkage results of the factors with the results of their component traits, evidence for linkage was observed for the TC-TG factor to a locus on chromosome 2p23 with a two-point LOD score 2.73 (marker GATA8F07) and a multipoint LOD score 1.81 (at 54 cM), while the LOD scores for TC and TG did not exceed 1 at this region.Our analysis showed a locus on chromosome 2 might have a pleiotropic effect on the cardiovascular disease-related traits TC and TG. High body mass index (BMI), systolic blood pressure (SBP), triglycerides (TG), total cholesterol (TC), blood glucose levels (GLUC), and low high-density lipoprotein cholesterol (HDL) are risk factors for cardiovascular disease (CVD). They are also the features of the metabolic syndrome (syndrome X) . The corIn this study, we examined the relationship between the CVD-related traits, identified the underlying factors, estimated their heritabilities, and performed a genome-wide quantitative trait linkage analysis for susceptibility loci influencing the factors by using the Framingham Heart Study families.2, age3, age4, number of cigarettes smoked per day, and grams of alcohol consumed per day using a multiple linear regression procedure, separately by cohort. The standardized residuals were used in the PCFA.We used subjects from both of the Framingham cohorts. For the original cohort, most of the traits are from Exam 11 (between 1969 and 1971) while the fasting lipids are from Exams 10 to 12 (between 1967 and 1973). For the offspring cohort, the traits are from Exam 1 (between 1971 and 1975). These particular exam periods were chosen because: 1) in the Original Cohort, TG was only measured once \u2013 either at Exam 10, 11 or 12; 2) the traits from the two cohorts were collected during the same period of time. If an individual was treated for hypertension at the selected time point, his/her SBP was set to be missing. BMI was calculated as the weight in kilograms divided by the square of the height in meters. HDL, TC, TG, SBP, and BMI were log transformed, and GLUC was reciprocally transformed to minimize non-normality. Each measure was then adjusted for the effects of age, sex, age*sex, agePCFA is a variable reduction procedure that extracts uncorrelated factors from correlated variables ,6. All aFor a valid factor analysis, the following assumptions should be met: 1) interval-level measurement; 2) random sampling; 3) linearity; and 4) normal distributions . Random Variance components method was applied for genetic analyses using SOLAR v 1.7.3 . HeritabSeparate correlations between the six traits  from the original cohort and the offspring cohort are shown in Table Table We also investigated whether there were any marked differences between the factor loading patterns of the whole data and the random samples . For the 20 random samples, the first three factors account for a mean of 69% of the total trait variance with range 66%-72%. The mean coefficients of congruence between the factors of the whole data and the factors of each of the random samples are 0.99 (range 0.98\u20131.00), 0.98 (range 0.94\u20131.00), and 0.98 (range 0.90\u20131.00). These results indicate that the factor structures of these random samples were very close to the structure of the whole data.P < 10-7).The heritabilities of the factors and individual traits are listed in Table The two-point linkage analysis results are summarized in Table The results from multipoint linkage analysis are similar to the two-point linkage analysis results. In particular, Figure In this study, we used PCFA to investigate the clustering features of six CVD-related traits. Three factors were generated: BMI-GLUC-SBP, HDL-TG, and TC-TG. All these factors and individual CVD-related traits are under significant genetic influences. Using two-point and multipoint variance components linkage analyses, we found suggestive evidence of linkage for these factors and individual traits.There has been another factor analysis study of the traits from the Framingham Heart Study offspring cohort . In thatComparing the linkage results of the factors to the results of individual traits, most of the loci that show suggestive evidence of linkage (LOD \u2265 2) to the factors likely reflect the strong linkage signal from one of its component individual traits. The exception is the region on chromosome 2p23.2, which shows evidence of linkage to the TC-TG factor but has no high LOD score for any of the individual traits. This is also a region (35 to 58 cM) that shows the evidence of linkage to TG in the Pima Indian population . This inFactor analysis has been criticized for being a subjective method due to the results being sensitive to the criteria used to determine the number of factors to choose, the factor loading threshold, and the number of variables to be included in the analysis. However, the factor loading patterns for traits related with metabolic syndrome appear to be relatively stable across demographic and metabolic subgroups . For a dBy using PCFA, we identified three independent factors underlying the intercorrelation of the CVD-related traits in a community-based study population. These factors are significantly influenced by genetic effects. Our analysis also shows suggestive evidence of linkage to chromosome 2p23.2 for a lipid factor. Since none of the results for individual traits and factors reaches genome-wide significance in this study, the improvement of the LOD score for the TC-TG factor compared to the LOD scores of its component traits may be due to chance or real pleiotropic effect. Further study is needed to explore the application of PCFA in genetic studies for closely correlated traits of common complex diseases."}
+{"text": "We performed multipoint linkage analysis of the electrophysiological trait ECB21 on chromosome 4 in the full pedigrees provided by the Collaborative Study on the Genetics of Alcoholism (COGA). Three Markov chain Monte Carlo (MCMC)-based approaches were applied to the provided and re-estimated genetic maps and to five different marker panels consisting of microsatellite (STRP) and/or SNP markers at various densities. We found evidence of linkage near the GABRB1 STRP using all methods, maps, and marker panels. Difficulties encountered with SNP panels included convergence problems and demanding computations. Our aims were to investigate 1) the utility of single-nucleotide polymorphisms (SNPs) versus microsatellites (STRPs), and 2) the impact of map assumptions on linkage analysis. We chose to focus our analyses on the COGA ECB21 trait and chromosome 4 because previous studies [A multivariate polygenic model was used to obtain maximum likelihood estimates of the heritabilities and genetic correlations of ECB21 and 12 other EEG measurements . On the All 275 Illumina SNPs on chromosome 4 and 550 consecutive Affymetrix SNPs spanning STRPs 2\u201312 on chromosome 4 were selected. Among SNPs with identical meiotic map positions, the SNP with the largest minor allele frequency was retained, leaving a relatively sparse panel of 140 Illumina SNPs with an average spacing of ~1.5 cM (ILMN_1.5) and a dense panel of 476 Affymetrix SNPs with an average spacing of 0.3 cM (AFFY_0.3) for further analysis. A subset of 97 Affymetrix SNPs (AFFY_1.5) was selected by requiring an empirically determined minimum distance of 1.1 cM between SNPs, starting from the first SNP, to achieve a similar average density as ILMN_1.5. SNPs were interpolated onto the COGA STRP map by pegging the two flanking SNPs to each STRP and interpolating the intervening SNPs based upon the proportional distances in the corresponding intervals on the COGA and provided SNP maps.Genetic maps were re-estimated from the COGA data using a hybrid algorithm, based on MCMC-EM (expectation maximization) and stochastic approximation for STRPs and MCMC-EM for SNPs, to find the maximum likelihood estimates of the recombination fractions. Sex-averaged and sex-specific maps were re-estimated using all 17 STRPs on chromosome 4, and a sex-averaged map was estimated using STRPs 2\u201312 plus AFFY_0.3. Haldane map distances were used in all analyses and figures.pairs [Linkage analyses of the ECB21 traits on chromosome 4 used three MCMC-based methods from the MORGAN and Loki software packages . First, pairs  and detepairs  was usedpairs was computed at each marker in each replicate using lm_ibdtest.To evaluate the effects of the real chromosome 4 STRP data and provided map on type I error, 1,000 replicates of an unlinked quantitative trait, based on the ECB21_Q model, were simulated on the COGA pedigrees. The simulated trait was then dichotomized using the same cut point as for ECB21_D. For comparison, true null datasets were created by pairing each of the 1,000 unlinked trait replicates with a single set of unlinked markers, simulated based on the chromosome 4 STRP allele frequencies and map. Saa = -1.22, \u03bcAa= -1.14, \u03bcAA = 5.79, and residual variance of 22.0. Penetrances for ECB21_D were 19%, 19%, and 73% for the aa, Aa, and AA genotypes, respectively.The polygenic analysis estimated a narrow-sense heritability of 0.61 for ECB21_Q and genetic correlation of 0.47 between ECB21 and TTTH3. Oligogenic segregation analysis of ECB21_Q indicated the existence of at least one QTL. The estimated parameters for the most likely QTL model were: frequency of 0.411 for the minor allele \"A\", genotype means \u03bcThe re-estimated maps based on STRPs were similar to those provided and published, but there was substantial map inflation when SNPs were included Figure . The sexp-value for Spairs was an increase from p = 0.007 with the COGA map to p = 0.023 with the sex-specific map for ECB21_D at STRP 10. The empirical distribution of Spairs, based upon 1,000 replicates of a simulated unlinked trait and the real chromosome 4 STRPs, showed an excess of allele-sharing at STRPs 4, 16, and 17, whereas little excess sharing was observed with the true null replicates (Figure We observed a strong linkage signal near STRP 4 that was insensitive to the STRP map estimate. Whereas the COGA and re-estimated sex-averaged maps provided similar linkage results, small differences resulted from use of the estimated sex-specific map. The largest change in the permutation-based Multipoint STRP scans with three different MCMC-based methods all showed evidence of linkage of the ECB21 traits to chromosome 4 Figure . The strMCMC multipoint analyses with STRPs versus SNPs yielded similar results in the chromosome 4 60\u201380 cM region, but also gave important differences. AFFY_0.3 results were noisy compared to STRP results Figure , and numThree different MCMC-based multipoint methods gave evidence in the COGA STRP data for linkage of ECB21 to STRP 4 on chromosome 4. We also found weaker evidence of linkage near STRP 10. Comparison of sex-averaged and sex-specific STRP maps suggested that results may be robust to map-misspecification in the presence of strong evidence for linkage. However, the investigation of map assumptions may be important in elucidating weak linkage signals, especially in chromosomal regions with substantial male-female map differences. Map estimation using SNP data led to substantial expansion of genetic distances compared to maps estimated from STRP data, suggesting possible undetected SNP genotype errors or effects of linkage disequilibrium. Our analyses of simulated null datasets with an unlinked trait and real STRP data indicated that some regions of chromosome 4, including STRP 4, may be prone to false-positive linkage signals, and that this tendency persists even using maps estimated from the data. Possible explanations for false-positive results include genotype error or allele frequency misspecification.Multipoint analyses using STRPs, SNPs, or a combination of STRPs and SNPs yielded comparable evidence of linkage to the chromosome 4 region with the strongest signal. The signal strength was not greater for the dense versus sparse SNP panels. Furthermore, localization and interpretation of linkage signals for the dense SNP panel were complicated by noisy results, which could reflect MCMC mixing problems and/or genotype error. Multipoint analyses using sparse SNP panels produced smoother LOD score curves than the dense SNPs. These results suggest that increasing the density of SNP panels beyond an average spacing of 1.5 cM does not substantially increase the evidence for linkage in the COGA dataset, which consists of moderate-size pedigrees with relatively complete genotype data. Additional studies will be needed to determine the optimal density for SNP panels in other datasets. Our analyses with current MCMC approaches indicate that, while useable with dense SNPs in limited chromosome regions with medium-size pedigrees, long runs are needed to produce stable linkage analysis results. Run times may prohibit the use of dense SNP panels for whole-genome scans with current MCMC analysis programs. MCMC-based methods are among the best tools now available for the analysis of large pedigrees, numerous markers, and complex traits. Further development of these methods in order to accommodate dense SNP panels in the context of large pedigrees would be of value.COGA: Collaborative Study on the Genetics of AlcoholismEEG: ElectroencephalogramEM: Expectation maximizationGAW: Genetic Analysis WorkshopMCMC: Markov chain Monte CarloQTL: Quantitative trait locusSNP: Single-nucleotide polymorphismSTRP: Short tandem repeat polymorphism"}
+{"text": "We combined the results of whole-genome linkage and association analyses to determine which markers were most strongly associated with Kofendrerd Personality Disorder. Using replicate 1 from the Genetic Analysis Workshop 14 Aipotu, Karangar, Danacaa, and New York City simulated populations, we determined that several markers showed significant linkage and association with disease status. We used both SNP and microsatellite markers to determine patterns and chromosomal regions of markers. Three consistently associated markers were C01R0050, C03R0280, and C10R0882. Using generalized linear mixed models, we modelled the effect of the three predefined phenotypic categories on disease status and concluded that the phenotypes defining the \"anxiety-related\" category best predicted the outcome. Whole-genome linkage analyses involve looking for coinheritance of chromosomal regions with disease in families. Association studies seek to determine differences in the frequency of genetic variants between individuals exhibiting or not exhibiting a phenotype of interest (commonly case-control status). Family-based association studies utilize the available pedigree genetic variations to determine whether the transmission of particular genetic variants is associated with disease status. The results of linkage and association studies have been successfully combined in many analyses to refine the location of disease genes and to test the involvement of candidate genes in disease. The aim of this contribution was to perform linkage analyses, in combination with association analyses, on replicate 1 of the simulated Genetic Analysis Workshop 14 (GAW14) data to determine which markers or regions of markers are associated with Kofendrerd Personality Disorder (KPD).a through e, is referred to as \"communally shared emotions\" (CSE). This was constructed in the data by assigning a positive affection status to an individual if they possessed at least one of phenotypes a through e, and assigning an individual a negative affection status otherwise. This procedure was similarly performed for the second category, consisting of phenotypes f through i, termed \"behavioral-related\" (BR) and for the third category, comprising phenotypes j through l, referred to as \"anxiety related\" (AR). This recoding procedure allowed us to assess affection status not only in terms of an overall status, but also in terms of the three different methods adopted by the different populations for deciding disease ascertainment.The GAW14 problem 2 description states because of the \"varied phenotypes\" for KPD, the \"nosology for KPD falls into three different classifications\", and that all three are used in diagnosis. The three main groups of phenotypes are indicative of three different methods used by each population for disease ascertainment. The different ascertainment methods and phenotypic categories suggest that complex interactions may be a key factor in identifying the causes and genetic determinants of KPD. Because we were blind to the simulated dataset answers, we chose to recode the data into these three additional grouped phenotypes to determine if complex combinations of phenotypes are of importance, in addition to examining the relationship between individual phenotypes and affection status. We chose replicate 1 as a representative data set for each of the four simulated populations. The first category, consisting of phenotypes To perform linkage analysis on the simulated datasets, we used the MERLIN  pedigreep-values. The QTDT result files were input to JLGRAPH to produce association graphs for each chromosome for each population.To perform association analysis on the binary traits for the simulated datasets, we used the computer program QTDT  to perfoThe results from the linkage and association analyses were collated to provide a list of potential regions of interest for further study. Each of the 917 SNPs and 416 microsatellites markers were examined to determine their significance (in terms of both linkage and association) for affection, CSE, BR, and AR, for each of the four populations. Marker regions that appeared to be significant for both linkage and association were closely examined. Candidate packets of markers consisting of potentially important SNPs were \"purchased\" in order to analyze chromosomal regions of interest in fine detail. The procedures outlined above for association and linkage analysis were then repeated, this time incorporating the new marker sets.To determine the effectiveness of each of the three newly defined categories for disease ascertainment, the data were modelled using a generalized linear mixed model (GLMM). Affection status was used as a binary outcome, with CSE, BR, and AR as factored predictor variables and family ID as a clustering variable. A mixed effects model was used so that a random intercept could be fitted, defining individuals within families to be correlated.p-value to determine those of highest significance. Table p-values for each marker represent the most significant score from affection status, CSE, BR, and AR).Using the compiled list of markers from the results of the linkage and association analyses, we produced chromosomal graphs for each population in terms of SNP markers and microsatellite markers. From these initial graphs, we proceeded to select regions that appeared to be of most significance for linkage and/or association. We ranked the markers by After \"purchasing\" more packets of markers and analyzing these in conjunction with the marker information already provided, several chromosomal regions showed significant linkage and association with disease status. The additional fine mapped packets available for download contained mostly SNP markers and we consequently determined that SNP markers would be of higher importance than microsatellites. In particular, regions surrounding the SNP markers C01R0050, C03R0280, and C10R0882 were examined. We describe the region surrounding marker C03R0280 as follows.p-values  forming peaks over relevant markers. Figure , provided very little evidence for linkage or association with KPD across all of the populations.SNP marker C03R0280 is located 2.94 M along chromosome 3 of the simulated population. Figure  and 1B s. Figure  and 1D rp-value < 0.05).Phenotypic data were modelled using GLMMs to determine how effective each of the three categories used for disease ascertainment were at predicting overall affection status for each population. For all four populations in the simulated data, AR was the only category that effectively predicted disease status  that contributed to significant linkage and association for affection status within markers for each population. This flexibility enabled us to locate markers such as C03R0280 and determine that this particular marker had significant Through linkage and association analyses, markers C01R0050, C03R0280, and C10R0882 and markers surrounding these were found to be associated with affection status and the three phenotypic categories we defined (to varying degree within each region). Given time and \"budgetary\" constraints imposed on GAW14 participants, we successfully identified three gene regions, one within each of the three regions examined. While we did not find all the disease-associated genes contained within the GAW14 simulated datasets, we were successful in locating genes in the regions we focused on, indicating that our linkage and association mapping approach can successfully identify genes.j-lAR: Anxiety related phenotypes f-iBR: Behavior related phenotypes a-eCSE: Communally shared emotions phenotype GAW: Genetic Analysis WorkshopGLMM: Generalized linear mixed modelKPD: Kofendrerd Personality DisorderSNP: Single-nucleotide polymorphismKWC performed the linkage and association analysis, generated the graphs and drafted the manuscript. PAM designed the GLMM framework, conducted the data modeling, and drafted the manuscript. LJP conceived of the study and participated in the design and coordination of the study."}
+{"text": "M for smokers to be the maximum number of cigarettes-per-day (MAXCIG) reported over the longitudinal course of the study. Adjustments were made for the significant covariates of gender and year of birth, and sib-pair based linkage analysis was performed.Using the Framingham Heart Study data set provided for Genetic Analysis Workshop 13, we defined the cigarette-use phenotype The primary analyses, in which individuals with MAXCIG = 0 were considered to have missing phenotype, resulted in modest linkage evidence, with LOD scores over 1 on chromosomes 5, 9, 13, 14, and 22.While the results reported here do not indicate definitive evidence for linkage to specific chromosomal regions, future studies may find it useful to include direct assessments of maximum and quantitative cigarette use. In defining and analyzing quantitative or \"maximum use\" phenotypes, the choice of how to handle individuals with MAXCIG = 0, or alternatively, individuals who are substance-naive, is a crucial one for genetic studies of nicotine and other substance use. In this study, the linkage results vary greatly depending on whether or not these \"unexposed\" individuals are included in the analyses. Cigarette smoking is a leading cause of premature death and is a serious public health concern. Past studies of smoking behavior have considered a variety of smoking phenotypes and found that while smoking-related traits are complex, there is evidence for significant genetic influences on smoking behavior ,2.The Framingham Heart Study data set provided for Genetic Analysis Workshop 13 (GAW13) includes longitudinal data on daily cigarette use. In the past the Framingham data have been used to study important cigarette smoking patterns such as cessation and resumption .The cigarette smoking data consist of the number of cigarettes smoked per day during periods of use in the year prior to each exam. We have defined cigarette-use phenotypes based on these available data. However, as the exams were  2 years apart for the original cohort and 4 years apart for the offspring cohort, it is possible that periods of smoking behavior could have been missed. Furthermore, no information was available on lifetime smoking, the duration of regular smoking over the past year, or whether the subject was still currently smoking at each exam.M, or MAXCIG, as the maximum cigarettes-per-day reported over all the available exams . For the purposes of the descriptive results below, the term \"smokers\" refers to individuals with M > 0, and \"non-smokers\" refers to individuals with M = 0. However, it is important to note that M is not the same as true lifetime maximum use, which cannot be determined from the longitudinal data provided.We defined a maximum number of cigarettes phenotype M = 0 were excluded by recording their phenotype as \"unknown.\" From past experience with alcohol phenotypes we expect that defining substance-naive individuals to have unknown phenotype is most appropriate, as individuals who have not been exposed to a substance have unknown response; however, we also performed parallel analyses that included M = 0 individuals for comparison. When M = 0 individuals are included, we obtained 714 nuclear families with at least two phenotyped offspring, providing 1545 non-independent phenotyped and genotyped sib pairs. For the primary phenotype (M = 0 considered unknown), there are 412 such nuclear families, containing 621 non-independent phenotyped and genotyped sib pairs.For genetic analyses, the primary phenotype was taken to be the maximum number of cigarettes phenotype where individuals with M = 0 individuals are included.Linear regression  was used to correct the primary maximum-cigarette phenotype for the significant covariate of gender in the initial linkage analyses. Additional regression models adjusted for both gender and year of birth , both with and without interaction terms. Each of the resulting phenotypes was used for linkage analysis. Note that a similar regression adjustment is not as appropriate in the case where The original and offspring cohorts have different time intervals between exams; this difference could lead to systematic differences in the resulting smoking phenotypes. Thus we have compared maximum cigarette use when sampling at 2-versus 4-year intervals in the original cohort to examine whether this difference in time interval significantly affects the resulting phenotype.n(n-1)/2 pairs for a sibship of size n) using Haseman-Elston regression as implemented in MAPMAKER/SIBS [Multi-point linkage analysis was carried out on all sib pairs  was subtracted from the starting year of the study (1948); for the offspring cohort, age at first exam was subtracted from 1971, the starting year for the offspring recruitment. Using these definitions, there were only five men and eight women in the sample who were born in the 1960s (and none later), so binned cohorts were defined by decades with the youngest cohort having a birth year in the 1950s or later.We also examined descriptive birth cohort effects on M ranged from 0 to 95. For this full sample, among smokers the mean for M was 20.4 for females and 27.6 for males.In the full sample of original cohort and offspring cohort, M for the original cohort to the alternative phenotype Mo obtained from 4-year exam intervals rather than 2-year intervals. Specifically, Mo is the maximum cigarettes-per-day reported over all available odd-numbered exams . Among men, 30.8% reported M = 0 versus 34.6% who reported Mo = 0. Among women, 52.8% reported M = 0 versus 55.0% with Mo = 0. Among smokers, a t-test showed no significant difference between the means for M and Mo in either gender. Hence the difference in the frequency of exams between the two cohorts does not appear to be a serious concern in defining M.We compared descriptive results for M, consistent with a familial trait. The correlations were 0.25 in male pairs (p < 0.0001), 0.33 (p < 0.0001) in female pairs, and 0.13 (p = 0.002) in male-female pairs. Lower correlation in male-female pairs compared to same-sex pairs has similarly been observed for maximum alcohol consumption traits [Sib-pair correlations were significant for n traits .M (with M = 0 recorded as missing) for covariate effects, gender was highly significant . Year of birth, tested with two degrees of freedom for the linear and quadratic terms, was also significant when added to the model with gender . Finally, including interaction terms was also significant at the 0.05 level ; this is consistent with the pattern of effects in Table Maximum cigarette use levels by gender and birth cohort are presented in Table M corrected for gender (and M = 0 recorded as missing) resulted in LOD scores of 1.10 at 200 cM on chromosome 5, 1.67 at 173 cM on chromosome 9, 1.18 at 41 cM on chromosome 13, 1.29 at 87 cM on chromosome 14, and 1.04 at 3 cM on chromosome 22. Results for birth-year-adjusted analyses were very similar, with a slight increase in LOD score for chromosome 9 (1.77 for the full-model adjusted trait).Linkage results are summarized in Table When nonsmokers were included in the analysis, linkage results were substantially different from those of the primary analyses above: none of the above regions gave a LOD above 1. Instead, LODs > 1 occurred on chromosomes 6, 8, 15, 17, and 20  indicated evidence for linkage to the region of the alcohol dehydrogenase gene cluster on chromosome 4 [The linkage results for these maximum use phenotypes are modest. These phenotypes were constructed from the available longitudinal data. The motivation for defining Additional corrections for age effects in addition to sex effects did not substantially affect the linkage analysis results, even though age is clearly a significant covariate for the maximum cigarette use phenotype studied. This minimal impact on results could be due to the fact that sib-pair based linkage analysis was performed; as siblings tend to be similar in age, age-adjustment would tend to have lesser effect on trait differences within pairs, although scaling differences between sib pairs of different cohorts still could have a meaningful effect.M = 0 individuals were set to missing compared with those for which M = 0 individuals were included. The two phenotype definitions provide very different sample sizes, and while there are a similar number of LOD scores over 1 for each phenotype, none occur in the same regions (across the phenotypes). There are at least two reasons why these differences in results may be expected. First, M = 0 is being used as a proxy to recognize substance-na\u00efve individuals, some of whom might nevertheless be susceptible to heavy cigarette use if suitably exposed; thus inclusion of these individuals with a trait value of zero could substantially alter the phenotype, in part from an effect similar to that of misdiagnosis, with individuals sharing genetic susceptibility having erroneously dissimilar phenotype. Second, we see from Table M = 0 is quite high, and including these subjects with M = 0 not only increases the sample size dramatically, but also forces a trait distribution with properties very different from those of the distribution for M > 0 individuals only. Specifically, there is a floor effect that is problematic for the distributional requirements of regression analysis.There were substantial differences in results of the linkage analyses for which M = 0 set to missing did result in LOD scores that were at least partially supportive of the Haseman-Elston findings  not meaningful due to the apparent violation of distributional assumptions.While Haseman-Elston regression is often described as a \"distribution-free\" linkage method, this property is usually invoked when pointing out protection from false positives; it is worth noting that changes in and rescaling of a trait's distribution will affect the power of an analysis to detect a particular effect. We used classical Haseman-Elston regression for our primary analyses due to concerns about the distributions of the quantitative traits studied. Besides Haseman-Elston regression, MAPMAKER/SIBS can also be used to perform ML (maximum likelihood) quantitative trait locus variance estimation on sib-pair data. However, this method makes a necessary assumption of normality in the distribution for sib pairs within each identity-by-descent (IBD) class at a locus. ML variance estimation of the sample with M studied here would be expected to differ from phenotypes obtained from direct questions such as \"How many cigarettes per day did you smoke during your heaviest period of use\" or \"What is the largest number of cigarettes you have ever smoked in a 24-hour period\". Maximum or quantitative cigarette-use phenotypes obtained from direct questions such as these may prove useful in other studies. It also appears useful to include questions that would permit direct identification of substance-naive or minimally exposed individuals.It should be noted that the maximum cigarette phenotype"}
+{"text": "Despite strong evidence for a genetic component to variation in high-density lipoprotein cholesterol levels (HDL-C), specific polymorphisms associated with normal variation in HDL-C have not been identified. It is known, however, that HDL-C levels are influenced in complex ways by factors related to age and sex. In this paper, we examined the evidence for age- and sex-specific linkage of HDL-C in a longitudinal sample of participants from the Framingham Heart Study.t1, t2, and t3, spaced approximately 8 years apart and corresponding respectively to visits 11, 15, and 20 for the original cohort and 1, 2, and 4 for the offspring and spouses. Additionally, to examine the effects of sex on linkage at each time point, we estimated the heritability and genetic correlation of HDL-C, performed linkage analysis of HDL-C, tested for genotype-by-sex interaction at a QTL, and performed linkage analysis of HDL-C in males and females separately.To determine if aging could influence our ability to detect linkage, we explored the evidence for linkage of HDL-C at three time points, In women, we found evidence for a QTL on chromosome 2q influencing HDL-C variation. Although the QTL could be detected in the combined sample of males and females at the first time point, the linkage was not significant at subsequent time points. Despite strong evidence for an additive genetic component to variation in high-density lipoprotein cholesterol (HDL-C) levels, specific polymorphisms associated with normal variation in HDL-C have not been identified. It is known, however, that HDL-C levels are influenced in complex ways by factors related to age and sex . With spThe study design and methods of the Framingham Heart Study have been detailed elsewhere ,5. Begint1, t2, and t3, spaced approximately 8 years apart and corresponding respectively to visits 11, 15, and 20 in the cohort and visits 1, 2, and 4 for the offspring and spouses. We chose the earliest observations to maximize our sample size, and considered only those individuals for whom complete data were available for age, sex, and cohort, various age-by-sex interactions, and cohort effects. Data were also cleaned for outliers  to reduce the effect of trait non-normality (kurtosis) on the analysis [t1, t2, and t3, respectively.We examined fasting HDL-C at time points analysis . The kurOur final sample size was 1562 participants, including in total 663 parent-offspring, 1273 sibling, 445 avuncular, which are aunt/uncle-niece/nephew pairs, and 717 pairs of first cousins. In all, the sample included information on nearly 3300 relative pairs.p = 0.10 level in the initial analysis were retained in subsequent analyses, even if the significance levels decreased after inclusion of other covariates. After the initial covariate screening, maximum likelihood methods were used to estimate the effects of covariates and additive effects of genes.Univariate quantitative genetic analysis was done to partition the phenotypic variance of HDL-C into its additive genetic and environmental variance components using maximum likelihood variance decomposition methods. The initial analysis screened for the following covariates: sex, age, age-by-sex interaction, cohort, body mass index (derived from height and weight), drinking status, and hypertensive status. Any covariates whose effects were significant at the Next, we generated genome-wide LOD scores for HDL-C at three time points, implemented in the program package SOLAR, using the methods detailed by Almasy and Blangero . A pair-The basic variance components approach can then be extended to a multivariate framework ,10. In ta and b can be trait 1 or 2 and \u03c1g is the additive genetic correlation between the two traits. This approach has been implemented in SOLAR version 2.0.where i,j is defined as:We next tested for linkage to HDL-C using a variance-components linkage model extended to include genotype-by-sex interaction at a QTL -13. The \u03c3ij g = 2\u03c6ij \u03c1ij g \u03c3gM \u03c3gF + \u03c0ij \u03c1ij q \u03c3qM \u03c3qF,\u03c6ij is the coefficient of kinship between the two individuals, \u03c1ij g is the additive genetic component of the correlation between the expressions of the trait in the two sexes, and \u03c3gM and \u03c3gF are the genetic standard deviations for males and females, respectively; \u03c0ij is the probability that individuals i and j are IBD at a quantitative trait locus tightly linked to a marker locus; \u03c1ijq is the marker-specific component of the trait correlation between the sexes; and \u03c3qM and \u03c3qF are, respectively, the marker-specific genetic standard deviations for males and females. Under the null hypothesis of no genotype-by-sex interaction at the QTL, the male and female marker-specific variances are equal (\u03c32qM = \u03c32qF).where Lastly, because a significant genotype-by-sex interaction was found, we generated genome-wide LOD scores for HDL-C at three time points in males and females separately in the program package SOLAR using the methods detailed by Almasy and Blangero .t1, t2, and t3, the mean ages of the sample members from the longitudinal sample were 38.3, 46.5, and 54.8 years, respectively. The residual heritability of HDL-C was estimated as 0.42 \u00b1 0.05 across all three time points (sexes combined). Age, sex, and age-by-sex interactions were significant between time points, where as cohort effects were significant only at the last observation, t3. The variance explained by covariate effects was nearly constant, varying from 18% at t1 to 20% at t3. In preliminary analyses we also considered body mass index (derived from height and weight), drinking status, and hypertensive status as covariates . The results were not significantly different from those presented below, and we did not interpret these analyses further.At The genetic correlations of HDL-C between time points, for males and females separately, are shown in t1 observation. At t2 the maximum LOD score was 0.6 at 120 cM, and at t3 the maximum LOD was 1.1 at 122 cM. Based on these results we chose to restrict further analysis to chromosome 2 and to examine males and females separately at each time point. The results of separate linkage analyses in males and females are summarized in Figure In a preliminary genome scan of HDL-C we obtained a maximum LOD score of 3.4 on chromosome 2 at 150 cM for the t1, however, males and females showed significantly different genetic variances (p = 0.01).We also considered the possibility of genotype-by-cohort and genotype-by-sex interactions on the HDL-C linkage, by formally modeling an interaction between sex and cohort in the linkage analysis . All estIn a longitudinal sample of women from the Framingham Heart Study we found evidence for a QTL on chromosome 2q influencing HDL-C variation. Although the QTL could be detected in the combined sample of males and females at the first time point, the linkage was not significant at subsequent time points.In sex-specific analyses, the QTL was detected consistently in females but not in males. Apart from the interaction with sex, our results are similar to those of Almasy et al. , who repUnfortunately, we did not have the desired covariate data to evaluate fully other possible sources of change across the time points, such as the influence of menopause, hormone therapy, oral contraceptive use, and nutrition. We therefore propose to examine further the evidence for sex-specific linkage of HDL-C in future studies of the Framingham Heart Study.D2S1326; these are the phospholipase A2 receptor 1 (PLA2R1) at 2q23\u20132q24 and the oxysterol binding protein-like 6 receptor (OSBPL6) at 2q32.1. Studies have demonstrated that estradiol affects PLA2R1 activity [A search of genome databases revealed two plausible candidate genes located on chromosome 2q near marker activity  and a reactivity ,17. Oxysactivity ,19.KEN and LJM performed statistical analyses and interpreted results. JW assisted in the interpretation of the results. TD calculated the mIBDs. AGC participated in the design of the study. All authors read and approved the final manuscript."}
+{"text": "Errors while genotyping are inevitable and can reduce the power to detect linkage. However, does genotyping error have the same impact on linkage results for single-nucleotide polymorphism (SNP) and microsatellite (MS) marker maps? To evaluate this question we detected genotyping errors that are consistent with Mendelian inheritance using large changes in multipoint identity-by-descent sharing in neighboring markers. Only a small fraction of Mendelian consistent errors were detectable . More SNP genotyping errors are Mendelian consistent compared to MS genotyping errors, so genotyping error may have a greater impact on linkage results using SNP marker maps. We also evaluated the effect of genotyping error on the power and type I error rate using simulated nuclear families with missing parents under 0, 0.14, and 2.8% genotyping error rates. In the presence of genotyping error, we found that the power to detect a true linkage signal was greater for SNP (75%) than MS (67%) marker maps, although there were also slightly more false-positive signals using SNP marker maps (5 compared with 3 for MS). Finally, we evaluated the usefulness of accounting for genotyping error in the SNP data using a likelihood-based approach, which restores some of the power that is lost when genotyping error is introduced. Genotyping errors occur in large datasets. Errors can arise for many reasons including data entry, technician, or assay errors. As we continue to genotype large numbers of microsatellite (MS) and single-nucleotide polymorphism (SNP) markers, we must consider the effect of these genotyping errors on our ability to detect or find genes. Although several previous studies have shown that genotyping error can reduce the power to detect linkage ,2, a comGenotyping error can be divided into two types, those that do and do not result in Mendelian inconsistencies. Genotyping errors that result in Mendelian inconsistencies can often be detected using a single marker, such as the segregation of five or more alleles in a nuclear family. Linkage programs that detect and remove Mendelian inconsistent genotyping errors are available . We combined the three populations to increase our power and used all 100 replicates. Parental information was removed to evaluate sib pairs without known parental genotypes. MS marker maps were simulated with 7-cM marker spacing, and SNP marker maps were simulated with 3-cM marker spacing. The genetic model for affected status is described elsewhere. Random genotyping error was simulated at error rates of 0.14% and 2.8%, which were selected to represent typical error rates for SNP and MS datasets, respectively. To simulate genotyping error for the SNP data, genotypes were randomly chosen for replacement at the specified error rate, and one of the alleles was selected randomly and changed to the other allele. To simulate genotyping error for the MS data, genotypes and alleles were randomly selected for replacement as above; however, the allele was replaced by one of the alleles adjacent in size  to mimic laboratory conditions.pre) and the current marker and its successor (\u03b4post) for each pair of individuals at each marker. If the absolute values of \u03b4pre and \u03b4post exceeded the same predetermined cutoff, \u03b4, for two or more sib pairs that included the same individual, the marker was deemed to be the site of a double recombinant for that individual. This means that double recombinants cannot be detected for pedigrees with a single sib pair or at the ends of the chromosome by our definition. The false- positive and false-negative rates were computed separately for the MS and SNP markers based on knowledge of the simulated errors. The Shannon information content (SIC) was computed using MLOD [Mendelian consistent genotyping error is not detectable based on information from a single marker. However, this type of genotyping error may appear as a double recombinant in multipoint analysis , which cing MLOD  to evaluTo evaluate the power and type I error rates, we performed model-free linkage analysis separately for the MS and SNP data using the w4 option in SIBPAL . As implThe loss in power that results from genotyping error can be minimized using likelihood-based approaches to account for the genotyping error in the linkage analysis ,12. We iFor the MS marker map, on average 35.6% of the genotyping errors were detected as Mendelian inconsistencies, and removed from the analysis, whereas none (0%) of the genotyping errors were identified as Mendelian inconsistencies for the SNP marker map. Therefore, under the most rigorous condition (\u03b4 = 0.9), 53.1% of the genotyping errors were detected for the MS marker map, whereas only 2.4% of the genotyping errors were detected for the SNP marker map. We then utilized sib-pair IBD sharing estimates to identify Mendelian consistent genotyping errors as double recombinants, detected as large changes in IBD sharing estimates from neighboring markers on both sides of a particular location. Figure The ability to detect Mendelian consistent genotyping errors may depend on factors such as the sibship size and the marker information content. Therefore, we explored the true- and false-positive rates of our method based on sibship size and family specific SIC Figure . As expep-value < 2.2 \u00d7 10-5) compared to the MS marker map (75% vs. 67%), while the false-positive rates are more similar (5 for SNP and 3 for MS).For all levels of genotyping error, the SNP marker map was more powerful than the MS marker map for detecting linkage Table . HoweverGenotyping error decreases the power to detect linkage for both types of marker maps. As shown in Figure After using the likelihood approach to account for genotyping error, we found that for the SNP marker map, there was very little improvement in either the true- or false-positive rate at the 0.14% error level. At the higher 2.8% error rate, while the true-positive rate remained fairly constant, there was a significant improvement in the false-positive rate. However, this error rate is probably much higher than is found in more recent genotyping.Mendelian consistent genotyping errors were more easily detected with our method for the MS marker map than the SNP marker map, even at the same marker information content. This suggests that when the error is corrected or accounted for using the likelihood-based approach, a greater amount of power will be restored for the MS marker map than the SNP marker map.The effect of genotyping error on linkage analysis for SNP vs. MS marker maps is similar if you compare the power for the SNP marker map at the 0.14% error rate to the MS marker map at the 2.8% error rate. The SNP marker map has slightly higher power to detect significant evidence of linkage compared to the MS marker map and similar false-positive rates.To make a fair comparison between the MS and SNP performances in our method to detect double recombinants, we need to compare the true-positive rate at similar false positive rates. A \u03b4 = 0.9 for the MS gives approximately the same false-positive rate (1.97%) as a \u03b4 = 0.2 for SNPs (1.70%). At these deltas, the true-positive rates are also very similar, 17.5% for MS and 16.9% for SNPs. From this, we can conclude that our method for detecting double recombinants performs very comparably for MS and SNP markers, but to achieve those error rates, different cutoffs for delta should be used depending on the type of marker.On the whole, our method to detect double recombinants resulted in more false positives than true positives. We conclude that it is not practical to detect genotyping errors as double recombinants through large changes in IBD sharing. Most of the situations that were identified through this method as a genotyping error were actually false positives. These results are consistent with the results of the method implemented in SIMWALK2 ,6. BadziAnother issue with this method of identifying double recombinants is that if a single recombinant occurs in both siblings, and the recombinant occurs on one side of a given marker in the first sibling and on the other side of that marker in the second sibling, the two siblings could in fact share an additional allele IBD for only that single marker. This method would have no way of distinguishing between a double recombinant and two single recombinants occurring in almost the exact same chromosomal region in two individual siblings and may account for some of the false positives that have been encountered. However, as the field moves toward using SNP marker maps, the marker density will increase and the chance of this happening will decrease. It would be interesting to look at the effect of the density of a map given the same type of markers on the false positive rate of this method.Genotyping errors have always been a problem in linkage analysis. In this paper we evaluated error levels of 0.14% and 2.8%, which are within the bounds of realistic genotyping error rates ,16. MethGAW14: Genetic Analysis Workshop 14IBD: Identity-by-descentMS: MicrosatelliteSIC: Shannon information contentSNP: Single-nucleotide polymorphismCLT ran some of the analyses, carried out a literature search, and the drafting of this manuscript. DB assisted with the initial design and wrote the program to detect errors. QL simulated the errors and assisted in the writing. GM ran some of the analyses and assisted in the manuscript revision. YS assisted with the analysis. SKI contributed to the initial conception and design and provided critical revisions of the manuscript. CG-M assisted with the initial conception and design, analysis, and statistical interpretation. KABG contributed to the initial conception and design, analysis interpretation and the writing of this article. All authors approved the final version of the manuscript."}
+{"text": "We have developed a recursive-partitioning (RP) algorithm for identifying phenotype and covariate groupings that interact with the evidence for linkage. This data-mining approach for detecting gene \u00d7 environment interactions uses genotype and covariate data on affected relative pairs to find evidence for linkage heterogeneity across covariate-defined subgroups. We adapted a likelihood-ratio based test of linkage parameterized with relative risks to a recursive partitioning framework, including a cross-validation based deviance measurement for choosing optimal tree size and a bootstrap sampling procedure for choosing robust tree structure.ALDX2 category 5 individuals were considered affected, categories 1 and 3 unaffected, and all others unknown. We sampled non-overlapping affected relative pairs from each family; therefore, we used 144 affected pairs in the RP model. Twenty pair-level covariates were defined from smoking status, maximum drinks, ethnicity, sex, and age at onset. Using the all-pairs score in GENEHUNTER, the nonparametric linkage tests showed no regions with suggestive linkage evidence. However, using the RP model, several suggestive regions were found on chromosomes 2, 4, 6, 14, and 20, with detection of associated covariates such as sex and age at onset. Alcohol abuse and alcohol dependence are psychiatric disorders with severe physiological and psychological ramifications including liver disease, heart disease, gastrointestinal disease, depression, suicide, and homicide. In addition, fetal alcohol syndrome is a leading cause of mental retardation. A 1992 estimate put the economic burden of alcohol use in Canada at $7.5 billion, or 40.8% of the costs of all substance use combined [We analyzed genome-wide microsatellite data from the Collaborative Study on the Genetics of Alcoholism (COGA), supplied by the Genetic Analysis Workshop 14. There were 1,614 individuals in 143 pedigrees. Probands, recruited from chemical dependency centers, and their families were invited to participate in the COGA study. All of the participants were assessed on several domains, including alcohol dependence; other psychiatric disorders, such as depression and other medical illnesses; the participant's family history of alcoholism; and other behaviors. Diagnoses of alcohol dependence and other psychiatric disorders were established using a structured, comprehensive, diagnostic interview called the Semi-Structured Assessment for the Genetics of Alcoholism, which was developed specifically for the COGA study.Most methods for nonparametric linkage (NPL) analysis require a fixed definition of affected status and can incorporate only a few covariates ,3. For aIn order to adapt the conceptual framework of a standard recursive partitioning (RP) (tree-based) model  for linkn informative affected relative pairs. The parameter \u03bbi measures the excess risk to an individual who shares, at the marker locus, i alleles identical by descent (IBD) with an affected relative compared to the population risk [\u03bb1 corresponds to IBD = 1, \u03bb2 corresponding to IBD = 2, and \u03bb0 = 1. fir(p) is the prior probability of sharing i alleles IBD for affected pair p of relative type r. For example, for sib pairs, the expected IBD sharing is  under the null hypothesis. gip represents the estimated probabilities of sharing i alleles IBD based on marker data for pair p. The parameters \u03bbi are estimated by optimizing the total likelihood ratio for all the affected relative pairs. This formulation unifies different types of relative pairs because expected allele sharing for any pair type can be expressed as functions of the same parameters \u03bbi. This leads to a test of linkage deviation from the null hypothesis (no linkage) based on two parameters \u03bb =  and 2 degrees of freedom.The likelihood ratio is summed over all the ion risk . \u03bb1 corrXp (Xp = 1 or 2), a likelihood ratio test of linkage in the presence of heterogeneity can be obtained by estimating two sets of parameters . We therefore define a splitting rule, in the spirit of regression trees, based on identifying the covariate that gives the largest likelihood ratio test statistic for linkage with heterogeneity. This is implemented recursively until the subgroups are too small for further splitting. Again following standard RP model concepts, we used 10-fold cross-validation [\u03bbkt represent the estimated relative risk parameters from cross-validation training set k, , and covariate-defined subgroup t  with tree size s. For s = 1, there is only one set of \u03bb estimates (corresponding to the root node of the tree), for s = 2 there are two sets, etc. Let p \u2208 t(k) denote the pairs in the tth subgroup of the kth cross-validation test set, where subgroups are defined by the tree grown on the kth training data set. A measure of deviance can therefore be constructed, based on the testing data:For each pair-defined binary covariate lidation  to estimkth cross-validation training test set. After choosing the optimal tree size, we used a bootstrap algorithm to determine the consistency of particular covariate selections. When one covariate clearly defines linkage heterogeneity, most bootstrap datasets will select the same covariate. When several covariates are associated with the disease gene, bootstrap datasets may choose a variety of tree structures (configuration of a tree).The optimal tree size is selected as the one with the largest deviance measure. The relative risk estimates used in the deviance calculation are those which optimized the likelihood ratio for splitting the tree in the p-values for tests of linkage and heterogeneity assuming an asymptotic chi-squared distribution. The RP model provides tests of linkage with and without covariate-induced heterogeneity, as well as tests of covariate effects on the linkage. As currently implemented, this model places no plausibility constraints on the \u03bb values. Hence deviation from the null hypothesis can show either excess allele sharing or decreased allele sharing.We calculated Use of the COGA data set was approved by the Hospital for Sick Children Research Ethics Board. We used primarily the ALDX2 (DSM-IV) criteria to define affection status. We treated category 5 (affected) as affected; categories 1 (pure unaffected) and 3 (unaffected with some symptoms) as unaffected; categories 0 (unknown) and 2 (never drank) as missing. Based on this definition, there were a total of 726 informative affected relative pairs. In order to avoid working with highly dependent affected pairs within a pedigree, we sampled non-overlapping affected relative pairs from the same family. Therefore, we used 144 affected pairs in the RP model.N = 914; non-smokers N = 467, missing N = 233). To differentiate between heavy and light smokers, we utilized a cut-point of 21.00 pack-years which represented the third quartile for all 1,381 individuals with available data. We then defined four pair-level covariates for smoking: 1) both smokers versus others, 2) both non-smoker versus others, 3) discordant smoking status versus others, and 4) both heavy smokers versus others. Note that \"others\" includes pairs with missing covariate information. A similar approach was taken to define binary covariates for other variables. Electrophysiological variables were not used in this analysis.We defined 20 pair-level covariates using smoking status, maximum drinks, ethnicity, sex, and age at onset. We defined smokers as those with non-zero pack-years (smokers ir(p), were obtained from GENEHUNTER [Multipoint NPL scores, the estimated IBD allele sharing, gip, and the null expected sharing for each affected relative pair, fNEHUNTER  using thp-value = 0.001; dashed lines in Figure 10 of the p-values) [p-values smaller than 0.001  . We then\u03bb estimates that violated the possible triangle constraints. Interpretation of the results can be difficult, especially when pairs in the subgroups are concordant for their covariate values. We are planning to implement constraints on the allele sharing parameters.Although NPL scores showed no linkage evidence on any of the 22 chromosomes despite a larger sample size , the RP data mining algorithm identified loci in regions that have been previously identified, which are on chromosomes 2  ,9, and 6The definition of \"affected\" is crucial for any linkage study. Expected patterns of allele sharing in linked regions vary with changes to these definitions. Our algorithm focuses on sharing between affected relative pairs, and hence, although we can find heterogeneity in linkage evidence, it is always predicated on the initial definition of affected status. It may be possible to construct better definitions of alcoholism from a combination of phenotypes.Our algorithm as currently implemented assumes independence of relative pairs, but this is violated when multiple pairs are constructed from the same pedigree. To reduce dependency, we selected non-overlapping pairs, but this excluded a large number of relative pairs. Therefore, we could expect the NPL scores based on the full pedigrees to have better power. However, the NPL method found no linked regions, whereas our approach identified several regions also identified by others. In the future, we plan to develop appropriate methods for dependent pairs.Despite the cross validation, any data mining algorithm is likely to find some false positive results. Therefore, additional strategies will be necessary to reduce false positive signals. For example, we might expect broader peaks to be associated with real linkage signals .We developed a recursive-partitioning model for linkage analysis to select covariates that are associated with the allele sharing in relative pairs. Cross-validation and bootstrapping are used to improve the properties of the model. In the COGA data, we were able to detect linkage signals involving covariate interactions that the NPL scores were unable to detect.COGA: Collaborative Study on the Genetics of Alcoholism\u2208: Is an element ofIBD: Identical by descentNPL: Nonparametric linkageRP: Recursive partitioningWX developed and implemented the RP algorithm under the supervision of CMTG. SBB and MC and drafted the manuscript. JV assisted with data manipulation and developed the plotting code. CT carried out the literature review under the supervision of MC. SBB and CMTG assisted in revising the manuscript."}
+{"text": "The goal of this study is to evaluate, compare, and contrast several standard and new linkage analysis methods. First, we compare a recently proposed confidence set approach with MAPMAKER/SIBS. Then, we evaluate a new Bayesian approach that accounts for heterogeneity. Finally, the newly developed software SIMPLE is compared with GENEHUNTER. We apply these methods to several replicates of the Genetic Analysis Workshop 13 simulated data to assess their ability to detect the high blood pressure genes on chromosome 21, whose positions were known to us prior to the analyses. In contrast to the standard methods, most of the new approaches are able to identify at least one of the disease genes in all the replicates considered. We consider various standard as well as new linkage analysis tools and methods. The Genetic Analysis Workshop 13 simulated complete data set gives an excellent opportunity to evaluate and compare these methods. First, we compare the standard nonparametric approach for sib-pair analysis as implemented in MAPMAKER/SIBS . We expeisolated observation(s) of high blood pressure or hypertensive treatment just by chance, e.g., if it happens to be a very stressful period for that person. That person should not be considered as  affected. Taking into account of all these factors, we label the following people as affected: a Cohort 1 person if he/she has three consecutive events, or a Cohort 2 person if he/she has two consecutive events, where event is defined as \"high blood pressure or hypertensive treatment at or before the age of 55\". Note that the difference in criteria for the two cohorts is due to the fact that the period between three consecutive events in Cohort 1 is roughly the same as two consecutive events in Cohort 2.We focus on a qualitative trait, high blood pressure. In addition to making use of the binary variable in the data, we also take into consideration several other factors. First, there is a related variable, hypertensive treatment, which is confounded with the presence or absence of high blood pressure. So, we combine observations on these two variables. Second, we have observations on each person over a range of time; this longitudinal feature of the data needs to be considered. Further, we note that by a certain (old) age, most people develop high blood pressure. This is most likely not attributable to a genetic effect. Also, a person may have one or more Instead of performing a whole-genome scan, we focus on chromosome 21, which has three genes, b37, s10, and s12, that directly affect high blood pressure. We note that the two high blood pressure genes, b37, and s12, are very close to each other. So we treat them as one gene and refer to it as s12. We compare and contrast methods in terms of their power and precision for finding these genes. We also study chromosome 20 to assess false-positive rates because it does not have any disease genes.For the confidence set approach, we randomly select five replicates from the first batch of 25 replicates of the simulated data, which yield estimated risk characteristics compatible with a single-locus model. For the Bayesian approach we analyze four randomly chosen replicates, one from each of the four batches provided. Because SIMPLE is computationally intensive, we analyze only one replicate for the comparison between GENEHUNTER and SIMPLE. To further save time, this replicate is randomly chosen from the four replicates used in the Bayesian approach because this method utilizes GENEHUNTER to compute LOD scores. We do not intend to make comparisons among the three new methods, as they are based on different types of data, and/or different model assumptions. Thus, we do not necessarily analyze the same replicates for all new methods.We use these popular standard packages for exact LOD score and IBD calculations. Both are based on the hidden Markov model approach.m, the hypotheses are Hom: \u03b8m \u2264 \u03b8o versus Ham: \u03b8m > \u03b8o, where \u03b8m is the recombination fraction between the disease and marker loci. We set \u03b8o to be the recombination fraction corresponding to a genetic distance of 10 cM, which is roughly half of the largest distance between any two adjacent markers in the simulated data. Note that this is a two-point approach. But the above formulation of hypotheses renders multiplicity adjustment unnecessary, no matter how many tests are performed. This approach needs population risk characteristics , which we estimate from the sample itself. We apply this approach to affected sib-pair (ASP) data, both with and without parental data. These results are compared with those from MAPMAKER/SIBS.This approach gives a confidence set of markers  for the locations of disease genes. We consider this method here in a nonparametric setting. The essence of this method lies in a new  formulation of linkage hypotheses. For each marker k families in the sample. Let \u03b1i = P(disease causing gene of the ith family is linked to the marker map), i =1,..., k, and let d be the position of disease gene on the chromosome. We formulate the mixture likelihood of the sample following Ott [i. We assume that the prior distributions of \u03b1i values are independent U. GENEHUNTER is used for the calculation of usual LOD scores at several distances, labelled 1 through N, on the chromosome. The prior distribution of d is defined over {1,..., N, \u221e} with probabilities 1/(22N) for the first N points and 21/22 for \u221e. This approach accounts for heterogeneity (if any) but is applicable to homogeneous samples as well.We describe this approach for the situation in which we assume there is only one disease gene on the chromosome of interest. Suppose there are wing Ott , but witi values and d. The chain is run for a burn-in period of 10,000 iterations, followed by an additional 90,000 iterations. The posterior distributions are estimated based on these 90,000 iterations. The proportion of the non-infinity distances, , can be interpreted as an estimate of the posterior probability for linkage, p. Hence, a value of  greater than 0.5 can be taken as an initial signal of linkage at that chromosome. In that case, the mean of the non-infinity distances, , gives an estimate of the position of disease gene. This approach has also been extended to the situation of two disease genes on a chromosome. With this extension, we want to see if we could detect the two genes on chromosome 21 simultaneously. We also evaluate our approach under various models to examine the effect of model specification.We use Markov chain Monte Carlo (MCMC) methods to sample from the posterior distributions of \u03b1all available information on all pedigree members. We compare the Spairs scores from SIMPLE with those from GENEHUNTER.SIMPLE is a Monte Carlo method based on sequential imputation and makes use of Before presenting the results, we note the following for ease in making comparisons. On chromosome 21, there are six markers. We use the sex-averaged distances in our analysis. The disease gene s12 lies between markers 3 (26.56 cM) and 4 (40.02 cM) at position 29.46 cM. The other gene, s10, is located at 53.59 cM between markers 5 (43.89 cM) and 6 (63.35 cM).This analysis is performed on five replicates. Table From Table For chromosome 20, the confidence set approach gives one false positive for Replicate 9. There are no other false positives by either method.This method is applied to Replicates 1, 32, 54, and 85. The chromosome 21 results for Replicate 54 are shown in Table Table Marginal analysis on chromosome 20 shows only one false positive  for the second model in Replicate 1. Since overall there is no signal of linkage found in the marginal analysis, it is doubtful that any joint analysis would yield positive result and hence no further analysis is carried out.pairs statistics of the pedigrees that GENEHUNTER skips or reduces, and add them to the Spairs scores of the pedigrees that GENEHUNTER analyzes without reduction. These are the total scores that SIMPLE would provide estimates for, if we analyze all pedigrees using it. We plot these scores in Figure pairs score of 2.42, which exceeds the customary threshold of 2.33 for suggestive linkage [pairs scores for GENEHUNTER, even after including the skipped pedigrees, do not exceed the same threshold. For chromosome 20, all the three curves indicate a false positive.We consider Replicate 85 for this comparison. For this replicate, GENEHUNTER drops individuals in 23 pedigrees. It also skips 27 pedigrees (15 of these pedigrees are informative for linkage) because these pedigrees become disconnected after GENEHUNTER drops individuals. In contrast, SIMPLE can analyze all pedigrees in their entirety. We use SIMPLE to calculate the SThe confidence set approach based on ASP data is more successful in identifying the disease genes in the replicates considered compared with the standard method. An attractive feature of this approach is that no multiplicity adjustment is needed. Also, unlike the traditional method, a confidence interval with known properties can be deduced. The reported confidence intervals are wide, though, due to the nature of single marker analyses. The results obtained by including parental information are similar.i values that are all close to 0.5 in the marginal analysis. It shows that none of the families are clearly linked or unlinked. It is encouraging to see that our approach yields positive results in this situation. Further we see that this method is reasonably robust to model specification as all the models we considered give similar results. Nevertheless, this procedure needs further evaluation and refinement.The Bayesian approach incorporating heterogeneity is able to identify s10 in all replicates and s12 in one out of four replicates. We note that we could have gained more linkage information by using SIMPLE to calculate LOD scores without dropping individuals. If the marginal analysis shows a signal for linkage, it seems worthwhile to explore the joint analysis. We note that the method used to simulate the data does not lead to a genetically heterogeneous sample. This is reflected in the \u03b1Comparison of SIMPLE with GENEHUNTER shows that by being able to handle large pedigrees as a whole, SIMPLE gives higher scores at positions in the vicinity of the disease locus. Since these results are based on a single replicate only that does not warrant general conclusions, we refer interested readers to the extensive simulation study that shows considerable power gains by using SIMPLE .In our search for disease genes, we identify s10 in a greater number of replicates than s12. This indicates that the effect of s10 is much greater than s12, consistent with the simulation model. We note that only the new methods are able to identify the gene s12. So, overall, our methods seem to be promising in the sense that there is an evidence of power gain without increasing false-positive rates considerably. However, we caution any generalization of these results, as the number of replicates studied is relatively small."}
+{"text": "Genomic imprinting, which is also known as the parent-of-origin effect, is a mechanism that only expresses one copy of a gene pair depending upon the parental origin. Although many chromosomal regions in the human genome are likely to be imprinted, imprinting is not accounted for in the usual linkage analysis. In this study, using a variance-components approach with a quantitative phenotype ttth-FP1, we found significant evidence of imprinting at two loci, D7S1790 and D1S1631, on chromosome 1 and chromosome 7, respectively. Our results suggest that allowing for the possibility of imprinting can increase the power to detect linkage for localizing genes for alcoholism. Genomic imprinting  is a mechanism by which only one copy of a gene pair is expressed, and this expression is determined by the parental origin of the copy. The deregulation of imprinted genes has been implicated in a number of human diseases. Expression of imprinted genes is regulated by allele-specific epigenetic modifications of DNA and chromatin. These modifications affect central regulatory elements that control the allele-specific expression of neighboring genes. Although many chromosomal regions in the human genome are likely to be imprinted, particularly those involved in developmental disorders, imprinting is not accounted for in the usual linkage analysis -8. In thAlcoholism is a complex disorder with involvement of genetic and environmental risk factors. Several studies have shown familial aggregation, segregation, and linkages to several regions . Therefoi be the phenotypic value for the ith individual in a pedigree:Quantitative variation in a trait often occurs because of the underlying variation in genetic factors. We recently developed a method to analyze quantitative traits using the variance components approach and allowing for imprinting as described by Shete and Amos  and Shetgi is the major-gene effect, Gi is the polygenic effect, \u03b2k values are covariate effects that are assumed to be uncorrelated with genetic and environmental factors, and ei is the environmental effect. The major gene effect has a mean value of a when individual's genotype is BB, d1 when the genotype is Bb, d2 when the genotype bB, and -a when the genotype bb. Here, we assumed that the first allele is derived from the father and the second allele is derived from the mother. Let d be the dominance effect and I be the imprinting effect. Then, d = (d1 + d2)/2 and I = (d1 - d2)/2. When d1 = d2, there is no imprinting. Shete and Amos [2af; an additive component due to the maternally derived allele, \u03c32am; and the usual dominance component, \u03c32d. These parent-specific additive components are:  where p and q are the frequencies of alleles B and b, respectively. Also, \u03c32af+\u03c32am = \u03c32a.where \u03bc is the overall mean, and Amos  decomposI = 0, \u03c32af and \u03c32am are equal to \u03c32a/2; and, when \u03c32af and \u03c32am are equal, I = 0. Hence, Shete and Amos [i and j\" asfollows:When the imprinting coefficient and Amos  proposedmf,ij and \u03c0mm,ij similarly. Then, the phenotypic covariance is given by [We define \u03c0given by ff,ij,\u03c0mm,ij, and  are equal if and only if \u03c32af and \u03c32am are equal, and \u03c32af and \u03c32am are equal if and only if the imprinting parameter I = 0 . Hence, the likelihood ratio test (LRT) for equality of these coefficients is a valid test for the null hypothesis of no imprinting. We do not estimate the parameters p, q, or I separately in the above equation, rather we estimate three parameters \u03c32af, \u03c32am, and (\u03c32a/2 - 2pqI2). Ordinarily, in a genome scan, one will test the joint null hypotheses of no linkage and no imprinting by testing \u03c32af = \u03c32am = 0.From the above equation, it can be seen that the coefficients of \u03c02 random variable with one and zero degrees of freedom. For joint testing of linkage and imprinting, we now have three parameters in the model. The two parameters \u03c32af and \u03c32am are independent; however, the third parameter (\u03c32a/2 - 2pqI2) is correlated with the first two parameters [2 distribution with 0, 1, 2, and 3 degrees of freedom with mixing parameters of 1/8, 3/8, 3/8, and 1/8 for joint testing of linkage and imprinting following the same rationale as in the standard linkage analyses using the approach of Self and Liang [2 distribution with 0, 1, and 2 degrees of freedom with mixing parameters of 4/8, 3/8, and 1/8. These asymptotic distributions can be used to obtain point-wise significance of the LRT test for testing linkage and/or imprinting.The asymptotic distribution of the LRT is complex. For testing linkage without imprinting the LRT test is assumed to be a half-and-half mixture of \u03c7rameters . Becausend Liang . Similari and j. The two alleles for individual i are denoted by a vector , where im and if are maternal and paternal alleles, respectively. Similarly, we define the vector for individual j. There are 15 possible ordered states of IBD between these two individuals [9 = , S10 = (if)(jf), S11 = (im)(jm),S12 = , S13 = (if)(jm), S14 = (im)(jf), and S15 = (im)(if)(jm)(jf). In these states, the pairs of alleles inside the parentheses are IBD. We used SIMWALK2 to obtain these 15 detailed states of identity sharing.Computation of multipoint parent-specific IBD is described by Shete et al. . There aividuals . Of thesividuals ,15, we dTo identify imprinted genes that affect the risk for alcoholism, we proposed to study 143 families consisting of a total of 1,614 individuals. We used the quantitative trait denoted by ttth1-FP1, far frontal left side channel, a quantitative measure of alcoholism. The empirical distribution of this quantitative trait is shown in the Figure p-value of 0.00129 for linkage. Furthermore, we obtained a negative log likelihood value of 129.95 using the variance components approach that tests for linkage allowing for imprinting. Using the LRT, the significance for joint testing of linkage and imprinting at this marker is 0.00003. When we compared the log likelihoods with linkage but no imprinting model with joint linkage and imprinting model, we obtain a p-value of 0.00057, which is significant evidence of imprinting. The evidence of imprinting is also evident from the lower p-value obtained using the imprinting model.For each of the markers listed, we calculated multipoint parent-specific IBDs using the methods described. On chromosome 7, for the marker D7S1790, we obtained a negative log likelihood value of 140.35 for the model in which the major gene variance component was fixed to zero, the model without linkage. The same value under the linkage without imprinting model was found to be 135.84. Using the LRT discussed above, we obtained a suggestive significant p-value of 0.00018 for joint testing of linkage and imprinting. When we compared the log likelihoods with linkage but no imprinting model with joint linkage and imprinting model, we obtain a p-value of 0.00143, which is a suggestive significant evidence for imprinting. Evidence of imprinting for marker D1S532 was not very significant, as shown in the Table On chromosome 1, for the marker D1S1631, we obtained a negative log likelihood value of 136.90 for the model with linkage without imprinting. This gives a significance value of 0.00417 for linkage. A negative log likelihood value of 131.90 was obtained for the linked model with imprinting. This leads to a significant p-values at the significant loci to confirm evidence of linkage. In conclusion, our results suggest that allowing for imprinting in the linkage analyses can increase the power to detect genes responsible for the alcoholism.Imprinting is not accounted for traditional linkage analyses. We found evidence of imprinting, even allowing for the multiple testing, on two loci. It may also be important to allow for other covariates of environmental exposures, such as smoking, in the model. In addition, the asymptotic distribution that we used may not be very accurate and recommend simulation-based COGA: Collaborative Study on the Genetics of AlcoholismLRT: Likelihood ratio testIBD: Identity by descentSS conceived the study, participated in the statistical analyses, and drafted the manuscript. RY participated in the statistical analyses and helped draft the manuscript."}
+{"text": "Using model-based two-locus methods for mapping genes, we analyzed the family data from the Collaborative Study on the Genetics of Alcoholism. Microsatellite data from 143 families ascertained through having three or more individuals affected with alcohol dependence were used for this investigation. Four regions showing evidence for linkage were identified using single-locus models from previous investigations. We investigated the genetic linkage, pattern of disease inheritance, and pair-wise genetic epistasis of these loci using the TLINKAGE program for two-disease-locus analysis. Many human diseases, such as cancers and psychiatric disorders, are caused by more than one gene. Analysis using two-locus methods is a logical method of adequately modeling genetically complex traits that are caused by two or more genes. Schork et al. compared the parametric one-locus and two-locus LOD-score analyses and found that linkage analysis with two-locus methods had higher power to detect linkage than did those with one-locus models for a trait governed by two genes . HoweverUsing segregation analysis, Yuan et al. suggested a multi-locus etiology for alcoholism . FurtherIn this work, we identified four regions on different chromosomes showing evidence for linkage using single-locus methods as described in the literature . On the We analyzed COGA microsatellite data for a 10-cM genome scan. The family data of COGA included 1,614 individuals of 143 pedigrees ascertained for having three or more family members affected with alcohol dependence. COGA criteria were used for diagnosis throughout the study for ascertainment. Marker allele frequencies were estimated from the data. We identified four markers showing evidence of linkage using single-locus methods as described in the literature [The TLINKAGE program was used for two-disease-locus analyses in this report . TLINKAG10 { L / L } ; 0 \u2264 \u03b8 \u2264 0.5.LOD(\u03b8) = logThe product of 2 ln(10) and the maximized LOD score is an asymptotically 50:50 mixture of a point probability mass at 0 and a chi-squared distribution with one degree of freedom under the null hypothesis of \u03b8 = 0.5.A loss of power due to incorrect specification of models and misspecification of disease transmission inheritance is always a concern for parametric approaches to linkage analysis. Despite its power loss, Risch et al.  and RiscIn this study, we considered five types of two-locus models: dominant/dominant, dominant/recessive, recessive/dominant, recessive/recessive, and modified dominant/dominant, in which the penetrance of double heterozygotes is half that of genotypes having three or four disease alleles. We assumed that two disease loci had the same effects on the trait; thus, the penetrance matrix was symmetric. The phenocopy rate was fixed at 0.05. A wide range of penetrance values and allele frequencies on the disease loci were carried out in the TLINKAGE program for each of the five models.Heterogeneity was considered a possible confounder for alcoholism . The simWe first performed TLINKAGE for two-disease-locus/one-marker-locus analysis for each of the four markers D1S1595, D6S1006, D7S1797, and D15S642. Markers D1S1595 and D6S1006 were selected on the basis of the high LOD score, D7S1797 on the basis of its z-score, and D15S642 on the basis of its nonparametric linkage score. No LOD scores for D1S1595, D6S1006, or D7S1797 were higher than 0.2 for any of the five models. All the LOD scores for these markers were close to 0 for the recessive/recessive models. D15S642 for the dominant/dominant models showed the strongest evidence of linkage among the four markers and five genetic models. Some of the LOD scores were 2.0 or more.It is noteworthy that, for D7S1797 and D15S642, some families had strong evidence of linkage, whereas others had negative LOD scores in the dominant/dominant models, which resulted in the smaller overall LOD scores. These phenomena suggest genetic heterogeneity. Therefore, we tested heterogeneity using the program HOMOG for these two markers. For D7S1797, the HLOD score was 1.8 with 15 linked families (10%). The HLOD score with 31 linked pedigrees (20%) was 4.47 for D15S642.We then calculated the corresponding HLOD scores using single-locus models. The HLOD scores were 2.0 and 4.29 for D7S1797 and D15S642, respectively. There were no significant discrepancies between the HLOD scores for D7S1797 and D15S642 using single-locus models and two-disease-locus models. We believe that neither D7S1797 nor D15S642 interacted with other postulated disease loci. A broad range of penetrance values and disease allele frequencies were carried out using TLINKAGE. In Table Results from our two-disease-locus model analysis using TLINKAGE did not support the evidence of linkage to the disease susceptibility loci on chromosomes 1 and 6 that had been previously identified using a single-locus method. This may be due to the program employed (GENEHUNTER versus TLINKAGE) or that there are more than two disease loci for alcohol dependence (neither single-locus models nor two-locus models are robust enough to detect linkage). Lin et al. suggested two-locus models might be too simple to analyze the underlying true model .The patterns of LOD scores by pedigrees for the markers D7S1797 and D15S642 revealed the evidence of genetic heterogeneity for the dominant/dominant models. Assuming heterogeneity, our findings suggest evidence of linkage to the disease susceptibility loci on chromosomes 7 and 15. There are no gene \u00d7 gene interactions between D7S1797 and D15S642 and other postulated disease loci on the basis of our findings.The results may suggest that the underlying true model for alcoholism is more complicated than the two-locus models are able to explain. More general multilocus methods may be required to adequately model the effects of major genes, polygenes, and environmental factors for alcohol dependence.Two-locus analysis is complex because of the specification of several parameters. However, it may not be powerful for traits with very complex genetic etiology. Previous studies ,13 suggeCOGA: Collaborative Study on the Genetics of AlcoholismHLOD: Heterogenecity LODBoth authors contributed equally to this work."}
+{"text": "Epidemiological studies have indicated that obesity and low high-density lipoprotein (HDL) levels are strong cardiovascular risk factors, and that these traits are inversely correlated. Despite the belief that these traits are correlated in part due to pleiotropy, knowledge on specific genes commonly affecting obesity and dyslipidemia is very limited. To address this issue, we first conducted univariate multipoint linkage analysis for body mass index (BMI) and HDL-C to identify loci influencing variation in these phenotypes using Framingham Heart Study data relating to 1702 subjects distributed across 330 pedigrees. Subsequently, we performed bivariate multipoint linkage analysis to detect common loci influencing covariation between these two traits.eq = 5.5) and appears to improve power to map the correlated traits to a region, precisely.We scanned the genome and identified a major locus near marker D6S1009 influencing variation in BMI (LOD = 3.9) using the program SOLAR. We also identified a major locus for HDL-C near marker D2S1334 on chromosome 2 (LOD = 3.5) and another region near marker D6S1009 on chromosome 6 with suggestive evidence for linkage (LOD = 2.7). Since these two phenotypes have been independently mapped to the same region on chromosome 6q, we used the bivariate multipoint linkage approach using SOLAR. The bivariate linkage analysis of BMI and HDL-C implicated the genetic region near marker D6S1009 as harboring a major gene commonly influencing these phenotypes (bivariate LOD = 6.2; LODWe found substantial evidence for a quantitative trait locus with pleiotropic effects, which appears to influence both BMI and HDL-C phenotypes in the Framingham data. The incidence rates of complex diseases such as obesity and type 2 diabetes have been increasing worldwide ,2. Obesith examination  of the original cohort, which occurred 1970 to 1971, was combined with information collected during the first examination of the offspring cohort, which occurred from 1971 to 1975. Of the more than 10,000 participants enrolled in either the Framingham Heart Study or the Framingham Offspring Study, genotypic information was available on 1702 individuals from 330 extended pedigrees. The pedigrees include from 2 to 29 genotyped individuals and the genotyped sample includes 394 individuals from the original cohort and 1308 individuals from the offspring cohort.For purposes of the current analysis, Framingham Heart Study participants from the original cohort were combined with participants from the offspring cohort to maximize the number of individuals per pedigree. Information collected as part of the 12We used BMI and HDL-C data collected as part of the Framingham Heart Study. BMI was calculated as weight (in kilograms)/height squared (in meters). HDL-C (mg/dl) was measured by automated enzymatic methods. BMI and HDL-C values were log transformed to minimize the problem of non-normality.A multipoint variance components linkage analysis was used to test linkage between marker loci and a given phenotype, which was based on specifying the expected genetic covariances between pairs of relatives as a function of their identity by descent (IBD) at a marker linked to a QTL . It allon QTLs and an unknown number of residual polygenes influence a given trait, the covariance matrix (\u03a9) for a pedigree is given byIn a simple additive model in which i is a matrix whose elements (\u03c0ijl) provide the expected proportion of genes that individuals j and l share IBD at a QTL (qi) that is linked to a genetic marker locus, \u03c32q is the additive genetic variance due to the major locus, \u03a6 is the kinship matrix, \u03c32g is the genetic variance due to additive genetic effects, I is an identity matrix, and \u03c32e is the variance due to random environmental effects.where \u03a02q), residual additive genetic effects (\u03c32g), random environmental effects (\u03c32e), covariate effects (age and sex terms), as well as the three associated correlations \u03c1q (correlation caused by a major gene), \u03c1g , and \u03c1e  are estimated simultaneously using maximum-likelihood estimates. Using this bivariate model, we tested the null hypothesis that \u03c32q = 0 for both traits by comparing the log likelihood of this restricted model to that of a model in which \u03c32q was estimated for the traits. In addition, tests for the hypotheses of complete pleiotropy  and coincident linkage  can be performed . Rec. Rec14]]Using a bivariate linkage approach, we have found strong evidence for a QTL, which appears to influence both BMI and HDL-C, in Framingham data. Our study suggests that the putative locus lies in the chromosomal region near marker D6S1009 on chromosome 6."}
+{"text": "In this analysis we applied a regression based transmission disequilibrium test to the binary trait presence or absence of Kofendred Personality Disorder in the Genetic Analysis Workshop 14 (GAW14) simulated dataset and determined the power and type I error rate of the method at varying map densities and sample sizes. To conduct this transmission disequilibrium test, the logit transformation was applied to a binary outcome and regressed on an indicator variable for the transmitted allele from informative matings. All 100 replicates from chromosomes 1, 3, 5, and 9 for the Aipotu and the combined Aipotu, Karangar, and Danacaa populations were used at densities of 3, 1, and 0.3 cM. Power and type I error were determined by the number of replicates significant at the 0.05 level.The maximum power to detect linkage and association with the Aipotu population was 93% for chromosome 3 using a 0.3-cM map. For chromosomes 1, 5, and 9 the power was less than 10% at the 3-cM scan and less than 22% for the 0.3-cM map. With the larger sample size, power increased to 38% for chromosome 1, 100% for chromosome 3, 31% for chromosome 5, and 23% for chromosome 9. Type I error was approximately 7%.The power of this method is highly dependent on the amount of information in a region. This study suggests that single-point methods are not particularly effective in narrowing a fine-mapping region, particularly when using single-nucleotide polymorphism data and when linkage disequilibrium in the region is variable. As the characterization of the human genome continues, increasingly dense marker maps are being created using single-nucleotide polymorphisms (SNPs). It has been suggested that the availability of these dense marker maps will make gene mapping by joint linkage and association preferable to tests for either linkage or linkage disequilibrium (LD) alone ,2 due to2 test is then conducted to compare the transmission of an allele in an affected child to the non-transmitted allele  = \u03c3E2 was estimated. The likelihood was maximized numerically both with and without the specified test covariate (in this case the transmitted indicator variable) and corresponding likelihoods calculated. Standard errors were determined by numerical double differentiation of the log likelihood and p-values, based on a Wald test, were calculated for the random, environmental variance and covariate coefficients. p-Values are two-sided for all covariate coefficients and one-sided for all variances.In this analysis we included only the residual individual random effect, which was assumed to be normally distributed with mean zero and variance \u03c3p-value less than 0.05 in unlinked regions greater than 10 cM away from the simulated disease locus. To determine power, we calculated the number of replicates for which the p-value for the marker locus nearest the simulated disease locus was less than 0.05. This was done for chromosomes 1, 3, 5, and 9. Adjustments for multiple comparisons were not performed.To calculate type I error rate, we identified the number of replicates with a In both the Aipotu and the combined populations, the type I error rate was approximately 7%. The power of this method to detect each of the disease loci on chromosomes 1, 3, 5, and 9 was very low when using the Aipotu population alone , possibly due to transmission distortion . In termInterestingly enough, the power of this method to detect linkage in a region where there was said to be no LD (chromosome 1) was actually higher (21%) than regions on chromosomes 5 and 9 where LD was said to have been simulated (<20%). McCaskie et al.  and SongBy using a transmitted allele as the test covariate in the regression model, the sample size was reduced substantially after excluding non-informative individuals . Nevertheless, we were able to explore the impact of increasing sample size by pooling populations. Surprisingly, tripling the average sample size  had a modest impact on power, but the high variability of the sample size makes interpretation of the effects of sample size less straightforward. The gains in power were highest on chromosomes 1 and 3, where presence of a signal was confirmed by other groups with association based tests. For example, power increased to 100% for the 0.3-cM density map on chromosome 3. Power also doubled for the 3-cM density map on chromosome 3 and the 1-cM density map on chromosome 1. Because all populations were simulated with the same disease loci, the modest gain in power due to larger sample size was not likely due to heterogeneity.To better understand our results, we performed a subsequent analysis, to characterize the amount of LD on chromosomes 1, 3, 5, and 9 by comparing the power at each of the SNPs in the 0.3-cM map to the informativity of those same SNPs (results not shown). There was indeed indication of LD between several of the markers on chromosomes 1 and strong LD on chromosome 3, particularly in regions of strongest signal. These results suggest that our method performed well in the presence of LD. However, even our strongest signal was not very precise. This is likely because it is a single-point method and therefore does not make use of all of the information available in the region.While our method performed reasonably well in regions where LD was confirmed, the power was highly dependent on the amount of information in a region, including density of markers and sample size. Certainly, the loss of sample size due to uninformative matings is a weakness of any transmission-based test, and the case in this study as well. Overall, this study suggests that single-point methods, particularly those based on transmitted alleles, are only marginally effective in narrowing a fine-mapping region, particularly when using SNP data containing varying degrees of LD in the region. Further assessment of this method will require detailed information about LD in regions containing the causal locus not available in this dataset.GAW14: Genetic Analysis Workshop 14KPD: Kofendred Personality DisorderLD: Linkage disequilibriumSNP: Single-nucleotide polymorphismsTDT: Transmission disequilibrium testEKL participated in the statistical analysis, interpretation of the data and drafting of the manuscript. CG-M conceived of the study, participated in the analysis, and interpretation of the data as well as drafting of the manuscript. KCC performed the statistical analyses and computer programming for this study. All authors read and approved the final manuscript."}
+{"text": "Four marker sets with varying information content and density on chromosome 3  were analyzed to detect two traits, the original affection status, and a redefined trait more closely correlated with D2. Multipoint parametric and nonparametric linkage analyses (NPL) were performed using programs GENEHUNTER, MERLIN, SIMWALK2, and S.A.G.E. SIBPAL. Our results suggested that the densest SNP map (0.3 cM) had the greatest power to detect linkage for the original trait (genetic heterogeneity), with the highest LOD score/NPL score and mapping precision. However, no significant improvement in linkage signals was observed with the densest SNP map compared with STR or SNP-1 cM maps for the redefined affection status (genetic homogeneity), possibly due to the extremely high information contents for all maps. Finally, our results suggested that each linkage program had limitations in handling the large, complex pedigrees as well as a high-density SNP marker set.Recent studies have suggested that a high-density single nucleotide polymorphism (SNP) marker set could provide equivalent or even superior information compared with currently used microsatellite (STR) marker sets for gene mapping by linkage. The focus of this study was to compare results obtained from linkage analyses involving extended pedigrees with STR and single-nucleotide polymorphism (SNP) marker sets. We also wanted to compare the performance of current linkage programs in the presence of high marker density and extended pedigree structures. One replicate of the Genetic Analysis Workshop 14 (GAW14) simulated extended pedigrees ( Previous studies have suggested that a high-density single-nucleotide polymorphism (SNP) marker set could provide equivalent or even superior information compared with currently used microsatellite (STR) marker sets for genome-wide scans by linkage -3. To daParametric and nonparametric linkage analyses (NPL) were used to map the D2 locus with chromosome 3 markers provided in the GAW14 simulated data. Because our goal was to compare the linkage results obtained by using different marker sets and different test statistics, we chose to know the true simulation model before the analyses were performed.n = 50) cohort because they contained 3 generation pedigrees with at least 4 affected individuals.Replicate 4 was identified as the largest dataset among the first 10 replicates and thus was chosen for all analyses. Analyses were also conducted using replicate 10 to make certain that our results were not biased due to selection of a non-representative replicate. We selected families from the New York City (NYC)  was modeled as a heterogeneous disease consisting of three phenotypes  with four genetic loci  involved. We chose the D2 locus as the major gene to be mapped in this study. The trait variable was analyzed in two ways. The first approach used the original affection status as the disease phenotype. Second, in an attempt to increase the underlying genetic homogeneity, we redefined affection status by classifying individuals who had all four subclinical traits e, f, h, and k as affected. Among these four subclinical traits, e, f, and h involved only D2, and trait k involved D2 and D4, as the major genetic susceptibility loci. Other trait combinations involving loci other than D2 were considered as unaffected.D2 was located at the telomeric end of chromosome 3. We analyzed all chromosome 3 STR markers (7-cM average spacing) and original SNPs (3-cM average spacing). In addition, we also \"purchased\" three 20-marker packets  containing 45 telomeric SNPs (B03T3021 to B03T3067) in a 12-cM region on telomeric chromosome 3, with an average spacing of 0.3 cM. To compare the linkage signal with SNP marker sets of different density, we created a 1-cM SNP marker set by only selecting every fourth marker on the dense (0.3 cM) SNP map. All genotype data from founders were removed to decrease the available linkage information content and to more closely resemble realistic situations. Information content of each marker set was measured using the entropy function in MERLIN [p-values) using all linkage programs and the precision of the peak as determined by the 1-LOD interval from multipoint analyses using GENEHUNTER.All families in the selected replicate were included in the analysis of the original trait. With the redefined affection status, 17 families became uninformative, consisting of either \u2264 1 affected individual within a family or containing only parent-offspring affected pairs. These families were removed when analyzing the redefined affection status and the remaining 33 families were included in all linkage analyses. We performed two-point LOD-score analysis using the MLINK program from the LINKAGE package , versionResults from the different linkage analyses are presented in Table We obtained similar results with a different replicate (replicate 10), thus our findings were unlikely to be caused by a replicate effect.In this study, we evaluated the use of SNP markers at different densities in linkage analysis involving large pedigrees and compared the results with those obtained using STR markers. Our results suggested that, for complex pedigrees provided in this simulated dataset, dense SNP marker sets did not provide significantly more information for gene mapping than STR markers at much lower density under the situation of genetic homogeneity. High-density SNPs might detect linkage signals with more precision, that is, with narrower linkage peaks, compared with STRs. However, the difference in 1-LOD intervals obtained from the STR and dense SNP maps (1 cM and 0.3 cM) was not significant (~1 cM) . Finally, although we did not evaluate the impact of linkage disequilibrium (LD) among SNPs on linkage findings due to the limited LD simulated in this region, previous work suggested that the presence of LD among SNPs on a dense SNP map might cause inflated LOD scores [In linkage studies involving high-density SNPs, one may face the challenge of analytical complexity when mapping genes in large pedigrees. GENEHUNTER and MERLIN, which both use the Lander-Green algorithm, cannot handle a large number of study subjects. GENEHUNTER and MERLIN dropped up to 15 and 9 genotyped individuals from one family in the linkage analyses, respectively. To minimize the problem associated with pedigree size, we also analyzed the data using SIMWALK2, which uses Markov chain Monte Carlo (MCMC) and simulated annealing algorithms in multipoint analyses. However, results from SIMWALK2 yield estimated statistics, in contrast to GENEHUNTER and MERLIN, which provide exact statistics. In addition, it may be difficult to guarantee the adequate convergence of the program; good approximations may require extensive computer processing time, especially when the marker spacing is very dense. In this study, we were unable to obtain a result when analyzing the SNP-0.3 cM marker set because of the failure of convergence. Compared with NPL D scores .Extremely dense SNP maps did not provide significant improvement in linkage signals compared with STRs with lower information content when phase information was easily reconstructed  and when there was genetic homogeneity. Further development and improvement of linkage programs are needed to accommodate the utilization of dense SNP markers in complex pedigrees.GAW: Genetic Analysis WorkshopKPD: Kofendrerd Personality DisorderMCMC: Markov chain Monte CarloNPL: Nonparametric linkageSNP: Single-nucleotide polymorphismSTR: Short tandem repeat polymorphismXY performed all linkage analyses and wrote the manuscript, KJ provided data management support and technical consulting, KFK and AWB participated in the design, analysis and result interpretation phases of this study and helped to draft the manuscript, and AMG and LRG provided analysis direction and recommendations at each phase of the analysis."}
+{"text": "The calculation of multipoint likelihoods is computationally challenging, with the exact calculation of multipoint probabilities only possible on small pedigrees with many markers or large pedigrees with few markers. This paper explores the utility of calculating multipoint likelihoods using data on markers flanking a hypothesized position of the trait locus. The calculation of such likelihoods is often feasible, even on large pedigrees with missing data and complex structures. Performance characteristics of the flanking marker procedure are assessed through the calculation of multipoint heterogeneity LOD scores on data simulated for Genetic Analysis Workshop 14 (GAW14). Analysis is restricted to data on the Aipotu population on chromosomes 1, 3, and 4, where chromosomes 1 and 3 are known to contain disease loci. The flanking marker procedure performs well, even when missing data and genotyping errors are introduced. The calculation of multipoint likelihoods on general pedigrees is computationally challenging. Factors influencing the complexity of multipoint calculations include family size, pedigree structure, marker number, and missing data. Efficient algorithms have been developed for handling large pedigrees with few markers  or smallIn this paper, the performance characteristics of CHROM-WALK, a computer program for calculating multipoint likelihoods on general pedigrees and many linked markers, is explored. Multipoint likelihoods are calculated using data observed only on markers flanking a hypothesized position of the trait locus. Calculating these three-point likelihoods is often feasible even on large complex pedigrees. Likelihood computations in CHROM-WALK are performed via VITESSE . The speThe CHROM-WALK computer program uses VITESSE to perform likelihood calculations needed to calculate three-point likelihoods across a chromosome on general pedigrees. Multipoint likelihoods are calculated on data on markers flanking a hypothesized position of the trait locus. Results are reported as either homogeneity LOD scores or HLOD scores at pre-specified positions of the trait locus. Functionally, CHROM-WALK is similar to GENEHUNTER . Only a rd and 24th marker locus. Chromosome 3 contained a disease locus between the 41st and 42nd marker locus. Chromosome 4 is unlinked to disease causing loci. There are 100 replicates of data.In this study, data generated on the GAW14 Aipotu population were chosen for analysis. This data consisted of 100 nuclear families, ranging in size from 2 to 10 siblings. For computational expedience, the other three GAW 14 populations were not analyzed. Marker and trait data were observed on each individual. The trait is dichotomous where an individual is either affected or unaffected for the disease. Microsatellite marker data on chromosomes 1, 3, and 4, containing 41, 42, and 44 linked markers, respectively, are selected for analysis. Inter-marker distances, on average, are 7.5 cM. Chromosome 1 contained a disease locus between the 23The accuracy of CHROM-WALK for detecting and localizing trait loci was examined through the analysis of simulated family data. A dominant trait model with incomplete penetrance  and a disease allele frequency of 0.01 was assumed. Here, three marker sets formed from the original GAW14 simulated data were considered: four linked markers closest to the disease locus (Mset4), 16 linked markers closest to the disease locus (Mset16), and all markers on a chromosome (MsetAll). Because chromosome 4 is unlinked to a disease locus, markers were selected from the beginning of the marker map. HLOD scores are calculated every 1 cM. Three-point HLOD scores are compared to multipoint HLOD scores calculated via GENEHUNTER. Multipoint scores on each data set are only calculated on markers available in that data set. Hence, the impact of increasing the number of markers incorporated into the multipoint calculation can be examined.Multipoint calculations on pedigrees are affected by missing data and genotyping error. To explore the utility of CHROM-WALK given imperfect data, missing data and Mendelian consistent genotyping errors were introduced. The marker phenotype at a locus for an individual was randomly removed with probability 0.01. Mendelian consistent genotyping errors were created, with probability 0.005, by randomly permuting with equal probability the transmitted allele from one of the parents. Note that this error model is simplistic since it cannot produce genotyping errors in the parents and does not make distinctions between types of genotyping errors, which are all equally likely in the present study. The assumed probability of Mendelian consistent errors was consistent with an overall (pedigree consistent and inconsistent) genotyping error rate of 1% [To examine the accuracy of calculating multipoint likelihoods using flanking markers, mean HLOD scores averaged over the 100 replicates are calculated at each hypothesized position of the trait locus for chromosomes 1, 3, and 4 for marker sets Mset4, Mset16, and MsetAll. There is close agreement between the CHROM-WALK and GENEHUNTER HLOD scores across hypothesized positions of the trait locus. The mean scores at the known location of the trait locus for chromosomes 1 and 3 and at an arbitrary chromosomal position for chromosome 4 are reported in Table To examine the utility of CHROM-WALK for detecting and localizing trait loci, differences in the peak GENEHUNTER and CHROM-WALK HLOD scores and differences in the chromosomal location of the peaks are investigated. Figure Figure Results are reported for the analysis of chromosome 1 in which both missing data and genotyping errors were randomly introduced. Results from the analysis of chromosome 3 and 4 data and data where either missing data or genotyping errors were introduced are not reported but are consistent with the analysis presented here. Mean CHROM-WALK and GENEHUNTER HLOD scores at hypothesized positions of the trait locus for data on chromosome 1 for the three markers sets  are plotted in Figure From Figure In this paper, the calculation of multipoint likelihoods using a new computer program CHROM-WALK is assessed through the calculation of HLOD scores on simulated data. By only considering data observed on flanking markers, the computational complexity of multipoint calculations are greatly reduced. For data simulated on nuclear families, there is little loss in accuracy using the proposed approximation procedure. Furthermore, CHROM-WALK produced multipoint results, on average, an order of magnitude faster than GENEHUNTER. Further exploration is warranted for extended families, differing amounts and patterns of missing data, differing amounts of genotyping error, and changes in marker informativeness.GAW14: Genetic Analysis Workshop 14HLOD: Heterogeneity LOD scoreAWG performed the analyses and wrote paper. LAM, AMS, and VJV developed CHROM-WALK software. MWL and CWB involved in the design of the simulation study, reporting of results and refining of the manuscript."}
+{"text": "However, the two highest densities of SNPs had unstable z score signals, and therefore were difficult to interpret. Analyzing a simulated trait with the same markers in the same pedigrees, we confirmed the higher power of all four densities of SNPs compared to the 10-cM microsatellites panel, although the existence of other confounding peaks was confirmed for maps that are denser than 1 SNP/cM. We further showed that estimating the gene position using SNPs is far less biased than using the usual panel of microsatellites (biases of 0\u20132 cM for SNPs vs. 8.9 cM for microsatellites). We conclude that using dense maps of SNPs in linkage analysis is more powerful and less biased than using the 10-cM maps of microsatellites. However, linkage signals can be unstable and difficult to interpret when several SNPs are genotyped per centimorgan. The power and accuracy of 1 SNP/cM or 1 SNP/2 cM may be sufficient in a genome-wide linkage scan while denser maps may be most useful in fine-gene mapping studies exploiting linkage disequilibrium.The efficacy of linkage studies using microsatellites and single-nucleotide polymorphisms (SNPs) was evaluated. Analyzed data were supplied by the Collaborative Study on the Genetics of Alcoholism (COGA). Alcoholism was analyzed together with a simulated trait caused by a gene of known position, through a nonparametric linkage test (NPL). For the alcoholism trait, four densities of SNPs  showed higher peaks of NPL  Most of these studies ,2 used dThe COGA definition of \"pure affecteds\" and \"pure unaffecteds\" was considered, i.e., individuals with only a few symptoms were considered as unknown phenotype. Because the COGA dataset is multiethnic, we analyzed only the \"White non-Hispanic\" group, following Sheffield et al.  and WindWe focused on chromosome 1 because previous studies pointed toward regions on this chromosome as potentially harboring genes for alcoholism . Two typThe software MERLIN  was useTo assess the accuracy and precision of NPL analysis using microsatellites versus SNPs, we simulated a susceptibility locus for a binary trait at a known position by choosing one SNP that lies at position 118.09 cM (Kosambi map) on chromosome 1 (the SNP tsc1596419). Only the trait was simulated; the real pedigree and all individual genotypes from COGA (Affymetrix SNPs) were kept. The minor allele of the trait locus (frequency of 0.04 in the White population) was considered as the susceptibility allele. A dominant model with complete penetrance and no phenocopies was simulated, thus the trait prevalence was 8%. We then localized this locus with the same mapping approach as with the real data. Though the genetic model simulated was simple, we believe that it can give a clear indication on the mapping accuracy using SNPs and microsatellites. Because we did not know the true location of an alcoholism gene, we could not address this question using the COGA data. The idea is that if one approach behaves poorly in a simple Mendelian disease, it should not be more powerful in complex diseases.z scores of NPL with microsatellites and different densities of SNPs are plotted on Figure p = 0.08) in the region 111\u2013122 cM. The z scores observed with SNPs are higher than those of microsatellites: the highest score varies between 1.76 and 2.14  but its position was at 109.2 cM, while the true position of the simulated gene was 118.09 cM , one additional peak was observed at position 121 cM  and GENEHUNTER-plus (GH+) , MERLIN  and GH+ . This isz scores and lower p-values with any of these densities of SNPs than with microsatellites . In fact, tests of linkage analysis assume the absence of LD and when it exists, it can affect the results of these tests. Figures z score is higher with all scenarios of SNPs that we tested than with microsatellites . Unlike with microsatellites, the one z score interval contains the real simulated gene position for all four tested SNP densities. Therefore, the four densities of SNPs give comparable results in this simulation.The simulation study confirms the higher efficiency of SNPs in linkage analysis compared with usual maps of microsatellites: the peak s Figure  and amonA range of SNP densities was shown to be more efficient in linkage studies than the usual 10-cM microsatellites panel. Densities of approximately 1 SNP per centimorgan or per 2 cM should be favored because they give a satisfying power, accuracy, and precision, while denser maps can be the most useful in fine-gene mapping exploiting linkage disequilibrium. Alternatively, developing new linkage methods that are adapted to very dense marker maps may allow better extraction of the information contained in the maps.COGA: Collaborative Study on the Genetics of AlcoholismGAW14: Genetic Analysis Workshop 14GH: GENEHUNTERGH+: GENEHUNTER+LD: Linkage disequilibriumNPL: Nonparametric linkageSNP: Single-nucleotide polymorphismJN performed data handling and analyses and drafted the manuscript. HR performed MERLIN acquisition and installation and contributed in data handling. DG supervised the work, gave advices on the analyses to perform and contributed in the interpretation of the results. All the authors read and approved the final version of the manuscript."}
+{"text": "Additional simulation studies of affected sib pairs assuming uniform LD throughout a marker map demonstrated inflation of significance levels at r2 values greater than 0.05. When genotypes are available only from two affected siblings in many families in a sample, trimming SNP maps to limit r2 to 0\u20130.05 for all marker pairs will prevent inflation of linkage scores without sacrificing substantial linkage information. Simulation studies on the observed pedigree structures and map can also be used to determine the effect of LD on a particular study.Dense SNP maps can be highly informative for linkage studies. But when parental genotypes are missing, multipoint linkage scores can be inflated in regions with substantial marker-marker linkage disequilibrium (LD). Such regions were observed in the Affymetrix SNP genotypes for the Genetic Analysis Workshop 14 (GAW14) Collaborative Study on the Genetics of Alcoholism (COGA) dataset, providing an opportunity to test a novel simulation strategy for studying this problem. First, an inheritance vector (with or without linkage present) is simulated for each replicate, i.e., locations of recombinations and transmission of parental chromosomes are determined for each meiosis. Then, two sets of founder haplotypes are superimposed onto the inheritance vector: one set that is inferred from the actual data and which contains the pattern of LD; and one set created by randomly selecting parental alleles based on the known allele frequencies, with no correlation (LD) between markers. Applying this strategy to a map of 176 SNPs (66 Mb of chromosome 7) for 100 replicates of 116 sibling pairs, significant inflation of multipoint linkage scores was observed in regions of high LD when parental genotypes were set to missing, with no linkage present. Similar inflation was observed in analyses of the COGA data for these affected sib pairs with parental genotypes set to missing, but not after reducing the marker map until  D' values greater than 0.6 [D' values increased between 0.4 and 0.8 [r2 measure of linkage disequilibrium (LD).Linkage genome scans using dense maps of single nucleotide polymorphism (SNP) markers have been shown to provide greater information content than 10-cM microsatellite scans . Howeverthan 0.6 ; and in The Collaborative Study on the Genetics of Alcoholism (COGA) datasets for Genetic Analysis Workshop 14 (GAW14) provided an opportunity to study this problem, because the Affymetrix SNP data revealed regions with high marker-marker LD. The main goal of the present analyses was to test a novel simulation strategy for studying the effect of LD on linkage scores with and without availability of parental genotypes.We selected the 116 European-ancestry pedigrees from the 143 families in the GAW14 COGA dataset. We focus here on analyses that used one pair of affected siblings per family plus parents, but additional analyses utilized the full pedigrees ; nuclear families ; or one sibship from each pedigree . In these additional analyses, comparisons of linkage results with different maps were similar to those reported in other GAW14 papers and so are not discussed in detail here; comparisons of information content for different maps, and analyses of the effects of LD on linkage scores, were consistent with those reported here for simulated data.P < 0.001), call rate < 0.8, or minor allele frequency < 0.05. We studied the remaining 176 SNPs (\"High-LD map\") and a subset of 109 SNPs (\"Low-LD map\") in which there was no pairwise r2 value > 0.05.Genotypes were available for a 10-cM microsatellite map, 4,752 Illumina SNPs and 11,560 Affymetrix SNPs. In the 66 Mb of chromosome 7 containing the largest linkage peak in these 116 pedigrees , there were 212 Affymetrix SNPs. We excluded 36 of these because of deviation from Hardy-Weinberg equilibrium . The r2 LD statistic was computed with HAPLOVIEW [Linkage analyses were carried out with ALLEGRO  .Data were simulated using SIM  and programs written for this study. For each replicate, SIM assigned 2 unique alleles to each founder , transmitted them to offspring by selecting locations of recombinations for each meiosis based on genetic distances, and then transmitted parental chromosomes . Then, for each replicate two different datasets were created by replacing each unique allele with an allele from a corresponding founder haplotype (gene-dropping). First, we assigned to each parent 2 haplotypes from among the 464 haplotypes inferred from COGA data as described above (LD condition), and then we assigned parental haplotypes created by random selection of alleles based on the allele frequencies in the COGA data, with no correlation between markers (No-LD).r2 values between marker pairs of 0\u20130.4 in steps of 0.05.In addition, to examine the effects of varying levels of inter-marker LD more systematically, 5,000 replicates were created for 650 pedigrees containing 920 affected sib pairs affected sib pairs (ASPs) with 30% of parents genotyped, for 200 SNPs (0.2 cM apart) with no linkage present, with uniform LD at r2 values for each marker with the marker to its left, which gives a reasonable indication of the variation in LD throughout the region, although the pattern for all possible pairs is more complex. There are regions around 43 and 60 Mb where LD is substantial, and where Zlr is inflated when parental genotypes are not available for the High-LD map. Table r2 values for the region around 60 Mb; values for all pairs of the 176 SNPs are available on request. In a third region, centered around 17\u201318 Mb, Zlr is inflated when parental genotypes are not available for both maps, suggesting that in this region the absence of parental data changes the scores.In analyses of the COGA dataset , if parental genotypes were available, then there were minimal differences between linkage scores for the High-LD and Low-LD maps for the full pedigrees, sibships, or ASPs (data not shown). Figure r2 between the SNP and the marker to its left; (b) distance (in Mb) between each pair of SNPs; (c) the average of 4 consecutive r2 values (computed for every fourth SNP to avoid overlap) such that for SNPs with order 1\u20132\u20133\u20134\u20135, the Avg4-LD is the average of r2 values for the pairs 1\u20132, 2\u20133, 3\u20134, and 4\u20135; and (d) the average of 4 consecutive distances, computed as described for Avg4-LD. The correlation coefficient was computed between the average Zlr difference (observed-true) at each marker position and either the r2 or distance measure for that SNP. The best predictor (r = 0.52) of the observed-true difference for ASPs (LD map) without parental genotypes was the Avg4 r2 measure. A multiple regression analysis was also computed, with Zlr difference for the ASP-LD dataset as the dependent variable, and Avg4-LD, Avg4-Distance and their interaction as independent variables. Only r2 was predictive of Zlr difference (P = 0.0063). Note that in 19% of replicates with no linkage present, the maximum Zlr score between 41 and 45 Mb was > 2.0, and in 2% it was > 3.0.Table r2) between each marker pair, for 650 pedigrees with 70% of parental genotypes. The red, blue and green lines show the proportion of replicates in which the largest linkage score exceeded the threshold observed 5, 1, or 0.1% of the time in the absence of LD (r2 = 0). For each threshold, false positive results increase with r2. Even at r2 = 0.10, the proportion of \"significant\" results was 0.0625 for the threshold associated with P = 0.05 for r2 = 0.Finally, Figure These analyses support the conclusion that when parental genotypes are missing and cannot be reconstructed from the constellation of genotyped individuals, the presence of marker-marker LD can substantially inflate linkage scores ,6. In thr2 threshold of 0.05 is probably desirable. Fortunately, as shown in Figure r2 < 0.05. Alternatively, it might be possible to correct for LD statistically, although it may prove difficult to account for patterns of LD that extend beyond the adjacent two markers.The data presented in Figure r2 values (such as the Avg4 measure described above).The simulation method described here can also be used to evaluate whether inflation of linkage scores is likely with a marker map and pedigree sample. One would first simulate replicates based on the pedigree structures in the real study as described above, with or without linkage present, assuming that all parental genotypes are available. Gene-dropping would then be carried out, using haplotypes inferred from the real data (and thus containing the observed LD pattern). After setting parental genotypes to missing, one would repeat the linkage analysis of each replicate for the \"true\" data  and the gene-dropping data that contain the LD pattern, compute the difference between these scores for each replicate, and determine whether the difference is correlated with ASP: Affected sib pairCOGA: Collaborative Study on the Genetics of AlcoholismGAW: Genetic Analysis WorkshopLD: Linkage disequilibriumSNP: Single nucleotide polymorphismDFL designed these studies and carried out the main analyses. PH participated in critical discussions of these ideas, reviewed the manuscript, and carried out the simulation study presented in Figure"}
+{"text": "This paper deals with the detection of significant linkage for quantitative traits using a variance components approach. Microsatellite markers were obtained for the Genetic Analysis Workshop 14 Collaborative Study on the Genetics of Alcoholism data. Ethnic heterogeneity, highly skewed quantitative measures, and a high rate of missing values are all present in this dataset and well known to impact upon linkage analysis. This makes it a good candidate for investigation.As expected, we observed a number of changes in LOD scores, especially for chromosomes 1, 7, and 18, along with the three factors studied. A dramatic example of such changes can be found in chromosome 7. Highly significant linkage to one of the quantitative traits became insignificant when a proper normalizing transformation of the trait was used and when analysis was carried out on an ethnically homogeneous subset of the original pedigrees.In agreement with existing literature, transforming a trait to ensure normality using a Box-Cox transformation is highly recommended in order to avoid false-positive linkages. Furthermore, pedigrees should be sorted by ethnic groups and analyses should be carried out separately. Finally, one should be aware that the inclusion of covariates with a high rate of missing values reduces considerably the number of subjects included in the model. In such a case, the loss in power may be large. Imputation methods are then recommended. The purpose of this paper is to illustrate the impact of three factors: robustness to non-normality, population admixture, and covariates with a high rate of missing values, on the linkage detected using a variance components approach. The effect of these factors has been well studied in linkage analysis. As we shall see, the Collaborative Study on the Genetics of Alcoholism (COGA) dataset offers a very good illustration of the dramatic changes observed when such aspects are not considered carefully.Variance components approaches determine whether genetic variation at a specific chromosomal location can explain the variation in the phenotype . This noPopulation admixture is another phenomenon that has been studied . It is kThe last aspect considered in this work deals with the inclusion of covariates in the model with a high proportion of missing values. Several methods of imputation for missing values are available, and we refer for example to Fridley et al.  regardinGenome-wide scan analysis was performed on 329 microsatellite markers obtained for 1,350 of the 1,614 individuals in the Genetic Analysis Workshop 14 (GAW14) COGA pedigree data.We first sought candidate traits that could be used as an illustration of the impact of the factors studied. After performing genome-wide scan analyses on different electrophysiological quantitative phenotypes, we selected three traits, named ttth1, ttdt3, and ttdt4 in the COGA data, that expressed significant linkage in some regions of the genome. These traits are also commonly referred to as event-related potential (ERP) traits.ethnicity = 6 . These last pedigrees are called the White pedigrees through this paper. Note that some pedigrees were truncated in order to preserve the unity of the self-reported ethnicity.In order to measure the influence of ethnicity of pedigrees on quantitative trait locus (QTL) linkage detection, we ran a genome-wide scan analysis on two sets of COGA pedigrees. The first set contained all pedigrees as in the GAW14 COGA data  and the second set contained 105 pedigrees extracted from the initial set of pedigrees, namely those whose members claimed All the statistical linkage analyses were carried out using the software SOLAR  ,7, whichFollowing the variance components method, a quantitative genetic analysis with covariate screening was performed. The best model was chosen by iteratively adding the covariates to the model, and by estimating the different parameters, such as the total additive genetic heritability (H2r) and the covariates regression coefficients, by maximum likelihood. Only significant covariates were kept in the model.Identity-by-descent (IBD) probabilities and multipoint identity-by-descent (MIBD) matrices  were computed using allele frequencies either provided with the COGA data (not ethnicity-specific) or estimated by SOLAR using a maximum likelihood approach (as in the White pedigree case). In multipoint linkage analysis, the Kosambi map function was used as supplied with the GAW14 data.Preparing the quantitative trait for QTL analysis is a crucial step. Normality of the trait is the basic requirement of the statistical method we used. It is important to ensure that the empirical distribution of the trait considered follows this requirement. Skewness and kurtosis are good measures that allow the identification of a potential violation of the normality assumption. Skewness is a measure of the asymmetry of the distribution while kurtosis is an indicator of how close the distribution matches a bell shape. If the distribution is normal, both measures should be zero. A common approach in regression-type models uses the logarithmic transformation. Such a transformation is often effective in reducing skewness and kurtosis. More generally, a Box-Cox transformation (see ) can be sex, erpage (age at ERP examination), ALDX11  for trait ttth11/4, erpage, ALDX12 for trait ttdt31/4 and erpage, ALDX12 for trait ttdt41/4.If not specified otherwise, the covariates included in the polygenic model  involving the transformed trait and the White pedigree were ALDX1, ALDX2 that were transformed into a number of indicator variables in order to use them as categorical covariates. For instance, ALDX12 represents ALDX1 = 5 (affected) and ALDX12 = 0 represents ALDX1<5.Covariates were taken as provided in the COGA dataset, except in the case of Pedigree-based analyses demonstrated three QTLs on chromosome 1 , chromosome 18 (between markers D18A535 and D18A877), and chromosome 7 . The first two QTLs appear to be linked to the traits labelled ttdt3 and ttdt4. As mentioned in Table Findings on chromosome 7 show a strong linkage signal for the other measure, ttth1, with a maximum LOD score of 4.08. However, this linkage seems to be strongly related to the self-reported ethnicity of the pedigrees as well as the skewness and kurtosis of the trait.1/4, on chromosome 7 for the White pedigree. Dramatic changes in the LOD scores can be observed when different transformations are applied. The trait ttth1 in its original form appears to be strongly linked to a QTL around 160 cM (LOD = 3.49). The logarithmic transformation of this trait gives only suggestive linkage to the same QTL (LOD = 2.67). However, the Box-Cox transformation of ttth1, i.e., ttth11/4, does not show any linkage result at this locus (LOD = 0.06). A maximum LOD score of 1.19 is obtained at location 112 cM. In this case, not only has the significance of the LOD peak has been dramatically reduced, but it has also been moved by 48 cM.Figure Clearly, such changes are not systematic across traits, chromosomes, and pedigrees. However, we illustrate here an extreme situation. Table Table 1/4. Figure erpage and ALDX12, with a total of 680 individuals. If the variable cigpkyrs (number of packs of cigarettes per day for one year) is also included in the model , we observe a deviation from the optimal model, regarding the significance of the LOD score, along the chromosome. If the variable cigpkyrs is replaced by the variable ageonset1, which contains 59.5% of missing values, the number of subjects included in the model drops to 348 individuals and we can observe extreme changes in the significance of the LOD score.As an illustration of the impact that the addition of covariates has in the linkage detected, we study the case of chromosome 1 using the White pedigrees and the trait ttdt3ethnicity.As we have seen, the COGA dataset provides a very clear illustration of the effects that ethnicity, covariates, and the normality of the trait have on the linkage observed. One should be aware that mixing different ethnic groups may introduce some noise, leading to the failure to detect strong linkage that may be present in one or more of the groups. As a result, populations admixtures should be avoided if no evidence suggests that the different ethnic groups behave similarly in terms of the trait and markers considered. Note that in the case studied, the preliminary multivariate analysis of the traits considered showed highly significant segregation by the self-reported When adding covariates in the model, one should pay special attention to the trade-off between the gain of information due to the covariate and the loss of information due to the reduction of the sample size. If possible, imputation methods should be considered.Finally, any statistical models are built on data assumptions, such as the normality of the trait. Again, being aware of these assumptions and trying to guarantee their validity is key in the success of an analysis. Note that many other factors could be discussed, such as the impact of a violation of some other model assumptions such as Hardy-Weinberg equilibrium and linkage equilibrium. The type of map used is also crucial in fine mapping studies. Some inconsistencies in the maps lead to great differences in the location of the linkage detected, especially when high density SNPs are used.COGA: Collaborative Study on the Genetics of AlcoholismERP: Event-related potentialGAW: Genetic Analysis WorkshopHWE: Hardy-Weinberg equilibriumIBD: Identity by descentLE: Linkage equilibriumMIBD: Multipoint identity by descentQTL: Quantitative trait locusBoth authors contributed equally to this work in all the aspects of the paper."}
+{"text": "This paper presents a method of performing model-free LOD-score based linkage analysis on quantitative traits. It is implemented in the QMFLINK program. The method is used to perform a genome screen on the Framingham Heart Study data. A number of markers that show some support for linkage in our study coincide substantially with those implicated in other linkage studies of hypertension.Although the new method needs further testing on additional real and simulated data sets we can already say that it is straightforward to apply and may offer a useful complementary approach to previously available methods for the linkage analysis of quantitative traits. Previously available methods of linkage analysis of quantitative traits applicable to complex pedigrees have consisted of variance components analysis -4, or reThe Framingham data set includes genotypic data and phenotypic measures related to traits including blood pressure, characterized in a large set of complex pedigrees. Previously it has been investigated for linkage using the variance components method, producing a number of interesting results including a multipoint LOD score of 4.7 using a treatment-adjusted measure for blood pressure with markers on chromosome 17 . Genome-The present study describes the application of the new method of model-free linkage analysis of a quantitative trait to a measure of blood pressure obtained from the Framingham data set.AA, Aa, and aa. The trait values for subjects having these genotypes have means MAA, MAa, and Maa, with a constant variance of the measure around each genotype-specific mean VAA = VAa = Vaa. A model of heterogeneity is assumed, whereby the susceptibility locus exerts its effect in a proportion \u03b1 of families while an unlinked locus exerts the same effect in the other families. For the purposes of analysis we assume equal frequencies for the two alleles and also constrain the genotype-specific means for the first two genotypes to be equal so that MAA = MAa. This might be thought of as saying that the A allele exerts a dominant effect. The genotype aa with mean Maa is therefore less common, with frequency 0.25. Defining the overall population variance as V and the standard deviation as S = \u221aV, then we set a variable T = (Maa - MAA)/S as a measure of the displacement between the genotype-specific means. There are limits for T at which the displacement between these means will be so large as to completely account for the overall population variance of the trait with zero variance around each mean (VAA = VAa = Vaa = 0), and between these limits it is straightforward to calculate a value for the variance around each genotype-specific mean (VAA = VAa = Vaa) that will produce the correct overall population variance, V. We allowed T to take a range of values ranging from -2 to 2. At the extreme values of Maa = MAA - 2S and Maa = MAA + 2S the fraction of the overall variance explained by the difference in genotype-specific means is 75%, while this fraction decreases towards zero as the absolute value of S approaches zero. We set further parameters \u03b8 and \u03b1 as follows. We write \u03b8 = t to imply the trait locus is at the test position, which may be on a multipoint map, and \u03b8 = 0.5 to indicate it is unlinked to any markers. We write \u03b1 to be the proportion of families in which the trait locus exerts an effect, with the assumption that in proportion 1 - \u03b1 of families an unlinked locus elsewhere exerts a similar effect. We then generate a range of transmission models by varying T from -2 through 0 to 2 and for each model we calculate three likelihoods which are functions of T, \u03b8, and \u03b1:The model-free method of linkage analysis that we present assumes that a susceptibility locus may have an effect on the trait in question such that a bi-allelic polymorphism exists with genotypes UNLINKED = LLLINKED = LLHET = L where LHET is maximized over \u03b1.LThree LOD scores are then defined as:10(max [LLINKED/LUNLINKED]) - maximized over TMLOD = log10(max [LHET/LUNLINKED]) - maximized over T and \u03b1MALOD = log10(max [LLHET]/max [LUNLINKED]) - numerator maximized over T and \u03b1, and denominator independently maximized over T.MFLOD = logAA = MAa = Maa or \u03b1 = 0). 2ln(10)MLOD is distributed as x 21; 2ln(10)MALOD is distributed as a 50:50 mixture of x 22 and x 20; and 2ln(10)MFLOD as a 50:50 mixture of x 21 and x 20. The new method has been implemented in a computer program called QMFLINK.These LOD scores are equivalent to those produced by the MFLINK program and the associated likelihood ratio statistics, calculated as 2ln(10)LOD (where ln(10) means the natural log of 10), are asymptotically distributed as chi-squared statistics as follows under the null hypothesis of the test locus exerting no effect on the quantitative trait  at 20 cM.We compared our results with those from other linkage studies of hypertension -16. Of pA number of markers that we find to show some support for linkage seem to coincide with regions implicated in other linkage studies of hypertension. The distal region of chromosome 17 highlighted by our analysis has provided evidence of linkage in an affected sibling pair study by Julier et al. . The posp = 0.02) at 63 cM on chromosome 16 correspond with a region implicated to contain a low blood pressure QTL [p = 0.02) may reflect the effect of the same locus as was implicated in a study by Krushkal et al. [p = 0.0001 in the interval 141\u2013153 cM.The MLOD of 1.3, MALOD of 1.4, and MFLOD of 0.9 (all sure QTL . Finallyl et al. , who fouWe are not aware of any other published papers that report evidence of linkage to the specific regions on chromosomes 4, 5, and 8 where we have positive results. It is possible that the results from chromosome 5 and 8 as well as the result previously discussed result on chromosome 6 correspond to those from the MRC BRIGHT study  but at tThe new method of analysis has been applied to a large sample of complex pedigrees, some containing loops. The results overlap to some degree with those obtained using a variance-components method to analyze the same data set. However we could not obtain an identical phenotypic measure because we were unable to allow for treatment effects. Thus we do not know the extent to which differences in the methods of analysis and differences in quantitative measures account for differences between our results and those obtained in the previous analysis. It is of interest that our analysis also provides some evidence for linkage in areas that were not highlighted in the previous analysis, some at least of which seem to be supported by the results of other studies. Of course, we cannot be sure which results are true- or false-positives until the genetic basis of hypertension has been fully elucidated.Analogously to the MFLINK program, the method can be described as \"model-free\" in that one does not need to specify in advance one particular inheritance model, but rather a range of models is considered. It is expected that if the true inheritance model is reasonably close to one of these then the method will be able to detect linkage when it is present, but if this were not the case then the method might lack sensitivity. Testing of MFLINK demonstrated that it had good ability to detect linkage under a variety of different conditions. The new method generates three different statistics, the MFLOD, MLOD, and MALOD. In general these will tend to be positively correlated but for a given marker one statistic may be more highly significant than the others. This could occur through chance or might happen when the test more accurately modelled the true biological situation. Having three partially independent statistics makes the overall interpretation of significance more problematic, but most users will probably wish to examine all three in order to avoid overlooking a potentially true positive result.In order to determine the utility of the new method it will need to be thoroughly tested on a large number of real and simulated data sets. For now, we can say that the method is straightforward to apply and may offer a useful complementary approach to previously available methods for the linkage analysis of quantitative traits."}
+{"text": "Body mass index (BMI) and adult height are moderately and highly heritable traits, respectively. To investigate the genetic background of these quantitative phenotypes, we performed a linkage genome scan in the extended pedigrees of the Framingham Heart Study. Two variance-components approaches (SOLAR and MERLIN-VC) and one regression method (MERLIN-REGRESS) were applied to the data.Evidence for linkage to BMI was found on chromosomes 16 and 6 with maximum LOD scores of 3.2 and 2.7, respectively. For height, all markers showing a LOD score greater than 1 in our analysis correspond to previously reported linkage regions, including chromosome 6q with a maximum LOD score of 2.45 and chromosomes 9, 12, 14, 18, and 22. Regarding the analysis, the three applied methods gave very similar results in this unselected sample with approximately normally distributed traits.Our analysis resulted in the successful identification of linked regions. In particular, we consider the regions on chromosomes 6 and 16 for BMI and the regions on chromosomes 6, 9, and 12 for stature interesting for fine mapping and candidate gene studies. In recent years, about 20 genome scans for obesity and obesity-related phenotypes have been published. Many of these focussed on obesity using the affected sib-pair design, which offers good power compared with the necessary recruitment effort. On the other hand, many epidemiological studies or genome scans for common diseases come up with large and well characterized samples. If a sufficient number of the recruited individuals is related and DNA or genotype information is also available, linkage analysis for several traits can be conducted. The Genetic Analysis Workshop 13 provides genetic and anthropometrical data from 330 general pedigrees of the Framingham Heart Study. Thus, we studied the genetics of body mass index (BMI) and height using a two-stage approach, which ensures that all individuals can be analyzed together. First, we built regression models for the phenotypes to obtain a single adjusted trait value for each individual. At the second stage we performed linkage analyses incorporating all genotyped individuals.. We used the sex-averaged positions converted to the Haldane mapping function. Phenotypic information is provided for 2885 persons . Detailed information about the Framingham Heart Study is given at .The individuals from the Framingham Heart Study were recruited at two time points  from the general population excluding those with cardiovascular diseases, heart attack, or stroke. Almost all participants were of Caucasian origin. From the 330 largest pedigrees with 4692 members, DNA was available for 1702 individuals, who were genotyped for 401 markers on the 22 autosomes. The positions of the markers were from the Marshfield website: We condensed and trimmed the given pedigrees to enable effective multi-point linkage analysis with MERLIN . CondensThe available longitudinal phenotypic information for each person was transformed into one specific value for each trait. For BMI, we defined an individual mean that accounts for all available BMI measures. This allowed us to circumvent the problem of missing values at single time points. The phenotype height was investigated as the maximum of the height measurements. Regression models for BMI and height were built for each sex in the original and the offspring cohort separately.th individual at time t is modelled as:BMI was log-transformed to account for the underlying skewed distribution and adjusted for age and smoking (cigarettes per day). To get an estimate of an overall mean for a person which accounts for the multiple measures, all available examinations of each person between the age of 20 and 70 years were considered and a class variable for each individual was incorporated. Thus, the observation for the iBMI)it = \u03bc + \u03c0i + \u03b21 ait + \u03b22 cit + eit,log , cit as the cigarette consumption at time t, and eit as the residual at time t. This model gives one value for the least squares mean \u03c0i for each individual. The standardized values of \u03c0i are approximately normally distributed and were used as phenotypic information in all linkage analyses.with \u03bc as the overall mean, \u03c0th individual is:For height, the maximum height of each individual older than 18 years was modelled, adjusted for age at first examination to account for the different years of birth. The model for the iheight)i = \u03bc + \u03b2 ai + eimax  models implemented in MERLIN  and SOLAFigures LOD scores greater than 1 were observed on chromosomes 1, 2, 4, 5, 6, 8, 9, and 16. The maximum LOD scores for BMI were found on chromosome 16 with 3.21 for SOLAR, 2.81 for MERLIN-VC, and 2.47 for MERLIN-REGRESS with a 1-LOD support interval reaching from 45 to 85 cM. A second interesting region was identified on chromosome 6 with LOD scores of 1.90 to 2.70 depending on the analysis method.For chromosomes 6p, 6q, 9, 12, 14, 18, and 22, LOD scores greater than 1 were obtained with at least one analysis method. The strongest evidence for linkage to height was found near the q-ter of chromosome 6, with a LOD score of 2.45 for MERLIN-REGRESS and a 1-LOD support interval spanning from 190 to 204 cM. The VC methods gave LOD scores of 1.83 and 1.67 at the same position.For this unselected sample both the VC methods and the regression method implemented in MERLIN are valid and showed remarkably close agreement over the whole genome  and one regression method (MERLIN-REGRESS). These linkage analysis methods were applied to the same data, thus allowing us to compare the results. All three methods can be used for this unselected sample with approximately normally distributed traits. The observed results are remarkably similar over the whole genome and show close agreement in the positions and magnitudes of the highest LOD scores. We cannot assess which method has the most power, since no functional relationships to BMI or height are proven for genes in the regions with high LOD scores, and therefore we cannot recommend a specific method from this application to real data. Using simulated data of normally distributed traits, Sham et al.  showed tConsidering the numerous studies for BMI and BMI-related phenotypes, annually reviewed in the human obesity gene map , we concFor the phenotype adult height, we were able to identify several regions that showed evidence for linkage in some of the six genome scans published to date. In particular on chromosome 6q we had a broad peak with a maximum LOD of 2.45 at 201 cM. Interestingly, Hirschhorn et al.  and Xu eThe power of linkage analysis was substantially reduced since for many founders no DNA was available. Nevertheless, this population-based and unselected sample has been a good example for the successful identification of linked regions. In particular, we consider the regions on chromosomes 6 and 16 for BMI and the regions on chromosomes 6, 9, and 12 for stature interesting for fine mapping and candidate gene studies. Our results indicate that for moderately to highly heritable traits the analysis of phenotypically well characterized but unselected and rather large samples of extended pedigrees is promising. Other such large epidemiological cohort studies, where many covariables are carefully collected, can be valuable and efficient tools in studying the genes and interactions between genes and environmental factors in common complex diseases."}
+{"text": "The largest separation in mean ages of onset of ALDX1 was 19.76 and 24.41 between male smokers who are carriers of the risk allele of tsc0041591 and the non-carriers, respectively. Hence, male smokers who are carriers of marker tsc0041591 on chromosome 2 have an average onset of ALDX1 almost 5 years earlier than non-carriers.A genetic analysis of age of onset of alcoholism was performed on the Collaborative Study on the Genetics of Alcoholism data released for Genetic Analysis Workshop 14. Our study illustrates an application of the log-normal age of onset model in our software Genetic Epidemiology Models (GEMs). The phenotype ALDX1 of alcoholism was studied. The analysis strategy was to first find the markers of the Affymetrix SNP dataset with significant association with age of onset, and then to perform linkage analysis on them. ALDX1 revealed strong evidence of linkage for marker  Past Collaborative Study on the Genetics of Alcoholism (COGA) results suggest that genetic linkage of alcohol dependence to markers on chromosomes 1, 2, 4, and 7 deserves further studies . For GenWe focused on ALDX1 and its age of onset. We used the given definition of alcoholism, ALDX1: classes 1, 2, 3, 4 were coded as unaffected and class 5 as affected. The data had the following characteristics: 143 pedigrees; 1,614 persons; 643 affecteds; 735 unaffected; 356 male smokers; 297 male non-smokers; 245 female smokers; and 470 female non-smokers.\u03b8, and the probability model for the penetrance function. The age-of-onset model of Elston [We adapted the well known Elston-Stewart  likelihof Elston  uses a mf Elston ,9, and Bf Elston , and ascf Elston , as need\u03bc. Using the subscript g to denote genotype at the unobserved disease locus coded by the dummy variable Xg, and subscript m for the marker locus, we applied one model for association and three different models for linkage analysis.The mean age of onset on the log scale is a linear function of the disease genotype (assuming single locus and coded as a dummy variable), marker locus, sex, and smoking, also coded as dummy variables. Products of these variables are included to study interactions. The versions of the model applied to ALDX1 differed only in the specification of the mean age of onset, \u03bc = \u03b20 + \u03b2m\u2022Xm\u03b2m = 0 and \u03b2m\u2260 0 using the likelihood ratio and its asymptotic chi-square with 1 degree of freedom. The analysis strategy first used marker association tests as a preliminary screening procedure to pick up markers for linkage study. Markers found significant at the 0.0001 level were tested further for linkage.Disease to marker association is tested by comparing the hypotheses The mean age of onset depends on the interaction of marker and disease genotype:\u03bc = \u03b20 + \u03b2g\u2022Xg\u2022Xm.The mean age of onset depends on the interaction of marker and disease genotype, and a linear sex effect:\u03bc = \u03b20 + \u03b2g\u2022Xg\u2022Xm + \u03b2sex\u2022Xsex.The mean age of onset depends on the interaction of marker and disease genotypes, sex, smoking, and the, interaction of gender and smoking, thus\u03bc = \u03b20 + \u03b2g\u2022Xg\u2022Xm + \u03b2sex\u2022Xsex + \u03b2smoker\u2022Xsmoker + \u03b2sex_smoking\u2022Xsex\u2022Xsmoking.Note that the association model does not include the unobserved disease genotype, but the linkage models do. Note also that the linkage models include disease locus and marker locus interaction but not their \"main effects\". Doing so led to horrible convergence problems due to \"over fitting\". For a test of linkage, LOD scores were calculated as usual from the formula10{max likelihood (0 \u2264 \u03b8 \u2264 1/2)}/{max likelihood (\u03b8 = 1/2)}.LOD score = LogThe significance of the effects of covariates, sex and smoking, were tested by usual comparison of the estimates of the coefficients with their standard errors, making the justified assumption that the sample was large enough for the likelihood estimates to have approximate normal distribution.p-value of 0.05 level. Females have a lower mean age of onset than males. The first (Model I) and second (Model II) linkage results revealed significant LOD scores for marker tsc0041591 on chromosome 2 and suggestive linkage for marker tsc0894042 on chromosome 3.Association and linkage results for observed marker genotypes are presented in Table tsc0041591, with the addition of smoking status and interaction of sex and smoking, the estimated regression coefficient for the interaction of sex and smoking was significant at the 0.05 level of significance. Male smokers have a lower mean age of onset than females. The linkage tests showed higher LOD scores for marker tsc0041591 on chromosome 2.For Model III, using chromosome 2 marker tsc0894042 are similar for Models I, II, and III. However, sex, smoking and their interaction are not significant at the 0.05 level. Hence the effect of chromosome 3 marker tsc0894042 on alcoholism appears to be purely genetic, while that of chromosome 2 marker tsc0041591 is significant by itself but is even more marked among smokers. Table tsc0041591 on chromosome 2. The mean age of onset of all ALDX1 cases is 22.18 for males and 23.18 for females. The mean age of onset of the risk allele carriers is lower, 20.39 for males and 22.78 for females; and that of non-carriers is higher, 23.63 for males and 24.41 for females. The lowest mean age of onset of ALDX1 occurs in smokers who are risk allele carriers; their onset of ALDX1 occurs almost 5 years younger on average.The LOD scores for chromosome 3 marker Furthermore, Figure Some general patterns are evident for Models I, II, and III. The markers that showed significant LOD scores without smoking as a covariate revealed higher LOD scores with smoking. The markers with non-significant LOD scores without smoking status as a covariate were also non-significant when smoking was added to the covariates.We reduced the number of markers and chromosomes by performing association tests and then determining the evidence of linkage on the selected markers. However, in performing this reduction we are assuming no association leads to no evidence of linkage.p-values of 0.05 for the association test. It is possible that we have missed some important markers in our conservative strategy of analysis.We selected markers that showed association at 0.01 significance level and tested them for linkage. There were many more markers significant at tsc0041591 and tsc0512083 on chromosome 2 and tsc0894042 on chromosome 3 revealed strong or suggestive linkage for alcoholism. LOD score values increased among smokers for markers tsc0512083 and tsc0512083 on chromosome 2 but not for marker tsc0894042 on chromosome 3. The effect of sex on the genetics of alcoholism was not as strong as that of smoking.In summary, the markers Concerning our tool, GEMs, we found the speed of computation to be very slow for the genome scan data. The version used did not utilize parallel processing. Moreover, the largest LOD scores sometimes only reflected local optimum conditions. The solution is to use several different initial estimates. This has now been automatically implemented in GEMs.COGA: Collaborative Study on the Genetics of AlcoholismGAW14: Genetic Analysis Workshop 14GEM: Genetic Epidemiology ModelsVA drafted the manuscript and performed statistical analyses. JA participated in the acquisition of data and analysis of data. JPH participated in the analyses and interpretation. RET participated in the data analysis and interpretation of results. GEB conceived of the study and help to draft the manuscript. All authors read and approved the final manuscript."}
+{"text": "Based on the results of the simulated GAW14 data, the WA test statistic showed good performance and could narrow down the region containing the susceptibility locus. However, the strength of the signal depends on both the strength of the linkage disequilibrium and the heterozygosity of the linked marker.We present a new method for fine-mapping a disease susceptibility locus using a case-control design. The new method, termed the weighted average (WA) statistic, averages the Cochran-Armitage (CA) trend test statistic and the difference between the Hardy-Weinberg disequilibrium test statistic for cases and controls (the HWD trend). The main characteristics of the WA statistic are that it improves on the weaknesses, and maintains the strengths, of both the CA trend test and the HWD trend test. Data from three different populations in the Genetic Analysis Workshop 14 (GAW14) simulated dataset  were first subjected to model-free linkage analysis to find regions exhibiting linkage. Then, for fine-scale mapping, 140 SNPs within the significant linkage regions were analyzed with the WA test statistic on replicates of the three populations, both separately and combined. The regions that were significant in the multipoint linkage analysis were also significant in this fine-scale mapping. The most significant regions that were obtained using the WA statistic were regions in chromosome 3 (B03T3056\u2013B03T3058,  It has been shown that fine-scale mapping of a susceptibility locus for a complex disease can be accomplished by evaluating the deviation from Hardy-Weinberg equilibrium (HWE). For example, Feder et al. , NielsenSong and Elston  develope and the HWD trend test statistic as . Then the WA statistic is given byFor a case-control study with a diallelic marker locus, write the CA trend test statistic as where Y is well approximated by a Gamma distribution, F, with mean \u03bc = 1.78 and variance \u03c32 = 3.45, i.e., \u03b8 = \u03c32 /\u03bc = 1.94 and \u03ba = \u03bc2 /\u03c32 = 0.92. The best value to take for w was problematic because it depends on details of the alternate hypothesis, which are usually unknown. Thus, they chose the value of w indicated above after performing several exploratory simulation studies. They modeled the empirical \u03b1  that would predict the probability of type I error from the \u03b1 corresponding to 1 - F(y\u03b1) using the regression equationSong and Elston  used sim\u03b1i  was estimated from a series of n simulation experiments. Based on extensive simulation results for various sample sizes > 50, they estimated a to bewhere e R \u00d7 loge S] + 0.1208[loge (R/S)] - 1.6929[1/(loge R \u00d7 loge S \u00d7 (0.5 - |M - 0.5|))] + 0.9250(M - 0.5)2 1.4922[1/log can be adjusted using the genomic control method of Devlin and Roeder [when there are R cases and S controls and different values of the marker allele frequency M. To adjust the test statistic by a factor that measures the amount of variance inflation caused by population stratification and cryptic relatedness, the variance of d Roeder  when thep-values for each marker location were calculated and pooled over the populations.We analyzed the binary trait affected/unaffected in replicates from the three different populations in the dataset . For each of the three different populations, we first used the microsatellite markers that were on average 7.5 cM apart to perform a linkage scan. We used SIBPAL , a model-free linkage program, to perform a multipoint linkage analysis. Evidence for linkage was evaluated by Haseman-Elston regression ). The emp \u2265 0.8), spread throughout the whole genome, were selected for this purpose. However, for the 300 cases and 300 controls, the estimated variance inflation factor was close to 1.00 for each of the 5 replicate samples, suggesting no significant stratification exists in this admixed population. Therefore, no adjustment for variance inflation was made for these data.After reviewing the first-stage genome-scan linkage results, we selected for a case-control study 140 SNPs 0.3 cM apart that encompassed the significant linkage regions. From each of three replicates (one from each population), we randomly sampled 100 affected probands as our cases and 100 unaffected probands as our controls. In an attempt to induce population stratification, we also pooled the replicates from the three populations to obtain 300 cases and 300 controls. Then the WA test statistic was calculated for each of the four different samples using the selected SNPs. We repeated the sampling from each population 5 times and recorded the percentage of times that the hypothesis of no disequilibrium was rejected. The potential confounding effect of population stratification could be allowed for by the genomic control method. Nineteen SNPs that were independent (>12 cM apart if on the same chromosome) and unlinked to the disease locus (p = 0.00028) and D01S0024 (p = 0.0016)), chromosome 3 (12-cM region that included D03S0126 (p = 0.0012) and D03S127 (p = 0.00022)), chromosome 5 (4-cM region that included D05S0172 (p = 0.0025)), and chromosome 9 (18-cM region that included D09S0348 (p = 0.000041) and D09S0349 (p = 0.0010)).In our analysis, we observed 8 microsatellite markers with a significance level of p \u2264 0.005 Evidence of linkage was found on chromosome 1  with a susceptibility disease gene in the region between markers B03T3056 and B03T3058 on chromosome 3 and weaker association at marker B09T8334 on chromosome 9 (p < 0.05). Using the sample size of 300 cases and 300 controls, the association analysis confirmed the linkage signals in all regions on chromosomes 1, 3, 5, and 9. In this analysis we found association signals between markers B01T0553 and B01T0555 on chromosome 1 (p = 0.00034)     against the marker heterozygosity, 2q(1-q), where q is the marker allele frequency. Figure pP, between 4 and 8, when the heterozygosity is about 0.5, i.e., when the marker is most informative. It was verified that markers with high values of pP but smaller values of 2q(1-q)  with a susceptibility disease gene in the region between markers B03T3056 and B03T3058 on chromosome 3 and weaker association at marker B09T8334 on chromosome 9 (p < 0.05). For a larger sample size (300 cases and 300 controls), as expected much more significant pP values were observed.Using the WA statistic, a sample size as small as 100 cases and 100 controls showed on average strong association (CA: Cochran-ArmitageHWD: Hardy-Weinberg disequilibriumHWE: Hardy-Weinberg equilibriumWA: Weighted averageKS participated in the design of the study, performed the weighted average analysis and was involved in drafting the manuscript. MSO participated in the design of the study, performed the linkage analysis, selected the unlinked SNPs, and helped in drafting the manuscript. QL participated in the design of the study and helped in the weighted average analysis and data manipulation. RCE was involved in critical revision of the manuscript for intellectual content. All authors were involved in the interpretation of the results and approved the final manuscript."}
+{"text": "We compared the results of quantitative linkage analysis using single-nucleotide polymorphisms and microsatellite markers and introduced a new screening test for multivariate quantitative linkage analysis using the Collaborative Study on the Genetics of Alcoholism data. We analyzed 115 extended non-Hispanic White families and tested for linkage using two phenotypes: the maximum number of drinks in a 24-hour period and the number of packs smoked per day for one year. Our results showed that the linkage signal increased using single-nucleotide polymorphisms compared with microsatellite markers and that the screening test gave similar results to that of the bivariate analysis, suggesting its potential use in reducing overall analysis time. The Collaborative Study on the Genetics of Alcoholism (COGA) is a multicenter research program to detect and map susceptibility genes for alcohol dependence and related phenotypes. Numerous behavior measures were collected, two of which we considered for our study. The first is the maximum number of drinks in a 24 hour period (drink24), which can be considered a surrogate to alcoholism diagnosis and provides a quantitative measure to grade non-alcoholic individuals . The secThe COGA data consisted of 143 extended families, a mixture of large and small families, with 1,350 family members with clinical and demographic data. Because these families consisted of different ethnicities, we analyzed the families that were white, non-Hispanic (WNH). A family was considered WNH if 75% of the reported ethnicity in the family was WNH; thus, our analyses were performed on 115 extended families. The phenotypes selected for the analysis were drink24 and pakyrs. Because drink24 and pakyrs measures have skewed distributions, a square root transformation (sqrt) was applied in both measures to normalize the distribution.2, and the cut-off value of 0.4, which from our experience removed the effects of LD without a great loss of information. After dropping the SNPs in LD, a total of 350, 258, and 161 SNPs on chromosomes 1, 4, and 9, respectively, were used in our analyses. Multipoint identity-by-descent (MIBD) sharing among pairs of relatives was calculated for microsatellite and SNP markers using the SIMWALK2 software program [The microsatellite markers and the Illumina SNPs were each used for our analyses. For the Illumina SNPs, we removed SNPs that were in linkage disequilibrium (LD) with another SNP. We based our criteria for LD using r program multic library. This is a new library based on the C++ multic program from ACT [For the quantitative linkage analysis, we used the locally developed SPLUS from ACT . For thefrom ACT  and de Afrom ACT . Sqrt, \u2265 2.87 as suggestive evidence , and \u2265 2.06 as tentative evidence of linkage (p \u2264 0.007). We inferred evidence of chromosomal regions with pleiotropic effects when the bivariate MLS met the criteria for at least tentative evidence of linkage and its nominal p-value was less than the univariate maxima at the same location.To test for genetic linkage, a likelihood ratio test (LRT) was applied. Under the null hypothesis, the linked gene parameter(s) is (are) restricted to equal 0. The distributions of the univariate and bivariate linkage tests are a mixture of 1/2 \u03c7ectively . In the k quantitative traits are represented by Y1, Y2, ..., Yk. For each trait a genome-wide scanning linkage analysis is performed using the VC quantitative trait approach. For each trait i, and genomic position j, the quantitative trait locus (QTL) variance component estimate (\u03c32ij) is estimated with its standard error. Our hypothesis for the proposed screening test is: if there is a gene with pleiotropic effects, its QTL VC should be incremented in an additive manner using combinations of correlated traits by simply adding its respective univariate QTL VC. Let \u03c32ijk be the QTL VC for trait i, position j on chromosome k. The null hypothesis is that there is no pleiotropic effect at position j on chromosome k, i.e., H0:  = 0 \u2200 i, j, k, which is equivalent to H0: . The alternative hypothesis is H1: \u03c32ijk > 0 for any i, j, k. The test statistic will be , where  is the maximum likelihood estimator (MLE) of \u03c32ijk. Under H0, E(\u03c32ijk) = 0, \u2200 i, j, k, Sijk ~ 1/2 N . By assuming the Sijk values are independent, , where T is the number of traits. Consequently by squaring and standardizing Tjk, [Let us assume ing Tjk, .p = 0.004, 201 cM) and 8 , and suggestive evidence of linkage on chromosomes 2  and 14 . For sqrt(drink24), there was tentative evidence of linkage on chromosome 10  and suggestive evidence of linkage on chromosome 13 . For SNP markers, we observed an increase in the LOD scores compared to microsatellite markers on chromosome 1 for sqrt(pakyrs)  and for sqrt(drink24) , and on chromosome 4 for sqrt(drink24) . Figure Genome-wide linkage analyses were performed for all autosomal chromosomes using microsatellite markers, and only on three chromosomes  using Illumina SNPs. These three chromosomes were selected because they contain regions of interest based on previous studies [The phenotypic correlation between sqrt(pakyrs) and sqrtdrink24) was 0.38 and the genetic correlation was 0.70, indicating that these two measures shared common genes. For the bivariate analyses no significant evidence of genes with pleiotropic effects on sqrt(pakyrs) and sqrt(drink24) was observed using either microsatellite or SNP markers. Our proposed screening test detected some genomic regions of interest, although not at the bivariate level of significance. Figure 4 was 0.3In our analyses using microsatellite markers, tentative and suggestive evidence of linkage were found on chromosomes 1, 2, 8, and 14 for sqrt(pakyrs) and on chromosomes 10 and 13 for sqrt(drink24). Bergen et al. identified several regions for sqrt(pakyrs) in the COGA sample, among chromosomes 2 (~10 cM) and 14 (~68 cM) . In our During our analyses several difficulties arose when SNPs were used in quantitative trait linkage analysis. First, the only software that could specifically handle pedigrees of large size was SIMWALK2 ; howeverWe observed evidence of linkage on chromosome 4 for alcohol consumption using SNPs; this linked region was in the same region previously identified by Saccone et al. . FurtherCOGA: Collaborative Study on the Genetics of AlcoholismLD: Linkage disequilibriumLRT: Likelihood ratio testMIBD: Multipoint identity-by-descentMLE: Maximum likelihood estimatorMLS: Maximum LOD scoreQTL: Quantitative trait locusSNP: Single-nucleotide polymorphismVC: Variance componentsWNH: White, non-HispanicAll the authors contributed equally in the analysis and in the preparation of the manuscript. All authors read and approved the final manuscript."}
+{"text": "The simulated dataset of the Genetic Analysis Workshop 14 provided affection status and the presence or absence of 12 traits. It was determined that all affected individuals must have traits E, F and H (EFH phenotype) and they must also have either trait B (B subtype) or traits C, D, and G (CDG subtype). A genome screen was performed, and linkage peaks were identified on chromosomes 1, 3, 5, and 9 using microsatellite markers. Dense panels of single-nucleotide polymorphism (SNP) markers were ordered for each of the four linkage peaks. In each case, association analyses identified a single SNP that accounted for the linkage evidence. The SNP on chromosome 1 appeared to primarily influence the B subtype, while the SNPs on chromosomes 5 and 9 primarily influenced the CDG subtype. The chromosome 3 SNP had the strongest effect and influenced both subtypes, as well as the requisite EFH phenotype. Recognizing the two subtypes prior to linkage analysis was key to identifying these loci using only a single replicate. This highlights the need in real life situations for careful examination of the phenotypic data prior to genetic analysis. Studies designed to identify genes contributing to complex diseases have been ongoing for many years, utilizing different study designs and methods with varied success. The simulated dataset of the Genetic Analysis Workshop 14 (GAW14) provided an opportunity to evaluate the standard analysis techniques used to identify genes underlying complex phenotypes. We have also attempted to predict disease status given the presence of apparent \"high risk\" or deleterious alleles  identified in our analyses.All analyses were performed using the simulated dataset without knowledge of the underlying model, including number of genes or their location. Analyses were performed with complete genotypic and phenotypic data from replicate 1. The true model was obtained only at GAW14, and was used in this report to verify our findings. No additional results or figures were generated after learning the simulation model.As the effect of genes may differ across subpopulations, each was analyzed separately as well as together. The linkage methods employed did not allow for large pedigrees; therefore, only the isolated populations \u2013 Aipotu (AI), Karangar (KA), and Danacaa (DA) \u2013 were used for linkage analysis. The large families recruited from the heterogeneous urban setting (NYC) were only used in secondary association analyses. The simulated dataset included a number of discrete traits as well as the affection status for the simulated disease. Presence or absence of specific traits was tabulated for each population among affecteds and unaffecteds. Disease subtypes based on these traits were assigned to each individual for future analyses.A genome screen was performed for affection status, as well as for each of the disease subtypes, using the nonparametric linkage analysis methods implemented in ALLEGRO . To assiAssociation analyses were performed for each of the SNPs contained in these panels, using the transmission disequilibrium test and discordant sib-pair test implemented in the pedigree disequilibrium test (PDT) . Tests wA single individual was randomly chosen from each family, and for each SNP the percentage of individuals with the corresponding phenotype was tabulated for those carrying 0, 1, and 2 deleterious alleles. This process was repeated 10,000 times and the bootstrapped mean penetrance is reported.Logistic regression was used to quantify the effects of the apparent deleterious alleles identified in these genetic analyses. Affection status and the subtypes were each used in turn as the dependent variable. The number of copies for each of the deleterious alleles was used as an independent variable. Interactions between genes and with sex were also added. Those variables without a significant contribution were dropped from the final predictive model.Upon close inspection of affected individuals, it was discovered that all affected individuals had traits E, F, and H (the EFH phenotype). It was discovered that two subtypes existed. In addition to the EFH phenotype, affected individuals had to have either trait B (the B subtype) or they had to have traits C, D, and G (the CDG subtype). This rule held true for all populations; however, different populations had different ratios of the two subtypes. All affected individuals in the DA population had the B subtype, and all of the affected individuals in the KA population had the CDG subtype  (see Table 2 = 0.16).Data for additional markers in these regions were obtained for follow-up studies. Packets 27, 28, and 29 were ordered for chromosome 1, packet 153 for chromosome 3, packet 207 from chromosome 5, and packet 417 for chromosome 9. Association studies were performed on all additional markers using the program PDT. A SNP on chromosome 3 (B03T3056) yielded the most significant result . Similarly, the deleterious SNPs on chromosome 5  and chromosome 9  were identified using the CDG phenotype.The deleterious allele could not be identified in the 3 packets directly under the chromosome 1 linkage peak when only affection status was examined. However, when the B subtype was used , B01T0561 was identified (Chromosome 3 (B03T3056) had the strongest contribution to disease risk, and its effect appeared additive Figure . ChromosUsing these four identified SNPs, logistic regression was able to classify individuals as either affected or unaffected with 65.3% accuracy. When logistic regression was instead used to predict the subtypes and the predicted subtypes used to infer affection status, accuracy of disease classification remained the same. When the SNPs on chromosomes 1, 5, and 9 were modeled as additive  instead of dominant/recessive , several odds ratios decreased, but the predictive accuracy did not change. Interactions between genes or with sex were always non-significant. Similar levels of significance \u2013 63.4% and 63.5% \u2013 were noted for replicates 2 and 3, when using the same beta values from the model obtained with replicate 1.Analysis of the individual traits identified two subtypes of the simulated disease. All affected individuals had the EFH phenotype , but they could be classified into the B subtype (presence of trait B) or the CDG subtype . Three genome screens were performed, substituting the presence of these three phenotypes for affection status. Evidence of linkage differed depending on which subtypes were included as affected in the analysis. This observation matched closely to the true underlying disease model, unknown at the time, in which there were three ways an individual could become affected. The B subtype corresponds to the P1 phenotype. The CDG subtype corresponds to the P2 phenotype. And the third type detailed in the model (P3) required traits B, C, D, and G; however, since a subset of these traits were sufficient to cause disease, this was not recognized, and was not necessary to identify the underlying genes.We identified what we thought was the underlying disease polymorphism for each of the linkage regions on chromosomes 1, 3, 5, and 9. The SNP on chromosome 3 (B03T3056) had by far the strongest effect and exhibited an additive effect for all forms of the disease . The SNP on chromosome 1 (B01T0561) appeared to act in an autosomal dominant manner, because there was no additional increase in risk if an individual had two copies of the deleterious allele versus just one. This SNP also appeared to affect only the B subtype and did not influence the CDG subtype. The SNPs on chromosomes 5 (B05T4136) and 9 (B09T8333) both appeared to follow an autosomal recessive mode of inheritance and both affected only the CDG subtype and not the B subtype. The disease model is summarized in the schematic of Figure The accuracy of classifying individuals as affected or unaffected based on these genotypes did not exceed 65.3%. This was originally interpreted as consistent with the existence of additional genes, environment and/or phenotypic variation not accounted for in the model. In fact, the major reason why the accuracy of disease classification was not greater was because the causative SNPs used in the simulation parameters to determine affection status were not provided in the fine mapping dataset. However, the \"high-risk\" haplotypes were identified, as well as the SNP that tagged these haplotypes most efficiently. Two additional modifier genes on different chromosomes were not identified (D5 and D6) due to their weak effect on the diagnostic phenotype.Only five GAW14 studies correctly identified the major loci and haplotypes without having the \"answers\" or without grouping together all 100 replicates. Part of the reason for this is that a good understanding of the phenotype, namely identifying the two subtypes, was critical to reaching significance in any one replicate. Only two other groups correctly detailed these subtypes and all three discovered them in a different way. Our group tabulated the frequencies of the traits between affected and unaffected individuals, and performed genetic analyses after stratifying by subtype. Alternatively, MacGregor et al. incorporated qualitative covariates into their linkage analysis , while LThe ability to increase power to detect linkage based on minor phenotypic differences in a heterogeneous disease is a particularly important lesson to apply to real-world studies. A few groups were successful in identifying all four major disease loci because the ratio of subtypes was skewed in certain populations and because they analyzed these populations separately. However, this was only a proxy for the underlying subtypes and the same genes are not usually as influential among different isolated and admixed populations. The most important lesson from these results, obtained from simulated data, is that because phenotypic heterogeneity is often due to genotypic heterogeneity, as was the case here, it is vital that all available phenotypic information be analyzed thoroughly before genetic analyses are begun if the goal is to identify as many causal genes as possible.AI: AipotuDA: DanacaaGAW14: Genetic Analysis Workshop 14KA: KarangarLD: Linkage disequilibriumPDT: Pedigree disequilibrium testSNP: Single-nucleotide polymorphismNP designed the study, carried out the genetic analyses, and drafted the manuscript. EE helped with the genetic analyses. TF helped with the design of the study and helped to draft the manuscript. All authors read and approved the final manuscript."}
+{"text": "Although current methods in genetic epidemiology have been extremely successful in identifying genetic loci responsible for Mendelian traits, most common diseases do not follow simple Mendelian modes of inheritance. It is important to consider how our current methodologies function in the realm of complex diseases. The aim of this study was to determine the ability of conventional association methods to fine map a locus of interest. Six study populations were selected from 10 replicates (New York) from the Genetic Analysis Workshop 14 simulated dataset and analyzed for association between the disease trait and locus D2. Genotypes from 45 single-nucleotide polymorphisms in the telomeric region of chromosome 3 were analyzed by Pearson's chi-square tests for independence to test for association with the disease trait of interest. A significant association was detected within the region; however, it was found 3 cM from the documented location of the D2 disease locus. This result was most likely due to the method used for data simulation. In general, this study showed that conventional case-control association methods could detect disease loci responsible for the development of complex traits. Current linkage and association methods have proven to be extremely successful in identifying genetic loci responsible for the development of diseases shown to have simple Mendelian modes of inheritance. However, most inherited diseases do not follow simple Mendelian patterns. The etiologies of these diseases are considered to be complex in nature, consisting of the interaction of multiple genetic loci as well as possible environmental factors. Given the complex nature of these diseases, how do we adequately identify genetic loci responsible for their development? Are the current methods sufficient to tease out one of the possibly many loci responsible for the development of a complex trait?The phenotype modelled in the simulated data set is a wonderful example of a complex trait. The behavioral disorder, Kofendrerd Personality Disorder (KPD), is characterized by the presence of any one of 12 clinical characteristics, which may be divided into three diagnostic groups . Each of these phenotypic groups is the result of simultaneous mutations in at least 2 loci with varying modes of inheritance. For our analysis we have selected to isolate locus D2 by association methods.In a complimentary paper presented at the workshop, Yang et al. used linkage analysis to map the D2 locus to the telomeric region of chromosome 3, which is in keeping with the expected result . AssociaGiven the goals of our study, we needed to know the location of the susceptibility locus of interest, D2. We therefore looked at the answers before analysis. The selected locus D2 was described as dominant with a disease allele frequency of 0.15. This locus was implicated in the development of 4 of the 12 clinical characteristics traits e, f, h, and k. To increase genetic homogeneity, and thus power to detect an association, we redefined the affection status so that individuals were classified as affected only if they possessed these four clinical characteristics.Disease locus D2 is located at the telomere of chromosome 3. We thus purchased 3 SNP sets , which included the linkage peak observed froma 7-cM map of chromosome 3[surveyselect function, taking care that only one affected individual from a family was selected as a case. For each case population a control population consisting of twice the number of cases was randomly selected, also with the SAS surveyselect function. The details of each case population are as follows: populations 1\u20133 contained 50 cases each. These three replicates provided at least 80% power  to detesion 0.5 .p-values were compared between study populations. This analysis was considered to be the most general analysis for a case-control association study. Given that the description of the simulation model indicated the presence strong linkage disequilibrium (LD) in the region of interest, we assayed LD using the program HAPLOVIEW (Haploview v2.05). We calculated D'  for all pair-wise marker comparisons. We also used HAPLOVIEW to compare haplotype frequencies between the case and control populations.For all study populations 2 \u00d7 3 contingency tables were generated based on genotype counts in the case and control populations. Pearson's chi-square tests for independence were performed on the tables and 10. As expected from the number of tests performed, a p-value of 0.05 or even 0.01 was not stringent enough to identify a true significant association, as many false positives were observed. Therefore, results with a p-value of 0.001 or less were considered to be significant as determined by Bonferroni correction. A significant association was detected between markers B03T3056, B03T3057, B03T3058, and C03R0281 and the disease trait. This result was confirmed in population 5; the replicate selected using the original disease definition. However, tests with this case population produced lower significance levels. No association was detected for SNPs closer to the disease locus, which was reported to be linked to SNP B03T3067.Genotypes from all SNPs (B03T3021 to B03T3067) in a 12-cM region at the telomere of chromosome 3 were tested for association with the disease trait by chi-square test for independence. The results obtained from all populations are shown in Figure The control population was used to assess the degree of LD present within a narrowed region of chromosome 3, from marker B03T3053 to B03T3067. This region was reported in the simulated dataset to contain significant LD. Figure n = 50) were slightly underpowered given the large number of loci tested. However, we were most interested in determining whether a linkage peak could be narrowed down using the association tests so we focused on the pattern of results across the region rather than the actual significance level.With the knowledge of the answers for the simulated dataset, we decided to isolate the disease locus D2 with standard methods, i.e., linkage analysis followed by association analysis. A complementary analysis presented from our group showed a significant linkage peak in the telomeric region of chromosome 3 . This peWithout prior knowledge of the disease locus one would feel that this study was highly successful. Linkage analysis identified a minimal disease region of approximately 3 cM linked to the disease trait . We showSeveral groups using various analytical methods at Genetic Analysis Workshop 14 observed the same association peak. The association peak 3 cM from the actual disease locus most likely resulted from the methods used for the data simulation. For this reason our results and conclusions may not be directly applicable to real datasets. With this in mind we are able to make some general conclusions. Case selection can greatly influence both power and locus detection. As shown in the analysis in which MERLIN was used to select the 250 cases from linked families, power increased dramatically over that observed from 250 randomly selected cases. Power may be increased by refining the phenotype definition, although this was not necessary for locus detection in this particular example. As expected, an increase in the number of selected cases not only increased the significance of association, but also decreased the variability in observed significance. However, even the slightly under-powered replicates were able to detect the correct location of association. We were able to show that a case-control approach could detect an association in a linked region but because there was very little LD present in the simulated dataset, we could not determine how well this approach could fine map an actual disease locus.LD: Linkage disequilibriumKPD: Kofendrerd Personality DisorderKFK participated in study design, performed the statistical analyses, and drafted the manuscript. KJ performed data management and provided technical consulting. XY and AWB participated in study design, results interpretation, and aided in drafting the manuscript. LRG and AMG aided in the establishment of study goals, analysis direction, and preparation of manuscript. All authors have read and approved the final manuscript."}
+{"text": "We performed quantitative trait locus (QTL) analysis of one of the most heritable human complex traits, adult stature (body height) using genome-wide scans performed for 3,817 families  derived from twin cohorts from Australia, Denmark, Finland, Netherlands, Sweden, and United Kingdom with an approximate ten-centimorgan microsatellite marker map. The marker maps for different studies differed and they were combined and related to the sequence positions using software developed by us, which is publicly available (https://apps.bioinfo.helsinki.fi/software/cartographer.aspx). Variance component linkage analysis was performed with age, sex, and country of origin as covariates. The covariate adjusted heritability was 81% for stature in the pooled dataset. We found evidence for a major QTL for human stature on 8q21.3 (multipoint logarithm of the odds 3.28), and suggestive evidence for loci on Chromosomes X, 7, and 20. Some evidence of sex heterogeneity was found, however, no obvious female-specific QTLs emerged. Several cohorts contributed to the identified loci, suggesting an evolutionarily old genetic variant having effects on stature in European-based populations. To facilitate the genetic studies of stature we have also set up a website that lists all stature genome scans published and their most significant loci (http://www.genomeutwin.org/stature_gene_map.htm).Twin cohorts provide a unique advantage for investigations of the role of genetics and environment in the etiology of variation in common complex traits by reducing the variance due to environment, age, and cohort differences. The GenomEUtwin ( http://www.genomeutwin.org). We performed quantitative family-based genetic linkage analysis for one of the most heritable human complex traits, adult stature (body height), using genome-wide scans derived from twin cohorts from Australia, Denmark, Finland, Netherlands, Sweden, and United Kingdom. Age, sex, and country were adjusted for in the data analyses. Human stature was found to be very heritable across all the cohorts and in the combined dataset. We found evidence for a shared genetic locus accounting for human stature on Chromosome 8, and suggestive evidence for loci on Chromosomes X, 7, and 20. Since twins from several countries contributed to the identified loci, an evolutionarily old genetic variant must influence stature in European-based populations. To facilitate the research in the field we have also set up a website that lists all stature genome scans published and their most significant loci (http://www.genomeutwin.org/stature_gene_map.htm).Twin cohorts provide a unique advantage for research of the role of genetics and environment behind common complex traits by reducing the variance due to environment, age, and cohort differences. The GenomEUtwin consortium consists of eight twin cohorts with the total resource of hundreds of thousands of twin pairs ( Human adult stature (body height) has been the target of numerous genetic quantitative trait linkage studies in the past few years \u201317. DespOne obvious strategic decision in addressing a quantitative trait regulated by multiple QTLs, each potentially with a minor effect, is to maximize the sample size. Although this could introduce multiple challenges, including an increase in genetic and phenotypic heterogeneity, robust QTL analysis of large cohorts of families is the natural first choice. Based on a meta-analysis of linkage studies, the only factors independently associated with successful locus identification are an increase in the number of individuals studied and a study sample drawn from one ethnic group . Our appWe performed QTL analysis of stature using data from genome-wide scans performed for 3,817 families  collected within GenomEUtwin from Caucasian populations in six different countries: Australia, Denmark, Finland, Netherlands, Sweden, and United Kingdom. This study sample extracts its information from twin pairs sharing early environment throughout the critical period of human growth and although we are pooling sibpairs from various populations, they are all of European origin.n = 2,609), Denmark (n = 628), Finland (n = 851), Netherlands , Sweden , and United Kingdom . The United Kingdom, Danish, and Swedish cohorts consisted of only DZ twin pairs with no additional family members. The covariate-adjusted heritability of adult height was 82% in the pooled sample, being 93% for the males and 98% for the females when analyzed separately, while it was considerable lower, 80%, for the opposite-sex pairs. The average map density was <10 cM.There were a total of 1,945 female\u2013female dizygotic (DZ) twin pairs and 661 male\u2013male DZ twin pairs and 695 opposite-sex twin pairs in the pooled material. The overview of the primary results from the pooled genome-wide linkage scan for stature can be seen in p-value from 100 replications = 0.08, Wilson's 95% confidence interval 0.04\u20130.15) was observed at 97 cM on 8q21.3. This result was mainly contributed by three cohorts, Australia, Finland, and Sweden  consisted of females, the females-only analyses revealed no LOD scores >2 in the pooled sample. The highest female-only signal was observed on the 8q21.3 region at 98 cM, with an MLOD (multipoint logarithm of the odds) value of 1.50. The highest male-specific signal was also observed at 8q21 with an MLOD 3.33 at 79 cM; these two sex-specific peaks on Chromosome 8 nicely overlapped each other, suggesting that this locus is not sex-specific. The second-highest male signal was observed at 20q21.2 at 21 cM with a suggestive MLOD of 2.70. The same region was previously linked with stature in a study sample of both Finnish and Australian families ,19.To minimize environmental heterogeneity, we stratified the sample by excluding all other family members except twins from the variance components analyses; however, all available genotypes were retained for phase information. This stratified sample consisted of 3,301 pairs with pheno- and genotype data. No genome region provided a signal of genome-wide significance in the DZ twin-only sample, probably because of decreased power due to smaller sample of individuals with phenotypes, in statistical analyses. However, in the DZ twins\u2013only sample, a LOD score of 2.54 was identified on Xq25 at 127 cM, slightly distal to a previously linked region in a United States sample . Both thA proper cohort-specific analysis is hampered by the different sample sizes and was thus not performed. However, two relatively significant linkage signals were observed that were mostly contributed by a single cohort; one with the Dutch sample of 366 families  on 10q (MLOD 3.04) and one on 15q24.1 with the Australian cohort of 1,091 families . Interestingly, these loci have not previously been reported to be linked to adult stature and did not provide significant evidence in the pooled sample.http://www.ncbi.nih.gov) and bibliography reviews (as of December 2006) to identify all eligible studies and have provided the essential data for them as well as the references on the website. The criteria for the eligibility for inclusion on this web site are: (1) the study is a family-based study utilizing genome-wide markers and quantitative linkage analysis on the adult human height and (2) the families are unselected for adult height. To date, about 20 genome scans have been published that investigate human adult body height \u201319. SincTo our knowledge, this is by far the largest genetic linkage study performed regarding human height and on any trait in DZ twins. The nuclear families originated from six different countries of European origin. The method of data pooling used here includes the assumption of some locus homogeneity across the study samples. Silventoinen et al.  have shoGrowth in twins differs to some degree from that in singletons, especially during fetal life. Dizygotic twins share the same intrauterine conditions, albeit with separate placenta and fetal membranes. Twins grow at the same rate as singletons during the first half of pregnancy, but exhibit slowing growth rates in the third trimester of pregnancy, primarily due to space restriction in utero. This results in lower birth weights than in singletons on average, mostly due to shorter gestation time . Twins sAlthough the most evident height differences among humans appear between the sexes, and nutrition and infections are critical factors for final adult height across the global populations , there iPHF6, and fibroblast growth factor 13, FGF13, [SLIM 1, which shows elevated expression levels in skeletal muscle during postnatal growth, and glypican 3, GPC3. Variants in GPC3 cause Simpson-Golabi-Behmel syndrome, a condition characterized by pre- and postnatal overgrowth (gigantism) with visceral and skeletal anomalies [GPC3 seems to be involved in the suppression/modulation of growth in the mainly mesodermal tissues and organs and also may interact with the insulin-like growth factor II (IGF2), thus regulating growth [However, in light of the large difference in stature between males and females, it would be surprising to find no evidence at all for QTL on a sex chromosome in such a large dataset. The potential role of the X-chromosomal locus was identified through analyses based on data from several cohorts, and this locus would not have been apparent in any stand-alone cohort analysis. This lends support to the pooling strategy used here to identify such minor-to-moderate QTLs. This X-chromosomal locus, Xq25, has previously shown suggestive linkage to stature in pedigrees of European origin  and it h, FGF13,  lie withnomalies . GPC3 seg growth .There are no previous QTLs linked to stature on Chromosome 15 . Here, tWe identified several other potential QTLs, which were not statistically significant, but nevertheless triggered some interest. For example, the loci for which data from many cohorts seem to contribute to and increase the overall LOD score (20p and Xq) might mirror minor loci either needing larger sampling or some kind of dissection of the sample to produce a more homogeneous sample in relation to the locus in question. Loci that have previously been reported to be linked to stature, such as on Chromosomes 8q, 20q, 21p, and Xq25, might well present a real QTL, but due to multiple testing of genome-wide scans here we cannot apply the more relaxed linkage thresholds that are often used in replication studies, and, thus, they remain suggestive. Several previous loci were not replicated in this study, maybe because stature is a very heterogeneous phenotype, or possibly due to type I or II errors in this or earlier studies. We think that our findings support the essential argument of quantitative genetics, that of the infinitesimal model. From the results we can deduce that, most probably for stature, the ultimate causation of variance components is segregating alleles at many underlying loci, each of which has a very small individual impact on the character in question. One of Fisher's several important contributions to evolutionary theory was to provide a statistical outline for such a complex inheritance model in a Mendelian framework that could account for continuous variation , giving http://www.genomeutwin.org), formed to explore genetic influences on common traits using large population and twin cohorts. For these analyses, genome-wide microsatellite scan data were available from six twin cohorts: The Australian Twin Registry [GenomEUtwin is a research consortium of 12 partners, including eight twin cohorts from Europe and Australia  and partly at Uppsala Rudbeck laboratory . A total of 487 Swedish twin pairs were genotyped by deCODE Genetics (http://www.decode.com), using the Applied Biosystems automated DNA sequencing system .While the twin studies served as the recruitment basis for the participants, additional siblings and/or parents/descendants of the twins were also recruited in some cohorts and included in the analysis set here where available. The total number of families included in the analysis was 3,817, and the number of full DZ sib pairs with genome-wide marker and phenotype data was 3,301. A federated database with open-source code has been created to share the data across the study partners ,45. Genohttp://www.sph.umich.edu/csg/abecasis/GRR/index.html) [http://www.sas.com). Measured height was available from the Dutch and United Kingdom cohorts as well as from large parts of the Australian and Danish cohort, while the Swedish and Finnish height data was acquired from questionnaires. Genotypes were checked for Mendelian inconsistencies using the PedCheck program [The program GRR,   was used program  and the  program  genotypiHaving access to raw data of all the genome-wide scans performed, we decided to pool all raw data (genotypes and phenotypes) instead of applying meta-analytic strategy. Since the original genome scans differed in their selection of genetic markers, we first harmonized the genetic marker maps using the in-house-developed Cartographer program . CartogrAfter harmonization of the maps across the cohorts studied, the cohorts were pooled together such that all identical markers that were genotyped in several populations were renamed for all of them and these renamed markers were located at 0.001 recombination fractions from each other. A total of 253 markers were genotyped in all six populations . To circumvent the problems produced by this partial overlap of markers among the cohorts when interpreting two-point LOD scores, only multipoint linkage results are shown here. All data were then analyzed together using the variance component method in the program Merlin . People Age, sex, and country of origin were used as covariates in the variance component analysis. The distribution of height was not notably skewed or kurtotic, so no transformations of any of the variables were necessary. Sexes were also analyzed separately with age and country of origin as covariates. The LOD scores are presented unadjusted for the analyses on these six subcohorts . No monozygotic twin pairs were identified in the analyses.p-values for the obtained results, a total of 100 replicates was created and analyzed using Merlin's simulate option. The same analyses that were conducted with the actual data were repeated for each of the simulated genomes. Since any evidence of linkage found in the simulated genomes is due to chance, these simulations allowed us to evaluate the false-positive rate. We determined the empirical genome-wide significance of a given LOD score as the fraction of simulated genome scans in which this LOD score was reached or exceeded. The Wilson confidence intervals were estimated as described in [To estimate empirical ribed in .Table S1Click here for additional data file.Marker Location of Multipoint LOD Scores(2.4 MB PDF)The Online Mendelian Inheritance in Man (OMIM) database accession numbers for the syndromes discussed in this paper are Borjeson-Forssman-Lehmann syndrome, 301900; Nijmegen breakage syndrome, 251260; and Simpson-Golabi-Behmel syndrome, 312870."}
+{"text": "The r2 estimates were then used to correct the multipoint identity by descent matrix (MIBD) calculation to account for LD and LOD scores on chromosomes 3 and 18 were calculated for COGA's ttdt3 electrophysiological trait using those MIBDs. Extensive LD was observed throughout both marker sets, and it was higher in Affymetrix's more dense SNP map. However, SNP density did not solely account for Affymetrix's higher LD. MIBD estimation procedures assume linkage equilibrium to construct genotypes of non-genotyped pedigree founder individuals, and dense SNP genotyping maps are likely to contain moderate to high LD between markers. LOD score plots calculated after correction for LD followed the same general pattern as uncorrected ones. Since in our study almost half of the pedigree founders were genotyped, it is possible that LD had a minor impact on the LOD scores. Caution should probably be taken when using high density SNP maps when many non-genotyped founders are present in the study pedigrees.Linkage disequilibrium (LD) content was calculated for the Genetic Analysis Workshop 14 Affymetrix and Illumina single-nucleotide polymorphism (SNP) genome scans of the Collaborative Study on the Genetics of Alcoholism samples. Pair-wise LD was measured as both D' and  Single nucleotide polymorphisms (SNPs) are ubiquitous throughout the human genome. SNPs are more closely spaced than microsatellites and they permit the construction of very high-density genome screening maps. Methods that rely on SNPs are very easy to automate , and will probably be more cost effective than microsatellites for performance high-throughput genotyping.Yet, several properties of SNP maps remain unclear. Kruglyak  evaluateThe impact of linkage disequilibrium (LD) on linkage studies has also been the subject of some discussion  and analAffymetrix and Illumina SNP genotyping of samples from the Collaborative Study on the Genetics of Alcoholism (COGA) for the Genetic Analysis Workshop 14 (GAW14) provide an excellent opportunity to investigate certain properties of SNP genome screening maps. It also leads to the comparison of several aspects of both company products, one of the most obvious being the performance of the highly dense Affymetrix in comparison with the more sparse Illumina array.Here, our main objective is to explore empirically the extent of LD that is present in the two complete SNP genome scans of real DNA samples and its effect on the LOD score.n = 505; 240 genotyped, 265 not genotyped) were chosen as a representative sample of unrelated individuals, and only their genotypes were included in the LD analysis.SNP genotypes from the clean GAW14 COGA datasets were selected from Affymetrix's GeneChip Mapping 10 K Array (AMA) and Illumina's Linkage Panel III (ILP). A total of 11,120 Affymetrix and 4,720 Illumina SNP markers (SNPs) were present in the final clean datasets, but only 10,084 and 4,599 of those SNPs, respectively, were used for this analysis. One thousand and thirty-six Affymetrix and 121 Illumina markers were not included in the first dataset that was distributed for GAW 14. Those markers became available after a substantial part of the work presented here was done and therefore were not included in the analysis. COGA pedigree founders  -9 was alr2 as described previously) above three arbitrary thresholds: 0.2, 0.4, and 0.6. A total of 12 (2 SNP sets*2 chromosomes*3 thresholds) \"corrected\" MIBDs were constructed as described, using all genotyped individuals (not only founders). Four (2 SNP sets*2 chromosomes) additional \"uncorrected\" MIBDs were used as the comparison standard for the LOD scores. Multipoint LOD scores were then calculated for the ttdt3 trait with SOLAR [For AMA and ILP SNP markers on chromosomes 3 and 18, MIBD matrices were calculated using LOKI . Chromosth SOLAR , using tp-values are uncorrected for multiple testing.Reported Overall, pair-wise haplotype frequencies were estimated for a median of 488 Illumina (range = 486\u2013520) and 490 Affymetrix (range = 276\u2013520) SNP haplotypes per chromosome. Nineteen AMA SNPs had to be discarded from the analysis because they were not polymorphic in the sample. Except for chromosomes 19 and 22 AMA had a more dense marker coverage of each chromosome.r2 and D', in ILP  as well as in AMA . But variance was greater in AMA's r2 and D' . Only 0.26% (N = 380) ILPSnP showed |D'| \u2265 0.5, while 6.85% (n = 759) AMASnP did. All ILPSnP and AMASnP r2 values for unlinked markers were below 0.06.Random LD between unlinked markers was found to be low, for both 10064,4597 = 24.56, p < 0.0001) between AMA (3.08 \u00b1 0.07 \u00d7 10-1 cM), and ILP (7.48 \u00b1 0.16 \u00d7 10-1 cM) pairs. Variance in distance between AMASnP SNPs was significantly lower  than between ILPSnP SNPs.We analyzed 10,065 AMA and 4,598 ILP linked SNP pairs. Of all AMASnP, 8,096 (80.44%) were pairs in which the distance between the SNPs was \u2264 1 cM, while only 1,242 (27.01%) ILPSnP were formed by markers at that spacing. The mean distance between the two SNPs in a pair was significantly different  of all AMASnP had measures of |D'| \u2265 0.5, while only 37 (2.98%) of all ILPSnP did. At the same \u2264 1 cM spacing, 960 (11.87%) AMASnP and 8 (0.64%) ILPSnP pairs had an r2 \u2265 0.5 measure.Figure 10064,4597 = 90.18, p << 0.0001) for AMA (0.2855 \u00b1 0.0006) than for ILP (0.3536 \u00b1 0.0003) SNPs. PIC was also significantly lower  for AMA (4.92 \u00b1 0.01 \u00d7 10-1) than for ILP (6.41 \u00b1 0.01 \u00d7 10-1) haplotypes. Total PIC variance was significantly higher in AMA than in ILP for both SNPs  and haplotypes .Mean PIC values were found to be significantly lower  in the location of the maximum LOD when AMA's 0.2 MIBD was used; and both, a 8-cM shift from 58 cM to 66 cM (chromosome 18) and a shift from 213 cM to 215 cM (chromosome 3) when ILP's 0.2, 0.4, and 0.6 MIBDs were used.Using AMA uncorrected MIBDs, the ttdt3 trait phenotype maximum LOD peaks were 2.94 at 58 cM from 18per se is not surprising, given that AMA's higher marker density causes SNPs to be more closely spaced. What's striking is that for markers under the same < 1-cM marker spacing, AMA shows a much higher proportion of LD than ILP . This suggests that there might be reasons, other than marker spacing, that affect LD content. For instance, Affymetrix and Illumina certainly have different protocols to select the SNPs that are in their AMA and ILP products. Differences in the SNP selection procedure might explain why we observed significantly more LD in the Affymetrix product.As both the AMA and ILP genome scans show, extensive LD is present throughout the genome. Yet it is more extensive within AMA haplotypes, something which The AMA and ILP genome scans also differ in the quantity of SNP and haplotype information. PIC was much more variable in the AMA dataset, and it was consistently lower for both SNPs and haplotypes. One possible explanation for this is that closely spaced markers became redundant because of the high LD present between them.What seems to be clear is that LD is a common feature of the genome. Tsunoda et al.  focused r2 > 0.4. John et al. [John et al.  also recr2 thresholds), when creating the MIBD matrices. We found \"modest\" LOD score changes in magnitude, as John et al. did. But those modest changes in magnitudes were able to shift the location of the maximum LOD score even by a great genetic distance  whole-genome scans. But as marker map density increases, so does LD content. We showed that considerable LD exists between markers in both the Affymetrix and Ilumina SNP genotyping sets, and it is more pronounced in Affymetrix's denser map. Since all methods used to calculate MIBDs assume LE when estimating haplotypes of non-typed founder individuals, the effect of violating this assumption using highly dense SNP maps in which LD is more the rule than the exception needs to be considered. We observed modest changes in LOD score magnitude and shifts in the position of the maximum LOD after correcting for LD in the MIBDs. But the effect of LD on LOD scores might not always be this subtle and it may be adverse in studies where a large number of founder individuals are not genotyped.AMA: Affymetrix GeneChip Mapping 10 K ArrayCOGA: Collaborative Study of the Genetics of AlcoholismEM: Expectation maximizationGAW14: Genetic Analysis Workshop 14ILP: Illumina Linkage Panel IIILD: Linkage disequilibriumLE: Linkage equilibriumMIBD: Multipoint identity by descentQTL: Quantitative trait locusPIC: Polymorphism information contentSNP: Single-nucleotide polymorphismJMP performed the statistical analysis and drafted the manuscript. TD did the data cleanup and helped with the preparation of the MIBD matrices. LA designed the study, participated in its coordination, and helped to draft the manuscript. DMW selected the chromosomes, the trait, and the model for the linkage analysis. JB participated in the design of the study."}
+{"text": "The basic idea of affected-sib-pair (ASP) linkage analysis is to test whether the inheritance pattern of a marker deviates from Mendelian expectation in a sample of ASPs. The test depends on an assumed Mendelian control distribution of the number of marker alleles shared identical by descent (IBD), i.e., 1/4, 1/2, and 1/4 for 2, 1, and 0 allele(s) IBD, respectively. However, Mendelian transmission may not always hold, for example because of inbreeding or meiotic drive at the marker or a nearby locus. A more robust and valid approach is to incorporate discordant-sib-pairs (DSPs) as controls to avoid possible false-positive results. To be robust to deviation from Mendelian transmission, here we analyzed Collaborative Study on the Genetics of Alcoholism data by modifying the ASP LOD score method to contrast the estimated distribution of the number of allele(s) shared IBD by ASPs with that by DSPs, instead of with the expected distribution under the Mendelian assumption. This strategy assesses the difference in IBD sharing between ASPs and the IBD sharing between DSPs. Further, it works better than the conventional LOD score ASP linkage method in these data in the sense of avoiding false-positive linkage evidence. Alcohol dependence  is a highly familial disorder that is a leading cause of morbidity and premature death. Several lines of evidence suggest a substantial genetic component to the risk for alcoholism . The ColIn this analysis, 315 microsatellite markers located on the autosomal chromosomes were used in the genome scan. Affected sibs were defined to be sibs who met both DSM-III-R Alcohol Dependence and Feighner definite alcoholism criteria , and unaffected sibs were defined to be \"pure\" unaffected. Sibs who reported some symptoms but did not meet the diagnostic criteria, or who had never consumed alcohol, were assumed to have unknown phenotypes.First we conducted a 2-cM genome scan using the ASP method with and without constraints  as implei is the relative risk to an individual who shares i allele(s) IBD with an affected sib; and  and  are the IBD distributions estimated at a given location from the observed marker data for ASPs and DSPs, respectively. With the DSPs' IBD sharing distribution serving as a control based on the above rationale, the modified LR statistic will be close to one under the null hypothesis of no linkage whether or not there is overall deviation from Mendelian inheritance, so that the false-positive linkage signals would be reduced. Finally, we compared the results of this new ASP/DSP method with those from the original Haseman-Elston (HE) regression analysis [10, with the p-value corresponding to the LOD score, i.e., computed by assuming the asymptotic chi-squared distribution with 1 d.f. for the 1-parameter model.where \u03bbanalysis , giving analysis , as implanalysis  program analysis . BecauseWe first conducted a genome-wide linkage scan by the ASP method (results not shown here) and any linkage signal close to 0 cM or the q end of the chromosome was ignored, because in multipoint linkage analysis IBD estimation around these 2 points is often unstable, with the result that the corresponding linkage information would not be reliable. This indicated 3 peaks on chromosome 7 (see dotted line in Figure Previous study  of the CASP: Affected sib pairCOGA: Collaborative Study on the Genetics of AlcoholismDSP: Discordant sib pairHE: Haseman-ElstonIBD: Identity by descentLR: Likelihood ratioP-YS carried out the linkage analysis, participated in the genome scan using the LODPAL program and the identification of the linkage regions on chromosome 7 using the SIBPAL program, and drafted the manuscript. TW contributed in modification of the test statistic as implemented in LODPAL to the new test statistic as implemented in nLODPAL and drafted the initial manuscript presented at the GAW meeting. CX carried out the linkage analysis on chromosome 7 using nLODPAL. MS helped to obtain the estimated IBD distributions of sib pairs using the S.A.G.E. program GENEIBD. YS developed the program nLODPAL. RCE conceived the study, participated in its design and coordination, and helped to draft the manuscript."}
+{"text": "Elevated resting heart rate has been shown in multiple studies to be a strong predictor of cardiovascular disease. Previous family studies have shown a significant heritable component to heart rate with several groups conducting genomic linkage scans to identify quantitative trait loci.We performed a genome-wide linkage scan to identify quantitative trait loci influencing resting heart rate among 3,282 Caucasians and 3,989 African-Americans in three independent networks comprising the Family Blood Pressure Program (FBPP) using 368 microsatellite markers. Mean heart rate measurements were used in a regression model including covariates for age, body mass index, pack-years, currently drinking alcohol (yes/no), hypertension status and medication usage to create a standardized residual for each gender/ethnic group within each study network. This residual was used in a nonparametric variance component model to generate a LOD score and a corresponding P value for each ethnic group within each study network. P values from each ethnic group and study network were merged using an adjusted Fisher's combining P values method and the resulting P values were converted to LOD scores. The entire analysis was redone after individuals currently taking beta-blocker medication were removed.P = 0.0013) on chromosome 5p13-14. We assessed heterogeneity for this locus between networks and ethnic groups and found significant evidence for low heterogeneity (P \u2264 0.05).We identified significant evidence of linkage (LOD = 4.62) to chromosome 10 near 142.78 cM in the Caucasian group of HyperGEN. Between race and network groups we identified a LOD score of 1.86 on chromosome 5 (between 39.99 and 45.34 cM) in African-Americans in the GENOA network and the same region produced a LOD score of 1.12 among Caucasians within a different network (HyperGEN). Combining all network and race groups we identified a LOD score of 1.92 (We found replication (LOD > 1) between ethnic groups and between study networks with low heterogeneity on chromosome 5p13-14 suggesting that a gene in this region influences resting heart rate. Heart rate has been implicated as a risk factor in cardiovascular disease (CVD) -9, canceSingh et al.  and moreDue to its importance in predicting cardiovascular and all-cause mortality, we have used data from Caucasians and African-Americans enrolled in the NHLBI Family Blood Pressure Program to evaluate genetic factors associated with resting heart rate. We report a meta-analysis of genome scans within the GenNet, GENOA, and HyperGEN Networks, using genotyping carried out by the Mammalian Genotyping Service.Subjects were recruited by four multicenter networks investigating the genetics of hypertension/blood pressure (BP) known collectively as the Family Blood Pressure Program (FBPP). Data frThe present analyses were based on a total of 7,271 subjects. There were 542 Caucasians (from 203 families) and 776 African-Americans (from 281 families) in GenNet; 1,400 Caucasians (from 481 families) and 1,695 African Americans (from 547 families) in GENOA; and 1,340 Caucasians (from 583 families) and 1,518 African Americans (from 684 families) in HyperGEN who had both genotyping and data on resting heart rate and other covariates for adjustment of the phenotype. Each network has been approved by the appropriate local Institutional Review Board and all participants provided informed written consent.Prior to examination, participants were told to avoid caffeinated products such as tea, coffee, and chocolate as well as any other food and heavy physical activity for twelve hours. Clinical examinations all took place in the morning. After being seated for five minutes, heart rate and blood pressure were measured by arterial pulsations measured from the upper arm using either a Dinamap or Omron automatic blood pressure monitor. A measure of heart rate derived from 24 hour monitoring would be ideal; however, the feasibility of such a measure on this number of participants is limited. GENOA and HyperGEN networks exclusively used the Dinamap monitor, whereas the GenNet network used both blood pressure monitors, with 71.3% of patients measured with a Dinamap monitor and 28.7% of patients measured with an Omron monitor. Each device was turned off and allowed to recalibrate between participants. Mean heart rate was calculated either from three (for the Dinamap monitor) or two (for the Omron monitor) measurements taken in a six minute interval to control for intra-individual variability.Blood was drawn into three 10 ml vacutainer tubes containing EDTA. Genomic DNA was isolated using standard protocols. The Mammalian Genotyping Service (MGS) in Marshfield, WI completed each genome screening using the same set of 368 highly polymorphic microsatellite markers for each network. These markers have an average heterozygosity of ~80%, an average intermarker distance of 10 cM, and cover ~95% of the human genome. Specific details on gel preparation, PCR conditions, and the genetic map were reported by Weber and Broman  and are Questionnaires were used to collect information on potentially confounding factors for heart rate including age, sex, smoking, alcohol use, and medication usage. Medication use was also assessed by inventory of the participant's current prescriptions. Height and weight for the calculation of body mass index (BMI) were ascertained during the participant's clinical examination. Smoking habits were defined by three categories: non-smoker, current smoker and past smoker and, if the participant answered yes to smoking currently or in the past, total pack years were calculated. Alcohol use was assessed by inquiring whether the person \"presently drinks alcoholic beverages\". Hypertension was designated for participants if they met any of three criteria: (1) currently taking anti-hypertension medication (2) average diastolic blood pressure \u2265 90 mmHg or (3) average systolic blood pressure \u2265 140 mmHg. In addition, normotensive individuals were defined as not being hypertensive or hypotensive.Previous reports of the effect of different hypertension medication use on heart rate within the networks of the FBPP have shown that \u03b2-blocker medication users had, on average, a significantly lower heart rate than non-users. Therefo2, BMI, pack-years, currently drinking alcohol (yes/no), hypertension status and \u03b2-blocker use. We then calculated the standardized residuals of heart rate for each gender and racial group within each network . A second standardized residual was generated after excluding \u03b2-blocker medication users from the sample and removing the \u03b2-blocker medication use covariate from the regression model. Residuals from the models described above were used in subsequent linkage analysis.Mean values of resting heart rate were calculated from all available measurements; for the Dinamap monitor three readings were used and for the Omron monitor two measurements were used. We used a regression model to evaluate the relationship of covariates to heart rate within each gender and racial group. Variables that were significant predictors of heart rate were included in the race and gender-specific regression model for adjustment. They included age, ageLinkage analyses were performed using variance component (VC) models as implemented in MERLIN version 0.10.2 . Multipon independent tests are made about the same hypothesis, resulting in the P values P1, P2... Pn then 2 with 2n degrees of freedom. Combined P Values where converted to LOD scores using the equation P = 1 - \u03a6 [sign (LOD) P values obtained from MERLIN's VC linkage were combined using Fisher's method of combining P values . Brieflyfunction . In addiP \u2264 0.05) was estimated from a null distribution of the Q statistic calculated from 10,000 rank permutations within each study network and ethnic group.A test for heterogeneity was performed using the program Heterogeneity and Genome Search Meta-analysis (HEGESMA),36. The Table Heritability estimates for adjusted heart rate within each network and ethnic group are shown in Table In Caucasians, the maximal LOD score of 2.06 was seen on chromosome 10 at 142.78 cM within the HyperGEN Network. In African-Americans, the maximal LOD score was seen in the GENOA Network, where chromosome 13 produced a LOD of 3.07 across a region from 55.31 cM to 63.9 cM. Excluding participants taking \u03b2-blocker medication had a minimal impact in LOD scores (average increase of 0.002). In the HyperGEN network the multipoint LOD score of 2.06 detected for Caucasians on chromosome 10 at 142.78 cM increased to 4.62  and the maximal LOD score seen in African-Americans in GENOA network on chromosome 13 decreased from 3.07 to 1.48 after removal of individuals using \u03b2-blocker medication. In African-Americans, the maximal LOD score in the sample not taking \u03b2-blocker medication was on chromosome 16 at 43.89 cM in HyperGEN with a LOD of 2.48, an increase from 1.86. Two networks (GENOA and HyperGEN) showed replication (LOD score > 1.0) across different ethnic groups on chromosome 5 between 39.99 and 45.34 cM at the microsatellites GATA145D09 and GATA7C06. Table P \u2264 0.05) for chromosome 5 between 39.99 and 45.34 cM.Figure Resting heart rate is a clinically significant variable that a large number of studies have shown to be associated with CVD, atherosclerosis, cancer, and all-cause mortality,11,37-39The pathogenesis of the connection between tachycardia-associated disease and death is not well understood. However, it has been postulated that tachycardia is a marker of abnormal autonomic control and is caused by shift in sympathovagal balance towards relative sympathetic dominance . Given tWe estimated the heritability of resting heart rate across all networks for all participants to be 32.62 % (SE = 2.53) for Caucasians and 27.41 % (SE = 2.11) for African-Americans. These values are slightly higher than previous estimates of 26%  and 21% In our genome scan for resting heart rate, we found suggestive evidence for linkage at several loci for both Caucasian and African Americans, but only one locus in Caucasians not taking \u03b2-blockers reached the level of genomewide significance with a LOD score of 4.62 . Combining all races and networks we found suggestive linkage at several loci including overlapping regions of linkage across races in two separate networks, GENOA and HyperGEN, implicating chromosome 5 with LOD scores of 1.86 and 1.12 respectively.The largest meta-analysis peak was seen on chromosome 10 at 142.78 cM and seems to be driven entirely by Caucasians with a nominal contribution of the African-American cohort. The second largest meta-analysis peak was seen on chromosome 5 between 39.99 and 45.34 cM and is driven by two completely separate networks and ethnic groups. This linkage peak between 39.99 and 45.34 cM overlaps with a linkage peak in a recent genome scan for neonatal atrial fibrillation . ContrarThe major strengths of our analysis include that it was carried out in multiple networks, each with multiple sites across the country, using a large population of Caucasian and African Americans. Moreover, adjustments for factors known to affect heart rate  were made prior to analysis.Our finding of linkage in several locations in the genome strongly suggests that heart rate is a polygenic phenotype and additional study of the implicated loci is needed. Notably, the overlapping linkage region (LOD > 1) across ethnic groups and study centers on chromosome 5p13-14 with low heterogeneity provides strong support for a QTL influencing elevated resting heart rate.The author(s) declare that they have no competing interests.JML assembled the genotype and phenotype data from the multiple networks and carried out the genome-scans and meta-analysis and drafted the manuscript. JBW conceived the study and assisted in assembling the genotype and phenotype data from the multiple networks and helped to draft the manuscript. SCH is the director of HyperGEN network and helped to finalize the manuscript. RCE is the director of the Framingham field center for HyperGEN data collection and helped to finalize the manuscript. AC is the main contact for the GenNet network and helped to finalize the manuscript. EB is director of the GENOA network and helped to finalize the manuscript. RHM assisted in data analysis and helped draft the manuscript. All authors have read and approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:Multipoint LOD scores for all study participants Each network is shown by race (CA for Caucasian and AA for African-American). Distance is given in cM. This is an Excel\u2122 file and Microsoft\u00ae Excel\u2122 should be used for viewing.Click here for file"}
+{"text": "However, each additional covariate analyzed will increase the degrees of freedom for the linkage test, and therefore can also increase the type I error rate. Use of a propensity score (PS) has been shown to improve consistently the statistical power to detect linkage in simulation studies. Defined as the conditional probability of being affected given the observed covariate data, the PS collapses multiple covariates into a single variable. This study evaluates the performance of the PS to detect linkage evidence in a genome-wide linkage analysis of microsatellite marker data from the Collaborative Study on the Genetics of Alcoholism. Analytical methods included nonparametric linkage analysis without covariates, with one covariate at a time including multiple PS definitions, and with multiple covariates simultaneously that corresponded to the PS definitions. Several definitions of the PS were calculated, each with increasing number of covariates up to a maximum of five. To account for the potential inflation in the type I error rates, permutation based Results suggest that the use of individual covariates may not necessarily increase the power to detect linkage. However the use of a PS can lead to an increase when compared to using all covariates simultaneously. Specifically, PS3, which combines age at interview, sex, and smoking status, resulted in the greatest number of significant markers identified. All methods consistently identified several chromosomal regions as significant, including loci on chromosome 2, 6, 7, and 12.These results suggest that the use of a propensity score can increase the power to detect linkage for a complex disease such as alcoholism, especially when multiple important covariates can be used to predict risk and thereby minimize linkage heterogeneity. However, because the PS is calculated as a conditional probability of being affected, it does require the presence of observed covariate data on both affected and unaffected individuals, which may not always be available in real data sets. Alcohol dependence has been shown to cluster in families. Multiple linkage analyses have been performed for phenotypes related to alcoholism, identifying phenotype-specific linkage evidence -5. To inThe study population consisted of families ascertained by the COGA. The COGA study and the data available for the Genetic Analysis Workshop 14 (GAW14) have been previously described in this issue. This study specifically uses the microsatellite genotype and covariate data that were released as part of GAW14.Covariate-based affected relative pair linkage analysis using single-point identity-by-descent (IBD) probabilities and a general conditional logistic model was performed as implemented in GENIBD and LODPAL of S.A.G.E. v4.6 ,11 on thThese PS values were derived from a logistic regression of affection status on the covariate data, using the model described below: where xj = the jth covariateThe affection status was coded as 1 for affected and 0 for unaffected. This logistic regression was performed in STATA (v8.2)  on the ep-value of a test statistic was calculated as the proportion of permutations whose statistic was equal to or greater than the observed value. Two types of statistics were computed. The first was a LOD score for each marker. The second was the sum of LOD scores across all markers, selected to simultaneously capture multiple regions of significant linkage evidence. Because the statistics were compared to their reference permutation distribution in the calculation of the p-values for each method, the relative proportion of significant tests between methods is an indication of relative power.Significance was determined by permutation testing. Affection status coupled with its covariate values was permuted within families generating 1,000 replicates, and single-point linkage analysis was performed on the observed data and on each replicate. The p-value and the number of significant markers across the genome according to the analysis method used . Including individual covariates did not necessarily lead to more significant loci identified as linked, and could even result in fewer significant findings of linkage compared to analyses with no covariates. However including a propensity score (such as with PS1 and PS3) can greatly increase the number of significant linkage results. Additionally, including the PS (except for PS2) did result in more significant regions of possible linkage compared with its corresponding multiple covariates method.Table p-value as well as the largest number of different markers yielding some significant evidence for linkage. The most significant individual markers were GATA193 (p = 0.0044) on chromosome 17, D2S200, D6S477, and D15S644 . In the logistic regression for PS3  appear to be important covariates that can be used to account for heterogeneity associated with alcoholism. The inclusion of PS3 in the linkage analysis led to both the most significant overall p-value that can be attained is p < 0.001, representing the situation in which none of replicate LOD scores was more extreme than the observed LOD score.To examine whether the markers yielding significant evidence for linkage were consistent across the methods, Figure p-values to determine significance. Having corrected for the inflation in the type I error rate, the use of a propensity score (except for PS2) compared with the use of all the covariate simultaneously does lead to the identification of more linked loci in this study. Even though several regions of significant linkage were consistent across the analysis methods, the location of the most significant regions was not consistent. Thus it is also important to emphasize that despite the power increase, the selection of covariates to include into the analysis method must be done carefully and the identification of the significant linkage regions can vary based on the covariates used. However, defining a PS that results in the covariates having the largest OR away from the null may be a means to identify important covariates for the PS, and the use of that PS may result in the greatest overall power gain to detect linkage.The incorporation of covariate information into a linkage analysis can potentially increase the power to detect linkage by identifying more loci with linkage evidence and also increased statistical linkage evidence for identified loci. Because the addition of each covariate into the analysis inflates the type I error rate in this likelihood model, it is important to use empirically derived COGA: Collaborative Study on the Genetics of AlcoholismGAW14: Genetic Analysis Workshop 14IBD: Identity by descentOR: Odds ratioPS: Propensity scoreAll authors participated in the discussions of the study design and statistical methodology and helped draft the manuscript. BQD performed the statistical analysis and CEF assisted with programming. All authors have read and approved the final manuscript."}
+{"text": "Longitudinal data often have multiple (repeated) measures recorded along a time trajectory. For example, the two cohorts from the Framingham Heart Study (GAW13 Problem 1) contain 21 and 5 repeated measures for hypertension phenotypes as well as epidemiological risk factors, respectively. Direct modelling of a large number of serially and biologically correlated traits in the context of linkage analysis can be prohibitively complex. Alternatively, we may consider using univariate transformation for linkage analysis of longitudinal repeated measures.We evaluated the utility of three conventional summary measures  for genetic linkage analysis of longitudinal phenotypes by analyzing the chromosome 10 data of the Framingham Heart Study. Except for the temporal slope, all of the summary methods and the multivariate analysis identified the previously reported region, marker GATA64A09, for systolic blood pressure or high blood pressure. Further analysis revealed that this region may harbor gene(s) affecting human blood pressure at multiple stages of life.We conclude that mean and principal components are feasible alternatives for genetic linkage analysis of longitudinal phenotypes, but the slope might have a separate genetic basis from that of the original longitudinal phenotypes. The Genetic Analysis Workshop 13 (GAW13) for longitudinal hypertension phenotypes, provided by the Framingham Heart Study group , and havFramingham Heart Study data sets for GAW13 Problem 1 contain up to 21 and 5 longitudinal systolic blood pressures (SBP) and the derived high blood pressure  measures as well as measures for numerous risk factors or related traits with cardiovascular diseases, respectively. We first considered analyzing the individual longitudinal measures separately. Although linkage analysis of individual repeated measures separately may lose some important loci that presumably have pleiotropic effects on multiple repeated measures, most of the major genes that turn on and off at different temporal stages should be detected via marginal analysis of the individual phenotypes.2)) and antihypertensive treatment (coded as 1 if the participant took medication and 0 otherwise) were obtained. Then, the residuals were analyzed using the new Haseman-Elston regression [P < 0.0001) for both the five longitudinal phenotypes of SBP (SBP1-SBP5) and HBP (HBP1-HBP5) in the offspring cohort, respectively.A particular characteristic of the data set is that a large proportion of members in the original cohort did not have genotype data although almost the same amount of phenotype information was available as for the offspring cohort. The number of informative sib pairs  is too small to render a reliable sib pair linkage analysis. Because of this and the significant difficulty in merging the two cohorts, we dropped Cohort 1 from this analysis. To make consistent comparisons with the previously published results [gression . SAS genP < 0.0001) for the mean summaries of longitudinal SBP and HBP phenotypes for both cohorts, respectively.We essentially repeated the analysis of Levy et al. . First, P < 0.0001) for the temporal slope for the offspring cohort, and the importance of BMI and antihypertensive treatment  was decreased.The subject-specific temporal slopes were obtained separately for each cohort and for each subject, by fitting a regression of the continuous SBP on the actual age at which the item had been measured. The estimated slopes were then adjusted for sex, mean BMI, and antihypertensive treatment. Next, the adjusted slopes for the two cohorts were merged and were used in the following linkage analysis. In contrast with the above two kinds of longitudinal phenotypes, sex was an important factor  with measures on all five time points were included. The eigenvalues and coefficients (loading matrix) are shown in Table For the same reasons as in the first approach, we removed the Cohort 1 from this analysis. To make consistent comparisons among these longitudinal measures, we first adjusted for the effects of the four covariates , as was done in the first approach. All five longitudinal SBPs and HBPs were standardized before obtaining the principal components. For the purpose of obtaining the principal components, all individuals in this analysis were considered to be independent, and only those individuals (P < 0.01) for SBP and HBP identified using different longitudinal measures is shown in Table P values) for linkage analysis of principal components is higher than that for individual measures [The summary of linked regions P < 0.0 for SBP measures  is suppot-tests. We defined the overall multivariate statistic to be the sum of those t2 statistics for which t > 0. The multivariate critical values at \u03b1 = 0.05, 0.01, 0.005, and 0.001 are 7.49, 11.20, 12.75, and 16.33, respectively. Figure t2 statistic profiles for the five principal components of longitudinal SBPs and HBPs, respectively. We identified four multivariate linkage regions (P < 0.01), two for each trait. The two regions for SBP correspond to markers GATA87G01 (P = 0.0014) and GATA64A09 (P = 0.0052). One region for HBP spans a 31-cM interval containing five markers (GATA87G01-GATA64A09), with the peak at marker GATA115E01 (P = 0.0020) and the second region is at the terminal marker .To test the joint effects of a putative gene(s) on multiple longitudinal SBPs and HBPs, we applied a multivariate statistical testing procedure to the derived principal components. We set the negative estimates of the Haseman-Elston regression slope to zero to account for the one-sided nature of the In this study we have evaluated the utility of three summary measures for genetic linkage analysis using the chromosome 10 data from the Framingham Heart Study. Our study supports the feasibility of mean and principal components as alternative phenotypes for longitudinal measures. The mean summary is analogous to principal components in that both are a linear function of the original traits, but the principal component approach is clearly superior because of its mathematical soundness and the ability to test more complicated genetic hypotheses . The temWe adopted a two-step approach to longitudinal linkage analysis. It has an advantage of simplicity and the resultant summaries are easily understood. Biologically and genetically, mean and slope summaries can be used to study genes varying in the course of life or genes having significant differential effects on hypertension phenotypes over time. The principal component analysis here is essentially the trend analysis in the repeated measures modelling. Not surprisingly, the results for the PRIN1 correspond closely to those for the mean summary. PRIN2, approximately the linear trend, identified two linkage signals for HBP phenotype, but they were not detected (or at least not significantly) with slope. As we expected and as was suggested by this study, the genes that influences trends of higher orders  are difficult to detect. Interestingly, several groups for GAW13 took hierarchical modelling approaches, which can be considered a systematic way to the two-step approach. Under the assumption of homogeneous with-subject variability over time, the two approaches are identical. However, if there is marked heteroscedacity of variance for the summary measures resulted from whatever reasons , a unified hierarchical analysis of the two steps that takes this into account automatically is desirable.P values might be interpreted differently. If we are willing to accept the notion that multiple longitudinal hypertension phenotypes have the same (or similar) genetic basis, then, the multivariate test reveals which gene(s) were active during the multiple stages of life. The facts that the marker GATA64A09 attained a multivariate significance (P < 0.01) for both longitudinal SBP and the derived hypertension and a univariate significance (P < 0.01) for SBP at multiple stages of life would strongly support the joint (pseduopleiotropic) effects of the putative gene(s). However, we should point out that the multivariate approach based on principal components was developed to handle pleiotropic effects of a gene and it cannot detect interactions between genes or between genes and environments, for which a sophisticated method such as step-wise discriminant analysis used in our separate GAW13 paper [The multivariate approach used in this study for evaluating the joint actions of gene(s) for hypertension phenotypes was originally proposed to handle multiple disease-related phenotypes . Here, w13 paper  is needeSelections of covariates and adjustment strategies for this study were made in accordance with the previously published paper . They arThe linkage analysis using three summary measures  supports the utility of univariate transformation from multiple longitudinal measures as an alternative for direct multivariate modelling, but interpretations of different summary measures in the context of genetics are different."}
+{"text": "An exclusion analysis suggests that further genes of effect size \u03bbsib > 1.24 are unlikely to exist in these populations of European ancestry. To our knowledge, this is the first genome-wide linkage analysis to map, and replicate, a CAD locus. The region on Chromosome 17 provides a compelling target within which to identify novel genes underlying CAD. Understanding the genetic aetiology of CAD may lead to novel preventative and/or therapeutic strategies.Coronary artery disease (CAD) is a leading cause of death world-wide, and most cases have a complex, multifactorial aetiology that includes a substantial heritable component. Identification of new genes involved in CAD may inform pathogenesis and provide new therapeutic targets. The PROCARDIS study recruited 2,658 affected sibling pairs (ASPs) with onset of CAD before age 66 y from four European countries to map susceptibility loci for CAD. ASPs were defined as having CAD phenotype if both had CAD, or myocardial infarction (MI) phenotype if both had a MI. In a first study, involving a genome-wide linkage screen, tentative loci were mapped to Chromosomes 3 and 11 with the CAD phenotype , and to Chromosome 17 with the MI phenotype (739 ASPs). In a second study, these loci were examined with a dense panel of grid-tightening markers in an independent set of families . This replication study showed a significant result on Chromosome 17 , which presents clinically as a heart attack  or angina, is a leading cause of death world-wide. The aetiology of CAD is complex with a substantial heritable component. Although there is a huge knowledge-base detailing many aspects of the underlying pathophysiology of CAD, it is likely that undiscovered pathways exist. Positional cloning projects can identify novel susceptibility genes; in the first step genome-wide linkage screens are used to assign loci to specific chromosomes.The authors have collected 2,036 CAD families from four European countries, in order to maximise the power of detecting genes that confer modest risks. A genome-wide linkage scan identified three promising regions for intensive study; one of the linked regions (Chromosome 17) was confined to families with multiple cases of myocardial infarction and was replicated in a second independent series of families. In addition the linkage scan confirmed a previously identified locus on Chromosome 2. These results demonstrate that novel CAD susceptibility genes are tractable to positional cloning which promises to lead to the identification of new molecular insights into this condition, and hopefully, new treatments. Coronary artery disease (CAD) is the most common cause of death in industrialised countries, and the prevalence is increasing dramatically in developing countries. The various clinical diagnoses that comprise CAD  are caused by atherosclerosis, a pervasive degenerative condition in which lipid and fibrous matrix is deposited in arterial vessel walls to form atheromatous plaques. The fibrous caps of some of these plaques sited in coronary arteries may be unstable and rupture. This will release thrombogenic material into the lumen of the vessel leading to coronary thrombosis, vessel occlusion, and subsequent infarction of the myocardium, a critical condition with high mortality. While much is known about CAD, and about some aspects of the underlying pathology of atherosclerosis, new strategies for risk predication and intervention are still needed. There thus remains considerable importance in increasing our understanding of CAD pathophysiology.Familial clustering of CAD has long been recognised , suggestALOX5AP gene (encoding 5-lipoxygenase activating protein) associated with CAD in the two countries, and the Icelandic haplotype was also associated with stroke in Iceland and in Scotland [Published genetic linkage studies have implicated several loci for CAD. The first such linkage study was from Finland and showed linkage of premature CAD to loci on Chromosomes 2q21.2\u201322 and Xq23\u201326 . SubsequScotland ,13. The Scotland  was alsoScotland ,12, and The status of CAD loci which have not been replicated is less clear. Differences in the population studied and/or in the selection criteria used might result in selection for different genetic effects in this heterogeneous disorder. However, the lack of replication might also be expected given that most of these linkage studies had rather low power relative to the anticipated effect size of CAD susceptibility genes. The published linkage screens have ranged in size from 156\u20131,933 families  and the linkage statistics reported have sometimes been suggestive rather than of firm genome-wide significance, suggesting that some postulated loci may be false positives. None of the published studies have had the statistical power to replicate a specific locus. Rather, the signal-to-noise ratio is such that these studies have had borderline power to detect individual susceptibility genes, and so each will tend by chance to identify a different selection.The PROCARDIS study was founded as a European collaborative project to assemble a sufficiently large number of families to map the modestly sized susceptibility genes that might plausibly be hypothesised to contribute to CAD risk. We present here the results of the first phase of the study, a linkage study of 2,658 ASPs from 2,036 families with multiple CAD siblings.Affected sibling pairs (ASPs) were identified in the families by applying two phenotypic criteria: 1) a broad CAD definition of disease status in the affected sibling(s), , with each ASP containing at least one proband with MI or SACS, or 2) a narrow definition of disease based on a clinical history of MI at age < 66 y. p = 0.81 , p = 0.71  and p = 0.08 . These results were supported by an alternative multipoint linkage analysis method using the Merlin programme , with a peak LOD = 0.7 mapping 11.9 cM outside the \u22121 LOD support interval identified in the genome-wide screen (p = 0.003) at location 67 cM (p = 1 \u2013 (1 \u2013 0.003)3 = 0.009) [sib) associated with the linked region on Chromosome 17 for MI phenotype. The linked region was further investigated in the replication cohort with CAD ; there was weak support for linkage between Chromosome 17 and the broad CAD phenotype (p = 0.156). Finally, an analysis of the combined data from the genome-wide screen and replication cohorts showed linkage to the MI phenotype on Chromosome 17 .In order to further test the potential importance of the loci identified in the linkage analysis, these regions were tested in an additional sample of 1,194 CAD ASPs and 344 MI ASPs (details shown in e screen . There won 67 cM . After a= 0.009) . Table 2sib at each genetic location associated with a LOD of \u22122 [sib = 1.24 (CAD phenotype) and \u03bbsib = 1.42 (MI phenotype).An exclusion analysis  was perfOD of \u22122 . Using tp = 0.08) was replicated in an independent cohort of families  is important and provides a target for further positional cloning studies.Susceptibility genes for human complex diseases are believed to confer modest risks and hence genetic linkage to such loci may be difficult to replicate. So our finding that a region of tentative linkage to the MI phenotype identified on Chromosome 17 in a genome-wide screen . The LOD \u22121 support interval for localising the MI susceptibility gene in the genome-wide screen was broad (42\u201379 cM) which is typical for complex diseases [sib at this position was 1.29 in the replication cohort).It is well known that estimates of genetic effect sizes from genome-wide screens are frequently upwardly biased  and thatdiseases . The peap > 0.156), it is important to consider the possible reasons for this phenomenon. Current knowledge of the pathophysiological processes that lead to CAD indicate that they are highly diverse, and to a large extent not understood in full detail. In addition the CAD disease entity is heterogeneous, consisting of four major diagnostic outcomes: MI, angina, unstable angina, and coronary revascularisation events [As the linked region on Chromosome 17 appears to be specific for the MI phenotype, with no evidence for linkage to the broad CAD phenotype in the replication set of ASPs  and D17S787 at 17q21 , spans the centromere and includes over 36 megabases of DNA containing over 300 genes of known function and about as many again predicted genes. This interval contains numerous genes that are plausible candidates for involvement in CAD, therefore our future research is focused on refining the region genetically by means of high density SNP coverage, using the PROCARDIS case-control collection and our trio families, and by an analysis of quantitative trait loci (QTL) for CAD intermediate phenotypes measured in the affected sib-pair families. There are published QTLs close to this region that may provide clues. For example, two QTLs for low-density lipoprotein cholesterol map just outside the PROCARDIS region to 17q23.2\u201325.3  is withiA region of linkage using ordered subset analysis in 26 young-onset CAD families has been identified between markers D17S787 and D17S944   which adIn conclusion, the results from the PROCARDIS genome-wide linkage analysis are important because they identify a novel replicated locus for MI on Chromosome 17. In addition, our CAD linkage results are consistent with a CAD susceptibility locus mapping to Chromosome 2. The lack of evidence for other linked loci suggests that the heritable component of coronary artery disease and MI may be composed of many genes, with few of them having sufficient effect to permit mapping by genetic linkage even in studies of this size involving thousands of affected sibling pairs.Ascertainment criteria for PROCARDIS probands were MI or SACS, on the assumption that the latter represents a similar pathological process according to modified World Health Organisation diagnostic criteria ,29 befor\u00ae Green. DNA was plated and genotyped for the ABI MD10  set of microsatellite markers using ABI 3700 capillary sequencing equipment [DNA was extracted from 9 ml EDTA-anticoagulated samples of frozen whole blood using Gentra Systems PUREGENE kits  according to the manufacturer's instructions, and its concentration determined fluorimetrically using SYBRquipment . All micquipment . After aAdditional grid-tightening markers were genotyped for the linked regions on Chromosomes 3 (eight markers), 11 (seven markers), and 17 (25 markers); details are included in Genetic relationships between cognate relatives were confirmed in an analysis using the RELPAIR program  with allsib) of the genetic effect size for this susceptibility gene; 95% CI for this \u03bbsib estimate were calculated using a bootstrapping technique with 1,000 replicates [sib associated with a MLS = \u22122 (a strict exclusion threshold) at each genetic location. This analysis is able to show in regions that show little or no evidence of linkage, that there is sufficient identity-by-descent information to confidently exclude susceptibility genes of modest effect sizes. The statistical significance of the MLS statistics were established by a computer simulation which modeled the family structures , marker density and informativity [Non-parametric linkage analysis was undertaken using the computer program MLSix, to compute single-point and multi-point maximum LOD score (MLS) statistics ,35 for aplicates . Exclusimativity ; 1,000 rThe authors had full access to the data and take responsibility for its integrity. All authors have read and agree to the manuscript as written.Figure S1http://www.sph.umich.edu/csg/abecasis/Merlin/index.htmlA genome-wide scan of CAD\u2014broad CAD phenotype, 1,464 ASPs (A) and MI\u2014narrow phenotype, 739 ASPs (B). Genetic location (abscissa) is scaled in Kosambi M. The ordinate denotes the Kong & Cox LOD score . The sol(586 KB TIF)Click here for additional data file.Table S1(62 KB DOC)Click here for additional data file."}
+{"text": "We assessed the linkage and correct linkage rate using deterministic record linkage among three commonly used Canadian databases, namely, the population registry, hospital discharge data and Vital Statistics registry.Three combinations of four personal identifiers  were used to determine the optimal combination. The correct linkage rate was assessed using a unique personal health number available in all three databases.Among the three combinations, the combination of surname, sex, and date of birth had the highest linkage rate of 88.0% and 93.1%, and the second highest correct linkage rate of 96.9% and 98.9% between the population registry and Vital Statistics registry, and between the hospital discharge data and Vital Statistics registry in 2001, respectively. Adding the first name to the combination of the three identifiers above increased correct linkage by less than 1%, but at the cost of lowering the linkage rate almost by 10%.Our findings suggest that the combination of surname, sex and date of birth appears to be optimal using deterministic linkage. The linkage and correct linkage rates appear to vary by age and the type of database, but not by sex. Record linkage techniques are used widely in epidemiological studies to obtain comprehensive information and conduct more robust analyses -13. For The evolution from manual to computerized record linkage has helped to answer the first question. There are two commonly used computerized record linkage approaches: deterministic and probabilistic ,9. The dUsing both deterministic and probabilistic linkage approaches, Roos, Wajda and Nicol  assessedIn our study, we selected four common identifiers  from a set of personal identifiers and composed three different combinations of four identifiers: (1) a combination of surname, sex and date of birth, (2) first name, sex, and date of birth, and (3) surname, first name, sex and date of birth. Then we assessed the deterministic linkage between the Vital Statistics registry and the population registry in one scenario, and between the Vital Statistics registry and the in-hospital death records in hospital discharge data in the second scenario for the fiscal years 1998/99 through 2001/02 for each combination. We assessed these three databases because they are widely used in population and health services research to determine death status, cause of death and medical history.Three administrative health databases were employed. We restricted our study population to residents of Calgary Health Region (CHR), Alberta, Canada during fiscal years 1998/99 through 2001/02. As of March 2002, CHR's population was approximately 1.1 million. Infants less than one year were excluded because of missing or inaccurate variables such as name. In Vital Statistics individuals were defined as CHR residents based on Standard Geographical Classification (SGC) for assessment of the linkage between Vital Statistics and the population registry. The SGC was used to classify residential areas based address or community . Of 2167The first database was the Alberta Health Care Insurance Plan Registry  for the CHR for the fiscal years 1998/1999 to 2001/2002. This registry contained demographic information of health care recipients. Canada has a government-financed universal health insurance system. All permanent residents are covered by the provincial health insurance plan except for Registered First Nations, prison inmates, and members of the military and the Royal Canadian Mounted Police all of which are the responsibility of the federal government. All eligible Alberta residents are assigned a unique lifetime Personal Health Number (PHN). Therefore, PHN is an ideal variable for performing record linkage. The Alberta insurance registry is nearly complete and consistent, and is used as a proxy for the population of Alberta.The second database was the Alberta Vital Statistics registry for fiscal years 1998/99 to 2001/2002. Information in the death registry was derived from the Death Registration form, medical certificate of death, and the medical examiner's certificate of death (where appropriate). The registry captured deaths that occurred within Alberta but misses Alberta residents who died out of Alberta. Provincial and territorial Vital Statistics registries on all deaths were submitted to Statistics Canada annually.The third database was the hospital discharge abstracts for the fiscal years 1998/1999 to 2001/2002. Abstracts are filed for all inpatient discharges from all hospitals in the CHR. Professional coders reviewed inpatient charts and extracted data on PHN, demographics, diagnoses, procedures, physician specialty and status of alive or death.We selected surname, first name, sex, and date of birth as our common identifiers because they were less likely to be changed over time, compared to other identifiers like address. We assessed three different combinations of four identifiers: (1) surname , sex and date of birth ; (2) first name, sex, and date of birth; (3) surname, first name, sex and date of birth.To perform record linkage, the common identifiers were formatted in the same way across the three databases, capitalizing letters, removing blanks and dashes. One common linkage problem with using the name variable as a common identifier was that an individual's name could be represented in many different ways, with alternate spellings, initials, abbreviations and shortened forms of names making the linkage difficult. To deal with this common linkage issue, a Soundex coding method was employed to identify linkage between names that fail to match due to variant spellings of the names in the two databases . The SouCorrect linkage among the linked records was assessed by checking whether the unique PHN from various sources was identical within the matched record. We excluded matched records without PHN in both files. Although this identifier is complete both in the hospital discharge data and population registry, only 70% of the records in the Vital Statistics registry had a valid PHN, because it was not a mandatory variable.We linked the Vital Statistics registry with the population registry three times, using each of the three approaches , their PHNs would not be recorded in the Vital Statistics if they died in Alberta. However, such cases account for a small proportion of all deaths in Alberta.The linkage rates between the Vital Statistics registry and hospital death do not vary much by age, but the linkage rate between the population registry and the Vital Statistics registry depends on age group. The linkage rate was significantly lower for the 1 to 9 year age group (ranging from 34.9% to 39.7% in 2001/02) than for 10 and over year age group (ranging from 47.0% to 90.0% in 2001/02). The difference in linkage rates may reflect less accurate information recorded for out-of-hospital deaths in younger persons. Records with the PHN may have more complete and accurate information on common identifiers than those without the PHN. In fact, we found the linkage rate between the population registry and Vital Statistics was higher for Vital Statistics records with PHN than for records without PHN (89.5% versus 81.0% in 2001/2002). There were more PHNs missing for children aged 1 to 9 years (42.9% in 2001/2002) than for individuals aged 10 or older (13.8% in 2001/02).A successful deterministic linkage relies not only on the completeness of data, but also on choosing an appropriate combination of common identifiers. In our study, a combination of surname, sex and date of birth has the highest linkage rate and second highest correct linkage rate. The combination of first name, sex and date of birth generated relatively low linkage and correct linkage rates. The combination of all four identifiers  resulted in an even lower linkage rate with little increase in the correct linkage rate. One possibility is that alternate spellings, initials, abbreviations and shortened forms of first names are common in the database. Therefore we recommend the use surname, sex and date of birth as common identifiers in linking databases deterministically when a unique identifier is not available.To further improve correct record linkage, other potential indicators, such as residence address or postal code, should be considered. In our study, only 67% to 77% of records in the Vital Statistics registry during 1998 and 2001 contained information on the unique identifier of personal health number, while 84% to 96% had information on postal codes. The linkage rates might be increased taking the strategy of first matching records using unique identifiers, and then matching remaining records using a combination of surname, sex and date of birth.This study has four major limitations. First, we assessed record linkage using administrative databases in a Canadian health region. The results might not be conceptually generalizable to other Canadian regions or other countries because the quality of administrative data may vary across geographical areas and institutions. Secondly, records without PHN may have less complete and accurate information on common identifiers than those with PHN. Therefore the higher the rate of missing PHNs is, the more likely the linkage rate is to be lower. We selected linked records with PHNs only to assess correct linkage rate. The potential selection bias may cause the correct linkage rate to be overestimated, particularly for children aged 1 to 9 since they have more missing PHNs than those aged 10 or older in Vital Statistics data. Thirdly, we excluded deaths with unknown residence area information and missed residents of the region who died out of Alberta, possibly leading to overestimates of our linkage rate if personal information on these deaths was less complete than those we analyzed. Fourthly, our linkage rate may be applicable to linking nested databases; one database contains all records of other database. In our study, all Vital Statistics Registry records are expected to appear in the Population Registry, and all in-hospital death records are expected to be present in the Vital Statistics Registry. Correct linkage could be assessed by four measures: true-link, false-link, true-nonlink and false-nonlink. Assessment of these four types requires a unique identifier present in both databases to establish the \"gold standard\". Our study addressed one question: what is correct linkage rate among links through deterministic record linkage?Our study findings suggest that deterministic record linkage using three basic indicators  appears to generate the highest linkage rate among three commonly used databases in health service research, namely the population registry, hospital discharge data and the Vital Statistics registry. The matched records appear to be highly accurate. However, the linkage and correct linkage rates appear to be influenced by type of database and age, but not by sex.The author(s) declare that they have no competing interests.BL contributed to the study design, statistical analysis, interpretation and writing of the manuscript. HQ contributed to the study design, the interpretation, and writing of the manuscript. AF helped data-analysis and interpretation and editing and proving the manuscript. ML contributed to the data interpretation and writing of the manuscript.The pre-publication history for this paper can be accessed here:"}
+{"text": "We address the question of whether statistical correlations among quantitative traits lead to correlation of linkage results of these traits. Five measured quantitative traits , and one derived quantitative trait  are used for phenotype correlation studies. Four of them are used for linkage analysis.We show that although correlation among phenotypes partially reflects the correlation among linkage analysis results, the LOD-score correlations are on average low. The most significant peaks found by using different traits do not often overlap.Studying covariances at specific locations in LOD scores may provide clues for further bivariate linkage analyses. If the same gene (pleiotropy) caused two quantitative traits, linkage analyses of these two traits would lead to a peak at the same region, and there would therefore be a statistical correlation of two sets of LOD scores in a specific region. On the other hand, if different genes caused two traits, no correlation is expected at the LOD score level unless there are tightly linked loci influencing both traits. In the case of pleiotropy, there should be a correlation between the two traits caused by the same gene. If two traits are highly correlated, the corresponding LOD scores from the linkage analysis would also be expected to be highly correlated, and it may therefore not be necessary to carry out linkage analysis twice. If the correlation between two traits were perfect then the correlation in LOD scores would also be perfect. Here we argue that any less-than-perfect correlation between the two traits may lead to quite different linkage analysis results, and that linkage analysis is therefore necessary for both traits.The Framingham data  providesThe Cohort 1 and Cohort 2 files contain trait information for the older and younger generations, respectively, in the Framingham Heart Study. There is a huge difference in the amount of missing data between the two files. In Cohort 1, measurements were taken 21 times, though for some traits they were only measured a few times (e.g. three times for TG). In Cohort 2, measurements were taken five times and there are rarely missing data. For our analysis, for simplicity as well as for the purpose of removing certain environmental effects, we do not study the time sequence of these measurements, so the average of each trait is used.It is well known that TG fluctuates wildly. Even measured on the same person, TG value may change during a day and depends on whether one eats or not. The distribution of TG is highly skewed. To make the distribution more Gaussian-like, we apply a logarithm transformation (log(TG)).Pair-wise Pearson's correlation coefficient was calculated between six traits and the age : TC, GLU, HDL, BLP, TG, and CR. For Cohort 1, one or a few trait values may not be available for some people. These persons are ignored in the corresponding correlation calculation. We also carried out a hierarchical cluster analysis of the six traits, using the Euclidean distance and average linkage. The traits BLP and GLU comprise one branch, which is separated from other branches and traits.The male vs. female difference of a particular trait can be tested by an analysis of variance (ANOVA). Note that ANOVA for two categories is equivalent to a t-test. If the correlation between the age variable and another trait is significant, there is also an age effect on that trait. Such correction analysis is carried out by two separate, gender-specific, regressions:yg = cg 0,+ cg 1,* AGE, \u00a0\u00a0\u00a0 g = {f, m}.The computer program MERLIN  is used Because of the limitation on the pedigree size when running MERLIN, we manually removed all untyped individuals who were deletable . Large pedigrees were also split into two or more sub-pedigrees so that all had \"bit\" value less than 20 , 55 (ped 24619), 39 (ped 26671), 38 (ped 27992), 37 (ped 31116), etc. A total of 31 pedigrees were split into smaller pedigrees. After simplifying the pedigrees, the number of individuals was reduced to 4095 from the original number of 4692. A program RECODE  was used to relabel (\"downcode\") allele values so that they started from 1.i, i = 1,2,...,398). Besides the correlation coefficients, scatter plots of a pair of LOD score sets are provided in order to discern any \"outliers\" (markers that behave very differently from the rest of markers).The Pearson correlation coefficient is calculated for two sets of LOD scores obtained for the two traits. Each set of LOD scores consist of LOD scores on 398 markers,averaged over all families (LODTable Table  for details) were also carried out, but the results were not consistent with the variance-component linkage analysis runs and are not shown here.Cluster analysis shows that the two traits, BLP and GLU, are on a separate branch from the other four traits. For this reason, we decided to focus on the four more closely related traits, TC, HDL, log(TG), and CR, for linkage analysis. The LOD scores obtained from single-marker variance component linkage analysis  are showFigure for each peak in LOD score for linkage analysis of one trait, there is also a matching peak in the linkage analysis of another. The example of perfect correlation  does not generalize to situations of less than perfect correlations. This observation has direct practical implication on linkage analyses of a few correlated quantitative traits, because the presence of one linkage signal for one trait may not lead to a linkage signal for another related trait . The most striking example is the marker Mfd232 on chromosome 19: even though CR is derived from TC and HDL, the linkage signal for CR at this marker is much stronger than those for the TC or the HDL trait alone. Finally, we note that there is a possibility that two LOD score peaks at the same location may be caused by two closely linked genes instead of the same gene. In other words, we may not be able to distinguish the situation of pleiotropy and the situation of two linked genes. For all practical considerations, such distinction is minor.It is clear from Figure"}
+{"text": "Complex diseases are multifactorial in nature and can involve multiple loci with gene \u00d7 gene and gene \u00d7 environment interactions. Research on methods to uncover the interactions between those genes that confer susceptibility to disease has been extensive, but many of these methods have only been developed for sibling pairs or sibships. In this report, we assess the performance of two methods for finding gene \u00d7 gene interactions that are applicable to arbitrarily sized pedigrees, one based on correlation in per-family nonparametric linkage scores and another that incorporates candidate loci genotypes as covariates into an affected relative pair linkage analysis. The power and type I error rate of both of these methods was addressed using the simulated Genetic Analysis Workshop 14 data. In general, we found detection of the interacting loci to be a difficult problem, and though we experienced some modest success there is a clear need to continue developing new methods and approaches to the problem. The topic of gene \u00d7 gene interaction (epistasis) has recently elicited great interest ,2 and evMany approaches to detecting genetic interactions using linkage analysis, both parametric and nonparametric, have been proposed. For reasons discussed by Cox et al. , we willA slightly different scenario is that in which a candidate marker has been identified and one wishes to incorporate that marker into the linkage analysis. One approach that can be used for such an analysis in large multiplex pedigrees is a re-parameterized version of Risch's  LOD scorWe investigated the correlation-based approach suggested by Cox et al.  and the In order to investigate the power and type I error of both approaches, we used the simulated GAW14 data. For a complete description of the simulated datasets, see . In brieType I error for each method was indicated by the proportion of times a significant interaction was found with marker loci on the null chromosomes (chromosomes that lacked any disease loci), while the power for each method was assessed by the proportion of times a significant interaction was found with the marker locus closest to the interacting disease locus.Multipoint NPL scores were calculated using MERLIN for the KPD disease phenotype and the three latent traits in the KA, DA, and AI populations. Single-point calculations were performed for the NY population. In all cases the microsatellite data were used. For candidate loci, the four microsatellite markers nearest to the disease loci D1, D2, D3, and D4 were selected. The correlations in NPL scores between these candidate loci and the remaining microsatellite markers were then calculated. A significant positive correlation (\u03b1 = 0.05) indicated a potential epistatic interaction between the two loci, while a significant negative correlation indicated potential heterogeneity. When a significant correlation was detected, NPL scores were then re-calculated using the per-family NPL scores of the candidate locus.p-value < 0.05) with the four candidate loci was estimated using all 100 simulations. The power of the method was assessed using the proportion of times a significant positive correlation was detected between two interacting candidate loci (again using a 0.05 significance level).All of the markers on chromosomes 4, 6, 7, and 8 were used to estimate the empirical type I error rate. These chromosomes (the null chromosomes) were selected because they lacked any disease loci. For each combination of phenotype and population, the proportion of markers on the null chromosomes having significant positive correlations  simulation algorithm [In this method, the marker data were used to estimate the proportion of alleles identical by descent (IBD) at each polymorphic marker locus for pairs of affected relatives. Multipoint IBD-sharing estimates for the NY pedigrees were obtained using the GENIBD program from the S.A.G.E. package, release 4.3 . Exact elgorithm  was usedFor these analyses we used a modified one-parameter model of the conditional logistic model , with cath percentile of the empirical null distribution.Linkage parameters were first estimated under the base model without any covariates and then recalculated with each candidate locus included as a covariate, i.e., the genome scan was repeated for each covariate. Regions in which the LOD score with a covariate was significantly increased versus the base model were presumably regions with genes that interacted with the candidate locus (covariate). To address concerns that the behavior of the test statistic, when applied to large multiplex families, was influenced by both family and marker characteristics, we performed the analysis for each model (each SNP covariate and phenotype) on the null chromosomes in the simulated dataset to determine the null distribution of the test statistic when no disease locus was present. Empirically, the mean (SD) inflation of the LOD difference statistic under the null ranged from 0.28 (0.39) to 0.42 (0.59) across all models tested. The power of the method was determined by the proportion of times the change in LOD score was above the 95The results for the empirical type I error rates of the correlation method are given in Table Table r = 0.703) with the degree of correlation between the two loci. Also, the effectiveness of re-calculating the NPL score is highly dependent upon which locus is conditioned upon. In this case, conditioning upon the D4 locus is more effective than conditioning on the D1 locus due to the stronger LD in the D4 region.To illustrate, we show results from the D1\u2013D4 interaction in the P3 phenotype. When restricting attention to replicates with a significant positive correlation between D1 and D4 (otherwise the conditioning is pointless), the average increase in NPL scores was 0.549 and 0.638 for the AI and KA populations, respectively, when conditioning on the D4 locus and 0.354, and 0.444 when conditioning on the D1 locus. This increase in the NPL score is highly correlated KPD: Kofendrerd Personality DisorderMCMC: Markov chain Monte CarloNPL: Nonparametric linkageSNP: Single-nucleotide polymorphismAll authors participated in the design of the study. GNB, BSM, THG, MEC, performed the statistical analyses. MLM acted as a supervisor for the design and statistical analyses. GNB and BSM drafted the manuscript. All authors read and approved the final manuscript."}
+{"text": "The Framingham Heart Study has contributed a great deal to advances in medicine. Most of the phenotypes investigated have been univariate traits . The aims of this study are to derive multivariate traits by identifying homogeneous groups of people and assigning both qualitative and quantitative trait scores; to assess the heritability of the derived traits; and to conduct both qualitative and quantitative linkage analysis on one of the heritable traits.Multiple correspondence analysis, a nonparametric analogue of principal components analysis, was used for data reduction. Two-stage clustering, using both k-means and agglomerative hierarchical clustering, was used to cluster individuals based upon axes (factor) scores obtained from the data reduction. Probability of cluster membership was calculated using binary logistic regression. Heritability was calculated using SOLAR, which was also used for the quantitative trait analysis. GENEHUNTER-PLUS was used for the qualitative trait analysis.-6) and had characteristics consistent with atherogenic dyslipidemia. We found both qualitative and quantitative LOD scores above 3 on chromosomes 11 and 14 . There were two Kong & Cox LOD scores above 1.0 on chromosome 6 (6p21) and chromosome 11 (11q23).We found four phenotypically distinct groups. Membership in the smallest group was heritable (38%, p < 1 \u00d7 10This approach may be useful for the identification of genetic heterogeneity in complex phenotypes by clarifying the phenotype definition prior to linkage analysis. Some of our findings are in regions linked to elements of atherogenic dyslipidemia and related diagnoses, some may be novel, or may be false positives. Contemporary advances in medicine are due, at least in part, to the long history of research conducted in Framingham. To date, the majority of the outcome measures have been univariate qualitative or quantitative traits. The objectives of the present analyses were to derive multivariate qualitative and quantitative traits empirically, to examine the heritability of the traits, and to conduct genome-wide linkage analyses with a trait that demonstrated some heritability. The analyses were conducted in the families collected by the Framingham Heart Study made available to participants in the Genetic Analysis Workshop 13.This study was conducted in the sample from the Framingham Heart Study distributed to participants in Genetic Analysis Workshop 13. The most extreme measurement category across all of the measures for an individual was used to create the multivariate phenotypes. For example, if, over the course of the available measurements, the maximum triglyceride level reached the fourth quartile, the summary measure was the fourth quartile. Continuous measures were categorized according to classes commonly used in clinical practice as follows: body-mass index (BMI) ; tobacco use ; alcohol use ; systolic blood pressure (sbp) ; cholesterol ; glucose ; atherogenic dyslipidemia . High density lipoproteins (HDL) and triglycerides were characterized in age- and gender-specific quartiles as observed in the Framingham Heart Study data. An individual was classified as having high blood pressure if they were being treated for hypertension, regardless of the clinical measurement.The strategy for the development of qualitative and quantitative traits included nonparametric data reduction, iterative two-staged clustering on the observed dimensions, and the assignment of probability of cluster membership in each cluster for each individual.Principal components analysis (PCA) is a method commonly used for data reduction. PCA is based upon a Pearson product-moment correlation which assumes a pair-wise Gaussian structure. The original continuous data were not pair-wise normal and did not meet the assumptions for this method. Multiple correspondence analysis (MCA) is a nonparametric data reduction method free of the assumptions underlying PCA. The only requirement for MCA is a non-negative rectangular data matrix. MCA uses a singular value decomposition (SVD) of the matrix. Eigenvalue (vector) decomposition is a special case of SVD. The objective of MCA is to identify a low-dimensional subspace that comes closest to all of the data points. It is analogous to graphing the results of a factor analysis in a multidimensional Euclidean space. However, the space identified in MCA is not Euclidian. The coordinates of each individual in the identified multi-dimensional space served as the basis for the identification of subgroups or clusters .Each study participant with phenotype data was assigned a score on each of the eight retained dimensions (data not shown). Next, a multistaged clustering strategy was used to identify distinct subgroups . It is nIt is possible to represent cluster membership as both qualitative and quantitative traits. The qualitative trait is membership in the cluster, which is binary. The quantitative trait represents the degree of affiliation with the cluster, distance from the cluster centroid, or probability of membership. To compare the utility of the measures and the consistency of the linkage results, both traits were constructed and linkage analyses were conducted on each.Binary logistic regression was used to estimate the probability of cluster membership for each study participant in each of the clusters. The natural logarithm of the probability of membership in Group 4 (described below) was the dependent measure in the quantitative trait analyses. Categorical cluster membership was used in the qualitative trait analyses. Two-point variance components linkage analysis was conducted using SOLAR . Multipon = 1030 (35.7%), n = 670 (23.22%), n = 881 (30.54%), and n = 304 (10.54%).Coordinates on eight axes  were retained and used for clustering. Four clusters were identified. The cluster sizes were An index measure of the prevalence of each of the independent variables within each of the clusters was calculated by dividing the observed category proportion in a cluster by its expectation, the marginal proportion. If the prevalence in a cluster did not differ from the sample, the index would be unity. Group 1 had indices higher than 1.25 for the first quartile triglyceride measure (2.02), high blood pressure (1.53), high cholesterol (1.44), hyperglycemia (1.43), fourth quartile HDL (1.39), and heavy alcohol use (1.27). Group 2 was characterized by high HDL (1.62) and lower rates of all other measures. This was a particularly healthy group. Group 3 was characterized by low HDL, obesity, and high triglycerides . The last group (Group 4) contained all of the individuals in the sample who met the criteria for atherogenic dyslipidemia as defined by lowest quartile for HDL and highest decile for triglycerides. They had high indices for atherogenic dyslipidemia (8.39), top decile for triglycerides (5.24), lowest quartile HDL (3.82), obesity (1.51), and smoking (1.49).Figure p < 1 \u00d7 10-6) for Group 1, 19% (p < 1 \u00d7 10-6) for Group 2, 39% (p < 1 \u00d7 10-6) for Group 3, and 38% (p < 1 \u00d7 10-6) for Group 4. Linkage analysis was conducted for the probability of membership in Group 4 and for a binary qualitative trait representing membership in the Group 4.The heritability of the probability of group membership was computed using SOLAR. The heritability of each of the quantitative traits is 20% (n = 3), 4q22, 4q28, 5q34, 6p12, 6p21, 8p16, 8q24 (n = 2), 9q34, 10q26, 11q13, 15q21, 17p11, 17q24, 18q22 (n = 2), 19p13, 22p11.Table In the multipoint NPL analysis, there were two K&C LOD scores above 1.0 Table , one on We found four empirically derived, phenotypically distinct subgroups. One group was very healthy, two groups had mild to moderately elevated lipid levels, and one group had lipid levels characteristic of atherogenic dyslipidemia. The profile of the latter group resembled atherogenic dyslipidemia and atherogenic dyslipidemia. Grundy  identifiThree loci were common across our qualitative and quantitative analyses. One of the three LOD scores above 3 in the quantitative trait was observed on 11q23. The highest NPL score in the qualitative trait analysis was observed in the same region. Similarly, there were consistent findings on 6p21 and 18q22 in both the qualitative and quantitative analyses.Several of our results are close to those reported by Aouizerat et al.  in a genLindsay et al.  reportedOur results are also close to those reported by Soro et al. . In a stFor the qualitative trait analyses, one location on chromosome 14p was common across this analysis and another done by this group examining a trait for atherogenic dyslipidemia . Both anSome of the present linkage findings are in regions linked to elements of atherogenic dyslipidemia and related diagnoses, some may be novel, or may be false positives. It is also possible that the number of LOD scores above 2 in the quantitative trait analysis is due to the clustering of distinct traits with distinct genetic etiologies rather than a single trait with an oligogeneic or polygenetic etiology."}
+{"text": "Genomic screens generally employ a single-locus strategy for linkage analysis, but this may have low power in the presence of epistasis. Ordered subsets analysis (OSA) is a method for conditional linkage analysis using continuous covariates.We used OSA to evaluate two-locus interactions in the simulated Genetic Analysis Workshop 14 dataset. We used all nuclear families ascertained by Aipotu, Karangar, and Danacaa. Using the single-nucleotide polymorphism map, multipoint affected-sibling-pair (ASP) linkage analysis was performed on all 100 replicates for each chromosome using SIBLINK. OSA was used to examine linkage on each chromosome using LOD scores at each 3-cM location on every other chromosome as covariates. Two methods were used to identify positive results: one searching across the entire covariate chromosome, the other conditioning on location of known disease loci.Single-locus linkage analysis revealed very high LOD scores for disease loci D1 through D4, with mean LOD scores over 100 replicates ranging from 4.0 to 7.8. Although OSA did not obscure this linkage evidence, it did not detect the simulated interactions between any of the locus pairs. We found inflated type I error rates using the first OSA method, highlighting the need to correct for multiple comparisons. Therefore, using \"null chromosome pairs\" without simulated disease loci, we calculated a corrected alpha-level.We were unable to detect two-locus interactions using OSA. This may have been due to lack of incorporation of phenotypic subgroups, or because linkage evidence as summarized by LOD scores performs poorly as an OSA covariate. We found inflated type I error rates, but were able to calculate a corrected alpha-level for future analyses employing this strategy to search for two-locus interactions. Such an approach is quite effective for detecting linkage to loci with strong effects, but may have low power in the presence of epistatic or heterogeneous effects. Two-locus  and condanalysis ,4.Ordered subsets analysis (OSA)  is a metWe used all available nuclear families ascertained by three groups: Aipotu, Karangar, and Danacaa. Affection status was determined using the criteria defined by each site; to better approximate a real world post-hoc pooled analysis of three genomic screens, we made no attempt to impose standardized diagnostic criteria. Given there was no genetic heterogeneity in the dataset other than that defined for the three phenotypes, this decision should have introduced some variability in the strength of the main effects to our combined dataset. Introduction of this variability should thereby have attenuated the main effects, allowing weaker epistatic interactions to be detectable. We analyzed each of the 100 replicates. We used the single-nucleotide polymorphism (SNP) linkage map . No follow-up markers were ordered.i a matrix of linkage statistics Zi is required as input, where d represents the disease location parameter and represents the genetic model, and the maximum ordered subset statistic for each family is calculated at a set of values for d and \u03b3. OSA begins by ordering N number of families by the covariate chromosome family-specific LOD score value xi, both in an ascending and a descending order, where Zj) is the linkage statistic matrix for ordered family j. The maximum LOD score is calculated for the jth family, as well as the estimates of dj) (and \u03b3j)  to the matrix for family 1 through j. In summary, the jth partial sum is created by adding each element of the linkage statistic matrix for each family up to and including ordered family j. Addition of each of the N families results in a set of maxima for each partial sum of the linkage statistic . . . ZN), ordered by the family-specific covariate value. The final OSA output includes an overall LOD score calculated using all families, a maximum subset LOD score (representing the highest LOD score using subsets of families with the highest covariate chromosome LOD scores), and an estimate of the disease location on the analysis chromosome.Multipoint affected sib pair (ASP) linkage analysis was performed using SIBLINK . A grid p-values for the increase in the LOD score in the subset of families identified by OSA over baseline LOD scores for the entire dataset were generated, using a minimum of 20 and maximum of 1,000 permutations for pair-wise comparisons of chromosomes with disease loci, and a maximum of 10,000 permutations for the null chromosome pairs. Two methods were used to identify positive OSA results. The first method searched over the entire analysis chromosome for the single most significant OSA LOD score . From the 100 replicates, the number of times that the minimum p-value at any position on the analysis chromosome was below 0.01 and 0.05 was calculated. Because we observed a highly inflated type I error rate for the 0.01 and 0.05 significance levels, we calculated a corrected alpha-level for controlling the global type I error rate using OSA results for the null chromosome pairs. The second method conditioned on the exact location of the disease locus on the covariate chromosome and did not suffer from severe type I error inflation. From the 100 replicates, we again calculated the number of times that the OSA empirical p-value was below 0.01 and 0.05, and the mean position of the maximum subset-based LOD score on the analysis chromosome.OSA was used to examine linkage on each chromosome with disease loci  using LOD scores at each 3-cM location on every other chromosome with disease loci (\"covariate\" chromosome) as covariates . We also performed these same analyses on \"null chromosome pairs\", using four pairs of chromosomes in which one or both members of the pair did not harbor any disease loci. Families were rank-ordered by decreasing LOD scores on the covariate chromosome in order to highlight potential epistatic interactions. Empirical a priori knowledge of the disease gene location or of epistatic relationships between loci, we first used the OSA strategy of searching across the entire covariate chromosome for evidence of heterogeneity in the family-specific LOD scores using p-value thresholds of 0.01 and 0.05. The proportion of replicates with empirical p-values less than 0.01 ranged from 0.19 to 0.52, suggesting a grossly inflated global type I error rate. A Bonferroni correction based on approximately 100 covariate positions analyzed per analysis chromosome  resulted in a more reasonable proportion of replicates with significant results, ranging from 0.02 to 0.18. There appeared to be no relationship between the proportion of replicates with significant results and chromosome pairs reported to harbor epistatically interacting loci . There was likewise no relationship between the true disease locus position on the covariate chromosome and the position highlighted as significant when using the above p-value thresholds. There was little deviation between the position on the analysis chromosome where the OSA LOD score was maximized and the true analysis chromosome disease locus position.To our great surprise, nonparametric multipoint ASP analysis with SIBLINK generated very high LOD scores for D2 on chromosome 3 (297 cM), ranging from 3.2 to 18.1 across the 100 replicates with a mean LOD score of 7.8. Single-locus LOD scores for D3 on chromosome 5 (at 6 cM) were also high, ranging from 1.3 to 9.0, with a mean of 4.1. LOD scores were likewise high for D1 on chromosome 1  and D4 on chromosome 9 . As expected, the presence of such strong single-locus results made it difficult to detect significant LOD score increases in family subsets. Lower overall LOD scores were obtained for D5  and D6 , both of which are disease-modifying genes affecting penetrance of the disease. To simulate a real-world scenario, in which one has no p-values. For D1 and D2 , the OSA LOD score was maximized at (or very close to) the true location of the disease locus, regardless of the covariate chromosome used. For D3, D4, and D5, there was larger variability in the analysis chromosome position, with OSA maximizing the LOD score in general 10\u201320 cM distal to the actual disease locus. For locus D6, OSA failed to maximize the LOD score at or near the actual disease locus. This is not surprising because D6 is a modifying locus that affects penetrance, and our analyses were restricted to affected individuals only.Table p-value threshold  to properly correct for testing multiple chromosome pairs with many OSA covariates. To this end, we analyzed four pairs of chromosomes in which one or both members of the pair did not harbor any disease loci  are presumably correlated with each other.Because our targeted analysis is not feasible for real data in which the true disease gene locations are unknown, we focused on calculating a ci Table . This reConditional linkage analysis did not identify any significant two-locus linkage effects between chromosomes modeled to have epistatic relationships. As shown in Figure p-value for use in future analyses.There are several potential reasons for our failure to detect the epistatic interactions in this dataset. First, family-specific LOD scores as a statistical measure of linkage evidence are likely to be poor surrogates for underlying genotypes with epistatic interactions, and probably have much less variability than measured phenotypic covariates, especially in small nuclear families. Second, we chose to combine the three definitions of Kofendrerd Personality Disorder used by the respective ascertainment sites, but this apparently did not introduce sufficient heterogeneity for OSA to identify genetically  more homogeneous subsets of families. It may have been more appropriate to incorporate the phenotypic heterogeneity into our analyses and test whether OSA had been able to detect it, or to analyze P1, P2, and P3 separately in order to detect the phenotype-specific epistatic interactions. Third, the strong single-locus effects modeled in the data, the magnitude of which was unknown to us when we embarked on this study, may have diminished our ability to detect the weaker epistatic effects. Table p-value threshold for controlling the global type I error rate.In summary, OSA as a method for conditional linkage analysis did not obscure the readily detected linkage evidence on chromosomes known to harbor disease loci. However, it did not detect any of the simulated epistatic interactions in the GAW14 dataset, nor did it refine the previously well defined locations for the disease genes, primary due to the strong single-locus effects and little genetic heterogeneity modeled in the dataset. OSA remains an important potential tool to evaluate epistatic interactions. Our analyses were useful in allowing us to comment on type I error. In a genome-wide OSA analysis, type I error rates are inflated, indicating that a correction for multiple testing is very important in order to avoid follow-up of false-positive results. By analyzing simulated null chromosomes, we found our corrected alpha level to be 0.0006, which was very similar to a Bonferroni correction for approximately 100 covariate positions evaluated on each analysis chromosome. Thus, if OSA were used on real genome screen data to identify epistatic interactions, this would be a reasonable Although we failed to identify epistatic interactions using OSA for conditional linkage analysis, potentially due to strong single-locus effects and failure to incorporate KPD endophenotypes, we were able to calculate a corrected alpha level to control the global type I error when conducting such analyses in a genome screen.ASP: Affected sib pairGAW14: Genetic Analysis Workshop 14OSA: Ordered subsets analysisSNP: Single-nucleotide polymorphismSHS, WKS, ERH, SS contributed to study design, manuscript drafting and revision, and statistical analysis. MAS, HM contributed to statistical analysis. All authors read and approved the final manuscript."}
+{"text": "The multipoint single-locus model identified four regions of the genome with LOD scores greater than one. These regions were on 7p near D7S1790 (LOD = 1.31), two regions on 7q near D7S1870 (LOD = 1.15) and D7S1799 (LOD = 1.13) and 21q near D21S1440 and D21S1446 (LOD = 1.78). Jointly modeling these loci provided stronger evidence for linkage in each of these regions . The evidence for linkage tended to increase among pedigrees with earlier mean age of onset at 8q23 ( The Collaborative Study on the Genetics of Alcoholism (COGA) is a study designed to identify the genetic influences of alcoholism. Although alcoholism itself and the corresponding risk factors are heritable, they are strongly believed to be complex genetic traits. Thus, in the search for genes that influence these traits we expect significant genetic heterogeneity, gene \u00d7 gene, and gene \u00d7 environment interactions. Statistical methods that have the flexibility to simultaneously consider multiple loci and environmental factors are potentially valuable tools in the search for putative disease-predisposing loci. The purpose of this paper is to examine the evidence for linkage using multilocus nonparametric linkage regression modeling and explore whether the evidence for linkage varies by the age of onset of alcoholism ,2.The genotyped sample provided by COGA to the Genetics Analysis Workshop 14 consists of 102 Caucasian pedigrees  and 41 non-Caucasian pedigrees . Given the limited number of pedigrees of non-Caucasian ethnicity, this paper focuses on the self-reported Caucasian sample genotyped on 315 microsatellite markers and 15,406 autosomal single-nucleotide polymorphisms (SNPs). The alcohol dependence diagnosis required that an individual have DSM-III-R alcohol dependence and Feighner alc definite. This yielded four affection status classifications: 1) unaffected, 2) never drank, 3) unaffected with some symptoms, and 4) affected. The primary focus of these analyses will use affection status 4 only.p-value should be interpreted as a point-wise p-value and was not adjusted for the number of comparisons across the genome. All analyses are based on multipoint IBD estimates.The initial genome scan linkage analyses were computed using the nonparametric linkage (NPL) (pairs) and NPL  statistics under 1) the exponential allele-sharing model implemented in GENEHUNTER PLUS  and 2) ap-value (\u0394p). MERLIN [Ordered subset analyses (OSAs)  were com. MERLIN  was also. MERLIN  examinedn = 5), three (n = 30), four (n = 32), five (n = 21), six (n = 8) and seven or greater (n = 6) individuals diagnosed with alcoholism.Using the strictest criteria of affection status, there were 643 affected individuals from 102 families. Among these families there were 656 relative pairs including: 404 full-sib pairs, 9 half-sib pairs, 8 grandparent-grandchild pairs, 178 avuncular pairs and 19 other relative pairs. The families consisted of pedigrees with two , 7p21 , 7q11 , and 7q22 . Chromosome 11q23 near D11S1998 (120 cM) provided modest evidence for linkage in the single-locus model (LOD = 0.81). Figure The results of the multilocus NPL regression model building using the microsatellite data are summarized in Table p = 0.0016), 14q21 (p = 0.0079), and 18q12 (p = 0.0021). Conversely, the evidence for linkage tended to be greater among those pedigrees with later age of onset of alcoholism at 4q35 (p = 0.0067) and 9p22 (p = 0.0008). The difference in mean age of onset tended to be about two to three years among those pedigrees that linked to these regions versus those that did not link (Table p = 0.0350). A similar result was also found with OSA, subsetting on the families with the later age of onset of alcoholism increased the LOD score on 7q21 (LOD = 2.62 \u0394p = 0.11). Subsetting on later age of onset yielded a significant increase in the LOD score on 17q23 . Subsetting on earlier mean age of onset only significantly increased the evidence for linkage at 9q33 . The result on 7p21 was within 10 cM of a similar result for earlier age of onset of alcoholism found with the NPL regression interaction analysis (p = 0.02586).Table p-value of 0.009. The 7q peak is a series of peaks from about 100 cM to 160 cM; this region was found in the original COGA analyses [p-value of 0.02. The 21q22 result showed up in both the ASM analysis and the NPL regression analysis, but completely disappeared when the SNP analysis was done.Upon reviewing two previously published genome scans of alcohol dependence, two of our regions were identified in these published studies. The strongest evidence of linkage when looking across both the microsatellites and SNPs in this set of COGA data was on chromosome 7. The region on 7p was also identified in an American Indian population  within 1analyses  within 1Alcoholism is a genetically complex disease and therefore requires sophisticated consideration of multigenic and phenotypic influences. In this study methods that consider genetic heterogeneity, gene \u00d7 gene interactions, gene \u00d7 age-of-onset interactions, and joint modeling of multiple loci increased the evidence for linkage at three chromosomal locations, two of which had been previously identified as being associated with alcohol dependence. These methods reduced the linkage support interval at all three loci. In addition, testing for a dependence of the evidence for linkage on age of onset identified five additional regions of interest. These results suggest the potential utility of incorporating characteristics of complex genetic traits in the analysis.COGA: Collaborative Study on the Genetics of AlcoholismIBD: Identity by descentLD: Linkage disequilibriumNPL: Nonparametric linkageOSA: Ordered subset analysisSNP: Single-nucleotide polymorphismAHW, WMB and CDL contributed to the identification of the question and wrote the manuscript. AHW conducted all analyses."}
+{"text": "The cross-sectional measures included BMI at each of the four selected time points and the longitudinal measure was the within-subject mean of BMI at the above four time points.To evaluate linkage evidence for body mass index (BMI) using both cross-sectional and longitudinal data, we performed genome-wide multipoint linkage analyses on subjects who had complete data at four selected time points  was at time point 1, while the lowest (1.9) was at time point 4. We also observed other suggestive linkages on chromosome 6, 10, and 18 at time point 1 only.The longitudinal measure we studied (mean of BMI) did not provide greater power to identify a positive linkage than some of the cross-sectional measures . The changing of linkage evidence over time provided some insights on the variation of genetic effect on BMI with aging. There may be a QTL on chromosome 16 that contributes to BMI and this locus, and maybe others, is more likely to affect BMI during early adulthood. Obesity, which is influenced by both genetic and environmental factors, is an independent risk factor for coronary heart disease (CHD). However, the reported quantitative trait loci (QTLs) for body mass index (BMI), which is one of the obesity measures, are not consistent. The discrepancies among various studies represent the difficulties in identifying susceptibility genes for common complex traits and may be caused by any of the factors affecting the ability to detect linkage. These factors include, but are not limited to, sample sizes, ascertainment methods, long-term or short-term environmental influences, and genetic heterogeneity. In addition, variable age-dependent expression of a trait can also affect our ability to identify corresponding susceptibility genes. A longitudinal study design will provide a unique opportunity to evaluate such age related effects.However, due to their relative feasibility, cross-sectional designs using phenotype data collected at one time point are used in most linkage studies. As compared with the cross-sectional design, longitudinal data may be able to answer certain questions that cross-sectional data can not. For example, genes that determine change of a trait over time  can only be identified through a longitudinal design. Furthermore, serial observations over time of the same trait may allow more accurate partitioning of genetic and environmental components than would a single observation . Thus, lThe Framingham Heart Study is a very successful longitudinal study of cardiovascular diseases that was started in 1948. This study has obtained vast longitudinal cardiovascular-related phenotypic measures in two generations of participants. A 10-cM density genome-wide scan in subjects from 330 families was finished in the late 1990s. This provided a unique opportunity to identify QTLs for CHD-related traits using longitudinal phenotypes.Our goals in this study were 1) to identify the QTLs for BMI using multipoint linkage analyses and 2) to assess the consistency of QTLs identified over time using the Framingham Heart Study data.th, 12th, 16th, and 20th years after their initial visit, for a total of five visits. A total of 330 extended pedigrees consisting of 4692 subjects were selected for the genome scan in the Framingham Heart Study. Longitudinal phenotype measurements were available for 2885 subjects; 1712 had genotype data.The original cohort in the Framingham Heart Study was recruited in 1948: 5209 subjects were followed up every 2 years, with a total of 21 visits. In 1971, the study enrolled an additional 5124 adult children of the original participants and spouses of these children as the offspring cohort. This offspring cohort was followed up at 8To make the linkage analysis results from different time points comparable, we analyzed only those subjects with complete phenotypic data at all four selected visits. In addition, only families with at least two individuals (non parent-offspring relationship) who met such criteria were included in the analysis.2], was selected as the quantitative trait in our analysis because of its completeness and uniform measurement method over time. Data collected in year 1971, 1979, 1983, and 1987 from both the original cohort  and the offspring cohort  were denoted as BMI1 \u2013 BMI4, respectively. The within-subject mean of the four visits over 16 years, denoted as MEAN, was also generated for analysis.BMI [weight (kg)/ height (m)Linkage analyses were conducted using the variance components analysis method as implemented in the SOLAR program . This meMultipoint linkage analysis for quantitative traits was performed on BMI1 \u2013 BMI4 and the MEAN. The covariates evaluated in the linkage model were age, gender, and cohort. A covariate was retained in the linkage analysis model only if it reached significance at a level of 0.05.Table Figure We performed genome scan linkage analyses for BMI measured at four different time points (over 16 years) and the mean of BMI over the four time points in a select sample from the Framingham Heart Study. The strongest evidence of linkage was observed on the short arm of chromosome 16 near centromere (16p11.2-12) at time point 1 LOD = 3.0). Although this LOD score does not quite reach the genome-wide significant linkage level (3.3), we observed the same maximum peak with all other time points and the MEAN, with the lowest LOD score at time point 4 (LOD = 1.9). Using the same Framingham Heart Study data, Atwood and colleagues detected two linkage regions on chromosome 6 and 11 [.0. Althosibling) at time 1 was higher (44%) than 12 years later (36%). These authors also suggested that some familial factors affect BMI consistently overtime and some additional familial factors affect BMI at different times. If such age-related heritability variations are true, and they are determined by different genes, it is conceivable that certain genes can only be identified during certain periods of life, for example suggestive linkage at 6q23-25 was only observed at time point 1. The consistent location of the maximum LOD score for BMI over time on 16p11.2-12 in our study indicates that this putative QTL has a relatively lasting effect on BMI, while such effect may be veiled by environmental factors over time. The chromosome 16 locus may be a true susceptibility locus for BMI, but this is the first report of this locus so an independent confirmation would increase its validity.It is intriguing that the highest LOD score at 16p11.2-12 was observed at time point 1, while the lowest was at time point 4, and that three other suggestive linkages were all observed at time point 1. Since all subjects have data on all time points, the only difference between each time point is age and BMI values. Time point 1 captured a younger stage of the same population as compared with later time points. The observations that the maximum LODs decreased over time, and that other suggestive linkages were observed only at time point 1 suggest that these QTLs may have greater influence on BMI during the early years of life. Such variations of genetic influence with age may be further supported by a few available heritability studies of BMI. In a study of the National Heart, Lung, and Blood Institute male veteran twins, Fabsitz et al. estimated a heritability of 82% at age 20 and observed a somewhat reduced heritability after age 48 (72\u201378%) , maximalUsing longitudinal observations of the same trait, BMI, we derived MEAN as a longitudinal phenotype that represents an overall status of BMI across 16 years. However, MEAN did not improve LOD score at either the previously reported 6q23-25 location or at our best peak on chromosome 16. One explanation could be that the effects of these QTLs were reduced at later years of life so that the composite measure, MEAN, from all age periods averaged out the effect of these QTLs. Although in this study, this longitudinal design did not provide more power to identify QTLs for BMI than a single cross-sectional data at time point 1, without longitudinal data, we would not be able to observe variations of genetic effect overtime. With such knowledge, in order to increase power to identify QTLs for BMI, we may design future linkage studies by ascertaining families with young adults. However, keep in mind that the effects of different QTLs and environmental factors may be different during a lifetime. When we restrict sample selection, we may gain power to identify certain genes but may not be able to identify others.In summary, BMI varies with age, and different genes may determine variations in the population at different age periods. For example, genes that influence childhood obesity may be different from genes that influence adulthood obesity. Nevertheless, when we analyzed cross-sectional BMI data at different time points and the mean of all time points, we observed a consistent linkage with BMI at 16p11.2-12 across all time points, and three other suggestive linkages, including the previously reported 6q23-25. In addition, we observed variation in LOD score over time with the highest at time point 1 and the lowest at time point 4 (16 years later). These results indicate that there may be a QTL on chromosome 16 that contributes to BMI and this locus, and maybe others, is more likely to affect BMI during early adulthood."}
+{"text": "An empirical comparison between three different methods for estimation of pair-wise identity-by-descent (IBD) sharing at marker loci was conducted in order to quantify the resulting differences in power and localization precision in variance components-based linkage analysis. On the examined simulated, error-free data set, it was found that an increase in accuracy of allele sharing calculation resulted in an increase in power to detect linkage. Linkage analysis based on approximate multi-marker IBD matrices computed by a Markov chain Monte Carlo approach was much more powerful than linkage analysis based on exact single-marker IBD probabilities. A \"multiple two-point\" approximation to true \"multipoint\" IBD computation was found to be roughly intermediate in power. Both multi-marker approaches were similar to each other in accuracy of localization of the quantitative trait locus and far superior to the single-marker approach. The overall conclusions of this study with respect to power are expected to also hold for different data structures and situations, even though the degree of superiority of one approach over another depends on the specific circumstances. It should be kept in mind, however, that an increase in computational accuracy is expected to go hand in hand with a decrease in robustness to various sources of errors. We s. We s5]]"}
+{"text": "Common human disorders, such as alcoholism, may be the result of interactions of many genes as well as environmental risk factors. Therefore, it is important to incorporate gene \u00d7 gene and gene \u00d7 environment interactions in complex disease gene mapping. In this study, we applied a robust Bayesian genome screening method that can incorporate interaction effects to map genes underlying alcoholism through its application to the data of the Collaborative Studies on Genetics of Alcoholism provided by Genetic Analysis Workshop 14. Our Bayesian genome screening method uses the regression-based stochastic variable selection, coupled with the new Haseman-Elston method to identify markers linked to phenotypes of interest. Compared to traditional linkage methods based on single-gene disease models, our method allows for multilocus disease models for simultaneous screening including both main and interaction (epistatic) effects. It is conceptually simple and computationally efficient through the use of Gibbs sampler. We conducted genome-wide analysis and comparison between scans based on microsatellites and single-nucleotide polymorphisms. A total of 328 microsatellites and 11,560 single-nucleotide polymorphisms (by Affymetrix) on 22 autosomal chromosomes and sex chromosome were used. Alcohol dependence is a complex disorder that is influenced by many genetic and environmental factors. Identifying genes associated with alcohol dependence is critical to understand its etiology and to develop efficient methods for prevention and treatment. However, this effort has been hampered by the complexity underlying alcohol dependence: rather than there being one or a few major genes affecting alcohol dependence, it is likely that multiple genes interact with each other, together with environmental factors, to affect susceptibility to alcohol dependence. In this paper, we describe analyses of the Collaborative Study on the Genetics of Alcoholism (COGA) data Problem 1), using self-reported \"maximum number of drinks consumed in a 24-hour period\" (denoted by M) as a quantitative trait, to map genes underlying alcohol dependence. The measure M is closely related to alcoholism diagnosis and provides a quantitative measure for alcohol dependence. For genome screens for this trait, we use the modified Haseman-Elston regression method [, using sThe Haseman-Elston method and its derivatives allow one to apply linear regression methods in linkage analysis. For each sibling pair, these methods use the number of alleles identical by descent (IBD) at each marker as the explanatory variable and a statistic measuring similarity of the quantitative traits in the sibling pair, squared difference, or cross-product, as the response variable.In practice, the number of markers and their possible epistatic effects are often larger than the number of observations (patients or sib-pairs), where the design model is referred as being \"supersaturated\". As we often have hundreds of markers to consider, we must deal with the problem of multiple testing in this context. Besides, if one would like to take epistasis into account, the number of tests can easily exceed tens of thousands. Performing hypothesis tests for linkage for all of these possibilities without appropriate adjustment of multiple comparisons can lead to the identification of spurious genetic effects or the masking of real effects. The supersaturated nature of the design model also makes the conventional best subset model selection methods  practicaIn this study, we apply the method developed by Oh  to the CD2 = (X1 - X2)2) in pairs of siblings on the number of alleles shared IBD between each sib pair at a given marker. Although the original Haseman-Elston method is simple, robust, and computational inexpensive, it may ignore information contained in the observed bivariate data. In fact, the squared mean corrected trait sum of sib pairs (S2 = (X1 + X2 - 2\u03bc)2) may provide additional information on the genetic effect [D2 and S2 in linkage analysis to improve statistical power. One of the simplest methods was proposed by Elston et al. [CP = (S2 - D2)/4 = (X1 - \u03bc)(X2 - \u03bc) as the response variable in the regression. It essentially averages both regression coefficient estimates (of D2 and S2) with equal weights. An additional advantage of using CP as the response variable is that it may be more normally distributed than D2 and S2.The original Haseman-Elston method  is a genc effect . This obD2 = X - X22) im markers along the genome. Among these m markers, some may display some evidence of tight linkage to genes with large effects and others only with weak effects. They may exhibit main effects and/or epistatic effects. In our regression setup, the CPs are treated as the responses and the IBD status at each marker Xj is the candidate explanatory variable. Then the observed CP value of sib-pair i can be described by the following linear modelAssume that we observe n is the number of sib pairs and the errors \u03b5~N are assumed to be independent. A subset model is represented by a binary vector \u03b3 = , where \u03b3j = 1 or \u03b3j = 0 represents the presence or absence of variable j in the model. A large effect of \u03b1j indicates that marker j has the evidence for linkage and  indicates the epistatic effect between markers j1 and j2. The marker effects \u03b1j  are given the prior of the mixture of normal and point-mass distributions conditional on the indicators \u03b3j, \u03b1j|\u03b3j~\u03b3jN + (1 - \u03b3j)\u03b4o. Similarly, the epistatic effects  with j1 <j2 are given . Hence, if the effect is absent in the model choice (\u03b3j = 0), we can simply omit that factor when building the model. If \u03b3j = 1, the magnitude of the effect is large and then a nonzero estimate should be included in the model and its posterior distribution is largely determined by the data. The prior \u03b3 should represent beliefs about the relationship between factors. If only main effects but no epistatic effects are considered, the prior of \u03b3 can be simply set as prob(\u03b3) = pk, where k is the number of ones in \u03b3. However, when epistatic effects are considered in model selection, the model space becomes enormous and the independent relationship may not be the proper assumption anymore. In this case, we incorporate Chipman's [ is present  may take on four different values, depending on the values of the pair ,where hipman's  hierarchp00,p01,p10,p11) represents the belief of the relationship between factors  (main effects), and  (epistatic effects). If we believe that an epistatic effect without main effects is quite unlikely, we can simply set the prior as  = . Alternatively, we can use  =  to relax these conditions.The choice of the values,  for microsatellites and MERLIN for SNPs for each sib pair at each marker. Because it is impractical to track the complete posterior of \u03b3, only the marginal posterior of each marker is obtained. In our analysis, we use  =  to give all the factors an equal opportunity to be included in the final model as long as one of main effects is in the model.In microsatellites and SNPs, there are 328 and 7,826 markers considered, respectively. Therefore, to consider epistatic effects, we needed to include 53,957 factors for microsatellites and about 30 millions factors for SNPs in the models. We set the prior as In the full sample of cases, the quantitative trait M ranges from 0 to 160. Five individuals are in the highest threshold class (M > 128 drinks). The highest reported M is 160. The use of the log transformation minimizes their impact on the regression analysis, which can be inflated by self-report . As repoIn each analysis, the Markov chain Monte Carlo (MCMC) sampler was run for 100,000 cycles after discarding the first 2,000 cycles for the burn-in period. Because MCMC samplers arise from recursive draws, they produce correlated samplers from the posteriors. Therefore, the chains are thinned (one iteration in every 10 cycles is saved) to reduce serial correlation in the stored samples. The total number of samples kept in the post-Bayesian analysis is 10,000. It takes ~2 hours for microsatellites and ~6 hours for SNPs to generate each sample with JAVA programs on a Linux cluster using 2.4-GHz Intel processors. Table In this study, we have compared the genome-wide linkage analyses based on microsatellites and SNPs. Our methods located the main effects of markers both from microsatellites and SNPs and produced similar patterns between them. However, the results for epistatic effect screening are less consistent and revealing. This might be purely because these epistatic effects are weak in nature and further research in this area is warranted.Bayesian genome screening methods provide a powerful and efficient tool in identifying potential markers and their epistatic effects. They are very effective because they are able to conduct searches over the entire model space; while the frequentist's best subset model selection procedure is constrained by computing power required to examine all candidate models. In addition, Bayesian genome screening methods can work on problems with many more candidate variables, which is essential to consider when epistatic effects are studied. When one tries to locate the epistatic effects, the number of covariates (factors) easily far outnumbers the sample size. Most traditional linkage methods do not work under this condition because they often assume a single-gene model and test effects one at a time. By using the prior structures that reflect the relationship among the candidate variables, our general approach can accommodate a large number of candidate markers as well as their epistatic effects by evaluating all factors simultaneously. We were able to locate markers on chromosome 4 that show the strong evidence of linkage with alcoholism related to quantitative phenotype, \"maximum number of drinks consumed in a 24-hour period\", both from microsatellite and SNP scans and weak evidence for epistatic effects.COGA: Collaborative Study on the Genetics of AlcoholismGAW14: Genetic Analysis Workshop 14IBD: Identical-by-descentMCMC: Markov chain Monte CarloSNP: Single-nucleotide polymorphismSSVS: Stochastic search variable selectionCO participated in the design of the study, performed the analysis, and drafted the manuscript. SW helped to obtain IBD values for linkage analysis. SW, NL, LC, and HZ participated in the design and the discussion of the study, and the preparation of the manuscript. All authors read and approved the final manuscript."}
+{"text": "Genome scans using dense single-nucleotide polymorphism (SNP) data have recently become a reality. It is thought that the increase in information content for linkage analysis as a result of the denser scans will help refine previously identified linkage regions and possibly identify new regions not identifiable using the sparser, microsatellite scans. In the context of the dense SNP scans, it is also possible to consider association strategies to provide even more information about potential regions of interest. To circumvent the multiple-testing issues inherent in association analysis, we use a recently developed strategy, implemented in PBAT, which screens the data to identify the optimal SNPs for testing, without biasing the nominal significance level. We compare the results from the PBAT analysis to that of quantitative linkage analysis on chromosome 4 using the Collaborative Study on the Genetics of Alcoholism data, as released through Genetic Analysis Workshop 14. The rapid advance of genotyping technology has resulted in a wealth of new, high-quality data that may hold promise for the further elucidation of the genetic determinants underlying complex disease. The ultimate utility of such rich data may be limited in scope by existing methods of linkage and association analysis. For example, it is somewhat unclear as to whether increasingly dense single-nucleotide polymorphism (SNP) genome scans will provide the necessary boost in power and/or information to uncover genes of modest effect size. Further, association methods will be subjected to extreme multiple comparison issues, as the number of statistical tests balloon with the vast number of available SNPs. To address the issue of multiple comparisons, recently developed screening tools implemented in PBAT  have theThe data provided for Problem 1 in the GAW14 dataset (COGA Study) includes genotypes from the Affymetrix GeneChip\u2122 Human Mapping 10 K array (Affymetrix), comprises 11,555 SNPs as well as quantitative trait information for approximately 1,614 subjects from 143 families of varying size and structure. Here, we focus on the quantitative trait data from the Eyes Closed Resting electroencephalogram experiment, and in particular the measure that corresponds to the first component of a trilinear singular value decomposition of the beta2 band and bipolar electrode data (ECB21). ECB21 was shown to be approximately normally distributed with a mean of 14.53 (standard deviation = 5.5) and ranged from 4.43 to 36.06. There was no substantial skewness or kurtosis found with the ECB21 trait. We restricted our analysis to genotypes from the 786 Affymetrix SNPs on chromosome 4. We chose chromosome 4 because it has been proposed to harbor a region of linkage to the ECB21 phenotype . Figure p-value) using the MERLIN LOD score p-value. We sequentially removed markers that were determined by HAPLOVIEW to be in LD both in the linkage region and outside the linkage region and found no difference in the resulting LOD score (data not shown).Variance component linkage analysis as implemented in MERLIN resulted in a LOD score of 3.55 . The SNP showing the strongest association was TSC0053776 (p = 0.0011), located at around 100 cM . After Bonferroni-adjustment for multiple comparisons (number of tests = 786), none of SNPs achieved statistical significance at the 5% significance level. We also assessed significance using the false discovery rate (FDR) method [q-value for SNP TSC0053776 = 0.63). Further, the highest LOD score among the 10 most significant SNPs was 2.18, for SNP TSC0286605. This SNP also had the lowest PBAT p-value among all SNPs that had LOD scores of greater than 2.0. Other PBAT p-values from SNPs with LOD scores over 2.0 were modest, with only three of them having p-values of less than 0.05. Using PBAT's screening utility, we focused our analysis on the five most powerful SNPs. Table p-values for the five most powerful SNPs as identified through PBAT's screening utility (note: two symbols overlap at around 36 cM). The horizontal dotted line refers to the 5% significance level after Bonferroni-adjusting for five comparisons. The black dots are the p-values for all other SNPs tested using PBAT.Table ) method . Consist p = 0.001, locateUsing Affymetrix SNPs from the COGA dataset, we identified a region that is linked LOD = 3.55) to the ECB21 phenotype at approximately 70 cM. This region had been previously identified in the COGA dataset by Reich et al. [.55 to thp = 0.0081 and 0.0085, respectively). As expected, testing each of the 786 SNPs for association resulting in a severe multiple-testing issue, as none of the SNPs across chromosome 4 was found to be statistically significant using either a Bonferroni correction or FDR methods. However, using PBAT's screening strategy allowed us to reduce the number of tests. We chose to test the top five most powerful SNPs as identified by PBAT, and found two SNPs significantly associated with ECB21 at the 5% significance level (after Bonferroni-adjustment for five tests). These two SNPs are physically very close to each other (~2 kb), and when tested together using PBAT's haplotype analysis function, the resulting 2 SNP haplotype maintained its significance and relative power (data not shown).Using the screening, we also identified two SNPs that are potentially associated with the ECB21 phenotype at approximately 35 cM  it is possible that the SNPs (in relatively low LD) displaying linkage are not associated with the underlying causal locus. It should also be noted that the linkage analysis conducted in the present study was not optimally performed and therefore, we did not maximize the linkage signal. However, the intent of the present study was to compare the two strategies by highlighting key similarities and differences, and not necessarily providing evidence in support of one strategy over the other. Furthermore, we propose that collectively, both strategies may prove useful in high-density genome-wide scans.Interestingly, the selected SNPs were not found to be located directly in the linkage region, as the significant SNPs are approximately 30\u201340 cM from the maximum LOD score. Furthermore, the LOD scores corresponding to the selected SNPs were 0. The discrepancy in these findings may be explained in a number of ways, particularly when one considers that the alternative hypothesis of the FBAT strategy is the presence of linkage ct sizes . TherefoWe compared the similarities and differences between linkage analysis and PBAT's approach to association analysis, using the same quantitative trait and using the same marker set. In this brief exploration, we did not find that linkage and association necessarily provided concordant results. Nonetheless, in the context of the high-density SNP scans, we feel that utilizing new strategies for association testing may provide additional information not otherwise discovered using linkage analysis alone.COGA: Collaborative Study on the Genetics of AlcoholismFDR: False discovery rateGAW: Genetic Analysis WorkshopLD: Linkage disequilibriumSNP: Single-nucleotide polymorphismMBM carried out the linkage analysis and drafted the manuscript. AM carried out portions of the association analysis and aided in drafting the manuscript. PK participated in the design and coordination of the study. JS participated in the design and helped with the analysis. RL participated in the design of the study. NL helped conceive of the study and participated in coordination of the study. CL helped conceive of the study and conducted the association analysis. KVS conceived the study and participated in the coordination of the study."}
+{"text": "Current statistical methods for sib-pair linkage analysis of complex diseases include linear models, generalized linear models, and novel data mining techniques. The purpose of this study was to further investigate the utility and properties of a novel pattern recognition technique  using the chromosome 10 linkage data from the Framingham Heart Study and by comparing it with step-wise logistic regression and linear regression.The three step-wise approaches were compared in terms of statistical significance and gene localization. Step-wise discriminant linkage analysis approach performed best; next was step-wise logistic regression; and step-wise linear regression was the least efficient because it ignored the categorical nature of disease phenotypes. Nevertheless, all three methods successfully identified the previously reported chromosomal region linked to human hypertension, marker GATA64A09. We also explored the possibility of using the discriminant analysis to detect gene \u00d7 gene and gene \u00d7 environment interactions. There was evidence to suggest the existence of gene \u00d7 environment interactions between markers GATA64A09 or GATA115E01 and hypertension treatment and gene \u00d7 gene interactions between markers GATA64A09 and GATA115E01. Finally, we answered the theoretical question \"Is a trichotomous phenotype more efficient than a binary?\" Unlike logistic regression, discriminant sib-pair linkage analysis might have more power to detect linkage to a binary phenotype than a trichotomous one.We confirmed our previous speculation that step-wise discriminant analysis is useful for genetic mapping of complex diseases. This analysis also supported the possibility of the pattern recognition technique for investigating gene \u00d7 gene or gene \u00d7 environment interactions. The search for efficient and powerful statistical methods and optimal mapping strategies for complex human diseases that are categorical in nature continues to be one of the main tasks faced by genetic epidemiologists. Sib-pair linkage analysis is one of the most popular methods (designs). The possible statistical methods for sib-pair linkage analysis of categorical human diseases include linear regression , generalized linear models , and the novel pattern recognition techniques  diagnosed to be hypertensive. This type of phenotypic partition is analogous to a clinical scoring system [P = 0.05)) were kept identical.We used the summary method proposed by Levy et al. . Two hypg system  to assesThe methodological details were described previously ,3. The fP-value for each (selected) independent variable was obtained from the final model fitting with only selected variables included via an asymptotic chi-squared statistic.The group variable was considered as an ordinal (or binary) dependent variable and the feature vector (multiple marker IBD estimates and covariates) as the independent variables. A nonlinear relationship (logit) between the dependent and independent variables was taken. For binary data (collapsing concordantly affected or unaffected sib pair into one group), a conventional logistic regression was used. For ordinal data, the SAS LOGISTIC  procedurP-values for the finally included predictors, determined by an F statistic, were reported.We extended the new version of Haseman-Elston regression by including multiple marker information and the analysis proceeds in a step-wise manner. The binary disease phenotypes were taken as if they were continuous, i.e., by giving \"affected\" and \"unaffected\" different quantitative scores-without loss of generality, 1 and 0, respectively. Then, the centered cross-product of two sibs' phenotypic values was linearly regressed onto the proportion of alleles that the sibs shared IBD at markers. The covariates were coded similarly to that for the dependent variable and fitted in the regression for adjustment. The SAS REG  was usedThe summary of linkage analysis of longitudinal SBP and HBP, with and without adjusting the hypertension risk factors as well as assessment of these risk factors using three different approaches, is given in Tables Due to the sequential nature of the partial F statistics used in STEPDISC, we can easily infer the joint actions of two effects (interactions), as demonstrated in the following. Table P = 0.0414).It is a generally accepted notion that an ordinal (with more than two categories) phenotype contains more information than binary data and thereafter is more efficient for linkage analysis if a generalized linear model is used . HoweverP-values might be liberal and might deviate from the true chromosome-wide P-values, as suggested by our simulations [Although most findings for the utility, performance and properties of STEPDISC in this study were also confirmed through a large simulation, and an application to a simulated disease for GAW12 , we viewulations . FurtherWe have compared three typical sequential statistical methods for genetic mapping of complex diseases. Genetic analyses for categorical traits are known to be difficult because phenotype cannot be described by a linear function of genetic and environmental effects. In the sib-pair-based linkage analysis, the very act of taking a quadratic form of sibs' phenotypic values has changed the relationship between the model components and renders the relationship unclear. Several issues for sib-pair based linkage analysis deserve our attention. First, what kind of relationship, linear or nonlinear, should be taken to describe the relationship between the new phenotype and its determinant, IBD values? Fortunately, our proposed discriminant sib-pair-linkage analysis does not require explicitly specifying this relationship and thus tactically avoids this difficulty. Second, how do we rank affection groups? Is the order important? To answer this question, we phenotyped the binary diseases (binary SBP and HBP) by giving \"affected\" a value of 0 and \"unaffected\" a value of 1 so that for logistic regression based linkage analysis, concordantly affected sib pair was coded as 0 and concordantly unaffected sib pair was coded as 2. Identical results were obtained for all the three methods (data not shown), indicating that interchanging the positions for two concordant groups has no effect for sib-pair linkage analysis of binary diseases. It can be easily shown that the new coding for marginal phenotypes does not change the numerical values of the centralized cross-product for the three affection groups, but a rigid mathematical proof is needed for logistic modelling. Finally, we conducted additional analysis to investigate the effects of collapsing two concordant groups into a single group. Using the collapsed two-group data, we did not identify a single marker to be significantly linked to hypertension phenotypes using all the three methods (data not shown), suggesting that this common collapsing practice lead to loss of statistical efficiency.Step-wise linear regression, logistic regression, and discriminant analysis are three representatives of sequential statistical methods that are potentially useful for sib-pair linkage analysis of complex human diseases. All the methods successfully identified the previously reported linked region, marker GATA64A09, at the chromosome-wide significance level of 0.05. However, from both theoretical and applied views, step-wise discriminant analysis appears to be the most efficient for sib-pair linkage studies. This conclusion was supported by this and the previous studies ,3. Furth"}
+{"text": "This paper summarizes the ease of detection for each of the simulated Kofendrerd Personality Disorder (KPD) genes across all of the replicates for five standard linkage statistics. Using the KPD affection status, we have analyzed the microsatellite markers flanking each of the disease genes, plus an additional 2 markers that were not linked to any of the disease loci. All markers were analyzed using the following two-point linkage methods: 1) a MMLS, which is a standard admixture LOD score maximized over \u03b8, \u03b1, and mode of inheritance, 2) a MLS calculated by GENEHUNTER, 3) the Kong and Cox LOD score as computed by MERLIN, 4) a MOD score , and 5) the PPL, a Bayesian statistic that directly measures the strength of evidence for linkage to a marker. All of the major loci (D1\u2013D4) were detectable with varying probabilities in the different populations. However, the modifier genes (D5 and D6) were difficult to detect, with similar distributions under the null and alternative across populations and statistics. The pooling of the four datasets in each replicate ( In this study we used the simulated the Genetic Analysis Workshop 14 (GAW14) data using the Kofendrerd Personality Disorder (KPD) affection status as our phenotype. We did this with full knowledge of the generating model. We chose to examine the performance of the statistics by comparing markers flanking a known disease gene location to a pair of markers from a chromosome containing no disease genes. Our data consist of 13 markers: two markers flanking D1, D3, D4, D5, and D6, a single marker flanking D2 , and our arbitrarily chosen unlinked markers, D04S128 and D04S129, which we refer to as markers flanking unlinked locus U1. We analyzed the data from all 100 replicates in each of the four populations as well as creating a pooled dataset of 350 pedigrees created by combining the data from all four populations.The first statistic we examined was the maximized maximum LOD score (MMLS) -3 that iRisch's maximum LOD score statistics (MLS scores) ,6 were cTwo-point Kong and Cox LOD scores (KCLS) , were coWe examined the performance of a Bayesian statistic, the posterior probability of linkage (PPL) -13. The Finally, we present the results of the MOD  score, wThe mean and maximum scores for flanking markers at each disease locus and each of the methods for each of the populations and the pooled data are contained in Table In general, MMLS and MOD scores are larger than MLS and KCLS scores. However, the MMLS and MOD scores were also higher for the unlinked locus than the other two methods, so that the increase in score does not necessarily indicate an increase in power. Nonetheless, there are a few things that can be determined from Table Table We have compared the performance of five statistics, the MMLS, the MLS, the KCLS, the MOD, and the PPL, by examining markers flanking the known disease locations in the GAW14 simulated data. By computing P, which is an empirical measure of the power, we are able to compare statistics that have different scales. We find that none of the statistics emerges a clear victor, with different statistics having greater power depending on which disease locus and population were examined. However, it is surprising that the MMLS and MOD score, which make use of the entire pedigree structure , and whose scores were calculated without any trimming or dropping of large pedigrees, were not able to utilize this information to their advantage in the NY population, where the sample consists of extended pedigrees. This is due to the high values of these statistics obtained under the null. The PPL performs better in the NY dataset, using data from the entire pedigree, without a similar inflation of null values. D1\u2013D4 appear detectable, with maximum scores in the range that would indicate linkage. However, values of P were surprisingly low for these loci, especially since the maximum values under the null, presented in Table GAW14: Genetic Analysis Workshop 14HLOD: Heterogeneity LODIBD: Identity by descentKCLS: Kong and Cox LOD scoresKPD: Kofendrerd Personality DisorderMLS: Maximum LOD score statisticMMLS: Maximized maximum LOD scorePPL: Posterior probability of linkageMWL performed analyses and prepared a draft of the manuscript. MWL, MAS, and VJV contributed computing resources. All authors contributed to study design and editing, and approved the final manuscript."}
+{"text": "We used ALDX1 and ALDX2, the COGA definitions of alcohol dependence, as well as electrophysiological measures TTTH1 and ECB21 to detect alcoholism susceptibility loci. Many chromosomal regions were found to be significant for each of the phenotypes at a p-value of 0.05. The most significant region for ALDX1 is on chromosome 7, with a maximum LOD score of 2.25 for Affymetrix SNPs, 1.97 for Illumina SNPs, and 1.72 for microsatellites. The same regions on chromosome 7 (96\u2013106 cM) and 10 (149\u2013176 cM) were found to be significant for both ALDX1 and ALDX2. A region on chromosome 7 (112\u2013153 cM) and a region on chromosome 6 (169\u2013185 cM) were identified as the most significant regions for TTTH1 and ECB21, respectively. We also performed linkage analysis on denser maps of markers by combining the SNPs datasets from Affymetrix and Illumina. Adding the microsatellite data to the combined SNP dataset improved the results only marginally. The results indicated that SNPs outperform microsatellites with the densest marker sets performing the best.The Collaborative Study on the Genetics of Alcoholism (COGA) is a large-scale family study designed to identify genes that affect the risk for alcoholism and alcohol-related phenotypes. We performed genome-wide linkage analyses on the COGA data made available to participants in the Genetic Analysis Workshop 14 (GAW 14). The dataset comprised 1,350 participants from 143 families. The samples were analyzed on three technologies: microsatellites spaced at 10 cM, Affymetrix GeneChip Alcoholism is a complex disorder in which multiple genes may contribute to the risk . To addrALDX1, the primary COGA definition of alcohol dependence, requires a person to meet both DSM-III-R criteria and the Feighner criteria . LinkageThe microsatellite-based screening approach has been used successfully for mapping Mendelian diseases. However, this technique has been proven to be unreliable for complex genetic diseases ,9. It ha\u00ae Human Mapping 10 K Array (HMA10K), and Illumina SNP-based Linkage III Panel. To identify susceptibility regions for alcoholism, we performed a genome-wide multipoint linkage analysis using alcohol dependence phenotypes ALDX1, ALDX2 (diagnosed by DSM-IV criteria), and quantitative traits TTTH1 and ECB21. The performance of microsatellites, Affymetrix HMA10K Array, and Illumina Linkage III Panel were compared in terms of information content, identified linkage regions and the 1-LOD support interval of the regions.The COGA dataset provided to participants at GAW14 included data from 1,350 participants from 143 families. The genotype dataset included data produced by a 10-cM map of microsatellites, Affymetrix GeneChipThe 10-cM microsatellite maps contained 328 microsatellites of which 309 have unique locations on the deCode high-resolution genetic maps. To map the SNPs, we first obtained the physical locations from build 34 of the human genome dbSNP database at the National Center for Biotechnology Information (NCBI). We then interpolated the genetic map locations using the microsatellite with unique physical locations in deCode genetic maps. 11,050 Affymetrix SNPs and 4,700 Illumina SNPs with unique sex-averaged genetic map locations were used in our study. We also created an even denser map of markers by combining Affymetrix and Illumina SNPs (Comb2). In addition, we combined Affymetrix and Illumina SNPs with the microsatellite data (Comb3) to determine the contribution of microsatellite markers.The datasets were prepared with PEDCHECK  to removInformation content (IC) measures how much of the inheritance information can be extracted from available genotype data. It closely predicts the power of a map to detect linkage . We usedall [p-values and can be used to construct 1-LOD support intervals [Alcohol dependence phenotypes ALDX1 and ALDX2 include five categories: no information; pure unaffected; never drank; unaffected with some symptoms; affected. We treated \"never drank\" as \"no information\", and combined \"pure unaffected\" and \"unaffected with some symptoms\" as \"unaffected.\" We performed nonparametric linkage (NPL) analysis based on the identity-by-descent (IBD) sharing among affected individuals in a pedigree. We used MERLIN to calculate NPLall  and the all . NPL scoall ,18. The ntervals . For thiWe conducted variance components analyses on the log transformed quantitative traits TTTH1 and ECB21, adjusting for age and sex. Heritability of the traits and the LOD scores at every marker locus were calculated by using MERLIN.MERLIN is a software package designed for dense genetic maps in pedigree data. It efficiently implements the Lander-Green algorithm  by usingThe summary statistics for the data are shown in Table Microsatellites had the lowest mean and highest standard deviation (SD) of genome-wide IC due to the limited coverage of the genome Table . With thBoth ALDX1 and ALDX2 phenotypes showed significant linkage on chromosome 7 (96\u2013106 cM) and chromosome 10 149\u2013176 cM) in all the datasets. In the SNP datasets, both phenotypes detected the same region on chromosome X (30\u201346 cM), although the LOD scores for ALDX2 were much less significant , 7 (96\u2013106 cM), 10 (141\u2013176 cM), and 17 (30\u201353 cM) were significant at a level of 0.05 in all the three datasets. Other significant regions on chromosome 2, 3, 6, 7, 9, 10, and X were detected in SNP data sets but were not present in the microsatellite data  overlaps with the one reported by Jones et al. [The estimate of heritability for ECB21 is 55.54% after adjusting for age and sex. One of the linkage regions on chromosome 4  overlaps with the highly significant linkage region found in previous studies . The mosBased on the COGA data provided to participants at GAW14, we have presented a NPL analysis for alcohol dependence phenotypes ALDX1 and ALDX2, and a variance component analysis for EEG measures TTTH1 and ECB21. Our results confirmed some of the linkage findings in previous studies -7. The ip-value of 0.05 as the significance level to report linkage regions. However, in order to define true linkage and explain the inconsistencies among the results of different datasets, it is important to choose a level of genome-wide significance. Commonly used resampling-based and gene-drop simulation approaches are computationally intensive and do not lend themselves to the analysis of the large amount of data in this study. We are investigating a more efficient Monte Carlo procedure to assess genome-wide significance in linkage analysis [In our study, we used a analysis .Our results show that a denser map can be more powerful for linkage analysis. IBD sharing based linkage analysis algorithms usually assume linkage equilibrium between the markers and the strong linkage disequilibrium between closely adjacent markers could potentially introduce false linkage results ,22. A stThis study represents an extensive performance comparison of three different platforms  in a series of linkage analyses for alcoholism. The high density and the robust performance of SNPs make the whole-genome scan a desirable approach for linkage analysis. This new approach may bring a renewed power to IBD sharing based linkage analysis.COGA: Collaborative Study on the Genetics of AlcoholismERP: Event-related potentialGAW: Genetic Analysis WorkshopIBD: Identity by descentIC: Information contentNPL: Nonparametric linkageSNP: Single-nucleotide polymorphismCZ carried out the statistical analysis and drafted the manuscript. SC assembled the Affymetrix linkage data, conducted qualify control and provided useful comments for the study. GL constructed unique genetic maps for all the data sets. MC generated data from the Affymetrix HMA10K array. HG modified MERLIN program to reduce the memory usage. GCK conceived of the study and participated in its design and coordination."}
+{"text": "This paper explores the decay of linkage disequilibrium (LD) on the autosomes and chromosome X. The extent of marker-marker LD is important for both linkage and association studies. The analysis of the Caucasian sample from the Collaborative Study on the Genetics of Alcoholism study revealed the expected negative relationship between the magnitude of the marker-marker LD and distance (cM), with the male and female subgroups exhibiting similar patterns of LD. The observed extent of LD in females was less across the pseudoautosomal markers relative to the heterosomal region of chromosome X. Marked differences in LD patterns were also observed between chromosomes X and the 22 autosomes in both males and females. In the human genome, alleles at two loci on the same chromosome often show stochastic dependence. This nonrandom pair-wise allelic association, or linkage disequilibrium (LD), is both interesting evolutionarily in its own right and a powerful tool in the mapping of genes for complex genetic traits. The development of the technology to genotype large numbers of single-nucleotide polymorphisms (SNPs) provides the means to explore the extent and patterns of linkage disequilibrium in the genome. The two pseudoautosomal regions on chromosomes X and Y PAR1 and PAR2) are approximately 2.6 and 0.4 Mb respectively, and are located at the tips of the sex chromosomes. Within these regions pairing and recombination may occur between X and Y as it naturally does in the autosomes or between two X chromosomes in females. Dense genetic linkage maps have revealed a high rate of recombination in PAR1 [ and PAR2Data used for our analyses were obtained from the Collaborative Study on the Genetics of Alcoholism (COGA). COGA is a large-scale family study designed to identify genes that contribute to the risk for alcoholism. Our analyses used data genotyped by both Affymetrix and Illumina. Four hundred and thirty-four genetic markers on chromosome X were analyzed (310 SNPs by Affymetrix and 124 SNPs by Illumina). SNPs genotyped and analyzed on the 22 autosomes by Affymetrix and Illumina totaled 15,371. Given that the proportion of self-reported non-Caucasian individuals in the COGA sample was small, our results are based on 102 Caucasian pedigrees. These 102 pedigrees were composed of 430 genotyped male and 446 genotyped female participants; 9 genotyped participants whose sex was not reported were not included in these analyses.r2 = D2/(p1p2q1q2) was calculated for each adjacent pair of markers across the genome. Here, D is defined as D = x11 - p1q1  [r2 results to the exponential, Weibull, Gamma and log-normal distributions, adjusting for distance (cM). Among these distributions, the Weibull distribution provided the best fit and will be reported. Inferences based on the other distributions are comparable. Note that these models do not account for the fact that each individual contributes multiple observations to the analysis  Chi-squared tests were used to compare the sex-specific Weibull distributions of r2 1) within the X chromosome, 2) between the pter pseudoautosomal region (PAR1) on X and the heterosomal region on X, 3) between PAR1 and the autosomes, and 4) between chromosome X and the autosomes.A sample of 172 genotyped unrelated participants was derived from the COGA pedigrees in order to compute LD statistics. To form the sample of unrelateds, a hierarchical approach was used. In pedigrees in which founders were genotyped, all available founders were chosen for analysis. Where founders were not genotyped, the proband (index case) was used if a proband was declared. Otherwise, the individual with the smallest COGA identification number within each family who had been genotyped was chosen. SNPs with minor alleles <0.05 were removed from analysis. The statistic ctively) . For adj-coefficients for distance are  = -0.1005,  = -0.6457, respectively). Very few adjacent SNP markers exhibited significant LD (r2 > 0.7), generally supporting the use of these markers in linkage analyses.Sex-specific LD analyses across the genome revealed the anticipated rapid decay of LD with increased distance (cM) for both males and females (r2 > 0.7). A comparison of the r2 measures for adjacent SNPs showed that the LD distribution for males and females on chromosome X were comparable   on chromosome X did not exhibit significant LD (3) Table .r2 < 0.1). The chi-squared test showed a significant difference in LD for the two regions on X  . The first 7 SNPs lie in the PAR1 region, while 265 SNPs lie in the heterosomal region of X (46\u2013130 cM). PAR2 was not analyzed due to lack of SNPs in that region. The 7 PAR1 SNPs exhibited very low LD (5) Table .r2 > 0.9) or very weak LD (r2 < 0.1)), while LD in the autosomes decreased gradually with distance (Tables p = 0.0019) for females  of chromosome X compared with the heterosomal region of chromosome X. This observation of low LD across PAR1 is consistent with previous results that suggest higher recombination rates on PAR1 when compared with the heterosomal region .COGA: Collaborative Study on the Genetics of AlcoholismEM: Expectation maximizationLD: Linkage disequilibriumSNP: Single-nucleotide polymorphismMEC, JKC, and CDL contributed to the formulation of the question and the writing of the manuscript. MEC and JKC conducted the analyses."}
+{"text": "Large-scale mutagenesis screens in the zebrafish employing the mutagen ENU have isolated several hundred mutant loci that represent putative developmental control genes. In order to realize the potential of such screens, systematic genetic mapping of the mutations is necessary. Here we report on a large-scale effort to map the mutations generated in mutagenesis screening at the Max Planck Institute for Developmental Biology by genome scanning with microsatellite markers.We have selected a set of microsatellite markers and developed methods and scoring criteria suitable for efficient, high-throughput genome scanning. We have used these methods to successfully obtain a rough map position for 319 mutant loci from the T\u00fcbingen I mutagenesis screen and subsequent screening of the mutant collection. For 277 of these the corresponding gene is not yet identified. Mapping was successful for 80 % of the tested loci. By comparing 21 mutation and gene positions of cloned mutations we have validated the correctness of our linkage group assignments and estimated the standard error of our map positions to be approximately 6 cM.By obtaining rough map positions for over 300 zebrafish loci with developmental phenotypes, we have generated a dataset that will be useful not only for cloning of the affected genes, but also to suggest allelism of mutations with similar phenotypes that will be identified in future screens. Furthermore this work validates the usefulness of our methodology for rapid, systematic and inexpensive microsatellite mapping of zebrafish mutations. Large-scale mutagenesis screens in the zebrafish employing the mutagen ENU have isolated several hundred mutant loci that represent putative developmental control genes ,2. In orThe established reference map for the zebrafish genome is the MGH map -7 which Two sets of microsatellite markers for scanning the genome were developed in parallel with the mutant mapping effort. The starting point was the testing of 314 markers for polymorphism in T\u00fc \u00d7 WIK crosses . 72 markThe average distance between markers of the G4 set is 11.6 cM, and all distances are smaller than 36 cM, except for a 71.1 cM interval on LG21 (between Z4425 and Z1497). Within this particular interval few MGH markers are available, and no suitably polymorphic marker could be identified in our reference crosses. For the H2 set the average distance is 11.5 cM, and all distances are smaller than 53.8 cM, except for a 83.3 cM interval on LG21. The more uneven chromosomal distribution of markers in the H2 set reflects the fact that frequently the best markers available were already used in the G4 set.Our mapping methodology as described below can theoretically detect significant linkage over a distance of approximately 36 cM . However, since the LOD score is proportional to the number of individuals scored, this range can be easily increased by adding more mutant individuals if a linkage is questionable. Our marker sets therefore cover the genome adequately to detect significant linkage with the great majority of mutant loci. All the mutant loci mapped in this work have confirmed linkage to at least one G4 or H2 marker (not shown if the closest flanking markers were selected from outside the sets).We report here on the mapping of 319 mutant loci identified in the ENU-based T\u00fcbingen I mutagenesis screen  and subsFor each mutation we crossed mutant carriers against the polymorphic reference line WIK which was established in our lab for this purpose . BrotherA potential linkage was considered confirmed if it had a two-point LOD score equal or greater than 3. The individuals were then genotyped for all polymorphic markers from the same marker set and chromosomal region in order to identify, if possible, a pair of markers flanking the mutation, and if that was not possible, the two closest markers on one side of the mutation. Occasionally additional markers not in the chosen marker set were also included in the genotyping. Decisions on whether or not a mutation was flanked by two markers were based on whether recombinations with the markers occurred independently. For details of the mapping procedure and the calculation of map positions see the Methods section and .spt), approaching the theoretical cutoff of 36 cM.In total, mapping was attempted for 486 mutations from the T\u00fcbingen I screen and subsequent screens of the mutant collection and successful for 389, giving a success rate of 80 %. 12 of these could be mapped only with the H2 set. Unsuccessful mapping experiments were due to difficulties in obtaining sufficient F2 individuals and to PCR problems as well as to a lack of polymorphic markers in our marker set. Among the mutations to be mapped, a group of 63 was prioritized based on interest in their phenotypes. For each of these several additional mapcrosses were set up (data not shown). 56 mutations of this group, or 89 % were successfully mapped, providing a lower limit for the percentage of mutations that our marker sets and methodology is capable of mapping if sufficient F2 individuals are available. The biggest distance to markers on either side at which we could confirm linkage was 31.9 cM  or with the number of Ensembl genes per chromosome in the Ensembl Zv6 assembly [2 = 0.19); by comparison, the values of Amsterdam et al. have a slightly stronger correlation to the number of Ensembl transcripts (R2 = 0.28). Because mapping with our methodology was successful for 80% of all mutations for which it was attempted, possible deficiencies of the mapping method cannot fully account for this low correlation. Rather, it probably reflects an uneven distribution of genes with specific, visible phenotypes in embryos or early larvae as identified in ENU mutagenesis screening, and the absence of such selectivity in the insertional mutagenesis experiment, demonstrating that both types of mutagenesis experiments complement each other in their coverage of their genome. Moreover, we cannot rule out region-specific differences in ENU mutagenesis efficiency.The number of mapped loci assigned to each chromosome is between 6 and 32 (on average 12.8 \u00b1 5.8)  are assigned to a different linkage group by ZMAP, in both cases based on results from the Heat Shock (HS) panel [frs/slc25a [ovl/ift88 is supported by the T51 panel (as shown on the ZFIN website) and by the latest version of the HS map [A comparison of the linkage group assignments shows that two of the 21 genes (S) panel -16. Howes/slc25a  while oue HS map . In concNext we compared the map positions of the mutations with those of the genes on ZMAP (using the median of the ZMAP positions if a gene was placed on more than one mapping panel). If we assume the gene positions to be correct, we obtain a standard error of our mutant map positions of 6.1 cM. Further assuming a normal distribution of errors, we can predict that approximately 95 % of the genes should be within 12.2 cM (two standard errors) of the rough mapping position of the mutation. Indeed, 17 out of the 19 genes mapped on the same chromosome (90 %) are within two standard errors of the mutation, and 16 out of 19 (84 %) within one standard error. Actually both mutation and gene mapping contribute to the observed errors to an unknown degree, so that 6.1 cM merely represents an upper limit for the standard error of our mapping procedure.We have obtained rough map positions for over 300 zebrafish mutants with an accuracy of approximately 6 cM and thereby validated the usefulness of our methodology for rapid, systematic and inexpensive microsatellite mapping of zebrafish mutations. The dataset that we have produced is a first step towards identification of the genes affected by the 277 mutations that are not yet cloned.In candidate gene approaches, our data can substantially narrow down the number of candidate genes, since on the order of 99 % of the genome are outside the two-standard-errors confidence limit of our map positions. Positional cloning approaches in the absence of obvious candidate genes will still require fine mapping by genotyping of additional individuals and identification of more closely linked markers, using the flanking markers identified by us as starting points. Particularly thorough fine-mapping is required in centromeric regions because the genetic recombination rate is often several-fold reduced in such regions , an effeWe have found that a relatively small number of microsatellite markers is sufficient to scan almost the entire genome and that the experimental procedures are robust and easy to perform. Other methods that have been proposed for the mapping of mutant loci in the zebrafish include half-tetrad analysis with microsatellite markers, genome scanning with SNPs and microarray based SNP mapping. While half-tetrad analysis requires only 25 markers to obtain a linkage group assignment -22, it hMapcrosses were set up between mutant carriers and the laboratory reference line WIK  and brot2O was added to the remainder. Plates were stored at -20\u00b0C.F2 embryos were sorted by phenotype and stored in Eppendorf tubes with 100 % MeOH at -70\u00b0C until use. Single embryos were arrayed on a 96-well microtiter plate with a glass Pasteur pipette. The MeOH was evaporated on a PCR block at 70\u00b0C and 25 \u03bcl of 1.7 mg/ml Proteinase K in 1 \u00d7 TE was added to each well. The plate was covered with sealing film and heated to either 55\u00b0C or 70\u00b0C for 240 min and to 94\u00b0C for 10 min in a thermocycler. 10 \u03bcl of each of the sibling and mutant lysates was pooled and 45 \u03bcl sterile ddH2 and 0.1 % (w/v) gelatin. All pipetting was done with a Biomek 2000 robot. Cycling was carried out by initially denaturating at 94\u00b0C for 2 min, 35 cycles of denaturation at 94\u00b0C for 30 sec, annealing at 60\u00b0C for 30 sec and extension at 73\u00b0C for 1 min, and a final extension at 73\u00b0C for 5 min. 5 \u03bcl of 6 \u00d7 loading buffer were added to each sample, and electrophoresis was carried out at 200 V for 45 min in 1 \u00d7 TBE buffer, on 2 % agarose gels. Gels were imaged and scored semi-automatically with NIH Image and a set of custom-designed macros.PCR was initially performed on mutant and sibling pools for genome scanning, and subsequently on the individuals that had been used for the pooling in order to confirm potential linkages to specific markers. 20 \u03bcl PCR reactions were set up from 14.28 \u03bcl of reaction mix , 0.16 \u03bcl each of 20 mM forward and reverse primer, 0.4 \u03bcl of 5U/\u03bcl Taq polymerase, and 5 \u03bcl of template DNA. 10 \u00d7 PCR buffer contained 100 mM Tris-HCl (pH 8.3), 500 mM KCl, 15 mM MgClDistances between mutations and markers were calculated by determining the recombination fraction in the mutant F2 individuals and applying the Kosambi mapping function. Linkages with a two-point LOD score equal or greater 3 were regarded as significant.In order to place a mutation in the genetic interval between the closest marker and another linked marker we determined whether recombinations for both of them were correlated. For this purpose we considered only single recombinants for the closest marker, i.e. heterozygotes. If the majority of these were heterozygous for the second marker we regarded the recombinations as uncorrelated and placed the mutation in the interval between the markers. Otherwise we placed the mutation outside the interval in the direction opposite from the second marker.Assuming complete meiotic interference, i.e. only a single recombination event per chromosome, all recombinants for the first marker should be either non-recombinant for the second marker if the markers flank the mutation, or heterozygous if both markers are on the same side of the mutation. In our data approximately half of the mutations gave results in between these extremes. This may be due to occasional contaminations of the PCR assays but also to less than complete meiotic interference, which would allow a second recombination in the same individual. We therefore did not eliminate any contradictory individuals from the calculation of genetic distances as they may represent a genuine second recombination.If a mutation could be placed in an interval between two markers, a map position was calculated by scaling the observed distances between the mutation and the markers so as to fit into the published distance between the markers. In the remaining cases only the distance to the closest marker was used to calculate the map position. A FileMaker Pro 5 database was used to store the scoring data and perform the calculations . The latRG implemented the mapping approach, supervised the project, analysed the results and drafted the manuscript. CNV initiated and supported the project. The remaining authors contributed equally to fish breeding and sorting by phenotype, PCR reactions and electrophoresis and scoring of gel images. GJR, BR and ER also evaluated the polymorphism of microsatellite markers for the selection of marker sets. All authors read and approved the final manuscript.For the following mutations, still listed as uncloned in Mapping results. Listed are 319 mutant loci identified in the ENU-based T\u00fcbingen I mutagenesis screen [unm\" for unnamed mutant). LG: linkage group. Pos: map position deduced from the two markers listed further to the right, in cM from the top of the linkage group. Gene: symbol of the corresponding gene as listed by ZFIN (or \"n.d.\" if not identified). Marker1: closest marker. Marker2: closest marker on the other side of the mutation (if available) or second closest marker on the same side. Pos1, Pos2: marker position on the MGH map, in cM from the top of the linkage group. Dis1, Dis2: distance between mutant locus and marker, in cM. LOD1, LOD2: LOD score for two-point linkage between the mutant locus and marker (\"inf\" for infinite if no recombinants were found). n1, n2: number of F2 individuals successfully scored. * Marker 2 was determined to be on the same side of the mutant locus as marker 1, since the majority of recombinants for marker 1 is also recombinant for marker 2. However, no usable marker was found on the other side. In these cases only the distance from marker 1 is used to calculate the map position. ** One of the F1 individuals is homozygous for marker 2, allowing only recombinations in the other individual to be scored. Such linkages are listed in order to support chromosomal assignments if no other usable marker was found. However, the distances are not comparable to the sex-averaged map because male and female recombination rates differ to an unknown extent. In these cases the mutation is placed at the position of marker 1. *** ali and bxe have ambiguous positions because marker 1 and marker 2 are on the same side of the mutant locus and the distance to both markers is the same. Thus, ali can be placed either at 107.5 or 134.0 cM from the top of LG21, and bxe either 34.7 or 78.7 cM from the top of LG13.s screen  and subss screen . The lisClick here for fileGenotypes. This file lists the scoring results of individual F2 fish that were used to determine the map positions of the mutations. Row: template plate row, identifies the individuals scored; one row of the file corresponds to two rows of wells on a microtiter plate. Abbr: mutant abbreviation. Allele: mutant allele. Marker: marker name. Genotypes: 24 genotypes, encoded as follows: \"1\", homozygous for the upper band , \"2\", homozygous for the lower band, \"3\", heterozygous, \"0\" or \"-\", not determined .Click here for file"}
+{"text": "Current linkage analysis methods for quantitative traits do not usually incorporate imprinting effects. Here, we carried out genome-wide linkage analysis for loci influencing adult height in the Framingham Heart Study subjects using variance components while allowing for imprinting effects. We used a sex-averaged map for the 22 autosomes, while chromosomes 6, 14, 18, and 19 were also analyzed using sex-specific maps. We compared results from these four analyses: 1) non-imprinted with sex-averaged maps, 2) imprinted with sex-averaged maps, 3) non-imprinted with sex-specific maps, and 4) imprinted with sex-specific maps. We found four regions on three chromosomes  with LOD scores above 2.0, with a maximum LOD score of 3.12, allowing for imprinting and sex-specific maps, at D18S1364 on 18q21. While we obtained significant evidence of imprinting effects in both the 18p11-q21 and 19q13 regions when using sex-averaged maps, there were no significant differences between the imprinted and non-imprinted LOD scores when we used sex-specific maps. Our results illustrate the importance of allowing for gender-specific effects in linkage analyses, whether these are in the form of gender-specific recombination frequencies, or in the form of imprinting effects. Genomic imprinting is the inactivation of a genomic segment depending on the sex of the parent from which it was inherited. Imprinting may play an important role in the inheritance of complex traits in humans. It has been shown by previous studies that incorporation of imprinting into linkage analysis may improve the power of variance component methods for detecting quantitative trait loci ,2. Howevh2 values ranging from 0.5 to 0.8 [We decided to analyze linkage of normal adult height because imprinted genes are involved in growth and development . Further5 to 0.8 -8. Altho5 to 0.8 , we seleThe Framingham Heart Study, which started in 1948, is a longitudinal study designed to evaluate the multifactorial nature of cardiovascular disease. The original cohort, collected in 1948, contained adult members from about two-thirds of the homes in Framingham, Massachusetts. In 1971, a second offspring cohort was included in the study, about three-fifths of which are the biological offspring of the original cohort. A more detailed description of these two cohorts is available in previous publications . We analFor both cohorts, observers measured and recorded multiple anthropomorphic measurements, at periodic examinations over the duration of the study, at two-year intervals for the original cohort, and at four-year intervals for the offspring cohort. Height measurements were taken with participants standing erect, with heads in the Frankfort plane. For the original cohort, height was measured at biennial examination 1 (in 1948), 5, 10, 13, and then at every subsequent examination onwards. For the offspring cohort, there was approximately an 8-year gap between the first exam (in 1971\u20131974) and the second exam; subsequent exams were given every 4 years.We examined the 2885 individuals who had multiple height measurements. Each individual's height measurements were compared in a pair-wise fashion for deviations greater than 2.0 inches. Where a single unique measurement was deviant from all others, this measurement was removed, along with its corresponding examination age. There were 22 such individuals. If we were unable to identify a unique deviant, we set that individual's height and age to unknown for the rest of the analysis. Only 12 individuals were zeroed out in this fashion.For each individual with known height measurements, we first computed a mean adult height measurement between the ages of 20\u201360 years, and a mean adult age of examination. Linear regression was performed to eliminate gender and age effects. Since there is evidence of change in the population distribution of height in the last few decades, we also regressed mean height against decade-of-birth for each individual. The residuals resulting from the linear regression of height against gender, mean-age, and decade-of-birth were converted into z-scores, which were used as the trait phenotype for linkage analysis.In order to incorporate imprinting, we have to include separate major-gene components for each parent. If we assume purely additive variance, the variance-covariance matrix \u03a9 is:MO\u03c32aMO + \u03a0FA\u03c32aFA + 2\u03a6\u03c32G + I\u03c32e,\u03a9 = \u03a0MO is a matrix containing the estimated proportion of maternal alleles shared identically by descent (IBD), \u03a0FA is a matrix containing the estimated proportion of paternal alleles shared IBD, \u03a6 is the kinship matrix, and I is an identity matrix. Here, we are partitioning the variance into four components: an additive component (\u03c32aMO) due to the effect of a maternally derived allele linked to the region of interest, an additive component (\u03c32aFA) due to the effect of a paternally derived allele, a polygenic component (\u03c32G), and an environmental effect (\u03c32e). As a simplifying assumption, we ignore the effects of shared environment. We computed five different likelihoods on our pedigree data:where \u03a02aMO = 0, \u03c32aFA = 0, \u03c32G, \u03c32e): No major gene effect. \u00a0\u00a0\u00a0 (1)L: Non-imprinted, where \u03c32aMO and \u03c32aFA are constrained to be equal. \u00a0\u00a0\u00a0 (2)L: Complete maternal imprinting, \u03c32aMO = 0 \u00a0\u00a0\u00a0 (3)L: Complete paternal imprinting, \u03c32aFA = 0 \u00a0\u00a0\u00a0 (4)L: Imprinted, i.e. \u03c32aMO, \u03c32aFA\u2265 0 \u00a0\u00a0\u00a0 (5)L(\u03c3We tested for linkage at each marker for equations (2\u20135) by comparing its log-likelihood against that of the polygenic equation (1) using the likelihood ratio test. LOD scores were also computed by dividing the likelihood ratio by 2ln(10). We also examined evidence for imprinting by comparing the log-likelihood of the non-imprinted model to that of the imprinted model.Linkage analysis was performed on 330 extended pedigrees containing 4692 individuals. SIMWALK2 version 2.83  was usedgender = -5.40, \u03b2decade-of-birth = 0.045, and \u03b2age = -0.01, with R2 = 0.5521. Gender and decade-of-birth effects are significant at the 0.01 level. The residuals are distributed normally with a mean of 0.00, standard deviation of 2.58 inches, skewness 0.094, and kurtosis = -0.174. The Shapiro-Wilkes test statistic is 0.99 with a p-value of 0.08. After conversion to z-scores, there are only five outliers, two individuals with z-scores less than 3.0, and three with scores larger than 3.0. These individuals were excluded from the linkage analysis.Height is dependent on gender, decade-of-birth, and age as follows: \u03b2p-value of approximately 0.0012, while for imprinted LOD scores, a LOD of 2 has an asymptotic p-value of approximately 0.0037.) Conventional analyses  identify two regions, 14q32.2 and 18q21.3-22.1  gene is close to this region.Several genome-wide linkage analysis studies have attempted to pinpoint the genetic factors that control height. A study on Pima Indians showed evidence of a locus on chromosome 20 . Other sm) is not equal to the female recombination fraction (\u03b8f), then a test that assumes \u03b8m = \u03b8f will be invalid. Furthermore, in a region of true linkage, such map misspecification results in loss of power to detect linkage [m = \u03b8f can increase the type I error rate of the imprinting LOD score, but that these increases are modest if the female:male map-distance ratio is \u2264 5:1. In our study, using sex-averaged maps, we obtained significant evidence of imprinting in precisely those regions with large differences between the male and female genetic distances  that teIt may not be appropriate to ignore dominance effects as we have in our variance components models. In addition, Spencer  shows thMost previous variance components-based analyses ,24 testi"}
+{"text": "The prevalence of tobacco usage in Native American adults and adolescents is higher than any other racial or ethnic group, yet biological risk and protective factors underlying tobacco use in this ethnic group remain unknown. A genome scan for loci associated with tobacco use phenotypes was performed with data collected from a community sample of Mission Indians residing in Southwest California.A structured diagnostic interview was used to define two tobacco use phenotypes: 1) any regular tobacco usage (smoked daily for one month or more) and 2) persistent tobacco usage (smoked at least 10 cigarettes a day for more than one year). Heritability was determined and a linkage analysis was performed, using genotypes for a panel 791 microsatellite polymorphisms, for the two phenotypes using variance component methods implemented in SOLAR.Analyses of multipoint variance component LOD scores for the two tobacco use phenotypes revealed two scores that exceeded 2.0 for the regular use phenotype: one on chromosomes 6 and one on 8. Four other loci on chromosomes 1,7,13, and 22 were found with LOD scores between 1.0 and 1.5. Two loci of interest were found on chromosomes 1 and 4 for the persistent use phenotype with LOD scores between 1.3\u20131.5. Bivariate linkage analysis was conducted at the site on chromosome 4 for persistent tobacco use and an alcohol drinking severity phenotype previously identified at this site. The maximum LOD score for the bivariate analysis for the region was 3.4, however, there was insufficient power to exclude coincident linkage.While not providing evidence for linkage to specific chromosomal regions these results identify regions of interest in the genome in this Mission Indian population, for tobacco usage, some of which were identified in previous genome scans of non-native populations. Additionally, these data lend support for the hypothesis that cigarette smoking, alcohol dependence and other consumptive behaviors may share some common risk and/or protective factors in this Mission Indian population. Tobacco use by North American Indians was documented by the first European explorers to the continent who described it as being consumed by smoking, chewing or in salves ,2. EarlySmoking has been described as particularly prevalent in Native American youth. No differences have been found between rural and urban Native American adolescents in one study of smoking prevalence, and in that study Native American youth reported more exposure to peers who smoked and greater access to cigarettes than other racial and ethnic groups . AmericaPsychosocial variables such as death, loss, and other stressful life events have been demonstrated to be risk factors for American Indian adolescents initiating smoking , howeverThe present report is part of a larger study exploring risk factors for substance dependence among Native American Mission Indians -34. The Participants, known collectively as Mission Indians, were recruited using a combination of a venue-based method ,38 and aOne hundred pedigrees containing 885 individuals were used in the analyses. Of these, 244 individuals have both genotype and phenotype data and 222 additional individuals have only phenotypic data . Fifty-nine families have only a single individual with phenotype data. These individuals were included within some analyses to the extent that they contribute information about trait means and variance and the impact of co-variants. The family sizes for the remaining families ranged between 4 and 38 subjects (average 13.5 \u00b1 10) with between 2 and 15 individuals having both genotype and phenotype data (average 5.4 \u00b1 4.2). Forty-one families were genetically informative. The data includes: 77 parent-child, 212 sibling, 26 half sibling, 8 grandparent-grandchild, 151 avuncular, and 245 cousin relative pairs where both genotype and phenotype data were available for both pair members. These data analyses have been described previously ,36.DNA was isolated from whole blood using an automated DNA extraction procedure, genotyping was done as previously described . Genotyp2) and its standard error were estimated for the regular and persistent tobacco use phenotypes using SOLAR. Variance component estimate methods were used to calculate LOD scores using SOLAR v2.0.4 [Genotypes were ultimately determined for 243 subjects. The total additive genetic heritability  or 0 (complete coincident linkage).Bivariate analyses were conducted with data on chromosome 4 for persistent tobacco use where a loci was identified in a location at @102 cM where we had previously identified a linkage signal for alcohol drinking severity . DrinkinThe demographic characteristics of the sample are virtually equivalent to the U.S. census data for the tribe, and have been presented previously . ParticiTwo hundred seventy-one out of a larger sample of 466 participants (58%) met the criteria for regular tobacco usage and (30%) met criteria for persistent usage. Men were not significantly more likely to be regular users or persistent users. Nor were those participants having a native heritage of 50% or greater likely to be regular or persistent users compared to those with lesser degrees of Native heritage. The mean age of onset of regular use was 17 yrs and the mean quantity and frequency of usage was 12 cigarettes a day for 9 years. Persistent and regular smokers did not differ from each other or non-smokers on age, education or income. The prevalence of alcohol dependence in the larger sample was 60% and in the linkage sample it was 61%. Alcohol dependence was co-morbid with both the persistent  and regular  smoking phenotypes.2) for the regular tobacco usage, treating the phenotype as a continuous variable, was 0.37 \u00b1 0.11 and persistent tobacco usage, 0.34 \u00b1 0.12. Estimated heritability nominally increased to 0.53 \u00b1 0.17 and 0.46 \u00b1 0.18 when a latent threshold model was used.The first aim of the study was to determine the heritability of the two tobacco phenotypes. The estimated heritability  dataset for habitual smoking and alcohol dependence . Loci onOne of the most consistent findings between several genetic linkage studies conducted in Native American and other mixed heritage populations is evidence for a protective association for alcohol dependence and related behaviors in a region on chromosome 4 near the Alcohol dehydrogenase gene cluster. In a genetic linkage study evaluating large families who were members of a Southwest Indian tribe, three loci in this region on chromosome 4q showed evidence for linkage . EvidencThere is one additional locus of interest in this Mission Indian study that was also identified in previous linkage analyses for alcohol-related phenotypes. Evidence for linkage was found on chromosome 6 for the regular tobacco use phenotype in a support interval previously identified for an alcohol withdrawal severity phenotype in this Mission Indian population . AdditioAdditional loci of interest were found on chromosome 7, 13 and 22 for regular smoking that have been identified in other studies ,25. OnlyThe results of this study should be interpreted in the context of several limitations. First, the findings may not generalize to other Native Americans or represent all Mission Indians. Second, comparisons of linkage findings to non-Indian populations of drug abusers may be limited by differences in a host of potential genetic and environmental variables. Third, the underlying assumption that these phenotypes are normally distributed, an assumption of variance component analyses, may not be warranted. Finally, because this population has significant admixture estimates of allele frequencies may produce biased LOD scores. The linkage analyses do not give us any information on whether the genes within the loci identified are coding for protective or risk factors. Despite these limitations, this report represents an important first step in an ongoing investigation to understand the genetic determinants associated with the development of substance use disorders in this high risk and understudied ethnic group.These data represent the first family-based genome-wide chromosome segregation analyses for tobacco use phenotypes in Native Americans. The results corroborated the possible importance of several chromosomal regions highlighted in prior linkage studies for substance abuse phenotypes and identify new regions of the genome for this ethnic group and/or Mission Indians. A replicate study is underway to test the reliability of the findings. Additionally, these data lend support for the hypothesis that cigarette smoking, alcohol dependence and other consumptive behaviors may share some common risk and/or protective factors in this Mission Indian population.2 total additive genetic heritability; LOD, log of the odds; COGA, collaborative study genetics of alcoholism; ADH, Alcohol Dehydrogenase.SSAGA, Semi-Structured Assessment for the Genetics of Alcoholism; HThe author(s) declare that they have no competing interests.Cindy L Ehlers contributed to the recruitment, collection and analysis of the clinical and genetic data on the subjects. Kirk C. Wilhelmsen contributed to the analyses of family structures, genome scan, linkage and heritability analyses.The pre-publication history for this paper can be accessed here:"}
+{"text": "Alcoholism is a complex disease in which genomic imprinting may play an important role in its susceptibility.To conduct a genome-wide search for loci that may have strong parent-of-origin linkage effects in alcoholism; to compare the linkage results between the microsatellites and the two single-nucleotide polymorphism (SNP) platforms.Nonparametric linkage analyses were performed using ALLEGRO with the three sets of markers provided by the Genetic Analysis Workshop 14 for the Collaborative Study on the Genetics of Alcoholism Problem 1 data. Both sex-averaged and sex-specific genetic maps were used. We also provided a valid statistical test to determine whether the parental allele sharing differed significantly.Significant maternal linkage effects  were observed on chromosome 12 using either the microsatellite markers or the two SNP panels. The two SNP sets did not improve the linkage signals compared to the results from the microsatellite markers on chromosome 12. Possible paternal linkage effects  on chromosome 7 and maternal linkage effects  on chromosome 10 were found using the two SNP panels.For diseases which may have parent-of-origin effects, linkage analysis looking at parental sharing separately may reduce locus heterogeneity and increase the ability to identify that which can not be identified with usual linkage analysis. Genomic imprinting (a class of parent-of-origin effects) occurs when the expression of a gene is dependent on the parent from which it was inherited. It has been suggested that genomic imprinting plays a role in the susceptibility to alcoholism  . In the In this study, we performed nonparametric linkage analyses using the microsatellite markers and single-nucleotide polymorphisms (SNPs) from Illumina and Affymetrix to search for loci with parent-of-origin effects in alcoholism. We also proposed a new test to determine whether the paternal and maternal allele sharing were significantly different from each other.The data from COGA provided for the GAW14 were used. The original dataset contains 143 extended pedigrees. We focused our study on the 112 Caucasian families. Due to computational limitations, 52 unaffected individuals who were least genotyped were removed from the 8 largest pedigrees. We used the COGA definition of alcoholism (ALDX1) as the phenotype. The affection status was coded as affected if ALDX1 was 5 (affected). All the other individuals were coded as unaffected.All three sets of markers  were used for two-point linkage analyses. Multipoint analyses were applied to all the autosomes for the microsatellite markers, 7 chromosomes  for the Illumina SNPs, and segments of 10 chromosomes  for the Affymetrix SNPs based on the two-point linkage signals. Instead of using the provided marker allele frequencies, we generated them with the computer program PEDMANAGER v0.9 .The sex-averaged and sex-specific genetic maps for the microsatellite markers were obtained from the Rutgers map  based onpair  scoring function was applied in all cases. The evidence for paternal and maternal imprinting was investigated with an imprinting-based score function [ALLEGRO v1.2c  was applfunction  that conTo test the null hypothesis that the maternal and paternal allele sharing are the same, we conducted a likelihood ratio test:Zlrm2 + Zlrp2) - Zlrb2 ~ \u03c7(1)2,(wheresign(dhat) is the sign of dhat, the measurement of the size of genetic effect [LOD is the allele sharing LOD score. Zlrm, Zlrp, and Zlrb are the Zlr values for maternal sharing, paternal sharing, and both parents' allele sharing, respectively. This likelihood ratio test has an asymptotic \u03c72 distribution with one degree of freedom under the null hypothesis assuming that the maternal identity by descent (IBD) is independent of the paternal IBD or in the absence of linkage. This assumption of the independence of maternal and paternal allele sharing is valid under an additive genetic model.c effect , and LODp \u2264 0.05) with one parental LOD score \u2265 2.5 are listed. Of these markers, one microsatellite marker on chromosome 12, two SNPs on chromosomes 2 and 13 from Illumina, and one SNP on chromosome 2 from Affymetrix showed significant excess maternal allele sharing, while four SNPs on chromosomes 1, 4, 7, and 11 from Affymetrix showed significant excess paternal allele sharing.Table p = 0.0007). However, it was not significantly different from the paternal effects . Five more loci on chromosomes 2, 7, 10, and 13 showed signals of paternal effects but none of their LOD scores was higher than 2.In the multipoint linkage analyses using the sex-averaged genetic maps, the same region that showed significant maternal effects on chromosome 12 using the microsatellite markers (at 165.9 cM) in the two-point analysis also showed significant maternal effects using the SNPs from both Illumina and Affymetrix  Table . Excess p = 0.0007) was observed at the same region (154.4 cM) on chromosome 12 using the SNPs from Affymetrix (Table p < 0.0001). This observation was also true for all the other tested chromosomes. Because information content depends on the genetic maps through multipoint allele sharing estimation, a possible explanation for the observation is that ALLEGRO incorporates the sex-averaged maps differently from the sex-specific maps in parental effect linkage analysis.In the multipoint linkage analyses using the sex-specific genetic maps, a LOD score of 2.16 (ix Table . The peaix Table . These rp = 0.1). For the region on chromosome 10, there was a maternal effect peak at 57.7 cM (LOD = 1.46 with p = 0.005) from the microsatellite markers. However, it was not significantly different from the paternal effects .In addition to the consistent results across the three panels of markers on chromosome 12, one region on chromosome 7 (at 1.2 cM for Illumina and at 13.7 cM for Affymetrix) and one region on chromosome 10 (at 57.0 cM for Illumina and at 62.5 cM for Affymetrix) also showed consistent signals of paternal and maternal effects, respectively, using both SNP panels (Table p = 0.0001) using the sex-averaged map and 2.14 (p = 0.0008) using the sex-specific map at 165.9 cM (closest marker D12S1045). The paternal results were close to zero at this location. The parental effect p-values were 0.002 and 0.02 for analyses using the sex-averaged and sex-specific maps, respectively. The female/male genetic map ratio around this 30-cM region was 1.7 (ranges from 1.56 to 1.85), which did not differ from the genome-wide mean of 1.65 [Because all the three sets of markers showed linkage signals of excess maternal allele sharing for alcoholism on chromosome 12, we selected 3 microsatellite markers, 19 SNPs from Illumina, and 28 SNPs from Affymetrix around the most significant region. These 50 markers covered about 30 cM of the chromosome. We repeated the linkage analyses using this combined dataset. The results did not improve very much compared to the results from the individual marker set Figure , and 1B.dhat values (the size of genetic effect) can help in this situation.Given the large number of tests we conducted, some of the significant parental effects could happen by chance. In addition, the test for parental effect assumes independence of paternal and maternal allele sharing, which is true under an additive genetic model. If the genetic model is not additive, the maternal and paternal sharing will be dependent and the parental test statistic will have less than one degree of freedom. In this case, our test will be conservative and will have low power. The test for parental effect can also be affected by marker informativity. An examination of the paternal and maternal Comparing our study with the previous studies from GAW11 -5, our rIn this study, we conducted a genome-wide scan of loci that might have significant parent-of-origin linkage effects in alcoholism. Evidence of excess maternal sharing  on chromosome 12 was shown in analyses using the microsatellite markers and the two SNP panels. In addition, the results from the two SNP panels showed evidence of excess paternal sharing on chromosome 7 and excess maternal sharing on chromosome 10. The significance was similar using the microsatellite markers versus the SNP sets due to their similar information content on chromosome 12. We also observed a drop of information content when the sex-specific maps were used in imprinting linkage analysis compared to when the sex-averaged maps were used, which might explain the lower LOD scores from our analysis using the sex-specific maps.COGA: Collaborative Study on the Genetics of AlcoholismGAW: Genetic Analysis WorkshopIBD: Identity by descentSNP: Single-nucleotide polymorphismTDT: Transmission disequilibrium testX-QL designed the study, performed the statistical analysis, and drafted the manuscript. CMTG provided the novel statistical method used in this study and assisted with the interpretation. K-SW helped with the revision of the draft. ADP conceived of the study and helped to draft the manuscript. All authors read and approved the final manuscript."}
+{"text": "The genome of each population was divided into 20-cM bin regions, and each bin was rank-ordered based on the most significant linkage p-value for that population in that region. The bin ranks were then averaged across all four studies to determine the most significant 20-cM regions over all studies. Statistical significance of the averaged bin ranks was determined from a normal distribution of randomly assigned rank averages. To narrow the region of interest for fine-mapping, the meta-analysis was repeated two additional times, with each of the 20-cM bins offset by 7 cM and 13 cM, respectively, creating regions of overlap with the original method. The 6\u20137 cM shared regions, where the highest averaged 20-cM bins from each of the three offsets overlap, designated the minimum region of maximum significance (MRMS). Application of the GSMA-MRMS method revealed genome wide significance  at regions including or adjacent to all of the simulated disease loci: chromosome 1 , chromosome 3 , chromosome 5 , and chromosome 9 . This GSMA analysis approach demonstrates the power of linkage meta-analysis to detect multiple genes simultaneously for a complex disorder. The MRMS method enhances this powerful tool to focus on more localized regions of linkage.In order to detect linkage of the simulated complex disease Kofendrerd Personality Disorder across studies from multiple populations, we performed a genome scan meta-analysis (GSMA). Using the 7-cM microsatellite map, nonparametric multipoint linkage analyses were performed separately on each of the four simulated populations independently to determine  After a genome scan, fine-mapping of the most promising regions proceeds. Identification of the regions must be as accurate as possible to minimize time and expense. In complex diseases, there are often many research groups working independently but cooperatively. A meta-analysis of the genome scans from diverse research groups can reveal the appropriate areas for fine-mapping. We proposed to use the results from the individual genome scans of the Genetic Analysis Workshop simulated populations in a meta-analysis to assess the optimal chromosomal region(s) to target for second stage fine-mapping. The genome scan meta-analysis (GSMA) ,2 methodLinkage between traits and markers was assessed via nonparametric multipoint linkage methods. For the multigenerational New York families, we used the descent graph approach, utilizing computer program SIMWALK V2.89 , and MEGR, with values 1\u2013144) according to the most significant p-value of any markers within that bin. Any ties were assigned equal ranks on the basis of the mean of the sequential ranks for those bins. Higher values of R represented the most significant p-values.For the GSMA procedure, the genome was divided into 20-cM regions, with bin width selected such that there were at least 2 bins on each chromosome and at least one marker in each bin. For each of the 4 scans, bins were assigned a rank  [Analysis proceeded without knowledge of the simulated disease loci.p-values less than 0.001. Many more markers had raw p-values < 0.05. There were 7 markers on 4 chromosomes that met the Bonferroni adjusted significance requirement, yet 6 of these markers were significant in only one population  and 1 marker (D03S0127) was significant in 2 populations (Aipotu and Karangar).Multipoint results in the four populations Figure  indicateThe bin-shifting procedure and the MRMS method Figure  identifip-values from the multipoint analysis, 3 regions varying from 14 to 33 cM would have been considered for fine-mapping on chromosomes 1, 3, and 5. Using p-values < 0.001 from the multipoint analysis, even larger regions varying from 24 to 44 cM would have been considered for fine-mapping on chromosomes 1, 3, and 5. The GSMA-MRMS enhanced method, in comparison to the alternative methods presented above, would be the most cost effective method for identifying regions for second stage fine-mapping.The GSMA-MRMS procedure correctly identified the 3 disease regions on chromosomes 1, 3, and 5. The fourth disease region on chromosome 9 revealed by GSMA-MRMS was directly adjacent to the simulated disease region. We believe that the GSMA-MRMS method is superior to other methods that might be used to identify localized regions of linkage. Without the shifting of the bins (MRMS method), the GSMA alone would have indicated a 20-cM region on each of the chromosomes 1,3, 5, and 9, effectively tripling the cost and time of the fine-mapping procedure. Using just the Bonferroni-corrected The GSMA method alone identified 20-cM regions while the GSMA method followed by the MRMS narrowed the regions to consider, leading to more efficient use of time, resources and funds for follow-up fine-mapping studies. With many investigators focusing on complex diseases with sometimes conflicting findings from study to study, and with the necessity to combine data across studies with potentially different study designs, the GSMA-MRMS methodology would expedite the discovery of a complex disease's genetic basis.GSMA: Genome scan meta-analysisMRMS: Minimum regions of maximum significancep-values. THG completed the simulations for the empiric p-values of the weighted data. BSM devised the methodology for the simulation of the weighted data. MLM created the method of bin shifting to narrow the regions of maximum significance to address the concerns of MEC, THG and BSM that the GSMA methods alone might lead to misleading results, depending on the placement of the bins.MEC completed the genetic analysis, identified the bins, and calculated the weights and the normal distribution of ranks used for the"}
+{"text": "We used the FBAT (family-based association test) software to test for association between 300 individual single-nucleotide polymorphisms and P1  in 100 simulated replicates of the Aipotu population. Using the Genetic Analysis Workshop 14 dataset, we calculated the power of FBAT to detect linkage disequilibrium on chromosome 3 (D2). Also, we calculated the false-positive rate on chromosome 1, which contains a true locus (D1) but no linkage disequilibrium was simulated between the trait and all the surrounding single-nucleotide polymorphisms.p-value = 0.0002), B03T3057 , and B03T3058  with power of 98%, 87%, 71% on chromosome 3, respectively. The overall false positve rate to detect association was 0.06 on chromosome 1.We were able to detect the associations between phenotype P1 and three adjacent markers B03T3056 . In the future, we will compare the performance of FBAT with alternative approaches, such as using FBAT-generalized estimating equations methods to test for association in families affected with complex traits. For complex diseases such as Kofendred Personality Disorder (KPD), linkage analysis using microsatellite markers may not be able to provide adequate resolution to identify the genes underlying phenotypic variation . Fine maThe aim of this study is to use FBAT to test for association between single-nucleotide polymorphism (SNP) markers and the P1 phenotype  using SNPs on chromosomes 1 and 3. We evaluated the power to detect association using FBAT in a simulated dataset of 100 replicates of the Aipotu population.FBAT has been used to test for genetic association by some investigators ,6. It buOur goal is to test the hypothesis of no association using genotype data in 100 nuclear families, each with different sibship size, provided by Genetic Analysis Workshop 14 (GAW14). We focused on two regions with known disease loci: chromosomes 1 and 3. For chromosome 1, we analyzed 230 SNPs (with average density of 0.3 cM), covering the region from 117 cM to 191 cM) containing the true disease locus D1, located at 167 cM. For chromosome 3, we analyzed 84 SNPs with the same average SNP density as chromosome 1 (covering the region from 274 cM to 299 cM), and containing the true disease locus D2, located at 299 cM.As described by Greenberg et al. , Aipotu p-value for SNPs on chromosome 1 over 100 replicates was not significant  region of chromosome 3 and located 2.3 cM proximal to the \"true\" disease locus D2  than the maximum possible . The estimated false-positive rate was 0.06. The proportion of SNPs out of 100 replicates p-values less than 0.05 is presented in detail in Figure p-values are described in Figure We also calculated the number of SNPs on chromosome 1, in which no association with the trait was simulated, that would meet the significance threshold of 0.05. We tested all 230 SNPs on chromosome 1 individually in 100 replicates using FBAT and then we calculated the proportion of significant markers among all tested SNPs using different cut-off s Figure . First, For complex diseases, such as KPD here, we need new statistical tools such as FBAT to detect associations between marker loci and disease genes where the disease phenotype is multivariate. In this study, we used 100 simulated replicates of the Aipotu population to calculate the power to detect association and evaluate the false-positive rate.), the results obtained by using \"-e\" to test genetic association do not differ greatly from the result obtained from not using \"-e\" in nuclear families, unless there are a few very large pedigrees that contribute most of the information. In addition, when we calculated the false-positive rate, we did not take into account the fact that some SNPs are correlated. These two limitations could bias the estimated false-positive rates. In this study, if we set the significance level to be 0.05, the proportion of observed \"significant\" results was 6%, which is slightly higher than the expected 5%. However, given the limitation we discussed above, we cannot conclude that this result suggests an inflated type I error.We would like to point out two limitations of this study. We were interested in testing for association with a latent trait containing 4 phenotypes. Therefore, we conducted multivariate analysis using FBAT. Given the fact that we used 100 nuclear families each with a single sibship to test association, a more appropriate method would have been to use the \"-e\" option implemented in FBAT to calculate the empirical variance of the test statistic to test for association in the presence of linkage ,9. HowevTo summarize, our results indicated the best power of 98% at the SNP B03T3056, within the designated LD region of chromosome 3, and for adjacent markers B03T3057 and B03T3058, the power was 87% and 71%, respectively. None of the other markers within the designated LD region revealed significant results. We conclude that FBAT provides another powerful approach to detect association in the presence of linkage.FBAT: Family-based association testsKPD: Kofendrerd Personality DisorderLD: Linkage disequilibriumSNP: Single-nucleotide polymorphismYYS and M-HW both participated in study design and data analyses. MG and M-HW provided bioinformatics support to speed up the data analyses. YYS, M-HW, and MG contributed to data interpretation and manuscript preparations."}
+{"text": "We used the LOKI software to generate multipoint identity-by-descent matrices for a microsatellite map (with 31 markers) and two single-nucleotide polymorphism (SNP) maps to examine information content across chromosome 7 in the Collaborative Study on the Genetics of Alcoholism dataset. Despite the lower information provided by a single SNP, SNP maps overall had higher and more uniform information content across the chromosome. The Affymetrix map (578 SNPs) and the Illumina map (271 SNPs) provided almost identical information. However, increased information has a computational cost: SNP maps require 100 times as many iterations as microsatellites to produce stable estimates. Traditionally, the mainstay of linkage has been use of highly polymorphic microsatellite markers. The ultimate goal would be completely polymorphic markers \u2013 each parent would have two uniquely occurring alleles. A highly polymorphic microsatellite provides a great deal of segregation information at a particular locus. At the other extreme, single-nucleotide polymorphisms (SNPs) usually have only two alleles  and alone provide much less information for segregation. Because SNP typing is less expensive, and available at a finer density than microsatellites, the use of dense SNPs in the place of microsatellites for linkage analysis is being investigated using data from the Collaborative Study of the Genetics of Alcoholism (COGA). Because segregation is at the heart of any linkage analysis, we examined IBD (identity by descent) matrices to compare the information content of SNPs versus microsatellites for linkage. We used the LOKI software ,2 to creApproximately 1,300 individuals previously typed with a microsatellite screen were typed with 4,720 SNPs from the Illumina linkage panel and 11,120 SNPs from the Affymetrix mapping array. These individuals were from 143 pedigrees with an average of 9.5 individuals typed per pedigree (range: 5\u201327). The sample was 77% White, 13% African American, and 10% from other ethnicities.In the presence of non-genotyped founders, allele frequency estimates are of paramount importance for IBD estimation. The large differences in allele frequencies between Whites and African Americans for microsatellites have been well established. Our group identified similar differences for allele frequencies of SNPs . BecauseThe generation of IBD matrices with microsatellite markers has been addressed through a number of different techniques. We are using multipoint IBD (mIDB) as our standard because of the low information provided by a single SNP. Although a new version of GENEHUNTER  has receLOKI uses Markov chain Monte Carlo (MCMC) methodology to repeatedly sample possible segregation patterns. However, determining appropriate run length (number of iterations) is important for the accuracy of the IBD estimates. Using three separate sets of markers:  we compared the average standard deviation of \"phi2\" (twice the kinship coefficient) between each pair of individuals in each pedigree at each centimorgan position on chromosome 7 for 10 replicates (from 10 different starting seeds) with 10,000, 100,000 and 1,000,000 iterations per replicate. To compare this information on different maps, we translated the genetic positions of the markers to the physical position based on NCBI build 34.3  at position x, we computeUsing the resulting matrices, information was first computed on sibships (regardless of phenotype) using the method presented in Kruglyak and Lander : for N sAt each chromosome position for each sibling pair, the variance in IBD 0, IBD 1, and IBD 2 estimates is divided by the variance in the absence of marker information . The mean of this measure is subtracted from 1. If the posterior IBD status is known with certainty for all pairs, the variance is 0 and the information is 1. This measure was then computed on all relative pairs except for parent-offspring, where the prior variance is 0 (since Pr(IBD = 1) = 1.0), notwithstanding a new mutation. The results for the sibling pairs are presented in Figure Finally, we note that LOKI assumes the markers are in linkage equilibrium (LD). However, substantial pair-wise LD exists between the SNPs, especially in the Affymetrix map. Considering all pairs of Affymetrix SNPs on chromosome 7, 59 pairs have a correlation greater than 0.9. For Illumina, 13 pairs have a correlation greater than 0.9. Recent work  suggestsDue to the large size of some of these pedigrees, software which computes all possible inheritance vectors (such as GENEHUNTER or Merlin ) has excIn general, MCMC software trades these memory requirements for substantially greater CPU usage. We thus chose LOKI for IBD matrix generation, but this software requires a choice of run length (number of iterations). The initial tests to determine an appropriate number of iterations for the generation of IBD matrices with LOKI shows that the higher density of the SNP maps greatly slows the MCMC process. In particular, while 10,000 iterations for microsatellites shows only slightly higher average variance of phi2 than one 100,000 iterations, the variance for SNPs is still quite high with 100,000 iterations. We ultimately used 1,000,000 iterations for the SNP datasets. Figures p = 0.3). We also note that the \"sparse\" Illumina map (with an average intermarker spacing of 1.1 cM) contains substantially more information for sibships than the microsatellites and nearly as much information as the full Illumina map (with an average intermarker spacing of 0.69 cM). The information for relative pairs is uniformly lower than sibling pairs for all four map sets (results not shown). This may be due to the greater number of missing founders when considering the extended pedigrees as opposed to sibships.The information content results for microsatellites show substantial dips between markers. Both the Affymetrix and Illumina map provide a higher and much more uniform level of information. There are several sharp dips in the Affymetrix map, but this may be due to the IBD generation failing to converge; the locations of reduced information correspond to the locations of higher variance in Figures Our results show that the information provided by dense SNP maps is generally higher and more uniformly distributed than with standard microsatellite panels composed of about 400 markers. This increased information comes at a cost of increased computational complexity. At least 100 times as many iterations are required and each iteration took 10\u201320 times longer for the SNP maps as for the microsatellite. For example, 100,000 iterations took 3.4 hours for the microsatellites, 30 hours for the Illumina SNPs, and 68 hours for the Affymetrix SNPs. While the increased time for each iteration is likely due to the increased number of markers, the increase in required iterations may be due to the reduced information of the SNP markers. This could be tested by comparing convergence with a dense microsatellite map.The Affymetrix map contains regions of reduced information, corresponding to the same locations where variance of phi2 is high. We examined the Affymetrix SNPs around the largest peak (at 15 Mb) and found that they were not significantly different from other Affymetrix SNPs on chromosome 7 in terms of density, heterozygosity, LD, or missing data rate. Other possible explanations include an increase in Mendelian-compatible genotyping errors or incorrect maps (either spacing or marker order). These possibilities could be tested in additional datasets to see if convergence problems existed at the same location.Although the Affymetrix map consists of more than twice as many SNPs as the Illumina map, increased density of SNPs in the Affymetrix map does not appear to provide more information. However, many of the SNPs in the Affymetrix map have fairly low heterozygosity . We alsoThese data suggest that SNPs are a cost effective and informative replacement for microsatellites for linkage analysis. Although the computational burden is substantially greater for IBD computations, the resulting information is higher and more uniform. Although estimates of IBD and information content may be elevated when markers are in linkage disequilibrium and parents are untyped, further tests also suggest that a less dense map would provide nearly the same level of information.COGA: Collaborative Study on the Genetics of AlcoholismIBD: Identity by descentLD: Linkage disequilibriumMCMC: Markov chain Monte CarloMLE: Maximum likelihood estimatorsSNP: Single-nucleotide polymorphismALH formatted the data files, analyzed the data, and drafted the manuscript. JSKK provided analyses identifying White subgroups. SB, LJB, GD, CHJ, and BKS participated in the design and coordination of the study. All authors read and approved the final manuscript."}
+{"text": "Piebald, Ticking, and Flecking loci interact to produce the Dalmatian's classic pigmented spots on a white background. The color of the pigmented spots in purebred Dalmatians can either be black or liver, but the locus responsible for color determination is unknown. Studies have been conducted to determine the underlying genes involved in coat color determination in the dog, e.g., in the Labrador Retriever, but none to date have addressed black versus liver in the Dalmatian.The distinctive coat pattern of a Dalmatian is the result of the interaction of several loci. While the encoded function of these genes is not fully understood, it is known the A genome scan was conducted in a multi-generational kindred of Dalmatians segregating black and liver spot color. Linkage analysis was performed using a total of 113 polymorphic microsatellite markers from the kindred. Linkage was found between spot color and a single microsatellite marker, FH2319 (LOD = 12.5) on chromosome 11.TYRP1 (Brown) locus is located at position 50.1 Mb on chromosome 11, which is approximately 0.4 Mb from marker FH2319. Given the recent characterization of TYRP1 genetic variations in the dog and the linkage evidence reported here, TYRP1 is likely responsible for the spot color variation of black versus liver seen in the Dalmatian.The  Specifically, it is known the ckground . The colckground , but theckground .Agouti, Extension, and Brown, have been characterized at the molecular level. The Brown locus describes tyrosinase related protein 1 (Tyrp1), which controls the production of eumelanin in melanocytes [wild-type allele results in black eumelanin while the recessive brown allele results in brown eumelanin [Extension locus has been found to be the melanocortin 1 receptor (Mc1r) protein [Agouti locus [Agouti and Extension acts as a switch for melanocytes to produce either phaeomelanin or eumelanin, with certain alleles resulting in a red or reddish-brown color [Several classic pigmentation loci, such as anocytes . In the  protein , which hwn color .Piebald, Ticking, and Flecking loci (or they would not display the classic spotting pattern) complicating characterization of these loci through standard techniques, such as linkage analysis, since there is not a segregating phenotype to detect. However, black and liver spot color is a detectable phenotype that segregates in Mendelian fashion. The objective of this study was to utilize a previously described multi-generational kindred of Dalmatians [All Dalmatians are homozygous for the lmatians  to conduTwopoint linkage analysis for spot color was performed using SOLAR v2.1.4  and 113 TYRP1 (Brown) locus is at position 50.1 Mb, which is approximately 0.4 Mb from FH2319. Given the recent characterization of TYRP1 genetic variations in the dog [TYRP1 is likely responsible for the spot color variation of black versus liver seen in the Dalmatian.A genome scan was performed on a multi-generational kindred of Dalmatians, previously analyzed for heritability and segregation of deafness . Spot co the dog  and the Brown locus for the depicted dogs. Dog 3 is heterozygous for the dominant B allele of the Brown locus (producing black spots) while dog 4 is homozygous for the recessive b allele of the Brown locus (producing liver spots). Progeny of dogs 3 and 4 reveal marker FH2319 allele 289, in this family, is linked with the B allele of the Brown locus. Additional alleles of marker FH2319 were also linked with the B allele in the Dalmatian kindred (data not shown). Due to this fact, linkage alone is not enough to establish if a dog with an unknown Brown locus genotype  was included. Based on examination of the kindred reported here, the TYRP1 gene should be further characterized in the Dalmatian to determine the specific genetic variations associated with the dominant and recessive alleles of the Brown locus.A recent study  examinedTYRP1 locus, supporting evidence that its gene product is responsible for the black or liver color of a Dalmatian's spots. Further characterization of TYRP1 in the Dalmatian is warranted to determine the specific genetic variations causative for color variation.Statistically significant linkage was found between marker FH2319 and spot color in a kindred of Dalmatians. FH2319 is 0.4 Mb from the Spot color was recorded from the Dalmatian kindred as previously described . There wGenomic DNA was isolated from 117 dogs  of the Dalmatian kindred reported [Microsatellite markers were amplified and resolved in multiplexed sets as described . A linkaTwopoint linkage analysis was performed using the program SOLAR v2.1.4  accordinEJC participated in design of the study, collected samples, performed the genome scan and linkage analysis, and drafted the manuscript. TRF was involved with the linkage analysis and review of the manuscript. RDS was involved with the linkage analysis and review of the manuscript. GMS was involved with collection of samples and review of the manuscript. KEM conceived of the study, participated in its design and coordination, and review of the manuscript."}
+{"text": "Over the past decade, genetic analysis has shifted from linkage studies, which identify broad regions containing putative trait loci, to genome-wide association studies, which detect the association of a marker with a specific phenotype. Because linkage and association analysis provide complementary information, developing a method to combine these analyses may increase the power to detect a true association. In this paper we compare a linkage score and association score test as well as a newly proposed combination of these two scores with traditional linkage and association methods. One advantage of association analysis is it can be carried out in samples of unrelated individuals, which may be easier to recruit. On the other hand, family samples provide extra information about segregation of the phenotype, and both linkage and association analysis may be performed when genotype and phenotype data are available on family members.Improvement in genotyping technologies has led to great advances in the quest to map genes influencing complex traits. In the late 1980s came linkage studies in family samples that identified broad regions containing putative trait loci. Recently, dense single-nucleotide polymorphism (SNP) chip technology has resulted in genome-wide association analysis, where the genome is queried for association with a specific phenotype. The high number of SNPs run  enables relatively thorough coverage of the genome, but also greatly increases the chance of false-positive results. A low Variance-component analysis  is a comPopulation-based methods can be applied to family samples provided that familial correlation is accommodated and there is no population stratification . Linear mixed-effect models are particularly well suited for association analysis of quantitative traits in family samples. The genotype effect and additional covariates are modeled as fixed effects while familial correlation is accommodated with a random effect with the covariance structure depending on the degree of relatedness between individuals. LRTs may be applied to determine the genotype-phenotype association. Alternatively, an efficient score statistic for association analysis with the variance computed conditional on the observed phenotype may offer a robust option against departure from the normality assumption .The objectives of this paper are two-fold: first, we compare the LRT with conditional score statistics for both linkage and association analyses using the Genetic Analysis Workshop 16 simulated Framingham Heart Study dataset (Problem 3). Second, we explore whether a combination of the linkage and association score improves power to detect associated SNPs.The subset of simulated Framingham Heart Study samples analyzed in our report consists of 736 families and 381 unrelated individuals. The families range in size from 2 to 291 participants with available genotypes and phenotypes, for a total of 6372 individuals.Initial data cleaning was performed using the software PLINK ,6 based Lipid-related phenotypes were available at three exams for each participant. To evaluate methods robust to departure from normality, we concentrated on low-density lipoprotein (LDL) and triglyceride levels (TG) levels because of their skewed distributions. We analyzed each trait measured at the first exam, but also averaged each trait over the three exams.Chromosome 11 was chosen because a group of 39 polygenes influencing high-density lipoprotein (HDL) clustered in the range of 110 Mb to 134 Mb and because of the presence of two major genes, one influencing LDL and the other influencing TG. Chromosome 22 was selected for ease of computation and because it harbors a major gene explaining 1% of the LDL variance.p-value \u2265 0.05, and a r2 < 0.04. Mendelian transmission errors were detected and corrected using the program PEDCHECK [For linkage analysis, we selected markers with low linkage disequilibrium, which may bias identity-by-descent (IBD) estimates when parental information is unavailable . We usedPEDCHECK .kth family, Y, has a multivariate normal distribution with mean E(Y|X) = \u03bcX = \u03bc + \u03b2X and covariance matrix: \u03c0tij is the proportion of alleles shared IBD by relatives i and j at genome location t; and \u03d5ij is the kinship coefficient. The covariance matrix unconditional on the IBD (\u03a3) is obtained by setting \u03c0tij = \u03d5ij in the expression for \u03a3\u03c0.The basic variance-component model assumes that the vector of phenotype in the t is N pedigrees. To test the null hypothesis of no linkage (H0: A\u03c0 is the matrix of centered IBD and W = (\u03a3-1(Y - \u03bcX)(Y- \u03bcX)'-I)\u03a3-1 with elements wij. The squared of the efficient score is standardized by an estimate of the variance conditional on the observed phenotypes to make it robust to violation of the normality assumption [The log likelihood for a QTL at sumption ,8.E = \u03bc + \u03b2X + \u03b3g, where g is the coded genotypes. The covariance unconditional on the IBD proportions, \u03a3, is typically used, although one could easily substitute \u03a3\u03c0 at the expense of added computation complexity. The null hypothesis of no association H0: \u03b3 = 0 is typically tested using a LRT. Alternatively, the efficient score test may be constructed to test for association. The efficient score statistic for association reduces to The linear mixed-effects model to test for association analysis is very similar to the variance-component model, with the exception that the genotype effect is included in the mean rather than in the covariance matrix: ociation .The conditional association score and our implementation of the linkage score are uncorrelated under the null hypothesis of no linkage and no association . Therefop-value above 10-6, and a low r2 (< 0.01) with all polygenes and major genes on chromosomes 11 and 22. We calculated power as the proportion of significant major gene association detected over all 200 phenotype replicates.We computed the LRT and association-conditional score statistics using an additive genetic model and a general 2-df genetic model for each SNP. For all models we adjusted for sex and age, or average age in the case of averaged phenotypes. We compared the association statistics in terms of type I error and power. To determine type I error, we selected SNPs with a call rate above 95%, a HWE We computed multipoint IBD probabilities using the software LOKI , making Figure As seen in Table \u03b1 = 5% is presented in Figure \u03b1 = 5% to detect this association as shown in Figure \u03b1 = 10-8 (data not shown). Power to detect association between rs901824 and LDL ranged between 60% and 80% at \u03b1 = 5%, but power was 0 for all statistics at \u03b1 = 10-8 (results not shown). All methods achieved 100% power to detect SNP rs2294207, an additively acting SNP on chromosome 22 explaining 1% of the LDL variation with either LDL phenotypes (data not shown).Power to detected major SNPs at Despite selecting phenotypes that violated the normality assumption, type I error rates for the additive association-conditional score and LRT statistics were not inflated, but slight type I error inflation was observed for the 2-df LRT statistic and moderate inflation for the 2-df association-conditional score. The type I error rate inflation for the 2-df statistics is most likely due to a violation of asymptotic assumptions caused by SNPs with low cell counts. Note that filtering by MAF less than 10% lowers the false-positive rate for both 2-df statistics.In our analyses, all additive statistics failed to detect the association between TG levels and rs603446, a SNP with a non-additive effect. This result suggests, as do Lettre et al. , that th\u03b1 = 0.05 to accommodate the extra degree of freedom used by the combined conditional score is 0.467 for the additive model and 0.396 for the general 2-df model. The percent of iterations that reached the minimum threshold ranged from 4.0% for the additive model for visit 1 TG levels at SNP rs603446 to 31.5% for the general 2-df model for average LDL at SNP rs901824. This suggests that a larger linkage signal or a more efficient combination of the conditional association and linkage scores may ultimately increase power.Including the information from the linkage analysis using the combined conditional score did not consistently increase the power to detect an associated SNP. However, despite the lack of a large linkage signal and even though the combined conditional score statistic uses an extra degree of freedom, the power remained fairly constant when using the combined conditional score compared with the LRT and the association-only conditional score. The minimum LOD score needed at A notable difference between the LRT and the conditional score for association was computation time. Performing the analysis for a total of 35,979 SNPs on chromosomes 11 and 22 using a single processor would take more than 16 hours using the LRT and less than an hour using the association-conditional score. The computation times for the linkage analysis were more comparable. Once the IBD was computed, the LRT using SOLAR took about 45 minutes to scan chromosome 11, while the linkage-conditional score took about 3.25 hours. Despite the longer linkage computation time, the combined conditional score was computed much faster than the LRT.Although more research is needed, conditional score analysis provides an interesting possibility of a gain in power by combining linkage information using the combined conditional score. Regardless, the conditional score for association provides a fast and comparable alternative to LRTs for analysis of family data.HDL: High-density lipoprotein; IBD: Identity by descent; LDL: Low-density lipoprotein; LOD: Logarithm of the odds; LRT: Likelihood ratio test; SNP: Single-nucleotide polymorphism; TG: TriglycerideThe authors declare that they have no competing interests.AEH performed the initial data cleaning, carried out the LRT association analysis, and wrote and revised the manuscript. YZ carried out the variance-component linkage analysis and revised the manuscript. JD conceived the study, implemented the conditional score analysis, and drafted and revised the manuscript. All authors read and approved the final manuscript."}
+{"text": "Atopic dermatitis develops as a result of complex interactions between several genetic and environmental factors. To date, 4 genome-wide linkage studies of atopic dermatitis have been performed in Caucasian populations, however, similar studies have not been done in Asian populations. The aim of this study was to identify chromosome regions linked to atopic dermatitis in a Japanese population.We used a high-density, single nucleotide polymorphism genotyping assay, the Illumina BeadArray Linkage Mapping Panel (version 4) comprising 5,861 single nucleotide polymorphisms, to perform a genome-wide linkage analysis of 77 Japanese families with 111 affected sib-pairs with atopic dermatitis.P = .0012) and weak linkage to 1q24 .We found suggestive evidence for linkage with 15q21 (LOD = 2.01, NPL = 2.87, We report the first genome-wide linkage study of atopic dermatitis in an Asian population, and novel loci on chromosomes 15q21 and 1q24 linked to atopic dermatitis. Identification of novel causative genes for atopic dermatitis will advance our understanding of the pathogenesis of atopic dermatitis. Atopic dermatitis (ATOD) is a hereditary, pruritic, inflammatory, chronic skin disease that occurs most commonly in early childhood but can persist or start in adulthood. The prevalence of ATOD has been studied in a wide variety of populations , and itsATOD is associated with cutaneous hyperresponsiveness to environmental triggers that are innocuous to healthy individuals . In the ATOD is the result of complex interactions between multiple genetic and environmental factors. Sixty-nine percent of patients with ATOD have one or both of parents affected by ATOD , and chiIL4), IL13, IL5, IL12B, and serine protease inhibitor Kazal-type 5 (SPINK5) . -12. 9-12Examining SNPs, instead of microsatellite markers for linkage is unlikely to yield different results because it has been reported that SNP-based genome-wide linkage study has the potential to be as powerful as traditional microsatellite-based analysis and offers good identification of specific locations for further fine-mapping association analysis . We perfThe 15q21 linkage region has not been reported previously as a region associated with ATOD. However, linkage of 15q21 to several other inflammatory diseases including osteoarthritis  and macuCD3Z) and chemokine ligand 2 (XCL2). CD3Z plays an important role to recognize the coupling antigen to several intracellur signal-transduction pathways [The 1q24 linkage region includes candidate genes such as T-cell receptor zeta chain isoform 2 precursor  declare that they have no competing interests.HE carried out molecular genetic study, participated in the study design and coordination and wrote the draft of the manuscript. SI, TT, KH, MI, TK, TA, YS, MK, MT, TS, and FO carried out molecular genetic studies. EN and TA participated in the design of the study and performed the statistical analysis. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:"}
+{"text": "Bovine whole genome linkage disequilibrium maps were constructed for eight breeds of cattle. These data provide fundamental information concerning bovine genome organization which will allow the design of studies to associate genetic variation with economically important traits and also provides background information concerning the extent of long range linkage disequilibrium in cattle.2 among all pairs of syntenic markers within eight breeds of cattle from the Bos taurus and Bos indicus subspecies. Bos taurus breeds included Angus, Charolais, Dutch Black and White Dairy, Holstein, Japanese Black and Limousin while Bos indicus breeds included Brahman and Nelore. Approximately 2670 markers spanning the entire bovine autosomal genome were used to estimate pairwise r2 values. We found that the extent of linkage disequilibrium is no more than 0.5 Mb in these eight breeds of cattle.Linkage disequilibrium was assessed using rLinkage disequilibrium in cattle has previously been reported to extend several tens of centimorgans. Our results, based on a much larger sample of marker loci and across eight breeds of cattle indicate that in cattle linkage disequilibrium persists over much more limited distances. Our findings suggest that 30,000\u201350,000 loci will be needed to conduct whole genome association studies in cattle. D'. The results indicated high levels of LD not only between closely linked markers but for markers located as much as 40 cM (~40 Mb) apart. Two subsequent studies examined the extent of LD in cattle although both used fewer animals and microsatellites [D' value was 44% with significant LD reported only for distances less than 10.3 cM. Linkage disequilibrium among non syntenic loci was not significant.Linkage disequilibrium (LD) maps are fundamental tools for exploring the genetic basis of economically important traits in cattle. Likewise, comparative LD maps enable us to explore the degree of diversity between breeds of cattle and to detect genomic regions that have been subject to selective sweeps within the different dairy and beef breeds which represent different biological attributes . The currently available information regarding LD in cattle is primarily based on microsatellite studies performed in dairy cattle. The most extensive of these studies used 284 genome-wide microsatellites in a population of Dutch Black and White Dairy cattle  to show tellites . Vallejotellites . Tenesa tellites  genotypeMore recently Khatkar et al.  scored 2D' was 16.3% for Japanese Brown and 25.1% for Japanese Black. Characteristic of D' as a measure of LD, significant LD was observed for marker pairs separated by as much as 40 cM in both breeds.Only recently has the extent of LD been examined in beef cattle populations. A sample of 162 half-sib progeny from a Japanese black sire and 406 half-sib Japanese brown cattle were genotyped with 246 and 156 autosomal microsatellite markers, respectively [Quantifying the extent of LD in the bovine genome is a necessary first step for determining the number of markers that will be sufficient for QTL mapping by linkage disequilibrium. The previous studies which used microsatellite markers were either too narrowly focused on particular chromosomes, or were of insufficient resolution to precisely estimate genome-wide LD and almost certainly were unable to precisely estimate short-range LD. The high density and low inherent rates of mutation of SNPs relative to microsatellites within mammalian genomes allows for the identification of ancestral haplotype blocks and the estimation of identity by descent probabilities which are crucial for haplotype-based association studies . In thisThe program GENOPROB 2.0 ,9 which Bos taurus. This ascertainment bias resulted in the SNP minor allele frequencies being substantially lower in the two Bos indicus breeds than in the Bos taurus breeds , consistent with the earlier reports  and not necessarily the physical distance between loci [2 is the preferred measure of LD, because it quantifies the amount of information that can be inferred about one (perhaps nonobservable quantitative trait or disease) locus from another [2 as the primary measure of LD in this study.It has been suggested that when large differences exist between marker allele frequencies, due to the presence of a rare allele, these two measures of LD are divergent . D' estieen loci . In the  another ,23, and  another . For thi2 values between breeds is evident in Figures 2 values between these breeds  and BTA14 in Holstein (0.91), respectively. The average r2 values across the Bos indicus breeds was 0.37 with minimum and maximum r2 values on BTA20 in Nelore (0.06) and BTA22 in Nelore (0.69). The relatively low level of LD at short inter-marker distances contrasts with previously published reports in cattle [2 values drop below 0.1 by 1 Mb , 20 Canadian Angus, 40 Charolais (Canada), 40 Brahman (USA), 97 Dutch Black and White Dairy cattle (Belgium), 48 Holstein (USA), 65 Japanese Black (Japan), 43 Limousin (USA) and 97 Nelore (Brazil) cattle. In order to phase the chromosomes using linkage information, we selected small families where members within the families were closely related but the families themselves were not closely related. Family structure and the number of individuals per family varied between the breeds but the general family structure consisted of a male grandparent, male parent and three or more progeny .Chromosomal coordinates for each SNP were obtained by aligning approximately 250 bp flanking each SNP by BLAST to the latest release of the bovine genome sequence assembly, Btau_3.1. These physical coordinates were compared to the linkage and RH maps of McKay et al. Thirty 2 values using GOLD [GENOPROB V2.0 ,9 was using GOLD  independThe author(s) declares that there are no competing interests. SDM conceived the study and participated in its design, data collection, analysis and manuscript preparation. RDS participated in study design, data analysis and manuscript preparation. BM participated in study design, genotyping and data analysis. LKM provided bioinformatics support. JA provided bioinformatics support. WC organized collection of Dutch Black and White samples, DC provided Charolais samples, EDN organized collection of Nelore samples, CAG provided bioinformatics support and organized collection of Brahman samples, CG provided bioinformatics support, HM organized the collection of Japanese Black samples, PS provided bioinformatics support, ZW provided statistical support, CVT made intellectual and bioinformatics contributions, JW made intellectual contributions and help in the manuscript preparation, JT participated in study design, provided Angus, Limousin and Holstein samples and helped draft the manuscript, SSM intellectual contributions. All authors read and approved the final manuscript.Genoprob summary. Proportion of genotypes reaching a given level of confidence where pGmx is the probability that the indicated genotype is correct and oGmx is the probability that the phase order of the two alleles of the genotype is correct.Click here for fileAverage measures of linkage disequilibrium for each breed.Click here for file2 values by breed and chromosome.Average rClick here for file2 values for inter-marker distances of 5\u2013100 kb for each breed and chromosome.Average rClick here for file2 values for inter-marker distances of 100\u2013500 kb for each breed and chromosome.Average rClick here for filePedigrees for each breed. Pedigrees for each breed include a Unique animal IDs, sex of each animal and relationship of each animal genotyped. All genotyped animals were shaded in gray.Click here for fileSNP information.Click here for file"}
+{"text": "Performing linkage and association analyses on a large set of correlated data presents an interesting set of problems. In the current setting, we have 3554 expression levels from lymphoblastoid cell lines in 194 individuals from 14 three-generation Utah CEPH (Centre d'Etude du Polymorphisme Humain) pedigrees. We formed multivariate expression phenotypes from six sets of genes. These consisted of a set of genes identified by the data providers as showing common linkage to a region of chromosome 14, as well as five other sets suggested by ontological evidence. Using principal-component analyses, we generated seven quantitative phenotypes for expression levels from these six sets of genes. We performed quantitative genome linkage screens on these traits using the expression traits from the third generation of each pedigree. As expected, the strongest linkage signal was achieved when the trait under analysis was the composite of the expressions of genes previously showing linkage to chromosome 14. In particular, this trait produced a LOD score of 5.2 on chromosome 14. The trait also produced LOD scores over 3.5 on chromosomes 1, 7, 9, and 11; this suggests that these genes may be controlled by additional genetic factors on the genome. Subsequent association analyses on the first two generations of these pedigrees identified two polymorphisms on chromosome 11 as significant after correcting for multiple tests. These results suggest that principal-component analyses are useful for the analysis of pleiotropic loci. Furthermore, we have identified two single-nucleotide polymorphisms that may influence the expression of multiple genes linked to chromosome 14. The Genetic Analysis Workshop 15 data set consists of 3554 expression levels from lymphoblastoid cell lines in 194 individuals from 14 three-generation Utah CEPH (Centre d'Etude du Polymorphisme Humain) pedigrees. In earlier work [Principal-components analysis (PCA) is a technique for reducing multi-dimensional data sets into lower dimensions for analysis. The goal is to capture as much variation as possible of the higher dimensional data set in a lower dimensional set. Because microarray expression data typically produces thousands of correlated observations for each array, PCA is a natural analytic technique. Several other groups have shown the utility of PCA for the decomposition of expression data -4; here In microarray data, PCA provides orthonormal bases for the expression array profiles and the gene transcriptional responses. The details are well described elsewhere .Our goal was to perform PCA on several different subgroups of expression phenotypes and then perform linkage analysis on the component scores. We performed the PCA in the R language . In ordeWe investigated the set of expression phenotypes linked to chromosome 14 by Morley et al. . The celFor the strongest linkage signals, we downloaded data from the HapMap project for the CEPH families for SNPs within the two-LOD support . We perfFor a number of phenotypes, SOLAR reported an estimated heritability of 1.0. Further investigation revealed that the individual expression levels as well as the component scores often showed an intraclass correlation (ICC) of more that 50% in sibships. Heritability is often estimated as twice the intraclass correlation in sibships; this would imply a heritability greater than one. This is clearly nonsensical, so we suspect that this indicates a shared environment with an effect on multiple expression levels. We performed a PCA on the 471 genes that had ICC in sibships greater than 0.5 (the \"high heritability\" genes). We also performed further tests to determine whether these genes contained an over-representation of ontological categories. In particular, we examined the 10 most represented ontological categories in the high heritability genes, and then performed 10,000 re-samplings of 471 genes from all 3554 to determine whether any of these 10 categories were over-represented.We first examined the reliability of the PCA. Using the entire set of phenotypes, we found that the components generated from 14 unrelated individuals had a 98.7% correlation with the components generated from all 110 third-generation individuals. Furthermore, 10,000 repeated samplings of 14 unrelated individuals produced an average correlation of 96.1% with the 14 individuals initially chosen. We took this as evidence that the components were reliable, even using a relatively small number of individuals; we then used the same 14 individuals throughout. The PCA analysis of each of the six categories of gene expression then produced 14 principal components. Recently, Raiche et al. [The VC analysis of the separate phenotypes linked to chromosome 14 yielded fewer significant linkage peaks than originally reported; this was not unexpected because the methodology differed. In particular, the original analyses used a modification of the Haseman-Elston (HE) method ; under sp = 9.8 \u00d7 10-6), so we included age in all SNP analyses. We identified 761 nonsynonymous SNPs in these regions, so we set our initial significance threshold to a conservative level of 0.05/761 = 6.6 \u00d7 10-5. One SNP, rs10458896, is significant at this threshold . The corresponding q-value is 0.034; this is the only q-value less than 0.05. This is a non-synonymous SNP in KIF18A, a kinesin family member on chromosome 11. In the analysis of all the SNPs, we found 143,798 SNPs in these regions. We set our significance threshold to 0.05/143798 = 3.5 \u00d7 10-7. One SNP in an intergenic region on chromosome 11, rs10768321, is significant at this threshold with a p-value of 5.8 \u00d7 10-8 and a q-value of 0.008; again, this is the only q-value less than 0.05.For the association analysis, we focused on the five regions with linkage signals over 3.5 for the traits linked to chromosome 14, including the linked region on chromosome 14. We found the first principal component of these traits was strongly associated with age , \"nucleotide binding\" (p < 0.0001), \"ATP binding\" (p = 0.0001), and \"RNA binding\" (p = 0.0002).For the analysis of the top ten ontological categories represented by the 471 \"high heritability\" genes, we computed empirical As expected, PCA of traits with common linkage to a region on chromosome 14 does show a very strong signal in this region. Several other novel linkage signals appear, including two for the ontological category \"cell-cycle.\" In principle, one would expect some genes from the same ontological category to be controlled by the same master regulators; this has been demonstrated in yeast . HoweverWe also note that the association analyses were performed on a set of individuals related to, but distinct from, the individuals used in the linkage sample. In particular, some individuals from the first two generations of the Utah CEPH pedigrees have been genotyped by the HapMap project. For the linkage analyses, we used only the phenotypes of the third generation. Because the linkage analysis was restricted to a single generation, we only used age as a covariate in the association analyses. A combined linkage and association analyses would be possible if the significant SNPs, rs10458896 and rs10768321, were genotyped on the third generation of the CEPH pedigrees. We would expect that the linkage signal on chromosome 11 would be reduced in a SOLAR analysis including these SNPs as covariates if the SNPs \"explain\" some of the linkage signal.The \"high heritability\" genes present a conundrum. We speculate that there may be shared environmental factors that influence many expression phenotypes; these factors would elevate the correlation in sibships and produce high heritability estimates. Because four ontological categories  are significantly over-represented in these high heritability genes, further investigation into potential environment factors modifying these functions may be of interest. These high heritability estimates do not occur when the other generations are included in the analyses; the inclusion of parents and potentially cohort effects reduce the heritability estimates.Performing PCA on a set of genes linked to chromosome 14 increases the LOD score; this cannot be considered stronger evidence of linkage because we anticipate a similar result if the clustering was due to chance. However, the linkage on chromosome 14 is supported by the analysis of all 3554 expression phenotypes where the largest peak is in the same region. We also find strong evidence for other loci on chromosomes 1, 7, 9, and 11 that control the genes linked to chromosome 14.Association analyses were successful in identifying two polymorphisms on chromosome 11 regulating the set of genes displaying linkage to a region of chromosome 14. The association analysis was performed in two steps; first, analysis of only non-synonymous SNPs and then analysis of all SNPs in the regions of interest. The analysis of non-synonymous SNPs yielded a polymorphism on chromosome 11 in KIF18A significant after correcting for the number of non-synonymous SNPs. The analysis of all SNPs in the region yielded a SNP in an intergenic region on chromosome 11 significant after correcting for the total number of SNPs in the regions of interest. Further investigation into these SNPs may show a role in the regulation of a large number of genes.We identified a set of genes that apparently had heritability greater than one. Shared environmental factors may increase the intraclass correlation within sibships. We found four ontological categories  that are significantly over-represented in these high-heritability genes; further investigation into potential environment factors modifying these functions may be of interest.The author(s) declare that they have no competing interests."}
+{"text": "HLA haplotypes that include DR3 or DR4 alleles.A genome-wide search for genes that predispose to type 1 diabetes using linkage analysis was performed using 900 microsatellite markers in 70 nuclear families with affected siblings from Finland, a population expected to be more genetically homogeneous than others, and having the highest incidence of type 1 diabetes in the world and, yet, the highest proportion in Europe of cases (10%) carrying neither of the highest risk HLA region on 6p21 , significant evidence of linkage in other chromosome regions was not detected with a single-locus analysis. The two-locus analysis conditional on the HLA gave a maximum lod score (MLS) of 3.1  on chromosome 9p13 under an additive model; MLS of 2.1  on chromosome 17p12 and MLS of 2.5  on chromosome 18p11 under a general model.In addition to the evidence of linkage to the HLA genes also in the high-risk Finnish population, and suggest that non-HLA genes also contribute to the familial clustering of type 1 diabetes in Finland.Our genome scan data confirmed the primary contribution of the  Type 1 diabetes is the third most prevalent chronic disease of childhood, affecting 0.4% of the general population by age of 30 years and has a lifetime risk of nearly 1% . The incThe observation of familial clustering of type 1 diabetes suggests that genetic factors are involved in the etiology of type 1 diabetes. For people of European ancestry, the frequency of type 1 diabetes in siblings of affected individuals is about 6% by the age of 30 ,11, whilHLA-encoded susceptibility to type 1 diabetes ; and also excluded the possibility of a locus with an effect equivalent to HLA. The individual impact of other susceptibility genes is, therefore, much smaller than that of HLA. Nevertheless, statistically significant and suggestive evidence of linkage of type 1 diabetes to at least ten chromosome regions has been published, although association studies at INS [CTLA4 have been required to confirm with fine map loci. These newly identified susceptibility loci showed evidence for linkage in some studies but could not be replicated in others. The discrepancies between the studies may be due to a number of factors, including sample size, genetic and phenotypic heterogeneity between data sets, genotyping methods, gender-specific effects and genetic epistasis [HLA haplotypes (approximately 10%), suggesting the possibility that non-HLA loci, perhaps with a reasonable penetrance and population allelic frequency due to founder effects exists in Finland.Following the lead from gene identification studies in rare Mendelian diseases and the clear evidence of linkage of the MHC in human and mouse to type 1 diabetes, genome-wide scans for linkage to type 1 diabetes were undertaken -26. Thess at INS  and CTLApistasis ,28. We aHLA region where the major type 1 diabetes susceptibility gene(s) locates. For the two-locus analysis, we fixed markers with highest MLS at the HLA region to adjust for the effect of HLA. Then the joint IBD sharing at the HLA locus and at a second locus was considered, which was placed at an increment of 1.0 cM across the genome. The MLS for the effect of HLA was subtracted so that the curves in figures represent the additional contribution of locus 2. As expected, at positions unlinked to HLA such as those on different chromosomes, the multiplicative curves were identical to those curves obtained by use of a single-locus model for locus 2 and therefore were not presented in the figures.A total of 868 loci with a length of 3518 cM were actually genotyped. Figures -6) at chromosome 6p21 (HLA region) was observed. Another region, on 9p13 shows a MLS of 3.09 under either a two-locus additive model  or general model . Two other regions on 17p12 and 18p11 showed positive findings under two-locus general model  have become available. 18p11 has been reported to have robust association in the first genome-wide association study in type 1 diabetes along with several other loci showing a significant association . The evis we did -26. TakiHLA genes and suggest that a linkage to chromosome 18p11 region might exist in this population with the highest risk of type 1 diabetes in the world.Current genotyping data originally was used to map loci for nephropathy selecting for statistical analysis only sib-pairs discordant (DSPs) for diabetic nephropathy . For DSPHLA genes also in the high-risk Finnish population, and suggest that non-HLA genes also contribute to the familial clustering of type 1 diabetes in Finland.Our genome scan data confirmed the primary contribution of the DNA from 70 Finnish nuclear families with at least two siblings affected with type 1 diabetes, including six families with three and one family with four affected children was collected from Finland. MODY families were not included. A total of 207 individuals (147 sibs and 60 parents) were genotyped, providing with 81 sib-pairs affected with type 1 diabetes for linkage analysis. The original study design was to collect DSPs, siblings affected with type 1 diabetes but discordant for diabetic nephropathy, to map loci for diabetic nephropathy. Therefoet al.[DNA was extracted from peripheral lymphocytes, according to standard procedures. A genome wide scan was performed using 900 microsatellite markers and using protocols described by Gretarsdottir et al. at the get al..-4 were considered a statistically significant threshold for linkage [A generalization of maximum lod score (MLS) method proposed by Risch  was used linkage . Thresho linkage .HLA region, where the single-locus MLS was greatest. The null hypothesis for a two-locus analysis is that the locus 2 is not involved in disease, and the results are given for a variety of two-locus models, each fitted with the second locus placed at increments of 1.0 cM across the genome. The multiplicative model estimates the conditional MLS at locus 2, taking account of any effect at locus 1. If loci 1 and 2 are unlinked, the multiplicative conditional MLS for locus 2 will be identical to the single locus MLS for locus 2. The additive and general models calculate the conditional MLS at locus 2 taking account of any effect at locus 1, assuming an additive model for the joint action of loci 1 and 2, or allowing for arbitrary epistasis between loci 1 and 2 in the general model. Significance levels  for a single-locus MLS were calculated according to the possible triangle constraints described by Holmans [Two-locus analysis with Risch's method developed via an extension of the method by Cordell ,38 was t Holmans  and for To calculate the two-locus MLS, a prior as well as a posterior probability that each affected sib-pair shares i alleles IBD  at particular positions on the genome is required. For unlinked loci, the IBD probabilities were obtained from the output of Genehunter (version 2.0) ,41 and fWe also estimated the power of our sample to test for linkage using the mean IBD sharing test  by compuQQ participated in the data analysis and drafting of the manuscript; AM\u00d6 and BH took part in the genotyping, managed and analyzed the data and approved the final version of the manuscript; JP and HJC participated in the data analysis and drafting of the manuscript; CS collected the data and approved the final version of the manuscript; LK collected the data and approved the final version of the manuscript; ETW participated in the concept and design of the study and approved the final version of the manuscript; KT and JT were responsible for the conception, funding, design and coordination of the study, approved the final version of the manuscript."}
+{"text": "Although rheumatoid arthritis has been shown to have moderately strong genetic component, both linked loci identified in linkage analyses and susceptibility variants from association studies are short of adequately accounting for a comprehensive catalogue of the molecular factors underlying this complex disease. The objective of this study was to use supplementary phenotype based on cumulative hazard of rheumatoid arthritis to identify linkage evidence for new and additional rheumatoid arthritis loci in a genome-wide linkage analysis of 342 affected sibling pair families from the United Kingdom.Using proportional hazards model, we estimated cumulative hazard of rheumatoid arthritis and then used it as a quantitative trait in a non-parametric multipoint variance component linkage analysis with 353 microsatellite markers distributed across the 22 autosomal chromosomes.We identified 3 new loci with genome-wide suggestive linkage evidence for rheumatoid arthritis on 9q21.13, 15p11.1 and 20q13.33. Our results also confirmed previously reported linkage evidence in the HLA-DRB1 region on chromosome 6 and on locus 1q32.1.This study demonstrates the potential for information gain through the use of supplementary phenotypes in genetic study of complex diseases to identify new and additional potential linked loci that are not detected by linkage analysis of traditional phenotypes; and our results provide further evidence of the involvement of multiple loci in the genetic aetiology of rheumatoid arthritis. Rheumatoid arthritis (RA) is a chronic inflammatory condition that belongs to the family of diseases referred to as autoimmune. The population prevalence of RA is about 0.01 , and theAs with many other complex human diseases, the difficulty in achieving a clear understanding of the role of genetics in disease can partly be attributed to limitations in phenotype definition and characterization. RA is a complex disease condition with variation in onset age, severity, and accompanying presence or absence of clinical features such as nodules, erosions, and rheumatoid factor. In addition, phenotypic, clinical and genetic heterogeneity exists between studies. To address the problem of heterogeneity associated with RA phenotype, John et al.  and Eyrehttp://www.medicine.manchester.ac.uk/epidemiology/research/arc/). Details of family recruitment and assessment protocols have been described elsewhere [et al [The RA affected sibling pair families used in this study were part of the UK National Repository of Multicase Rheumatoid Arthritis Families maintained by the Arthritis Research Campaign ; where S(t) is the survival function at time 't' estimated from the fitted proportional hazards model.We restricted all analyses to RA affection status, age at onset and sex since other measures such as severity of RA, erosion status and RF status are measures of post-RA affection symptoms. Our objective was to study only those variables that influence an individual's hazard to RA. Survival analysis was performed using RA status, sex, and onset age data on 750 affected subjects from 364 families. Since first symptoms of RA can begin at any point in time, it is reasonable to suppose that observed ties in event times (age at onset) among subjects are likely results of approximation or convenience in measurement and that there is a true but unknown ordering for the tied event times. This supposition appears more plausible for event as onset of RA and as such tied event times were treated according to the method described by Kalbfleisch and Prentice , and DeLFor the linkage analysis, 22 families with singletons were excluded because these were uninformative for linkage analysis. The linkage analysis was therefore based on 342 families with a total of 728 affected siblings. There were 353 microsatellite markers across the 22 autosomal chromosomes. For subjects not genotyped for some markers, their genotypes were set to missing for such markers. Allele frequencies for each marker were calculated using maximum likelihood estimation method. The marker-specific identity by descent (IBD) for sibling pairs within each family, and the multipoint IBDs were computed using the software SOLAR . Since tP = 0.8360)  Table . InfluenP \u2264 2 \u00d7 10-5) for linkage on chromosome 6. The linkage signal spanned the HLA-DRB1 region  were identified on chromosomes 1, 9, 15 and 20 and are displayed in Figure P < 0.05) was identified on eleven other chromosomal regions , Mackay et al  families confirmeIt is worth noting that in this study we did not include clinical covariates such as rheumatoid factor status and erosion status that were included in some of the previous studies because these covariates measure symptoms resulting from RA affection. It is therefore not illogical to assume that an individual's cumulative hazard to RA could not be influenced by such post-RA onset phenotypes or clinical symptoms. We therefore chose not to include in the model variables that we think do not influence an individual's hazard of being affected by RA. Whether all the identified linkage regions in the present study are true positives requires further studies. We declare that the reproduction of linkage evidence at previously reported potential RA loci provides support for the validity of the definition of supplementary RA phenotype and modelling that we have used in the present study. We recognise that supplementary phenotype as defined and treated as quantitative trait in the present study may not be practicable for every complex human disease phenotype. However, for complex diseases for which this approach is applicable, this can lead to identification of new additional genetic loci that could explain part of the missing heritability  observedThis present study demonstrates the potential information gain through the use of supplementary phenotypes in genetic study of complex diseases such as RA to detect new and additional linked loci that are undetected in linkage analysis of the traditional phenotypes. Also, our results provide further evidence of the involvement of multiple loci in the genetic aetiology of RA.The authors declare that they have no competing interests.BOT conceived of the study, performed all statistical analyses, interpreted results and wrote the manuscript. YL participated in the design, statistical analyses, and preparation of manuscript. AK, AM, MT and RSC participated in the design and preparation of manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2350/10/142/prepub"}
+{"text": "The trait locus is estimated at approximately 45.51\u201345.82 cM, with standard errors of the estimates range from 0.82 to 1.26 cM, depending on whether and which quantitative variable is incorporated. The standard error of the estimate of trait locus decreased about 28% to 35% after incorporating the additional information from the quantitative variables. This mapping technique helps to narrow down the regions of interest when searching for a susceptibility locus and to elucidate underlying disease mechanisms.Rheumatoid arthritis is a complex disease in which environmental factors interact with genetic factors that influence susceptibility. Incorporating information about related quantitative traits or environmental factors into linkage mapping could therefore greatly improve the efficiency and precision of identifying the disease locus. Using a multipoint linkage approach that allows the incorporation of quantitative variables into multipoint linkage mapping based on affected sib pairs, we incorporated data on anti-cyclic citrullinated peptide antibodies, immunoglobulin M rheumatoid factor and age at onset into genome-wide linkage scans. The strongest evidence of linkage was observed on chromosome 6p with a  Several biomarkers, including anti-cyclic citrullinated peptide (anti-CCP) antibodies and immunoglobulin M rheumatoid factor (IgM RF), are used to characterize rheumatoid arthritis (RA). Anti-CCP antibodies and IgM RF are important surrogate markers for diagnosis and prognosis in RA. The genetic mechanism of these biomarkers might directly underlie disease status. If not, the quantitative trait loci (QTL) might be linked to the loci responsible for RA, or the quantitative trait might interact phenotypically with RA. Hence, incorporating the quantitative trait into analyses will increase our ability to map the genes that predispose to RA . In addi\u03c4, along with sampling uncertainty to help investigators to narrow down chromosomal regions putatively harboring disease locus. The genetic effect is denoted by \"C\", and the value, (1 + C)/2, characterizes the probability of an affected sib pair sharing the same allele at \u03c4 from the parent. Chiou et al.  on chromosome 6 . Hence, the probability of an affected sib pair sharing the same allele from a parent is estimated to be 0.61 at \u03c4. Other regions showing evidence of linkage included the region at around 91.6 cM on chromosome 5 (p = 0.04), 7.09 cM on chromosome 8 (p = 0.0027), 40.11 cM on chromosome 9 (p = 0.02), 84.21 cM on chromosome 10 (p = 0.047), 49.83 cM on chromosome 16, and 70.22 cM on chromosome 18 (p = 0.043) ; joint involvement, according to the joint alignment and motion (JAM) score; the presence and extent of erosive disease on hand/wrist radiographs; functional status according to the health assessment questionnaires (HAQ) scores, age and calendar year of RA onset; the presence of nodules or other extra-articular manifestations; and the presence of other autoimmune diseases . Hence, We demonstrated that the efficiency of the disease locus localization was greatly improved by the incorporation of quantitative variables related to RA. By applying this approach, the investigators would be able to narrow down the regions of interest when searching for disease susceptibility loci. The significance of the improvement in the location estimate could be assessed by a bootstrap method. We are currently conducting systematic simulation studies to assess the improvement, the results will be reported elsewhere.The estimated standard error of  studies . The estThe author(s) declare that they have no competing interests."}
+{"text": "Iron-sulfur clusters are ubiquitous structures which act as prosthetic groups for numerous proteins involved in several fundamental biological processes including respiration and photosynthesis. Although simple in structure both the assembly and insertion of clusters into apoproteins requires complex biochemical pathways involving a diverse set of proteins. In yeast, the J-type chaperone Jac1 plays a key role in the biogenesis of iron sulfur clusters in mitochondria.Jac1 yeast knockout mutant suggesting a role for AtHscB in iron sulfur protein biogenesis in plants. In contrast to mitochondrial Jac1, AtHscB localizes to both mitochondria and the cytosol. AtHscB interacts with AtIscU1, an Isu-like scaffold protein involved in iron-sulfur cluster biogenesis, and through this interaction AtIscU1 is most probably retained in the cytosol. The chaperone AtHscA can functionally complement the yeast Ssq1knockout mutant and its ATPase activity is enhanced by AtHscB and AtIscU1. Interestingly, AtHscA is also localized in both mitochondria and the cytosol. Furthermore, AtHscB is highly expressed in anthers and trichomes and an AtHscB T-DNA insertion mutant shows reduced seed set, a waxless phenotype and inappropriate trichome development as well as dramatically reduced activities of the iron-sulfur enzymes aconitase and succinate dehydrogenase.In this study we demonstrate that AtHscB from Arabidopsis can rescue the Our data suggest that AtHscB together with AtHscA and AtIscU1 plays an important role in the biogenesis of iron-sulfur proteins in both mitochondria and the cytosol. Besides harbouring a bacterial SUF-like (mobilization of sulfur) system encompassing the SufA, SufB, SufC, SufD, SufS and SufE proteins Iron-sulfur proteins were first identified in plants and several excellent reviews have summarized both early and recent updates in the field of plant [Fe-S] biogenesis et al pioneered this field by identifying the Arabidopsis Sta1 as an Atm1p-like ABC transporter of yeast supporting the maturation of [Fe-S] protein in mitochondria iron sulfur cluster) system Compared to chloroplasts, [Fe-S] biogenesis in plant mitochondria has attracted much less attention. Kushir In the plant cytosol, [Fe-S] biogenesis is much less well understood. However, recent work by Balk and colleagues In bacteria and yeast, the HscA/Ssq1 chaperones and the HscB/Jac1 co-chaperones are important elements of the ISC-like system. HscA/Ssq1 are ATPases, stimulated by the J-type co-chaperone HscB/Jac1 and have been shown to interact with the scaffold protein IscU/Isu, which is regulated by HscB/Jac1 by binding to IscU/Isu to assist [Fe-S] delivery to the chaperone Here we demonstrate that Arabidopsis contains a functional AtHscA1/AtHscB/AtIscU1 protein cluster involved in [Fe-S] protein biogenesis. In contrast to yeast, the AtHscA1/AtHscB/AtIscU1 protein cluster is localized to both mitochondria and the cytosol of Arabidopsis suggesting a dual action between these two spatially separate compartments.E. coli HscB and yeast Jac1 showing 30% and 24% identity, respectively (A full-length cDNA (759 nt) encoding the At5g06410 open reading frame was cloned and its predicted amino acid sequence compared to ectively . The At5jac1  marked plasmid pRS316, with pGADT7-AtHscB or pGBKT7-AtHscB and positive transformants were screened on synthetic dropout media SD/-Leu (minus L-leucine) or on SD/-Trp (minus L-trypothan), respectively. Once scored the wild-type Jac1 cDNA was removed by streaking \u0394jac1/pGADT7-AtHscB and \u0394jac1/pGBKT7-AtHscB colonies onto YPD media containing 1 mg/ml 5-FOA (5-Fluoroorotic Acid). As URA3 encodes orotine-5\u2032-monophosphate dicarboxylase, which convert 5-FOA to toxic fluorodeoxyuridine, colonies will only grow where pRS316 has been removed and functional complementation has occurred. On YPD media, \u0394jac1/pRS316-Jac1, \u0394jac1/pGADT7- AtHscB and \u0394jac1/pGBKT7-AtHscB showed clear growth  and transiently expressed this transgene in tobacco cells  whose products are predicted to be mitochondrial pLysS by auto-induction and affinity purified the protein to >95% purity  directly upstream of the AtHscB start codon was PCR-cloned into the \u03b2-glucoronidase (GUS) binary vector pBADG and transformed into wild type Arabidopsis. GUS staining of T2 lines showed that AtHscB is universally expressed at low levels but with relatively high levels of expression in anthers and trichomes .AtHscB-specific primers LP585159 and RP585159. Two PCR fragments were obtained and sequenced revealing the presence of two T-DNAs inserted 0 and 5 nt downstream of the AtHscB stop codon (AtHscB transcript and undetectable AtHscB protein (CUT1 sense suppressed plants AtHscB followed by phenotypic characterization. More than 95% (20 out of 21) of transformed resistant plants showed a wild type phenotype (AtHscB-deficiency is responsible for the observed mutant phenotypes. Due to the high level of AtHscB expression in trichomes (AtHscB gene expression patterns homozygous mutants not only have fewer trichomes than wild-type (An Arabidopsis T-DNA insertion line (SALK_085159) was identified and analyzed by PCR with the T-DNA specific primer LBb1 and the op codon . Althougop codon  several op codon  showing  protein . Phenoty protein , a similhenotype  confirmirichomes , we moniild-type  but thesild-type .To test whether AtHscB indeed has an effect on iron sulfur proteins in Arabidopsis, we assayed both aconitase and succinate dehydrogenase (SDH) activities in wild type plants and in homozygous N585159 mutant plants. jac1 mutant  AtHscB can rescue the yeast \u03941 mutant , (ii) At1 mutant  and . Based on our findings it is reasonable to suggest that AtHscB, AtHscA1 and AtIscU1 may function in both mitochondria, and through retention of AtIscU1 by AtHscB  were grown at 21\u00b0C with 16 h of light (100 \u00b5mol photons m\u20132 s\u20131) per day and 60% humidity unless otherwise stated. The AtHscB T-DNA insertion mutant Salk_085159 (N585159) Wild-type Arabidopsis, transgenic Arabidopsis and the Pac1 and SacI of pBA002a AtHscB ATG was amplified with primers P06410-AscI-L/P06410-PacI-R, cloned into AscI and PacI of pBADG and transformed into wild-type Arabidopsis for GUS expression analysis. AtHscB and AtIscU1 were PCR amplified using primers AtHscB-GBK-L/AtHscB-GBK-R and AtIscU1-GBK-L/AtIscU1-GBK-R, and cloned into NdeI and BamHI of pGBKT7 or pGADT7 (Clontech). AtHscB, AtIscU1 and AtHscA1 were amplified using primers AtHscB-XhoI-L/AtHscB-KpnI-R, AtIscU1-XhoI-L/AtIscU1-KpnI-R and AtHscA1-KpnI-L/AtHscA1-KpnI-L R, digested with XhoI/KpnI or with KpnI and inserted into pWEN18, pWEN-N-YFP (YFP amino acids 1\u2013154) and pWEN-C-YFP (YFP amino acids 155\u2013238). AtHscB, AtIscU1, and AtHscA1 were PCR cloned into pET28a using primers AtHscB-ET-L/AtHscB-ET-R AtIscU1-ET-L/AtIscU1-ET-R and AtHscA1-ET-L/AtHscA1-ET-R. AtHscB was PCR amplified with primers AtHscB-XhoI-L/AtHscB-SpeI-R, digested with XhoI/SpeI and cloned into pBA002 for N585159 complementation experiments. Primers are shown in A GFP-GUS DNA fragment was PCR amplified using primers GFPGUS-L/GFPGUS-R  and pWEN-C-YFP (pWEN-CY) containing AtHscB and AtIscU1 and control vector controls were transformed into HF7c and tested for His auxotrophy restoration following the Matchmaker two-hybrid system III manual (Clontech).pGBKT7 and pGADT7 containing jac1 complementation, \u0394jac1-pRS316-Jac1 was transformed with pGBKT7-AtHscB or pGADT7-AtHscB and screened on SD/-Trp (minus L-trypothan) or SD/-Leu (minus L-leucine). Colonies were streaked onto 5-FOA media  to screen for complementation. Isolated colonies were verified by RT-PCR using primers Jac1-L/R and AtHscB-L/R.For \u0394ssq1 complementation, \u0394ssq1 was transformed with pGBKT7-AtHscA1 and positive transformants screened on SD/-Trp. Overnight cultures were spotted onto YPD or H2O2 plates  and incubated at 34\u00b0C for 4 days.For \u0394AtHscB, AtIscU1 and AtHscA1 were transformed into E. coli Rosetta(DE3)pLysS (Novagen) and expression performed for 20 h in auto-induction ZYM-5052 media 2, 100 mM NaCl, 1% Triton X-100 and 10 \u00b5M/ml Benzonase\u00ae Nuclease (Novagen) and the proteins purified using TALON affinity resin (BD Biosciences). Purity was assessed by SDS\u2013PAGE analysis.pET28a containing 32P] ATP (1 Ci\u200a=\u200a37 GBq) (specific activity 10 mCi/mmol). All reactions were terminated using 1 \u00b5l of 1 M formic acid. Samples were applied to TLC plates (Macherey and Nagel), developed in 0.5 M LiCl and 0.5 M formic acid buffer, and visualized by autoradiography. The hydrolyzed ATP was quantified using phosphorimager.ATPase assays were performed as described previously et al2, 0.1% Triton X-100), 200 \u00b5M cis-aconitate, 1.3 mM NADP+, 400 \u00b5U IDH (isocitrate dehydrogenase), and a reference cuvette lacking cis-aconitate and IDH was prepared. To each cuvette, protein extraction (\u224840 \u00b5g protein) was added and absorption increase at 340 nm in a double-beam spectrophotometer was measured (\u03b5340nm\u200a=\u200a6220 M\u22121 cm\u22121). SDH assay: Two cuvettes containing 950 \u00b5l SDH buffer , 0.25% succinate, 70 \u00b5M dichlorophenol-indophenol, and 60 \u00b5M decylubiquinone were prepared. 0.25% malonate was added to the reference cuvette and the assay was initated by adding cell suspension (\u224840 \u00b5g protein) to the second cuvette. The absorption decrease was measured at 600 nm in a double-beam spectrometer .Aconitase and succinate dehydrogenase (SDH) assays were performed following Stehling Table S1Primers used in this study(0.04 MB DOC)Click here for additional data file.Figure S1A. Amino acid sequence alignment of AtHscB with HscB of E.coli and Jac1 of yeast. Accession number of HscB is NP_417022 and Jac1 NP_011497. Filled arrow indicates the possible cleavage site of the signal peptide. * underlines the important motif conserved for all the three proteins. B. Stable expression of AtHscB-YFP in Arabidopsis. C. Stable expression of AtHscA1-YFP in Arabidopsis. D. Western blot showing the specificity of the AtHscB antibody. Lane a) represents wild-type E. coli cell extract, lane b) represents cell extract from E. coli expressing AtHscB and lane c) represents total cell extract from wild-type Arabidopsis. The slight size increase in lane b) is due to the presence of a 6xHis affinity tag on the protein. E. Immunogold labelling using pre-immune serum. F. Western blot to check the AtHscB expression in AtHscB-CY /AtIscU-NY  double transformed plants. WT. wild type plants. OX-1 and OX-2 are two double transformed plants. The upper panel used anti-AtHscB. The lower panel is a loading control stained with coomassie.(9.04 MB TIF)Click here for additional data file.Figure S2Amino acid sequence alignment of HscA-like proteins. Accession number: HscA of Ecoli, NP_417021; Ssq1 of yeast, NP_013473; At4g37910 (AtHscA1), NP_195504; At5g09590 (AtHscA2), NP_196521.(4.85 MB TIF)Click here for additional data file.Figure S3A. Conditional sterile phenotype of N585159. Mut (humid): mutant grown under humid condition; Mut: mutant grown under normal condition; Wild-type plant as positive control. Inserted icon: Siliques of WT (wild-type), Mut(h) (mutant plants grown under humid condition) and Mut . B. SEM of N585159 C. SEM of N585159 complemented with AtHscB.(5.79 MB TIF)Click here for additional data file."}
+{"text": "During this recent decade, microarray-based single nucleotide polymorphism (SNP) data are becoming more widely used as markers for linkage analysis in the identification of loci for disease-associated genes. Although microarray-based SNP analyses have markedly reduced genotyping time and cost compared with microsatellite-based analyses, applying these enormous data to linkage analysis programs is a time-consuming step, thus, necessitating a high-throughput platform.SNP High Throughput Linkage analysis system). In this system, SNP chip data of the Affymetrix Mapping 100 k/500 k array set and Genome-Wide Human SNP array 5.0/6.0 can be directly imported and passed to parametric or model-free linkage analysis programs; MLINK, Superlink, Merlin and Allegro. Various marker-selecting functions are implemented to avoid the effect of typing-error data, markers in linkage equilibrium or to select informative data.We have developed SNP HiTLink (The results using the 100 k SNP dataset were comparable or even superior to those obtained from analyses using microsatellite markers in terms of LOD scores obtained. General personal computers are sufficient to execute the process, as runtime for whole-genome analysis was less than a few hours. This system can be widely applied to linkage analysis using microarray-based SNP data and with which one can expect high-throughput and reliable linkage analysis. Recent technological development of high-density SNP chips has made it practical to genotype more than a million SNPs. Because microarray-based dense SNP typing requires less time and typing cost and can provide much more information than PCR-based microsatellite markers, it is now widely recognized as a powerful tool for linkage analysis -3. To apWe have herein developed SNP HiTLink that directly accepts Affymetrix SNP CHP files and perform parametric/nonparametric linkage analyses with quite flexible marker selection functionalities.SNP HiTLink works under Windows XP SP2 or later/Vista (Use only 32-bit versions of Windows) and unix (supporting perl 5) OS [Additional files Figures SNP HiTLink can run four standard linkage analysis programs, MLINK ,5, Super2, which are indexes of LD, can be defined by users.1) To eliminate markers with typing errors, HWE, call rate, and confidence score are used as the effective indexes because deviations from HWE, lower call rates and higher confidence scores at particular markers sometimes suggest problems with genotyping. 2) To select informative markers useful for linkage analyses, the 'MAF zero test' and 'No call test' will be performed because these markers are totally uninformative. 3) To avoid employing markers in LD in the multipoint analysis, appropriate intermarker distances or D' and r\u2022  HWE test: the user sets p-value which is calculated from genotype frequencies in control samples. SNPs with a p-value below the settings are eliminated.\u2022 Minimum call rate: the user sets the minimum call rate, which is calculated from \"no call/call\" ratio in all control samples, to avoid markers with lower call rates suggesting difficulties in genotyping.\u2022 MAF zero test: markers where MAFs are zero can be eliminated.\u2022 NoCall test : markers that are not called in any samples analyzed will be eliminated.\u2022 Maximum confidence: confidence scores that are reliabilities of signal calling from hybridization can be set here. When the user skips this setting, the default value : minimum intermarker distances will be set. There are two marker-selecting methods, the min-max method and min MAF and interval method. In the min-max method, the user sets minimum and maximum intervals, then SNP with the highest MAF in the region defined by these intervals will be adopted. On the other hand, the min MAF and interval method select SNPs with MAFs higher than defined, and one SNP locating nearest to the minimum interval from the former SNP will be adopted.2 scores to eliminate neighboring markers in LD with D' or r2 scores higher than the threshold. The reference LD data file containing all D' and r2 data obtained from the Hapmap database [\u2022 LD: the user sets the maximum D' and rdatabase  can be dSNP HiTLink produces a binary file (.lkin file) containing the marker and pedigree information with parameter settings, and this file is transported from Windows OS to Unix OS. Perl programming (run_linkage.pl) performs MLINK, Superlink, Merlin or Allegro against a specified '.lkin' file. Whole genome analysis will be carried out automatically but the user can also specify a chromosome number by option when analyzing only the chromosome of interest. Outputs of haplotype prediction by Allegro in a specific text format are easily visualized on the windows system by using the haplotype viewer implemented in this system. Data are shown in columns and can be copied to an Excel sheet for further use [see also the manual of Additional file \u00ae Linkage Mapping Set) data. SNPs and microsatellite markers showed similar results in both pairwise and multipoint analyses but a higher resolution and a clearer border of regions where comparably high LOD scores were expected were achieved using SNP markers. These results indicated that SNP data were comparable or even superior to those obtained from microsatellite markers. The maximum LOD scores of pairwise analysis using microsatellite and SNP markers, were 1.7 and 1.5, respectively. In multipoint analysis, maximum parametric LOD score of 1.8, and nonparametric allele sharing LOD and NPL scores of 1.8 and 2.4, respectively, were obtained using both microsatellite and SNP markers.Figure 2 and about 28000 SNP markers were retained when D' = 0.2 and r2 = 0.2, indicating that there are many neighboring markers that are in LD from each other , and the runtime of whole genome linkage analysis of a pedigree performed using general personal computer was about 4 hours for pairwise analysis, when using all of approximately 1 million markers on Genome-Wide Human SNP array 6.0. For multipoint analysis less than 1 hour was required even in the case of a family including consanguineous loops when intermarker distances were set to be varied from 300 bp to 100 kbp. These results show that extremely dense markers that are now mainly utilized for the genome wide association study (GWAS) can also be utilized for high-throughput linkage analysis.We have developed the SNP HiTLink, system for executing parametric/nonparametric linkage analysis using SNP data. This is the first and unique system that directly accepts recent 100 K, 500 K and 1 M markers of Affymetrix SNP CHP files and prepares very flexible marker-selecting implementations for linkage analysis, although some convenient pipelines that pass the SNP data to a linkage analysis program ,19 or toYF, HA and EN dealt with the computational aspects in development of the system, and YF carried out analyses of the data. YN, YT performed SNP genotyping, and AM, RK performed microsatellite genotyping. HD, JG contributed to general planning and interpretation. ST provided overall guidance for this project. All authors read and approved the final manuscript.SNP HiTLink. The SNP HiTLink main program.Click here for filerun_linkage.pl. The run_linkage.pl program for executing linkage analysis program in Unix OS.Click here for filesample_data. Sample file set including a map file, pedin.dat and pedin.pre file.Click here for fileSNP HiTLink manual. The manual for SNP HiTLink.Click here for file"}
+{"text": "Ankylosing spondylitis (AS) is a chronic, potentially crippling, spondyloarthropathy with strong genetic components affecting approximately 0.3% of the population. Its exact genetic mechanism and mode of transmission, however, remains obscure.The authors conducted a genome wide scan on 75 individuals across multiple generations of three Han Chinese families affected with AS. Segregation analysis and pedigree investigation suggested an autosomal dominant inheritance. Pairwise logarithm of odds (LOD) scores were calculated using LINKAGE package for the obtained genotypes. High resolution mapping was then performed based on markers with significant LOD scores. To minimise the number of crossovers in each family, haplotype were constructed and assigned. Two of the pedigrees shared one candidate region for AS on 2q36.1\u20132q36.3 spanning 6-cM (maximum heterogeneity LOD score of 12.41 at marker D2S2228), while the other showed strong linkage to the HLA-B region.This is the first report which proposes one of the new genetic models of autosomal dominant transmission in AS. The breakthrough in the identification of linkage to chromosome 2q36.1\u20132q36.3 and the HLA-B region highlights the future potential of more comprehensive genetic studies of determinants of disease risk. Ankylosing spondylitis AS; MIM 106300) is a chronic, debilitating, inflammatory disorder that primarily affects the axial skeleton and frequently involves the peripheral joints, the attachments of ligaments and tendons to joints (the entheses), and also extra-articular structures. The overall prevalence of AS in the world population has been reported to be 0.2\u20130.9%.06300 is 2The precise cause of AS is still unclear. The aetiology of the disease is predominantly determined by genetic factors, and environmental factors also play a role. Previous twin based studies estimate that disease heritability exceeds 90%.7Genome-wide linkage scans have also implicated several additional loci outside the major histocompatibility complex (MHC) region.Peripheral blood samples were obtained from a total of 75 individuals (25 AS patients and 50 unaffected subjects) representing three to four generations of three large Han Chinese families affected with AS. Each patient was assessed by at least two qualified rheumatologists, and the diagnosis was made according to the 1984 modified New York criteria of AS.Before the model based genome wide linkage analysis of the three pedigrees with AS, segregation analysis was performed to investigate the presence of major type effects and segregation patterns within the pedigrees using the SAGE program SEGREG for binary phenotypes.where yi is the trait value for the ith individual and is 1 for an affected individual and 0 for an unaffected individual; and \u03b8i is the logit of the susceptibility for the ith individual, which depends on the major type (u\u200a=\u200aAA or AB or BB) and covariates xi1, xi1,\u2026, xip:2 distribution.15The nuclear familial residual association parameter (r), which is analogous to the correlation parameter in regressive models for continuous traits,Genomic DNA was extracted from venous blood according to established protocols.We conducted both parametric and non-parametric linkage analysis in all three pedigrees . For parametric linkage analysis, pairwise two-point (logarithm of odds) LOD scores were calculated using the LINKAGE package. An autosomal dominant model was assumed and, in view of the prevalence of the disorder, the frequency of the abnormal allele was set at 0.003 with an assumed penetrance.High resolution mapping was performed in all three pedigrees . Marshfield markers were selected according to marginal negative markers with priority given to marker heterozygosity based on the initial genome scan results. We constructed and assigned the shared haplotype in families for potential region.The distribution of affected AS patients among the three pedigrees and their family structure are shown in Complex segregation analysis was employed to investigate the inheritance pattern within the pedigrees. Parameter estimates for six different models of family C are shown in the 2) has three degrees of freedom (df). The null hypothesis of no transmission of major gene effect was tested by comparing the commingled model (H02: qA\u200a=\u200a\u03c4AA\u200a=\u200a\u03c4AB\u200a=\u200a\u03c4BB) with the model in which all transmission probabilities were estimated . The null hypothesis of Mendelian transmission was tested by comparing the mixed Mendelian model  with a model where all transmission probabilities were estimated . The null hypothesis of dominant Mendelian inheritance (H04) was tested by comparing the model of the mixed Mendelian model  with the mixed model . The null hypothesis (H05: SuscAA\u2260SuscAB\u200a=\u200aSuscBB) of recessive Mendelian inheritance was tested in similar way. In family C, the hypothesis of no major effect was rejected , and the hypothesis of no transmission of the major effect was also rejected . As expected, the hypothesis of Mendelian transmission was not rejected . Both dominant and recessive Mendelian inheritance models were not rejected with p\u200a=\u200a0.81 and 0.19, respectively.The null hypothesis of no major gene effect was assessed by comparing the model containing multifactorial inheritance alone (H01: qA\u200a=\u200a1) with that containing both a Mendelian major gene and multifactorial inheritance . The model with both effects has three additional parameters (qA and two susceptibilities), so the likelihood ratio statistic , but both the hypothesis of dominant and recessive Mendelian inheritance models were not rejected with p\u200a=\u200a0.44 and 0.35, respectively. This discrepancy between the three tests might due to the fact that the two models for H01 are not hierarchical with each other and thus the test for H01 is less powerful, although such a test was extensively used in the segregation analysis. The results of the later two tests clearly support the conjecture that a putative major gene was segregating in these two pedigrees, and maybe it is more likely to be segregating in the manner of autosomal dominant Mendelian inheritance. All the LOD scores were evaluated at recombinant fraction from 0.0 to 0.4 with the penetrance from 0.56 to complete penetrance  in family A, while a negative LOD score of \u22124.72 at this marker was obtained in pedigree C. Additional markers within 10 cM around marker D2S126 (221.13 cM) from the Marshfield website were selected to extend fine mapping in pedigree A and B . The hetBased on linkage to chromosome 6 at marker D6S422  in initial genome scan in pedigree C, we selected five Marshfield markers located between 42.27 cM and 50.75 cM around the HLA-B region, including D6S2439, D6S273, D6S1666, D6S1583 and D6S1051, to extend fine mapping in pedigrees A, B and C. In pedigree C, the highest LOD score 4.02 was obtained at D6S273 (\u03b8\u200a=\u200a0). In pedigree A, the highest LOD score 2.67 was obtained at D6S1583 (\u03b8\u200a=\u200a0). In pedigree B, no linkage was observed for these fine mapping markers.The inheritance mode of AS tested by sibling recurrent risk studies has been considered as oligogenic and multiplicative interaction among loci.et al conducted a genome scan in 1998 and revealed nominal linkage with a LOD score of 0.8 at marker D2S126,Brown Previous genome-wide scans have suggested that the susceptible locus of AS is in the HLA region. Strong associations with HLA-B region have been identified by non-parametric linkage analysis and case\u2013control association studies. However, the role of HLA-B as the principal genetic determinant of AS has been questioned and a handful of non-MHC loci have recently been evaluated and proven to be associated with AS.In conclusion, by parametric linkage analysis, we have confirmed linkage to the HLA-B region in one AS pedigree and reported a new locus in two AS pedigrees located on 2q36 for the first time with autosomal dominant transmission."}
+{"text": "We carried out an analysis of the Genetic Analysis Workshop 15 simulated Problem 3 data. We restricted ourselves to the present/absent phenotype. Linkage analysis revealed a very strong signal on chromosome 6. Association analysis revealed additional susceptible loci located on chromosomes 11 and 18. The latter two signals were subsequently verified with linkage analysis \u2013 but only after 20 replicates were pooled. Analysis of linkage disequilibrium patterns, in concert with family-based association tests, led us to infer the presence of a second chromosome 6 locus located in the vicinity of single-nucleotide polymorphisms 160\u2013162. These analyses were carried out without knowledge of the model used to generate the simulation. In the last few decades the genes responsible for hundreds of simple Mendelian phenotypes have been identified and their variants characterized. Progress on complex diseases, however, has been much slower. With the advent of new technologies capable of quickly genotyping millions of single-nucleotide polymorphisms (SNPs), the prospect of making substantial inroads in understanding complex phenotypes has improved. Nonetheless, to characterize the genetic architecture of complex phenotypes fully, a researcher will need to employ the full armamentarium of traditional methods, including linkage analysis, association analysis, and family-based transmission tests.s of 9.03. A total of 100 replicates were simulated. Each replicate consisted of 1500 nuclear families with a pair of offspring with RA and a random sample of 2000 unrelated individuals each drawn from the offspring generation of families containing no RA cases. Further details can be found in Miller et al. [We chose to investigate the simulated Problem 3 data set. Briefly, the simulation was designed to mimic the familial pattern of rheumatoid arthritis (RA), including the effect of the DR ideotypes at the MHC on chromosome 6, a lifetime prevalence of 1.07%, a 3:1 female:male affection ratio, and a \u03bbN = 9187) SNP map. The evidence for linkage was evaluated with MERLIN software [We performed linkage analysis on the affected sib-pair nuclear families. We restricted our attention for the preliminary analysis to the \"sparse\"  by selecting the most severely affected sib. If both sibs were equally affected, we selected the first sib. This case sample was compared to the N = 2000 controls supplied by the data providers. Ordinary chi-squares were computed for all SNPs  in the sparse map.We carried out an association analysis on the same sparse SNP map. From each of the first 50 replicates, we formed a group of unrelated cases  that interfaces with the TRANSMIT package . The sofp-value of 0.003). By contrast, the linkage signal on chromosome 6 attained a maximum LOD of 90.33 at SNP 152. Moreover, the LOD scores were positive over the entire length of the chromosome. Figure Linkage analysis of Replicate 1 revealed a very strong signal on chromosome 6 and little else. Only one other chromosome attained a LOD score > 1.0 , we decided to pool the first 20 replicates and repeat the analysis. Figure LD analysis suggests that SNP 153 on chromosome 6 and HLA-DR locus are very close to one another and the effects attributed to SNP 153 are likely due to its nearly complete LD with HLA-DR  the following counts were observed: 0, 114, 2525, 357, 0, 4 and 2, 293, 938, 2767, 0, 0, respectively).2 coefficients between SNP 153 and the suspected proximal and distal association signal regions. We first estimated the haplotypes from the genotypes ignoring the phase information given by the data providers and then we used the phase information. When estimated from the genotypes of cases in Replicate 1, we found no compelling evidence of LD between SNP 153 and SNP 160 or 162 . The phase-known haplotypes also give no evidence of LD. For the SNPs 138/139 vs. SNP 153 estimates, however, we find evidence of significant LD by both estimates (the phase-known chi-square is 31.4 and the phase-unknown maximum likelihood estimates yield a chi-square of 25.6).The linkage analysis of chromosome 6 in the first replicate produced a massive signal. We were, nonetheless, surprised that for a chromosome that is modeled to have a gender-averaged genetic length of 197.5 cM, the LOD scores were positive at every SNP position. The case/control association analysis gave a hint that other susceptibility loci may be located under the large LOD peak show in Figure Z-scores are significant \u2013 especially SNPs 152\u2013155 \u2013 it is noteworthy that the next four most deviant SNPs  are the same SNPs that gave a strong association signal.To further scrutinize the pattern of significant associations on chromosome 6, we performed an analysis of SNPs 120\u2013165 with FBAT to determine whether there is preferential transmission at any of the sites that showed association. Figure This long-range LD \u2013 over a simulated distance of >3.7 MB \u2013 was unexpected and suggests that the association and FBAT signals at SNPs 138/139 are a consequence of the LD rather than an independent risk locus. Accordingly, if there is a second risk locus on chromosome 6, a location in the vicinity of SNPs 160\u2013162 would seem to be favored.R2 < 0.1. For chromosome 6 this involved the removal of 506 (75%) of the 674 SNPs. The resulting LOD distribution (not shown) is virtually identical to that produced with all 674 SNPs . This is not the case if parental genotypes are removed. For chromosome 6 using all 674 SNPs, but no parental genotypes, the linkage is still detected although the maximum LOD score is only 20.9 and is incorrectly positioned at SNP 121. The trimmed map  attains higher maximum LOD score (112.2) but is also incorrectly positioned at SNP 502.To evaluate the influence of LD on the linkage analysis, we selectively removed SNPs so that all pairs within one Mb had a It is instructive, and perhaps sobering, to realize that there are occasions in which linkage analysis is essentially powerless to detect a signal  that is readily detected by association analysis in a sample approximately one-tenth the size. Cox and Bell  providedThe presence of LD is known to inflate linkage statistics and the problem is exacerbated when parental genotypes are unavailable . Our anaExcept to determine whether trimming SNPs in LD could decrease type I error in the absence of parental genotypes, as noted above, all of the work reported here was undertaken without knowledge of the generating model.True to what is known about rheumatoid arthritis, the major genetic effect appears to be due to HLA-DR alleles on chromosome 6. Three additional signals were detected in our blind analysis; a second chromosome 6 signal in the vicinity of SNP 160\u2013162, a signal on chromosome 11 in the vicinity of SNP 389, and a signal on chromosome 18 in the vicinity of SNP 269. These latter two signals were best detected with association analysis. To be detected by linkage analysis, a prohibitively large sample size of families would be required.The author(s) declare that they have no competing interests."}
+{"text": "Angiotensin I-converting enzyme (ACE) plays an important role in cardiovascular homeostasis. There is evidence from different ethnic groups that circulating ACE levels are influenced by a quantitative trait locus (QTL) at the ACE gene on chromosome 17. The finding of significant residual familial correlations in different ethnic groups, after accounting for this QTL, and the finding of support for linkage to a locus on chromosome 4 in Mexican-American families strongly suggest that there may well be QTLs for ACE unlinked to the ACE gene.A genome-wide panel of microsatellite markers, and a panel of biallelic polymorphisms in the ACE gene were typed in Nigerian families. Single locus models with fixed parameters were used to test for linkage to circulating ACE with and without adjustment for the effects of the ACE gene polymorphisms.max = 3.5); other nearby markers on chromosome 17 also showed modest support. After adjustment for the effects of the ACE gene locus, evidence of \"suggestive linkage\" to circulating ACE was found for D4S1629 (Zmax = 2.2); this marker is very close to a locus previously shown to be linked to circulating ACE levels in Mexican-American families.Strong evidence was found for D17S2193 (ZIn this report we have provided further support for the notion that there are QTLs for ACE unlinked to the ACE gene; our findings for chromosome 4, which appear to replicate the findings of a previous independent study, should be considered strong grounds for a more detailed examination of this region in the search for genes/variants which influence ACE levels.The poor yields, thus far, in defining the genetic determinants of hypertension risk suggest a need to look beyond simple relationships between genotypes and the ultimate phenotype. In addition to incorporating information on important environmental exposures, a better understanding of the factors which influence the building blocks of the blood pressure homeostatic network is also required. Detailed studies of the genetic determinants of ACE, an important component of the renin-angiotensin system, have the potential to contribute to this strategic objective. Angiotensin I-converting enzyme (ACE) plays an important role in the maintenance of cardiovascular homeostasis -3. Compl2+ metallopeptidase; the circulating form is derived from tissues by cleavage of the C-terminal transmembrane stalk [ACE is a membrane-bound Znne stalk . ACE levne stalk -21. The ne stalk -24. Simine stalk  suggest ne stalk ; a possiIn addition to effects localised to the ACE gene locus, there is evidence which suggests that there are other loci, unlinked to the ACE gene, which influence circulating ACE levels. In analyses of black Jamaican families , it was Given that there is strong evidence of genetic determination of circulating ACE levels we were interested in using model-based analysis to explore whether we could identify a QTL for ACE levels on chromosome 17 and, if that were possible, to use the same approach to explore whether we could identify additional QTLs for ACE levels unlinked to chromosome 17. In an effort to do this we have conducted an autosomal genome-wide search for loci linked to plasma ACE levels in a dataset comprising 2,079 members of 289 Nigerian families. Two-point linkage analysis under a model with fixed parameters was used to evaluate support for linkage to plasma ACE levels with and without adjustment for the effects of the ACE-linked QTL.The recruitment, phenotyping, and measurement of ACE levels in these families have been described in detail previously. Briefly, the sampling frame for this study was provided by the International Collaborative Study on Hypertension in Blacks (ICSHIB) ,28. NuclThe protocol was approved by the IRB at Loyola University and the Ethics Committee, University College Hospital, Ibadan. Informed consent was presented in Yoruba or English and was obtained from participants by local staff.DNA was extracted from buffy coats and submitted to the NHLBI Mammalian Genotyping Service, Marshfield, WI. Tandem repeat markers from Marshfield \"Set 10\" , with anHigh-resolution mapping of a putative ACE QTL linked to the ACE gene has been carried out in a subset of families recruited as part of the ICSHIB project. Briefly, genotypes were determined for 35 biallelic markers in or near to the ACE gene. Twenty-two markers were typed by DNA re-sequencing ; the remSerum ACE level was treated as a continuous trait since none of the participants were receiving consistent antihypertensive treatment at the baseline exam. Linear regression was used to adjust ACE level for age and sex . In the subset of families who had genotype data for the ACE gene markers described above, linear regression was used to adjust ACE level for age, sex, and for the effects of the ACE gene-linked QTL ; biallelic ACE gene markers were sel\u03bcAA = \u2013 1.9, \u03bcAa = 0.5, and \u03bcaa = 2.5, frequency of allele A  = 0.75, and a common within-genotype variance, \u03c32 = 0.5. A model with widely spaced genotype means, and a relatively low allele frequency corresponds to a high penetrance ratio between genetic and non-genetic cases for discrete traits [Two-point linkage analyses for QTLs influencing either QT1 or QT2 were conducted under a fixed major gene model which assumed a two-allele QTL , genotype-specific means, e traits . The mode traits . Likelihe traits -40.We used merlin  to simulThe dataset available for this analysis included 2,079 persons in 289 pedigrees. The median pedigree size was six , the median number of sibships of at least size two per pedigree was one IQR 1 \u2013 2), and the median number of siblings per sibship was four (IQR 2 \u2013 6). The analysis of QT1 was conducted on families with ACE levels that were also typed for the Marshfield markers. The analysis of QT2 was conducted on a subset of these families that were also typed for ACE gene markers. The characteristics of the QT1 sample and of the QT2 subset are shown in Table  \u2013 2, andmax) by chromosome for each of the autosomal markers tested for linkage to QT1. The strongest evidence for linkage was found on chromosome 17 where one marker (D17S2193) achieved a Zmax value of 3.51 and three other markers had Zmax values greater than one including one marker (D17S2195) with a Zmax value greater than 1.5  with Zmax values greater than 1.5  and QT2 .The Z18 Table . Chromos.5 Table . Chromosmax thresholds observed in our study. For the QT2 dataset (i.e. the subgroup typed for both genome-wide microsatellite markers as well as ACE gene SNPs) we found that Zmax values of 1.0, 1.5, and 2.0 would be expected to be associated with 5.8, 1.5, and 0.2 false positives per genome screen. A Zmax threshold of 1.6, for this dataset, represents 'suggestive linkage' [max of 1.75 . Using this empirically-derived set of thresholds for QT2, we then have at least 2 markers (D4S1629 and D21S1411) which show 'suggestive linkage', and one marker (D11S1392) which just fails to meet that standard. Furthermore, it should be noted that the Zmax value of 2.18 for D4S1629 is associated with a false positive rate of 0.1 (95% CI 0.00 \u2013 0.24) per genome screen which is an order of magnitude beyond the threshold for suggestive linkage and even begins to approach the false positive rate for 'significant linkage' .We conducted a limited simulation exercise in order to make a preliminary, indicative estimate of the genome-wide false positive rates associated with various Zlinkage' . That ismax value of 2.18 at which we estimated the genome-wide false positive rate to be consistent with \"suggestive linkage\"; as previously proposed [max values but in each case there was another marker nearby with Zmax > 1.0.There is strong evidence of genetic determination of circulating ACE and there is also evidence that this genetic determination may include loci unlinked to the ACE gene on chromosome 17. After adjustment for the effect of the ACE gene locus we found that there was modest support for linkage between circulating ACE levels and microsatellite markers on chromosomes 4, 11, and 21. D4S1629 had a Zproposed . The marmax > 3.0 for the adjusted trait (i.e. QT2); this is unsurprising and is likely to be due to the relatively small number of families typed for both microsatellite markers and ACE gene SNPs. In order to examine the power of the QT2 dataset, we have performed simulations using slink [max \u2265 2.0 and \u2265 2.5 in only 46% and 29% of 1000 replicates respectively. It is also possible that the use of a single, possibly mis-specified, model may have had an impact on the evidence for linkage; the use of model-free or non-parametric methods is motivated primarily by this concern [max ~ 3.5) between microsatellite markers near the ACE gene and circulating ACE levels, the first such report using FEML with microsatellites from a sparse map, suggests that models with widely-spaced genotype means, and a relatively low allele frequency (corresponding to high penetrance ratios between genetic and non-genetic cases for discrete traits) should not lead to incorrect inferences regarding detection of linkage signals for circulating ACE. With all of these considerations in mind, it seems a reasonable proposition that our finding of \"suggestive linkage\" to D4S1629 represents a replication of the identification of a putative QTL for ACE on chromosome 4 in Mexican-American families [prima facie evidence of linkage even under the conservative proposals, for multifactorial disease, that have previously been made [In our analysis we adjusted circulating ACE levels for SNPs in the ACE gene  and then performed linkage analysis using fixed effects maximum likelihood (FEML); adjustment for known sources of familial correlations is known to improve power for detection of QTLs -49. We dng slink ,51; for  concern ,53. Neve concern ,54-58. Ieen made ; when onAlthough there is a considerable amount of information available about the physiology of blood pressure regulation, the search for genetic variants which influence risk of high blood pressure in the general population has, on the whole, been disappointing. This has been true even where investigators have moved away from classical family-based designs; in a recent large-scale genome-wide association study , not onlIn this report we have shown that there is strong evidence that there may be loci, unlinked to the ACE gene, which influence circulating ACE levels. One of the QTLs identified in this analysis of Nigerian families appears to be the same as a QTL identified previously in Mexican-American families. Given the importance of the renin-angiotensin system in long-term blood pressure regulation, further characterisation of the genetic determinants of circulating ACE has the potential to improve our understanding of the mechanisms underlying the development of high blood pressure.The authors declare that they have no competing interests.This report is a sub-study within the International Collaborative Study of Hypertension in Blacks (ICSHIB). CAM conceived and designed the sub-study, analysed the data and drafted the manuscript. XZ advised on the statistical analyses. TEF, AL, and RSC were involved in the conception, design, and supervision of the parent study. AAA was involved in the design of, and recruited participants for the study. NB conducted the genotyping. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:Relative pairs. A listing of the numbers of the different types of relative pairs found in the QT1 (\"All family members\") and QT2 (\"ACE markers typed\") datasets.Click here for file"}
+{"text": "AGT gene. We conclude that multivariate analysis is appropriate for the analysis of multiple correlated phenotypes, and our findings suggest that it may yield new linkage signals undetected by univariate analysis.Complex traits are often manifested by multiple correlated traits. One example of this is hypertension (HTN), which is measured on a continuous scale by systolic blood pressure (SBP). Predisposition to HTN is predicted by hyperlipidemia, characterized by elevated triglycerides (TG), low-density lipids (LDL), and high-density lipids (HDL). We hypothesized that the multivariate analysis of TG, LDL, and HDL would be more powerful for detecting HTN genes via linkage analysis compared with univariate analysis of SBP. We conducted linkage analysis of four chromosomal regions known to contain genes associated with HTN using SBP as a measure of HTN in univariate Haseman-Elston regression and using the correlated traits TG, LDL, and HDL in multivariate Haseman-Elston regression. All analyses were conducted using the Framingham Heart Study data. We found that multivariate linkage analysis was better able to detect chromosomal regions in which the angiotensinogen, angiotensin receptor, guanine nucleotide-binding protein 3, and prostaglandin I2 synthase genes reside. Univariate linkage analysis only detected the  Many common diseases are characterized by several correlated factors. These may be the results of a battery of test scores or they may be series of serum lipid levels or anthropometric measures. It is likely that these correlated traits are influenced by common genes (pleiotropy) or at least genes in common pathways. Eaves et al.  point ouAGT) [TNFR2) [ECE1) [AGTR1), beta-3 subunit of guanine nucleotide-binding protein (GNB3) [PTGIS) [Hypertension (HTN), defined by consistent, elevated blood pressure (systolic (SBP) and/or diastolic (DBP)) is an example of a multifactorial trait correlated with multiple other phenotypes. Certainly, environmental factors such as diet and exercise are important determinants of HTN, but the influence of genetic factors is also well supported. In fact, there are a small percentage of HTN cases with monogenic forms of the disease . The resAGT) , tumor n [TNFR2) , endothe) [ECE1) , angioten (GNB3) , and pro [PTGIS) .This study compares a univariate and multivariate method for linkage analysis using a measure HTN, specifically SBP, and then a set of correlated phenotypes influencing SBP as examples and using the location of established candidate genes as our metric. It is our contention that by using information from multiple factors correlated with SBP levels and each other (rather than either the single continuous or dichotomous trait), we will be more effective in identifying regions of the genome previously demonstrated to be linked to SBP levels without as great a penalty for multiple testing.We analyzed the Framingham Heart Study data including observations for Original, Offspring, and Generation 3 cohorts as long as data for all the traits of interest were present. Data were obtained and used in compliance with the data use agreement and Case Western Reserve University Institutional Review Board approval. Low-density lipoprotein (LDL) values were derived using high-density lipoprotein (HDL) and total cholesterol values as required by the Friedewald equation. We used data from the last visit for the Original and Offspring cohorts where all variables of interest were measured. There was only one observation available for the Generation 3 cohort, so that is what we used. The choice of using the latest time point was made in an effort to obtain the most extreme values in our phenotypes of interest (because the study participants would be older).Based on preliminary model-fitting statistics, we adjusted for age at exam, sex, and the interaction of age at exam by sex by including them as covariates in all analyses. We adjusted for possible HTN treatment by adding a constant of 10 to SBP . FinallyAGT (204 cM), TNFR2 (13 cM), and ECE1 (21.5 cM); AGTR1 (150 cM); GNB3 (6.8 cM); and PTGIS (47.5 cM), respectively. Our final marker list comprised 611 SNPs.Because the purpose of our study is to demonstrate the utility of a multivariate linkage method, using the full set of 500 k genome-wide single-nucleotide polymorphisms (SNPs) would lead to far too much redundancy in the data (due to linkage disequilibrium). Therefore, we selected markers every 1000 kb  on which to perform linkage analysis. We further reduced the size of the dataset of analyses by choosing only chromosomes on which there were both previously published linkage signals and candidate genes, including chromosomes 1, 3, 12, and 20, containing candidate genes Prior to linkage analysis, mendelian inconsistencies were identified in the data using MARKERINFO (S.A.G.E. v5.4.1) and the genotypes of all individuals in a family with an inconsistency were set to missing for the given marker. GENIBD was used to estimate the proportion of alleles shared identically by descent (IBD) between sibling pairs using information from individual and neighboring markers . Parental genotypes from the original cohort were used where available in the estimation of IBD sharing. Four pedigrees with complex structure and more than 200 members were removed before IBD sharing estimation.The univariate phenotype of interest was the quantitative trait SBP, a measure of hypertension. The multivariate traits comprised three phenotypes highly correlated with SBP: TG, HDL, and LDL Table . Thus, wWe used performed Haseman-Elston regression  on the tTo conduct multivariate linkage, we used the new S.A.G.E. program RELPAL, which implements a test similar to the multivariate Haseman-Elston . This mop < 0.0001 , while the former gives credence to the multivariate analysis because the subphenotypes appear to co-vary within a family more than does the univariate trait SBP.Cross-trait correlations, both within individual and sibling pair, are shown in Table AGT gene (204 cM) and therefore could be representative of this effect [Of the four chromosomal regions analyzed, we found a few regions of note linked to SBP using the univariate analysis at the \u03b1 = 0.01 level. These regions were on chromosome 1 between 159 and 172 cM, at 186 cM, and between 195 and 198 cM  and TNFR2 is located at 13 cM, very near to the first SNP analyzed. On chromosome 12 (Figure GNB gene. At the \u03b1 = 0.05 level, we observed linkage on chromosome 3 (Figure AGTR1.Using the multivariate model, we detected two regions significant at \u03b1 = 0.01. On chromosome 1 Figure  we detec2 Figure , we obse3 Figure  between a priori weights for association analysis [AGT, TNFR2, AGTR, and GNB3 genes reside. Though the univariate results were near the AGT gene, the multivariate results identified this genomic region more precisely. There were also univariate linkage findings in the vicinity of GNB3 and PTGIS, but not nearly as significant as the multivariate findings (Figures In this study, our objective was to compare univariate and multivariate linkage results of four chromosomal regions known to contain mendelian genes linked to HTN. Linkage analysis remains a relevant approach for the analysis of rare and/or mendelian genetic effects ,20, as wanalysis , so we e Figures  and 1D. In summary, we observed linkage to chromosomal regions containing candidate genes for HTN. Our multivariate analysis identified more such regions than our univariate analysis. These findings support the use of multivariate linkage analysis when analyzing a number of correlated phenotypes that together predispose to a complex trait like HTN.DBP: Diastolic blood pressure; HDL: High-density lipoprotein; HTN: Hypertension; IBD: Identical by descent; LDL: Low-density lipoprotein; SBP: Systolic blood pressure; SNP: Single-nucleotide polymorphism; TG: TriglycerideThe authors declare that they have no competing interests.CG-M and CMS conceived of and designed the study and drafted the manuscript. NJM helped conduct the study and draft the manuscript. YS performed the statistical analysis. All authors read and approved the final manuscript."}
+{"text": "Winter chill is one of the defining characteristics of a location's suitability for the production of many tree crops. We mapped and investigated observed historic and projected future changes in winter chill in California, quantified with two different chilling models .st century, and 90\u2013100% by late century.Based on hourly and daily temperature records, winter chill was modeled for two past temperature scenarios (1950 and 2000), and 18 future scenarios . For each scenario, 100 replications of the yearly temperature record were produced, using a stochastic weather generator. We then introduced and mapped a novel climatic statistic, \u201csafe winter chill\u201d, the 10% quantile of the resulting chilling distributions. This metric can be interpreted as the amount of chilling that growers can safely expect under each scenario. Winter chill declined substantially for all emissions scenarios, with the area of safe winter chill for many tree species or cultivars decreasing 50\u201375% by mid-21st century that will no longer support some of the main tree crops currently grown in California, with the Chilling Hours Model projecting greater changes than the Dynamic Model. The tree crop industry in California will likely need to develop agricultural adaptation measures  to cope with these projected changes. For some crops, production might no longer be possible.Both chilling models consistently projected climatic conditions by the middle to end of the 21 Cool temperatures in the winter are essential for successful cultivation of many tree crops Insufficient winter chill can severely reduce crop yields and crop quality. When chilling requirements are not completely fulfilled, trees display irregular and temporally spread out flowering, leading to inhomogeneous crop development. This process ultimately results in varying crop sizes and maturity stages at the time of harvest, which can substantially reduce yield amount and value Agricultural scientists have developed mathematical models that are used by growers to select tree cultivars with chilling requirements that correspond to available chilling at a specific location. However, a grower's understanding of available winter chill is likely to reflect conditions of the past rather than those expected for a warmer future. Since orchards often remain in production for decades, consideration of future expected winter chill is necessary in times of imminent climatic changes. Without such considerations, many orchards might receive inadequate chilling by the time they reach physiological maturity, even though at the date of planting, climatic conditions were optimal for the chosen cultivars. Depending on the pace of winter chill decline, the consequences for California's fruit and nut industries could be devastating.While a few studies have investigated the impact of climate change on winter chill For generating the hourly temperature records needed for quantifying winter chill from daily records, which are more readily available, we correlated short-term hourly with long-term daily temperature records by Partial Least Squares regression , only 4% of the area in the Central Valley was suitable in 2000, and virtually no areas remained suitable by 2041\u20132060 under any emissions scenario. In interpreting these range estimates, it should be noted that the currently used chilling requirements (in Chilling Hours) might not be valid in a warmer climate.Winter chill decline strongly affected the spatial extent of areas suitable for the cultivation of tree crops with chilling requirements. For cultivars requiring 200 Chilling Hours, such as low-chill almonds, winter chill conditions are unlikely to become critical by the end of the 21 century . For culst century. For cultivars with chilling requirements above 1000 Chilling Hours, such as apples, cherries and pears, very few locations with safe chilling levels were found to exist today, and our modeling results project that virtually none will exist by mid century.Observed historic and future projected temperature increases in California strongly decreased the availability of winter chill under all greenhouse gas emissions scenarios, regardless of the model used to quantify this important climatic parameter for fruit production. On a global scale, it is likely that most other growing regions of subtropical fruit and nut trees with chilling requirements will be similarly affected by declining winter chill. Our projections showed that for many tree crops that now cover large areas within the Central Valley, climatic conditions will become less suitable and in many cases potentially prohibitive for production. Areas where safe winter chill exists for growing walnuts, pistachios, peaches, apricots, plums and cherries (>700 Chilling Hours) are likely to almost completely disappear by the end of the 21The resulting reductions in crop yield and quality could severely impact California's tree crop growers. According to the USDA Agricultural Census of 2002, the state had 38,693 fruit and nut orchard farms, covering 1.2 million hectares of land and driving a US$ 8.7 billion industry Given the long life spans of orchards compared to annual crops and the substantial investments required for orchard establishment, tree crop growers will be much more vulnerable to the long and medium term effects of climate change than growers of annual crops, making the development of predictive temperature models for tree crop yields crucial for strategic planning of orchard operations.Improved orchard management might have potential for alleviating winter chill decline, since planting density, pruning practices and irrigation regime can influence orchard microclimate Research on chilling models in many subtropical regions has indicated that the Chilling Hours Model is not very precise in this climatic zone While we are confident of the general trend of declining winter chill, some locations within the Central Valley might remain suitable even for crops with high chilling requirements. Locations with cooler microclimatic conditions might be found along major rivers, in the foothills of Sierra Nevada and Coastal Range, where cold air tends to drain, as well as close to the Sacramento Delta and in those parts of the Central Valley, where frequent fogs reduce temperatures during the winter. On the other hand, it is likely that warmer temperatures will reduce the incidence of fog in many places, leading locally to stronger deterioration in winter chill than projected in this study.The high sensitivity of the commonly used Chilling Hours Model to climate change While this study focused only on winter chill, climate change may have other (positive and negative) effects on tree crop production. Rising summer temperatures can be expected to be beneficial to some crops, while having a negative impact on others Hourly temperature records are required for estimating winter chill with all common methods without resorting to idealized daily temperature curves. We obtained records of hourly temperatures for all 205 (active and inactive) stations of the California Irrigation Management Information System Since the CIMIS network was only established in 1982, it is not very suitable for analyzing long-term climatic changes. We therefore obtained daily measurements of minimum and maximum temperatures and precipitation from all 113 weather stations in California that belong to the cooperative weather station network administered by the National Climatic Data Center For using daily measurements to estimate hourly temperatures, each CIMIS weather station was associated with a nearby NCDC station. Using the Euclidean Allocation function of a Geographical Information System , each CIMIS station was assigned the closest weather station of the other network, resulting in pairs of weather stations that were on average 20 km apart (max. distance was 76 km). The daily and hourly datasets of these station pairs were then joined. To remove records that were considered faulty, all hourly temperature records that were more than 5\u00b0C above the daily maximum or below the daily minimum of the NCDC record were eliminated from the dataset.When analyzing observed or modeled weather records, long-term trends are often obscured by interannual variation. Temperatures observed during a particular year are often substantially warmer or cooler than the long-term running average for that year. This constraint can be overcome by generating synthetic weather records, which allow correction for interannual variation and facilitate statistical evaluation of weather records We used the LARS-WG stochastic weather generator min), daily maximum temperature (Tmax) and daylength (DL), where Tmin and Tmax were measured at the weather stations, and DL was modeled for each day of the record Since all common winter chill models require hourly temperatures as inputs, such records had to be derived from the daily datasets. To establish a relationship between daily and hourly temperatures, we performed separate Partial Least Squares In order to achieve the most accurate predictive equations, we used a cross-validation procedure  to identify the most appropriate dimension for the regression models. This procedure splits the dataset into two or more groups and fits a regression model to all groups except one. The resulting model is then used to predict the values in the omitted group. This process is repeated for all groups and errors are quantified, providing an estimate of overall model accuracy. The number of latent factors is then chosen to maximize the overall accuracy in estimating hourly temperatures.Two climate scenarios representing 1950 and 2000 conditions were based on temperatures observed during the historic record. To obtain representative conditions for these two years, we calculated separate linear regression analyses for each month of the year from the entire daily temperature record for each NCDC weather station that was used to estimate hourly temperatures. Regressions were calculated for minimum and maximum daily temperatures, as well as for daily precipitation. Based on the resulting equations, representative values for all three parameters were obtained for both 1950 and 2000, and converted into separate climate scenario input files for LARS-WG for each weather station pair.http://www.prism.oregonstate.edu) to calibrate the downscaling. Then the average minimum daily and maximum daily temperatures for each month during 2041\u20132060 and during 2080\u20132099 were calculated for each of the CIMIS weather station locations using the ClimateWizard climate-change analysis toolbox . A twenty-year period was averaged to give a robust estimate of temperatures around 2050 (mid 21st century) and around 2090 (late 21st century) that is not influenced by year-to-year fluctuations in the projected climate.Future winter chill conditions were estimated based on statistically downscaled climate projections for minimum and maximum daily temperatures (averaged monthly) from three General Circulation Models\u2014UKMO-HadCM3, CSIRO-MK3.0, and MIROC3.2(medres)\u2014each run under the SRES A2, A1B, and B1 greenhouse gas emissions scenarios from the Intergovernmental Panel on Climate Change AR4 st and Mar 1st of each winter season.We calculated winter chill according to two methods that are currently used in California. The most common chilling model used in the state is the Chilling Hours Model . In this model, chilling is quantified by simply adding up all hours, during which temperatures range between 0 and 7.2\u00b0C [refer to ref. 8 for equations describing both models]. As commonly practiced in California, we quantified accumulated winter chill between Nov 1In recent years, growers of cherries in California have adopted the Dynamic Model, developed in Israel For each time period analyzed , we calculated winter chill for 100 replications of each year, which allowed statistical evaluation of winter chill estimates. That is, rather than simply producing one value representing the winter chill accumulated during a given year, we used the variability produced by the stochastic weather generator to evaluate the distribution of winter chill over 100 replications of that year. This provided the ability to estimate the percentage of years, during which particular amounts of winter chill are likely to be available to fruit and nut growers.While trends in winter chill are often analyzed using the mean of the chilling distribution, this measure is of subordinate importance to growers, because they economically depend on obtaining good yields in most (e.g. 90%) years rather than in an average year. Inadequate winter chill as often as once in ten years can threaten the economic sustainability of a farming operation. Here we present a novel climate change metric called \u201csafe winter chill\u201d, which we define as the 10% quantile of the winter chill distribution. This metric specifies the maximum chilling requirement that will be fulfilled in 90% of all years at a given site. In addition to calculating the mean of the winter chill distribution, we also calculated this safe winter chill metric.Using the procedure outlined above, we estimated safe winter chill for all twenty climate scenarios at all suitable CIMIS weather stations in California. This procedure provided point estimates of safe winter chill, which needed to be interpolated to cover all of the state. We used the Kriging interpolation technique  to create winter chill surfaces at a 20 arc-minute spatial resolution. While the resulting surface should fairly accurately describe safe winter chill in the relatively flat Central Valley, its validity in more mountainous terrain is limited, because elevation has a strong effect on winter chill and many locations are at substantially lower or higher elevations than the closest CIMIS station. To adjust for this effect, we estimated the elevation error of the interpolated surface by generating a Kriging surface through all point elevations of the weather stations. This surface was then subtracted from a Digital Elevation Model of California We then estimated the rate at which safe winter chill increases with increasing elevation separately for each climate scenario by calculating simple linear regressions between estimated safe winter chill and elevation across all weather stations. While the resulting regression equations were relatively poorly defined (coefficients of determination of <0.1 in many cases), all regressions were statistically significant at p<0.05, and their slopes should represent a reasonable estimate of the additional effect of elevation on safe winter chill. Multiplying the resulting rates with the elevation error surface and adding the results onto the original interpolated chilling grids produced error-adjusted safe winter chill surfaces for the entire state for each climate scenario.For easier interpretation of the results, we finally created average surfaces for the chilling estimates resulting from the different General Circulation Models. This process resulted in eight winter chill surfaces, representing climatic conditions in 1950, 2000 and in 2041\u20132060 and 2080\u20132099 under the B1, A1B and A2 greenhouse gas emissions scenarios, respectively. To facilitate data processing, we implemented most analysis steps in JSL, the scripting language of JMP 7 and in the ArcGIS ModelBuilder.Supporting Information S1Accuracy estimate of winter chill projections. This text file describes the methods used to assess the accuracy of interpolated winter chill surfaces. Results are displayed in (0.05 MB PDF)Click here for additional data file.Figure S1Error estimates of projected winter chill. Qualitative error estimates of winter chill projections caused by elevation differences between the interpolated location and the closest CIMIS station (a) and by distance to the closest station (b).(1.54 MB TIF)Click here for additional data file.Figure S2Safe winter chill throughout California (in Chilling Hours). Safe winter chill (10% quantile of distribution over 100 modeled repetitions for each year) in California, quantified with the Chilling Hours Model for eight climate scenarios, representing climate conditions observed around 1950 and 2000, and projected for 2041\u20132060 and 2080\u20132099 under the B1, A1B and A2 IPCC greenhouse gas emissions scenarios.(4.70 MB TIF)Click here for additional data file.Figure S3Safe winter chill throughout California (in Chill Portions). Safe winter chill (10% quantile of distribution over 100 modeled repetitions for each year) in California, quantified with the Dynamic Model for eight climate scenarios, representing climate conditions observed around 1950 and 2000, and projected for 2041\u20132060 and 2080\u20132099 under the B1, A1B and A2 IPCC greenhouse gas emissions scenarios.(4.63 MB TIF)Click here for additional data file."}
+{"text": "Modern linkage-based approaches employing extended pedigrees are becoming powerful tools for localizing complex quantitative trait loci. For these linkage mapping methods, it is necessary to reconstruct extended pedigrees which include living individuals, using extensive pedigree records. Unfortunately, such records are not always easy to obtain and application of the linkage-based approaches has been restricted. Within a finite population under random mating, latent inbreeding rather than non-random inbreeding by consanguineous marriages is expected to occur and is attributable to coalescence in a finite population. Interestingly, it has been revealed that significant random inbreeding exists even in general human populations. Random inbreeding should be used to detect the hidden coancestry between individuals for a particular chromosomal position and it could also have application in linkage mapping methods. Here we present a novel method, named finite population based linkage mapping (FPL) method, to detect linkage between a quantitative trait and a marker via random inbreeding in a finite population without pedigree records. We show how to estimate coancestry for a chromosomal position between individuals by using multipoint Bayesian estimation. Subsequently, we describe the FPL method for detecting linkage via interval mapping method using a nonparametric test. We show that the FPL method does work via simulated data. For a random sample from a finite population, the FPL method is more powerful than a standard pedigree-based linkage mapping method with using genotypes of all parents of the sample. In addition, the FPL method was demonstrated by actual microsatellite genotype data of 750 Japanese individuals that are unrelated according to pedigree records to map a known Psoriasis susceptible locus. For samples without pedigree records, it was suggested that the FPL method require limited number of individuals, therefore would be better than other methods using thousands of individuals. Identifying susceptible loci of complex common disease is an important challenge in human statistical genetics. Whole genome association scans among unrelated individuals is a popular tool for localizing susceptible genes of complex common diseases. The crux upon which association-based approaches rests is the common disease common variant hypothesis. In cases where this hypothesis does not hold true, e.g. a disease caused by multiple rare variants that have small effects, association-based approaches may lose their power Many complex common diseases are evaluated and diagnosed directly on a quantitative scale. Modern linkage-based approaches employing extended pedigrees are becoming powerful tools for localizing quantitative trait loci, and they had led to the successful identification of human quantitative trait loci Sewall Wright introduced F statistics, which measure degree of coancestry between pairs of homologous genes within a finite population Here, we propose a novel method, named finite population based linkage mapping (FPL) method, to detect linkage between a quantitative trait and a marker via random inbreeding in a finite population without pedigree records. Considering IBD status among four homologous genes of a pair of individuals, we devise a consistent population genetic model for random inbreeding. Then, using computer simulations assuming usage of microsatellite and SNPs data, we illustrate significance and power of the test. Finally, we demonstrate the linkage mapping method to the microsatellite genotype data of Japanese psoriasis patients and healthy controls that are unrelated according to pedigree records but are expected to be distantly related.j-th marker of the i-th pair of individuals be i-th pair of individuals be Consider a random mating population which consists of j-th marker of the i-th pair of individuals , D6S2427, D6S1017, and D6S2410, and their positions from the telomere in the Marshfield genetic map were 0, 11.11 25.08, 40.14, 46.81, 53.81, 63.28 and 73.1 in cM, respectively, with the positions of F13A1 and D6S2931 were estimated on the basis of their physical positions in the human genome . The genotypes of D6S2931 are provided in We investigated the anti-conservativeness of the standard parametric regression. We set the trait to be unlinked. For each case of population demography, 1,000 populations with microsatellite maps with 0, 1, 5, and 10 cM intervals. From each simulated population 500 individuals were randomly sampled, and p-values by the Mantel test and the t-test of the regression were computed. The proportion of populations for which the p-value was less than 0.05 is shown in Standard pedigree-based linkage analysis is prone to large increase in type I errors when the markers are tightly linked and there is large linkage disequilibrium among them. It comes from assuming linkage equilibrium among markers in linkage disequilibrium when there is missing phase information Subsequently, we investigated the power of the FPL method in various models of a trait locus. The broad sense heritability, proportion of the trait variance determined by the trait locus: In addition, we investigated the significance and power and of the new linkage mapping method by assuming the usage of SNP markers. SNP maps of 0.01 and 0.1 cM interval were generated for each parameter set, the models and the samplings. One of the strategies employed involved the direct use of these SNPs as biallelic markers. The results for the significance are shown in It is important to note that the results obtained for specific parameters, as presented here, does not provide any general rule regarding the power. The parameter sets considered here were chosen solely as an illustration. The issue of power should be carefully considered for each specific study design, depending on the population demographic history, sample size, marker spacing, properties of the marker and trait locus, etc. see .The estimate of mall see . This biSince the estimate of thod see , inaccurWith the FPL method, it is expected that only a few false positives may be triggered by population stratification, since the mapping method relies not on the allele frequencies but on the IBD status between individuals, as other linkage-based approaches do. However, the robustness of the FPL method against population stratification is not trivial, since the estimate of coancestry depends on the allele frequencies. We considered the robustness by examining a model of population amalgamation, in which the founder population consisted of two equal sized populations with no allelic types of markers being shared between them. We set the trait to be unlinked. For each case of population demography, we simulated 1,000 populations with microsatellite maps of 1 cM interval. From each simulated population 100 individuals were randomly sampled, and p-values were computed by the Mantel test. The proportion of the populations for which the p-value smaller than 0.05 was 0.043, 0.047, 0.046, and 0.043, for Case 1\u20134, respectively. Inflate of the significance by population stratification seems to be absent.The properties of the trait locus affect power of the FPL method. We considered the model where two alleles are segregating in the trait locus in the founder population . Here, two other models of allelic heterogeneity in the trait locus were considered. First, we considered the ten segregating sites model, whereby the ten sites have an equal effect and are segregating in the founder population. Compared to the single segregating site model, the ten segregating sites model exhibited no significant power reduction (data not shown). The second model was the infinite sites mutation model with a mutation rate of 0.001 for each site and mutation should occur at a non-segregating site. We assumed that none of the mutations existed in the founder population and that all the segregating sites have equal effects. Under the infinite site mutation model, a large number of low frequency alleles appear and the locus becomes highly heterogeneous. We set the heritability to be 50%, and the additive model was assumed. For each case of population demography, we simulated 100 populations with microsatellite maps of 1 cM interval. From each population 500 individuals were sampled randomly, and p-values were computed by the Mantel test. The proportions of the populations for which the p-value smaller than 0.05 at the trait locus were 33%, 93%, 33%, and 84%, for Case 1\u20134, respectively. Compared to the single segregating site model, the infinite site mutation model exhibited a significant power reduction.Using simulations, we compared the power of the FPL method with that of a standard pedigree-based linkage mapping method, which is known as the variance component approach and implemented in the SOLAR computer software package To demonstrate the applicability of the FPL method, we determined the genotypes of 8 microsatellite markers on the chromosome 6 of 375 Japanese patients with psoriasis vulgaris and 375 healthy controls. All the participants are unrelated according to pedigree records. The average marker spacing was 10.4 cM. A marker D6S2931 is located in PSORS1 at 6q21.3 near the HLA-C gene, which has been shown to be significantly linked and associated with psoriasis First, using the 375 healthy controls, we estimated coancestry for these markers. The distribution of Subsequently, using the 375 healthy controls and the 375 psoriasis patients , we conducted linkage mapping by using the new method. In the present paper, we presented a new linkage mapping method within a finite population under random mating, without pedigree records. In addition, we assessed the feasibility of this mapping by using simulated and actual data. This method is based on the estimation of coancestry for a chromosomal position between individuals, with the use of multipoint Bayesian estimation. Association-based approaches are commonly used for unrelated individuals, when it is difficult to obtain an extended pedigree for a population. We hope that our linkage mapping method may provide a basis for linkage mapping approach of quantitative trait within such populations, since linkage-based approaches have several advantages when compared to association-based approaches. First, the linkage-based approaches remain powerful even in case of allelic heterogeneity. Second, only a few false positives are triggered by population stratification, since these approaches rely not on the allele frequency but on the IBD status. Third, as mentioned above, the significance of the new linkage mapping method does not increase with population stratification, and its power is retained in cases of mild allelic heterogeneity. Finally, in contrast to standard pedigree-based linkage analysis, the new linkage mapping method described here is robust against the increase in type I errors caused by linkage disequilibrium among markers.Remarkably, for random samples the FPL method would be generally more powerful than a standard pedigree-based analysis, even if we additionally determine genotypes of parents of sample for the pedigree-based analysis. It might seem counter-intuitive that the FPL method is powerful than a standard pedigree-based linkage analysis in the respect of sample size, but it is a reasonable consequence because we assumed that we correct the sample without caring the pedigrees. Standard pedigree-based linkage analyses use known kinship among individuals, but such close kinship is rare in randomly sampled individuals. In contrast, the FPL method uses all kinship, which might be remote but would be in abundance within a finite population. Another interesting question is how extensive pedigree information is needed for standard pedigree-based linkage approach to over perform the FPL method, when we do not know genotypes of the ancestors. Unfortunately, pedigree-based linkage analyses are so computer intensive that we could not address this issue, but three generation pedigree information was not enough to over perform the FPL method.Understanding the relatedness between individuals is important for many aspects of genetics and ecology. Many different estimators have been developed for kinship coefficient in infinitely large populations The new linkage mapping method in itself does not require any of the parameters detailing population demographic history. However, the power of the new linkage mapping method is a complicated function of these parameters. For example, the power is not necessarily gained by use of a population with high mple see . SubsequAppendix S1(0.23 MB DOC)Click here for additional data file.Table S1(0.11 MB DOC)Click here for additional data file.Table S2(0.06 MB XLS)Click here for additional data file.Figure S1The moment estimates of Fst as a function of number of allelic types. The horizontal lines show the actual values of Fst. This figure shows bias, especially for small number of alleles.(1.12 MB TIF)Click here for additional data file.Figure S2Posterior expectation of coancestry see , which i(1.35 MB TIF)Click here for additional data file."}
+{"text": "Using single-nucleotide polymorphism (SNP) genotypes and selected gene expression phenotypes from 14 CEPH (Centre d'Etude du Polymorphisme Humain) pedigrees provided for Genetic Analysis Workshop 15 (GAW15), we analyzed quantitative traits with artificial neural networks (ANNs). Our goals were to identify individual linkage signals and examine gene \u00d7 gene interactions. First, we used classical multipoint methods to identify phenotypes having nominal linkage evidence at two or more loci. ANNs were then applied to sib-pair identity-by-descent (IBD) allele sharing across the genome as input variables and squared trait sums and differences for the sib pairs as output variables. The weights of the trained networks were analyzed to assess the linkage evidence at each locus as well as potential interactions between them.Loci identified by classical linkage analysis could also be identified by our ANN analysis. However some ANN results were noisy, and our attempts to use cross-validated training to avoid overtraining and thereby improve results were only partially successful. Potential interactions between loci with high-ranked weight measures were also evaluated, with the resulting patterns suggesting existence of both synergistic and antagonistic effects between loci.Our results suggest that ANNs can serve as a useful method to analyze quantitative traits and are a potential tool for detecting gene \u00d7 gene interactions. However, for the approach implemented here, optimizing the ANNs and obtaining stable results remains challenging. Complex traits are often hypothesized to be influenced by multiple interacting loci. Detecting gene \u00d7 gene interactions remains a challenge. Artificial neural networks (ANNs), however, are suited for pattern recognition involving combinations of loci.ANNs were first applied for human linkage analysis in Genetic Analysis Workshop 10 (GAW) . Others The GAW15 Problem 1 data set, from Morley et al. , include) and variance-components (VC) linkage analysis using Merlin [To select phenotypes for ANN analysis, we carried out Haseman-Elston (H-E) regression  using thg Merlin . The forg Merlin .[[We used two different genetic maps. We compiled Map 1 from the SNP Consortium map data . Map 2 w[. Compari[.[We used the Stuttgart Neural Network Simulator (SNNS), Version 4.2 . ANN tra,3; after.We used a network architecture with two hidden layers. We found that two hidden layers performed better than a single layer, attaining lower error and more appropriate output values. Note that the phenotype coding allows the squared trait sums and differences to range freely. Thus, the activation function at the output nodes was the identity function rather than the usual logistic sigmoid function, since the latter would restrict outputs to range between 0 and 1. We believe the second hidden layer is therefore performing some rescaling needed to obtain appropriate output values. Thus, the primary ANNs contained 362 input nodes, two hidden layers of 50 nodes each, and two output nodes.I input nodes, two hidden layers with H nodes each, and O output nodes, such that each node in a given layer is connected to every node in the next layer. Suppose the ith input xi is connected to the jth hidden node via a weight uij, the jth hidden node is in turn connected to the kth hidden node in the next layer via a weight vjk, and this kth hidden node is connected to the lth output node via wkl. Then we calculate an \"importance measure\" for the ith input: To identify input loci that are important in determining phenotypic status, we used an algorithm similar to those in our previous work . ConsideI inputs xi forms the argument for the activation function fj of the jth node of the next layer, with weights uij as the coefficients, as follows: aj is an intercept term. We hypothesized that if two inputs have positively correlated weights leading to the next layer, similar input values will have cumulative effect on the arguments of the activation functions; for two inputs with negatively correlated weights, similar input values will have antagonistic effect. A simplified example, in which the weights from one input node are a fixed multiple of those from a second, unlinked input node and thus are perfectly correlated with those latter weights, demonstrates this most clearly. Letting c be the scaling factor for the weights and indexing these two inputs with 1 and 2, then uj 2= cuj 1for all j, and the jth activation function value is: c| = 1, we see that when c is positive, similar input values will have cumulative effects across all activation functions in the hidden layer, and when c is negative, subtraction will cancel out the effects of two similar input values.For our novel analysis of the interaction between a given pair of loci, we considered the weights for each pair of connections leading from the two loci to the same node in the next, hidden layer. We calculated the Pearson correlation for the resulting data set of 50 pairs of weights. A strong positive correlation is interpreted as cumulative or synergistic action of the loci, and a strong negative correlation may be interpreted as either antagonistic or complementary action. The rationale for this correlation analysis relies on the following details of an ANN's operation. For the generic network described above, a linear combination of the To further evaluate potential interactions detected by our correlation analysis, we used both VC (SOLAR with the \"-epistasis\" option ) and a sTGIF, TGFB-induced factor) and 210910_s_at . These traits gave at least two distinct H-E signals with p-values less than 0.0001, and had consistent VC results , and strong negative correlation between the chromosomes 1 and 9 signals (r = -0.896)  using Map 2 are given in Figure ANN results for 210910_s_at , the highest ratio of dissimilar pairs (56%) was in the bin corresponding to low sharing at the first locus and high sharing at the second. Thus, while from H-E we expect low trait similarity among low IBD sib pairs at each locus, considering the IBD status jointly reveals an interaction. The negative correlation was observed in the presence of an underlying pattern in which high sharing at one locus together with low sharing at the other led to reduced trait similarity.We subjected the most interesting locus pairings from our weight correlation analysis of For the computationally intensive cross-validated analyses, we targeted only TGIF. We completed three five-fold cross-validated runs and observed whether the H-E/VC verified loci were supported. The first run supported the loci at chromosome 1, 40 cM (rank = 7); chromosome 9, 110 cM (rank = 4); and chromosome 15, 20 cM (rank = 8). The second supported only the chromosome 9 locus (rank = 4), and the third supported chromosome 9 (rank = 6) and chromosome 15 (rank = 5), with evidence on chromosome 1, 90 cM (rank = 1). Other high-ranking loci were not always in common across the three runs. Thus these results were not as consistent as expected; however, they did provide some support for the key loci.We performed secondary analyses of rescaled data (inputs and outputs), but results did not notably improve. Based on our results, we did not further develop those approaches.Currently, genome-wide linkage scans are typically carried out one chromosome at a time. Approaches that can analyze all markers simultaneously and detect patterns of locus interactions are a desired alternative. We applied ANNs to map QTLs and calculated correlations between the ANN weights to evaluate potential interactions.Training and testing ANNs on all sib pairs  did detect linkage evidence at loci highlighted by traditional methods. We favor the use of cross-validation when possible . We applTGIF) gene expression phenotype, Map 1, and a randomly selected training subset of four-fifths of the sib pairs (data not shown). This preliminary analysis was promising because it detected the expected linkage peaks on chromosomes 1, 9, and 15, and only one additional unconfirmed peak. Furthermore, the correlation analysis was even more striking (r = 0.985 and -0.917), with these being the two strongest correlations between distinct loci across the genome. When updating to Map 2, analyzing all sib pairs gave what appear to be noisier results  declare that they have no competing interests."}
+{"text": "The pathogenesis of atherosclerosis involves both hemostatic and inflammatory mechanisms. Fibrinogen is associated with both risk of thrombosis and inflammation. A recent meta-analysis showed that risk of coronary heart disease may increase 1.8 fold for 1 g/L of increased fibrinogen, independent of traditional risk factors. It is known that fibrinogen levels may be influenced by demographic, environmental and genetic factors. Epidemiologic and candidate gene studies are available; but few genome-wide linkage studies have been conducted, particularly in minority populations. The Strong Heart Study has demonstrated an increased incidence of cardiovascular disease in the American Indian population, and therefore represents an important source for genetic-epidemiological investigations.The Strong Heart Family Study enrolled over 3,600 American Indian participants in large, multi-generational families, ascertained from an ongoing population-based study in the same communities. Fibrinogen was determined using standard technique in a central laboratory and extensive additional phenotypic measures were obtained. Participants were genotyped for 382 short tandem repeat markers distributed throughout the genome; and results were analyzed using a variance decomposition method, as implemented in the SOLAR 2.0 program.STAT3), were identified within 2 and 8 Mb of this 1 LOD drop interval respectively. A LOD score of 1.82 on chromosome 17 between 68 and 93 cM is supported by reports from two other populations with LOD scores of 1.4 and 1.95.Data from 3535 participants were included and after step-wise, linear regression analysis, two models were selected for investigation. Basic demographic adjustments constituted model 1, while model 2 considered waist circumference, diabetes mellitus and postmenopausal status as additional covariates. Five LOD scores between 1.82 and 3.02 were identified, with the maximally adjusted model showing the highest score on chromosome 7 at 28 cM. Genes for two key components of the inflammatory response, i.e. interleukin-6 and \"signal transducer and activator of transcription 3\" (In a minority population with a high prevalence of cardiovascular disease, strong evidence for a novel genetic determinant of fibrinogen levels is found on chromosome 7 at 28 cM. Four other loci, some of which have been suggested by previous studies, were also identified. Hypotheses concerning the pathogenesis of atherosclerosis have frequently included both hemostatic and inflammatory mechanisms. Fibrinogen levels are positively associated with risk of thrombosis  and are Fibrinogen levels are increased by many non-genetic factors, e.g. advancing age, smoking, obesity, oral contraceptive use and estrogen replacement therapy, whereas moderate alcohol intake has been related to lower fibrinogen levels ,9,10. AcModerate heritabilities of 34% and 37% were found in two primarily Caucasian populations ,14. In oCardiovascular disease accounts for a large proportion of mortality and morbidity in American Indian (AI) communities . Since aThe Strong Heart Study (SHS) is a population-based, cohort study of cardiovascular disease among American Indians. The participating communities, study design, survey methods and laboratory techniques have been described previously ,27. In 1The protocols for collection of all phenotypic data were briefly described in previous publications . FibrinoThe procedures for genotyping in the SHFS have been described previously . In brieversion 8.0, was used to screen covariates for statistical significance using stepwise linear regression by center. Univariate quantitative genetic analyses were used to partition the phenotypic variance into its' additive genetic and environmental components, using maximum likelihood variance decomposition methods [SAS,  methods . This ap methods  and alloThe use of the variance component approach requires an estimate of the identity-by-descent (IBD) matrix. We used the Loki package , which eA total of 3535 SHFS participants were considered for analysis (Arizona (AZ) = 1195, Dakota (DA) = 1158, Oklahoma (OK) = 1182) after excluding those individuals with missing covariate data and as indicated below to normalize the phenotypic trait distribution. Because variance components methods are sensitive to kurtosis , all pheThe power of this study for each center was estimated by simulations, assuming fully informative markers. The simulations were based on a total heritability of the fibrinogen phenotype either 25% or 50%. An estimate of the power for all centers combined was determined by calculations based on the results from the individual centers, due to the otherwise extremely high computational demands for simulations.The statistical descriptors of fibrinogen and other covariates are stratified by center and displayed in table Table Further suggestive evidence of linkage to fibrinogen was observed in the Oklahoma center on chromosome 3 at 166 cM. The 1-cM LOD unit support interval spanned 15 centimorgans from 157 \u2013 172 cM . In addition, we found suggestive evidence of linkage to fibrinogen within all centers combined to chromosome 6 at 122 cM. Of the suggestive LOD scores, the only one that declined with adjustment was at chromosome 6, 122 centimorgans, in the analysis including all centers. In a post-hoc analysis, the only available measure of inflammatory state, white blood cell count (WBC), was included with previously described models to adjust for inflammatory state. LOD scores in these analyses showed essentially identical values for OK, model 2, and DK, chromosome 7, model 1 of 2.51 and 2.81 respectively. The LOD score for DK, chromosome 7, model 2 was reduced slightly to 2.41 and became insignificant (0.51) for DK, chromosome 17, model 2.While the \u03b1,\u03b2,\u03b3 fibrinogen genes are on chromosome 4, the present study shows LOD scores for the DK center only at 4q28, 155cM, ranging between 1.36 for Model 2 to 1.70 for Model 1; none of the other centers exhibited LOD scores over 0.3 in this region.Assuming a genome-wide heritability of 25%, power calculations indicated an 80% power to detect a locus with a LOD score > 3 for a locus contributing a minimum of between 15 and 20% of total variance for each of the three centers and 10% for all centers combined.Evidence for a pathophysiologic role of both thrombotic and inflammatory influences on atherosclerosis continues to accumulate. The fact that fibrinogen was shown in 31 prospective studies involving over 150,000 participants (without apparent disease at baseline) to predict future CVD events is compelling, but not absolute, support for a primary role in the development of atherosclerosis.  The inflIn addition to multiple environmental influences on plasma fibrinogen levels ,9 the efWhile genetic polymorphisms in the fibrinogen genes are found in diverse populations around the world ,39, and AGTR1, 149.4 Mb-150.1 Mb) and this gene is thus well within the 1 LOD interval (144.8 Mb-169.8 Mb). The angiotensin converting enzyme I (ACE1) gene is also located within the 1 LOD interval of our suggestive locus at chromosome 17 (68 cM to 93 cM) [AGTR1, ACE1, and fibrinogen all have relevance to CVD, there is no readily apparent physiologic connection between fibrinogen levels and the renin-angiotensin-system (RAS). While the RAS is an important factor in the regulation of blood pressure, and fibrinogen is often elevated in the presence of hypertension, we are unaware of any evidence that increased fibrinogen preceeds or influences the development of hypertension. It is possible that a genetic variant in this locus simultaneously controlling expression of both fibrinogen and RAS genes.Suggestive evidence of linkage to novel loci was identified in this study on chromosomes 3 (OK center), 6  and both 7 and 17 (DK center). Our linkage results at 3q22.2  are within 0.5 Mb of the angiotensin II receptor, type 1 gene (o 93 cM)  AlthoughFindings of Ding et al  support ITGB8) gene is located at 31 cM. This gene is related to ITGB3 (see below) and shares similar important relationships with transforming growth factor, beta 1 (TGFB1) and other elements of the immune system [IL6) is located 2 Mb beyond the 1 LOD drop interval on 7p21 at 22.7 Mb, closest to deCODE markers at 41.69 cM. Zhang et al [IL6 and fibrinogen levels . Not all [IL6 polymorphisms are known to affect fibrinogen levels [IL6 in inflammatory physiology would make this a strong candidate for effects on fibrinogen as an acute phase reactant, although the fact that the DK LOD score at this locus was essentially unchanged with adjustment for an inflammatory marker (WBC) suggests inflammatory stimuli are not the sole determinants of fibrinogen levels.Within the 1 LOD drop interval at 7p21.1 , the integrin beta-8  lies within the 1 LOD drop interval and exerts an influence on fibrinogen levels [ITGB3) and \"signal transducer and activator of transcription 3\" (STAT3) genes both reside within 3 and 8 Mb respectively of this 17q23.3 interval and both are known to affect baseline and acute phase fibrinogen expression [Our findings at chromosome 17, 76 cM (nearest marker D17S1607) are in close proximity to the LOD of 1.4 found by Yang et al  at 96 cMn levels . The intpression ,49.These current findings derive from specific subsets of the North American Indian population and each center of the study analyzes a group essentially the same size as that reported from the Framingham Heart Study . One migStrengths of this study include the population-based recruitment with large numbers of participants analyzed and the large, multi-generational families recruited. Our calculations indicate an 80% power to identify a QTL with a LOD score of more than 3 contributing at least 20% of the total phenotypic variance in any center. In spite of the above-mentioned diversity of findings, the genetic background of the populations are likely to be more uniform than population-based studies from urban areas. The prospective collection of a very well characterized phenotype, including many demographic, environmental and physiologic parameters is another strength.Although the large size of the families in this study increases the power to detect genetic effects, it may also magnify the effects of uncommon polymorphisms in a particular family. This should be minimized by the fact that recruitment for the family study was not based on the presence or history of CVD and the fact that 13.6% of the family study participants were also members of the original, population-based cohort.In a minority population with a high prevalence of cardiovascular disease, strong evidence for a novel genetic determinant of fibrinogen levels is found on chromosome 7 at 28 cM. Four other loci, some of which have been suggested by previous studies, were also identified. Fibrinogen is of particular interest since it may act through two independent pathophysiologic mechanisms, i.e. thrombosis and inflammation. A better understanding of the genetic determinants of fibrinogen levels and how they influence the development of atherosclerosis and its complications will assist in fashioning better preventive and treatment strategies for this very serious public health problem.The authors declare that they have no competing interests.All authors have read and approved the final manuscript. LGB: Originated and conducted the majority of the analysis, drafted the manuscript and is the corresponding author. KEN: Provided key oversight and direction of analysis and played a major role in editing the manuscript. XL: Provided initial linear regression analyses, residual files for linkage analysis and editing suggestions. VP: Offered important editorial suggestions for the manuscript. JGU: Supervised laboratory determinations of fibrinogen and made edits to the methods section. JM: Supervised genetic data collection and preparation. SL: Evaluated pedigrees, provided the final pedigree structure and performed the genotypic data cleaning. KH: Provided extensive quality control and data management of the genotyping. HG: Evaluated pedigrees and provided the final pedigree structure VPD: Provided \"identical by descent\" genotype files for analysis. Evaluated pedigrees and provided the final pedigree structure. LA: Guidance related to statistical linkage analysis. ETL: Phenotypic data collection and quality assurance. RPT: Guidance related to laboratory methodology. SC: Supervised the genotyping and provided important editorial comments.The pre-publication history for this paper can be accessed here:"}
+{"text": "Our findings reiterate the necessity of properly handling dense markers in linkage analysis, especially when parental genotypes are unavailable.The presence of linkage disequilibrium violates the underlying assumption of linkage equilibrium in most traditional multipoint linkage approaches. Studies have shown that such violation leads to bias in qualitative trait linkage analysis when parental genotypes are unavailable. Appropriate handling of marker linkage disequilibrium can avoid such false positive evidence. Using the rheumatoid arthritis simulated data from Genetic Analysis Workshop 15, we examined and compared the following three approaches to handle linkage disequilibrium among dense markers in both qualitative and quantitative trait linkage analyses: a simple algorithm; SNPLINK, methods for marker selection; and MERLIN-LD, a method for modeling linkage disequilibrium by creating marker clusters. In analysis ignoring linkage disequilibrium between markers, we observed LOD score inflation only in the affected sib-pair linkage analysis without parental genotypes; no such inflation was present in the quantitative trait locus linkage analysis with severity as our phenotype with or without parental genotypes. Using methods to model or adjust for linkage disequilibrium, we found a substantial reduction of inflation of LOD score in affected sib-pair linkage analysis. Greater LOD score reduction was observed by decreasing the amount of tolerable linkage disequilibrium among markers selected or marker clusters using MERLIN-LD; the latter approach showed most reduction. SNPLINK performed better with selected markers based on the D' measure of linkage disequilibrium as opposed to the  With rapid development of high-throughput genotyping technologies, more researchers have begun genome-wide searches for genes associated with complex diseases using dense single-nucleotide polymorphisms (SNPs). These dense SNPs create clusters of SNPs in linkage disequilibrium (LD) along each chromosome. The underlying assumption of linkage equilibrium (LE) between markers in many multipoint linkage methods is violated among dense SNPs where LD may exist. Such violation can lead to incorrect pedigree haplotype inference , especiar2 were applied in handling LD: 0.1, 0.3, 0.5, and 0.7 for D' and 0.01, 0.1, 0.3, and 0.5 for r2. Using varying cut points served our goal to show a gradual change or trend of LOD score inflation along all ranges of LD measures. We performed linkage analysis over all 100 replicates of the simulated data at different LD thresholds and three pedigree data sets, with zero, one or two ungenotyped parents, and compared our results to the unadjusted linkage signal obtained using the complete marker data with no ungenotyped parents.Rheumatoid arthritis (RA) simulated data provided by GAW15 include 1500 pedigrees  with 6000 individuals genotyped with a very dense set of 17,820 SNPs on chromosome 6. We focused our analysis on a sub-region containing 937 SNPs along chromosome 6 (170 to 185 cM) where there is no evidence for linkage. Our selection was based on the availability of dense SNPs on chromosome 6 combined with our attempt to find a null region that was farthest from the strong linkage peak at 49 cM to evaluate false-positive evidence for linkage by inflated LOD scores. Each pedigree data set was modified to create two additional data sets with one and both parental genotypes missing for the purpose of evaluating false-positive linkage with ungenotyped parents. According to the distribution of the LD measures, the following sets of LD thresholds by D' and The RA simulated data consist of families with one ASP. We used nonparametric linkage (NPL) analysis implemented in MERLIN  to evalur2) was calculated using Haploview [Based on the selected sub-region, pairwise LD  by organizing markers into clusters using pre-specified r2 . MERLIN Using the original data with complete parental genotypes, we observed no substantial linkage evidence  over the selected sub-region in ASP linkage analysis of the RA dichotomous phenotype . Of the 438,516 SNP pairs formed by 937 SNPs, there were 28,703 SNP pairs on average with r2 greater than 0.01. Consequently, this resulted in most SNPs being omitted from the analysis after applying the algorithms at such threshold. In general, the number of SNPs included in each analysis decreased as the cut points decreased. In some cases, especially with the lower cut points, only 20 to 50 SNPs remained to be analyzed within our selected 15-cM region; however, this still offered a fairly dense coverage (1 to 3 SNPs per cM) and served our attempt to present a trend and magnitude of LOD score inflation due to LD among dense SNPs.As a note, in this selected sub-region, the mean, median, and mode of D' are 0.13, 0.06, and 1.0, respectively. On the other hand, most pairwise r2 cut points  with 446 clusters at r2 = 0.5 evaluated in linkage analysis. Although the lowest average maximum LOD score was below 3, it was still slightly higher than the value of 1.9 obtained with full parental information shown in Figure Figure We examined and compared three approaches of handling LD using dense SNPs on chromosome 6 from the GAW15 simulated data. We performed ASP and QTL linkage analyses on a selected sub-region with low evidence of linkage (170 to 185 cM) that contains 937 SNPs. We observed inflation of LOD scores with missing parental genotypes only in ASP linkage analysis of the RA phenotype. In QTL linkage analysis, we observed no inflation in LOD scores even with missing parental genotypes using the unascertained data with respect to severity categories.r2), all three methods showed a similar pattern of less LOD score inflation as the LD cut points decreased. Using r2 threshold, MERLIN-LD outperformed other approaches across all cut points; however, the reduction by the lowest threshold did not quite reach the LOD score with full parental genotypes available. With D' threshold, both SNPLINK and simple algorithm performed at a similar level; however, SNPLINK showed further reduction of LOD scores, especially with lower cut points. SNPLINK and the simple algorithm performed better with D' with low r2, but this result may not be generalizable to regions with high r2.In ASP analysis across the LD thresholds  declare that they have no competing interests."}
+{"text": "To evaluate whether there is evidence for two rheumatoid arthritis (RA) susceptibility genes on chromosome 6, we applied new robust methods for two-locus multipoint identical-by-descent mapping to the rheumatoid arthritis data of the Genetic Analysis Workshop 15. The software GEEARP was used to estimate the locations and the corresponding genetic effects for one locus or two linked loci in a region on chromosome 6, on the basis of affected relative pairs. These methods were applied to the data sets from the North American Rheumatoid Arthritis Consortium, Canada, France, and the first screen of United Kingdom. From the resultant 95% confidence intervals given by a robust variance estimator, a linked region, other than the well-known HLA region, was at 54.7\u201369.6 cM, providing evidence for a second rheumatoid arthritis susceptibility locus on chromosome 6. DRB1 alleles located in the HLA region of chromosome 6 affect susceptibility to rheumatoid arthritis (RA), and linkage to HLA has been confirmed . BecauseFor the 757 families in the NARAC data, genotypes of 404 single-nucleotide polymorphisms (SNPs) and 27 microsatellites on chromosome 6 were combined for the estimation of IBD. There were 1019 ARPs with genotypes, including 877 full siblings (FS), 45 HS, 13 first cousins (FC), and 84 AP. Only FS, HS, and AP were used in our analyses because each had reasonable sample size. For the Canada-Illumina data, there were 59 ASPs genotyped from 59 families with 404 SNPs on chromosome 6. The French data contained 88 families with 119 ASPs, with 64 microsatellite markers on chromosome 6. We used the first screen of the United Kingdom (UK) data, combining 18 microsatellite markers genotyped on 175 families with 667 SNPs genotyped on 157 families. There were 237 ASPs in the UK data. RA affection status was the only phenotype analyzed.r2 > 0.5 and all intervening markers were joined into clusters to estimate IBD scores, using the software Merlin (version 1.0.1) [To avoid biased estimates of IBD from linkage disequilibrium (LD) among high-density SNPs, markers with pair wise n 1.0.1) ,10.1 and \u03c42) of two susceptibility loci, and genetic effects for each locus . When the two disease susceptibility loci are linked, Ck 1and Ck 2do not represent the marginal effects of each locus, but rather they incorporate the effect of each other, and thus they change with the recombination fraction between them. When there are several kinds of ARPs, this method involves a large number of parameters , which can cause considerable variability in parameter estimates, especially when the numbers of the different types of ARPs are small. To reduce the number of parameters, we also fit a constrained model, which constrains the genetic effect of each locus across the different types of ARPs. That is, based on results from Risch [ki) can be formulated as functions of only one parameter for each locus, \u03bbi, i = 1 or 2. A score test proposed by Schaid et al. [The unknown parameters in the mean function are the locations . Similarly, for the two-locus model (Table p = 0.77). Because the constrained model provided shorter confidence intervals, this model might provide more precise confidence in gene locations. The two-locus model provides suggestive evidence for two linked regions on chromosome 6, with 95% CIs of 45.6\u201352.7 (the HLA region) and 54.7\u201369.6.The estimated gene locations and risk ratios from the one-locus models confirmed linkage at HLA  it is robust in the sense that a genetic model need not be specified, 2) it summarizes information across all types of ARPs , 3) it estimates gene locations and genetic effects (or transformed to recurrence risk ratios) simultaneously, and 4) it provides 95% CIs of relevant parameters. Although gene location is estimated, the precision can be low because of the nature for coarse mapping in linkage analyses, and if the linkage information content is not high. To identify causative loci, follow-up association analyses in the confidence intervals are necessary. Some cautions about the use of our method based on estimating equations are: 1) it suffers from downward bias in genetic effects (or recurrence risk ratios), which is absent in Kong-Cox LOD scores , with a Comparing the one- and two-locus models, the one-locus model performed well when the linkage peaks were far apart (close to the situation of unlinked genes) and there is no dominating peak. So, the results of one- and two-locus models were similar in the Canadian data. However, if there is a gene with much larger genetic effect than others, the one-locus model may fail to find other minor linked loci. The results of NARAC-FS and the unconstrained and constrained models showed this. In contrast, the two-locus model allows us to find the second gene in the presence of a much stronger signal. Note, however, that if there is only one gene in a region, the two-locus model can be over-parameterized and lose statistical efficiency. With these considerations, we suggest fitting both one- and two-locus models to evaluate linkage of multiple genes in a region, and our GEEARP software provides both of these analyses.AP: avuncular pairARP: affected relative pairASP: affected sib pair CIs: confidence intervalsFC: full cousinFS: full siblingGAW15: Genetic Analysis Workshop 15GEE: generalized estimating equationHS: half siblingIBD: identical by descentLD: linkage disequilibriumNARAC: The North American Rheumatoid Arthritis ConsortiumRA: rheumatoid arthritisSNP: single-nucleotide polymorphismThe author(s) declare that they have no competing interests."}
+{"text": "After genetic linkage has been identified for a complex disease, the next step is often fine-mapping by association analysis, using single-nucleotide polymorphisms (SNPs) within a linkage region. If a SNP shows evidence of association, it is useful to know whether the linkage result can be explained in part or in full by the candidate SNP. The genotype identity-by-descent sharing test (GIST) and linkage and association modeling in pedigrees (LAMP) are two methods that were specifically proposed to address this question. GIST determines whether there is significant correlation between family-specific weights, defined by the presence of a tentatively associated allele in affected siblings, and family-specific nonparametric linkage scores. LAMP constructs a pedigree likelihood function of the marker data conditional on the trait data, and implements three likelihood ratio tests to characterize the relationship between the candidate SNP and the disease locus. The goal of our study was to compare the two approaches and evaluate their ability to identify disease-associated SNPs in the Genetic Analysis Workshop 15 (GAW15) simulated data. Our results can be summarized as follows: 1) GIST is simple and fast but, as a test of association, did not perform well in the GAW15 data, especially with adjustment for multiple testing; 2) as a test of association, the LAMP-LE test performs best when the linkage evidence is strong, or when there is at least moderate linkage disequilibrium between the candidate SNP and the trait locus. We conclude that LAMP is more flexible and reliable to use in practice. The goal of a gene mapping study is to identify genetic variants that predispose to human diseases. For a complex disease, investigators often map the locus of interest first by linkage analysis, which typically results in a large candidate genomic region up to 40 Mb in size. To localize the susceptibility allele more precisely, disease-marker association analyses are performed, using a much denser map of genetic markers within the linkage region. One particular method of association analysis is based on comparing marker allele frequencies between unrelated cases and controls. In this design, only a subset of the samples originally collected for linkage analysis can be used. As an alternative, family-based association methods have been developed. The classic family-based transmission/disequilibrium test was proposed to test for association in the presence of linkage in family trios containing two parents and one affected offspring . This apGIST considers one particular marker allele as tentatively associated with the disease variant, and calculates family-specific weight variables defined by the presence of this allele. The variable has to be defined in such a way that the weight variable and IBD sharing configuration among affected family members at that same locus are uncorrelated if there is no disease-marker association, also called the \"unbiased selection scheme\". If there is a significant correlation between family-specific weights and family-specific linkage evidence, this suggests that the SNP allele could account in part for the observed linkage signal. GIST calculates three kinds of family-specific weight variables, corresponding to dominant, recessive, or additive inheritance models .W has been defined, the sample correlation coefficient between family weights W and family-specific nonparametric linkage (NPL) scores Z is computed. Under no disease-marker association, this correlation is expected to be zero and a one-sided test may be performed. A transformation of the correlation coefficient  that is asymptotically standard-normally distributed is used as the test statistic. When we do not know the underlying disease model, an alternative to carrying out all three tests in GIST is to use Xmax = max. The distribution of Xmax under no disease-marker association is estimated empirically by simulating a large number of ASPs under no linkage for various allele frequencies [Xmax should be the most appropriate test because we usually do not know the true genetic model for a complex disease.Once a weight variable quencies . The tesLAMP quantifies the degree of linkage disequilibrium (LD) between the candidate SNP and the putative disease locus through joint modeling and estimation of linkage and association parameters. LAMP constructs a likelihood of the marker data conditional on the trait data for a sample of families with disease penetrances and disease-SNP haplotype frequencies as parameters. Model parameters are estimated by maximum likelihood.0: r2 = 0). The third test is an indirect association test, which assesses whether there are other variants that can explain the linkage signal . If the null hypothesis of complete LD between the SNP and the disease allele is rejected, the SNP does not fully explain the observed linkage signal, and there may be multiple disease variants in this region [Three likelihood ratio tests are proposed to characterize the relationship between the candidate SNP and the disease locus. The first test assesses whether the candidate SNP and the disease locus are linked (LAMP linkage test). The second test is the direct association test, which assesses whether the candidate SNP and the disease locus are in partial LD so that the SNP may account in part for the linkage signal ).All SNP markers on chromosome 6, 7, 8, 9, 11, 16, and 18  were analyzed by GIST and LAMP. We also analyzed genotypes at all eight trait loci with LAMP. For GIST, we used the family-specific NPL scores at the location of the maximum multipoint LOD score on each chromosome, using the microsatellite markers . LAMP wap-value above a threshold value. If we only considered the SNP closest to the trait locus as the candidate SNP, the power was estimated by the proportion of replicates in which the p-value at this SNP was above a threshold value.The power to reject the null hypothesis of GIST and LAMP-LE was defined in two different ways: if we considered all SNP markers in the region defined by the true trait locus \u00b15 cM as the correct candidates, the power was estimated by the proportion of replicates with at least one p-values) obtained for this chromosome across the 100 replicates define a null distribution for the hypothesis of \"no linkage and no association\". Because it was previously shown that linkage and association test statistics are independent under this null hypothesis [p-value distribution for each test was set as the threshold value .The threshold value was set differently for each test. Because chromosome 7 does not contain any trait loci, the analysis results (r2 = 1), we analyzed genotypes at the actual trait loci. Due to the complexity of the GAW15 simulation model, we do not have the perfect null situation. Because a basic assumption of LAMP is the presence of only one disease variant in the region, we decided not to use the trait loci on chromosome 6 and 9. Despite the fact that none of loci A, B, E, and F are \"pure\" disease susceptibility loci, we used these loci as the best guess approximation for the null distribution for the LAMP-LD test.To derive a null distribution for the LAMP-LD test , even with adjustment for multiple testing, but, as expected, has low power when there is no LD, e.g., locus A, B, G, and H, as in Table r2 increases.The power comparison of GIST and LAMP-LE across a \u00b15 cM region is shown in Table r2 increases, and also depends on the magnitude of the linkage evidence. For locus C and D, both located on chromosome 6 and showing very strong evidence of linkage, the power to reject the the null hypothesis of complete LD with a single susceptibility variant is high, even though there is almost complete LD with the closest SNP (r2 = 0.94). Locus G and H are also located on the same chromosome, but they show little evidence of linkage and hence the power to reject complete LD is low. For locus A and B, LAMP-LD has low power to reject complete LD, presumably also because of little linkage evidence. Because locus E has a lower r2 value with the closest SNP than locus F, and they both have moderate linkage evidence, it makes sense that the power to reject complete LD is higher for locus E than locus F.The pattern of the power estimates for the LAMP-LD test is similar in Tables We compared the results from the LAMP linkage test with a standard linkage analysis using MERLIN, and also compared results from the LAMP-LE association test with PDT (data not shown). These comparisons suggest that the linkage and association tests from LAMP for these family structures are very similar to the linkage test implemented in MERLIN and the family-based association test implemented in PDT, respectively.We have completed GIST and LAMP analysis on eight totally different trait loci for rheumatoid arthritis simulated in GAW15 using SNPs that mimic a 10 K SNP chip set and microsatellite markers. Our results from applying linkage and association tests to the data are consistent with the genetic effects and levels of LD for each chromosome.GIST can only be applied to affected sibship data and considers only genotypes at the tentatively associated SNP, without incorporating flanking markers. In theory, GIST can detect association in regions with little overall evidence for linkage. Our results show that the GIST combined test performs poorly even in cases which would seem to be favorable for detecting an associated SNP (both linkage and LD). A possible reason for the failure of GIST to detect associated alleles is the multiple loci interacting to affect the RA hazard, and the overwhelming genetic heterogeneity, perhaps even within-family heterogeneity. Because the linkage evidence is very modest on chromosomes 11, 16, and 18 (average LOD scores < 1), there is also little power to detect association to the disease alleles.r2 values are as low as 0.145. The major disadvantage of LAMP is the speed. For large and sparsely genotyped pedigrees, LAMP can be painfully slow and such pedigrees must be trimmed or discarded in practice.LAMP can be applied to general pedigree data, including affected and unaffected individuals, and can incorporate flanking markers. Our study shows that LAMP-LE works well when there is at least moderate LD between marker and disease variant, even when GIST is simple and fast once family-specific NPL scores and weight variables have been computed, but it did not perform well in the GAW15 simulated data. LAMP is more flexible and more powerful, but much slower. LAMP seems to work particularly well when there is at least moderate LD between the candidate SNP marker and the trait locus.The author(s) declare that they have no competing interests."}
+{"text": "Anthocyaninless (anl) mutants of Brassica rapa fail to produce anthocyanin pigments. In rapid-cycling Brassica rapa, also known as Wisconsin Fast Plants, the anthocyaninless trait, also called non-purple stem, is widely used as a model recessive trait for teaching genetics. Although anthocyanin genes have been mapped in other plants such as Arabidopsis thaliana, the anl locus has not been mapped in any Brassica species.Anthocyanins are flavonoid pigments that are responsible for purple coloration in the stems and leaves of a variety of plant species. Brassicas and identified 37 that amplified a product in rapid-cycling Brassica rapa. We then developed three-generation pedigrees to assess linkage between the microsatellite markers and anl. 22 of the markers that we tested were polymorphic in our crosses. Based on 177 F2 offspring, we identified three markers linked to anl with LOD scores \u2265 5.0, forming a linkage group spanning 46.9 cM. Because one of these markers has been assigned to a known B. rapa linkage group, we can now assign the anl locus to B. rapa linkage group R9.We tested primer pairs known to amplify microsatellites in Brassicas. It also connects a classical mutant frequently used in genetics education with molecular markers and a known chromosomal location.This study is the first to identify the chromosomal location of an anthocyanin pigment gene among the  Brassica anthocyaninless mutants show decreased tungsten accumulation [Anthocyanins are flavonoid pigments that are responsible for purple coloration in the stems and leaves of a variety of plant species. They have been cited as contributing to protection from photoinhibition , protectmulation  and failmulation .AN1 and AN2 encode transcription factors that regulate pigment production in petunia [Pp1, Pp2 and Pp3 contribute to purple pigment in bread wheat [A, orthologs of which have been mapped in the related Solanaceae species tomato and potato [Arabidopsis thaliana, study of anthocyaninless mutants led to the discovery of ANTHOCYANINLESS1 (TAIR locus ANL1), responsible for anthocyanin production; ANTHOCYANINLESS2 (ANL2), a homeobox gene that affects anthocyanin distribution [ANTHOCYANIN11 (TAIR locus AT1G12910), which contributes to anthocyanin production; TRANSPARENT TESTA 9 (TAIR locus TT9), which is involved in flavonoid biosynthesis, and three other pigmentation genes .Anthocyanin-related genes have been isolated in diverse species.  petunia . Pp1, Ppad wheat . Anthocyd potato . In the ribution ; ANTHOCYBrassica rapa (RBr), also known as Wisconsin Fast Plants, completely lack the purple coloration.  RBr are strains of Brassica bred for short life cycle, early flowering, and ease of cultivation, and are used in science education and research [anthocyaninless (anl) locus. The anthocyaninless trait, also called non-purple stem, makes an excellent model trait in monohybrid crosses in genetics education because the trait is easily scored and expressed at all stages of the life cycle [B. rapa anthocyaninless phenotype has also been used as a marker to assess honey bee pollen deposition patterns [Anthocyaninless mutants of rapid-cycling research . Absencefe cycle . In addipatterns  and to epatterns .ANL allele, the intensity of anthocyanin coloration is a quantitative trait described as purple anthocyanin (0\u20139) (Pan(0\u20139)), which is controlled by multiple modifying alleles [RBr are a valuable tool for \"hands on\" genetics teaching. Cultivation is simple and inexpensive, and genetic crosses are easy to perform because they are self-incompatible for pollination. Many easily scored Mendelian traits have been identified including anthocyaninless, yellow-green , and hairless (lack of trichome on stems and leaves), to name a few. In addition to Mendelian traits, quantitative and polygenic traits are also available. For example, in those plants that possesses a wild type  alleles ,15. Likeanl locus using molecular markers.Despite these strengths, the RBr genetic repertoire lacks some important elements. All of the reported RBr mutations have been found to segregate independently of each other, so linkage analysis cannot be done with RBr. Also, none of the RBr loci used for education have been characterized at the molecular level. Therefore, we have sought to add such capabilities to RBr genetics, and our first step is to map the Brassica and have been mapped in the related Arabidopsis, the anl locus has not been mapped in any Brassica species. In this study we create a microsatellite marker-based linkage map of anl in RBr. The linkage group may be integrated into known B. rapa linkage groups and used for comparative mapping among related species. Our findings open the door for experiments in linkage analysis and the use of molecular markers with RBr in science education.Although anthocyanin mutants are studied in B. rapa, but a few from other Brassica species were tested since the sequence homology between Brassicas in the U Triangle allowed for analysis of microsatellites first identified in B. napus, B. oleracea and B. nigra [anl.We tested a total of 138 microsatellite markers for amplification and polymorphism in the RBr test population. Most (122 out of 138) were from B. nigra -18. Of tBrassica rapa strains are outbred, when a cross is conducted between two different strains, a given marker may be polymorphic between some pairs of parental generation plants but not others. Even for markers that are polymorphic between strains, some alleles may be shared. Therefore, after we had identified microsatellite markers that were polymorphic in the Standard Brassica rapa strain (Table 1 generation plants were grown and siblings were mated (inbred sib-pairs). The F1 sib-pairs were surveyed, and those exhibiting a high degree of marker heterozygosity were chosen for further analysis. These 44 F1 plants (22 F1 sib-pairs) produced 699 F2 plants, of which 177 (25.32%) were anthocyaninless. This ratio is consistent with monogenic inheritance of this recessive trait. The 177 anthocyaninless F2 plants constituted the RBr test population for determining linkage between anl and the 22 polymorphic microsatellites . ThuBRMS-024 [anl linkage group . Given such results, it is likely that sequences that can be amplified with BRMS-024 primers are present more than once in the B. rapa genome due to extensive intragenomic duplications [BRMS-024b.Our data strongly indicate that a polymorphic microsatellite that is amplified with primers for BRMS-024  is part B. rapa map data in other crucifers. B. rapa and Arabidopsis thaliana are related by a common ancestor from which they differentiated ~14.5 to 20.4 million years ago [B. rapa genome [B. rapa map are observed in Arabidopsis [Brassica species is currently under way , including the sequencing of B. rapa linkage group R9 . Linkage group R9, tentatively referred to as chromosome 5 [Arabidopsis chromosomal map [Bn9A locus on B. rapa Chr1 is located within a region that is conserved on Arabidopsis Chr1 . Additionally, a small region just below the Bn9A locus in B. rapa is collinear with another region on Arabidopsis Chr1 . While we cannot yet be certain that anl lies precisely within these conserved boundaries on B. rapa Chr1, the presence of the orthologous anthocyanin pigment gene AN11 on Arabidopsis Chr1 (TAIR accession number 2010356) supports the idea that the Arabidopsis ortholog of anl is located within the conserved regions.Comparative mapping studies allow utilization of ears ago . Althouga genome , the twobidopsis . A multimosome 5 , is now anl locus in RBr and identified three molecular markers linked to it. This linkage map of anl in B. rapa represents the first localization of an anthocyanin pigment gene in the Brassicas, and may be used for B. rapa map enhancement and comparative mapping of related species.We have found the chromosomal location of the Brassica microsatellites and their PCR primer sequences were identified from published sources and primer pairs were produced by custom synthesis  , 1.5 mM MgCl2, 200 uM dNTPs, 0.05 U/uL Accuprime Taq DNA polymerase (Invitrogen), 40 ng Brassica rapa DNA, 10 pmol forward primer and 10 pmol reverse primer in a total reaction volume of 10 uL. The PCR program was as follows: 94\u00b0C for 2 minutes; 24 cycles at 94\u00b0C for 30 seconds; 61\u00b0C for 1 minute; 72\u00b0C for 1 minute; finished at 72\u00b0C for 4 minutes.138 DNA was purified from frozen leaf tissue using Plant DNAzol Reagent (Invitrogen) with the manufacturer's recommended protocol, including the use of polyvinylpyrrolidone to remove polyphenolics. The concentration of DNA samples was assayed using Quant-iT PicoGreen Reagent (Invitrogen).Microsatellite alleles were resolved by non-denaturing polyacrylamide gel electrophoresis. Most PCR products were resolved using minigels ) consisting of 8% acrylamide/bis (24:1) run at 150 V for 60 to 90 minutes. When marker alleles could not be resolved on minigels, they were resolved in large (18 cm) gels (Protean II (Bio-Rad Laboratories)) which consisted of 8% acrylamide/bis (19:1) run at 150 V for 1350 Volt-hours. Gels were stained with SYBR Green Stain (Invitrogen) and visualized with a Molecular Dynamics Storm 860 Scanner .anl locus and microsatellite markers. The rapid-cycling Brassica rapa for the parental generation were strains of Wisconsin Fast Plants obtained from Carolina Biological Supply Company . The parental generation consisted of a true breeding anthocyaninless strain  and a true-breeding purple strain . One anthocyaninless and one purple plant constituted a mating pair. F1 sibling pairs were then crossed to produce the F2 generation.Three-generation pedigrees were used to assess linkage between the Brassica rapa plants. Each mating pair in the parental generation was then tested for polymorphism for those markers. When a given marker was found to be polymorphic in a pair of parental generation plants, their F1 progeny were genotyped for that marker. Finally, for each pair of mated F1 siblings in which a given marker was polymorphic and informative, their F2 generation offspring with anthocyaninless phenotype were genotyped for that marker. This inbred sib-pair mating design ensured that alleles from the F2 anthocyaninless test population could be traced to their parental generation ancestor, so that linkage phase of the marker and anl loci would be known.Before genotyping families, microsatellite markers were screened for usefulness by testing each pair of primers for the ability to amplify a product and identify polymorphism in DNA of a panel of several Standard 2 offspring were assigned a parental or recombinant designation for segregation between anl and the marker. The parental:recombinant ratio was then assessed for deviation from the expected 1:1 ratio for unlinked loci by the chi-square test. Markers presenting significant chi-square values (p < 0.05) were identified as candidates for linkage to anl, and two-point LOD analysis was performed between the marker and anl. Those markers with preliminary LOD scores greater than 3.0 were further analyzed with Mapmanager QTX  to determine marker arrangements and multi-point LOD scores [For each marker that was polymorphic within a family, anthocyaninless FD scores ,27.The author(s) declares that there are no competing interests.CB participated in the design of the study, carried out all breeding and genotyping, conducted analysis of the data, and drafted the manuscript. DW participated in the conception of the project and design of the study, assisted with analysis of the data, and edited the manuscript.Both authors read and approved the final version of the manuscript."}
+{"text": "Peromyscus maniculatus) and congeneric species are the most common North American mammals. They represent an emerging system for the genetic analyses of the physiological and behavioral bases of habitat adaptation. Phylogenetic evidence suggests a much more ancient divergence of Peromyscus from laboratory mice (Mus) and rats (Rattus) than that separating latter two. Nevertheless, early karyotypic analyses of the three groups suggest Peromyscus to be exhibit greater similarities with Rattus than with Mus.Deer mice .These data suggest that: 1. the  Peromyscus constitutes the most abundant and speciose group of North American mammals. Though superficially similar in appearance to rats and mice, deer mice represent a more distantly related lineage. Mouse and rat are thought to have diverged from each other ~10\u201312 million years ago (mya) while they last shared a common ancestor with the deer mouse (P. maniculatus) lineage ~25 mya [P. maniculatus species complex is a series of semi-interfertile populations spanning nearly every habitat on the continent and is consequently an emerging tool for the study of natural mammalian genetic variation. Facilitating such research is the existence of captive stocks derived from individual populations. Utilizing two of these stocks, we have developed a comparative genomic map for the deer mouse to further research of this genus and to provide insight into the genome rearrangements seen in rats, mice, and other mammals.The cricetid genus Comparative genomic analyses can reveal substantial amounts of information about the biology and evolution of species and are one of the keys to deciphering the roles that genomic structure and organization play in areas such as development, gene expression, and speciation. These analyses, however, are limited to portions of the genomes that have been mapped in all the species being compared and may be compromised by uncertainty of gene orthology and order between any two species.Although whole-genome sequences are available for many species, proper genome annotation is difficult and typically requires additional resources (e.g. meiotic linkage and radiation hybrid cell maps) . As a reRattus norvegicus) and mouse (Mus musculus), which both belong to the rodent family Muridae. Rodentia is the largest mammalian order, containing > 2000 of the ~4600 recognized species and the murids constitute the majority of these [Two of the most complete mammalian genomic maps are associated with the most used biomedical models, the rat  and P. polionotus stock derived from Ocala Nat'l forest in Florida (PO). While neither population is completely inbred, both originated from a limited number of founders and have been maintained as closed colonies. Thus, identifying fixed differences between the two  was typically not difficult.We employed a standard backcross design for these studies utilizing P. maniculatus was conducted using both the Rattus and Mus maps as references [Rattus chromosomes on our backcross panels. Table Mus and Rattus genomes that are informative for this comparative analysis.Map construction for ferences ,16 and uferences . In all,Our backcross panels facilitated linkage of markers at distances ranging from 1.2 cM to 35.8 cM. There are a few instances, however, where marker co-segregation occurs. These may be due to recombination \"cold-spots\", segmental inversions between the BW and PO strains, or simply the interval distance may be below our mapping panel resolution.Peromyscus genome using loci from Mus Chr 11 [Rattus Chrs 10 and 14 and the resulting chromosomal breakpoint is shared with the Rattus genome relative to the Mus genome  indicating rat genome homology. However, all three markers co-segregated. At a distance of only 1.9 Mb in the Rattus genome, these markers are likely to be closer in the deer mouse than our panel is able to resolve.To explore this possibility, we expanded the existing linkage groups using loci whose orthologs lie on Rattus Chr 14 homology group using two loci from Mus Chr 5 that have orthologs on Rattus Chr 14, Afp and Hip2. Similar to both Rattus and Mus genomes, Afp and Hip2 are linked in the deer mouse at a distance of 33.6 cM (LOD = 4.4)   of Rattus Chr 14 in the deer mouse with only a few markers. Our results again show a greater similarity between Peromyscus genome organization and the Rattus genome than either to the Mus genome.Using a similar strategy, we also expanded the Rattus genome similarity that we found for these two deer mouse linkage groups warranted examination of additional informative regions where synteny breakpoints occur between the Mus and Rattus genomes. While not comprehensive for every chromosome, this strategy accelerated our examination of chromosome evolution for the deer mouse genome and helped determine the best reference genome for future deer mouse genome mapping.The high degree of Rattus Chr 1, focusing our efforts within the regions surrounding the breakpoints between Mus Chrs 7, 17, 10, and 19 . This linkage conservation is again consistent with the Rattus genome but not with Mus.We next chose markers that spanned 9 Figure . Within Mus Chr 17 and Mus Chr 7 homology segments of Rattus Chr 1 and found no linkage in Peromyscus between any Mus Chr 17 markers and the Mus Chr 7 marker Usp29, which is only 17.8 Mb away on Rattus Chr 1. We made no further efforts to extend the Mus Chr 7 linkage group, as Usp29 is only 5.93 Mb from the centromeric end of Mus Chr 7 and additional markers in this region were unlikely to yield a different result. We also tested for linkage between the Mus Chr 17 marker Mas1 and the Mus Chr 7 markers Usp29 and Myod1 on a PO \u00d7 PO/BW backcross panel. This was to ensure that our negative linkage results were not a result of aberrant interspecific chromosomal recombination in the BW \u00d7 BW/PO panel. Again, Mas1 did not link to either locus. The lack of linkage between Mus Chr 17 and Mus Chr 7 homology segments in the deer mouse genome constituted the first example of a common breakpoint between the deer mouse and Mus genomes when compared to Rattus.We next tested for linkage between the Mus Chr 7 section of Rattus Chr 1 using five markers: Usp29, Q8C5Y2, Ube3a, Rab6ip1, and H19. Althoughthe RIKEN cDNA marker Q8C5Y2 has not been accurately mapped in either Rattus or Mus, BLAST results indicated intervals between Q8C5Y2 and Usp29 at 26.4 Mb for the Rattus genome and 37.3 Mb in the Mus genome. In support of our placement, the Mus Chr 7/Rattus Chr 1 marker Ube3a co-segregated with Q8C5Y2 in the deer mouse. Our data for these five Mus Chr 7 markers showed high conservation of linkage and gene order with both Rattus and Mus genomes  Rattus and the deer mouse genome by markers spanning the breakpoint between the Mus Chr 7 and Chr 19 regions of Rattus Chr 1. The Mus Chr 7 marker H19 is linked to the Mus Chr 19 marker Prdx5 at a distance of 6.5 cM (LOD = 12.2). Prdx5 is also linked to a second Mus Chr 19 marker Prpf19 at a distance 4.9 cM (LOD = 13.4 cM).We also found genome homology between Rattus Chr 1 loci show that the two deer mouse linkage groups span two Mus genome breakpoints but only one Rattus genome breakpoint, which shows a continued bias towards similarity with the Rattus genome. Our results also imply that the Mus genome has been more rearranged in this region.Overall, our data for Rattus chromosomes. Rattus Chrs 4 and 6 were priority candidates because of the simple breakpoint arrangements and well-conserved gene orders between Rattus and Mus.To broaden the scope of the deer mouse map and help reduce bias resulting from any localized phenomenon such as segmental inversions, we acquired data for markers from multiple Rattus Chr 4 is represented by the entirety of Mus Chr 6 and approximately 30 Mb of the centromeric end of Mus Chr 5. To test for a conserved organization in the deer mouse genome, we typed two markers from each side of the breakpoint on our backcross panel  and the two Mus Chr 6 markers, Ccdc132 and 1700016G05Rik, are linked to each other at a distance of 7.6 cM (LOD = 14.8). Spanning the breakpoint, we found that Sri and Ccdc132 are linked to each other at a distance of 13.3 cM (LOD = 10.3). Overall, our results span about 30% of Rattus Chr 4 and show conservation of the Rattus gene order. However, the linkage between Sri and Srpk2 was shorter than expected and may be due to a recombination cold-spot, an interspecific inversion, or a deletion.Rattus Chr 6 loci consisted of ten markers: Sos1, Ppp1cb, Preb, Dnmt3a, Allc, 493504H06Rik, Pygl, Clec14a, Pcnx, and 1700001K19Rik. These markers span Mus chromosomes 17, 5, and 12 and our results yielded two separate linkage groups . Within the Mus Chr 5 segment, Ppp1cb is linked to Preb at a distance of 6.1 cM (LOD = 16.8). A second Mus breakpoint is spanned by the linkage between Preb and the Mus Chr 12 marker Dnmt3a at a distance of 5.2 cM (LOD = 16.4). Also from Mus Chr 12, Allc represents the terminal marker and links to Dnmt3a at an interval of 16.5 cM (LOD = 7.6).Our mapping of Mus Chr 12 form a second linkage group in Peromyscus. Although synteny with both Rattus Chr 6 and Mus Chr 12 is conserved, we discovered an inversion with respect to both Mus and Rattus involving markers Clec14a and Pygl  while Clecl4a is linked to the more telomeric marker Pcnx at a distance of 18.2 cM (LOD = 6.3). We also found that Clec14a and Pygl have smaller distance interval at 1.3 cM (LOD = 21.2) than would have been expected from the physical intervals in Mus (11.9 Mb) and Rattus (13.4 Mb). Forming the end of the linkage group, Pcnx was linked to 1700001K19Rik at a distance of 25.3 cM (LOD = 3.9).The five remaining markers from Mus genome breakpoints by the deer mouse linkage groups again indicates a more Rattus-like genome organization. However, the breakpoint between the two Peromyscus linkage groups flanked by the two Mus Chr 12 markers Allc and 493504H06Rik represents a unique rearrangement that differs from both the Rattus and Mus genomes. ZOO-FISH results from Mlynarski et al.  also concur that Rattus Chr 6 is indeed represented by two separate chromosomes in the deer mouse.The spanning of two Rattus genome similarity, we also selected markers to span Rattus synteny breakpoints in relation to the Mus genome. This involved markers that span approximately 89% of Mus Chr 1 but are located separately in Rattus on Chrs 5, 9, and 13  but without segregation to Oprk1 and Tram1, despite a distance 35.3 Mb in the Rattus genome between Ube2j1 and Oprk1.For the Mus Chr 1, we detected linkage between the Rattus Chr 13 markers Acbd3 and Glul at a distance of 21.2 cM (LOD = 9.6) and between Glul and Bcl2 at 35.8 cM (LOD = 3.8) . However, Col3a1 is surprisingly not linked to Col9a1, which represents a deviation from the Rattus genome. To confirm these results, we also tested Mut, a marker that is closely linked to Col9a1 on Rattus Chr 9 , thus identifying a linkage similarity between the Rattus and deer mouse genomes. The breakpoints present in the deer mouse and Mus maps for this region are offset and may represent breakpoint area re-usage and a rearrangement hotspot [Col9a1 and Col3a1 on Rattus Chr 9 are needed to refine the breakpoint location. We discovered additional Rattus Chr 9 similarity using the marker Lama, which represents a second and separate region of Mus Chr 17 than that of Mut .Amongst the 9 Figure . Mut is t Figure . We founPeromyscus genome homology to Mus, we performed an analysis using six loci from Mus Chr 8 that form two linkage groups in the Rattus genome  and Mtus1 is linked to Spata4 at a distance of 16.9 cM (LOD = 9.3). Spata4 is also linked to the terminal marker 9130404D08Rik at a distance of 7.0 cM (LOD = 16.4). The two markers from Rattus Chr 19, Elmod2 and Pmfbp1, are linked 10.3 cM (LOD = 12.5).As with the Rattus Chr 17 and Mus Chr 2, Sec61a2 links to the Rattus Chr 19/Mus Chr 8 marker Pmfbp1 at a distance of 30.1 cM (LOD = 3.7). This linkage indicates a clear deviation from both the Rattus and Mus genomes by the deer mouse genome and highlights the benefit of performing cytogenetic analyses in tandem with meiotic linkage mapping.Based on suggestions from ZOO-FISH results , we also examined an additional chromosomal segment for linkage. Representing Mus genome similarity within the deer mouse using loci from Mus Chrs 17, 5, and 13  were tested for linkage in all possible arrangements despite having already been assigned to other deer mouse linkage groups. No linkage was found other than that which already existed amongst the Rattus Chr 1 markers Tcp10b, Pacrg, and Mas1 . The strong organizational similarity of the deer mouse genome with the Rattus genome rather than the more morphologically similar Mus musculus suggests that a significant amount of rearrangement occurred in the Mus genome after the divergence of the cricetid and murid lineages. Concomitantly, our results suggest that the genomic organizations of Rattus and Peromyscus are more representative of the ancestral muroid genome than the Mus genome, which is in agreement with previous literature that indicated a higher rate of genome rearrangement for Mus [Mus genome is extraordinary with approximately 200 homology blocks. However, the Mus genome is not unique in having higher relative rearrangement rates, as the canine and gibbon genomes have approximately twice the average number of homology blocks [There are three instances, however, where the deer mouse map differs from both the y blocks .Mus and Rattus shared a common ancestor significantly more recently than either have with Peromyscus. Our data does not propose to change this phylogeny but rather merely highlights that DNA sequence variation and chromosome rearrangement are independent processes and greater understanding of both processes can provide different insights into the evolution of the structure and function of the eukaryotic genome.Our results also show that genome rearrangement can act independently from other forms of genome evolution, such as sequence mutation. Although rodent sequence mutation rates are higher compared to other mammals, such measurements of genome evolution show P. maniculatus bairdii; BW) and the old field mouse  and were obtained from the Peromyscus Genetic Stock Center at the University of South Carolina [We chose interspecific backcross analysis in order to maximize genetic polymorphism and for the ease of linkage analysis . The twoCarolina . We set Carolina .plt BW \u00d7 PO) F1 animals with 12 unrelated plt BW animals to generate 116 backcross progeny. Backcrosses to BW can be performed using both female and male F1 hybrid animals, as both matings will give viable offspring. However, all but one of the matings used for this panel were F1 \u00d7 BW (\u2640 \u00d7 \u2642).We created two separate backcross panels, BX116 and BX2, for the linkage analysis. For the BX116 panel, we bred twelve hybrid . Genomic DNA for the BX2 panel animals was extracted using a phenol/chloroform/isoamyl alcohol extraction method to increase yields.Il23a, Mas1, H19, Sos1, and Grb10 were developed or obtained from published Peromyscus data [Trp53 primers were developed from P. maniculatus sequence and were kindly provided by Michael McLachlan. We developed all other primers from P. maniculatus EST sequences . Deer mouse EST clones were used for marker design because of greater PCR amplification success (>80% versus ~60% for the published sets).We obtained primer sequences for some Type I markers from published sets of orthologous gene markers. These are termed UMPS, CATS, and TOASTs -33. Primcus data -36. Trp5Mus musculus genomic sequences using cross-species megaBLAST (NCBI). Some critical markers however, spanned larger introns and resulted in amplicons slightly larger than the ideal size parameters.We designed all the markers to be ~400 bp\u20131500 bp and to span an intron to increase polymorphism detection. This was done by aligning deer mouse DNA sequences to P. maniculatus and P. polionotus DNA using a MJ Research PTC-200 DNA Engine gradient thermal cycler and are defined as follows: 1) Standard: 95\u00b0C for 14.5 min followed by 35 cycles of , 72\u00b0C for 10 min, 4\u00b0C hold. 2) Touchdown65 (TD65): 95\u00b0C for 14.5 min followed by 20 cycles of  followed by 15 cycles of , 72\u00b0C for 10 min, 4\u00b0C hold. If no product was obtained using Touchdown65, the starting annealing temperature was changed to 60\u00b0C or 55\u00b0C, with the final annealing temperature remaining 10\u00b0C lower than the starting temperature.PCR cycling conditions for all Type I markers were optimized for 2), 200 \u03bcM each dNTP, 0.4 \u03bcM forward primer, 0.4 \u03bcM reverse primer, and 1 unit Qiagen HotStar Taq polymerase. Some difficult templates required the use of Qiagen Q-solution at either 1\u00d7 or 0.5\u00d7 strength. Four markers, Sparc, Xbp1, Grb10, and Ugp2 required 2.0 mM MgCl2.PCR was performed using 20 ng of genomic DNA in a 10 \u03bcl reaction containing 1 \u03bcl 10\u00d7 Qiagen HotStar buffer  and incubated at 37\u00b0C for 15 minutes followed by heat-inactivation at 80\u00b0C for 15 minutes. For sequencing reactions, 2.0\u03bcL of purified PCR product was direct sequenced with BigDye (v3.1) (ABI) on an ABI 3130 \u00d7 l according to the manufacturer's protocol.Mus musculus genome. Any predicted simple size polymorphisms between BW and PO were tested by gel electrophoresis using amplification products from BW, PO, and BW/PO mix  DNAs. Markers not showing size polymorphisms were further analyzed for species-specific RFLPs by sequence comparison using Sequencher software (Gene Codes Corporation), the TCAG program available as part of the Biology Workbench software utilities provided at the San Diego Supercomputer Center [Sequence identities were verified by cross-species megaBLAST or BLASTN search to the r Center , or withr Center . CandidaGbl to Hba and Hba to Canx, of which both only used 39 animals due to the small usable data set for Hba. For the BX2 panel, no fewer than 60 animals were typed between any two markers with the exception of Mut and Col9a1, for which only 40 animals could be genotyped in common.We tested the backcross panel parental mice with each marker prior to use on the backcross panel. We typed then typed the markers on all possible backcross animals whose parents exhibited the expected genotype. On the BX116 panel, no fewer than 73 animals were used for data to establish linkage between any two markers with exception of We performed backcross linkage analysis using Map Manager QTX software . A minimBLAST Basic Local Alignment Search ToolPeromyscus maniculatus bairdiiBW CATS Comparative Anchor Tag SequencescDNA complementary DNAChr chromosomecM centimorgan20 distilled deionized waterddHdNTP dinucleotide triphosphateEDTA ethylenediaminetetraacetic acid-disodium saltEST expressed sequence tagLOD logarithm of the odds (to the base 10)Mb megabasemya million years agoNEB New England BiolabsPCR polymerase chain reactionplt platinum coat-color mutationPeromyscus polionotus subgriseusPO RFLP restriction fragment length polymorphismSAP shrimp alkaline phosphataseSNP single nucleotide polymorphismSSLP simple sequence length polymorphismTBE Tris-Borate EDTATE Tris-EDTA TLE Tris-low EDTA TOAST traced orthologous amplified sequence tagsUMPS universal mammalian primer sequenceZOO-FISH cross-species fluorescence in-situ hybridizationCR was the lead researcher, conceived the study, participated in its design and coordination, developed markers and assays, participated in the design of the backcross panel, performed linkage analysis, performed molecular genetic experiments, performed sequence alignments, and drafted the manuscript. AL developed markers and assays, performed molecular genetic experiments, and performed sequence alignments. JG developed markers and assays, participated in the design of the backcross panels, performed molecular genetic experiments, performed sequence alignments, and edited the manuscript. PV developed markers and assays, performed linkage analyses, participated in the design of the study, and contributed to and edited the manuscript. RO participated in the design and coordination of the study and helped to draft the manuscript. MD was the principle investigator on the project, participated in its design and coordination, performed sequence alignments, participated in the design of the backcross panels, and contributed in drafting the manuscript and figures. All authors read and approved the final manuscript."}
+{"text": "HLA locus on chromosome 6 as a susceptibility locus. Other regions of interest include loci on chromosomes 11, 2, and 12.We have used the genome-wide marker genotypes from Genetic Analysis Workshop 15 Problem 2 to explore joint evidence for genetic linkage to rheumatoid arthritis across several samples. The data consisted of four high-density genome scans on samples selected for rheumatoid arthritis. We cleaned the data, removed intermarker linkage disequilibrium, and assembled the samples onto a common genetic map using genome sequence positions as a reference for map interpolation. The individual studies were combined first at the genotype level  prior to a multipoint linkage analysis on the combined sample, and second using the genome scan meta-analysis method after linkage analysis of each sample. The two approaches were compared, and give strong support to the  Problem 2 of Genetic Analysis Workshop 15 (GAW15) includes genome-wide genotyping of marker sets for linkage studies in rheumatoid arthritis (RA). Four research groups contributed sets of markers across the genome genotyped in four independent pedigree samples. NARAC (North American Rheumatoid Arthritis Consortium) held by far the largest sample, consisting of multiplex families genotyped for 10-cM linkage mapping set and a panel of single-nucleotide polymorphisms (SNPs) genotyped by Illumina. A Canadian group provided pedigrees genotyped for the same Illumina marker panel as well as a dense 100 k Affymetrix SNP map. ECRAF (European Consortium on Rheumatoid Arthritis Families) genotyped a dense microsatellite panel. There was also a United Kingdom (UK) data set, comprising both microsatellite and SNP markers with a two-stage design.p-value), with a concomitant loss of spatial accuracy. The GSMA statistic is calculated for each bin as the mean rank across studies, with significance levels determined by permutation of the observed ranks within each study.The diversity of these samples and marker maps presents a complex problem to anyone seeking to merge them in order to achieve the greater power of a combined sample. In particular, we were interested in judging whether there is a \"best\" way to merge marker maps, or if available information would force one particular solution. We combined the data by placing markers on a common genetic map and performing linkage analysis on the whole sample jointly , which We were also interested in determining the evidence for linkage across the genome in each of the four samples compared with mega-analysis and meta-analysis. The NARAC sample is considerably larger than the others , and might be expected to outweigh the others in any joint analysis.Four data sets were analyzed for autosomal genetic linkage to RA Table . The CanWe assembled all markers from all studies onto a common centimorgan (cM) genetic map using a procedure described in Hamshere et al. . MarkersThe UK microsatellite positions were judged to be on the same map as NARAC, on the basis of 19 (out of 20) microsatellites common to both sets, which had identical centimorgan positions. Because the UK SNP marker map was of unknown provenance and the marker names cryptic, it was assumed to be comparable if not identical to the UK microsatellite map.r > 0.99). Of note are three markers which differed considerably in genetic position between the two maps: D9S144, D12S43, and D16S289 were originally positioned at 0 cM, and shifted by between 20 and 100 cM in the standard map, due to their current position in the sequence database. These instances were excluded from our analysis, and should be followed up in order to be sure of the identity and location of the typed marker.Because all other marker sets could be positioned relative to the NARAC/UK genetic map with a minimum number of assumptions, this was used as a standard map, and the NARAC microsatellites were used as reference markers (RMs). NCBI base-pair positioning permitted the interpolation of all other markers into each RM interval. Markers positioned outside of this standard map on each chromosome were removed from the analyses to prevent the possibility of negative map positions or overinflated chromosomal lengths. A total of 517 markers were removed from the NARAC and Canada SNP maps, and 74 markers from the ECRAF microsatellite map. A high level of correlation was observed between the original map and our standard map (interpolated on the basis of sequence positions) for the ECRAF microsatellites  was eliminated from each data set in order to prevent artificial inflation of the multipoint linkage statistics arising from incorrect allele frequency estimation in cases of missing parental data . In the Multipoint inkage analysis of the RA binary trait was performed on a 2-cM grid using MERLIN , with thA GSMA was performed as described previously ,9. The rThe maximum LOD score on each chromosome from the four individual samples are presented in Table High linkage scores were also detected on chromosomes 11 , 2 , and 4 . Loci showing low or modest linkage with some concordance across studies included chromosomes 9 (136\u2013146 cM), 12 (104\u2013136 cM), and 21 (30\u201352 cM). Five chromosomes  showed no LOD score > 1.The results of the meta-analysis Table  implicatThree of these samples  have been used in previous meta-analyses ,12. As iHLA-DRB1 locus in bin 6.2. CTLA, which may be a susceptibility locus for RA [PTPN22 locus maps to bin 1.6, which was the 25th highest ranked bin in the meta-analysis, and was not significant. This locus did not feature in any of the individual analyses, although the mega-analysis shows a small peak over the gene locus at 156 cM, with a maximum score of 0.99 .Several of these bins span candidate gene loci, including the s for RA  is locatThe conduct of our study was largely focused on the details of combining samples for mega-analysis in a rigorous fashion, and we conclude that the process adopted will often be dictated by the characteristics of the samples, and the information available on each. Researchers should follow common sense in including the data that provide the maximum information for each sample. The use of commercial high-density SNP maps, and the full-genome physical maps have made this process easier and probably more accurate than in the recent past. Meta-analysis techniques such as GSMA are more straightforward to perform than a mega-analysis, since they do not require access to the genotype data. Both the meta- and mega-analyses detect the strong linkage to chromosome 6. In addition, regions on chromosomes 2 (194 cM) and 12 (42 cM) are highlighted by both analyses. However, the mega-analysis showed evidence for linkage to chromosomes 10q and 11q, which was not observed in the GSMA. This is because chromosomes 2 and 12 show consistent linkage evidence across the four samples, whereas the linkages of chromosomes 10q and 11q are due almost entirely to high LOD scores from the NARAC sample, with negligible linkage evidence in the other three samples. The GSMA assigns significance to regions where linkage statistics are consistently highly-ranked across studies, but does not take into account the magnitude of the linkage statistics. Thus, it will not detect linkages based on a very high linkage statistic from one sample.This is the largest combined sample analyzed so far for linkage to RA. Both meta- and mega-analysis detected a highly significant linkage to chromosome 6p, with weaker linkages to chromosomes 6q, 2q, and 12p. In each of these regions there was consistent linkage evidence across the four samples. The mega-analysis also detected linkage to chromosomes 10q and 11q. In these regions, the linkage evidence came almost entirely from the NARAC sample, so they were not picked up by the meta-analysis. When performing an analysis of combined samples, it is important to ensure that marker maps are compatible, and that as many genotyping errors as possible are removed. It is also important to keep inter-marker LD to a minimum.The author(s) declare that they have no competing interests."}
+{"text": "Myopia and its extreme form, high myopia, are common vision disorders worldwide, especially in Asia. Identifying genetic markers is a useful step toward understanding the genetic basis of high myopia, particularly in the Chinese population, where it is highly prevalent. This study was conducted to provide evidence of linkage for autosomal dominant high myopia to a locus on chromosome 5p13.3-p15.1 in a large Chinese family.After clinical evaluation, genomic DNA from 29 members of this family was genotyped. A genome-wide screen was then performed using 382 markers with an average inter-marker distance of 10 cM, and two-point linkage was analyzed using the MLINK program. Mutation analysis of the candidate genes was performed using direct sequencing.Linkage to the known autosomal dominant high myopia loci was excluded. The genome-wide screening identified a maximum two-point LOD score of 3.71 at \u03b8=0.00 with the microsatellite marker D5S502. Fine mapping and haplotype analysis defined a critical region of 11.69 cM between D5S2096 and D5S1986 on chromosome 5p13.3-p15.1. Sequence analysis of the candidate genes inside the linked region did not identify any causative mutations.A genetic locus was mapped to chromosome 5p13.3-p15.1 in a large Chinese family with autosomal dominant high myopia. Myopia, the most common eye disease worldwide, is also the leading cause of visual impairment . The preGenetic and environmental factors both contribute to the development of myopia. Environmental factors such as work at close range and prolonged reading are suggested to be involved in the progression of myopia ,12,13 anThe inheritance of high myopia is equivocal. It may be inherited as an autosomal dominant, autosomal recessive, or X-linked recessive trait. Genetic mapping studies have identified at least 18 chromosomal regions suspected of harboring a myopia gene. X-linked recessive inheritance myopia has been mapped on Xq28 (MYP1) and Xq23\u201325 (MYP13) ,18. In aIn this study, we recruited a four-generational Chinese family with autosomal dominant high myopia. Through genome-wide screening and linkage analysis, we mapped the disease to a locus on chromosome 5p13.3-p15.1.A large family with autosomal dominant high myopia was identified in Zhejiang province, China. This family contained 11 affected individuals over four generations. Participating in this study were 29 individuals (aged 11 to 80 years): 10 affected and 19 unaffected . The stu2 (25\u00a0mM) and 0.4 U HotStar Taq DNA polymerase  in a \ufb01nal volume of 10\u00a0\u03bcl. Six to eight primer pairs were multiplexed in the ampli\ufb01cation reaction. Samples were incubated in a PTC-225 DNA Engine Tetrad  for 15 min at 95\u00a0\u00b0C to predenature, followed by 35 cycles of 30 s at 94\u00a0\u00b0C, 40 s at 55\u00a0\u00b0C, 40 s at 72\u00a0\u00b0C, and a \ufb01nal extension at 72\u00a0\u00b0C for 10 min. Ampli\ufb01cation products were appropriately pooled into prescribed panels, diluted, and denatured for 5 min at 95\u00a0\u00b0C, then incubated on ice for 2 min. Subsequently, the products were run in an automated DNA sequencer .Genomic DNA was isolated from peripheral blood leucocytes by the standard proteinase K digestion and phenol-chloroform extraction. The genome-wide screen was conducted on ABI 3700 sequencer by using PRISM Linkage Mapping Set MD-10  that have 382 highly polymorphic fluorescent markers with an average spacing of 10 cM. The markers were ampli\ufb01ed by polymerase chain reaction (PCR) under the following conditions: 50 ng genomic DNA, 2 pmol each primer, 0.2\u00a0\u03bcl dNTP (10\u00a0mM each), 1\u00a0\u03bcl 10\u00d7 buffer, 0.6\u00a0\u03bcl MgClMLINK program from the Linkage software package (version 5.2). For fine mapping, additional microsatellite markers spanning the chromosome 5p region were selected from the genetic map of the Marshfield Center for Medical Genetics . The myopia in the family was analyzed as an autosomal dominant trait with 90% penetrance and with a disease-gene allele frequency of 0.01. Recombination frequencies were assumed to be equal between males and females. Haplotype analysis was performed with Cyrillic software (version 2.0) and confirmed by inspection.Data were analyzed using GeneScan 3.7NT and Genotyper 3.7NT software . Two-point LOD scores were calculated using the SNP database was also referenced to determine whether the sequence variant was a polymorphism.The identi\ufb01ed genes located in the linkage region were proposed as candidate genes on the basis of their functional information. Mutations of these genes were screened by direct sequencing. Using the soft Primer Express 2.0 (Perkin Elmer), primers were designed to amplify each exon including exon-intron boundaries regions of the candidate genes from genomic DNA , \u22122.43; D1S218 (juvenile glaucoma), \u22122.5; D12S85 (Stickler syndrome type 1), \u22126.44; D1S206 (Stickler syndrome type 2), \u221210.77; and D6S276 (Stickler syndrome type 3), \u22124.15. Linkage to all of the known loci for non-syndromic autosomal dominant high myopia showed no statistically significant or suggestive evidence of linkage in this family (data not shown). Through subsequent genome-wide screening, a two-point LOD score of 3.02 (\u03b8=0.00) was initially obtained with the microsatellite marker D5S419, suggesting that the causative locus for the family with high myopia was mapped to a region adjacent to D5S419 on chromosome 5. For fine mapping, an additional 13 closely flanking microsatellite markers were tested, and the linkage analysis resulted in a significant LOD score at 5p13.3-p15.1. Seven microsatellite markers displayed positive LOD scores, with D5S502 having the highest LOD score, 3.71 at \u03b8=0.00 .Haplotype analysis of the affected individuals revealed recombination events that narrowed the region containing the gene, as shown in The critical region was found to be between the markers D5S2096 and D5S1986. A telomeric recombinant event occurred between markers D5S2096 and D5S2074 in the affected individual III:17, which defined the distal limit of the region to marker D5S2096. Affected individual IV:9 displayed evidence of a centromeric recombinant event between markers D5S1986 and D5S1470. Another centromeric recombination event was observed between markers D5S819 and D5S1986 in two affected individuals, III:7 and III:11. These defined the proximal limit of the region to marker D5S1986. Ultimately, we mapped high myopia to a locus on chromosome 5p13.3-p15.1, covering an approximately 11.69 cM (14.14 Mb) region between D5S2096 and D5S1986.CDH6), cadherin 10, type II (CDH10), cadherin 12, type II (CDH12), PDZ domain-containing protein 2 (PDZD2), Golgi phosphoprotein 3 (GOLPH3), zinc finger RNA binding protein (ZFR) were selected as candidate genes on the basis of their function: cell adhesion, intracellular signal transduction, protein trafficking, and DNA/RNA binding activities, which we thought were the functions most likely to be associated with myopia. A description of these genes was provided in Within the linkage region, the six genes cadherin 6, type II  have proven difficult to identify. The likely explanation for this difficulty is that the gene-environment interplay affects even Mendelian patterns of myopia ,12,13. TCDH6, CDH10, CDH12, PDZD2, GOLPH3, and ZFR at this high myopia locus were selected on the basis of their function to screen for gene mutations by re-sequencing. The classical cadherins mediate homophilic cell\u2013cell adhesion and are key regulators of many morphogenetic processes [Six candidate genes rocesses .CDH6 regulates the differentiation of retinal ganglion cells, amacrine cells, and photoreceptors in Zebrafish [CDH10 and CDH12 were detected in the mouse eye during the first postnatal week when several developmental processes, such as cell migration and formation of synaptic connections, occur simultaneously [PDZD2 is a ubiquitously expressed multi-PDZ-domain protein [GOLPH3 is a peripheral membrane protein of the Golgi stack. It is required for trafficking from the Golgi to the plasma membrane and for the normal extended Golgi ribbon. Depletion of GOLPH3 alters the Golgi ribbon, changing its normal appearance of extending partially around the nucleus, to condensing at one end of the nucleus [ZFR contains three widely spaced zinc finger domains. Zinc finger proteins with a similar pattern of zinc finger motifs are known to bind RNA, DNA, and DNA/RNA hybrids [ZFR can be involved in DNA repair and chromosome organization [ZFR knockout mice indicate that ZFR is essential for at least some developmental pathways, as embryonic death occurs at 8\u20139\u00a0days gestation in these mice. In homozygotes, genetic ablation of ZFR causes increased embryonic cell death and/or decreased cell proliferation rates [Loss-of-function studies demonstrate that the classical cadherins play a crucial role in vertebrate retinogenesis. They have multiple morphoregulatory functions in retinal proliferation, migration, differentiation, and layer formation, as well as axonal outgrowth, pathfinding, target recognition, and synaptogenesis ,35. CDH6ebrafish . CDH10 aaneously . PDZD2 i protein . PDZ dom protein . GOLPH3  nucleus . ZFR con hybrids . ZFR cannization . Analyseon rates . In the A previous study revealed an autosomal dominant high myopia locus mapped to chromosome 5p15.33-p15.2 with an interval of 17.45 cM between D5S1970 and D5S1987 in three Chinese pedigrees originating from Hong Kong (HK) . In our In summary, we have mapped a genetic locus for autosomal dominant high myopia in a large Chinese family. Myopia is the most common eye disease. Identification of the mutant gene(s) for myopia potentially would advance the understanding of the causes of this common eye disorder, and may thus lead to methods for preventing or slowing its progression."}
+{"text": "Bombyx mori, the domesticated silkworm, is a well-studied model insect with great economic and scientific significance. Although more than 400 mutations have been described in silkworms, most have not been identified, especially those affecting economically-important traits. Simple sequence repeats (SSRs) are effective and economical tools for mapping traits and genetic improvement. The current SSR linkage map is of low density and contains few polymorphisms. The purpose of this work was to develop a dense and informative linkage map that would assist in the preliminary mapping and dissection of quantitative trait loci (QTL) in a variety of silkworm strains.Through an analysis of > 50,000 genotypes across new mapping populations, we constructed two new linkage maps covering 27 assigned chromosomes and merged the data with previously reported data sets. The integrated consensus map contains 692 unique SSR sites, improving the density from 6.3 cM in the previous map to 4.8 cM. We also developed 497 confirmed neighboring markers for corresponding low-polymorphism sites, with 244 having polymorphisms. Large-scale statistics on the SSR type were suggestive of highly efficient markers, based upon which we searched 16,462 available genomic scaffolds for SSR loci. With the newly constructed map, we mapped single-gene traits, the QTL of filaments, and a number of ribosomal protein genes.The integrated map produced in this study is a highly efficient genetic tool for the high-throughput mapping of single genes and QTL. Compared to previous maps, the current map offers a greater number of markers and polymorphisms; thus, it may be used as a resource for marker-assisted breeding. Bombyx mori, which was domesticated over the past 5,000 years from the wild progenitor Bombyx mandarina. Cocoon quality is very important because it can influence the yield of sericulture and determines whether a silkworm line can be used in silk production. Through the efforts of silkworm breeders over several thousands of years, many silkworm strains have been collected and conserved. Moreover, the different properties of these conserved silkworm strains, such as filament length, cocoon weight, cocoon shell weight, cocoon shell ratio, and cocoon color, have distinctive applications. Until now, crossbreeding was the only method of enriching loci that control cocoon quality to enhance the yield from a silkworm cocoon. Modern techniques involving gene cloning and marker-assisted breeding are now widely considered to be the most effective way of improving silk properties.Silk fibers are derived from the cocoon of the silkworm Genetic linkage map is an essential tool for mapping traits of interest and are used in positional cloning and marker-assisted breeding. Some genetic maps for the silkworm have been reported, including various genetic markers such as restriction fragment length polymorphisms RFLPs; ,2), rand, rand2])In our previous SSR linkage map , the 518A general approach for increasing the marker density in genetic linkage maps involves the identification of more markers and the integration of several linkage maps. Xia et al.  construcThe choice of using the parental population of Dazao and C108 was based mainly on its internationally consistent use in silkworm genetic research. However, neither strain is applied widely for economic production of silk-related products. In China, more than 70% of silkworm breeders use the Jingsong strain for practical applications. Jingsong has properties that are advantageous for silk production, such as an average filament length of 1,200\u20131,500 m. In contrast, L10, which has poor silk-producing properties, possesses high stress resistance. Additional matings between strains of different origins may increase the mapping efficiency of markers due to the increased potential for genetic diversity.Herein, we report an improved method for constructing silkworm SSR genetic maps with more informative loci based on new mapping populations Figure . Using tApproximately 2,670 SSRs isolated from our genomic libraries were subjected to polymorphism detection, including 518 SSRs that had been mapped  and JL [Lan10 \u00d7 (Jingsong \u00d7 L10)] were integrated with the previous DC data set  . We corrSince MAPMAKER only handles one cross at a time, we could not directly pool scoring in different crosses to construct a consensus map. Instead, we first merged the single data sets into a consensus data set. Given the similar sizes of three mapping populations , we made up a large population of 567 individuals that could include three sets of scoring. From this, the first 189, the next 190, and the last 188 represented individuals came from DC, JL, and FN, respectively. If the individuals were not genotyped in the corresponding cross, they were considered as missing data loci (designated 0). Fortunately, we could treat many common markers, typed in two or three crosses with little or no missing data loci, as anchors for groups from different mapping populations. The resulting consensus data set was used to perform a linkage analysis using MAPMAKER. The framework generated by a single cross was accounted for if the result of ordering was dramatic. JoinMap was also employed for reference, which automatically constructs an integrated consensus map.SSR: simple sequence repeats; QTL: quantitative trait loci; CAPS: cleaved amplified polymorphic sequences; CW: whole cocoon weight; CSW: cocoon shell weight; CSR: cocoon shell ratio; PW: pupal weight.YH, ML, XM, and SZ planned the work. ML arranged the strain mating. QG and WL prepared the DNA samples. SZ and XM designed the markers. SZ, QG, YZ, and WL carried out the genotyping. SZ, JH, and ML performed the data analysis. SZ and ML wrote the draft. YH, MRG, and JH commented on an earlier draft. All authors have read and approved the final draft of the manuscript.NF linkage maps, part I. The most likely linkage maps for seven chromosomes  generated by the NF data set are shown.Click here for fileNF linkage maps, part II. The most likely linkage maps for seven chromosomes  generated by the NF data set are shown.Click here for fileNF linkage maps, part III. The most likely linkage maps for seven chromosomes  generated by the NF data set are shown.Click here for fileNF linkage maps, part IV. The most likely linkage maps for six chromosomes  generated by the NF data set are shown.Click here for fileJL linkage maps, part I. The most likely linkage maps for ten chromosomes  generated by the JL data set are shown.Click here for fileJL linkage maps, part II. The most likely linkage maps for 13 chromosomes  generated by the JL data set are shown.Click here for fileInformation on the markers used in this study. This table contains the work name, map name, forward primer sequence, reverse primer sequence, labeled type, and population source.Click here for fileIntegrated maps of three mapping populations, part I. The most likely linkage maps for the chromosomes integrated with three data sets are shown in this file and the following four additional files  integrated with three data sets are shown. The sites that contained confirmed neighboring markers are indicated by a star. An additional star indicates that a polymorphism was detected. Chromosome 11 and 11' shared one common marker; however, their order could not be determined. The map of chromosome 7 is not included here because there were not any new identified markers on it.Click here for fileIntegrated maps of three mapping populations, part III. The most likely linkage maps for six chromosomes  integrated with three data sets are shown. The sites that contained confirmed neighboring markers are indicated by a star. An additional star indicates that a polymorphism was detected.Click here for fileIntegrated maps of three mapping populations, part IV. The most likely linkage maps for five chromosomes  integrated with three data sets are shown. The sites that contained confirmed neighboring markers are indicated by a star. An additional star indicates that a polymorphism was detected.Click here for fileIntegrated maps of three mapping populations, part V. The most likely linkage maps for six chromosomes  integrated with three data sets are shown. The sites that contained confirmed neighboring markers are indicated by a star. An additional star indicates that a polymorphism was detected. Chromosome 25 and 25' shared one common marker; however, their order could not be determined.Click here for fileResult of 100 replicates of multi-marker joint analysis in sample JL. We included the major and epistatic QTL that were detected at least once in 100 replicates by multi-marker joint analysis (LOD = 2). The position information, power value, mean LOD score, mean effect value, and individual value of each replicate for the filtered loci are listed in the file.Click here for filePutative epistatic QTL and their interaction effects (100 replicates). We performed multi-marker joint analysis to estimate the association between markers and phenotypes. This table contains the significant interacting pairs with their statistical power value (at least 20%), mean LOD score, and mean interaction effect in 100 replicate tests.Click here for fileInformation on all confirmed neighboring SSR markers. The marker name, original site, forward primer sequence, reverse primer sequence, and detection results are shown.Click here for fileComparison of the efficiency of different mapping populations and marker types. This table contains the origin of three populations and the counting information on markers used in each population.Click here for file"}
+{"text": "In order to identify regulatory genes, we determined the heritability of gene transcripts, performed linkage analysis to identify quantitative trait loci (QTLs), and evaluated the evidence for shared genetic effects among transcripts with co-localized QTLs in non-diseased participants from 14 CEPH (Centre d'Etude du Polymorphisme Humain) Utah families. Seventy-six percent of transcripts had a significant heritability and 54% of them had LOD score \u2265 1.8. Bivariate genetic analysis of 15 transcripts that had co-localized QTLs on 4q28.2-q31.1 identified significant genetic correlation among some transcripts although no improvement in the magnitude of LOD scores in this region was noted. Similar results were found in analysis of 12 transcripts, that had co-localized QTLs in the 13q34 region. Principal-component analyses did not improve the ability to identify chromosomal regions of co-localized gene expressions. There is a breadth of information being generated by the Human Genome Project and the interpretation of these data has been a major area of research. For simple Mendelian disorders, the identification of genetic effects is fairly straightforward due to understanding the biology that drives these disorders. However, for complex oligogenic or polygenic disorders, understanding all the interconnections between genes influencing a trait is a difficult task because the understanding of the biology of many of these disorders is still evolving. Multiple gene \u00d7 gene and gene \u00d7 environment interactions can influence the expression of phenotypes. Genes can interact by modifying the expression of other genes and therefore function as regulatory genes .In an effort to dissect some of these complexities, we performed linkage analysis of gene expression transcripts of members of Centre d'Etude du Polymorphisme Humain (CEPH) Utah families to determine the heritability of transcripts and the evidence for regulatory quantitative trait loci (QTLs) and we performed pairwise bivariate linkage analysis and principal-component analysis (PCA) for data-reduction to evaluate the evidence for shared genetic effects. The ability to assess gene expression traits simultaneously and to link them to QTLs offers the possibility of identifying previously unknown underlying molecular processes for future investigation.We used the Genetic Analysis Workshop 15 (GAW 15) Problem 1 microarray gene expression profiles for the analyses. Data were available for 14 three-generation CEPH Utah families. Expression levels of genes were obtained from lymphoblastoid cells using the Affymetrix Human Focus Arrays . We were. Genetic map positions were obtained using the SNP Mapping web application developed by the University College Dublin Conway Institute of Biomolecular and Biomedical Research , which uses the Rutgers Combined Linkage-Physical Map of the Human Genome and data taken from the NCBI dbSNP Build 123 (in Kosambi centimorgans). This information was used to calculate multipoint identity by descent matrices (MIBDs) with Merlin and Minx [Family members were genotyped for 2882 autosomal and X-linked single-nucleotide polymorphisms (SNP) generated by the SNP Consortium and Minx . MIBDs w2, sex, age \u00d7 sex and age2 \u00d7 sex interaction using predictive linear regression models in SAS 9.1 . We generated these residuals as part of our processing of the transcripts for linkage analyses.Transcript distributions were normalized using an inverse normalization transformation of z-scores of individual transcripts regressed on the mean individual transcript level. We further adjusted for the effects of age, ageSOLAR [G) and correlations caused by random environmental effects (\u03c1E) [G is constrained to 0  or \u03c1G = 1  is compared to that of a model in which \u03c1G is estimated for the traits. Significant differences among the models (\u03c1G \u2260 0) suggest that some of the same genes influence both traits. We also performed linkage analysis using the factors obtained from the PCA in a sample of transcripts with co-localized QTLs.Heritability was estimated using maximum likelihood variance decomposition methods in SOLAR ,6. GenomSOLAR . For tracts (\u03c1E) . To testtrans-regulatory genes). Because of the small number of individuals in the study and concerns of overfitting the model, a maximum sample of 50 transcript values were considered at one time [PCA was used to reduce the number of expression profiles into statistically meaningful groups while retaining the original total variance using all the expression profiles ,10. We sone time . The numone time . Factorsone time . The pritrans-regulatory sites. In addition, the location and LOD scores of QTLs identified in single individual transcript analysis  were compared with the location of the QTLs identified using bivariate analysis or factors of the PCA. This allowed a determination of whether the bivariate analysis or PCA data reduction analysis improved our evidence for linkage, and if so, a more in-depth examination of the transcripts included in the principal components needs to be examined for biologic interactions on complex disorders.Linkage signals of individual transcript expressions were recorded and the location of QTLs was compared to the location of the transcript gene in order to identify n = 2688) of the transcripts had significant heritability (p < 0.05) and were considered for the linkage analysis. Of this, 1448 (54%) transcripts displayed suggestive evidence of linkage (had a maximum LOD score \u2265 1.8 [trans-regulatory sites). We used two different chromosomal regions with co-localized transcript QTLs, chromosomes 4q28.2-q31.1 and 13q34, for more in-depth analyses.Among 194 individuals from 14 families, 17 individuals with missing information on age were excluded. Seventy-six percent (re \u2265 1.8 ). The QTMX2, NUCB2, and SNX4 genes. Using PCA, we obtained five factors from the 15 transcripts with eigenvalues greater than 1. Only one factor, with a high positive loading for the MX2 gene transcript, had a significant heritability and a LOD score \u2265 1.8. The linkage analysis using this factor identified the chromosome region for the MX2 gene, but the LOD score was lower than the one obtained by single linkage analysis of the MX2 transcript.Table SNX4, YWHAQ, ASS, MX2, and ISGF3G gene transcripts). Both factors had significant heritability; however, only Factor 1, loading heavily on the MX2 gene, localized to the 4q28.2-q31.1 region . The heritability of one factor obtained using PCA for Group 2 transcripts was not significant and further analysis was not performed.We then performed bivariate analysis of all pairwise co-localized transcripts on 4q28.2-q31.1 and found evidence for genetic correlation of co-localized genes, although without much increase in the magnitude of the LOD score , and therefore they may provide redundant information that may reduce the power for detection of the bivariate signal [G is a test of the overall additive genetic correlation among two traits and not the QTL-specific pleiotropy, it is possible that the co-localized linkage signals are not in fact genetically correlated. Further analysis is required to address these issues.We also performed pairwise bivariate genetic analysis on those transcripts that co-localized to the same genomic region, presumably because this area of the genome harbored genes involved in the regulation of these transcripts . We detee signal . In addiThe chromosome regions selected for detailed analyses were arbitrarily chosen as we identified multiple other regions with co-localized linkage of gene expressions. The results from our univariate genome scan differ markedly from those reported by Morley et al.  because trans-regulatory genes with significant heritability. Some of these regulatory genes displayed strong additive genetic correlations, and may be part of genetic networks. However, when compared to univariate analysis, linkage analysis of bivariate phenotypes and factor scores obtained from PCA did not improve the ability to identify chromosomal regions of co-localized gene expressions.We identified several chromosomal regions of co-localized CEPH: Centre d'Etude du Polymorphisme HumainGAW: Genetic Analysis Workshop2: heritabilityHLOD: logarithm of the oddsMIBD: multipoint identity-by-descent matricesN/A: not applyNCBI: National Center for Biotechnology InformationPCA: principal-component analysisQTL: quantitative trait lociSE: standard error of the meanSNP: single-nucleotide polymorphismSOLAR: Sequential Oligogenic Linkage Analysis RoutinesThe author(s) declare that they have no competing interests."}
+{"text": "Microarray technologies allow the measurement of the expression levels of thousands of transcripts at the same time. As part of Genetic Analysis Workshop 15 (GAW15), we analyzed a data set that measured the expression of more than 3000 genes in 14 families. Our goal was to identify genomic regions that regulate the expression of several genes at the same time. We tried two different approaches: one was maximum likelihood-based variance-component linkage analysis and the other was a new linkage regression approach. We detected some loci that were linked with the expression level of more genes than would be expected by chance. These loci are candidates for master regulators of transcription (MRT). Finally, for each candidate MRT, we did a gene ontology (GO) analysis to test whether the genes linked to it were biologically related. One common feature observed in studies of transcript expression is the presence of hot spots, that is, individual loci that affect large number of transcripts . Hot spoIn this study we applied two different linkage strategies to localize hot spots of transcript expression in the Genetic Analysis Workshop 15 (GAW15) Problem 1 data. On the one hand, we used the maximum likelihood-based variance components linkage method and on the other, we tried a new linkage regression approach that implements a feature selection algorithm. Finally, we tried to identify commonalities among the different genes that seem to be regulated by each MRT using the information given by the gene ontologies (GO) .We used the Problem 1 data of GAW15 as described by Mosley et al. . These d. We estimated multipoint identity-by-descent (IBD) matrices using all of the genotyped SNPs with Merlin  was used as a measure of phenotypic distance and the IBD estimates between pairs were used as measures of the genetic similarity at each genetic location. In the standard Haseman-Elston regression, the phenotypic distance is regressed on the genetic similarity, for a set of locations along the genome . The result is a linkage test for each location. Here we tried a different approach: given a phenotype we tried to select the set of genetic locations that best explained the phenotypic distances. Instead of models that test linkage at only one location, we allowed models that combined locations. The linkage analysis result for a phenotype is the combination of genetic locations that best explains the phenotypic distances between sib pairs.We present a regression method based on the Haseman-Elston approach . Given an where n is the number of locations. In these cases, sub-optimal algorithms can be used to find a selection. This sub-optimality presents the risk of falling to local minima in the solution.The selection of genetic locations related to a specific phenotype based on exhaustive search is computationally impractical with current computer capabilities: the number of combinations rises up to 2n features as the initial subset, and applies the following steps: 1) evaluate a quality criterion for the current model; 2) try dropping each of the features in the subset and compute the corresponding criterion; 3) select the best candidate model; 4) iterate until the quality criterion is not improved.The sequential backward selection (SBS) algorithm  begins wThe number of combinations needed for this solution will obviously be less than for an exhaustive search. In the symmetric algorithm, namely sequential forward selection (SFS), the initial subset contains zero elements and step two (above) includes a feature instead of dropping it.Note that when one feature is dropped in SBS, there is no possibility of including it again in the subset, although it could provide further information and improve the criterion. To avoid this nesting effect, a variant family of the above algorithms called \"floating search methods\" has been proposed . For seqA SFFS algorithm has been built for determining genetic locations related to a phenotype. The floating search algorithm targeted the minimization of the root mean square error (RMSE) of a partial least squares (PLS) model when predicting a given phenotypic distance from the complete set of loci. For each model constructed, the number of PLS optimal latent variables was obtained from the training set (one-third of the available pairs) by means of four-fold cross-validation. RMSE was computed on the validation set, covering two-thirds of the available pairs.We applied this algorithm to the 3554 phenotypes. Because the SFFS algorithm is computationally intensive, instead of using all the available genetic locations (one for every centimorgan), we used only 800 locations evenly spaced, one every 5 cM. Results are given as a vector of the locations (expressed in cM) selected for each phenotype.We performed tests to evaluate statistical over-representation of gene ontology (GO)  categoriWe followed a classical variance-components approach and we found 11 candidate MRT (hot spots), see Table We performed a GO analysis of the groups of genes controlled by the same MRT. Over-representation of GO terms in 9 of the 11 sets of genes was found at a significance level of 0.01, either in the biological process or in the molecular function ontologies  declare that they have no competing interests."}
+{"text": "Colorectal cancer is one of the most common causes of cancer-related mortality. The disease is clinically and genetically heterogeneous though a strong hereditary component has been identified. However, only a small proportion of the inherited susceptibility can be ascribed to dominant syndromes, such as Hereditary Non-Polyposis Colorectal Cancer (HNPCC) or Familial Adenomatous Polyposis (FAP). In an attempt to identify novel colorectal cancer predisposing genes, we have performed a genome-wide linkage analysis in 30 Swedish non-FAP/non-HNPCC families with a strong family history of colorectal cancer.Statistical analysis was performed using multipoint parametric and nonparametric linkage.Parametric analysis under the assumption of locus homogeneity excluded any common susceptibility regions harbouring a predisposing gene for colorectal cancer. However, several loci on chromosomes 2q, 3q, 6q, and 7q with suggestive linkage were detected in the parametric analysis under the assumption of locus heterogeneity as well as in the nonparametric analysis. Among these loci, the locus on chromosome 3q21.1-q26.2 was the most consistent finding providing positive results in both parametric and nonparametric analyses Heterogeneity LOD score (HLOD) = 1.90, alpha = 0.45, Non-Parametric LOD score (NPL) = 2.1).The strongest evidence of linkage was seen for the region on chromosome 3. Interestingly, the same region has recently been reported as the most significant finding in a genome-wide analysis performed with SNP arrays; thus our results independently support the finding on chromosome 3q. Colorectal cancer is a major problem in the Western world, ranking as the second most common cause of cancer-related death with a 5% lifetime risk. One of the strongest associated risk factors for colorectal cancer is a family history of the disease. From twin studies, Lichtenstein has estimated that heritable factors account for 35% of the risk . EpidemiWe have recently published a genome-wide study in 18 Swedish familial colorectal cancer families suggesting candidate loci on chromosomes 11 and 14 . We now hMLH1, hMSH2, hMSH6 or hPMS2 in cases fulfilling Amsterdam criteria [In total, 30 colorectal cancer families with 275 typed individuals were included in the study. All families were of Swedish origin and were collected through the Family Cancer Clinic at the Karolinska Hospital, Stockholm, Sweden. In all cases, the diagnosis was confirmed by medical and pathological reports and informed consent was obtained from all participants. The study was undertaken in accordance with the Swedish legislation of ethical permission 2003:460) and according to the decision in the Stockholm regional ethical committee (Dnr: 2005/566-31/1). FAP and MUTYH were excluded clinically since only one of the individuals with CRC or under surveillance had more than 4 polyps. This was a family with a dominant inheritance pattern and the APC gene was screened negative. Although none of the other families (22 with a dominant pattern of inheritance) fulfilled the criteria for screening, the APC gene had been screened in 20 of the families and the MUTYH gene in 15 of the families without any clearly pathogenic mutations found 03:460 an. This prcriteria . The 12 Genomic DNA was extracted from peripheral blood by standard procedures. Genotyping and a first-quality check of the additional 12 families were done at deCODE Genetics . A total of 545 fluorescently labeled microsatellite markers located on the 22 autosomes and the X chromosome with an average marker density of 7.25 cM were analyzed. Overall, 97.1% of the genotypes were successfully determined. Genetic map was used as provided by deCODE.\u00ae and Genotyper\u00ae software programs . In the combined analyses, the genetic map was constructed using the Marshfield linkage maps [For fine mapping of the locus on chromosome 3 and 6 an additional set of eleven and seven markers were typed, respectively. Each marker was amplified separately according to a standard PCR protocol (conditions are available upon request). The PCR products were pooled and separated on an ABI 377 DNA sequencer. Electrophoretic data were analyzed using the Genescanage maps .All genotyping data were reanalyzed concerning the Mendelian inheritance and the relationship patterns of the families using the Pedcheck program . Any marIndividuals with positive colorectal findings were coded as affected only if they had colorectal cancer or adenomas with a high degree of dysplasia. Non-related family members (spouses) were coded as unaffected and any other family members as unknown. In addition, the data for chromosome 3 and 9 obtained from combined analysis from all thirty families were recoded using recently published criteria for affected status and reanalysed using age-dependant penetrance as described by Kemp et al. .An autosomal dominant mode of inheritance with the disease allele frequency of 0.0001 was used while the penetrance and phenocopy rate were assumed as 80% and 5%, respectively. In addition, single point LOD score analysis on chromosome X was performed using the Fastlink program .Database analysis revealed 340 genes (NCBI build 36) within the 65 cM interval between markers D3S1558 and D3S3592. Based on their known or hypothesized function we sequenced 20 genes in the region on chromosome 3 with a positive LOD score. Sequencing of 14 genes was done as described in Sj\u00f6blom et al. . For the2O, 2.5 \u03bcl TaqMan Universal PCR Master Mix  and 0.125 \u03bcl Assay. The SNP genotyping assay contains two primers for amplifying the sequence and two probes for detecting alleles. Each probe contains a reporter dye at the 5'-end; specifically, the VIC dye is linked to the allele 1 probe and the FAM dye is linked to the allele 2 probe. After the initial denaturation step at 95\u00b0C for 10 min there were 40 cycles of denaturation at 92\u00b0C for 15 sec, annealing and extending at 60\u00b0C for 1 min. The fluorescence was measured after the last PCR cycle was completed by the ABI PRISM 7900 HT instrument  in which the fluorescence intensity of each well was read. Negative and positive controls were included in all analyses as a quality control measure. Fluorescence data files from each plate were analyzed using the SDS 2.2.1 software . We used Haploview [Genotyping of 10 SNPs on chromosome 3 was performed using TaqMan SNP Genotyping Assay  in 190 familial colorectal cancer cases and 190 healthy controls matched to patients according to their gender and age. A standard PCR was carried out in a 384-well format, with a total reaction volume of 5 \u03bcl using 6 ng genomic DNA diluted in 2.375 \u03bcl Haploview  to deterIn the combined data set of the 30 families, genome-wide linkage analysis did not reveal any locus with a statistically significant LOD score. However, genome-wide HLOD scores over 1 and NPL scores over 1.3  providing evidence for suggestive linkage were obtained in both parametric and nonparametric linkage analyses. Overall, four regions with p-value < 0.05 were identified. The results are summarized in table Eight families with possible linkage to chromosome 3q were included in subsequent fine mapping. Positive LOD scores were detected for the region between D3S1558 and D3S3592 spanning 65 cM , on the basis of their known or predicted function and position near the markers that showed the highest LOD score. We sequenced the exons, the intron/exon boundaries, 5' and 3' UTR and, for some genes, the promoter region. No deleterious mutations were found, but instead many variants , already reported in the databases, were recorded. We were particularly interested in family 242 because it gives the largest contribution to the LOD score values on chromosome 3. Ten SNPs  were present on the disease haplotype in family 242. Association studies were performed to test these 10 SNPs using 190 familial colorectal cancer cases and 190 controls without a family history. Eight SNPs had a frequency above 5%, were tested and did not show significant differences between the two groups; 2 SNPs (rs2291382 and rs3762802) had a frequency below 5%, 8 cases and 7 controls were found in the two groups respectively and no further tests were performed.In the 12 new families, 545 microsatellite markers were used for genotyping, compared to 400 markers in our first screening with 18 families. For this reason we analyzed the 12 new families separately as well. The strongest evidence of linkage was found on chromosome 6q22.3-24.3 with an NPL score of 2.06  for D6S1656. Furthermore, a p-value < 0.05 was obtained for three additional loci on chromosomes 1p36.22, 3q24-25.1, and 13q33.2. Under the assumption of locus heterogeneity, linkage was most evident in the region on chromosome 6q22-23. The highest HLOD score was obtained for marker D6S270 (HLOD = 1.68 for \u03b1 = 0.4). Positive HLOD scores of 1.59 (\u03b1 = 0.4) and 1.39 (\u03b1 = 0.45) were seen in this analysis for both flanking markers, D6S1656 and D6S1009, respectively and supported the results obtained for this region in the nonparametric analysis. Out of the 12 families, seven provided positive LOD scores for this region. Fine mapping on chromosome 6q using additional 11 markers confirmed the results and determined the region of linkage between markers D6S1712 and D6S1637 (30 cM) with a NPL peak of 2.3 for 2 closely located markers (250 kbp), D6S976 and D6S270. No common shared haplotype was found among the linked families.We have recently presented results from a genome-wide screen in 18 Swedish colorectal cancer families. In order to further evaluate and narrow down regions of suggestive linkage, we extended our family material with 12 additional families, and combined the results from the new genome-wide analysis in the 12 families with our previously published findings.In our combined analysis containing 275 subjects from 30 non-FAP/non-HNPCC colorectal cancer families, we did not find any support for the two loci on chromosomes 11 and 14 suggested from the previous analysis of 18 of the families. Instead, we now found suggestive linkage to chromosome 3q, and the same region was also suggested from the nonparametric analysis of 12 families alone. Interestingly, our candidate region of approximately 65 cM between markers D3S1558 and D3S3592 on chromosome 3q13.31-q27.1 overlaps that recently identified as the most significant finding by Kemp et al. . Their rIn the analysis using the 12 new families alone, the strongest linkage (NPL = 2.3) was observed for the markers D6S976 and D6S270, and the candidate region was identified between D6S1712 and D6S1637 (30 cM). This locus was further supported in the pooled analysis using all 30 families where suggestive NPL scores for these markers were provided in nonparametric analysis. The same locus has previously been suggested from the linkage analysis using sib pairs affected with colorectal adenomas and carcinomas [No confirmation of the previously published region on chromosome 9q22.2-q31 was found. Neither was any support of linkage found when the analysis was performed using a criteria for affected status and age dependant penetrance as described by Kemp et al . In the Our data support the idea of a genetic heterogeneity among colorectal cancer families, and indicate that a further subdivision of families into groups sharing similar phenotypic and molecular features is needed. Several loci with suggestive linkage were detected in the parametric analysis performed under the assumption of locus heterogeneity as well as in the nonparametric analysis. The strongest evidence of linkage was seen to the region on chromosome 3. Interestingly, the same region has recently been reported as the most significant finding in the genome-wide analysis performed with SNP arrays by Kemp et al; thus, oThe author(s) declare that they have no competing interests.SP carried out the sequencing of the genes, performed the association studies, participated in the linkage analysis and drafted the manuscript. JV performed the linkage analysis and drafted the manuscript. SJ participated in the sequencing of the genes. TD participated in the linkage analysis. JS participated in the linkage analysis. XLZ participated in the linkage analysis. VV contributed to the study design. BV contributed to the study design. AL conceived the study, participated in its design and coordination and drafted the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:"}
+{"text": "Adiponectin is inversely associated with obesity, insulin resistance, and atherosclerosis, but little is known about the genetic pathways that regulate the plasma level of this protein. To find novel genes that influence circulating levels of adiponectin, a genome-wide linkage scan was performed on plasma adiponectin concentrations before and after 3 weeks of treatment with fenofibrate (160 mg daily) in the Genetics of Lipid Lowering Drugs and Diet Network (GOLDN) Study. We studied Caucasian individuals (n = 1121) from 190 families in Utah and Minnesota. Of these, 859 individuals from 175 families had both baseline and post-fenofibrate treatment measurements for adiponectin. Plasma adiponectin concentrations were measured with an ELISA assay. All participants were typed for microsatellite markers included in the Marshfield Mammalian Genotyping Service marker set 12, which includes 407 markers spaced at approximately 10 cM regions across the genome. Variance components analysis was used to estimate heritability and to perform genome-wide scans. Adiponectin was adjusted for age, sex, and field center. Additional models also included BMI adjustment.Baseline and post-fenofibrate adiponectin measurements were highly correlated (r = 0.95). Suggestive (LOD > 2) peaks were found on chromosomes 1p35.2 and 3q28 (near the location of the adiponectin gene).IL22RA1 and IL28RA, lie under the chromosome 1 peak; further analyses are needed to identify the specific genetic variants in this region that influence circulating adiponectin concentrations.Two candidate genes,  Adiponectin is an adipokine that is inversely related to both adiposity and many chronic disease risk factors in several populations. Adiponectin increases insulin sensitivity  when administered intravenously to rats  and has Given the relations of adiponectin with chronic disease risk factors, there is much interest in learning about the pathways through which adiponectin itself is regulated. Several studies have found circulating adiponectin to be heritable, with heritability estimates ranging from 0.42 to 0.93 -15, suggThe Genetics of Lipid Lowering Drugs and Diet Network (GOLDN) Study is a genetic family study that included a three-week trial of fenofibrate, a drug that significantly decreases triglycerides and increases high-density lipoproteins without increasing low-density lipoproteins . AlthougDetailed methods can be found in Additional File The study design and general population for the GOLDN Study have been previously described . A total2, sex, and field center. Some models were additionally adjusted for body mass index (BMI) in an attempt to reduce the proportion of variance in adiponectin due to environmental exposures. Two single-nucleotide polymorphisms  in the adiponectin gene (ADIPOQ) were genotyped using a TaqMan assay with allele-specific probes on the ABI PRISM 7900 HT Sequence Detection System  according to standardized laboratory protocols [Plasma adiponectin was quantified using an ELISA assay from R & D Systems . Comparison of 58 blind replicates embedded in study samples showed the adiponectin assay had a reliability coefficient of 0.95. All participants were genotyped using the Marshfield Mammalian Genotyping Service screening set 12, which included 407 markers spaced at approximately every 10 cM across the genome. Heritability and linkage analyses were performed using SOLAR  . All mod2). In the subset of the study population with baseline and post-trial adiponectin measurements, there was large variability in the change in adiponectin over the three-week trial of fenofibrate. The mean change in adiponectin was a decrease of 0.4 \u03bcg/ml (median decrease 0.3 \u03bcg/ml) with a standard deviation of 1.4 \u03bcg/ml. Changes over the three-week trial ranged from an increase of 5.8 \u03bcg/ml to a decrease of 6.2 \u03bcg/ml. Both transformed and non-transformed measurements of baseline and post-trial adiponectin , additional genome scans were run adjusting for the two polymorphisms. Adjustment for ADIPOQ SNP genotypes (modeled additively) attenuated the peak on chromosome 3 only slightly; adjustment for SNP rs17300539 decreased the peak LOD from 2.04 to 1.91, adjustment for SNP rs2241766 decreased the peak LOD to 1.94, and adjustment for both SNPs decreased the peak LOD to 1.90.Figure IL22RA1, at 24.3 Mb) and interleukin 28 receptor, alpha (IL28RA at 24.4 Mb), were identified. For the peak on chromosome 3q28, the -1 LOD support interval was 195 cM -217 cM (180.8 Mb \u2013 196.0 Mb) and this region contained 152 genes. One candidate gene, adiponectin (ADIPOQ at 188.0 Mb), was identified.Table In the subset of the population (n = 859) where both baseline and post-trial adiponectin were measured, the correlation of the two adiponectin measurements was high (r = 0.95). On average, adiponectin decreased slightly over the course of the trial. GOLDN is the only trial we are aware of in which adiponectin concentrations fell following treatment with fenofibrate. Earlier trials either reported non-significant increases in adiponectin ,21 or siCross-sectional correlations between adiponectin and measures of adiposity have been reported in many other studies. The correlations between adiponectin and BMI in GOLDN were somewhat low, but still within the range of correlation values reported previously (r = -0.3 to -0.5) ,25.The heritability of adiponectin has been examined previously in several large family studies -15. The ADIPOQ SNP shown to significantly attenuate an adiponectin linkage signal on chromosome 3 in an Amish population [ADIPOQ  may contribute to this linkage signal. This result mirrors the results of a linkage analysis in the IRAS Family Study were SNP rs17300539 was not found to be a major determinant of the linkage peak found on chromosome 3 [No areas of (genome-wide) significant linkage were detected in this analysis. Two areas of suggestive linkage LOD > 2.0 were detected on chromosomes 1 and 3, and several areas of modest linkage (2.0 > LOD > 1.5) were also detected. Several genome-wide linkage scans of adiponectin have been published previously -16. Comppulation ) as covamosome 3 .The suggestive peak observed on chromosome 1p35.2 and the modest peaks observed on chromosomes 6p23 and 7p14.2 are novel. The peak observed on chromosome 1p35 in GOLDN has not been reported in other adiponectin linkage scans, but peaks have been found in that location in linkage scans of BMI and type 2 diabetes. Both the Quebec Family Study and a study in the Old-Order Amish detected significant areas of linkage (p = .05 and p = .0099) on chromosome 1p35.1 for BMI ,28. As tThere are several explanations for the novel modest linkages observed on chromosomes 6p23 and 7p14.2 in this study. It is possible that there are some genetic variants that impact circulating adiponectin in GOLDN, a Caucasian group of families from Utah and Minnesota, but not in other populations in which linkage scans have been conducted. It is also possible that some genes impact concentrations of circulating adiponectin in all populations, but that the effect of the genes was not detectable in some populations due to environmental variation, small effect sizes, and/or limited statistical power. In GOLDN, participants were not permitted to take lipid-lowering drugs or dietary supplements; this uniformity in the study population may have reduced environmental variation caused by these agents which was likely present in other linkage scans. Finally, given the modest size of the LOD scores for these linkages, these signals may represent false positive findings.in vivo and in vitro by cytokines and their receptors such as IL-6 and TNF-\u03b1 [Identifying candidate genes for adiponectin is relatively difficult as little is known about upstream molecular regulators of adiponectin. Some studies have suggested that adiponectin production and secretion is regulated nd TNF-\u03b1 . Anothernd TNF-\u03b1 . FinallyIL22RA1 encodes a portion of the receptor for interleukin 22. Interleukin 22 is a cytokine involved in the acute-phase inflammatory response. The second gene, IL28RA, also encodes a portion of a cytokine receptor; IL28RA is believed to be a subunit in the receptors for interleukin 28A, interleukin 28B, and interleukin 29. If interleukin 22, 28A, 28B, or 29 regulate adiponectin, then variations in genes involved in the receptors for these cytokines may impact circulating concentrations of adiponectin. The most likely explanation for the linkage peak observed on 3q28 is the adiponectin gene itself, as it has been shown that variation in this gene influences circulating concentrations of adiponectin in many populations. No other candidate genes were found under the peak on 3q28.Two cytokine receptor genes were identified as candidate genes on chromosome 1p35. A primary strength of this study was that the study population likely had less variability in environmental exposures than other observational studies, as no individuals participating in the trial took lipid-lowering drugs or certain dietary supplements during the study period. Homogenous environmental exposures reduce the impact of gene by environment interactions which can mask linkage signals.There were limitations to this study. The results may not be widely generalizable because of the homogeneity of the study population. Additionally, no household information was collected in the GOLDN Study which prevented the use of a household (shared environment) matrix in heritability and linkage analyses. As a result, heritability may have been overestimated if variance due to common environment was mistakenly attributed to variance due to additive genetic effects.ADIPOQ.This study performed whole-genome linkage scans for circulating adiponectin before and after a three-week trial of fenofibrate. There was a very high tracking correlation for adiponectin before and after the fenofibrate trial, and it was hypothesized that any meaningful difference in the heritabilities or linkage scans for the baseline and post-fenofibrate measurements of adiponectin was due to differences between the baseline sample and sub-sample that completed the trial of fenofibrate. Adiponectin was found to be moderately heritable, and the heritability increased after adjustment for BMI. No areas of significant (LOD > 3) linkage were discovered, but there were two suggestive (LOD > 2) peaks on chromosomes 1p35.2, and 3q28. The peak on chromosome 1 corroborates areas of significant linkage for BMI and type 2 diabetes mellitus. The peak on chromosome 3 contains BMI: body mass index; GOLDN: The Genetics of Lipid Lowering Drugs and Diet Network; LOD: logarithm of the odds.The authors declare that they have no competing interests.LJR-T analyzed and interpreted the data and drafted the manuscript. JSP helped in the analysis and interpretation of the data. JSP, JMP, IBB, JEH, MYT, EKK, and DKA were involved in the design of the study and the acquisition and preparation of genetic and phenotypic data used for this manuscript. All authors participated in critical revisions of the manuscript and all have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:Detailed Materials and Methods. The text provides details on the design of the GOLDN study and additional description of the methods used in this analysis.Click here for file"}
+{"text": "Left ventricular mass (LVM) is an important risk factor for stroke and vascular disease. The genetic basis of LVM is unclear although a high heritability has been suggested. We sought to map quantitative trait loci (QTL) for LVM using large Dominican families.Probands were selected from Dominican subjects of the population-based Northern Manhattan Study (NOMAS). LVM was measured by transthoracic echocardiography. A set of 405 microsatellite markers was used to screen the whole genome among 1360 subjects from 100 Dominican families who had complete phenotype data and DNA available. A polygenic covariate screening was run to identify the significant covariates. Variance components analysis was used to estimate heritability and to detect evidence for linkage, after adjusting for significant risk factors. Ordered-subset Analysis (OSA) was conducted to identify a more homogeneous subset for stratification analysis.LVM had a heritability of 0.58 in the studied population (p < 0.0001). The most significant evidence for linkage was found at chromosome 12p11  with peak marker at D12S1042. This linkage was significantly increased in a subset of families with the high average waist circumference .We mapped a novel QTL near D12S1042 for LVM in Dominicans. Enhanced linkage evidence in families with larger waist circumference suggests that gene(s) residing within the QTL interact(s) with abdominal obesity to contribute to phenotypic variation of LVM. Suggestive evidence for linkage (LOD = 1.99) has been reported at the same peak marker for left ventricular geometry in a White population from the HyperGEN study, underscoring the importance of this QTL for left ventricular phenotype. Further fine mapping and validation studies are warranted to identify the underpinning genes. Stroke and cardiovascular diseases are of major public health concern, particularly among the rapidly growing Hispanic population . StudiesLVM is an important risk factor for stroke. In the Framingham Heart Study, the highest quartile of LVM was associated with a 2.7-fold increased risk of stroke when compared to the lowest quartile of LVM, after adjusting for age, sex, systolic blood pressure, treatment of hypertension and other established stroke risk factors . We haveAs a quantitative trait, LVM is highly heritable. In the Framingham family study the heritability of LVM was estimated to be 0.24\u20130.32.  In the HPreviously, we have published a comprehensive heritability analysis on LVM as well as adjusted LVM measurements: LVM divided by body surface area or height; relative wall thickness; LV end-diastolic diameter; LV end-systolic diameter; interventricular septum thickness; and posterior wall thickness. The estimates of heritability for these LV measurments ranged from 0.23 to 0.65 after different adjustment for covariates, with LVM demonstrating the highest heritability (0.47 to 0.65 in different models) . TherefoThe details of the family study design have been described elsewhere.  In briefDemographic, socioeconomic and risk factor data were collected through direct interview of each family member based on instruments developed in NOMAS . Extensi3 - (LVDD)3] +0.6.  +0.6. .DNA was sent to the Center for Inherited Disease Research (CIDR) for genotyping. A set of 405 microsatellite markers were genotyped at an average interval of 10 centimorgan (cM) across the genome. Autosomal microsatellite genotypes were used to verify and adjust family structure using the program PREST.  In orderHeritability was evaluated using a pedigree-based maximum-likelihood method implemented in SOLAR.  VarianceTo decide the covariates that should be included in estimating heritability and calculating LOD scores, a polygenic covariate screening implemented in SOLAR was run to screen age, sex, smoking, diabetes, dyslipidemia, hypertension, and body mass index (BMI). Interaction between age and sex was automatically included in SOLAR. A permissive threshold of p < 0.1 was used for this evaluation to allow for inclusion of any potentially significant covariates. Hypertension was defined as reported history of high blood pressure, systolic blood pressure \u2265 140 mmHg, diastolic blood pressure \u2265 90 mmHg, or use of antihypertensive medication. Smoking was defined as never versus ever. Dyslipidemia was defined as a history of hyperlididemia or total cholesterol greater than 240 mg/dL.For the OSA, trait-related quantitative covariates were used to rank families for a more homogeneous subset . LOD scoOverall, 110 families with high vascular disease burden  were enrolled in our family study. Seventy percent of the subjects were enrolled in Northern Manhattan and 30% in the Dominican Republic. In order to reduce population stratification, we restricted our QTL mapping to the 100 Dominican families, which was composed of 2182 individuals. The mean family size was 22 \u00b1 11 with median of 20, and range of 4\u201387. LVM measurements and DNA samples were available for 1360 subjects from the 100 Dominican families. Among the 1360 individuals, there were 1207 sib pairs, 362 half-sib pairs, and 1713 avuncular pairs. With our dataset, we had over 80% power to detect QTLs for traits with heritability estimates greater than 0.18 at a LOD score threshold of 2.0. The mean age of the study cohort was 46 years. The mean LVM was 174.85 \u00b1 56.14 , and BMI as significant covariates. Heritability and LOD scores were calculated adjusting for these covariates. The significant covariates together explained 0.33 of the total LVM variance. The heritability estimate of LVM is 0.58 (p < 0.0001) for the remaining variance of LVM after adjusting for the significant covariates.To localize the genetic loci affecting LVM variations, we completed a QTL mapping on LVM. This analysis revealed a significant region on chromosome 12p11 with peak marker at D12S1042   as the covariates to order families because high blood pressure and obesity, especially elevated waist circumference (WC), are major risk factors that lead to an increase of LVM in general population -24. We oUsing well-characterized, extended Dominican Republic families, we mapped a novel QTL for LVM on chromosome 12p11. This QTL influences LVM independent of other risk factors such as gender, age, hypertension, BMI, and smoking. By implementing OSA to reduce phenotype heterogeneity, we have greatly strengthened the evidence for linkage, narrowed the linkage region and identified a sub-phenotype that has concentrated genetic effects from chromosome 12p11. Our findings provide a well-defined chromosome region and phenotype for future validation and fine mapping studies to localize genes controlling LVM.The majority of genetic studies on LVM phenotype to date have focused on candidate genes derived from biological pathways that have impact on the LVM phenotype, such as genes implicated in myocardial cell growth/remodeling, calcium homeostasis, extracellular matrix, hemodynamic load, and blood pressure regulation. Polymorphisms in several genes have been associated with LVM, such as the 825C/T polymorphism in the G protein beta subunit (GNB3) gene,  polymorpa priori knowledge on the molecular basis of phenotypic variations and have advantages in identifying unknown/unexpected genes and pathways for complex traits. Previously, two genome-wide studies of LVM as a quantitative trait have been reported. Both of them were conducted in Caucasian populations [-5 were found on chromosomes 2, 6, and 11. One SNP is located within a heat shock protein gene HSPA8; the other two SNPs are not located in or near known genes. Linkage analysis revealed strong evidence for linkage on chromosome 5q (MLOD = 4.4) and suggestive evidence for linkage on chromosomes 1q and 8q (MLOD = 2.4).Other approaches to identify the underpinning genes for a disease-associated trait are unbiased genomic approaches, such as genome-wide linkage and association studies. These studies do not require ulations ,35. One ulations . In the There is no overlap among the QTLs delimited in the British, the Framingham Heart, and the current studies. One possible explanation is that the unique ethnic population in our study may have a different genetic basis for LVM than Caucasians analyzed in the other two studies. However, the lack of replication even between the British and Framingham studies as well as the conflicting candidate gene studies, underscore the challenges in genetic analysis of complex traits: the genetic basis is likely to be complex and heterogeneous, involving multiple genes each with moderate effect and interaction with environmental factors. Within a given dataset, only one or two loci with the strongest effect in that population can be detected. In our study, the 12p11 QTL only accounts for less half of the total heritability of LVM. Other loci that have smaller effect sizes are still at large due to the limited power of the current dataset. Future studies in a similar population, including meta analyses are needed to catalogue all the genetic causes for the inter-individual LVM variations.To reduce heterogeneity, stratification analysis based on a trait-related variable has been proposed in genetic studies of complex traits. The OSA ranks families by a trait-related variable and sequentially introduces the families to the linkage analysis until the maximum evidence of linkage is observed . The subUsing OSA, we found that the linkage signal on chromosome 12p11 is mainly from families with high WC. To exclude the possibility that we mapped a locus for WC instead of LVM, we performed a linkage analysis on WC and found no evidence suggesting that the signal on 12p11 is driven by WC (LOD = 0 at D12S1042 for WC). Therefore, the 12p11 linkage is a bona fide QTL for LVM and genes under this QTL interact with visceral obesity to influence inter-individual LVM variations. Adipose tissue is an active organ that secretes a variety of bioactive peptides. Several proteins of the renin angiotensin system, an essential regulatory axis for blood pressure, are expressed in adipose tissue.  Among thThe OSA based on BMI did not significantly enhance the linkage evidence despite that BMI and WC are correlated measurements, suggesting that WC provided additional information to BMI in the analysis of LVM. Consistently, recent reports from Rodrigues et al.  and TsioIt is worth noting that in the HyperGEN study suggestive evidence for linkage (LOD = 1.99) for LV geometry was found at the same peak marker (D12S1042) for LV mass in our study . LV geomOur success in mapping a strong QTL relies on the strength of our study design. We focused on one ethnic group and used a family study design to minimize the effects of population substructure. All the families have high vascular disease burden and detailed systematic measurements of the quantitative phenotype. The extended family structure (average family size of 20) in our dataset provides unprecedented information content and statistical power. The QTL mapping approach also offers additional statistical advantages over discrete traits. A major limitation of our study is that we did not pinpoint the causal genetic variations that lead to increased LVM. This study is only the first stage to elucidate the genetic basis for LVM. Looking through all the genes under the 12p11 linkage peak did not find any gene that would have an obvious effect on LVM based on the current knowledge. However, the molecular basis of increased LVM is not clear and many genes' functions are not fully understood. We plan to perform fine mapping study across the linkage peak to narrow the critical region and identify the candidate gene in future studies.Most genetic studies of cardiovascular disease have focused on non-Hispanic populations -46 with With the unbiased genome-wide approach, we mapped a novel QTL for LVM, a significant risk phenotype for stroke and vascular events. The strong evidence for linkage on chromosome 12p11 warrants further validation and fine mapping efforts. In addition, our family study provides unique data on the genetics of cerebrovascular risk phenotypes in an understudied minority population.The authors declare that they have no competing interests.LW: planned the analysis, interpreted data, draft the manuscript. AB: analyzed the data and participated in the interpretation of data. MRD: ascertained and phenotyped the subjects, assisted in study design, revised the manuscript for important intellectual content. SS: designed the analysis, analyzed the data, and participated in the interpretation of data. SHB: designed the analysis, interpreted data, and critically revised the manuscript. TR: ascertained and phenotyped the subjects, assisted in study design, and critically revised the manuscript. RS: conceived the overall study, secured funding, and critically revised the manuscript. All authors have read and approved the content of the manuscript.The pre-publication history for this paper can be accessed here:"}
+{"text": "Here, we have investigated the inheritance of the scurs phenotype in the French Charolais breed and examined whether the previously proposed localisation of the scurs locus on bovine chromosome 19 could be confirmed or not.Polled animals are valued in cattle industry because the absence of horns has a significant economic impact. However, some cattle are neither polled nor horned but have so-called scurs on their heads, which are corneous growths loosely attached to the skull. A better understanding of the genetic determinism of the scurs phenotype would help to fine map the polled locus. To date, only one study has attempted to map the scurs allele (Sc) reaches 69.9% in the French Charolais population. Eleven microsatellite markers on bovine chromosome 19 were genotyped in 267 offspring . Both non-parametric and parametric linkage analyses suggest that in the French Charolais population the scurs locus may not map to the previously identified region. A new analysis of an Angus-Hereford and Hereford-Hereford pedigree published in 1978 enabled us to calculate the frequency of the Sc allele in the Hereford breed (89.4%) and to study the penetrance of this allele in males heterozygous for both polled and scurs loci (40%). This led us to revise the inheritance pattern of the scurs phenotype proposed for the Hereford breed and to suggest that allele Sc is not fully but partially dominant in double heterozygous males while it is always recessive in females. Crossbreeding involving the Charolais breed and other breeds gave results similar to those reported in the Hereford breed.Our results indicate that the inheritance pattern of the scurs phenotype in the French Charolais breed is autosomal recessive with complete penetrance in both sexes, which is different from what is reported for other breeds. The frequency of the scurs locus in double heterozygous Hereford and Angus males. The specific inheritance pattern of the scurs locus in the French Charolais breed represents an opportunity to map this gene and to identify the molecular mechanisms regulating the growth of horns in cattle.Our results suggest the existence of unknown genetics factors modifying the expression of the  To explain this situation, the authors propose the putative existence of a new gene that would be epistatic to the scurs gene. Kr\u00e4u\u00dflich and R\u00f6hrmoser ). IDVGA46 was discarded since it could not be amplified in our PCR conditions. PCR primers were designed with Primer 3 [Eleven microsatellites located on BTA19 were genotyped in this study. BMS1920, CSSME070, BMS2142, IDVGA46, BP20 and BMS2389 were chosen based on their chromosomal localization in a region 25.6 cM long and containing the etic map ,18 , using the Multiplex PCR kit  according to the manufacturer's recommendations, on a PTC-100 thermocycler . The resulting PCR products were purified on Sephadex G50 columns  before running on a MegaBACE 96 capillaries sequencer . Raw data were then analyzed using Genetic Profiler v1.5 (Molecular Dynamics).For linkage analysis, we used a map based on the bovine sequence assembly Btau_4.0, assuming 1 Mb equivalent to 1 cM. Marker order and map distances were validated using CRIMAP 2.4 software . The FLINon-parametric linkage (NPL) analysis and parametric linkage analysis  were performed using GENEHUNTER software . In caseAC participated in the design of the study and in data acquisition, performed animal genotyping and statistical analysis, interpreted the results and drafted the manuscript. CG extracted the DNA samples, participated in data acquisition and animal genotyping and revised the manuscript. MG participated in statistical analysis and revised the manuscript. AE conceived and coordinated the study and revised the manuscript. All authors read and approved the final manuscript."}
+{"text": "The incorporation of disease-associated covariates into studies aiming to identify susceptibility genes for complex human traits is a challenging problem. Accounting for such covariates in genetic linkage and association analyses may help reduce the genetic heterogeneity inherent in these complex phenotypes. For Genetic Analysis Workshop 15 (GAW15) Problem 3 simulated data, our goal was to compare the power of several two-stage study designs to identify rheumatoid arthritis-related genes on chromosome 9 (disease severity), 11 (IgM), and 18 (anti-cyclic citrinullated protein), with knowledge of the answers. Five study designs incorporating an initial linkage step, followed by a case-selection scheme and case-control association analysis by logistic regression, were considered. The linkage step was either qualitative-trait linkage analysis as implemented in MERLIN-nonparametric linkage (NPL), or quantitative-trait locus analysis as implemented in MERLIN-REGRESS. A set of cases representing either one case from each available family, one case per linked family (NPL \u2265 0), or one case from each family identified by ordered-subset analysis was chosen for comparison with the full set of 2000 simulated controls. As expected, the performance of these study designs depended on the disease model used to generate the data, especially the simulated allele frequency difference between cases and controls. The quantitative trait loci analysis performed well in identifying these loci, and the power to identify disease-associated alleles was increased by using ordered-subset analysis as a case selection tool. There are many possible mechanisms by which environmental or clinical covariates may either influence the risk of complex human diseases directly, or partially account for genetic heterogeneity. For example, they may act as independent environmental risk factors, or increase the disease risk in concert with genetic susceptibility via gene \u00d7 environment interaction, or define a more homogeneous subgroup of patients in which the main effect of a particular susceptibility gene is more apparent. The purpose of our analysis of the Genetic Analysis Workshop 15 (GAW15) simulated data was to evaluate the power of several two-stage study designs consisting of separate linkage and association analysis steps. These study designs incorporated disease-associated continuous covariates in several different ways. Power comparisons were focused on two distinct factors: 1) thresholds used for the linkage analysis step, which determined the subset of markers included in a subsequent case-control association analysis, and 2) criteria for selecting cases (one per family) to include in this association analysis.With knowledge of the answers, we analyzed the simulated GAW15 microsatellite and SNP data on chromosomes 9, 11, and 18 in an attempt to detect the loci responsible for three disease-associated covariates: disease severity, IgM, and anti-cyclic citrinullated protein (CCP) values, respectively. To investigate Type I error, we also analyzed the relationship of anti-CCP values and genotypes on chromosome 15, which does not harbor any disease-associated loci (\"null chromosome\"). We analyzed covariate and genotype data from all 1500 nuclear families, and genotype data from all 2000 unrelated controls. We used the MERLIN package  and the p-value threshold 0.05, 10-cM region centered on the linkage peak) and loose . Each design consisted of two stages. If the first-stage linkage analysis of 1500 families using the microsatellite marker map met the linkage threshold, it was followed by a second-stage association analysis of the SNPs in the linkage region in unrelated cases (one per family) and 2000 controls, using logistic regression with an additive allele coding. The case selection strategies are summarized in Table p-value from the logistic regression survived the Bonferroni correction for the number of analyzed markers.We examined five distinct study designs Table , each ofTable p-value thresholds affect the type I error rate of the Stage 2 association analysis.It was previously shown that linkage and association test statistics are statistically independent under the null hypothesis of i) no linkage and no association; ii) linkage and no association; iii) association and no linkage . ConsistOur study demonstrates that the incorporation of disease-related covariates into a combined linkage and association analysis can help identify genes that contribute directly or indirectly to the risk of RA. Specifically, results for chromosome 18 show that the efficiency of a case-control association analysis can be greatly increased when linkage and covariate information are used to select the cases. For the simulation models used to generate the GAW15 data, the OSA method worked particularly well in this regard because it uses both the family-specific identify-by-descent (IBD) sharing information, and the relationship between covariate distribution and IBD sharing across families to enrich the case sample for the disease allele of interest. For the data sets simulated here, the \"linked best\" strategy (Design B in our study) was able to achieve the exact same power as Design A with a 34% reduction in the number of analyzed cases, even though it ignored covariate information  difference between cases and controls and almost complete LD with the causal allele  resulted in improved power for the association analysis (from near 0 to 20\u201330%). However, because OSA used family-specific NPL scores for the binary affection status as input, regardless of disease severity, the overall power of Design E remained low (16% at best). A family-based association analysis of disease severity with the QTDT (quantitative transmission-disequilibrium test) package  or a logp-value threshold, improved power for chromosome 18, and to a lesser extent chromosome 11, since it identified a subgroup of cases with reduced allelic heterogeneity, even though the linkage evidence in this subgroup continued to be low. In real data sets, a SNP map of the density simulated here is unlikely to include SNPs in high enough LD with susceptibility or quantitative trait loci to detect strong association signals, and the two-stage approach presented here continues to be of practical importance.The GAW15 data provided very weak linkage signals for the three loci considered here, presumably due to substantial within-family heterogeneity with respect to the simulated disease loci Table . This maThe author(s) declare that they have no competing interests."}
+{"text": "Chronic diseases affecting the inner ear and the retina cause severe impairments to our communication systems. In more than half of the cases, Usher syndrome (USH) is the origin of these double defects. Patients with USH type II (USH2) have retinitis pigmentosa (RP) that develops during puberty, moderate to severe hearing impairment with downsloping pure-tone audiogram, and normal vestibular function. Four loci and three genes are known for USH2. In this study, we proposed to localize the gene responsible for USH2 in a consanguineous family of Tunisian origin.Affected members underwent detailed ocular and audiologic characterization. One Tunisian family with USH2 and 45 healthy controls unrelated to the family were recruited. Two affected and six unaffected family members attended our study. DNA samples of eight family members were genotyped with polymorphic markers. Two-point and multipoint LOD scores were calculated using Genehunter software v2.1. Sequencing was used to investigate candidate genes.Haplotype analysis showed no significant linkage to any known USH gene or locus. A genome-wide screen, using microsatellite markers, was performed, allowing the identification of three homozygous regions in chromosomes 2, 4, and 15. We further confirmed and refined these three regions using microsatellite and single-nucleotide polymorphisms. With recessive mode of inheritance, the highest multipoint LOD score of 1.765 was identified for the candidate regions on chromosomes 4 and 15. The chromosome 15 locus is large (55 Mb), underscoring the limited number of meioses in the consanguineous pedigree. Moreover, the linked, homozygous chromosome 15q alleles, unlike those of the chromosome 2 and 4 loci, are infrequent in the local population. Thus, the data strongly suggest that the novel locus for USH2 is likely to reside on 15q.Our data provide a basis for the localization and the identification of a novel gene implicated in USH2, most likely localized on 15q. MYO7A, USH1C/USH1C, USH1D/CDH23, USH1F/PCDH15, USH1G/SANS, USH2A/USH2A, USH2C/VLGR1, USH2D/WHRN, and USH3A/USH3A (Usher homepage). Mutations in USH2 genes can also manifest as atypical USH [Usher syndrome (USH) is an autosomal recessive disorders characterized by sensorineural hearing impairment (HI), retinitis pigmentosa (RP), and variable vestibular dysfunction . It is cical USH , or as nIn this study, we investigated a Tunisian family with USH2. This family originates from centre of Tunisia. Two affected  and six healthy family members  attended our study. We also recruited 45 controls  from different regions of Tunisia. Written informed consent was obtained from both parents, in accordance with the ethics committee of the University Hospital of Sfax. The pedigree was obtained upon interviews with parents . ClinicaUsher homepage) at least three microsatellite markers were selected on the basis of their map position (UCSC Genome Browser) and heterozygosity coefficient . Fluorescent dye-labeled microsatellite markers were genotyped for all the participating family members. Furthermore, a genome-wide scan was performed using 400 fluorescent dye-labeled microsatellite markers with an average spacing of approximately 10 cM . We used the True Allele PCR Premix (Applied Biosystems) for PCR reactions according to the manufacturer\u2019s instructions. Fluorescently labeled alleles were analyzed on an ABI Prism 3100-Avant automated DNA sequencer (Applied Biosystems).For each gene and locus responsible for USH  spanning the ceramide kinase-like gene (CERKL) to check their cosegregation with the disease before proceeding to mutation screening. Primers and conditions were previously described [We also analyzed by direct sequencing three single nucleotide polymorphism (SNP) markers , D4S402 (4q26), D15S978 (15q21.1), and D15S117 (15q22.1). Additional markers were genotyped in all three regions to define the critical intervals. The homozygous region in chromosome 2 was delimited by two informative markers rs1002207 and D2S311; the chromosome 4 region was bordered by D4S1572 and D4S2938; and the chromosome 15 region stretched from D15S1039 to D15S120 -stimulated blood culture using standard procedures. Chromosome analysis of patient BT189 showed normal karyotype (data not shown).To localize the causative gene, we performed linkage analysis using polymorphic microsatellite markers bordering all described  D15S120 . Analysie family . InvestiGenehunter software. Maximum LOD scores (1.765 at \u03b8=0) were identified for the candidate regions on chromosome 4 between D4S2989 and D4S2975, and chromosome 15 between D15S978 and D15S1036. On chromosome 2, a maximum LOD score of 1.51 was found for D2S117 microsatellite marker.We genotyped markers located in the three candidate regions in 40 healthy unrelated Tunisian individuals for more accurate estimation of allele frequencies and to determine the best candidate region. In the first region, we analyzed four markers . In the first region, homozygous alleles were predominantly present in the population and the allele frequencies were 0.35 (D2S148), 0.125 (D2S384), 0.311 (D2S364), and 0.203 (D2S117). For the second region, three markers were analyzed and the frequencies of linked alleles were as follows: 0.025 for D4S2989, 0.122 for D4S402, and 0.125 for D4S2975. In contrast, the homozygous alleles of the chromosome 15 region were not frequent in controls. Allele frequencies of the polymorphic markers D15S992, D15S978, D15S117, and D15S1036 were assumed to be 0.05, 0.05, 0.1, and 0.075, respectively. These results suggest that the disease locus is most probably on chromosome 15. Multipoint LOD scores were calculated for the family data using UCSC Genome Browser). More than 100 candidate genes in these three regions are expressed in the inner ear and in the retina. Although the region on chromosome 2 was not the best candidate locus , we chose to investigate the CERKL gene, encoding a ceramide kinase, as candidate since it has been described to cause nonsyndromic autosomal recessive RP (RP26) [USH2A are responsible for USH2 as well as nonsyndromic recessive RP [rs1157595 and rs155100) genotyping was compatible with linkage of the CERKL gene by cosegregation and homozygosity criteria (rs1002207 (C/T), which was located at 0.8 Mb from CERKL gene. We screened this gene for mutations. Two affected children (BT188 and BT189) were compound heterozygous for two novel variants  . The basssive RP . SNP  mapped to 15q23-q25.1 in five large Pakistani families [MYO1E, was selected. Myosins are motor proteins that hydrolyze ATP and translocate along actin filaments [MYO7A have been reported essentially in families with USH1 but also can lead to atypical USH [MYO1E is a member of a Myosin I isozyme which are essential for hair cells, the sensory cell of inner ear. All eight Myosin I isozymes are expressed in rodent auditory and vestibular epithelia. Three Myosin I isosymes Myo1b, Myo1c, and Myo1e, are expressed at birth in cochlea and vestibular organs. In mouse, Myo1e is expressed in hair cell of the auditory and vestibular epithelia. [MYO1E gene were sequenced in patients and were found to be negative for functional sequence variants.This consanguineous Tunisian family displayed no evidence of linkage to any known USH locus. A genome-wide genotyping was performed and revealed three homozygous regions on chromosomes 2q31.3\u201333.1, 4q24\u201328.2, and 15q21\u201315qter. The highest LOD scores were identified for the regions on chromosome 4 and 15. The determination of population frequencies of the homozygous alleles of each linked marker in these three regions showed that only the homozygous alleles of chromosome 15 were rarely present in 40 control Tunisian individuals. More controls (45) were used to check for the novel variant on families . Among iilaments . Indeed,ilaments . Mutatioilaments -23. MutaAs the chromosome 15 interval is large and no more information can be obtained from this family to reduce the size of this locus, other families with USH2, even if small, would be useful to identify the novel gene."}
+{"text": "Pedigree studies of complex heritable diseases often feature nominal or ordinal phenotypic measurements and missing genetic marker or phenotype data.We have developed a Bayesian method for Linkage analysis of Ordinal and Categorical traits (LOCate) that can analyze complex genealogical structure for family groups and incorporate missing data. LOCate uses a Gibbs sampling approach to assess linkage, incorporating a simulated tempering algorithm for fast mixing. While our treatment is Bayesian, we develop a LOD (log of odds) score estimator for assessing linkage from Gibbs sampling that is highly accurate for simulated data. LOCate is applicable to linkage analysis for ordinal or nominal traits, a versatility which we demonstrate by analyzing simulated data with a nominal trait, on which LOCate outperforms LOT, an existing method which is designed for ordinal traits. We additionally demonstrate our method's versatility by analyzing a candidate locus (D2S1788) for panic disorder in humans, in a dataset with a large amount of missing data, which LOT was unable to handle.LOCate's accuracy and applicability to both ordinal and nominal traits will prove useful to researchers interested in mapping loci for categorical traits. Many heritable traits, from pathogen resistance in plants et al.et al.Most previous work done on family-based mapping of categorical traits has been restricted to particular types of pedigrees; these include backcross et al., which is designed for ordinal traits. We further demonstrate the versatility of our method by reanalyzing a study of panic disorder in humans previously analyzed as a binary trait In this paper, we develop a Bayesian statistical framework for linkage analysis of a categorical trait with a user-specified penetrance function of arbitrary form. We implement this framework in the software LOCate . Our method can analyze an ordinal or nominal trait with any number of categories, can handle missing genotype and phenotype data, and can analyze pedigrees with inbreeding loops. We demonstrate the versatility of our method's user-specified penetrance function through analysis of simulated pedigrees with a nominal trait, and find that our method outperforms LOT X consists of individuals' phenotypes and unphased marker genotypes, and the unobserved data Y consists of all individuals' disease locus and phased marker genotypes, as well as any unobserved phenotypes and unphased marker genotypes. As the number of individuals in the family increases, the sum over all possible genotype assignments Y can grow unwieldy. Instead of considering all possible values of Y, Gibbs sampling is used to randomly explore the space of genotype configurations, emphasizing those configurations Y which have the highest values of In our linkage analysis framework, we seek the probability of a pedigree conditional on X) and unobserved data (Y), conditional on the recombination rate i received from its father and mother; i received from its father and mother; i received the grandpaternal or grandmaternal allele from each parent at the disease locus and the marker, respectively; i's observed, unphased marker genotype; i's phenotype; and penetrance refers to the matrix of HWE refers to the genotype frequencies assuming the founders are drawn from a population under Hardy-Weinberg Equilibrium.Y in proportion to the probability in Equation 1. In our Bayesian implementation, we used a uniform prior on the marker genotypes of individuals with missing data. We also used We derived a Gibbs sampler to sample genotype configurations i's marker alleles and selectors i's father in the current iteration.The Gibbs sampler updates each set of variables conditional on its Markov blanket Here,i's offspring is analogous to this. If individual i's parents are not included in the pedigree, then i is a founder, and m is the number of distinct marker alleles.Also,Slow mixing is a chronic problem in Gibbs samplers for linkage analysis To ameliorate this problem, we implemented simulated tempering ency see . This grency see  and timeency see  of our GY. The LinReg estimator of While results of an analysis using our framework may be interpreted entirely from a Bayesian perspective by assuming a prior over the grid values of We assessed the performance of our method using two sets of simulated data. First, we tested the accuracy of LOD score estimation for single, small simulated pedigrees. Since any errors that occur in the analysis of one pedigree will be multiplied when multiple pedigrees are aggregated in a typical linkage analysis study, it is important that our method perform accurately when only a small amount of data is available. The simulated pedigrees included from 4 to 18 individuals; some examples are shown in For our second set of simulations, we assessed the ability of our method to detect linkage in cases where the pedigree(s) may be reasonably broken into a large number of small family groups or where the study includes a large number of small families. For these simulations, we considered linkage studies of 100 families, each family consisting of 2 parents and 2 offspring. We simulated a trichotomous trait with penetrances as given in et al.et al. analyzed these data using ANALYZE Panic disorder is a common illness in humans, characterized by periods of intense anxiety. Because individuals exhibit varying degrees of symptoms of panic disorder, this psychiatric illness is a natural choice for analysis as an ordinal trait. We used LOCate to perform trichotomous linkage analysis on the panic disorder data set of Fyer is model .et al. We varied et al., to  in model D, to represent a disease which is much more penetrant when individuals with \u201cany symptoms\u201d are included as affected.In each of the variations, we used a low (.01 or .1) phenocopy rate, similar to the .01 rate used in Fyer et al. study. Nine additional pedigrees had some observed genotypes, but were uninformative due to lack of variation in the observed phenotypes or genotypes. These pedigrees were dropped from our analysis, leaving 1332 individuals in 104 families. Of these, 35 families, ranging in size from 4 to 10 individuals, could be analyzed in LOCate on 1.7 GB-memory instances on the Amazon cloud Seven pedigrees had no observed phenotypes, due to having been collected for a different phase of the Fyer n is the number of penetrance models; in this case, the threshold is Using LOCate, we first analyzed a reduced set of 96 subfamilies to compare 4 trichotomous penetrance models , and theWe also attempted to analyze the cut pedigrees using LOT, but found that LOT froze during this analysis. Test analyses with simulated phenotypes on the same pedigree structures revealed that this was due to the large proportion (32%) of individuals with unobserved phenotypes.We compared our LinReg estimator to the Reverse Logistic Regression (RLR) estimator of Geyer ed error . Given tinverseNormal option was also not effective in improving the fit of the continuous model.LOCate accurately estimated LOD curves for individual simulated pedigrees with binary traits  and tricWe present the results of our analysis of simulated 100-family linkage studies in The LOD scores produced by LOCate under the binary and trichotomous analyses are shown in et al.'s binary HLOD(.2)\u200a=\u200a3.20 on the uncut pedigrees. However, the very negative LOD scores under the trichotomous model are surprising, and give evidence that the trichotomous penetrance matrix in It is clear that the necessary pedigree cutting had an effect on our results, as we found a binary HLOD(.2)\u200a=\u200a1.85, compared to Fyer Bayesian methods for linkage analysis are useful because they allow for incorporation of prior information about allele frequencies, meiotic drive, and other factors important to linkage calculations. This, along with LOCate's versatility for ordinal and nominal traits, makes our method a valuable complementary tool to existing frequentist methods.K, the number of populations represented by an observed sample of genotypes.Even in a Bayesian framework, it is desirable to have a means of computing LOD scores, as they are commonly used to assess linkage. We developed a new, linear-regression based estimator for The choice of a penetrance model is an important question in any parametric linkage analysis, and this choice becomes even more challenging when analyzing categorical traits, as the number of possible penetrance matrices increases with the number of levels of the trait. An important distinction in the choice of penetrance matrices for categorical traits is whether the model should be ordinal or nominal. LOT estimates penetrances according to an ordinal model; this gives it an advantage for researchers who are confident their trait follows an ordinal model, but who do not wish to estimate the penetrances in advance. In contrast, LOCate is flexible to both ordinal and nominal penetrance models, but requires the penetrances to be estimated in advance. As we have done in this paper, these can be estimated on the basis of previous estimates of the phenocopy rate and overall penetrance of the trait. As our simulations demonstrate, LOCate exhibits better power than LOT when used to analyze a nominal trait, even when the input penetrance matrix is only a rough estimate. This robustness mitigates the importance of exactly estimating the penetrance matrix, and makes LOCate a valuable alternative method for researchers who wish to test penetrance models that do not have the ordinal proportional-odds property.Due to LOCate's computational intensiveness, our simulation study was limited in scope. We believe our simulations establish LOCate as a valuable complementary approach for linkage analysis of categorical traits, particularly nominal traits. We are currently developing extensions to increase the computational speed of LOCate, which will enable a more extensive range of simulations to compare LOCate's performance to LOT on a variety of ordinal and nominal traits with varying amounts of missing data and inbreeding.et al., demonstrates that modeling genetic contributions to categorical traits is not a simple matter of applying a few modifications to existing binary models. Further investigation of panic disorder as an ordinal trait is needed, to establish more complete bounds on the range of possible penetrance models. In addition, further methods development, such as a Bayesian treatment of the penetrance matrix, would enable us to analyze categorical traits without specifying the penetrance matrix in advance.We further demonstrated the versatility of our method through a trichotomous linkage analysis of a dataset of humans affected by panic disorder with a large proportion of missing data. By splitting the most memory-intensive pedigrees into nuclear families, we were able to analyze the dataset using LOCate, while LOT was unable to process the large proportion of individuals with missing phenotypes. In this particular application, it was interesting to note the very negative LOD scores produced in the trichotomous analysis, while the binary analysis on the same set of subpedigrees had positive LODs. This demonstrates that the trichotomous model in https://sourceforge.net/projects/categorical. LOCate is an effective and versatile approach for single marker analysis of nominal, ordinal, and binary traits on arbitrary family-sized pedigrees, including those with inbreeding loops and missing phenotypes and/or genotypes. While our method currently has scaling limitations for larger pedigrees, we are developing extensions for LOCate that make use of variable elimination to make the method available for multimarker analysis as well as the analysis of arbitrarily sized linkage studies.We have implemented our method in the software LOCate, available at Text S1Equations used in variable updates, details of simulated tempering, and parameters used in other software.(0.06 MB PDF)Click here for additional data file.Figure S1Examples of simulated pedigrees. Black\u200a=\u200aaffected; white\u200a=\u200aunaffected; gray\u200a=\u200amoderately affected. Each individual's unphased marker genotype is listed below the individual. A, B, and D are examples of simulated pedigrees with binary traits; C shows a simulated pedigree with a trichotomous trait and an inbreeding loop. Question marks in B indicate missing genotype data.(1.74 MB TIF)Click here for additional data file.Figure S2Estimated LOD curves for simulated pedigrees with binary traits. Our method (red) and Superlink (black) give nearly identical results.(7.32 MB TIF)Click here for additional data file.Figure S3Treating trichotomous traits as binary. When our method is run on simulated pedigrees with a 3-level categorical trait, the LOD curve estimate (red) is a good fit to the theoretical LOD curve (black). When the categorical trait is treated as binary, the LOD curve estimates (from Superlink) are a much poorer fit (blue).(7.70 MB TIF)Click here for additional data file.Figure S4LOD scores from simulated linkage studies. Red bars show the frequency of LOD scores for simulations with a linked QTL; black bars show the frequency for simulations with an unlinked QTL. Both penetrance models have good distinguishing power, but the LOD scores under the inaccurate model C have a smaller range.(0.40 MB TIF)Click here for additional data file.Figure S5i)P and i+k)P. Without simulated tempering (black line), distantly separated iterations of the Gibbs sampler remain highly correlated. With simulated tempering, the autocorrelation reaches near-independence  for k>15, demonstrating improved mixing of the Gibbs sampler.Lag-k autocorrelation with and without simulated tempering. We show the correlation between (0.21 MB TIF)Click here for additional data file.Figure S6Gelman-Rubin statistics for the likelihood of a simulated pedigree. Without simulated tempering (blue bars), the Gelman-Rubin statistics are significantly greater than 1, indicating that the chains have not reached stationarity, at a burn-in of 64,000 iterations. With simulated tempering (red bars), a burn-in of 1,000 iterations is sufficient to achieve Gelman-Rubin statistics very close to 1.(3.49 MB TIF)Click here for additional data file.Table S1Additional trichotomous penetrance models used to analyze Panic Disorder data. We tested each of these models on 96 subfamilies, as discussed in the (0.01 MB PDF)Click here for additional data file."}
+{"text": "In a previous study of the Hypertension Genetic Epidemiology Network (HyperGEN) we have shown that metabolic syndrome (MetS) risk factors were moderately and significantly associated with echocardiographic (ECHO) left ventricular (LV) phenotypes.The study included 1,393 African Americans and 1,133 whites, stratified by type 2 diabetes mellitus (DM) status. Heritabilities of seven factor scores based on the analysis of 15 traits were sufficiently high to pursue QTL discovery in this follow-up study.cadherin 13 gene, implicated in heart and vascular remodeling.Three of the QTLs discovered relate to combined MetS-ECHO factors of \"blood pressure (BP)-LV wall thickness\" on chromosome 3 at 225 cM with a 2.8 LOD score, on chromosome 20 at 2.1 cM with a 2.6 LOD score; and for \"LV wall thickness\" factor on chromosome 16 at 113.5 with a 2.6 LOD score in whites. The remaining QTLs include one for a \"body mass index-insulin (BMI-INS)\" factor with a LOD score of 3.9 on chromosome 2 located at 64.8 cM; one for the same factor on chromosome 12 at 91.4 cM with a 3.3 LOD score; one for a \"BP\" factor on chromosome 19 located at 67.8 cM with a 3.0 LOD score. A suggestive linkage was also found for \"Lipids-INS\" with a 2.7 LOD score located on chromosome 11 at 113.1 cM in African Americans. Of the above QTLs, the one on chromosome 12 for \"BMI-INS\" is replicated in both ethnicities, (with highest LOD scores in African Americans). In addition, the QTL for \"LV wall thickness\" on chromosome 16q24.2-q24.3 reached its local maximum LOD score at marker D16S402, which is positioned within the 5th intron of the Our previous study and this follow-up suggest gene loci for some crucial MetS and cardiac geometry risk factors that contribute to the risk of developing heart disease. Metabolic Syndrome (MetS), a cluster of obesity, insulin resistance and glucose intolerance, dyslipidemia, and high blood pressure, is related to echocardiographic (ECHO) measurements of the heart. For example, left ventricular hypertrophy (LVH) is a complex trait that is a major manifestation of target organ damage in hypertension . MetS anUntil recently, different studies have reported QTLs for MetS or ECHO. Teran-Garcia and Bouchard  provide Another recent publication of Mayosi et al  performeThe present study extends previous investigations by focusing concurrently on both MetS risk components and ECHO phenotypes. In addition, we expect an increase in power for QTL discovery by utilizing FA factors, because they capture inter-variable correlations. In this study we aimed to identify QTLs that control genetic variability of combined MetS-ECHO factors, as well as individual MetS and ECHO factor domains in the Hypertension Genetic Epidemiology Network Study (HyperGEN).HyperGEN is part of the NHLBI Family Blood Pressure Program (FBPP), which studies the genetic aspects of high blood pressure and related conditions . FurtherEchocardiograms followed standard protocols as described by Devereux and Roman  to obtai2.7 (LVMI), aortic root diameter (ARD), arterial stiffness defined by pulse pressure/stroke volume (PP/SV), and LV midwall shortening (MWS) underwent appropriate distribution transformations and removal of only a few outliers to achieve normality. Log transformation was applied to INS, HDLC, TG, BMI, RWT, LVMI, PWT, and PP/SV; the reciprocal of squared GLUC (1/GLUC2) and cubic power transformation for MWS (MWS3) were applied in both African Americans and whites  variables named factors. The latent factors help to identify inter-correlations among primary variables. Two categories of factor analysis, with and without Varimax rotation, were performed, as previously described [The 15 traits that follow constitute the original traits which through multivariate factor analyses were transformed into factor scores. The factor scores served as phenotypes in our linkage analyses. Fasting glucose (GLUC) and insulin (INS); triglycerides (TG) and high-density lipoprotein cholesterol (HDLC); body mass index (BMI); medication-adjusted systolic and diastolic blood pressure (SBP and DBP); heart rate (HR), diastolic LV internal dimension (LVID), posterior wall thickness (PWT) and relative wall thickness (RWT); LV mass indexed to heightg et al.  and applR), and a residual non-familial variance component (r). The null hypothesis of no linkage was tested by restricting the QTL heritability (h2g) at the putative locus at h2g = 0. The linkage test becomes a likelihood ratio of the alternative hypothesis against the null hypothesis, in every location throughout the genome where h2g is estimated. This test turns out to be a \u03c72 with 1 degree of freedom [The HyperGEN study genotyped 391 microsatellite markers at the National Heart, Lung, and Blood Institute Mammalian Genotyping Service , of which 370 autosomal markers were used for this genome-wide linkage analysis. Software used to insure the marker' quality control included ASPEX, GRR, GENEHUNTER and PEDCHECK -19. The  freedom .Table Table In the present study we examined a group of latent factors identified by performing factor analyses on 15 MetS or ECHO traits. The seven identified latent factors reduced the complexity of this large number of phenotypes. \"Lipids-INS\" factor was mainly contributed to by HLDC, TG and INS. \"BMI-INS\" was another factor identified from MetS domains. From the ECHO variables two latent factors were identified, \"LV wall thickness\" with main contributions from LVMI, PWT, RWT and MWS, and \"LV geometry\" with main contributors LVMI, LVID and RWT. \"BP\" factor, primarily a combination of SBP and DBP, was strongly evidenced in African Americans. However, when factor analysis with no rotation was applied in the whites, BP combined with \"LV wall thickness\" and \"LV geometry\" to form two new latent variables \"BP-LV geometry\" a combination of SBP, DBP, LVMI, PWT and LVID and \"BP-LV dimension wall thickness\" a combination of SBP, DBP, LVID, RWT and PP/SV. We look at these factors as MetS, ECHO, and a combination of MetS-ECHO latent factors. The presence of BP as a connector between MetS and ECHO is consistent with the increased risk of CV disease associated with hypertension. Chinali et al  found thACACB gene located on 12q24.1 may be a candidate. This gene reported in murine studies, has been implicated in controlling mitochondrial fat oxidation and is considered a regulator of energy expenditure [Two QTLs, one from MetS domains, and one from ECHO were prominent. The chromosome 12 QTL for the \"BMI-INS\" factor was replicated across ethnic groups, although the LOD scores were larger in African Americans. Its location coincides with linkage reports at 12q24.2 for non-insulin dependent DM, rheumatoid arthritis, and multiple sclerosis ,24. In tenditure . Althougenditure  reportedCDH13) gene. CDH13, which is also called heart cadherin, is believed to be a calcium dependent mediator of cell-cell interaction in the heart and acts as negative regulator of neural cell growth. Joshi et al [cadherin may have multiple signaling functions in vascular remodeling and may protect endothelial cells from oxidative stress-induced apoptosis. The CDH13 gene is about 1.2 M bp long, has 14 exons and encodes for a protein with 713 amino acids. This gene is most highly expressed in the heart. The AFM031XA5 marker represents a sequence between 81,851,181\u201381,851,356 (bp), precisely in the non-coding 5th intronic region. We hypothesize that the CDH13 polymorphism, involved in calcium mediated cell-cell adhesion, can influence calcium deposition in the heart. Such a hypothesis is supported also by the Bella et al [Of particular note are the results in the region around the marker AFM031XA5 (D16S402) at 16q24.2-q24.3 for the \"LV wall thickness\" in whites. This location marked by D16S402, has been found to be linked with cadherin 13  which binds to the C terminus of the cardiac voltage-gated sodium channel Na(v)1.5 and modulates the properties of the channel [Other genetic linkage findings of interest included the MetS and ECHO factors, for \"BP-LV wall thickness\" and \"BP-LV geometry\" in whites Table . For theh muscle ,30. A LO channel .MetS related factors showed potentially important QTLs for African Americans. For example, \"BMI-INS\" factor showed highly significant results on 2p22-2p21 and on 12q24.2, as did the \"BP\" factor on 19q13.1 , the suggestive QTL found in the previous study on chromosome 11q24, is replicated in this study with a similar LOD score, but with a shift of the peak location (from 131.3 cM to 113.1 cM). Combined traits analysis can discover QTL locations that affect more than one trait, i.e. with pleiotropic effects. The 11q23-24 human genome location is well known for a cluster of genes , which effect the lipid levels as well as is associated with DM and heart disease [Our new findings differ in several regards with the previous analysis of MetS in the HyperGEN study . First,  disease . In cont disease  showed t disease . Another disease  have shoWe expected not only that factor scores to provide an opportunity to detect pleiotropic gene effects. In addition, when traits closely related such as SBP and DBP form a separate factor they provide an increased power to detect putative QTLs compared with single trait analysis. For example, analyzing separately SBP and DBP produced weak LOD scores in the peak found on chromosome 19. When analyzed with the factor scores in African Americans we found a LOD score of 2.67, 2.18, 2.97, and 1.98 depending on the rotation method used and including or excluding T2D subjects. Nevertheless we do not know if the BP factor findings are true or type I error results. Consequently, further elucidation of the precise location of the causative polymorphisms of the identified linkage peaks is being processed with an abundant number of SNPs.cadherin 13 gene and the one on 12q24 which replicated in both ethnicities.This study is the first genome-wide linkage study that utilizes factors scores derived simultaneously both from domains of MetS and of cardiac geometric and functionality phenotypes. They provide some insight into the genetic relationship underlying MetS and cardiovascular traits. Refined SNPs genotyping with a million SNPs platform will soon enhance the ability to discover causative genes of the cardiac- and MetS related QTLs on chromosome 16q24.2-q24.3 that coincided with linkage within 2.7; MetS: metabolic syndrome; MWS: LV midwall shortening; PP/SV: arterial stiffness defined by pulse pressure/stroke volume; PWT: posterior wall thickness; QTL: quantitative trait loci; RWT: relative wall thickness; SBP: medication-adjusted systolic blood pressure; SNPs: single nucleotide polymorphisms; TG: fasting triglycerides.ARD: aortic root diameter; BP: blood pressure; BMI: body mass index; cM: centiMorgan; CV: cardiovascular disease; DBP: medication adjusted blood pressure; DM: diabetes mellitus; ECHO: echocardiography; FA: factor analysis; GLUC: fasting glucose; HDLC: high-density lipoprotein cholesterol; HR: heart rate; HyperGEN: Hypertension Genetic Epidemiology Network Study; INS: fasting insulin; LOD: lod score; LV: left ventricular; LVH: left ventricular hypertrophy; LVID: diastolic LV internal dimension; LVMI: LV mass indexed to heightThe authors declare that they have no competing interests.All authors contributed equally.The pre-publication history for this paper can be accessed here:Figure 1. Linkage analysis of factors in African Americans, all data, no rotation. The graph represents the linkage analysis results.Click here for fileFigure 2. Linkage analysis of factors in African Americans, excluding DM, no rotation. The graph represents the linkage analysis results.Click here for fileFigure 3. Linkage analysis of factors in African Americans, all data, Varimax rotation. The graph represents the linkage analysis results.Click here for fileFigure 4. Linkage analysis of factors in African Americans, excluding DM, Varimax rotation. The graph represents the linkage analysis results.Click here for fileFigure 5. Linkage analysis of factors, whites all data, no rotation. The graph represents the linkage analysis results.Click here for fileFigure 6. Linkage analysis of factors, whites excluding DM, no rotation. The graph represents the linkage analysis results.Click here for fileFigure 7. Linkage analysis of factors, whites all data, Varimax rotation. The graph represents the linkage analysis results.Click here for fileFigure 8. Linkage analysis of factors, whites excluding DM, Varimax rotation. The graph represents the linkage analysis results.Click here for file"}
+{"text": "The sequential nature of gel-based marker systems entails low throughput and high costs per assay. Commonly used marker systems such as SSR and SNP are also dependent on sequence information. These limitations result in high cost per data point and significantly limit the capacity of breeding programs to obtain sufficient return on investment to justify the routine use of marker-assisted breeding for many traits and particularly quantitative traits. Diversity Arrays Technology (DArT\u2122) is a cost effective hybridisation-based marker technology that offers a high multiplexing level while being independent of sequence information. This technology offers sorghum breeding programs an alternative approach to whole-genome profiling. We report on the development, application, mapping and utility of DArT\u2122 markers for sorghum germplasm.PstI+BanII complexity with a subset of clones obtained through the suppression subtractive hybridisation (SSH) method. The genotyping array was used to analyse a diverse set of sorghum genotypes and screening a Recombinant Inbred Lines (RIL) mapping population. Over 500 markers detected variation among 90 accessions used in a diversity analysis. Cluster analysis discriminated well between all 90 genotypes. To confirm that the sorghum DArT markers behave in a Mendelian manner, we constructed a genetic linkage map for a cross between R931945-2-2 and IS 8525 integrating DArT and other marker types. In total, 596 markers could be placed on the integrated linkage map, which spanned 1431.6 cM. The genetic linkage map had an average marker density of 1/2.39 cM, with an average DArT marker density of 1/3.9 cM.A genotyping array was developed representing approximately 12,000 genomic clones using Sorghum bicolor and have demonstrated that DArT provides high quality markers that can be used for diversity analyses and to construct medium-density genetic linkage maps. The high number of DArT markers generated in a single assay not only provides a precise estimate of genetic relationships among genotypes, but also their even distribution over the genome offers real advantages for a range of molecular breeding and genomics applications.We have successfully developed DArT markers for  Sorghum is a major staple food and fodder crop grown worldwide, with an annual average production of 61 million tonnes over the past decade [Investment in sorghum breeding and genomic resources has been less than for the other major cereals rice, wheat, maize and barley. Interest has focused on the crop due to its drought resistance and small genome size (~760 Mb) compared to close relatives maize ~2500 Mb) and sugarcane 2550 to 4200 Mb) [500 Mb an200 Mb [5Numerous studies have demonstrated that sorghum is very diverse crop, with cultivated sorghums exhibiting great phenotypic variability. The cultivated germplasm has been classified into five major races  and 10 intermediate races based on panicle and spikelet morphology . In ordeThe current molecular marker technologies have characteristics which additionally affect the level of genome coverage, their discrimination ability, reproducibility and technical and time demand. A number of the limitations associated with the different marker technologies can be overcome by utilising specialised hardware such as high throughput capillary electrophoresis machines, which can impact on discrimination ability, reproducibility and speed. However, the majority of the limitations are related to the sequential nature and high assay costs of the marker technologies, in addition to reliance on DNA sequence information. Diversity arrays technology (DArT) can over come these limitations and has been developed as a hybridisation-based alternative to the majority of gel-based marker technologies currently in use. The DArT genotyping method was developed originally for rice  and has This paper reports the results of a study designed to (1): develop a sorghum diversity array for DArT genotyping, (2): determine linkage map positions of polymorphic DArTs and (3): assess utility of DArT technology in diversity analyses on a set of diverse sorghum lines, including selected lines from the Department of Primary Industries and Fisheries (DPI&F) sorghum breeding program. In order to evaluate the discriminatory ability of DArTs, efforts have been made to include the same genotypes used in genetic diversity studies based on other molecular marker technologies.PstI-based genomic representations [PstI with different frequently cutting restriction enzymes (RE) as a complexity reduction approach for sorghum. DNA samples from the eight sorghum genotypes were digested with PstI and several frequently cutting RE , ligated to a PstI adapter and then amplified with the PstI-0 primer. Gel analysis of the PCR products showed that a uniform smear (without major bands) only appeared in the PstI+BanII combination. Other RE combinations gave a smear with one or more dominant bands. Such strong bands represent highly amplified restriction fragments and correspond to abundant repetitive sequences in the representation; a feature which is highly detrimental to DArT performance [The initial tests of DArT performance in sorghum were done on eight sorghum genotypes  method [PstI+BanII Library C were used for all genotyping work reported here.Based on the extended ) method , the besThe selected DArT clones were tested for their ability to resolve genetic relationships among a set of 90 lines. The reproducibility of the DArT genotyping array was successfully validated by independent assays from the same DNA. The genotypes selected represent a significant proportion of the genetic variation in sorghum with all 5 races represented and 5 intermediate races also represented. In addition, the germplasm set included elite lines from breeding programs some of which had high levels of co-ancestry.DArTsoft analysis  identified 508 markers polymorphic among 90 genotypes typed on the array. The PIC values of these 508 markers were very high with over 69% of the markers having a PIC value between 0.4 and 0.5 Table . The aveS. propinquum and the weedy subspecies, S. bicolor subsp. verticilliflorum (formerly S. arundinaceum). Cluster 9 consisted of 2 caudatum genotypes (IS 12656C and IS 10302). Cluster 10 consisted of 11 genotypes in total, of which 8 were caudatum or caudatum-derived. Cluster 11 consisted of a tight cluster of restorer (R) lines predominately from Australian breeding programs; R9990066, R999017 and R999003 are all progeny of the line R31945-2-2. Cluster 12 is a looser cluster consisting of 15 lines of which 7 were caudatum or caudatum-derived. Finally cluster 13 consisted of 4 genotypes of which 2 are bicolor or bicolor intermediate and S. bicolor subsp. drummondii which is very similar morphologically to the bicolor race.Cluster analysis based on the DICE dissimilarity index and the unweighted neighbour-joining method was performed on the 508 DArT markers for 90 genotypes Figure . This clTo confirm that the sorghum DArT markers behave in a Mendelian manner, we constructed a genetic linkage map for a cross between R931945-2-2 and IS 8525. We selected 370 DArT markers with Q-values greater than 80% and merged with the segregation data for 286 markers, consisting of 55 SSRs, 229 AFLPs and 2 morphological markers derived from the original map .PstI+BanII sub-libraries. Of the 358 DArTs included on the map, 172 were SSH-dervied and of these 50 (29%) were redundant, whereas the 186 non-SSH derived DArTs exhibited a reduced level of redundancy (26%). Overall redundancy in the map dropped from 33.7% to 24% when SSH-derived DArT markers were excluded. Interestingly, the difference in apparent redundancy levels between SSH-derived and \"normal\" DArT markers was smaller compared to what we observed for tomato and sugarcane  and in fern species Asplenium and moss species Garovaglia .In total, 596 markers could be placed on the integrated linkage map, which spanned 1431.6 cM Table , suggestThere was no statistically significant difference between DArT and non-DArT markers in the distribution of parental alleles across the genome. Twelve DArTs were removed during the course of map construction either because of lack of linkage to other markers, or as they formed small linkage groups containing only DArTs which did not link to the known chromosomes.This is the first report of the use of DArT technology in sorghum and our results demonstrate that the sorghum DArT markers are high quality, as assessed by their call rate, scoring reproducibility and PIC values. The DArT marker quality parameters measured for the sorghum array are comparable to those obtained for pigeonpea , barley S. bicolor subsp. drummondii, in cluster 13  and R  lines was also noted. In this study, there was less variation among the B-lines than the R-lines, with the B-lines clustering tightly together in only 2 of the 13 clusters, whereas the R-lines grouped more loosely in 5 clusters. It has also been noted  that theComparisons of the discrimination ability for DArT markers and their ability to reflect pedigree backgrounds in contrast to other marker types is made much simpler when over-lapping sets of germplasm are used in a number of studies. Seventeen genotypes included in this study were examined by Menz et al. , 54 genoThe separation of \"wild\" sorghums from cultivated germplasm seems to be less pronounced compared to the results based on SSR data . This isThe integrated genetic linkage map comprising DArTs, AFLPs and SSRs clearly demonstrates that the new sorghum DArT markers behave in a Mendelian manner. In total, 358 DArTs were mapped to 257 unique loci. The higher level of redundancy of the DArT markers is reflected by the higher number of AFLP and SSR markers having unique segregation patterns. However, the markers on the DArT array used in this study were not filtered for redundancy, whereas the SSR/AFLP data set had previously undergone curation , to remoThe total length of the integrated genetic linkage map was 1431.6 cM, with an average DArT marker density of 1 per every 3.9 cM. The total map length is comparable to other recently reported sorghum genetic maps, being slightly shorter than the 1713 cM high-density genetic linkage map based on 2926 AFLP, RFLP and SSR markers , and sliSorghum bicolor and have demonstrated that DArT provides high quality markers that can be used for diversity analyses and to construct medium-density genetic linkage maps. The high number of DArT markers generated in a single assay not only provides a precise estimate of genetic relationships among genotypes, but also their even distribution over the genome offers real advantages for a range of molecular breeding and genomics applications. Additionally, the availability of the sorghum whole genome sequence by the end of 2007 offers very exciting opportunities for assessing the colinearity of the DArT markers on the genetic linkage maps with the markers on the sequence map. As DArT assays are performed on highly parallel and automated platforms the cost of datapoint (a few cents per marker assay) is reduced by at least an order of magnitude compared to current, gel-based technologies.We have successfully developed DArT markers for S. bicolor subsp. bicolor) with all 5 races and 5 intermediate races represented, two weedy subspecies (S. bicolor subsp. drummondii and subsp. verticilliflorum) and a wild species, S. propinqum . The best markers from the initial experiments were \"cherry picked\" to assemble the \"re-array library\" with 768 polymorphic clones. Clones from this library together with 5367 clones from PstI+BanII Library C were used for all genotyping work reported here. The re-array library was created using the PstI+BanII and the subtraction (SSH) libraries. Details of the re-array library and other libraries used for sorghum genotyping are included in the supplementary material  by the total variance of hybridisation level of the marker, in the experiment.A group of 90 sorghum lines  population derived from a cross between the two lines were typed using the genotyping array. Clones with Q > 80% and a call rate of at least 80% were selected for mapping. DArTs markers were merged with an existing mapping data set consisting of 286 markers including 55 SSRs and 229 AFLPs [s value of 0.40 was used to initially group the markers into ten linkage groups. Multipoint linkage analysis of loci within each LG was then performed and marker order was further verified through re-sampling for quality control via jack-knifing [The lines R931945-2-2, a commercially accepted restorer line in Australia and IS 8525, an Ethiopian line (kafir race), in addition to 92 lines of a F29 AFLPs . A genet29 AFLPs . The RIL-knifing . Markers-knifing . The Kos-knifing  mapping ESM carried out the diversity and mapping analyses and drafted the manuscript. LX co-developed the DArT technology for sorghum. DRJ conceived of the study, and participated in its design and coordination, germplasm development and selection and helped to draft the manuscript. KH was involved in the optimisation of the methodology and participated in the mapping analysis. DKP participated in the mapping analysis. EH participated in the development of the DArT technology and editing of the manuscript. PW contributed to ongoing improvements of the DArT technology and the quality assessment of sorghum clones. AK supervised development of the DArT technology, participated in the study's design and coordination and helped to draft the manuscript.List of sorghum genotypes used for the development of the sorghum DArT array and the diversity analyses. The table includes details of genotype IDs and aliases, race and origin and inclusion status in both the methodology developmental stages and diversity analyses.Click here for fileSummary of sorghum libraries. The table includes details of the number of genotypes used in the development of each sorghum library and the number of clones identified.Click here for fileLibraries used for generation of the genotyping array. The table details the barcode for each sorghum library.Click here for file"}
+{"text": "To detect association of the DR1 allele with rheumatoid arthritis (RA) given linkage in the affected sibling pairs of the replicates of Problem 3 of Genetic Analysis Workshop 15 (GAW15), we propose a new score statistic that takes into account the linkage information. We knew the answers. Linkage studies are often followed by case-control association studies of candidate genes located under the peak to identify the causes of a linkage peak. One strategy is to type the affected sibling pairs from the original linkage study and a set of unrelated controls for single-nuclear polymorphisms describing the genetic variation of these genes. For this affected sibling pair-control design, we propose a relative-risk model for the relationship between the disease outcomes of sibling pairs and their genotypes and identity-by-descent status at the locus of interest. From this model, we derive a score statistic to analyze genetic association given linkage. We compare the performance of the new statistic to the method of Li et al. and to a standard association analysis that neglects the information on the identity-by-descent status of the sibling pair. We conclude that for the GAW15 data the new method performs well and that methods that use the linkage information may be more efficient than standard comparisons of genotypes in cases and controls. Genome-wide linkage analyses are often followed by association studies of candidate genes located under the linkage peak using a case-control design. With these genetic association studies one hopes to identify candidate genes whose variation causes the excess identical-by-descent (IBD) sharing of marker alleles in the linkage study. Single-nucleotide polymorphisms (SNPs) describing the genetic variation of the studied genes are typed in a set of controls and a set of cases and the genotype distributions are compared between cases and controls. Often a part of or all cases originates from the linkage study. Li et al.  studied We first derive a relative-risk model for the disease outcomes with a term modeling association between the candidate SNP and the outcome and a term modeling excess IBD sharing at the SNP locus. This is a model for joint linkage and association. From the likelihood of this model, we derive a score statistic for testing association given linkage. The new score statistic appears to be a stratified analysis of the genotype distributions according to the IBD status of the sibling pair at the SNP locus. If the SNP is one of the causes of excess IBD sharing in the region, the stratified analysis should be more efficient than an unstratified analysis, which neglects the IBD information. The unstratified analysis compares the genotype frequencies of the affected sibling pairs and the controls.p-values. A disadvantage of the method is that it models one single gene under the peak, which may not be realistic for complex genetic traits studied in sibling pairs who share a relatively large region. Further likelihood ratio tests may be more sensitive for model deviations than score statistics [Recently, a model linking disease outcomes in sibling pairs to linkage and association information measured at a candidate SNP was proposed by Li et al. . The relatistics .We apply the new score statistic, the unstratified comparison of genotype frequencies between the affected sibling pairs and controls, a statistic which combines the new score statistic and the unstratified statistic and the likelihood ratio method of Li et al.  to the AG measure the total effect of the unobserved genes under the identified linkage peak. Without loss of generality, we assume that G has mean value of zero. Further, we assume that the disease is rare. The relative effect RR of G is modelled by RR = exp(G). By using second-order Taylor approximations around G = 0, the conditional probability of being affected given G is proportional to (1 + G + 0.5G2) and the probability that two siblings are affected given their genetic effects G1 and G2 is proportional to (1 + G1 + G2 + 0.5 G12 + 0.5 G22 + G1G2).Let G is for each sibling pair, the pair of genotypes at the candidate SNP of interest  and the IBD status at the candidate locus IBDS. The conditional probability that two siblings are affected given  and IBDS is proportional to (1 + E(G1|S1) + E(G2|S2) + 0.5 Var). By applying Bayes rule and assuming Var = Var(G1 + G2|IBDS), we obtainThe information available on \u03bc equal to E(S1). By using Var = Var(G1 + G2|IBDS), we assume that given the IBD status, the variance and covariance of the genetic effects do not depend on the SNP genotypes. The parameter \u03b4 measures linkage at the SNP location and the parameter \u03b2 measures association of the SNP to the disease. The model extends the model of Kong and Cox [with  and Cox  by also Now the log likelihood function is given byc is a constant independent of \u03b2. The corresponding score statistic U to test the null hypothesis H0: \u03b2 = 0 given IBDS is given bywhere \u03b4 can be obtained by applying Kong and Cox method [\u03bc can be obtained from the controls. Under the null hypothesis the statistic U has mean value of zero. The variance of U can be empirically estimated or computed based on the genotype frequencies under the null hypothesis.The parameter x method  to the AG1 + G2|S1, S2, IBDS) = Var(G1 + G2|IBDS) is violated the stratification according to the IBD groups will not be optimal. The test statistic is still valid, but the gain in power compared to the unstratified test statistic will be smaller. Therefore, we propose also the statistic U*, which combines the unstratified statistic and the new score statistic by pooling the one and two IBD groups.The unstratified version of this statistic tests the null hypothesis of no association without accounting for linkage information. Also, for this statistic the genotypes of sibling pairs are not independent. The variance of the statistic can be empirically estimated. If the assumption of Var of 9.03 was used. The marker information was high due to a dense map (average spacing of 5 cM), a high marker heterozygositiy (above 0.7), and the availability of parental genotypes. For association, we studied the DR locus with two risk variants (DR1 and DR4). The DR1 allele has a frequency of 0.1 and a genotype relative risk for homozygous carriers versus homozygous carriers of the DRx allele of 1.5. The other variant DR4 has a frequency of 0.25 and increases the risk for RA enormously. The genotype relative risk for homozygous carriers DR4 versus homozygous carriers of the DRx allele is 30. Our aim was to identify the DR1 allele at the DR locus.\u03b4 and to compute the multipoint IBDS at the DR locus, assuming an additive model for each sibling pair. For almost all sibling pairs, the IBD status was observed. When the IBD status was uncertain, the most likely IBD status was assigned to the sibling pair. In the 100 replicates, the parameter \u03b4 varied from 0.13 to 0.36 with a mean of 0.25. The genotype Sk for k = 1 or 2 was defined as the number of DR1 alleles carried by sibling k and its expectation \u03bc was computed from the controls. In the replicates, the parameter \u03bc varied from 0.28 to 0.37 with a mean of 0.32.We first removed the sibling pairs and controls who are homozygous carriers of the DR4 allele. The number of affected sibling pairs used for analysis varied from 211 to 270 with a mean number of 238 sibling pairs. The number of homozygous carriers in the controls was small, and around 2000 controls were available for association analysis. For each replicate we used Merlin  to estim\u03bc in the computation of the p-value for the score statistics. To be able to compare the performance of the score statistics with the performance of the Li method, we made the uncertainty in the estimated allele frequencies in the controls negligible by multiplying each control record four times.We applied the new score statistic U, the unstratified test statistic and the statistic U*, which combines the sibling pairs who share two alleles IBD and one allele IBD. We estimated the variances of the three statistics empirically. Finally, for the method of Li et al.  we used p-values of the various statistics applied to the first five replicates are given. Table In Table G1 + G2|S1, S2, IBDS) = Var(G1 + G2|IBDS). This assumption does not hold when multiple disease variants or disease loci are in linkage disequilibrium (LD) with the candidate SNP. Thus the statistic U allows for multiple disease loci as long as they are not in LD with the candidate SNP. When this assumption is violated, the stratification is not optimal and the statistic loses power. An ad hoc solution is the modified statistic U*, which combines the unstratified statistic and the new statistic. Probably due to the presence of the DR4 allele in the reference category and the LD between the DR locus and locus C, U* and the unstratified statistic perform better than the score statistic U. The method of Li et al. models a disease locus in LD with the candidate SNP and is therefore more flexible to deal with multiple disease variants. Extended simulations are needed to study the performance of these statistics under multiple disease loci models.The new statistic U* performs as well as the likelihood-ratio statistic of Li et al . The metSk variables in the model corresponding to the different risk alleles. When these other variants are unknown, the parameter of the linkage term should depend on the genotypes Sk, for example by inclusion of interaction terms between the association and linkage information in the model.When multiple variants exist or multiple disease loci are present in the LD block around the candidate SNP, the statistic U is still valid, but loses power. In this paper, we used the ad hoc U* to deal with the presence of the DR4 allele in the reference category. More sophisticated solutions are warranted. When multiple variants are observed, the model could be extended by including more The version of the score statistic presented in this paper assumes observed IBD for the sibling pairs. The statistic can easily be adapted to deal with uncertainty in IBD status by using a weighted statistic with weights equal to the IBD probabilities. Another extension is to allow for haplotype ambiguity. However this is not straightforward because the haplotypes of sibling pairs are not independent. The control population is rather large in the GAW15 data, we assumed known population frequencies of the DR1 genotypes. For smaller populations the uncertainty in the estimates can be taken into account by computing the variances of the statistic under the null hypothesis.Similar to the models of Li et al. , the relWe conclude that the relative-risk model provides a new framework for joint linkage and association analysis. When testing the null hypothesis of no association, more efficient statistics may be obtained when the linkage information is used. To test the null hypothesis of no association given linkage, the modified score statistic U* performs as well as the method of Li et al. in detecting the effect of the DR1 allele in the replicates of Problem 3.The author(s) declare that they have no competing interests."}
+{"text": "The combination of gene expression profiling with linkage analysis has become a powerful paradigm for mapping gene expression quantitative trait loci (eQTL). To date, most studies have searched for eQTL by analyzing gene expression traits one at a time. As thousands of expression traits are typically analyzed, this can reduce power because of the need to correct for the number of hypothesis tests performed. In addition, gene expression traits exhibit a complex correlation structure, which is ignored when analyzing traits individually.Saccharomyces cerevisiae. Both methods decompose the data into a set of meta-traits, which are linear combinations of all the expression traits. The meta-traits were enriched for several Gene Ontology categories including metabolic pathways, stress response, RNA processing, ion transport, retro-transposition and telomeric maintenance. Genome-wide linkage analysis was performed on the top 20 meta-traits from both techniques. In total, 21 eQTL were found, of which 11 are novel. Interestingly, both cis and trans-linkages to the meta-traits were observed.To address these issues, we applied two different multivariate dimension reduction techniques, the Singular Value Decomposition (SVD) and Independent Component Analysis (ICA) to gene expression traits derived from a cross between two strains of These results demonstrate that dimension reduction methods are a useful and complementary approach for probing the genetic architecture of gene expression variation. Saccharomyces cerevisiae [cis and trans, with most trans-linkages being due to a few regulatory \"hotspots\".Recently, the combination of gene expression profiling and classic quantitative trait locus (QTL) mapping has emerged as an important tool in dissecting the genetic basis of gene expression variation -5. Usingrevisiae . LinkageCurrent statistical methods that analyze high-dimensional phenotypes, such as expression traits, one trait at a time suffer from low power because of the challenges associated with multiple hypothesis testing. In addition, such approaches fail to take advantage of the potentially informative correlation structure of high-dimensional phenotypes. In order to exploit the correlation structure among genes, various data reduction techniques can be used to reduce the overall dimensionality of the data. For example, in the context of eQTL studies, hierarchical clustering has been performed followed by linkage mapping of the average expression of cluster members . Such anSingular Value Decomposition (SVD) and Independent Component Analysis (ICA) are popular dimension reduction techniques with different operating characteristics. Briefly, SVD is a factorization method that decomposes the data into a set of mutually orthogonal \"eigentraits\" that are sorted according to variance explained ,10. ICA Both SVD and ICA have been previously applied to gene expression data ,16-19. SIn this study, SVD and ICA were applied to a well studied eQTL data set in yeast. The resulting meta-traits, which are mutually uncorrelated, can be thought to represent independent trends in expression variation . We usedSaccharomyces cerevisiae strains BY and RM. The expression level of each gene was treated as a quantitative trait (which we will refer to as gene expression trait) and eQTL were identified by linkage analysis using 3312 genetic markers distributed across the genome.We used data previously described by Brem et al  who measOur goal was to investigate the use of data reduction techniques for mapping eQTL and to compare it with traditional single trait analyses. To this end, we first performed a genome-wide linkage analysis on each of the 6216 gene expression traits by standard regression techniques . Each trSVD analysis was performed to reduce the dimensionality of the data from the original 6216 expression traits to 112 eigentraits, where each eigentrait is a linear combination of all gene expression traits. The proportion of variance explained for each eigentrait relative to total variation in the data set is shown in Figure Independent modes from an ICA based decomposition were sorted by the Liebermeister criterion  (see Metp < 0.0001) gene expression traits range from 5 to 1919 . As shown in Tables To identify general biological themes, we performed a Gene Ontology (GO) analysis for each set of significantly correlated expression traits across the top 20 meta-traits Table . In addip < 0.05, Table For the top 20 meta-traits, we performed a genome-wide linkage analysis using 3312 genetic markers that were genotyped in each segregrant. Linkage analysis was performed by regressing marker genotypes on trait values for each meta-trait and significance was determined by permutations. We considered markers to be significant according to a permutation based genome-wide error rate of 5% as significant . The gentrans-regulatory hotspots [Ten of 21 unique eQTLs identified map to previously described regions of trans-acting effects, which is consistent with the fact that both SVD and ICA were able to capture the major sources of variation by its top ranked components. Of the eleven new eQTL, four showed evidence for cis-linkage, which will be discussed in more detail below. The other linkages map to regions in the genome that either show no significant enrichment of linkages from the single trait analysis or there is no obvious gene to explain the enrichment of GO annotation for that meta-trait.The genome-wide linkage results of meta-traits derived from SVD and ICA show considerable overlap Figure . OverlapFigure cis-linkage is characterized as an expression trait showing linkage to its own genomic location. In the setting of meta-trait linkage scans, we define a cis-linkage of a meta-trait as linkage to the genomic position of a trait that is also significantly correlated with this meta-trait. In the following sections we describe in depth analyses of four novel cis-linkages that were not described in previous eQTL studies. These include eigentraits 4 and 19, and ICAtraits 7 and 9, which show significant enrichment of GO annotation terms.In traditional linkage scans where traits are analyzed one at a time, a cis-linkage on chromosome 12  and the top four correlated genes make up the tandem array of asparaginase (ASP) genes. The nine remaining significantly correlated genes are strong candidates for participating in the asparaginase metabolism network. Comparative sequence analysis with the published draft RM genome sequence [ASP gene cluster in the RM strain, which is consistent with the eQTL being supported by the highest F-statistic among all linkages and the small number of genes that are principal contributors to this particular eigentrait.Eigentrait 4 has a strong 2 Figure . The regsequence  revealedcis-linkage on chromosome 4. This eigentrait is enriched for genes involved in sodium transport. Genes with the highest correlation to eigentrait 19 are the group of ENA genes ENA5, ENA2 and ENA1, which are members of the sodium efflux ATPase family and span the chromosome 4 region that surrounds the linked eQTL  and one significant eQTL was mapped on chromosome 5 . Thus cross-hybridization among these genes may be influencing this eigentrait. Note, linkage analysis of eigentrait 8 identified two closely linked cis-eQTL on chromosome 12  traits in 112 (M) segregants was mean centered for each trait to remove the baseline level of expression to focus on levels of expression variation. SVD was performed on the centered data to result in U, a N \u00d7 M \"eigensegregant\" matrix, D a M \u00d7 M diagonal \"eigenweight\" and VT, the M \u00d7 M \"eigentraits\" matrix:The expression matrix v(ei) was calculated as:The proportion of variance explained by each eigentrait, ei denotes the eigenvalue of the ith eigentrait.where The Shannon entropy of the data was calculated as:To determine the significance threshold for eigentraits, we used Horn's procedure . A null fastICA algorithm implemented in the fastICA R package [X, where each trait was mean centered. Independent Component Analysis decomposed X into its constituent source matrix S, and mixing matrix A.The  package ,42 was uA, which are linear combinations of the expression traits form the ICAtraits. The default Liebermaster contrast value from mlica R package [The modes of  package ,43 was uTo identify genes that make a significant contribution to a meta-trait, we calculate the correlation of each of the 6216 gene expression traits to each of the 20 meta-traits. To estimate the significance threshold for the correlation measure, 1000 permutations of the segregants are performed and correlation with each eigentrait is estimated as described previously. The absolute value of the null correlations are pooled and threshold set at a p-value < 0.0001.\u03c00 = 0.152 of the expression traits showing no linkage [Genome-wide linkage analysis was initially performed on each expression trait separately. Briefly, for each trait we tested for linkage across all 3312 genetic markers using standard linear regression. To estimate null statistics, the gene expression of 112 segregants were permuted 10 times and linkage tested as before with all markers. The maximum statistic in each permutation was retained and used to calculate the p-value for each trait. The 5013 significant linkages at a FDR cut-off of 0.05, with an estimated upper bound of Similar single marker analysis was used to perform genome-wide linkage scans for each of the 20 meta-traits. The genome-wide error rate (GWER) of 0.05 was determined by estimating p-values from pooling together the maximal statistic across 3312 markers from 10,000 permutations of each meta-trait . As therSB, JDS, and JMA conceived and designed the experiments. SB analyzed the data. SB and JMA wrote the manuscript with contributions from JDS.Linkage hotspot information. List of all significant genes from the single trait linkage analyses where the linkage results were binned into 20 kilobase bins across each chromosome to identify \"hotspots\". Using a poisson distribution, the probability of having 20 linkages present in a bin by chance is < 2.1E-4. 17 such \"hotspots\" were identified.Click here for fileEigentrait composition. List of all transcripts that are significantly correlated with each of the top 20 eigentraits.Click here for fileICAtrait composition. List of all genes that are significantly correlated with each of the top 20 ICAtraits.Click here for fileMultipoint linkage profile of Eigentrait \u2013 4 and Eigentrait \u2013 19. The multipoint linkage profile of Eigentrait 4 and 19 are plotted in the upper and lower half of the figure, respectively. In each case, only a section of the chromosome spanning the maximum LOD score is plotted with the 1 LOD support interval denoted by solid black bar. For Eigentrait 4, the position of the tandem array of three genes that are involved in asparaginase catabolism are represented by blue vertical bars. Similarly, for Eigentrait 19 the position of the tandem array of sodium ion efflux genes are denoted by blue vertical bars.Click here for fileMultipoint linkage profile of ICAtrait \u2013 7 and ICAtrait \u2013 9. Similar to S4, the multipoint linkage profile spanning the maximum LOD score for ICAtrait 7 and 9 are plotted in the upper and lower half of the figure, respectively. In both plots, the position of a subset of genes that lie in the 1 LOD support interval represented by solid black bar is shown. For ICAtrait 7, only YERCTy1-1, a retrotransposon, shows significantly correlation while for ICAtrait 9, only ZAP1 shows a significant correlation with the ICAtrait.Click here for fileMultiple sequence alignment of YJL056C. CLUSTALW was used to create a sequence alignment of the protein encoded by YJL056C/ZAP1 from two strains of Saccharomyces cerevisiae and two related species Saccharomyces mikatae and Saccharomyces paradoxus. The alignment output was then run through BOXSHADE to generate a colored output based on the conservation and degree of identity of the aligned residues. Nineteen SNPs were detected in the protein alignment, of which 10 were non-synonymous.Click here for fileDifferential expression of two Eigentraits showing cis-linkage. The top panel represents Eigentrait 4 and the bottom panel represents Eigentrait 19. Scatter plot of eigentrait values on the y-axis against the parental genotypes at the marker with the highest linkage statistics on the x-axis shows marked differential expression in the segregants (p < 0.0001).Click here for file"}
+{"text": "We applied nonparametric quantitative trait linkage analysis to two rheumatoid arthritis quantitative phenotypes, IgM rheumatoid factor (RF) and anti-cyclic citrullinated peptide autoantibody titer measurements, using 5700 genome-wide Illumina single-nucleotide polymorphism genotypes on 658 Caucasian North American Rheumatoid Arthritis Consortium families. Peak LOD scores for both quantitative traits were located in the human leukocyte antigen region 6p21  followed by 11p12 (3.2 and 3.6). In addition, there were LOD scores of 3.2 on 2q32 for RF and 3.6 on 4q24 for anti-cyclic citrullinated peptide. The resulting linkage signals for both phenotypes are very similar to previous results for rheumatoid arthritis as a qualitative variable, with rheumatoid factor measurements being most closely aligned. Interestingly, anti-cyclic citrullinated peptide exhibits a stronger linkage peak on 2p14 than rheumatoid factor and rheumatoid arthritis, and stronger linkage on 4q24. Finally, we used ordered subset analyses to determine if sub-ranges of these two traits increased rheumatoid arthritis linkage signals; however, our analyses did not reveal significant effects of the quantitative traits on rheumatoid arthritis linkage signals in this population. Rheumatoid arthritis (RA) is a chronic autoimmune disease with heterogeneous phenotypes exhibited among affected individuals. While it is known to have a strong genetic component, studies attempting to identify chromosomal regions influencing RA have had mixed results, except for the human leukocyte antigen (HLA) region of chromosome 6 . It is tRecently, a genome-wide single-nucleotide polymorphism (SNP) analysis of North American Rheumatoid Arthritis Consortium (NARAC) families  implicatFamilies were selected for analysis that had at least two affected members with Illumina SNP genotyping and anti-CCP and/or RF titers. We also analyzed the Caucasian subgroup of families to limit genetic heterogeneity. PEDSTATS  was usedR2 value of 0.1 was used to define high-LD clusters.Because linkage disequilibrium (LD) of tightly linked loci can lead to artificial inflation of linkage scores in the presence of some missing parental genotypes , regionsBoth quantitative traits were truncated \u2013 RF values below 11 (16%) were set to 11, and anti-CCP values above 210 8%) were set to 210 \u2013 because measurement of data in these ranges is not reliable (GAW15 Problem 2 notes). QT linkage analyses were performed using the MERLIN \"--deviates\" option because both traits are not normally distributed and the NARAC sample is selected (in particular to contain multi-case RA families). This option necessitates specification of a population mean; in the absence of population data, we chose 11 as the mean for RF and 4.6 as the mean for anti-CCP, as done previously [% were seIn order to directly compare the linkage results for RF and anti-CCP in regions of interest, we repeated the LD-modelled analyses using the subset of affected subjects having measurements for both quantitative traits.Finally, anti-CCP and RF titers were evaluated for their influence on RA linkage on chromosomes 1 to 22 using the FLOSS  implementation  of ordern = 580) had a single sib pair meeting this criterion. In addition, there were 60 families with 3 affected members, 12 families with 4, 5 families with 5, and 1 family with 6 affected members. PEDSTATS reported a total of 814 sib pairs, 34 half-sib pairs, 11 cousins, 8 parent-child pairs, and 27 avuncular pairs. Of the affected individuals, 40% had one parent genotyped, and 10% had both parents genotyped.A total of 1419 affected individuals from 658 families met our selection criteria, with 1415 having RF titers and 1341 having anti-CCP titers. Most families  with p < 0.001. Filtering of unlikely genotypes by MERLIN eliminated 5254 person-markers, 0.05% of the available genotypes.PEDSTATS did not encounter any Mendelian errors in the available genotypes of these families. There were no HWE R2 value = 0.1). The results of these peak locations with and without LD modeling are shown in Table With the resulting pedigrees, MERLIN (--deviates) was first run for all of chromosomes 1 through 22. MINX was used to analyze the X chromosomes. Then all chromosomes having peak LOD score > 3 were rerun with MERLIN's LD cluster modeling  was for the effect of RF maximum difference on linkage to chromosome 17, but with consideration for multiple testing (two traits for 22 chromosomes) this result does not provide convincing evidence for a non-random association with RA linkage.Finally, we explored whether there were RF or anti-CCP trait-ordered subsets  that proOur quantitative trait linkage results are similar to the RA linkage results using the same SNP set , with RFDifferences in linkage evidence compared with an earlier microsatellite quantitative trait locus (QTL) analysis of a subset of these families  \u2013 in parThe MERLIN deviates method was chosen for this analysis due to the non-normal trait distributions and because the trait sample was selected to only include RA subjects. We did not pursue variance-components  methods,Using ordered subset analyses, we did not identify any subset of families, based on quantitative trait ranges, which significantly increased the RA linkage signal. However, it is possible that this could be more successfully applied in a less selected population.The author(s) declare that they have no competing interests."}
+{"text": "Venturia inaequalis is an economically-important disease of apple causing annual epidemics of scab worldwide. The pathogen is a heterothallic ascomycete with an annual cycle of sexual reproduction on infected apple leaf litter, followed by several cycles of asexual reproduction during the apple growing season. Current disease control is achieved mainly through scheduled applications of fungicides. Genetic linkage maps are essential for studying genome structure and organisation, and are a valuable tool for identifying the location of genes controlling important traits of interest such as avirulence, host specificity and mating type in V. inaequalis. In this study, we performed a wide cross under in vitro conditions between an isolate of V. inaequalis from China and one from the UK to obtain a genetically diverse mapping population of ascospore progeny isolates and produced a map using AFLP and microsatellite (SSR) markers.MAT. A linkage map was constructed consisting of 294 markers , spanning eleven linkage groups and with a total map length of 1106 cM. The length of individual linkage groups ranged from 30.4 cM (Vi-11) to 166 cM (Vi-1). The number of molecular markers per linkage group ranged from 7 on Vi-11 to 48 on Vi-3; the average distance between two loci within each group varied from 2.4 cM (Vi-4) to 7.5 cM (Vi-9). The maximum map length between two markers within a linkage group was 15.8 cM. The MAT locus was mapped to a small linkage group and was tightly linked to two AFLP markers. The map presented is over four times longer than the previously published map of V. inaequalis which had a total genetic distance of just 270 cM.Eighty-three progeny were obtained from the cross between isolates C0154 (China) \u00d7 01/213 (UK). The progeny was screened with 18 AFLP primer combinations and 31 SSRs, and scored for the mating type locus V. inaequalis such as virulence factors, aggressiveness and mating type. The linkage map presented here represents a significant improvement over currently published maps for studying genome structure and organisation, and for mapping genes of economic importance on the V. inaequalis genome.A genetic linkage map is an important tool for investigating the genetics of important traits in  Venturia inaequalis is a fungal pathogen of major economic importance, causing annual epidemics of apple scab [ple scab . The funple scab  and is aV. inaequalis, it is essential to understand pathogen virulence structures and the extent to which evolutionary forces may alter such structures. Analysis of microsatellite profiles of V. inaequalis samples from five continents suggested that the fungus originated in Central Asia, but it is now well established worldwide displaying high within-population diversity [For breeding apple cultivars with durable resistance to iversity -5. Isolaiversity  as well iversity . More inV. inaequalis [V. inaequalis genome. The mating-type (MAT) locus was mapped in that population and was flanked by two RAPD markers at 28.9 and 18.9 cM.Genetic linkage maps are essential for studying genome structure and organisation, and are a valuable tool for locating genes controlling important traits of interest such as avirulence, toxin production, host specificity and mating type. Molecular linkage maps can be used to develop molecular markers linked to such traits and ultimately permit the positional cloning of the genes that control them. Genetic linkage maps of other fungal genomes have been constructed using restriction fragment length polymorphisms (RFLPs), random amplified polymorphic DNA (RAPDs), amplified fragment length polymorphisms (AFLPs), microsatellite or simple sequence repeat (SSR) markers, and diversity array technology (DArT) in ascomycetes -13, basiaequalis , composein vitro conditions between an isolate of V. inaequalis from China and one from the UK to obtain a genetically diverse mapping population of ascospore progeny isolates. We scored this mapping population with AFLP and SSR markers and the mating type (MAT) locus and generated a more extensive genetic linkage map than the previously published map of V. inaequalis. The map we present covers a genetic distance of 1106 cM over eleven linkage groups and is the first map of V. inaequalis to contain transferable SSR loci.In this study, we performed a wide cross under in vitro on cellophane discs following previously published methods [A population of ascospore progeny isolates from a cross between isolates C0154 (from cv. Qinguan in China) and 01/213 (from cv. Worcester in the UK) was raised following published methods , and 83  methods . DNA wasMseI + 1/EcoRI + 2 primer combinations  according to the manufacturer's protocol and screened with 18 Twenty-eight published SSR primer pairs ,22 were Each isolate (including the two parent isolates) was manually crossed with four additional isolates, two from each of the two mating types \u2013 inferred from a previous study . The sucMAT locus was coded a/b for a haploid population and data were analysed using JOINMAP 4.0  applying the Kosambi mapping function. Marker placement was determined using a minimum LOD score threshold of 4.0, a recombination fraction threshold of 0.35, ripple value of 1.0, jump threshold of 3.0 and a triplet threshold of 5.0. Only linkage groups containing more than five markers were included in the linkage map presented. The map was constructed using MAPCHART2.2 for Windows [Segregating molecular markers and data for the  Windows .Eighty-three progeny were obtained from the cross between isolates C0154 \u00d7 01/213 (Q \u00d7 W). Table P = 0.41).Of the 518 polymorphic bands, 280 were present in parent C0154 (Q) and 238 in parent 01/213 (W). In total, 295 molecular markers deviated from the expected 1:1 segregation ratio in the progeny at the 5% level of significance (Table MAT) resulted in a genetic map of V. inaequalis, consisting of 11 linkage groups, covering a total genetic distance of 1106 cM  were mapped onto the 11 linkage groups. Of the 283 AFLP markers mapped, 120 exhibited transmission distortion at the 5% level of significance , flanked by two AFLP markers at 0.2 cM and 3.9 cM  to 166 cM (Vi-1). The number of molecular markers per linkage group ranged from 7 on Vi-11 to 48 on Vi-3; the average distance between two loci within each group ranged from 2.4 cM (Vi-4) to 7.5 cM (Vi-9). The maximum map length between two markers within a linkage group was 15.8 cM. The number of mapped AFLP markers ranged from 5 to 27 per primer pair (average = 15.7). Ten of the 12 SSR loci mapped to 5 linkage groups . The 1 to 166 V. inaequalis composed of 294 markers, the majority of which are AFLPs. The total map length is 1106 cM, spanning eleven linkage groups, one of which contains the mating type locus, MAT. The map presented is over four times longer than the previously published map of V. inaequalis which had a total genetic distance of 270 cM [V. inaequalis genome.We have constructed a genetic linkage map for f 270 cM  and was In this study, approximately 42% of AFLP markers mapped exhibited significant deviations from expected 1:1 segregation ratio at the 5% level of significance. Several genomic processes could be responsible for distorted segregation, including the expression of linked lethal genes, non-disjunction during meiosis due to the parents having divergent genomes and chromosome complements , or an aP < 0.01) have been excluded from mapping studies of Mycosphaerella fijiensis [Fusarium [Heterobasidion annosum [Bremia lactucae [In previous studies, markers with highly distorted segregation (ijiensis , but incFusarium , Heterob annosum , and Brelactucae . DespiteV. inaequalis from these two continents differed significantly in AFLP markers and virulence characteristics [F. circinatum and F. subglutinans [The two parent isolates used in the present study were from two continents and populations of eristics . They weeristics ,27. Overlutinans , howeverVenturia linkage map further.Whilst 518 markers were scored in the progeny produced, just 283 mapped to groups containing more than five molecular markers. Many of the 235 'unmapped' markers were contained in groups of five markers or less and it is probable that, with the addition of further markers through continued mapping efforts, these smaller groups will become linked to the eleven groups presented here, extending and enhancing this MAT locus was mapped to a small linkage group and was tightly linked to two AFLP markers. The availability of markers linked to the MAT locus will enable us to study the relative importance of conidia and ascospores as primary inoculum by comparing the ratio of two mating types in the autumn and early spring. Understanding the relative importance of conidia and ascospores as primary sources of inoculum may enable appropriate sanitation measures to be taken. The two parent isolates chosen in this study were known to differ in their virulence characteristics against several commercial apple cultivars [The ultivars . Severalultivars ,28. FurtV. inaequalis genome as it is composed of eleven linkage groups, more than would be expected for a fungus with a base chromosome number of x = 7. However, this map provides a foundation for further genome characterisation using transferable markers such as SSRs and gene-specific markers, and for the development of sequence-characterised markers linked to virulence characteristics and the MAT locus in V. inaequalis.A genetic linkage map is important for investigating genetics of important traits of plant fungal pathogens such as virulence factors, aggressiveness and toxin production and mating type. The map presented here does not span the entire The authors declare that they have no competing interests.XX project leader, provided overall planning for the research, performed data analysis and co-wrote the manuscript. TR maintained fungal isolates and carried out AFLP analysis. DB project consultant, mainly in fungal molecular biology. NH developed six new SSRs and participated in screening isolates for SSRs. LG Assisted in developing new SSR markers. DJS carried out PCR reactions, performed linkage analysis and co-wrote the manuscript. All authors read and approved the final manuscript."}
+{"text": "For Genetic Analysis Workshop 15 Problem 2, we organized data from several ongoing studies designed to identify genetic and environmental risk factors for rheumatoid arthritis. Data were derived from the North American Rheumatoid Arthritis Consortium (NARAC), collaboration among Canadian researchers, the European Consortium on Rheumatoid Arthritis Families (ECRAF), and investigators from Manchester, England. All groups used a common standard for defining rheumatoid arthritis, but NARAC also further selected for a more severe phenotype in the probands. Genotyping and family structures for microsatellite-based linkage analysis were provided from all centers. In addition, all centers but ECRAF have genotyped families for linkage analysis using SNPs and these data were additionally provided. NARAC also had additional data from a dense genotyping analysis of a region of chromosome 18 and results from candidate gene studies, which were provided. Finally, smoking influences risk for rheumatoid arthritis, and data were provided from the NARAC study on this behavior as well as some additional phenotypes measuring severity. Several questions could be evaluated using the data that were provided. These include comparing linkage analysis using single-nucleotide polymorphisms versus microsatellites and identifying credible regions of linkage outside the HLA region on chromosome 6p13, which has been extensively documented; evaluating the joint effects of smoking with genetic factors; and identifying more homogenous subsets of families for whom genetic susceptibility might be stronger, so that linkage and association studies may be more efficiently conducted. Rheumatoid arthritis (RA) is a complex disease with a moderately strong genetic component. The recurrence risk ratio for siblings is typically estimated at around six in Caucasians, but with a broad range of values, primarily because the prevalence in the population is not well characterized . The pre0401, 0404, 0405, 0408, 0409, 1001, 1402, and 1406, with highest risk alleles being bolded [The HLA region on 6p21 has been implicated by numerous studies, and there is consistent evidence that DR alleles contribute to disease risk. The 'shared epitope' hypothesis was proposed by Gregersen et al. [g bolded . This moPTPN22 locus have been shown to confer an increased risk for RA [PTPN22 have been implicated as causing increased risk for RA; with the R620W allele in rs2476601 (hCV16021387) conferring 1.7- to 1.9-fold increased risk to heterozygotes and higher risks to homozygous carriers. Increased risk was also noted for either hCV8689108 or hCV25762283 [PTPN22 alleles to affected offspring in families [Specific autoantibodies are noted to co-occur with rheumatoid arthritis. Rheumatoid factor (RF) IgM is a measure of active disease correlated with erosive arthritic disease. However, a more newly identified autoantibody, anti-cyclic citrullinated peptide (anti-CCP), is more specific for the disease and is a better predictor of erosive outcome . Elevatik for RA . At leasfamilies .PADI4, which encodes the enzyme catalyzing citrullination in macrophages (on chromosome 1p), intron 1 of SLC22A4 on chromosome 5q, RUNX1 (on chromosome 1q), and a locus on chromosome 17 possibly predisposing to psoriasis. Marker data for these other loci could not be obtained, but have generally not shown consistent increases in risk for Caucasian populations. The CTLA4 locus on chromosome 4p has been associated with mildly increased risk for rheumatoid arthritis [Additional loci that have been implicated include rthritis .Aside from identified genetic factors and sex, few environmental cofactors have yet been identified as affecting risk for rheumatoid arthritis. However, current smoking confers about a two-fold increased risk . KlareskThe primary goal of the studies that were submitted for the Genetic Analysis Workshop 15 has been to identify genetic factors that predispose for rheumatoid arthritis. Four independent academic groups and one company have provided data for the workshop. In addition, given some previously identified evidence for effects of smoking on rheumatoid arthritis risk and difference in risk according to gender, there is considerable interest in identify gene \u00d7 environment and gene \u00d7 gene combinations that yield particularly high risks to individuals for rheumatoid arthritis.Data for the workshops were provided by five centers. Two centers (Canada and NARAC) had SNP genotyping performed jointly. The data were transmitted from each center to the University of Texas M.D. Anderson Cancer Center, where the data sets were checked to assure the availability of data definitions and to evaluate the formatting and completeness of the data transfer. Subsequently, data were transmitted to the Southwest Foundation for Biomedical Research for integration and transfer to GAW15 participants. Questions about data integrity or meaning were transmitted to the University of Texas M.D. Anderson Cancer Center, which then interacted with the data providers to obtain answers. All affected subjects in all of the studies met the standard ACR criteria for affection with rheumatoid arthritis [The familial clustering patterns, association with extra-articular findings, and correlation in ages of onset in most of the NARAC collection have been described by Jawaheer et al. . AffecteIllumina performed analysis of about 5600 genome-wide SNPs on all families including 66 families from Katherine Siminovitch, a collaborator in Canada. Results of the analysis of the NARAC Caucasian families were published and indicate previously unreported linkages on chromosomes 2, 4, and 11 along with the known linkage on chromosome 6p .A dense panel of 2719 SNPs were genotyped by Illumina for an approximately 10-kb region of chromosome 18q that showed evidence for linkage in the U.S. and French linkage scans. Of these, 2300 met quality control criteria and have been retained and distributed for analysis. These markers were individually genotyped on 460 cases and 460 controls. Controls were recruited from a New York City population and cases have been recruited from multiple U.S. centers. As a part of the data release process, we also distributed the estimated Northern versus Southern European ancestry of cases and controls [Two quantitative phenotypes that are used for identifying RA-affected individuals include anti-CCP and RF-IgM. The heritability of these measures is hard to obtain from the selected sib pairs we are studying. After proband correction, the heritability estimates are 11% and 30%; before correction the heritabilities are 15% and 67%. Linkage analysis of Rf-IgM and anti-CCP phenotypes with microsattelites  showed sClinical measures that are available include age at onset, sex, ethnicity, presence of erosions, and duration of disease. Data were also provided concerning the physician-reported severity of disease (JAM scores) as well as the patient's functional status (HAQ scores). Smoking increases risk for rheumatoid arthritis, and limited smoking data are available for families and controls. Digitized hand radiographs are available on the NARAC website. The currently available X-ray scores were derived by a single radiologist at the Bethesda Naval Medical Center. Jawaheer et al. studied clinical characteristics of the study subjects .PTPN22 genotypes are available from this collection. All affected subjects from this study met ACR criteria [ECRAF provided high-density microsatellite data from 88 families, including 75 affected sib pairs, 12 affected sib trios, and 1 affected sib quaternion typed with 1089 microsatellite markers . PTPN22 criteria .The UK group led by Jane Worthington and Sally John provided data from analysis of 10,156 SNP markers that were genotyped and passed quality control filters on 157 families . In addin = 86) were recruited from large clinical populations in the Toronto area in collaboration with academic-based rheumatologists. Sibships with affected pairs were also recruited from academic centers in Nova Scotia (n = 72). All affected patients met 1987 revised criteria for RA [The Canadian group, led by Katherine Siminovitch, provided 60 families that have been genotyped using the Illumina platform used by NARAC (performed at the same time as the NARAC study) as well as 79 families (one sib pair had only one affected sibling and is excluded from tabulations) that were genotyped using an Affymetrix 100 K platform. Patients  Does smoking influence severity or age to onset of disease? What are the best procedures for using known covariates such as sex, anti-CCP levels, and shared epitope status to identify genetic loci influencing disease susceptibility?3. Is there evidence for gene \u00d7 environment interactions? Do 4. Do the quantitative variables provide any increased power to identify genetic loci? Although microsatellite data have been analyzed for the quantitative traits, at this time the SNP data have not yet been analyzed.PTPN22 locus on chromosome 1 be identified by linkage?5. Meta-analysis: What are the best ways to combine data across the studies? Is there any strong evidence for gene \u00d7 gene interactions? Is there more than one locus on chromosome 6 influencing disease risk? Can the 6. Association data: Are there any loci on chromosome 18 that reliably predict disease risk? Are there any subsets with particularly high risks for disease?The data that were provided is composed largely of affected sib-pairs. Efforts were made to collect extended relatives when they were available. However, the aggregation of rheumatoid arthritis in families usually occurs in siblings and parents of the proband and only rarely occurs in extended pedigrees. Due to fiscal constraints, only a few of the families from NARAC included unaffected relatives, and none of the other sites provided data from unaffected relatives. A variety of methods are required to unravel the complex genetic and environmental interactions that cause this complex disease. The value of the genetic analysis workshop has been that it brings together analysts with a wide variety of skills and approaches. The data providers were thankful for the opportunity to have the extensive data that have been developed and studied in detail by a wide range of analysts.The authors report the following competing interests relating to this publication. Dr. Lindsey Criswell reports receiving consulting fees from Celera Diagnostics. Dr Ann Begovich is an employee of Celera which funded some of the genotyping work included in this manuscript."}
+{"text": "Genome-wide significant evidence for linkage was observed on chromosome 6. Genome-wide suggestive evidence for linkage was observed on chromosomes 13 and 20 when conditioning on age at onset, chromosome 15 conditional on gender, and chromosome 19 conditional on RF IgM after allowing for multiple testing of covariates.Rheumatoid arthritis is the most common systematic autoimmune disease and its etiology is believed to have both strong genetic and environmental components. We demonstrate the utility of including genetic and clinical phenotypes as covariates within a linkage analysis framework to search for rheumatoid arthritis susceptibility loci. The raw genotypes of 1302 affected relative pairs were combined from four large family-based samples . The familiality of the clinical phenotypes was assessed. The affected relative pairs were subjected to autosomal multipoint affected relative-pair linkage analysis. Covariates were included in the linkage analysis to take account of heterogeneity within the sample. Evidence of familiality was observed with age at onset ( Rheumatoid arthritis (RA) is the most common systematic autoimmune disease and is believed to have both strong genetic and environmental components in its etiology. Females are at a higher risk than males and their age at presentation shows considerable variability . Here weAutosomal microsatellite data for the NARAC, UK, and ECRAF samples were combined with the Illumina single-nucleotide polymorphism (SNP) sample from Canada. The genotypic data were stored in sample-specific files and their marker maps were aligned to improve map correspondence between samples. The Canada and ECRAF loci were placed on the NARAC and UK genetic map using the NCBI physical positions of NARAC, ECRAF, and Canada loci \u2013 see Segurado et al.  for morer2 > 0.05 and separated by less than 5 cM were thinned further until no LD remained.We removed evidence of LD to minimize the chance of excessive false positives . MicrosaHLA-DRB1 on chromosome 6 was also investigated. We defined a binary measure for HLA to represent whether an individual carried a high risk allele, as described in the GAW15 Problem 2 data description [The genetic and clinical phenotypes analyzed were gender (binary), age at onset , definite erosion (binary), and rheumatoid factor (RF) IgM . The ECRAF and Canada phenotype information available was limited to gender and RA status. The RA susceptibility locus The familiality of the phenotypes AAO, definite erosion, and RF in the individuals affected with RA was assessed in a mixed-effects regression framework by taking the phenotype of interest as the dependent variable, implemented in the software packages MIXOR  and MIXRpr, can be modelled in a logistic regression framework and can be written as logit(pr) = O + \u03b1 + \u03b2x, where O is a fixed offset that depends on the relationship between the pair, \u03b1 is a measure of divergence of IBD from the null in the sample as a whole, and \u03b2 incorporates covariate x into the model. Because the parameters pr, O, and \u03b1 are based on pairs of individuals, so must be the covariate parameter. When considering a continuous measure, covariates were constructed for the mean and difference for each pair. A binary measure (- or +) was resolved into either -/-, -/+, or +/+ pairs of individuals. For further information on including covariates in the model and constraining the parameters, see Hamshere et al. [pr, given particular covariates, and then to obtain ARP linkage statistics. Because HLA resides on chromosome 6, no HLA covariate analysis was performed on chromosome 6.Multipoint model-free affected relative-pair (ARP) linkage analysis was performed with the raw phenotypes AAO, definite erosion, RF, HLA, and gender included as covariates . Sample-specific allele frequencies and pair-wise IBD (identity-by-descent) allele sharing probabilities using information from the full pedigree were estimated by MERLIN  at 2 cM e et al. . The IBDFor each chromosome and covariate, two multipoint LOD scores were produced at each 2-cM position: i) the covariate LOD score and ii) a univariate LOD score, in only the ARPs included in the covariate analysis, i.e., excluding those with missing covariate data. An increase in the maximum LOD score over the chromosome  in excess of 2.0 was taken to indicate a potential covariate effect. Empirical significance levels for each LOD score peak in the observed data were obtained as follows: 10,000 replicates of chromosome 22 were simulated in the absence of linkage, using the same pedigree structures, marker locations, marker allele frequencies and missing genotype patterns as the original data. The average number of peaks per chromosome reaching the required height was calculated from these replicates . The number of peaks per genome was approximated by multiplying by 60 . This procedure gives similar results to those obtained by simulating replicates of all 22 chromosomes (data not shown), and is considerably easier computationally. Correction for the multiple testing of six non-independent genome scans was applied as follows. First, criteria were chosen for each covariate to give the same significance level  as the test peak. Then, for each replicate chromosome, the locations and heights of all the peaks from all six covariates were combined into a single list, and the total number of peaks greater than their corresponding criterion was obtained. The distance criterion of 30 cM for defining separate peaks ensured that peaks from several covariates that are close together  were counted only once. The expected number of peaks per genome was calculated as before. Following Lander and Kruglyak [The sample comprised 1302 ARPs informative for linkage from 982 pedigrees, originating from 633 (466) NARAC, 494 (370) UK, 117 (88) ECRAF, and 58 (58) Canada samples of ARPs (pedigrees). The NARAC and UK samples contributed a total of 61 non-sibling ARPs. Estimates of the ICCs indicate the familiality of each phenotype and are presented in Table The highly significant clustering of AAO and RF within pedigrees replicates the evidence observed in monozygotic twins , suggestHLA, which is thought to contribute 30 to 50% of the total genetic component of RA [HLA. Evidence for all four loci was also observed by Etzel et al. [HLA (tested as a binary covariate). This suggests that the regions on chromosomes 12, 16, and 18, if true linkages, may harbor genes that act independently of HLA. Our HLA measure excluded individuals who carried medium but no high risk alleles. Two additional HLA measures were created, where the seven medium risk alleles contribute to 1) the HLA+ group and 2) the HLA- group. The linkage results were unchanged. It is possible that we have lost important HLA allelic information when creating our binary HLA measure. Also, if HLA is not the actual disease gene, but in LD with a disease-causing mutation of relatively large effect, as suggested by the very large LOD on chromosome 6, a covariate analysis with the binary HLA measure may be less powerful than using a covariate based on the IBD information [HLA genotypes. To investigate this further we performed covariate analyses conditional on the chromosome 6 IBD information (30 to 80 cM), producing two regions with an ILOD > 2.0, both having chromosome-wide significance (see Table p = 0.323).The maximum LOD score of 20.73 at 46 cM on chromosome 6 is by far the most convincing evidence for linkage that we observe. This peak is at the location of the genetic locus nt of RA ,13. We al et al.  and in tormation , especiap = 0.04; IBD estimates of NARAC: 0.46, UK: 0.47, ECRAF: 0.61, Canada: 0.58 at 104 cM), suggesting that heterogeneity is minimal. Each of the covariate regions of interest were analyzed in the separate samples, e.g., NARAC only. All regions showed similar effects in the separate samples as in the combined sample. The inclusion of gender, AAO, and RF as covariates produced some potential regions of interest. We observed evidence for an AAO covariate effect on chromosome 1 (expected to occur by chance 1.467 times per genome scan) within 7 cM of an effect observed by analyzing only NARAC and UK individuals with an AAO < 40 years [A benefit of combining the raw genotypes of samples is that the power to detect disease susceptibility loci of small effect is increased. However, it is possible that pooling samples introduces heterogeneity. Incorporating origin of sample as a covariate in the analysis detected evidence of allele sharing heterogeneity only on chromosome 12  the distribution of the continuous covariate is not important and ii) the chromosome 19 RF result may be an artifact of creating an ordinal measure from continuous data and analyzing it as a continuous measure.The RF data we analyzed were ordinal (four levels). This was the highest common form of the data as the original data were a combination of ordinal (UK) and continuous (NARAC). We subjected chromosome 19 to further scrutiny by analyzing the NARAC sample with RF as continuous data. We did not replicate the results. The distribution of the continuous RF data is very highly positively skewed, with no obvious outliers. Of the total sample, 45% of the continuous RF data was coded as 4 (high positive) in the ordinal RF measure. We transformed the data with logr, p << 0.001). Family-wise ILODs > 2 were observed on chromosomes 13 and 20, with both mean and maximum (the minimum measures gave ILODs > 1). OSAs also identified these two regions; chromosome 13 , chromosome 20 . The regions identified with family-wise measures lie within 10 cM and with the same direction of effect as those identified to be genome-wide suggestive (after adjusting for six scans) with the pair-wise measure. The similarity of the results from the three covariate methods is likely to reflect minimal heterogeneity of AAO within pedigrees, corresponding to the highly significant ICC (p << 0.001) presented in Table We also considered two family-based methods that use a single covariate defined for each pedigree: a family-wise ARP method in which the covariate  is assigned to all ARPs from that family and an ordered subset analysis (OSA) in which pedigrees are ranked on their covariate values and sequentially added to the analysis with FLOSS . We focuWe provide evidence to support the familiality of AAO and RF IgM. Incorporating covariates in the linkage analysis allowed us to identify novel regions that may harbor RA susceptibility loci. This information may be useful in conducting future population based case-control analyses, where it will be important to take into account the particular covariate. Replication in independent samples will be important to determine whether our findings are owing to chance alone.The author(s) declare that they have no competing interests."}
+{"text": "Genetic factors make an important contribution to the aetiology of congenital talipes equinovarus (CTEV), the most common developmental disorder of the lower limb. WNT7A was suggested as a candidate gene for CTEV on the basis of a genome-wide scan for linkage in a large multi-case family. WNT7A is a plausible candidate gene for CTEV as it provides a signal for pattern formation during limb development, and mutation in WNT7A has been reported in a number of limb malformation syndromes.We investigated the role of WNT7A using a family-based linkage approach in our large series of European multi-case CTEV families. Three microsatellite markers were used, of which one (D3S2385) is intragenic, and the other two  are 700 kb 5' to the start and 20 kb from the 3' end of the gene, respectively. Ninety-one CTEV families, comprising 476 individuals of whom 211 were affected, were genotyped. LOD scores using recessive and incomplete-dominant inheritance models, and non-parametric linkage scores, excluded linkage.No significant evidence for linkage was observed using either parametric or non-parametric models. LOD scores for the parametric models remained strongly negative in the regions between the markers, and in the 0.5 cM intervals outside the marker map. No significant lod scores were obtained when the data were analysed allowing for heterogeneity.Our evidence suggests that the WNT7A gene is unlikely to be a major contributor to the aetiology of familial CTEV. Congenital talipes equinovarus (CTEV), colloquially known as \"clubfoot\", is a common developmental disorder of the lower limb, with an incidence of 1 \u2013 7 per 1000 births in various populations . CTEV isWnt genes encode a family of highly conserved cysteine rich glycoproteins that play an important role in the normal developmental processes during embryogenesis ,7 and inChildren affected by clubfoot and their parents were recruited through the United Kingdom support group for children with lower limb deformities, STEPS, and through the Dutch support group VOK Fig . All famThe study was approved by the Grampian Research Ethics Committee and the Medical Ethical Committee of the Academic Medical Center in Amsterdam.DNA was extracted from the cheek smears and mouthwashes by using Instagene matrix  and sodium hydroxide, respectively. Three short tandem repeat markers were selected for DNA amplification. D3S2403 and D3S1252 are linked to the gene WNT7A. D3S2403 is 700 kb 5' to the start of the gene while D3S1252 located 20 kb from the 3' end of the gene. D3S2385 is within an intron :D3S2403: 0.0001, 0.0001, 0.0173, 0.0878, 0.0216, 0.0001, 0.0043, 0.0288, 0.1685, 0.0894, 0.5763, 0.0057;D3S2385: 0.0155, 0.2795, 0.472, 0.1988, 0.0342;D3S1252: 0.69, 0.0001, 0.3099.For the heterogeneity analysis, we used the facility of GENEHUNTER to optimise the value of alpha (fraction of linked families) to maximise the LOD score.Genotyping of the three markers was performed in 476 individuals from 91 families, which included 211 affected cases. In the linkage analysis, we used both a simple recessive model of inheritance (genotype penetrances 0/0/1) and an incomplete penetrance dominant model (penetrances 0/0.33/0.33) since both models have been suggested as plausible by previous studies . The datet al. [WNT7A encodes a secreted protein that stimulates LMX-1 to confer dorsal patterning in the developing limb ectoderm . The linet al.  suggesteSamples from the 91 families studied represented 168 affected, and 92 unaffected meioses. It is very unlikely that our ability to detect linkage was comprised by inadequate power, as if inheritance were autosomal dominant with perfectly informative markers, these meioses, could yield a lod of 50 or so at theta = 0. Analyses were performed using both plausible models of inheritance. Heterogeneity analysis gave no evidence that the linkage might be present in a sub-set of families. Such a linkage study cannot exclude genetic variation in WNT7A as a low penetrance risk factor for CTEV, nor can it exclude linkage in rare families or in populations other than those European populations studied. However, this candidate gene was proposed on the basis of such linkage analysis, and this study does exclude it as a major contributor to familial congenital talipes equinovarus.et al. [NAT1 and NAT2 were reportedly associated with CTEV [To date, a small number of genes have been implicated in a small proportion of CTEV families. A mutation (R279W) in the diastrophic dysplasia sulphate transporter gene (DTDST) was reported as the aetiology of CTEV in two sets of siblings of western French ancestry , but thiet al.  identifiet al. . We receet al. . Also reith CTEV . DespiteOur evidence suggests that the WNT7A gene is unlikely to be a major contributor to the aetiology of familial CTEV.The authors declare that they have no competing interests.ZM and LS designed the ECCE study and led its conduct. ZM had the idea to study Wnt-7a. GL, JI and SS selected genetic markers for analysis, designed and performed the molecular assays. ZM, AC, RH and LS designed and implemented the sample collection. DS performed and interpreted the linkage analysis. GL and ZM drafted the paper with input from all authors.The pre-publication history for this paper can be accessed here:"}
+{"text": "Left ventricular (LV) mass and wall thickness are closely associated with measures of body size and blood pressure and also correlated with systolic and diastolic function, suggesting a contribution of common physiologic mechanisms, including pleiotropic genes, to their covariation.Doppler echocardiography was performed in 434 African-American  and 284 white families . We conducted a genome-wide linkage scan for LV mass, LV structure and function, and composite factors derived from a factor analysis of LV structure and function in the HyperGEN Study population.Factor analysis identified (i) a LV wall thickness factor correlated strongly with interventricular septal thickness (IVSTd) and posterior wall thickness (PWTd) and (ii) a LV diastolic filling factor strongly correlated with early and atrial phase peak transmitral filling velocities. The LV phenotypes and composite factor scores were analyzed in multipoint variance components linkage model implemented in SOLAR with 387 microsatellite markers. In whites, the two highest LODs were 3.42 for LV atrial phase peak filling velocity at 144 cM on chromosome 1 and 3.12 for the LV wall thickness factor at 160 cM on chromosome 7. The peak LODs of the component traits (IVSTd and PWTd) clustered at the same region as the composite factor. Adjusting the factor score for body mass index (BMI) substantially reduced the peak LOD at this region (LOD = 1.92). Bivariate linkage analysis of the composite factor with BMI improved LOD to 3.42 at 158 cM. Also in whites, suggestive linkage was observed on chromosomes 2 and 4 for LV mass, chromosomes 3, 5, 10, and 17 for LV atrial phase peak filling velocity, and chromosome 10 for LV diastolic filling factor. In African Americans, suggestive linkage was observed on chromosome 12 for LV mass, chromosome 21 for IVSTd, and chromosome 3 for LV internal diameter at end-diastole.Our study suggests that a region on chromosome 7 contains pleiotropic genes contributing to the variations of both LV wall thickness and BMI in whites. Left ventricular hypertrophy (LVH), due to thickening of LV walls and/or LV chamber enlargement, is a strong predictor of cardiovascular mortality and morbidity . IncreasGenetic influences have been demonstrated for measures of LV mass, wall thickness, and function -10. In tFactor analysis is a multivariate data reduction technique that derives latent phenotypes by identifying variation common to a set of correlated variables. It has been shown to be a useful tool in genetic studies to localize genomic regions contributing to clustering of correlated traits, such as metabolic syndrome-related risk variables . In the HyperGEN is one of four Family Blood Pressure Program networks supported by the NHLBI to identify genetic contributions to hypertension . HyperteTwo-dimensionally (2D) guided M-mode, 2D and Doppler echocardiograms were performed following a standardized protocol developed at the Echocardiography Reading Center at the New York Hospital-Cornell Medical Center . MeasureLV systolic function was assessed by stress-corrected midwall fractional shortening (MWS). LV diastolic filling parameters were measured by pulsed Doppler in apical four chamber view. MVE and MVA obtained at the mitral valve leaflet tips were used in the analysis.The reading center has reported good reproducibility of LV measurements by a protocol identical to that used in the HyperGEN  .A total of 387 polymorphic markers (Cooperative Human Linkage Center Screening set 8) spaced an average of 9.7 cM apart were genotyped by the Marshfield Clinic's Center for Medical Genetics-Mammalian Genotyping Service in Marshfield, WI. A high-throughput scanning fluorescence detector system was used to genotype short tandem repeat polymorphisms. The average heterozygosity of the markers was 0.76. Marker consistency with Mendelian expectations within families was tested using ASPEX , and onl2, and gender in race- and generation-specific linear regression models. Residuals from the linear models were standardized to zero mean and unit variance in each of the four race-by-generation groups. The standardized residuals were included in linkage and factor analyses stratified by race. Participants were excluded from factor analyses if their standardized residuals were \u2265 5 standard deviations (SD) from the means. For linkage analysis, a more stringent exclusion (> 4 SD) was used since linkage analysis is more sensitive to the influence of outliers.Comparison of distributions of demographic and clinical characteristics between race groups was made by generalized estimating equations, which account for familial relatedness. Prior to linkage and factor analyses, each LV variable was adjusted for age, ageA maximum likelihood-based factor analysis was applied to the residuals of PWTd, IVSTd, LVIDd, stress-corrected MWS, MVA, MVE, and isovolumic relaxation time (IVRT). Details of factor analysis modeling can be found in a supplement  . Details2, and gender prior to the factor analysis, the linkage model without covariate adjustment is referred to as minimally adjusted model. Secondly, the following covariates were adjusted for in linkage analysis and this model is referred to as maximally adjusted model: BMI, SBP, diabetes, and current smoking. Heart rate was included in the maximally adjusted model for MVA, MVE, and the factor related to LV diastolic filling parameters. In addition, when a LOD score from the maximally adjusted model was substantially reduced compared to the minimally adjusted model at a position where the minimally adjusted LOD score was above 3.0, each of the covariates was adjusted for separately to delineate the influence of individual covariate adjustment on the linkage signal.In the linkage analysis, LV phenotypes and factor scores were first analyzed without covariate adjustments. Since these variables have been adjusted for age, ageIn the above analysis, if adjustment for an individual covariate had important influence on the LOD score for a LV phenotype, it is possible that this indicates pleiotropic genetic effects between the phenotype and covariate. To test this hypothesis, bivariate linkage analyses  were conFinally, the SOLAR \"lodadj\" procedure was used to estimate empirical single-point p-values for the observed univariate and bivariate LODs. Details on this procedure can be found in the supplement  and 6.8% , respectively. LOD plots from bivariate and univariate linkage analyses on chromosome 7 are shown in Figure \u03c1Q = 0 was rejected (p = 0.0004) and that of \u03c1Q = 1 could not be rejected (p = 1.0). In the bivariate linkage analysis, further adjustment for SBP, smoking, and diabetes slightly increased the peak LOD to 3.57  at the same location.Bivariate genetic analysis between LV wall thickness factor and BMI found significant genetic  and environmental  correlations between the two phenotypes. Based on these correlations, the proportion of total additive genetic variance and environmental variance that is due to shared genes and shared environment was estimated as 7.8%  than the single-trait LOD for MVA. Heart rate alone did not show linkage signal at this region (LOD = 0), suggesting that the strong linkage signal observed for MVA in the minimal adjustment model was likely confounded by loading condition measured by heart rate.There are also significant genetic  and environmental  correlations between MVA and heart rate after adjusting for age, ageTo our knowledge, our study is the first to apply a factor analysis approach to investigate the multivariate correlation architecture underlying LV wall thickness, dimension, and systolic and diastolic function. Furthermore, we conducted a multipoint genome-wide scan for the composite factor scores derived from the factor analysis models to localize genetic loci contributing to the clustering of these traits. We observed measures for LV wall thickness (PWTd and IVSTd) strongly loaded on a factor that was also associated with LV systolic function measured by stress-corrected MWS; LV diastolic filling parameters loaded on a different factor. In the linkage analysis, we identified regions on chromosomes 7q36 and 1p13 that showed strong linkage for the LV wall thickness factor and MVA, respectively. The linkage evidence for LV wall thickness factor is further strengthened by the fact that both individual measures of wall thickness strongly mapped to the same region. This locus had pleiotropic effects on body size as measured by BMI. Based on the genome-wide significance criteria (p = 0.000049 and LOD \u2265 3.3) proposed by Lander and Kruglyak , the LODAt the region on chromosome 7 where the LV wall thickness factor showed strong linkage in whites, LV mass peaked at approximately the same location but with weaker signal in the same population: peak LOD was 0.44 at 156 cM and 0.14 at 155 cM in the minimally and maximally adjusted models, respectively. The difference in magnitude of linkage signal between LV mass and the LV wall thickness factor is likely due to the fact that the locus at this location is mainly for LV wall thicknesses, which is captured at a larger extent by the wall thickness factor than LV mass. Mathematically, the formulae for LV mass  includesIn addition to the strong linkage signals for the LV composite factor scores, we also observed suggestive linkage for LV mass and other structure phenotypes on other chromosomes Table . Mayosi Our observation that LV diastolic filling parameters mainly loaded on a different factor than LV wall thickness and systolic function may reflect differences in the underlying physiologic mechanisms regulating LV diastolic and systolic function. Specifically, the primary physiologic determinant of systolic function is myocardial contractility, whereas it is myocardial relaxation and effective chamber compliance that influence LV diastolic early and late filling, respectively -26. At t2, and gender. These data agree with evidence from other genetic studies of LV mass with body size in twins and families [Since body size is an important correlate of LV mass, it is possible that there are pleiotropic genes contributing to these two phenotypes. In our study, the genetic correlation between LV mass and BMI was 0.35 \u00b1 0.08 (p < 0.05) in African Americans and 0.57 \u00b1 0.09 (p < 0.05) in whites after adjustment for age, agefamilies . For exafamilies . Our stufamilies  and 2.91families . In the families . In our There are 161 annotated genes in the 1-LOD-unit support interval (154 cM to 167 cM) for the peak LOD region on chromosome 7. Within this interval, we identified two possible candidates which may contribute to the variation of LV wall thickness factor and its correlation with BMI: nitric oxide (NO) synthase 3  and AMP-activated non-catalytic protein kinase \u03b32 . NOS3 synthesizes NO and is constitutively expressed in the cardiac myocytes . NO is iWe also searched the Framingham 100K genome-wide association study  databaseIn general, we observed different loci for the LV phenotypes between African Americans and whites. The overall discrepant results may be explained by differences in population characteristics. Specifically, as shown in Table We have observed a locus on chromosome 7 that showed strong linkage signals for the clustering of LV wall thicknesses. This locus also had pleiotropic effects on BMI and height. There are plausible candidate genes in this region that may contribute to the increase of LV wall thickness and body size. If the linkage findings are replicated in other populations, fine-scale genotyping of the identified region, including the candidate genes, is needed to identify the specific genes and variants that are responsible for the observed linkage. Identification of the responsible genes offers promise in understanding novel mechanisms and developing therapeutic strategies in the prevention and treatment of LVH.AA: African American; BMI: body mass index; cM: centimorgan; DBP: diastolic blood pressure; df: degrees of freedom; IVRT: isovolumic relaxation time; IVSTd: interventricular septal thickness at diastole; LOD: logarithm of the odds; LV: left ventricular or left ventricle; LVH: left ventricular hypertrophy; LVIDd: left ventricle internal diameter at diastole; MVA: left ventricular transmitral atrial phase peak filling velocity; MVE: left ventricular transmitral early peak filling velocity; MWS: stress-corrected midwall fractional shortening; PWTd: posterior wall thickness at diastole; RWT: relative wall thickness; SBP: systolic blood pressure; SD: standard deviation; SR: sarcoplasmic reticulum.The authors declare that they have no competing interests.WT conceived and designed the study, performed the statistical analyses, and drafted the manuscript. RBD conceived and designed the study and produced the echocardiographic data. NL interpreted the results. AO conceived and designed the study. DCR interpreted the results. SAC interpreted the results and participated in the revision of the manuscript. DWK, PNH, and DKA conceived and designed the study and interpreted the results.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2350/10/40/prepubSupplemental text and tables. This file provides additional statistical methods, results, and legends for supplemental figures.Click here for fileSupplemental figure 1. This file provides multipoint linkage plots for LV mass and LV structure and function phenotypes in whites.Click here for fileSupplemental figure 2. This file provides multipoint linkage plots for LV mass and LV structure and function phenotypes in African Americans.Click here for fileSupplemental figures legend. This file provides legend for Supplemental figures 1 and 2.Click here for file"}
+{"text": "The Mediterranean island of Sardinia has a strikingly high incidence of the autoimmune disorders Type 1 Diabetes (T1D) and Multiple Sclerosis (MS). Furthermore, the two diseases tend to be co-inherited in the same individuals and in the same families. These observations suggest that some unknown autoimmunity variant with relevant effect size could be fairly common in this founder population and could be detected using linkage analysis.To search for T1D and MS loci as well as any that predispose to both diseases, we performed a whole genome linkage scan, sequentially genotyping 593 microsatellite marker loci in 954 individuals distributed in 175 Sardinian families. In total, 413 patients were studied; 285 with T1D, 116 with MS and 12 with both disorders. Model-free linkage analysis was performed on the genotyped samples using the Kong and Cox logarithm of odds (LOD) score statistic.In T1D, aside from the HLA locus, we found four regions showing a lod-score \u22651; 1p31.1, 6q26, 10q21.2 and 22q11.22. In MS we found three regions showing a lod-score \u22651; 1q42.2, 18p11.21 and 20p12.3. In the combined T1D-MS scan for shared autoimmunity loci, four regions showed a LOD >1, including 6q26, 10q21.2, 20p12.3 and 22q11.22. When we typed more markers in these intervals we obtained suggestive evidence of linkage in the T1D scan at 10q21.2 (LOD = 2.1), in the MS scan at 1q42.2 (LOD = 2.5) and at 18p11.22 (LOD = 2.6). When all T1D and MS families were analysed jointly we obtained suggestive evidence in two regions: at 10q21.1 (LOD score = 2.3) and at 20p12.3 (LOD score = 2.5).This suggestive evidence of linkage with T1D, MS and both diseases indicates critical chromosome intervals to be followed up in downstream association studies. IL7R gene are associated with disease [T1D and MS are common inflammatory disorders which result from an autoimmune attack on the pancreatic beta-cells and the central nervous system, respectively. Both disorders are complex, multifactorial traits resulting from the interplay of largely unidentified predisposing genetic variants, in the presence of unknown environmental factors. Aside from the HLA region, only a few susceptibility variants have been detected in T1D, mainly using candidate gene, and more recently, genome wide association (GWA) strategies . Until r disease ,6.A number of whole genome linkage scans have been performed for both T1D and MS. These have mainly been based on the analysis of affected sib-pairs (ASPs), and have provided, overall, weak and conflicting results. The low power to detect small-size effect variants with low penetrance  represents the most likely explanation for these failures. The likely presence of locus and pathogenic heterogeneity might have further complicated previous efforts. Contrast these disappointing results with the very recent successes of GWA studies ,7 that aP values detected in GWA studies, thus helping to filter false positive results [An alternative, still largely unexplored approach is to concentrate on large, genetically-isolated populations, such as Sardinia, where the diseases of interest are common and where there is evidence of powerful founder effects for all genetic systems so far studied. Notably, Sardinia represents the major exception to the general North-South gradient of both T1D and MS incidence in Europe. In Sardinia, T1D and MS not only have a much higher frequency compared with surrounding Mediterranean regions but they also show an increased probability of co-occurrence, in the same individuals and in the same families, which is only partially explained by shared genotype variation within the HLA complex . The two results .Here, we used a collection of Sardinian multiplex families with T1D and MS (including some in which both disorders were present in the same pedigrees) to perform a whole genome linkage scan.All families selected for this study had Sardinian origins dating back at least three generations from both the maternal and paternal side, as established by interviewing the parents when samples and informed consent were collected. The full dataset included 175 families with at least two affected individuals, which we refer to here as multiplex families, consisting of 413 affected individuals  and a total of 954 samples (pedigrees of the collected families are provided in the online supplement). Since linkage analysis is informative only when there are at least two affected relatives in each pedigree, families with just one case affected by either T1D or MS (simplex) are effective only in the combined analysis of both diseases. As decomposition of the family dataset into the various subsets is complex, with some families containing both MS and T1D cases, a Venn diagram presenting the relationships is shown in Additional file Blood samples and clinical data for T1D patients were collected in the following Clinical Units: Divisione Pediatria, Ospedale G. Brotzu, Cagliari; Istituto di Clinica Medica, Servizio di Diabetologia, Universit\u00e0 di Sassari; Diabetologia dell'Adulto, Ospedale Zonchello, Nuoro; Divisione Pediatria, Ospedale S. Francesco, Nuoro; Divisione Pediatria, Ospedale Crobu, Iglesias; Prima Clinica Pediatrica, Unit\u00e0 Operativa di Diabetologia dell'Et\u00e0 Evolutiva, Cagliari; Centro Diabetologico, Ospedale G. Brotzu, Cagliari.Blood samples and clinical data for MS families were collected at the Multiple Sclerosis Centres in Cagliari. Forty-nine of these families had already been analysed in a previous linkage study . The ranGenomic DNA was extracted from blood using standard salting-out procedures, after which it was coded and stored at -20\u00b0C until used.IDDM12 and IDDM13 loci, the IDDM5 region of 6q16.1-q25.3, and 16q21-23.3 indicated in the large combined linkage study of Cox et al. [A genome linkage scan was conducted for the whole sample set, initially using 474 microsatellite markers  \u00d7 10 times, (30\" at 89\u00b0C/30\" at 55\u00b0C/30\" at 72\u00b0C) \u00d7 20 times, 10' at 72\u00b0C, 15\u00b0C ad infinitum. PCR was performed separately for each microsatellite marker which were then pooled in panels of 10\u201320 markers. After electrophoresis, the PCR fragments were sized and genotyped using the Genetic Profiler software (Amersham Biosciences). Alleles at each microsatellite were then given a numerical value  starting with the allele with the lowest number of base pairs.Genotyping of microsatellites was carried out using a semi-automated method: fluorescently-tagged PCR products were separated on a polyacrylamide polymer using a MegaBACE 1000 DNA capillary sequencer. Microsatellites were amplified by PCR using fluorescently-labelled primers in a reaction volume of 7.5 \u03bcl  in a 2.5 mM MgClBefore performing linkage analysis, genotype data were checked using the following procedures for quality control: 1) Mendelian inheritance was monitored using Pedstats  both forTo calculate the locus-specific sibling risk ratio for disease , identity by descent sharing was estimated using the MapMaker/SIBS programme. For the purpose of this analysis, to estimate the proportion of alleles shared identical by descent in all available affected sib-pairs, complex pedigrees were decomposed into nuclear pedigrees using the Pedmanager programme . To obtaP values were calculated using the Kong and Cox linear model. Information content at various positions along the genome was estimated using Merlin. In order to perform genetic linkage analysis of markers on the X chromosome, we used the X-specific version of Merlin, Minx. Finally, exclusion mapping was performed at the canonical -2 LOD score value, for the hypothesis of \u03bbs = 2 and 3 by using the Genehunter software, version 2.1, release 3 [Linkage of microsatellite markers with T1D and MS was evaluated in the individual disease sample sets and in the two datasets merged together  using the Merlin software. Model-free multipoint linkage analysis was performed using the Kong and Cox modified version of the non-parametric linkage score to identify regions showing evidence for genetic linkage between markers and the presence of disease in families . Allele elease 3 .IDDM1), with a LOD score of 8.3 (P < 1.0 \u00d7 10-5). Estimation of the locus-specific disease risk for siblings of affected patients (\u03bbs) for IDDM1 was 2.81 (CI 1.56\u20135.07). Aside from the HLA region, we found four additional regions showing a LOD score \u22651: 1p31.1 , 6q26 , 10q21.2  and 22q11.22 .The mean distance between markers in the primary map of 474 microsatellites was 7.42 cM; the minimum distance was 0.01, while only three pairs of markers were separated by a gap of >20 cM. Results of the model-free linkage analysis with T1D, MS or both disorders on the whole genome, performed using the Merlin software  are showP = 2.0 \u00d7 10-2), 18p11.21  and 20p12.3 .In the MS_SCAN , we found three regions showing a LOD score \u22651; 1q42.2, , 10q21.2 , 20p12.3  and 22q11.22 . In this T1D-MS_SCAN, evidence of linkage in the HLA region was less significant  than in the T1D_SCAN. This result reflects the modest genetic effect of the HLA region in the familiar clustering of multiple sclerosis.In the combined T1D-MS_SCAN (testing for shared autoimmunity loci) in which all families were analysed jointly, aside from the HLA, four regions showed a LOD >1 for shared autoimmunity loci, including 6q26 .Based on these results, we increased the density of markers in all intervals showing evidence of linkage \u2265 1, aside from chromosome 6q, due to the already high marker density in that region; results are shown in Table P = 1.0 \u00d7 10-3) at marker D10S589. Estimation of the relative risk for 10q21.1 provided a \u03bbs of 1.84 (CI 1.16\u20132.92). For the 22q11.21 region, the maximum LOD score was unchanged, 1.5, but the position of the peak score moved to the nearby marker D22S446 (P = 4 \u00d7 10-3), while in the interval on chromosome 1p31.1 the maximum LOD score decreased to 1.5 at marker D1S207 (P = 4 \u00d7 10-3).In the T1D_SCAN, suggestive evidence of linkage was obtained after increasing the marker density in the 10q21.1 region with a maximum LOD score of 2.1  and at 18p11.22 at marker D18S1158 . The \u03bbs were 4.03 (CI 1.44\u201311.3) and 4.27 (CI 1.48\u201312.32) in these two intervals respectively. For the 20p12.3 region, we found a LOD score of 1.6 at marker D20S194 (P = 4.0 \u00d7 10-3) in these families.In the MS_SCAN, suggestive evidence of linkage was obtained using the enriched map at 1q42.2 at marker D22S446) in 22q11.21 when all T1D and MS families were analysed together, two regions showed suggestive evidence of linkage in this combined T1D-MS_SCAN. Notably, the 10q21.1 region showed a LOD score of 2.3 (P = 6.0 \u00d7 10-4) with a \u03bbs of 1.58 (CI 1.13 \u2013 2.24) at marker D10S589 and the 20p12.3 region gave a LOD score of 2.5 (P = 4.0 \u00d7 10-4) with a \u03bbs of 1.77 (CI 1.22 \u2013 2.56) at marker D20S194.Following analysis of the individual diseases, we then used the enriched marker maps in the intervals showing some initial evidence of linkage to a shared autoimmunity locus to conduct a joint T1D and MS scan. While there was a decrease in the LOD score  declare that they have no competing interests.MP performed microsatellite genotyping of T1D families and participated in microsatellite genotyping of MS families, performed statistical analysis on the data, revised the manuscript and designed all figures and tables. PZ helped in sample collection, supervised and participated in the microsatellite genotyping of T1D and MS families, collaborated in the statistical analysis and revised the manuscript. RM performed microsatellite genotyping of MS families, ED performed, with MP, the bulk of microsatellite genotyping of T1D families, MZ participated in microsatellite genotyping of T1D and MS families, DM collected blood samples and clinical data for T1D families, LM helped in sample collection, extracted DNA from peripheral blood and participated in microsatellite genotyping, CM helped with dataset construction and assisted in automating microsatellite genotyping. VO helped with the sample collection and DNA extraction, DC constructed the dataset of T1D families. GC, ED, ES, EF, LS, MCM, CM, ST, MR and DC participated in microsatellite genotyping of MS families. ML collected blood samples for MS families. MAS and SC extracted DNA from peripheral blood of MS families, RL participated in DNA extraction from peripheral blood, AN participated in DNA extraction. GP, AP, MM, PF, MC, RR, SL, AMM, AFM, NL and MAZ collected blood samples and clinical data for T1D patients. MW assisted in manuscript revision. FS helped with statistical analysis. MGM participated in the study design, led sample and clinical data collection from all MS patients and organised lab activities on the MS samples. MD participated in the study design, assisted in manuscript revision and supervised statistical analysis. FC designed and supervised the study and wrote the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:Family dataset. Set diagram of the families examined in this study. Because the two diseases may occur in the same family, and even in the same patient, there is overlap between the various family sets. The designation \"Two generation\" means that the families are nuclear families with at least two siblings affected by the respective disease. Multigenerational families are families in which there are at least two cases of the disease but the cases occur in different generations. Simplex families have only one case of a given disease (either MS or T1D) and are included here only when they present more than one affected child for the other disease or when considering both diseases together in the same family (searching for shared autoimmunity loci). For each designation, the minimal family structure with respect to each disease is considered. For example, 101 nuclear families with at least 2 siblings with T1D and no siblings with MS were studied, shown as Two generation T1D, as well as 1 nuclear family with at least 2 T1D and 2 MS patients  MS case.Click here for file"}
+{"text": "Idiopathic epilepsy in the Belgian shepherd dog is known to have a substantial genetic component. The objective of this study was to identify genomic regions associated with the expression of generalized seizures in the Belgian Tervuren and Sheepdog.DNA from 366 dogs, of which 74 were classified as epileptic, representing two extended families were subjected to a genome-wide linkage scan using 410 microsatellite markers yielding informative coverage averaging 5.95 \u00b1 0.21 Mb. Though previous studies based on pedigree analyses proposed a major gene of influence, the present study demonstrated the trait to be highly polygenic. Studies of complex disorders in humans indicate that a liberal composite evaluation of genetic linkage is needed to identify underlying quantitative trait loci (QTLs). Four chromosomes yielded tentative linkage based upon LOD scores in excess of 1.0. Possible QTLs within these regions were supported also by analyses of multipoint linkage, allele frequency, TDT, and transmission of haplotype blocks.Taken together the data tentatively indicate six QTLs, three on CFA 2, and one on each of CFA 6, 12, and 37, that support fine mapping for mutations associated with epilepsy in the Belgian shepherd. The study also underscores the complexity of genomic linkage studies for polygenic disorders. Belgian Tervuren (BT) and Belgian Sheepdogs (BS) (collectively referred to as Belgian shepherd dogs outside of the United States) have a long history of idiopathic epilepsy ,2. An eaAs in other breeds -8, the pDue to the similarities of seizure activity between humans and dogs , the useIdentification of a locus linked to the expression of epilepsy would assist in reducing the prevalence of idiopathic epilepsy in the Belgian shepherd and may also provide clues to mutations contributing to epilepsy in other breeds. Genetic testing currently is used by dog breeders seeking to improve the health of their breeds as evidenced by participation in the Canine Health Information Center  while coDog owners who participated in the study were recruited with cooperation from the American Belgian Tervuren Club, the Belgian Sheepdog Club of America, interested breeders, and individual owners. Research protocols were approved by the Institutional Animal Care and Use Committee of University of California, Davis. The present study included purebred Belgian Tervuren (BT) and Belgian Sheepdogs (BS) affected with epilepsy, as well as unaffected dogs. Genomic DNA was obtained by owners submitting three buccal swabs from each dog. Along with the DNA sample, owners provided specific seizure information , veterinary reports, and a three-generation pedigree. The dogs were designated as phenotypically epileptic based on information provided by the owner and veterinarian. Dogs were defined as epileptic if they exhibited repeated \"epileptic seizure phenomenology\" common to generalized seizures based upon the classification system used for humans and applied in dogs .The BT data set consisted of 355 dogs, including 177 dogs for which buccal swab derived DNA was available for analysis and 178 additional dogs included to construct the appropriate pedigrees for analysis  [Genomic DNA was extracted from the buccal swabs  and used/STRand) .Several linkage analyses were undertaken; the first making use of the variance components strategy introduced by Almasy and Blangero . Two-poiij is the observed age at first seizure , \u03bc is an unknown constant, sex is the effect of the i-th sex  and eij is an unknown residual. Computation of the Cox proportional hazards model and the ensuing Martingale residuals was facilitated through the survival package of the public domain language R (version 2.9.1) [where yn 2.9.1) .Epilepsy was evaluated with or without correction for age at onset as well as sex of dog. Algebraically, this yields a model ofij is the observed epilepsy phenotype (yes/no), \u03bc is an unknown constant, sexi is the effect of the i-th sex , \u03b2 is the regression coefficient for age at first seizure and eij is an unknown residual. When age at onset was evaluated as a dependent variable, the effect of the sex of dog was included as an independent variable. For either phenotype, a two-point analysis was done, moving to the multipoint analysis for chromosomes suggested to play an important role in the expression of phenotype.where yAn additional two-point linkage analysis was undertaken with CRI-MAP v2.4 according to published instructions . ApplicaFor the chromosomes of interest, the simple technique, sib-pair analysis, proposed some time ago by Haseman and Elston  was usedRelationships between disease status and the presence or absence of various marker alleles  were evaluated by the Fisher exact test using published criteria for linkage between the marker and a genetic locus for epilepsy  though tHaplotypes were generated with Mendel 8.0 (Haplotype option 3) . The magOf the 436 microsatellite markers used, 410 consistently amplified the genomic DNA  . The linIn some cases single LOD scores were potentially interesting but unsupported by adjacent markers, multipoint, or Fisher exact test. Based upon multiple assessments of linkage and association, six genomic regions on four chromosomes were found to tentatively harbor QTL associated with epilepsy in the Belgian shepherd. The linkage blocks, although large, perhaps represent the best available evaluation using a linkage association based on microsatellites and their incumbent limitations. For example, 13% of the microsatellites specifically chosen for their allele diversity were uninformative for use in the BT and BS populations. In contrast, microsatellites seem to be very useful in the exclusion of potential candidate genes . If applThe rather limited utility of the sib-pair analysis was not unexpected given the design of the experiment as a familial linkage study and the incomplete availability of littermates from a given breeding. However, all the data taken together do provide compelling evidence of suggestive QTLs in influencing the expression of epilepsy in the Belgian shepherd dog. From the present study microsatellites appear to lack resolution to clearly identify possible causal mutations for a complex, variable phenotypic disorder. Fine mapping of these regions will require greater saturation of markers to generate greater haplotype diversity using the genetic structure of the dog .There are numerous positional candidate genes, based upon their involvement in human epilepsy, possible for these six regions. Syntenic to CFA 2 are the human genes BAFME2   and EIG5The absence of robust association may be due to the polygenic nature of the disease as noted before. Further aspects that confound the study of epilepsy are the accuracy of seizure characterization, the subtle differences in seizure presentation that while appearing to be common in terms of expression represent differences in underlying genetic causality, and the classification as idiopathic epilepsy in the absence of a positive diagnosis. Recent reports of canine epilepsy suggest that the genetics involved in seizures may be distinctly different within the different lines of a single breed . AdditioThe composite data presented here suggests six chromosomal regions on four chromosomes may tentatively harbor QTLs involved in generalized idiopathic epilepsy in the Belgian shepherd dog. Fine mapping is necessary using single nucleotide polymorphisms which should enable the identification of genes and mutations in predisposing a dog to seizure activity.AMO conceived the study, study design, coordination, and drafted the manuscript. JMB participated in coordination, carrying out methods including choosing microsatellites, constructing pedigrees, genotyping, manuscript revision and statistical analyses. DIG participated in carrying out methods including choosing microsatellites, constructing pedigrees, and genotyping. KRR participated in carrying out methods including choosing microsatellites, constructing pedigrees, genotyping and statistical analyses. TRF participated in conceiving the study, study design and statistical analyses. All authors read and approved the final manuscript.Subset of Belgian Tervuren pedigree used in linkage analysis. An example of the dogs used in the genome-wide linkage analysis. Filled in symbols reflect dogs classified as epileptic, open symbols reflect dogs known to be unaffected, and symbols labeled with a \"?\" reflect dogs classified as having an unknown status.Click here for fileAge Distribution for age at onset of Epilepsy in the Belgian Tervuren and Belgian Sheepdogs. The data present the characteristic ages of epilepsy diagnosis.Click here for fileGenome-wide association of 410 microsatellite markers with epilepsy in Belgian Tervuren dogs. This table lists the details of all markers used in the microsatellite linkage evaluation.Click here for file"}
+{"text": "We conducted a linkage analysis in high myopia families to replicate suggestive results from chromosome 7q36 using a model of autosomal dominant inheritance and genetic heterogeneity. We also performed a genome-wide scan to identify novel loci.Genehunter 2.0 and Merlin 1.0.1. Two autosomal recessive, two autosomal dominant, and four autosomal additive models were used in the parametric linkage analyses.Twenty-six families, with at least two high-myopic subjects  in each family, were included. Phenotypic examination included standard autorefractometry, ultrasonographic eye length measurement, and clinical confirmation of the non-syndromic character of the refractive disorder. Nine families were collected de novo including 136 available members of whom 34 were highly myopic subjects. Twenty new subjects were added in 5 of the 17 remaining families. A total of 233 subjects were submitted to a genome scan using ABI linkage mapping set LMSv2-MD-10, additional markers in all regions where preliminary LOD scores were greater than 1.5 were used. Multipoint parametric and non-parametric analyses were conducted with the software packages No linkage was found using the subset of nine newly collected families. Study of the entire population of 26 families with a parametric model did not yield a significant LOD score (>3), even for the previously suggestive locus on 7q36. A non-parametric model demonstrated significant linkage to chromosome 7p15 in the entire population . The interval is 7.81 centiMorgans (cM) between markers D7S2458 and D7S2515.The significant interval reported here needs confirmation in other cohorts. Among possible susceptibility genes in the interval, certain candidates are likely to be involved in eye growth and development. Myopia is currently divided into low to moderate myopia  and high myopia (beyond \u22125 D). The prevalence of myopia varies moderately in Western countries, ranging from 16% in Australia and 18% in the Netherlands, to an average value of 25% in the USA in adults between 40 and 80 years old  are affeHGF (hepatocyte growth factor) in mouse [Increasing prevalence and associated health costs ,6 make min mouse  and man in mouse .Our previous studies analyzed the inheritance of high myopia, suggested an autosomal dominant model with weak penetrance , and fouVolunteers received information about the study in agreement with the Helsinki Declaration principle. Participants were included according to the French laws governing participation in biomedical research and with approval of the local ethics committee. Each participant provided written, informed consent. Families of high myopia were enrolled through a proband affected with non-syndromic high myopia . The family was retained for the study if it included at least two high myopic members and if at least three members on two generations agreed to participate. Exclusion criteria were myopia in prematurity retinopathy, Marfan\u2019s syndrome, Stickler\u2019s syndrome, Wagner\u2019s disease, retinitis pigmentosa, corneal dystrophy, keratoconus, myopia secondary to cataract, or any developmental genetic disorder. In addition, families with unilateral high myopia and families with X-linked compatible high myopia were excluded.Twenty-six families were included in this study (nine new families and 17 previously studied families ). The neRefractive values (RVs) were measured using standard autorefractometry after dilation. In patients under 16 years old, three instillations of 1% cyclopentolate at 0 min, 5 min, and 10 min were performed and RVs were measured 45\u201360 min after the last instillation. In patients over 16 years old, dilation was induced with 1% tropicamide at 0 min, 5 min, and 10 min, and the RV measured 30\u201345 min later. Tropicamide (1%) was instilled every 5 min three times, and then measurement is performed. Axial length was evaluated by A-scan ultrasonography. A total of three readings were taken for each eye, and the average value was recorded. Astigmatism was assessed by autorefractometer.  The included patients had minimal astigmatism, indicating that bias was avoided. Each proband had a complete ocular examination including visual acuity, intraocular pressure, and fundus, to confirm that the high myopia was a primary finding and in completion of a general physical examination to avoid recruitment of syndromic myopia patients. For the probands\u2019 relatives, ophthalmologists of their choice were asked to examine subjects according to the standard protocol above. In the case of cataract surgery, only the ocular measurement before surgery was considered.NCBI) and GDB databases.DNA extraction from venous blood was performed according to standard phenol-chloroform extraction procedure . For gen2, 200\u00a0\u00b5M each dNTP, 250 nM specific primer to each marker, 0.28 U/\u00b5l GoTaq DNA polymerase (Promega), and 25 ng of genomic DNA. Fluorescent labeled amplification products were electrophoresed on an ABI PRISM\u00ae 3100 Genetic Analyzer with GeneScan 500HD ROX standard size label (Applied Biosystems) and analyzed with Genescan 3.5 software (Applied Biosystems). Alleles were analyzed with Genotyper 3.6 program (Applied Biosystems).Amplifications of microsatellites were performed in 10\u00a0\u00b5l total volume, with 1X GoTaq PCR buffer , 2.0\u20132.5\u00a0mM MgClMerlin 1.0.1 program [Marshfield Center for Medical Genetics. The order consistency was compared to the NCBI physical map (Build 36.2). The effects of ethnic origin, sex, and age were compared between affected and unaffected individuals using a Fisher\u2019s exact and student\u2019s t-tests with a level of significance below the p value of 0.01. In this study, RV is a spherical equivalent . The RV correlation between right and left eye was equal to 0.95. The RV mean was used for status qualification and as the trait in quantitative trait loci (QTL) analyses. Subjects with a RV mean of less than or equal to \u22125 D were considered to be affected. The others persons were classified as unaffected.Familial relationship inconsistencies and genotyping error checking and cleaning were performed with the  program ,45. MarkGenehunter 2.0 [Merlin 1.0.1 programs [Multipoint parametric linkage (PL) and non-parametric linkage (NPL) analyses were performed under multiple models with nter 2.0  and Merlprograms . A totalMerlin 1.0.1 using the regression option. The sex, age, and ethnic origin were included as covariates.Myopia was also treated as a quantitative trait. The RV mean was transformed to reach normality . A QTL aA total of 26 families and their DNAs were collected from all over France. Twenty-five families were of French origin and one family (of 13 members) was of Algerian origin. The entire cohort included 347 individuals. The population characteristics are shown in Clinical data were collected for the 233 genotyped individuals. Among them, 45% were men and 55% women. Age distribution was similar in both genders (78% of men and 75% of women were older than 20 years). Eighty-one adults over 20 years old (32 men and 49 women) and 17 patients under 20 years old (6 boys and 11 girls) had high myopia. The unaffected group was formed by 97 adults over the age of 20 years (49 men and 48 women) and 38 individuals under the age of 20 years (16 boys and 14 girls). No significant effect of sex (p=0.02), age (p=0.03), or ethnic origin (p=1) between affected and unaffected persons was observed.No significant or suggestive linkage was observed in parametric linkage (PL) and non-parametric linkage (NPL) analyses of the nine novel families in any model tested. No significant  or suggestive  [The PL analysis did not show any suggestive or significant linkage to any locus. Results of the NPL analysis are summarized in The definition of high myopia threshold is to some extent arbitrary. Thus, we examined the influence of the threshold used to define high myopia on the non-parametric results. Moderate myopic persons (with RV between \u22123 and \u22125 D) were considered affected in PL and NPL analysis. A suggestive linkage in 7p15.2\u201315.3 region was observed when the myopic threshold was equal to \u22126 D (p=0.0006), \u22123 D (p=0.00003), \u22122 D (p=0.00004), and \u22121.5 D (p=0.0006).Merlin 1.0.1 gave an estimation of heritability of 10% for the RV mean. The axial length was only available for 122 subjects with clinical examination, thus this trait was not used in the QTL analysis.The RV mean for the entire cohort was considered for a QTL analysis but did not give any significant or suggestive linkage. The aim of this study was to confirm previously reported linkage positions. The results of the genome wide scan (GWS) linkage study on the nine new families or on all 26 families together do not support the suggestive linkage to 7q36 region. The inclusion of new families and the enlargement of previously recruited families considerably modified the structure of the cohort. Although these data do not formally exclude the linkage of a high myopia locus on chromosome 7q36, it is likely that other myopia susceptibility loci play a more important role in the families that we studied. Our inability to confirm the previously identified position probably reflects the inclusion of more families with a larger sample size and of additional markers. Genotyping errors in the samples might vary between the study conducted by Naiglin et al.  and the Another interesting finding is on chromosome 7p15 because linkage signal was detected in NPL analysis even when applying different definitions of the myopic phenotype. However, no significant linkage result in this region was found in any of the PL models used even under the hypothesis of genetic heterogeneity. Even if the NPL approach is robust in the face of uncertainty about the mode of inheritance ,49, the The qualification of quantitative RVs as myopia, emmetropia, or hyperopia is to some extent arbitrary. A quantitative trait analysis was performed. This approach is generally more informative and powerful because continuous data on refractive errors  provide more information than discrete trait data . In our cohort, the QTL analysis did not confirm the NPL result. The weak signal of the NPL analyses of binary traits may be due to low power. This paper highlights the complex heredity of high myopia and the great difficulty of replicating mapping results.NPVF) lies in our candidate region on 7p15.2-p15.3. The molecule encoded by this gene belongs to the family of neuropeptides with the Arg-Phe-amide motif at their C-termini (RF-amide peptides) involved in various neurotransmission/neuromodulation processes and muscle contraction control. It is expressed in the rat and human central nervous system [NPY) are encoded by genes present within the interval [HGF) polymorphism with high myopia. The overexpression of SNX15 leads to decreased cleavage of both insulin and HGF receptors [SNX10. Further studies will be needed to determine putative association between such genes and the myopia phenotype within these families.Sixty-seven genes or putative coding sequences are contained in the significant 7p15 region detected in this study (about 7.81 cM long). Candidate genes could be similar in function or structure to genes in other loci intervening in high myopia. Neurotransmitters can modulate experimental myopia . A neuros system  and in ts system . Interess system . Gamma-as system  have deminterval . Recentlinterval  demonstreceptors , thereby"}
+{"text": "In contrast, CHROMSCAN evades heavy Bonferroni correction and auto-correlation between SNPs by using composite likelihood to model association across all markers in a region and permutation to assess significance. Analysis by CHROMSCAN identifies a 36-kb interval that includes the most significant SNP (msSNP) observed in a 10-Mb target suggested by linkage. Unexpectedly, stratification by gender and age of onset shows that association evidence comes almost entirely from females with age of onset less than 40. Combining evidence from a meta-analysis of linkage studies and three subsets of the NARAC data provides significant evidence for a determinant of rheumatoid arthritis in a 36-kb interval and illustrates the principle that estimates of location and its information are more powerful than estimates of p-values alone.Although single chi-square analysis of the North American Rheumatoid Arthritis Consortium (NARAC) data identifies many single-nucleotide polymorphisms (SNPs) with  Recent developments of linkage disequilibrium units (LDU), composite likelihood, control of auto-correlation, and meta-analysis are incorporated into the CHROMSCAN program [Initially, linkage mapping dealt with rare and highly penetrant genes. Without cytogenetic assignment, the preferred strategy was segregation analysis to determine all relevant parameters except recombination, followed by linkage analysis to determine recombination frequency . Complex program ,4 to incThe data, provided by NARAC (North American Rheumatoid Arthritis Consortium) consist of 2300 single-nucleotide polymorphisms (SNPs) in a 10-Mb region of 18q21 with linkage evidence in U.S. and French scans . Illuminerg test  were remith SNP interval is given by \u03b5idi, where \u03b5i describes the exponential decline of association with physical distance di in kb. Values of \u03b5i are estimated by composite likelihood that fits the Malecot model [M), and residual association at large distance (L) to predict rho (\u03c1), the probability of association.The theory for constructing LDU maps has been described . Brieflyot model  to multi\u03b5\u0394(Si - S) to estimate the location (S) of a disease gene, where Si is the location of the ith marker in kilobases or LDU. The Kronecker \u0394 is used for map direction and assures a correct sign, with \u0394 = 1 if Si \u2265 S or -1 if Si <S. To calculate the expected association with distance, zi, the model becomes M is diminished by complex inheritance and L is the association at large distance. The observed association ad - bc \u2265 0 and b \u2264 c is ensured by rearrangement of columns and rows [zi are the observed and expected association values, respectively, at the ith SNP. Their squared difference is weighted by information (Ki) which is estimated as: The CHROMSCAN program  uses a mand rows . Given tM = 0 with predicted L [H0 in which there is no association between affection status and SNPs. Model D estimates M, S, and L. Therefore the \u039bA - \u039bD comparison tests for a disease determinant at location S. For both models, \u03b5 is fixed to 1 for the LDU map and to a value of \u03b5 determined from pairwise marker-by-marker association data for the kilobase map. In order to account for autocorrelation between SNPs as a result of LD, the significance of evidence is determined by a rank-based permutation test [Sub-hypotheses of the Malecot model are used to test for a causal polymorphism. Model A, which estimates none of the parameters and uses dicted L , is takeion test .H1 is reliable. To minimize computation time, the initial analysis was restricted to 100 replicates. Significant regions identified by the initial screen were re-analyzed separately using 1000 and 5000 replicates in order to verify convergence. To demonstrate the power of LDU maps, this analysis was repeated using the kilobase map and two estimates of the exponential decline \u03b5 derived from the significant region and the 10-Mb region [Three separate analyses of the data were performed by CHROMSCAN. The first is a preliminary screen of the entire 10-Mb bin, which is divided into 18 nonoverlapping regions, each with at least 30 SNPs and covering at least 10 LDUs. To determine accurate levels of significance, the number of permutation replicates must approach the actual level of significance so that interpolation of the variance under b region . The risb region . Our thip < 0.05, none of which reach significance after Bonferroni correction (0.05/2293). The initial screen by CHROMSCAN divides the 18q21 bin into 18 nonoverlapping regions. Although the most significant SNP  occurs in region 6, the next msSNP in region 11 is deceptively close in terms of significance, and several other regions contain suggestive SNPs (Table p = 0.01259). The intensive screen of region 6 identified a large increase in significance between 100 and 1000 replicates, which is attributed to the relationship between number of replicates and significance, while the small decrease in significance between 1000 and 5000 replicates suggests that convergence has been achieved (Table S) at 53308 kb.Single chi-square analyses of the 10-Mb region identifies 125 SNPs with \u03b5 [\u03b5, corresponding to the 10 Mb interval (0.021) or region 6 alone (0.031), were investigated. Despite the large difference between \u03b5 values for the kilobase map, the significance level and location were almost identical. However, the ratios of The CHROMSCAN analysis of region 6 was repeated using the kilobase map so that its performance can be compared with the LDU map. Using a kilobase map requires specification of the exponential decline \u03b5 . Two valerval 0.01 or regiBecause King et al.  demonstrp-values without estimates of location or standard errors. Without this information, the power for meta-analysis is reduced because the sum of two 1 instead of weighting estimates of location by their information. Perhaps because of this inefficiency, the combined LOD1 from this meta-analysis is 1.542, well below the conventional value of 3 for asserting significance. The corresponding p-value in large-sample theory is 0.007714, providing strong but inconclusive evidence for localization in the 18q21 region. Despite its limitations, linkage contributes evidence that should not be ignored.Choi et al.  reportedn independent samples, the ith of which contributes a Pi value that on the null hypothesis is uniformly distributed. Then -2 ln Pi would be distributed as Si and information Ki, but has three disadvantages; first, equal weight is given to samples with different standard errors; second, there is no test of homogeneity; and third, there is no point estimate to become more precise as n increases. As a consequence, much information is lost. Accepting these limitations and assuming accuracy of the P estimates, Table 1 with no Bonferroni correction is 2.676 for linkage and pooled association. When location and information weight are available, the evidence for association is combined by determination of the difference between n - 1 degrees freedom where 1 = 3.401, p = 0.000076). Even with conservative adjustment of the p-value to account for the 18 regions tested by association (18*0.000569), and despite strong although not formally significant, evidence from linkage for at least one causal gene in the 18 regions, the meta-analysis is supportive . We conclude that evidence for region 6 is probative, with linkage and association both providing critical evidence despite lack of a point estimate and information weight for linkage.The simplest meta-analysis is based on S \u00b1 1.96 (SE), covers 36 kb between 53296 and 53332 kb and includes the msSNP rs3745064. Although no described genes are within this region, it does include four human mRNAs from GenBank: CR590917, AK021217, AK124558, and BC01314, all to the left of point estimate (S). Of these, CR590917 appears to be the most interesting because it is expressed within T cells and could therefore conceivably affect risk for rheumatoid arthritis. Finally, geneid [S). However, nothing is known about the function of this gene and its reliability is questionable. The fascinating directions revealed by these findings have yet to be explored. Ultimately, interaction with other contributing loci and environmental factors will be recognized and, more importantly, locus-specific treatment will be found.This application demonstrates that CHROMSCAN is a powerful approach for gene mapping in complex inheritance, which is applicable to meta-analysis. Obvious extensions include identification of a causal locus and more precise definition of the phenotype associated with it. The 95% confidence interval, given by , geneid  predict S and its information K. One manuscript presented in GAW15 that used meta-analysis without those estimates failed to detect the strong signal on chromosome 18q demonstrated by composite likelihood [Recent papers testify to growing interest in meta-analysis, looking backward to linkage rather than forward to association mapping. Rank permutation provides a valid significance test, but the genome search meta-analysis (GSMA) that uses regional assignment with arbitrary weights cannot give a reliable estimate of effect and therefore has low power for estimating point location and detecting heterogeneity ,17. MostThe author(s) declare that they have no competing interests."}
+{"text": "The search for susceptibility genes in autism and autism spectrum disorders (ASD) has been hindered by the possible small effects of individual genes and by genetic (locus) heterogeneity. To overcome these obstacles, one method is to use autism-related subphenotypes instead of the categorical diagnosis of autism since they may be more directly related to the underlying susceptibility loci. Another strategy is to analyze subsets of families that meet certain clinical criteria to reduce genetic heterogeneity.In this study, using 976 multiplex families from the Autism Genome Project consortium, we performed genome-wide linkage analyses on two quantitative subphenotypes, the total scores of the reciprocal social interaction domain and the restricted, repetitive, and stereotyped patterns of behavior domain from the Autism Diagnostic Interview-Revised. We also selected subsets of ASD families based on four binary subphenotypes, delayed onset of first words, delayed onset of first phrases, verbal status, and IQ \u2265 70.When the ASD families with IQ \u2265 70 were used, a logarithm of odds (LOD) score of 4.01 was obtained on chromosome 15q13.3-q14, which was previously linked to schizophrenia. We also obtained a LOD score of 3.40 on chromosome 11p15.4-p15.3 using the ASD families with delayed onset of first phrases. No significant evidence for linkage was obtained for the two quantitative traits.This study demonstrates that selection of informative subphenotypes to define a homogeneous set of ASD families could be very important in detecting the susceptibility loci in autism. To meet the criteria for a diagnosis of autism (Mendelian Inheritance in Man [MIM] 209850), a neurodevelopmental disorder, a child must exhibit impairments in social interaction and communication, as well as show restricted and stereotyped patterns of behavior and activities . These sIn comparison, autism subphenotypes may have advantages over simply employing the diagnosis of autism for genetic mapping. Subphenotypes  are defiet al. ; Silverman et al.  and repetitive behaviors and stereotyped patterns domain total scores ; and Kolevzon et al. (p < .0005) and communication domain total scores  were also familial using data from affected monozygotic twins. In addition, Sung et al.  also provides evidence of genetic heterogeneity in ASD. Studies have shown that some evidence for linkage for ASD was obtained only in the subsets of ASD families defined by certain subphenotypes, such as the language-related subphenotypes , developAlthough the above genetic analyses using the autism subphenotypes have shown some promising results, the susceptibility genes for autism and the subphenotypes remain evasive, possibly due to the low statistical power of modest numbers of families. Previously, we reported linkage results for the categorical diagnosis of autism (defined in a broad and narrow way) using 1181 families from the Autism Genome Project (AGP) Consortium data . In the et al. . Even though our linkage analyses used autism subphenotypes rather than the categorical diagnosis, to reduce genetic (locus) heterogeneity, families were included in the linkage analysis if they had at least two individuals diagnosed with ASD. Based on the results of Risi The following six subphenotypes were used for the linkage analyses: a best estimate IQ and five measurements from the ADI-R, including reciprocal social interaction domain total scores (SOC); restricted, repetitive, and stereotyped patterns of behavior domain total scores (BEH); age of first words; age of first phrases; and verbal/nonverbal status. These subphenotypes were only available for the individuals with ASD.The ADI ages of first words and phrases were specified in months for most individuals, but some individuals had been coded into groups. For example, code 993 was used for an individual who had some words then lost them. Due to the difficulties of imputing these \u201c99x\u201d codes into exact ages, these two variables were categorized into binary traits. Individuals who had delayed onset of first words (>24 months or the ADI-R code = 994 or 997) or first phrases (>36 months or the ADI-R code = 994 or 997) were coded as affected for the traits DelayedWord and DelayedPhrase, respectively. Associations between the pairs of the autism subphenotypes were tested. Chi-square tests were used for the pairs of categorical variables; nonparametric Kruskal-Wallis tests were used for the pairs of categorical and continuous variables, while Spearman rank correlation tests were used for the pairs of continuous variables. The effects of the six potential covariates on SOC and BEH were tested using mixed linear models with family as a random effect . There were four categorical covariates: AGP site, gender, best estimate IQ , and verbal/nonverbal status . There were two continuous covariates: the age and calendar year of ADI-R completion. These covariates were chosen based on the literature and our preliminary review of the data. Due to nonnormality, rank transformation was applied to SOC and Box-Cox transformation to age of ADI-R completion.http://www.affymetrix.com/products/arrays/specific/10k.affx, and details of quality control can be found in http://compgen.rutgers.edu/maps/) (http://www.sph.umich.edu/csg/abecasis/Merlin/index.html) assumes a no-interference model, the Kosambi map was converted into the Haldane map for linkage analyses while all results were reported on the Kosambi scale. The marker allele frequencies were calculated using the founders from the inferred Caucasian families by Haploview (http://www.broad.mit.edu/mpg/haploview/index.php)  10K single nucleotide polymorphism (SNP) arrays at the Translational Genomics Research Institute . Detaileu/maps/)  was usedu/maps/) . Becausedex.php) .p values were reported for these linkage analyses. For the most significant linkage results from the subset analyses, the FLexible Ordered SubSet  software was used to generate empirical p values  . Multipop values . More deThere were 976 families selected for final analyses . The associations between the two quantitative traits and the binary traits were different. The subphenotype SOC was positively associated with DelayedWord and DelayedPhrase but negatively associated with Verbal and IQ \u2265 70 . In other words, the mean scores for SOC were higher in the affected groups defined by DelayedWord and DelayedPhrase than the mean scores in the unaffected group but were lower in the affected groups defined by Verbal and IQ \u2265 70 than the mean scores in the unaffected group. On the other hand, BEH was not associated with DelayedWord (p = .7), DelayedPhrase (p = .6), and IQ \u2265 70 (p = .9) but was positively associated with Verbal . As expected, DelayedWord and DelayedPhrase were positively associated with each other  and Verbal and IQ \u2265 70 were positively associated with each other, but the two sets of traits were negatively associated with each other .p < .05) using a mixed linear model. These covariates were AGP site, verbal/nonverbal status, age of ADI-R completion, and best estimate IQ and together they accounted for 23% of the total variance in the model. The covariates AGP site, verbal/nonverbal status, age of ADI-R completion, and gender were significantly associated with BEH (p < .05) and together accounted for 12% of its total variance. The final set of covariates was selected based on both the strength of association with the subphenotypes and the number of missing values for a particular covariate. For example, best estimate IQ was a very important covariate for SOC in the simple mixed linear model. When covariates verbal/nonverbal status, AGP site, and age of ADI-R completion were included in the multiple model, the association between best estimate IQ and SOC was still significant but with IQ accounting for less than 1% of the total variance of SOC. Since the inclusion of best estimate IQ would reduce the sample size by approximately one quarter due to missing values in IQ, it was not included as a covariate for SOC. p = 5 \u00d7 10\u22129) for SOC and .52 (p = 5 \u00d7 10\u221217) for BEH.Four of the six potential covariates were significantly associated with SOC  (p = .003). The next strongest linkage signal was on chromosome 11p15.4-p15.3 for DelayedPhrase  (p = .007) for DelayedWord, 1.89 (p = .002) for Verbal, and 2.15 (p = .0008) for IQ \u2265 70. There was also a LOD score of 2.18 (p = .0008) at this region when all the ASD families were used. The linkage results for individual chromosomes are provided in The genome-wide linkage results for the seven traits are illustrated in ] = .25) . Interes\u03b4 = .19) . In thisp = .00001 for the ASD families with IQ \u2265 70 and LOD score = .08, p = .3 for all the ASD families), the probability that a subset of 313 families randomly selected from all the ASD families could reach a LOD score of 4.01 at this locus was .0006 with a 95% confidence interval of .0004 to .001. For the most significant linkage result on chromosome 11 , the probability that a subset of 412 families randomly selected from all the ASD families could reach a LOD score of 3.40 at this locus was .03 with a 95% confidence interval of .02 to .05.Using permutation tests, for the most significant linkage result on chromosome 15  in a study by Duvall et al. (p = .0002) for the locus on chromosome 11 using the ASD families with DelayedPhrase and from 4.01 to 2.59 (p = .0003) for the locus on chromosome 15 using the ASD families with IQ \u2265 70.The two most significant chromosomal regions  in this study were also identified in several previous studies even though their signals were not as strong. Spence et al.  showed el et al.  used famp = .00007) and the most significant rs1454985 at 24.8 cM .Our linkage signal at the 15q13.3-q14 region  is about 10 cM (or 6\u20137 Mb) telomeric to the 15q11-q13 region, which has been a focus of many association studies due to the interests in the \u03b3-aminobutyric acid receptor \u03b1, \u03b2, and \u03b3 subunit genes in this region . Most inAutism spectrum disorders and schizophrenia are two distinct diseases according to DSM-IV  and ICD-Caution should be taken when interpreting the results of this study. First, the linkage signals on chromosomes 11 and 15 may be due to either a subset of ASD cases with delayed onset of first phrases and normal or high IQ or more general language and IQ loci, which also exist in the absence of ASD. Second, the results are based on a total of seven genome-wide scans for seven traits. Because of the correlations between the traits and the subset analyses, the final number of tests is equivalent to about 5.75 independent genome-wide scans . StrictlUsing the quantitative subphenotypes, SOC and BEH, we did not find significant linkage signals, even though we found reasonably high heritabilities for both traits (.35 for SOC and .52 for BEH). Ascertainment bias, where a pedigree was selected only if there were at least two individuals above thresholds for both the social and communication total scores, may have a large impact on heritability estimates . No asceIn addition, in a multisite study of this nature, variation in ascertainment by site may add further complexity to the analysis. To identify loci that specifically affect SOC and BEH, the effects of age of ADI-R completion, gender, verbal/nonverbal status, and AGP site on the phenotypes were removed in the linkage analyses . HoweverEven though the analysis method using subsets of ASD families may be limited by chance findings due to reshuffling of the families and by decreased power due to reduction of sample size, the two most significant linkage signals on chromosomes 11 and 15  show that it is still a potentially useful method to overcome genetic heterogeneity. Autism spectrum disorder families with IQ \u2265 70 may represent a more genetically homogenous group, while the families that have rare single gene disorders, undetected chromosomal abnormalities, or de novo copy number variations tend as a whole to have lower IQ  and inclThis study is our first attempt at using the pooled multisite data to localize genetic locations using autism subphenotypes. It is apparent that pooling data from multiple sources to increase sample size is not a panacea due to the possible presence of genetic heterogeneity . HoweverThe Autism Genetics Cooperative: Canagen: SEB, JB, X-QL, ADP, WR, PS, SWS, APT, and LZ; AGC Data Coordinating Center : CBa and VJV; Miami Institute for Human Genomics: MLC, JRG, and MAP-V; Paris Autism Research International Sibpair (PARIS) Study: CBe, CG, and MLe; Seaver Autism Research Center: JDB and KLD; Stanford University: JH and LL; Vanderbilt University: JLH and JSS; University of North Carolina/University of Iowa: JP, VS, and THW.The Autism Genetic Resource Exchange Consortium: RMC, DHG, and JM.The Collaborative Programs of Excellence in Autism (CPEA) and Autism Centers of Excellence (ACE): HC, BD, GD, AE, OK, JM, GDS, EMW, and WMM.The International Molecular Genetic Study of Autism Consortium (IMGSAC): France: CM, BR, and KW; Germany: SB, CMF, SMK, AP, and FP; Greece: KP and JT; Italy: AB and EM; The Netherlands: HVE and MdeJ; UK: AJB, GB, PFB, TB, ALeC, JG, JAL, MLa, APM, HM, AP, JRP, KR, MLR, EW, and SW; USA: CC, EHC, SJG, VH, BLL, CL, JS, and FV; Canada: EF.Other AGP sites: Dublin: SE, AG, LG, and MG; Indiana (USA): CJM, JIN, and DP; Portugal: GO and AMV.Drs. Xiao-Qing Liu, Andrew D. Paterson, Peter Szatmari, and all participating members of AGP reported no biomedical financial interests or potential conflicts of interests."}
+{"text": "Music perception and performance are comprehensive human cognitive functions and thus provide an excellent model system for studying human behaviour and brain function. However, the molecules involved in mediating music perception and performance are so far uncharacterised.To unravel the biological background of music perception, using molecular and statistical genetic approaches.Methods: 15 Finnish multigenerational families  were recruited via a nationwide search. The phenotype of all family members was determined using three tests used in defining musical aptitude: a test for auditory structuring ability  commonly used in Finland, and the Seashore pitch and time discrimination subtests (SP and ST respectively) used internationally. We calculated heritabilities and performed a genome-wide variance components-based linkage scan using genotype data for 1113 microsatellite markers.The heritability estimates were 42% for KMT, 57% for SP, 21% for ST and 48% for the combined music test scores. Significant evidence of linkage was obtained on chromosome 4q22 (LOD 3.33) and suggestive evidence of linkage at 8q13-21 (LOD 2.29) with the combined music test scores, using variance component linkage analyses. The major contribution of the 4q22 locus was obtained for the KMT (LOD 2.91). Interestingly, a positive LOD score of 1.69 was shown at 18q, a region previously linked to dyslexia (DYX6) using combined music test scores.Our results show that there is a genetic contribution to musical aptitude that is likely to be regulated by several predisposing genes or variants. Music is an ancient and universal feature across all human societies. The ability to appreciate music requires no explicit training. The universality of musical behaviour and validity of common rules such as use of octave-based scale systems and preference for consonance over dissonance in nearly all types of music can be seen as evidence for innateness. Rules have arisen independently in isolated cultures, and some of them also apply to the music perception of non-human species. This implies that these rules have their basis in brain organisation rather than in culture.1Observations on fetuses and infants have revealed that basic auditory abilities, such as pitch discrimination and also more complex capabilities such as melody recognition, are already present in the early stages of development.Neuroimaging and neurophysiological studies have shown that listening to and/or playing music has multiple effects on brain structure and function, suggesting a biological effect. Neurophysiological studies have shown that musical stimuli activate specific areas of the brain.It has been observed that professional musicianship clusters in families. How much this aggregation is due to genetic or environmental factors has been the subject of debate.Musical ability varies between individuals, and seems to be expressed at the population level in such a way that both extremes  are rare, and hence most individuals express moderate ability.The ethics committee of Helsinki University Central Hospital approved the study, and informed consent was obtained from all participants.In total, 234 people from 15 families of Finnish origin with some professional musicians and/or active amateurs were recruited for the study via a nationwide search by sending information leaflets or letters to the families whose members had studied or were studying at Sibelius Academy or music institutes in Finland . The famAll of the tests used a continuous scoring scale , with one point obtained for each correct answer. The reliabilities (\u03b1 coefficients) of the tests were: KMT 0.88, SP 0.91 and ST 0.78. Calculating reliabilities is a standard procedure within the behavioural sciences. This index is an estimate of the share of systematic, non-random variance in measurements (maximum\u200a=\u200a1). The correlations between the three tests were: KMT/SP 0.64, KMT/ST 0.41 and SP/ST 0.41. These results show that the tests are reliable and only partially overlapping.The results of the three music tests were given to the participants if they were interested. Instructions for carrying out the tests are available on request.To get a rough idea of the age where musical aptitude stabilises, we analysed the relationship between the musical aptitude test scores and age in three groups ANOVA, . In all et al,We also characterised the relationship between the music test scores and training status . A questWe conducted statistical genetic analyses  on the separate scores of the three music tests and also on a combined music test score, as we hypothesised that there would be a common denominator for the three tests. In preparation, we made the following adjustments and transformations. A few people  scored below the expected value for random guessing of answers (50% of answers correct), and their scores results were raised to these values, under the assumption that any differences in scores below the guessing limit are solely due to chance, rather than being a reflection of differences in ability. The combined phenotype was subsequently computed as the sum of the z-scores of the three individual test results. We summed the z-scores  to weight all three tests equally. This would not be case if we had summed the raw scores, as the tests have slightly different ranges. An exact inverse normal transformation was subsequently used on all phenotypes  to ensure a normal distribution. Sex, age, and ageWe performed a simulation study to estimate the power to detect linkage in our pedigree sample. In agreement with the small sample size, the study had only modest power to detect linkage. Assuming a heritability of 48% , we estimated that the study had 50% power to detect linkage with a LOD score of 3 for a locus explaining \u223c55% of the phenotypic variance.Family members >12 years of age were allowed to give a DNA sample for molecular genetic analyses. After obtaining informed consent, venous blood samples were collected from the participants and their DNA was extracted using standard procedures. Altogether, genotype data was obtained for 205 participants. Genotyping was conducted with 1113 microsatellite markers with an average marker density of 3.3 cM. The genetic locations of the genotyped markers were based on the genetic map developed by deCODE genetics.18Genotyping errors were \u201ccleaned\u201d using the multipoint approach implemented in the software package SimWalk2, eliminating inconsistent genotypes and those that appear unlikely to be correct.The heritability estimates are shown in The results of the multipoint linkage analysis are illustrated in http://www.ncbi.nlm.nih.gov). In the highest peak region, between markers D4S423 and D4S2986, an interesting candidate gene appeared: the netrin receptor UNC5C precursor interacts with netrins that direct axon extension and cell migration during neural development. Netrins interact with robo family receptors.ROBO1 is known to be a candidate gene for dyslexia.To unravel the biological basis for musical aptitude, we performed genome-wide linkage analysis using microsatellite markers spanning the genome at \u223c3 cM density and were able to show significant evidence for linkage at 4q22. In silico analysis revealed of a total of 50 genes between markers D4S2986 and D4S2361  and several putative genes have been assigned in the human genome.There has been discussion about the common origin of music and language faculties.Our study is based on multigenerational families, which is expected to reduce the number of genes or alleles associated with musical aptitude compared with case\u2013control studies. The study was performed in families originating from an isolated population of Finland, where founder effect and genetic drift have shown to restrict the number of alleles in human diseases.Our study represents the first systematic molecular genetic study to attempt identification of candidate genes and genetic  variants associated with musical aptitude. The results of our study suggest that musical aptitude is an innate ability that is associated with several predisposing genetic variants. The current work represents a starting point for further mapping, isolation and characterisation of genes that predispose to musical aptitude. The identification of genes or genetic variants involved in mediating music perception and performance should offer new tools to understand the role of music in human brain function and human evolution, and its relationship with language faculty.We showed that three tests of musical aptitude, an auditory structuring ability test , Seashore test for pitch (SP) and for time (ST) showed substantial heritability in 15 Finnish familiesSignificant evidence of linkage was obtained for chromosome 4q22 (LOD 3.33) and suggestive evidence of linkage for 8q13-21 (LOD 2.29), with the combined music test scores using variance component (VC) linkage analyses in the Finnish familiesOur results show that there is a genetic contribution to musical aptitude that is likely to be regulated by several predisposing genes/variants."}
+{"text": "We performed multipoint linkage analyses with multiple programs and models for several gene expression traits in the Centre d'Etude du Polymorphisme Humain families. All analyses provided consistent results for both peak location and shape. Variance-components (VC) analysis gave wider peaks and Bayes factors gave fewer peaks. Among programs from the MORGAN package, lm_multiple performed better than lm_markers, resulting in less Markov-chain Monte Carlo (MCMC) variability between runs, and the program lm_twoqtl provided higher LOD scores by also including either a polygenic component or an additional quantitative trait locus. Our aims were 1) to compare results from several multipoint linkage analysis programs that are available for quantitative traits and 2) to investigate the performance of MCMC-based programs on the GAW15 expression data in 14 three-generation CEPH families genotyped for clustered SNP markers  for Bayeh2). We chose six traits that showed high VC LOD scores and h2 \u2265 0.31: CHI3L2, GSTM1, PSPH, VAMP8, PPAT, and TM7SF3. The first two of these had only a single peak with VC LOD > 3, representing potentially simple traits, and the latter four had multiple peaks, representing potentially complex traits. For these six traits, we performed Bayesian oligogenic joint segregation and linkage analyses using Loki and parametric LOD score analysis with a 1Q model using lm_markers and lm_multiple. For the first four traits only, we also performed parametric LOD score analysis with more complex models using lm_twoqtl.For 62 traits previously reported to show evidence of linkage ,6, we peWe used the Rutgers map  for linkh2 for these 62 traits with a VC polygenic model [For the 62 traits, we performed genome-wide VC linkage analysis with Merlin for both the jittered and original nonjittered maps. VC LOD scores were computed only at the marker positions. We also obtained MLEs of ic model . Using MFor the six traits, we performed Bayesian oligogenic segregation analysis and oligogenic joint segregation and linkage analysis using Loki. For segregation analysis, we used every fourth iteration in a 50 k iteration run to estimate QTL models. For linkage analysis, we used every fourth iteration in a 999 k iteration run to compute Bayes factors for presence versus absence of a QTL in each 2-cM bin. We used QTL models estimated from Bayesian segregation analysis in all our LOD score analyses.We recently developed three programs in MORGAN: lm_markers, lm_multiple, and lm_twoqtl. The first two programs compute LOD scores for the 1Q model, and lm_twoqtl computes LOD scores for more complex models . In addi2(a) + \u03c32(e) as the environmental variance. For the first four traits, we also used lm_twoqtl with one linked plus one unlinked QTL (1Q + UQ) and one QTL plus a polygenic component (1Q + P) models. In addition, for VAMP8, we used lm_twoqtl with a two-linked-QTL (2Q) model. For the first three traits, the secondary QTL model was from oligogenic segregation analysis, whereas for VAMP6, the secondary QTL model was the same as the first QTL model. LOD scores at the marker positions as well as midway between two markers were evaluated for all MORGAN programs. We obtained initial starting configurations by using sequential imputation for all MORGAN programs and the locus sampler for Loki. Burn-in iterations were 150 for all MORGAN programs and 1000 for Loki. We used a 50:50 ratio of locus to meiosis sampler for all MCMC-based analyses. For lm_multiple, the probabilities for updating meioses from random subsets, individuals, full sibships, and full three-generation families were 0.2, 0.3, 0.3, and 0.2. For lm_twoqtl, we used every tenth scan in a 30 k scan run for computing LOD scores. For lm_markers and lm_multiple, we used every scan.We performed parametric linkage analysis using these three MORGAN programs. For the six traits, we obtained ten estimates of LOD scores using MCMC and both lm_markers (3 k and 30 k scans) and lm_multiple (3 k scans), to compare their performance. For comparison, we also computed exact LOD scores for the 1Q model, also using lm_markers. Parameter values for the trait model were almost identical to those for the mixed model in Table h2 ranging from 0.13 to 0.86. Five traits had a maximum VC LOD score < 1, with h2 ranging from 0 to 0.11. Most traits had only a single peak in the genome with VC LOD \u2265 3, suggesting a simple mode of inheritance. Two traits (PSPH and DDX17) had three peaks with VC LOD \u2265 3, and three traits  had two peaks with VC LOD \u2265 3. The jittered and nonjittered maps yielded virtually identical VC LOD scores, except for VAMP8 on chr 2, where the largest peak was slightly narrower with the nonjittered map.Of the 62 traits, 24 had a VC LOD score \u2265 3, with CHI3L2, GSTM1, PPAT, PSPH, TM7SF3, and VAMP8 for further analysis. The actual locations of these genes were at the maximum VC LOD scores , 10 cM away (VAMP8), or 25 cM away (PPAT). Bayesian oligogenic segregation analysis for these traits provided posterior mean numbers of QTLs ranging from 2 to 3.5. Estimation of the primary QTL model was relatively straightforward  declare that they have no competing interests."}
+{"text": "We explored the utility of population- and pedigree-based analyses using the Framingham Heart Study genome-wide 50 k single-nucleotide polymorphism marker data provided for Genetic Analysis Workshop 16. Our aims were: 1) to compare identity-by-descent sharing estimates from variable amounts of data; 2) to apply each of these estimates to a case-control association study designed to control for relatedness among samples; and 3) to contrast these results to those obtained using model-based and model-free linkage analysis methods. The study of quantitative traits has led to the development of tools of varying complexity designed to identify chromosomal regions associated with disease. This has been coupled in recent years by the increasing availability of data sets that include hundreds of thousands of markers. We investigated the utility of using more data and more sophisticated analyses by applying analytical methods of variable complexity to a single data set and comparing their results. We also estimated the same statistics using variable amounts of marker data to investigate the influence of the amount of marker data on the estimates. Generally speaking, we asked whether we really benefit from these new tools and large data sets, or do they simply increase the complexity of our research? More specifically, we: 1) compared estimates of identity-by-descent (IBD) sharing from increasing amounts of marker data; 2) evaluated how well different IBD estimates corrected a case-control association study for relatedness; and 3) compared the results of our case-control study and various linkage analyses to contrast any signals of association between genotype and phenotype.We focused exclusively on the Genetic Analysis Workshop (GAW) 16 50 k marker data, and primarily analyzed only chromosome 7. We matched the position of each chromosome 7 marker to the sex-averaged Kosambi map sequence position on the Rutgers map [We filtered markers with >3% missing data and with minor allele frequency <0.05. We used chi-square tests to test the null hypothesis of Hardy-Weinberg equilibrium, and removed markers yielding the largest 1% of the test statistics, leaving 2132 markers on chromosome 7. A \"thinned\" marker panel was obtained by selecting approximately every tenth marker from this \"dense\" filtered marker set, preferentially selecting markers with higher rare allele frequencies among founders because they are more suitable for linkage analysis. The final thinned data set included 214 markers on chromosome 7 (and 3465 genome-wide markers) with a marker density of ~1 per cM.To ensure compatibility with the linkage analysis programs in the MORGAN package , we mergFor linkage-based analyses, we focused on high-density lipoprotein level (HDL) and chromosome 7 due to previous evidence of linkage within the Framingham Heart study (FHS) -8. We usWe performed Bayesian oligogenic segregation analysis using the software package Loki 2.4.7  to identk-coefficients, where ki is the probability that i alleles are shared IBD, using the thinned chromosome 7 and genome-wide panels of markers, as well as subsets of 214 and 1000 genome-wide markers. We estimated k-coefficients for all possible pairs of independent people (n = 1827), using all founders in the pedigrees and other unrelated individuals, and for all pairs of individuals within each pedigree. Kinship coefficients, \u03a6, were subsequently computed as \u03a6 = 0.25k1 + 0.5k2 and pairs of individuals with \u03a6 > 0.2 were noted.Without reference to the pedigree structures, we estimated We selected four pairs of individuals showing high apparent relatedness as estimated from the thinned chromosome 7 markers while differing with respect to pedigree numbers. For each pair, the dense chromosome 7 markers were used to detect IBD segments using the model of Thompson . We usedth percentiles of the trait distribution in the full data set. The correction for relatedness essentially eliminates inflated test statistics resulting from inclusion of related individuals. We corrected the na\u00efve chi-square statistic p-values using three types of kinship coefficients: pedigree-based prior, pedigree-based posterior, and population-estimated kinship coefficients. The pedigree-based prior was computed based on pedigree structure alone, while the pedigree-based posterior was based on the gl_auto results (described below) that used both pedigree structure and marker data. The dense chromosome 7 marker panel was used for the case-control study, while the thinned chromosome 7 marker panel was used for estimation of kinship coefficients. The population-estimated kinship coefficient was a maximum-likelihood estimate based on the thinned chromosome 7 marker data.We used a new \"case-control\" study design that corrects for relatedness (both known and estimated as cryptic kinship) within the sample , choosinTwo MORGAN  programsw-score [w-score analyses were performed only on the size 4-9 pedigrees, while VC analysis was performed on this subset as well as on all pedigrees for comparison. We summarized the results using randomized p-values for the conditional test [p-values for the VC analysis through trait simulation and the inheritance vectors described above [th out of 500,000 iterations were used to compute Bayes' factors for the presence of a QTL within each 2-cM bin.Genetic linkage can be detected with pedigree data using inheritance vector realizations. We used the inheritance vectors obtained from gl_auto for two linkage analysis methods: 1) standard variance-components (VC) analysis using SOLAR, and 2) a novel conditional inheritance vector test using the w-score , which inal test  and empied above , where ek1 or k2 values for chromosome 7. This indicates there is non-negligible relatedness among independent individuals across pedigrees.We found several pairs of independent individuals that share non-zero average IBD Figure , but thek1 decreased as the number of markers used increased for two of the four pairs of individuals. The IBD segments analysis estimated the full nine-state IBD probabilities along the chromosome, with the average of these values reported in Table Detailed analysis of four such pairs of independent individuals with unique pedigree numbers are summarized in Table Figure p-value = 0.0087). All linkage analyses detected one or more peaks in the region between 20 and 40 cM. A single Loki run is represented in Figure Linkage analysis results are summarized in Figure p-values summarize test significance as well as uncertainty of the test results. For example, although we find significant evidence for linkage near 38 cM using VC analysis and trait resimulation, the conditional test p-values in the same region are uncertain, as indicated by the range of p-values estimated at that position. To resolve these uncertainties, we would need to use markers at greater density or with greater polymorphism levels to infer the inheritance vectors in this region.Randomized k1 > 0.8 than 214 markers from chromosome 7.As a rule, more marker data increases the stability and apparent accuracy of IBD estimates Figure , as the k-statistics estimated from 214 vs. 1000 genome-wide markers, little difference is observed between estimates from 1000 vs. 3465 genome-wide markers. This suggests that we can thin our marker panels to avoid the effects linkage disequilibrium without strongly altering our inferred k-statistics. This also allows for the generation of multiple, equivalent, thinned data sets from a single dense data set, to be used for replication purposes.However, a relatively modest number of markers were needed to achieve this stability. Although we see a dramatic difference between the Even with thousands of markers, relationships between some pairs of independent people were detected by several approaches. Some of these pairs shared pedigree numbers but could not be connected in the pedigree file. This suggests that our methods were able to detect real relatives even when they were not labelled as such. However, some pairs had unique pedigree numbers, suggesting some cryptic relatedness among the FHS participants and raising the possibility that adjustment for such relationships might be necessary in some analyses.p-values in the case-control study. Fortunately, our corrections using pedigree- and marker-estimated relatedness worked well. Because many case-control studies do not have access to pedigree data, this suggests that our method may be applied to genome-wide association studies without the need for additional pedigree information. The pedigree-posterior estimate of relatedness overcorrected our test statistic, although as the analysis used only the thinned chromosome 7 markers, this is not surprising given the results in Figure Relatedness, known or cryptic, clearly inflated uncorrected w-score and MCMC-based oligogenic linkage analyses. The w-score also identified a possible linkage signal near 95 cM, although the confidence in actual p-values varied across the chromosome. Analysis of the size 4-9 pedigrees emphasized the peak near 20 cM at the expense of the peak near 40 cM, while analyses of all pedigrees identified a modest signal near 180 cM. This signal was detected in a previous GAW [The number and strength of linkage signals varied across methods and by the amount of data used. Linkage analyses, but not the case-control analyses, provided evidence for HDL loci on chromosome 7. The bimodality of the linkage signal between 20 and 40 cM was more clearly defined with the more computationally intensive and trait-model-based w-score provided both localization and confidence of information in linkage analysis. Although encompassing a wide range of approaches, these methods show clear promise for future work.Our novel methods were useful in a variety of situations. Inheritance vectors generated by gl_auto were used in the VC analyses and in empirical significance testing. Analysis of IBD segments identified wide swathes of shared chromosomal regions between pairs of independent people with patterns not visible in a single summary statistic. The method to correct case-control studies for relatedness was practical and effective, and the The use of additional data and analytical methods of increasing complexity appears to have paid dividends. However, there are clearly limits. More markers provide better IBD sharing estimates, but a marker density greater than between 1 and 3 cM would likely give only a slight improvement. Correcting case-control studies for relatedness is effective, relatively simple, and can be done using marker data alone. Linkage analyses of greater complexity identified more, albeit weak, linkage signals than simpler analyses. It would appear that all association and linkage methods are capable of detecting strong and clear signals. Because not all studies are fortunate enough to have strong signals, sophisticated analytical tools and large, but not too large, data sets may deliver additional results along with their complexity.BMI: Body mass index; FHS: Framingham Heart Study; GAW: Genetic Analysis; Workshop; HDL: High-density lipoprotein; IBD: Identity by decent; MCMC: Markov-chain Monte Carlo; QTL: Quantitative trait locus; SNP: Single-nucleotide polymorphism; VC: Variance components.The authors declare that they have no competing interests.All authors participated in study design and approved the final manuscript. EAT, FB, and YC participated in the IBD analyses, and YC performed the case-control study. EEM, YD, CC, MS, and EMW participated in the linkage analyses. EEM drafted the manuscript."}
+{"text": "Daphnia magna is a well-established model species in ecotoxicology, ecology and evolution. Several new genomics tools are presently under development for this species; among them, a linkage map is a first requirement for estimating the genetic background of phenotypic traits in quantitative trait loci (QTL) studies and is also very useful in assembling the genome. It also enables comparative studies between D. magna and D. pulex, for which a linkage map already exists.D. magna. We generated 214 F2 (intercross) clonal lines as the foundation of the linkage analysis. The linkage map itself is based on 109 microsatellite markers, which produced ten major linkage groups ranging in size from 31.1 cM to 288.5 cM. The total size of this linkage map extends to 1211.6 Kosambi cM, and the average interval for the markers within linkage groups is 15.1 cM. The F2 clones can be used to map QTLs for traits that differ between the parental clones. We successfully mapped the location of two loci with infertility alleles, one inherited from the paternal clone (Iinb1) and the other from the maternal clone (Xinb3).Here we describe the first genetic linkage map of D. magna linkage map presented here provides extensive coverage of the genome and a given density of markers that enable us to detect QTLs of moderate to strong effects. It is similar in size to the linkage map of D. pulex.The  Daphnia magna has been used in biological research since the 18th century 29]. For 2*(n+1)) ,30, wherGenotyping error was estimated from a random set of twice genotyped markers, 11 loci and 50 clones genotyped by two different people. The first set was genotyped with a ABI 310 Sequencer , and the second set was genotyped with a AB3130xl Sequencer .JR genotyped EST markers, analysed the data and wrote the manuscript. BJ developed and genotyped the microsatellite markers and contributed to data analysis. IC generated the F2 mapping population, developed and optimized EST markers, and initialized the infertility trait analysis. DE and LDM conceived the study, designed the F2 panel and edited the manuscript. The F2 panel was established in the Ebert lab. All authors read and approved the manuscript.EST VNTR markers predicted functions. Name of the marker, EST id in wFleaBase, homolog in protein databases', E-value, predicted biological process and molecular function (excel file).Click here for fileDetails of the marker loci. Name, linkage group (LG), distance based on recombination fractions in Kosambi centiMorgans (cM), chi square test of linkage distortion (\u03c72), corresponding p value, accession number, forward primer, and reverse primer. The asteriks *** indicate markers that have significant associations with infertility phenotypes (excel file).Click here for file"}
+{"text": "The Atlantic salmon is a species of commercial and ecological significance. Like other salmonids, the species displays residual tetrasomy and a large difference in recombination rate between sexes. Linkage maps with full genome coverage, containing both type I and type II markers, are needed for progress in genomics. Furthermore, it is important to estimate levels of linkage disequilibrium (LD) in the species. In this study, we developed several hundred single nucleotide polymorphism (SNP) markers for the Atlantic salmon, and constructed male and female linkage maps containing SNP and microsatellite markers. We also investigated further the distribution of male and female recombination events across the genome, and estimated levels of LD between pairs of markers.2 increased with decreasing inter-marker distance in a bimodal fashion; increasing slowly from ~60 cM, and more rapidly more from ~12 cM. Long-ranging LD may be consequence of recent admixture in the population, the population being a 'synthetic' breeding population with contributions from several distinct rivers. Levels of r2 dropped to half its maximum value (above baseline) within 15 cM, and were higher than 0.2 above baseline for unlinked markers ('useful LD') at inter-marker distances less than 5 cM.The male map had 29 linkage groups and was 390 cM long. The female map had 30 linkage groups as was 1983 cM long. In total, the maps contained 138 microsatellite markers and 304 SNPs located within genes, most of which were successfully annotated. The ratio of male to female recombination events was either close to zero or very large, indicating that there is little overlap between regions in which male and female crossovers occur. The female map is likely to have close to full genome coverage, while the majority of male linkage groups probably lack markers in telomeric regions where male recombination events occur. Levels of rThe linkage map presented here is an important resource for genetic, comparative, and physical mapping of the Atlantic salmon. The female map is likely to have a map coverage that is not far from complete, whereas the male map length is likely to be significantly shorter than the true map, due to suboptimal marker coverage in the apparently small physical regions where male crossovers occur. 'Useful LD' was found at inter-marker distances less than 5 cM. Salmo salar) is a species of worldwide significance as a prized species in recreational fishing and a major contributor to the world's aquaculture production. The genomes of the Atlantic salmon and other salmonids are purported to be derivates of an autotetraploidisation event that occurred in their common ancestor 25 to 100 million years ago . Furtheiewed in ,9-13.Two low-density maps have been published for Atlantic salmon ,14, in aHere, we provide an update of an ongoing project aiming at detecting, testing, and mapping large numbers of EST-derived SNP markers in Atlantic salmon ,19. We ain silico detection of a large number of putative SNPs for Atlantic salmon, and the subsequent experimental testing of 86 of these SNPs in a diverse validation panel. In the present study, another set of 1369 SNP markers were tested in a similar validation panel  > 0.2 and heterozygosity (microsatellite) > 0.5. The average r2 for marker pairs with markers located on different linkage groups was 0.16. The average r2 for physically, but not genetically linked marker pairs (markers located more than 50 cM apart on the same linkage group) was 0.20. Levels of r2 increased with decreasing inter-marker distance from ~60 cM, increased more rapidly more from ~12 cM  within 15 cM.Levels of LD, measured through the correlation coefficient between pairs of loci rThe male map contained 29 linkage groups, corresponding well with the most common karyotype in European Atlantic salmon, which has 2n = 58 . Most liThe lack of markers in at least two segments of the female map shows that the female map is shorter than the true female genetic map. At the same time, the low number of female-informative markers that could not be mapped, in conjunction with the relative good fit of the map length with the length of the SALMAP map , indicaMap distances should be expected to be slight overestimates since any genotype errors would tend to inflate genetic distances. Although practically all genotypes resulting in double crossovers were re-checked in detail and corrected if necessary, genotype errors cannot be ruled out, in particular not for markers located at the end of linkage groups .Contrary to expectation, we did not observe any instances of pseudolinkage in the data set. Pseudolinkage describes the apparent linkage in male mapping parents of markers that are not linked in female mapping parents, with an excess of non-parental genotypes in offspring, and is believed to be caused by the non-random breaking up of tetravalent complexes formed during male meiosis . PseudolIn salmonids, male recombination rates are much reduced compared to female recombination rates -11,14. IThe most common karyotype of European Atlantic salmon consists of 16 metacentric and 42 acrocentric chromosomes . If one The site-specific distribution of male and female recombination events must be taken into account when QTL experiments are designed. In Atlantic salmon, it has become quite common to first perform a coarse genome scan using male segregation only and one or a few markers per linkage group, on the assumption that there is practically no recombination in males  offspring were assumed to have inherited the chromosome segment without recombinations from their respective fathers. In this way, alleles inherited from sire and dam could be deduced, and the markers could be re-coded as fully informative. It should be pointed out that this strategy was only used if no male recombinations had been observed in a priori informative offspring, meaning that no recombination had been observed in a minimum of 92  meioses. The strategy was therefore conservative.In this study, we exploited the lack of male recombination in large parts of the genome to draw more information out of the data. More specifically, for markers located in such regions, when both parents were heterozygous and identical within a given family, heterozygous  using microsatellites. LD was also computed between SNP-SNP pairs (results not shown), and found to be much lower than for microsatellite-SNP LD. Levels of microsatellite-microsatellite LD were comparable to those of microsatellite-SNP LD, though slightly higher (results not shown). Lower LD values for SNP-SNP pairs when compared to microsatellite-SNP or microsatellite-microsatellite pairs is likely to be, at least in part, a consequence of differences in heterozygosity between marker types ,36. Howe2 between markers located on different linkage group was higher than expected (r2 = 0.16). This could be caused by limited effective population size and/or by relatedness between individuals in the sample (there were three pairs of full-sibs in the sample). However, removing closely related animals did not decrease, but rather increased r2 between unlinked markers, indicating that the small sample size may have been the main reason for high LD between unlinked markers. Levels of r2 is biased upwards when sample sizes are small, although much less so than the LD statistic D' [The average ristic D' . At the LD between linked markers seemed to increase with decreasing inter-marker distance in a bi-modal manner. LD first increases slowly from ~60 cM, then more rapidly from ~12 cM. This finding may reflect upon the fact that the population from whence the haplotypes were derived is a 'synthetic' population, formed seven generation ago from individuals from different Norwegian rivers . Due to 2 is equal to the amount of information provided by one locus about the other, meaning that for a gene-trait association to be detected, sample size must be increased by 1/r2 if a marker in LD with the trait-affecting gene is used rather than the gene itself. Levels of r2 from 0.1 to 0.3 have been proposed as minimum values of 'useful ld' [2 > 0.2 as the threshold, 'useful LD' could in this study be found at inter-marker distances below ~5 cM, indicating that ~400 fully informative, evenly spaced markers would be sufficient to at least start capturing inherent LD information. Thus, currently available maps  were more than 0.2 above the baseline for unlinked markers at inter-marker distances less than 5 cM. At inter-marker distances larger than 15 cM, r2 decreased slowly, possibly reflecting LD due to population admixture that have had limited time to be broken down by recombination. The map presented here will serve as a framework map onto which a larger number of SNP markers, currently being identified from alignment of EST sequences and from DNA re-sequencing [In this study, we constructed male- and female genetic linkage maps of Atlantic salmon, elucidated further the distributions of recombination events in males and females, and provided initial data on levels of linkage disequilibrium. The female map presented here is likely to represent the true genetic map well, whereas the male map is probably incomplete due to male recombination being localised to narrow telomeric regions. There appear to be little overlap between regions in which male and female recombination events occur. Levels of LD  MSV multiple sequence variant) [PSV  = duplicated SNP without homozygotes; 4) all homozygous = all animals were homozygous; and 5) failed assay = poor clustering of genotypes and/or unreliably scored genotypes. Sequences of SNPs and contigs can be found in Additional File From among the 2507 putative SNP discovered lable at  by usingvariant)  = SNPs w-10. Matches to hypothetical proteins and genomic sequences were filtered out.Sequences containing SNPs were clustered into contigs using a two stage Phrap assembly process. The first stage assembly of 434,384 Atlantic salmon EST sequences (parameters 100 minmatch and 0.99 repeat_stringency) resulted in 119,912 contigs which were then reassembled  into 81,398 contigs . CompletThe mapping families were provided by the breeding company Aqua Gen AS, and were used also for QTL mapping for resistance against the viral disease Infectious Pancreatic Necrosis (IPN) . Hence, the offspring had been challenge tested for resistance to IPN. The 20 mapping parents came from two different year populations of Aqua Gen salmon; 16 from year class 2001, and 4 from year class 2000. Both populations were formed in the yearly 1970's from salmon from different Norwegian rivers , and hav2, 0.25 \u20131 pmol of each primer (depending on amplification efficiency of each marker in multiplex), 0.25 \u03bcl DMSO, and 5 ng DNA template. PCR cycling conditions were 95\u00b0C for 10 minutes, 35 cycles of 94\u00b0C for 30 seconds, 54\u00b0C for 1 minute, and 72\u00b0C for 1 minute, followed by a final extension of 60\u00b0C for 45 minutes. The lengths of the fluorescent PCR products were determined relative to the LIZ500 size standard (Applied Biosystems) on a 3730 DNA Analyzer (Applied Biosystems), using GeneMapper 4.0 (Applied Biosystems) software for allele calls.Most of the microsatellite markers used in this study were chosen from the SALMAP microsatellite map of Atlantic salmon (as it was in 2006) , and werdouble haploid format), containing information on alleles inherited from that parent only. Marker grouping was done at a minimum LOD score of 4.0. Following marker grouping, homologous linkage groups from each sire and each dam were integrated into single sex-specific maps. The marker orders determined by Joinmap 3.0 were tested and corrected using the flips function of CRIMAP, with a moving window of 7 markers (flips7). Using the final marker orders as calculated by CRIMAP, the data was examined for unlikely double recombinants, for inconsistencies in marker order between parents, using a custom Visual Basic for Applications (VBA) for Excel program. Segregation distortion was tested for using the same program, by incorporating a Pearson's goodness-of-fit test for 1:1 segregation of alleles from individual parents to offspring. Double recombinants occurring over small distances were checked for genotyping error. Markers displaying segregation distortion (P < 0.01) were also inspected. After marker orders and potential genotype errors had been verified, the final maps were constructed using the fixed function of CRIMAP. The Kosambi mapping function was used. Map drawings were made using Joinmap 3.0 [Since recombination rates in salmonids have been shown to differ dramatically between sexes, separate male and female maps were constructed. Marker grouping and initial marker ordering was done in Joinmap 3.0 . A Joinmnmap 3.0 .Since SNPs are bi-allelic, there were frequent occurrences of both parents of a family being heterozygous for the same two alleles of a SNP. In such cases, all heterozygous offspring were initially uninformative for mapping. We exploited the complete lack of male recombinants in most parts of the genome to deduce the inheritance of alleles in situations where i) both parents were heterozygous for the same two alleles of a SNP (marker A), and ii) the SNP was linked without any observed male recombinants to another marker (marker B); the latter marker being fully informative in the male. The steps of the deduction process were 1) determine the linkage phase between A and B in the male parent, 2) use the linkage phase to deduce which allele was inherited from the father at A, to offspring heterozygous at that marker, and 3) assign the other allele at A (of heterozygous offspring) to the female parent. This strategy was applied during the process of checking the data, automated through a VBA program.Before map construction, SNP markers located within the same contig were combined to produce one marker point (i.e. a haplotype).vice versa for unlinked male segments corresponding to a single female homologue).Linkage groups were numbered according to the SALMAP map, using shared microsatellites to infer homologies. Whenever two or more markers were shared between a linkage group and its SALMAP homologue, the linkage group was also oriented in the same way as the SALMAP linkage group. One linkage group did not share any markers with any linkage group on the SALMAP map, and was termed \"A\". In the cases where two unlinked (in this data set) female segments corresponded to a single male homologue X, the segments were designated Xa and Xb to indicate that they were likely to be part of the same linkage group . Instead, a 6th degree polynomial was fitted to the data.The animals used for calculation of LD were a subset of the mapping parents, more specifically the 16 parents that came from the breeding population of major representation. Thus, the data set consisted of 32 haplotypes. Only microsatellite-SNP pairs were used, to mimic the mapping of a QTL using microsatellite markers. SNPs with minor allele frequencies < 0.2 and microsatellites with heterozygosities < 0.5 were culled. Composite SNP markers (haplotypes of SNPs within the same contigs) were grouped with microsatellites. The haplotypes of the mapping parents were deduced at every linkage group using a custom-made VBA program. Briefly, the program performed these steps (for every linkage group within every mapping parent): 1) Start at the first informative marker from one of the linkage group; 2) find the linkage phase between that marker and the next informative marker, minimising the number of recombination events in the offspring; 3) proceed in this manner to find the linkage phase between all informative marker, and thus to build the two haplotypes; and 4) for monomorphic markers, insert the same allele in both haplotypes. Measures of LD were calculated using the function ram GOLD . The LD amer's V . The samr's V [2 . Sved's r's V [2  was fittin silico SNP detection. MB coordinated microsatellite genotyping and took part in SNP genotyping. PB coordinated SNP genotyping. SK organised and carried out tissue sampling. BFK took part in annotation and provided the bulk of EST sequences. WSD took part in annotation. SWO provided laboratory facilities and took part in planning the study. SL provided laboratory facilities, did manual inspection of SNP validation data, and took part in SNP validation and data collection. All authors read and approved of the manuscript.TM took part in microsatellite genotyping and SNP validation, did data analyses except for SNP detection and annotation, and wrote a draft of the manuscript with contributions from BH, MB, PB, BK, WSD, BFK, and SL. BH did Properties of SNP markers used in the study. Excel workbook containing IDs of SNPs, contigs sequences w/SNP sequence and position, allele frequencies, heterozygosities, and BLASTX hitsClick here for fileAtlantic salmon linkage map. Linkage map in Excel format.Click here for fileMarker pairs with male-female order discrepancies. Marker pairs for which marker order were inverted in one map relative to the other.Click here for file"}
+{"text": "Comparative genomic studies suggest that the modern day assemblage of ray-finned fishes have descended from an ancestral grouping of fishes that possessed 12\u201313 linkage groups. All jawed vertebrates are postulated to have experienced two whole genome duplications (WGD) in their ancestry (2R duplication). Salmonids have experienced one additional WGD (4R duplication event) compared to most extant teleosts which underwent a further 3R WGD compared to other vertebrates. We describe the organization of the 4R chromosomal segments of the proto-ray-finned fish karyotype in Atlantic salmon and rainbow trout based upon their comparative syntenies with two model species of 3R ray-finned fishes.Evidence is presented for the retention of large whole-arm affinities between the ancestral linkage groups of the ray-finned fishes, and the 50 homeologous chromosomal segments in Atlantic salmon and rainbow trout. In the comparisons between the two salmonid species, there is also evidence for the retention of large whole-arm homeologous affinities that are associated with the retention of duplicated markers. Five of the 7 pairs of chromosomal arm regions expressing the highest level of duplicate gene expression in rainbow trout share homologous synteny to the 5 pairs of homeologs with the greatest duplicate gene expression in Atlantic salmon. These regions are derived from proto-Actinopterygian linkage groups B, C, E, J and K.Danio rerio and Oryzias latipes (descendants of the 3R duplication) can, in most instances be related to at least 4 whole or partial chromosomal arms in the salmonid species. Multiple arm assignments in the two salmonid species do not clearly support a 13 proto-linkage group model, and suggest that a 12 proto-linkage group arrangement  may have occurred in the more basal soft-rayed fishes. We also found evidence supporting the model that ancestral linkage group M underwent a single chromosome duplication following the 3R duplication. In the salmonids, the M ancestral linkage groups are localized to 5 whole arm, and 3 partial arm regions . Thus, 3 distinct ancestral linkage groups are postulated to have existed in the G/H and M lineage chromosomes in the ancestor of the salmonids.Two chromosome arms in  Salmonid fishes are known to have descended from an autopolyploidization event that occurred within the ancestral grouping that gave rise to the Salmoniformes sometime within the early Tertiary or late Cretaceous periods . SupportOncorhynchus mykiss), and Atlantic salmon  up to 14 Hox paralogons or chromosomal locations (up to 16 are expected) have been identified supporting the model that salmonids are 4R derivative lineages [Actinopterygians are also now largely recognized as being descended from ancestral forms that underwent a WGD event. Two current models exist, which suggest that the ray-finned fishes arose from an ancestral form possessing either 12  or 13 anlineages . Thus, iDanio rerio) and medaka (Oryzias latipes), and thus, comparative synteny analyses may be extremely valuable in identifying genes of importance regulating physiological, developmental, and behavioural modalities in salmonid fishes.The most recent model on vertebrate evolution postulates that 10 different linkage group blocks existed in a proto-chordate ancestral form [Currently there are 1855 markers located onto the L25 and L44 rainbow trout mapping panels. These include: 727 AFLP markers; 102 Type I genes; 337 EST markers; 665 SSR markers; 21 BAC-end sequence markers; and the phenotypic marker SEX. There are presently 1671 markers localized to the Br5 and Br6 Atlantic salmon mapping panels. These markers are distributed as: 275 AFLP markers; 43 Type I genes; 174 Type I-SNP markers; 187 EST markers; 410 SSR markers; 582 BAC-end sequence markers; and the phenotypic marker SEX. Information on the distribution of variation in these markers within the four mapping parents used for each species is given in Additional File et al. model [As expected, the assignment of linkage groups within the two salmonid species to the ancestral ray-finned fish karyotype indicates that most ancestrally derived 3R linkage groups are represented by approximately 4 whole or partial arm affinities in the salmonids Table , 2 and 3l. model , linkageAlthough many large synteny blocks were detected in the salmonids among the various 3R teleost linkage groups  Figures . AdditioGiven the fact that salmonids have undergone a more recent WGD compared to most extant teleosts, it is expected that whole chromosome arms should retain duplicated marker signatures of this event. The fact that most salmonid species retain between 96 to 104 chromosome arms is also strong support that a WGD has occurred within this lineage given the observation that the modal chromosome number in teleosts is 48\u201350 acrocentric based chromosomes yielding 24\u201325 linkage groups. Thus, in salmonids we might expect a similar number of homeologous affinities to be observed , dependent upon the number of extant chromosome arms in the species and whether individual chromosome arms may have experienced pericentric inversions during their evolutionary history. Multiple arm re-arrangements and translocations may also increase the number of arm homeologies observed, as a single chromosome arm may have affinities to more than one other chromosome arm in the genome. Such a situation is evident for the 9p arm and centromeric region of RT9, wherein multiple single marker putative homeologies have been observed. When considering syntenic block homeologies within rainbow trout, 20 linkage group arm pairs have been observed to possess either more than one duplicated marker, or in the case where only a single duplicated marker has been observed there is supporting evidence from the 3R syntenic blocks that these regions are homeologous , and one partial arm (RT25q). A fourth region of homology may reside on RT24p, but this has not been confirmed through synteny mapping within the medaka genome. Greater than 4 whole arm affinities exist for homologies detected with ancestral groupings G/H and M. If G/H do in fact represent two separate ancestral linkage groups as postulated by the Kasahara et al.  model thMultiple partial arm affinities may be expected through time, following translocation and genome re-arrangement events within each species. While it can be postulated that as more comparative data is accumulated the chromosome arm affinities currently assigned to an ancestral grouping will be represented by a more mosaic association pattern among chromosome arms, the current data allows us to observe certain whole arm affinities within linkage groups that appear to have arisen by the fusion of arms within the same ancestral groupings. For example, RT30 and its homologous region AS17qa show extensive homology to group D throughout the length of the currently identified linkage group markers. Using the comparative medaka genome as a template, it can be observed that both Olat15 and Olat19 markers form two large synteny blocks within RT30, suggesting a possible fusion of group D 3R duplicates within this region of the salmonid genome. Conversely, Olat19 contributes most extensively to the RT9p/RT17q/RT22p affinities, while Olat15 underlies the RT6q and RT17p affinities Figure  derived Tetraodon nigriviridis) genomes [Two ancestral linkage groups (H and I) appear to share a propensity in forming adjacent synteny blocks within chromosome arms, as indicated by their partial arm affinities within RT1/AS9qa, RT9p/AS5qb, and the whole arm fusions represented by AS25qa and AS25qb. A small region near the centromere on RT12q also possesses a synteny block derived from ancestral grouping H. These observations are of interest given the finding by Kasahara et al.  that anc genomes . This obSimilar to the single ancestral chromosome doubling within the M linkage group that was postulated to account for the retention of large synteny blocks among 3 modern day teleost linkage groups ,20, the Ancestral groupings G/H/I share segments from 3  of the 10 linkage group blocks of the postulated proto-chordate genome clusters , may be Salvelinus alpinus) possessing a few metacentric chromosomes) [Far fewer duplicated markers were identified in Atlantic salmon (9.5%) compared to rainbow trout (26%), despite both genetic maps currently having approximately the same number of informative type I and type II marker distributions . A greater percentage of type I gene and EST-based markers were analyzed in rainbow trout, however, compared to Atlantic salmon which may indicate that duplicate expression is retained to a greater degree within coding vs. anonymous DNA. Another complementary explanation for this phenomenon may be the lack of recombination experienced within the Atlantic salmon genome . mosomes) . While cmosomes) , or evenWithin Atlantic salmon, the five homeologous pairs of linkage groups exhibiting the highest level of duplicated marker expression (\u2265 5 pairs)  are all localized within either metacentric or larger fused acrocentric chromosomes . SimilarIncreased recombination among homeologs will lead to increased frequencies of multivalent formation during meiosis which often generates pseudolinkage affinities among ancestral homeologues involved in the pairing . InteresMore extensive arm re-arrangements within the genome of Atlantic salmon may limit the occurrence of multivalents in this species. Strain background may also influence the propensity to express pseudolinkage given that recombination rates appear more elevated in inter-strain hybrid versus pure strain genomic backgrounds . TherefoThe concept that the surrounding genetic structure within an ancestral genomic region may be extremely important in defining the propensity to retain duplicated gene copies can be observed within the current dataset. All five of the regions retaining duplicate gene expression patterns in Atlantic salmon, share affinity to the same ancestral teleost regions within rainbow trout genome, and are syntenic with the same chromosome arm in rainbow trout  spanning several kb often surround important regulatory gene regions in the genome such as early development regulating genes ,32. Stro-6, and bp length of \u2265 100. The first 50 highest ranked fragments meeting or exceeding these criteria, for each family member were retained for the analysis. While the results for zebrafish were mixed with respect to the distribution of Tc1 elements across chromosomes, within the medaka genome, there was evidence that chromosomes belonging to ancestral groupings B, C, D, E, and K, had somewhat lower distributions of Tc1-like fragments scattered throughout their length  families in salmonids, a BLASTN search was performed using the recently documented sequences from several family members in this grouping from Atlantic salmon  and rainSalmonids, due to their 4R polyploid ancestry retain the genetic signatures of past gene duplication events. Most extant duplicated DNA copies map to the chromosome arms that are immediate descendants of the 4R duplication. However, up to 4 different chromosomal arms in their genome can be related through comparative synteny searches, to two different chromosome arms in model teleost fishes such as zebrafish and medaka . Based upon models of vertebrate chromosome evolution ,20, we mDescriptions and characteristics of the two rainbow trout mapping panels, and two Atlantic salmon mapping panels used for the comparative studies have previously been provided . Genetic and  with BAC end primer sequence information located exclusively at the former website. Information on the majority of the markers used in this study can also be obtained directly from the NCBI website . All SNP genotyping was performed using the MassARRAY system from Sequenom , and details on the protocol, EST sequences, and SNP polymorphisms are given in Moen et al. [Primer sequences for the rainbow trout and Atlantic salmon type I markers, SSR and EST markers can be found at the cGRASP websites: n et al. . v47 \u2013 v49. The most recent assemblies for the zebrafish genome  and medaka  were the subjects of query searches using the various type I, SSR, EST, and BAC end sequences from the salmonids. Sequences having expectation values lower than 10-6 and identity values greater than 70% were accepted as possessing acceptable homology matches to the salmonid genomes. In instances where multiple matches to several different linkage groups were detected, all with relatively low e-values, the marker assignments were ignored. In many instances, however, matches to only 1 or a few alternate linkage groups were evident in the subject searches. If the identified linkage groups were consistent with expected duplicated 3R linkage group affinities, the marker assignments were reported in Additional Files Homology assignments to the 3R teleost genomes were performed using the Distant Homologies BLASTN default options in ENSEMBL th linkage group (M) present in triplicate (following a single chromosome duplication) shortly following the origin of the ray-finned fishes. Each ancestral grouping should exhibit extensive homology to a minimum of two linkage groups within extant 3R derivative teleosts. Due to rearrangements within the genomes of these species, multiple smaller linkage group affinities are also evident for certain ancestral groups. The available data summarized in Table Information on the assignment of ancestral proto-Actinopterygian linkage groups within the genomes of zebrafish and medaka were obtained from the Kasahara et al. study . ThirteeData for the salmonid Tc1/Mariner elements were obtained from the de Boer et al. study  along wiWithin metacentric chromosomes we adopt the notation that short arms are designated as 'p' arms, while the long arm within each chromosome is designated as the 'q' arm. For fused Atlantic salmon chromosome arms, the segment closest to the centromere is designated as 'a' and more distal segments are designated as 'b' and 'c' . These oRGD carried out the bioinformatics analysis and wrote the manuscript, and along with MMF and WSD helped organize the conceptual framework of the manuscript. EAD, KG, KL, HKM and JP helped with development and genotyping of EST and BAC-end sequence markers, along with BH for many of the SSR markers. RBP helped with the interpretation of chromosome arm assignments related to short/long arm designations. BFK and SL contributed information on the Atlantic salmon SNP markers. All authors read and commented on the manuscript.s_table1_rt_lgs.xls. Genetic marker assignments to rainbow trout linkage groups.Click here for files_table2_as_lgs.xls. Genetic marker assignments to Atlantic salmon linkage groups.Click here for files_table3_l25f.map. Female genetic map for rainbow trout Lot 25 mapping panel.Click here for files_table4_l44f.map. Female genetic map for rainbow trout Lot 44 mapping panel.Click here for files_table5_l25m.map. Male genetic map for rainbow trout Lot 25 mapping panel.Click here for files_table6_l44m.map. Male genetic map for rainbow trout Lot 44 mapping panel.Click here for files_table7_br5f.map. Female genetic map for Atlantic salmon Br5 mapping panel.Click here for files_table8_br6f.map. Female genetic map for Atlantic salmon Br6 mapping panel.Click here for files_table9_br5m.map. Male genetic map for Atlantic salmon Br5 mapping panel.Click here for files_table10_br6m.map. Male genetic map for Atlantic salmon Br6 mapping panel.Click here for files_figure1-rtvsas_homology.pdf. Depiction of homologous Atlantic salmon linkage group blocks within the rainbow trout genome. Merged rainbow trout female maps are used as a template.Click here for files_figure2-asvsrt_homology.pdf. Depiction of homologous rainbow trout linkage group blocks within the Atlantic salmon genome. Merged Atlantic salmon female maps are used as a template.Click here for files_table11_rt-vs-dr&olat_synteny.xls. Database of zebrafish and medaka synteny blocks within the rainbow trout genome.Click here for files_table12_as-vs-dr&olat_synteny.xls. Database of zebrafish and medaka synteny blocks within the Atlantic salmon genome.Click here for filesupplementary figure 3.ppt. Regions of shared homology among zebrafish chromosomes 5, 10 and 21, and their shared affinities with zebrafish chromosome 8.Click here for files_table13_tc1_transposon-hits-medaka.xls. Location of salmonid mariner/Tc1 elements in the medaka genome.Click here for files_table14_salmonid_transposon-hits-vs-zf&medaka.xls. Counts of Tc1 element salmonid transposon BLASTN hits to all zebrafish and medaka linkage groups.Click here for filesupplementary figure 4.ppt. Number of Tc1/mariner transposon hits to zebrafish and medaka linkage groups.Click here for filesupplementary figure 5.ppt. Average bp interval among all DNA/LINE/SINE/LTR transposons detected among medaka linkage groups.Click here for file"}
+{"text": "We performed linkage and family-based association analysis across chromosomes 1\u201322 in Replicates 1\u20135 of the Genetic Analysis Workshop 15 simulated data. Linkage analysis was performed using the Kong and Cox allele-sharing test as implemented in the program Merlin. Association analysis was performed using the transmission/disequilibrium test (TDT). A region on chromosome 6 was consistently highlighted as showing significant linkage to and association with the disease trait. We focused in on this region and performed fine-mapping using stepwise regression approaches using the case/control and family-based data. In this region, we also applied several new methods, implemented in the computer programs LAMP and Graphminer, respectively, that have recently been proposed for association analysis with family and/or case/control data. All methods confirmed the highly significant associations previously observed. Differentiating between potentially causal single nucleotide polymorphisms (SNPs) and other non-causal loci (associated with disease merely due to linkage disequilibrium) proved to be problematic. However, in most replicates we did identify two SNPs  that together explain most of the observed disease association in the DR/C locus region, and an additional SNP (3931 or 3933) that accounts for the association 5 cM away at locus D. We analyzed Replicates 1\u20135 of the Genetic Analysis Workshop 15 (GAW15) simulated data using both linkage and association methods. The analyses were performed without knowledge of the 'answers', however we subsequently obtained the 'answers' to inform our discussion. Using 1500 fully genotyped affected-sib-pair (ASP) families (parents and two children) in each replicate, we first tested for linkage across the genome using the Kong and Cox . We thenp-value of 10-3 to enter/exit the model) with the subset of SNPs showing a TDT \u03c72 > 30. We repeated the regression analyses using logistic  regression in a case/control data set constructed by taking the first affected sib from each ASP together with the 2000 fully genotyped population controls provided.Given the highly significant results obtained on chromosome 6, we attempted to fine-map this region using first the original non-dense SNP set, then the dense chromosome 6 SNP set, and finally the combined non-dense/dense SNP set. We used the stepwise conditional logistic regression (case/pseudocontrol) approach for family data proposed by Cordell and Clayton . We inve and can be used to analyze either the full sample of ASPs together with the unrelated controls, the ASPs alone, a single affected sibling drawn from each ASP, or a single affected sibling together with the unrelated controls. Finally, we also used a recently proposed Bayesian approach for case/control association analysis [In addition to fine-mapping, we also investigated the use of a new likelihood-based association analysis approach in this region  in whichanalysis  implemenanalysis .In each replicate, regardless of whether microsatellite or non-dense SNP markers were used, the most significant region of linkage was found on chromosome 6, centered around the 50 cM location where the 'true' DR and C loci reside. The Kong and Cox  LOD scorp < 10-4) across all five replicates were at SNP 389 on chromosome 11 , and at SNP 269 on chromosome 18 . It is interesting to note from the 'answers' that the position of the chromosome 11 result corresponds to 'true' locus F which has an indirect effect on disease susceptibility through IgM, while the position of the chromosome 18 result corresponds to 'true' locus E.When using the non-dense SNPs, highly significant TDT  results p = 4 \u00d7 10-8) was still seen at 'true' locus D  was found when using the maximum sample size . Strong evidence for association was also found using the Bayesian approach implemented in Graphminer. Both the non-dense and the dense SNP sets Figures , 6 provid Figure : the resNot surprisingly, given the large sample size and strong simulated effects at the DR, C and D loci (mimicking known effects at HLA in diseases such as RA and type 1 diabetes), this chromosome 6 region was consistently significantly implicated in disease susceptibility via both linkage and association analysis. No other regions were consistently implicated by linkage analysis, but genome-wide association analysis detected SNPs associated with disease susceptibility at locations corresponding to 'true' loci E and F on chromosomes 18 and 11, in all five simulation replicates examined.Fine mapping of the chromosome 6 region using Graphminer [The author(s) declare that they have no competing interests."}
+{"text": "Parental consanguinity is a risk factor for congenital heart disease (CHD) worldwide, suggesting that a recessive inheritance model may contribute substantially to CHD. In Bangalore, India, uncle-niece and first cousin marriages are common, presenting the opportunity for an international study involving consanguinity mapping of structural CHD. We sought to explore the recessive model of CHD by conducting a genome-wide linkage analysis utilizing high-density oligonucleotide microarrays and enrolling 83 CHD probands born to unaffected consanguineous parents.HOMEZ gene, a ubiquitously expressed transcription factor containing leucine zipper as well as zinc finger motifs. Re-sequencing of HOMEZ exons did not reveal causative mutations in Indian probands. In addition, genotyping of the linked allele (G) in 325 U.S. CHD cases revealed neither genotypic nor allele frequency differences in varied CHD cases compared to 605 non-CHD controls.In this linkage scan involving single nucleotide polymorphism (SNP) markers, the threshold for genome-wide statistical significance was set at the standard log-of-odds (LOD) score threshold of 3.3, corresponding to 1995\u22361 odds in favor of linkage. We identified a maximal single-point LOD score of 3.76 (5754\u22361 odds) implicating linkage of CHD with the major allele (G) of rs1055061 on chromosome 14 in the HOMEZ may harbor recessive mutations leading to CHD in the Indian population. Further research involving large multinational cohorts of patients with specific subtypes of CHD is needed to attempt replication of the observed linkage peak on chromosome 14. In addition, we anticipate that a targeted re-sequencing approach may complement linkage analysis in future studies of recessive mutation detection in CHD.Despite the statistical power of the consanguinity mapping approach, no single gene of major effect could be convincingly identified in a clinically heterogeneous sample of Indian CHD cases born to consanguineous parents. However, we are unable to exclude the possibility that noncoding regions of  EVC or EVC2PDA1 as a candidate locus for patent ductus arteriosus in an Iranian population Congenital heart disease (CHD) affects approximately one percent of newborn infants The consanguinity mapping approach has not yet been widely applied to nonsyndromic CHD, in part because parental consanguinity is uncommon in places where research efforts have historically been most intense. However, interest is growing in the statistical power of this approach for genetic investigations of other complex phenotypes, such as autism, as demonstrated by the Homozygosity Mapping Collaborative for Autism CHD presents major public health challenges in India, due to the large annual number of births in the Indian population and the limited resources for specialized care of children with CHD HOMEZ on chromosome 14 at 22,814,772. The LOD score was 3.76 (5754\u22361 odds), and the optimal theta value was 0.05. The genotype frequencies of the probands were GG\u200a=\u200a0.83, GA\u200a=\u200a0.13 and AA\u200a=\u200a0.04. The second SNP, rs12433225, yielded a LOD score of 3.65 (4467\u22361 odds) and maps to chromosome 14 at 25,596,730, more than 250 kb away from any known gene in either direction. As a supplemental analysis, we also conducted a TDT test of association using WASP, and obtained a significant p-value of 0.0495 for rs1055061 with the major allele (G) overtransmitted and a non-significant p-value of 0.8185 for rs12433225.The phenotypic distributions of the cohorts are described in Each of the linked SNPs was examined in the context of autozygosity of the probands. SNP rs1055061 resided in a region of autozygosity in 13 patients, while SNP rs12433225 localized to a region of autozygosity in 14 patients. Analysis of 39 other SNPs randomly selected from the Affymetrix platform resulted in a mean of 6.6 probands  with autozygosity surrounding the region of interest. Additionally, the minimum region of autozygosity shared by the 13 patients surrounding rs1055061 is Chr14:21,071,230\u201324,393,433, and the minimum region of autozygosity shared by the 14 patients surrounding rs12433225 is Chr14:24,952,386\u201325,774,000.HOMEZ in 54 probands. On sequence analysis of the two coding exons, four previously known SNPs We re-sequenced the coding regions of HOMEZ SNP rs1055061 was also associated with CHD in an American population, we genotyped cases and controls from this population to assess allele frequency differences using an ABI TaqMan assay. Frequencies were 282 GG, 42 GA, and 1 AA in 325 patients with CHD and 548 GG, 57 GA and no AA in 605 control patients. The difference in the frequency of the GG genotype in cases versus controls was not statistically significant by Chi-squared analysis (P\u200a=\u200a0.074). Secondary analysis of an allele frequency difference between cases and controls was not statistically significant by Chi squared analysis (P\u200a=\u200a0.062).The population in our linkage analysis was comprised of children of consanguineous Indian parents. To determine if the We have presented statistically significant linkage evidence for a potential CHD susceptibility locus on chromosome 14 in a South Indian population selected for consanguinity. However, the linkage finding was not robust in a genetic association follow-up study of CHD in the general United States population. Utilizing the extensive genotyping information in oligonucleotide arrays, linkage analysis revealed two markers with LOD scores above 3.3. Because approximately 2.8 Mb separates these markers, a distance far in excess of typical linkage disequilibrium values in the Indian population HOMEZ, a vertebrate specific homeobox gene with an interesting structural organization. This transcription factor harbors three atypical homeodomains, two leucine zipper-like motifs, and an acidic domain. The protein is ubiquitously expressed in human tissues, and has been further studied in a murine developmental model HOMEZ have not been described Of the two markers, only rs1055061 is intragenic, causing a non-synonymous amino acid substitution. This SNP lies within The rs1055061 SNP identified by linkage induces an amino acid substitution from the polar, positively charged arginine to the polar, but neutral glutamine. The allele with the higher population frequency was overtransmitted in the TDT analysis, whereas one might expect a rare recessive allele to confer susceptibility to an uncommon phenotype such as CHD. Linkage analysis resulted in a theta value of 0.05, indicating that a causative mutation most likely lies within 50 kb of this SNP. Additionally, an increased number of probands exhibited a region of autozygosity surrounding each of the linked SNPs, supporting our hypothesis of autosomal recessive inheritance due to parental consanguinity. We do acknowledge that the LOD score of 3.76 indicates a 1\u22365754 odds of this finding being due to chance alone, but argue that the convergence of multiple methods of inquiry support rs1055061 as a true finding.HOMEZ in a subset of the Indian probands with the hypothesis that a deleterious mutation in linkage disequilibrium with rs1055061 would be discovered. We detected previously reported SNPs and three novel sequence variations. None of the novel variations were found to be homozygous, as would be expected for causative mutations in offspring of a consanguineous relationship. Thus, we were unable to identify any causative homozygous mutations in the coding regions of HOMEZ.We thus proceeded with sequence analysis of the coding regions of We also studied the rs1055061 SNP in an American cohort of patients affected with various types of CHD and compared them to population controls. No significant difference was detected in the genotype or allele distribution between cases and controls, suggesting that our linkage finding in South Indians does not extend to genetic association in an American population of mixed European ancestry.Although this study did not reveal definitive evidence of causal mutations leading to autosomal recessive CHD, a number of future directions and refinements are suggested. A limitation in the application of consanguinity mapping to CHD is that the fraction of this heterogeneous group of diseases attributable to recessive inheritance and the approximate number of loci involved is not known. Ascertaining consanguineous families with multiple affected children (suggesting recessive inheritance) would theoretically reduce genetic heterogeneity and increase power for linkage. If only multiplex pedigrees are enrolled, the proportion of probands with sporadic or autosomal dominant forms of CHD will be minimized.a priori which forms of CHD share a common genetic etiology. This may have resulted in diffusion of the linkage signal because of both clinical and genetic heterogeneity. Enriching the study population for predefined, specific phenotypic classes would likely decrease genetic heterogeneity in the study population.We did not restrict our analysis to specific CHD phenotypes because it is impossible to select Recent work in massively parallel whole exomic Written informed consent was provided by the parents of all children assenting to participate in the study, using consent documents approved by the ethics board of Narayana Hrudayalaya Heart Hospital. Dr. Sunita Maheshwari, Principal Investigator at Narayana Hrudayalaya Heart Hospital, and Dr. Amit Misri, co-investigator, obtained consent from participating families. Genotyping was performed on de-identified samples without knowledge of any personal identifiers under IRB approval from Yale University, Washington University in St. Louis, and Vanderbilt University.We enrolled children affected with congenital heart disease and born to consanguineous parents in order to identify candidate regions for CHD by linkage analysis. The families were ascertained in a cardiology clinic at Narayana Hrudayalaya in Bangalore, India, from 07/2004 to 07/2007. The inclusion criteria were the presence of structural congenital heart disease and parental consanguinity. Exclusion criteria were isolated conduction system defect, cardiomyopathy, or extracardiac syndromic features. The patients had an echocardiogram performed in the clinic and interpreted by pediatric cardiologists. In all, 83 probands were recruited from 81 families. Probands were not known to be related to each other with the exception of one family that enrolled three affected siblings. Both parents were recruited in 71/81 families, and one parent in 10/81 families. The phenotypic distribution of the probands' cardiac lesions is described in A replication cohort of 325 probands affected with congenital heart disease was ascertained at Vanderbilt University Medical Center via therapeutic clinical trials of oral Genomic DNA for probands 1\u201326; 39\u201350 and relatives was isolated from blood spots on Whatman filter paper using GenSolve blood spot DNA recovery kit according to the manufacturer's protocol . DNA from probands 27\u201338 was isolated from peripheral blood samples using Gentra PureGene Blood Kit . For probands 51\u201383 and relatives, saliva was collected with Oragene kits, and DNA was extracted per manufacturer's protocol . We performed genome wide scans utilizing Affymetrix SNP 5 microarrays for probands 1\u201330 and relatives with genotyping reactions performed at Precision Biomarker Resources . Probands 39\u201383 and relatives were genotyped on Affymetrix SNP 6.0 microarrays, and genotyping reactions were performed according to the manufacturer's instructions . Genotypes were generated using the BRLMM-P for Affymetrix SNP 5 arrays and the Birdseed V-2 algorithm for Affymetrix SNP 6.0 arrays as released in Genotyping console v3.0.1 . The SNP 5 arrays had an average QC call rate of 94.5% with SD of 4.8%. The SNP 6 arrays had an average QC call rate of 91.2% with SD of 5.6%. The genotype data was exported as .chp files and further analyzed with Merlin,v1.1.2 Assessment of regions of autozygosity were conducted in Partek software, V6.4 Copyright 2009  with the algorithms for loss of heterozygosity (LOH). Unpaired analysis parameters included max probability 0.99, genomic decay 0, genotype error 0.02, and default frequency 0.3. All genotype calls from the probands were used to generate array specific baselines. Random markers were selected from the Affymetrix SNP5 & SNP6 platforms using a random number generator. Each result from the LOH analysis overlapping the genomic location of interest was visually inspected to determine true autozygosity.HOMEZ with bidirectional sequencing in a subset of Indian probands (n\u200a=\u200a54). Four primer sets covering the coding exons and their flanking regions were designed with Primer3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). Fragment amplification was performed under standard PCR conditions. In brief, 50 ng of DNA was used in the reaction; PCR program started with 94\u00b0C for 5 min, 35 cycles of 94\u00b0C, 57\u00b0C and 72\u00b0C, a final extension cycle of 72\u00b0C for 6 minutes and stored at 4\u00b0C. PCR products were purified using Exo-SAP (Fermentas) and sequenced with the ABI 3730xl DNA Analyzer at the DNA Sequencing Facility, Vanderbilt University.We carried out a mutation screening of For the replication cohort, a proprietary TaqMan genotyping assay for rs1055061 was obtained from Applied Biosystems (Assay ID: C___2484687_10). After lyophilizing 10 ng genomic DNA per sample, the genotyping assay and TaqMan Universal PCR Master Mix (Applied Biosystems 4371355) were then prepared and run as per manufacturer's protocol. Applied Biosystems SDS\u00ae 2.3 was used to analyze the results of the genotyping assay. Genotypes were assigned without knowledge of the affected status of the samples."}
+{"text": "We conducted linkage only, linkage and association, and association only tests under this framework. We also compared these results with variance-component linkage analysis and regression analyses. The results indicate that the method shows some promise, but finding genes that have very small (<0.1%) contributions to trait variance may require additional sources of information. All methods examined fared poorly for the smallest in the simulated \"polygene\" range ( Both linkage analysis and association analysis provide useful, but slightly different, forms of information in identifying genetic contributions to complex traits. Frequently, either one or the other type of analysis is employed, which can result in information being overlooked: linkage does not account for the association frequently seen between disease alleles and nearby markers, while association (on family data) may account for family structure, but does not make use of the location information that the meiotic events provide. When both are used, the analyses are typically done under different frameworks. Here, we consider an integrated analysis of both linkage and association under an oligogenic model  and comp\u00ae Human Mapping 500 k Array Set and the 50 k Human Gene Focused Panel) from actual Framingham data, also provided to GAW16. Traits were simulated on to these SNPs by selecting several SNPs as oligogenes (h2 of 0.01 to 0.001) and 1000 SNPs as polygenes (h2 of 0.0015 to 0.0002) for each trait. Some of the \"polygenes\" have effects as large as those of the \"oligogenes.\" As in real data, the boundary between the two is fuzzy. The polygenes were selected more randomly, although some were selected as clusters. We focused our analysis on chromosome 11 and simulated low-density lipoprotein (LDL) and high-density lipoprotein (HDL) at the first visit. For linkage, we selected two subsets of 1-cM spaced markers with a simple algorithm that traversed the chromosome at 1-cM distances given two starting points. Linkage information is captured almost completely by markers at this density. Thus, adding additional markers only provides a marginal increase in information, while increasing the computational burden [For Genetic Analysis Workshop (GAW) 16, a simulated data set was provided based on the Framingham Heart Study. Multiple replicates of the simulation were provided to help assess methods and we used several of these replicates for this purpose. This simulation used ~550,000 single-nucleotide polymorphisms (SNPs) (GeneChipWe used Markov-chain Monte Carlo (MCMC) methods to produce Bayesian statistics for segregation, linkage, and association. These methods are implemented in the computer program Loki . Covaria\u03bc is the \"reference\" trait value, X is the incidence matrix for covariate effects, \u03b2 is the vector of covariate effects, Qi is the incidence matrix for the effects of quantitative trait locus (QTL) i, \u03b1i is the vector of effects for QTL i, e is the normally distributed residual effect, k is the number of QTLs currently estimated (k \u2265 0), Sj is the incidence matrix for the effects of SNP j, \u03b3j is the vector of effects for SNP j, and l is the number of SNPs being tested for association in the analysis run. The MCMC process samples \u03bc, \u03b2, \u03b1i, \u03b3j, i, and e as well as parameters such as unobserved marker genotypes and QTL genetic position. All of these parameters are sampled from the space of model values consistent with the data observed. Values are sampled proportional to their posterior probability. After the number of sampling iterations is sufficiently large, the sampled values provide an estimate of the posterior probability distribution over the parameter space. The difference between this and previous applications of the method is in the number of SNPs we test. Previously, we have included select candidate genes as genetic covariates. Here we will test all the SNPs available on chromosome 11 for association with this method to examine how the method scales up to genome-wide association testing.where l = 0). Subsequently, single SNPs and sets of SNPs were added to test for association. When testing SNPs for association, we considered 1) z-scores for each of the elements of \u03b3j being non-zero, which assessed the strength of the association, and 2) whether adding SNP j reduced any linkage signal found in the region, which indicated that SNP j was associated with the mutation causing the linkage signal. If a single SNP in a region produced the linkage signal, including it for association testing should eliminate the linkage peak, by moving the effect from the segregation term  led to insufficient power. These results indicate that this method can be useful in candidate gene studies.First, we compared the MCMC linkage s Figure . Some SNh2 < 0.001, was marginal and so different replicates may have had different signal strength at different locations.In comparison of the variance-component linkage results and MCMC oligogenic linkage, we found that within a simulated replicate for this data set, the two methods produce similar results Figure . Howeverr2 explained within 2-cM intervals. In Figure r2 between replicates and neither seems to reflect the concentration of SNPs around 110 cM. These results suggest that both linkage and association tests alone might fare poorly within gene clusters with a multitude of mutations with each h2 < 0.001.To further explore between-replicate variation, we computed z-scores| > 2. This is slightly greater than the 5% expected under the null hypothesis. With many simulated \"causative\" SNPs, nearly all z-scores could be argued to be \"near\" a causative SNP, and thus there was no empirical null in this data. These single-SNP tests were computationally intensive, and probably not practical on a genome-wide scale with current technology. There were some positive signals here, so an alternate methodology may facilitate these tests with dense SNPs. As currently implemented, these methods are best suited to combined linkage and candidate gene association studies.Finally, in single-SNP association tests under the MCMC oligogenic model, ~1,700 out of ~26,000 SNPs had |h2 on the order of 0.01. We had issues detecting some of the \"polygenes\" in this simulation (h2 of 0.0015 to 0.0002), but all methods appear to lack power to detect the smallest of these \"polygene\" effects. At the margin of such effects, it is important to extract as much information from the data as possible and our motivation here was to examine the benefits of combining both linkage and association.Oligogenic analysis of combined linkage and candidate gene association appears to work for genes with an r2 explained in regression computed over 2-cM intervals are discouraging because of the poor correspondence between gene locations and r2 peaks. However, there does appear to be room for improvement.Here, we saw that there was some benefit to examining both types of information simultaneously. However, the lack of power to detect the very smallest \"polygenes\" is cause for concern. If the genes in this simulation are not unrealistically smaller than those that exist in real traits, these results suggest that many true positives could be due to random reinforcing of true signals, and replication will be difficult. It could be that the very smallest effects in the simulation may be undetectable. The results of computed z-scores with an absolute value >2, they do not survive multiple testing corrections at the genome-wide association study level. Incorporating additional sources of information, such as that on gene networks, might help with this issue. While our success here was more limited than we hoped, incorporating information from multiple sources in a single framework may help detect marginal genetic signals.The methods used here, in their current state, are very useful for testing of candidate gene associations. Fully incorporating genome-wide association study data will require algorithmic improvements. In particular, while there were many GAW: Genetic Analysis Workshop; HDL: High-density lipoprotein; LDL: Low-density lipoprotein; MCMC: Markov-chain Monte Carlo; QTL: Quantitative trait locus; SNP: Single-nucleotide polymorphismThe authors declare that they have no competing interests.EWD conceived and coordinated the study, participated in its design, carried out the MCMC analyses, and drafted the manuscript. JP performed the variance-component linkage analysis and plotted the results. MF participated in the design and helped with the variance-component linkage. XG participated in the design. DM carried out the association analysis. JC helped prepare the data and design algorithms for marker selection. MAP participated in the design. IB participated in the design."}
+{"text": "Autism is a common heritable neurodevelopmental disorder with complex etiology. Several genome-wide linkage and association scans have been carried out to identify regions harboring genes related to autism or autism spectrum disorders, with mixed results. Given the overlap in autism features with genetic abnormalities known to be associated with imprinting, one possible reason for lack of consistency would be the influence of parent-of-origin effects that may mask the ability to detect linkage and association.GH\u200a=\u200a3.79, empirical p<0.005 and LODAspex\u200a=\u200a2.96, p\u200a=\u200a0.008), 15  and 20 .We have performed a genome-wide linkage scan that accounts for potential parent-of-origin effects using 16,311 SNPs among families from the Autism Genetic Resource Exchange (AGRE) and the National Institute of Mental Health (NIMH) autism repository. We report parametric  and allele-sharing linkage (Aspex) results using a broad spectrum disorder case definition. Paternal-origin genome-wide statistically significant linkage was observed on chromosomes 4 (LODThese regions may harbor imprinted sites associated with the development of autism and offer fruitful domains for molecular investigation into the role of epigenetic mechanisms in autism. Autism is a neurodevelopmental disorder that is manifested in early childhood and is characterized by impairments in reciprocal social interactions and language, and a restricted range of behaviors and interests. Autism is considered a spectrum disorder (ASD) with heterogeneity in symptom presentation. Inheritance clearly plays a major role in susceptibility to autism Epigenetic factors, which are often heritable, yet not part of the DNA sequence, are one element which may contribute to this etiologic complexity. Imprinting is an epigenetic modification that is parental origin specific, leading to preferential expression of a specific parental allele in somatic cells of the offspring CNTNAP2 gene (contactin-associated protein-like 2) using genome-wide SNP analyses If imprinting plays a role in the heritable etiology of ASD, the power of linkage analyses to identify susceptibility loci may be improved by accounting for allelic parent-of-origin. This has been observed for specific autism-implicated genomic regions such as 7q, where both paternal and maternal allele sharing have been observed to account for the linkage to an autism locus in this region To date, however, no genome-scale parent-of-origin-specific linkage analysis has been reported for ASD. Here we apply parent-of-origin linkage analysis to the genome-wide SNP data recently reported by Weiss et al. in a common set of multiplex autism families www.agre.org). From AGRE, we considered children with autism, \u201cnot quite autism (NQA),\u201d or \u201cbroad spectrum\u201d as affected family members to encompass those with related disorders such as Aspergers syndrome and PDD-NOS. Information on participant recruitment and study procedures for the NIMH sample is available on the program website (www.nimh.nih.gov). We selected NIMH families with a child diagnosed with an Autism Spectrum Disorder based on evaluation by the Autism Diagnostic Interview-Revised (ADI-R) and ADOS instruments. The combined data set, consisting of 1,216 nuclear families, was used for genetic analyses. All families used in our analyses had at least one genotyped parent; 89.4% had genotypes for both parents.The samples used here were previously described by Weiss et al. All samples used in this study arose from investigations approved by the appropriate Institutional Review Boards for institutions where participants were recruited, evaluated, or where genotype data were generated. Written informed consent was obtained for all adult study participants; for children under age 18, both the consent of the parents or guardians and the assent of the child were obtained. This secondary analysis of de-identified data was considered to be exempt from IRB review.2 >0.1, providing a marker density of 0.25 cM. Genetic distances were taken from the Affymetrix Genetic Map (www.affymetrix.com/estore/browse/products.jsp?productId=131459&categoryId=35906#1_3)SNP genotyping was previously described i.e., penetrances of 1, 0.8, 0.6, 0.4, 0.2) and by increasing phenocopy rates at similar increments. Empirical p values for these sensitivity results were estimated similarly to the initial genome-wide empirical p values (see below), but with the optimized parametric model applied for all locations.Parametric and non-parametric parent-of-origin linkage methods were applied. Parametric linkage analysis was conducted using GENEHUNTER-IMPRINTING 2.1 (GHI) For non-parametric linkage analysis, maximum likelihood estimates of allele sharing at each locus were computed using the ASPEX \u201csib_ibd\u201d command. The \u201csex_split\u201d option was implemented to evaluate evidence for linkage based on maternal and paternal sharing separately. 1070 affected sibling pairs were informative  for these analyses. LOD scores indicate the log ratio of the likelihood of the marker data at this position with the MLE estimate of the sibling recurrence risk ratio, versus the likelihood assuming a sibling risk ratio of 1.Empirical genome-wide p values were calculated via simulation. The program Merlin Genome-wide results for maternal and paternal linkage analyses via both parametric and non-parametric methods are shown in Although significant maternal peaks were observed in both parametric and allele-sharing methods, no consistency in signal was seen. A significant maternal peak was observed on chromosome 14 in parametric analyses , although this was not observed in allele-sharing analysis. Maternal allele sharing peaks were observed on chromosomes 5, 6, 7, and 9 , althougGiven the vital role of imprinted genes in development, the fact that many known imprinted genes are expressed in the brain, and evidence of overlapping features in autism and imprinting disorders, we investigated the effect of incorporating allelic parent-of-origin into an autosomal linkage scan for autism. To our knowledge, this is the most extensive linkage analysis for parent-of-origin effects in autism to date. We found the strongest evidence for parent-of-origin effects on chromosomes 4, 20 and 15, implicating sites where imprinted loci related to autism may reside.\u22126) located on this region and autism using the same data without regard to parental origin. The strongest candidate gene in this region is CLOCK, which codes a protein regulating circadian rhythm and whose involvement in ASD was first proposed by Wimpory et al. The section of chromosome 4 located between markers rs6826933 and rs17088473 showed several significant results in our analysis and spans the region between 4q12-4q13.2. Recently, Weiss et al. found an association between one SNP . The location of this microsatellite peak also showed linkage in the SNP data, but it was not the highest SNP linkage peak on chromosome 4.Analyses of a panel of microsatellite markers in 348 AGRE families from previously reported linkage analysisRASGRF1 gene, a homologue of the imprinted rasgrf1 in mouse NRG4 (neuregulin 4) and CHRNA3/B4 . Genes in the neuregulinMTHFS  gene which is implicated in DNA methylation cycle and may be particularly important in an epigenetic mechanism of autism risk.A region of chromosome 15 (15q23-15q25.3) also shows paternal linkage. This region was previously implicated using traditional linkage analysis in these SNP data SNPH (Syntaphiliyn) gene. SNPH interacts with the synaptic vesicle-associated protein synaptobrevin/VAMP and the plasma membrane-associated protein SNAP25 to form the SNARE complex, which is required for synaptic vesicle docking and fusion. Expression of this gene appears to be brain specific.We have also reported a strong paternal linkage on chromosome 20p, which was previously implicated in the linkage analyses reported by Weiss et alet al report a male child with autism having a maternal uniparental disomy (UPD) of chromosome 1 et al. also found a linkage between the 1q23-1q24 region and autism using an AGRE sample Other suggestive parent-specific linkage regions are located throughout the genome . The paternally linked region on chromosome 1 (1q23-1q24.2) was previously associated with autism. Wassink www.geneimprint.com) lists four predicted imprinted genes on chromosome 5; however the regions do not directly overlap the location detected by our analysis.A maternally linked region was observed on chromosome 5, with the peak at 5p13.1. Recent genome-wide association studies have reported risk loci for autism at 5p14.1 PARK2 gene located on 6q25.2-6q27 was significantly enriched for CNVs and observed in the ASD cases only PARK2 allele inherited from father. PARK2 is an ubiquitin-protein ligase, mutations of which cause autosomal recessive juvenile Parkinson's disease The signal found on 6q25.3-6q27 region was previously linked to autism CNTNAP2 gene, a member of the neurexin superfamily, that is significantly associated with autism susceptibility Two other groups have previously reported parent-of-origin linkage with autism for closely located loci on chromosome 7; one was a paternal contribution in the region 7q31.33-7q34 Few previous studies have considered parent-or-origin effects in autism. Those that have used previous-generation marker sets and much smaller samples than the results presented here. Two previous studies observed parent-or-origin linkage on chromosome 7, but with different regions and types of parental sharing In an attempt to detect loci with possible parent-of-origin effects, we used multiple statistical approaches, rather than relying on a single strategy. Consistent evidence of linkage across multiple methods increases support for a true linkage. However, the appropriate interpretation of inconsistent results across parametric and non-parametric analyses is not entirely clear. These may be due to chance findings in one analysis, or they may be true linkage that only one method was sufficiently powered to detect. For example, the chromosome 1 peak was significant only in the parametric analysis, a method which is more powerful given that the parameters are correctly specified. While it is unrealistic to believe that we could actually have specified the \u201ccorrect\u201d parameters given the complex nature of autism, those selected may have been sufficiently close. Peaks on chromosomes 6 and 9 were significant in the non-parametric ASPEX analysis; however, the parametric GHI analysis did not find significant peaks on these chromosomes, which may be due to selection of \u201cincorrect\u201d parameters for the models run.These analyses considered as affected all children with an ASD, as defined by the ADI-R and ADOS in the NIMH sample. However, in AGRE, we included those with autistic disorder, as well as those with \u201cnot quite autism\u201d and \u201cbroad spectrum\u201d to encompass Asperger's and PDD-NOS. This may have contributed to some heterogeneity or misclassification in our data, but was considered more appropriately inclusive and comparable to the NIMH ASD families than excluding a larger number of AGRE families with an ASD other than autistic disorder.Our results suggest the usefulness of genome-wide analysis with evaluation of parent-of-origin effects, although future studies are necessary to determine if these results can be replicated. Given the potential role for imprinting and other epigenetic mechanisms in neuropsychiatric disorders such as autism Figure S1Parent-of-Origin Linkage Analysis for Microsatellite Markers in 384 AGRE families. A: Parametric results. Panel B: Allele sharing results. Maternal scores are shown in red; paternal scores are in blue.(0.68 MB TIF)Click here for additional data file."}
+{"text": "It has been suggested that efforts to identify genetic risk markers of autism spectrum disorder (ASD) would benefit from the analysis of more narrowly defined ASD phenotypes. Previous research indicates that 'insistence on sameness' (IS) and 'repetitive sensory-motor actions' (RSMA) are two factors within the ASD 'repetitive and stereotyped behavior' domain. The primary aim of this study was to identify genetic risk markers of both factors to allow comparison of those markers with one another and with markers found in the same set of pedigrees using ASD diagnosis as the phenotype. Thus, we empirically addresses the possibilities that more narrowly defined phenotypes improve linkage analysis signals and that different narrowly defined phenotypes are associated with different loci. Secondary aims were to examine the correlates of IS and RSMA and to assess the heritability of both scales.A genome-wide linkage analysis was conducted with a sample of 70 multiplex ASD pedigrees using IS and RSMA as phenotypes. Genotyping services were provided by the Center for Inherited Disease Research using the 6 K single nucleotide polymorphism linkage panel. Analysis was done using the multipoint linkage software program MCLINK, a Markov chain Monte Carlo (MCMC) method that allows for multilocus linkage analysis on large extended pedigrees.Genome-wide significance was observed for IS at 2q37.1-q37.3 (dominant model heterogeneity lod score (hlod) 3.42) and for RSMA at 15q13.1-q14 (recessive model hlod 3.93). We found some linkage signals that overlapped and others that were not observed in our previous linkage analysis of the ASD phenotype in the same pedigrees, and regions varied in the range of phenotypes with which they were linked. A new finding with respect to IS was that it is positively associated with IQ if the IS-RSMA correlation is statistically controlled.The finding that IS and RSMA are linked to different regions that only partially overlap regions previously identified with ASD as the phenotype supports the value of including multiple, narrowly defined phenotypes in ASD genetic research. Further, we replicated previous reports indicating that RSMA is more strongly associated than IS with measures of ASD severity. Diagnostic and Statistical Manual of Mental Disorders 4thed. (DSM-IV)[Although it is generally accepted that genetic factors play a major role in the etiology of autism spectrum disorders (ASDs), identif (DSM-IV) diagnost (DSM-IV)-5. Thus, (DSM-IV),6.et al., Genome-wide linkage using the Social Responsiveness Scale (SRS) in Utah autism pedigrees, submitted). Although each of these methods has merit, it should be noted that the first method attempts to reduce heterogeneity of the diagnostic phenotype by stratification on a specific trait, whereas the second and third approaches seek to identify risk markers for the trait itself.Specific ASD/AD traits have been employed in genetic studies most often either to stratify pedigrees for linkage analysis or as the dependent variable in association tests for specific alleles. For example, the first approach has found stronger ASD linkage signals in pedigrees with more abnormal levels of phrased speech delay ,8, repetRepetitive and stereotyped behavior is a promising candidate for further genetic study because it probably comprises at least two even more specific phenotypes that differ in their behavioral correlates, familiality, and relation to genetic linkage with ASD. The 'restricted and repetitive stereotyped behavior' (RRSB) domain of the Autism Diagnostic Interview--Revised (ADI-R) ,19 is a It is well established that IS and RSMA have different patterns of relationship with other ASD traits. Specifically, RSMA, but not IS, has been reported to be associated with lower IQ, less adaptive behavior, and later age of appearance of first words and phrases ,20,21, wet al. [There is more empirical support for a genetic effect on IS than on RSMA. Whereas modest evidence of familial concordance occurs for IS, no reported concordance occurs for RSMA ,25. Thuset al.  reportedet al.  over scoet al. , which sWe are not aware of previous genetic linkage studies with either IS or RSMA as the phenotype. The primary aim of the present study was to perform a genome-wide linkage analysis with both IS and RSMA as phenotypes using large extended ASD pedigrees. Thus, our goal was to identify genetic risk regions for IS and RSMA in ASD cases rather than to stratify on IS and RSMA to reduce ASD heterogeneity. Because IS and RSMA data were available only for ASD cases rather than for all pedigree members, we focused our analyses on these specific phenotypes in ASD cases and did not include clinically unaffected family members in this study. Signals obtained with these phenotypes were compared with those found in the same set of pedigrees using ASD diagnosis . ContrasThis study has ongoing approval from the University of Utah institutional review board (IRB). All adults participating in the research signed informed consent documents. All subjects under the age of 18 signed assent documents and their parents or guardians signed parental permission documents. These documents were approved by the University of Utah IRB.Subjects were members of 70 pedigrees having at least two family members with ASD. In total, 653 subjects were genotyped, 192 of whom had a study diagnosis of ASD. Study diagnosis was based in almost all instances on both the ADI-R ,19 and tRSMA and IS scales were derived from the RRSB domain of the ADI-R, which was available for 183 subjects with a study diagnosis of ASD. RSMA and IS items were ADI-R items that reliably loaded on one scale or the other in previous factor analytic studies ,20-25. FItems from the ADI-R ('age of first words' and 'age of first phrases') were used to assess language delay in ASD cases. For parents who indicated normal onset but who could not remember the exact ages, values were set to 23 months for words and 32 months for phrases . For parents who indicated delayed onset but could not remember the exact ages, values were set to 1.5 standard deviations above the mean. For subjects who never acquired language, values were set to 3 standard deviations above the mean.IQ was measured in subjects with ASD using an assessment instrument appropriate for the subject's age and developmental level. IQ measures included the Wechsler Intelligence Scale for Children, 3rd revision (WISC-III) , the WecThe SRS is a quantitative measure of social ability ranging continuously from significantly impaired to above-average social abilities . Althoug2 value of 0.05 between SNPs has been supported with extensive simulation studies [2 of 0.33 regressed simultaneously over all SNPs in the selected window. This r2 considers not only the correlations between SNPs but also between linear combinations of SNPs [2 value of approximately 0.05. This screening for LD removed 1,207 SNPs. As part of the validation procedure, we also removed 115 SNPs with a minor allele frequency < 0.10 and 4 SNPs that were not in Hardy-Weinberg equilibrium (standard 1 degree of freedom test failed at the 0.05 level). The total number of SNPs left after this phase was 4,718.Genotyping services were provided by the Center for Inherited Disease Research (CIDR), using the 6 K single nucleotide polymorphism (SNP) linkage panel. Methods and quality control procedures have been described in detail previously . After q studies . Therefo studies , which r of SNPs , and corThe heritability (proportion of variance in the trait due to genetic influences) of IS and RSMA was estimated using SOLAR software . For disWe used the genetic map provided by CIDR based on the deCODE genetic map . Base paWe performed nonparametric and general parametric model-based analyses. Although nonparametric methods are the standard analytic approach for complex psychiatric disorders, parametric methods have some advantages in the analysis of a complex trait such as ASD, particularly when using large extended pedigrees. Parametric models, which are based on assumptions about the genotype-phenotype relationship, simplify the parameter space and allow for more powerful and efficient analyses without leading to false-positive results ,52. We dFor both IS and RSMA, the phenotype was coded as unknown if the measure was not available, unaffected if the score was in the lowest tertile for the scale, and affected if the score was in the upper two tertiles. This approach re-codes affection status for all subjects rather than selecting a subset of subjects with high values on the traits. For IS, raw score tertile bins were 0-1, 2-3 and > 3; for RSMA, they were 0-3, 4-6 and > 6. The tertiles were given different liability classes (penetrances) to weight those in the upper tertile more strongly. Our recessive model assumed a disease allele frequency of 0.05 and penetrances of each of the three genotypes of 0.0014, 0.0014 and 0.8 in the upper tertile, and 0.01, 0.01 and 0.5 in the middle tertile. For the dominant model, the disease allele frequency was 0.0025. The penetrances were 0.0014, 0.8 and 0.8 in the upper tertile, and 0.01, 0.5 and 0.5 in the middle tertile. These model parameters roughly reproduce the reported population frequency of ASDs [Linkage analyses were repeated on the basis of residual scale scores to determine whether signals could be replicated using measures of IS and RSMA phenotypes that were statistically independent of each scale's correlation with the other. Thus, for each scale, residual scores were computed using the other scale as a covariate . Then, residual scores were divided into tertiles, and phenotype and liability values were coded in the same manner as were raw scores, that is, the lowest tertile was coded as unaffected and the top two tertiles were coded as affected, and the penetrance of the highest tertile was greater than that of the lower two tertiles.For HLOD scores, results are presented using the Lander and Kruglyak  genome-wP < 0.001), indicating that they share 10% of their variance (r2 = 0.322 = 0.10). Consequently, residual scores were closely correlated with the raw score of the same scale , and 90% of the variance of each scale was unique (r2 = 0.952 = 0.90).The zero-order correlation between RSMA and IS was r = 0.32 (P < 0.01) only for non-verbal IQ. IS-Adj was positively correlated with IQ measures even though raw score IS was not, and IS-IQ correlations were significantly higher with residual than with raw scores. Thus, the unique variance of both IS and RSMA was less strongly associated with ASD but more strongly associated with IQ, although the direction of the relations with IQ was opposite , and for RSMA, H2 was 0.51 . Because the scales were significantly correlated, we also estimated the heritability of each with the other as a covariate. With RSMA as a covariate, IS was still significant  and RSMA was a significant covariate (P = 0.003). By contrast, when IS was entered as a covariate for RSMA, RSMA was not significantly heritable , but IS was a significant covariate (P < 0.0001).The heritability of both scales was significant. For IS, HP < 0.005 for nonparametric tests). There was strong correspondence between regions for which there was evidence of linkage with the recessive model and nonparametric linkage (NPL), which suggests that these linkage findings are resistant to model mis-specification. Fewer tests of the dominant model, compared with the recessive model, were suggestive or significant. Thus, to simplify presentation of genome-wide results, Figures Table Evidence of linkage reached genome-wide significance levels HLOD > 3.30) for two regions, 2q37.1-q37.3 and 15q13.1-q14  may be examples of the hoped-for outcome of identifying susceptibility loci that are specific to narrowly defined phenotypes . Given tFurther, our findings provide an example of increased knowledge of the nature of genetic effects that may be possible with more homogeneous phenotypes. Previously, we reported possibly distinct ASD regions with evidence of linkage at 15q13.1-q14, 15q14-q21.1 and 15q21.1-q22.2 . We now The variability observed in this study in the phenotypic scope of linkage regions leads us to suggest that multiple ASD phenotypes should be used in future genetic studies to characterize the nature and breadth of the phenotypic linkage or association of risk variants. It is possible that variants with broad phenotypic effects may affect the root causes of ASD, whereas variants with narrow effects contribute to phenotypic heterogeneity among individuals with ASD. The use of multiple phenotypes emphasizes the importance of additional research aimed at developing an empirical model of the relations and interactions between specific features of ASD. Such a model should lead to identification of a set of phenotype measures that assess all the key specific features of ASD. The work of previous investigators to identify IS and RSMA as distinct features of repetitive behavior is a substantial contribution to this goal.We note that our results are again consistent with the well-replicated finding of complexity and heterogeneity in ASD genetics. Our lod scores showed inter- and intra-family heterogeneity. For extended pedigrees, the scores expected under an assumption of a shared haplotype across all affected members exceeded by several lod units those actually found, depending upon the pedigree and model assumptions. Homogeneity clearly did not exist across all pedigrees in our sample; for any given region, multiple pedigrees showed no evidence of linkage.et al. [et al. did not find significant evidence of linkage in the 15q11-q13 region across all 81 families they studied but they did find significant evidence of linkage in the region of marker GABRB3 in the subset of 23 families with the highest mean IS scores. Stratifying families by RSMA or RRSB did not enhance the signal. GABRB3 is located at 24.4 Mb, which is upstream of the lower boundary (27.94 Mb) of 15q13.1-q14. We did not choose subsets of our sample, but rather re-defined affection status based on IS or RSMA phenotypic information, using information from all ASD members of the pedigrees. The methodological differences between our study and that of Shao et al. preclude firm conclusions about why they found that stratifying on IS but not RSMA enhanced the AD linkage signal, whereas we found both RSMA and IS, but particularly RSMA, to be associated with a region just downstream.Shao et al.  reportedet al. [Studies that stratified pedigrees by other repetitive behavior measures, including individual RRSB items and the 'compulsions' factor examined by Tadevosyan-Lefer et al. , report et al.  and at 1et al. . Signifiet al.  and the et al. . None ofThe suggestive evidence of IS linkage that we observed on chromosome 9 for IS spans a region implicated as a susceptibility locus for OCD in two studies ,67. This10(4) = 0.6 lod score units. Our thresholds would then be 2.26 for suggestive evidence and 3.9 for significant evidence. With this adjustment, results on chromosome 15 remain significant and many other results remain suggestive, but other results would be considered as nominal.Our sample was a cohort of multiplex ASD pedigrees, and IS and RSMA data were collected only on subjects thought to have ASD. We believe our method is appropriate to the valid aim of uncovering susceptibility loci for ASD and related phenotypes within extended families containing multiple members with ASD. However, we acknowledge that our method limits the generalizability of our findings to other research aims. For example, the absence of repetitive behavior phenotype data for family members without ASD limits our ability to answer the question of whether repetitive behavior is a broader autism phenotype that occurs in unaffected relatives ,69. FurtIS and RSMA, two factors within the ADI-RRSB domain, were found to be linked to largely non-overlapping chromosomal regions. Genome-wide significance was observed for IS at 2q37.1-q37.3 (dominant model HLOD = 3.42) and for RSMA at 15q13.1-q14 (recessive model HLOD = 3.93). Regions varied in the range of phenotypes with which they were linked. These findings support the value of including multiple, narrowly defined phenotypes in ASD genetic research.http://www.lineagen.com from 12/1/07 to 12/31/08. This salary support is not ongoing.HC, WMM and JMM received partial salary support from Lineagen Inc. DSC conducted the statistical analyses and drafted the manuscript. JMM confirmed research diagnoses of ASD, supervised collection of all phenotype data and made significant contributions to the interpretation of results. RJR and KAB assisted with interpretation of results and helped draft the manuscript. MEV and NKW verified the accuracy of data extracted from a research database and contributed to the interpretation of results. WMM participated in the design of the study and helped draft the manuscript. HC conceived of the study, participated in its design, directed the statistical analyses and helped to draft the manuscript. All authors read and approved the final manuscript."}
+{"text": "In affected sibling pair linkage analysis, the presence of linkage disequilibrium (LD) has been shown to lead to overestimation of the number of alleles shared identity-by-descent (IBD) among sibling pairs when parents are ungenotyped. This inflation results in spurious evidence for linkage even when the markers and the disease locus are not linked. In our study, we first theoretically evaluate how inflation in IBD probabilities leads to overestimation of a nonparametric linkage (NPL) statistic under the assumption of linkage equilibrium. Next, we propose a two-step processing strategy in order to systematically evaluate approaches to handle LD. Based on the observed inflation of expected logarithm of the odds ratio (LOD) from our theoretical exploration, we implemented our proposed two-step processing strategy. Step 1 involves three techniques to filter a dense set of markers. In step 2, we use the selected subset of markers from step 1 and apply four different methods of handling LD among dense markers: 1) marker thinning (MT); 2) recursive elimination; 3) SNPLINK; and 4) LD modeling approach in MERLIN. We evaluate relative performance of each method through simulation.2) above 0.3 for or 2 SNPs per cM using MT.We observed LOD score inflation only when the parents were ungenotyped. For a given number of markers, all approaches evaluated for each type of LD threshold performed similarly; however, RE approach was the only one that eliminated the LOD score bias. Our simulation results indicate a reduction of approximately 75% to complete elimination of the LOD score inflation while maintaining the information content (IC) when setting a tolerable squared correlation coefficient LD threshold (rWe have established a theoretical basis of how inflated IBD information among dense markers overestimates a NPL statistic. The two-step processing strategy serves as a useful framework to systematically evaluate relative performance of different methods to handle LD. With rapid development of high throughput genotyping technologies, researchers have begun genome-wide searches for genes associated with complex diseases using dense single nucleotide polymorphisms (SNPs). With increased marker density, there is an increase in the likelihood that SNPs will be in linkage disequilibrium (LD), where some combinations of alleles or genetic markers inherently occur more frequently than would be expected at random. However, most multipoint linkage methods assume linkage equilibrium (LE). Among dense SNPs, the assumption of LE in linkage analysis may lead to incorrect pedigree haplotype inference ,2 with u\u00ae 500 K set of SNPs using information from the subset of SNPs included in The HapMap Project to evaluate the relative performance of these various approaches in handling LD.In our study, we evaluate methods to handle LD in order to make recommendations on the best methods and tolerable LD thresholds to use in qualitative linkage analysis. We first present a theoretical evaluation of IBD sharing estimation under the incorrect assumption of LE and of the resulting inflation in linkage evidence in affected sib pair (ASP) analysis using a nonparametric linkage (NPL) statistic. We then describe different methods to handle LD achieved through a two-step processing strategy. The first step is an initial filtering where we applied three techniques: 1) removal of uninformative markers; 2) removal of redundant markers; 3) a combination of 1 and 2. In the second step, we examine and compare the following four approaches of handling LD in ASP linkage analysis: 1) a marker thinning (MT) algorithm; 2) a recursive elimination (RE) algorithm to select the most informative markers in LE; 3) SNPLINK ; and 4) Spairs and Sall, which were implemented by Kruglyak et al[Spairs is based on the number of distinct alleles shared IBD by affected members. If there is more than one affected sibling pair in a family, Sall assigns increased score values based on the joint patterns of genetic transmission in all affected individuals in a family. For ASP, both scoring functions are equivalent; thus we consider Spairs in our investigation of the effect of LD on NPL statistics.There are several methods available to perform multipoint linkage analysis using ASP, including maximum likelihood-based methods and the NPL analysis. The maximum likelihood-based approach is a powerful method but is sensitive to misspecification of the true mode of inheritance ,10. On tyak et al. Spairs 0, \u03b11 and \u03b12 as the probabilities of sharing 0, 1 or 2 alleles IBD. When the markers are in LE and under H0, both \u03b12 and \u03b10 are equal to 1/4; thus 2 (0 (n) increases. When accounting for LD between markers, there is no inflation and E(LOD) = 0.11; note that the expected value under H0 is greater than 0 because the NPL LOD score cannot be negative. However when we ignore the presence of LD in the linkage analysis, E(LOD). Therefore, if LD is not properly accounted for in the linkage analysis, it results in LOD score bias and hence the excess type-I error. We next propose and implement a strategy to minimize the excess type-I error in ASP analysis with markers in LD.To be more explicit, we denote \u03b1Step 1 allows us to study the degree of reduction in LOD score bias and IC by systematically filtering markers. In practice, working with a more manageable number of markers increases computational efficiency when performing multipoint linkage analysis. In an attempt to filter the dense set of markers, we apply three techniques: 1) removal of uninformative markers; 2) removal of redundant markers; and 3) a combination of 1 and 2. These techniques have been widely used in practice; however all three have not been systematically and formally evaluated together.2 \u2265 0.95, where r2 is a pairwise LD measure between two markers. A perfect correlation (r2 = 1) indicates that a single SNP from the pair is sufficient to capture all of the information provided by the SNP pair. We omit one of the SNPs in each pair using the r2 threshold. The third technique is a combination of the first two techniques. We apply 5% MAF threshold in conjunction with removal of redundant SNPs.The first technique removes SNPs which are not very informative for linkage analysis, as measured by the minor allele frequency, MAF. A subset of SNPs with MAF above a pre-defined threshold is used in linkage analysis. We apply three MAF thresholds: 5%, 10% and 20% to the dense set of markers. The second technique removes SNPs with redundant information, as defined using rThe three techniques together produce a total of 5 subsets of markers to compare: 3 subsets removing SNPs with low MAF ; 1 subset without redundant SNPs; and 1 subset removing SNPs with 5% MAF and without redundant SNPs. We perform linkage analysis using these 5 subsets in families with 2, 3 or 4 affected siblings with and without genotyped parents. For each marker subset, we evaluate the LOD scores and the IC to measure relative performance in terms of reducing the LOD score bias and loss of IC compared to using the full set of markers.In step 2, we use the selected baseline subset of SNPs from step 1 with a reduced number of markers but with close to full IC to evaluate relative performance of four different methods to handle LD using varying LD cut points in the ASP linkage analysis. The four approaches are: 1) MT algorithm, 2) RE algorithm, 3) SNPLINK and 4) MERLIN-LD. Two of the four approaches, SNPLINK and MERLIN-LD, are currently available approaches to handle LD and have been used in other studies. The MT algorithm has been suggested as a simple way to deal with LD in datasets -15. We pMT algorithm utilizes various pre-specified marker densities to select SNPs over the entire chromosome of interest. This approach thins out dense mapping by decreasing the SNP density to be analyzed. Depending on the density level and the chromosome length, one SNP is chosen at each interval creating a smaller subset of SNPs. We apply four different density thresholds: 1, 2, 4 and 8 SNPs per 1 cM region.2) to select a subset of markers to perform linkage analysis. Markers with a higher MAF are more informative in linkage analysis because individuals are more likely to carry heterozygous genotypes; segregation from homozygous individuals cannot be determined unambiguously. Using a user-specified LD threshold, this approach first takes a pair of SNPs above the given LD threshold and removes the less informative marker from the pair. This procedure is repeated iteratively until no pairwise LD measures exceed the threshold. The resulting subset of SNPs contains a reduced number of SNPs without pairwise LD above a given threshold. The resulting subset may have reduced information content despite the attempt to keep the most informative markers from each pair of marker in LD.We propose the RE algorithm to combine the informativeness of each marker with a measure of LD , where applicable, to show a gradual change or trend of LOD score inflation along the ranges of LD measures. The following sets of LD thresholds by D' and r2 are applied in handling LD: 0.1, 0.3, 0.5, and 0.7.In our investigation of the effects of LD, we use varying thresholds or cut points of two widely used measures of LD (D' and r2) in terms of reducing the LOD score bias. In general, with complete data where both parents are genotyped, there was no LOD score inflation in all study designs. Thus our discussions to follow mainly focus on those results where both parents are ungenotyped.We evaluated different methods to handle LD in the framework of our proposed two-step processing strategy. In step 1, we performed NPL analysis over 1,000 replicates of the simulated data at different LD thresholds for families with 2, 3 or 4 affected siblings and zero or two ungenotyped parents to determine which subset of SNPs retained full IC while reducing the LOD score bias. In step 2, we performed NPL analysis over 1,000 replicates of the simulated data at different LD thresholds using the subset of SNPs obtained from step 1 for all 9 study designs, with zero or two ungenotyped parents. We evaluated different approaches at varying degrees of LD . As the number of unaffected siblings increased in a family the inflation of LOD scores diminished further and in some cases were eliminated completely.In Table For the four MT cut points with 8, 4, 2 or 1 SNPs per 1 cM region, we observed the average MLS of 1.20 (SD = 0.96), 0.65 (SD = 0.67), 0.66 (SD = 0.68) and 0.76 SD = 0.73), respectively  shown in Table ly Table . When co2 thresholds, we observed only moderate reduction of the LOD score bias , 3 (n = 200) or 4 (n = 100) affected sibs. We analyzed the data with and without genotyped parents. Additional study designs were examined by adding 1 or 2 unaffected sibs to the first three scenarios. Through a simulation study, we investigated the relative performance of our two-step processing strategy to handle LD among dense markers against the baseline LOD score inflation and the level of IC.2 0.3 or 2 SNPs per cM using the MT algorithm. In addition, fewer markers were selected using D' compared to using r2, and we observed more reduction of the LOD score bias using r2 compared to using D' in our data for RE and SNPLINK approaches. For a given number of markers, RE, SNPLINK and MT methods performed at a similar level using D' threshold; however, RE approach was the only one that eliminated the LOD score bias. As a note, we found that the estimates become unstable when the number of makers analyzed falls below a certain level. This is apparent using D' threshold, where we observed that average MLS for both MT (1snp1cM) and RE (0.1) thresholds show elevation compared to the higher thresholds  in reducing the LOD score bias. As the cut points decreased, we observed increased reduction of bias; nevertheless, at lower cut points, some loss of information was observed.The two-step strategy has enabled us to evaluate methods to handle LD in terms of reducing the upward bias. Step 1 filters the marker map while keeping the IC which reduced a burden of computation intensity and time. Step 2 systematically evaluates different approaches to handle LD. In general, we reduced approximately 75% to complete elimination of the LOD score inflation while maintaining the IC when setting a tolerable LD threshold above r2 cut points. Moreover, Boyles et al [2was superior in terms of predicting inflation compared to D' in their study. Different samples appear to have slight variations in thresholds of LD measures and in the number of tolerable SNPs per cM that eliminate the inflation of LOD score [Through our investigation of the effect of LD in qualitative trait linkage analysis, we learned that inflation of LOD score depends on the following factors: ungenotyped parents, sample size, study design and the structure of the underlying LD in the dataset. In addition, the inflation increases as more markers in LD are considered in multipoint linkage analysis ,5. In tees et al  concludeOD score . Therefo2 0.3 or 2 SNPs per cM using the MT algorithm. Our results encourage further research in development of methods that combine the flexibility in handling LD with ease of application.Overall in our study, we have established the theoretical basis for the effect of LD between markers on linkage statistics for qualitative traits. We have proposed and implemented a two-step processing strategy to systematically minimize the impact of LD in linkage analysis and to evaluate different methods in handling LD. Finally, we have made recommendations on appropriate methods and LD thresholds that minimize the impact of LD among dense SNPs. In summary, for a given number of markers, all approaches evaluated for each type of LD threshold performed similarly; however, RE approach was the only one that eliminated the LOD score bias. In general, approximately 75% to complete elimination of the LOD score inflation can be reached while maintaining the IC when setting a tolerable LD threshold above rWith rapidly advancing genotyping techniques and decreasing cost, the field of genetic mapping is moving towards genotyping even denser markers than currently used 500 K set of markers. With increasing volume of available genotypes, there is also an increasing demand and interest in dealing with marker selection and linkage disequilibrium among dense markers in linkage analysis. The need and efforts in developing and investigating new algorithms and approaches to appropriately and manageably handle all markers on the finest scaled mapping will continue to grow in the field of genetic mapping and analyses.KC participated in the design of the study, interpretation of results, performed the simulation studies and drafted the paper. JD contributed substantially to the conception and design of the study, to the interpretation of results and revised the manuscript. Both authors read and approved the final manuscript."}
+{"text": "Rheumatoid arthritis (RA) is a multifactorial disease with complex genetic etiology, about which little is known. Here, we apply a two-stage procedure in which a quick first-stage analysis was used to narrow down targets for a more thorough and detailed testing for gene \u00d7 gene interaction. Potentially interesting regions were first identified by testing for major gene effects using non-parametric linkage methods. To select regions of interest, we first tested for linkage to three different RA-related traits one at a time: RA affection status and the quantitative phenotypes rheumatoid factor IgM and anti-cyclic citrullinated peptide levels. These linkage analyses identified regions on chromosomes 3, 5, 6, 8, 16, 18, 19, and 20. We subsequently analyzed the selected regions in a pairwise manner to detect gene \u00d7 gene interactions influencing RA using a recently developed two-dimensional linkage method. We found evidence of interacting loci on chromosomes 5, 6, and 18. HLA-DRB, PTPN8, NFKBIL1, RUNX1, and SLC22A4.Rheumatoid arthritis (RA) is a complex inflammatory disease primarily affecting the joints. Research on the genetic basis of RA has identified several loci with potentially significant effects, including Whole-genome scans typically focus on identifying single genes with major effects and often lack power to detect linkage to sets of interacting genes. A two-dimensional genome scan has recently been used to simultaneously detect linkage to two interacting genomic regions in the study of hypertension by Bell et al. .In this study, we attempt to identify gene \u00d7 gene interactions influencing RA by employing a two-stage linkage analysis procedure. In the first step, one-dimensional linkage analyses of each of the three phenotypes-RA status, rheumatoid factor IgM and anti-cyclic citrullinated peptide levels-were carried out using genome spanning microsatellite markers. We then selected single-nucleotide polymorphims (SNPs) (from a panel of 5 K SNPs), which were located in the chromosomal regions showing elevated linkage signals for any of these three traits, which were analyzed in a pair-wise manner for linkage to RA in the two-dimensional (2D) genome scan step. We present the results of the 2D genome scan on 5-cM regions flanking each marker that showed a one-dimensional (1D) trait-specific LOD score above 1.5. Because traditional LOD score thresholds are not applicable to a 2D genome scan, we determined significance levels for the 2D genome scan results by comparing them against their empirical distributions (obtained using simulated sets of unlinked markers).The genotype and phenotype data are from the North American Rheumatoid Arthritis Consortium (NARAC) . We analOur data consisted of 472 multiplex pedigrees containing 1164 genotyped persons, 1076 phenotyped for the anti-CCP trait, and 1094 phenotyped for the RF trait. We used all 402 autosomal microsatellite markers for the 1D linkage analysis of the three traits. For two-locus analysis, we selected a subset of SNPs from the 5 K SNP scan of the NARAC data [Merlin version 1.01  was usedA 2D genome scan was performed for RA affection status to test for gene \u00d7 gene interactions using the Merloc program . Merloc For the 2D linkage analysis, we used 746 phenotyped pedigrees for whom 5 K SNP scan data was also available. These were divided into 1724 nuclear families because Merloc currently only handles sibship data); subsequently uninformative pedigrees were trimmed using Merlin, leaving 724 informative nuclear families with 3053 individuals.We selected a subset of loci from the 5 K SNP data, which were located in the significant regions identified from the microsatellite scans. In this step, each marker locus from one region is paired with each marker locus from a second region in turn, and analyzed as a pair. For selecting the subset of SNPs we first identified microsatellite markers with LOD scores >1.5  and then selected SNPs within a 10-cM region flanking these markers. Where two or more loci with LOD score >1.5 occurred in close proximity, we selected a single region of 10 cM centered on the marker with highest LOD score. In instances in which more than two sites were included on the same chromosome, we confirmed there was reasonable separation between the regions. Such a constraint on the distance between regions on the same chromosome was necessary because the 2D genome scan method assumes locus pairs are unlinked. The 11 separate regions selected finally are shown in Table Preliminary analysis indicated that similar results are obtained with closely spaced SNPs using the two-locus method (data not shown), which could possibly be due to LD between closely spaced SNPs within a region. So, we estimated LD between markers within each of the 11 regions identified, but not across region pairs because these were selected to be reasonably distant from each other. LD was estimated using H-clust [We simulated 1000 replicates of genotype data for 24 locus pairs contained within the 14.69\u201323.45 cM region on chromosome 5 and the 30.33\u201338.32 cM region on chromosome 6. Unlinked genotype data for SNPs were simulated using the program Simulate by Terwilliger et al. , while mWe observed LOD scores >1.5 on chromosomes 6, 16, and 18 ). Chromosomes 3, 6, 8, 18, 19, and 20 had elevated linkage signals for the anti-CCP trait. Both RF and anti-CCP showed a strong linkage signal on chromosome 3 as well.Figure Table Evidence of interaction can be inferred in this table by comparing the maximum of the MLS over all locus-pairs within a pair of regions, then comparing it in turn to the maximum of the MLS of the respective single-locus models within those two regions. For example, in the second row of Table We pooled the LOD scores from all 24,000 locus pairs from the 1000 replicates to derive an empirical distribution of the two-locus LOD scores. The threshold values observed at 1% and 0.5% significance levels for the additive, multiplicative, and epistatic models are all around 1.85 and 2.07, respectively. The general model has higher threshold values than the more restricted models, 1.93 and 2.13, respectively. Single-locus threshold values were close to their expected values, 1.28 and 1.68, respectively. Threshold values for 0.1 level, used to assess significance of the 2D genome scan results, were all similar ranging from 2.42 for the single-locus model to 2.57 for the general model.The bulk of the NARAC data consists of affected sib pairs, and because IgM and anti-CCP are both strongly related to RA, family members are expected to be phenotypically concordant. It has been suggested  that regressing traits on IBD data alone has very low power to detect linkage in the presence of highly concordant relative pairs, and, in such a situation, the allele-sharing statistic contains most of the information regarding linkage. Variance-components analysis was not appropriate for this data because both quantitative traits show a highly non-normal distribution within the NARAC pedigrees. The method proposed by Sham et al. , which rWe chose not to include two-locus analysis on syntenic loci. It has been previously noted by Bell et al.  that theThe locus pairs within each simulation replicate are correlated, so the effective number of independent observations is smaller than 24,000. Thus, our simulation size may not be large enough to accurately estimate the extreme quantiles. Moreover, SNP genotypes have limited variation within the replicates, which further imposes limits on the range of possible LOD scores that can be obtained.Searching for gene \u00d7 gene interactions over a dense genome-scan data poses many difficulties, such as demanding computations, and may result in high false-positive rates. Thus, we decided to limit the number of SNPs based on certain predefined criteria. Rather than relying on the affection status alone, we used linkage of two quantitative traits for selecting the regions of interest. Subsequent 2D genome scanning showed promising evidence of linkage as well as gene \u00d7 gene interactions between chromosomal regions. Interestingly, these interacting regions have also recently been identified as being linked with RF and anti-CCP on the same data set  and thisThe author(s) declare that they have no competing interests."}
+{"text": "We performed linkage analysis on families with rheumatoid arthritis, stratifying by ethnic origin. We compared results using either Kong and Cox nonparametric LOD scores or MOD score analysis using the software GeneHunter MODSCORE. We first applied SNPLINK to remove markers showing excess linkage disequilibrium from the SNPs in the Illumina IV SNP Linkage panel. In this analysis there were 659 self-reported Caucasian families and 29 self-reported Hispanic families in the NARAC collection. Chromosome 19 yielded MOD scores > 3.00 in the Hispanic group, while chromosomes 2, 6, 7, 11, and XY had MOD scores > 3.00 in the Caucasian group. We performed simulation studies to evaluate the empirical distribution of the MOD score for autosomal loci separately in Hispanics and Caucasians. Results showed genome-wide significant evidence for linkage in Caucasians for chromosomes 2q and 6p, but no significant evidence for any linkages in the Hispanics, including little evidence for linkage to chromosome 6p in this group. An examination of the difference of phenotypes in two ethnic groups suggested significantly earlier mean age of onset, higher percentage of anti-cyclic citrullinated peptide positive people, and lower percentage of affected people carrying shared epitopes in Hispanics than those in Caucasians. A larger sample size of the Hispanic group is needed to identify linkage regions. Rheumatoid arthritis (RA) is a complex genetic disease with possible genetic heterogeneity among different ethnic groups . There iPreviously we performed a genome-wide single-nucleotide polymorphism (SNP) analysis of NARAC (North American Rheumatoid Arthritis Consortium) Caucasian families and identified two new loci at 2q33 and 11p12, in addition to confirming evidence for linkage in the HLA region (Kong and Cox LOD score of 16.14) [p-values.Here we applied standard linkage analysis methods as well as the MOD score approach to this same set of Caucasian families as well as to a set of Hispanic families to compare evidence of linkage to RA in these two groups. In addition, the MOD score method provides estimates of the penetrance for putative disease-susceptibility loci, while conditioning on the disease status, thus adjusting for ascertainment of the families. However, the distribution of the MOD score test statistic is complex, and we therefore have performed extensive simulations to obtain empirical p-values. Significant differences of phenotypes between these two groups were found in age of onset, proportion of anti-CCP positive people, and percentage of affected people carrying the shared epitopes.Evidence of linkage on chromosomes 2 and 6 was confirmed by MOD score analysis for the Caucasian group, and weak evidence of linkage to chromosome 6 was found to be not significant in Hispanic group using empirical R2 value, a measure of linkage disequilibrium (LD), of 0.05 was used as cut-off to remove markers of linkage disequilibrium (LD) using SNPLINK [An  SNPLINK ,4. The d SNPLINK ,5 and by SNPLINK .To assess significance in the Hispanic sample, we performed simulations of the families for all chromosomes (excluding the X chromosome) using 10,000 replicate samples with the computer program Allegro . To derit-test, binomial test, or survival modeling, since segregation analysis cannot be done because of the complex ascertainment used for selecting these families. We know who the primary proband is but not who the obligatory additional proband is. Therefore, we have chosen to apply a MOD score approach, which conditions on the disease status in the family and subsequently performs a segregation analysis to estimate parameters describing penetrance.Distributions of phenotypes, including age of onset, anti-CCP, RF-IgM and shared epitopes, were also compared between two groups. The significance of the differences were formally assessed using a The results presented in Table p-value of 0.42, suggesting that evidence on chromosome 19 was a false-positive finding for Hispanics.To better characterize the results that we obtained from MOD score analysis suggesting linkage on chromosome 19 in Hispanics, we performed a genome-wide simulation study with 10,000 replicates. Figure p-values of ~0.0, 0.08, 0.17, and 0.42, respectively. The empirically derived MOD scores corresponding to p-values of 0.05 in Hispanics and Caucasians deviated somewhat. For Caucasians, the empirical MOD score for 5% significance is 4.39 while for Hispanics it is 4.08.We also performed a similar simulation study for Caucasians using only 1000 replicates because of the computational burden \u2013 more than 20 days of CPU time per chromosome would be needed and therefore a genome-wide study of 10,000 replicates is computationally prohibitive, since the sample size for the Caucasian group is more than 20 times larger than that of Hispanic group. The results are summarized in Figure p-value = 0.02 in Cox regression analysis after correction). The means of anti-CCP and RF-IgM of two groups were compared using t-test and was found not significant . However, the difference in proportion of anti-CCP-positive individuals in Hispanics and Caucasian is highly significant using the binomial test . The difference of percentage of shared epitopes in affected people is also significant .Comparison of the distributions of phenotypes including age of onset, anti-CCP, RF-IgM, and shared epitopes between two groups were included in Table The mean number of affected siblings in the families is about the same in both groups . The proportion with parents available might affect MOD score calculations, but is actually higher in Hispanics (65.52%) than in Caucasians (41.09%). Allele frequencies for the SNPs giving high Kong and Cox LOD scores or MOD scores on chromosomes 6 and 19 did not reveal significant difference between the two ethnic groups (data not shown here). There were no detectable genotyping errors in these ethnic groups.The results may reflect underlying genetic variations between Caucasian and Hispanic groups useful for diagnosis and treatment of disease. The sample size of Hispanics available for study strongly limits our ability to generalize our findings. Using the Caucasian sample as standard, the expected LOD score in Hispanics from 29 families is 0.71, so the evidence for linkage to chromosome 6 is comparable to its expectation. However, we note that chromosomes other than 6 yielded higher LOD scores in the Hispanics, suggesting that non-HLA-region genes may play a stronger role in this population than in Caucasian populations. The weaker linkage to HLA and lower percentage of people carrying shared epitopes in Hispanics are interesting because the associations in that group with the shared epitopes are quite weak and tend to yield lower associations in general . There ap-values obtained from genome-wide simulations, only chromosome 6 HLA region showed very significant linkage, and chromosome 2 showed suggestive linkage to RA in the Caucasian group. MOD score analysis did not yield evidence for any new linkages and failed to provide significant evidence of linkage to chromosome 11, which has been suggested based upon standard linkage analysis.Based on empirical Further research with a larger sample size of the Hispanics group is needed to confirm our findings, which suggest phenotypic and linkage heterogeneity in this group compared to Caucasians.The author(s) declare that they have no competing interests."}
+{"text": "The CEPH samples are an invaluable resource for mapping genes that contribute to traits that can be measured in cell lines. With the many markers that have already been genotyped for the Centre d'Etude du Polymorphisme Humain (CEPH) pedigrees and are readily available, one need only obtain phenotypes to conduct a linkage analysis. For Genetic Analysis Workshop 15 (GAW15), over 3000 expression levels of genes in lymphoblastoid cells in 14 of the CEPH pedigrees were provided. For this study, eight of these expression levels were selected to obtain a spectrum of heritabilities, three were selected based on linkage results with traditional LOD scores >3, and one trait was selected at random. A Bayesian Monte Carlo Markov chain oligogenic segregation and linkage analysis was conducted on each of these 12 traits using the genome-wide single-nucleotide polymorphism linkage markers provided for GAW15. Our goal was to assess the ability of these methods to map genes in the CEPH pedigrees. Surprisingly, positive linkage signals were found for all 12 traits, even those with a very small traditionally calculated heritability. However, the portion of the variance attributed to genetic sources by the oligogenic segregation analysis differed substantially in some cases from the traditional heritability. It appears that genetic variance estimated from oligogenic segregation analysis may be a better indicator of whether genes can be mapped for complex traits than traditional heritability. The Centre d'Etude du Polymorphisme Humain (CEPH) reference families were originally collected as a resource for constructing linkage maps. Cell lines from these families are available to the research community and have been used in many studies. As a result, many genetic markers have been typed and are available. This resource has the potential to be used to map genes for any phenotype that can be measured in cell lines.For Genetic Analysis Workshop 15 (GAW15), gene expression levels were available for thousands of genes measured in 14 CEPH pedigrees . These levels were obtained via the Affymetrix Human Focus Arrays and selected as described in Morley et al. [MCMC oligogenic combined segregation and linkage analysis has been implemented in the program Loki . These mTraits were selected on several differing criteria, including heritability, linkage found with other methods, and random selection. Heritabilities were computed by Yu et al.  for all The meiotic map assembled by Sung et al.  guided t\u03bc is the \"reference\" trait value, X is the incidence matrix for covariate effects, \u03b2 is the vector of covariate effects, Qi is the incidence matrix for the effects of QTL i, \u03b1i is the vector of effects for QTL i, e is the normally distributed residual effect, and k is the number of QTL currently estimated (k \u2265 0). The MCMC process samples \u03bc, \u03b2, \u03b1i, i, and e as well as parameters such as unobserved marker genotypes. All of these parameters are sampled from the space of model values consistent with the data observed. Values are sampled proportional to their posterior probability. After a number of sampling iterations, the sampled values provide an estimate of the posterior probability distribution over the space of possible parameter configurations. Genome-wide analyses of each trait were run for 1,000,000 iterations with a LM sampler ratio of 0.2. In each analysis, all chromosomes were analyzed simultaneously. The raw (untransformed) traits were analyzed without covariates. Graphical analysis was used to assess MCMC mixing.To estimate the number, effects, and location of loci contributing to each phenotype, we applied the MCMC segregation and linkage analysis methods described by Heath . These mTo evaluate evidence for linkage, we considered L-scores estimated over 1-cM wide bins along the chromosomes. An L-score is simply the posterior probability divided by the prior probability. In the absence of any data, the posterior probability should be equal to the prior probability. Thus, a L-score of 1 indicates that the data contains no information for or against linkage, while a L-score >1 indicates evidence for linkage, and an L-score <1 might be considered evidence against linkage.h2), and a basic summary of the oligogenic segregation analysis are provided in Table h2 had the largest percentage of genetic variance (%gv) in the oligogenic segregation analysis and also the largest L-scores we have encountered to date. In addition to these two, the three traits selected for LOD scores >3 also had L-scores >100, and in all five of these traits, the high L-score and LOD occur on the chromosome with the structural gene. In two other traits, the highest L-score is on the structural gene chromosome, but no others have a LOD >1.5 near the structural gene. In the five traits with no linkage to the structural gene, 27 L-scores >7.3 and 5 LOD scores >1.5 were found, but only two chromosomes had both. Overall, 42 L-scores >7.3 and 24 LOD scores >1.5 were found, including nine chromosomes with both. On these nine, the plausible interval for the L-score and the LOD-1 interval overlap on eight, and are ~35 cM apart on the ninth (chromosome 11 for 218264_at). In general, the L-score intervals are similar or smaller than the LOD intervals: on chromosome 1 for 204418_x_at, the two intervals are nearly identical, while on chromosome 7 for 210910_s_at, the L-score interval is ~6 cM in the middle of a ~17-cM LOD interval. No L-scores >7.3 were found for the simulated null. While some small h2 values were selected, the smallest percentage of genetic variance for these traits from the oligogenic model was 24%. Consequently, it may not be surprising that evidence of linkage was found for all 12 phenotypes.The 12 traits and simulated null, selection criteria, heritabilities  in a sample of 14 families comprising 194 individuals. The LOD scores and the L-scores identified some regions in common and some individually. The L-score appears to do better than the nonparametric LOD by some measures , but the individual results may indicate both types of analyses can be useful.These results indicate that MCMC oligogenic segregation and linkage analysis may be useful in localizing genes for a variety of traits in a sample like that provided by the CEPH pedigrees. The percentage of trait variance estimated to be due to genes in an oligogenic segregation analysis may be a better predictor of the ability to map genes for that trait than a traditionally computed The author(s) declare that they have no competing interests."}
+{"text": "Genome-wide linkage studies for Alzheimer's disease have implicated several chromosomal regions as potential loci for susceptibility genes.et al. , with ARPs from Sweden and Washington University. In this total sample collection of 397 ARPs, we have analyzed linkage to chromosomes 1, 9, 10, 12, 19 and 21, implicated in the previous scan.In the present study, we have combined a selection of affected relative pairs (ARPs) from the UK and the USA included in a previous linkage study by Myers APOE locus increased considerably as compared to the earlier scan. However, linkage to chromosome 10q21, which provided the strongest linkage in the previous scan could not be detected.The analysis revealed that linkage to chromosome 19q13 close to the The present investigation provides yet further evidence that 19q13 is the only chromosomal region consistently linked to Alzheimer's disease. APP) on chromosome 21 [PSEN1) on chromosome 14 [PSEN2) on chromosome 1 [APOE) on chromosome 19q13 is so far the only identified susceptibility gene with consistently demonstrated association [Alzheimer's disease (AD) is the most common form of dementia and the number of affected individuals rises dramatically with an aging population. Age is the most prominent risk factor, but genetics is also important for the risk of developing AD. Three genes are known to cause autosomal dominant early-onset AD: the amyloid beta precursor protein  from the UK and the USA included in an earlier linkage study by Myers et al. . We haveA total of 580 individuals from 261 families affected by late onset AD (family mean age at onset \u226560 years) divided into 397 ARPs were analyzed in this study. Out of these, 116 ARPs were collected in Sweden, 87 ARPs in the UK and 194 ARPs in the USA   that were also genotyped in the study by Myers et al. but with another microsatellite marker set. Twelve of the families from Sweden were analyzed in Giedraitis et al. 2006 [et al. [Samples from the UK and the Indiana Alzheimer Disease Center National Cell Repository were also included in the study by Myers al. 2006 . There i [et al. ,14, but et al. [APOE was included as a genetic marker. Data from an additional 170 microsatellite markers located on other chromosomes and with an average genotyping success rate of <80% were included in the analysis of family structure, but not in the linkage analysis.A total of 100 microsatellite markers on chromosomes 1, 9, 10, 12, 19 and 21 also used in a study by Blacker et al.  were incAmplification of the microsatellite markers was performed by multiplex PCR and the resulting fragments were separated according to size on an ABI PRISM 3700 . For quality control, each run included two CEPH samples (1331-01 and 1331-02)  and two APOE genotyping was performed at the respective research center.The Genotyper software v3.7 (Applied Biosystems) was used for allele calling. Marker order and intermarker distances were obtained from the Marshfield reference map . APOE geAPOE \u03b54+ , and APOE \u03b54- . File conversion was performed using Mega v4.0 [Family structures were verified through the Graphical relationship representation software (GRR) . Mendeliega v4.0 .pairs and family weighting option \"power: 0.5\" was used. Significance levels of detected MLSs in the total sample and the analyzed subgroups were simulated through 1000 replications using the actual data set from the selected chromosomes.Allele sharing multipoint LOD scores (MLS) and two-point LOD scores (TLS) were calculated for all groups using the Allegro v2.0 software . As suggIn order to ensure high quality of the data included in the analysis, the GRR program was used on all available genotypes, including data from 270 microsatellite markers. This prompted us to exclude samples which displayed deviations from the expected average allele sharing between sibs or other family members. Having performed this quality check, 580 affected individuals from 261 pedigrees remained and were included in the analyses  in the whole sample. Blacker et al. also found linkage in the region, to chromosome 10q22 (92 cM) in their total collection of NIMH samples. In the present study, we could not detect linkage to chromosome 10q21, even though a suggestive linkage of MLS 1.8 was detected to chromosome 10q22 (105 cM) in the APOE \u03b54- sample. Although the sample size in the present study is smaller than in the study by Myers et al. , the previous study by Kehoe et al. [APOE \u03b54- subsample coincides with the previously detected linkage to chromosome 10q21-22, although the positions of these peaks differ by 13-23 cM. Certain caution is also called for as the APOE \u03b54- subsample is rather limited in size (42 ARPs).In the original whole genome scan by Myers me 10q22 2 cM in tet al., suggests that finding significant linkage to both chromosome 19q13 and additional regions in the same sample is uncommon.Inconsistent results between linkage studies might reflect heterogeneity in sample cohorts, including age at onset, ethnic background and diagnostic criteria. Our finding of significant linkage to chromosome 19q13, but to no other regions in the total sample in combination with the results presented by Blacker APOE is so far the only locus demonstrating strong association to AD [In the past few years, whole genome association studies have successfully identified susceptibility loci for a number of complex conditions. However, on to AD -28. Sampon to AD . Increason to AD .et al. [APOE gene. This linkage was extensively contributed by the Swedish samples, which has a lower average age at onset than the other subgroups. There was no evidence of the previously demonstrated linkage to chromosome 10q21 in the whole sample collection, and the relevance of the suggestive linkage within the APOE \u03b54- subgroup to chromosome 10q22 is somewhat uncertain due to the altered position of the peak and the restriction to this subgroup only. Our study demonstrates that chromosome 19q13 including APOE, at this point, is the only consistently linked locus for AD. This is also supported by genome wide association studies, demonstrating that APOE is the major susceptibility gene for AD [In this linkage study, we have analyzed a sample collection of AD ARPs from Sweden, the UK and the USA for linkage to chromosomes 1, 9, 10, 12, 19 and 21, implicated in the previous study by Myers e for AD . Any addThe authors declare that they have no competing interests.ESB carried out the genotyping, compiled data and participated in drafting the manuscript; VG carried out the statistical analyses; SA participated in the genotyping; MLH participated in creating the pedigree file; OA participated in the genotyping; A. Goate and JW participated in the design of the study; LL conceived the study and participated in its design and coordination; JH conceived the study, participated in its design and coordination, and revised the manuscript; FWDV participated in the genotyping and in designing and coordinating the study; A. Glaser participated in designing and coordinating the study and in drafting the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2350/10/122/prepubChromosomal positions of markers included in the scan. This word DOC contains a table displaying chromosomal positions of markers included in the scan.Click here for file"}
+{"text": "Rheumatoid arthritis (RA) is a complex disease that involves both environmental and genetic factors. Elucidation of the basic etiologic factors involved in RA is essential for preventing and treating this disease. However, the etiology of RA, like that of other complex diseases, is largely unknown. In the present study, we conducted autosomal multipoint linkage scans using affected sib pairs by incorporating the smoking status into analysis. We divided the affected sib pairs into three subgroups based on smoking status . Interactions between the susceptibility genes and smoking could then be assessed through linkage mapping. Results suggested that the genetic effect of chromosome 6p21.2-3 in concordant current smoker pairs was about two-fold greater than that of the concordant non-current smoker pairs or discordant pairs. With incorporation of smoking status, additional regions with evidence of linkage were identified, including chromosomes 4q and 20q; while evidence of linkage remained in the regions of chromosomes 6p, 8p, and 9p. The interaction effects varied in different regions. Results from our analyses suggested that incorporating smoking status into linkage analyses could increase the statistical power of the multipoint linkage approach applied here and help elucidate the etiology of RA. Numerous epidemiologic studies have shown that both genetic and environmental factors contribute to the development of rheumatoid arthritis (RA). Few studies have proceeded further to study how environmental and genetic factors might interact in individuals with RA . To previ)/2 (where the genetic effect for stratum i is denoted by \"Ci\") characterizes the probability of an affected sib pair in stratum i sharing the same allele at \u03c4 from the parent. The genetic effects from the susceptibility locus for all stratified groups could be estimated, and the significance levels of these genetic linkage effects could be assessed at the estimated putative disease locus. Further, the significance of the interaction between a gene and smoking can be assessed through testing the null hypothesis, where all C values are considered equal. Hence, we adopted this multipoint linkage approach to assess the gene \u00d7 smoking interaction for RA in the present study.Recently, Liang et al. -5 proposA total of 1096 affected sib pairs from 757 multiplex families in the North American Rheumatoid Arthritis Consortium (NARAC) study were included in the study. Only 615 or 627 sib pairs  had genotype information, and thus these were used for our analysis. The NARAC multiplex families contain 8017 individuals, most of whom are Caucasians (90.6%). We performed the analyses using the entire data set and the subset of Caucasians and found that the results from both data sets were virtually identical. We therefore reported only the results from the entire data set here. A total of 375 microsatellite markers were used in the analyses. There were 615 affected sib pairs available for chromosomes 1\u201311, 13\u201316, and 19\u201322, and 627 affected sib pairs available for chromosomes 12, 17, and 18. The smoking variables included \"ever smoker\" and \"current smoker.\" Due to the missingness of smoking variables, the total number of affected sib pairs included in the analysis varied from 585 to 597, depending on which smoking variable was used, and which chromosomal region was studied.Several studies have shown that the association between current heavy smokers and RA was striking, while the association between \"ever smokers\" and RA was modest . To undC0, C1, and C2 were the genetic effects for the three groups stratified by one of the smoking statuses, respectively. The GeneHunter program was used to calculate identity-by-decent (IBD) sharing of affected sib pairs. The GeneFinder program was applied to obtain the estimates of \u03c4 and Ci, i = 0, 1, 2, and their 95% confidence intervals, as well as to calculate the p-values of the genetic effects to test whether C values were all equal . In addition, we compared these results with the results from analyses excluding environmental factors.The parameters 0 = 0.21 (p = 0.00012), 1 = 0.20 (p = 0.000031) and 2 = 0.22 (p = 2.32 \u00d7 10-6). Therefore, the interaction between the susceptibility locus and \"ever smoked\" status was not statistically significant (p = 0.95). Nevertheless, the interaction of the gene by \"ever smoked\" status was observed at 25.84 cM on chromosome 8 (p = 0.0055), at 23.9 cM on chromosome 13 (p = 0.026), at 55.12 cM on chromosome 15 (p = 0.029), and at 44.85 cM on chromosome 17 (p = 0.017). The  and  pairs showed significant genetic effects  at the susceptibility locus identified on chromosome 8, but not on chromosomes 13, 15, or 17, indicating that the genetic effect of \"ever smoked\" status varied from region to region, and the interaction could still exist when the genetic effect for each stratum in the region was not statistically significant.For comparison, we demonstrated the results from the autosomal-wide scan in which smoking status was not incorporated in Table p = 0.023). The genetic effect from the  group was estimated to be 0.43 (p = 0.0018), about two-fold higher than those from the   and   groups as illustrated in Figure When stratified by \"current smoking\" status Table , the susp = 0.0086) and 9 (p = 0.00052). Among them, the  group showed statistically significant genetic effects on chromosomes 8p and 9p with 0 = 0.12 (p = 0.00095) and 0.11 (p = 0.0060), respectively. The  group showed a statistically significant genetic effect on chromosome 4q , and the  group showed a statistically significant genetic effect on chromosomes 20q .Other regions showed a significant interaction between the susceptibility locus and current smoking status, including the locations of 160.2, 26.3, 38.8, 26.9, 38.0, 83.0, 74.5, 81.1, 88.5, and 38.4 cM on chromosomes 4, 8, 9, 10, 13, 14, 18, 19, 20, and 21, respectively. The gene \u00d7 smoking interactions were most striking on chromosomes 8  declare that they have no competing interests."}
+{"text": "The recent development of new high-throughput technologies for SNP genotyping has opened the possibility of taking a genome-wide linkage approach to the search for new candidate genes involved in heredity diseases. The two major breast cancer susceptibility genes BRCA1 and BRCA2 are involved in 30% of hereditary breast cancer cases, but the discovery of additional breast cancer predisposition genes for the non-BRCA1/2 breast cancer families has so far been unsuccessful.In order to evaluate the power improvement provided by using SNP markers in a real situation, we have performed a whole genome screen of 19 non-BRCA1/2 breast cancer families using 4720 genomewide SNPs with Illumina technology (Illumina's Linkage III Panel), with an average distance of 615 Kb/SNP. We identified six regions on chromosomes 2, 3, 4, 7, 11 and 14 as candidates to contain genes involved in breast cancer susceptibility, and additional fine mapping genotyping using microsatellite markers around linkage peaks confirmed five of them, excluding the region on chromosome 3. These results were consistent in analyses that excluded SNPs in high linkage disequilibrium. The results were compared with those obtained previously using a 10 cM microsatellite scan (STR-GWS) and we found lower or not significant linkage signals with STR-GWS data compared to SNP data in all cases.Our results show the power increase that SNPs can supply in linkage studies. Genomewide linkage scans have traditionally been performed using low-density maps of microsatellite markers with a spacing of about 10 cM across the genome -7. AlthoGenomewide linkage scans have become a widely used tool in the effort to unravel the genetic bases of human hereditary diseases. One example of this is the search for high-penetrance genes involved in breast cancer. The two major breast cancer susceptibility genes, BRCA1 and BRCA2, have been shown to be involved in a significant proportion of families affected with breast and ovarian cancer, but it is clear that about 70% of familial breast cancer is not caused by mutations in these genes -13. ThesTherefore, we have conducted a linkage study with 4.720 SNPs across the genome in nineteen BRCAX families to identify candidate regions containing BRCAX gene(s). We show the existence of different candidate regions linked to a small number of families, and a power improvement by using SNPs instead of microsatellite markers.Results of multipoint non parametric linkage analysis (NPLA) in all chromosomes for our SNP data (SNP-GWS) are shown in Figure Six regions on six different chromosomes were selected according this criteria to contain susceptibility genes involved in breast cancer using both CEPH and ALL frequencies: chromosome 2, with a maximum NPLOD score of 2.26 and 1.70 ; chromosome 3 with a maximum NPLOD score of 2.29 and 2.19 ; chromosome 4 with a maximum NPLOD score of 2.29 and 2.01; chromosome 7 with a maximum NPLOD score of 2.56 and 2.44 ; chromosome 11 with a maximum NPLOD score of 2.21 and 2.15  and chromosome 14 with a maximum NPLOD score of 2.25 and 1.89  for these regions using ALL frequencies are summarized in Table Only a small fraction of families showed significant linkage values (p < 0.05) in these regions when NPLOD score per Family was calculated. Three families were selected in candidate regions in chromosome 3, 4 and 7 with moderate linkage values  and a single family with very high NPLOD score value  in chromosome 2, 11 and 14 was found (see Table 2 > 0.2/0.5/0.8); the same regions are shown in Figure 2 values (0.2/0.5/0.8), that is, almost all candidate regions are maintained with significant p values (p < 0.05) in all analyses . The other method to discard possible markers in LD was to take the genetic distance between them into consideration. We found a more drastic decrease in the NPLOD scores when this approach was used. The information content (IC) value was also calculated and compared for both analyses. In additional information [see Additional file p-values and IC in both methods are shown].The inflation of the nonparametric multipoint LOD score due to inter-marker linkage disequilibrium (LD) has been recently described ,18, and We have calculated how many regions with similar NPLOD scores could be expected by chance, in order to examine the false positive rates in our data. Gene dropping simulations were performed. This analysis replaces our real data with simulated chromosomes, maintaining the original pedigree structure, allele frequencies and recombination fraction, and retaining the original missing data patterns. These datasets are generated under the null hypothesis of no linkage or association to observed phenotypes. Using Merlin software, we generated 1000 random genomewide scan replicates of the data and those regions with an NPLOD score with p-values \u2264 0.01 using CEPH frequencies and were still significant  when more conservative analysis using ALL frequencies was done, were computed (according the criteria applied in our candidate region selection).p-value of 0.015. The results found with the simulated data suggest more allele sharing in our data than would be expected by chance.The distribution of the number of candidate regions identified according these criteria from 1000 randomly simulated genomewide scans is shown [see Additional file et al. [Wiltshire et al.  demonstrMaximum NPLOD score comparison in individual families with and without fine-scale markers in our candidate regions are shown in Table Firstly, the data quality was extremely high with successful genotyping obtained for 98.9% of SNP markers compared to STR call rate of 96.7%.Secondly, in order to evaluate whether a map of closely spaced SNPs offers equal or higher power to detect linkage compared with the traditional approach based on STR-GWS, we compared the linkage values obtained along these six candidate regions. Parametric and nonparametric analyses were performed using 16 of the 19 families with both types of data available, and the nonparametric NPLOD scores obtained with SNP-GWS and STR-GWS are shown in Figure In all cases, the lowest NPLOD score values were found when microsatellite markers were analyzed. When STR markers were used, the maximum NPLOD scores obtained with SNPs dropped from 2.25 (p = 0.012) to -0.21 (p = 0.6) in chromosome 2, 1.90 p = 0.03) to 0.42 (p = 0.3) in chromosome 3, 2.22 (p = 0.013) to 1.65 (p = 0.05) in chromosome 4, 2.59 (p = 0.005) to 1.77 (p = 0.04) in chromosome 7 and from 2.51 (p = 0.006) and 2.41 (p = 0.008) to 0.53 (p = 0.3) and 0.98 (p = 0.2) in chromosomes 11 and 14, respectively. Also, our results suggest that SNP mapping allows loci to be defined more precisely than STR marker due to the higher marker density  and nonparametric analyses (NPA). Using PA, no exceptionally strong linkage signals were found, since this value depends on the assumed genetic model being correct. This means that evidence for linkage might be missed if we only perform PA. On the other hand, using NPA we identified a region in chromosome 3 that was later discarded when fine mapping genotyping data were considered, but this false positive evidence of linkage was not found when PA was performed. These results highlight the importance of performing both parametric and non-parametric analyses in order to avoid false negative and false positive results.et al. [et al. [et al. study and in our study, we have also identified the same region on chromosome 4p14q12, but while in Smith et al. [et al. [et al. [et al. [Regarding our candidate regions, in a previous paper Huusko et al.  found a et al. . This st [et al. , even thh et al.  this res [et al. . Similar [et al. . We also [et al.  found noet al. [et al. [Smith et al.  reported [et al.  have perInflation of the linkage signals may arise especially when intermarker LD is present and pedigree founders are not available. Two analyses were performed to assess whether LD affected the linkage results from our data, each using a different approach to exclude SNPs in order to remove correlated markers. We found that LOD scores were maintained when SNPs were excluded based on observed LD patterns, but when genetic distance was the criterion for exclusion, it resulted in much lower IC values, suggesting that it was too severe. Our results demonstrate that modelling marker-marker LD to eliminate redundant SNPs is sufficient to avoid false positive signals but at the same time limits the decrease in IC and consequent loss of power, and therefore seems to be the best strategy for the optimal use of SNP linkage panels. Besides, the small number of markers discarded in LD modelling analysis indicates that only modest LD was present in the Illumina SNP panel used. Therefore, we can conclude that LD between loci does not significantly affect the overall detection of linkage regions in our SNP genome scan.We have compared results obtained from SNP markers using the Illumina panel (version III) to those from a traditional 10 cM microsatellite scan, and we have demonstrated that dense SNP maps can provide higher power, identifying regions suggestive of linkage that would be missed by a standard microsatellite scan. Using this strategy, we have observed a clear improvement in the power of linkage signal detection because it is noteworthy that while five regions were consistently identified performing both parametric and non-parametric analysis of SNP-GWS data, only one of these was identified when traditional STR-GWS data were analysed.In conclusion, our strategy of two-stage linkage mapping, therefore, has allowed us to identify five new putative loci related to breast cancer with moderate values that suggests the existence of genetic heterogeneity among these non-BRCA1/2 breast cancer families. We also found two regions (11q13.5 and 14q21.1\u201314q21.3) linked to the same family (FAM153) and confirmed these by fine mapping analysis. This result is consistent with the hypothesis of a polygenic model as has been previously suggested.Therefore, our results show that genomewide scans using SNP markers, followed by fine mapping using STRs to confirm the veracity of the primary scan, appears to be an optimal strategy for future linkage analyses. In addition, this approach provides more and stronger linkage signals than those using traditional STR markers and allows both to screen for well-defined positive areas in some regions, and to filter any false positive signals.Nineteen families with non-BRCA1/2 hereditary breast cancer from the USA, the Netherlands and Spain were selected for this study. Sixteen of them were previously genotyped using a low density genomewide scan with microsatellite markers and most of them were included in the recent Breast Cancer Linkage Consortium Study .Families had to satisfy the following criteria: a) at least three women diagnosed with breast cancer below age 60 years, b) no case of ovarian cancer or male breast cancer in a blood relative, c) DNA samples available for genotyping from at least three women affected with breast cancer, or from children of affected individuals such that the genotypes might be inferred . DNA samples were available from 81 female family members with breast cancer. The nineteen families were recruited by three groups. The Spanish families 5 families) were ascertained by the Familial Cancer Unit at The Spanish National Cancer Centre (CNIO) in Madrid; the American families (7 families) were ascertained by Henry Lynch at Creighton University in Omaha Nebraska and originally genotyped at the International Agency for Cancer Research (IARC); and the Dutch families (7 families) were ascertained by the Clinical Genetic Centres in Leiden and Rotterdam and through the Netherlands Foundation for the Detection of Hereditary Tumours (STOET) [see Additional file  familiesSNPs markers were genotyped using the Illumina BeadArray linkage mapping panel (version III). Oligonucleotides were designed and synthesized by Illumina, Inc. Details of the GoldenGate assay have been previously described ,23. A toFor sixteen of the nineteen families, a total of 400 polymorphic microsatellite or STR markers from the ABI Prism Linkage Mapping Set-MD10 (Applied Biosystems) were analysed on ABI 3700 DNA sequencers at the Welcome Trust Sanger Institute. The average interval between the markers was 10 cM. Genotypes were called automatically with Genotyper software.In addition, in order to narrow down potential regions of interest based on the analysis of the genomewide SNPs (GWS-SNPs), we selected STRs with high heterozygosity across candidate regions (one every 2\u20133 cM). Fine density genotyping around linkage peaks (8 STRs/region on the average) was performed in families that showed suggestive linkage. Genotyping was performed at the CNIO using the ABI 3700 DNA sequencer platform and data analysis was carried out using Genescan software.To control the quality of experimental variables such as plate orientation, and to provide the opportunity to test genotyping reproducibility, inter- and intra-plate duplicate genotyping was performed. The PEDSTATS software was used to determine the genotyping success rate, to confirm the pedigree structure, and to correctly specify the relationships between individuals in each family . The PEDThe analysis of all families combined was done assuming that all families had the same genetic background, coming from the same homogeneous (European) population. Due to the limited number of families (19 families) and individuals , the allele frequencies were not estimated separately in each of the three populations , nor among the founders . We therefore estimated allele frequencies across all individuals pooled. In order to demonstrate that the estimated allele frequencies derived from the full data set (ALL frequencies) were appropriate for the analysis, we compared them with the frequencies provided by Illumina for each marker, estimated from 82 unrelated CEPH individuals (CEPH frequencies). We found no statistically significant differences after Bonferroni corrections (data not shown). Focusing on the highest non parametric LOD score using NPL all statistics (NPLOD score) values identified, we compared the results obtained using both types of frequencies and we found no important differences (the average change was just 0.13) [see Additional file In order to obtain more conservative results and avoid false positives due to specific population differences, all analyses with SNP markers have been performed using allele frequencies estimated from the full data set.et al. [Frequency estimation for genomewide scan data with microsatellites was carried out according to Smith et al. . For theet al. , and thiet al. .et al. [Multipoint nonparametric linkage analysis  for SNP markers was performed using Merlin and MINX (Merlin in X) programs ,29 to caet al. , where ret al. . We alsoet al.  under doCandidate linkage regions were defined as those with NPLOD scoreswith associated p-values < 0.01 using CEPH frequencies, but that were still significant  when more conservative analysis (using ALL frequencies) was done. The genomewide scan with microsatellite data and additional fine-mapping microsatellite data were also analysed using the Merlin program and parametric and non parametric analyses were performed.We compared the linkage of a genomewide scan using SNPs alone (SNP-GWS) with a genomewide scan using microsatellites alone (STR-GWS) in sixteen families for which data from both types of scan were available. Parametric and non parametric analyses were performed.The power of linkage analysis and consequently the expected LOD score is related to the amount of information extracted from the map . The inf2 between adjacent markers across the genetic map was 0.11 \u00b1 0.003 in the Illumina III panel (SNP-GWS), based on 82 CEPH individuals. To determine the effect of LD between marker loci, two approaches were taken. Under the first, SNPs in strong LD with other SNPs  were removed and the data set reanalysed. Under the second, NPLOD scores (ALL frequencies) were calculated using an approach for modelling LD between markers during multipoint analysis [The average ranalysis . We alsoTo evaluate our linkage results, we used Merlin's simulation option to empirically estimate the probability that the observed proportion of the genome that showed excess allele sharing could be observed by chance. One thousand genomewide replicates were analysed under the null hypothesis of no linkage to breast cancer, and the percentage of the genome with an NPLOD score over the specified threshold was determined in each genomewide replicate.SNP- Single Nucleotide Polymorphism; STR- Short Tandem Repeat; LD- Linkage disequilibrium; GWS- Genomewide scan; NPLOD score- Non parametric LOD score; PLA- Parametric linkage analysis; NPLA- Non parametric linkage analysis; HLOD score- Heterogeneity LOD score; DM- Dominant model; RM- Recessive model.EG, JMR, MS carried out the genotyping. AO, JB and AGN participated in the design of the study. AGN, GP, JMR participated performing the statistical analysis. JB, DG and PD have helped to draft the manuscript. MS, OS, HR, RO, CA, NH and JB have provided the samples and DG and PD have provided the microsatellite data to allow the SNP-STR comparisons. All authors read and approved the final manuscript.List of candidate regions. List of candidate regions selected and LOD score comparison using full data set (ALL) frequencies and Illumina frequencies (CEPH).Click here for fileSNP markers used in all the analysesClick here for filep-values for both methods used to modelling marker-marker LD. NPLOD score, IC and p-values were calculated considering, different measures of LD between marker loci (left table) and, different measures of genetic distance between marker loci (right table)Values for NPLOD score, IC and Click here for fileNumber of candidate regions observed from simulated data. Distribution of the number of candidate regions with an NPLOD score with p-values \u2264 0.01 using CEPH frequencies and still significant  when more conservative analysis using ALL frequencies, identified from 1000 times random genomewide scan data.Click here for fileSummary of families by population group.Click here for file"}
+{"text": "Future research is needed to develop rigorous methods in mapping of genes affecting the expression of a group of transcripts.We explored approaches to using multiple related traits (gene expression levels) in linkage analysis. We first grouped mRNA transcripts according to their functions annotated in biological process of gene ontology (GO). We then compared using sample average, principal-components analysis (PCA), and linear discriminant analysis (LDA) to derive a univariate composite trait. Our results showed that PCA generally yielded stronger evidence for linkage, through the LDA component had the highest heritability. We also developed an algorithm to search for clusters of linkage peaks from multiple traits in the same group and a heuristic method for calculating  Our aim is to explore approaches to collecting information from multiple sub-clinical traits  in search for loci responsible for complex diseases. Given a particular regulatory/metabolic pathway that is important in the development of a disease, we use the expression levels of genes in the pathway to map the loci affecting the pathway, thus affecting the clinical outcome. To avoid confusion between the genes we are trying to map and the genes whose expression levels are used as traits, we will call the expression levels \"traits\" of \"transcripts\", as we will talk about functions of the transcripts. Because these traits reflect more immediate effects of the gene in question, we may reduce the confounding effects of environmental factors and the disease genetic heterogeneity.Because there is no clinical trait or covariate information in Problem 1, we defined a group of transcripts as those sharing some common biological functions, and explored whether we can gain more linkage information by combining multiple expression traits in a group.The 3554 transcripts were grouped based on the biological processes in which they are involved according to the gene ontology (GO) via Onto-Express . On the prcomp in R. The PCA did not take into account the fact that the subjects came from several distinct families. As an alternative, we sought to find a linear combination of the original data that maximized the ratio of inter-family variance to within-family variance, for which we used LDA with the family as the class label. The LDA was carried out using function lda in R.All traits were standardized to have sample mean of 0 and sample variance of 1. We used three approaches to producing a univariate summary of the expression traits of individual genes in each group: a sample average, principal-components analysis (PCA), and linear discriminant analysis (LDA). All three approaches derive a single or multiple univariate \"composite\" traits by using a linear combination of the individual traits. The components in PCA are orthogonal linear combinations of original data and are ordered by decreasing sample variances . In partMultipoint variance-component LOD scores for each transcript and composite traits were calculated using Merlin .C . The threshold was set to be relatively high such that the chance of type I error was low. 2) The peak locations were defined to be where the local maximum LOD scores were. 3) Using a sliding window with width W , we defined a \"cluster\" as the window inside which more than one distinct gene had one or more peaks.In linkage studies in which multiple related traits  are analyzed, it is often of interest to see if several of the traits have linkage signals around a common region, often done by simply visualizing the LOD scores along a chromosome. We developed a heuristic algorithm for identifying the clustering of linkage peaks. 1) Linkage \"peaks\" were defined as LOD scores greater than a particular threshold p-value, assuming that linkage peaks were independent and uniformly distributed along a chromosome, conditional on the observed number of peaks for each gene. Let L denote the total chromosome length. For gene k, conditional on observing nk peaks, the probability of observing at least one peak in a window is To assess whether a cluster was due to chance, we calculated a simple K transcripts in a group, the probability that there are at least K0 peaks in a window isFor the total Figure We calculated the LOD scores for individual transcripts and composite traits for the ten functional groups. Generally the first principal component (PC1) captured the common LOD peaks in individual traits well, and sometimes the PC1 had higher LOD scores than any of the individual traits but it might miss a high LOD score that would be obtained from just one trait. The sample average performed similar to or slightly worse than PC1, while LD1 (first discriminant in LDA) was the least capable of recovering and enhancing the single-trait LOD score peaks, despite the fact that LD1 always had the highest heritability, as expected, as a result of its construction Fig. .The LOD score curves for individual transcripts and composite traits for Groups 2 and 5 are shown in Figure Table Li et al.  used theWe selected the ten groups for linkage analysis based on the heritability. There is substantial difference in the correlation structure within each group as evidenced in Figure Although using PCA does not necessary yield higher LOD scores than a single expression trait, different thresholds for \"significant\" LOD scores are necessary due to multiple testing when multiple traits are analyzed individually. A dimension reduction approach necessarily reduces the number of tests conducted. We could, for example, use PCA to screen for functional groups that are more likely to be co-regulated by a common gene among all GO groups.p-value calculation was based on perhaps over-simplified assumptions, such as the independence of peak locations under the null hypothesis. Much further research is needed.The way we searched for clustering of linkage peaks among related traits was just a proof-of-concept exercise. In particular, information such as the width or height of a linkage peak was not used. Our Finally, given an overwhelmingly large set of variables , how to define functional groups will likely be the most critical part of the analysis. We realized that the GO functional groups do not necessarily imply the transcripts in a group belong to the same metabolic pathway or are regulated by common genes. Without sufficient biological knowledge and no clinical outcomes, we could not address the problem of how to select traits for joint analysis and opted to simply use GO information. Solutions to this difficult problem are highly context dependent and close collaboration between statisticians and subject-area experts is needed.The author(s) declare that they have no competing interests."}
+{"text": "There has been a growing interest in developing strategies for identifying single-nucleotide polymorphisms (SNPs) that explain a linkage signal by joint modeling of linkage and association. We compare several existing methods and propose a new method called the homozygote sharing transmission-disequilibrium test (HSTDT) to detect linkage and association or to identify SNPs explaining the linkage signal on chromosome 6 for rheumatoid arthritis using 100 replicates of the Genetic Analysis Workshop (GAW) 15 simulated affected sib-pair data. Existing methods considered included the family-based tests of association implemented in FBAT, a transmission-disequilibrium test, a conditional logistic regression approach, a likelihood-based approach implemented in LAMP, and the homozygote sharing test (HST). We compared the type I error rates and power for tests classified into three categories according to their null hypotheses: 1) no association in the presence of linkage , 2) no linkage adjusting for the association , and 3) no linkage and no association. For testing association in the presence of linkage, we found similar power among all tests except for the homozygote sharing test that had lower power. When testing linkage adjusting for association, similar power was observed between LAMP and HST, but lower power for the conditional logistic regression method. When testing linkage or association, the conditional logistic regression method was more powerful than FBAT. The availability of high throughput single-nucleotide polymorphism (SNP) genotyping technologies at more affordable costs has generated increasing enthusiasm for genome-wide association study (GWAS) for a wide range of disorders . How to 0: no association, in the presence of linkage or the tested SNP is in linkage equilibrium (LE) with all disease loci (denoted T1); 2) H0: no linkage adjusting for the association, or the tested SNP is in complete linkage disequilibrium (LD) (r2 = 1) with all disease loci (denoted T2); 3) H0: no linkage and no association (denoted T3). It is vital to understand the differences among these hypotheses and the relative efficiencies of valid tests for each hypothesis. In what follows, we classify the tests we considered into the three categories and compare the power and type I error among them.There are three major types of family-based association tests categorized by their null hypotheses: 1) H1. Although the LOD scores range from 3 to 6, these 947 SNPs are far away from the disease loci  and should be in LE with the disease loci. For T3, type I error rates were evaluated using all 16 chromosomes that do not harbor any disease loci. Available data from controls were used to determine LD, as measured by r2 between each of 2102 dense SNPs and the disease loci. For the tri-allelic DR locus, a generalized R2 was calculated for each SNP and tested using the LOGISTIC procedure in SAS version 8 , the expected identical-by-decent (IBD) sharing between g1 and g2 given the observed marker genotypes and e1* is the expected IBD sharing between g1 and \u03b2 = 0 is a test of T1 (denoted Millstein-b), a test of \u03b3 = 0 is a test of T2 (denoted Millstein-c), and the two degree of freedom likelihood ratio test (LRT) of \u03b2 = 0 and \u03b3 = 0 is a test of T3 (denoted Millstein-a).where g* represents the four possible offspring genotypes; 1 and LAMP-LD is a test of T2. The statistical significance of these two tests is assessed empirically by comparing the observed statistic with simulated null distributions. We exclude flanking short tandem repeat (STR) markers in LD [Li et al.  proposedrs in LD  with tes0:1/2 <\u03b1homo = \u03b1het vs. H1: 1/2 \u2264 \u03b1homo <\u03b1het, where \u03b1homo and \u03b1het are the probabilities that an affected sib-pair shares one allele IBD with respect to homozygous and heterozygous parents, respectively. The HST is defined asThe HST statistic ,12 is coj\" allele IBD from homozygous and heterozygous parents respectively . This HST statistic (denoted HST-LE) is a test of T1. Once subsets of SNPs explaining some of the linkage evidence have been identified, one can then test H0: 1/2 = \u03b1homo <\u03b1het vs. H1: 1/2 <\u03b1homo <\u03b1het with the following HST statistic:where 2. Both HST-LE and HST-LD are LRTs under independent parental transmissions, equivalent to assuming a multiplicative model of transmission. Under the null hypothesis of LE between the tested SNP and disease loci, both HST statistics asymptotically follow a chi-square mixture distribution of Rejection of the null hypothesis indicates that the tested SNP does not explain fully the linkage evidence. This HST statistic (denoted HST-LD) is a test of TM1 and M2, they showed that for a marker in LE with the disease loci, the probability that both affected siblings receive M1  into two allele-specific IBD sharing probabilities , the HSTDT asymptotically follows a chi-square mixture distribution of Similar to HST-LE, HSTDT is a LRT under the assumption of independent parental transmission. Under the null hypothesis of LE between the tested SNP and disease loci (T1 or T2 is that the former can be used to test if a SNP explains the linkage peak (by using IBD information at the linkage peak). When assuming no recombination between the tested SNP and the presumed disease locus at the linkage peak, testing association in the presence of linkage is equivalent to testing whether a SNP partially explains the linkage peak; while testing no linkage, adjusting for association is equivalent to testing whether the tested SNP fully explains the linkage peak. However, when the assumption is violated, for tests of T1 and T2 other than HST and HSTDT, one may not be able to claim that the tested SNP explains the peak linkage evidence but rather that it explains the linkage evidence at the location of the tested SNP. When a linkage signal is identified in a linked region, the LOD score at the linkage peak should be of greatest interest and is the usual quantity reported. In this report, HST and HSTDT are applied to identify SNPs explaining the peak linkage evidence.The major distinction between homozygote sharing tests (HST and HSTDT) and other tests of Tr2 = 0) included 947 dense SNPs between 130 and 140 cM on chromosome 6 that were used to assess type I error rates for T1. In Table r2 = 0, \u03b8 = 0.5) included 6597 SNPs from all 16 chromosomes that did not harbor any disease loci and were used to assess type I error rates for T3. The remaining SNPs were grouped by the maximum of the three mean r2 values with the three disease loci . For example, the group 0.1 <r2 <= 0.3 included SNPs with mean r2 between 0.1 and 0.3 with at least one of the disease loci. Power and type I error rates were assessed at significance levels of 5%, 1%, and 0.1%.SNPs were classified into five groups according to their LD with the disease loci. In Table 1. All methods have appropriate type I error rates (r2 = 0), except that HSTDT has slightly inflated type I error rates. TDT is the most powerful among these six methods, followed closely by HSTDT, FBAT-e, Millstein-b and then LAMP-LE. HST-LE appears to be less powerful.Table 2. These tests are used to examine if a SNP explains all the observed linkage evidence. LAMP-LD and HST-LD test whether a SNP is in complete LD with the disease loci. Equivalently, Millstein-c tests if there is residual linkage when conditioning on the genotype covariate of the tested SNP and is essentially a binary-trait version of the test proposed by Almasy and Blangero [Table Blangero . Because3. The type I error rates  of FBAT and Millstein-a are appropriate. FBAT is less powerful than Millstein-a for SNPs in low LD with the disease loci. Note that Millstein-a directly uses IBD between sib pairs in the model, while FBAT uses allelic transmission from parents to children, possibly explaining the difference in their ability to pick up the linkage signal.Table With a dense SNP map, it is natural to speculate whether all disease loci under a linkage peak have been identified or their contributions to the phenotypic variation are fully explained by a small subset of SNPs in association with these disease loci. Recently, there was much interest in testing whether a SNP can partially or fully account for all the observed linkage evidence.We examined several methods for joint linkage and association analysis and identifying SNPs that explain the linkage evidence. For testing association in the presence of linkage and for testing linkage or association, all methods have appropriate type I error rates. For testing association in the presence of linkage, TDT is most powerful . For testing whether a SNP explains all the linkage evidence, HST and LAMP have similar power and are more powerful than Millstein-a; the difference in power may be explained by the difference in type I error, which we were unable to assess in the GAW15 data set because there was no single disease locus explaining all of the linkage evidence. For testing linkage or association, we found that Millstein-c was more powerful than FBAT. These conclusions may not extend to study designs other than nuclear families each with two affected children with parental genotypes available, a requirement for HST, HSTDT, and Millstein et al.  FurthermIn this study, there are three disease loci in the linked region and they do not contribute equally to the linkage signal, with the DR locus having a major effect on affection status. A different scenario may lead to different results for methods that use linkage peak information to identify SNPs, explaining the linkage evidence. However, for a complex disease, there may be multiple disease loci acting interactively, so methods that do not assume a single causal variant would be most helpful in identifying SNPs associated with disease loci. There is a great need for developing methods suitable for multiple disease loci.The author(s) declare that they have no competing interests."}
+{"text": "Bombyx mori has been fully sequenced, but comparative lepidopteran genomics has been hampered by the scarcity of information for other species. This is especially striking for butterflies, even though they have diverse and derived phenotypes (such as color vision and wing color patterns) and are considered prime models for the evolutionary and developmental analysis of ecologically relevant, complex traits. We focus on Bicyclus anynana butterflies, a laboratory system for studying the diversification of novelties and serially repeated traits. With a panel of 12 small families and a biphasic mapping approach, we first assigned 508 expressed genes to segregation groups and then ordered 297 of them within individual linkage groups. We also coarsely mapped seven color pattern loci. This is the richest gene-based map available for any butterfly species and allowed for a broad-coverage analysis of synteny with the lepidopteran reference genome. Based on 462 pairs of mapped orthologous markers in Bi. anynana and Bo. mori, we observed strong conservation of gene assignment to chromosomes, but also evidence for numerous large- and small-scale chromosomal rearrangements. With gene collections growing for a variety of target organisms, the ability to place those genes in their proper genomic context is paramount. Methods to map expressed genes and to compare maps with relevant model systems are crucial to extend genomic-level analysis outside classical model species. Maps with gene-based markers are useful for comparative genomics and to resolve mapped genomic regions to a tractable number of candidate genes, especially if there is synteny with related model species. This is discussed in relation to the identification of the loci contributing to color pattern evolution in butterflies.Lepidopterans (butterflies and moths) are a rich and diverse order of insects, which, despite their economic impact and unusual biological properties, are relatively underrepresented in terms of genomic resources. The genome of the silkworm  Butterflies and moths  are a large and diverse group of insects that has long captured the attention of biologists and laymen. The colorful patterns on the wings of butterflies, in particular, offer an ideal system to investigate which genes and developmental mechanisms contribute to evolutionary diversification. Genetic analyses that try to find the position of genes along chromosomes are invaluable for such efforts, also because they allow researchers to compare chromosome content between species. Here, we report on a study which built a gene-based map for the chromosomes of a butterfly \u201clab rat\u201d and identified chromosomes carrying color pattern genes. We compare our map to that of the reference lepidopteran species, the silkworm. Despite these species having diverged some 100 million years ago, there is much conservation in terms of which genes are found together in chromosomes and even how genes are ordered within chromosomes. However, because we were able to compare positioning of many more genes than had ever been reported before for this group, we also found evidence of several large- and small-scale chromosomal rearrangements. We discuss the advantages of gene-based maps in understanding the genetic basis of color pattern evolution. With the need for a wider sampling of biological diversity Bombyx moriB. mori genome have been hampered by the relative scarcity of relevant genomic information. Dipterans, the closest insect lineage with available sequenced genomes, diverged from lepidopterans more than 200 MYA, and there is relatively little genomic information within the Lepidoptera. This is especially striking for butterfly species (derived from moths some 100 MYA), despite much interest in their diverse, derived, and ecologically-relevant wing patterns.The Lepidoptera (butterflies and moths) are a diverse order of insects with an abundance of species, including many agricultural pests, and one of two species of domesticated insects. Lepidopterans have some unusual genetic properties, such as holocentric chromosomes, heterogametic females, and male-restricted meiotic recombination, whose underlying mechanisms and consequences for genome evolution remain to be fully explored. However, lepidopteran species are relatively under-represented in terms of genomic resources with little available outside the model silkworm Drosophila melanogaster have been implicated in formation . With the largest collection of anchor loci mapped to date for any butterfly species, we were able to do a broad-coverage comparison of gene co-segregation and gene order between B. anynana butterflies and the lepidopteran reference species, Bombyx mori. Our analysis confirmed previous reports of conserved synteny in the Lepidoptera and also detected several small- and large-scale chromosomal rearrangements separating B. anynana butterflies and B. mori moths.We used a biphasic linkage mapping method We selected 768 SNPs in expressed genes to genotype in a mapping panel composed of 288 individuals from 12 F2 families . These mB. anynana linkage groups (cubitus interruptus (ci), Ecdysone receptor (EcR), engrailed (en), APC-like (Apc), naked cuticle (nkd), cinnamon (cin), Henna (Hn), echinus (ec), Catalase (Cat), and Heat-shock protein 70 (Hsp70). Failure to map the other four candidate genes was due to a failed assay , Mendelian inconsistencies , or the SNP being fixed in the mapping panel .Absence of recombination in lepidopteran females can be exploited to construct genetic linkage maps via \u201cbiphasic mapping\u201d e groups \u20134, posSpotty mutation for which a 2 LOD support interval included positions at either end, but excluded the middle region of LG10 , and 2) the Goldeneye mutation which mapped to LG28 whose validity we are uncertain of (see below). Poor mapping resolution for the visible markers likely reflects the fact that: 1) any given mutation was typically only segregating in 1\u20134 families  in tHeliconius melpomene  and \u223c122 cM (LG19) . This maApc (LG6) and EcR (LG10). For Apc two of the three ordered markers overlap and the third maps at a distance of 5.4 cM from them, while for EcR all three markers map to positions within 2.6 cM from each other  is a measure of potential map expansions due to errors. About 26% (70 of 269) of the \u201cfemale distances\u201d between neighbor ordered markers were greater than 0 cM . The avech other \u20134. Thede novo SNP identification. This approach involved: 1) focusing on SNPs identified in EST collections  and for which the minor allele was seen at least twice , and, once assigned to a linkage group, we maximize the chance of identifying at least a second family in which that SNP is  male informative. Of the 508 mapped markers, 11 corresponding to nine Z-linked loci and to two autosomal markers , we will be able to choose from mapped, intermediate-frequency, informative SNPs, to design assays for larger mapping panels derived from a smaller number of founders. For this, having a large number of gene-based markers (even if mapped with limited resolution) and knowledge of SNP frequency is more useful than having a very accurate map of sparse markers (which may not be informative in another context).In downstream uses of this map  with material coming from two chromosomes and two additional scaffolds (2.2%) with material derived from three chromosomes, suggesting that there are errors with the current assembly. A visual inspection suggests that those assembly errors tend to be associated with the very ends of scaffolds. So, although the fraction of large scaffold with such errors is significant, very little of the assembly is affected. We next fitted local regressions for each scaffold that allowed for predictions of genetic positions (cM) given a physical position (bp) on the scaffold , will be crucial to put the gene collections and genetic maps, growing for a variety of butterfly species, into phylogenomic context.The current B. anynana  mapped in other lepidopteran species: the butterfly Heliconius melpomene , and the silworm Bombyx mori . Of the 508 B. anynana markers, 29 (18 ordered and 11 unordered) had an ortholog among the 101 anchor loci mapped in H. melpomeneB. mori scaffold. Of the remaining 46 mapped B. anynana markers . Both B. mori and B. anynana have 28 pairs of chromosomes, while basal lepidopterans have 31 pairs B. anynana LGs are made up of syntenic blocks from different B. mori LGs suggest that the two lineages have undergone independent karyotype reductions, via non-homologous chromosomal fusions.Despite the ca. 100 MY that separate butterflies and moths ori LG28 \u20134. Of ori LG28 . The 37 ori LG28 , blasts Heliconius melpomene) and the reference lepidopteran (Bombyx mori) and concluded that extensive synteny existed B. mori and B. anynana (this paper) and that for B. mori and H. melpomeneH. melpomene and B. mori did not detect any of the chromosomal rearrangements we document , and must thus have undergone further, or independent, chromosomal fusions relative to B. anynana. It is, however, noteworthy that the proposed fusions separating H. melpomene and B. mori are based on few pairs of mapped orthologous markers , with a mapped B. mori ortholog on a syntenic block, only LG10 and LG21 have fully conserved marker order , inferred marker order in B. anynana was at least twice as likely than B. mori order in 20 cases (and at least 630 times more likely for 18 of the comparisons). For the four situations where the B. mori order was better supported than the one originally inferred for B. anynana , and for LG13 , we used the B. mori order as a starting point in CRI-MAP and further improved it  and 34.8 for LG11 (i.e. inferred order \u223c10\u222734 times better)  . BecauseH. melpomene and B. moriB. anynana, and extending comparative analysis to additional species will be crucial to rigorously quantify the extent of inversions separating different lepidopteran lineages. Unfortunately, the number of shared ordered markers in the H. melpomene and B. anynana maps prevents evaluating the consistency of gene order within Nymphalid butterflies. We have a single B. anynana LG (LG15) with greater than two ordered markers with mapped orthologs in H. melpomene. However, of those four markers, only one has a resolved genetic map position in H. melpomeneOur data suggest that previous conclusions of highly conserved gene order between H. melpomene - B. mori marker pairs were much more localized than the study presented here. They either focused on one individual chromosome , and comparisons of marker order at the level of single BACs can only infer conservation at sub-centimorgan scales. Here, we extended the analysis of gene order to many more markers in many more linkage groups and alert for the fact that, even though we have syntenic blocks and broad conservation of gene order , we also have clear evidence of multiple rearrangements . These intra-chromosomal rearrangements do not mean that B. mori cannot serve as a pan-macrolepidopteran reference, but they do argue that marker order is likely conserved over tens of centimorgans as opposed to entire linkage groups. Our observations are remarkably similar to the emerging consensus view in the Drosophila clade (including species diverged some 40 MYA), that the assignment of genes to Mullerian elements is highly conserved but gene order within those elements is variable Previous studies that analyzed order of more than three ordered B. mori ortholog to the pigmentation gene black localizes to a B. mori scaffold , and identify candidate genes among available markers, but also because conservation of gene grouping and gene order in related species with dense linkage maps might allow identification of extra candidate genes within the implicated genomic regions. For example, the henotype .Bigeye and Chocolate , at present we have only mapped our collection of B anynana visible mutants to entire linkage groups  biology. We looked for H. melpomene and B. mori color pattern loci mapping to orthologous LGs to those where we mapped visible markers in B. anynana . The F2 families were obtained by using single-pairs of P grand-parents that were either from \u201coutbred\u201d, or 1\u20133 generation inbred  populations. DNA from thorax or head of freshly frozen butterflies (killed in liquid nitrogen and stored at \u221280\u00b0C until processed) was extracted using the QIAGEN tissue kit following manufacturer's recommendations. Genomic DNA was checked for quality and yield on agarose gel and NanoDrop spectrophotometer. From each of the 288 individuals in the mapping panel, 1.7 \u00b5g of genomic DNA in 100 \u00b5l of QIAGEN kit elution buffer was dried down (SpeedVac), re-suspended in 20 \u00b5l water, and sent to Southern California Genotyping Consortium - Illumina Genotyping Core Laboratory at UCLA We used different B. anynana genes , programmed cell death (ec), lifespan (Cat), and stress response (Hsp70).We selected 768 SNPs to genotype using the Illumina Golden Gate platform na genes . These cna genes  . The coB. anynana homologs of developmental candidate genes, we designed two or more assays. These were: two SNPs in the pigmentation gene yellow (BaC4163), in ci (BaG15), en (BaG21), and nkd (BaG24 and BaG25); and four SNPs in Apc (BaG14 and BaG16), and EcR (BaG19 and BaG20). Many criteria went into choosing the target SNPs, including: the estimated frequency of the SNP , absence of secondary polymorphisms in the \u223c100 bp up- and down-stream of it, the contig annotation (preference given to markers in genes with sequence similarity to genes in public databases), and score for Illumina \u201ctype-ability\u201d. Sequences associated with the 768 markers we attempted to genotype are available in Genbank's sequence or EST archive; accession numbers in We attempted to choose only one high quality SNP for each gene but, in the case of a minority of putative A large fraction of the SNPs assayed converted into working assays and \u223c75% had a call rate of greater than 95%. The poorest 15% of SNP assays had a call rate of 0%, whereas the best 80% had a minimum call rate of 89%. The individuals in our genotyping panel consistently generated good data; a 95% Confidence Interval on the number of called SNPs over individuals was 626 to 644 with the poorest and second poorest individuals yielding 527 and 600 called SNPs, respectively. Consistent with this narrow confidence interval, we did not consider removing any individuals from the study because of poor quality DNA. The vast majority of SNPs were ascertained from an EST project so the observation that 15\u201325% of the attempted SNPs did not convert to a useful assay was not unexpected. Reasons for failure to convert include factors such as: SNPs having a low allele frequency in the mapping panel, some SNPs being falsely identified due to over assembly problems, introns resulting in non-functioning Golden-Gate assays, and errors in flanking regions that the Golden-Gate oligonucleotides anneal to We visually examined the dataset for clear genotyping errors that resulted in a SNP showing a pattern of inheritance inconsistent with Mendelian expectations . SNPs feall the SNPs in that group. Unpublished simulations suggested that this algorithm rarely results in \u201cover-clustering\u201d. At our CRIMAP inclusion LOD score of 11 we identified 27 segregation groups, with the smallest number of markers in any given group being three, the largest 28 and the mean 13.6 SNPs. Increasing the LOD score for inclusion to values as high as 16 resulted in fewer SNPs assigned to segregation groups, and never split a segregation group identified at an inclusion value of 11 into two. Whereas decreasing the LOD score resulted in the merging of segregation groups (and fewer than 27 clusters).We used marker-pairs that were female fully-informative  to initially assign markers to segregation groups. For all possible pairs of SNPs in the 513 putative autosomal SNPs, we calculated a LOD score summarizing the evidence for complete linkage /L]). For any given pair of SNPs that LOD score could be missing  or summarize linkage information from 1 to 12 female fully-informative families. We then grouped SNPs connected by LOD scores of greater than eleven. As a result, a SNP could be assigned to a segregation group despite not having a LOD score of greater than 11 with For all the SNPs assigned to a given segregation group we used CRIMAP We manually inspected the resulting maps. In cases where the two \u201cordered-loci\u201d used to initialize the Expectation Maximization algorithm underlying CRIMAP were loosely linked we re-ran the build option with a different set of random starting loci. In other cases where we observed SNPs that were completely linked in males we re-ran the \u201cbuild\u201d using the \u201chap_sys\u201d option for those SNPs. We then used the \u201cflips4\u201d option on the ordered loci to confirm that our maps had the highest possible likelihood, creating a new order when necessary, and re-running the \u201cbuild\u201d and \u201cflips4\u201d analysis until the order stabilized.We then used the \u201ctwo-point\u201d option in CRIMAP in an attempt to assign to segregation groups the remaining 146 putative autosomal markers, not initially assigned. This resulted in our being able to: 1) assign 134 markers to pre-existing segregation groups, 2) merge two pairs of pre-existing linkage groups in single groups, and 3) identify two novel small linkage groups (one having three and the other four SNPs). Typically, added markers displayed a high LOD score for linkage with several members of a pre-existing linkage group and below background LOD scores with members of any other group. The few SNPs that could not be assigned to any segregation group were typically only informative in a single family and/or showed segregation patterns that were unlikely under Mendelian inheritance. Based on the newly defined segregation groups and starting with the markers ordered in the previous round, we carried out another round of \u201cbuilds\u201d, followed by another round of \u201cflips4\u201d, and iterating until we achieved an ordering for which the \u201cflips4\u201d command could no longer identify orders with higher likelihoods. Details about the mapping of the ordered and unordered markers are found in comet and Missing, to linkage groups. For the seven mutants mapping to linkage groups we used the \u201call\u201d option of CRIMAP separately for each mutant and its respective linkage group in an attempt to localize that mutation within a linkage group.A total of nine Mendelizing visible mutants affecting adult or larval coloration were segregating in six of the twelve full-sibs families used for mapping . All offB. anynana map, we used blastn and tblastx analysis  against the scaffolds from the B. mori genome assembly  did not have a significant direct blast hit to any of the target H. melpomene markers  to estimate its position in the B. mori map. Details of the blast analysis with B. mori and H. melpomene can be found in B. anynana ordered and unordered markers, respectively.For all genes in the B. mori scaffolds and used blastn to query all the mapped B. mori SNPs from (\u201cDE\u201d accessions from B. mori map position for any B. mori gene. For B. mori scaffolds having greater than four mapped markers this predictor is simply the slope and intercept obtained from a linear regression of map position on base position (of the midpoint of the highest scoring blast hit). For B. mori scaffolds with one to four markers this predictor is simply the average position of the markers mapping to that scaffold. For scaffolds with no mapped markers the predictor is undefined. This heuristic seemed reasonable, as a large fraction of the genome is contained in scaffolds with more than four mapped markers, and scaffolds harboring four or fewer markers are typically small enough that returning a single map position for the midpoint of that scaffold is acceptable. During this annotation effort we discovered a small number of B. mori scaffolds with termini mapping to different chromosomes, we assumed these are mis-assembly errors and removed these sections of scaffold from further consideration.We downloaded the new collection of B. anynana were different from those in B. mori. To do this we used the \u201cfixed\u201d option of CRIMAP to compare the inferred (non-haplotype system) order in B. anynana to that in B. mori for the subset of markers having orthologs. This analysis allowed us to obtain log10 likelihoods for both orders, and identify instances where the B. mori order was more highly supported . This final build went though additional \u201cflips\u201d rounds and then \u201cfixed\u201d was run on the final order to obtain the map displayed in We wished to ask if within linkage group, inferred marker orders in B. anynana genetic map, and of synteny between B. anynana and B. mori chromosomes .(0.54 MB TXT)Click here for additional data file.Table S2Genotypes of 288 panel individuals for each of the 533 converted SNPs.(1.04 MB XLS)Click here for additional data file.Table S3B. mori and H. melpomene.Mapping details for ordered markers including comparisons to (0.03 MB TXT)Click here for additional data file.Table S4B. mori and H. melpomene.Mapping details for unordered markers including comparisons to (0.01 MB TXT)Click here for additional data file.Table S5B. mori markers in the unpublished scaffold sequences of B. mori.Location of mapped (0.07 MB TXT)Click here for additional data file.Table S6B. anynana and that in B. mori.Statistical comparison of marker order inferred for (0.01 MB TXT)Click here for additional data file.Text S1Readme: text file explaining contents of each of the supplemental tables.(0.01 MB TXT)Click here for additional data file."}
+{"text": "A multiplicity of study designs such as gene candidate analysis, genome wide search (GWS) and, recently, whole genome association studies have been employed for the identification of the genetic components of essential hypertension (EH). Several genome-wide linkage studies of EH and blood pressure-related phenotypes demonstrate that there is no single locus with a major effect while several genomic regions likely to contain EH-susceptibility loci were validated by multiple studies.We carried out the clinical assessment of the entire adult population in a Sardinian village  and we analyzed 16 selected families with 62 hypertensive subjects out of 267 individuals. We carried out a double GWS using a set of 902 uniformly spaced microsatellites and a high-density SNPs map on the same group of families.Three loci were identified by both microsatellites and SNP scans and the obtained linkage results showed a remarkable degree of similarity. These loci were identified on chromosome 2q24, 11q23.1\u201325 and 13q14.11\u201321.33. Further support to these findings is their broad description present in literature associated to EH or related phenotypes. Bioinformatic investigation of these loci shows several potential EH candidate genes, several of whom already associated to blood pressure regulation pathways.Our search for major susceptibility EH genetic factors evidences that EH in the genetic isolate of Talana is due to the contribution of several genes contained in loci identified and replicated by earlier findings in different human populations. Essential hypertension (EH MIM 145500]), defined as chronically elevated blood pressure (BP) occurring in the absence of other predisposing conditions, has a worldwide prevalence of nearly 27% . All 99 selected affected individuals were clustered in a smaller pedigree including 2,246 people in order to reduce the number of subjects that was not directly connected to the samples used in the present study  software  to perfo2>0.4 as parameter.Linkage analysis assumes linkage equilibrium between markers, but when dense maps are used, this assumption is not respected and it can lead to inaccurate results, especially when parental genotypes are missing. We corrected marker-marker linkage disequilibrium using the methods implemented by MERLIN. This algorithm clusters tightly linked markers and uses haplotypes frequencies to model LD within each cluster. Carrying out STRs analysis, we used the distance k as standard that inserts a cluster breakpoint between markers that are less than k cM apart. We used k = 2 cM. To evaluate LD values using SNPs, we created clusters using rThe Talana-specific STRs genetic maps were constructed for all autosomal chromosomes using CRI-MAP  that was. Number of markers, inter-marker distances and marker allele frequencies were simulated according to the typed marker set. We performed linkage analysis using Merlin in the 16 sub-pedigrees setting to \"missing\" the genotypes of individuals not sampled. For each genome screen the highest LOD score was stored. Cumulative density function of the obtained maximum LOD scores approximates the distribution of the type I error rates. We performed 1000 simulations for STRs map and the results showed that LOD score of 2.42 corresponds to a genome-wide type I error of 5% and that a LOD score of 1.77 corresponds a genome-wide type I error of 50%  and informativeness (MAF > 0.02) in the Talana population. The mean information content (IC) across 22 autosomal chromosomes for the full SNP set was uniformly higher than that for the microsatellites. The mean genome-wide IC, over all chromosomes, was 0.65  for the microsatellites while for the SNPs was 0.716 .Using MERLIN, we performed two genome wide search (GWS): the first used 902 uniformly spaced microsatellites while the second used about 16,300 SNPs from the GeneChipA total of nine regions showed significant linkage for the STRs analysis (LOD SCORES>2.42) and eight suggestive loci (LOD SCORES>1.77) were founded, while in the SNPs analysis, the standard threshold of significance do not allow us to identify significant chromosomic regions (LOD SCORES > 3.3), but only three suggestive loci (LOD scores> 1.9) . SNPs maximum LOD score was 2.03 at position 163.5 Mb.Both GWS show a similar result  on chromosome 11q23.1\u201325. Microsatellite analysis identified a significant locus from 114.7 to 123.1 Mb with a max peak for D11S1998 (117.2 Mb). SNPs analysis detected a region from 112.8 to 126.6 Mb with maximum LOD of 2.45 at 119 Mb. The inspection of peaks position obtained with the 2 analyses reveals that they are in a region where microsatellites coverage and information content is lower than average compared to the rest of the chromosome. The mean information content for SNPs was 0.70 (range 0.63\u20130.75) and for microsatellites was 0.65 (range 0.55\u20130.68). This led us to consider the SNPs localization as more precise.Our double analysis identified an extensive region on chromosome 13q14.11\u201321.33. SNPs analysis showed a locus spanning about 30 Mb (44.9\u201370.4 Mb) with max LOD score of 2.09 at 56.3 Mb. The region identified with STRs is smaller  but it is not well defined because there are no markers covering the region between 60 and 74 Mb.On chromosome 19q13.41\u201313.42 STRs and SNPs analyses show a perfect overlap in a region of about 4 Mb in size (58.4\u201362.3 Mb) but the results showed only a suggestive threshold for STRs analysis. This result is strengthened by informative sets of markers for both analyses: at 60.2 Mb with STRs analysis (LOD score 1.96) and at 60 Mb with SNPs analysis (LOD score 1.60).For chromosome 3p24.1\u201314.3, the results we obtained were suggestive for STRs analysis, given the overlap and pattern similarity but the statistical significance was lower what obtained for other loci described in this article. Therefore we considered the microsatellites IC not satisfactory for the entire region.Similar results were obtained on chromosome 2 where linkage was confirmed both with microsatellites and SNPs with similar patterns but where the peak values were low (D2S367 = 1.14 and rs2710605 = 1.44).The microsatellite NPL analysis found several additional loci with significant LOD SCORE>2.42 not confirmed by the biallelic variants analysis. The microsatellites analysis identifies significant results on the following regions: 1p36.32\u201336.21, 6p24.1\u201312.3, 10q25\u201326, 14q11.2, 15q22\u201324 and 18q21.1  in which genetic heterogeneity and environmental noise are likely to be reduced.We carried out a careful epidemiological investigation of Talana's population in order to assess the environmental and clinical features of the selected object of our study. This section of the study is important because there are very few epidemiological studies in particular in Sardinia where we have limited epidemiological data coming primarily from the main hospitals in Cagliari (the main city of the island). The first phase of the study had the purpose of determining the prevalence of common diseases and quantitative traits presenting familial aggregation. One of the aims was to verify if the essential hypertension clinical and epidemiological features found in this peculiar isolate are comparable with those observed in large out-breeding populations. Prevalence, determinants, awareness and treatments of hypertension were comparable and similar in trend to those reported in large epidemiological surveys. We found that the prevalence of hypertension in the village was about 26%, pretty close to the 27% prevalence worldwide. Hypertension prevalence for Talana was lower than that published by the Italian Istituto Superiore di Sanit\u00e0 for Sardinia as a whole they report a prevalence of EH of 29% in females and 33% in males considering as hypertensive a subject with a SBP/DBP > 160/95 mm Hg or under pharmacological treatment, while for Italy as a whole, they report a prevalence of hypertension of 31% in females and 33% in males. Although we used less restrictive criteria we found a lower prevalence of EH in these isolated villages. This result could be due to the substantially rural nature of these communities, characterized by a healthier environment, nutrition and life style. Similar conclusions have been suggested in studies carried out in several countries, where it has been found that the mean blood pressures of subjects living in transitional and urban areas were significantly higher than those of subjects in rural areas ,33. OgliWe performed a non parametric linkage analysis, the most common approach used when, as in the case of EH, it is not possible to define a clear transmission pattern within families. To gain in power in order to detect linkage we performed two genomewide search, the first using microsatellites and the second using SNPs to benefit from a more dense map and from higher IC . We deciWe identified several loci associated to EH. We considered particularly relevant the loci with significant e/o suggestive LOD scores obtained both with SNPs and STRs scans. Further support to consideration of positive loci came from their previous association to hypertension or to cardiovascular disease phenotypes in the published literature.The potential importance of the identified locus on chromosome 2q24 in the control of blood pressure is supported by previous genome-wide scans and association studies in other human populations. Specifically, 5 genome-wide linkage screens detected a signal overlapping with our region and we can make a direct comparison of the chromosome 2q locus identified in Talana families with the other studies. Zhu et al,  found liFurthermore, we have recently published a linkage study, that identified a locus for total serum cholesterol and LDL cholesterol on chromosome 2q21.2-q24.1 . In lighOur SNPs analysis restricted the candidate region to the first part of the identified region. The current release of the human genome assembly, indicates that the 2q24.3 to q31.1 region between markers rs526540 and rs3914147 (157.081 to 167.624 Mb) encompasses 70 genes of known or predicted function. None of the genes responsible for the monogenic forms of hypertension are found within this region. However, we have identified 5 well-characterized sodium channels, voltage-gated alpha subunit genes  that could be considered biologically plausible candidates. Particularly, SCN7A codes for the a-subunit of a cardiac and skeletal muscle sodium channel protein. A polymorphism in exon 18 of this gene was associated with essential hypertension among the Chinese Han population of Shanghai, China .Furthermore, 4 biallelic markers with the highest LOD score were placed on the potassium voltage-gated channel, subfamily H (eag-related), member 7 gene (KCNH7). Two markers are located on the gene  and the others lie in the strong linkage disequilibrium section of the 3' region of the gene . The voltage-gated potassium channels represent the most complex class of voltage-gated ion channels from both functional and structural standpoints. Their diverse functions include regulating neurotransmitter release, heart rate, insulin secretion, neuronal excitability, epithelial electrolyte transport, smooth muscle contraction, and cell volume. Murine models proved that ion channels encoded by ether-\u00e0-go-go-related genes (eag) have been implicated in re-polarization of the cardiac action potential and also as components of the resting membrane conductance in various cells .We also confirmed the association of hypertension related phenotypes to the 11q region detected by 2-stage genome screen on Caucasian in United Kingdom . Sib-paiRegarding the 13q14.11\u201321.33 locus, the genomic region identified in our study is not well enough defined to justify a gene search since both STRs markers and SNPs analyses do not consent to focus on a restricted area. But also in this case, we find the same location associated to hypertension in meta-analysis and other genome scans reported in literature ,48.On chromosome 19 we found a STRs suggestive region with a precise overlap between SNPs and STRs analysis in a region of only 4 Mb in size. As previously, we replicated a suggestive result obtained on a cohort of 1174 individuals  that were classified as having hypertension from the Framingham Heart Study. This genomic interval includes several potential candidate genes and in particular a cluster of three gamma subunit genes: CACNG6, CACNG7 and CACNG8.Finally, particular attention was focused on the first part of chromosome 2 where, in a previous study, we identified an hypertension associated locus carrying out GWS on a single Talana family . In thatThe genome-wide screens published to date for hypertension genes are diverse in phenotype, ethnic origin, selection criteria, numbers and structures of families and range from analysis of single large pedigrees to various hundreds of sib-pairs or families. Our genome-wide scan differs from many others because to gain in power in order to detect linkage we performed two genomewide search, the first using microsatellites and the second using SNPs to benefit from a more dense map and from higher IC in the same group of families from a little village. The loci we identified in this study are clearly unlikely to reflect all the potential genetic effects on EH. However, our results prove that our population possesses genetic characteristics comparable to outbreeding population since the four loci identified in this study confirm previously identified genomic regions in association with blood pressure disease phenotypes. We are going to replicate our positive results in a large scale cohort of subjects from other villages in the same area in order to study a population with a highly homogenous genetic background as is the people from Ogliastra and it is useful to conduct further analysis to restrict the regions of interest and identify the genes involved in the etiology of EH.The research group was employed by Shardna Life Sciences while carrying out this work.EM, MPC contribute to study design, data analysis and drafted the manuscript. MF and NP performed part of the statistical analyses. VC, CF, IP, AS, AP and DP performed the marker analyses. MA and DS established the cohorts and provide the clinical data. GB performed the epidemiological analyses. MP provided concepts and ideas. AA provided concepts, ideas, study design, data analysis and write the manuscript. All authors have read and approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:"}
+{"text": "Overall, we found little agreement between linkage results from the different pre-processing methods: most of the linkage signals were specific to one pre-processing method. However, agreement rates varied according to the criteria used to select the traits. For instance, these rates were higher in the set of the most heritable traits. On the other hand, the pre-processing method had little impact on the relative proportion of detected cis and trans-regulating loci. Interestingly, although the number of detected cis-regulating loci was relatively small, pre-processing methods agreed much better in this set of linkage signals than in the trans-regulating loci. Several potential factors explaining the discordance observed between the methods are discussed.We evaluate the impact of three pre-processing methods for Affymetrix microarray data on expression quantitative trait locus (eQTL) mapping, using 14 CEPH Utah families (GAW Problem 1 data). Different sets of expression traits were chosen according to different selection criteria: expression level, variance, and heritability. For each gene, three expression phenotypes were obtained by different pre-processing methods. Each quantitative phenotype was then submitted to a whole-genome scan, using multipoint variance component LODs. Pre-processing methods were compared with respect to their linkage outcomes (number of linkage signals with LODs greater than 3, consistencies in the location of the trait-specific linkage signals, and type of  Evidence for heritability of mRNA levels has been observed in several organisms like the mouse, yeast, and human -3. ThereDifferent DNA chip pre-processing methods have been shown to influence measures of gene expression ,8,9. TheThe study is based on all 14 three-generation CEPH (Centre d'Etude du Polymorphisme Humain) Utah families. Gene expression levels in lymphoblastoid cells of 194 individuals have been obtained using the Affymetrix Human Focus Arrays that contain probes for 8792 transcripts. Details regarding the microarray experiments are given in Morley et al. .2-transformed values.Transcript expression data were obtained using the three methods described above: MAS5, RMA, and GCRMA. Expression levels in 82 individuals with technical replicates were averaged over replicates. All subsequent analyses were performed on logIt may be appropriate to restrict linkage analysis to the traits that are expressed in the target tissue and show detectable variation between individuals. Clearly, this is rarely the case for all the transcripts analyzed on a microarray. For example, Morley et al.  chose toThe loci controlling expression levels (the quantitative traits) of these 650 genes were localized using all autosomal marker data provided in the Problem 1 GAW15 dataset. Multipoint LOD scores were computed using the variance-component linkage test with the software Merlin . In thiscis- or trans-acting loci). A gene was assumed to be cis-regulated when the locus controlling its expression level mapped within 10 Mb of the gene itself, and trans-regulated otherwise. Finally, results obtained using two different pre-processing methods were considered consistent when the distance between their linkage peaks was less than 20 Mb. Concordance rates between two or more methods were defined as the ratio of the number of consistent linkage peaks between methods over the total number of linkage peaks detected by any of the three methods.Several measures were defined in order to compare the three pre-processing methods: 1) the number of linkage signals, 2) the number of traits with at least one linkage signal as well as the number of linkage signals per trait, and 3) the location of the loci controlling gene expression with respect to the position of the gene itself , concordance rates between the three methods were 44% (4/9) for the group of 350 traits chosen at random, 100% (1/1) in the group of non-expressed genes, 73% (11/15) in the group of the most heritable genes, and 60% (3/5) in the group of the most variable traits. Further, concordance rates between GCRMA and RMA were especially high and always greater than 75%. On the other hand, in the remaining set of signals (trans-regulators), much lower concordance rates were obtained .The number of cis-acting than for trans-acting regulators.As previously suggested by other studies ,8,10, ouSeveral factors may explain partly why the three methods produce different results. First, as already stated in the Background, the underlying models converting probe level data to expression values are different from one method to another. Although the normalization step is not the same for the three methods, previous work has shown that these differences have little effect relative to that of the background correction, which entails a variance/bias trade-off. Especially, it has been shown that background correction decreases the bias but that na\u00efve background correction procedures, such as MAS5 and RMA, increase the variance . GCRMA iAnother potential factor for explaining differences between methods is departure from normality of the phenotypic distribution, especially when using a variance-component approach. We found that, in general, GCRMA led to the highest rate of traits failing the Shapiro normality test. Among expressed genes, these rates were 5.4, 0.8, and 1.7% for GCRMA, RMA, and MAS5, respectively (using a Bonferroni correction for multiple testing at level 5%). These rates might explain the large number of linkage signals observed for a few traits with GCRMA. However, it seems unlikely that conflicting eQTL mapping results are mainly due to differences in the gene expression distributions per se.In conclusion, the true genetic determinants of the studied traits in the GAW Problem 1 data are unknown, preventing us from drawing definite conclusions on the best and more robust pre-processing method. Further, in the context of a genome-scan, high agreement rates across experiments are not expected because most of the linkage signals are likely to be false positives. It is unclear whether it would be sound to use several pre-processing methods in a systematic manner. Such guidelines were proposed recently but remain controversial -12. In oThe author(s) declare that they have no competing interests."}
+{"text": "Nature 2004, 430:743\u2013747) detected significant linkages to the expression levels of 142 genes (of 3554) at a reported threshold of genome-wide p = 0.001 (LOD \u2248 5.3), using 14 three-generation Centre d'Etude du Polymorphisme Humain pedigrees. Most of the linkages (77%) were trans, i.e., more than 5 Mb from the expressed gene. However, the analysis did not account for the expected anti-conservative effect of the skewed distribution of score- or regression-based statistics in large sibships, or for the possible variance distortion due to correlations among tests. Therefore, we re-analyzed their data, using a robust score statistic for the entire pedigrees and correcting the p-values for skewness. We found that a LOD of 5.3 had a skewness-corrected genome-wide p-value of 0.016 instead of 0.001 (a result that we confirmed using simulation), with around 50 expected false positives. We then further corrected for correlation among the (skew-corrected) p-values by using Efron's method for obtaining the empirical null distribution. Setting a threshold of FDR = 10% , we detected linkage for the expression levels of 22 genes, 19 of which are cis. Limiting the analysis to cis regions, linkage was detected to the expression levels of 46 genes with 4.6 expected false positives (FDR = 10%).Morley et al. ( However, Tang and Siegmund ij = \u03bdij(t). Define \u03a3 to be the phenotypic covariance matrix. Assuming no dominant genetic effect, then according to Tang and Siegmund 1/2. Here,From the working assumption that at a trait locus ect at \u03c4  in the fl\u03b1(t) are replaced by their conditional expectation given the genotypic data, while their variances are estimated from multipoint genotypic data. To make the test robust to the normality assumption of the traits, we use Z(\u03c4) = l\u03b1(\u03c4)/[E0(\u03c4) | Y)]1/2.In practice, unobserved values of the IBDs in \u03c11 for grandparent-grandchild and \u03c12 for sibs were estimated by maximum-likelihood estimation (MLE) with the genetically natural constraint \u03c11 \u2264 \u03c12/2. The sex-average genetic map provided to Genetic Analysis Workshop 15 by Sung et al.  for x >"}
+{"text": "Am J Hum Genet 2005, 76:934\u2013949; Am J Hum Genet 2006, 78:778\u2013792]. Analyses were conducted on the simulated rheumatoid arthritis (RA) data in Genetic Analysis Workshop 15 (GAW15), using single-nucleotide polymorphisms (SNPs) on chromosome 6 over the 100 simulated replicates. We found that the LOD score for testing association in the presence of linkage dramatically increased when unrelated controls were added to affected sib pairs (ASPs), and that choosing a sufficient number of flanking markers is critical in order to distinguish between perfect linkage disequilibrium  and incomplete linkage disequilibrium .This study evaluated the utility of unrelated controls and flanking markers when performing joint modeling of linkage and association by the LAMP software (version 0.0.6) [ D'| and r2 [D'| = 1 when the deviation of a haplotype frequency from randomly associated alleles attains its maximum value, given the marginal allele frequencies; r2 = 1 when two single-nucleotide polymorphisms (SNPs) are perfectly correlated, sometimes called \"perfect LD\". This can arise when two SNPs arose on the same branch of the genealogy and remain undisrupted by recombination. In contrast, r2 can have a value less than 1 when SNPs arose on different branches, or if an initially strong correlation has been disrupted by crossing over [r2 = 1) and complete LD (|D'| = 1).The DRB1 alleles located in the HLA region of chromosome 6 have been found to affect susceptibility to rheumatoid arthritis (RA), and linkage at HLA was confirmed by several studies . However| and r2 ,6: |D'| ing over . Here weTo identify genes perfectly associated with disease, Li et al.  proposedTo study the power of association tests in a linked region with different study designs, we used the SNP data on chromosome 6 for all 100 simulated replicates, and evaluated designs that either use only ASPs or combine ASPs with controls. All analyses were conducted with the software LAMP (version 0.0.6). We studied the simulated RA data with answers, to compare the LOD scores provided by LAMP under the two designs.Using RA affection status as a binary trait, there were 1500 families with one ASP and their parents, all genotyped on 674 SNPs along chromosome 6. Additionally, 2000 unrelated controls were included in each replicate. There were no missing data or genotype errors. For the LAMP analyses, we specified the lifetime prevalence of RA to be 0.0107 as stated in the introduction of the simulated RA data. The sex-averaged map locations  were used for the maps.L(\u03b8 = r2 = 0), where \u03b8 is the recombination fraction and r2 is the measure of LD. LAMP estimates marker allele frequencies with the assumption of Hardy-Weinberg equilibrium, and there is only one fitted parameter for each SNP; 2) a linkage equilibrium model (LE) for linkage without association, L. LAMP estimates disease and marker allele frequencies, as well as the penetrances for disease genotypes. The estimation of these parameters is constrained by the assumed disease prevalence; 3) a general model (GM) for linkage with any level of association, L. This most general model estimates three marker-disease haplotype frequencies and the penetrances for disease genotypes; 4) a linkage disequilibrium model (LD) for linkage with complete association, L. For this model, one of the marker alleles is assumed to directly affect disease susceptibility. The marker allele frequency and the penetrances for disease genotypes are estimated. The four models are summarized in Table The software LAMP was used to fit four models by maximum likelihood: 1) a base model (BM) for no linkage and no association, r2 threshold to 0.4 in Merlin  [The four models were used to create three likelihood ratio tests, and hence LOD scores: 1) a test for linkage, 2) a test for association in the presence of linkage, and 3) a test for other linked variants. In contrast to traditional parametric linkage analyses that specify a genetic disease model, LAMP calculates parametric maximized LOD scores by maximizing over all assumed model parameters. The method allows LD between the measured candidate SNP and an unobserved disease allele, but assumes linkage equilibrium between the flanking markers and the measured candidate SNP . Note th-cfreq\") ,11. To aWe considered two study designs: 1500 ASPs alone (ASPs), and 1500 ASPs with unrelated 2000 controls (ASPs+controls). The combined data should be more powerful to detect association, but not linkage. Our aims were to evaluate the increase in LOD scores provided by controls for the test for association (in the presence of linkage), and to evaluate the benefit of flanking markers for the test for other linked variants.r2 = 1) with the causal locus.Results from the simulated replicates were very consistent, as summarized in Table In addition to the impact of unrelated controls, flanking markers can have a large impact on LOD scores. Flanking markers smooth the LOD scores of the test for linkage, as we can see from the first column of Figure The joint modeling of linkage and association proposed by Li et al.  uses genIn our analyses, we used sex-averaged maps without considering the sex differences in genetic map distances. The results of testing for linkage, testing for association in the presence of linkage, and testing for other linked variants were consistent with the simulation answers. However, ignoring sex differences in genetic maps may not lead to accurate inferences when genotypes for only one parent of each ASP are available . A drawbThe author(s) declare that they have no competing interests."}
+{"text": "Maintenance of high muscular fitness is positively related to bone health, functionality in daily life and increasing insulin sensitivity, and negatively related to falls and fractures, morbidity and mortality. Heritability of muscle strength phenotypes ranges between 31% and 95%, but little is known about the identity of the genes underlying this complex trait. As a first attempt, this genome-wide linkage study aimed to identify chromosomal regions linked to muscle and bone cross-sectional area, isometric knee flexion and extension torque, and torque\u2013length relationship for knee flexors and extensors.In total, 283 informative male siblings (17\u201336 years old), belonging to 105 families, were used to conduct a genome-wide SNP-based multipoint linkage analysis.\u22125). Suggestive evidence for linkage was found at 14q32.2  for muscle and bone cross-sectional area, at 2p24.2  for isometric knee torque at 30\u00b0 flexion, at 1q21.3, 2p23.3 and 18q11.2  for the torque\u2013length relationship of the knee extensors and at 18p11.31  for muscle-mass adjusted isometric knee extension torque.The strongest evidence for linkage was found for the torque\u2013length relationship of the knee flexors at 14q24.3 (LOD \u200a=\u200a4.09; p<10We conclude that many small contributing genes rather than a few important genes are involved in causing variation in different underlying phenotypes of muscle strength. Furthermore, some overlap in promising genomic regions were identified among different strength phenotypes. From a general health perspective, muscular fitness is associated with independently performing activities of daily living.isom), concentric (Fconc), and eccentric (Fecc) muscle strength are under moderate to high genetic control, with heritability rates of 60\u201395% for MBA, 44\u201378% for Fisom, 31\u201361% for Fconc, and 65\u201377% for Fecc.8\u2013et al.Several studies suggest that muscle and bone cross-sectional area (MBA) and isometric , p21 , MyoD  and retinoblastoma  are potentially interesting regions for further genetic studies . Strengthened by these preliminary single-point linkage results and the physiological evidence for the entire myostatin pathway, a multipoint linkage analysis was performed on a larger and partially independent sampleMYF5; 12q21.31) and Myf6 , insulin-like growth factor-1 , cyclin-dependent kinase-2 , and titin .CDK2 , RB1  and IGF1 .The human gene map for performance and health-related fitness phenotypesTo date, only 5% of the total genome has been scanned for linkage with muscle strength characteristics. We therefore performed a genome-wide linkage scan using 6008 SNP markers, aiming to identify genomic regions harbouring candidate genes that cause variation in MBA, isometric torque and the torque\u2013length relationship of the knee flexors and extensors.We identified several promising chromosomal regions harbouring a set of candidate genes, with some overlapping regions for different strength characteristics, suggesting pleiotropic gene action.The procedures used in this study were approved by the medical and ethical committee of the Katholieke Universiteit Leuven. Before participation, the purpose and procedures of the study were explained in detail and the subjects gave their written informed consent.From the total sample of the LGfMS, 283 male siblings aged 17\u201336 years in 105 families were selected; this group was composed of 13 quads, 47 trios and 45 pairs of brothers, resulting in 309 pairwise comparisons. The sibling pairs were selected based on their level of discordance for different strength phenotypes. The recruitment procedures and subject characteristics have been described in detail previously.18et al.18A detailed overview of the anthropometrical and muscle strength measurements in the LGfMS project can be found in Huygens MBA of the mid thigh was estimated based on the circumference of the mid thigh corrected for skinfold thickness at the mid thigh:2)/(4\u00d7\u03c0).MBA \u200a=\u200a (circumference mid-thigh\u2212(skinfold thickness mid thigh\u00d7\u03c0/10)Measurements were taken by experienced anthropometrists and are described in more detail elsewhere.19The Cybex NORM isokinetic dynamometer  was used to assess the maximum isometric and torque\u2013length knee strength characteristics. After a 5\u201310 minute warm-up period on an ergometer cycle and light stretching exercises, subjects were positioned on the dynamometer, following the manufacturer\u2019s instructions. Anatomical zero was set at full extension of the knee and the rotation axis of the joint was aligned with the mechanical axis of the dynamometer. Two isometric and four concentric submaximal trials preceded the actual tests, to allow familiarisation with the testing procedure . Maximal isometric knee strength was measured at two angles (30\u00b0 and 60\u00b0). At each angle, highest torque values (Nm) during a 6-second isometric contraction of three maximum flexion and extension contractions were retained for further analysis. Between each contraction, subjects rested for 30 seconds. In accordance with the torque\u2013length relationship of a muscle, optimum strength is generated at longer muscle length\u2014that is, at an angle of 60\u00b0 for knee extension (quadriceps) and 30\u00b0 for knee flexion (hamstrings). The torque\u2013length relationship for knee flexion and extension was quantified by calculating the ratio of torque at the joint angle in which the lowest mean isometric force is produced  over torque at the joint angle with the highest mean isometric force output , multiplied by 100:(isometric flexion torque at 60\u00b0/isometric torque at 30\u00b0)\u00d7100.The Baecke Physical Activity QuestionnaireDNA was extracted using the Chemagic DNA blood kit on an automated Chemagic Magnetic Separation Module I  and a Multiprobe I  robotic station. An Oragene saliva kit   was provided to siblings who were not able to deliver a blood sample. DNA from these saliva kits was extracted following the guidelines of the manufacturer.The Illumina SNP-based Linkage Panel IVb was used for genotyping. The panel includes 6008 SNP markers distributed evenly across the genome. The average and median intervals between markers were 482 kb (0.64 cM) and 298 kb (0.35 cM), respectively. The largest interval between successfully genotyped markers was 5.02 cM on chromosome 8. The Illumina markers were typed with the Illumina Beadstation 500GX, in accordance with the manufacturer\u2019s standard recommendations. The genotype success rate was 99.6% . Only autosomal SNPs were included for further analyses.2) were estimated using the variance-components (VC) analysis procedure in QTDT.Upper-limit heritability of the traits  outlined by Sham \u2013Simulated data for 100 genome scans were generated using MERLIN26\u2013It is now a widely accepted practice to obtain empirical p values for reported LOD scores.29et alTests for joint linkage and association in regions with suggestive and significant evidence for linkage were carried out using a quantitative transmission disequilibrium test (QTDT) with MBA as a covariate.SNPs in a \u22121 LOD region around the SNP showing significant evidence for linkage and SNPs in overlapping chromosomal regions between phenotypes were tested for association with the MERLIN likelihood-ratio test.2) estimated on the total LGfMS sample. Subjects had normal weight and height; the mean (SD) body mass index (BMI) of 22.9 (2.9) kg/m2 indicates that they were, on average, rather lean.2 estimates of 90% and 85%, respectively. In this sample, isometric torque at 30\u00b0 flexion and 60\u00b0 extension were under strong genetic control , whereas genetic control of the torque\u2013length relationships for flexion and extension was only moderate . At most, only 50% of variability in the Sport Index could be accounted for by genetic factors.As expected, the upper-limit heritability estimate of stature was high (92%), and genetic determination of body mass and BMI were somewhat lower . Fat-free mass and MBA mid thigh showed hCorrelations between the different strength phenotypes ranged from \u22120.26 to 0.72 . Moderat\u22125. Suggestive evidence for linkage (LOD between 2.2 and 3.3) was found for MBA mid thigh on 14q32.2 , isometric knee flexion at 30\u00b0  and the torque\u2013length relationship of the knee extensors . No suggestive evidence for linkage was found for isometric knee extension at 60\u00b0. Overlapping or closely neighbouring chromosomal regions of interest were observed for torque\u2013length flexion and extension (10q26 and 12q24), and for isometric torque flexion and torque\u2013length extension (2p23-24) .39DIO1 (1p32p33), GDF8 (2q32.2), MYLK (3q21), NR3C1 (5q31), TNF (6p21.3), CFTR (7q31.2), CNTFR (9p13), IGF2 (11p15.5), CNTF (11q12.2), ACTN3 (11q13q14), VDR (12q13.11), IGF1 (12q22q23), COL1A1 (17q21.33) and ACE (17q23.3). Some of these genes are located in the vicinity of linkage regions reported in the present study: region 6p25.2 near to TNF (6p21.3), linked to torque\u2013length for the knee flexors; regions 12q24.32 and 12q24.31 close to IGF1 (12q22q23), linked with torque\u2013length for the knee flexors and extensors, respectively; and region 17q25.3 near to COL1A1 (17q21.33) and ACE (17q23.3), linked to torque\u2013length for the knee extensors.Several candidate gene association studies for muscles strength phenotypesHIF1A), which has been reported to be associated with maximum oxygen consumption before and after exercise training,et al;TRIM54) gene (2p23.3) is a member of the muscle ring finger proteins, which are identified as myogenic regulators of the microtubule network of striated muscle cells and reveal a link between microtubule organisation and myogenesis.ADAM12) gene (10q26.3) has high expression in muscle development and regeneration, but its specific role in skeletal muscle is not yet understood.46\u2013AKT1, located at 14q32.32 (close to the suggestive linkage region for MBA at 14q32.2), codes for the Akt-1 protein, which has previously been associated with hypertrophy, and also has a key role in atrophy through the phosphorylation of a transcription factor, Foxo.  Further fine mapping analysis will be required to assess the potential contribution of each of these candidate genes to explain muscle strength variation.Apart from these \u201cusual suspects\u201d, other candidate genes can also be found in or near to the regions identified in our autosomal linkage analysis. For example, the hypoxia-inducible factor 1\u03b1 gene . Several candidate genes that might be relevant from a physiological point of view are located within the identified regions, and warrant further fine-mapping analyses by additional linkage and/or association studies. Although we selected the largest families from the LGfMS project, the relatively small sample size limits the possibility to detect chromosomal regions where genes with small effect sizes are harboured. Fine-mapping and replication studies of the current findings should therefore focus on highly informative samples (families) of sufficiently large size, and preferably extend to females and other age groups. The chromosomal regions identified within this healthy population might have implications beyond the field of muscular fitness and be directive in muscular disease research.The National Center for Biotechnology Information (NCBI) Entrez database accession numbers for the genes discussed in this paper are:ACE), 1277Angiotensin 1 Converting Enzyme (ACTN3), 89Actinin 3 (ADAM12), 8038A disintegrin and metallopeptidase domain 12 (AKT1), 207Protein kinase B (CDK2), 1017Cyclin-dependent kinase 2 (CFTR), 1080Cystic fibrosis transmembrane conductance regulator (CNTF), 1270Ciliary neurotrophic factor (CNTFR), 1271Ciliary neurotrophic factor receptor (COL1A1), 403651Collagen type 1 alpha-1 (DIO1), 1733Deiodinase iodothyronine type I (GDF8), 2660Myostatin , 3091IGF1), 3479Insulin-like growth factor 1 (IGF2), 3481Insulin-like growth factor 2 (MYLK), 4638Myosin light chain kinase (NR3C1), 2908Nuclear Receptor Subfamily 3 Group C Member 1 (RB1), 5925Retinoblastoma (TNF), 7124Tumour necrosis factor (VDR), 7421Vitamin D receptor ("}
+{"text": "Otitis media (OM) is a common worldwide pediatric health care problem that is known to be influenced by genetics. The objective of our study was to use linkage analysis to map possible OM susceptibility genes.Using a stringent diagnostic model in which only those who underwent tympanostomy tube insertion at least once for recurrent/persistent OM are considered affected, we have carried out a genome-wide linkage scan using the 10K Affymetrix SNP panel. We genotyped 403 Caucasian families containing 1,431 genotyped individuals and 377 genotyped affected sib pairs, and 26 African American families containing 75 genotyped individuals and 27 genotyped affected sib pairs. After careful quality control, non-parametric linkage analysis was carried out using 8,802 SNPs.SFTPA2 (10q22.3), 48 kb from IFNG (12q14), and 870 kb from TNF (6p21.3).In the Caucasian-only data set, our most significant linkage peak is on chromosome 17q12 at rs226088 with a p-value of 0.00007. Other peaks of potential interest are on 10q22.3 (0.00181 at rs1878001), 7q33 (0.00105 at rs958408), 6p25.1 (0.00261 at rs554653), and 4p15.2 (0.00301 at rs2133507). In the combined Caucasian and African American dataset, the 10q22.3 peak becomes more significant, with a minimal p-value of 0.00026 at rs719871. Family-based association testing reveals signals near previously implicated genes: 513 kb from AP2B1, CCL5, and a cluster of other CCL genes, and in 10q22.3, SFTPA2.Our scan does not provide evidence for linkage in the previously reported regions of 10q26.3 and 19q13.43. Our best-supported linkage regions may contain susceptibility genes that influence the risk for recurrent/persistent OM. Plausible candidates in 17q12 include  Otitis media (OM MIM 166760]) is a worldwide pediatric health care problem. Nearly all children experience at least one episode of acute OM (AOM). Otitis media, particularly in children who experience recurrent/persistent disease, in addition to the short- and long-term effect on the child places considerable financial burden on the families  and on the health-care system . In 1996, Gates [66760) isEpidemiologic studies using various methodologies have demonstrated evidence that genetics plays an important role in recurrent and persistent OM: population studies -10; adopWhile there is substantial evidence that genetics plays a role in OM, there has been only one genome-wide linkage scan to date by Daly et al  that proOtitis media is a complex disease with contributions from immunocompetence, inflammatory regulation and craniofacial abnormalities that could lead to Eustachian tube dysfunction; therefore, the underlying genetic determinants are likely to be complex and involve several loci. Ilia et al  in a recOur present study aims to localize genes that contribute to susceptibility to recurrent/persistent middle ear disease by carrying out a genome-wide screen for linkage using families containing at least two affected siblings. The identification of susceptibility genes may enable us to better understand the pathogenesis of OM leading to development of better and more innovative methods for prevention and treatment, which is especially important in this era of multi-drug resistant bacteria.Full siblings, two or more, who had a history of tympanostomy tube insertion due to a significant history of OM, their parent(s) and other full sibling(s) with no history of tympanostomy tube insertion were eligible for the study. There was no exclusion of subjects based on gender or race. The study was explained in detail to the parent(s) and children, and informed consent was obtained prior to enrollment. This study was approved by the Human Rights Committee of the Children's Hospital of Pittsburgh (CHP) and by the University of Pittsburgh Institutional Review Board.The study required only one visit to the Ear, Nose and Throat (ENT) Research Center at CHP. A detailed history regarding recurrent/persistent OM was obtained for each enrolled family member, as well as history regarding risk factors such as breast-feeding, day care attendance, siblings, and exposure to smoking for the enrolled children. Medical records of enrolled children were obtained for review whenever possible. When feasible, children and parent(s) had an ear examination using pneumatic otoscopy by the study physicians (MC and EM) who are validated otoscopists . TympanoIn order to assure a history of significant ear disease, two or more full siblings who both or all had undergone tympanostomy tube insertion were enrolled. While still recording each subject's medical history, the need for tympanostomy tube insertion established that the subject's history of middle ear disease was truly significant, resulting in the need for a surgical procedure. A subject was only considered \"affected\" if he/she had undergone tympanostomy tube insertion at least once for recurrent/persistent OM, while a subject was considered \"unaffected\" if he/she had never had tympanostomy tubes and had no known history of recurrent/persistent OM. The remaining subjects were considered as having \"unknown\" disease status.EDTA anticoagulated whole blood  was collected from an arm vein in the Pediatric General Clinical Research Center or in the ENT Research Center or in satellite clinics and transferred to the Human Genetics Laboratory within 48 hours. High molecular weight DNA was isolated by the method of Miller et al . Followi\u00ae Human Mapping 10K Array in two different versions: 1) the original GeneChip\u00ae Human Mapping 10K Array Xba 131 with 11,560 SNPs with a mean intermarker distance of 210 kb and an average heterozygosity of 0.37, and 2) the newer GeneChip\u00ae Human Mapping 10K Array Xba 142 2.0 with 10,204 SNPs with a mean intermarker distance of 258 kb and an average heterozygosity of 0.38. These two chips are very similar to each other: the newer SNP chip was created by removing 1,400 SNPs from the original SNP list and adding approximately 70 new SNPs. Samples were processed according to the GeneChip Mapping Assay Manual (Affymetrix) hybridized at 48\u00b0C for 16 hours in an Affymetrix GeneChip Hybridization Oven. After 16 hours the probe arrays were washed and stained according to the GeneChip Mapping Assay Manual using the DNAARRAY_WS2 protocol on the Affymetrix Fluidics Station 400. Arrays were scanned once with the Agilent GeneArray 2500 scanner and analyzed with Affymetrix GeneChip DNA Analysis Software (GDAS) to generate genotype calls for each of the SNP probes on the array. In this study we used the older Xba 131 SNP chip to genotype 515 people, and the newer Xba 142 2.0 SNP chip to genotype 1,216 people.Genotyping was carried out using Affymetrix's GeneChipStarting with the available annotation from Affymetrix, we carefully updated it, as a fair number of SNPs are actually bad, hybridizing to multiple positions in the genome. To detect these, we took the primer sequence for each SNP and used BLAST to find its position(s) in the genome, carefully identifying poor multi-hit SNPs, as well as verifying the current best physical position for each good SNP in the data set. After careful resolution of reference sequence (rs) numbers, current physical map positions were retrieved from Ensembl and used to linearly interpolate genetic map positions based on the Rutgers Combined Linkage-Physical Map .To verify the stated pedigree structures, we carried out relationship testing using a subset of 1,534 well-typed informative autosomal SNPs (with MAF > = 0.40 and > = 99% genotyping rate) and then made a number of changes to the structures to minimize the number of relationship errors while keeping as many people as possible.PREST identified 73 within-family pairs with p-values < 0.001 ,27. SimiGender checking based on PLINK's algorithm led to the removal of genotypes for 6 people because of unresolved gender errors . We replA number of conservative changes were made in response to the relationship testing results, including the following: To resolve the 10 putative monozygotic (MZ) twin pairs identified, from the larger families, we removed the least typed member of an MZ pair 6 times, and we completely removed 2 families that each contained only a single inferred MZ pair. To resolve putative half-sib (HS) families, we removed 6 families containing only a single inferred HS pair, and from larger families, we removed a single person to eliminate the putative HS pair 4 times. To resolve the two families that had participated twice in our study, in both cases, only the more completely typed family was kept.After resolving the relationship errors, we then removed all genotypes for the 250 individuals with a per person genotyping success rate less than 90%.Identity-by-state cluster analysis based on PLINK's hierarchical clustering algorithm was used to verify that the self-reported ethnicities were consistent with the clusters generated from the SNP marker data.Consistent with the ethnicity distribution in our sampling area, the majority of our families, 403, were Caucasian, while there were only 26 African American families. For the purposes of analyses, the larger Caucasian data set was first analyzed by itself. Here, 147 SNPs were excluded due to a HWE p-value < = 0.001 (computed by PLINK using only founder genotypes); 1,936 SNPs were excluded because their genotyping success rate across all individuals was less than 90%; 999 SNPs were excluded due to an MAF < 0.05; leaving 8,802 SNPs that were used in the linkage analyses.For the initial analyses using Merlin , allele all statistic using sex-averaged genetic maps and allele-counting allele frequency estimates; Minx was used to analyze the X-linked data. Summary statistics were computed using Pedstats [We used PedCheck  to detecPedstats . File-foPedstats .To assay the effects of SNP-SNP linkage disequilibrium, we recomputed the LOD scores using Merlin's LD modeling (--rsq 0.1) option . To examAfter the initial analyses using the Caucasian-only data set, we carried out additional linkage analyses of the combined Caucasian and African American data set. For these analyses, we used Mendel's options for estimating and using ethnic-group specific allele frequencies. We did not analyze the African American families by themselves, as the sample size was too small by itself to have adequate power: only 26 African American families containing 27 genotyped affected sib pairs.We used the GRAIL \"Gene Relationships Across Implicated Loci\" software to search our linkage peaks for possible candidate genes based on similarity to a set of previously implicated OM candidate genes . SimilarUsing the Caucasian-only data set, we tested for association of 8,585 autosomal SNPs  using the gamete competition statistic implemented in Mendel . This stall LOD scores > 2.0 with a maximum LOD of (a) 2.83 on chromosome 17q12 at rs226088 and (b) 2.25 on 6p25.1 at rs554653  . Initial linkage analyses with Merlin found two regions with linkage peaks with linear SMendel-based linkage results, which use better allele-frequency estimates, sex-specific maps, and empirical P-values, are summarized in Table When we carried out analyses of the combined Caucasian and African American sample, using group-specific allele frequency estimates, three of the peaks  became a bit less significant, while the chromosome 7 peak significance level stayed essentially the same and the chromosome 10 peak became more significant, with a minimal p-value of 0.00026 at rs719871 . For the chromosome 17 linkage peak, the most significant candidate genes with p-values < 10-13 are CCL5, CCL18, CCL3, and CCL4. For the chromosome 10 peak, the most significant candidate genes with p-values < 10-13 are SFTPA1B, SFTPA2, SFTPD, and PLAU. For the chromosome 7 peak, the most significant candidate gene EPHB6 had a p-value of 2.6 \u00d7 10-6. For the chromosome 6 peak, the most significant candidate genes with p-values < 10-11 are LY86, SERPINB9, and F13A1. For the chromosome 4 peak, the most significant candidate gene with p-values < 10-11 was SOD3.We used the GRAIL \"Gene Relationships Across Implicated Loci\" software to search our linkage peaks for possible candidate genes based on similarity to a 'seed' set of previously implicated OM candidate genes . To do tSFTPA2 on chromosome 10. Two SNPs  are 48 kb from IFNG on chromosome 12. Two SNPs  are 870 kb from TNF on chromosome 6. Note that the gamete competition test should detect association signals at much larger distances than a regular case/control association test in unrelateds because the gamete competition test relies on measuring transmission distortion within families.Using the Caucasian-only data set, we tested for association of 8,585 autosomal SNPs  using the gamete competition statistic implemented in Mendel . The mosWhile there is fairly strong evidence that otitis media has a strong genetic component, to our knowledge there has been only one prior family-based linkage study of otitis media. This first study provided evidence of linkage of COME/ROM to chromosome 10q26.3 at marker D10S212 and to chromosome 19q13.43 at marker D19S254 . We repoThe rate of tympanostomy tube insertion in second siblings may be increased due to the parents' wishes to have the procedure performed earlier or for less significant disease than the first child because of prior positive experiences in the first sibling. This could potentially make it difficult to separate the genetic effect of OM from the parents' influence on the treatment. However, we have reason to believe such an effect, if it exists, will be small in our sample. Firstly, the majority of our sample was treated at the Children's Hospital of Pittsburgh, where our criteria for insertion for tubes are consistent and very stringent. Secondly, at entry we obtained, for each subject, a history of middle-ear disease prior to tube insertion to assess the subject's eligibility.Since the majority of our families were Caucasian, we present two linkage analyses, one where we analyzed only the 403 Caucasian families by themselves, and a combined analysis where we jointly analyzed both the 403 Caucasian and 26 African American families Table . The CauAP2B1 , which plays a role in Nef-mediated CD8 down-regulation [CCL (chemokine C-C motif ligand) genes, several of which were highlighted as possible candidates by the GRAIL analyses. CCL5, also known as RANTES, is 18 kb from the linkage peak, and has been previously associated with otitis media [CCL5 is an eosinophil chemoattactrant that is thought to play a role in the accumulation of eosinophils often observed in middle ear effusions of OM with allergy.The very tip of our 17q12 linkage peak occurs in gulation , and chigulation . Howeveris media -52. CCL5SFTPA2. This peak also contains a strongly associated SNP rs1437803  is expressed in the Eustachian tube, plays a role in innate host defense, upregulates phagocytosis of many OM risk pathogens , and consists of two very similar functional genes SFTPA1 and SFTPA2 located 5 kb apart on chromosome 10. Ramet et al [The combined 10q22.3 linkage peak is 1.2 Mb from a previously implicated candidate gene et et al  reportedet et al  also fouIn the remaining linkage peaks, the majority of the genes highlighted by GRAIL as possible candidates do not have prior evidence of involvement with OM.SFTPA2 mentioned above, it is noteworthy that we have associated SNPs 48 kb from interferon-\u03b3 (IFNG) on chromosome 12. Genetic variants at IFNG are associated with risk for otitis media in infants infected with the respiratory syncytial virus (RSV) [TNF) on chromosome 6. A TNF -308 polymorphism was associated with both otitis media susceptibility and placement of tympanostomy tubes [TNF were associated with being otitis-prone [While none of the association results  . When RSus (RSV) . We alsomy tubes . Variantis-prone .The gamete competition test of association used here relies on transmission distortion within families, and so should detect association signals at larger distances than the conventional case/control association test in unrelateds. Even so, the proximity of some association signals to previously implicated candidate genes mentioned above may not be that meaningful, as these regions contain many other plausible candidate genes.SFTPA2, IFNG, and TNF). Our hope is that our genetic results will help contribute to an enhanced understanding of the etiology of otitis media that ultimately may help lead to improved treatment and prevention of this extremely common childhood disease.Our linkage scan, the largest to date, has identified two strong linkage peaks, on 17q12 and 10q22.3 that are likely to contain genes which influence risk for severe OM. While both of these peaks contain intriguing and plausible candidate genes, further fine-mapping, replication, and functional studies are required to reach more firm conclusions. We also find association signals near previously implicated risk genes , WGA Viewer, GRAIL, The authors declare that they have no competing interests.MLC, EMM, REF, and DEW conceived of the study and obtained funding; KT, EMM, and MLC carried out the clinical work; REF supervised the genotyping; JPS, JJ, AR, and DEW carried out the statistical analyses; DEW, MLC, REF, and EMM wrote the initial draft of the manuscript; all authors read and approved the manuscript.Current affiliations: JJ: Department of Medical and Molecular Genetics, Center for Computational Biology and Bioinformatics, Indiana University, School of Medicine, Indianapolis, IN, 46202, USA; JPS: Eating Disorders Program, Department of Psychiatry, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA; AR: Statistics, reporting, and analysis (Cardiac Rhythm Management division), St. Jude Medical, Sunnyvale, CA, 94086, USA.The pre-publication history for this paper can be accessed here:Supplemental Figure S1. This file provides multipoint linkage curves as computed by Merlin with and without linkage disequilibrium.Click here for fileSupplemental Table S1. This file provides the gamete competition results in the Caucasian families for those SNPs with p-values < 0.01.Click here for file"}
+{"text": "High blood pressure or hypertension is a major risk factor involved in the development of cardiovascular diseases. We conducted genome-wide variance component linkage analyses to search for loci influencing five blood pressure related traits including the quantitative traits systolic blood pressure (SBP), diastolic blood pressure (DBP) and pulse pressure (PP), the dichotomous trait hypertension (HT) and the bivariate quantitative trait SBP-DBP in families residing in American Samoa and Samoa, as well as in the combined sample from the two polities. We adjusted the traits for a number of environmental covariates such as smoking, alcohol consumption, physical activity and material life style.We found suggestive univariate linkage for SBP on chromosome 2q35-q37 (LOD 2.4) and for PP on chromosome 22q13 (LOD 2.2), two chromosomal regions that recently have been associated with SBP and PP, respectively.We have detected additional evidence for a recently reported locus associated with SBP on chromosome 2q and a susceptibility locus for PP on chromosome 22q. However, differences observed between the results from our three partly overlapping genetically homogenous study samples from the Samoan islands suggest that additional studies should be performed in order to verify these results. High blood pressure or hypertension is a major risk factor involved in the development of cardiovascular diseases (CVD) , which tThe population on the Samoan islands, including both American Samoa and Samoa, has a common evolutionary history of approximately 3,000 years with homogeneity of allele frequencies and linkage disequilibrium (LD) structure . HoweverBlood pressure levels and hypertension rates increase with adult age, are greater in more modernized American Samoa than in Samoa, have increased in the last 30 years, and are strongly related to adiposity measures and psychosocial stress ,10-12. HDuring the last decades many genome-wide linkage investigations have been conducted for qualitative and quantitative blood pressure related traits  but the In the present study we performed genome-wide variance-component linkage analyses, as implemented in LOKI  and SOLAThe study samples from the population on the Samoan islands have been extensively described elsewhere ,3,5. FamSystolic (SBP) and diastolic blood pressures (DBP) were measured using mercury sphygmomanometers and the auscultatory method following American Heart Association (AHA) standard protocols. The research staff was trained using AHA audiotapes. Before the measurements were made, participants refrained from cigarette smoking for 30 minutes and emptied the bladder. Participants were seated in a quiet undisturbed place. Blood pressure was measured three times for each participating individual and the average of all measurements was used in the statistical analysis.To include the individuals who currently were treated for hypertension, we followed the example of Hottenga et al , that prQuestionnaires were used to collect self-reported information on environmental factors including education (years), moderate to heavy physical activity (hours/week), alcohol consumption (yes/no), smoking (yes/no) and material life standard. As described in our previous study  the infoGenotyping was performed as previously described ,4 using Since falsely assuming normality may lead to excessive type I errors and to a biased estimate of the major gene effect when using a variance component analysis  we appliAs described previously -4 we extWe have used the \"set correct_errors 1\" option in LOKI  to remov2, age2 *sex, body mass index (BMI), cigarette smoking, alcohol consumption, education, physical activity and material life style index (MLSI). To further control for any unmeasured differences in the environment between American Samoa and Samoa that might influence the traits in the combined data set, we also screened for significance of polity of residence.To carefully take advantage of the environmental information collected from the study samples we used the \"polygenic screening\" command in SOLAR to investigate the significance of the covariate for each of the studied traits. Covariates with p-values \u2264 0.1 were considered as significant and only the covariates that were significant in all three study samples were included in the polygenic model. All traits from all study samples were screened for inclusion of age, sex, age*sex, ageIn this investigation of three study samples from the Samoan islands we have applied the same statistical strategy for univariate and bivariate genome-wide linkage analysis as we previously used when investigating serum lipid levels in these study samples . As doneWe used the multipoint variance component linkage analysis as implemented in SOLAR ,19 to seSince the current version of SOLAR does not correctly carry out X-linked variance component analysis we used Mendel v7.0.0  to analyi.e. the residual) were used as the traits in the bivariate linkage investigation. The residuals appeared to be normally distributed and no further transformation was performed. We used the \"loddf\" command in SOLAR to transform the bivariate LOD score to 1 degree of freedom , which is comparable to univariate LOD score.We performed genome-wide bivariate multipoint linkage analysis using SOLAR to simultaneously test for linkage to SBP and DBP (SBP-DBP). The bivariate analysis tests for linkage of two phenotypes to a single genetic region. Prior to the bivariate linkage analysis, we regressed the covariates that were significant in the univariate analysis onto the traits using a linear model. The difference between the observed and the fitted values  were observed for DBP (h2 = 0.08) in Samoa and for PP (h2 = 0.00) in American Samoa (Table Heritability estimates significantly different from zero (p < 0.05) for the studied traits range from 0.11 to 0.29, where the higher values were observed for SBP and HT (Table The strongest quantitative trait loci (QTL) detected in this study was observed for SBP LOD = 2.41) on chromosome 2q35 in the American Samoan study sample  was detected for any of the traits on the X chromosome in any of the three study samples.In this study, we have searched for susceptibility loci for variation in five blood pressure related traits including the quantitative traits SBP, DBP, PP, the dichotomous trait HT, and the bivariate quantitative trait SBP-DBP in three study samples from the Samoan islands.The phenotypes used for this study were collected by trained personnel in the participant's home environment. By collecting data in an environment familiar for the participant, we attempted to minimize the risk of a \"white coat effect\", where the initial blood pressure measurements are higher than the later measurements due to the stress for the individual of being in an unfamiliar environment. Our data do not indicate any overall trends in the three replicates of the blood pressure measurements and thus we have no reason to believe that a \"white coat effect\" influenced our data sets. Furthermore there is no \"zero bias\" of the measured blood pressures, meaning the last digit of the measures do not end in zero more often than expected, indicating good quality measurements without any excessive rounding.To maximize the power of our study samples we have included all phenotyped individuals regardless of whether they were treated with antihypertensive medication or not. We adjusted for the use of antihypertensive medication by adding a factor of 14 mm Hg to the SBP and 10 mm Hg to the DBP. This is an efficient way to ensure that all available phenotypes are included in the study. However, this strategy assumes that all individuals who reported to be under antihypertensive treatment are actively using their medication and furthermore it assumes that all individuals have an equal response to their medication.The heritability estimates for SBP and DBP detected for our study sample are similar to those previously reported in an earlier Samoan study population . In TokeIn this study we identified three susceptibility loci for blood pressure related traits that reached the level of suggestive linkage. On chromosome 2q35-q37 we detected two closely placed linkage peaks for SBP with LOD score 2.4 in American Samoa and 2.2 in the combined study sample Table . FollowiRecently a number of genome-wide association studies -48 on blThe fact that the linkage signals do not overlap between the two polity-specific studies and further change when we investigate the more powerful combined study sample could be due to many factors. First, despite the homogenous population on the Samoan islands, founded by a limited numbers of individuals and fairly isolated for approximately 3,000 years, the power to detect linkage for complex traits within this study sample is limited. Thus the lack of overlap between the study samples suggests that studies of such complex traits as blood pressure related traits require a further extended study sample to detect significant linkage. Furthermore, although the study samples are from a genetically homogenous population, the environmental exposure is different between the American Samoan and the Samoan study sample as well as within the two polity-specific samples. Substantial variation in environmental influences might cause fluctuations in the investigated traits that are far larger than the effect of the genetic component/s and therefore no highly significant genetic susceptibility can be detected. In an attempt to control for alterations of environmental factors we have screened for inclusion of different environmental covariates including cigarette smoking, alcohol consumption, physical activity, education and material life style index. Somewhat surprisingly, none of the environmental factors showed significant effects on SBP, PP or HT and only cigarette smoking and alcohol consumption were of significant effect for DBP in all three study samples, which may suggest that physical covariates such as age, sex and BMI are of greater importance for blood pressure related traits than social and behavioral environmental factors.The overlap in genomic regions identified between our previous studies on adiposity- and lipid-related phenotypes and our current study is negligible -6. TakenIn this study we have detected additional evidence for a recently reported susceptibility loci for SBP on chromosome 2q and a susceptibility locus for PP on chromosome 22q. However, the differences observed in the results from the three investigated study samples from the Samoan islands suggest additional studies should be performed in order to further verify these results.K\u00c5, mentored by DEW, carried out the statistical analyses of the data and wrote the manuscript. FD, under the supervision of DEW, performed checking and correction of reported pedigrees and wrote the original scripts for data checking and bivariate linkage analysis. SV and JT provided support and advice for the fieldwork in Samoa and American Samoa, respectively. GS and SRI conducted genotyping of all samples. STM, RD, and DEW collaboratively designed and led this study and obtained funding for it. All the authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:Plots of genome-wide LOD scores of blood pressure related traits.Click here for file"}
+{"text": "The power of genome-wide association studies can be improved by incorporating information from previous study findings, for example, results of genome-wide linkage analyses. Weighted false-discovery rate (FDR) control can incorporate genome-wide linkage scan results into the analysis of genome-wide association data by assigning single-nucleotide polymorphism (SNP) specific weights. Stratified FDR control can also be applied by stratifying the SNPs into high and low linkage strata. We applied these two FDR control methods to the data of North American Rheumatoid Arthritis Consortium (NARAC) study and the Framingham Heart Study (FHS), combining both association and linkage analysis results. For the NARAC study, we used linkage results from a previous genome scan of rheumatoid arthritis (RA) phenotype. For the FHS study, we obtained genome-wide linkage scores from the same 550 k SNP data used for the association analyses of three lipids phenotypes . We confirmed some genes previously reported for association with RA and lipid phenotypes. Stratified and weighted FDR methods appear to give improved ranks to some of the replicated SNPs for the RA data, suggesting linkage scan results could provide useful information to improve genome-wide association studies. In particular, genome-wide linkage scans can provide complementary information to genome-wide association studies (GWAS). The weighted false-discovery rate control (WFDR) method incorporates genome-wide linkage study results by converting the linkage scores into SNP-specific weights then re-scaling the association each SNP . The streach SNP ) prioriteach SNP . SFDR iseach SNP . We appleach SNP . We alsoThe NARAC data provided for GAW 16 included a set of 868 cases and 1,194 controls with information on a binary outcome (RA affection status) and on 545,080 genome-wide SNP genotypes from the Illumina 550 k chip as well as the physical locations for the SNPs. In our association analysis, RA affection status was defined as positive for anti-cyclic citrinullated peptide antibody (anti-CCP).N1 = 2,584) and the Generation 3 Cohort  for association with each of the blood lipid measures, high-density lipoprotein (HDL), low-density lipoprotein (LDL), and triglyceride (TG), and included all family members in the sample who had been genotyped and phenotyped. The mean of lipid measures over multiple exams was adjusted for age, sex, body mass index (BMI), alcohol intake, and cigarette smoking. The phenotype measures of treated people were imputed using the methods in Kathiresan et al. [SNP genotyping data were provided based on the GeneChip Human Mapping 500 k Array and 50 k Human Gene Focused Panel. We analyzed a combined sample of the Offspring Cohort  [We performed a genome-wide linkage scan for each of the three lipid phenotypes values , using 8,545 individuals from 1,349 FHS families . We selected 5,102 SNPs for the linkage scan according to the criteria of MAF > 0.45, HWE test Zi, i = 1, ..., M, was interpolated from the linkage scores of the available neighboring markers according to the relative distance between markers.The linkage score corresponding to each of the ~550 k GWA SNPs, q-values using the method suggested by Storey [q-value of a single SNP analysis was less than the chosen FDR control threshold value, the hypothesis of no association between the SNP and the disease was rejected.False-discovery rate (FDR) control was performed by computing y Storey . If the C = 1.64 for the NARAC RA data and LOD threshold value C = 0.5 for the FHS data. SNPs that fell into a high-linkage region were grouped as Stratum 1 (Zi \u2265 C) and SNPs that fell into a low-linkage region were grouped as Stratum 2 (Zi <C). FDR control was applied separately for the SNPs in Stratum 1 and Stratum 2 [Based on the linkage scan results, high and low linkage regions were determined using a NPL threshold value of tratum 2 .wi = exp(B\u00b7Zi)/v, where B = 1 . To focus on results outside regions of established importance, Table CTLA4 was 285 using FDR, which changed to 28 and 96 using SFDR and WFDR, respectively. In some weak linkage regions , WFDR retained similar ranks as FDR, whereas the SFDR ranks increased. However, q-values for WFDR were generally much higher than those of FDR and SFDR (results not shown). These analyses suggest several new associations with RA .The SNPs in chromosome 6 (MHC region) showed very strong association with Table The SFDR and WFDR methods can improve power of genome-wide association analyses when linkage scans are informative ,3. In thThe threshold value for Stratum 1 and 2 in SFDR was somewhat arbitrarily set as 1.64 for the RA data (where the linkage results were NPL scores) and 0.5 for the FHS data (where the linkage results are LOD scores) to maintain the proportion of Stratum 1 to be about 5% when the power of linkage scans is relatively low. The effect of small differences in threshold values for SFDR was insignificant on average in a simulation study . In the B parameter) for the WFDR method.The power under FDR control depends on the proportion of true alternative hypotheses  in a family of tests. By preserving most of the potentially true causal SNPs in a small-sized Stratum 1, we can improve study power . HoweverSFDR and WFDR control methods using previous linkage results can be extended to multi-marker analysis with fixed or sliding windows, for example, by using the average linkage score within a window as a measure of prior information. Further study is warranted to evaluate extensions to multi-marker analysis settings.Anti-CCP: Anti-cyclic citrinullated peptide antibody; BMI: Body mass index; FDR: False-discovery rate; GAW: Genetic Analysis Workshop; GWAS: Genome-wide association studies; HDL: High-density lipoprotein; IBD: Identical by descent; LD: Linkage disequilibrium; LDL: Low-density lipoprotein; NARAC: North American Rheumatoid Arthritis Consortium; NPL: Nonparametric linkage; RA: Rheumatoid arthritis; SFDR: Stratified false-discovery rate control method; SNP: Single-nucleotide polymorphism; TG: Triglyceride; VC: Variance-component; WFDR: Weighted false-discovery rate control.The authors declare that they have no competing interests.YJY contributed to the design and writing of the paper; association analysis of FHS data; and SFDR and WFDR analysis of RA and FHS data. DP contributed to the design and writing of the paper, association analysis of RA, data and linkage analysis of FHS data. DW contributed association analysis of FHS data and QC analysis of RA and FHS data. SBB contributed to the critical revision of the paper for important content. LS contributed to the design of the paper and critical revision of the paper for important content."}
+{"text": "HLA-DRB1 and PTPN22. We conducted genome-wide linkage analysis with 128 affected individuals from 60 families in a Canadian cohort that were genotyped using the Illumina linkage panel and genome-wide association analysis with 158 affected individuals from the same cohort that were genotyped using the Affymetrix 100 K platform. Multipoint nonparametric linkage scan revealed three linkage peaks with LOD scores greater than 1.5. We also identified 13 significantly associated SNPs at the genome-wide level of 0.05 after Bonferroni adjustment for multiple testing. Several of the significantly associated SNPs are located close to previously identified linkage regions, but not in the linkage peaks identified in the same cohort. We could not replicate association with HLA-DRB1 and PTPN22. Our results indicate that high coverage and sufficient sample size are crucial for the success of genome-wide association studies.Rheumatoid arthritis (RA) is an autoimmune disease with a moderately strong genetic component. Previous linkage and candidate gene studies have identified several regions that predispose to RA, including the  Rheumatoid arthritis (RA) is a complex autoimmune genetic disorder in which the immune system attacks normal tissues as if they were invading pathogens. Twin and family studies have suggested that the heritability of RA is ~60%. A well established RA susceptibility locus is the HLA region located on chromosome 6p, which is estimated to account for one-third of the genetic component of RA etiology. Apart from the HLA region, a number of other chromosomal regions have been replicated among various genome-wide linkage scans in which the leading regions include chromosome 1p13, 1q41\u201343, 6q16, 16p, and 18q , where 1 and 0 are the likelihoods maximized under the alternative and the null models, respectively. Under the null hypothesis of HWE, T is approximately distributed as a \u03c72 distribution with one degree of freedom. This test assesses departure from HWE after adjusting for possible association with the disease, therefore minimizing the chance that an important marker is flagged as problematic.Assume a homogeneous sample of ASPs is collected and genotyped at a diallelic marker with two alleles PAIR statistic [PAIR statistic combines information from pairs of affected individuals and can detect regions of excess IBD sharing.We performed genome-wide, nonparametric multipoint linkage analysis using the Statistic  as impletatistic  on the 6We performed single-marker association analysis using LAMP ,6, whichp-value) > 2.37 , chromosome 6 , and chromosome 9 . We did not observe evidence of linkage in the HLA region, despite the fact that approximately one-third of the total genetic contribution in RA is attributed to genes in the HLA region. Because the Affymetrix 100 K platform includes a denser set of SNPs in the HLA region and more ASPs in the CRAGS were genotyped, we also conducted nonparametric linkage analysis on chromosome 6 with the Affymetrix SNPs. The analysis was conducted using MERLIN [Our multipoint nonparametric linkage analysis revealed three linkage signals at a LOD score threshold of 1.5, corresponding to a -log10 declare that they have no competing interests."}
+{"text": "We used the Genetic Analysis Workshop 15 Problem 1 data set to search for expression phenotype quantitative trait loci in a highly selected group of genes with a supposedly correlated role in the development of the enteric nervous system. Our strategy was to reduce the level of multiple testing by analyzing at the genome-wide level a limited number of genes considered to be the most promising enteric nervous system candidates on the basis of mouse expression data, and then extend the analysis to a larger number of traits only for a small number of candidate linked regions. Such a study design allowed us to identify a \"master regulator\" locus for several genes involved in the enteric nervous system, located in 9q31. In particular, one of four traits included in the genome-wide analysis and 2 of 57 from the follow-up single-chromosome analysis showed LOD scores above 2 around position 109 on chromosome 9 by univariate variance-component linkage analysis. Bivariate linkage analysis further supported the presence of a common regulatory locus, with a maximum multipoint LOD score of 5.17 and five additional LOD scores > 3 in the same region. This region is particularly interesting because a susceptibility locus for Hirschsprung disease, a disease characterized by enteric malformation, was previously mapped to 9q31. The proposed strategy of limiting the genome-wide analysis to a small number of well characterized candidate expression phenotypes and following up the most promising results in a larger number of correlated traits may prove successful for other groups of genes involved in a common pathway. However, the extremely large number of phenotypes tested resulted in a severe multiple testing problem, and thus necessitated a rigorous correction with extremely small p-values to avoid inflation of type I error [The Genetic Analysis Workshop 15 (GAW15) Problem 1 data set is based on the proposed approach of considering natural gene expression variation as a quantitative trait influenced by genetic determinants. The resulting phenotypes can thus be used in linkage and association analyses to map loci involved in expression regulation in the genome [a priori on the basis of a hypothesized common biological role. Prompted by our interest in Hirschsprung disease (HSCR), a congenital gut malformation characterized by absence of ganglia in the colon [RET proto-oncogene, expressed throughout enteric neurogenesis, is required for normal ENS development and is the major HSCR gene. We based our trait selection on a recent article that described a microarray comparison between RNA from normal  and Ret mutant (aganglionic) gut tissue in embryonic mouse and that has identified hundreds of candidate ENS-expressed genes [Here we carried out a genome-wide linkage analysis on a small number of traits, chosen he colon , we chosed genes .The GAW15 data provided the expression phenotypes for the human orthologues of several of these genes. We conducted genome-wide variance-component linkage analysis on a highly selected group of four traits to search for candidate linked regions. We then extended the analysis to a larger number of traits focusing only on the region identified as most significant in the genome-wide screen, thus limiting the problem of multiple test significance. Because bivariate analysis has been shown to increase power to detect linkage of related traits to a common QTL, we used bivariate linkage analysis to test for the presence of genes with pleiotropic effects on the selected traits.in situ hybridization for 47 genes selected for further analysis, representing diverse functional classes and either uncharacterized or partially characterized or known ENS marker genes [By using RNA from WT and aganglionic gut tissue and DNA microarrays, Heanue and Pachnis  conducteer genes . The GAW[Data from 194 individuals belonging to 14 three-generation CEPH (Centre d'Etude du Polymorphisme Humain) Utah families have been used for quantitative genetic analysis by means of the variance-component approach. Narrow sense heritability of the traits with its significance, and genetic and environmental correlations between pairs of traits were estimated using SOLAR version 2.1.4  and mark10 of the likelihood ratio of these two models.Multipoint identity-by-descent (IBD) probabilities were estimated by GENEHUNTER and imported into SOLAR to estimate the genetic variance attributable to a hypothetical quantitative trait loci (QTL) linked to any given location. A test for linkage was carried out by testing whether the QTL additive variance was significantly different from 0 by comparing the likelihood of this model with that of a restricted model in which the genetic variance at the same location was fixed at 0. LOD scores reported in the text and the tables are calculated by SOLAR as the logGenome-wide univariate and bivariate linkage analyses were carried out on four highly selected traits presenting non-null heritability. To follow up on the genome-wide results, univariate and bivariate analyses were also performed on a larger number of selected phenotypes only for markers located on chromosome 9..The marker maps used for each chromosome were obtained by a marker position file released with the data and considering 1 cM \u2248 1 Mb. The linkage analysis on chromosome 9 was carried out with both the physical map and the Rutgers genetic map 2 and age2 \u00d7 sex as covariates. Information about age was taken from the CEPH website .No covariates were taken into account in the genome-wide analysis, while analysis for chromosome 9 was also repeated including sex, age, age \u00d7 sex, agep-values of the univariate LOD scores were calculated by means of an empirical null distribution obtained by simulation of 10,000 replicates of an unlinked marker. Asymptotic p-values for the bivariate LOD scores were calculated based on a 1/4 p-values.The ormation . No corrh2) , and for trait 202154_x_at at position 99 on chromosome 11 . All other LOD-scores were \u2264 2. There was no linkage evidence of cis regulators for any of the traits  Table  and were Table  ap-value = 0.0005), suggesting the presence of a common regulator for the two traits in this genomic region. Two other traits (203440_at and 218501_at) in combination with 201387_s_at yielded LOD scores > 2 at the same location; however the bivariate LOD scores were lower than the univariate LOD score observed for 201387_s_at alone. Two additional LOD scores > 2 were observed on chromosome 11 in bivariate analyses that included trait 202154_x_at, but were lower than the maximum univariate LOD-score observed for trait 202154_x_at alone.LOD-scores from bivariate genome-wide linkage analysis were similar to those from the univariate analysis Table . The maxTo follow up on the most significant finding, we extended the analysis of chromosome 9 to the other traits potentially involved in the ENS development included in the data set. Among these, 5 presented null heritability and were excluded. Of the remaining 57 traits, 10 had maximum chromosome 9 LOD scores at position 107 or 109 (data not shown). In particular, 2 traits had LOD scores > 2 at these positions .We obtained similar results using the Rutgers chromosome 9 genetic map (data not shown). For instance, trait 209034_at  was mapped at 140 cM with LOD score of 3.52, and trait 201387_s_at (LOD score = 2.66 at position 109) was mapped at 139 cM with LOD score of 2.60. The region from position 107 to 110 in the physical map that we initially used corresponds to the region from 130 to 151 cM in the Rutger's genetic map, and in particular position 109 corresponds to 139 cM.2 and age2 \u00d7 sex) on variability of all the traits. Overall, at least one of the covariates showed significant effects for 42 of the 67 traits (p < 0.1). Age was more frequently found to have a significant effect on trait variation (35/42), while sex was rarely significant (3/42). After inclusion of the significant covariates, only slight differences were found in the results of the chromosome 9 linkage analyses. Two traits gave the most discordant results (difference in maximum LOD scores > 1): 209267_s_at, for which the highest LOD score at position 116 went from 2.12 to 1.06; and 212120_at, for which the LOD-score at position 11 went from 3.06 to 1.33. Results for the traits that showed LOD scores > 2 around position 109 were consistent with and without covariates.The quantitative analysis was also repeated to estimate the effect of several covariates  declare that they have no competing interests."}
+{"text": "Three LOD score statistics are often used for genome-wide linkage analysis: the maximum LOD score, the LOD score statistic proposed by Kong and Cox, both based on the allele-sharing between affected sib pairs, and the maximization of the LOD score function of Morton on two genetic models and an heterogeneity parameter.p-values: comparison and interpretation of several linkage statistics cannot be done on the observed value. The Kong and Cox LOD score statistic had slightly better power to detect the HLA region involved in rheumatoid arthritis compared to the other methods. In a second step, we show that performing the analysis under a greater number of genetic models in the hope of better scanning the space of models, does not increase the power of detection.Using only identity-by-descent sharing between affected sibs as linkage information, we studied the behavior of these three statistics under the null hypothesis in the rheumatoid arthritis simulated data . Distributions under the null hypothesis show that identical values of the statistics correspond to very different genome-wide  Genome-wide linkage studies are often performed on affected sib pairs to detect disease susceptibility genes in multifactorial diseases. Many statistics have been proposed to achieve such a goal.Two methods, the LOD score statistic proposed by Kong and Cox (KC-LOD) , which iAn alternative strategy, proposed by Greenberg et al. , maximizThese statistics are all LOD scores, i.e., the decimal logarithm of the ratio of two likelihoods (linkage versus no linkage). They are computed at each marker of a given chromosome, taking into account the multipoint information provided by the entire set of markers. The maximum value observed for each chromosome is then retained to perform the linkage test. However, since these statistics differ on the parameters on which the maximization is achieved, they are likely to have different statistical properties.In this work, we study the behavior of these statistics under the null hypothesis, and then evaluate their performance for detecting the HLA risk factor in the rheumatoid arthritis (RA) simulated data . The simulating model was known prior to the analysis.The segregation of 730 microsatellite markers, spaced on 22 chromosomes with an average inter-marker distance of about 5 cM, was simulated on 100 replicates of 1500 families with at least two affected sibs.Preliminary linkage analyses showed that it was not possible to make any power comparison with such sample sizes: all linkage statistics were highly significant for detecting the role of HLA, while their power was very low (less than 5%) for the other loci. Therefore, we decided to focus on the detection of the susceptibility factor in the HLA region and to split each replicate into smaller family samples in order to have a lower, but not too low, power of detection. A sample size of 60 seemed appropriate. Each replicate was split into 25 sub-samples. The study was thus performed on 2500 replicates of 60 families each. Parental status was considered unknown in all replicates, so that linkage information consists of the identity-by-descent (IBD) sharing between affected sibs.The data were analyzed by four LOD score statistics, MLS, KC-LOD, HLOD-S1, and HLOD-S2.The MLS  maximizeThe KC-LOD proposed by Kong and Cox  is maximHLOD-S1 was calculated as initially proposed by Greenberg et al.  under a p = 0.002 per chromosome) or to determine the genome-wide p-value corresponding to a given value of the statistics.We first studied the distribution of these four statistics under the assumption of no linkage by analyzing the 16 chromosomes that did not harbor a susceptibility gene. The maximum value on each chromosome was recorded for each statistic, leading to 40,000 values (2500 replicates \u00d7 16 chromosomes). This provides the distribution of the maximum of each statistics for an average chromosome. Thus, for a full genome scan, one may apply a Bonferroni correction for 22 chromosomes. This procedure can be used either to determine the threshold for a genome-wide type I error of 5% .p-values. For example, when a value of two is observed, the false-positive rate is 31% for KC-LOD, while a value of two attains a false-positive rate of almost 50% for the MLS and HLOD-S1 . This shows that the comparison and interpretation of several linkage statistics cannot be done on the observed value.Identical values of observed KC-LOD and MLS (or HLOD-S1) give rise to very different genome-wide The thresholds corresponding to a genome-wide type I error of 5% are 2.89, 3.23, and 3.19 for the KC-LOD, MLS, and HLOD-S1, respectively.The power of linkage detection of each statistic was determined for 2500 replicates of 60 families on chromosome 6, using the thresholds above. The power is 48.5%, 45.9%, and 44.6% for KC-LOD, MLS, and HLOD-S1, respectively, showing the slight advantage of the KC-LOD.r2 > 0.97) between the HLODs obtained for the two dominant models (q = 0.2 and q = 0.01) and for the two recessive models, respectively.In a second step, we compared the impact of four versus two genetic models in the HLOD analysis. Both statistics have the same the 5% genome-wide threshold. The power of the four-model HLOD-S2 is very slightly, but not significantly, increased (from 44.6 to 45.8%). This increase may be explained by the very strong correlation  declare that they have no competing interests."}
+{"text": "We conclude our series of articles on the \u201cAnatomy Charts\u201d (http://en.wikipedia.org/wiki/Anatomy_Charts_of_the_Arabs)To the Editor: The table and references below summarize available information on 17 sets of very similar anatomy figures that I have been able to glean from the current published literature and put at the disposal of the readers. It is fair to assume that there are a few more similar figures, the locations of which are not yet known. I urge the reader and expressly enlist his kind cooperation in the task of completing this collection, which, at present, stands at exactly 100 colored hand-painted anatomy figures.There are several disputed points and questions about these figures. Here are some of them:Why did a few historians assume that these figures might be of Scythian or Tibetan origin?Which set is the original and which are copies?Who is the author of these figures?Why is the head always represented in a Mongolian perspective and why was the squatting position chosen?Why do these figures usually (with the exception of three sets) appear in mas \u2018wd's text on anatomy?Why aren't these figures always mentioned or referred to in the text in which they are found?Why do they, sometimes, seem to have been just bound within the volume containing them and do not seem to form an integral part of it?Are they all from the same hand or drawn by different scribes?What is their relation to the similar Latin figures?Once we have the answers to these questions, one can hope to study the available data with a comparative and critical analysis so that some of the existing mysteries can be resolved."}
+{"text": "We performed a multipoint linkage analysis for rheumatoid arthritis (RA) using high-density single-nucleotide polymorphism (SNP) data for chromosome 6 and chromosome 21 using Genetic Analysis Workshop 15 (GAW15) data. These regions were previously shown to have high LOD scores, not accounting for linkage disequilibrium (LD). We propose three novel methods to control for LD in a linkage analysis: allow for LD between markers using graphical modeling, eliminate high-LD markers by principal-component analysis (PCA) using haplotype data, and eliminate high-LD markers by PCA using genotype data. All three novel methods were compared to the previously published SNPLINK high-LD elimination method. Although all four methods verified the previous results, differences in linkage peak height and position were observed across methods. Additional work is required to further understand the effects of LD on linkage results and explore LD control methodology. The recent availability of rapid, accurate, and relatively low-cost genome-wide, high-density single-nucleotide polymorphism (SNP) panels is changing the study of many complex diseases. Linkage analysis to track inheritance of chromosomal regions in pedigrees no longer must rely on highly informative but sparsely spaced conventional microsatellite markers. Rather, SNPs, which are far more abundant than microsatellite markers, have the capacity to yield a higher information content, and hence an improved potential for localizing disease genes .Classical linkage analysis assumes linkage equilibrium between markers. Applying classical linkage packages to genome-wide SNP panels may generate biased results, if parental genotypes are missing, as SNP panels will include markers that are in linkage disequilibrium (LD). LD between markers may inappropriately weight shared haplotypes in the likelihood calculations, which can lead to inflated LOD scores, resulting in false-positive evidence of linkage . To overHere we propose three novel methods to control for LD between markers in a linkage analysis that do not require a contiguous LD structure: one method that allows for LD between markers using graphical modeling and two methods that eliminate markers in high LD using principal components. Our three novel methods were compared to the previously published analysis generated by the SNPLINK  program.Details regarding NARAC subject enrollment, phenotype designation, and genotype information are published elsewhere . Using t, which we will refer to as McLink-LD in this manuscript. For the three LD elimination methods, we used both the original MCLINK [n = 1991 individuals) the estimated allele frequencies from all genotyped individuals provides reasonable estimates of the founder allele frequencies, even without adjustment for familial relationships. The original pedigree structure for all 757 pedigrees was used for all analyses, except for the Merlin analyses, in which 56 large pedigrees were removed because of memory limitations. For the genetic map, we assumed that 1 Mb was equivalent to 1 cM. Although this is a simplistic assumption, when inter-marker distances are relatively short and when a dense marker map is used, the assumption has been shown to produce nearly identical linkage results as a more detailed genetic map [The four methods  were each applied to the \"Complete\" SNP data files. For the graphical modeling of LD combined with linkage analysis method, we used the new McLink software l MCLINK  and Merll MCLINK  softwareetic map . We perfetic map ..Thomas and Camp  and ThomThrough application of graphical modeling to the haplotypes of founders in a pedigree, we are able to obtain valid linkage statistics for dense SNP loci without having to discard any data. The new McLink-LD software, made available by Alun Thomas, incorporates the LD model obtained from graphical modeling and computes LOD score statistics using MCMC methods similar to those described by Thomas et al.  The progAlthough McLink-LD can estimate an LD model using a large subset of unrelated individuals and it can also model genotype errors, for consistency with our other LD elimination methods, LD assessment was performed on 100 unrelated, random individuals with genotype data selected from the 757 rheumatoid arthritis families. LD modeling using 100 individuals has been shown previously to be a sufficient sample size to capture the underlying genetic variation . All SNPPrincipal-component analysis (PCA) has been used for selection of tagging-SNPs for candidate gene studies ,5. AdvanFor the PCA-Genotype method, we also used the same 100 unrelated, random individuals described above to characterize LD. For each SNP we recoded all of the genotype data from the \"Complete\" SNP data sets as -1, 0, 1 ; correspr2 between consecutive marker pairs for all individuals in a data set, ignoring relationships. Only one marker from each high-LD pair of SNPs, based on a high-LD threshold defined by the user, is retained. We defined high LD to be D' \u2265 0.7, consistent with our PCA-Haplotype method. We successfully analyzed the chromosome 6 \"Complete\" SNP data using SNPLINK, but found that SNPLINK halted unexpectedly when running the chromosome 21 data. Therefore, using the SNPLINK protocol, we manually identified which SNPs to eliminate because of high LD in both chromosomes 6 and 21. Our results for chromosome 6 compared well to the SNPLINK output. Four markers differed between the two marker lists; we selected from among two equal markers the opposite marker as that selected by SNPLINK. Hence, we feel confident that our analysis of the chromosome 21 data set would be similar to the output of SNPLINK had we been able to obtain it.SNPLINK  is a freDescriptions of the peak region including the number of markers studied and the median and interquartile distance between markers for the chromosome 6 and 21 regions are displayed in Table Graphical illustrations of the linkage results are provided for McLink-LD and the LD elimination methods analyzed in MCLINK in Figures HLA-DRB1 gene, the best characterized single genetic risk factor contributing to rheumatoid arthritis, maps between two SNPs included in our data sets at 28.96 cM and 33.1 cM, which is the peak location obtained from analysis of the Complete data set and all LD elimination methods. Although here we reported TLOD results as this is currently the only output of McLink-LD, rheumatoid arthritis is a complex disease, and heterogeneity LOD (HLOD) scores may more accurately reflect the true position of a peak. However, it should be noted that the HLOD scores from Merlin were reported at identical  peak position values for the three LD-elimination methods using MCLINK at both the chromosome 6 and 21 regions.The linkage results after controlling for LD all consistently verified the conclusions of Amos et al. , that isWhile a method that incorporates the underlying LD structure should more accurately reflect the true linkage peak height and position for a chromosomal region, it may be too early to consider McLink-LD the 'gold standard'. MCMC methods can be problematic due to computational requirements and poor mixing properties and it may not be suitable for use in all cases. We note that an additional feature of the new McLink-LD program is maximization of LOD scores across recombination fractions on the  interval. The  interval may better be able to incorporate model errors and improve mixing properties of the graphical LD model. Maximizing over the  interval, we observed a peak TLOD score of 13.75 at 37.04 cM, which is more similar to the results for the high-LD SNP elimination methods. However, because the high-LD SNP elimination methods all used the traditional  interval, the comparison is not ideal. We also note that maximization of LOD scores over the  interval resulted in general symmetry of the LOD function about a recombination fraction of 0.5. This is most likely due to a heavy reliance on two-generation pedigrees in the GAW15 NARAC data resource with no phase information. Any asymmetry in the chromosome 6 and 21 regions may be due to inadequate modeling of parameters [The three high-LD elimination methods all peaked at the same position, but with different peak heights. We examined residual LD between all pairs of SNPs for each of the LD elimination methods. Using a threshold for high LD of D' > 0.7, the number of pairs exceeding the threshold was less than 0.7% compared to the total number of pairwise comparisons for each method across both chromosomes, suggesting that the majority of high-LD SNPs pairs were captured. However, we do note that for chromosome 6, although the percentages were small, the number of pairs of SNPs exceeding the threshold tended to follow the same pattern as the peak TLOD results . Thus, differences in peak height most likely are due to residual LD in each of the methods. Although not investigated here, it is also possible that the PCA-genotype method, which is the most conservative, may have diminished power compared to the other two LD elimination methods as more SNPs were eliminated. The PCA methods provided a logical approach to elimination of SNPs that were not based on a contiguous LD structure or random elimination of SNPs from a high-LD set as does SNPLINK. Because an automated PCA package for removing high-LD SNPs is yet to be developed, we recommend that PCA be used only for follow-up of regions with at least suggestive linkage evidence.Control for LD is an essential component of analyzing high-density SNP linkage data, and inflated linkage peaks may result if some method for controlling for LD is not implemented. Because we observed differences in linkage peak height and position across the four methods studied here, further work is needed to explore these and other LD control methods.The author(s) declare that they have no competing interests."}
+{"text": "A previous genome-wide study in Orthodox Ashkenazi Jewish pedigrees showed significant linkage of ocular refraction to a Quantitative Trait Locus (QTL) on 1p34-36.1. We carried out a fine-mapping study of this region in Orthodox Ashkenazi Jewish (ASHK) and Old Order Amish (OOA) families to confirm linkage and narrow the candidate region.Families were recruited from ASHK and OOA American communities. The samples included: 402 individuals in 53 OOA families; and 596 members in 68 ASHK families. Families were ascertained to contain multiple myopic individuals. Genotyping of 1,367 SNPs was carried out within a 35cM (~23.9 Mb) candidate QTL region on 1p34-36. Multipoint variance components (VC) and regression-based (REG) linkage analyses were carried out separately in OOA and ASHK groups, and in a combined analysis that included all families.Evidence of linkage of refractive error was found in both OOA  and ASHK families . Combined analyses showed three highly significant linkage peaks, separated by ~11cM (or 10 Mb), within the candidate region.In a fine-mapping linkage study of OOA and ASHK families, we have confirmed linkage of refractive error to a QTL on 1p. The area of linkage has been narrowed down to a gene-rich region at 1p34.2-35.1 containing ~124 genes. OMIM] database for a complete list, and Tang et al. ) have been reported in human association studies [Using a 1-LOD drop as a cutoff, the area of linkage in the present study spans from  studies , three o studies  \u2013though In order to follow-up our findings and identify causal polymorphisms for refractive error variation, we are conducting family-based association analyses of SNP markers. These analyses will help identify either causal polymorphisms or markers strongly correlated with causal genetic variations . In the future, association analyses of additional SNP and copy-number polymorphisms in the region may help to identify the DNA variants responsible for this linkage signal.In a fine mapping study in Orthodox Ashkenazi and Old Order Amish families, we have confirmed linkage of refractive error to a quantitative trait locus on 1p and have narrowed the region of interested to a ~10 Mb area spanning 1p34.2-35.1. Given that linkage was found in two independent, culturally and genetically isolated, groups, it is probable that the underlying genetic cause is a single genetic locus with variant alleles of large effect that can be detected in both these ethnic groups. It is reasonable to expect that such a locus would also have variant alleles that influence refractive error in other European-derived populations."}
+{"text": "NFKB1, NFKB2, REL, RELA, and RELB) and variable expression levels of 15 NF-\u03baB regulated proteins with heritability greater than 0.40: BCL2A1, BIRC2, CD40, CD44, CD80, CFLAR, CR2, FAS, ICAM1, IL15, IRF1, JUNB, MYC, SLC2A5, and VCAM1. SNP genotypes and expression phenotypes from pedigrees of Utah residents with ancestry from northern and western Europe were provided by Genetic Analysis Workshop 15 and supplemented with additional genotype data from the International HapMap Consortium. We conducted association, linkage, and family-based association analyses between each candidate gene and the 15 heritable expression phenotypes. We observed consistent results in association and linkage analyses of the NFKB1 region (encoding p50) and levels of FAS and IRF1 expression. FAS is a cell surface protein that also belongs to the TNF-receptor family; signals through FAS are able to induce apoptosis. IRF1 is a member of the interferon regulatory transcription factor family, which has been shown to regulate apoptosis and tumor-suppression. Analyses in the REL region (encoding c-Rel) revealed linkage and association with CD40 phenotype. CD40 proteins belong to the tumor necrosis factor (TNF)-receptor family, which mediates a broad variety of immune and inflammatory responses. We conclude that variation in the genes encoding p50 and c-Rel may play a role in NF-\u03baB-related transcription of FAS, IRF1, and CD40.The nuclear factor-kappaB (NF-\u03baB) family of transcription factors regulates the expression of a variety of genes involved in apoptosis and immune response. We examined relationships between genotypes at five NF-\u03baB subunits ( NFKB1 encoding p50, NFKB2 encoding p52, REL encoding c-Rel, RELA encoding p65, and RELB encoding Rel-B). The NF-\u03baB pathway is a critical candidate gene pathway for numerous cancers and cardiovascular endpoints.The nuclear factor-kappaB (NF-\u03baB) family of transcription factors regulates the expression of hundreds of genes including pro-inflammatory and apoptosis genes -3. Trans2 [Genetic Analysis Workshop 15 (GAW15) Problem 1 included data on 14 three-generation pedigrees  consisting of Utah residents with ancestry from northern and western Europe . Pedigree members had genotypes on ~2882 single-nucleotide polymorphisms (SNPs) spread throughout the genome and ~3554 phenotypes consisting of expression levels from lymphoblastoid cells hybridized onto Affymetrix Genome Focus Arrays [2 .NFKB1 (90.6 cM to 117.5 cM on chromosome 4), NFKB2 (94.5 cM to 119.8 cM on chromosome 10), REL (45.7 cM to 73.4 cM on chromosome 2), RELA (44.7 cM to 78.4 cM on chromosome 11), and RELB (41.2 cM to 58.0 cM on chromosome 19). Denser genotypes from HapMap within 5 kb of each gene were also used: NFKB1 , NFKB2 , REL , RELA , and RELB .Genotypes from 21 GAW15-provided SNPs surrounding ~20 cM of each candidate gene were analyzed: N = 165) were compiled from review of the literature [h2) using the Splus/R library multic [h2 greater than 0.4  were included in the current analysis  were performed using the program FBAT  to examiHaploStat [Using data on 28 unrelated individuals, analysis of variance (ANOVA) tested associations between 15 heritable expression levels and genotypes at dense HapMap SNPs surrounding the five candidate genes. With the Splus library NFKB1 (FAS and IRF1 expression), NFKB2 (IRF1 and SLC2A5 expression), REL , and RELA regions . Linkage regions and maximum LOD scores for each gene are presented in Tables Linkage analysis showed elevated LOD scores in the NFKB1 suggested an association between rs721412 at 111.3 cM and FAS, IRF1 expression. Haplotypes containing this SNP were also associated with FAS and IRF1 expression , and haplotype rs2306365-rs732072-rs11820062 was associated with all phenotypes .Analyses using GAW15-provided genotypes surrounding NFKB1 and REL  had suggestive haplotype associations. Among six phenotypes associated with REL SNPs, all six phenotypes had suggestive haplotype association  receptor family; signals through FAS are able to induce apoptosis. IRF1 is a member of the interferon regulatory transcription factor family, which regulates apoptosis and tumor-suppression. CD40 proteins also belong to TNF protein family, which is essential in mediating a broad variety of immune and inflammatory responses. Based upon our results, we concluded that variation in the NFKB1 and REL genes may play a role in downstream regulation of FAS, IRF1, and CD40 expression.We utilized a variety of methods  in an attempt to understand the relationship between variation in NF-\u03baB genes and expression levels of 15 proteins. We consider this to be an exploratory analysis of publicly available data with a limited sample size. We sought to reveal avenues for future study within the NF-\u03baB pathway. As an assessment of these methods, we concluded that haplotype analysis combined with single-SNP analysis, family-based association tests, and linkage analysis has helped inform our understanding of the NF-\u03baB pathway. Analyses revealed association and linkage between There are several limitations to this study, including lack of adjustment for multiple tests on multiple loci and use of a small sample size; interpretation of tests on a sample of 14 warrants caution. No results were statistically significant after taking into account the multiple comparisons. Nonetheless, these exploratory analyses provide clues for further large scale studies.We make three general conclusions. First, single-SNP association testing was less conservative than haplotype and FBAT analysis, where haplotype analyses indicated association, results of single-SNP association testing were also significant; however, association found by single-SNP testing was not always revealed by haplotype analysis. Because this is not simulated data, we do not know whether the single-SNP results represent true or false positives. Second, because haplotype analysis requires two or more SNPs, for those genes with only one or very few SNPs, haplotype analysis might not be an appropriate analysis to perform. Third, FBAT analysis was relatively conservative compared to single-SNP and haplotype association analyses. FBAT found fewer SNPs and haplotypes with point-wise significance. In summary, we suggest that single-SNP and haplotype association analyses be used in first-stage analysis to generate a smaller set of candidate SNPs; FBAT and linkage analysis can then narrow down the list of potentially important loci.The author(s) declare that they have no competing interests."}
+{"text": "The APOA1-C3-A5 gene cluster plays an important role in the regulation of lipids. Asian Indians have an increased tendency for abnormal lipid levels and high risk of Coronary Artery Disease (CAD). Therefore, the present study aimed to elucidate the relationship of four single nucleotide polymorphisms (SNPs) in the Apo11q cluster, namely the -75G>A, +83C>T SNPs in the APOA1 gene, the Sac1 SNP in the APOC3 gene and the S19W variant in the APOA5 gene to plasma lipids and CAD in 190 affected sibling pairs (ASPs) belonging to Asian Indian families with a strong CAD history.2 48% \u2013 70%; P < 0.0001). A subset of 77 ASPs with positive sign of Logarithm of Odds (LOD) score showed significant linkage to CAD trait by multi-point analysis  and to Sac1 (LOD score 4.49) and -75G>A (LOD score 2.77) SNPs by single-point analysis (P < 0.001). There was significant proportion of mean allele sharing (pi) for the Sac1 (pi 0.59), -75G>A (pi 0.56) and +83C>T (pi 0.52) (P < 0.001) SNPs, respectively. QTL analysis showed suggestive evidence of linkage of the Sac1 SNP to Total Cholesterol (TC), High Density Lipoprotein-cholesterol (HDL-C) and Apolipoprotein B (ApoB) with LOD scores of 1.42, 1.72 and 1.19, respectively (P < 0.01). The Sac1 and -75G>A SNPs along with hypertension showed maximized correlations with TC, TG and Apo B by association analysis.Genotyping and lipid assays were carried out using standard protocols. Plasma lipids showed a strong heritability (hThe APOC3-Sac1 SNP is an important genetic variant that is associated with CAD through its interaction with plasma lipids and other standard risk factors among Asian Indians. Lipids and lipoproteins have been traditionally associated with high risk of incident coronary artery disease (CAD). The Apolipoprotein A1-C3-A4-A5 gene cluster on chromosome 11q23 (Apo11q) is among the most well characterized regions of the human genome with reference to their dynamic association with plasma lipids and lipoproteins ,7 as welThe SNPs in the Apo11q region, particularly the 3238C>G, Sac1 SNP in the 3' untranslated region (UTR) of the APOC3 gene , as wellAssociation studies have been previously reported on the Asian Indian population, mostly on healthy adult volunteers, to elucidate the role of specific genetic variants in the APOA1 gene , APOC3 gA total of 523 families comprising 2318 individuals were enrolled in Phase1 of the Indian Atherosclerosis Research Study [IARS], an ongoing genetic epidemiological study that aims to identify specific genes and regulatory pathways associated with CAD in the Asian Indian population. Families were identified through a proband with a strong family history of CAD and/or stroke, from select hospitals/clinics in Bangalore and Mumbai. Probands were defined as patients with angiographically proven ischemic heart disease with age at onset \u2264 60 years for men and \u2264 65 years for women. Other affected/unaffected family members  were recruited when available and willing. None of the participants had a history of past major illness or concomitant infection. The institutional ethics committee approved the IARS, which was conceived according to the guidelines defined by the Indian Council of Medical Research for research conducted on human subjects. A signed informed consent was obtained from all participants in the study.A detailed case record form pertaining to information on demographics, anthropometry, medical history and coronary risk factors such as presence of diabetes, hypertension, smoking, lifestyle and current medication was completed for each participant through personal interviews and through perusal of their medical records.\u00ae, Greiner Bio-One GmbH, Vienna, Austria). Serum, EDTA and citrate plasma samples were separated by centrifugation and aliquots were preserved at -80\u00b0C until analysis. Cell pellets were stored at -20\u00b0C and used for extraction of genomic DNA at a later date.Venous blood was collected in evacuated tubes after an overnight fast of 12 to 14 hours , Triglycerides (TG), Lipoprotein (a) , High Density Lipoprotein-cholesterol (HDL-cholesterol) , Apolipoprotein A1 (Apo A1) and Apolipoprotein B100 (Apo B)  were estimated on the Cobas Fara II Clinical Chemistry Auto analyzer , following the manufacturer instructions. Serum Low Density Lipoprotein-cholesterol (LDL-cholesterol) was calculated using the Friedwald's formula. Three commercial controls purchased from Randox Laboratories, one from Orion Diagnostica for apolipoproteins and a normal in-house serum pool were run with every batch of assay. The inter-assay coefficients of variation (CV) for the commercial controls and normal serum pool ranged from 4.9% to 7.0% for TC, 6.1% to 7.7% for TG, 7.1% to 12.2% for HDL-cholesterol, 9.9% to 14.2% for ApoA1 and 10.7% to 13.9% for ApoB levels.Heritability refers to the proportion of phenotypic variance attributable to genetic variance. Heritability estimates were performed for the various atherothrombotic phenotypes in 508 IARS families consisting of 2305 individuals. Circulating levels of lipid markers were measured in family units comprising proband, spouse and offspring. Age- and sex-adjusted heritability was estimated by variance component analysis using the sequential oligogenic analysis routine (SOLAR) (SOLAR v 1.4.1) program. Spouse pair correlations were determined to account for the household effects.Genomic DNA was extracted by the salting-out procedure . Based oThe present study consisted of 194 ASPs selected from 131 IARS families. Of these, there were 114 families containing 1 ASP, 14 with 3 ASPs and two families with 6 ASPs, respectively, while one family consisted of 15 ASPs. Taking into account the genotyping failures in some cases, only data on 190 ASPs for whom genotyping was performed for all the four SNPs, was used in the final analysis.Non-parametric linkage analysis was performed by the ASP analysis method  to test For the preliminary study, linkage between the Sac1 SNP and plasma TG levels was tested using the deviation in the proportions of 0-, 1- or 2- alleles shared, identity by state (IBS) between the 190 ASPs, against an expected distribution of 25%, 50% and 25%, respectively. Association of the Sac1 genotypes with plasma TG levels was tested by analysis of variance (data unpublished).all and MERLINpairs options under MERLIN [Three additional SNPs  were used for the subsequent linkage study. Appropriate input files for linkage analysis were created with the MEGA2 program . Multipor MERLIN  as well r MERLIN : 0 = no On the basis of the individual sign of LOD score obtained from linkage results on the entire dataset (N = 287), families where all ASPs showed a positive LOD score (LOD score \u2265 0) (N = 104) were selected for further analysis . AdditioAnalysis of QTL was performed on families with positive sign of LOD score and 'hyper TG families' using the 'QTL' option in MERLIN program. All lipid variables  were independently tested against the four markers for evidence of linkage. Age, gender, smoking, diabetes, hypertension and statin use were used as covariates.2 test (P > 0.05). The major allele was referred to as 1 and the minor allele as 2; the corresponding genotypes were assigned as 1 1, 1 2 or 2 2 for the homozygous normal, heterozygotes and homozygous variants, respectively. Genetic variants with the 2 2 genotypes, having a frequency of less than 10 (Apo A1 +83C>T and Apo AV S19W) were grouped with the heterozygotes for analysis purpose. Haplotype frequency was estimated using the DECIPHER program of SAGE.Routine statistical analysis was performed using the SPSS v.15  package. Mean and standard error of mean (SEM) were calculated for the quantitative variables. Genotype frequencies were determined by direct counting. Conformity to Hardy-Weinberg equilibrium was determined by \u03c7P value of 0.05 or less was considered as statistically significant.Correlations among the plasma lipids and lipoproteins were tested using the Pearson's correlation coefficients estimate. Student t-test or ANOVA and multivariate analyses were used to test for association between the Apo11q SNPs and plasma levels of lipids and lipoproteins before and after adjusting for confounding factors such as age, gender, BMI, smoking, presence of diabetes or hypertension and statin use. Step-wise linear regression analysis was employed to understand the extent of contribution of individual SNPs to the variation in plasma lipid and lipoprotein levels. The non-parametric Kolmogorov-Smirnov test was used as test of normality for the quantitative variables and the values were subsequently log-transformed to normalize distribution. CAD severity was defined on the basis of age at CAD onset, the number of diseased vessels and the event score  classified according to the clinician's diagnosis. The Chi-square test was used to test for association between genotypes and CAD severity. Other discrete variables such as presence/absence of diabetes, hypertension and smoking were also tested against the 4 Apo11q SNPs. Presence of diabetes (Type 2 DM) and hypertension (HTN) was ascertained based on self-report of physician's diagnosis and/or use of prescription medications along with proof obtained from their medical records. A nominal In order to determine the contribution of SNPs to the variation in lipid markers in a cohort of subjects with premature CAD, a two-stage procedure was adopted comprising of correspondence analysis in the first stage followed by canonical correlations . The 'R'The set of discrete variables consisted of the four SNPs in the Apo11q cluster, hypertension and diabetes. In the first stage, these categorical data were subjected to correspondence analysis in order to obtain the scores for each row category represented by the individual subjects in the CAD group that were derived as linear functions of the column profiles characterized by the respective genotypes for the 4 SNPs, hypertension and diabetes. These provide the least square estimates obtained as a linear function, which act as weightages of each individual.st stage (Set 2 variables). Canonical correlation analysis is a multivariate data analysis technique, which maximizes the correlations between the linear functions of two sets of variables. Since canonical correlations are best defined when both the sets are continuous in nature, we adopted this two-stage procedure.In the second stage, Canonical correlation analysis was performed between the quantitative variables namely the lipid markers, body mass index (BMI) and waist hip ratio (WHR) (Set 1 variables) and the coordinates derived by correspondence analysis carried out in the 1P = 0.046) and ApoA1 (P = 0.013). Diabetes (P = 0.006) and hypertension (P = 0.004) were significantly higher among females  when compared to the corresponding proportions in males . While 47.3% of males were smokers, there were no female smokers in our cohort. Mean age of CAD onset was 50.3 \u00b1 8.4 years for males and 53 \u00b1 8.8 years for females. The number of diseased vessels and event score showed similar distribution in both sexes. More than 93% of females were postmenopausal but none of them were receiving hormone replacement therapy. Over 62.6% of males and 78.3% of females were on the lipid lowering drug, statin, respectively.A total of 531 families were recruited in the Phase 1 of the IARS, of which 23 incomplete families were excluded from analysis. Of these, 131 families having 190 ASPs were selected for the present study. Gender-wise distribution of lipid profile, CAD severity and coronary risk factors among the affected siblings is shown in Table P < 0.0001) was observed for TC (h2 = 0.65 \u00b1 0.06), TG (h2 = 0.53 \u00b1 0.06), HDL-cholesterol (h2 = 0.48 \u00b1 0.06), LDL-cholesterol (h2 = 0.55 \u00b1 0.07), Apo A1 (h2 = 0.71 \u00b1 0.04) and Apo B (h2 = 0.70 \u00b1 0.05). There was minimal spouse-pair correlation for most lipid traits  except HDL-cholesterol, which showed a higher correlation coefficient value .Significant heritability (P < 0.0001), suggesting tentative linkage of the Sac-1 locus with CAD  across the APOC3-Sac1 genotypes, namely S1S1, S1S2, and S2S2, respectively  by multipoint analysis for the CAD trait, which was confirmed using the LODPAL program of SAGE. Single point analysis showed strong evidence of linkage for the Sac 1 SNP  and a suggestive evidence of linkage for the -75G>A SNP . The mean proportion of alleles shared IBD was significant for the Sac-1 , -75G>A  and +85C>T  SNPs using the GENIBD program of SAGE. , gave highly significant LOD score of 7.42 , HDL-cholesterol  and Apo B  only in families with positive LOD score  levels. Other features such as CAD severity, BMI, WHR, and frequency of Type 2 DM and HTN showed similar distribution across the two groups (Data not shown).When the various coronary risk factors as well as CAD severity were compared between the group with positive sign of LOD score (N = 104) and negative sign of LOD score (N = 181), the mean levels of the various lipid markers were marginally higher among the former group than the latter group. The difference was statistically significant only for plasma ApoA1 (P > 0.05). The distribution of minor allele frequencies (MAF) and genotypes across the four SNPs is given in Table All the four SNPs in the Apo11q cluster were in Hardy Weinberg equilibrium (P < 0.001) were positive, there was significant negative correlation between the HDL-cholesterol and TG levels . This trend was retained following covariate adjustment for age, gender and BMI.Association between the Apo11q genotypes and quantitative phenotypes were performed on the whole dataset of 190 ASPs comprising of 287 CAD subjects. Significant correlation was observed among the lipid and lipoprotein biomarkers using the Pearson's correlation coefficient estimates Table . While cP = 0.007), with mean LDL-cholesterol levels across the -75G>A genotypes (P = 0.043) and with ApoA1 levels across the S19W genotypes (P = 0.026). Age was a common confounding factor that showed significant interaction with the lipids while gender and BMI did not make any significant impact in this dataset.Multivariate analysis showed significant differences in the mean TG levels across the Sac-1 genotype (P = 0.005) of the variation in plasma TG levels. With regard to the effect of conventional risk factors on lipids, HTN contributed to the variation in TC, TG, LDL-cholesterol, ApoA1 and ApoB levels , while waist hip ratio (WHR) contributed to over 1.5% (P = 0.037) of variation in HDL-cholesterol levels. Age was the other significant contributory factor to the variation among the plasma lipids .Results from linear regression analysis showed that APOC3-Sac1 SNP was the only significant contributory genetic marker among the 4 SNPs and was able to account for about 2.8% , which prompted us to expand by including additional markers in this region. Our hypothesis was strengthened by the findings of linkage analysis, albeit in a subset of the cohort. Application of sophisticated statistical analysis tool such as canonical correlations assisted in identifying and segregating the specific variables, namely the Sac1 SNP, -75G>A SNP and hypertension, that showed maximum contribution to the variability in our dataset of affected sibling pairs. The present study is to our knowledge, the first report to simultaneously look into the linkage and association of at least 4 markers in the APOA1-C3-A5 gene cluster among Asian Indians with premature CAD. However, our findings will benefit from the sequencing of this region in a small group of probands in order to understand the genetic variability of this highly heterogeneous chromosomal region in a population that does not have a representation, as yet, in the HAPMAP database. We plan to embark on this study in the near future. Additionally, we hope to confirm these preliminary, yet interesting observations, on a larger cohort of over 2000 CAD patients and age and gender- matched controls.In conclusion, the Sac 1 and the -75G>A SNPs could serve as useful tools for the direct quantitative measure of variation in lipid and lipoprotein levels and an indirect assessment of CAD risk among the Asian Indian population.ApoA: Apolipoprotein A; APOA1: Apolipoprotein A1 gene; APOA5: Apolipoprotein A5 gene; ApoB: Apolipoprotein B; APOC3: Apolipoprotein C3 gene; ASPs: Affected sibling pairs; BMI: Body mass index; CAD: Coronary Artery Disease; FCHL: Familial Combined Hyperlipidemia; HDL-c: High-Density Lipoprotein cholesterol; HTN: Hypertension; IBD: Identity By Descent; IBS: Identity By State. LDL-cholesterol: Low Density Lipoprotein cholesterol; LOD score: Logarithm of Odds score; MI: Myocardial Infarction; SAGE: Statistical Analysis for Genetic Epidemiology; SOLAR: Sequential Oligogenic Analysis Routine; SNPs: Single Nucleotide Polymorphism; TC: Total Cholesterol; TG: Triglyceride; Type 2 DM: Type 2 Diabetes Mellitus; UTR: Untranslated region; WHR: Waist Hip Ratio; QTL: Quantitative Trait Loci.The authors declare that they have no competing interests.JS was involved in the study design, molecular studies, data analysis, and drafting of the manuscript and takes full responsibility for the accuracy and integrity of the data. GP was responsible for carrying out the genotyping assays while NBK performed the lipid analysis. VSR supervised the data quality of the phenotypic assays and gave useful insights while preparing the manuscript. SJ carried out the statistical analysis while SH designed the association analysis and helped in data interpretation. MM conceptualized the study. VVK provided critical intellectual content while preparing the manuscript and gave the final approval for the publication of this article. All authors read and approved the final manuscript."}
+{"text": "Taeniopygia guttata) chromosomes  using two types of marker, Single Nucleotide Polymorphisms (SNPs) and microsatellites. To assess the effectiveness and accuracy of each kind of marker we compared maps built with each marker type separately and with both types of marker combined. Linkage map marker order was validated by making comparisons to the assembled zebra finch genome sequence.Genetic linkage maps are essential tools when searching for quantitative trait loci (QTL). To maximize genome coverage and provide an evenly spaced marker distribution a combination of different types of genetic marker are sometimes used. In this study we created linkage maps of four zebra finch  rather than the most informative should be used to create framework maps before the addition of other potentially more error prone markers (microsatellites). This raises questions about the accuracy of marker order and predicted recombination rates in previous microsatellite linkage maps which were created using the conventional building process, however, provided suitable error detection strategies are followed microsatellite-based maps can continue to be regarded as reasonably reliable. Linkage maps are fundamental tools in many genetic studies and have been created using various types of polymorphic markers since their conception by Sturtevant in 1913 -4. HowevTwo key components to consider when constructing a linkage map are the number and type of markers to use. Phenotypic (i.e. visible) markers were the first to be utilized, but now the choice has been extended to include a range of molecular markers including allozymes, Random Amplified Polymorphic DNA (RAPDs), Restriction Fragment Length Polymorphisms (RFLPs), Amplified Fragment Length Polymorphisms (AFLPs), Sequence-Tagged Sites (STSs), microsatellites (Simple Sequence Repeats) and SNPs. Each of these exhibit slightly different advantages and disadvantages but there are three main considerations when deciding which genetic markers to use in linkage map construction: (i) the markers need to be polymorphic, (ii) they need to be evenly spread across the genome or region of interest and provide dense marker coverage, and (iii) they must have a low genotyping error rate.Microsatellites are obvious candidates for linkage mapping; they are highly polymorphic, relatively easy and cheap to score (once a library is established), and can exhibit cross-species utility in closely related species -13. As aMicrosatellites and SNPs also differ with respect to consideration (iii); the error rate. Markers with lower error rates obviously produce more accurate linkage data. A trade-off associated with the highly polymorphic nature of microsatellites is that they can have relatively high genotyping error rates ,27. MethIn a recent simulation study, where markers were compared for mapping accuracy, a difference in power between bi-allelic markers and polymorphic markers was reported, with a higher density of SNPs required to accurately produce similar results to the polymorphic microsatellites . HoweverTaeniopygia guttata) genome using SNP markers has recently been released [The aim of this study was to compare linkage maps built with microsatellites, SNPs and a combination of both markers to determine the ability of each method to produce accurate linkage maps. A linkage map of the zebra finch  public databases were assessed ,43. By phttp://abajian.net/sputnik/) to search two zebra finch sequence databases for microsatellites. The first approach took advantage of the zebra finch EST sequences located on the National Centre for Biotechnology Information (NCBI) website (described in [We attempted to increase our marker density on these 4 chromosomes using the program SPUTNIK (Sputnik ribed in ), and thribed in ,49. Therribed in .-5). The chromosome position with the lowest e-value was considered a good hit if the e-value of the hit was less than 1e-10 and the difference between it and the next best hit was 10 decimal places. In general, the length of the query sequence significantly affects whether a reliable BLAST hit can be found [A zebra finch homologous location for each microsatellite marker was found by directly comparing its sequence against the zebra finch genome assembly. Chromosomal locations were identified by Basic Local Alignment Search Tool (BLAST) analysis to compare the avian microsatellite or the zebra finch sequence (Whole Genome Shotgun (WGS) tracefiles or ESTs) against the zebra finch genome sequence . BLAST analyses were carried out using Standalone BLAST version 2.2.17  using thbe found . HoweverThere are three minor distinctions between the way loci were identified and primers designed in this study. These can be discerned by the names used for each primer pair, with the prefixes ZF, TG and ZEST used to discriminate between the different design methods Table .First, to increase the probability of microsatellite co-amplification in other species, consensus sequences were used to identify the most conserved regions for primer design. For these markers, prefixed \"TG\", the primer sites are in regions of 100% identity between the zebra finch and chicken (their design is described elsewhere ). Secondhttp://frodo.wi.mit.edu/primer3/). The PRIMER3 default parameters were used, except that a CG clamp was required at the 3' end of each primer and the maximum consecutive number of the same bases (Max-polyX) was lowered to 3. The maximum primer melting temperature (Tm) was set at 60\u00b0C, the minimum was set at 54\u00b0C and the maximum Tm difference between the forward and reverse primer was lowered to 0.5\u00b0C. By using the settings stated above we attempted to ensure all primers would amplify with a Polymerase Chain Reaction (PCR) annealing temperature of 56\u00b0C.Primers were designed using the web interface of the program PRIMER3 2SO4, 75 mM Tris-HCl pH 9.0, 0.01% (w/v) Tween), including 2.0 mM MgCl2 and 0.2 mM of each dNTP. PCR amplification was performed in a TETRAD DNA Engine  or a Touchdown thermal cycler (Hybaid). The following PCR program was used for these singleplex test reactions, 3 minutes at 94\u00b0C, followed by 35 cycles of 30 seconds at 94\u00b0C, 30 seconds at 56\u00b0C and 30 seconds at 72\u00b0C, then 10 minutes at 72\u00b0C. PCR products were then separated and visualized using an ABI 3730 DNA Analyzer (Applied Biosystems) using Prism set DS-30 and the ROX size standard  and allele sizes assigned using GENEMAPPER 3.7 (Applied Biosystems).The forward primer was labeled with a fluoro-dye  to enable allele size assignment on an ABI 3730 DNA Analyzer. The initial tests for amplification were each carried out in 10 \u03bcl PCR reactions. These contained about 50 ng of genomic DNA, 1.0 \u03bcM of each primer and 0.25 units Taq DNA polymerase  in the manufacturer's buffer . Any markers which had more than 2 alleles were then tested on 24 birds in the mapping population. However, the 38 primers with prefix \"ZF\" were initially tested in all 24 individuals. After this, observed and expected heterozygosity and predicted null allele frequencies were estimated using CERVUS v3.0 . A totalThe majority of the pedigree individuals were of known phenotypic sex . All individuals were PCR sex-typed using the Z-002 sex-typing primers  during tWhen genotyping the whole pedigree, a Qiagen multiplex mix was used during PCR amplification. Four multiplex sets were designed, each containing between 6-9 markers Table . All mar2 = 23.8, p < 0.005, d.f. = 2). This was also the case for ZF01-025 with set 2 containing 61 ungenotyped samples compared to 24 in set 3 . The lower call rate in Set 2 is probably due to the increased number of markers incorporated into this set, as increased competition between primers could lead to reduced amplification of some loci. In total, 12 of the 273 individuals, which could be genotyped at locus ZF01-196 in all 3 sets, exhibited 2 different genotypes. The genotype that was exhibited in 2 of the sets was assumed to be the correct one, which gave a 1.46% error rate at this locus. The repeatability of locus ZF01-025 was better with only one genotyping inconsistency within the 289 samples that were genotyped in both sets giving an error rate of 0.17%. The errors were distributed across all multiplex sets, making it difficult to identify the reasons for these anomalies, but suggesting that they were not due to consistent differences between the quality or reliability of the multiplexes.Two markers (ZF01-196 and ZF01-025) were included in multiple multiplex sets to verify repeatability between the different PCR sets. Marker ZF01-196 was multiplexed in sets 2, 3 and 4 and ZF01-025 in sets 2 and 3. For ZF01-196 the number of samples that did not amplify was significantly greater in set 2 (42) than in sets 3 (10) and 4 (18)  and used only the most informative marker in each haplogroup for map assembly. Using the remaining informative markers, framework maps were created using the BUILD command. The BUILD command was implemented by entering two linked, informative markers with recombination fractions of between 0.1-0.2 as ordered loci, and inserting the remaining loci. The likelihood threshold was set at 5, so the marker order with a likelihood of LOD \u2265 5 better than the next most likely order was chosen. The process was repeated twice, each time starting with a different pair of markers. Of the three maps, the one with the most markers was chosen as the first framework map. The FLIPS5 function was used to shuffle marker orders and identify the most likely marker order. Using this framework map, we added the remaining markers by progressively dropping the likelihood threshold to LOD = 3, 1, 0.1, 0.01 and then 0.001. This results in a comprehensive map of all markers, with the final order determined by the best likelihood given the data available. The positions of markers mapped at the lower LOD thresholds are less well supported. In most studies a LOD of \u22653, i.e. marker order 1000 times more likely than any other marker order is considered a conservative (or framework) map . The FLIUsing CHROMPIC, one microsatellite marker (ZF01-054) was excluded from Tgu1A as even when positioned in its location with highest likelihood it was consistently flagged, suggesting it had a high error rate. This was also supported by the fact that its presence increased map length by a substantial margin (20 cM) . A subseafter the SNPs (hereafter this is termed the 'SNPs preceding microsatellites' map).Linkage maps were constructed for the microsatellites and the SNPs separately using the approach outlined above. We also built combined linkage maps including both the microsatellites and SNPs. We constructed these combined maps via two different methods. The first approach followed the conventional method as described above and elsewhere . Typicalhttp://genome.wustl.edu/genomes/view/taeniopygia_guttata/. We also compared SNP-only maps with maps that contained SNPs and microsatellites, in order to test whether the inclusion of microsatellites caused map length inflation.To validate the accuracy of the maps we compared marker order on each linkage map with the order on the zebra finch genome assembly Microsatellite genotyping error has been highlighted as a major cause for concern in population genetic analyses, including parentage assignment (reviewed in ). ParticThe greater number of alleles of microsatellite markers, along with a less automated method of genotyping leads to a higher error rate relative to SNPs . Within Although genotyping errors are not uncommon, most can be easily identified. In this study the pedigree was known, and due to the number of markers typed in the entire pedigree (25), it was possible to detect any allele calls that caused departures from Mendelian inheritance. Any individuals involved in a parent-offspring mismatch were re-examined on the program GENEMAPPER. Eleven individuals were inconsistent with Mendelian inheritance at greater than 5 microsatellite loci and therefore were assumed to have been contaminated or incorrectly labelled. The results from these samples were subsequently excluded from all analysis. The high variability of microsatellites aids error detection as most errors will result in Mendelian incompatibilities. However, with SNPs this error-checking procedure will be less successful as they are only biallelic. Some studies have predicted that up to 30% of errors in SNP genotyping will remain undetected because they will not cause Mendelian inheritance incompatibilities ,62.de novo mutations in the germline. Given that we have excluded loci with high error rates, identified mis-labelled or mis-pedigreed individuals and re-scored individuals that cause mismatches or unlikely double recombination events, it seems reasonable to assume that the subsequent impact of typing error on map construction that we report is conservative. Of course, it should be regarded good practice to remove as much of the detectable error as possible. It is important to note, and perhaps underappreciated, that some genotype errors will not cause Mendelian inconsistencies, but can still lead to the inference of spurious recombination events. For example, suppose an offspring with a true genotype of A/A was miss-scored as A/B. If it has parents with genotype A/A (mother) and A/B (father) both the true and incorrect genotype are compatible with Mendelian inheritance. However it would be wrongly assumed that allele B was inherited from the father, which would lead to the inference of at least one, but more likely two, additional recombination events. Therefore, even with careful error checking, loci with high error rates are likely to cause map inflation.After re-examining the allele calling any remaining mismatches could be due to allelic dropout, scoring error, more cryptic biochemical artefacts or The linkage maps generated by the 4 different methods are presented in additional file The greatest difference between the SNP and microsatellite maps is the number of markers on each, with all chromosomes containing substantially more SNPs. The linkage groups Tgu1, Tgu1A, Tgu2 and Tgu9 contain 73, 71, 82 and 24 SNPs respectively compared to only 3, 5, 6 and 6 microsatellites. However, every microsatellite was assigned to the correct linkage group, even on the low-density microsatellite only maps. The ability to detect linkage between the microsatellites when so few are used is due to the relatively high number of informative meioses of these markers  more than 7 times greater than the SNP map (0.02 cM Mb-1). The Tgu2 maps are more difficult to compare as both comprehensive maps exhibit rearrangements compared to the genome sequence  was ten times greater than that predicted by the SNP map (0.02 cM Mb-1). This degree of inflation is worrying, especially considering the attempts to control for the influence of microsatellite typing error. For Tgu9 we also estimated the recombination rates using the distal microsatellites according to the genome assembly. The maps lengths were more similar but the recombination rate of the microsatellite maps was still inflated . In general, the difference between SNP and microsatellite recombination rates seems to be most pronounced in the central parts of macro-chromosomes. This is logical, as recombination in these regions is relatively rare, and therefore the relative impact of spurious recombination events caused by typing error will be greater than in regions where true recombination events are more frequent.The microsatellite maps consistently estimated higher recombination rates than did the SNP maps. For example, on Tgu1A the microsatellites span a predicted 24.4 Mbp and the linkage distance is 15.8 cM, whereas a similar region covering 21.7 Mbp is only 0.6 cM on the SNP map. On linkage group Tgu1 (region spanning ~31.4 Mbp to 94.5 Mbp) the microsatellite map exhibits a recombination rate  than SNPs (5%). This is unlikely to lead to map inflation of the microsatellite maps as there is no reason why missing genotypes should be biased with respect to whether they affect recombinant or non-recombinant meiotic events. To test this further we simulated 10 replicates of the LG1 SNP map (63.7 cM) with 15% missing data and then rebuilt the maps. The simulated datasets had a mean map length almost identical to the original . In other words there was no evidence that map inflation of the microsatellite maps was caused by a higher genotyping failure rate. It is also worth noting that the greater variability of microsatellites means that they resulted in a much higher number of informative meioses than the SNPs . However, the degree to which this has influenced the order of markers on the physical map is thought to be negligible. As a precaution we have also used assembled supercontigs as a reference against which to compare orders of the different linkage maps (see below). These supercontigs were created independently of any linkage map and so provide a robust test of the accuracy of map order, without any potential influence of the linkage map on the assembled zebra finch sequence.To validate the marker order of SNP and microsatellite maps, the zebra finch genome sequence assembly was used as a template against which all linkage maps were evaluated. It is important to note that the SNP map has been used as a reference during the latter part of the zebra finch sequence assembly process  with an average length of ~10 Mb. For each framework marker Figure  the supeIt should be acknowledged that some discrepancies between the linkage maps and the physical genome sequence could be indicative of mistakes in the assembly. Genome assemblies are an ongoing process, and it is likely that the zebra finch assembly will be improved as more information becomes available, and possibly as the chicken genome assembly improves. Limitations of the zebra finch sequence assembly were apparent when performing BLAST searches; four SNP markers  had hits on the chicken genome assembly to the chromosomes predicted by the linkage maps, yet they fail to produce a hit on the zebra finch genome sequence. As these SNPs have been isolated and obtained from zebra finch DNA, it suggests that the zebra finch genome assembly remains relatively incomplete. Putative assembly errors however, cannot explain the much greater recombination rates of the microsatellite maps, and there is no a priori reason to expect inaccuracies in the assembly to affect marker order of microsatellites but not SNPs.Another key point to consider when assessing these maps is that the greater marker density of the SNP maps, make the detection of 'unusual' double recombinants indicative of typing error more obvious. The problems associated with microsatellites highlighted in this paper may be less pronounced if more markers were typed, although they are unlikely to disappear completely as evidenced by the map inflation of the high density combined SNP and microsatellite maps, and by the previously undetected map inflation observed on the high density microsatellite-based chicken linkage maps .When combined, the microsatellites and SNPs were assigned to the predicted linkage groups based on their known chicken and zebra finch assembly positions and their earlier linkage map positions . InteresIt is likely that the combination of greater informativeness and higher errors of the microsatellites cause them to be added first but in the wrong order. Subsequent addition of the SNPs fails to rectify the errors as the relatively large number of SNPs (compared to microsatellites) renders the FLIPS5 command unable to resolve errors in marker order between microsatellites separated by large numbers of SNPs. FLIPS takes consecutive markers, reorders them and produces likelihood scores for these different orders. Due to computational constraints it is impractical to use FLIPS for greater than five markers. What can happen in this situation is that a local likelihood maxima is reached between a group of closely linked markers such that no changes in the order of these will give a better likelihood. However, if it was possible to flip the order of more than 5 markers then a more accurate order may be revealed.One of the outcomes of this investigation is that the conventional method of building linkage maps has to be modified when using markers that differ significantly in their number of informative meioses and error rates. Markers with the lowest error rates should be included first to provide an accurate foundation, to which other markers can then be added. This may be especially important for larger chromosomes as Tgu1, Tgu1A and Tgu2 exhibited the largest differences between the two construction methods, whereas the smaller chromosome Tgu9 did not differ as substantially. It is possible that those regions of macro-chromosomes with very low recombination rates are most sensitive to typing error. However this could also be due to the markers on Tgu9 exhibiting lower error rates, and investigation of additional smaller chromosomes would be needed to confirm this observation.This study highlights potential problems, as well as some solutions, of linkage mapping construction with moderately error prone markers. Although microsatellites are informative, they can provide misleading results because they have greater error rates than SNPs. Even with the usual methods of error reduction and detection in microsatellite genotyping there is still potential for map inflation and incorrect marker orders. The results of this study emphasise the importance of careful examination of CHROMPIC outputs to identify possible genotype errors, which can create spurious double recombinants. We also suggest that the least error prone markers (SNPs) should be mapped before adding the microsatellites, at least until it is computationally practical to FLIP greater than 5 markers at a time.This study has highlighted the importance of knowing how much effect error rates have had on the final outcome of a linkage map. With map inflation likely to be present in other microsatellite-based studies, it remains to be seen whether subsequent SNP mapping projects will reveal previously undetected errors in map order or length. However, although microsatellites are more error prone than SNPs, their widespread availability, ease of typing, high polymorphism and their cross-species utility will ensure that they remain useful in linkage-mapping studies, especially for comparing the genomes of non-model organisms. In summary, we are not suggesting that microsatellites should be abandoned in mapping studies, but we do urge that appropriate error checking precautions are employed.AB participated in the microsatellite discovery, carried out the microsatellite genotyping and linkage map construction and drafted the manuscript. JStapley participated in the SNP linkage map construction, provided the SNP genotyping and co-drafted the manuscript. DAD participated in the microsatellite discovery and primer design. TRB developed and managed the zebra finch mapping pedigree. TB participated in the conception and design of the study. JSlate conceived of the study and participated in its design and coordination, as well as co-drafting the manuscript. All authors read and approved the final manuscript.Table S1. Characterisation of microsatellite markers. Characterisation of 26 zebra finch (Taeniopygia guttata) microsatellite markers used to create the linkage maps for the zebra finch chromosomes 1, 1A, 2, and 9.m, melting temperature TEST, expressed sequence tag\u2020, all primer sequences were identical to zebra finch sequence (obtained either via trace files or assembled genomic sequences)a, Excluded after CHROMPIC revealed an excess of double recombination events with adjacent markers, indicative of high error rate.b, Excluded from linkage maps as parent-offspring mismatches estimated the error rate > 0.02c, Could not be accurately scored after multiplexingClick here for fileTable S2. Results of linkage mapping. Summary of the linkage map results for four zebra finch (Taeniopygia guttata) chromosomes . The predicted positions of all markers on the zebra finch genome sequence after a BLAST analysis are included. Linkage map positions highlighted in bold are the framework loci that are supported at a LOD score \u2265 3 for that linkage map.Click here for file"}
+{"text": "Bicyclus anynana, have received little attention, despite the scientific importance of this species. This study presents the characterization of chromosomes in this species by means of cytogenetic analysis and linkage mapping.The chromosome characteristics of the butterfly B. anynana were examined by karyotype analysis and construction of a linkage map. Lepidoptera possess a female heterogametic W-Z sex chromosome system. The WZ-bivalent in pachytene oocytes of B. anynana consists of an abnormally small, heterochromatic W-chromosome with the Z-chromosome wrapped around it. Accordingly, the W-body in interphase nuclei is much smaller than usual in Lepidoptera. This suggests an intermediate stage in the process of secondary loss of the W-chromosome to a ZZ/Z sex determination system. Two nucleoli are present in the pachytene stage associated with an autosome and the WZ-bivalent respectively. Chromosome counts confirmed a haploid number of n\u200a=\u200a28. Linkage mapping had to take account of absence of crossing-over in females, and of our use of a full-sib crossing design. We developed a new method to determine and exclude the non-recombinant uninformative female inherited component in offspring. The linkage map was constructed using a novel approach that uses exclusively JOINMAP-software for Lepidoptera linkage mapping. This approach simplifies the mapping procedure, avoids over-estimation of mapping distance and increases the reliability of relative marker positions. A total of 347 AFLP markers, 9 microsatellites and one single-copy nuclear gene covered all 28 chromosomes, with a mapping distance of 1354 cM. Conserved synteny of Tpi on the Z-chromosome in Lepidoptera was confirmed for B. anynana. The results are discussed in relation to other mapping studies in Lepidoptera.Physical genomic features in the butterfly This study adds to the knowledge of chromosome structure and evolution of an intensively studied organism. On a broader scale it provides an insight in Lepidoptera sex chromosome evolution and it proposes a simpler and more reliable method of linkage mapping than used for Lepidoptera to date. Bicyclus anynana  is among the most extensively studied Lepidoptera species. It has been established as an emerging model organism to address many evolutionary questions with a particular focus on genetic and environmental effects on wing pattern formation The butterfly Agrodiaetus, with haploid chromosome numbers that vary between 10 and 134 Samia cynthia show, besides different chromosome numbers, a high polymorphism of sex chromosomes Philaethria dido species complex, which is no longer regarded as single species, since no evidence of hybrids between individuals of sympatric populations with different chromosome numbers was found Bicyclus is less spectacular . The two sets of homologous LGs are incompatible and can only be combined with anchoring markers. Male informative markers, allelic AFLPs and microsatellites can act as such anchors and there are various approaches to integrate the two sets of dominant markers. For example, Lepidoptera specific software was designed to create a linkage map for Here we report on a novel approach for the final step in Lepidoptera linkage maping by using the option in JOINMAP to join maps, i.e. to present the two opposite phased homologous maps as different mapping populations and use the software to integrate them based on the anchoring markers. The advantages are that the female-derived component can be removed instead of presented as missing data, and the same software combines the two phases automatically. To compare our mapping distance with that of other species of butterfly, we also performed a MAPMAKER analysis because Mapping distances generated by the two programs can differ substantially th instar larvae were dissected in physiological solution, then fixed for 20 min in Carno\u1ef3s fixative (6 : 3 : 1 ethanol-chloroform-acetic acid), macerated in 60% acetic acid, spread on a slide at 45\u00b0C, dehydrated by three washes in increasing concentrations of ethanol , and dried at room temperature, leaving the preparations suitable for different types of staining. Some preparations were stained for 5 min and mounted in 2.5% lactic acetic orcein. Others were stained with YOYO-1 fluorescent dye  under the following conditions: the dry preparations were first soaked for 5 min in PBS , then stained with 50 \u00b5l of 100 nM YOYO-1 in PBS for 20 min, briefly washed in tap water, air-dried and mounted in 20 \u00b5l of antifade based on DABCO -octane; Sigma-Aldrich, St. Louis, MO, USA)  for 15 min, and then fixed in Carnoy's fixative for 15 minutes. The testes were subsequently squashed in 20 \u00b5l of 50% acetic acid using a siliconised cover slip, followed by dehydration in an alcohol series as described above. Staining involved a 5 min incubation in PBS/1% Triton-X, followed by 15 min in PBS/1% Triton-X with 0.25 \u00b5g/ml DAPI . The slides were then rinsed for 5 min in PBS/1% Triton-X with 1% Kodak PHOTO-FLO, followed by 10s rinsing in H2O containing 1% Kodak PHOTO-FLO. Finally, the preparations were mounted in 20 \u00b5l of antifade.Male metaphase I and II chromosomes were obtained from testes of the 5th instar larvae. The tubules were dissected in physiological solution, fixed in Carnoy's fixative for 2 min, and then stained with 1.5% lactic acetic orcein for 4 min.To determine the sex chromatin status see , prepara1 crosses were set up by combining random brothers and sisters to produce segregating F2 offspring. The larvae were raised on maize plants and the adults were fed with banana. They were reared at 23\u00b0C to minimize the effect of temperature on eyespot size, since this temperature is an intermediate between the temperature that would produce small (20\u00b0C) and large (27\u00b0C) eyespots as a result of phenotypic plasticity. The cross that produced the largest amount of F2 adults was selected to produce the linkage map. All procedures have been performed following our institutional animal husbandry guidelines. From a total offspring of 71 males and 113 females, 23 individuals from both ends of the phenotypic extremes of the F2 generation were genotyped in each sex . DNA was extracted from half a thorax using DNeasy tissue spin columns .The linkage analysis was based on a cross between individuals from divergent selection lines for eyespot size on the ventral hindwing, designated High (H) and Low (L) for large and small eyespots respectively MseI and EcoRI , 0.612 \u00b5M Mse-adapter (5\u2032-GACGATGAGTCCTGAG-3\u2032+5\u2032-TACTCAGGACTCAT-3\u2032), 0.068 \u00b5M Eco-adapter (5\u2032-CTCGTAGACTGCGTACC-3\u2032+5-AATTGGTACGCAGTCTAC-3\u2032), 0.6 Weiss units T4 Ligase, 2.5 \u00b5g BSA and 5 \u00b5l DNA extract from the 2nd Qiagen DNeasy tissue kit elution (corresponding to approximately 125 ng DNA).We followed a modified procedure of the AFLP technique 5\u2032-GACTGCGTACCAATTCA-3\u2032), 0.92 \u00b5M Mse+C primer (5\u2032-GATGAGTCCTGAGTAAC-3\u2032), and 2 \u00b5l undiluted restriction-ligation product as template. Preamplification PCR cycle was 120s 72\u00b0C, 120s 94\u00b0C, followed by 20 cycles of 10 s 94\u00b0C, 30 s 56\u00b0C, 120 s 72\u00b0C.Preamplification was performed in 15 \u00b5l 1\u00d7AFLP Amplification Core Mix Module  supplied with 0.12 \u00b5M Eco+A primer , 120 s 72\u00b0C, with Ta decreasing 1\u00b0C per cycle from 65\u00b0C down to 57\u00b0C. Then 25 cycles of 10 s 94\u00b0C, 30 s 56\u00b0C, 120 s 72\u00b0C, and a final extension of 30 min at 72\u00b0C. Twelve of the combinations were genotyped on an ABI 377 automated sequencer with 3 different dyes and ROX500 size standard, and an additional 21 on an ABI 3100 with 4 dyes and LIZ500 size standard. The ABI377 data output was analyzed with GENOGRAPHER 1.6.0 Selective amplifications with 33 different primer combinations were processed in 10 \u00b5l 1\u00d7Core Mix with 0.05 \u00b5M fluorescently labeled Eco+ANN primer, 0.25 \u00b5M Mse+CNN primer and 1 \u00b5l 10\u00d7diluted preamplified product as template. For sequence and fluorescent labels of the primers see The microsatellite markers available for this species were processed under the conditions described in 17 primer under standard conditions. A section of the Tpi (Triose-phosphate isomerase) gene was amplified with arthropod-specific degenerate primers 197fin1F and 197fin2R TTCGGCTGAGATGATAAAGG) and Ba_TPI_473L (AGTACCAATGGCCCACACTG) were designed within the Tpi sequence to amplify an intronic region, using the same genomic template as for the AFLP reactions. PCR conditions were as described above, except for using Ta 52\u00b0C instead of 50\u00b0C. The F1 parents were screened for SNPs (single nucleotide polymorphisms) by means of sequencing the intron. Genotyping the F2 offspring was based on PCR amplification (as above), the amplicons were subsequently treated with 1 unit of AluI restriction enzyme (NEB) for 2 h at 37\u00b0C, which either cuts a 230 bp fragment into 30 bp and 200 bp or leaves it intact, depending on the genotype. The restriction pattern was screened on a 3% agarose gel. The Tpi partial cds and intron sequence are submitted to GENBANK under accession numbers EU675861 and EU675862.RNA was extracted from ground thorax with TRIZOL  following the methods suggested by the manufacturer. cDNA was synthesized with SUPERSCRIPT III (Invitrogen) with 50 ng template and a T1 genotypes. Female informative (FI) markers are present in the F1 female and absent in the F1 male and segregate 1\u22361, male informative (MI) markers are present in the F1 male and absent in the F1 female and segregate 1\u22361 as well. BI (both informative) markers segregate with a 3\u22361 ratio, resulting from F1 male and female that are both heterozygous peakpresent. Z-linked markers were identified by a peakpresent in all male offspring and a 1\u22361 ratio in the female offspring (representing an F1 WZ+ (\u2640)\u00d7Z+Z\u2212 (\u2642) F1 cross, with \u201c+\u201d\u200a=\u200apeakpresent allele and \u201c\u2013\u201d\u200a=\u200apeakabsent allele). All F2 female MI-markers were compared with this Z-specific 1\u22361 pattern in JOINMAP to reveal the WZ\u2212\u00d7Z+Z\u2212 crosses in which both male and female offspring have a 1\u22361 ratio.The AFLP markers were divided into different groups, depending on the F2 genotypes, which is identical for all loci on the same chromosome and which displays the exact opposite pattern in all markers in repulsion. This fixed set of genotypes has been named the \u201cchromosome print\u201d B. anynana was carried out as described in 1) chromosome the peakpresent of a marker lies and from which grandparent (P-generation) it came. If the marker is present in the grandmother and absent in the grandfather, the linkage phase is \u201c0\u201d, the reverse gives linkage phase \u201c1\u201d. When the marker is present in both grandparents, the linkage phase is determined by the software based on co-segregation in the F2 with markers for which linkage phases are known. Linkage phases consist of a maternal and a paternal component, indicating marker orientation (and P-origin) in the F1 mother and the F1 father.Due to the absence of meiotic recombination in females, syntenic FI-markers are transmitted to the offspring in complete association, independent of their relative position. Consequently, they cannot be positioned within LGs. A cluster of syntenic FI-markers displays a chromosome-specific pattern of FThe chromosome prints were numbered based on the output order of the software. It is important to reduce the number of errors in chromosome prints to a minimum because they are subsequently used for error detection and identification and removal of uninformative markers. With multiple FI-markers defining a chromosome print, inconsistencies were rescored and when persistent, the chromosome print was based on the most common genotype in the inconsistent individual.Chromosome prints for chromosomes without FI-markers were reconstructed based on BI and MI-markers as described in BI and microsatellite markers were grouped by screening them against the 21 chromosome print patterns in JOINMAP with a LOD threshold of 3. This mapping step also established the linkage phase of the markers. Markers in the six LGs for which chromosome prints were initially not available were assigned to LG22-LG27.The markers were subsequently screened by a \u201cforbidden genotype\u201d analysis to confirm or reject correct LG assignment and to detect scoring errors Part of the observed variation in AFLP data is caused by indels (insertions or deletions) between the two restriction sites at a single locus, resulting in amplicons of different sizes. To determine whether two BI loci are in fact different alleles of the same locus, we applied the following criteria: (1) they must have the same primer combination; (2) they must group together in the same LG when presented as independent loci in the initial uncensored BI screening; and (3) linkage phases of markers with 3\u22361 ratio must be opposite for both the maternal and the paternal component. Either one or both peaks present in an individual would be a prerequisite for codominance in species with recombination in both sexes, but with non-recombining females, that same condition is already covered by forbidden genotype restrictions.MI alleles were detected as well, but they do not provide more analytical power when combined together into one codominant marker as is the case in the BI-markers. Their opposite paternal linkage phases produce fully complementary peak patterns that hold the same mapping information.1 mother and the other half from the F1 father. A female derived peakpresent obscures the male-derived allele in dominant markers, so that it is impossible to distinguish between F2 homozygotes and heterozygotes. This is not an issue when mapping species with recombination in both sexes, because mapping software can treat these unknown allele combinations as \u201ceither heterozygous or homozygous\u201d. However, without recombination in the females, genotype scores that have a positive F1 female signal have to be excluded from analysis, which means that part of the paternal information is also lost. What remains are scores for those individuals that obtained a peakabsent from the female and either a present or absent from the male in a 1\u22361 ratio. The criteria for filtering out the female component is straightforward because the female BI peakpresent is always fully linked to either a positive or negative chromosome print value, depending on their relative maternal linkage phases (The BI-markers (with a 3\u22361 ratio in the offspring) obtain half their peakpresent alleles from the Fe phases . MarkersThe censored BI genotypes are initially replaced with \u201cmissing data\u201d. The BI and microsatellite markers with their LG designations are then analyzed together with the MI and microsatellite markers in JOINMAP to establish to which LGs they belong.Microsatellites were translated to their male informative component as described in 2 offspring. The female F2 offspring have a 1\u22361 ratio for all markers, while the F2 males have 100% peakpresent when the F1 female is also peakpresent. These 100% male scores were excluded from analysis and all the female markers and the remaining male markers were separately mapped and then joined as described above.The Z chromosome markers were divided into male- and female FBesides the linkage map construction with JOINMAP, we followed the procedures described in All steps except the \u201cFinal map construction\u201d were identical to the procedures described above, since B. anynana in our stock from Malawi  sex chromosome system, typical for the majority of advanced Lepidoptera  fragment, or both of them within the same individual, thus representing both homozygotes and the heterozygote. Female F2 offspring had either the 230 bp or the 200 bp fragment (but not both) per individual, thereby showing a hemizygous (Z-linked) pattern.A total number of 458 polymorphic segregating markers was generated with AFLPs. The effective number was smaller because the female informative markers do not contribute to mapping, a small number of markers failed the forbidden genotype screening, and 52 markers that behaved as alleles were merged to form 26 single locus codominant markers. This resulted in 347 AFLP loci that could be used for the construction of the linkage map. The markers cover all chromosomes except for the W chromosome, which cannot be mapped even if markers were available because this chromosome not involved in recombination. Additionally, there were seven polymorphic microsatellites that could be positioned on the map and another two that could only be assigned to specific LG's by their female informative component because they were homozygous in the FChromosome prints based on FI-markers were available for 21 of the 27 autosomes, another three were reconstructed from BI and MI-markers  and the remaining three were based on BI-markers alone , with random 1\u22361 designation for the unassigned values as described in Tpi gene. The mapping lengths of the chromosomes range from 8 to 84 cM, but we assume that the smaller linkage groups have insufficient coverage rather than representing chromosomes that are relatively small. Therefore, the estimated map length does not necessarily reflect the actual chromosome length.The final linkage map is shown in The mapping order in MAPMAKER was similar to the JOINMAP output for most chromosomes. However, in some LG's with low proportions of anchoring markers vs. BI markers, or unevenly distributed anchoring markers, large rearrangements sometimes occurred. This is caused by the fact that MAPMAKER compares small subsets of markers rather than all representatives of an LG at the same time. MAPMAKER initially uses a maximum of eight markers, and subsequently positions additional markers within the initial (eight marker) map. Finally, the mapping order is fine-tuned by using a sliding window of five markers (ripple command). The use of a subset of markers  that is made up of BI markers of both maternal linkage phases and less than two anchoring markers, results in an unreliable suggested marker order. The reliability of the initial (eight marker) map can be improved by including all available MI and codominant markers, but with the ripple command the representative markers cannot be hand-picked because their grouping depends on the provisional marker order suggested by MAPMAKER. Similar to the ripple command that is used to determine marker order, a sliding window analysis also reveals the reliability of the marker order, by comparing the likelihood of the most likely marker order with alternative orders (flips test). This test is confronted with the same bi-phasic incompatibility problems and cannot be used on a censored data set with missing data. The consequences of comparing only subsets of markers within a linkage group (i.e. sliding window) are illustrated with an example based on LG21, which is characterized by codominant anchoring markers close to both ends and ten dominant markers of both phases in between them . JOINMAPThe marker order suggested by JOINMAP (following the procedures used for the present linkage map) is far more reliable than the MAPMAKER approach because it does not attempt to map incompatible BI markers relative to each other directly. The ripple test, which can cause serious problems with missing data analysis, strongly increases the reliability of the marker order when analyzing markers of each maternal linkage phase separately in JOINMAP. Instead of reporting a flips test value, JOINMAP simply excludes markers that do not meet the criteria for reliable neighboring markers . MAPMAKER on the other hand always suggests a mapping order and will always produce a linkage map that includes all presented markers.B. anynana is 0.49 pg The mapping distances given by MAPMAKER were larger than those produced by JOINMAP under all circumstances. The mapping distances decreased substantially with error detection activated in MAPMAKER, but were on average still 38% larger than in JOINMAP, ranging from 1.02 to 2.14 times in size for the different LGs . The totB. anynana correspond to those generally found in Lepidoptera. Female heterogamety is confirmed by the presence of a WZ bivalent in pachytene oocytes and the presence of a heterochromatic W-body in female somatic interphase nuclei, which are absent in males. The chromosomes are indistinguishable in different stages of both mitotic and meiotic divisions, except for orcein stained pachytene, where different bivalents can be differentiated to a certain degree. We regularly identified two distinctive bivalents that were associated with two different nucleoli in female pachytene spreads. One of these nucleoli is associated with the WZ bivalent and the other with an autosome bivalent. The autosome bivalent carried a terminally located NOR that was associated with small but clear heterochromatin. The presence of heterochromatin at the NORs is common in animals , in which the W chromosome was shortened by irradiation A. subflava, in which the W chromosome comprises about half of the Z chromosome but shows still a conspicuous heterochromatic mass , or that the female BI component segregation is 1\u22361 (BI only reconstruction). Empirical tests based on LGs with available chromosome prints showed that this approach creates chromosome prints that are identical or nearly identical to the available ones, and linkage maps that are very similar to those based on conventionally censored datasets. The stochastic deviations from the 1\u22361 segregation have a negligible effect on the mapping distance and no effect on the mapping order. This validates the BI censoring approach for LGs without FI-markers.One effect of having just one set of grandparents is that BI markers carry information in only one of both paternal linkage phases for most LGs . AnotherThe selective genotyping approach was chosen to avoid genotyping intermediate eyespot phenotypes in the offspring, since they provide hardly any additional information in QTL mapping compared to that of the extreme phenotypes B. anynana with other Lepidoptera linkage maps, since this is the first species in this taxon for which the final mapping step was performed in JOINMAP. This software has not been used before for Lepidoptera linkage maps, presumably because it is less able to deal with a substantial portion of non-overlapping genotypes than MAPMAKER. Our approach avoided this problem by adapting that of Using MAPMAKER with censored BI-markers as missing data resulted in a map that was 38% larger in size than the one produced from two subsets per chromosome with JOINMAP. This size difference is caused by two factors. Firstly, there is a software effect  that is revealed by analyzing only the (uncensored) MI-markers, that accounts for 17% of the difference in this study. The rest of the difference is caused by the treatment of the incompatible bi-phasic censored BI-markers. The main purpose of the MAPMAKER analysis was to allow comparison of mapping distance in Tpi gene of B. anynana is consistent with all (distantly related) Lepidoptera species for which this gene has been mapped to date Click here for additional data file.Supplement S2Reconstruction of the chromosome print(0.05 MB DOC)Click here for additional data file.Supplement S3Forbidden genotype screening(0.05 MB DOC)Click here for additional data file.Supplement S4Censoring of BI markers and map integration with anchoring markers(0.12 MB DOC)Click here for additional data file.Supplement S5Microsatellite censoring(0.02 MB DOC)Click here for additional data file.Supplement S6Implications of sliding window analysis of a \u201cmissing data\u201d-censored dataset based on an example.(0.24 MB DOC)Click here for additional data file.Supplement S7Linkage group sizes of Bicyclus anynana produced with MAPMAKER and with JOINMAP(0.09 MB DOC)Click here for additional data file.Supplement S8Implications of a full-sib design(0.08 MB DOC)Click here for additional data file."}
+{"text": "Actinidia (kiwifruit) consists of woody, scrambling vines, native to China, and only recently propagated as a commercial crop. All species described are dioecious, but the genetic mechanism for sex-determination is unknown, as is the genetic basis for many of the cluster of characteristics making up the unique fruit. It is, however, an important crop in the New Zealand economy, and a classical breeding program would benefit greatly by knowledge of the trait alleles carried by both female and male parents. The application of marker assisted selection (MAS) in seedling populations would also aid the accurate and efficient development of novel fruit types for the market.The genus A. chinensis have been constructed with 644 microsatellite markers. The maps consist of twenty-nine linkage groups corresponding to the haploid number n = 29. We found that sex-linked sequence characterized amplified region (SCAR) markers and the 'Flower-sex' phenotype consistently mapped to a single linkage group, in a subtelomeric region, in a section of inconsistent marker order. The region also contained markers of expressed genes, some of unknown function. Recombination, assessed by allelic distribution and marker order stability, was, in the remainder of the linkage group, in accordance with other linkage groups. Fully informative markers to other genes in this linkage group identified the comparative linkage group in the female map, where recombination ratios determining marker order were similar to the autosomes.Gene-rich female, male and consensus linkage maps of the diploid species A. chinensis. As all Actinidia species are dioecious, we suggest that the sex-determining loci of other Actinidia species will be similar to that region defined in our maps. As the extent of the non-recombining region is limited, our result supports the suggestion that the subtelomeric region of an autosome is in the early stages of developing the characteristics of a sex chromosome. The maps provide a reference of genetic information in Actinidia for use in genetic analysis and breeding programs.We have created genetic linkage maps that define the 29 linkage groups of the haploid genome, and have revealed the position and extent of the sex-determining locus in  Actinidia, being the country which commercialized the fruit of Actinidia deliciosa (A. Chev.) C.F. Liang et A.R. Ferguson var. deliciosa, as kiwifruit, and which recently released Actinidia chinensis Planch. var. chinensis 'Hort16A', the gold-fleshed kiwifruit, as an alternative cultivar. While New Zealand was instrumental in bringing these fruits to commercial attention, the genus is native to China and neighbouring countries where more than 60 species are known. This germplasm is relatively unexplored in terms of horticultural development of new and novel cultivars and offers a huge range of fruit characters and 'eating attributes', and plants suited to a wide range of climatic conditions. The diversity of flavours, fragrances, colours, and health factors are also of interest in genomic studies, offering the possibility of defining chemical pathways and identifying gene function.New Zealand has a long history of interest in the genus Actinidia species present challenges to research and breeding. All known species in the genus are dioecious. Female plants bear flowers that are hermaphroditic in appearance but produce only empty pollen grains, while male plants have flowers that are unisexual with numerous stamens surrounding a rudimentary pistil whose growth is suppressed before style elongation or ovule initiation. Full dioecism is shown by about 4% of seed plants, and a second group display a variety of sub-dioecious conditions , where O and E are the observed and expected values, and the corresponding degrees of freedom is equal to one less than the number of linkage groups.Distribution of markers among linkage groups was assessed by comparing observed marker numbers in linkage groups with expectations under a random Poisson process. The expected number of markers for linkage group LGF conceived the study, carried out SSR discovery, marker polymorphism discovery and population genotyping, and drafted the manuscript. GKT, CFH, and GPG performed SSR and marker polymorphism discovery and population genotyping, GKT also assisted in preparing the manuscript. PMD performed genotyping analyses and map construction. MAM recommended the most suitable mapping population and performed genotyping analyses. HNDS assisted with map construction, carried out all statistical analyses and wrote the statistical methods section for the manuscript. RNC developed the bioinformatics system used for SSR discovery. All authors read and approved the final manuscript.Actinidia chinensis.Genetic linkage map  of  The markers prefixed 'Ke' were from the kiwifruit EST database and represent expressed genes. Those prefixed 'udk' were from enriched genomic libraries, while all other prefixes relate to the bud libraries, and various markers as described in materials and methods. A number in brackets following a marker name indicates that a single primer pair amplified more than one locus. In the female map 29 linkage groups were defined. Incipient sex chromosomes were identified in Linkage Group 17 where the sex-determining locus was located in the subtelomeric region.Click here for fileActinidia chinensis.Genetic linkage map  of  The markers prefixed 'Ke' were from the kiwifruit EST database and represent expressed genes. Those prefixed 'udk' were from enriched genomic libraries, while all other prefixes relate to the bud libraries, and various markers as described in materials and methods. A number in brackets following a marker name indicates that a single primer pair amplified more than one locus. In the male map 29 linkage groups were defined. Incipient sex chromosomes were identified in Linkage Group 17 where the sex-determining locus was located in the subtelomeric region.Click here for fileMarker distribution in the female and male maps. A statistical programme that assumes markers are randomly distributed gave an estimate of intra-marker distance. Markers were estimated to be within 10 cM of each other in over 96% and 94% of the female and male genomes respectively.Click here for file"}
+{"text": "Recently, gene expression levels have been shown to demonstrate familial aggregation, suggesting a direct role of heritable DNA variation. We studied the gene expression levels in lymphoblastoid cells of the Centre d'Etude du Polymorphisme Humain Utah families made available to Genetic Analysis Workshop 15 (GAW15), using genome-wide linkage analyses.Heritability was estimated for the expression levels of each individual phenotype. Genome wide linkage analysis was then performed using the 2819 SNPs for the expression levels of all the genes.Heritability exceeded 0.21 for 50% of the expressed phenotypes. Genome-wide linkage analysis demonstrated that 19 of them reached significance after correcting for multiple comparisons, only 4 of which were reported previously. We did not identify any hot spots of transcriptional regulation when assuming LOD score > 5.3 for significant linkage evidence.Our analysis suggests that inconsistent results in comparison to the previous report may be due to the different approaches, phenotype transformation, and different pedigree data used in the analyses. Genetic diseases are the ultimate manifestation of pathological genetic variation, although under some circumstances they may also reflect the influence of environmental factors. Gene expression at the transcript level  is considered an intermediate stage between DNA sequence variation and complex traits. Recently, Cheung et al.  studied The human gene expression data in lymphoblastoid cells included 14 three-generation CEPH Utah families. The expression levels of 3554 of the 8500 genes tested were available for GAW15. In addition, 2819 autosomal SNPs were genotyped and provided by GAW15. The linkage map of the SNPs was calculated based on the deCode map using interpolation . LiUGT2B17 and PYGB was no longer statistically significant. 3) Morley et al. [Further analyses suggested several factors that might contribute to the inconsistencies, as summarized below. 1) We used different analysis approaches. In our multipoint genome-wide linkage analysis we used the variance-components approach implemented in Merlin while Morley et al. applied SIBPAL, which is robust to the normality assumption . Using tWe failed to observe the hot spot of transcriptional regulation on chromosome 14 reported by Morley et al. . This inThe author(s) declare that they have no competing interests."}
+{"text": "Genomic scan analyses have suggested that the chemokine receptor cluster  on the short arm of chromosome 3 may contribute to susceptibility to HIV-1 infection and to the expression of a number of inflammatory diseases. Two single nucleotide polymorphisms (SNP) and a deletion in these chemokine receptors have also been found in case-control studies to be associated with susceptibility for asthma and related phenotypes. We extended these case-control studies by establishing whether these polymorphisms were in linkage and linkage disequilibrium with asthma and related phenotypes using linkage and haplotype analyses.We genotyped 154 nuclear families identified through two child probands with physician-diagnosed asthma  including 303 unrelated parents and 150 unrelated children. Atopy was defined as a positive skin prick test (SPT 3 mm) to a panel of common inhaled allergens.From a panel of ten known SNPs, only three polymorphisms: \u2013G190A in CCR2, \u2013T51C in CCR3, and a 32 bp deletion in CCR5 were found to occur at clinically relevant frequencies. All 154 families were used for haplotype analysis but only 12 nuclear families were eligible for linkage analysis. Both analyses confirmed that the mutations were in linkage with asthma, but not with atopy.The chemokine receptor genes on 3p21.3 are significantly plausible candidate genes that can influence the expression of asthma. The previous association of the CCR5\u039432 deletion with protection from childhood asthma appears to be explained by linkage disequilibrium with the \u2013G190A mutation in the CCR2 receptor gene. Asthma is a complex disease which in childhood is frequently, but not invariably, associated with atopy,Chemokines, or chemotactic cytokines, are secretory proteins produced by leucocytes and tissue cells, either constitutively or after induction.Chemokines have been implicated in both inflammatory and homeostatic leukocyte migrations.5It has been suggested that there may be some contribution of the functional mutations in the chemokine pathway in several diseases including rheumatoid arthritis7Most of the genes encoding CC chemokine receptors including CCR1 to CCR9 have been mapped to chromosome 3p21.3-p24, and of these, CCR2, 3, and 5 are clustered within a total distance of 350 kb on chromosome 3p21.3.In view of the complex interdependencies, redundancies, and in particular, the close genomic distance between three of the strongest and most studied candidates in human disease expression, we assessed the linkage and the linkage disequilibrium between SNPs in CCR5, CCR2, and CCR3 in families at high risk for asthma and atopy.We analyzed numbers of SNPs within the chemokine receptors CCR2, CCR3, and CCR5 using DNA pooling and allelic quantification (AQ) to identify two most common SNPs in our population. Three highly allelic single nucleotide polymorphisms were observed: \u2013G/A substitution at position 190 of the CCR2 gene changes the amino acid at position 64 from isoleucine to valine of its protein, \u2013T/C substitution at position 51 of the CCR3 gene results in a silent mutation at position 17 of its protein, and a 32 base pair deletion within the CCR5 gene clustered in chromosome 3p21.4 within a 300 kbp span were examined by the linkage disequilibrium and linkage analysis for asthma and atopy.1 of 20% (PC20).Population characteristics have been described in detail elsewhere.14Alternaria) referenced to a negative control. These five allergens were selected according to our own clinical survey and patient questionnaire for atopic patients. This survey confirms that these five allergens are the most common in our populations. DNA was isolated from EDTA-treated whole blood using the phenol-chloroform method.Atopy was defined as the presence of at least one positive skin prick test (SPT) with a wheal size of \u22653 mm according to the European guidelinesConsent was obtained from each participant and approval was awarded for all the studies by the Grampian Research Ethics Committee, our local IRB.2; 10 mM dNTPs; 10 pmol/\u03bcL of each primer   were gen2; 10 mM dNTPs; 10 pmol/\u03bcL of each primer . The Fisher exact test statistical analysis was applied to calculate Haplotype association and asthma status. Cross-tabulation statistical analyses were applied to calculate haplotype association and asthma status. These analyses suggested that haplotype 2 was associated with a high risk of asthma (P=.01), and haplotypes 3 and 4 were associated with controls (P=.0001).An EH algorithm computer program was used to enumerate all possible haplotypes of adjacent markers by considering all haplotype lengths. For each haplotype, we counted the frequency of its occurrence among trait-associated and controls. Visual inspection suggested a clustering of apparently identical segments between distinct haplotypes in the region of these chemokine receptors, possibly reminiscent of a conserved founder haplotype. Analysis was done of the relationship between the genotypes defined by the combination of CCR5\u039432, CCR2-G190A, and CCR3-T51C polymorphisms in relation to asthma with or without atopy. The frequencies of individuals and haplotypes meeting for unrelated parents and children, cases, and controls are shown in Genetic linkage analyses have been successful in the identification of genes involved in many single gene disorders such as cystic fibrosis and have also been used with limited success in disorders involving many genes and environmental factors such as hypertension29In the current study, an attempt was made to use genetic linkage analysis to assess the role of the chromosome 3p21.3 region in asthma with or without atopy. Computer simulation revealed the limited power of the pedigrees used in this study by showing that there was only 10% probability of obtaining a lodscore >3.0 for a marker located at \u03b8=0.01 from the hypothetical trait locus. Actual linkage analysis performed on the 12 pedigrees showed positive values for lodscores across the entire region of chromosome 3p21.3 with a maximum value of 3.90 at the CCR5 gene (\u03b8=.00). High LOD scores for the \u039432 allele, compared to the theoretical maximum, might have been in part due to the layout of the parameter files and the fact that the \u039432 allele had a high allele frequency in the present population. Although a dominant effect was assumed in the analysis, other models, including recessive or partial penetrance, would have been unlikely to change the overall results because of the high allele frequency and the large number of affected individuals in the families studied. This might limit the detection of linkage, especially for a complex disease such as asthma.Haplotype analyses with the EH program suggested significant linkage disequilibrium between hypothetical SNPs within chemokine receptors and asthma and supported the suggestion that the -G190A polymorphism of CCR2 was protective against asthma.13In summary, the present study demonstrated the utility of linkage analysis in asthma. However, the availability of large multigenerational pedigrees remains a major hurdle, especially in complex diseases such as asthma or atopy. Heterogeneity among different populations for genetic factors contributing to complex traits such as asthma or atopy also complicates linkage analysis.The linkage and haplotype analyses using a panel of SNPs in three adjacent chemokine receptor genes as markers in this study suggested that there was an association between chromosome 3 and asthma. However, a larger data set would be required to confirm the suggestions from these haplotype analyses due to the low number of individuals in some of haplotype fields."}
+{"text": "Dissecting the genetic complexity of ASD may require phenotypic data reflecting more detail than is offered by a categorical clinical diagnosis. Such data are available from the Social Responsiveness Scale (SRS) which is a continuous, quantitative measure of social ability giving scores that range from significant impairment to above average ability.Autism Spectrum DisorderWe present genome-wide results for 64 multiplex and extended families ranging from two to nine generations. SRS scores were available from 518 genotyped pedigree subjects, including affected and unaffected relatives. Genotypes from the Illumina 6 k single nucleotide polymorphism panel were provided by the Center for Inherited Disease Research. Quantitative and qualitative analyses were done using MCLINK, a software package that uses Markov chain Monte Carlo (MCMC) methods to perform multilocus linkage analysis on large extended pedigrees.When analysed as a qualitative trait, linkage occurred in the same locations as in our previous affected-only genome scan of these families, with findings on chromosomes 7q31.1-q32.3 [heterogeneity logarithm of the odds (HLOD) = 2.91], 15q13.3 (HLOD = 3.64), and 13q12.3 (HLOD = 2.23). Additional positive qualitative results were seen on chromosomes 6 and 10 in regions that may be of interest for other neuropsychiatric disorders. When analysed as a quantitative trait, results replicated a peak found in an independent sample using quantitative SRS scores on chromosome 11p15.1-p15.4 (HLOD = 2.77). Additional positive quantitative results were seen on chromosomes 7, 9, and 19.The SRS linkage peaks reported here substantially overlap with peaks found in our previous affected-only genome scan of clinical diagnosis. In addition, we replicated a previous SRS peak in an independent sample. These results suggest the SRS is a robust and useful phenotype measure for genetic linkage studies of ASD. Finally, analyses of SRS scores revealed linkage peaks overlapping with evidence from other studies of neuropsychiatric diseases. The information available from the SRS itself may, therefore, reveal locations for autism susceptibility genes that would not otherwise be detected. Compelling evidence exists to suggest that autism spectrum disorder (ASD) has a complex heterogeneous genetic aetiology . HoweverRelatives of individuals with ASD have subclinical traits related to autism more frequently than the relatives of controls -8. TheseThe social, behavioural and communication characteristics of ASD are common and may be viewed as on a continuum with possible cutoffs for affected individuals ,14. The We report genome-wide SRS linkage results using single nucleotide polymorphism (SNP) data genotyped on a unique resource of large extended and smaller multiplex ASD families from Utah. We have previously reported linkage evidence in these families using clinical diagnostic status and only including information from affected pedigree members. The current study has two advantages. First, we include additional phenotype information above and beyond our previous affected-only genome scan by using the quantitative SRS measure: clinically unaffected relatives can contribute information to this analysis. The SRS phenotype therefore adds to our previous study in our extended pedigrees. Second, we add to other previous studies of the SRS which focus on nuclear families. The likely heterogeneity of ASD increases the value of studies that include extended pedigrees ,22. Collhttp://www.hci.utah.edu/groups/ppr/. Previous studies have shown low rates of inbreeding within the UPDB [et al. [et al. [Subjects were initially ascertained from 70 pedigrees that had at least two family members with an ASD. Six families did not have sufficient SRS phenotype information (defined as at least two family members with SRS scores) and were therefore dropped from this analysis. A total of 629 genotyped subjects from 64 families were used here, 181 of whom were defined as having either a strictly defined autistic disorder (AD) or a more broadly defined ASD. Table the UPDB ,26. Usin [et al. . In addi [et al. .Families interested in participating were asked to give questionnaire consent, to give initial information regarding possible exclusion criteria and to complete the Social Communication Questionnaire (SCQ) . The SCQAll available subjects were given the SRS. Completed questionnaires were obtained from a total of 523 family members who were genotyped. Completed questionnaires were obtained for 92.1% of index cases, 83.9% of siblings (affected and unaffected), 87.2% of fathers, 78.8% of mothers and 73.7% of more extended relatives . The child version of the SRS was completed for 205 subjects and the adult version was completed by 318 subjects. The child version was filled out by a parent for almost all cases . The adult version was usually filled out by a spouse  but parents were raters for adult siblings over age 18 . Other informants filled out the remainder of the adult SRS forms . No children were rated by a parent with ASD. However, after surveys were returned, five adult raters were found to have ASD. Scores from these five raters (fathers) on their spouses (mothers) were not used for subsequent analyses, leaving 518 subjects with scores. There were an additional four children whose parent was coded as a poor rater on the ADI for under-reporting of behaviours. While no data regarding rater quality were available for the SRS directly, the similarity of content across the ADI and SRS made ratings done by these parents was suspect. Scores for these subjects were therefore not used in the quantitative analyses of data. For qualitative SRS analysis, data on these subjects were not deleted because possible under-reporting on the SRS did not impact upon the SRS category.Genotyping services were provided by the Center for Inherited Disease Research (CIDR) using the 6 K single nucleotide polymorphism (SNP) Illumina Linkage Panel 12, which includes 6090 SNP markers, with an average genetic coverage of 0.65 cM. There were 46 SNPs who were dropped due to poor performance, leaving 6044 SNPs. Data from 653 pedigree subjects passed stringent quality control criteria for initial inclusion in the study . Of the r2 value of 0.05 between SNPs has now been supported with extensive simulation studies [R2 of 0.33 regressed simultaneously over all SNPs in the selected window. This R2 considers not only the correlations between SNPs but also between linear combinations of SNPs [r2 of approximately 0.05. This screening for LD removed 1207 SNPs. Also, as part of the validation procedure, we removed 115 SNPs with a minor allele frequency less than 0.10 and 4 SNPs out of Hardy-Weinberg equilibrium. The total number of SNPs left after this phase was 4718.Eliminating linkage disequilibrium (LD) between markers in linkage studies has been strongly recommended, as false positive results can occur in the presence of LD -36. As t studies . Therefo studies , which r of SNPs  and corrWe used the genetic map provided by CIDR based on the deCODE genetic map . Base paThis qualitative phenotype was tested using MCLINK, a Markov chain Monte Carlo (MCMC) method that allows for multilocus linkage analysis on large extended pedigrees . Using bP-values from the non-parametric qualitative analyses were converted to LOD scores for ease of interpretation and comparison with the parametric results. We present results in the commonly used framework of significance thresholds proposed by Lander and Kruglyak [P = 0.0017) represents suggestive evidence and LOD score \u2265 3.30 (P = 0.000048) represents significant evidence. We recognize that our study design does not match perfectly with the design that generated these thresholds but they do provide reasonable benchmarks which enable us to organize and present our data. All P-values are theoretical, not empirical.We performed a non-parametric analysis and a general parametric model-based analysis using simple dominant and recessive model parameters that reproduced the reported population frequency of ASD . For theKruglyak : LOD scoIn order to analyse the SRS as a quantitative trait, we first applied a square root transform the SRS data to adjust for non-normality in the distribution, as has been done with previous SRS analyses . The effIn summary, we note that use of the SRS results in three important differences between this analysis and our previous affected-only analysis using clinical diagnosis on these families. First, this analysis includes information from an additional 337 clinically unaffected relatives. Second, for the qualitative analysis, affection status was defined using SRS data rather than clinical measures. Third, a quantitative analysis uses a full range of scores on autism-related traits in all measured pedigree members.Diagnostic and Statistical Manual of Mental Disorders (DSM-IV-TR criteria). One hundred and thirty-five affected subjects met criteria for strictly defined autistic disorder (AD) and 46 met criteria for an ASD. There was a 7.4:1 male:female ratio among the subjects with strictly defined autism, which fell to 3.6:1 among the subjects with an ASD. For all subjects combined, the male:female ratio was 5.96:1. As expected, ADI-R scores were significantly higher for the autism group compared to the ASD group . The nonverbal total cannot be compared because no ASD subjects were nonverbal. Quantitative scores on the ADOS are not compared because of differences between modules but significantly more individuals in the autism group were administered Modules 1 and 2 compared to the ASD group.Table t = 3.71, P = 0.0003) and between ASD and other relatives . Among affected subjects, substantial correlations were observed between ADI domain scores and the SRS total score. The highest correlations were observed with the ADI social domain score, with a correlation of 0.60 among ASD subjects and a correlation of 0.46 among subjects with AD. For ASD subjects, 17.1% scored in the normal range on the SRS, 39.0% scored in the spectrum range and 43.9% scored in the affected range. Among subjects with a clinical diagnosis of autism, 5.6% scored in the normal range, 19.1% scored in the spectrum range and 75.4% scored in the affected range. The 95 genotyped spouse pairs showed a significant spouse correlation of 0.33 (P = 0.001) using the square root transformed adjusted scores.Regression adjustment of the SRS for age and gender effects resulted in parameter estimates of -0.074 for age and 1.6 for gender . Figure et al. [The genome-wide analyses of the data are summarized in Table et al. .Figure We also saw evidence for linkage in other locations  in the same region as the evidence from the qualitative analyses. Our next most significant peak was on chromosome 11p15.1-p15.4 spanning a region from 7,300,000 bp to 22,400,000 bp, with a maximum LOD of 2.77 at two adjacent SNPs (rs214101 and rs4757574) from a parametric analysis. The base pair location of our peak  is well within one of the most significant peaks previously reported by Duvall et al.  in theiret al. ,57.The present genome-wide linkage scan of SRS scores was performed using 518 relatives from a unique resource of large extended and smaller multiplex ASD families from Utah. We found genome-wide significant linkage on chromosome 15q13.3, and several other suggestive findings. Peaks on chromosomes 7q, 13q and 15q were also found in our previous genome scan of affected-only subjects in these families based on clinical diagnosis . SRS quaThe SRS was developed as a continuous measure of autistic traits, with variation intended to extend across affected subjects and unaffected relatives. Among affected subjects, our data suggest substantial correlations between clinical measures of autism from the ADI and SRS total scores, particularly for the social behavioural domain. ADI and SRS are expected to differ to some degree because ADI scores are based on information from the developmental period between ages 4-5 and also on the most severe presentation through the individual's life, while the SRS focuses on current behaviour. In addition, most clinically affected subjects scored in the spectrum or affected ranges on the SRS, though as expected, we observed a substantial range of scores. Most of the clinically unaffected relatives scored in the normal range on the SRS (87.4%), but considerable variation also occurred in these scores.N = 14) scored within the normal range on the SRS and therefore also contributed different information to the current scan. Our families also replicated previous evidence for significant SRS spouse resemblance  [In our previous genome-wide scan, all relatives with no clinical diagnosis were considered unknown, so results were based on phenotypic information from 192 genotyped affected cases. Using the SRS in the present study, we added information for 337 clinically unaffected relatives, though 11 clinically affected subjects from the previous scan were missing SRS scores and were not included here. A small percentage of clinically diagnosed relatives (= 0.001) .10(5) = 0.7 LOD score units [The new information provided by the SRS in these families revealed additional findings beyond our previous affected-only clinical diagnosis scan . Not surre units  for theset al. [Our genome-wide significant result on chromosome 15q13.3 was supported by the qualitative NPL and recessive models and some lesser evidence also by the quantitative analysis  has been implicated previously by other studies -63. Our Other peaks were observed on chromosomes 6 and 10. The regions on chromosome 6p25.3 and 6p22.1 have appeared in schizophrenia studies -68 and aet al. [A quantitative analysis also revealed findings of interest. While results using the quantitative range of the SRS often paralleled the qualitative findings, there were interesting exceptions. We note that while we used the same genetic analysis software, the variation in the trait was treated quite differently, so direct overlap of findings would not necessarily be expected. For the quantitative analysis, we also note that adjusting for age and gender may have been a conservative adjustment, as clinical affection status is associated with age and gender. As a result of this adjustment, our quantitative results are unlikely to be due to behaviours that change with age or behaviour specific to gender. Our highest quantitative peak was on chromosome 7 in the same region as described above. Our next highest quantitative peak was on chromosome 11p15.5-p15.4, spanning a region from 7,300,000 bp to 22,400,000 bp (12.11 - 37.63 cM). This peak replicates a previously reported SRS peak from 15-45 cM found in an analysis of quantitative SRS scores . Liu et et al.  also fouet al. . On chroet al.  and alsoet al. .Finally, we observed peaks in two other regions previously identified in our families in scans using affection status. We observed a quantitative peak on chromosome 9p24.3, from the telomere to about 4,330,000 bp. This peak has been previously reported for obsessive compulsive disorder (OCD) ,81. The While it is important to seek replication of these findings in independent samples, we intend first to pursue these findings within our sample families. It is possible that findings may be unique to our family sample; indeed, a rare genetic finding for autism would be our expectation. However, if we can find genetic variation responsible for these statistical findings, the results may still provide relevant evidence regarding biological mechanisms and genetic pathways implicated in autism. Our family data will allow us to investigate segregation of the genetic variants responsible for these peaks and to determine more detailed associations with phenotypes related to autism. The extended family design allows a more powerful tool to investigate rare genetic variants in relation to phenotype than the genome-wide association study design.et al. study [In summary, our results highlight three important points. First, using a quantitative analysis, we replicated a previously reported SRS peak on chromosome 11 peak found in the AGRE family sample . This rel. study  includedl. study . These pAD: autistic disorder; ADHD: attention deficit and hyperactivity disorder; ADI-R: Autism Diagnostic Interview-Revised; ADOS-G: Autism Diagnostic Observation Schedule-Generic; ASD: autism spectrum disorder; BAP: broader autism phenotype; CIDR: Center for Inherited Disease Research; CNV: copy number variation; HLOD: heterogeneity logarithm of the odds (LOD); IRB: Institutional Review Board; LD: linkage disequilibrium; MCMC: Markov chain Monte Carlo; NPL: nonparametric linkage; OCD: obsessive compulsive disorder; SCQ: Social Communication Questionnaire; SNP: single nucleotide polymorphism; SRS: Social Responsiveness Scale; UPDB: Utah population database.HC, WMM, and JSM received partial salary support from Lineagen Inc. (www.lineagen.com) from 12/1/07 to 12/31/08.  This salary support is not ongoing.HC conceived the study, participated in its design, directed the statistical analyses and drafted the manuscript. MEV verified the accuracy of the phenotype data and made contributions to the manuscript. RJR assisted with the interpretation of the results and helped draft the manuscript. NJC, DSC, and KAB helped with the statistical analyses and helped draft the manuscript. JSM confirmed the research diagnoses of ASD, supervised the collection of all phenotype data and made significant contributions to the interpretation of results. WMM participated in the design of the study and helped draft the manuscript. All authors read and approved the final manuscript."}
+{"text": "Salmo salar) from North America and Europe are genetically distinct. These observations are supported by karyotype analysis, which revealed that North American Atlantic salmon have 27 pairs of chromosomes whereas European salmon have 29 pairs. We set out to construct a linkage map for a North American Atlantic salmon family and to compare this map with the well developed map for European Atlantic salmon.Several lines of evidence including allozyme analysis, restriction digest patterns and sequencing of mtDNA as well as mini- and micro-satellite allele frequencies indicate that Atlantic salmon  whose parents were derived from the Saint John River stock in New Brunswick, Canada. As large differences in recombination rates between female and male Atlantic salmon have been noted, separate genetic maps were constructed for each sex. The female linkage map comprises 218 markers in 37 linkage groups while the male map has 226 markers in 28 linkage groups. We combined 280 markers from the female and male maps into 27 composite linkage groups, which correspond to the haploid number of chromosomes in Atlantic salmon from the Western Atlantic.A comparison of the composite NB1 and SALMAP linkage maps revealed the reason for the difference in the chromosome numbers between European and North American Atlantic salmon: Linkage groups AS-4 and AS-32 in the Scottish salmon, which correspond to chromosomes Ssa-6 and Ssa-22, are combined into a single NB1 linkage group as are linkage groups AS-21 and AS-33 (corresponding to chromosomes Ssa-26 and Ssa-28). The comparison of the linkage maps also suggested some additional chromosomal rearrangements, but it will require finer mapping, potentially using SNPs, to test these predictions. Our results provide the first comparison of the genomic architecture of Atlantic salmon from North America and Europe with respect to chromosome organization. Salmo comprises two main species: brown trout (S. trutta) and Atlantic salmon  [The genus . salar) . Brown t. salar) ,3. This . salar) .Tf) [1 Tfis present in all populations, whereas Tf2 is only present in European Atlantic salmon and the 3 Tfand 4 Tfalleles are restricted to North American salmon. This substantial difference prompted Payne et al. [Salmo salar europaeus and Salmo salar americanus, respectively. Allozyme analysis gave similar results for NAD+-dependent malic enzyme [MDH-3,4 [Ssa-A45/2/2 [The first study that indicated that there is a genetic difference between Atlantic salmon from Europe and North America involved four electrophoretic alleles of the serum protein transferrin (Tf) . Tf1 is e et al.  to suggec enzyme  and mala[MDH-3,4 . Restric[MDH-3,4  and dire[MDH-3,4  of mitoc[MDH-3,4  and the -A45/2/2 , have alPerhaps the most convincing genetic evidence that North American and European Atlantic salmon are genetically distinct comes from karyotype analysis. Chromosomal rearrangements, particularly Robertsonian fissions and fusions, are common in salmonids ,13. The Salmo trutta; 2n = 80) produce hybrids in the wild [Despite the genetic and chromosomal differences between Atlantic salmon from Europe and North America, it has been possible to produce fertile hybrids and viable back-crosses; however, no information about relative survival of the offspring was given in this report . The viathe wild  and undethe wild . Therefothe wild .Our goal was to compare the genomic architecture of Atlantic salmon from North America and Europe with respect to chromosome organization. We expected that the chromosomal differences between North American and European Atlantic salmon would be reflected in their genetic maps. As the integration of the genetic map and the karyotype of European Atlantic salmon has recently been completed , we firsThe parents of the NB1 family were part of a broodstock development program based on Saint John River stock. The mitochondrial genomes of the parents were analyzed, and we found that the mitochondrial DNA of both parents had sequences  and haplThe transmission of alleles from each parent to the offspring was used to construct female-specific and male-specific genetic maps for family NB1   corresponding to AS-10 in European Atlantic salmon corresponds to two chromosomes in North American Atlantic salmon. Chromosome Ssa-9 contains two blocks of intra-chromosomal heterochromatin which divides the chromosome into three sections, each of which correspond to a chromosome/linkage group in rainbow trout . This haThe simplest explanation for the difference in the number of chromosomes between the Atlantic salmon from either side of the Atlantic Ocean is that two Robertsonian fissions or fusions having occurred in the European Atlantic salmon (29 chromosome pairs) or North American Atlantic salmon (27 chromosome pairs), respectively. A comparison of the SALMAP composite female linkage map and the composite NB1 genetic map identified two pairs of linkage groups in the SALMAP composite female linkage map that correspond to single linkage groups in the NB1 family. Microsatellite markers in the SALMAP linkage groups AS-4 and AS-32 mapped to a single NB1 male linkage group, NB1-4/32 m  precludes making accurate predictions of this and other rearrangements. This will have to await the production of denser genetic maps, which will become available using an Atlantic salmon SNP microarray .Salmo salar europeaus and Salmo salar americanus, respectively [Here we report the first genetic map for North American Atlantic salmon. A comparison of this map with the corresponding genetic map for European Atlantic salmon led us to propose testable predictions about the evolution of chromosome number and a rationale for the gross chromosomal differences seen between Atlantic salmon from both sides of the Atlantic Ocean. It is somewhat surprising that, despite their chromosomal differences, North American and European Atlantic salmon produce viable, fertile hybrids . The difectively ,5, althoectively .The NB1 mapping family consists of two parents, derived from the Saint John River, New Brunswick, Canada and 40 offspring. The family, which was part of a broodstock development program, was produced in the fall of 2005, and was maintained at Fisheries and Oceans Canada, St. Andrews Biological Station, St. Andrews, New Brunswick. Tissue samples from the parents were obtained at time of spawning. Blood samples were collected from the offspring in the fall of 2006. The offspring were not euthanized at the time of blood collection, and unfortunately it was not possible to determine their gender from external morphological characteristics. DNA was isolated from the blood samples using the PUREGENE\u2122 DNA isolation kit protocol for \"DNA isolation from 2 \u03bcL Non-Mammalian Whole Blood\" .Microsatellite analysis was carried out according to the methods used to construct the European SALMAP Atlantic salmon linkage group . Informahttp://www.uoguelph.ca/~rdanzman/software/LINKMFEX/[et al. [The genotypes for the parents and offspring were entered into the LINKMFEX software LINKMFEX/, which wLINKMFEX/. Genome /[et al. .WSD and BFK conceived and designed the project. SJ, EAD and JP carried out the genotyping. SJ and KPL constructed the NB1 linkage maps and made the comparisons with the SALMAP maps. SW was responsible for producing and rearing the NB1 family. KPL, SJ and WSD prepared the manuscript and the figures. All authors commented on drafts of the manuscript and approved the final version.Table S1 Information on the genetic markers used in this project.Click here for fileFigure S1 Comparison of the merged SALMAP female linkage groups with the corresponding male-specific and female-specific linkage groups from the NB1 family.Click here for fileFigure S2 Continuation of the comparison of the merged SALMAP female linkage groups with the corresponding male-specific and female-specific linkage groups from the NB1 family.Click here for fileFigure S3 Continuation of the comparison of the merged SALMAP female linkage groups with the corresponding male-specific and female-specific linkage groups from the NB1 family.Click here for fileFigure S4 Continuation of the comparison of the merged SALMAP female linkage groups with the corresponding male-specific and female-specific linkage groups from the NB1 family.Click here for fileTable S2 Phase files for NB1 female-specific linkage groups.Click here for fileTable S3 Phase files for NB1male-specific linkage groups.Click here for file"}
+{"text": "P=0.01). This relation, which was also observed in an earlier Australian twin study, could be because of the linkage disequilibrium between D9S942 and a neighbouring functional locus. Further investigation of this region is warranted in large-scale linkage or association studies.Rare mutations in the CDKN2A gene are highly penetrant for melanoma. Density of nevi is under strong genetic control and high density is a potent risk factor for melanoma. We used linkage and association analysis in adolescent twins from the UK to examine the hypothesis that the region containing the CDKN2A gene also contains a quantitative trait locus influencing normal nevus development. Five markers in the CDKN2A region were genotyped in 115 dizygotic twin pairs, and one marker (D9S942) was genotyped in 103 monozygotic twin pairs, all of whom had been phenotyped for nevus density. Linkage analysis showed no evidence of a quantitative trait locus influencing nevus density in this chromosomal region. A model partitioning the variation in phenotype into within- and between-twin pair components showed weak evidence of association between higher nevus density and longer mean length of the two D9S942 alleles ( They found some evidence for linkage. Correlations in log mole counts between dizygotic (DZ) twins sharing 0, 1 and 2 alleles at this locus identical by descent (IBD) were estimated as 0.44, 0.64 and 0.74. As would be expected if a QTL for mole count exists close to this marker, the correlations were heterogeneous , increasing as the number of alleles shared IBD increased. The authors went on to fit a path model to further investigate this locus, and they estimated that the QTL accounted for 27% of the variance in log-transformed mole count. Phenotype was also regressed on the mean length (in base pairs) of the two D9S942 alleles. Results were described for total flat moles, and although this explained less than 1% of the variation, there was significant evidence of association (P=0.004).Rare mutations in the CDKN2A gene at chromosome 9p21 underlie disease susceptibility in up to 40% of multiply affected melanoma families  spanning a 5.7\u2009Mb region round CDKN2A were genotyped in the 115 DZ twins. The three central markers are in linkage disequilibrium and span a region of less than 7\u2009kb within the CDKN2A gene . Nevus counts and densities were log transformed to produce a less skewed phenotype distribution. They were regressed on age and sex, and residuals from the regression were used in further analyses. Intraclass correlation coefficients were then calculated for each of the three haplotype-sharing groups among the DZ twins and for the MZ twins. The Haseman\u2013Elston method, regressing the squared difference in phenotype on the number of haplotypes shared IBD, was applied to test for linkage  using thTo confirm or refute the findings of yij = \u03b1+\u03b3i+\u03b2xij+\u025bijyij is the phenotype for twin j from pair i, \u03b1 is the overall mean, \u03b3i is a random effect term for twin pair i and is assumed to be normally distributed with mean 0 and variance \u03c3u2, xij is the mean allele length for twin j from pair i and \u025bij is the individual level error assumed normally distributed with mean 0 and variance \u03c3e2. This is the simplest form of multilevel model, as described by \u025bij) are assumed to be independent between observations and of the other terms in the model, and the level 2 residuals (\u03b3i) are assumed to be independent across twin pairs. The parameters \u03b2, \u03c3u2 and \u03c3e2 were estimated here by maximum likelihood (xtreg with the mle option in Stata). Under the null hypothesis of no association, \u03b2=0. The analysis was performed using DZ twin pairs only and then using all twin pairs, since the assumptions of a single distribution for the level 1 residuals may be violated by grouping together the MZ and DZ twins.where Descriptive statistics are presented in Table 1Overall for these five markers genotyping was 99% complete on the 115 DZ twin pairs, and a total of 162 parents were also typed. For D9S942, genotyping was complete for 103 (97%) twin pairs. Allele frequencies for D9S942 are shown in Figure 2Among the DZ twins, 32 (28%) twin pairs shared 0, 59 (52%) shared 1 and 22 (19%) shared 2 haplotypes IBD, which was consistent with expected proportions (1\u2009:\u20092\u2009:\u20091) under Mendelian segregation. Correlations in nevus density between co-twins are shown in Table 2\u03b2 estimated to be 0.012, 95% confidence interval , P=0.04). Note that this is a regression coefficient relating phenotype to the individual twin's mean allele length having allowed for the (random) effect of the twin pair and is based on both the within- and between-twin pair information. Longer allele lengths were thus associated with greater nevus density. In keeping with the findings in the Australian study, this mean allele length only accounted for 1% of the overall variation in phenotype. It explained 4% of the within-twin pair variation and less than 1% of the between-pair variation.Based on DZ twins, the random effect model showed weak evidence of association between nevus density and mean D9S942 allele length (regression coefficient P=0.01). Since there is no variation in mean allele length between the two MZ twins in a pair, the MZ pairs contribute no within-pair information but do contribute to the estimate of \u03b2.Including MZ twin pairs in the analysis, the estimated coefficient is 0.013 .As (a), but for all twin pairs.The mean phenotype for a twin pair plotted against the mean allele length for the twin pair in DZ twins .As (c) but for all twin pairs.Figure 3The linkage analysis in this study offers no support for the existence of a QTL influencing nevus density in this region of chromosome 9. The observed pattern of correlations in twins sharing 0, 1 and 2 haplotypes IBD is in fact opposite to that expected in the presence of such a locus, and our data are only consistent with a very modest negative slope in the Haseman\u2013Elston regression. In contrast our association analysis provides some support for a relation between mean D9S942 allele length and nevus density, and the fact that this relation is found within twin pairs indicates that the result cannot be attributed to population stratification.The discrepancy between our linkage results and those of posthoc power calculations on the association analysis using the methods and program of Alternatively, the finding of linkage in the sample of Australian twins and the weak evidence for association in both studies could be false-positive results. The evidence for association, although present in both studies, is not altogether convincing. In both cases only a modest effect on phenotype was found, and Zhu and colleagues estimated that this accounted for less than 6% of the variation in phenotype attributable to the linked QTL. We have done some et al for their choice of analyses, and the analyses they present are all based on regression of phenotype on allele length in one form or another. We found, like Zhu et al, that alternative models regressing phenotype on the length of the shorter allele, the longer allele or both alleles fit the data less well than using mean length. It is possible that the observed association is because of linkage disequilibrium between D9S942 and another functional locus. No association was found between nevus density and the other four markers studied.A further consideration is that an association with mean number of CA repeats is surprising, since the length of such markers is unlikely to affect gene function. However, D9S942 is so polymorphic that some sort of structure or grouping of alleles must be imposed before association can be investigated. No detailed justification was presented by Zhu The age matching inherent in a twin study is advantageous, since nevus development is age-dependent, but it is difficult to achieve very large sample sizes using twins. This region clearly warrants further investigation in much larger association and linkage studies using alternative study designs."}
+{"text": "Dystocia, difficult labour, is a common but also complex problem during childbirth. It can be attributed to either weak contractions of the uterus, a large infant, reduced capacity of the pelvis or combinations of these. Previous studies have indicated that there is a genetic component in the susceptibility of experiencing dystocia. The purpose of this study was to identify susceptibility genes in dystocia.OXT) on chromosome 20 and oxytocin-receptor (OXTR) on chromosome 3.A total of 104 women in 47 families were included where at least two sisters had undergone caesarean section at a gestational length of 286 days or more at their first delivery. Study of medical records and a telephone interview was performed to identify subjects with dystocia. Whole-genome scanning using Affymetrix genotyping-arrays and non-parametric linkage (NPL) analysis was made in 39 women exhibiting the phenotype of dystocia from 19 families. In 68 women re-sequencing was performed of candidate genes showing suggestive linkage: oxytocin (We found a trend towards linkage with suggestive NPL-score (3.15) on chromosome 12p12. Suggestive linkage peaks were observed on chromosomes 3, 4, 6, 10, 20. Re-sequencing of OXT and OXTR did not reveal any causal variants.Dystocia is likely to have a genetic component with variations in multiple genes affecting the patient outcome. We found 6 loci that could be re-evaluated in larger patient cohorts. Dystocia, defined as prolonged and difficult labour, is a common obstetric problem affecting 6-8% of all deliveries . It is aParturition is regulated by many factors and the mechanisms regulating labour and the expulsion of the child are incompletely understood . Animal We used the Swedish Medical Birth Registry  that covFollowing the telephone interviews and the study of the medical records we concluded that the phenotype is heterogeneous since not all of the women had dystocia as an indication for their caesarean section. Given that the primary focus of the study was uterine dysfunction rather than other reasons for dystocia and to enable us to pick the affected sib pairs with the most uniform phenotype for genetic analysis we divided the diagnosis of dystocia into four subgroups: certain dystocia, likely dystocia, unlikely dystocia and no dystocia. These subgroups are described below:1. Certain dystocia: Definite diagnosis , all subsequent deliveries by caesarean section or instrumental delivery and no other indications.3. Unlikely dystocia: No induction of labour, short delivery time (< 20 h), other indication for caesarean i.e. relative disproportion, child \u2265 5000 g, subsequent deliveries without caesarean section or instrumental delivery.4. No dystocia: Absolute disproportion, breech presentation, caesarean section before start of labour, delivery misclassified as caesarean but was actually vaginal.Additional sisters and cousins were invited to participate in families where several women were identified with delivery-related problems. Of the initial 150 women, 56 were excluded and 10 new cases added making the study population a total of 104. Of these 104 women, 83 provided a blood sample for the genetic analysis. Only families with women in group 1 and 2 were used in the genetic analysis with non-parametric linkage (NPL). See Table \u00ae Mapping 10 K 2.0 array containing approximately 10,000 SNP markers following the manufacturer's instructions .Single nucleotide polymorphism (SNP) genotyping was performed for 18 affected sib-pairs and one family with three affected siblings using the Affymetrix GeneChipMERLIN  was usedOXT) and oxytocin receptor (OXTR) genes. Cases were not matched with the reference group, but were used to establish an estimate of the population frequency of potential mutations. Exons including 100 bp flanking sequence on both sides and 1 kb upstream of the first exon were amplified using polymerase chain reaction (PCR). Purified PCR products were sequenced using DYEnamic ET dye terminator kit following manufacturer's instructions  and electrophoresed using a MegaBACE 1000 instrument . Sequence analysis was performed using the MegaBACE Sequence Analyser 3.0 software (Amersham Biosciences) and Staden package computer programs [Genomic DNA from 68 affected individuals with dystocia and a historic reference material consisting of 107 healthy women without adverse obstetric history who had given a written consent to participate in studies on complications of pregnancy were used to sequence oxytocin  exhibiting a phenotype of certain or likely dystocia. Since many of the subjects were post-term, 53 (51.0%) of them had undergone induction of labour. In 16 of the 47 families (34.0%), the mother had had dystocia or some other obstetric problem such as instrumental or breech delivery. Thirty-eight (36.5%) women said that the delivery had been a bad or very bad experience and 22 (21.2%) said that their first delivery had negatively influenced the number of children they had born. Clinical characteristics have been collected in Table .1% exhibAccording to simulations performed with Merlin, significant P-value (identified by an NPL-score reached in 5% of simulations) would have corresponded to an NPL-score of 3.64, while suggestive P-value (identified by an NPL-score reached at least once per simulation) would have corresponded to and NPL-score of 1.98. Our best linkage peak was located on chromosome 12p12 (NPL score 3.15), and other peaks with suggestive linkage were found on chromosomes 3, 4, 6 and 20. Linkage results are summarized in Table OXT and OXTR exons including splice sites and putative promoter regions in five individuals with dystocia and one control to detect common variations. One known polymorphism was detected within the OXT locus, but did not differ in allele frequency from that expected from dbSNP data. We were unable to sequence exon 2 due to high (> 70%) GC content. Sixteen variations were identified within the OXTR locus, of which four were novel and the remaining 12 were included in dbSNP with rs numbers. Eleven OXTR polymorphisms were selected for sequencing in 68 dystocia cases  and oxytocin receptor (OXTR), both of which are obvious candidate genes, did not allow us to identify any potential causal mutations. However, it is possible that we have overseen regulatory variants affecting gene expression, mRNA stability or localization of protein product. It is also possible that we have overlooked true candidate genes on regions showing suggestive linkage on chromosomes 4, 6, 10 and 12. We chose comparison with a healthy historic reference material for the re-sequencing of OXT and OXTR. Evidently this group differs from our cases regarding age, BMI, gestational length and birth weight but we did not manage to find any mutations. If we actually had found any mutations a comparison with matched controls would of course have been preferable.According to our knowledge, this is the first paper assessing the genetic origin of dystocia through non-parametric linkage analysis. There is strong suggestive evidence of linkage at chromosome 12p12 and we found several possible genes in the areas of interest but none that struck as being solely responsible for this condition. The re-sequencing of oxytocin  declined to participate directly at the telephone interview and no case of offending a woman by this direct approach was encountered. On the contrary, the majority of women were willing to donate blood samples without any compensation. Thus, the direct approach appears feasible and has great potential to increase the scientific value of medical registers.This study indicates that dystocia is a complex disease that is probably not caused by a single locus disease allele. Knowledge of the genetic architecture of complex diseases is still incomplete and it is possible that the risk for any common disease is dependent on a large number of loci, each with a number of low frequency disease-predisposing alleles . The stuDuring evolution there is balance between mutation and selection and since dystocia-causing mutations would not be brought on to the next generation in the absence of the possibility of caesarean section, there is most likely genetic heterogeneity responsible for the phenotype. The disease-causing alleles can of course also be propagated by male offspring, but we have so far not studied any possible male phenotype.Since the occurrence of dystocia is likely to involve allelic variations in several different loci we think that it might be very cumbersome and costly to collect a large enough sample set to reach significance in any single locus with genome-wide linkage analysis. An alternative and complimentary approach would be to focus on extensive re-sequencing of candidate genes in affected individuals and controls to assess genetic variability within candidate loci and identify possible causal variants. We have focused on the maternal genotypes but during pregnancy it is probably of interest to take the genetics of the child into account as well. For example, our study does not assess whether dystocia risk is affected by the properties of the fetus, but this might be worthwhile to pursue further.In the overall assessment of a woman giving birth the obstetrician might find the knowledge that epidemiological studies have shown an increased risk of dystocia in a primiparous woman if her mother or sister has experienced the same problem to be useful. Our study indicates that there actually might be a genetic background for dystocia through a strong suggestive evidence of linkage at chromosome 12p12 and also at 5 other loci that might be of interest. We believe that larger studies including patients with a well defined phenotype of dystocia might lead to new insights into the aetiology and physiology of this important condition.The authors declare that they have no competing interests.MA participated in planning the study, collected the material, participated in the genetic and statistical analysis and wrote the main part of the manuscript. HP and KK participated in designing the study, and made the main part of the NPL-analysis and re-sequencing as well as the statistical analysis. JK and MW originated the study, participated in its design and helped in writing the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2350/11/105/prepub"}
+{"text": "ACTANE  Consortium. Parametric and non-parametric linkage (NPL) analyses were performed. Two-point LOD scores failed to show evidence of linkage at any marker (maximum two-point LOD score = 0.40 at recombination fraction \u03b8 = 0.2 with marker D1S2850). Using a multipoint heterogeneity analysis, the estimated proportion of families linked to this putative locus (\u03b1) was 0% (95% CI = 0.00\u20130.33). Non-parametric linkage analysis also found no evidence of linkage . This analysis of 131 ACTANE families does not support the presence of a locus for a prostate cancer susceptibility gene at 1q42.2\u201343. Although we cannot rule out the existence of such a locus, analysis indicates that less than 16% of families could be linked to this region. These findings may be a reflection of the locus heterogeneity involved in this disease indicating that there are still other major susceptibility loci to be identified. \u00a9 2000 Cancer Research Campaign http://www.bjcancer.comGenetic linkage studies worldwide have proposed various chromosomal localizations for prostate cancer susceptibility genes. A recent study found evidence for linkage to chromosome 1q42.2\u201343. The aim of our study was to attempt to confirm these findings by performing linkage analysis in 131 families with multiple prostate cancer cases selected from the"}
+{"text": "To account for systematic variation known to exist in microarray data, it is critical to properly normalize gene expression traits before performing genetic linkage analyses. However, imposing equal means and variances across pedigrees can over-correct for the true biological variation by ignoring familial correlations in expression values. We applied the robust multiarray average (RMA) method to gene expression trait data from 14 Centre d'Etude du Polymorphisme Humain (CEPH) Utah pedigrees provided by GAW15 . We compared the RMA normalization method using within-pedigree pools to RMA normalization using all individuals in a single pool, which ignores pedigree membership, and investigated the effects of these different methods on 18 gene expression traits previously found to be linked to regions containing the corresponding structural locus. Familial correlation coefficients of the expressed traits were stronger when traits were normalized within pedigrees. Surprisingly, the linkage plots for these traits were similar, suggesting that although heritability increases when traits are normalized within pedigrees, the strength of linkage evidence does not necessarily change substantially. Genetical genomics  integratRMA adjusts for systematic variation by performing a quantile normalization procedure, which assumes that the data for the variable considered (such as study sample) all are sampled from the same or similar distributions and the values for that variable are then normalized to a standard distribution. However, it is not yet known which standard distribution is the best to use for family data in genetical genomics studies. Linkage analysis utilizes data from pedigrees, which usually have more homogeneity within pedigrees for the trait under study than between pedigrees, both biologically and environmentally. Thus, the increased background sharing within pedigrees can result in increased correlation between related individuals for expression levels. Consequently, the familial correlation within each pedigree due to biological similarity is of interest in linkage studies. Therefore, we hypothesized that linkage analysis that uses expression trait data normalized by ignoring pedigree membership could improperly 'correct' the trait values by imposing equal means and variances across pedigrees. A conceptual graph Fig.  plots thOur aim is to maintain the individual familial distributions with normalization. We address this problem of normalization of expression data within pedigrees by comparing two possible standard distributions for normalization: 1) applying RMA across all arrays as a pool, which assumes all individuals are independent and share the same distribution of trait values, and 2) applying RMA to arrays within pedigrees assuming family members within a pedigree share the same distribution.Study subjects consisted of 194 individuals from 14 Centre d'Etude du Polymorphisme Humain (CEPH) Utah pedigrees, and 2882 autosomal and X-linked single-nucleotide polymorphism (SNP) genotypes were available from The SNP Consortium .t-tests using 8793 gene expression values of four founders of one family in two ways: comparing the values of the four founders after normalizing using themselves as the pool to paired gene expression values after normalizing 1) using all family members including themselves as the pool, and 2) using independent individuals as the pool (other grandparents from other families) [p-values for these paired t-tests if only random variation is removed by each method. We assumed that these t-tests indicated a significant difference in the normalization methods if the p-value was less than a conservative p-value of 0.001 . However, we also evaluated this using a p-value of 0.05 as the significance threshold.In order to examine the effect of using different normalization pools consisting of different types of individuals, we performed paired cis-acting transcriptional regulator phenotypes with previous evidence of cis-acting linkage to the known location of each corresponding structural gene [p-values of the nonparametric linkage score [To examine the effect of normalization methods on linkage results, we selected 18 ral gene . Nonparage score . Based op-value < 0.001 and 91% had a p-value < 0.05. Interestingly, 95% of the genes had a p-value < 0.001 and 99.5% had a p-value < 0.05 when comparing data normalized using the four individuals as their own normalization pool versus using independent individuals as the normalization pool. This suggests that using independent individuals as the normalization pool may be removing biological variation in addition to removing random error. Figure When comparing the gene expression data normalized using four individuals as their own normalization pool versus using their family members as a pool, 39% of traits had a CPNE1, CSTB, ICAP_1A, and TM7SF3 have negative familial correlation coefficients . The difference in maximum LOD scores between the methods ranged from 0.7 to 0.01. When we compared the negative log p-values of the NPL scores, the patterns of most genes normalized within-pedigree (red solid line) and using all individuals in the normalization pool (black dashed line) were similar except for RPS26 and CTSH  when traits were normalized within pedigrees. This is not surprising because the increased correlation of a quantitative trait within a family may decrease evidence for linkage. It is also possible that linkage signals are inflated when normalizing using all individuals as the pool. Because we did not evaluate linkage of these 18 traits to all markers on other chromosomes in this data set, we cannot evaluate possible inflation of linkage signals. However, the pattern of the chromosomal linkage plot for these genes (15/18) did not differ across the two normalization strategies. This lack of difference suggests that normalization within pedigrees may not be necessary, at least in studies with small pedigree sizes. However future linkage studies on expression data with larger pedigree sizes and/or large sample sizes may benefit from normalization by pedigree.The author(s) declare that they have no competing interests."}
+{"text": "The influence of certain alleles of the HLA-DRB1 locus on risk for rheumatoid arthritis has been well established through linkage and association studies. In addition, other loci in the HLA region on 6p21 may also affect an individual's risk profile. Here, we used a method to detect excess identity-by-descent sharing between affected sib pairs conditional on the observed genotypes at the hypothesized causal locus to test for the presence of additional arthritis risk loci in the linked region. We used affected sib pairs from two different studies. Because the test depends heavily on specifying accurate allele frequency estimates at the proposed causal locus, we used HLA-DRB1 allele frequency estimates from a large, population-based sample. We also discuss an alternate form of the test in which we could condition on parental genotypes, thereby eliminating the need for actual allele frequencies. The test showed no evidence for the presence of additional arthritis risk loci in the region in the British or North American samples made available for Genetic Analysis Workshop 15. Given the prior knowledge that there likely are arthritis risk loci other than HLA-DRB1 in the region, it appears the tests may have inadequate power to detect the presence of these loci in certain cases. There is substantial evidence from linkage and association studies for a locus contributing to rheumatoid arthritis (RA) risk on chromosome 6p21. The HLA-DRB1 locus may be the sole cause of this linkage signal, but given the complexity of the HLA region and autoimmune function, other polymorphisms in the region could also influence RA risk. In addition, previous research suggests the presence of other RA risk loci in the region . The anaFor these analyses we used data from two RA studies, the North American Rheumatoid Arthritis Consortium (NARAC), and The Arthritis and Rheumatism Council's UK National Repository of family material. Details about the designs of these studies are published elsewhere -7, but b.We initially used Merlin  to confiWe used the method described by Sun et al.  to deterH0 is the probability that the hypothesized causal marker is the sole cause of the linkage signal, I is the IBD sharing for a sib pair at the candidate locus, and GC indicates the sibs' genotype configuration at the locus. Sun et al. use the distribution of IBD sharing between affected sibs given the sibs' genotypes at the candidate locus, GC,  to obtain the null conditional mean sharing, \u03bcG, which is equal to EH0[S], and variance, \u03c3G2, which is equal to VarH0[S] for some IBD sharing statistic S. A variation of the usual NPL score statistic or the linear or exponential likelihood of Kong and Cox [G}/\u03c3G is then used to assess evidence against H0. We used population allele frequencies from published results by Klitz et al. [G. To obtain the actual sharing between sibs, we used Merlin [where Pr and Cox  based onz et al.  from a sd Merlin  to calcuC in Eq. (1) is replaced by the parental and ASP genotypes, {GP, GC}, and \u03bcG and \u03c3G are based on the IBD distribution given the ASP and parental candidate SNP genotypes.We also considered a modified version of the method of Sun et al. proposed by Biernacka et al. . In thisp = 4.28 \u00d7 10-16), and the peak score in the UK families was at marker D6S276 . We also confirmed the association of HLA-DRB1 alleles with RA in the NARAC data set. In NARAC families, the odds ratio for each additional risk allele was 18.2, (p < 0.0001). One SNP, rs910516, is located approximately 5 Mb from D6S1629 and suggests association with RA in the NARAC data (p = 0.01). There is significant LD between D6S1629 and HLA-DRB1 alleles; only one high-risk allele (1402), however, is in significant LD with an allele (3) of D6S1629 (D' = 0.64).Using both the NARAC and UK data, we confirmed the evidence for linkage to RA on chromosome 6. The peak LOD score in NARAC families was at marker D6S1629 , suggesting that the HLA-DRB1 locus may be the sole contributor to the linkage signal. We got a similar result in the 309 ASP from the UK data . Table The Sun et al. test in the HLA-DRB1 region, computed in 452 Caucasian ASPs from the NARAC data failed to reject the null  declare that they have no competing interests."}
+{"text": "Despite the current trend towards large epidemiological studies of unrelated individuals, linkage studies in families are still thoroughly being utilized as tools for disease gene mapping. The use of the single-nucleotide-polymorphisms (SNP) array technology in genotyping of family data has the potential to provide more informative linkage data. Nevertheless, SNP array data are not immune to genotyping error which, as has been suggested in the past, could dramatically affect the evidence for linkage especially in selective designs such as affected sib pair (ASP) designs. The influence of genotyping error on selective designs for continuous traits has not been assessed yet.We use the identity-by-descent (IBD) regression-based paradigm for linkage testing to analytically quantify the effect of simple genotyping error models under specific selection schemes for sibling pairs. We show, for example, that in extremely concordant (EC) designs, genotyping error leads to decreased power whereas it leads to increased type I error in extremely discordant (ED) designs. Perhaps surprisingly, the effect of genotyping error on inference is most severe in designs where selection is least extreme. We suggest a genomic control for genotyping errors via a simple modification of the intercept in the regression for linkage.This study extends earlier findings: genotyping error can substantially affect type I error and power in selective designs for continuous traits. Designs involving both EC and ED sib pairs are fairly immune to genotyping error. When those designs are not feasible the simple genomic control strategy that we suggest offers the potential to deliver more robust inference, especially if genotyping is carried out by SNP array technology. Linkage analysis of family data have been extensively used in the past in the search for genetic determinants. Nowadays, investigators favor large epidemiological studies of unrelated individuals, however several family datasets are currently being re-analyzed and/or pooled (e.g. ). The peIn the search for genetic determinants of complex traits by linkage, the use of selective designs appears to be an efficient way to gain adequate power for detection of typically small gene effects. A few authors have shown by simulation that the impact of genotyping error on evidence for linkage could be particularly severe in affected sib-pair (ASP) designs -6, virtupopulation frequency error model and false homozygosity model), we show analytically what effects such error generating processes (occurring at rate \u03f5 per sib pair) induce for an idealized fully informative marker. It is shown that it results in a reduction of the slope estimate (i.e. of the estimated linkage parameter) by a factor 1 - A method of choice is now emerging for the analysis of quantitative traits arising from selected sib pairs. This method is essentially a regression through the origin of excess identical by descent (IBD) sharing on a function of the trait value, whose slope is an estimate of the linkage parameter. It was first proposed by Sham et al.  and turnpopulation frequency error model and the false homozygosity model. In those two models, we consider a single marker with m alleles and further assume that a maximum of one allelic error per sib pair can be made and that this happens with probability \u03f5. This restriction to 'one error per sib pair' is just a first order approximation, for small \u03f5, of a process where all four alleles would be allowed to be independently erroneous and does not restrict the generalizability of our results.We consider two mechanisms for the generation of errors in marker data, namely the population frequency error model re-assigns the erroneous allele (chosen at random among the four forming the sib-pair genotype) to one of the possible m alleles with probability equal to population allele frequency. One mathematical advantage of this model is that the marginal distribution of alleles and genotypes is unaltered. The false homozygosity model keeps homozygotes unchanged but re-assigns heterozygotes to homozygotes with alleles equal to one of the two original alleles chosen according to probabilities proportional to population allele frequencies.The false homozygosity is a common type of error: fairly rare alleles go un-reported in samples. The population frequency error model provides an approximation to a process whereby alleles are misread. Errors at the two alleles of a marker's genotype might be correlated, we do not consider this type of process in details here although the effect on linkage will be qualitatively the same as in the two other models. We refer the reader to Sobel et al. [To our knowledge, l et al.  for a del et al. ,11.\u03c0 the proportion of alleles shared identical by descent (IBD) at a certain locus by two siblings. Tests for linkage are based on the IBD sharing distribution and although errors as described earlier are made at the genotype level (G is read as G\u03f5), the effect of errors on linkage will be entirely mediated via the distortion of the IBD distribution (the true IBD status \u03c0 of two siblings may be incorrectly inferred as \u03c0\u03f5). We are therefore interested in deriving the probability distribution P(\u03c0\u03f5|\u03c0), this is done by conditioning on both the true and observed genotypes as follows:Let's denote by ii/ii, ii/ij, ii/jj, ii/jk, ij/ij, ij/ik and ij/kl depending on the number of homozygous sibs in the pair and the number of distinct alleles in the sib-pair genotype. Sharing 0 alleles IBD corresponds to a sib-pair genotype of the ij/kl class, should an error occur according to the population frequency error model then one of the four alleles would be transformed into yet another type , therefore the sib pair genotype will remain in the ij/kl class and the observed IBD status \u03c0\u03f5 will still be 0. For the same starting genotype, an error according to the false homozygosity model produces an ii/jk class and \u03c0\u03f5 also equals 0 therefore P(\u03c0\u03f5 = 0|\u03c0 = 0) = 1 whatever the genotyping error mechanism considered previously. The same line of reasoning leads to P(\u03c0\u03f5 = 0.5|\u03c0 = 0.5) = 1 - P(\u03c0\u03f5 = 0|\u03c0 = 0.5) = P(\u03c0\u03f5 = 1.0|\u03c0 = 1.0) = 1 - \u03f5, P(\u03c0\u03f5 = 0.5|\u03c0 = 1.0) = \u03f5. Those results can be summarized by the transition matrix below, where the  element is equal to P(\u03c0\u03f5 = (j - 1)/2|\u03c0 = (i - 1)/2)Let us consider the case of complete information. This can be conceptualized by means of an idealized marker whose number of alleles is infinite, in particular identity by state (IBS) status is equivalent to IBD status. The unordered genotypes of a sib pair can be partitioned into seven exclusive classes denoted E(\u03c0\u03f5|\u03c0) = (1 - \u03f5/2)\u03c0 and E(\u03c0\u03f5) = The overall effect of genotyping error is thus to reduce the observed IBD sharing, indeed x = ' have been adjusted for any relevant covariates  and have been standardized so that the (known) population mean, variance and sib-sib correlation are 0, 1 and \u03c1 respectively. Under the additive variance components model, x given IBD information p follows a bivariate normal distribution with zero mean and variance-covariance matrix given byWe assume that the sib pair phenotypic data \u03b3 \u2265 0 denotes the proportion of total variance explained by the putative locus. Under this model, an optimal testing strategy first advocated in [\u03c0 - C function of the trait values:where cated in  (and som\u03b3 [This test turns out to be a score test for the linkage parameter \u03b3  and is b\u03b3 :0(\u03c0). In a set of sibships indexed by i, an efficient estimate of the linkage parameter \u03b3 is E(\u03b3 and has variance var0(-1(\u03b1) + \u03b31/2). Fisher's information where \u03b3\u02c6=8\u2211i\u03c0i\u2212Ci\u2211iCi2. ize only .P = \u2211\u03c0P(\u03c0\u03f5|\u03c0) P, using the transition probabilities P(\u03c0\u03f5|\u03c0) derived earlier, while the P's are given in [By conditioning on the true IBD sharing values, we can compute given in . This pe\u03b3 using As mentioned earlier, the corresponding variance under the null hypothesis is only slightly altered. The effect of genotyping error is thus to shrink the regression line by a factor 1 - \u03b3 = 0 in this formula gives the type I error rate. Since C variable) has average 0, or takes values far away from 0. The further away the X-variable C is from 0, the smaller A, hence the smaller the bias.Note that taking \u03b3 is typically small, the distortion of the usual linkage test in presence of genotyping error heavily depends on the design-specific quantity C (hence about the distribution of A) in the whole population or in a selected sample. Nevertheless, Monte Carlo simulations can be used to determine the characteristics of the C and A distributions in the whole population or for a specific ascertainment scheme. In random samples and under the variance components model, C is a score function hence E(C) = 0 therefore its sample estimate \u03c1 = 0). The result is that the bias will be small for random samples. The same finding would hold for any ascertainment scheme where C| \u2265 C0) does not warrant that C. In EC designs , Since n scheme  that wouA and \u03c1 and the degree of selection. One obvious way to correct for the shift in the intercept induced by genotyping error would be to leave the regression unconstrained, this would correct for most of the bias. Unfortunately, in selected designs where the variance of C is reduced, this results in a very inefficient estimator of the linkage parameter \u03b3. The right-hand side of Table In the left-hand side of Table -4). The most visible impact is on type I error rates in ED design which is up to 7 times its nominal value. The In Table a. In this section, we propose a completely data-driven strategy for doing this.As we have seen in previous sections, the main effect of genotyping error is to modify the intercept in the regression used to test for linkage. Although an unconstrained regression would correct most of the bias due to genotyping error, the inefficiency of this strategy makes it impractical. In order to obtain an efficient and robust inference, it therefore seems natural to try and constrain the regression through its correct origin n where n is the number of sib pairs available. If we knew that the position is unlinked or if the sample of sib pairs was random then the deviation of this mean from a in the linkage regression.At any position, the sample mean IBD sharing has variance 1/84, a number that is almost never reached in linkage studies. In order to obtain an intercept estimate Unfortunately, detection of a position-specific intercept corresponding to typical error rates would require a sample size of order 10e robust .\u03c0 is computed at a series of approximately regular positions indexed by t across the whole genome. Let yt be the sample mean (among families) excess IBD at position t i.e. \u03b3, equation (3) implies thatLet's assume that the proportions of alleles shared IBD yt across positions provides an estimate of a. In selected samples, we can use a trimmed version of the mean of y, for example a 20%-trimmed mean of the (yt)t series  will provide a robust genomic estimate a. Because a \u2264 0 and yt values respectively before taking the mean. Of course, how much we trim is arbitrary but 20% can safely be taken as a conservative value for oligogenic traits  from the sample used to estimate intercept a.). An ad-hoc implementation of the concept of genomic control is then to plug in the estimate of the intercept In random samples or in any sample where Under two basic error models, we were able to predict quantitatively the consequences of genotyping error on inference in linkage analysis. In the idealized situation of complete IBD information, both error models have the same impact on linkage analysis. As we have seen, the effect is due to a decrease in IBD sharing. A contrario, an error process which would increase IBD sharing would produce opposite results. The true error processes involved in practice are complicated mixtures of the models alluded to here. In our experience however, it seems that processes which lower IBD sharing are predominant. Because genotyping error tends to decrease the estimated number of alleles shared IBD, the effect on evidence for linkage is opposite in EC (reduced power) and ED (increased type I error) designs, it can be dramatic in typical designs and paradoxically less severe for more extreme ascertainment schemes. By analogy, for a dichotomous trait, this means that the effect of genotyping error is less severe in ASP designs for rare diseases than for common diseases. Remarkably, in designs combining both ED and EC pairs like the population frequency error model (error rate = 0.01). The microsatellites map (MS) had 13 equi-frequent ten-allele markers (heterozygozity = 90%) located 10 cM apart  and the SNP map had 41 equi-frequent SNPs (heterozygozity = 50%) spanning the 50\u201370 cM chromosomal region . The resulting average reduction in IBD sharing for an error rate of 0.01 was measured every 2 cM in the 50\u201370 cM region, it ranged from 0.4974 to 0.4976 in the MS map and from 0.4945 to 0.4955 in the SNP map. For these two maps which mimic the two most widespread genotyping paradigms nowadays, those simulations confirm results derived under the complete marker information assumption with a reduction in IBD sharing from 0.5 to 0.5 \u2013 0.01/4 = 0.4975. Our results therefore appear to be applicable to real-life situations where IBD information is incomplete.Our study used an idealized model where IBD information is assumed to be complete. In practice, IBD is uncertain and it is inferred using marker data and multipoint algorithms as implemented in publicly available software ,17, the The genomic-control strategy that we have proposed, although triggered by the specific issue of genotyping error, potentially offers a general robust method for carrying out linkage analysis. It is nonetheless important to recognize its limitations. Firstly, if the trait is highly polygenic with contributing genes scattered across the genome, the high correlation between linkage positions will make it impossible to estimate the IBD sharing at null positions. The genomic control strategy should therefore only be considered with oligogenic traits. Secondly, the concept of genomic control relies on the assumption that the genotyping error rates are similar across markers. For markers with a similar degree of polymorphism , this assumption might be acceptable. In a multipoint setting, an additional assumption required to ensure the validity of a genomic control strategy is that inter-marker distances be approximately equal. With microsatellite markers, both these assumptions might fail resulting in differences in the IBD sharing reduction across markers. The 'regression-based linkage testing' view allows one to qualitatively assess how deviation from these assumptions will impact linkage testing. For example, in ASP or EC designs, wrongly assuming that IBD is uniformly reduced across markers will result in inflated type I error at marker positions with low genotyping error rate compared to other markers. The advent of SNP chips in linkage mapping holds the promise of regular marker maps with less variable information content than in classical microsatellites maps ,3. The mElston et al.  have poiUnder realistic genotyping error scenarios, power losses observed in extremely concordant designs are modest but the effect on type I error in extremely discordant designs can be dramatic. Our analytic approach provides some understanding of the differences in influence of genotyping errors across study designs. The advent of SNP arrays does not eliminate the impact of genotyping errors but it makes genomic control a feasible option with the potential to deliver more robust inference in linkage analysis data subject to genotyping errors or other mechanisms distorting the IBD signal.ASP: affected sib pair; EC: extremely concordant; ED: extremely discordant; EDAC: extremely concordant and extremely discordant; IBD: identical-by-descent; QTL: quantitative trait locus; SNP: single-nucleotide-polymorphism.JJPL participated in the method development, carried out the simulations summarized in Table"}
+{"text": "Focusing on chromosome 1, a recursive partitioning linkage algorithm (RP) was applied to perform linkage analysis on the rheumatoid arthritis NARAC data, incorporating covariates such as HLA-DRB1 genotype, age at onset, severity, anti-cyclic citrullinated peptide (anti-CCP), and life time smoking. All 617 affected sib pairs from the ascertained families were used, and an RP linkage model was used to identify linkage possibly influenced by covariates. This algorithm includes a likelihood ratio (LR)-based splitting rule, a pruning algorithm to identify optimal tree size, and a bootstrap method for final tree selection.p-values, obtained by simulating marker data under null hypothesis of no linkage. Two suggestive linkage regions on chromosome 1 were detected by the RP linkage model, with identified associated covariates HLA-DRB1 genotype and age at onset. These results suggest possible gene \u00d7 gene and gene \u00d7 environment interactions at chromosome 1 loci and provide directions for further gene mapping.The strength of the linkage signals was evaluated by empirical  Rheumatoid arthritis (RA) is a chronic, inflammatory autoimmune disease in which the patient's immune system attacks primarily the joints. The mean age of onset is between 45 and 50 years of age, although it can occur at any age; its prevalence may be as high as 1% in adults ,3. This Our primary goal is to improve the understanding of the etiology of RA through more detailed linkage analysis. It is interesting to know to what extent covariates such as smoking, sex, or age at onset can influence the identification of genetic loci that predispose for RA. Previous studies have found linkage evidence on chromosome 1 -6, with Some existing model-free linkage analysis methods that allow covariates can incorporate only one or few covariates and rely on an assumption of linear covariate effects -9. HowevWe applied the method of Xu et al.  for the The RP algorithm uses log-likelihood ratio (LLR) statistics for constructing a splitting rule based on a likelihood-ratio (LR)-based linkage model . Pair-leN affected sib pairs. The parameter \u03bbi measures the excess risk to an individual who shares, at the marker locus, i alleles IBD with an affected sibling compared to the population risk. \u03bb1 corresponds to IBD = 1, \u03bb2 corresponds to IBD = 2, and \u03bb0 = 1\u00b7fi(p) is the prior probability of sharing i alleles IBD for affected sib pair p\u00b7gip represents the estimated probabilities of sharing i alleles IBD based on marker data for pair p. The parameters \u03bbi are estimated by optimizing the total LR for all ARPs.where the LLR is summed over all the Xp (Xp = 1 or 2), the LLR of sub-nodes can be used to test linkage in the presence of heterogeneity by estimating two sets of parameters . The splitting rule is defined by identification of the covariate that gives the largest LLR statistic over the sub-nodes, that is identifying the strongest linkage heterogeneity. This is implemented recursively to grow a full tree.For each pair-defined binary covariate The next step consists of a pruning algorithm that trims the full tree, which may otherwise overfit the data. In this study, we used a bootstrap algorithm to estimate the deviance function for choosing the optimal tree size . The optimal tree size is selected as the one with the largest estimated deviance function .After choosing the optimal tree size, we used an independent bootstrap algorithm to choose the final tree structure. Across the trees generated in bootstrap samples that have the same tree size as the optimal tree size, for each locus, the proportion of the trees with particular covariate splits can be calculated. When one covariate clearly defines linkage heterogeneity, most bootstrap data sets will select that covariate. When several covariates are associated with the disease gene, bootstrap data sets may choose a variety of tree structures and covariates. The RP model chooses, as the final tree, the tree structure that appears most frequently among all the bootstrap sampling trees .The linkage test statistic of a final tree is a global linkage test that reflects marginal linkage and genetic heterogeneity. For each marker, this statistic is calculated as:s is the final tree size, the summation is over all the terminal nodes, and the parameters where Use of the North American Rheumatoid Arthritis Consortium (NARAC) data set  was apprAfter creating all covariates, we examined their frequencies and excluded from analysis covariates in which the rare category occurred with a frequency of 10% or less, since power would be insufficient in such cases; therefore, 19 covariates were considered by the RP model. Genehunter  was used\u03c72 distribution [p-values.The global linkage test statistic does not follow an asymptotic ribution . In orderibution . Then we10 of the p-values from the NPL scores and the RP model. Neither test provided strong evidence for linkage to chromosome 1, although both methods had minimum p-values near 0.01 somewhere on the chromosome. Using the NPL score, a suggestive region was identified near marker 132.66 cM, and using the RP model, we found two other suggestive regions on this chromosome. The first region ranges from 102.02 cM to 125.51 cM, and the second region contains only the F27b marker at 239.66 cM. Table Figure p-value is 0.0001; hence this subgroup shows very strong linkage.The HLA-DRB1 genotype was detected as an associated covariate for all four markers in the first RP-detected region; the high-risk subgroup consisted of relative pairs not carrying HLA-DRB*04. Age at onset was found to be associated with the marker at 239.66 cM (marker F27b). The final tree structure at marker F27b is shown in Figure We applied a recursive-partitioning model for linkage analysis to select covariates that are associated with the allele-sharing patterns in relative pairs. A pruning algorithm based on the bootstrap method and a final tree structure selection algorithm was used to improve the performance of the model. In the NARAC data, we identified two linkage signals involving covariate interactions in regions distinct from the region with the maximum NPL score. However, the NPL peak at 132.66 cM was not identified by the RP model global linkage test. One possible reason is that the necessity of controlling \"false\" splits in the RP model  may reduce the power to detect marginal linkage signals.p-values of the detected markers to their asymptotic p-values based on the \u03c72 distribution, and noted, as expected, that the asymptotic p-values were smaller  declare that they have no competing interests."}
+{"text": "Data linkage is a technique that has long been used to connect information across several disparate data sources \u2013 most commonly for medical and population health research. Often the purpose is to connect data for individuals over extended time periods or across different service settings, and so person-based linkage using detailed personal information is preferred. Linkage which aims to link connected events, on the other hand, requires information about the time and place of the event as well as the person or persons involved in that event in order to make high quality linkages.This paper describes the detailed process of event linkage and compares directly an event-based linkage method for identifying transition events between two care sectors in Australia with a well-established high quality longitudinal person-based linkage which facilitates identification of event data for individuals.Direct comparisons are made between transition events identified using an event-based linkage and an existing person-based linkage for people moving from hospital into aged care in Western Australia. Several aspects of event-based linkage are examined: refinement of the strategy to reduce false positives, causes of false positives and false negatives, quality of the linked event dataset, and utility of the linked event dataset for transition analysis.Over 97% of the event-based links were among those selected using the person-based linkage and over 90% of the latter were identified by the event-based method, with the remainder missed mostly due to differences in reported event date or residential region. Consequently the two linked datasets were sufficiently similar to give very similar results for analyses, but the actual volume of movement from hospital to RAC was underestimated by about 10% by the event-based method.This project has allowed a 'preferred event' event-based linkage strategy to be selected and deployed across Australia to study movements from hospital to residential aged care facilities using databases in which only limited personal information is available, but event dates and details can be readily accessed. The utility of this approach in other transition situations depends on the volume of movement and the accuracy of recording information in each setting. Data linkage is a technique that has been used since the 1940s to connect information across several disparate data sources, most commonly for medical and population health research. The technique uses as much information as is available in each data source to join records or other data structures that are thought to belong to the same person, place or event. Person-based linkages use personal information such as name, address and date of birth, or a personal identification number, to connect data for individuals over extended time periods or across different service settings. However, linking events for people requires information about the time and place of the event as well as the person or persons involved in that event to make high quality linkages. Additional details that might be required for an event linkage are the location of the event (i.e. the establishment or address) as well as the time and date when the event occurred.The quality of any linkage relies on the amount of information available for linkage as well as that information's accuracy, consistency and completeness. For instance, person-based linkages across a long time period require access to a universal, unique identifying numbering system or extensive personal details such as name, address and date of birth. However, within a narrow period of time or limited population group, fewer details may be needed to achieve links of the same quality.Event linkages may be achieved through reference to the event details in isolation, or may be achieved by selecting specific events from a sequence of events within, say, a person's linked health history. Some events, such as environmental accidents and road crashes, can involve more than one person and rely heavily on the time and place of the event to make the connection between affected individuals. On the other hand, a person may have several similar related 'transition' events occurring within a limited period of time, for example movement between one health setting and another. Identification of these related events requires information about the individual as well as the time and place of the event. An event sequence may occur over a period of time that may be seconds, minutes, hours or days.An important example of a transition event is when a person moves from hospital into an aged care facility. In Australia, where 13% of the population of 20.5 million is aged 65 or over and 5% of these are in residential aged care  movementAustralia-wide administrative data are available separately for hospital and RAC episodes. These data provide little information on the movements of clients between the residential and acute care sectors, and the two datasets contain neither a common person identifier nor detailed identifying information (such as name). However, a range of data items is common to both datasets, and event linkage, without access to comprehensive personal information, could be used to provide insight into transitions between hospital and RAC. Such linkage has been used successfully in a number of other scenarios -12. ThesTwo earlier studies suggested that there is adequate personal demographic information in conjunction with event information available for linking the transition events on the two Australian datasets. Theoretical investigations have shown that the expected rate of false matches is low  and, takWhile the earlier studies point convincingly towards the utility of the event-based linkage strategy, the absence of validation of the resulting links against a reference standard still leaves room for doubts. Investigations of linkage validity are rare , and comIn the current analysis, we compare two distinct record linkage strategies for unidirectional linking of discharges from hospital with the corresponding admissions into or returns to care in a RAC facility. The scope of the comparisons is limited to movements from hospital to RAC by people aged 65 and over in Western Australia during 2000\u201301. Western Australia was chosen for this study because of its unique position in Australia of having a well-established health data linkage system that includes both hospital and RAC data . OverallTwo independent linkage processes were used in this study. The first linkage strategy was a selection process from an existing person linkage \u2013 here termed N linkage \u2013 carried out by the Data Linkage Branch of the Western Australian Department of Health. The second was the event-based E linkage strategy developed within the Australian Institute of Health and Welfare (AIHW) ,13 whichThis project was performed as part of the Western Australian Cross Jurisdictional Data Linkage Project covered by a Memorandum of Understanding between the Western Australian state Department of Health and the Australian Government Department of Health and Ageing. The person-based linkage was covered by approvals from the two agencies' confidentiality and ethics committees . Before the event-based data linkage was undertaken, ethics approval was also obtained from the AIHW Ethics Committee  and permission to use the required data was obtained from the relevant bodies.The person-based linkage was undertaken using the Western Australian Data Linkage System (WADLS), a dynamic ongoing system used to create and store a web of links across population and research health data collections in Western Australia. The system is updated continuously by a small number of dedicated staff. The WADLS probabilistic linkage process, which follows the 'Two-Stage Protocol' to protect privacy , uses asThe linked data represented within the WADLS varies from one single record to more than 3000 for persons either resident in Western Australia or receiving a service in Western Australia. As stated above, the linked records in the WADLS provide name and address reporting histories across a range of health service events over four decades resulting in linkage accuracy and completeness levels that are further improved with each new dataset that is incorporated. The N-linkage between hospital and RAC records also benefited from detailed clerical review using these resources.The underlying quality of the person-based linkage in the WADLS has previously been measured by manually checking a sample of linked hospital admission and death records. The measured error rate of this section of the WADLS in 2001 and 2002 was less than 0.1% of persons having at least one incorrect event linked within their medical history. An earlier estimate 1998) of false negative errors estimated these at approximately the same level (0.1%) [98 of falIn order to select the events of interest for this study, all relevant hospital and RAC event records were retrieved separately from their original sources and then joined using relevant WADLS hospital-RAC person 'linkage keys'. From among this subset of linked records, the most appropriate hospital-RAC event link was chosen by measuring the closeness in time of the hospital and RAC event dates. For those individuals where there was a choice between different RAC event types, the match to the earlier RAC event was selected.Overall, this N strategy resulted in 8106 links between hospital separations and entry (or re-entry) into RAC during the period of interest. Because of the high level of the accuracy of person links, these links were used as the reference linkage in this study.Event-based E linkage strategies link records by using event dates and event characteristics information in conjunction with limited demographic data. The purpose of this strategy is to allow identification of related events for people when there is insufficient non-event information to establish person links as a precursor to identifying related events. The resulting linked dataset may vary depending both on how much of this information is used when identifying links, and how particular data items are used to specify the linkage process.To make the most of the availability of a reference dataset (i.e. N links) a step-wise approach was taken to comparing match results and refining the E linkage strategy:\u2022 Match using a specific version of E linkage\u2022 Compare results to N linkage\u2022 Identify shortcomings\u2022 Consider options for improvement\u2022 Test proposed improvement.Basic E linkage \u2013 as used in the feasibility study  \u2013 was inThe initial E linkage (as above) was applied using a single transition date and without taking into account how the two data collections were derived or whether there was other information that could be used to improve the matching process. The two datasets did in fact contain a range of information about their respective events. Variables on the datasets that could assist in isolating preferred transitions were identified by considering the actual relationship between the hospital and RAC events and ascertaining which, if any, variables on the two datasets might help to describe this relationship. Questions considered to identify variables that could assist in the linkage process were as follows:(a) A hospital episode has a start and an end date, with the latter being the transition date for hospital-RAC transition events. Are there additional event date data on the RAC dataset that relate to the hospital episode dates?(b) Is there any information on the hospital data which indicates how the hospital event started or which sector the patient came from?(c) Is there any information on the hospital data concerning where the patient went after discharge?(d) Is there any information on the RAC data concerning where a resident was just prior to admission?(e) Is it possible for there to be more than one event validly reported for the same person on the same day \u2013 either for hospital discharge events or RAC entry events?(f) It is not clear from data definitions whether address of usual residence as reported in the hospital data is meant to refer to before the period in hospital or after. Is there any additional address-type information on either dataset that could assist in identifying events for people who change their usual residence on leaving hospital?Answers to these questions led to identifying several additional data items on both the hospital and RAC datasets that could be used to support the E linkage Table . The adjWebSphere\u00ae software. This statistical package contains a suite of tools for identifying duplicate records for individuals (or entities) within datasets and for matching records for the same individual (or entity) across datasets; WebSphere\u00ae was previously known as Automatch\u00ae and Integrity\u00ae [For both E strategies, matching was undertaken using probabilistic methods via tegrity\u00ae . ProbabiWebSphere\u00ae matching process. In general, smaller regions were used in earlier passes to ensure that matches based on the most detailed information were identified first. Broader match regions were then used to allow for change of address within the neighbourhood . Match passes were undertaken in the order indicated in the table. If exact duplicate matches occurred in a pass, one was selected at random (this rarely happened). When event dates were used as match variables, match criteria were set within WebSphere\u00ae specifying that a gap of no more than two days between hospital exit and RAC entry event dates was allowed when identifying matches. This approach was taken both to reduce the number of person-level false positives (i.e. matching events for different people) and to reduce within-person false positives (i.e. linking the wrong two events for the same person). A small gap was tolerated to allow for inconsistencies in reporting date on the two administrative systems. After matching using WebSphere\u00ae, a number of deterministic rules were applied to ensure that allowable differences in the values of the match variables were not violated.For the current project, basic event characteristics were used to subset the datasets prior to matching to improve the likelihood of identifying appropriate links , no link (true negative), a mis-link  and a missed link . In the event-based strategy, false positive links can be caused by several individuals \u2013 either leaving hospital or entering RAC \u2013 having the same basic demographic data so that events for the wrong people are linked together. In addition, errors or inconsistencies in the event information for the same person may cause both false positives and false negatives.Given the high quality of the WADLS person-based links, N-linkage was used here as the reference standard against which the E linkage results were compared to determine whether an E link (or lack thereof) was 'true' or 'false'. Comparisons between N and E linkage results were undertaken both to measure the quality of E links and to identify causes of false positives and false negatives, thereby allowing refinement of the method for the current application.In this paper, two key measures are used when comparing matches. Using terminology originating in epidemiology and clinical trials , these aWhen comparing linkage strategies we can easily identify whether a record has been linked and whether it should have been linked. However, in the current context of identifying transition events it is also important that an event record in one dataset matches with the correct event record in the other dataset. That is, as well as knowing whether or not an event should have been linked, we need to know which event in the other dataset it should have linked to. Overall, as illustrated in Figure Direct comparison with a reference linkage provides a rare opportunity to refine a particular strategy by identifying those match passes that lead to too many false positives relative to true matches. By making such comparisons those passes that lead to an acceptably high likelihood of making a true match can be retained and those that have an unacceptably high likelihood of making a false match can be excluded. At the same time, passes whose exclusion would result in a high number of false negatives  can also be retained. It needs to be remembered, however, that the final strategy needs to be reproducible when there is no reference standard. One way of identifying an acceptable blocking/match pass strategy is to use the reference linkage to estimate the range of the actual PPV for a particular pass given the observed PPV. It is then assumed that in similar studies without a reference linkage similar PPVs would apply.For the current study, from the point of view of ensuring a low rate of false positives, we wanted to be confident that, at the very least, a particular match pass resulted in more true matches than false matches. To this end, using standard statistical tests  p82), th, th2 witThe results of comparisons between matches identified through N linkage and those found using several E strategies are presented below. In addition, causes of false positives and false negatives are examined. Finally, to establish the overall utility of the E-linked data, we compare the results for three example analyses based on E and N linked datasets to see whether patterns within the various transition groups as identified through E linkage reflected those seen when using N linked data.The high quality of links in the N linked dataset was re-affirmed when reviewing the person links implied by the E-only links (i.e. those only found by the event-based method): E linkage found just two additional links between hospital and RAC clients that had previously been missed using the WADLS.Selection of transition events from existing person links relies on two factors \u2013 the identification of the correct person (i.e. the assumption that the person links are correct) followed by selecting the correct pair or sequence of events for that person.It may be possible to estimate false negative (or missed) links in some situations if there is an expectation of finding a matching transition record. Recorded hospital leave as a data item in the RAC dataset provided this opportunity: wherever the RAC facility recorded a visit to hospital, it would be expected that the person-based matching would result in hospital matches for (nearly) all such recorded RAC hospital leave events.The N linkage strategy did not identify a hospital-RAC link for just over 4% of all RAC hospital leave events. This suggested that either\u2022 the N strategy does not identify some people who really had used both RAC and hospital services, possibly due to insufficient or erroneous demographic details. The lack of event data on the RAC records for WADLS linkage may have contributed to this.\u2022 the RAC or hospital administrative data systems contained reporting errors in event types or event dates preventing appropriate selection of event records among person records; or\u2022 people may have left RAC to go to hospital but some were never actually admitted but were treated in an emergency department and so did not appear in the hospital 'admission' dataset.A close examination of all the WADLS-linked events for emergency, hospital admission and death records for a sample of people with a link to RAC hospital leave missed by N linkage showed that all three of the above scenarios had occurred, with no one single cause dominating.Taken together, the above results confirm the use of the N linkage as the reference linkage for this study.For the initial basic E linkage strategy, only two match passes were used  were considered for matching to allow for reporting differences and transcription errors. Inconsistencies in reported region of usual residence were considered highly likely as a sizeable proportion of the transitions relate to people in the process of changing their usual residence (i.e. being admitted into permanent RAC for the first time) \u2013 nearly 20% of links found using basic E linkage were for permanent admissions. A number of region definitions were tested, based on two main definitions which differed primarily in the size of the region used when establishing links. The first was based on linking within SLGs (as per basic E linkage). In the second variant, matching was restricted to simple postcode-based regions, and so generally used smaller match regions.Overall, six different versions of constrained E linkage were considered, with three based on SLG (termed SLG strategies) and three based on postcode (termed PC strategies) (see Table After dropping poorly performing match passes (PPV < 60%), the E linkage constrained strategies resulted in identifying between 7078 and 7595 transitions from hospital into RAC, depending on the regions used in matching Table  increaseAs can be seen in Table In the current application, choice of region used when matching had a significant effect on the sensitivity of the linkage, but not on the PPV Table . The impChoice of region had a limited effect on matches to RAC hospital leave. This is because the RAC facility is already the person's usual residence so that for RAC hospital leave the v1 and v3 variants of E linkage are the same. The difference between the sensitivity for the v1 and v2 linkages for RAC hospital leave \u2013 an increase of 42 true positives out of 43 matches for the SLG linkage \u2013 is most likely due to people going to a different, but nearby, RAC facility on discharge from hospital, with the hospital reporting the new RAC facility as the place of usual residence . The postcode of the (new) receiving RAC facility was not available for this study to confirm this.Using N linkage as the reference standard, N-only links represent false negatives, while E-only links represent false positives. All link types shown in Figure Overall, looking at SLG v2 linkage, there were 61 link pairs where an event in one dataset matched to a different event in the other dataset under the N and E matching strategies (before dropping the poorly performing match passes). Manual examination of these led to the following rule: if an E link matched to an event for the same person as the N link, then for the purposes of match comparison the E link was classified as a true positive. If, on the other hand, the E-link event was for a different person, then the E link was classified as a false positive.The majority of false positives were for admissions rather than leave events (ratio of 2:1). Many of the E-only links to RAC admissions had exact matches on both date of birth and event dates, suggesting that the false matches were caused by similar hospital and/or RAC activity by similar people . Comparisons indicated that one of the most effective ways of reducing the number of false matches made under the E strategy would be to reduce the size of the geographic region used in matching. However, data quality and consistency issues mean that overly narrowing the geographic matching criteria would result in dropping many more true positives than false positives. For example, dropping three- and two-digit postcode matching from the SLG v1 strategy  and including matching which replaces the person's postcode with that of the RAC facility (v3 versus v2).Poor region matching was a less important, but still significant, reason for missing matches among RAC leave events. However, missed matches to RAC leave events  were primarily the result of inconsistent event dates recorded for related hospital and RAC events. The large gaps between the recorded dates for the N-linked events in these false negatives (commonly 3 or more days) indicated that it would not be possible to adjust the E matching strategy to allow capture of these matches without risking the introduction of large numbers of false positives.Modelling the propensity of E linkage to miss N links within RAC event type, only a small number of variables were found to have statistically significant effects. Variables with significant effects in the fitted models included outlet region, hospital care type, reported post-hospital destination and whether there were leave events from hospital. Different variables were significant in the models for the three RAC event types, and in fact no statistically significant effects were found when fitting models for respite admissions. Overall, small regional differences and/or differences in basic hospital episode characteristics in the profile of N and E links to permanent RAC admission and RAC hospital leave events were detected , consistently high sensitivities within RAC event type and would be easier than its SLG-based counterpart to apply to national data.The 91% sensitivity of the E linkage strategy is reflected in the absolute numbers of people identified making the various transitions following a period in hospital , and subsequently to find ways to overcome these differences, is also key.The accuracy of the constrained E linkage used in this study compares favourably with those achieved for other studies. For example when linking New Zealand census and mortality records without using name or person identifier, Blakely and colleagues reported a PPV 'in excess of 95%', with 77% of eligible mortality records being linked . In addiTheoretical calculations of the expected number of false matches due to different people having similar demographic and event data led to PPV estimates in a similar range to those observed for the constrained linkage strategies . For exWhen choosing the preferred E linkage strategy to use in other studies two issues need to be considered in addition to the levels of accuracy found in the test linkage. In particular, the ease of applying the method and whether there are differences in the new population under study that need to be taken into account. In the current context, having identified several possible E linkage strategies for linking Australian hospital and RAC data we have to choose one to apply to the full national datasets for investigation into the hospital-RAC interface across Australia. In terms of ease of use, the PC methods are easier to apply than the SLG variants as derivation of SLG from postcode can be quite complex. In addition, the most accurate SLG and PC strategies had similar PPV and sensitivity; however, the SLG strategies use larger match regions and so could lead to lower PPVs in the more populous regions in other Australian states. Finally, using a particular data item (RAC facility postcode) to overcome a particular data matching issue  is preferred over an approach which simply relaxes match criteria (going to two-digit postcode). Taking the above into account, overall the PC v3 strategy is preferred for future analysis of the hospital-RAC interface in Australia.All the E linkage strategies underestimated the flow of people from hospital into RAC  did not use any name or unique identifier information, but relied on event details in conjunction with limited demographic data. Implementation of both these methods showed that when identifying service transition events, detailed knowledge of both the service systems and the data collection practices within those systems is essential.Using comparisons with the name-based reference linkage, a PPV of 98% was achieved using event-based linkage and the sensitivity was greatly increased \u2013 from 81% using the simplest strategy to 91% for the preferred strategy. The event-based strategies resulted in several times more false negatives than false positives, and the volume of flow from hospital to RAC was therefore underestimated. Nevertheless, illustrative examples examining patterns of use and characteristics of people making particular types of transitions between the two sectors showed that analyses using output from the person-based N linkage and event-based E linkage strategies led to very similar conclusions. Consequently, event-based linkage provides a useful tool for examining transitions, provided there is an understanding of the limitations and caution in applying analytical techniques.This detailed comparison of event linkage methods has also demonstrated two important roles for high quality population-based reference linkages, such as those available through the WADLS, in assisting in the development of strategies using reduced amounts of information. The first is as a tool for refinement during strategy development and the second is as a 'gold standard' against which to measure accuracy and completeness of the set of links produced and the utility of subsequent analyses.Importantly the work reported here has allowed a 'preferred event' event-based strategy to be selected and deployed across Australia to study the national situation in movements from hospital to residential aged care facilities using databases in which detailed personal information is not available, but dates and event details can be readily accessed. More generally, the results presented here suggest that such an event-based linkage approach could be used in other areas where relatively small populations are moving across single interfaces within a narrow time window.AIHW Australian Institute of Health and Welfare. CHIC Department of Health Western Australia Confidentiality of Health Information Committee. DEC DoHA Departmental Ethics Committee. DOB date of birth. DoHA Australian Government Department of Health and Ageing. E linkage event-based event linkage (name data not used). ICD-10-AM International Classification of Diseases 10th revision Australian Modification, based on the World Health Organization's internationally accepted classification of diseases and related health conditions. N linkage name-based event linkage. NPV negative predictive value. PC postcode. PPV positive predictive value. RAC residential aged care. SLG Statistical local group. WADLS Western Australian Data Linkage SystemThe authors declare that they have no competing interests.RK undertook the event-based data linkage of transition events, refined the E strategies based on linkage comparisons, and led the analysis. DR undertook the name-based data linkage of transition events, and assisted in the analysis. Both authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:"}
+{"text": "P. falciparum infection, like the intensity and seasonality of malaria transmission, the virulence of parasite and host characteristics like age or genetic make-up. Although admitted nowadays, the involvement of host genetic factors remains unclear. Discordant results exist, even concerning the best-known malaria resistance genes that determine the structure or function of red blood cells. Here we report on a genome-wide linkage and association study for P. falciparum infection intensity and mild malaria attack among a Senegalese population of children and young adults from 2 to 18 years old. A high density single nucleotide polymorphisms (SNP) genome scan (Affimetrix GeneChip Human Mapping 250K-nsp) was performed for 626 individuals: i.e. 249 parents and 377 children out of the 504 ones included in the follow-up. The population belongs to a unique ethnic group and was closely followed-up during 3 years. Genome-wide linkage analyses were performed on four clinical and parasitological phenotypes and association analyses using the family based association tests (FBAT) method were carried out in regions previously linked to malaria phenotypes in literature and in the regions for which we identified a linkage peak. Analyses revealed three strongly suggestive evidences for linkage: between mild malaria attack and both the 6p25.1 and the 12q22 regions , and between the 20p11q11 region and the prevalence of parasite density in asymptomatic children . Family based association analysis pointed out one significant association between the intensity of plasmodial infection and a polymorphism located in ARHGAP26 gene in the 5q31\u2013q33 region . This study identified three candidate regions, two of them containing genes that could point out new pathways implicated in the response to malaria infection. Furthermore, we detected one gene associated with malaria infection in the 5q31\u2013q33 region.Multiple factors are involved in the variability of host's response to  Plasmodium falciparum  malaria represents one of the most important causes of mortality and morbidity in tropical areas. Despite the strong involvement of international and national institutions to enhance the effective level of coverage of malaria interventions, the goals defined by Roll Back Malaria in 2000 seem difficult to reach The great majority of genetic epidemiology studies used candidate genes approach which is restricted to the examination of specific genes, based on their presumed functional relevance to malaria (e.g. genes involved in the control of immunity). These studies led to numerous but frequently discordant results. For example association between polymorphisms within the Major Histocompatibility Complex (MHC) region on chromosome 6p21\u2013p23, that contains the Tumor Necrosis Factor alpha (TNF-\u03b1) gene, and severe To our knowledge three genome wide studies have been performed in malaria area P. falciparum infection intensity and mild malaria attack among a Senegalese population of children. The population belongs to a unique ethnic group and was closely followed up during 3 years. Genotyping of SNPs was performed using the high density Affimetrix GeneChip Human Mapping 250K-nsp .Here we report on a genome-wide linkage study for Individuals included in the follow-up were aged from 2 to 18 years at the beginning of the study (mean age (SD)\u200a=\u200a8.97 years (3.57)). Four children with less than 8 thick blood smears (TBS) during the follow-up were excluded from analyses. Other children had on average 14 TBS. Distribution of raw data and final phenotypes are presented on st, 2nd or 3rd year), consistent with a different transmission level between the 3 years, and the transmission season. HbS and G6PD polymorphisms had no effect on MLPD, IntPI and PrevPI phenotypes but we observed a strong effect of HbS polymorphism (p\u200a=\u200a3.56\u00d710\u22126) on MMA phenotype. Note that no effect of sex or village was detected on any of the phenotypes. From 364 to 370 children were genotyped, depending on phenotype, and included in linkage analyses. The estimations of heritability were 0.08, 0.26, 0.26 and 0.40 respectively for MLPD, IntPI, PrevPI and MMA.Statistical approaches used to define phenotypes and the correlations between phenotypes are summarized in The variance component (VC) and regression-based (RB) linkage analysis were applied to the data. Results for RB analysis are shown in \u22125) and 3.96  for RB and VC respectively. According to Lander and Kruglyak LYRM4 (LYR motif containing 4) interacts as an adaptor between frataxin and NFS1/ISCU, an essential component of iron-sulfur cluster biogenesis that could connect with immune response.Two evidence of linkage signals were obtained for MMA. The first one on chromosome 6p25.1 showed a logarithm of odds (LOD) score of 3.50  and 3.40  for RB and VC respectively. In this case too, the linkage signal can be considered as strongly suggestive . The maximum asymptotic LOD score with RB was located at a cluster of markers from rs4275668 to rs10777556 corresponding to 104.11cM (92.8Mb). The 1-LOD support interval defined a 1.34cM/1.18Mb chromosomal distance (from rs4590917/rs10859418 to rs11107437/rs7316309) containing 12 genes as well. Several ones can be considered as functional candidates: PLXNC1 (Plexin C1) involved in the up regulation of ICAM1 (intercellular adhesion molecule 1); UBE2N (Ubiquitin-Conjugating Enzyme E2N) involved in cytokine signalling pathways (MAPK and IKK) directly implicated in the modulation of immune response SOCS2  involved in the control of the proinflammatory cytokines production The second region with evidence of linkage for MMA phenotype was obtained on chromosome 12q22 with a LOD score of 3.49  and 2.78  for RB and VC respectively. This linkage signal is highly suggestive as well since average number of peak per replicate was 0.09 for RB analysis. Interestingly the same chromosomal region showed a suggestive linkage signal with the phenotype MLPD with a LOD score of 3.63  and 2.20  for RB and VC analyses respectively . For both phenotypes, the maximum asymptotic LOD with RB was located at a cluster of markers from rs6138473 to rs2073077 corresponding to 54.76cM (24.96Mb). The 1-LOD support interval encompassed an 11.44cM/13.5Mb, and a 3.33cM/9.5Mb chromosomal distance for respectively the PrevPI and MLPD phenotypes. The first region contains 149 genes and the second one 129. Possible functional candidates include the following genes: PROCR that could interact in an immunomodulatory process; RIN2 encoding a small GTPase involved in membrane trafficking in the early endocytic pathway that can reduce the endothelial integrity; HCK encoding a tyrosine kinase that could play a role in nitric oxide and TNF alpha secretion in macrophages DEFB123, belonging to a beta-defensin gene family that play a role in innate immunity For asymptomatic infection, evidence of linkage was obtained for the phenotype prevalence of infection (PrevPI) on chromosome 20p11q11 with a LOD score of 3.58  and 1.63  for RB and VC respectively . The maximum LOD score was achieved for rs10819510, corresponding to 137.7cM/131.19Mb. The 1-LOD support interval encompassed a 2.55cM/933Kb chromosomal distance, containing 18 genes. Among them, ASB6 (for ankyrin repeat and SOCS box-containing 6) could constitute a candidate gene. The other genes in this region seem not to be involved either in immune response or in red blood cell physiology.Finally, a suggestive linkage signal was obtained with the 9q34 chromosomal region for IntPI phenotype, showing a LOD score of 3.08  between SNPs is taken into proper account by the method implemented in MERLIN.P. falciparum density (PD), association was tested with the 5q31q33 region and respectively, with the 9q34 region for IntPI, and the 20p11q11 for both PrevPI and MLPD. From 1339 to 2062 SNPs were tested per phenotype. Following Li and Ji procedure Finally, to explore the chromosomal regions we identified during linkage analysis  but also the 5q31q33 and 10p15 regions, which have been described as involved in the control of parasitemia and malaria fever episodes respectively ARHGAP26 gene . ARHGAP26 (Rho GTPase activating protein 26) codes for a protein acting in particular as a negative regulator of Rho A. It is interesting to note that even non significant, a borderline association and linkage evidence to MLPD  was observed for a SNP (rs1056189) located in 5q31.1 region within a gene which has also been proposed to participate in the regulation of Rho proteins (FLJ41603).Taking into account multiple testing, no linkage/association was found within the regions we identified in this study neither within 10p15 region. In contrast a significant association and linkage was found in 5q31.3 region between the phenotype IntPI and SNP rs830532 located in Haplotypes analyses (results not shown) did not improve the results obtained with single SNP association.ARHGAP26 gene in the 5q31\u2013q33 region.To our knowledge, this is the first genome-wide study applied to non severe malaria that used a high density GeneChip array. We identified three strongly suggestive evidences for linkage: between mild malaria attack and both the 6p25.1 and the 12q22 regions, and between the 20p11q11 region and the prevalence of parasite density in asymptomatic children (PrevPI). Although not strictly significant, our linkage peaks are located in chromosomal regions containing relevant candidate genes involved in the control of immunity. The more relevant ones will be presented latter in the discussion. Suggestive linkages were also shown between the mean level of non zero PD measurements (IntPI) and the 9q34, and between the Mean Level of P. falciparum Density (MLDP) and the 20p11q11, regions respectively. The family based association analysis pointed out one significant association between the intensity of plasmodial infection (IntPI) and a polymorphism located in Our study was originally designed for a genome-wide linkage analysis. The high density SNPs map used here allows to provide higher information content compared to microsatellite maps and to define smaller intervals around linkage peaks. Furthermore our family sample allows us to perform an association analysis as well, which provides non-redundant and complementary information. In linkage analysis with dense SNP maps particular attention must be paid to the LD between SNPs, because it can be responsible for a bias. To deal with this point, we used the method implemented in Merlin which performs well in our particular case: relatively few parents missing and a low LD between markers. However this low LD characteristic of African populations constitutes in counterpart a handicap for association analysis. As LD between SNPs and causal variants are lower in African than in European populations, it is more difficult to achieve significant threshold for association analysis Several environmental and behavioural factors, that can be shared by groups of individuals within (or not) families, can influence both the risk of disease and the response to infection Among these factors, a very particular attention must be paid for transmission intensity and anti-malaria medicine intake. Data on transmission level in Niakhar area are consistent with a strong homogeneity of transmission rates www.ird.sn/activites/niakhar/), uncontrolled circulation of drugs existed and, at this time, concerned essentially chloroquine Medicine intake was taken into account because the children received their treatment within our research program framework. Nevertheless, it is well known that despite the intensive presence of health workers in area . This linkage is detected for two different phenotypes, malaria attacks in our study, the maximum of PD in Skuntabhai et al., but in two neighbouring Senegalese populations.Our study shows very little overlap with the preceding genome-wide linkage studies. However for all the regions identified by genome scan except one (10p15), linkage did not reach statistical significance. The discrepancy between results could be also explained by parasite genetic variability and/or by the genetic heterogeneity in the susceptibility/resistance mechanisms to malaria in host. About this last hypothesis it is interesting to note that the only result in common with other studies is the evidence of linkage in 12q22 region. This region is located close (1.5 Mb) to the linkage peak obtained by Sakuntabhai Among the regions identified in this linkage analysis, two of them can be considered particularly interesting because they contain genes involved in pathways related to immunity, the 12q22 and the 6p25.1 ones.PLXNC1 encodes plexin C1 (VESPR/CD232), one of the receptors of semaphorins which are a large family of secreted and transmembrane signalling proteins  is also strongly related to the control of immunity. Indeed, SOCS proteins negatively regulate receptor signalling via the Janus kinase/signal transducer and activation of transcription pathway (JAK/STAT pathway) SOCS2 deficient mice had, during intracellular infection, uncontrolled production of proinflammatory cytokines, aberrant leukocyte infiltration and elevated mortality. Very recently, the differential roles of some SOCS proteins during coinfection with P. falciparum and helminth in children have been shown P. chabaudi infection. Consistent with STAT1 and STAT2 activation, they also observed an increased abundance of genes encoding downstream targets of interferon signalling in response to infection, including the interferon regulatory factor 1 gene, located in the 5q31q33 chromosomal region. It is also interesting to note that another member of STAT family (i.e. STAT6) could be involved in mediating erythropoietic suppression during acute blood-stage malaria and that interleukin-4 (in 5q31\u2013q33 region) and possibly interferon-\u03b3 could also play a role in this mechanism In 12q22 region we noticed three candidate genes. LYRM4). The product of this gene interacts with the cystein desulfurase (Nfs1) and its scaffold protein (IscU), and is required for the initial step of Fe-S cluster assembly. Fe-S clusters are involved in multiple cellular processes, including macrophages activity. It has been demonstrated that Nfs1 and IscU were strongly down-regulated at both mRNA and protein levels in IFN-\u03b3 stimulated macrophages. As immune response against P. falciparum infection is initiated by early interferon response, these data suggest a connection between the immune responses and the biogenesis of Fe-S clusters 6p25.1 chromosomal region contains a gene involved in iron-sulfur (Fe-S) cluster biogenesis  are different In the present study, no evidence of linkage was obtained for parasite density to the 5q31\u2013q33 chromosomal region but we demonstrated a significant association between the intensity of plasmodial infection (IntPI) and a polymorphism located in P. falciparum density and the D5S636 marker IL3, IL4, IL5, IL9 and IL12B) and for other genes involved in immunity: IRF1 for immune regulatory factor 1; CSF2 for colony stimulating factor 2; c-fms, the gene encoding the receptor of the colony-stimulating factor 1 (CSF1R). Secondly, linkage between the 5q31 locus and other parasitic diseases, such as schistosomiasis We were the first group to underline the potential interest of this chromosomal region in a small sample of 9 Cameroonian nuclear families by detecting a borderline significant linkage between the mean level of ARHGAP26, which is involved in the integrin signalling transduction pathway. Although the role of cytoadherence in malaria is clearly admitted, little is known of signalling triggered by this cytoadherence. The protein encoded by ARHGAP26 acts as a negative regulator of RhoA, a member of Rho family, which cooperates in Rho kinase pathways that reduce the endothelial integrity, and is activated by the adhesion of P. falciparum to endothelial cells in vitro. Members of the Rho family are known to play an important role in the signal transmission of various receptors, including ICAM-1, one of the endothelial receptors of P. falciparum infected erythrocytes. The importance of the integrity of endothelial cells ARHGAP26 as a functional candidate gene.Nevertheless, previous studies did not allow identifying the causative gene(s) and variant(s) possibly involved in this complex control. The SNP identified here (i.e. rs830532) is located in the 5q33.1 region, where are located the three markers for which the linkage with the intensity of infection was demonstrated . We did not regenotype this SNP to discard possible genotyping errors and confirm our result. Anyway it seems unlikely that genotyping errors occurred systematically according to the phenotype IntPI and then bias our result in favour of association All together, this work underlines several chromosomal regions which could play a role in resistance mechanism to malaria infection: 12q22 which have already been pointed out by a previous linkage analysis and three new regions . Two of them contain very few candidate genes and, if confirmed in association analysis, could point out new pathways implicated in the response to malaria infection.An. gambiae s.l. of which 97% were represented by An. ArabiensisThe study took place in the Niakhar area located 150 km south-east from Dakar, the capital city. Niakhar area is composed of 30 villages, regrouping 30,000 persons, the great majority of them belonging to the Sereer ethnic group. Malaria is endemic in the area and its transmission is seasonal and estimated between 9 and 12 infective bites per person per year, occurring almost exclusively between September and December, following the rainy season from July to September. Transmission is due exclusively to the complex Two villages of the area (Diohine and Toucar) were included in the study because of the existence of a dispensary with which we collaborated for several years. In January 2001, 1,202 children and young adults from 2 to 18 years old lived in these villages. A cohort of 504 individuals living with their two parents, and who agreed to take part in our study (informed consent signed by parents) was constituted. Neither age nor sex differed significantly between children included and not included in the cohort .P. falciparum; P. malariae and P. ovale) and leucocytes were counted. The PD was defined as the number of parasites per 100 leucocytes. This mode of calculation allowed avoiding misestimations of the parasitemia resulting from its determination on the basis of an assumed count of white blood cells per microliter of blood A TBS was collected to measure the PD in June, September, November and December 2001, in January, June, September, October and November 2002 and in January, April, June, September, October and December 2003. TBS were stained with Giemsa and asexual parasites  with a PD above 2500 trophozoites/\u00b5L. We only considered malaria attacks due to All participants were recruited, and human experimentations were conducted, in Senegal. This protocol was previously submitted and accepted by the ethic committee of the Health Minister of Senegal (N\u00b0 000526/MS/DERF/DER). At the time the study took place, all the programmes managed by the Unite de Recherche 010 from the Institut de Recherche pour le D\u00e9veloppement in Senegal followed this procedure. We obtained informed consent from all participants involved in our study. This consent was written, translated in Serer, and obtained from all the families included in the study.P. malariae since co-infections could influence the level of PDs For each child the following information was collected: (1) age (in years); (2) sex; (3) ethnic group ; (4) village of residence. We also noticed the presence of co-infection with P. falciparum asymptomatic infection nor MMA (cf. infra in the phenotype definition section) differed significantly (p>0.10) between children declaring to use a bed net and other children. Two ways were used to take into account medicine intake. Firstly, if a child was treated, by our team, for malaria attack during the follow-up, his PD measurement was not included in the mean level of P. falciparum infection during the following three weeks. Secondly, although most of the time children received their treatment within our research program framework, the uncontrolled circulation of drugs in Niakhar remains an important problem  declared the use of a bed net during the preceding night . Neither the measurements of P. falciparum infection and mild malaria disease. Concerning asymptomatic infection we defined three phenotypes of interest: (1) the mean level of P. falciparum density (MLPD) taking into account all the measurements performed during the follow-up, including negative TBS. This mean parasite density correspond to the phenotype for which the 5q31q33 chromosomal region were initially described P. falciparum infection (IntPI) that took into account only the positive TBS during the follow-up. This phenotype emphasizes the ability of an individual to tolerate parasite density without clinical disease; (3) the prevalence of asymptomatic P. falciparum infection (PrevPI). This prevalence reflects the acquisition of a non-sterilising immunity, occurring after repeated infections, but also dependent on human genetic factors involved in immune control, and acting independently of exposure. Analyses were realised on log transformed PD values (LDP) using log (PD+1) transformation to allow for 0 count. As some children may have not been present at each visit, we considered only the children with at least height measurements out of the fifteen performed during the follow-up.We were interested by asymptomatic P. falciparum infection. To deal with a unique variable accounting for the intensity of malaria infection, a mean log parasite density was computed for each subject, based on all their TBS, positive or negative. During the follow-up, the LPD varied significantly with time  consistent with the variation of malaria transmission. Then, before computing the mean level of infection, individual LPD were adjusted for this variability, by subtracting the mean LPD of the corresponding visit. Once computed, the mean level of infection was adjusted for the other risk factors  using a multiple linear regression. MLPD corresponds to the residual of the multiple linear regression, and was referred as the mean level of P. falciparum density during the follow-up.We used the same statistical method as in Garcia et al. P. malariae, year, sex and village) as well as one random intercept per individual in order to take into account the repeated measurements. We associated a residual density to each individual by considering the normalized sum of the model residuals  for each individual.The intensity of parasite infection was obtained through a linear model with mixed effect. We only considered here positive measurements . A stepwise model selection leaded to adjust for fixed effects  as well as one random intercept per individual in order to take into account the repeated measurements. A Pearson residual was then derived for each individual by considering the normalized difference between the observed and expected number of positive blood smear for each individuals (each measurement contribution being the inverse logit of the fixed effects part of the model).For the prevalence, the residual risk of having a positive blood smear was estimated through a logistic regression model with mixed effect, to take into account repeated measurements. A classical stepwise model selection leaded to adjust for several fixed effect  deficiency A- were determined on children. Frequencies of these variants in this Sereer ethnic group are 0.13 for HbS and 0.16 for G6PD A- \u22123). However, there was no difference concerning the 4 phenotypes under study between the two groups of children.Overall, a high density SNP genome scan was performed for 626 individuals: i.e. 249 parents and 377 children out of the 504 ones included in the follow-up. Among the 127 children for whom no genotyping data was available, 48 were absent or refused blood sampling and, for the 79 remaining children, technical problems occurred at the time of DNA extraction or DNA quality control. Children for whom DNA was not available were significantly younger than other  with a single primer set. After purification of the PCR products, amplicons were quantified, fragmented, labeled, and subsequently hybridized to the 250K SNP mapping array. The genotype of each SNP was determined by the BRLMM algorithm. A call rate (percentage of SNPs genotyped by sample) of 98.6% was obtained across the entire sample. Mapping order, physical and genetic distances of markers were obtained from Affymetrix. Note that physical distances for this GeneChip are based on NCBI Reference Genome Sequence Build 36.3.Genotyping was done with Affymetrix GeneChip Human Mapping 250K Rigorous quality control was performed before statistical analyses to ensure reliable genotyping data. Six samples with a call rate<95% were deleted. Gender of each sample was checked with the number of heterozygosities at SNPs on chromosome X and the relatedness of individuals was analysed using the GRR program \u22124. Then we examined the relationship between the number of Mendelian errors detected for each SNP and the deviation from HWE. We observed a high inflation of the percentage of SNPs out of HWE for SNPs with more than 8 Mendelian errors, indicating a genotyping of poor quality, and removed 3529 SNPs with more than 8 Mendelian errors. For remaining Mendelian errors, genotypes were deleted in members of the respective families. Non mendelian errors, i.e. unlikely genotype, were also investigated with MERLIN 2\u200a=\u200a1) with another proximate SNP in our data to avoid large block of SNPs. Exclusion of these SNPs was done using the Tagger tag SNP selection algorithm Allele frequencies and tests for Hardy-Weinberg equilibrium (HWE) were calculated from founders. Pedstats We performed two multipoint genome-wide linkage analyses on each phenotype using MERLIN program, a variance components linkage analysis 2\u200a=\u200a0.05 was used to join SNPs in clusters. However clusters defined by MERLIN are limited in size (21 SNPs) and the model assumes no LD between clusters. Thus we used a second method in the regions where a linkage peak was identified to check that LD was taken into proper account. This method consists in selecting from the whole SNPs available a subset of SNPs with a minimum of LD before realising linkage analysis. We used MASEL algorithm which selects a subset of SNPs with a minimum of LD (here r2<0.05) trying to optimise the number of informative SNPs and the SNP distribution along region Dense SNP maps have progressively replaced traditional panels of microsatellite markers for genome-wide linkage analyses because they provide more accurate genotyping and higher information content. However the presence of LD between SNPs is known to increase type I error in non-parametric multipoint linkage analyses, especially when founder genotypes are missing The genome-wide significance of linkage peaks was assessed by 10,000 gene dropping simulations on chromosome 22. This procedure enables to determine how many peaks of similar height might arise by chance conditional on the set of phenotypes being analysed, the marker map and the family structure. Quantitative trait linkage analyses were performed on each of these replicates. Then the average number of linkage peaks per replicate with a LOD score greater than or equal to a given threshold was calculated . As the genetic length of chromosome 22 is approximately 1/44 of the total length of the autosomal genome, the average number of peaks per replicate on the whole genome was obtained by multiplying the previous average number by 44. Following Lander and Kruglyak Finally, we performed an association analysis using FBAT method Regions around the maximum LOD score were defined using the 1-LOD support method. For 5q31q33 region, we tested association with SNPs of the whole region from 5q31.1 to 5q33.3 . For 10p15 region analysis was done on a 11 Mb region (as defined by Timman et al. FBAT method was applied to test association in the presence of linkage and we used the approach using the empirical variance-covariance estimator"}
+{"text": "The use of molecular genetic technologies for broodstock management and selective breeding of aquaculture species is becoming increasingly more common with the continued development of genome tools and reagents. Several laboratories have produced genetic maps for rainbow trout to aid in the identification of loci affecting phenotypes of interest. These maps have resulted in the identification of many quantitative/qualitative trait loci affecting phenotypic variation in traits associated with albinism, disease resistance, temperature tolerance, sex determination, embryonic development rate, spawning date, condition factor and growth. Unfortunately, the elucidation of the precise allelic variation and/or genes underlying phenotypic diversity has yet to be achieved in this species having low marker densities and lacking a whole genome reference sequence. Experimental designs which integrate segregation analyses with linkage disequilibrium (LD) approaches facilitate the discovery of genes affecting important traits. To date the extent of LD has been characterized for humans and several agriculturally important livestock species but not for rainbow trout.We observed that the level of LD between syntenic loci decayed rapidly at distances greater than 2 cM which is similar to observations of LD in other agriculturally important species including cattle, sheep, pigs and chickens. However, in some cases significant LD was also observed up to 50 cM. Our estimate of effective population size based on genome wide estimates of LD for the NCCCWA broodstock population was 145, indicating that this population will respond well to high selection intensity. However, the range of effective population size based on individual chromosomes was 75.51 - 203.35, possibly indicating that suites of genes on each chromosome are disproportionately under selection pressures.Our results indicate that large numbers of markers, more than are currently available for this species, will be required to enable the use of genome-wide integrated mapping approaches aimed at identifying genes of interest in rainbow trout. The use of molecular genetic technologies for broodstock management and selective breeding of aquaculture species is becoming increasingly more common with the continued development of genome tools and reagents for species of interest . Rainbowet al. 73]. Brieon 9.3.1 . The hapHWE analysis for each of the 49 loci typed in 96 unrelated individuals was performed using the ALLELE procedure of the software package SAS\u00ae, version 9.3.1 [HWE for each microsatellite loci  was estimated using 10,000 permutations.The on 9.3.1  using th\u00ae, version 9.3.1 [The LD analysis was performed between marker pairs within each linkage group using the ALLELE procedure of software package SASon 9.3.1 . We usedDuv between two alleles from marker loci M and N, respectively, was estimated using this expression [The linkage (or gametic) disequilibrium coefficient pression ,puv is probability that an individual receives the haplotype MuNv for marker loci M and N, pu is the probability of the u allele, and pv is the probability of the v allele.Where Duv's between two markers are zero as follows,The ALLELE procedure calculates the maximum likelihood estimates, k-1) and (l-1) degrees of freedom for markers with k and l alleles, respectively. A Monte Carlo estimate of the exact P-value for testing the hypothesis was calculated by conditioning on the haplotype counts. The significance level is obtained by permuting the alleles at one locus to form 2 n new two-locus haplotypes. We used 10,000 permutations to estimate the exact P-values.Which has (r2) as the LD measure for each pair of alleles Mu and Nv located at loci M and N, respectively [We used the squared correlation coefficient  was estimated by fitting the following equation ,LD for marker pair i in chromosome j, the constant k = 2 for sex chromosome and k = 4 for autosomes, dij is the recombination rate from two-point linkage analysis for marker pair i in chromosome j, bj is the estimate of effective population size for chromosome j, and eij is a random residual. The estimates of r2 for pairs of markers were adjusted for experimental sample size n is the chromosome sample size (n = 192). We performed the non-linear modeling with JMP\u00ae Genomics 3.1 .Where r2 - \u00ae, version 9.3.1 [We estimated LD adjusted for experimental sample size  and effective population size Ne,The analysis was based on the known relationship between c is the recombination rate between the microsatellite loci and n is the experimental sample size. The constant \u03b1 = 1 in the absence of mutation [\u03b1 = 2 if mutation is taken into account [k was set to k = 2 for sex chromosome and k = 4 for autosomes.Where mutation  and \u03b1 =  account -79. The r2 and c, we estimated Ne for each chromosome by fitting this nonlinear regression model,Given the formulae described in the linkage disequilibrium section, and knowing n) for marker pair i in chromosome j, cij is the recombination rate from two-point linkage analysis for marker pair i in chromosome j. The parameter \u03b2j is the estimator of effective population size for chromosome j where \u03b1j and \u03b2j were estimated iteratively using non-linear modeling with JMP\u00ae Genomics 3.1 .Where CR participated in design, marker selection, genotyping, and drafted the manuscript, RV participated in design, performed the calculations for LD and effective population size. Both authors read and approved the final manuscript."}
+{"text": "BRCA1 and BRCA2, indicating that still unidentified genes may explain relatively few families each or interact in a way obscure to linkage analyses. This has drawn attention to possible benefits of studying populations where genetic heterogeneity might be reduced. We thus performed a GWS for linkage on nine Icelandic multiple-case non-BRCA1/2 families of desirable size for mapping highly penetrant loci. To follow up suggestive loci, an additional 13 families from other Nordic countries were genotyped for selected markers.A significant proportion of high-risk breast cancer families are not explained by mutations in known genes. Recent genome-wide searches (GWS) have not revealed any single major locus reminiscent of GWS was performed using 811 microsatellite markers providing about five centiMorgan (cM) resolution. Multipoint logarithm of odds (LOD) scores were calculated using parametric and nonparametric methods. For selected markers and cases, tumour tissue was compared to normal tissue to look for allelic loss indicative of a tumour suppressor gene.The three highest signals were located at chromosomes 6q, 2p and 14q. One family contributed suggestive LOD scores  at all these regions, without consistent evidence of a tumour suppressor gene. Haplotypes in nine affected family members mapped the loci to 2p23.2 to p21, 6q14.2 to q23.2 and 14q21.3 to q24.3. No evidence of a highly penetrant locus was found among the remaining families. The heterogeneity LOD (HLOD) at the 6q, 2p and 14q loci in all families was 3.27, 1.66 and 1.24, respectively. The subset of 13 Nordic families showed supportive HLODs at chromosome 6q (ranging from 0.34 to 1.37 by country subset). The 2p and 14q loci overlap with regions indicated by large families in previous GWS studies of breast cancer.BRCA1/2 families. For genetic counselling it would seem important to resolve the mode of genetic interaction.Chromosomes 2p, 6q and 14q are candidate sites for genes contributing together to high breast cancer risk. A polygenic model is supported, suggesting the joint effect of genes in contributing to breast cancer risk to be rather common in non- BRCA1 or BRCA2 or other known genes [RAD51C [RAD51C was identified using a candidate gene approach, but if more high-penetrance genes are yet to be identified it might also be helpful to analyse families from populations where genetic heterogeneity might be reduced [BRCA1/2 families) [ESR1 gene is located [Increased susceptibility to breast cancer (BC) has been shown to be caused by germline segregation of three different classes of alleles: 1) high-penetrance genes with rare risk variants, 2) moderate-penetrance genes, also with rare variants and 3) low-penetrance alleles of common frequency [wn genes  and may wn genes -8. Howev [RAD51C , demonst reduced ,11. Receamilies) -16, togeamilies) -19, indiamilies)  and the  located ,16, whic located .BRCA1 and BRCA2 are tumour suppressor genes and most often involve wild-type loss of heterozygosity (wt-LOH) in mutation carriers' tumours [BRCA1 and BRCA2 have been found with recurrent mutations, one in each gene, with the BRCA2-999del5 mutation occurring in 8.5% of BC patients and 0.5% of the population [BRCA1/2 families for a GWS of high-risk genes under a parametric dominant linkage model. Regions considered suggestive (LOD \u2265 1.5 per family) were also subjected to wt-LOH analyses in tumours from putative gene carriers, and the same regions were genotyped in a collection of Nordic non-BRCA1/2 families, in order to estimate the possible proportion of linked families in Iceland and other Nordic countries.Two issues helped shape our current GWS study. First, both  tumours ,21, resu tumours . Any newpulation -25. Othepulation . This acBRCA1 and BRCA2 mutations in 438 BC cases diagnosed in Iceland in the years 1989 to 2001, the history of BC was evaluated in the pedigrees of both the mother's and the father's family side of the non-BRCA1/2 cases. Nine families were selected and subjected to a GWS of BC linkage by the selection criteria of (1) at least three women diagnosed with BC under age 60 years , (2) the availability of blood or paraffin-embedded normal tissue for isolation of DNA of sufficient quality from at least four affected cases (any age), and (3) evidence against linkage to BRCA1 or BRCA2 according to genotyped microsatellite markers flanking and within these genes. Each of the nine families consisted of descendents of a single pair of founders. In five families a DNA sample was available from six or more BC cases  in order to find possibly overlapping positions, which could then be further examined by manual comparison of haplotypes.After screening for recurrent Icelandic BRCA1/2 families at other Nordic centres, in line with the above criteria and in such a way that the genotyped affected family members would not be expected to share by descent more than approximately 6% of alleles by chance (through at least six meioses). Written informed consent was obtained with all blood samples and appropriate Institutional Review Board approvals were obtained. Characteristics of the 22 families are summarised in Table Thirteen additional Nordic families were used in follow-up studies on suggestive loci on chromosomes 2p, 6q and 14q. They were selected from available non-et al. [DNA was extracted from nuclei of lysed blood samples according to Miller et al.  or by stet al.  using thMerlin software   was usedlinked under the admixture model, the files prepared by Merlin software were reformatted to fit LINKMAP software , Bethesda, Maryland, USA) for sliding three-point linkage analysis of selected markers. Eighteen markers were analysed at 6q, 16 at 14q and 7 at 2p, using country-specific allele frequencies. Disease allele frequency and age dependent liability classes were as above, under the dominant model [In order to analyse conditional probabilities by family of being nt model ,30.a priori unknown positions), being simultaneously cosegregated with a trait through m meioses, in one out of N families with similar or greater total number of meioses (Bonferroni adjustment), was calculated as described , were genotyped for a set of 811 genome-wide distributed microsatellite markers for subsequent linkage analysis. As all pedigrees appeared high-risk and dominant, the main analysis was based on the parametric dominant model but other models were also considered in order to see if the LOD scores were sensitive to model misspecification. Figure BRCA1/2 linkage is shown in Figure Nine Icelandic BC families, unexplained by BRCA1/2 BC-affected women in this family and eight of these women also shared a haplotype (9 Mb) at 2p  the probability of observing cosegregation of two loci (6q and 14q) through 13 meioses in one of five families is P = 0.006. The corresponding probability for all three loci and 11 meioses is also P = 0.006 (formula (2)). We also used Merlin to simulate marker data in order to get an indication of the frequency of obtaining three separate dominant model LOD scores > 2.5. A total of 700 replicates of the per-family runs were generated using the same allele frequency estimates as in the original run. In none of the replicates did we obtain a family with three peaks of this magnitude, in two instances there was a family with two peaks, in 75 instances we obtained a family with one location with dominant LOD > 2.5 (in six instances a family exceeding LOD 3.0), and in the remaining simulated cases no dominant LOD > 2.5 was obtained.When analysing separate families, one named 70234) was seen to contribute high scores (LOD 2.63 to 3.03) at all three major positions, 2p, 6q and 14q Figure . At 6q a34 was seEight breast tumours from family 70234 were assayed at markers within the shared haplotypes, for loss of heterogeneity selective for losing wild-type alleles (wt-LOH) in harmony with Knudson's model of tumour suppressor genes. At 6q, LOH was seen at all informative markers in three tumours, with wt-LOH in two but the third lost all alleles from the risk-related haplotype (data not shown). Copy-number loss of the region was confirmed in the three tumours by array-CGH (data not shown) which also revealed amplification at 6q21 in a fourth tumour. At 2p and 14q, signs of allelic losses were confined to single markers and of inconsistent allelic phase (three tumours each chromosome), and not supported by array-CGH since intensities were generally within thresholds.P-values of < 0.005 . LOD scores of weaker indication were seen at multiple positions . An analysis of heterogeneity, using LINKMAP was performed on all 22 Nordic families (treated as 25 for the reasons given above) at the three chromosomal positions and the results are shown in Table To follow up suggestive linkage signals (HLOD > 1.5 by family), markers at 2p, 6q and 14q were genotyped in an additional 13 Nordic non-BRCA1/2 families, substantial linkage signals were observed at chromosomes 2p25.1 to p22.1, 6q15 to q22.31 and 14q21.3 to q31.3, and the strongest contribution to all three regions occurred in the same family (70234). On a single family basis, all three signals in the family are exceptionally high compared to previous studies [BRCA1/2 families. The position of the more centromeric signal in our 2p region (2p23.3 to p21) was in fact pinpointed on the basis of single families by two independent studies [In the present GWS for BC linkage in nine Icelandic non- studies -19, but  studies . The reg studies ,13. The  studies . The reg studies ,19 and dBRCA1/2 BC as mainly polygenic with decreasing possibility of finding new high-risk genes. Our results support this view in the following way: The current study was primarily designed to find whether dominant mutations of high penetrance exist in large Icelandic non-BRCA1/2 families with a Mendelian pattern concurrent with such genetic explanation. Most families failed to reveal evidence of any such locus. Although three chromosomes provided suggestive linkage signals, their coexistence in one family hardly supports the idea of a single causative gene. Some families have previously been reported with two or three suggestive chromosome regions [P = 0.006 of seeing any two loci that are not linked to each other cosegregate by chance with the disease through 13 meioses as here seen at 6q and 14q. This would argue for the existence of more than one causative gene in family 70234. Whether the third locus (2p), which also cosegregates to eight of the nine cases, plays a role in the risk is a little more ambiguous. Although the ninth case inherited some alleles identical with the common haplotype (and therefore contributed to the high LOD of 2.63) they were on closer scrutiny  seen to be interrupted by non-matching alleles and therefore she should be regarded as a phenocopy with respect to possible 2p-linkage. By formula (2), the probability of observing cosegregation of three loci through 11 meioses in one of five families is P = 0.006. Although this might be adjusted with respect to different possible ways of observing a phenocopy, and to the fact that the phenocopy did inherit two of the three haplotypes, the resulting value would still be on the same order as the compared value for two loci. The 2p-haplotype may therefore be irrelevant to the BC risk but it gains support from the two previous reports of a candidate BC susceptibility locus which overlaps with this 2p-region [Recent studies have indicated familial non- regions -13,16 an regions . We do nIt may be asked if the unique triple strong signals in family 70234 as compared to other families possibly reflect distinction by genetic linkage models. The absence of notable linkage peaks in the remaining families conforms to a polygenic model with frequent alleles, sometimes cosegregating and sometimes being replaced by variants of different lineage or of other genes. Family 70234 seems to differ in this respect, even if questioning the 2p-linkage, since the two haplotypes at 6q and 14q fully cosegregate with the disease in nine cases, with maximum LOD ranging from 2.74 to 3.03. Therefore a replacement of one haplotype by a new variant (if needed) would appear to be a rare event in this instance, and one might expect a low population frequency of the gene variants involved. This would have implications for genetic counselling, since continued cosegregation of the two (not to mention three) haplotypes would seem improbable for the descendants of family 70234. It would then also be important to resolve whether each of the genetic variants contributes an independent proportion of the disease risk (additive or multiplicative joint effects), or in an interdependent way, their risk being dependent on the presence of all cofactors. With such a scenario, one would view this family's cancer history mainly as a very rare chance result of cosegregation of limited consequence for later generations of the family. Alternatively, the families studied here may all comply with the same genetic model, with cooperative alleles of moderate or high frequency, and the strong linkage signals in family 70234 to be accounted for by the absence of phenocopies. We note, in this context, that by treating the nine families as 12 for linkage calculations, most were not as highly informative by the number of cases or meioses as 70234, but nevertheless they were expected to reveal clues of genomic position by pairwise comparison of separate parts of the larger pedigree.The additional Nordic families support the risk indication of chromosome 6q, but seem not to support the 2p- and 14q-linkage. One Finnish family may be of the linked type at the same 2p position as family 70234 but this is not higher than expected by chance from 13 families, each with up to 6% sharing, by descent, of genetic material between its affected members. The 6q-linkage gains some support from other Icelandic families ; LOD: logarithm of odds; LOH: loss of heterozygosity; non-The authors declare that they have no competing interests.RBB and AA designed the study and together with EG selected the Icelandic families after an initial power simulation performed by EG. HG and GJo carried out the DNA analysis and genotype data collection and AA did the processing and checking of the data. KJ carried out the linkage analyses and significance testing, helped with organising and writing statistical parts of the manuscript, and together with AA, shaped the statistical analysis of cosegregation of multiple loci. P-OB performed the heterogeneity analyses. HG and GJo carried out the array-CGH analyses. OThJ, BAA, RBB, AA, HN, RW, \u00c5B, PM, BM, KP, AM, TH, KA and CB provided samples and information on the families included in this study. AA, with the help of RBB, wrote the manuscript and all co-authors critically read and approved it. RBB conceived and coordinated the study.Figure S1, pedigrees of the families in the GWS. This is a jpg file showing pedigrees of the families included in the GWS. Pedigrees of nine Icelandic non-BRCA1/2 families with each showing BC cases traced to a single pair of founders but otherwise omitting relatives if not genotyped. Genotyped family members are marked with an asterisk. Circles denote females and boxes males, with red filling denoting diagnosis of BC and shaded red also ovarian cancer. Tan filling indicates cancer at other sites than breast, or of unknown origin. Information about the site and approximate age (in years) at diagnosis of cancer is shown below the symbols . Dotted vertical lines between family branches show how the family was separated in two parts for linkage calculations. Pedigrees are somewhat distorted in order to avoid recognition.Click here for fileSupplementary methods. A Word document containing a methodological description of intra-family significance testing for multiple loci.Click here for fileFigure S2, parametric LOD scores by family. A jpg file showing graphs of parametric LOD scores by chromosomal position, for the families in the GWS . LOD scores are shown for the dominant  and the recessive model (plum). Three families  were separated in smaller units for linkage analysis, as indicated by adding the letter a or b to the family name.Click here for fileFigure S3, NP-LOD scores by family. A jpg file showing graphs of NP-LOD scores and associated P-values by chromosomal position in individual families included in GWS, using different exponential scoring options in Merlin software: S-all (orange thick line) and S-pairs (indigo). Three families  were separated in smaller units for linkage analysis, as indicated by adding the letter a or b to the family name.Click here for fileP < 0.005Table S1, Maximum LODs by chromosome and family, for NP-LODs with . A Word file containing a table of per-family LOD signals , for consideration of whether any chromosomal positions may be indicated by more than one family.Click here for file"}
+{"text": "Genomic imprinting is a mechanism in which the expression of a gene copy depends upon the sex of the parent from which it was inherited. This mechanism is now well recognized in humans, and the deregulation of imprinted genes has been implicated in a number of diseases. In this study, we performed a genome-wide joint linkage and imprinting scan using two data sets provided by Genetic Analysis Workshop 15 (GAW15).The first data set was high-risk rheumatoid arthritis families collected by the North American Rheumatoid Arthritis Consortium. We used both model-based and model-free methods of joint linkage and imprinting analyses. Although a genome scan of rheumatoid arthritis families using GENEHUNTER-MODSCORE suggested regions that might be imprinted, further analyses using variance-components method failed to obtain significant signals of imprinting. The second data set was Problem 1 of GAW15, which included single-nucleotide polymorphism genotypes and gene expression data for Centre d'Etude du Polymorphisme Humain pedigrees. A previous genome-wide linkage scan identified loci that may be regulators of gene expression: our genome-wide joint linkage and imprinting scan using a variance-components approach found significant signals for linkage.TGFBR3 gene, we found a point-wise p-value of 0.03 for imprinting, but increase in the LOD score did not meet the required threshold to reliably identify imprinting as the correct mode of inheritance in genome-wide linkage scans.Our linkage scan results suggest that imprinted genes are unlikely to be involved in susceptibility to rheumatoid arthritis. However, for expression level of  Genomic imprinting is a phenomenon in which the expression level of a gene is determined by the parental origin of the chromosome on which it resides. In maternal imprinting, genes that are on the paternally inherited chromosome are expressed while their counterparts on the maternally inherited chromosomes are silenced. Several genes that affect development in mammals are known to be imprinted, and imprinted genes have been implicated in several human disorders. Currently, only 41 transcriptional units have been identified as imprinted in humans  recentlytrans-acting regulator loci in a linkage scan [ALG6) was also included because a trans-acting regulator of ALG6 was mapped to chromosome 19 in a previous genome-wide linkage scan [ALG6, we compared results obtained using our method with the previous linkage scan based on Haseman-Elston sib-pair based regression approach [In our analyses of North American Rheumatoid Arthritis Consortium (NARAC) data, we used measurements of anti-cyclic citrullinated peptide (CCP) antibody as quantitative trait values. Because these quantitative phenotypes are part of the diagnosis of rheumatoid arthritis, the variation in these phenotypes should be due to the same underlying variation in genetic factors as in rheumatoid arthritis. In the analyses of gene expression data Problem 1 of GAW15), we used single-nucleotide polymorphism (SNP) data of 14 Centre d'Etude du Polymorphisme Humain (CEPH) Utah pedigrees. The expression levels of 60 genes  \u00d7 LODReg) asymptotically follows a half-and-half mixture of \u03c72 random variable with 1 and 0 degrees of freedom. A LOD score of 3.0 is considered significant for genome-wide linkage scan, which gives an asymptotic p-value of 0.0001. For joint testing of linkage and imprinting, the likelihood ratio test (2 \u00d7 Loge(10) \u00d7 LODImp) asymptotically follows a mixture of \u03c72 distributions with 0, 1, and 2 degrees of freedom in the proportions of 1/4, 1/2, and 1/4. Therefore, to achieve an asymptotic p-value of 0.0001, a LODImp score of 3.4 is needed. Under the no imprinting variance-components model, expression levels of three genes had LOD scores greater than 3.0. These LOD scores were 4.08 on chromosome 2, 4.15 on chromosome 2, and 3.26 on chromosome 20, for transforming growth factor beta receptor III (TGFBR3), solute carrier family 25 member 32 (SLC25A32), and early growth response 2 (EGR2), respectively. Under the model that allows for imprinting, we obtained LOD scores of 4.86 on chromosome 2, 4.29 on chromosome 2, and 3.79 on chromosome 20, for TGFBR3, SLC25A32, and EGR2, respectively. No additional linkage signals were observed under the imprinting model. It is important to note that the maximum LOD scores either obtained by allowing for imprinting or obtained without allowing for imprinting were at the same or nearby markers. We used a statistic \u0394 = 2 \u00d7 Loge(10) \u00d7 (LODImp-LODReg) to obtain p-values for the test of imprinting where LODImp and LODReg are LOD scores using imprinting model and no imprinting model, respectively. The \u0394 statistic is a likelihood ratio test that is asymptotically a mixture of 0 and \u03c72, with one degree of freedom in the proportions of 1/2 and 1/2 under the null hypothesis. For TGFBR3, the difference between LODImp and LODreg was 0.78 at marker rs117261, where maximum LOD score was observed under the imprinting model. Based on \u0394 statistic, TGFBR3 showed p-value of 0.03 for imprinting. The estimated values of variance components suggested maternal expression. Greenberg et al. [We used two models in genome scans, one allowing for imprinting and one without. We obtained multipoint LOD scores for 22 autosomal chromosomes under both models. The highest multipoint LOD scores for each gene were listed in Additional file g et al.  proposedWe conducted simulation studies to evaluate power to detect linkage of an imprinted locus using the 14 CEPH families provided by GAW. We simulated a quantitative trait that had 20% heritability and was tightly linked to a marker locus (theta = 0) with eight equally frequent alleles (to simulate the nearly complete information about segregation provided by the dense SNP scan). The trait locus had two alleles  and was maternally imprinted. The means of trait values were set at 0.68 for genotypes AA  and Aa, and -0.68 for aA and aa. We also simulated a separate locus unlinked to the marker to represent the polygenic component of genetic variance. The unlinked locus had two equally frequent alleles (B and b) with means of 1.0 for genotype BB, 0.0 for genotypes Bb and bB, and -1.0 for genotype bb. The total residual variance was 1.0. Under these conditions, we observed an average LOD score of 3.38 from 100 replicates which suggest that an imprinted disease locus with 20% heritability can be detected using these families.ALG6, the highest LOD score of 2.18 was obtained at rs1019937 on chromosome 19 when imprinting was not allowed. This LOD score is suggestive for linkage but not significant in a genome-wide scan. Our result is different from previous reports by Morley et al. [trans-acting regulator of ALG6 on chromosome 19 . Furthermore, when we included imprinting in our linkage analyses, we obtained a LOD score of 2.34 at the same marker location. The difference in LOD scores, with imprinting and without imprinting, is not significant for concluding that imprinting is a mode of inheritance.For p-value > 0.5). Our results suggested that imprinted genes are unlikely to be involved in susceptibility to rheumatoid arthritis.Linkage analyses of NARAC data was conducted in two stages. We first performed multipoint MOD score analysis over the entire genome with GENEHUNTER-MODSCORE . We thenIn our analyses of NARAC data, we observed differences between Caucasian and Hispanic families that were pronounced in the distributions of age of onset, anti-CCP antibody, and other variables. These differences may be caused by the distinct genetic backgrounds of the two groups.p-value of 0.03 for imprinting for the TGFBR3 gene, which is of interest, but the increase in the LOD score did not meet the threshold of 1.5 to reliably identify imprinting as the correct mode of inheritance in genome-wide linkage scans. However, our search is exploratory and far from exhaustive. We only included genes that had shown evidence of linkage in previous studies. With proper transformation, expression levels of other genes can be analyzed in a similar fashion. Whether some other regulators of gene expression may be imprinted is still a question that deserves further investigation. Our simulation study indicates that our method has ample power to detect linkage under imprinting using pedigrees similar in size to the CEPH families.We identified three regulatory loci of gene expression in our genome scans. We found a point-wise ALG6 expression data yielded results less significant than those previously published, perhaps because of differences in the raw data (SNP density) and statistical methods that were used. Previous results were obtained using a modified version of Haseman and Elston method [ALG6, excluding parents and grandparents. We obtained much higher maximum LOD scores on chromosome 19 (3.61 without imprinting and 3.76 with imprinting), which are significant for linkage, but difference between the two were not suggestive for imprinting.Our analyses of n method , which iTGFBR3 gene, we found a point-wise p-value of 0.03 for imprinting, but the increase in the LOD score did not meet the required threshold to reliably identify imprinting as the correct mode of inheritance in genome-wide linkage scans.Our linkage scan results suggest that imprinted genes are unlikely to be involved in susceptibility to rheumatoid arthritis. However, for The author(s) declare that they have no competing interests.SS and XZ participated in project conception, and drafted the manuscript. XZ performed nonparametric linkage analyses of NARAC and expression data. WC performed MOD score analyses of NARAC data. RY suggested changes in the analyses. YL and RY participated in data reformatting and management. MDS, C-CW, and CIA contributed in discussions of results and revision of the manuscript.Genome scan of gene expression data using MULTIC-IMPRINTING.Click here for file"}
+{"text": "Our aim is to develop methods for mapping genes related to age at onset in general pedigrees. We propose two score tests, one derived from a gamma frailty model with pairwise likelihood and one derived from a log-normal frailty model with approximated likelihood around the null random effect. The score statistics are weighted nonparametric linkage statistics, with weights depending on the age at onset. These tests are correct under the null hypothesis irrespective of the weight used. They are simple, robust, computationally fast, and can be applied to large, complex pedigrees. We apply these methods to simulated data and to the Genetic Analysis Workshop 16 Framingham Heart Study data set. We investigate the time to the first of three events: hard coronary heart disease, diabetes, or death from any cause. We use a two-step procedure. In the first step, we estimate the population parameters under the null hypothesis of no linkage. In the second step, we apply the score tests, using the population parameters estimated in the first step. It is well known that heterogeneity results in loss of statistical power when studying genetic factors of complex genetic diseases. To deal with heterogeneity additional data such as covariates  are collected. In this paper we are interested in adjusting linkage for age at onset.Frailty models have been proposed for age-at-onset linkage analysis -5. GammaA second model for multivariate survival data is the log-normal frailty model. Using this model, Pankratz et al.  proposedTij be the random variable of age at onset for relative j in family i, i = 1, ..., N. Let  be the observed data where tij is the observed age at onset if dij = 1 and age at censoring if dij = 0. The conditional hazard for individual j in family i, with covariates xij and random effect Zij, is given by \u03bb = \u03bb0(tij | xij)Zij. Without loss of generality, we assume that E [Z] = 1. The baseline hazard \u03bb0(t) is the hazard for x = 0 and Z = 1. The frailty Z is decomposed into the sum of independent gamma distributed effects, namely a linkage effect, a residual additive effect, and a non-shared environment effect. The scale parameter is common to all of the effects and is defined as the sum of the shape parameters. When the proportion of alleles shared identically by descent (IBD) for a relative pair  is known (\u03c0jk), the marginal bivariate survival function can be derived from the additive gamma frailty model  with weight matrix W = \u03a31-1M(\u03a31-1M)'-\u03a31-1 and \u03a31 taken in \u03b3 = 0. In this paper we approximate the baseline cumulative hazard with the marginal cumulative hazard.Let \u03c1 = 0.46 and the variance estimated by the gamma frailty models was \u03c1 = 0.5 and the variance estimated by a log-normal frailty model [Three phenotype files were provided: Original Cohort participants, Offspring participants, and Generation 3 participants. We combined the three files and used this dataset as a random sample from the population. The total number of individuals considered was 6879. The number of disease-free survival events was 644 , with prevalence around 10%. We estimated the marginal survival functions stratified by sex using the Kaplan-Meier estimator. By age 60 years, 20% of males and 10% of females were affected. Using these estimated survival functions we fitted a marginal pairwise correlated gamma frailty model. The sib-sib marginal correlation was n = 1599 nuclear families. The number of nuclear families with at least one affected sibling was n = 488. Only 46 nuclear families were available with at least two affected siblings.In the Genetic Analysis Workshop (GAW) 16 Framingham Heart Study (FHS) data 765 pedigrees with 2 to 301 genotyped subjects were available. To simplify the IBD computation, large pedigrees were split into The GAW16 Framingham dataset included 550 k SNP genotype data. Using the nuclear families with at least one affected individual , we selected 15 k SNPs informative for linkage. First, markers with known physical position were selected (497 k). Second, 10 markers per centimorgan with minor allele frequency larger than 0.15 were considered (37 k). Finally, SNPs were simulated on 250 sib-pairs in order to select 15 k SNPs with the highest information content. The information content of the final set of SNP was around 85%.To assess power and type I error rates, we simulated data using a frailty model with parameter values estimated in the GAW16 FHS data. The random effect was gamma-distributed with a mean of one and variance of Table We performed a genome-wide linkage analysis using the unweighted NPL test (mean IBD test) with variance of the allele shared IBD estimated by simulations . Figure n = 1599), on the families with at least one affected siblings (n = 448) and on the subset of families with at least two affected siblings (n = 45). The maximum LOD-scores were obtained considering only families with at least two affected siblings. Figure We applied the proposed methods to the data of these two chromosomes. The linkage analysis was performed on all the nuclear families  studies, the proposed methods can be applied to ascertained families.For the two identified regions, association analysis in the presence of linkage may be the next step. The proposed models can be easily extended to study association in the presence of linkage by including the genotype of the siblings as a covariate.In this paper we computed IBD probabilities using MERLIN and we estimated the variance of the allele shared IBD using simulations . BecauseSoftware to apply the proposed methods is freely available .We proposed two new score tests for age of onset linkage analysis. Both methods are simple and can be applied to general pedigrees. Simulations showed that the proposed methods outperform the traditional affected-only NPL method. On the application to the GAW16 FHS data, adjusting for age at onset slightly increased the interesting linkage peaks.FHS: Framingham Heart Study; GAW: Genetic Analysis Workshop; IBD: Identical by descent; NPL: Nonparametric linkage; SNP: Single-nucleotide polymorphismThe authors declare that they have no competing interests.AC participated in method development, carried out data analysis, and drafted the manuscript. QH carried out the SNP data selection. JJH-D participated in method development. H-WU acquired the data. All authors read, critiqued, and approved the final manuscript."}
+{"text": "With the covariate 'anti-CCP level', statistically significant increases in NPL scores were observed in chromosomes 2 (p = 0.0001), 18 (p = 0.00007), and 19 (p = 0.0003). Once we identified the linked genomic region, we then attempted to identify the best plausible parametric model at that linked locus. Our results show significant improvement in evidence for linkage and demonstrate that OSA is a useful technique to detect linkage under heterogeneity.Rheumatoid arthritis (RA) is a chronic, complex autoimmune inflammatory disorder with poorly known etiology. Approximately 1% of the adult population is afflicted with RA. Linkage analysis of RA can be complicated by the presence of phenotypic and genetic heterogeneity. It is shown that the ordered-subset analysis (OSA) technique reduces heterogeneity, increases statistical power for detecting linkage and helps to define the most informative data set for follow-up analysis. We applied OSA to the family data from the North American Rheumatoid Arthritis Consortium study as part of the Genetic Analysis Workshop 15 (GAW15). We have incorporated two continuous covariates, 'age of onset' and 'anti-CCP level' (anti-cyclic citrinullated peptide), into our genome-wide ordered-subset linkage analysis using 809 Illumina SNP markers in 5713 individuals from 606 Caucasian RA families. A statistically significant increase in nonparametric linkage (NPL) scores was observed with covariate 'age of onset' in chromosomes 4 ( Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disorder of unknown etiology. Although environmental influences may trigger a response leading to the development of this autoimmune disease, both genetic and environmental factors are implicated in its pathogenesis . It affeRA is a clinically heterogeneous disease and most likely has complex genetic involvement. The presence of underlying genetic heterogeneity of a trait often masks the effect of genetic markers with disease predisposing variants; hence, there may not be linkage in families in which the marker is not involved in the disease etiology . One metThe aims of our present analysis are to: 1) identify homogeneous subset of families and assess linkage and its location, 2) rigorously analyze the homogenous subsets of families with statistically significant chromosomal locations to find a parsimonious genetic model.We analyzed data from the North American Rheumatoid Arthritis Consortium (NARAC) study as part of the Genetic Analysis Workshop 15 (GAW15). Only Caucasian families were used for these analyses. Initially, out of the original 637 families, 31 families were removed due to mixed ethnicity or because they were uninformative for linkage analysis (single affected member per family). In larger families, ungenotyped individuals were trimmed to facilitate computation that otherwise was not possible due to time and memory constraints on the computer hardware used. We performed genome-wide linkage analysis of 809 Illumina SNP markers in 5713 individuals from 606 Caucasian rheumatoid arthritis families. Analyses were performed using FLOSS , MERLIN, GeneHunter, GeneHunter-Modscore, and Genehunter-Plus with the ASM  module. We used several complementary programs to compare the accuracy of our results.k smallest or k largest covariate scores. Thus, the subset type used here was extreme. The FLOSS program was used to create a covariate file for family covariate scores for all families and all covariates, and to calculate nonparametric linkage (NPL) scores. Permutation tests were used to assess the null hypothesis of independence of family linkage scores at each locus and family covariate scores. Each subset of homogeneous families that generated a statistically significant linkage was analyzed with GeneHunter to further confirm the NPL score.To date, several clinical and epidemiological factors have been identified as potential trait-related covariates for RA. Among them, increasing age of onset has been associated with worse outcome in RA, with evidence that there has recently been a shift towards an older age of onset . There aOnce we identified the linked genomic region, we then attempted to identify the most plausible parametric model  at that linked location. For each subset, parametric LOD scores were maximized using GeneHunter-Modscore. These allele frequencies and penetrance values were utilized in GeneHunter/GeneHunter-Plus with ASM module, which provides NPL, nonparametric LOD, parametric LOD, and heterogeneity LOD (HLOD) scores. In addition, information content was provided, which gave an index of the inheritance information extracted at each point in the genome by the marker genotyped. The LIN function  of allep = 0.000003, peak at 102.03 cM, 472 families) and suggestive evidence was observed at chromosome 9 . With covariate 'anti-CCP level', statistically significant evidence of linkage was identified at chromosome 18 , and suggestive evidence of linkage was observed at chromosome 2  and chromosome 19 . The information content extracted at the linked region ranged from 46% to 80%.The results of OSA along with the other relevant statistics are provided in Table p < 0.05) when we used all families versus subset of families. Further, the results with the GeneHunter program using the ordered subset families produced a statistically significant linkage that confirms the nearly identical NPL score obtained by the FLOSS program. Table For each linkage region, the NPL score was significantly increased . The optimal range of 'anti-CCP level' in the ordered subset of families was greater in chromosomes 2 (133 to 413) and 18 (234 to 413), but lower for chromosome 19 (0.800 to 3.50). The values of peak maximized LOD (MOD) score and HLOD scores are nearly equal (which is expected). After using the allele-sharing model, not much difference was seen between the peak MOD score and nonparametric LOD scores produced by ASM except on chromosome 19.We have identified five linked chromosomal regions  that may harbor the susceptibility genes for RA. Previous studies ,14 had iad hoc correction procedure that raised the threshold of LOD score to 3.9. [This is calculated as: LOD(corrected) = LOD(conv) + log10(#test) [(conv) is conventional LOD score to be significant = 3.3.] Interestingly, all three linkages remain significant after correcting for multiple testing.We considered both nonparametric and parametric linkage analysis in this study. Both parametric and nonparametric results are very similar in terms of detecting the peak linkage locations. If we use the nonparametric LOD score then we have evidence for three statistically significant linkages at chromosomes 2, 4, and 18 that exceed the Lander and Kruglyak criteria (LOD score of 3.3) . However0(#test) ,17, wherFor a complex trait like RA, successful identification of genetic risk loci has relied on the ability to minimize disease and genetic heterogeneity to increase the power to detect linkage. One way to account for disease heterogeneity is by incorporating covariate data. Phenotypically similar families may be genetically more homogeneous as well, in which case OSA can greatly improve the power of linkage analysis. Our results clearly show that 'age at onset' and 'anti-CCP level' are potentially two clinical markers that can be useful to detect linkage for RA and that OSA is an important technique to identify the linkage in the presence of heterogeneity. Such linkage studies could now be used for candidate gene as well as and fine mapping studies to identify the actual RA susceptibility genes.A genome-wide OSA was performed to identify the linkage for RA. We used two continuous covariates, 'age of onset' and 'anti-CCP level' to identify a more homogeneous group. We have identified two statistically significant regions with evidence of linkage at chromosomes 4 and 18 and three regions with suggestive evidence of linkage at chromosomes 2, 9, and 19. Our results clearly demonstrated that OSA is a useful technique to detect linkage under heterogeneity.The author(s) declare that they have no competing interests."}
+{"text": "Parametric linkage methods for quantitative trait locus mapping require explicit specification of the probability model of the quantitative trait and hence can lead to misleading linkage inferences when the model assumptions are not valid. Ghosh and Majumder developed a nonparametric regression method based on kernel-smoothing for linkage mapping of quantitative trait locus using squared differences in trait values of independent sib pairs, which is relatively more robust than parametric methods with respect to violations in distributional assumptions. In this study, we modify the above mentioned nonparametric regression method by considering local linear polynomials instead of the Nadaraya-Watson estimator and squared sums of sib-pair trait values in addition to squared differences to perform a genome-wide scan of rheumatoid factor-IgM levels on sib pairs in the Genetic Analysis Workshop 15 simulated data set. We obtain significant evidence of linkage very close to the quantitative trait locus controlling for RF-IgM. We find that the simultaneous use of squared differences and squared sums increases the power to detect linkage compared to using only squared differences. However, because of all the sib pairs are selected for rheumatoid arthritis, there is reduced variance of RF-IgM values, and empirical power to detect linkage is not very high. We also compare the performance of our method with two linear regression approaches: the classical Haseman-Elston method using squared sib-pair trait differences and its extension proposed by Elston et al. using mean-corrected sib-pair cross-products. We find that the proposed nonparametric method yields more power than the linear regression approaches. Heritable quantitative characters are precursors of a clinical end-point trait. Because end-point traits are usually binary in nature (affected/unaffected) and hence contain minimal information on variation within trait genotypes, it may be statistically more powerful to use a correlated quantitative phenotype for identifying genes for the underlying complex trait. Unlike qualitative or binary traits, which can be characterized completely by allele frequencies and genotypic penetrances, quantitative traits require a stronger layer of modeling: the probability distribution of the underlying trait. Thus, compared to likelihood-based approaches like variance components ,2, whichFor our analyses, we used data on RF-IgM levels and genome-wide information on 730 microsatellite marker loci distributed over the 22 autosomal chromosomes. Our method utilizes marker genotype data on 1500 independent sib pairs and their parents for identity-by-descent (IBD) computations. The nonparametric regressions for the linkage scan are based on the RF-IgM and IBD data. We performed our analyses on all 100 available replicates.yij denotes the RF-IgM of the jth sib in the ith family, i = 1, 2,..., 1500; j = 1, 2; and ith sib pair at an arbitrary point p on the genome. We define Ui = (yi1 - yi2)2 and Vi = (yi1 + yi2)2. The classical Haseman-Elston method [Ui values) on estimated marker IBD scores  to consider a linkage finding to be statistically significant. Since the \"answers\" were available to us, we considered a linkage peak to be true positive only if both the following criteria were satisfied: it was within a 20 cM window (10 cM on either side) of the true position of a QTL and all other markers within this window provided significant evidence of linkage. Hence, we have assessed the empirical power and the false-positive error rate based on the proportion of replicates yielding significant linkage peaks.We performed our nonparametric regression analyses on all 22 autosomal chromosomes for all 100 available replications. We compared the results of the nonparametric regression with those of the classical Haseman-Elston regression using squared differences  using mep-value < 0.05) at marker STRP11_22 (113 cM) on chromosome 11 in 17 replications using squared differences only and in 31 replications using both the squared differences and the squared sums. All of the other markers within the 20-cM window of the position of the QTL have also provided evidence of linkage at level 0.05 for all these replications. Although given a threshold of p < 0.001 for a linkage peak to be statistical significant, the empirical power was only 0.1 when only squared differences were used and 0.23 when both squared differences and squared sums were used, we note that the linkage peak is close to Locus F (115 cM), the QTL controlling RF-IgM. However, the major aim of the study, that is, the belief that the combined use of squared differences and squared sums is more powerful than using only the squared sums is validated by our results. We also found that there was no other marker which provided a statistical evidence of linkage at level 0.05 in more than three replications.Based on the proposed nonparametric regression, we obtained a linkage peak  declare that they have no competing interests."}
+{"text": "For diagnosis of neuropsychiatric disorders, a categorical classification system is often utilized as a simple way for conceptualizing an often complex clinical picture. This approach provides an unsatisfactory model of mental illness, since in practice patients do not conform to these prototypical diagnostic categories. Family studies show notable familial co-aggregation between schizophrenia and bipolar illness and between schizoaffective disorders and both bipolar disorder and schizophrenia, revealing that mental illness does not conform to such categorical models and is likely to follow a continuum encompassing a spectrum of behavioral symptoms.We introduce an analytic framework to dissect the phenotypic heterogeneity present in complex psychiatric disorders based on the conceptual paradigm of a continuum of psychosis. The approach identifies subgroups of behavioral symptoms that are likely to be phenotypically and genetically homogenous. We have evaluated this approach through analysis of simulated data with simulated behavioral traits and predisposing genetic factors. We also apply this approach to a psychiatric dataset of a genome scan for schizophrenia for which extensive behavioral information was collected for each individual patient and their families. With this approach, we identified significant evidence for linkage among depressed individuals with two distinct symptom profiles, that is individuals with sleep disturbance symptoms with linkage on chromosome 2q13 and also a mutually exclusive group of individuals with symptoms of concentration problems with linkage on chromosome 2q35. In addition we identified a subset of individuals with schizophrenia defined by language disturbances with linkage to chromosome 2p25.1 and a group of patients with a phenotype intermediate between those of schizophrenia and schizoaffective disorder with linkage to chromosome 2p21.The findings presented are novel and demonstrate the efficacy of this approach in detection of genes underlying such complex human disorders as schizophrenia and depression. Emil Kraepelin's descriptions of psychiatric diagnoses at the turn of the 20th century were groundbreaking and remain influential to this day. Kraepelin's dichotomous classification of manic-depressive insanity (bipolar disorder) and dementia praecox (schizophrenia) provides a simple way for conceptualizing an often complex clinical picture and has been extended to include a categorical classification system utilized for a vast array of psychiatric illnesses Additionally, the categorical diagnostic model does not adequately accommodate atypical or sub-clinical casesThe limitations of the existing categorical model with arbitrary diagnostic boundaries and hierarchical diagnostic definitions that do not allow for presence of the spectrum of sub-clinical symptoms have impeded progress in psychiatric genetic research. An alternative model is the suggestion that psychiatric disorders are related as part of a continuum of psychosis We present an analytical framework for detection of genetic factors contributing to specific subsets of behavioral symptoms. This approach is a paradigm shift from traditional genetic analytic methods, where the genetic analyses are performed within given pre-defined diagnostic categories. In contrast, in this approach, individuals are grouped into a hierarchical network based on shared behavioral symptoms without regard to the diagnostic category to which they had originally been assigned. This form of behavioral clustering is highly flexible allowing for both separation and overlap of clinical symptoms within diagnostic categories. Genetic linkage and association tests can then be preformed using the individual groupings from clustering of individuals with an observed set of symptom profiles. In this way, the genetic determinants underlying a specific cluster of symptoms that define a clinical sub-phenotype may be detected. This framework was especially developed for analysis of data from genetic studies of neuropsychiatric disorders with a wide spectrum of clinical symptoms. With this in mind, we applied this method to analysis of schizophrenia data, where we demonstrate the efficacy of this approach in refining the findings from a previous schizophrenia genome scan The recruitment and diagnosis of patients and their family members are described elsewhere a-to-l. The genetic architecture of this disorder involves four disease gene loci D1, D2, D3, and D4 . A total of 300 families were analyzed, consisting of 2,077 individuals of which 781 were affected with KPD and 1,296 were unaffected. All genotype data for the 10 simulated chromosomes were examined, with 416 microsatellite markers approximately 7.5 centimorgans (cM) apart.Reviewing the findings from the genome scan, we identified those chromosomes with moderate to significant linkage evidenceThe hierarchical behavioral network representing phenotypic relatedness is obtained through the use of a character-based analysis. This approach optimizes the change of behavioral traits, where a change is defined by the loss (1\u21920) or gain (0\u21921) of the presence of a behavioral trait as the network is traversed from one individual to anotherThe character-based algorithm described here, which generates a behavioral network based on individual symptoms, accounts for phenotypic heterogeneity. The goal is to identify genetically meaningful phenotypic groups. Since an optimal arrangement of shared behavioral symptoms was used to build the phenotypic network, the symptoms defining a phenotypic group of interest can easily be determined. As phenotypic groups are analyzed at multiple, increasingly inclusive levels, genetic loci that show linkage or association within small or large groups of individuals with particular symptoms can be detected . This provides a direct means of detecting genetic loci that can potentially modulate behavioral symptoms.Specifically, our algorithm consists of four distinct components including (1) estimating a behavioral network, (2) nesting of the behavioral network into inclusive groups/clades0 and HA respectively) are straightforward, where 2\u200a=\u200ap3\u200a=\u200ap4p with corresponding maximum likelihood estimates of 3\u200a=\u200ap4p with corresponding maximum likelihood estimates of ix is the number of individuals with symptoms in group i and in is the total number of individuals in group i. Here p is the probability of the presence of a behavioral trait for any of four possible groupings, such that 2p is the probability of observing a given trait in the clinical diagnostic group that does not overlap with the clade, and 3p is that trait's probability in the clade of interest that does not overlap with the major diagnostic group. 4p is the probability of trait overlap between 2p and 3p, and 1p is the probability of the trait in individuals that are not in any of the previously noted groups. The null hypothesis (H0) assumes that the trait probability distribution for the individuals in the clade and the clinically defined diagnostic group (as well as the overlap) are the same . Whereas, the alternative hypothesis (HA) assumes that the probability of the trait is not the same . For the boundary conditions where one group is contained within the other, for HA2p is compared to 4p, while for H0 they are equal. The situation is similar in the case of no overlap between categories where 2p and 3p would be the key parameters. The likelihoods thus formulated are evaluated for each trait of interest and assessed via the likelihood ratio test at the parameter's maximum likelihood estimates. Furthermore, for assessment of statistical significance, empirical p-values were estimated using 10,000 permutations of the data.The likelihoods for the null and alternative hypotheses  and heterogeneity (HLOD)The behavioral networks thus generated were examined and nested groups/clades were identified for genetic analysis. Since the datasets considered consist of family data, we performed genetic linkage analysis using the program MILINK from the LINKAGE software package M) is evaluated by recording the number of times a maximum LOD score from a replicate exceeds the maximum LOD score from that of the observed data, across all genetic models within each clade (phenotypic model) and therefore corrects for the testing of multiple genetic models and marker loci tested for a particular phenotypic model. The global p value (pG) is calculated by making comparisons to the maximum LOD scores from randomized replicates across all genotypic and phenotypic models (clades) and marker loci tested. This approach accounts for multiple testing of all combinations of genetic-phenotypic models as well as genetic markers tested.To account for multiple testing, we estimated empirical p values, allowing for multiple phenotypic and genetic models, as well as multiple marker loci tested. To this end, we randomly assigned genotypes to the founders within each family while conditioning on the observed allele frequency and intermarker distances in the data and then mating individuals within families according to the pedigree structure using the program SIMULATE Behavioral traits from all family members were analyzed to generate a behavioral network. The network correctly grouped the three latent phenotypes, as shown in depression\u201d (clades 6_4 and 4_28) and two \u201cschizophrenia\u201d (clades 6_6 and 6_1). These clades can be represented in a behavioral network  with corresponding evidence for genetic linkage. The salient clinical features of these groups were similar to major depression and schizophrenia with two groups resembling \u201c network . In this network . In this network . The sig network . Those b network .depression6_4 is characterized by the symptom \u201cdifficulty concentrating\u201d . We also observed suggestive evidence for linkage on 2q13 for the second 4_28depression clade (HLOD of 2.86), where the dominating symptoms were those of sleep disturbances. Comparatively, analysis of families in the total dataset using the diagnosis of major depression to define affectedness yielded lower linkage signals. In fact, the maximal linkage signal overlapped with the same chromosomal region identified by the 4_28depression clade, with a lower score . This linkage peak is approximately 66 cM from the peak LOD score of 2.99 reported previously 6_1schizophrenia clade yielded maximal linkage signal on chromosome 2p21 , including paranoid, schizoid, schizotypal and borderline traits  contributing to these symptoms. With this method, we further refined previous linkage findings in this dataset, providing improvements in the linkage results by limiting potential phenotypic heterogeneity. Analysis of individuals with shared behavioral symptoms is a powerful and straightforward approach to identify the genetic factors underlying such symptom profiles and will lead to discovery of endophenotypes that are likely to be more biologically meaningful than standard diagnostic categories.In this study, we have developed an analytical framework for the characterization of behavioral symptoms with shared genetic contribution. This method is a departure from the traditional approaches to analysis of neuropsychiatric disorders in that it does not rely on Our results clearly demonstrate the separation of individuals with major depression on the basis of whether they exhibit sleep disturbance symptoms. These physiological symptoms may distinguish the etiological factors for the disorder in each group. Further, the separation of the two linkage peaks for major depression on chromosome 2 based on two primary behavioral symptoms  may indicate the identification of two groups of patients whose symptoms have two distinctly different genetic origins. The individuals in the group suffering from sleep disturbances appear to have more severe symptoms during childhood, indicating a possible prodrome or an earlier age of onset. This increases the likelihood that these symptoms may have high genetic loading, consistent with epidemiological studies, suggesting a strong correlation between the diagnosis of depression and sleep disturbances The most striking schizophrenia finding involved the group of individuals with language disorders, considered central to the symptom pathology of schizophrenia While we grouped individuals based primarily on shared behavioral symptoms, it is also possible to include physiological trait measurements, as well as non-genetic or environmental factors. In future analyses, the relative importance of traits can be weighted when building the network based on the heritability of the trait. Traits with high heritability would contribute more to the structure of the network defining phenotypic relationships. The analytical procedure is also easily extendable to association studies using a case-control study design by simply performing association tests on phenotypic groups instead of linkage analyses. This method is ideally suited for application to diseases where clinical heterogeneity of phenotypes is suspected and multiple symptoms are recorded for study subjects, as is typically the case for most neuropsychiatric disorders.Our study shows that reconsideration and refinement of phenotypic definitions will reveal a myriad of new phenotypic and genotypic relationships. Although the representation of behavioral symptoms among individuals is not likely to be hierarchical, with some genes affecting multiple symptoms and multiple genes having an effect on an individual symptom, our method aims to generate hypotheses regarding specific subgroups of individuals with shared symptoms and to delineate the genetic basis of such behavioral symptoms. In this way, individuals with shared symptom profiles will be grouped together and genes with effects on these symptoms will be detected, generating novel phenotype-genotype relationships.Figure S1Simplified example of a network representing relationships between patient's behavioral phenotypes. This shows how behavioral symptoms of individuals in the dataset are used to group patients together into a hierarchical network. (A) Depiction of eight patients each scored for the presence (1) or absence (0) of ten behavioral symptoms. (B) Network representing the grouping of patients based on phenotypic relatedness. Patients who share more behavioral symptoms in common are grouped together, where the closest neighbors in a network are binned together into a more inclusive group (referred to as nesting level).(8.21 MB TIF)Click here for additional data file.Figure S2Depression LOD score plots. This demonstrates the separation and amplification of linkage peaks in the two mutually exclusive depression groups as compared to the overall diagnoses of depression. The LOD score peak at approximately 122 cM circled for the depression diagnoses (panel A) is significantly amplified in the depression4_28 clade (panel B), which also lacks a LOD score peak at \u223c218 cM position. In contrast, the depression6_4 clade shows amplification of the peak at \u223c218 cM (panel C), corresponding to the second peak for the depression diagnoses (panel A). Interestingly, the depression6_4 clade lacks the peak at approximately 122 cM, present in the depression4_28 clade (panels B and C). These two depression clades are mutually exclusive, indicating a potential separation of two genetically distinct groups of individuals within the diagnostic category major depression who can be distinguished phenotypically by the presence/absence of sleep disturbance symptoms.(9.47 MB TIF)Click here for additional data file.Table S1Diagnostic and LOD score statistics for clade 5_15. For this clade, the number of individuals carrying each diagnosis is provided as well as the number of individuals and families and the relevant linkage statistics. The maximum LOD score is reported maximized over all genetic models and analysis schemes examined with corresponding model parameters. The term HLOD denotes the maximum heterogeneity LOD score and \u03b1 is the corresponding heterogeneity parameter. Marker refers to the genetic marker with the observed maximum HLOD. Model refers to the genetic model as described in the text. PM and PG are the estimated model-based and global empirical p-values respectively.(0.04 MB DOC)Click here for additional data file.Table S2Linkage Results of Simulated GAW14 Data. Clade refers to the clades under consideration, Phenotype refers to the latent phenotypes, HLOD is the maximum heterogeneity LOD score and \u03b1 is the heterogeneity parameter, Model refers to the genetic model as described in the text, and PM and PG are the estimated model-based and global empirical p-values respectively. Chromosome is the chromosome on which this peak occurs, position is the position on the chromosome at which the peak occurs in cM, and disease locus is the disease locus that it identifies. Details of the genetic models and analyses as well as the empirical p-value estimations are given in main text. (A) Linkage analysis of the clade (0_21) containing the latent phenotype P1 showed strong linkage to the disease gene D1 on chromosome 1, which together with D2 define the underlying genetic contribution for P1. Analysis of clade 0_4 also revealed significant evidence for linkage with the disease genes D3 and D4 contributing to the latent phenotypes P2 and P3. However, the disease locus D2 was not detected in linkage analysis of these clades, which included individuals who harbored the latent phenotypes of interest. This is likely due to the fact that the latent phenotypes P2 and P3 are caused by alternate epistatic interactions between the disease loci D2 and D3, where for P2 the disease loci D2 has a recessive mode of inheritance and D3 has a dominant mode of inheritance, and in contrast for P3 the disease loci D2 has a dominant mode of inheritance and D3 has a recessive mode of inheritance. As for the linkage analysis there was no resolution between the phenotypic groups P2 and P3. Thus, they were analyzed with these opposite modes of inheritance which coupled with the reduced sample size resulting from subdividing this group reduced the potential genetic signal for the disease loci D2. Interestingly, D2 was correctly localized when the clade containing the majority of the unaffecteds was examined. The disease locus D2 is the genetic determinant for traits e, f and h in unaffected individuals, which acts in a dominant manner with a penetrance of approximately 20%. (B) Whole-genome scan analysis on the entire dataset using Kofendred Personality Disorder as the major diagnosis . All the major disease loci were also identified in this analysis, yet with less significant linkage signals. The exception was locus D2 which had a more significant HLOD score in this genome scan. (C) The two clades separated by the presence (P3) or absence (P2) of trait b were examined via linkage analysis, and loci D1, D2 and D4 were successfully isolated within the separated phenotypic groups P2 and P3.(0.05 MB DOC)Click here for additional data file."}
+{"text": "A linkage study on autosomal recessive high myopia (arHM) has not been reported, although several loci for autosomal dominant high myopia (adHM) have been mapped. Data from a consanguineous Chinese family with arHM were collected to map the genetic locus associated with this condition.Phenotypic information and DNA samples were collected from family members. A genome-wide linkage scan combined with homozygosity mapping was performed by using 382 microsatellite DNA markers from the entire genome spaced at intervals of about 10 cM.The pedigree and clinical data of the family indicate that the high myopia is autosomal recessive. A genome-wide scan of chromosomes 1\u201322 gave a LOD score greater than 1.0 for 22 markers. Linkage to most of these markers was not supported by closely flanking markers except for three possible loci on chromosomes 11, 14, and 17. Fine mapping and haplotype analysis provide evidence for a locus at 14q22.1-q24.2 in a 25.23 Mb region between markers D14S984 and D14S999 with a maximum LOD score of 2.19. All 11 microsatellite markers inside the linkage interval as well as haplotype construction point to a gene at this locus. Linkage elsewhere on chromosome 11 and chromosome 17 could not be excluded due to the small size of the family.Pedigree and clinical data suggest that an autosomal recessive gene is responsible for high myopia in a consanguineous Chinese family. Genome-wide linkage analysis was used to map the gene for high myopia to a few limited loci. The resultant information should help future studies identify the gene for arHM. To our knowledge, this report is the first clinical and linkage study on a consanguineous family with arHM. Myopia is a leading cause of visual impairment worldwide, and Chinese people living in industrialized or urban/suburban areas have a significantly higher incidence of myopia than other populations -4. High MYP1 [Xq28]) ) ,15, MYP28p11.31) ,16, MYP3q21-q23) ,17, MYP4]) [MYP2 p11.31 [Mq22-q27) , MYP12 (q23-q25) ,19, and ) ,15, MYq23-q25) . Althougq23-q25) , the genq23-q25) . FurtherHere, we report a clinical and linkage study on a consanguineous Chinese family with arHM. A genome-wide linkage analysis combined with homozygosity mapping provides suggestive linkage of high myopia to a few loci including a novel locus in the q21-q24 region of chromosome 14 between markers D14S288 and D14S74.The consanguineous Chinese family with arHM was one of the 628 families with high myopia ascertained as part of a project to identify the genetic causes of high myopia in China. This family was from the Guangdong province of China. It includes one consanguineous marriage with three affected individuals . Three aLINKAGE program package [NCBI) map. Haplotypes were generated using the Cyrillic 2.1 program  and confirmed by inspection. The criteria for establishing linkage have been previously described [Genotyping for all participating family members was performed using 5\u2032-fluorescently labeled microsatellite markers as previously described , except  package ,32. The  package ,34. For escribed .The consanguineous family originates from a small town in northeast Guangdong province, which is about 400 km away from Guangzhou, China . Three oLinkage to all known loci for high myopia was initially excluded. A genome-wide scan excluded linkage to 313 of the 382 panel markers for chromosomes 1\u221222 by a LOD score equal to or lower than \u22122.0. Twenty markers yielding LOD scores between -2.0 and 0 were further excluded from linkage due to heterozygous alleles by homozygosity mapping. For the remaining 49 markers with LOD scores greater than \u22122.0, markers D7S513 and D12S310 were unsuccessful for genotyping, and 25 of these markers with positive LOD scores of less than 1.0 were not informative where the neighboring markers gave LOD scores of less than \u22122.0.Only 22 markers yielded two-point LOD scores greater than 1.0 in a genome-wide scan of chromosomes 1\u221222 . LinkageIn this study, we described a consanguineous Chinese family with three offspring affected with high myopia. All three affected family members presented with extreme high myopia in early childhood, which is characterized by a typical and similar myopic fundus and an obvious elongation of ocular axial length. Other ocular and systemic diseases were excluded. These similar characteristics together with pedigree and family information such as the comparatively normal refraction and fundus in parents suggest that the high myopia in this family is inherited as an autosomal recessive trait. Although arHM has been suggested by population and segregation analysis -38, arHMGNG2), G protein-coupled receptor 135 (GPR135), SIX homeobox 4 (SIX4), and regulator of G-protein signaling 6 (RGS6). Even though the exact locus and gene have not been defined, our study does provide useful clues for identifying the gene for arHM in further studies. Recruitment of additional families could possibly help to define the exact locus, which would lead to the cloning of the causative gene.It has been suggested that arHM represents a common form of hereditary high myopia -39. Unfo"}
+{"text": "We test for linkage, accounting for heterogeneity, and classify individual families as \"linked\" and \"unlinked\" on the basis of their contribution to the overall evidence of linkage.Heterogeneity poses a challenge to linkage mapping. Here, we apply a latent class extension of Haseman-Elston regression to expression phenotypes with significant evidence of linkage to  Microarray technology makes it possible to measure the expression levels of thousands of genes simultaneously. The abundance of expression data has prompted the study of natural variation among humans in baseline gene expression -3, inclutrans regulators of gene expression. More specifically, we use a latent class extension of Haseman-Elston (H-E) regression developed by Bastone et al. (unpublished work) that accounts for heterogeneity with respect to linkage. This method includes a classification procedure that is used to determine which individual families are segregating and therefore contribute to the overall evidence of linkage.Here, we use a statistical model that accounts for heterogeneity and apply our method to follow up linkage evidence of cis and trans regulators. The 142 expression phenotypes exhibiting linkage evidence that achieved genome-wide significance were further classified as cis and trans, based on the location of the linked SNP marker relative to that of the expressed gene. Here, we expand upon the findings of Morley et al.  = \u03b1s + \u03b2s *X. We assume the marginal density of the outcome variable is a mixture of the conditional densities within the latent classes, such that the marginal mean is E[Y|X]= \u2211s \u03c0s (\u03b1s + \u03b2s *X). Y is regressed on X within each class, and a test for linkage is based upon the magnitude of the regression slope in the segregating class. The null hypothesis that the regression slope in this class equals zero, is tested against the one-sided alternative that the regression slope was less than zero. Empirical Bayes estimates of the parameters in the latent class model were used in turn to estimated the posterior probability that a given family is in latent class s, P. We estimated the probability of membership in the \"linked\" class for each family and subsequently assign families to their more likely class.Latent class methodology assumes that the distribution of the outcome variable  is a mixture of two or more parametric distributions. Let Based on the results of the one-class model, we select the single SNP exhibiting the maximum evidence of linkage with the trait and obtain multipoint IBD sharing information using Merlin . The rawEvidence of heterogeneity was detected under the linkage model for all five singleton phenotypes. More specifically, in the two-class model, all phenotypes exhibited evidence of a zero slope in one class, and a statistically significant negative slope in the other (\"linked\") class  contribute to the overall evidence of linkage, despite the strong marginal effect. This suggests that for these five phenotypes, we detected QTLs with relatively rare alleles with strong effects.trans peak chromosome. The stratified scans were implemented in S.A.G.E. [We repeated the linkage scans for each class separately. Figure S.A.G.E. . These gp-value from H-E regression. The maximum new negative log p-value in \"unlinked\" families ranged from 3.3 for CBR1  to 4.1 for DSCR2 . In the original analysis, Morley et al. [p-value of 4.4 was used as a less stringent threshold. Thus, while the evidence of linkage to other regions in the genome did increase in the \"unlinked\" families, none of the new linkage peaks achieved genome-wide significance by this criterion.In addition to the chromosome-specific linkage scan, we performed a stratified, genome-wide linkage scan for each singleton phenotype. By omitting those families whose phenotypic variation is already explained (\"linked\" families above), we might have more power to detect additional QTLs. This reasoning assumes that, for example, when families in the sample are segregating for two distinct QTLs, individual families will be segregating for one or the other, but not both QTLs. Of course, this is not necessarily true. However, if the less common allele at each of the QTLs is rare, it may be a good working assumption. In the linkage scans, the evidence for linkage is measured by the negative log We next applied the latent class method to the chromosome 14 and 20 phenotypes. Specifically, we analyzed data for 29 phenotypes with significant evidence of linkage to a 5-Mb region on chromosome 14. Again, we found that a relatively small number of families contributed to the overall evidence of linkage . In fact, for 12 phenotypes, only one family is classified as \"linked\". Surprisingly, a single family (CEPH 1418) is classified as \"linked\" for all phenotypes. We repeated a linkage scan removing that family and found that only one phenotype continued to exhibit linkage to chromosome 14. We compared the expression values of Family 1418 to those of the other families and found the mean and range of expression values to be quite similar. Thus, there is nothing remarkable about this family with respect to phenotypic values. The strong dependence of the linkage findings on this family might indicate a relatively rare allele at a QTL with a very strong effect, detected primarily in one family.We repeated the analysis described above for 24 expression phenotypes with significant linkage to a 5-Mb region on chromosome 20. In contrast to the results for the chromosome 14 phenotypes, we found that different families contribute linkage evidence for different phenotypes. All families contribute evidence for at least two phenotypes and the maximum number of phenotypes to which one family contributes is 14. In general, somewhat larger numbers of families contributed to the evidence of linkage for the chromosome 20 phenotypes , as compared to the chromosome 14 phenotypes.trans regulators of gene expression. The family classification procedure under the latent class model allows us to estimate which families are contributing to the overall evidence of linkage. A key feature of the latent class approach is that it provides a means for classifying families by fitting a single model. One could imagine an alternative approach in which the linkage analysis was repeated for all possible combinations of families in order to find the subset of families for which the evidence of linkage was strongest. However, this alternative approach would introduce a substantial multiple-testing problem that is avoided by our unified latent class approach.We applied a latent class extension of H-E regression to evaluate heterogeneity and linkage of expression phenotypes to We used the family class assignments that follow from the latent class model to estimate the number of families contributing to the overall evidence for linkage. We also used the procedure to identify influential families (\"linked\"), thereby identifying a subset of families (\"unlinked\") in which to look for additional QTLs. For all five phenotypes considered here, we found that a small number of families were classified as \"linked\". The fact that for those genes, significant evidence of linkage was found in the one-class model is likely due to the large sibship size in the CEPH families and the fact that alleles at QTLs regulating gene expression may have relatively large effects. We applied the latent class model to target genes that were pre-selected based on the results of Morley et al. . Determicis and trans regulators has been detected in various studies. In a number of cases, the linkage findings for cis regulators have been supported by subsequent analyses. For example, Morley et al. [cis regulator. In contrast, validating evidence of linkage to trans regulators by association methods has been difficult and the genetic dissection of trans regulators remains a challenge. It is likely that genetic heterogeneity contributes to the difficulty of identifying trans regulators. Methods such as the one applied in this study that appropriately model genetic heterogeneity may improve our ability to map trans regulators and, ultimately, increase our understanding of the genetics of gene expression.Linkage between expression phenotypes and putative y et al.  found stThe author(s) declare that they have no competing interests."}
+{"text": "P. massoniana and P. hwangshanensis and their distribution ranges lead to the emergence of the two species as desirable organisms to study the genetic mechanisms for speciation.Exploring the genetic mechanisms underlying speciation is a hot topic in modern genetics and evolutionary studies. Distortion of marker transmission ratio is frequently ascribed to selection against alleles that cause hybrid incompatibility. The natural introgression between Using seeds sampled from trees at different elevations, we consistently detected sharp decreases in seed germination rates of trees in the hybrid zone, which might be due largely to the hybrid incompatibility. A genetic map was established using 192 megagametophytes from a single tree in the hybrid zone of the two species. Segregation distortion analysis revealed that the percentage of significant-segregation-distortion (SSD) markers was extremely high, accounting for more than 25% of the segregating markers. The extension range, the distortion direction, and the distortion intensity of SSD markers also varied dramatically on different linkage groups.P. massoniana and P. hwangshanensis. Our study provides a valuable platform for conducting genome-wide association of hybrid incompatible QTLs and/or candidate genes with marker transmission ratio distortion in the hybrid.In this study, we display the potential chromosomal introgression barriers between  A biological species is defined as a group of natural populations that mate and produce offspring with one another, but do not breed with other populations. Yet biologists have argued over the details of the definition since around 1900. Inter-sP. hwangshanensis and P. massoniana are desirable organisms to study the genetic mechanism triggering speciation. P. hwangshanensis is a native representative conifer that distributes in the subtropical mountainous areas in southeast of China, and it is found at higher elevation than P. massoniana. The ranges of the two species are frequently found to be immediately adjacent to each other, and overlapped with a narrow hybrid zone. The two species are different in morphological, cytological and timber anatomical characteristics, and show clear environmental and spatial separation [P. massoniana and P. hwangshanensis are closely related species and they both possess 12 haploid chromosomes. However, there might be some other chromosomal changes between the two species, including chromosomal rearrangement, genome expansion, differential gene expression and gene silencing. These changes may lead to selection for fertility and ecological traits that alter the genome structures of the alternate species, in return acting as introgression barriers [P. massoniana and P. hwangshanensis by building a genetic map using megagametophytes from a single tree sampled in the hybrid zone and to identify regions of the map displaying extreme segregation distortion.paration -13. Treeparation . The majparation . These eP. hwangshanensis was generally above 900 m, and that of P. Massoniana was generally below 700 m in the south of the Yangzi River in China, the hybrid zone roughly spanned a vertical range from 700 to 900 m [The germination rates of seeds collected from trees along the transaction lines at different elevations of two different locations were listed in table to 900 m -12. Germet al. [EcoRI; M; MseI; the digital numbers were the number of selective nucleotides). There was considerable variation in the number of segregating loci generated by different primer combinations, ranging from 11 to 33. Based on the Chi-square test, 82 (25.5%) markers were significantly distorted from the expected 1:1 segregation ratio at \u03b1 = 0.05 significance level (corresponding to Chi-square of 3.84). Among them, 37 skewed to more presence and 45 skewed to more absence. The highest Chi-square value of SSD markers is 26.39. Since segregation distortion demonstrates the uneven transmission ratio of alleles on the alternate chromosomes in the mapping parent and is related to hybrid incompatibility [Twenty-one AFLP primer combinations were selected for this study according to Li et al. . Segregaet al. , more setibility , once thP. sylvestris [P. taeda [EcoRI/MseI restriction deserts in the corresponding genomic regions. Tremendous effort might be needed to fill such gaps with randomly generated markers. The 14 major linkage groups consisted of 312 markers with an average distance of 5.18 cM between the adjacent markers. The established map provides a useful platform for demonstrating the potential chromosomal introgression barriers, and for exploring their expansion ranges on different chromosomes.Linkage analysis with 192 megagametophytes from the hybrid pine was performed with all the obtained 321 segregating AFLP markers. In this paper, all markers that significantly departured from Mendelian segregation ratio were included because these markers were hypothesized to reveal sites of hybrid incompatibility. Using LOD thresholds of 10.0, 7.0, 6.0, 5.0, 4.0, 3.0, 2.0, 321 markers were initially assigned to 19, 18, 16, 16, 16, 9, 3 groups respectively, each run left some ungrouped markers. When LOD\u2264 3.0, there were less linkage groups than the haploid chromosome numbers of pine; at LOD = 4.0, 5.0 or 6.0, the same grouping results were derived. Therefore LOD = 6.0 was used to assign markers into linkage groups. Under this criterion, markers were assigned to 14 major linkage groups, 1 triplet, 1 doublet, and 4 unlinked markers. The major linkage groups ranged from 26.9 to 177.9 cM in size  undergoes meiosis to produce four haploid cells, then three of those cells degenerate, results in one functional megaspore per ovule. The megaspore then undergoes megagametogenesis to give rise to the megagametophyte and to produce the female gamete. Thus, the megagametophyte is haploid tissue and has the same DNA as the female gamete that is fertilized by pollen to form embryo in each seed. As a result, marker distortion revealed by megagametophyte genotyping is closely related to selection of alleles on the alternate chromosomes in the maternal parent. Since megagametophytes are haploid, in mapping studies, they possess the similar characteristics as recombination inbreeding lines obtained by the single seed descent method that widely used in the establishment of mapping pedigree for crops.Seed germination rates reflect the reproductive ability of trees sampled at different elevations. In order to make seed germination rates comparable, seeds from each tree were randomly selected in the test. We did not cull out the empty seeds or seeds with congenital dysplasia embryos. Thus, the low germination rates of trees in hybrid zone could be the result of both pre- and post-zygotic barriers. Pre-zygotic barriers mainly include factors associated with pollination and flowering time, and also include factors affecting process of gamete union to form a zygote. Once a hybrid zygote is formed, it may have low viability or be sterile, and the underlying factors are known as post-zygotic barriers . To resoP. massoniana and P. hwangshanensis. We proposed that germination rate reflected the hybrid incompatibility of the alternate species. By using megagametophytes from a single tree in the hybrid zone, we built a nearly complete genetic map for pine genome. Based on the SSD markers on this map, we discovered the potential chromosomal introgression barriers between P. massoniana and P. hwangshanensis. This study provided the basis for associating SSD markers with their introgression behavior in natural stands, and established a useful platform for conducting genome-wide associations of hybrid sterility QTL and/or candidate genes with marker transmission ratio distortion in the progeny of a hybrid pine.In this paper, we consistently detected low germination rates of seeds collected from trees in the hybrid zone of One set of samples used in this study were collected from Huang-gang Chimney in Wuyi Mountain of Fujian province. The elevation range was from 445~1250 m. The other set of samples were collected from Kulongjiang Chimney at Qimen in Anhui province. The elevation range was from 350~1100 m. Linear transects were set up from the foot to the top of the mountains at both locations. To avoid significant geographic changes in horizontal direction, from foot to top, cones were collected from trees within 10 m from the transaction lines. Altogether, cones from 8 trees at 8 elevations were collected in Fujian, and cone from 12 trees at 12 elevations were collected in Anhui. Elevation of each tree was recorded by its GPS reading.Seed germination was conducted according to \"Woody plant seeds inspection standard\" in the Chinese Southern Forest Seed Inspection Center . For eacet al [et al [et al [EcorI, M: MseI), in addition to the number of selective oligonucleotides, following the approximate allele size of the segregating marker. In the name of each primer combination, E primer was before the \"/\", and M primer was after the \"/\".We selected a tree at 784 m in Wuyi Mountain and harvested the megagametophyte tissues from seeds of this tree that germinated into normal seedlings. DNAs from 192 megagametophytes were extracted as described by Yin et al . AFLP prl [et al . AFLP geet al [c was the proportion of the genome within d cM of a marker, m was the number of markers.The Chi-square test was performed to check whether a marker segregated in 1:1 ratio. The linkage analysis was conducted by MapMaker version 3.0 , and mapet al . Map chaet al . Genome et al , assuminS) that causes marker segregation distortion as S = |o - e|, then the Chi-square value of each marker in this study is o is the observed number of individuals with a visible allele, e is the expect number of individuals with the corresponding visible allele, and N is the number of megagametophytes genotyped by the corresponding primer combination. If we assume there is only one locus that causes marker segregation distortion in each cluster, the Chi-square value of a marker that has recombination rate r with the driven locus would be \u03c7d2) of the two loci, and M is the genetic distance in Kosambi centiMorgan. Under single driven locus assumption, the two-directional distortion range under the observed highest selection intensity  would be \u03c7d2 = \u03c7h2-3.84.If we define the selection intensity LSX, CY and GHD conducted most of the molecular work and data analyses. LSX drafted the manuscript. YTM conceived the work and edited the manuscript critically. All authors have read and approved the final manuscript.P. massoniana and P. hwangshanensisGenetic map for a natural hybrid of . This genetic map is determined by using megagametophytes of 192 normally germinated seeds from the mapping parent. Marker with name ending with 'r' was in repulsion linkage phase.Click here for file"}
+{"text": "Genome scan meta-analysis (GSMA) can prove very useful in detecting genetic effects too small to be detected in an individual linkage study and can also lead to more consistent results. In this paper, we propose a new kernel-based estimation procedure for GSMA. Instead of estimating identity by descent between markers, as performed in interval mapping approaches, we estimated directly the nonparametric linkage score between markers using a kernel procedure. The GSMA is then extended to take into account the kernel estimate of the nonparametric linkage score and its variance at a given chromosomal position. The method is applied to the rheumatoid arthritis genome scan data . Rheumatoid arthritis (RA) is a chronic inflammatory disease that primarily affects the synovial tissues of multiple joints in the body. The etiology of the disease remains unknown, but it appears to have a complex genetic component. Several genome scans for RA studies have been performed to identify susceptibility loci, but most of the results have not been replicated . These iWe have included three linkage studies in the meta-analysis of RA: NARAC (North American Rheumatoid Arthritis Consortium), ECRAF (European Consortium on Rheumatoid Arthritis Families) and United Kingdom (UK). Only microsatellite scans and RA affection status (binary outcome) were considered. NARAC has performed microsatellite scans for 511 mutliplex families, decomposed into 757 smaller families. ECRAF had high-density microsatellite data from 88 families, including 105 sib pairs typed with 1089 microsatellite markers. The UK group performed two screens: an initial screen of the entire genome using 369 markers analyzed on 175 families and a second screen performed on 197 families using 89 markers in regions showing evidence for linkage. The two screens were combined in our analyses. A summary of the data analyzed is given in Table We first performed a multipoint linkage analysis of each individual study and estimated the NPL score and marker information content (IC) at each marker location as well as at each systematic 2-cM interval using the program MERLIN . We thenZij is the NPL score from the ith study at the jth position, k is the number of studies, and wij is the weight given to each study. To perform the GSMA, we used three different strategies that differ in the way the NPL score and IC are estimated between markers and the definition of the weight.where wij is the product of the number of sib-pairs equivalents (SPE) from the ith study and the IC estimated from MERLIN for the ith study at the jth interval:Following Etzel et al. , the firThis approach is not based on marker alignment. Instead of using an interval mapping estimate of the NPL score and IC between markers, we used a kernel regression method. The statistic ZMA is then computed at all marker positions available after merging the three data sets . The weight is identical to Method 1 except that the IC is now replaced by its kernel estimate (ICK):SDnpl):The third approach is identical to Method 2 but now takes into account in the weight the precision of the kernel estimator, more precisely the inverse of standard deviation of NPL kernel estimator (Y) and the marker location (T) is modelled using a nonparametric model given by:In Methods 2 and 3, the relationship between the NPL score (or the information content) (m(\u00b7) and \u03b5 denote, respectively, the regression function to be estimated and the model error; term n is the number of observations. The stochastic distribution f(\u00b7) of \u03b5 is typically unknown and is unlikely to follow any familiar distribution such as the normal distribution. Hence, we decided to use nonparametric statistics. The random variable enables to characterize the variation of Y around m(t), the mean regression curve with:where m(\u00b7) depends on the joint and the marginal densities, which are both unknown. A density estimator allows the analysis of data sets that could exhibit skewness and multimodality due to different factors . A histogram type estimator is the most frequently used but could be strongly affected by the choice and number of classes chosen. So we decided to use a nonparametric kernel estimator that behaves much better statistically. A kernel estimator of a function f(\u00b7) is defined by:So, the regression function n is the sample size, h is the bandwidth (the smoothing parameter) to be determined, and K is the continuous fixed kernel function with finite variance generally satisfying K > 0, K(-t) = K(t), and \u222b K(t) dt = 1. Here we considered the Gaussian kernel. This raises the question of determining the bandwidth parameter. The estimator h is small and very flat when h is large. Different procedures have been previously proposed to determine h, for example cross-validation. The problem in our application is that we need to estimate the NPL score function at different marker locations using the kernel procedure but also its variance. Both the kernel estimator and the variance depend on the same smoothing parameter and an optimal choice for the kernel estimator might not be optimal for the variance. To our knowledge, there is no optimal procedure for this problem. For that reason, we could not apply the classical cross-validation procedure, so we decided to choose the bandwidth empirically. The bandwidth was chosen inversely proportional to the number of markers of each individual study on each chromosome. Therefore, h was not constant in our study but depended on the genetic background. More exactly, we chose: Mi is the number of markers of each individual linkage study on one particular chromosome and the constant was fixed to 4.0. Intuitively, we understand that a study with less markers yields more variable results. Applying a larger h leads to a smoother function and thus to a decreased variability. This determination of h provided a good estimation of both the NPL score function and its variance.where m(\u00b7) is:Using the kernel estimator of the marginal and joint densities function, the regression estimator of Kh(\u00b7) was chosen to be the Gaussian kernel function here. This form of the estimate of the regression curve was proposed by Nadaraya [m(t) and normally distributed when h tends toward 0. It is also shown [h tends toward 0, the variance of the estimator where Nadaraya  and WatsNadaraya . The estso shown ,10 that t).and R for Linux.In our method 3 above, we took MA Ztest statistic on the whole genome are presented in Figure MA Ztest statistic close to 8.0 on chromosome 6  might improve the consistency of linkage results by over-weighting studies with more precise estimate of the NPL score function. This could reflect, for example, a higher marker density. A larger weight will be given to a study that finds a linkage peak with many markers than to a study that finds the same peak with fewer markers. Therefore, the information about NPL score variability is very useful to weight each individual study. Our procedure can also down-weight thin peaks. Further simulation studies are needed to better understand its properties, in particular in terms of detection of true linkage peaks.The author(s) declare that they have no competing interests."}
+{"text": "It is well known that genetic components play an important role in the etiology of mandibular prognathism, but few susceptibility loci have been mapped.In order to identify linkage regions for mandibular prognathism, we analyzed two Chinese pedigrees with 6,090 genome-wide single-nucleotide polymorphism (SNP) markers from Illumina Linkage-12 DNA Analysis Kit (average spacing 0.58 cM). Multipoint parametric and non-parametric (model-free) linkage analyses were used for the pedigrees.The most statistically significant linkage results were with markers on chromosome 4 . Candidate genes within the 4p16.1 include EVC, EVC2.We detected a novel suggestive linkage locus for mandibular prognathism in two Chinese pedigrees, and this linkage region provides target for susceptibility gene identification, a process that will provide important insights into the molecular and cellular basis of mandibular prognathism. Mandibular prognathism (MP) is a common clinical problem all over the world. However, its prevalence varies relative to populations: the highest incidence has been observed in Asian populations (approximately 15%) and the lowest in Caucasian populations (1%). The uinque concave profile of mandibular prognathism not only seriously affects the masticatory function but also extremely endangers psychology to patients. Today, this type of disharmony remains difficult for dentists because of varied etiologies and limited understanding of the mandibular growthIt is well known that environmental and genetic components have both contributed to the etiology of mandibular prognathismRecent progress in molecular genetics has enabled the genetic determinant to be approached directly. Genetic linkage maps using various types of polymorphic markers are essential tools in many genetic studies. Short tandem repeat (STR) become popular genetic markers because of their polymorphism and hereditary. Yamaguchi et alAs ethnicity is a major risk factor for MP and Chinese pedigrees have never been studied, we speculated as to the existance of a special locus for Chinese Han People. In addition, technological advances in genotyping Single Nucleotide Polymorphisms (SNPs) has caused an increase in their application in linkage mapping studies because of high-throughput. In this study, we recruited two MP pedigrees of Chinese Han People in an attempt to discover a specific locus or the major gene that regulates mandibular growth by performing a genome-wide analysis with SNPs.When compared with normative cephalometric standards of ChinaPedigree analysis by visual inspection suggested an autosomal-dominant inheritance with incomplete penetrance. Multipoint parametric and non-parametric linkage scores obtained from the 0.58-cM resolution genome-wide scan revealed that only one chromosomal locus provided evidence of linkage: 4p16.1 . The multi-point linkage results on chromosome 4p16.1 are shown in http://www.ncbi.nlm.nih.gov). This search revealed that human genes EVC, EVC2 are within this region.These data substantiated the linkage signals of the susceptibility locus on 4p16.1 from our MP pedigrees. We futher identified candidate genes of biologic interest for the locus using biologic approaches  significance levels and genome-wide significance levels. As multipoint parametric and non-parametric (model-free) multipoint linkage analyses were performed, we identified susceptable locus according to both these results\u2014 the value of NPL and LOD. The linkage analyses results revealed a new locus\u2014 4p16.1 . Taking into account for both values, we considered 4p16.1 as a novel locus genetic linked to MP.There are 23 function genes in the region. It harbors positional candidate genes of interest such as EVC and EVC2. EVC2 encodes a protein that functions in bone formation and skeletal development. Mutations in this geneIn summary, when performing a genome-wide linkage analysis with two mandibular prognathism pedigrees of Chinese Han people, we detected a novel chromosomal region\u20144p16.1 potentially linked to mandibular prognathism. EVC2 and EVC were considered as candidate genes to influence the bone tissue. To efficiently identify the mutations that may be responsible for this disease, second-generation sequencing with Solelxa technology will soon be carried out within this candidate region. The findings in the current study can be combined with those from previous studies to further understand the genetic basis of mandibular prognathism.The protocols for the current study, as well as participant consent, were reviewed and approved by the institutional review board at Tongji University Dental School. Consent to participate in this study  was obtained from every adult participant or in the case of minors from a parental guardian. The consent was written.The family structure was as follows: pedigrees comprised of 4 generations with a total of 42 individuals containing 18 affected individuals. The age range was between 12 and 64 years, the average age being 38.7 years. All of the samples were drawn from China. These two pedigrees were not biologically related and were from different provinces. The pedigree charts were shown in The subjects were first diagnosed by lateral cephologram, in conjunction with orthodontic study models or visual inspection. Subjects were diagnosed as affected individuals if they had an ANB angle (Point A-Nasion-Point B) of centric jaw relationship under 0.0 degreesThe cephalometric tracing were performed by at least two orthodontists. Eighteen cephalometric parameters were used for diagnosis of the samples. Cephalometric tracing and major measurements were followed as The power to detect linkage was calculated using the program SLINK with 500 replicates. This pedigree showed an maximum LOD score of 4.5, assuming an autosomal-dominant model with incomplete penetrance of 0.95Genomic DNA was isolated from whole blood cells with the use of a QIAamp DNA Blood Kit . Genotyping was carried out with Illumina Linkage-12 DNA Analysis Kit (Human) . A Genome scan was performed with a total of 6090 single-nucleotide polymorphism (SNP) markers, having average 0.58 cM genetic map spacing and average 441 kb physical map spacing. The average minor allele frequency observed in the Asian sample group was 0.28.Multipoint parametric and non-parametric (model-free) multipoint linkage analyses were performed on the family of the subjects. Non-parametric linkage (NPL) analysis, which has been described as a powerful approach compared with the commonly used parametric methods"}
+{"text": "Human lifespan is approximately 25% heritable, and genetic factors may be particularly important for achieving exceptional longevity. Accordingly, siblings of centenarians have a dramatically higher probability of reaching extreme old age than the general population.P\u200a=\u200a0.037) and a locus on chromosome 9q31-34 with a highly suggestive LOD score of 3.89 . The empirical P value for the combined result was 0.002. A third novel locus with a LOD score of 4.05 on chromosome 12q24 was detected in a subset of the data, and we also obtained modest evidence for a previously reported interval on chromosome 4q22-25.To map the loci conferring a survival advantage, we performed the second genomewide linkage scan on human longevity and the first using a high-density marker panel of single nucleotide polymorphisms. By systematically testing a range of minimum age cutoffs in 279 families with multiple long-lived siblings, we identified a locus on chromosome 3p24-22 with a genomewide significant allele-sharing LOD score of 4.02 (empirical Our linkage data should facilitate the discovery of both common and rare variants that determine genetic variability in lifespan. APOE (MIM 107741) haplotypes FOXO3 (MIM 602681) and longevity are also quite promising Many common diseases of adulthood increase in prevalence with age. These morbidities accompany an exponential increase in mortality rate that is maintained until approximately age 90, whereupon it starts to decelerate Estimates of the heritability of normal human lifespan range from 10% to 58%, averaging about 25% Subjects were recruited through Elixir Pharmaceuticals, the New England Centenarian Study (NECS) now of Boston University Medical Center, Beth Israel Deaconess Medical Center (BIDMC), and Children's Hospital Boston (CHB), as described previously All subjects provided proof of age. There was a predominance of female subjects in our cohort, likely reflecting the original ascertainment criterion of having a proband of at least 98 years of age regardless of gender Since we could not predict what minimum age requirements would provide optimal power, ten sets of gender-specific minimum expected age at death requirements were applied to all subjects, with the hypothesis that as the cutoffs increased, the loss of power due to the decreasing number of families might be partially offset by an increase in genetic homogeneity and/or magnitude of effects in older subjects. As designated hereafter, Categories 1 to 10 cover the range from the upper 5% to the upper 0.2% tail of the birth cohort, corresponding to a minimum expected age at death of 90 to 100 for males and 95 to 104 for females . SubjectIn addition to analyzing the complete Categories 1 to 10 , we divided the sample set by two criteria into two subgroups for each. To explore differences between this study and the first scan P value below 10\u22123 among 282 unrelated subjects, and 267 SNPs that had a call rate of below 90% across all 642 samples. A total of 453 SNPs meeting one or more of these criteria was eliminated, resulting in a final panel of 9751 SNPs.Genomic DNA samples were purified from blood as described previously Linkage analysis was performed using MERLIN/MINX v1.1.2 all scoring function and the exponential allele-sharing model P values were determined solely on the basis of nonparametric LOD scores.We computed a multipoint nonparametric Kong and Cox allele-sharing LOD score with the S2) exceeded 0.16, above which LOD score inflation due to inter-marker LD becomes appreciable when parental genotypes are unobserved We used the --rsq option in MERLIN P value for linkage peaks from any category in the Total group that met the significance threshold of LOD \u200a=\u200a3.6 for a single genomewide scan with a fully informative marker panel To account for the multiple partially-dependent hypotheses represented by the ten age categories, we calculated an Overall empirical P\u200a=\u200a(r+1)/(n+1) to calculate empirical P values, where r is the number of replicates reaching the observed LOD score, and n is the number of replicates analyzed P value, but provides a more accurate estimate of the type I error rate than the conventional, unbiased (yet anticonservative) formula P\u200a=\u200ar/nP values were calculated using the conservative Clopper-Pearson exact method for a binomial distribution We used the formula P values of 0.037 [upper 95% confidence limit 0.048] and 0.054 [upper 95% confidence limit 0.067]. Only a single replicate achieved two LOD scores of at least 3.89 at distinct locations across all categories, yielding an empirical P value for the combined result of 0.002 [upper 95% confidence limit 0.006]. The stability of the chromosomes 3 and 9 linkage peaks was evaluated by dividing the marker panel into two independent subsets containing every other SNP, and both regions retained strong evidence for linkage in the halved panels (data not shown).In the Total group we identified a region on chromosome 3p24-22 with an Overall maximum LOD score of 4.02 in Category 4, and a region on chromosome 9q31-34 with an Overall maximum LOD score of 3.89 in Category 8. No other intervals achieved a LOD score above 3.0 , and givThe LOD scores for all linkage peaks varied considerably by group . The chrWe did not discover any additional linkage peaks in the gender-stratified analyses. The chromosome 3 linkage peak was somewhat stronger in the FO group  than in the MC group , whereas the chromosome 9 linkage peak showed a greater difference in the opposite direction . The subgroup characteristics are given in P values, and linkage peak locations for chromosomes 3, 9, 12, and 4 are given in The parametric analysis generally supported the results of the nonparametric analysis, showing all four linkage peaks in their respective groups and categories to be robust to different assumptions about the mode of inheritance. The chromosome 3 peak produced a higher maximum hLOD score under a recessive model (4.652) than a dominant model (3.334), whereas the maximum hLOD score for the chromosome 9 peak was higher under the dominant model (3.883) than the recessive model (2.680). The dominant model on chromosome 9 also yielded positive conventional parametric LOD scores of 1.047 in Category 8 and 2.840 in Category 9, suggesting reduced locus heterogeneity in the oldest sibships. The chromosome 12 peak achieved similar maximum hLOD scores of 3.915 and 3.875 under the dominant and recessive models, respectively, which likewise produced similar results on chromosome 4. We did not assess whether differences in hLOD scores under the dominant and recessive models were significant. Overall maximum nonparametric LOD and parametric hLOD scores, We performed the second genomewide linkage scan on families of exceptionally long-lived siblings, with substantial improvements in power over the first scan P\u200a=\u200a0.037), a highly suggestive peak on chromosome 9q31-34 , and a peak on chromosome 12q24 (LOD\u200a=\u200a4.05) in the newly recruited subset of our subjects. These linkage peaks are preliminary results that warrant replication studies, as several factors may influence their significance  as associated with longevity Future linkage studies may benefit in power from denser marker panels TOP2B (MIM 126431) in the chromosome 3 linkage peak encodes an isozyme of topoisomerase II, which is potently inhibited by resveratrol and related compounds from grape cell culture WRN (MIM 604611) BLM (MIM 604610) DBC1 (MIM 602865) in the chromosome 9 linkage peak directly interacts with SIRT1 (MIM 604479) and inhibits its activity SIRT1 is activated by resveratrol SIRT1 is stimulated by FOXO3TLR4 (MIM 603030) in the chromosome 9 linkage peak have been associated at least once, though often not reproducibly, with exceptional longevity in men ANK2 (MIM 106410), in the previously reported chromosome 4 linkage peak, cause long-QT syndrome ANK2 has been reported to regulate the QT interval Ank2+/\u2212 knockout mice displayed multiple signs of premature senescence and their lifespan was significantly reduced compared to wild-type littermates ALPK1 (MIM 607347) and both age at death and morbidity-free status at age 65 ANK2 and ALPK1 are within the 99% confidence (-2 LOD) MTTPANK2, ALPK1, or another gene besides MTTP could explain the previous linkage result. The positions of all the above genes within linkage peaks suggest they merit attention in future gene identification efforts.Several candidate genes in the linkage peaks discussed here warrant mention. The gene The linkage scans reported here should contribute to the analysis of both linkage and association studies on exceptional human longevity, which are underway in a massive data set Table S1Subject characteristics by age category for four subgroups, plus parametric analysis settings. Age, gender, and sibship composition statistics for the Previous, New, MC, and FO subgroups, plus parametric linkage parameters for all groups, are given across ten categories defined by gender-specific minimum requirements for expected age at death. The designations 2-ship, 3-ship, 4-ship, and 5-ship refer to sibships with two, three, four, or five siblings, respectively.(0.14 MB DOC)Click here for additional data file.Text S1Nonparametric linkage data for Total group.(10.21 MB TXT)Click here for additional data file.Text S2Nonparametric linkage data for Previous subgroup.(10.05 MB TXT)Click here for additional data file.Text S3Nonparametric linkage data for New subgroup.(9.74 MB TXT)Click here for additional data file.Text S4Nonparametric linkage data for MC subgroup.(10.03 MB TXT)Click here for additional data file.Text S5Nonparametric linkage data for FO subgroup.(9.75 MB TXT)Click here for additional data file.Text S6Supplemental discussion.(0.05 MB DOC)Click here for additional data file."}
+{"text": "INTRODUCTION: The aim of this study was to compare the antimicrobial effects of MTAD, sodium hypochlorite (NaOCl) and their combination on endodontic micro-organisms.MATERIALS AND METHODS: Zone of Inhibition (ZI) and Minimum Inhibitory Concentration (MIC) were the techniques used. In ZI technique blood agar plates were inoculated with organisms, paper discs were soaked with irrigants and maximum zones of bacterial inhibition were recorded. In the MIC technique the irrigants were serially diluted in TSB tubes and 0.1 mL of the tested microbe solutions were added. Results were obtained on the basis of turbidity and growth on agar plates. Statistical analyses were carried out using ANOVA and Tukey tests.RESULTS: In ZI technique, we investigated 120 specimens including 5 microbial species, 3 irrigants and their control groups, each with 6 repetitions. The results demonstrated MTAD greater antimicrobial efficacy compared to NaOCl, and their mixture (M+N) against Staphylococcus (S) aureus, Enteric (E) bacteria and Enterococcus (E) faecalis (P<0.001). NaOCl was more effective in eradicating Candida (C) albicans than the others (P<0.01). MIC method (155 tubes) showed MTAD to be more effective against E. bacteria and S. aureus. MTAD and NaOCl were equally effective against E. faecalis; however, NaOCl was more effective against C. albicans.CONCLUSION: Bacterial species were more susceptible to MTAD than NaOCl, C. albicans, however, was more susceptible to NaOCl. The advantage of NaOCl is that it has broad spectrum antimicrobial activity. The joint solution (M+N) did not prove to be more effective than their individual use. When the cultures were negative, the success rate was 94%; when bacteria were found however, success rate was reduced to 68% after 5 year follow up  are due to the presence of microbes in the root canal system . EliminaEnterococci (Actinomyces (Streptococci (Enterobacter (Fungi (Staphylococci (S) (Enterococcus (E) faecalis, the most prominent, is present in 30-70% of failure cases (Enteric (E) bacteria form approximately 5%  such as erococci -15, Actiinomyces , Streptoptococci , Enterobrobacter ,12,16,17r  ,12,15. Ere cases ,17,22,23fungi 7%  of the iE. faecalis and Candida (C) albicans is questionable (Various Sodium Hypochlorite (NaOCl) concentrations have been used over the years as an irrigant in RCT. The main benefits of NaOCl are its ability to dissolve necrotic tissue , its brotionable ,17,32-35faecalis  has been introduced as a final irrigant to be used after NaOCl for disinfecting the root canal into the market . The resfaecalis -40 thougfaecalis -44.in-vitro.We decided to compare the antimicrobial effect of NaOCl, MTAD, and their mixture against resistant microbes in RCT with Zone of Inhibition (ZI) and Minimum Inhibitory Concentration (MIC) E. faecalis, Pseudomonas (P) aeruginosa, Escherichia (E) coli, S. aureus and C. albicans. E. faecalis was incubated in Bile Scoline Agar, P. aeruginosa and S. aureus in Blood Agar, E. coli in Esoin Methylen Blue (EMB), and C. albicans in Sabouraud Dextrose Agar plates. After 24 hours a suspension was prepared from each microbial species with a concentration of 1 McFarland (i.e. 3\u00d7108 Colony of bacteria in the Trypticase Soy Broth environment) and used in the investigation.The investigation was carried out in Shahid Beheshti University Dental School and approved by the ethical committee. The microbes tested were \u00b0C to make sure sterilized conditions were met and bacteria were not introduced. The sterile paper discs  diameter of 6 mm were divided into three groups. Each group was soaked either with 4 drops (0.2 mL) of NaOCl, MTAD or an equal mixture of MTAD and NaOCl (M+N).To assess the antimicrobial power of each irrigant ZI and MIC was employed. In the ZI technique 30 blood agar plates  were prepared according to manufacture\u2019s instructions and the 125 mm Petri dishes were uniformly covered. These were then stored for 2 days in 37\u00b0C and 10% CO2. The maximum microbial ZI around each disc was then measured and recorded in millimetres.Five mL from each 1 McFarland suspension was removed with a sterile swab next to a flame and under the ventilation hood and placed on a blood agar plate in a criss-cross fashion. Each culture area was then divided into 4 sections in the following manner: the first section contained disc soaked with BioPure MTAD , the second section with NaOCl 5.25%  and the third section contained disc soaked with equal amounts of MTAD and NaOCl (5.25%), the final section consisted of normal saline . This was repeated 6 times for each of the microbes; altogether 120 samples were taken. Samples were placed in the incubator  for 48 hours at 37\u00b0C and 10% CO2).In the MIC technique 30 test tubes were prepared for each microbe. One mL of Trypticase Soy Broth (TSB)  was poured into each test tube and then autoclaved (sterilization). The tubes were then divided into three groups of 10 test tubes each. One mL of the selected irrigant was placed in the first test tube of each group and shaken with Cenco instrument MIJ  to homogenise the fluid. Subsequently, 1 mL of the solution was taken from the first test tube and transferred into the next tube; this was then repeated for all the ten tubes. The irrigant was gradually diluted from 1:2 to 1:1024 in the last test tube. An extra test tube was used with normal saline as the control. Exactly 0.1 mL of the selected microbial suspension (1 McFarland) was added to each of the test tubes, and this was performed for all the tubes and investigated irrigants. Test tubes were placed in the incubator for 24 hours  and placed in the incubator for 48 hours. One hundred and fifty five test tubes were used to test the 5 individual microbes, 5 of which were controls. The MIC of the irrigants for each individual microbe was recorded based on the degree of turbidity (with the naked eye) and the absence of microbial growth on the agar plates. The experiment was carried out in accordance with sterilisation principles .The relevant results of ZI were added into SPSS for Windows V 11.5. Descriptive criteria  were calculated and the data was analysed with ANOVA and Tukey tests and P<0.05 was considered statistically significant.S. aureus was statistically the most sensitive micro-organism (maximum ZI) against investigated irrigants (P<0.002) (The results for the ZI technique displayed that P<0.002) .E. faecalis, S. aureus and E. bacteria (P<0.001) (MTAD was more effective than 5.25% NaOCl and NaOCl was more effective than MTAD+NaOCl against P<0.001) .E. faecalis, S. aureus, E. bacteria and C. albicans: NaOCl>MTAD=MTAD+NaOCl (P<0.019)  .C. albicans was the most resistant micro-organism to MTAD (P<0.001).S. aureus and E. bacteria, however, NaOCl 5.25% and MTAD were equally effective against E. faecalis   . They uset al. has compared the effectiveness of MTAD and NaOCl (5.25%) using the ZI technique and discovered their similar antibacterial action against E. faecalis  was less than MTAD or NaOCl alone. A recent study showed that MTAD used alone has greater substantivity than other irrigants . NaOCl rThere have been reports that antioxidant such as ascorbic acid rinse following NaOCl irrigation will remove remnants of hypochlorite .C. albicans distorts the findings as NaOCl is stronger agent than MTAD and M+N; MTAD showed no antifungal effects. Ruff et al\u2019s study also supports our results; they demonstrated that Chlorohexidine 2% and NaOCl 6% were most effective in reducing the CFU of C. albicans  and types of C. albicans to have greater resistance to irrigation when compared to S. aureus  was effective when diluted 32 times against E. faecalis  with 150 mg of Doxycycline i.e. 3%. MTAD is naturally opaque and therefore measuring its turbidity may cause discrepancy in the results. MTAD instructions require NaOCl to be used as the final irrigant . The res of MTAD . The advWe suggest further investigations using irrigants in a polymicrobial environment to mirror endodontics infections.C. albicans and its substantivity may be altered when used in conjunction with NaOCl. C. albicans is often present in resistant and secondary endodontic infections as well as in peri-radicular lesions. NaOCl is an inexpensive and readily available chemical with broad spectrum action which has stood the test of time.MTAD is ineffective against"}
+{"text": "Knowledge of the linkage disequilibrium (LD) between markers is important to establish the number of markers necessary for association studies and genomic selection. The objective of this study was to evaluate the extent of LD in Nellore cattle using a high density SNP panel and 795 genotyped steers.2 and |D'| was 0.17 and 0.52, respectively. The LD (r2) decreased with increasing physical distance between markers from 0.34 (1 kb) to 0.11 (100 kb). In contrast to this clear decrease of LD measured by r2, the changes in |D'| indicated a less pronounced decline of LD. Chromosomes BTA1, BTA27, BTA28 and BTA29 showed lower levels of LD at any distance between markers. Except for these four chromosomes, the level of LD (r2) was higher than 0.20 for markers separated by less than 20 kb. At distances < 3 kb, the level of LD was higher than 0.30. The LD (r2) between markers was higher when the MAF threshold was high (0.15), especially when the distance between markers was short.After data editing, 446,986 SNPs were used for the estimation of LD, comprising 2508.4 Mb of the genome. The mean distance between adjacent markers was 4.90 \u00b1 2.89 kb. The minor allele frequency (MAF) was less than 0.20 in a considerable proportion of SNPs. The overall mean LD between marker pairs measured by rThe level of LD estimated for markers separated by less than 30 kb indicates that the High Density Bovine SNP BeadChip will likely be a suitable tool for prediction of genomic breeding values in Nellore cattle. Nellore is a beef cattle (Zebu) breed that originated in India. The first specimens of the breed arrived in Brazil at the end of the 18th century and Nellore animals rapidly became the predominant breed in the Brazilian herd. Over thThe breeding value of animals can be obtained from genomic data by marker-assisted selection covering the whole genome, also called genomic selection,5. Genom2 and |D'|[2 parameter represents the correlation between two loci and is preferred in association studies since an inverse relationship exists between r2 and the size of the sample needed for the same detection power. Linkage disequilibrium is necessary to detect associations between a QTL and a marker[The two measures most commonly used to evaluate LD between biallelic markers are rand |D'|-10. Thesa marker.2 = 0.2) for markers separated by less than 100 kb. Similar results have been reported by[2) between 2,670 markers in eight cattle breeds. Villa-Angulo et al., 2009[2) between adjacent markers of 0.21.The LD between markers has been studied in the genome of taurine breeds. In this respect, analyzinorted by who estil., 2009 studied l., 2009 genotypeThe first step necessary to determine the number of markers required for QTL mapping and genomic selection is the quantification of the extent of LD in the cattle genome. Therefore, the objective of the present study was to evaluate LD in Nellore cattle using a high density SNP panel (Illumina High Density Bovine SNP BeadChip\u00ae).2 and |D'|) between synthetic adjacent markers obtained for each autosome are shown in Table\u00a0The results of descriptive statistics of the SNP markers and LD  were estimated for chromosomes BTA1, BTA27, BTA28 and BTA29. This relatively low level of LD obtained for these chromosomes is in contrast to findings previously published for Zebu breeds[The mean LD between adjacent SNPs across autosomes ranged from 0.003 to 0.21 for ru breeds. Accordiu breeds, there i2 and |D'| were estimated for each bin per autosome  were observed at distances < 30 kb. When the distance between markers increased from 30 to 100 kb, the mean r2 value decreased from 0.20 to 0.11. A high variability in r2 estimates was observed for marker distances of more than 10 kb. Markers showing LD (r2) higher than 0.30 and 0.15 had an average spacing of 38.9 and 41.8 kb, respectively. However, not all markers with a spacing of 40 to 50 kb presented an r2 value higher than 0.3. For distances of less than 40 kb, the proportion of markers with an r2 > 0.15 and > 0.30 ranged from 35 to 57% and from 21 to 42%, respectively. This proportion was lower than that reported by[2). Recently,[2) > 0.25 was 29%.To analyze the decline in LD according to physical distance between markers, synthetic SNP pairs were classified into intervals (bins) based on the distance between markers and mean values of rFigures\u00a0 and for Figures\u00a0. Moderatorted by (68.34%)res\u00a02 0. to 0.34 2) was higher than 0.20 for markers separated by less than 20 kb, and higher than 0.30 for markers separated by less than 3 kb. For marker distances higher than 100 kb, the level of LD (r2) decreased from 0.11 (100 kb) to 0.05  (data not shown). McKay et al., 2007[Bos taurus and Bos indicus) and reported a mean LD (r2) ranging from 0.15 to 0.20 for a physical distance of 100 kb between adjacent markers.Except for autosomes BTA1, BTA27, BTA28 and BTA29, the level of LD  were excluded , a linear relationship was observed between chromosome length and LD (r2), i.e., the level of LD increased with increasing chromosome size. According to[Bos taurus cattle population and a much lower marker density.In the present study, certain autosomes presented higher LD than others. In addition, when autosomes with low levels of LD  between markers was higher when the MAF threshold was high (0.15), particularly when the distance between markers was short  between markers increased with increasing MAF threshold, especially in the case of very close SNP pairs (0\u201310 kb). For adjacent markers (< 10 kb), the |D'| remained unchanged for different MAF thresholds  observed in the present study was lower than that reported in other studies on pulation. Neverthpulation. Anotherstimates,24.The level of LD estimated for markers separated by less than 30 kb indicates that the High Density Bovine SNP BeadChip will likely be a suitable tool for prediction of genomic breeding values in Nellore cattle. Further studies investigating the magnitude of LD in a larger sample of animals from this population are needed to confirm the estimates obtained here.Seven hundred and ninety five Nellore bulls born in 2008 and 2009 from 117 sires, which belonged to the three Brazilian beef cattle breeding programs, were used in the present study. This research did not involve humans and the Animal Care and Use Committee approval was not obtained for this study because the data were from an existing database. Genotyping was performed by high density bead array technology using the Illumina Infinium HD Assay\u00ae and Illumina HiScan system\u00ae. The High Density Bovine SNP BeadChip contains 777,962 SNP markers spread across the genome at a mean distance of 3.43 kb between markers. The HiScan images and genotypes were first analyzed using the Genome Studio\u00ae software (Illumina). A total of 1,465 markers were excluded due to unknown genome position and 15,116 markers were monomorphic. For sake of the present study, only autosomal markers with minor allele frequencies (MAF) higher than 0.05, 0.10 or 0.15 were included in the LD analysis. In addition, only markers with a call rate > 0.90 and heterozygote excess < 0.30 were considered. A total of 11,785 markers were excluded because they showed low mean cluster intensity .longissimus dorsi muscle sample was removed and stored in a 2 ml Eppendorf tube. The tubes were identified with the identification of each animal and then stored in styrofoam boxes in a freezer at \u221220\u00b0C. Next, 25 to 30 mg of muscle tissue specimens were weighed on an aluminum sheet using an analytical balance and transferred to Eppendorf tubes (1.5 to 2 ml). DNA was extracted from the muscle samples using the DNeasy Blood & Tissue Kit  according to the manufacturer\u2019s instructions.For DNA extraction, about 5 g of 2 and the absolute value of D'. The r2 was calculated as follows:The LD between two SNPs was evaluated using rwhere,andfreq. A, freq.a, freq. B and freq.b are the frequencies of alleles A, a, B and b, respectively, and freq. AB, freq.ab, freq.aB and freq. Ab are the frequencies of haplotypes AB, ab, aB and Ab in the population, respectively. If the two loci are independent, the expected frequency of haplotype AB (freq. AB) is calculated as the product between freq. A and freq. B. A freq. AB higher or lower than the expected value indicates that these two loci in particular tend to segregate together and are in LD. The measures of LD (r2 and |D'|) were calculated for all marker pairs of each chromosome using the SnppldHD software .where 2 and |D'|). The exclusive use of maternal haplotypes is a common practice in studies estimating LD when the population consists of half-sib families, as was the case here. The reason is that the pedigree structure leads to the over-representation of paternal haplotypes in the sample since sires have multiple progenies in the dataset, which might increase the frequency of certain haplotypes and consequently overestimate LD[Only maternal haplotypes were considered for the estimation of LD measures (rimate LD.LD: Linkage disequilibrium; SNP: Single nucleotide polymorphism; MAF: Minor allele frequency; QTL: Quantitative trait loci.The authors declare that they have no competing interests.RE and FB participated in the design of the study, performed the genome studio analysis, statistical analysis and drafted the manuscript, AAB participated in the design of the study, helped with the genome studio analysis, statistical analysis and to draft the manuscript, FRPS and DFC participated in the DNA extraction, carried out the molecular analysis and helped to draft the manuscript, DMG, RLT and RE participated in the collection and preparation of the samples, HNO participated in the design of the study and to draft the manuscript, HT helped to draft the manuscript, MS participated in the design of the study, helped with LD analysis and to draft the manuscript, FSS participated in the design of the study, helped with LD analysis and to draft the manuscript, RC participated in the design of the study, helped the genome studio analysis, statistical analysis and to draft the manuscript, JAF carried out the molecular analysis and helped to draft the manuscript, LGA conceived the study and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript."}
+{"text": "To assess whether the value of CYFRA21-1 in the aspirates of ultrasonography-guided fine-needle aspiration biopsy (US-FNAB) can contribute to improving the performances of US-FNAB in the diagnosis of axillary lymph node (LN) metastasis in breast cancer patients.US-FNAB was performed in 156 axillary LNs in 152 breast cancer patients . Concentrations of CYFRA21-1 were measured from washouts of the syringe used during US-FNAB. Tumor marker concentrations, US-FNAB, intraoperative sentinel node biopsy (SNB), and surgical pathology results were reviewed and analyzed. For comparison, the values of CEA and CA15-3 were also measured from washouts.P<0.001). US-FNAB combined to CYFRA21-1 showed significantly higher sensitivity, NPV, and accuracy compared to US-FNAB alone . All diagnostic indices of US-FNAB combined to CYFRA21-1 were significantly higher compared to US-FNAB combined with CEA or CA15-3 . Of the 28 metastatic LNs which showed metastasis on SNB, CYFRA21-1 showed higher positive rate of 75.0% .Among the 156 LNs, 75 (48.1%) were benign, and 81 (51.9%) were metastases. Mean concentrations of CYFRA21-1 were significantly higher in metastasis compared to benign LNs (Measuring CYFRA 21-1 concentrations from US-FNAB aspirates improves sensitivity, NPV, and accuracy of US-FNAB alone, and may contribute to reducing up to 75.0% of unnecessary intraoperative SNB. Compared to CEA or CA15-3, CYFRA21-1 shows significantly higher performances when combined to US-FNAB in the preoperative diagnosis of LN metastasis in breast cancer patients. Presence of metastatic lymph nodes in the axilla is the most important prognostic factor in predicting outcome of patients diagnosed with breast cancer Recently, there had been some investigations using tumor markers during evaluation of axillary LN metastasis The Institutional Review Board (IRB) of Severance Hospital, Yonsei University approved of this study, and written informed consent was obtained from the patients included.in situ (n\u200a=\u200a13), papillary carcinoma (n\u200a=\u200a5), invasive lobular carcinoma (n\u200a=\u200a4), mucinous carcinoma (n\u200a=\u200a1), and tubular carcinoma (n\u200a=\u200a1).From March 2008 to July 2010, 152 consecutive women diagnosed with breast cancer who had undergone US-FNAB in 156 axillary LNs at our institution were included in this study . One-hunUS was performed by one of the seven board-certified radiologists specializing in breast imaging , each of whom had various experiences in breast imaging and biopsy (range: 1\u201311 years), using a 5-12-MHz  or 5-12-MHz  linear array transducer. Suspicious US features suggestive of metastasis in the axillary LNs were as follows; loss of fatty hilum, cortical thickening measuring more than 3 mm in thickness, irregular or round shape, extracapsular tumor extension, markedly hypoechogenicity of the cortex, and prominent peripheral blood flow on US Doppler US-FNAB was performed with a 23-gauge needle attached to a 2-mL disposable syringe using freehand technique. Each LN was aspirated at least twice. Material aspirated were immediately expelled onto glass slides and placed in 95% alcohol for Papanicolaou staining. The remaining contents in the needle and syringe from the first aspiration were rinsed with 1 mL of saline. Concentrations of CEA, CA15-3 and CYFRA 21-1 were measured individually from these washouts. Washouts from the second aspiration were sent for cell block processing.Five cytopathologists (with 1\u201315 years of experience) interpreted the FNAB specimen. The cytologic results were divided into two categories based on the reports. Cytologic results showing metastasis from breast cancer or atypical cells were considered positive. Reactive hyperplasia or benign lymphadenopathy, results that are non-related to breast cancer were considered negative. Reports showing insufficient material for specific diagnosis were considered negative, as proposed in other reports Tumor marker concentrations were analyzed with an automated immunoanalyzer system with chemiluminescent immunoassays for CEA , CA 15-3 , and CYFRA 21-1 . A noncompetitive immunometric sandwich assay format containing monoclonal antibodies for each tumor marker was used with the immunoassay reagents .Histopathologic results from subsequent surgery or clinical evidence during follow-up were regarded as standard reference. Among the LNs with surgical confirmation, aspirated LNs were correlated by the location seen during surgery.t-test was used in comparison of mean size between benign and metastatic LNs. Shapiro-Wilk test and Wilcoxon rank-sum test was used in comparison of the concentrations of each tumor marker between benign and metastatic groups.Student\u2019s Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy were assessed based on standard reference for US-FNAB alone, US-FNAB combined with CYFRA 21-1, and US-FNAB combined with CEA or CA15-3. Logistic regression method was used in estimation of cutoff concentration levels of tumor markers. Generalized estimating equation (GEE) method or weighted least square (WLS) method was used was used in comparison of diagnostic performances between US-FNAB alone and US-FNAB combined with CYFRA 21-1, and US-FNAB combined with CYFRA 21-1 and US-FNAB combined with CEA or CA15-3. Fisher\u2019s exact test was used in comparing performances among tumor markers in LNs which had undergone intraoperative SNB.P<0.05 was considered to indicate statistical significance.Statistical analyses were performed with SAS, version 9.1.3 for Windows . Of the 156 axillary LNs in the 152 patients included, 75 (48.1%) were benign, and 81 (51.9%) were metastases. Mean size of the 156 axillary LNs was 13.07\u00b16.38 mm, ranging from 1.00 to 35.00 mm. Among the positive LNs, 69 were diagnosed with surgery, while the remaining 12 were diagnosed with cytology alone, due to the presence of distant metastasis (n\u200a=\u200a11) or plans for chemotherapy or radiation therapy than surgery (n\u200a=\u200a1).Of the 69 metastatic LNs diagnosed with surgery, 32 (49.2%) underwent SNB prior to axillary LN dissection, among which 28 showed metastatic results . Of the P\u200a=\u200a0.001). Mean concentrations of CEA, CA15-3 and CYFRA 21-1 showed significantly higher levels in metastatic LNs compared to the benign LNs .Cytology results from US-FNAB in the 156 LNs included are as follows: inadequate or insufficient material for diagnosis in 6 (3.9%), benign in 86 (55.1%), and metastasis in 64 (41.0%).Diagnostic performances of CEA, CA15-3 and CYFRA 21-1 alone, US-FNAB, US-FNAB combined with CYFRA 21-1, and US-FNAB combined with CEA or CA15-3 are summarized in P\u200a=\u200a0.001), 91.1% to 81.5% (P<0.001), and 93.6% to 89.1% (P\u200a=\u200a0.001), respectively, but decreased PPV, 96.1% to 100.0% (P\u200a=\u200a0.002). Specificity of US-FNAB combined to CYFRA 21-1 was lower than US-FNAB alone, 96.0% to 100.0%, but without significance (P\u200a=\u200a0.085). For comparison with a previous study P<0.001).Optimal cutoff concentration values of the three tumor markers calculated from logistic regression methods were as follows: CEA 0.58 ng/mL, CA15-3 2.8 IU/mL, and CYFRA 21-1 1.93 ng/mL, respectively. US-FNAB combined to CYFRA 21-1 showed significantly improved sensitivity, NPV and accuracy compared to US-FNAB alone, 91.4% to 79.0% (P\u200a=\u200a0.076). All 4 metastatic LNs diagnosed as benign on SNB showed positive results in CEA or CA15-3 and CYFRA 21-1.P\u200a=\u200a0.021).Of the 70 benign LNs diagnosed as benign SNB, CYFRA 21-1 showed significantly higher negative results compared to CEA or CA15-3, 95.7% to 81.4%, respectively , the most sensitive tumor marker used in monitoring non-small cell lung cancer In addition to CEA and CA15-3, CYFRA 21-1  In conclusion, measuring CYFRA 21-1 concentrations from US-FNAB aspirates improves sensitivity, PPV, NPV, and accuracy of US-FNAB alone, and may contribute to reducing up to 75.0% of unnecessary intraoperative SNB. Compared to CEA or CA15-3, CYFRA 21-1 shows significantly higher performances when combined to US-FNAB in the preoperative diagnosis of LN metastasis in breast cancer patients."}
+{"text": "The genetic heterogeneity of sensorineural hearing loss is a major hurdle to the efficient discovery of disease-causing genes. We designed a multiphasic analysis of copy number variation (CNV), linkage, and single nucleotide variation (SNV) of whole exome sequencing (WES) data for the efficient discovery of mutations causing nonsyndromic hearing loss (NSHL).From WES data, we identified five distinct CNV loci from a NSHL family, but they were not co-segregated among patients. Linkage analysis based on SNVs identified six candidate loci (logarithm of odds [LOD] >1.5). We selected 15 SNVs that co-segregated with NSHL in the family, which were located in six linkage candidate loci. Finally, the novel variant p.M305T in ACTG1 (DFNA20/26) was selected as a disease-causing variant.Here, we present a multiphasic CNV, linkage, and SNV analysis of WES data for the identification of a candidate mutation causing NSHL. Our stepwise, multiphasic approach enabled us to expedite the discovery of disease-causing variants from a large number of patient variants. By virtue of the recent development of massively parallel DNA sequencing technologies, access to genomic composition has become easier than ever. With the advantage of exome sequencing, many studies have identified causal variants responsible for numerous disorders. Exome sequencing provides a particularly powerful method with which to identify disease-causing single nucleotide variations (SNVs) in Mendelian disorders-4. ThougFiltering patient data against normal populations and inferring identity-by-descent (IBD) in family studies can enrich candidate genes,6. GenetCopy number variation (CNV) has been implicated in both Mendelian diseases and commNonsyndromic hearing loss (NSHL) contributes to more than 70% of inherited cases of hearing loss. To date, approximately 50 genes have been shown to be causally related to NSHL. Many studies have identified more than 129 loci responsible for NSHL; however, 47 loci have not yet been mapped to proper genes,16. The Analysis of WES data can be expanded to obtain more information useful for identifying causative mutations in Mendelian diseases. In this paper, in order to analyze WES data from an entire family, we applied three different methods, namely, CNV, linkage, and segregation analysis. By combining the results obtained from these methods, we efficiently identified a causative mutation from the family data. We applied this approach to WES data from a NSHL family to identify candidate disease-causing variants.GJB2 is one of the most frequently detected genes in individuals with NSHL, and thus we first investigated the sequence of GJB2 in the NSHL patients. After failing to identify any mutations in GJB2, we performed WES on several members of the Korean family in order to identify a disease-causing mutation.We identified a Korean family with six members affected by NSHL and seven unaffected members Figure\u00a0A. Pure tSPAG11  , BNTL3 in 5q35.3 (II-1), and LILRB2 in 19q13.4 (I-2) (data not shown). We also applied Fisher\u2019s exact test for the LOD score per exon to detect co-segregated regions of CNVs, but there were no peaks with values reaching significance. We identified two groups based on the pattern of segregation of SPAG11, GSTM1, and beta-defensin genes to validate the relevance of this method  to validate the significance of peaks obtained from exome linkage analysis. The six hot spots detected from sequencing data were also detected in microarray analysis with a relatively high LOD score  was strictly conserved in 19 of 20 eukaryotes analyzed (HomoloGene:74402), with the M305 codon being conserved in 19 species. Protein damage prediction analysis identified p.M305T as \u201cpossibly damaging\u201d by HumDiv, \u201cprobably damaging\u201d by HumVar in Polyphen2. The mutWES is a powerful technique that can be used to discover causative genes in human diseases. Although WES has been integral in identifying more than 1,000 novel genes in Mendelian disorders[GSTM1 and UGT2B17, genes with frequently reported deletions[BNTL3 and LILRB2, exhibited CNVs. We used Fisher\u2019s exact test on the affected and unaffected family members after validating this method for 8p23.1 and GSTM1 groups to determine the amplification or deletion of multiple exons that matched the co-segregation pattern of the disease. Multiple statistically significant peaks at 8p23.1 and GSTM1 were identified, and were identical to plots from the first approach. However, there was only one statistically significant peak identified by testing the two groups that segregated with the disease, and this peak did not correlate with disease status. Thus, while WES may provide a method to identify CNV regions with highly similar sequences, determining accurate copy numbers can prove difficult.8p23.1, which contains a beta-defensin cluster, was detected as a region with high copy number (II-9) and low copy number (II-3 and II-7) , we may have lost critical information located outside of exons. In addition, potential genotyping errors in linkage analyses can reduce statistical power for detecting linkage peaks or result in false positive linkage peaks. Even soAfter applying linkage analysis results, the co-segregated variants were all found to be located in the loci of high LOD scores. However, linkage analysis can decrease the number of candidate variants, particularly in instances where candidate variants are widely distributed. Additional linkage analysis of WES data demonstrated a similar performance to that of SNP microarray data and simultaneously generated results during variant calling. Considering that CNVs could be also detected using this approach, the multiphasic analysis of WES data efficiently narrowed and identified candidate variants and was advantageous compared with established methods such as initial aCGH, variant calling according to WES data alone, or linkage analysis based on SNP microarray data.ACTG1)[In vivo and in vitro studies of ACTG1 indicate that it is required for reinforcement and long-term stability of actin filamentous structures of stereocilia, but not for auditory hair cell development, which is in line with the progressive nature of hearing loss related to ACTG1 mutations in humans[ACTB or ACTG1 have recently been reported to cause Baraitser-Winter syndrome. Interestingly, of the 11 mutations that cause DFNA20[Actin is a highly conserved cytoskeletal protein that plays important roles in eukaryotic cell processes such as cell division, migration, endocytosis, and contractility. Actin isoforms are classified into two groups based on expression patterns. ACTA1, ACTA2, ACTC, and ACTG2 are \u201cmuscle\u201d actins, predominantly expressed in striated or smooth muscle, whereas ACTB and ACTG1 are cytoplasmic \u201cnon-muscle\u201d actins. AutosomACTG1),28. In vn humans,30. Furte DFNA20,28,31-34ACTG1 and resultant NSHL[ACTG1 necessitates an early molecular genetic diagnosis and timely auditory rehabilitation.ACTG1 is predominantly expressed in intestinal epithelial and auditory hair cells. Detectiant NSHL. The sevTwo or more platforms  have previously been required to generate complex genetic information such as CNVs, linkages, SNVs, and indels. In general studies of Mendelian disorders, WES has primarily been utilized to obtain only SNVs and indels. Our study agrees well with other work demonstrating that analysis of WES data also allows for CNV and linkage determination due to its quantitative traits. Given the robust nature of WES data, it is clear that the full capabilities of this relatively new technology have not yet been fully realized. Our multiphasic WES analysis proved very powerful for the interpretation and narrowing of WES results, in particular when a large amount of family data is available.This study was approved by the Institutional Review Boards (IRBs) of Seoul National University Hospital (SNUH) and Seoul National University Bundang Hospital (SNUBH). Written informed consent for participation in the study was obtained from participants or from a parent or guardian in the case of child participants. A three generation pedigree was established for the family (SNUH3) Figure\u00a0A. Among Pure tone and speech audiometry and physical examinations were performed for nine members of the cohort Figure\u00a0B. Pure tEight of the 13 recruited subjects (four affected and four unaffected) were chosen for commercial WES  and analyzed as previously reported[CNVs were detected by CONTRA software using BEWe tabulated a 3 \u00d7 2 exon copy variation contingency table based on the whole per-exon CNV status of the eight subjects  =  and disease allele frequency = 1e-05. The penetrance parameters f0, f1, and f2 were also defined using conventional notation as below.Using WES data, we filtered out the following variants: those located on sex chromosomes, those with low coverage (<10X), and those with a low genotype quality score (<30) in any of the eight subjects with 17,498 SNVs. We used a Genome-Wide Human SNP Array 6.0 , which contains 328,125 SNP markers located on autosomal chromosomes. We performed parametric linkage analysis with the R package paramlink. The pedThe recombination fraction between the disease locus and markers was set to \u03b8=0 by default. We computed single-point LOD scores for all markers. We compared LOD scores from SNP microarray and WES. We matched the subjects and the markers that were common between both platforms using manual python and R scripts. Finally, single-point analyses were performed with all of the data.http://www.pdb.org)[Protein damage prediction analysis was performed using HumDiv and HumVar in Polyphen2, and alspdb.org). PDB entpdb.org) on P6071pdb.org) to obserThe authors declare that they have no competing interests.GP participated in the design of the study, analysis and interpretation of data; JG analyzed copy number variation from the exome data; AK validated variants by Sanger sequencing and checked segregation of the variant; KH collected samples from the family; HK analyzed clinical features; SO collected patients and initiated this study; TP have been involved in drafting the manuscript; WP designed the study, wrote the manuscript, revised it; BC participated in the design of the study, interpretation of data, and drafting the manuscript, revising it critically and have given final approval of the version to be published. All authors read and approved the final manuscript."}
+{"text": "There are some errors that occurred during uploading Figures 5(a), 6(b), 6(e), and 7(b). The following are the corrected figures."}
+{"text": "To identify the disease locus for autosomal recessive congenital cataracts in a consanguineous Pakistani family.All affected individuals underwent a detailed ophthalmologic examination. Blood samples were collected and genomic DNA was extracted. A genome-wide scan was completed with fluorescently-labeled microsatellite markers on genomic DNA from affected and unaffected family members. Logarithms of odds (LOD) scores were calculated under a fully penetrant autosomal recessive model of inheritance.Ophthalmic examination suggested that affected individuals have bilateral cataracts. Linkage analysis localized the critical interval to chromosome 8p with LOD scores of 3.19, and 3.08 at \u03b8=0, obtained with markers D8S549 and D8S550, respectively. Haplotype analyses refined the critical interval to 37.92 cM (16.28 Mb) region, flanked by markers, D8S277 proximally and D8S1734 distally.Here, we report a new locus for autosomal recessive congenital cataract mapped to chromosome 8p in a consanguineous Pakistani family. Congenital cataracts are the principal cause of visual impairment in children worldwide and are responsible for about one third of cases of blindness in infants ,2. The pEPHA2), connexin50 (GJA8), glucosaminyl (N-acetyl) transferase 2 (GCNT2), heat-shock transcription factor 4 (HSF4), lens intrinsic membrane protein (LIM2), beaded filament structural protein 1 (BFSP1), alphaA-crystallin (CRY\u03b1A), betaB1-crystallin (CRY\u03b2B1), and betaB3-crystallin (CRY\u03b2B3) have been identified [Approximately one-third of congenital cataract cases are familial , and to-entified ,16,18-22Here, we report a consanguineous Pakistani family with multiple affected individuals. Linkage analysis localized the critical interval to chromosome 8p with a significant Lod scores and haplotype analyses refined the critical interval to a 37.92 cM (16.28 Mb), flanked by markers D8S277 proximally and D8S1734 distally.A total of 125 consanguineous Pakistani families with nonsyndromic cataracts were recruited to participate in a collaborative study between the National Centre of Excellence in Molecular Biology, Lahore, Pakistan, and the National Eye Institute, Bethesda, MD, to identify novel loci associated with congenital cataracts. Institutional Review Board (IRB) approval was obtained from the both Institutes and all participating subjects gave informed consent consistent with the tenets of the Declaration of Helsinki. A detailed medical history was obtained by interviewing family members. Ophthalmic examinations were conducted with slit-lamp microscopy. Approximately 10\u00a0ml of blood samples were drawn from affected and unaffected members of the family and stored in 50\u00a0ml Sterilin\u00ae falcon tubes  containing 400\u00a0\u03bcl of 0.5 M EDTA. Blood samples were kept at \u221220\u00a0\u00b0C for long- term storage.DNA was extracted by a nonorganic method as described previously ,14. Brie2, and 0.2 U Taq DNA polymerase (Applied Biosystems). Initial denaturation was performed for 5 min at 95\u00a0\u00b0C, followed by 10 cycles of 15 s at 94\u00a0\u00b0C, 15 s at 55\u00a0\u00b0C, and 30 s at 72\u00a0\u00b0C and then 20 cycles of 15 s at 89\u00a0\u00b0C, 15 s at 55\u00a0\u00b0C, and 30 s at 72\u00a0\u00b0C. The final extension was performed for 10 min at 72\u00a0\u00b0C. PCR products from each DNA sample were pooled and mixed with a loading cocktail containing HD-400 size standards (Applied Biosystems). The resulting PCR products were separated in an ABI\u00a03100 DNA Analyzer (Applied Biosystems) and genotypes were assigned with GeneMapper software (Applied Biosystems).A genome-wide scan was performed with 382 highly polymorphic fluorescent markers from the ABI PRISM Linkage Mapping Set MD-10  having an average spacing of 10 cM. Multiplex polymerase chain reaction (PCR) was completed in a GeneAmp PCR System 9700 thermocycler (Applied Biosystems). Briefly, each reaction was performed in a 5\u00a0\u03bcl mixture containing 40 ng genomic DNA, various combinations of 10\u00a0mM dye-labeled primer pairs, 0.5\u00a0ml 10\u00d7 GeneAmp PCR Buffer (Applied Biosystems), 1\u00a0mM dNTP mix, 2.5\u00a0mM MgClLINKAGE Program Package, whereas the Maximum LOD scores were calculated with ILINK from the LINKAGE Program Package [Marshfield database and the National Center for Biotechnology Information (NCBI) chromosome 8 sequence maps. For the initial genome scan, equal allele frequencies were assumed, while for fine mapping allele frequencies were estimated from 96 unrelated and unaffected individuals from the Punjab province of Pakistan.Two-point linkage analyses were performed using the FASTLINK version of MLINK from the  Package ,24. AutoA large consanguineous family, PKCC144, consisting of four affected and four unaffected individuals was recruited from the Punjab province of Pakistan . The medInitially, linkage to all reported loci associated with autosomal recessive cataract loci were excluded by haplotype analyses with closely spaced fluorescently-labeled microsatellite markers (data not shown). Subsequently, a genome-wide scan was completed and during the genome-wide scan maximum two-point LOD scores of 3.19, and 3.08 at \u03b8=0 were obtained with markers D8S550 and D8S549, respectively . AdditioVisual inspection of the haplotypes supported the results of linkage analysis . There iHere, we report a new locus for autosomal recessive congenital cataracts localized to chromosome 8p in a consanguineous Pakistani family. The medical records available to us suggest a congenial onset, whereas the slit lamp examination was suggestive of bilateral cataracts. The genome-wide scan localized the critical interval to chromosome 8p, which was further supported by haplotype analyses and places the critical interval in a 37.92 cM (16.28 Mb) region, flanked by markers D8S277 proximally and D8S1734, distally.While the maximum Lod score of 3.19 is only slightly higher than the traditional limiting value of 3.0, it represents the maximum value obtainable with this family with real allele frequencies. In addition, the lack of evidence for linkage during the genome-wide scan, except with markers D8S549 and D8S550 provides additional support for localization of the cataract locus to chromosome 8p. This is the first report associating chromosome 8p with autosomal recessive congenital cataracts and illustrates the heterogeneity of the disease.The critical interval is a gene rich region that harbors more than 100 genes according to the UCSC database. We have prioritized all annotated genes based on their known function and available expression data in the eye, and currently are sequencing each candidate in one affected and one unaffected individual. Identification of the pathogenic mutations that lead to congenital cataracts will increase our understanding of lens biology at a molecular level and will help in development of better treatments and therapeutics."}
+{"text": "Adequate models are needed to reach this goal. The markers used to perform the statistical calculations can be linked and there may also be linkage disequilibrium (LD) in the population. The purpose of this paper is to present a graphical Bayesian Network framework to deal with such data. Potential LD is normally ignored and it is important to verify that the resulting calculations are not biased. Even if linkage does not influence results for regular paternity cases, it may have substantial impact on likelihood ratios involving other, more extended pedigrees. Models for LD influence likelihoods for all pedigrees to some degree and an initial estimate of the impact of ignoring LD and/or linkage is desirable, going beyond mere rules of thumb based on marker distance. Furthermore, we show how one can readily include a mutation model in the Bayesian Network; extending other programs or formulas to include such models may require considerable amounts of work and will in many case not be practical. As an example, we consider the two STR markers vWa and D12S391. We estimate probabilities for population haplotypes to account for LD using a method based on data from trios, while an estimate for the degree of linkage is taken from the literature. The results show that accounting for haplotype frequencies is unnecessary in most cases for this specific pair of markers. When doing calculations on regular paternity cases, the markers can be considered statistically independent. In more complex cases of disputed relatedness, for instance cases involving siblings or so-called deficient cases, or when small differences in the LR matter, independence should not be assumed. (The networks are freely available at  There are several areas of applications motivating this paper. The general problem is to determine the most likely pedigree and in this paper we discuss models to achieve this goal. It is well known that linkage analysis performed to locate disease mutations may be misguided if the pedigree is incorrectly specified as will be the case if for instance false paternities are not detected. Similarly, association analyses frequently assume that all individuals are unrelated and again deviations from this assumption may affect conclusions. In forensic cases, for instance paternity cases or identification following disasters, establishing the most likely pedigree is the main objective. Traditionally forensic applications have been based on unlinked markers in linkage equilibrium. For some applications however, these assumptions have been questioned The evidence is conventionally summarized by the LR (likelihood ratio) et al. recently published an overview of the commercial STR kits describing several pairs of markers separated by less than 50 cM A forensic example involving two short tandem repeat (STR) loci, D12S391 and vWa, will serve as a motivating case. These markers are located on chromosome 12 only 6.3 Mb apart, but the genetic distance has been estimated to be as large as 10.8 cM et al.http://genie.sis.pitt.edu) to create the Bayesian networks. One alternative is the commercially available Hugin (http://www.hugin.com).Object Oriented Bayesian Networks (OOBN) may provide an alternative solution with an appealing graphical interface. The object-oriented approach also provides a simple user-interface, hiding the complexities within the objects (nodes) http://arken.umb.no/~dakl/BayesianNetworks/. We present networks for some basic relationships, but the model can easily be extended to other pedigrees as well. In addition to previous investigations, this provides an alternative approach to the study of LD between D12S391 and vWa, but also more generally when studying pairs of linked STR markers. In contrast to other studies, which often measures the disequilibrium, or association of alleles, in terms of an 2r value or a p-value depending on a sample size, our intention was to investigate the effects of LD on actual cases.In this paper we model linkage, linkage disequilibrium, and mutations in a single Bayesian network (BN), freely available at et al.In order to model linkage disequilibrium (LD), haplotype probabilities must be estimated. A simplified model was constructed  per generation and hence,one generation will only have a minor impact on the disequilibrium.In order to obtain haplotype counts, we used data from regular trio paternity cases. When the parenthood is established and no mutations are present, the phase, i.e., the haplotypes can be deduced for the child using a simple algorithm. There are, however, ambiguous cases where the haplotypes cannot be determined for the child, e.g. when the parents and the child are all heterozygous for the same alleles. Out of 450 selected trios, 6 where discarded due to more than one possible haplotype configuration. As these ambiguous cases constitute only 1.3% of the total cases, it was not considered to bias the calculations enough to influence the conclusions. Notice that the phased haplotypes for the father and mother, based on the child's genotypes, are generally unknown since recombination might have occurred. Although reasonable estimates of the parents' haplotypes can be obtained, e.g. through the EM-algorithm or Gibbs sampling . http://arken.umb.no/~dakl/BayesianNetworks/. In addition we provide a short user manual as well as a software to generate the networks based on your own data.A simple Bayesian network describing a paternity case is illustrated in et al. for a paternity case Recombination node as well as an LD node. The Recombination node describes the probability for a cross-over to occur, i.e. the recombination rate. Also for each possible inheritance of a D12S391 allele, the P/M nodes transmit whether the Paternal or Maternal vWa allele have been passed on. The LD node is also connected to each possible inheritance of a D12S391 allele. If the LD node is instantiated to Yes, conditional allele probabilities will be used. The Mutation nodes contain a transition matrix. In this study a simple mutation model was used, where each transition has an equal probability of occurring, i.e. \u03bc/(n\u22121), where \u03bc is the mutation rate and n is the number of alleles. Mutation rates for each locus were obtained from a local database. The Child Paternal Allele (CPA) nodes are subject to the Hypothesis node (Either Is Father or Are Siblings depending on the network), with states Yes and No. The Hypothesis node will display the posterior probabilities for the given relationships. The tables for the CPA nodes are based on the Alleged father given that he is the father and the allele frequencies if he is not the father. Also, if the LD node is set to Yes, conditional allele probabilities for the D12S391 allele will be used. Two different scenarios were considered; a regular paternity case, et al.The networks were tested on a selection of real cases where the likelihood ratio (LR), assuming marker independence, had already been calculated using the software Familias To further test the method, we also created a network where instead of using data from D12S391 and vWa we used data from two other closely located markers, D5S818 and CSF1PO . A recomComparison are due to the use of slightly different allele frequency databases, where the Comparison LR has been calculated using a Norwegian population database utilized in routine casework. However, it is notable that the differences between the results using any of M1, M2 (10% recombination rate and LD not considered) or M3 (10% recombination rate and LD is considered) are in many cases comparable to the differences between M1 and the Comparison methods. Consequently, the differences between method M2 and M3, allele frequencies versus conditional allele probabilities, can perhaps be considered as merely a small bias in the estimation of allele frequencies.We have demonstrated the application of Object Oriented Bayesian Networks in modeling linkage, linkage disequilibrium and mutations in cases of disputed genetic relatedness. As an example, we present data from a pair of STR markers, vWA and D12S391, recently studied with regards to possible linkage disequilibrium. Two different networks were created to investigate a selection of actual cases as well as fictional, see Worst Case Scenarios in M2/LRM3 larger than 2, and for most of the cases the quotient is close to 1. Also, the Worst Case Scenarios do not display quotients larger than 4. We should of course always expect some differences since no data will indicate exact linkage equilibrium  where the haplotypes from the children can, in most cases, be unambiguously determined, as long as the possibility of mutation is disregarded. In our study we used 444 phased unrelated children, i.e. 888 haplotypes, to determine the observed as well as expected haplotype frequencies. We observed 100 of a total of 128 possible haplotypes. An important consideration is if this is enough material for a reliable estimation of population haplotype frequencies? In particular, can we reliably estimate the probability of observing a haplotype that has not been observed in the database? The same dilemma exists when previously unseen or new alleles are observed in regular genotyping, but for haplotypes one may use allele frequencies to construct a reasonable guess at a probability. Our formula contains a parameter \u03bb which loosely corresponds to the pseudo-counts often used in the estimation of population allele frequencies. Although a value for \u03bb might be estimated for data, we have simply used \u03bb\u200a=\u200a1. This gives the initial estimates, constructed as products of allele frequencies, the same weight as a single haplotype observation, leading to fairly small estimates of conditional probabilities for unobserved haplotypes.An imminent practical concern for forensic laboratories using closely located STR markers, such as the pair studied in this paper, is how computations should be performed with such data. One issue is whether linkage must be taken into account. Though statistical calculations in regular paternity cases is not affected by linkage and disputed paternities make up the majority of cases for most labs, we believe that in sibling cases and other more extended relationships, linkage should be taken into account. We recommend that forensic labs perform sensitivity calculations and/or simulations to investigate the effect of recombination rate, especially in kinship analyses and deficient paternity cases. The recently released software FamLink provides features to perform such analyses The other major concern, besides recombination, is whether to use conditional allele probabilities, i.e. to account for linkage disequilibrium. All calculations are affected by the use of such probabilities, even standard paternity and match probability calculations. The effect on the marker pair vWA/D12S391 is, according to our results, reasonably small. In addition, the marker pair D5S818/CSF1PO displays equal deviation from expectation, further corroborating results in previous studies. Moreover, our implementation heavily depends on the estimates of conditional allele probabilities, which are currently fairly uncertain. We have illustrated how estimates can be generated based on data from trios, but clearly much larger datasets are needed to reduce the uncertainty. Furthermore, other models to approach the problem with unseen haplotypes should be considered.http://arken.umb.no/~dakl/BayesianNetworks/, which can be used as prototypes for investigations of linkage and linkage disequilibrium for pairs of closely located STR markers.Nevertheless, this paper demonstrates how software implementing Object Oriented Bayesian Networks can be used to assemble and code models reasonably quickly, and how these models can subsequently be used to explore complex questions about the interplay between genetic phenomena such as linkage, LD, and mutations. The models can then in fact be used for relevant computations in actual cases. We present Bayesian networks for two basic relationships, available at Figure S1Are Siblings? contains the different hypotheses.Bayesian network describing a sibling case, where the children are known to share the same mother. The nodes P/M tell whether the vWa paternal or maternal allele is inherited. The P/M node connected to the D12S391 allele also contains the recombination frequency. The LD node is connected to the paternal and maternal allele nodes and decides whether or not to use conditional allele frequencies. Furthermore, the node (DOC)Click here for additional data file.Table S1Observed haplotype frequencies.(DOC)Click here for additional data file.Table S2Expected haplotype frequencies.(DOC)Click here for additional data file."}
+{"text": "Familial hypercholesterolemia (FH) is a heritable disorder that can increase the risk of premature coronary heart disease. Studies suggest there are substantial genetic heterogeneities for different populations. Here we tried to identify novel susceptibility loci for FH in a Chinese pedigree.P<0.00001, and simulated occurrence per genome scan\u200a=\u200a1.08) and 21q22.3 . In the interaction analysis with a trimmed version of the pedigree, we obtained a significantly increased joint LOD score (2.70) compared with that obtained when assuming the two loci uncorrelated, suggesting that more than one locus was involved in this pedigree. Exon screening of two candidate genes ABCG1 and LSS from one of the suggestive region 21q22 didn't report any causative mutations.We performed a SNP-based genome-wide linkage scan with the Chinese FH pedigree. Two suggestive linkage loci not previously reported were identified on chromosomes 3q25.1-26.1 (NPL\u200a=\u200a9.01, nominal These results confirm complex etiologies and suggest new genetic casual factors for the FH disorder. Further study of the two candidate regions is advocated. Familial hypercholesterolemia (FH) is a heritable disorder characterized by high concentration of total cholesterol and low density lipoprotein cholesterol in serums. Elevated levels of cholesterol can give rise to xanthomas, a deposit of cholesterol in peripheral tissues, accelerating atherosclerosis, and therefore increase the risk of premature coronary heart disease (CHD) LDLR) on chromosome 19 APOB) on chromosome 2 PCSK9) on chromosome 1 The majority of FH cases are transmitted in an autosomal-dominant manner, known as autosomal dominant hypercholesterolemia . Three genes identified in autosomal dominant FH disorder are the low density lipoprotein receptor gene  analysis was performed to identify linkage signals and potential locus interaction.To identify novel FH loci in Chinese individuals, we performed a genome-wide linkage analysis of a Chinese pedigree without the known FH mutations in Lipid levels and clinical features including xanthoma and coronary heart disease (CHD) for the pedigree members are shown in P<0.00001, and simulated occurrence per genome scan\u200a=\u200a1.08) and 8.95  on chromosomes 3q25-26 and 21q22, respectively  scores of 9.01  between rs1920395 and rs1369276 . On chroNotably, the disease linked haplotype on either locus was also shared among some unaffected individuals. On chromosome 3q25-26, those individuals were 27, 28, and 21 . On chroP\u200a=\u200a0.000565) that was higher than those from single-locus analysis on chromosomes 3q25-26 and 21q22 .The two-locus linkage analyses on 3q25-26 and 21q22 further improved the linkage scores in both parametric and nonparametric analyses over those from single-locus analyses. The two-locus linkage analysis using a trimmed version of the pedigree  under a multiplicative model yielded a maximum joint LOD score of 2.70. This was a significant increase over the single-locus LOD scores of 1.10 and 1.14 on chromosomes 3q25-26 and 21q22, respectively, with the same trimmed pedigree structure. It was also observed that, the parametric two-locus linkage analyses using a heterogeneous and an additive model yielded LOD scores of 1.17 and 1.19, respectively, which are no better than the single-locus LOD scores with the same trimmed pedigree structure. Therefore, the interaction between these two regions was better modeled by the multiplicative model. This interaction was further supported by a joint NPL score . The main reason for the insignificance of the parametric linkage score was the difficulty in defining an accurate mode of inheritance. Incomplete penetrances, phenocopies, or polygenic inheritance may also lead to loss of power in single-locus parametric linkage mapping Based on a genome-wide linkage scan of a Chinese pedigree using SNP markers, we identified two suggestive linkage loci for FH. One locus was mapped to 3q25.1-26.1 in a 7.42-cM interval (13.19 Mb) between rs1920395 and rs1369276, and the other was mapped to 21q22.3 in a 20-cM interval (4.54 Mb) from rs220271 to the q telomere. Both regions are distinct from the previously reported candidate regions for both autosomal dominant and recessive FH. Although the parametric linkage scores for both loci were not significant, the nonparametric scores were suggestively significant for both loci -2, 3 oxidosqualene to lanosterol. Exon screening of both genes failed to identify any mutation that co-segregated with the disease trait in this pedigree. This result was not unexpected, because more than one gene was implied in the pedigree according to our results. It is more probable that a few common variants, each with mild effect rather than a single causative mutation, may contribute to FH. Nevertheless, detailed screening for molecular variants in the candidate genes should be carried out, perhaps with increased screening size in a case-control design, and including extended screening regions beyond exons. Indeed, recently a novel ABCG1 \u2212257T>G promoter polymorphism that influences CHD severity in Japanese males has been found There are 92 and 113 genes in the candidate regions of chromosomes 3q25.1-26.1 and 21q22.3, respectively. We selected the two most likely candidate genes LDLR mutations can exhibit a more severe FH phenotype than their parents who carried only a single mutation In this study, the proband had a characteristic FH phenotype with extremely high levels of plasma TC (817.2 mg/dl) and LDL-C (761.1 mg/dl) and tendinous xanthomas, while the other affected members had a relatively mild phenotype. Because both parents were affected, one possibility was that the proband may inherit two sets of pathogenic mutations  in one gene or in different genes. Previous clinical observations revealed that patients with compound heterozygote ABCG1 and LSS were identified as plausible candidate genes in one of the candidate regions. Our findings revealed new and complex genetic etiology for the disease. Further research is advocated to identify the susceptibility genes in these newly discovered candidate regions.In summary, the SNP-based genome-wide linkage scan with a Chinese FH pedigree revealed two suggestive linkage signals on chromosomes 3q25.1-26.1 and 21q22.3. Both loci are distinct from previously reported FH regions. Interaction analysis assuming two disease loci suggested the involvement of more than one locus in this pedigree. The study complies with the Declaration of Helsinki. All participants provided written consent, and the ethics committee of the Beijing Anzhen Hospital of the Capital University of Medical Sciences approved all studies. Separate informed consent was obtained to publish the photographs in A 5-year-old boy with cutaneous vegetations on bilateral elbows, knees, and buttocks was identified as the proband. A further examination at the Beijing Anzhen Hospital found multiple cutaneous and tendinous xanthomas in the boy . The conAn extended four-generation pedigree of the proband with 17 available members from Henan Province, China was obtained for this study. All individuals had their lipoprotein measurements recorded and general information such as gender, age, history of smoking, dietary habits, personal history, and family history were collected at the same time . None ofVenous blood samples were drawn from the pedigree members after overnight fasting for 12 to 14 hours. The concentrations of total plasma cholesterol, triglycerides, and high-density lipoprotein cholesterol (HDL-C) were measured by applying standard enzymatic techniques. The LDL-C concentration was calculated by applying the Friedewald formula LDLR, APOB, and PCSK9, caused the FH phenotype of this pedigree. The LDLR gene was examined by sequencing, whereas the APOB and PCSK9 loci were examined by genotyping locus-linked microsatellite markers.Genomic DNA was extracted from venous blood of the 17 available pedigree members using a phenol-chloroform method. Prior to the linkage scan, we excluded the possibility that any known mutations from the three FH genes, The genome-wide linkage scan was then conducted using the Illumina Human Linkage-12 panel. The panel contains 6,090 single nucleotide polymorphism (SNP) markers that are well spaced. Reactions were performed according to the manufacturer's protocols. Fluorescent signals were scanned using Illumina's BeadStation; genotypes were called with Illumina BeadStudio Software v3.1.8.Mendelian inconsistencies of the genotype data were investigated with PEDCHECK v1.1 The pedigree used in our study contained 17 genotyped individuals with 11 affected and 6 unaffected members. Power calculation was performed with SLINK To determine the genome-wide significance level of the linkage signal, we performed simulation studies of 1000 replicates generated with the gene-dropping approach implemented in MERLIN. In each simulation, marker allele frequencies, genetic distances, pedigree structures, and missing data patterns were retained. The number of simulations exhibiting equal or greater linkage scores was used to calculate the frequency of the observed linkage signal occurring by chance.To test for an interaction between two putative loci on different chromosomes, GENEHUNTER-TWOLOCUS software was used to perform parametric and nonparametric linkage analysis under two disease loci ABCG1 (15 exons) and LSS (23 exons) were amplified with PCR and sequenced on ABI Prism 3730xl DNA Analyzer.Five affected and 3 unaffected pedigree members, including the proband, his parents, his maternal uncles and aunts, and his maternal grandparents, were selected for sequencing of the candidate gene. All exons and intron-exon boundaries of the two candidate genes,"}
+{"text": "Most genome linkage scans for autism spectrum disorders (ASDs) have failed to be replicated. Recently, a new ASD phenotypic sub-classification method was developed which employed cluster analyses of severity scores from the Autism Diagnostic Interview-Revised (ADI-R). Here, we performed linkage analysis for each of the four identified ADI-R stratified subgroups. Additional stratification was also applied to reduce intra-family heterogeneity and to investigate the impact of gender. For the purpose of replication, two independent sets of single nucleotide polymorphism markers for 392 families were used in our study. This deep subject stratification protocol resulted in 16 distinct group-specific datasets for linkage analysis. No locus reached significance for the combined non-stratified cohort. However, study-wide significant (P\u200a=\u200a0.02) linkage scores were reached for chromosomes 22q11 (LOD\u200a=\u200a4.43) and 13q21 (LOD\u200a=\u200a4.37) for two subsets representing the most severely language impaired individuals with ASD. Notably, 13q21 has been previously linked to autism with language impairment, and 22q11 has been separately associated with either autism or language disorders. Linkage analysis on chromosome 5p15 for a combination of two stratified female-containing subgroups demonstrated suggestive linkage (LOD\u200a=\u200a3.5), which replicates previous linkage result for female-containing pedigrees. A trend was also found for the association of previously reported 5p14-p15 SNPs in the same female-containing cohort. This study demonstrates a novel and effective method to address the heterogeneity in genetic studies of ASD. Moreover, the linkage results for the stratified subgroups provide evidence at the gene scan level for both inter- and intra-family heterogeneity as well as for gender-specific loci. Autism is a common early onset neurodevelopmental disorder belonging to a group of conditions known as autism spectrum disorders (ASDs), which include classical autism, pervasive developmental disorder-not otherwise specified and Asperger syndrome Table S1 in File S1 for detail on previously reported linkage).It is now generally accepted that multiple genes contribute to the etiology of autism, but the questions of how many susceptibility genes are involved and how they relate to respective subgroups of individuals remain unanswered. To date, several independent genome-wide linkage studies have been performed to investigate the genetic underpinnings of ASD, but with limited success, since the majority of the identified linked regions have not been replicated  found no genome-wide significant linkage peaks, but detected suggestive linkage at 11p and 15q chromosomal regions In the second largest autism linkage study reported in 2009, more than 800 families and 16,000 rigorously filtered SNPs were included The heterogeneous phenotype of autism suggests the need to employ strategies to identify homogeneous groups of subjects with common or more similar features. There have been attempts at phenotypic stratification that focus on different ADI-R criteria, such as language related phenotypes, by use of scores on ADI-R items corresponding to phrase speech delay Recently, Hu and Steinberg Two independent datasets of single nucleotide polymorphism (SNP) were utilized to perform the linkage analysis. SNP dataset-1 contains data on approximately 8,000 markers throughout the genome derived from the Affymetrix 10 K SNP array, generated from >1000 families in the phase one AGP Table S2 in File S1. Both parents were mostly genotyped which minimizes the impact of ethnic specific allele frequencies on linkage analysis.Our subject inclusion criteria were the availability of both the ADI-R related cluster assignment of the probands File S1 for detail on the clustering method. In this study, these four ADI-R subgroups are referred to as the following: (g1) severe, with language impairment, (g2) mild, with lower symptom severity across all items, (g3) moderate, with notable savant skills, and (g4) intermediate phenotype.Phenotypic subtyping of the probands was assigned using previously performed ADI-R cluster analyses methods The affected subject\u2019s ADI-R sub-phenotype  was used to create group-specific SNP datasets using a three-step stratification process as shown in Table S2 in File S1. Also shown is the number of families in the original group of combined cases (referred to as \u201cALL\u201d).Initially, only subjects with a strict classification of autism by AGRE were included (n1) in our analysis, and broad spectrum subjects were removed. Upon completion of our initial linkage scans, broad spectrum subjects were then added (n2) to each subgroup based on their ADI-R-determined sub-phenotypes Figure S2 in File S1 for detail on linkage analysis and permutation. Since the second SNP dataset has undergone a more rigorous filtration, the reported LOD scores in this study are based on the values obtained using this dataset. The AGP SNP dataset et al. paper See N/A.Table S3 in File S1. The applied multi-step stratification process provided a pipeline to further filter the original heterogeneous ASD pedigree data file (ALL) to more homogeneous datasets by first using ADI-R cluster analysis, followed by removal of sub-phenotypically discordant siblings, and finally by separation of male-only and female-containing pedigrees.Our stratification workflow and the resulting 16 subgroups for the linkage study are illustrated in To assess whether genotyping quality or artifacts contributes to our results, linkage analysis was performed at the discovery and validation phases, using two independent SNP datasets. We first ran linkage analyses using SNP dataset-1 . Next, the replication of suggestive linkage results was assessed by repeating genome-wide linkage, for the same subgroups, using SNP dataset-2 . The reported LOD scores represent values that have been generated by the second SNP dataset because the second SNP dataset has been subject to a more rigorous quality control filtration.Table S2 in File S1]. This addition of broad spectrum subjects increased sample sizes in all groups except female-containing subsets [denoted as n2 in Table S2 in File S1].To assess the impact of increasing sample size on linkage results, we added subjects described as \u201cbroad spectrum\u201d by AGRE to the initial cohort which included only subjects with a strict diagnosis of autism . Such a side-by-side comparison demonstrates that the highest linkage scores may be achieved at different levels of stratification . For example, locus 13q21 is potentially a shared region (LOD\u200a=\u200a4.37) for all affected siblings in G1 pedigrees (232 multiplex families) regardless of the sub-phenotype of siblings. After excluding discordant siblings, 169 of 232 G1 pedigrees (73%) are no longer multiplex and thus cannot contribute to linkage. This substantial reduction of the number of pedigrees (from 232 to 63) causes loss of linkage peak for this region in the G1s group (LOD\u200a=\u200a0), demonstrating a pattern best fitting with intra-family shared regions. On the other hand, removal of discordant sub-phenotypes within pedigrees, to generate Gs level families, resulted in significantly improved LOD scores for the three remaining loci listed in Despite the observed differences in linked regions among these ADI-R subtypes, several overlapping linkage signals were also seen for different subgroups. For example, two separate loci (5p15 and 22q11) with positive LOD scores were shared by G1Fc and G2Fc subgroups Table 3.Table S4 in File S2]. In this figure, LOD scores are displayed as a linkage heat map (using a supervised method) which shows improved linkage in at least one of the stratified subgroups relative to the undivided ALL group (see File S1 for detail on method). Chromosomal locations of the positive linked loci and their associated genes are summarized by subgroup in Table S5 in File S1. What is clear from this visual map of genome-wide LOD scores across the stratified subgroups is that reduction of phenotypic heterogeneity on the basis of cluster analyses of severity scores across a broad spectrum of ASD symptoms and behaviors greatly improves the ability to identify genetic linkage for specific sub-phenotypes of ASD. Unsupervised hierarchical clustering analysis and principal components analysis of this data further corroborate sub-phenotype dependent linkage results .Table S6 in File S1]. However, no associations were seen for either SNP when a total of 166 autism cases from all female-containing pedigrees  was analyzed (see File S1 for detail on TDT association method and result).In previous GWAS studies, the most significant associations for autism have been reported for the SNPs at 5p14 (rs10513025) and 5p15 (rs4307059) Disparity in linkage results for autism highlights the degree of genetic heterogeneity both within and among families. Studies of population isolates such as the Finnish et al. study To address this critical gap, we reanalyzed previously generated SNP data available from 392 AGRE families, a subset of samples included in both the first phase of the AGP Table S2 in File S1. Larger sample sizes, including sufficient number of subjects from different ethnic backgrounds, are required to assess if there exists ethnicity-related variations in the prevalence of the ADI-R subtypes in autistic populations.From the present study it appears that subsets representing intermediate phenotypes  are more likely to consist of multi-ethnicity groups, compared with the most severely language impaired subsets , as shown in DIAPH3 with suggested connections to both autism and language impairment. DIAPH3, an auditory neuropathy gene whereby affected subjects show impairment of speech perception DIAPH3 might be involved in synaptic activity and function downstream of SHANK3 (chromosome 22q13) SHANK3 in language development has also been suggested by its implication in cases with a severe speech and language delay The genes residing in the linkage intervals may provide some insight into the biology of ASD. The 13q21 region has been previously linked with autistic subjects ascertained for language impairment TBX1GNB1LCOMT, one of the autism susceptibility genes in this chromosomal region has also been investigated in correlation with language production and semantic verbal fluency Several lines of evidence have already documented associations of chromosome 22q11 with language related disorders DIAPH3, SHANK3, and COMT) for ASD individuals with this language-impaired phenotype. See Tables S5, S7, S8 in File S1 for more discussion on potential candidate genes in the linkage intervals.Given that the 13q21 and 22q11 regions both show the highest LOD scores for the subtypes of ASD with severe language impairment , the above-mentioned studies and our current linkage findings suggest that further evaluation of genes within these regions is warranted, especially candidate genes .In the two previous large genome-wide linkage studies et al. In our study, only the G1 and G1s subtypes showed significant linkage to 13q22 and 22q11, respectively. The location of 13q22 linked region is very close to the previously reported region by Bartlett Heterogeneity in ASD is also reflected at the family level. In multiplex families, autistic symptoms may vary among affected siblings. To explore the impact of this layer of heterogeneity on linkage analyses, we adopted a multi-step subject stratification approach, denoted by the G and Gs annotation, wherein intra-family phenotypic heterogeneity was included or reduced, respectively. The linkage data obtained by this stratification method supports the idea that some loci might be common in all affected siblings within a family, as shown by loci producing highest linkage peaks at the G level. On the other hand, some loci exhibited higher LOD scores after reducing intra-family heterogeneity, i.e., at the Gs level see Table 2.As observed with gene expression profiling MSN gene on chromosome X A suggestive linkage peak at 5p15 was found for the G1.2Fc combined group . This linkage score did not pass the study-wide significant estimated by permutation tests. However, this suggestive linkage is in agreement with the AGP report where linkage to 5p14.33 was also detected for female-containing families. This concordant finding further emphasizes that female-containing families might be more informative for linkage et alDespite small sample sizes, we also found a suggestive association with the G1.2Fc subjects for both of the previously reported SNPs on chromosome 5p. Such a positive trend for association was not detected when assessing all female-containing families, further demonstrating the positive impact of our stratification approach. Together with these recent linkage, GWAS, and noncoding RNA studies, the suggestive linkage and TDT findings in our G1.2Fc group suggest that studying pedigrees in this ASD subset may provide a greater chance of revealing other relevant information in the integrated model proposed by Kerin Our study demonstrates a novel and powerful stratification method to address the heterogeneity in autism spectrum disorders within and among families. Herein, we used ADI-R clustering subtyping for subject classifications to test the validity of our multi-step stratification strategy. ADI-R clustering is only one way of stratifying ASD subjects. Similarly, other ASD stratification measurements can be used when employing the present deep stratification method. Such multi-faceted methods  can be also applied to all genomic studies to improve the likelihood of uncovering previously undetected genetic factors masked by clinical heterogeneity. The number of families examined to identify suggestive linkage regions in the subgroups is considerably fewer than the total number of families in the undivided group. These findings thus illustrate the added likelihood to detect significant linkage when the heterogeneity of the ASD population is reduced by sample stratification. Finally, our present study provides evidence at the linkage level for both inter- and intra-family heterogeneity, reflecting both shared and distinct genetic makeup in the autism population.File S1Figure S1, Hierarchical clustering and principal components analyses, Figure S2, Workflow describing the applied permutation analysis, Table S1, A summary of previously reported linkage results for autism, Table S2, The number of multiplex families, in each subgroup, without (n1) and with (n2) BroadSpectrum subjects, Table S3, Overlap between the subgroups at the G level, Table S5, Chromosomal locations of the positive linked loci (LOD\u22652) and their associated genes per subgroups, Table S6, TDT result for two previously associated SNPs at chromosome 5p, Table S7, List of the genes associated with the SNPs with the highest LOD scores in 13q21 (G1 group), and Table S8, List of the genes associated with the SNPs with the highest LOD scores in 22q11 (G1s group).This file contains (PDF)Click here for additional data file.File S2Table S4, The SNPs and corresponding LOD scores \u22652.0 across all subgroups.This file contains (XLSX)Click here for additional data file.File S3Table S9A, Simulation data (17 groups).This file contains (XLSX)Click here for additional data file.File S4Table S9B, Simulation data (20 groups).This file contains (XLSX)Click here for additional data file."}
+{"text": "Record linkage techniques are widely used to enable health researchers to gain event based longitudinal information for entire populations. The task of record linkage is increasingly being undertaken by specialised linkage units (SLUs). In addition to the complexity of undertaking probabilistic record linkage, these units face additional technical challenges in providing record linkage \u2018as a service\u2019 for research. The extent of this functionality, and approaches to solving these issues, has had little focus in the record linkage literature. Few, if any, of the record linkage packages or systems currently used by SLUs include the full range of functions required.This paper identifies and discusses some of the functions that are required or undertaken by SLUs in the provision of record linkage services. These include managing routine, on-going linkage; storing and handling changing data; handling different linkage scenarios; accommodating ever increasing datasets. Automated linkage processes are one way of ensuring consistency of results and scalability of service.Alternative solutions to some of these challenges are presented. By maintaining a full history of links, and storing pairwise information, many of the challenges around handling \u2018open\u2019 records, and providing automated managed extractions are solved. A number of these solutions were implemented as part of the development of the National Linkage System (NLS) by the Centre for Data Linkage  in Australia.The demand for, and complexity of, linkage services is growing. This presents as a challenge to SLUs as they seek to service the varying needs of dozens of research projects annually. Linkage units need to be both flexible and scalable to meet this demand. It is hoped the solutions presented here can help mitigate these difficulties. Record linkage is the process of bringing together data relating to the same individual from within and between different datasets. When a unique person based identifier exists, this can be achieved by simply merging datasets on the identifier. When this identifier does not exist, some form of data matching or record linkage is required. Often, statistical or probabilistic matching processes are applied to records containing personally identifying information such as name and address.Record linkage techniques are widely used in public health to enable researchers to gain event based longitudinal information for entire populations. In Australia, research carried out using linked health data has led to numerous health policy changes ,2. The sThere are differing operational models for the provision of record linkage services; however, some elements of the current infrastructure are similar. For example, in Australia and Wales, record linkage is conducted by trusted third parties or specialised linkage units (SLUs). SLUs are usually located external to the data custodians and researchers. This provides an element of separation, which enhances privacy protection . Using sThe record linkage processes used by SLUs can be quite complex and involve many components e.g. data cleaning and standardisation, deterministic and/or probabilistic linkage, clerical review, etc. Many factors influence the consistency and quality of linkage results .Notwithstanding the complexity of record linkage, SLUs face additional technical challenges in providing linkage \u2018as a service\u2019 for research. The extent of this functionality, and approaches to solving these issues, has had little focus in record linkage literature. Few, if any, of the record linkage packages or systems in use by SLUs today include the full range of functions required of/by these entities.The purpose of this paper to identify and discuss some of the technical issues associated with the provision of record linkage services, and to propose solutions to these problems. Of particular interest is the array of challenges associated with on-going linkage (i.e. continuous linkage of changing datasets over time). These issues have not been previously addressed in the literature, and it is the aim of this paper to do so.per se, there are other technical challenges that present to SLUs. These include the general management of data, handling different linkage scenarios, the management of routine, on-going linkage (and the complexity of storing and handling changing data), the need for automation and the ever present need to accommodate larger sized datasets. In this section we discuss each of these emerging problems.The role of SLUs has become more prominent in the research infrastructure landscape and the level and complexity of demands placed on them for linkage services has increased. While there are a variety of techniques available to undertake record linkage such as deterministic rules-based methods, sort and match algorithms , and proAs the number of linkage projects increase, SLUs need robust, efficient methods of managing all forms of data. These include: incoming data from custodians . SLUs need the ability to handle various linkage scenarios including both project based (create and destroy) and ongoing linkage research projects.\u2018Project based linkage\u2019 is arguably the simplest scenario. This is where one or more datasets are required to be linked together for a single research project. These datasets are to be linked to each other, with the links only to be used for a specified research project. Based on the data agreements for the project; the datasets, and the links, often require to be deleted/destroyed after the project has completed.On-going linkage. As systems, processes and relationships mature, SLUs typically move from a \u2018project\u2019 based approach, where data is linked for each specific research project and then the links are discarded when no longer required, to an on-going approach, where a central core of links is created and maintained over time and re-used for multiple research projects. As new records are added to the system, the links are updated. This approach dramatically reduces effort and improves linkage quality, as the same data are not required to be re-linked over and over with the impact of quality intervention and clerical review is not lost [not lost ; howeverDespite the vast array of record linkage software packages available, most focus on linking files on a \u2018project\u2019 basis, that is, linking a single file to itself  or linking two files to each other at a single instance in time. Currently there are a range of desktop applications that perform this function and although these are usually easy to implement and use, they can struggle to handle medium (>1 million) and large scale (>10 million) linkages . Few, ifAlternative approaches to on-going incremental linkage have been developed in recent years, including those outlined by Kendrick ,28 in hiOther linkage scenarios. There are occasional scenarios where on-going linkage may not be possible, or the most appropriate solution. A SLU needs to understand requirements in both the long and short term, and how it can accommodate both \u2018project based\u2019 and \u2018on-going\u2019 linkage requests, if at all.bring your own\u2019 linkage. This is where a researcher who has collected information on a study cohort wishes to link this data to another dataset which may or may not already exist in the linkage system. While this researcher\u2019s data should link to the required dataset(s), there is no requirement that it should form part of the on-going system.Another linkage scenario often dealt with by SLUs is \u2018There are several considerations that need to be addressed before implementing an on-going linkage system; these issues typically do not appear in simpler, project based linkage operations. These differences are subtle and are mainly a result of the intricacies of managing data over time. Each of the approaches has their strengths and weaknesses and their applicability or suitability will depend on project requirements.routine, continuous linkage of (changing) datasets over time. In on-going linkage, previously created links are retained by the system, and added to on the arrival of new records from the same datasets. New records entering the system needed to link to other new records  as well as to existing records that are currently in the system .Similarly, the linkage system should have the ability to remove a record from the map. Ideally, this should occur in a way that removes any associations that may have been created by the existence of this record in the system.as they occur over time and for all records in the system, including those that are added or updated on an incremental basis.On-going linkage systems require the maintenance of a central linkage map (a list of each record and the group they belong to). As linkage processes are continuous, the map needs to reflect results Maintaining a linkage map and its history has utility for researchers, as well as for SLUs. Once researchers receive their data, they may have queries relating to how specific records were linked together. The linkage map is constantly being updated as new records arrive, and as the linkage map may no longer contain these records/links, it may be unclear how these records were brought together. The same problem can occur when a researcher requests a second extraction of their data, . When they receive their second extraction data, they find that the linkage map has changed  making it difficult to reconcile individual patient histories. For on-going linkage systems, a linkage unit must understand how it will accommodate project requests over time.The main goal of adopting on-going linkage is to reduce the amount of time and effort required in conducting a large amount of project linkages, which are routinely re-linking the same data. Taking steps to automate parts of the linkage process fits in naturally with the aim of reducing operator time and effort and increasing scalability.As on-going linkage systems typically contain a central linkage map which is used in every current and future linkage, the cost of an operator mistake can be very high. Systematic automation and reporting can be useful to ensure and control the quality of linkages over time.A SLU may employ one of a number of models to ensure that linkage is carried out efficiently and securely while satisfying the linkage needs of the research. Some approaches to automation, linkage scenarios and the creation, management and use of a linkage map are presented below.Linkage processes are made up of several discrete steps , while using an entirely different set of processes/tools for core, on-going linkage. The processes for project linkage may even include manual components.A more complicated option is to design a single system for all linkage projects but which accommodates differing linkage scenarios for each specific project. Under this option, a linkage project may be configured to be on-going. The associated linkage map would also be \u2018on-going\u2019. A linkage project may also be designated to be a hybrid of projects and on-going linkage, that is, a linkage in which new project datasets are linked to records drawn from an existing, on-going datasets. Linkage results from these project, may, or may not, be added to the on-going linkage map, depending on the requirements of the research project and the likely quality of results.The most appropriate option will depend, in part, on the number of different linkage scenarios facing SLUs. If requests for separate linkages and linkages to researcher datasets are common, then the first (simpler) option will require a large amount of operator time and resources, defeating the purpose of moving to on-going linkage, while the second may require a large amount of computational resources which may not be feasible.There are several possible methods for conducting on-going linkage and the linkage output will be influenced by a number of factors. One factor is the overlap of people between the files being matched i.e. how many new records have true matches in the existing linked file. Another influence is the size of the existing file, the larger the number of records involved in a probabilistic linkage the greater the likelihood that information will agree \u2018by chance\u2019 across records being compared.These factors have an influence on the number of records brought together for linkage, the matching strategy and in the post-linkage processes that convert pairs of matched records into groups of records that are stored in a linkage map.The relationship within and between files and the level of confidence in existing links/relationships are important considerations in the design and optimisation of linkage strategies.all records in the incoming dataset to all other records in the system. This method allows pairs to be created describing the relationships between all records in the system. With this approach, there are no expectations or assumptions made about how records match against each other or how they group together to become \u2018sets\u2019 of records that belong to the same individual. In terms of linkage strategy, this scenario represents a relatively unconstrained many-to-many linkage. If, however, the linkage task involves linking records to an authoritative record type , then a one-to-one or many-to-one linkage may be more appropriate and there is opportunity to adapt matching strategies to leverage this knowledge [For example, one approach is to link nowledge ,30.A related issue is whether or not to allow merging of groups in the linkage map. A linkage method known as \u2018best-link matching\u2019  makes usa.This method uses underlying knowledge of the quality of the population spine to make decisions about future linkage results. Most SLUs accept that a small percentage of matches will be incorrect. In the situation where one of these matches merges two groups, the error is compounded and all records within these two groups are now incorrectly linked togetherAn alternate approach is to allow the merging of groups to occur. This method does not rely on the existence of a high quality reference dataset (spine). For this reason this method may be useful in a much greater range of circumstances.There is an additional advantage to choosing strategies which allow merging of groups and which use all records in linkage. The advantage of this approach (and this approach only), is that the order of the incoming records does not affect system groupings. It is intuitive that this should be the case, as in practice the order of received records is typically highly dependent on contractual arrangements and other arbitrary preparations, which should not have an effect on the groups made by the system.In on-going linkage, the linkage map is constantly changing and there may be requests from researchers to access results from previous linkages. There are several ways in which a SLUs can manage changing linkage maps and accommodate requests for past information. One solution is to take snapshots of the linkage system at the point of extraction for all research projects. This allows researchers access to the data and linkage map at the time of extraction and will solve the majority of the researchers queries, although the system would not be able to determine exactly why things have changed. While multiple snapshots of the system would take up a large amount of space, these do not necessarily need to be stored on on-going infrastructure, and could be moved elsewhere until required.An alternative solution is to have a linkage map which stores the full history of groups, recording details of when additional records entered or left specific groups. This allows full understanding of how groups of records came together, as well as giving the ability to \u2018roll-back\u2019 to a point in time when an extraction for a researcher occurred  project linkage based approaches to complex on-going linkage. On-going linkage requires consideration of a number of additional time-sensitive issues which do not affect project based linkages. Despite the complexity, the advantages of moving to a more automated, efficient and sustainable way of conducting linkage far outweigh the intricacies of doing so. Table\u00a0Several themes run throughout the issues presented in this paper. One is the trade-off between automation and bespoke approaches. Bespoke approaches will always be more flexible, but will always suffer from issues of transparency, maintainability and replicability. A second theme is the focus on issues and processes that complement and support the specialised activities of record linkage units. As presented in this paper, there are a number of key technical issues which must be understood and overcome in order for SLUs to deliver efficient record linkage \u2018services\u2019 for researchers.There are several areas of further research required. To our knowledge, none of the options presented in this paper have been empirically compared against each other. However the employment of one option over another depends  on assumptions about linkage quality, a measurable trait. If empirical research investigated the effect on linkage quality of several of these options over time given different datasets and other parameters, linkage units would be better equipped to decide on the most appropriate option for their systems.A second area of research is related to the benefit of bespoke processes over automated processes. While it is assumed that automatic processes will likely produce lower quality results, the actual degradation in quality is not known. Research which tests and quantifies these effects is warranted. Until we know the true effect that automation has on linkage quality (if any), linkage units cannot make an informed decision about the benefit of this move.The process of conducting numerous linkages on a large scale is both complex and resource intensive. Linkage systems need to be both flexible and scalable to meet the future demands of enterprise-level record linkage. It is hoped the solutions presented here help reduce these difficulties.aIn this method false negatives found in the originating dataset used for the population spine will never be brought together no matter what additional information is found in other datasets. Additional records can provide new information which makes it clear that two records previously existing within the system actually belong to the same person. In these situations, \u2018best-link matching\u2019 will not be able to use this information to improve quality.The authors declare that they have no competing interests.Initial design and conception provided by JHB, AF and JS. Further technical design provided by AB, JKB and SR. First draft of manuscript provided by SR; subsequently edited significantly by JHB, AF and JS. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6947/14/23/prepub"}
+{"text": "Metriaclima zebra and M. mbenjii, that differ in several aspects of their body and fin color. We genotyped 798 SNPs in 160\u00a0F2 male individuals to construct a linkage map that was used to identify quantitative trait loci (QTL) associated with the pigmentation traits of interest. We also used the linkage map to anchor portions of the M. zebra genome assembly.Pigmentation patterns are one of the most recognizable phenotypes across the animal kingdom. They play an important role in camouflage, communication, mate recognition and mate choice. Most progress on understanding the genetics of pigmentation has been achieved via mutational analysis, with relatively little work done to understand variation in natural populations. Pigment patterns vary dramatically among species of cichlid fish from Lake Malawi, and are thought to be important in speciation. In this study, we crossed two species, M. zebra genome assembly. Within each QTL interval we identified several candidate genes that might play a role in pigment cell development.We constructed a linkage map consisting of 834 markers in 22 linkage groups that spanned over 1,933\u00a0cM. QTL analysis detected one QTL each for dorsal fin xanthophores, caudal fin xanthophores, and pelvic fin melanophores. Dorsal fin and caudal fin xanthophores share a QTL on LG12, while pelvic fin melanophores have a QTL on LG11. We used the mapped markers to anchor 66.5% of the This is one of a few studies to identify QTL for natural variation in fish pigmentation. The QTL intervals we identified did not contain any pigmentation genes previously identified by mutagenesis studies in other species. We expect that further work on these intervals will identify new genes involved in pigment cell development in natural populations. Most vertebrate species display a complex and species-specific pigment pattern that enhances organismal fitness by contributing to crypsis, signaling, or mate recognition. Variation in pigmentation arises through differences in development ,2, nutriEarly work to understand the genetic basis of variation in pigmentation focused on the analysis of mutant mice. Recently, fish have become an attractive model system due to their short generation times, large numbers of offspring, and the significant genetic resources available for some species. Zebrafish and medaka have been valuable models for identifying genes integral to pigment pattern formation via mutational analysis -9. AdditFishes display some of the most spectacular pigmentation observed in nature. Not only can a variety of colors be found, but also patterning including bars, stripes, spots, concentric rings, and blotches . These pThe cichlid fishes of East Africa provide an excellent example of fish pigment pattern diversity and evolution. Endemic radiations of cichlids have arisen in each of the three major Rift Valley lakes . Of the three radiations, Lake Malawi is of particular interest because it is thought that most of the 500+ species of cichlids in the lake have arisen over the last two million years . Diversi2 hybrid cross that suggested only a small number of genes underlie pigmentation differences between two Lake Malawi African cichlids, Metriaclima zebra and M. mbenjii[M. zebra genome sequence assembly to the linkage map, in order to identify candidate genes within each QTL interval.Because many of the species in Lake Malawi can be hybridized, it is possible to use a forward genetics approach to map genes underlying phenotypic diversity . We prev. mbenjii. In the 2 hybrid cross was generated by crossing a single male M. mbenjii to a single female M. zebra. This resulted in a single F1 family that had a single male intercrossed to sibling females to produce the F2 offspring. While both male and female F2 offspring were produced, only sexually mature, dominant male F2 were analyzed. The two grandparent species differ in several aspects of male pigmentation. Male M. mbenjii have a light blue body with orange dorsal and caudal fins. Their pelvic fins are clear with an iridophore streak on the leading edge , were sequenced in separate lanes of an Illumina HiSeq 1000. The F0 grandparents were sequenced in an additional lane with 9 other individually barcoded samples. Reads were quality filtered by requiring Sanger quality scores of at least Q20 across 90% of the read. Reads were processed for individual barcodes and then assembled de novo into loci using the software pipeline Stacks v. 0.996 [2 individuals were chosen for creating the linkage map.SNPs were identified and genotyped via restriction site associated DNA sequencing (RAD-seq) . Reducedv. 0.996 . A minimM. zebra genome browser (http://www.bouillabase.org). Information on these markers can be found in Additional file Additional microsatellite markers for the putative sex locus and selected candidate genes were added by selecting previously described microsatellite primer sequences from GenBank or by designing new primers to microsatellites identified in the 2 male progeny derived from a single F1 family. The grouping module of JOINMAP assigned 834 of these markers to 22 linkage groups using a logarithm of odds (LOD) score of 5.0. We built a genetic map with the mapping module of JOINMAP, using the Kosambi mapping function, a recombination threshold of 0.450, and a jump threshold of 5.0. Linkage group numbers were assigned based on homology to the existing linkage groups of tilapia [A linkage map was created using JOINMAP 3.0 . The loc tilapia ,25.M. zebra genome browser (http://www.bouillabase.org). Candidate genes were identified via literature searches using the gene names.QTL were detected using R/qtl ,27. FirsM. zebra genome assembly version 0 were determined via BLAST. Assembly scaffolds were placed into anchored linkage groups if there were at least two markers from the same linkage group that blasted to that scaffold. The order and orientation of these scaffolds within each linkage group was then determined based on the BLAST location of markers relative to one another.The locations of mapped markers on the 2 progeny. 662,570,635 (89.1%) passed our Q20 filter. A total of 624,492,015 reads (94.3% of the filtered reads) were successfully processed for barcodes by Stacks. This corresponds to approximately 7.7 million reads per F2 progeny. After filtering and barcode processing, there were 7,535,127 reads for the F0 female and 18,850,602 reads for the F0 male used for the cross.A total of 743,486,491 reads were produced from the 5 lanes of Illumina HiSeq for the 160\u00a0F2 progeny from the M. zebra x M. mbenjii cross. The average coverage of each genotype SNP was 49.9x (range of 6.9x-201.7x) in the F2 progeny, 254.9x in the male F0, and 105x in the female F0. The average genotype completeness was 77% and the frequencies of each genotype class were 27.7% AA, 45.5% AB, and 26.9% BB . 60 individuals had between 0\u2013100 genotypes missing, 42 individuals had 101\u2013200 genotypes missing, 25 individuals had between 201\u2013300 genotypes missing, 22 individuals had between 301\u2013400 genotypes missing, 7 individuals had between 401\u2013550 genotypes missing, and 4 individuals had greater than 500 genotypes missing. The average number of missing genotypes per individual was 182. High numbers of missing genotypes can be attributed to low coverage in some of the F2 individuals, with coverage ranging from 357,000 reads to 10,500,000 reads. We obtained a linkage map that contained 22 linkage groups and spanned over 1,933\u00a0cM. This agrees with previous work indicating that there are 22 chromosomes in cichlids [We scored 834 genetic markers in 160 male FM. zebra genome assembly. To be included in the anchored map, we required that scaffolds be anchored by at least two markers in the linkage map. We found 114 scaffolds (6.5 per linkage group) that met this criterion. The average size of these scaffolds was 3,918,467\u00a0bp for a total of 564,259,264 anchored bp. This represents 66.5% of the 848,776,495\u00a0bp\u2009M. zebra genome assembly. An additional 110 scaffolds had a single hit to a particular linkage group and were not included in the anchoring. If these single scaffolds were included in the anchoring, 92.3% of the assembly would become anchored. Additional file This linkage map was then used to order scaffolds of the 2 did not have enough power to detect QTL for traits for which the difference in parental means was smaller than 2.8 standard deviations.A genome wide scan resulted in the identification of three QTL. Phenotypes with significant LOD scores included dorsal fin xanthophores, caudal fin xanthophores, and pelvic fin melanophores Figure\u00a0. The faiM. mbenjii grandfather  on that linkage group, the scaffold and scaffold position (basepair) at which the marker is found is given beneath each marker name. The rows following provide the genotype for each F2\u2009individual at that marker is also given.Click here for fileNon-RAD primer sequences.Click here for fileMZebra_anchoredmap.xlsx Anchored M. zebra map.\u2009This file shows the linkage groups and the scaffolds contained within them. For each linkage group, markers that hit to the same scaffold are color-coded the same, with the exception of those with only one hit, which are color-coded light yellow. Linkage groups are separated by a black bar. Markers that were placed in the linkage group by JoinMap but excluded during the anchoring process are noted.Click here for fileCandidate genes for identified QTL regions.Click here for file"}
+{"text": "Rosa hybrida) represent most of the commercial cultivars of cut roses and form the basis for breeding programmes. Due to intensive interspecific hybridizations, modern cut roses are complex tetraploids for which the mode of inheritance is not exactly known. The segregation patterns of molecular markers in a tetraploid mapping population of 184 genotypes, an F1 progeny from a cross of two heterozygous parents, were investigated for disomic and tetrasomic inheritance. The possible occurrence of double reduction was studied as well. We can exclude disomic inheritance, but while our observations are more in line with a tetrasomic inheritance, we cannot exclude that there is a mixture of both inheritance modes. Two novel parental tetraploid linkage maps were constructed using markers known from literature, combined with newly generated markers. Comparison with the integrated consensus diploid map (ICM) of Spiller et al.  contains supplementary material, which is available to authorized users. Rosa L. of the family of the Rosaceae, comprising about 180 species and thousands of cultivars (Debener and Linde 3x & 4x) and Noisette roses (2x), which were then crossed with hybrid perpetual roses (4x) . These modern roses show vigorous growth and their large flowers are borne on stiff pedicels so that they look up (Marriott Roses belong to the genus ) Zlesak , in whicMarriott . Due to Tetraploid hybrid tea roses represent most of the commercial cultivars for cut roses currently available on the market, and they still form the basis of breeding programmes. In fact, the tea roses originate from about ten species, which is only a small part of the gene pool available for genetic improvement. Therefore, numerous other species could be used to exploit more of the genetic resources to introduce new desired traits like disease resistance. The creation of new cultivars is still mainly empirical, and new and interesting genotypes with attractive traits are fixed by vegetative propagation. If breeders want to make use of such traits in their breeding programme or if they want to enlarge the genetic basis of hybrid tea roses, a good understanding of the inheritance mode of tea rose is needed to implement an appropriately designed breeding programme. This will improve the efficiency and facilitate the transfer of novel traits into tetraploid cultivars such as disease resistances or new flower types  to unravel the mode of inheritance by studying the segregation patterns of molecular markers in this tetraploid cross and (2) to construct two parental tetraploid genetic linkage maps by combining AFLP and SSR marker data from Yan , with nePodosphaera pannosa  de Bary (syn. Sphaerotheca pannosa). After inoculation with a spore suspension, development of infection symptoms was scored on a scale from 0 to 6. The scores given were 0: no symptoms; 1: very small necrotic lesions with <1\u00a0% leaf area covered with mycelium; 2: 1\u20135\u00a0% leaf area with mycelium; 3: 6\u201320\u00a0% leaf area with mycelium; 4: 21\u201340\u00a0% leaf area with mycelium; 5: 41\u201360\u00a0% leaf area with mycelium and 6: >61\u00a0% leaf area with mycelium. Disease scores were recorded 11\u00a0days after inoculation.The tetraploid rose population K5 from Yan  investigEcoRI/MseI (E-M) and PstI/MseI (P-M). A prescreening for polymorphisms with different primer combinations, having either two (some PstI primers) or three  selective nucleotides, was done using DNA of the parents and a few individuals of the progeny. Amplified fragments of each primer-restriction enzyme combination were radioactively labelled ([\u03b3-33P]-ATP), separated on 6\u00a0% denaturing polyacrylamide gels and visualized by autoradiography. Polymorphic markers were coded and dominantly scored as described in Yan et al. , NBS3 (5\u2032-GTWGTYTTICCYRAICCISSCATICC-3\u2032), and NBS5a6 which is a 1:1 mixture of NBS5a (5\u2032-YYTKRTHGTMITKGATGAYGTITGG-3\u2032) and NBS 6 (5\u2032-YYTKRTHGTMITKGATGATATITGG-3\u2032). Amplified fragments of each primer-restriction enzyme combination were radioactively labelled ([\u03b3-33P]-ATP), separated on 6\u00a0% denaturing polyacrylamide gels, and visualized by autoradiography.NBS profiling is a multiplex screening technique, producing amplified resistance gene analogue fragments by using degenerate primers based on conserved motifs present in the NBS domain of resistance genes. NBS profiling was performed on 200\u00a0ng DNA as described in Van der Linden et al. . Twelve AluI in combination with NBS5a6 scored at position 12: A5a6-12).Polymorphic bands were manually scored as dominant markers. Marker codes correspond to the first letter of the restriction enzyme followed by the number of the NBS primer and finally followed by the position of the marker on the film  can be inferred, again assuming a single-locus situation. Hence, all six possible classes can be scored. Such markers are rarely found. Therefore, also the segregation of SSR markers with two unique single-dose alleles in one of the parents of the mapping population was studied. In this situation, not all six possible phenotypic classes in case of autotetraploidy can be distinguished. Instead, four phenotypic classes are expected with frequencies 1/6, 2/6, 2/6, 1/6 for tetrasomic inheritance, and, alternatively 1/4 each for disomic inheritance. Cases of disomic inheritance with only two phenotypic classes at equal frequencies did not occur.Double reduction is a phenomenon associated with multivalent formation in meiosis  and refers to the fact that parts of sister chromatids come together in the same gamete during the second meiotic division. The segregation data of SSR markers with three unique single-dose alleles in one parent were tested for the occurrence of double reduction. Assuming that the alleles correspond to a single locus, individuals of the progeny that displayed none of the unique alleles were assumed to have a double dose of the fourth allele (OO). Detection of a double dose of any of the three unique alleles was not possible since the marker phenotype is not different from the single-dose phenotype.2 [1] with a, b, c, d being the observed numbers of plants in the four marker genotype classes of the two loci  in the progeny. \u03c72 [1] was defined as (a\u2212b\u2212c\u00a0+\u00a0d)2/(a\u00a0+\u00a0b+c\u00a0+\u00a0d) (Mather r) under the assumption of coupling phase and under the assumption of repulsion phase for disomic inheritance: n\u00a0=\u00a0a\u00a0+\u00a0b\u00a0+\u00a0c\u00a0+\u00a0d.The inheritance mode was also investigated according to the procedure outlined by Wu et al. . Linkage) Mather  which war1\u00a0<\u00a00.5 and in repulsion phase if r1\u00a0\u2265\u00a00.5 . Under complete disomic inheritance, the expected numbers of detected coupling phase and repulsion phase linked marker pairs are equal. For the Chi-square test, the significance does not depend on which estimate of the recombination frequency is used, but just on the observed numbers of individuals in the marker classes.Marker pairs were considered to be in coupling phase if For each linkage group of each parental map, the ratio between the number of coupling phase pairs and repulsion phase pairs was calculated and tested against the expected ratio 1:1 under a disomic model with a Chi-square goodness-of-fit test at \u03b1\u00a0=\u00a00.05.All polymorphic bands from NBS profiling and SSR primers were scored as presence/absence. Chi-square goodness-of-fit tests were performed on the segregation data of all markers assuming simplex segregation ratios 1:1 and 3:1). Markers deviating significantly at \u03b1\u00a0=\u00a00.05\u20130.01 from the ratio expected for that marker (deduced from the parent genotypes and the segregation ratio in the progeny) were included on the genetic linkage maps and marked with a single asterisk Figs.\u00a0, 3, wher:1. MarkeThe newly generated uni- and bi-parental simplex markers were added to the tetraploid parental linkage maps of Yan  using Jo10log (p) value of 3.127 (p\u00a0=\u00a00.00075). Six additional uni-parental duplex markers were tested separately by single-marker ANOVA in Genstat, using the same threshold.Phenotypic data on prickle number on stem and petal number per flower were used for marker-trait studies. This study further includes powdery mildew resistance observed by Yan  and Yan Both parents and 184 offspring were genotyped for 619 markers, including those of Yan . Table\u00a02Twenty-six AFLP primer pairs Yan  generateRsa1). Table\u00a0From the NBS gels, 168 polymorphic markers were dominantly scored with a maximum of 24 polymorphic markers per combination  amplified well and showed clear bands on agarose gel Table\u00a0.Five markers out of the set of uni-parental markers were considered as duplex segregating in agreement with a 3:1 type of segregation. Three of them could be mapped on different linkage groups. Moreover, six markers were considered as simplex\u2013duplex with a 7:1 segregation ratio Table\u00a0. Such seEight markers not significantly deviating from duplex segregation ratios as expected in tetrasomic inheritance were also not significantly different from a 7:1 segregation ratio Table\u00a0, which idf\u00a0=\u00a05) for the uni-parental three-allelic marker RMS033 from parent P867. Offspring plants for this marker exhibited patterns in the six predicted phenotypic classes in a ratio not significantly different from expectations for tetrasomic inheritance  was tested with a Chi-square goodness-of-fit test  and 1/4 each in case of disomic inheritance. Here also, a Chi-square goodness-of-fit test was used  showed allelic combinations and a segregation pattern in agreement with tetrasomic inheritance . For all SSRs, all phenotypic classes expected in case of tetrasomic inheritance were present in the progeny but their frequencies were not always as expected. Besides, for RhCP521, the phenotypic classes OOOO and DOOO were observed for 39 individuals . Those classes are only possible in case of double reduction. The phenotypic class (OOOO) was also observed for seven individuals  of a nearby marker Rh98 (6.2\u00a0cM). Linkage groups B4 contain five markers  that show phenotypic classes that can be explained by occurrence of double reduction.For RMS082, where two models are possible , gametes BO and DO are not found and the phenotypic class ABCD is three times larger than expected, which suggest preferential pairing for this SSR. The phenotypic class ABCDE for RMS094 (ABCO\u00a0\u00d7\u00a0CDEO) was observed for eight (5.2\u00a0%) individuals of the progeny . Such a phenotype can only be explained if three alleles of one parent are transmitted to the progeny, which theoretically is not possible. Possibly the primers amplify a fragment at another locus in the genome, or two SSR loci on different locations. In the case of RMS094, five fragments were amplified from the two parents but only three fragments were mapped, one for each parent in linkage group 7 (A7-4 and B7-4) and one in linkage group A2-1. This indicates that here a second locus is amplified as well.Two ways of calculating recombination frequencies between pairs of markers were used. One set of estimates for coupling phase simplex\u00a0\u00d7\u00a0nulliplex and simplex\u00a0\u00d7\u00a0simplex marker pairs was calculated with JoinMap in order to generate linkage maps. JoinMap does not take into account tetrasomic inheritance, but estimates for simplex\u00a0\u00d7\u00a0nulliplex markers in tetraploids are identical to those of coupling phase markers in a diploid.2[1] test to determine significance of linkage in polyploids. The pairwise recombination fractions obtained with this method under the assumption of coupling phase (r1) and under the assumption of repulsion phase  allowed validation of the assignment of the linkage phases in the genetic linkage maps constructed with JoinMap since the method of Wu et al. , and a Chi-square goodness-of-fit test was performed \u03b1\u00a0=\u00a00.05) to test the hypothesis of a 1:1 ratio coupling: repulsion for the numbers of pairs per linkage group, which would correspond to disomic inheritance  and Rh76 for P867 which are also mapped to LG3 in the diploid ICM.The multi-allelic markers RhP507, RhCP521 and Rw5D11 enabled us to identify LG4 groups for both parents. This assignment was confirmed by markers Rh59, Rh65, Rh98, Rw55C6 and Rw55E12 because these were mapped to LG4 in the ICM as well. Two alleles of marker Rw22A3 were mapped to two subgroups of LG4, whereas they were mapped to LG6 on the diploid ICM. One of the allele was assigned to A4-2 on our maps because it shows a high LOD score (7.65) with Rw5D11, which is 0.3\u00a0cM from Rh98 that mapped on LG4 on the diploid ICM. Marker RhAB38 was assigned to B4-3 whereas in the diploid ICM, it was mapped on LG5.The multi-allelic marker RA023b enabled us to identify linkage groups of LG5 for both parents. Other groups could be assigned to this LG set from the markers Rh77, Rh99, Rw10J19 and Rw52D4 as in the diploid ICM.Linkage group 6 contains two marker groups for P540  and 1,225.4\u00a0cM (209 loci) for P867. Yan et al.  estimateYan\u2019s tetraploid parental maps (2005) covered 695\u00a0cM for P540 (102 markers) and 697\u00a0cM for P867 (110 markers), which correspond to about 35\u00a0% coverage. The number of groups per chromosome has been improved considerably since Yan\u2019s maps did not contain a complete set of four groups for any of the chromosomes.Alignment of individual diploid genetic linkage maps has been attempted within several mapping projects Supplementary material 2 (XLS 76 kb)Below is the link to the electronic supplementary material."}
+{"text": "To investigate quantitative trait loci linked to refractive error, we performed a genome-wide quantitative trait linkage analysis using single nucleotide polymorphism markers and family data from five international sites.Genomic DNA samples from 254 families were genotyped by the Center for Inherited Disease Research using the Illumina Linkage Panel IVb. Quantitative trait linkage analysis was performed on 225 Caucasian families and 4,656 markers after accounting for linkage disequilibrium and quality control exclusions. Two refractive quantitative phenotypes, sphere (SPH) and spherical equivalent (SE), were analyzed. The SOLAR program was used to estimate identity by descent probabilities and to conduct two-point and multipoint quantitative trait linkage analyses.We found 29 markers and 11 linkage regions reaching peak two-point and multipoint logarithms of the odds (LODs)>1.5. Four linkage regions revealed at least one LOD score greater than 2: chromosome 6q13\u20136q16.1 , chromosome 5q35.1\u201335.2 , chromosome 7q11.23\u20137q21.2 , and chromosome 3q29 . Among these, the chromosome 6 and chromosome 5 regions showed the most consistent results between SPH and SEM. Four linkage regions with multipoint scores above 1.5 are near or within the known myopia (MYP) loci of MYP3, MYP12, MYP14, and MYP16. Overall, we observed consistent linkage signals across the SPH and SEM phenotypes, although scores were generally higher for the SEM phenotype.Our quantitative trait linkage analyses of a large myopia family cohort provided additional evidence for several known MYP loci, and identified two additional potential loci at chromosome 6q13\u201316.1 and chromosome 5q35.1\u201335.2 for myopia. These results will benefit the efforts toward determining genes for myopic refractive error. Worldwide, individuals do not share the same myopic development risk, as the prevalence of myopia varies in different countries. Studies, primarily in adults but in some schoolchildren cohorts, have reported approximate prevalence rates of 17% in Australia, 26%\u201335% in the United States, and 27% in Western Europe  and SEnew=4.4xlog10[-(SE-0.25)].Before calculating the likelihood, locus-specific IBD information was computed for all pairs of relatives . Followim deCode . The facOf 254 families typed by the CIDR, the largest subset comprised 225 Caucasian families. This subset included 1,168 subjects. SPH data for only one eye were available for 164 subjects, and 256 subjects had SEM data for only one eye. These individuals were therefore not included in quantitative phenotype SPH or SEM analytical assessments. Summary descriptive statistics information for the samples included in the QTL analysis is provided in The heritability of SPH or SEM was highly significant , with heritabilities of 23.2% (SPH) and 33.9% (SE). We used a threshold level of LOD\u22651.50 to highlight promising initial linkage regions of interest for two-point and multipoint linkage analyses  40]..40].rs2000203 at 87.57 centiMorgans  and rs1457947 at 89.14 cM (two-point LOD=1.52) on chromosome 6 are in close proximity to each other. These markers also fall within a linkage region identified by the Caucasian subset in our data when treating SPH as a binary trait [Four markers across two chromosomes resulted in two-point LOD scores \u2265 1.5 . Graphicry trait . Howeverrs4868073 (183.33 cM) with a two-point LOD score of 1.27. The second highest multipoint peak is on chromosome 6q13\u20136q16.1 , which showed a LOD score over 2 when the SEM was analyzed (see next section). Other regions of interest include chromosome 1p31.1 (peak multipoint LOD=1.69 at 100 cM), chromosome 10p11.21\u201310q11.22 (peak multipoint LOD=1.68 at 64 cM), and chromosome 5p13.3  and chromosome 5q35.1\u20135q35.2 (peak two-point LOD=1.62 at 187.27 cM and peak multipoint LOD=1.80 at 188 cM). In particular, the chromosome 6q13\u201316.1 region is strongly supported by a set of seven markers with two-point LOD scores ranging from 1.19 to 2.13  and chromosome 7q11.23\u20137q21.2  chromosome 6q13\u20136q16.1 with multipoint peak LOD scores of 1.96 for SPH and 2.18 for SEM and (2) chromosome 5q35.1\u20135q35.2 with multipoint peak LOD scores of 2.05 for SPH and 1.80 for SEM. The consistency of the LOD score significance derived from two different but similar measures of refractive error, SPH and SE, for these two loci. The chromosome 5q35.1\u201335.2 locus overlaps with a region that we reported previously using high myopia as a qualitative disease phenotype . The chrOverall, our LOD scores are low (not achieving the standard significant threshold LOD \u22653) . This isIn conducting our analysis, we recognized that an investigation of axial length, a major determinant of axial myopia, in our data might be insightful. Unfortunately, axial length data were collected for fewer than a third of our study participants . Quantitative trait linkage analysis was conducted for the axial length data set, but no two-point or multipoint results exceeded a LOD threshold of 1.5. The power to detect a linkage signal for axial length was severely hampered by the low sample size.In our previous report of linkage regions for high-grade myopia, the linkage regions were largely inconsistent between the analyses of the disease states defined by the SPH and the SEM . When SPThe present study provides important information. First, two new loci on chromosomes 5 and 6 are likely to be new chromosomal regions that link to myopic refractive errors. Second, our linkage analysis again replicated suggestive significance of the MYP3 locus on chromosome 12q. Clearly, this locus should undergo additional scrutiny in other data sets. Third, our analysis underscores the benefits of using refractive error as a quantitative trait for linkage, and demonstrates the advantage of using an SNP-based linkage screening protocol with higher marker density than conventional microsatellite markers to map new loci. The results of the present study of a large family data set using high-density SNPs for linkage scanning should aid in triaging candidate genes and loci for future genome-wide association studies and deep sequencing efforts."}
+{"text": "Macular Telangiectasia type 2 (MacTel) is a relatively rare macular disease of adult onset presenting with distortions in the visual field and leading to progressive loss of visual acuity. For the purpose of a gene mapping study, several pedigrees were ascertained with multiple affected family members. Seventeen families with a total of 71 individuals (including 45 affected or possibly affected) were recruited at clinical centers in 7 countries under the auspices of the MacTel Project. The disease inheritance was consistent with autosomal dominant segregation with reduced penetrance. Genome-wide linkage analysis was performed, followed by analysis of recombination breakpoints. Linkage analysis identified a single peak with multi-point LOD score of 3.45 on chromosome 1 at 1q41-42 under a dominant model. Recombination mapping defined a minimal candidate region of 15.6 Mb, from 214.32  to 229.92 Mb , encompassing the 1q41-42 linkage peak. Sanger sequencing of the top 14 positional candidates genes under the linkage peak revealed no causal variants in these pedigrees. The cause of the disease is unknown and there is no treatment.Macular telangiectasia is a group of diseases characterized by Gass and Blodi in 1993 Clinical characteristics of MacTel include loss of retinal transparency, autofluorescence changes in the macula, macular edema, presence of intraretinal crystals, and disruption of macular pigment transport. Symptoms of advanced disease include the presence of a macular hole, dilated and tortuous vessels in the perifoveal region, leakage from retinal vessels and neovascularization arising from the intraretinal vessels While MacTel had been presumed to be a very rare disease, recent epidemiological studies suggest that it is under-diagnosed and, therefore, more common than previously thought. The Beaver Dam Eye Study recently reported a prevalence of 0.1% in a retrospective study of 4,790 individuals, aged 43\u201386 years of age MacTel was proposed to have a genetic component based on case reports of affected sibling pairs and concordant monozygotic twins The MacTel Project was established as a consortium of basic science researchers and clinicians in order to study the natural history, identify the cause(s) of the disease, and propose targets for treatment. Patients were screened and enrolled at 23 clinical centers in seven countries . Family members were actively recruited and given complete ophthalmic examinations. Seventeen multiplex families were identified that were informative for linkage analysis, together with additional parent-child duos Altogether, these data provided a basis for genome-wide linkage mapping that identified a significant linkage peak for this disease.Seventeen families with a total of 71 individuals (45 affected or possibly affected) were analyzed for linkage. The inheritance pattern in families with more than one affected individual was consistent with autosomal dominant transmission. MacTel exhibits reduced penetrance based on the observation that in some multiplex families neither parent is clearly affected with the disease. Variable disease expressivity is evident in many pedigrees in this cohort; while probands presented to the clinic experiencing vision loss, some relatives were given a diagnosis of MacTel only after a complete ophthalmic exam as a part of this study. Based on a masked analysis of images by a central reading center, not influenced by the initial diagnosis from a recruiting center, all subjects were categorized as definitely affected, possibly affected, probably not affected, or definitely not affected. This clearly illustrates the variable expressivity of MacTel, complicating genetic analysis. No gender bias was observed in patients, and male to female and female to male transmissions were both observed in the pedigrees. Most families are too small to make a reliable estimation of the ratio of affected offspring, however, four large families had ratios of affected offspring consistent with autosomal dominant inheritance.A total of 112 individuals in 33 MacTel families were screened on Illumina 1 M Duo arrays. Seventeen informative families were analyzed by multi-point, affected-only parametric linkage analysis under an autosomal dominant model using a subset of independent SNPs from the Illumina 1 M chip. Family members diagnosed as possibly and definitely affected were coded as affected. Relatives diagnosed as unaffected or probably not affected were coded as unknown. There was a single significant peak observed on chromosome 1, with a LOD score of 3.45 and HLOD of 3.54  over an interval of approximately 15 Mb  and 3. OIBD status was inferred along each chromosome with MERLIN Comparison of regions that were not excluded by either linkage analysis or by recombination breakpoint analysis revealed four regions that were not excluded by either analysis, including the significant linkage peak on chromosome 1. Three additional regions with LOD scores between 0 and \u22122 under autosomal dominant linkage analysis were not excluded by breakpoint analysis in strictly affected siblings: chromosome 7, 125.92\u2013145.41 Mb; chromosome 12, 5.38\u20137.03 Mb; and chromosome 14, 102.52\u2013106.38 Mb . Reviewing regions of exclusion in these two different ways serves to clarify whether a chromosomal segment is excluded based on information from definitely affected individuals or possibly affected family members where the phenotype is less strictly defined. A total of 32.378 Mb were not excluded by parametric linkage analysis, including the two regions of positive linkage. A total of 153.81 Mb were not excluded by the more stringent recombination breakpoint analysis based only on definitely affected siblings.DISP1, TLR5, SUSD4, BEND5, CAPN8, CAPN2, TP53BP2, FBXO28, DEGS1, NVL, CNIH4, WDR26, and CNIH3 genes and the micro-RNA, MIR320B2 were screened by Sanger sequencing in two affected family members, one from family 8 , the other from family 156 . Each of these families consists of an affected sib pair, unaffected siblings, and two parents, one of which is affected in each family. Altogether, sixteen variants  were detected in coding regions of these genes . Informed written consent was obtained at each participating clinical center in accordance with ethics protocols for human subjects approved by the appropriate governing body at each site in accordance with the Declaration of Helsinki. All protocols and records of consent were centrally managed by the EMMES Corporation . The following ethics boards granted approval for human subjects enrollment at each participating center. Quinze-Vingts, Paris, France: Comite De Protection Des Personnes Hopital Saint-Antonie; Centre for Eye Research, Victoria, Australia: The Royal Victorian Eye & Ear Hospital; Clinique Ophtalmolgie de Creteil, Paris, France: Comite De Protection Des Personnes Hopital Saint-Antonie; Hospital Lariboisiere, Paris, France: Comite De Protection Des Personnes Hopital Saint-Antonie ; Jules Stein Eye Institute, UCLA, Los Angeles, United States: The UCLA Institutional Review Board; Lions Eye Institute, Nedlands, Australia: Sire Charles Gairdner Group Human Research Ethics Committee; Manhattan Eye, Ear & Throat Hospital, New York, United States: Lenox Hill Hospital Institutional Review Board; Moorfields Eye Hospital, London, U.K.: National Research Ethics Service; Retina Associates of Cleveland, Inc., Cleveland, United States: Sterling Institutional Review Board; Save Sight Institute, Sydney, Australia: South Eastern Sydney Illawarra Area Health Service Human Research Ethics Committee \u2013 Northern Hospital Network; Scripps Research Institute, La Jolla, United States: Scripps Institutional Review Board; St. Franziskus Hospital, Munster, Germany: Ethik-Kommission Der Arztekammer Westfalen-Lippe Und der Medizinishchen Fakultat der Westfallschen Wilhelms-Universitat; The Goldschleger Eye Institute, Tel Hashomer, Israel: Ethics Committee The Chaim Sheba Medical Center; The New York Eye and Ear Infirmary, New York, United States: The Institutional Review Board of the New York Eye and Ear Infirmary; The Retina Group of Washington, Olympia, United States: Western Institutional Review Board; University of Bonn, Bonn, Germany: Rheinische Friedrich-Wilhelms-Universitat Ethik-Kommission; University of Chicago, Chicago, United States: The University of Chicago Division of Biological Sciences \u2013 The Pritzker School Institutional Review Board; University of Michigan, Ann Arbor, United States: Medical School Institutional Review Board (IRBMED); University of Wisconsin, Madison, United States: Office of Clinical Trials University of Wisconsin School of Medicine and Public Health; The Wilmer Eye Institute of Johns Hopkins University, Baltimore, Maryland: Johns Hopkins School of Medicine Office of Human Subjects Research; Scheie Eye Institute University of Pennsylvania, Philadelphia, United States: University of Pennsylvania Office of Regulatory Affairs; University of Bern, Bern, Switzerland: Kantonale Ethikkommission Bern; John Moran Eye University of Utah, Salt Lake City, United States: The University of Utah Institutional Review Board; Bascom Palmer Eye Institute University of Miami, Miami, United States: The University of Miami Human Subjects Research Office; Columbia University, New York, United States: Columbia University Medical Center Institutional Review Board Category 4 waiver for research involving specimens obtained from de-identified subjects.Participants were given a standardized ophthalmic examination, including best corrected visual acuity, fundus photography, fluorescein angiography, optical coherence tomography, and blue light reflectance. Images were adjudicated at the Reading Center at Moorfields Eye Hospital, London. Diagnoses were made in accordance with the criteria described by Clemons et al. Peripheral venous blood was drawn from each participant and used to isolate DNA . DNA concentration was determined using the NanoDrop spectrophotometer . DNA samples of low purity were subjected to column purification (Qiagen blood and tissue kit 69504).2 greater than 0.17. The final marker set consisted of 11,676 independent markers. An identity by state-based relatedness analysis was performed in PLINK using genome-wide marker sets to confirm family structures.Samples were genotyped on the Illumina 1 M chip. The linkage marker set was selected by pruning stringently for genotype quality in GenomeStudio using the following parameters: GenTrain threshold \u22650.50, cluster separation \u22650.16, number of no calls\u200a=\u200a0. The remaining markers were pruned based on LD in PLINK Parametric multipoint linkage analysis was carried out using MERLIN Recombinations were mapped by determining IBD allele sharing using the NPL algorithm in MERLIN. Only definitively affected sib pairs and trios were included in the initial analysis to mitigate the possibility of excluding chromosomal regions based on incorrect diagnoses. Allele sharing was determined by comparing NPL scores from siblings to scores from a simulated dataset, to determine the values associated with each allele sharing state. Where parental genotypes were missing, segments with NPL scores of \u22120.3  were excluded. Where parents were genotyped and one parent was affected, segments with NPL scores of \u22120.3  and \u22120.12  were excluded. For sib trios, segments with NPL scores of \u22120.12 were excluded . Chromosomal segments with intermediate values were classified as ambiguous. The results of this analysis were aggregated to compile a genome-wide map of chromosomal segments where at least one allele was shared IBD in siblings in all families. This result was compared to parametric linkage results from the entire cohort to examine allele sharing in the linkage interval on chromosome 1, to validate exclusion by negative parametric LOD score, and to search for regions that could be prioritized for gene screening by Sanger sequencing.Genewiz . Primers were selected using Primer3 (http://frodo.wi.mit.edu/primer3/). Sequences were compared to the hg19 reference sequence.Genomic DNA from two unrelated affected individuals was amplified using primers specific for exons in the genes of interest. PCR amplification and sequencing was performed as previously described"}
+{"text": "Salmonids are regarded as 4R derivative species, having experienced 4 whole genome duplication events in their ancestry. Many duplicated chromosome regions still share extensive homology with one another which is maintained primarily through male-based homeologous chromosome pairings during meiosis. The formation of quadrivalents during meiosis leads to pseudolinkage. This phenomenon is more prevalent within 5 of the 12 ancestral teleost linkage groups in salmonids.We constructed a genetic linkage map for brook charr and used this in combination with the genetic map from Arctic charr, to make comparisons with the genetic map of rainbow trout. Although not all chromosome arms are currently mapped, some homologous chromosome rearrangements were evident between Arctic charr and brook charr. Notably, 10 chromosome arms in brook charr representing 5 metacentric chromosomes in Arctic charr have undergone rearrangements. Three metacentrics have one arm translocated and fused with another chromosome arm in brook charr to a make a new metacentrics while two metacentrics are represented by 4 acrocentric pairs in brook charr. In two cases , an apparent polymorphism was observed with the identification of both a putative metacentric structure (similar to metacentric AC-4 = BC-4 and a joining of acrocentric AC-16 + one arm of AC-28 = BC-16), as well as two separate acrocentric linkage groups evident in the mapping parents. Forty-six of the expected 50 karyotypic arms could be inter-generically assigned. SEX in brook charr (BC-4) was localized to the same homologous linkage group region as in Arctic charr (AC-4). The homeologous affinities detected in the two charr species facilitated the identification of 20 (expected number = 25) shared syntenic regions with rainbow trout, although it is likely that some of these regions were partial or overlapping arm regions.Salvelinus) and a trout (genus Oncorhynchus) have identified that linkage group arm arrangements are largely retained among these species. Previous studies have revealed that up to 7 regions of high duplicate marker retention occur between Salmo species  and rainbow trout, with 5 of these regions exhibiting higher levels of pseudolinkage. Pseudolinkage was detected in the charr species  consistent with three of the five 'salmonid-specific' pseudolinkage regions. Chromosome arms with the highest number of duplicated markers in rainbow trout are the linkage group arms with the highest retention of duplicated markers in both charr species.Inter-generic comparisons among 2 species of charr (genus  Understanding the evolution of vertebrate genomes requires knowledge of the consequences of the whole genome duplications that have characterized their history . ComparaOncorhynchus mykiss) [Tetrasomic segregation is expected to prevail as a result of quadrivalent formations during meiosis following a whole genome duplication event. The gradual decay towards modes of disomic segregation from the increasing formations of paired sets of bivalents during meiosis is expected through time . Both mo mykiss) , and com mykiss) ,8.Salvelinus alpinus) and brook charr  have more acrocentric than metacentric chromosomes while Group B species  such as rainbow trout have more derived karyotypes with greater numbers of metacentric chromosomes [Structural divergence of homeologous chromosomes into homologous chromosomes during the diploidization process is thought to occur through centric fusions between non-homeologous chromosomes . The degomosomes . The Atlomosomes .Genetic linkage maps have been used to more fully investigate the patterns of chromosomal rearrangements that have taken place after the 3R and 4R WGD events. The ancestral linkage groups of ray-finned fishes share whole arm affinities with the homeologous chromosomal segments in Atlantic salmon and rainbow trout . CompariA more complete picture of genome evolution in salmonids requires a more detailed reference framework in which to evaluate chromosomal rearrangements. Initial studies have been based on comparisons between rainbow trout and Atlantic salmon to Arctic charr with its more basal karyotype. Unfortunately, the Arctic charr genetic linkage map is relatively incomplete relative to those of the more derived species resulting in a limited degree of comparison. The objective of the current research is to use an updated genetic linkage map for Arctic charr as a template to create a linkage map for brook charr, a second species of salmonid with a relatively basal karyotype. We predicted that individual brook charr linkage groups would share a high degree of homology to single Arctic charr linkage groups due to the apparent lack of major chromosomal rearrangements that have been observed in other salmonid species when comparative data has been utilized . FurtherOf the 103 primer sets utilized, 26 amplified two polymorphic loci and five amplified three polymorphic loci. A total of 35 linkage groups were identified  representative of their configuration in Arctic charr. Conversely, these markers were joined into a single LOD = 3.0 cluster in the HL7 female parent suggestive of a metacentric chromosome. The two clusters detected within the HL3 female may simply represent unjoined BC-16 groupings given the low numbers of markers genotyped, While these two arms appear to be separate linkage groups in Arctic charr, they also form a metacentric linkage group in rainbow trout . If these two regions do in fact represent separate linkage groups in some brook charr females, then this could represent a case of female-specific pseudolinkage. Such a configuration would, however, be extremely unlikely given that it would be female based, and most importantly, involve linkage group arms that are unrelated ancestrally. An interpretation that this represents a polymorphism involving either a metacentric or two separate acrocentrics is more likely. We have tentatively identified both of these linkage group arms as BC-16 but denote the second HL3 cluster as BC-16b in Additional File Linkage group BC-4 was made up of two clusters of markers in the HL7 male parent. Markers homologous to AC-4 were joined in the female mapping parents, while the linkage group region marked by SSOSL32 was unlinked in the HL7 male. This same marker was linked to other markers on BC-4 in the female mapping parents. Although SSOSL32 is duplicated in Arctic charr  this primer set amplifies only a single locus in both parents from the HL7 family.Significant differences in recombination rate were detected both within and between sexes , while average recombination rate did not differ between the HL3 and HL7 males    , and BC-4 (homologous the Arctic charr sex linkage group AC-4), as being the most likely genomic locations to possess the sex determining region in brook charr. The sex-linked marker Yp136 was reported ,17 to shThe sex determining region in brook charr is localized to the BC-4 linkage group , suggesting affinities to 38 possible linkage groups.Salvelinus linkage groups suggests that an interpretation of whole-arm rearrangements is more likely.The karyotype of brook charr purportedly has 34 acrocentric and 8 metacentric chromosomes . These lIt is likely that the recombination rate differences identified in this study are not representative of genome-wide differences in recombination rate given the small number of comparisons used to produce these estimates. Within brook charr, comparisons were limited to 9-17 marker intervals (14-27 pairwise comparisons) and thus much of the genome was not represented in the various estimates. When multiple intervals within a single linkage group were present for comparison, recombination ratios between the parents being compared were often variable , indicating the importance of complete genome coverage for accurate average recombination rate estimates. However, BC-16 is homologous to RT-8 in rainbow trout, and RT-8 has been reported to have extremely unusual recombination rate dynamics, in that both female and male recombination rates were observed to be greatly suppressed throughout most of the length of the linkage group . Thus, iIn the one case where recombination rate was significantly higher in the male mapping parent relative to the female, the loci (OMM5102/ii and BHMS465/i on BC-24) appear to be located near the telomere. Comparative mapping places OMM5102 distally  on the homologous Arctic charr (AC-3) and rainbow trout (RT-7q) linkage groups. BHMS465 has not been mapped in rainbow trout and the one copy mapped in Arctic charr maps distally on AC-24. Assuming that this pair of loci is located telomerically on BC-24, these results are not surprising in light of the work of Sakamoto et al. , who fouThe pairwise female: male recombination rates observed in this study among all four possible pairwise combinations of the mapping parents in brook charr  is somewhat higher than the levels observed in Arctic charr . This level of recombination is more similar to what has been observed in the rainbow trout mapping panels  , and mucEvidence exists that segregation distortion can influence marker order, estimates of map distances, and linkage relationships . While tAll mapping parents except the LN4F contained markers on BC-16 which exhibited significant segregation distortion prior to Bonferroni correction. This might have partially accounted for the variability in map distances observed among mapping parents. Interestingly, the homologous linkage group to BC-16 in rainbow trout  has a large degree of recombination suppression in females ,12. ThisLastly, it should be noted that segregation distortion rates might be elevated in the HL brook charr due to their hybrid history. In Arctic charr, segregation distortion is elevated in hybrid relative to pure strain families . Given tSalvelinus. Clearly, the examination of this sex linkage association in additional families of brook charr is needed. Ideally, this should be conducted across multiple strains of the species. In addition, a more complete genotyping survey of the markers located on this linkage group is needed in order to assess more precisely the 'break-points' in the SEX: marker associations, and define more accurately the recombination distances between SSOSL32 and Ots500NWFSC in brook charr.The observation that SEX is unlinked to the SSOSL32 marker in brook charr but appears tightly coupled to the Ots500NWFSC marker on BC-4 is intriguing given the polymorphisms with SEX linkage reported in Arctic charr . In ArctSalvelinus linkage maps. Markers from AC/BC-31 are only syntenic with those on RT-16q  identified in brook charr, have not been detected to date in Arctic charr. While most of the identified homeologies in brook charr appear to represent conserved, known homeologous affinities in Arctic charr, rainbow trout, and Atlantic salmon, two of these  appear to represent relationships which are not currently identified in other salmonine species. BC-14 shows homology to RT-19p (single marker) and RT-24q (multiple markers), while BC-30 shares homology to RT-3p and RT-6p (each with single marker affinities). Regions on RT-6p, and 19p are derived from the M ancestral karyotypic lineage in teleost fishes , suggestInterestingly, the majority of duplicated markers identified in brook charr correspond to linkage groups in rainbow trout and Atlantic salmon where the highest number of duplicated markers have been detected. Eight of the 12 conserved homologies between the duplicated homeologs in the two charr species and rainbow trout  are supported by a high number of duplicated markers in rainbow trout [The regions possessing the highest retention of duplicated markers are also those regions most likely to exhibit pseudolinkage in these species. These regions were homologous to RT-2p/29q; RT-2q/9q; RT-7q/15p; RT-12p/16p; RT-27p/31p in rainbow trout . Here weThe suppression of diploidization in these linkage groups due to the continued exchange of chromosomal segments  would ensure the continued retention of duplicated marker expression in populations exhibiting such phenomena. These meiotic processes would also shelter genetic markers towards the central parts of male metacentric linkage groups from recombination during meiosis . Thus, iExamination of genetic markers in brook charr, Arctic charr, and rainbow trout linkage group arms suggests that a high degree of retention in marker affinities exist among salmonid chromosome arms. Evidence was obtained for potential polymorhisms in chromosome structure suggesting that two brook charr metacentric chromsosomes  may also exist as two separate acrocentrics within individuals. BC-4 is the sex linkage group in brook charr and is homologous to the Arctic charr sex linkage group AC-4. Possible sex chromosome polymorphisms  have also been detected with the Arctic charr AC-4 linkage group. Brook charr linkage groups possessing more duplicated markers appear more likely to exhibit pseudolinkage, and in general, pseudolinkage regions appear to be conserved among salmonid species.Salvelinus-based map to make comparative synteny assignments to the more complete linkage map available for rainbow trout [Microsatellite markers were named as outlined in Sakamoto et al. . Microsaow trout . The refThe mapping panels consisted of two families (HL3 and HL7) from the Hill's Lake (HL) strain and one from the Lake Nipigon (LN) strain (LN4). The HL strain has been maintained for 20 generations at the Hill's Lake Fish Culture Station near Englehart, Ontario, and for an unknown number of generations in Pennsylvania prior to transfer to the Hill's Lake Fish Culture Station . This stth, 2007. The LN family was made on December 13, 2007 at the Codrington Fisheries Research Facility from gametes collected at this facility. Adipose fin tissue was taken from all parents and stored at -80\u00b0C for later DNA extraction. The families were raised in 16 compartment FAL (Heath-style) vertical flow incubating racks fed by water chilled to a constant 4\u00b0C. When the embryos had visible eye pigment, the families were reduced to 120 individuals by randomly selecting embryos from each family. Fish used for the genetic map construction, were killed at 1-3 months post hatching and stored at -80\u00b0C. Final family progeny numbers were 115 for HL3, 113 for HL7, and 110 for LN4.HL gametes were collected from the Hill's Lake Fish Culture Facility and taken to the Codrington Fisheries Research Facility where families were made on October 4Clock gene were screened for variation in the mapping panels. Of the markers which had detectable polymorphism in the parents, 101 microsatellites and two copies of Clock were selected to be used for the construction of the linkage map. All progeny from HL3, HL7 and LN4 were genotyped for the subset of 103 polymorphic markers for which they were informative. Counting all the duplicated locus positions that were amplified with some of the polymorphic markers, the final comparative maps generated from the three brook charr mapping panels consisted of 114 (HL3), 116 (HL7), and 54 (LN4) informative loci in each family  using a phenol - chlorophorm - isoamyl alcohol protocol . ParentsTo assess the putative location of the sex determining region in brook charr, additional progeny from the HL3 and HL7 mapping families were reared at the Codrington Fisheries Research Facility for an additional 2.5 years, when 48 fish from each of these families were sacrificed and internally sexed in September, 2010. DNA was then extracted from fin clips on these fish and genotyped for markers on the BC-18 (= homology to Yp136 marker region), and BC-4a; BC-4b linkage group regions (= homology to the AC-4 sex linkage group).http://www.uoguelph.ca/~rdanzman/) [http://www.joinmap.nl). To facilitate future cross comparisons of linkage groups within fishes of the genus Salvelinus, all brook charr linkage groups were designated according the their homologous chromosome arms in Arctic charr following the designations given in [Linkage analysis was performed using a number of programs  contained in the LINKMFEX software suite that is available in the Link to Computer Software tab at (anzman/) , and unlanzman/)  .Recombination rate differences along conserved chromosomal segments were calculated using the program RECOMDIF, which uses a two-way contingency G-test that compares parental versus recombinant genotypes inherited from each parent, for each pair of linked markers shared between the mapping parents being compared. Williams' correction was applied when the number of recombinants was less than five. Bonferroni correction for multiple comparisons was applied by dividing alpha (0.05) by the number of linkage groups tested in each comparison in order to determine the adjusted critical \u03c72 test (with the program LINKMFEX), which is an appropriate test for sample sizes between 25 and 200 [For all polymorphic loci  goodness of fit to the expected 1:1 segregation ratio was assessed using a log likelihood adjusted  and 200 . The proSalvelinus species, and therefore, more extensive comparisions are not reported in this study. The programs markerSORT and markerCOMP, were used to assist in the assignment of cross-species homologies and homeologies. Putative cross species homologies and homeologies were assigned based on conserved syntenic blocks of markers, and conserved syntenic blocks of duplicated markers (except where noted differently), respectfully. The construction of marker-specific synteny blocks was accomplished using the program BLOCKON with Arctic charr as the reference genome to depict assignments to brook charr and rainbow trout arm homologies. Using the composite female Arctic charr map, it was also possible to infer the more precise structure of the composite Salvelinus linkage groups by referencing each linkage group to the more complete rainbow trout linkage map. Salvelinus linkage groups showing homology to two different linkage group arms in rainbow trout were inferred to be metacentric in structure. As comparisons to a physical map have not been completed for either Arctic charr or brook charr at present, linkage group arms were arbitrarily designated as 'a' and 'b' arms within metacentrics.Linkage map comparisons between the brook charr and Arctic charr mapping parents .Click here for fileComposite female linkage maps derived from mapping data in the HL3 and HL7 mapping panel female parents.Click here for fileLinkage map based upon genotypic segregation data from the HL3 and HL7 mapping panel male parents.Click here for fileAssignment of polymorphic markers to the various identified Arctic charr linkage groups in two mapping panels (Family 2 and Family 3).Click here for fileWithin family comparisons (Hills Lake strain) of recombination rate differences between female and male brook charr mapping parents.Click here for fileIntra-sex comparisons of the recombination rates between the Hills Lake brook charr female and male mapping parents.Click here for fileObserved deviations from Mendelian expectations in the brook charr mapping parents of families HL3 and HL7.Click here for fileComparative genetic maps between Arctic charr, brook charr, and rainbow trout, using the combined female of Arctic charr as a template.Click here for fileSalvelinus (based primarily upon the Arctic charr) and rainbow troutOxford Grid of the linkage group arm assignments between .Click here for fileComplete listing of all the genetic markers screened in the brook charr mapping panels.Click here for file"}
+{"text": "Genetic variants are likely to contribute to a portion of prostate cancer risk. Full elucidation of the genetic etiology of prostate cancer is difficult because of incomplete penetrance and genetic and phenotypic heterogeneity. Current evidence suggests that genetic linkage to prostate cancer has been found on several chromosomes including the X; however, identification of causative genes has been elusive.Parametric and non-parametric linkage analyses were performed using 26 microsatellite markers in each of 11 groups of multiple-case prostate cancer families from the International Consortium for Prostate Cancer Genetics (ICPCG). Meta-analyses of the resultant family-specific linkage statistics across the entire 1,323 families and in several predefined subsets were then performed.Meta-analyses of linkage statistics resulted in a maximum parametric heterogeneity lod score (HLOD) of 1.28, and an allele-sharing lod score (LOD) of 2.0 in favor of linkage to Xq27-q28 at 138 cM. In subset analyses, families with average age at onset less than 65 years exhibited a maximum HLOD of 1.8 (at 138 cM) versus a maximum regional HLOD of only 0.32 in families with average age at onset of 65 years or older. Surprisingly, the subset of families with only 2\u20133 affected men and some evidence of male-to-male transmission of prostate cancer gave the strongest evidence of linkage to the region . For this subset, the HLOD was slightly increased (HLOD\u2009=\u20093.47 at 134 cM) when families used in the original published report of linkage to Xq27-28 were excluded.Although there was not strong support for linkage to the Xq27-28 region in the complete set of families, the subset of families with earlier age at onset exhibited more evidence of linkage than families with later onset of disease. A subset of families with 2\u20133 affected individuals and with some evidence of male to male disease transmission showed stronger linkage signals. Our results suggest that the genetic basis for prostate cancer in our families is much more complex than a single susceptibility locus on the X chromosome, and that future explorations of the Xq27-28 region should focus on the subset of families identified here with the strongest evidence of linkage to this region. Prostate cancer (PC) is the most common male cancer in developed countries. In the RNASEL gene was later implicated as harboring rare variant alleles that increase risk of prostate cancer and may account for this linkage signal[RNASEL as a prostate cancer risk locus, with several recent large case\u2013control and cohort studies and a very large meta-analysis all showing significant associations of prostate cancer risk with polymorphisms in this locus[In 1996, the first prostate cancer linkage report implicated chromosome 1q23-25. The RNAis locus.Several other susceptibility loci presumed to contain rare variants of large effect on individual risk of prostate cancer have been suggested,4,11-42 HPCX)[HPCX Xq27-28 region using 108 independent prostate cancer patients selected from families with multiple affected men (55 were from the linkage study above) and 257 controls  from the same Finnish population. Significant association was observed for two markers in the region, DXS1205 (p\u2009=\u20090.0003) and bG82i1.1 (p\u2009=\u20090.0006), with stronger association observed at DXS1205 in the subset of 60 cases from families with no evidence of male-to-male transmission (p\u2009=\u20090.0002)[In 1998, a study of 360 multiple-case families found evidence for a prostate cancer susceptibility locus on chromosome X in the region Xq27-q28 (HPCX). A subseHPCX). There hHPCX),51-53 wiHPCX), and oneHPCX). A fine-\u20090.0002). AssociaSPANX genes . The SPANX genes encode nucleus-associated sperm proteins and their expression has been detected in a variety of cancers. While they were originally suggested as candidate genes for the HPCX susceptibility locus[Under the Xq27-q28 linkage peak is a region of ~750 kb containing five ty locus, more rety locus. Howeveret al. conducted a genome-wide SNP association study of prostate cancer in over 23,000 Icelanders followed by a separate replication study. Of the two novel SNPs identified by this study, one, rs5945572, was found on XP11.22 (odds ratio (OR)\u2009=\u20091.23)[et al. also found association to this region in a large GWAS[Gudmundsson \u2009=\u20091.23). Eeles erge GWAS. HoweverThe aims of this study were to examine the evidence for linkage of prostate cancer to chromosome X using 1,323 multiple-case prostate cancer families from the ICPCG and genotyping a consensus map of 25 microsatellite markers and using both parametric and non-parametric allele-sharing linkage analyses. The pedigree subsets evaluated were presence/absence of male-to-male disease transmission (a surrogate for X-linked inheritance), Carter criteria of HPC,58, averThis analysis was performed on 1,323 families with hereditary prostate cancer ascertained by 11 groups participating in the ICPCG. The process of ascertaining families and confirming diagnosis of prostate cancer differed among the groups, but in all samples, men were considered to be affected with prostate cancer only if medical records or death certificates could confirm the diagnosis. The 11 groups that participated in this linkage analysis are described elsewhere.In the statistical analysis, all families were first analyzed together. In addition, several subsets of families were created based on pedigree characteristics. A pedigree was classified as satisfying the Carter criteria for hereditary prostate cancer,58 if atEach group had genotyped a different set of markers in the Xq27-q28 region. In order to use the available genotype data without re-genotyping a common panel of markers, a consensus map of the genetic markers from the different groups was created as follows. A total of 26 different markers on chromosome Xq  markers  marker set always resulted in the same or lower allele-sharing LODs (e.g. 1.22 at 125 cM in the complete dataset). The subsets that resulted in higher allele-sharing LODs for the GWS markers were the 732 families with 2\u20133 affecteds , the 627 families where mean age at onset was <65 years , and the subset of 288 families with 2\u20133 affecteds that appeared to exhibit male-to-male transmission . The subsets of families that appeared to exhibit patterns of prostate cancer consistent with X-linked inheritance did not have high positive allele-sharing LODs  families all gave positive HLODs, with a stronger signal observed in the latter group of families. When the FM marker set was used, the same pattern was observed: the subset of X-linkage transmission families gave a maximum HLOD\u2009=\u20090.246 at 132 cM, the unclear families gave a maximum HLOD\u2009=\u20090.142 at 143 cM, and the non-X-linkage families  yielded a maximum HLOD\u2009=\u20090.62 at 153 cM. The subset of 627 families with mean age at onset 65 years or younger gave HLOD\u2009=\u20091.8 at 138 cM using the GWS markers. The 696 families with mean age greater than 65 had maximum HLOD\u2009=\u20090.32 at 120 cM. Subdivisions based on the Carter criteria alone were not highly correlated with linkage evidence. Number of affected males in the family had a larger effect on linkage evidence, particularly when combined with pattern of transmission. The 732 families with 2\u20133 affecteds per family had maximum HLOD\u2009=\u20092.01 at 134 cM, whereas the 438 families with 4\u20135 affected males had HLOD\u2009=\u20090.1 at 168 cM and the 153 families with 6 or more affected males had HLOD\u2009=\u20091.4 at 153 cM. Consistent with the non-parametric analyses, the strongest evidence for linkage occurred in the subset of 288 families with 2\u20133 affected males and at least some evidence of male-to-male transmission: maximum multipoint HLOD\u2009=\u20093.24 at 134 cM  or those with evidence of male-to-male transmission when considering linkage evidence provided by those subsets of families for a PC susceptibility locus at Xq27-q28. Families with smaller numbers of affected men appeared to contribute the most evidence to linkage in this region. While classification of each family based on proportion of affected men out of total men old enough to be affected might provide more homogeneous subsets, this was not feasible for this study given the many sources of families with quite different ascertainment schemes and different degrees of completeness of pedigree data collection. In addition, given that prostate cancer is a late age at onset disease and is quite common, we are only able to assign \u201cunaffected\u201d status to men who are over the age of 75 years and who have a normal digital-rectal exam and normal PSA at that advanced age. There are small numbers of such well-characterized, elderly unaffected men in this set of families and most of the families do not have any of them, which would make a proportion misleading. Families with 2\u20133 affecteds per family had maximum HLOD\u2009=\u20092.01 at 134 cM. However, the strongest suggestion of linkage was observed in both the parametric and non-parametric analyses in families with 2\u20133 affecteds and possible male-to-male transmission. This pattern was observed whether we included the families from the initial linkage publication in the analysis or not. This subset of families, when excluding the families from the original linkage publication, had a multipoint HLOD of 3.47 at 134 cM. The HLOD in these new families was slightly over the Lander and Kruglyak threshold of 3.3 for genome-wide \u201csignificant\u201d linkage but this threshold does not account for our multiple testing of different subsets, which likely requires a larger threshold to claim robust statistical evidence of linkage. However, this level of significance would meet the Lander and Kruglyak threshold for replication of a previously significant linkage (p\u2009=\u20090.01 or a LOD of approximately 1.0) even after correction for the multiple analyses. One candidate locus, ge study and thesHPCX region, all maternally related affected males share a linked haplotype in this region and the one paternally-related affected male does not share this haplotype. Finally, it is possible that the true causal allele in this region lies within the pseudoautosomal regions PAR2, which is near this linkage peak. Since female carriers cannot become affected with prostate cancer and since no marker loci have been genotyped in the PAR2 region in our families, our current data are inadequate for resolving this. Our complete prostate cancer pedigree resource, with additional fine-mapping in the Xq27-28 region, provided, at best, modest suggestive evidence for linkage to this region. However, subsets of families with fewer affected individuals and paradoxically, families with some possible evidence of male-to-male disease transmission showed stronger linkage signals. This same result was observed in the analysis of very large Utah pedigrees, in whicAlthough our results do not provide strong evidence for a major prostate cancer susceptibility gene located in this region of Xq27-28, there is some evidence for a locus that may contribute to risk in families with 2\u20133 affecteds and in some larger families such as the one in FigureThe authors declare that they have no competing interests.JEB-W, ELC, DJS, RE, DE, DJS, JLS, EAO, DK, LM, GGG, JLH, GS, HG, FW, ME, OC, GC-T, KAC, NJC, LAC-A, ASW, WBI and JX contributed to the design of the study. JEB-W, ELC, DJS and JX contributed to the performance of the meta-analyses. JEB-W, ELC, and CDC wrote the manuscript. DJS, SJM, RE, DE, JLS, EAO, GGG, WDF, JLH, GS, JS, TLT, GC-T, KAC, NJC, LAC-A, ASW, IO-G and WBI contributed to critical revision of the manuscript. MG, SE, JLS, EAO, DK, LM, GGG, WDF, JLH, GS, JS, TLT, OC, LM, PM, AV, KAC, LAC-A, KEW, SDI, PCW and WBI collected and maintained samples and data. EAO, DMK, SNT, SH, SB, GC-T, AV, KAC and CH performed or oversaw laboratory-based studies of samples. JLS, EAO, DMK, LM, SKM, CM, JH, HG, FW, GC-T, EML, NJC, JF, ASW, IO-G and MDB contributed to the linkage analyses at an ICPCG site. KD built and maintains a genotyping database at an ICPCG site. JX and LD coordinated and stored the linkage results from the contributing sites and harmonized the data. All authors have read and approved the final manuscript.The members of the International Consortium for Prostate Cancer Genetics are as follows:ACTANE Group: + Principal InvestigatorsSutton: S. Bullock, Q. Hope, S. Edwards, S. Bryant, S. Mulholland, S. Jugurnauth, N. Garcia, M. Guy, L. O'Brien, B. Gehr-Swain, A. Hall, R. Wilkinson, A. Ardern-Jones, D. Dearnaley, The UKGPCS Collaborators, British Association of Urological Surgeons' Section of Oncology, R. Eeles +UK, ambridge: Chris Evans, M. Dawn Teare, Doug Easton\u2009+\u2009UK, CAustralia: John Hopper+, Graham Giles+, Dallas English, Gianluca SeveriCanada: William D. Foulkes+, Nancy Hamel, Steven Narod, Jaques Simard+Texas: Mike Badzioch+, Chris Amos : Ketil Heimdal, Lovise M\u00e6hle, P\u00e5l M\u00f8ller\u2009+\u2009Norway, Oslo: Nicolai Wessel, Tone Andersen\u2009+\u2009Norway, UllevaalEU Biomed: Tim Bishop+, The EU Biomed Prostate Cancer Linkage Consortium1, Richard Gallagher2, Jerry Halpern1, Chih-lin Hsieh3, Laurence Kolonel4, Ingrid Oakley5, Dee West1,5, Alice S. Whittemore1 and Anna Wu3 BC/CA/HI Group: Raymond N. BaliseCeRePP Group: G\u00e9raldine Cancel-Tassin, Antoine Val\u00e9ri, Philippe Mangin, Olivier Cussenot JHU Group: Kathleen E. Wiley, Sarah D. Isaacs, Marta Gielzak, Charles M. Ewing, Patrick C. Walsh, William B. Isaacs Mayo Group: Daniel J. Schaid, Shannon K. McDonnell, Gerald B. Christensen, Julie M. Cunningham, Scott Hebbring, Jennifer C. Guenther, Stephen N. Thibodeau 1, Cralen C. Davis1, W. Mark Brown1, Cathryn H. Bock2, Kathleen A. Cooney2 Michigan Group: Ethan M. Lange1, Danielle M. Friedrichsen2, Suzanne Kolb3, Elaine A. Ostrander2, Lee Hood1, Janet L. Stanford3 Fred Hutchinson Cancer Research Center Group (PROGRESS): Kerry Deutsch1, Henna Mattila1, Virpi Laitinen1, Riikka Nurminen1, Daniel Fischer1, Teuvo L.J. Tammela1, Asha George2, Joan Bailey-Wilson3, Johanna Schleutker1 Tampere Group:Tiina Wahlfors1, Thomas Paiss2, Josef Hoegel1, Florian Kurtz1,3, Manuel Luedeke1,2, Antje Rinckleb1,2, Kathleen Herkommer2,3, Walther Vogel1, Mark Schrader2, Christiane Maier1,2 Ulm Group: Ulm Group: Sylvia BochumUme\u00e5/Karolinska Group: Fredrik Wiklund, Anders Bergh, Monica Emanuelsson, Ingela G\u00f6ransson, Bj\u00f6rn-Anders Jonsson, Fredrik Lindmark, Elisabeth Stenman, Henrik Gr\u00f6nbergUtah Group: Lisa A. Cannon-Albright, Nicola J. Camp, James M. Farnham Data Coordinating Center: Jianfeng Xu, Deborah A. Meyers, Bao-Li Chang, Aubrey R. Turner, Latchezar Dimitrov, Tamara S. Adams Daniella Seminara The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2350/13/46/prepubTable S1. Markers genotyped and used in the linkage analyses by each data collection group[on group.Click here for file"}
+{"text": "High-density genetic linkage maps were constructed for the Japanese flounder . A total of 1624 microsatellite markers were polymorphic in the reference family. Linkage analysis using JoinMap 4.0 resulted in the mapping of 1487 markers to 24 linkage groups, a result which was consistent with the 24 chromosomes seen in chromosome spreads. The female map was composed of 1257 markers, covering a total of 1663.8 cM with an average interval 1.35 cM between markers. The male map consisted of 1224 markers, spanning 1726.5 cM, with an average interval of 1.44 cM. The genome length in the Japanese flounder was estimated to be 1730.3 cM for the females and 1798.0 cM for the males, a coverage of 96.2% for the female and 96.0% for the male map. The mean recombination at common intervals throughout the genome revealed a slight difference between sexes, i.e. 1.07 times higher in the male than female. High-density genetic linkage maps are very useful for marker-assisted selection (MAS) programs for economically valuable traits in this species and for further evolutionary studies in flatfish and vertebrate species. Furthermore, four quantiative trait loci (QTL) associated with growth traits were mapped on the genetic map. One QTL was identified for body weight on LG 14 f, which explained 14.85% of the total variation of the body weight. Three QTL were identified for body width on LG14f and LG14m, accounting for 16.75%, 13.62% and 13.65% of the total variation in body width, respectively. The additive effects were evident as negative values. There were four QTL for growth traits clustered on LG14, which should prove to be very useful for improving growth traits using molecular MAS. Genetic linkage mapping has become a critically important tool in many areas of genetic research. In order to perform a useful linkage study, it is necessary to genotype and map large numbers of the available genetic markers on the mapping families. Microsatellites comprise an excellent opportunity for genomic mapping due to their abundance in most vertebrate genomes, as well as their genomic distribution pattern, high polymorphism rate and ease of typing, all of which are determinable via PCR. Meanwhile, the simple sequence repeat (SSR) alleles are typically co-dominant, and their polymorphisms can be scored in either a simple polyacrylamide gel separation format or with high-throughput capillary arrays The traditional methods of genetic improvement of quantitative traits have relied mainly on phenotype and pedigree information Japanese flounder  is a marine fish which is economically important as a food, and has been widely cultured in Asian countries such as China, Japan and Korea. It is mostly distributed along the coast of China, where it has been cultured for approximately 20 years due to its favourable traits such a fast growth rate, good adaptability to temperature and disease resistance in a variety of cultivation conditions. With extensive cultivation, however, farming of the Japanese flounder has also been confronted with certain problems, including a high mortality rate as well as a decline in growth. In order to further increase the productivity of Japanese flounder farming, it is thus essential to carry out both classic selective breeding and MAS. Recently, a number of genetic studies in this species have been reported. Fuji et al. To obtain microsatellite markers useful for linkage analysis, we examined the segregation patterns of 4600 markers in the mapping family. Among the 4600 markers, 1624 (35.3%) were polymorphic in either the male or female. The sequence data of 1587 newly identified polymorphic microsatellites were deposited with the GenBank Data Library under the accession numbers: JN900500-JN902086. A list of the 1624 polymorphic microsatellite markers is presented in a supplementary file.Segregation distortion from expectation under Mendelian inheritance was found in 625 (38%) of 1624 microsatellite markers. Although these markers are normally excluded from linkage analysis, the independence LOD score, one of the grouping parameters provided by JoinMap4.0, allows these markers to be included. This test for independence is not affected by segregation distortion and leads to a less spurious linkage When the total of 1624 polymorphic microsatellite markers was analyzed, 1487 markers were located on the linkage maps, containing 24 linkage groups (LGs), at a LOD score threshold value of 4.0.Significant linkages were identified for 1624 genetic markers. However, 137 microsatellite markers went unmapped in this analysis. Consequently, the mapping ratio of these markers is 91.6%. The female and male maps contained 1257 and 1224 markers, respectively. Both maps were found to have 24 linkage groups. The total length of the female map was determined to be 1663.8 cM, with an average interval of 1.35 cM. The linkage group size ranged from 44.9 cM to 94.1 cM. The number of loci per genetic linkage group varied from 27 to 76. The male linkage map spanned a total genetic distance of 1726.5 cM. The length of each linkage group varied from 54.1 to 104.5 cM and contained 27\u201366 loci per group, with an average interval of 1.44 cM. The sex-specific genetic linkage maps are presented in Either bridge markers or homologous loci were used to identify the co-linear regions in the female and male maps. The resulting integrated map contained 1487 microsatellite markers with 1466 unique positions. Twenty-four LGs were found, with an average of 62 microsatellites per LG. The total length of the map was 1763.3 cM with an average interval of 1.22 cM. The genome length of the Japanese flounder was therefore estimated to be1798.0 cM, and a coverage of 96.7% was observed. The linkage group length varied from 50.2 cM to 101.3 cM, and the number of markers on the linkage groups varied from 30 to 81.The sexes have slight differences in recombination rate, both in general and for specific pairs of linked markers. The availability of SSR markers in the male and female maps allowed an evaluation of the respective rate of meiotic recombination. The recombination rates obtained from 24 linkage groups were on average 0.0135 in females and 0.0144 in males. Therefore, the relative recombination ratio  in these pairs was 1\u22361.07, which was slightly higher in males than females.The result of test of normality for body length, weight and width were presented in Genetic maps provide critically important genomic information and allow the exploration of QTL, which can be used to maximize the selection of target traits. The availability of a large number of genetic markers is essential for constructing a useful linkage map and for QTL mapping of genetic traits of interest. In this study, we constructed high-density microsatellite genetic linkage maps using 1624 microsatellites in the Japanese flounder. These markers will serve as an important tool for future comparative map studies and to establish the underlying correspondence with the linkage groups of other closely related species. The current maps span a total length of 1663.8 cM for the females and 1726.5 cM for the males, with 24 LGs. There were no small (doublet or triplet) linkage groups, indicating that this linkage map is complete. Only 12 of the 1624 markers studied remained unlinked to any other marker. This degree of completeness supports the utility of the genetic map as a reference tool for future genetic analysis in this species.In humans, mice, cows and pigs, and indeed in most vertebrates studied so far, the recombination rates exhibit significant differences between the sexes. In contrast to most teleosts and mammals, but similar to previous report in this species In the mapping family, segregation distortion was observed for 625 markers and the distortion rate was approximately 38%. A higher distortion rate has been reported in previous studies, such as 40.5% for the Pacific white shrimp Information on genetic markers associated with quantitative trait loci (QTL) can be used in breeding programs to identify and select individuals carrying desired traits. In a previous study, one possible QTL was detected associated with resistance to lymphocystis disease The flatfishes (Pleuronectiformes), which include flounder, plaice, sole, turbot and halibut, are a broad taxonomic group comprising 11 families and approximately 500 species worldwide. And they are a group of teleosts having considerable commercial and environmental interest in terms of both fisheries and aquaculture. The increase in the genome sequencing data has allowed the construction of genetic linkage maps in a variety of flatfish species, such as the Japanese flounder The first genetic linkage map of the Japanese flounder was reported by Coimbra et al. The present study developed a high-density genetic linkage map for Japanese flounder. The improved linkage maps consisted of 1487 markers. The female and male maps have 1242 and 1215 unique positions, respectively, which afford sufficient marker density for fine mapping of the QTL. Meanwhile, mapping the QTL for growth traits (body weight and body width) in the Japanese flounder will provide a basis for identifying marker for growth-related genes and provide a powerful tool for building a foundation for genetic improvement programs using MAS.The Japanese flounder linkage map provides a basis for a thorough exploration of the entire genome, and for identifying genomic regions related to productivity and of evolutionary interest in the Japanese flounder. Since QTL are often conserved across closely related species All the experimental animal programs involved in this study were approved by the Yellow Sea Fisheries Research Institute\u2019s animal care and use committee, and followed the experimental basic principles. A slight fin tissue from the parents and F1 offspring was sheared under MS222 anesthesia, and all efforts were made to minimize suffering.In May 2011, a full-sib family of Japanese flounder was constructed and used for the development of a genetic linkage map. The male parent was selected from a group of fish derived from wild populations. The female parentwas selected from a cultured population. The induction of the maturation of broodstock and artificial fertilization of sperm and eggs were carried out as described previously A total of 4600 Japanese flounder Microsatellite markers were tested for segregation across a set of eight individual progeny. These microsatellite markers were recruited from two sources: The first set of 4000 microsatellite markers was obtained from different scaffold by genome sequencing of Japanese flounder in our laboratory. (2) The remaining 600 markers had been previously reported in this species by Casta\u00f1o-S\u00e1nchez et al. 2), 0.6 \u00b5M of each primer and 10\u201330 ng template DNA. The final volume was adjusted with sterile distilled water. The PCR products were separated on 8% polyacrylamide gels (PAGE) and visualized by silver staining The primers flanking the microsatellite regions were designed using Primer 5. All primers were designed for a 57.5\u00b0C annealing temperature, a 100\u2013300 bp total amplification product size and 40\u201360% GC content. All of the microsatellite markers were used to genotype two parents and six progeny for screening the segregation markers in the mapping population. The microsatellite markers that produced polymorphic fragments were used in the subsequent genotyping of the parents and 92 progeny to construct the linkage maps. Amplifications were performed in an ABI Veriti 96 well thermal cycler, BIO-RAD MyCycler thermal cycler and Fisher Scientific LabServ LS-P96G thermal cycler. The PCR amplifications were performed under the following conditions: 95\u00b0C for 5 min, followed by 32 cycles at 95\u00b0C for 30 s, a specific annealing temperature of a specific primer pair for 30 s and 72\u00b0C for 30 s, and the final extension was 72\u00b0C for 10 min. Amplification reactions were carried out on a 15-\u00b5l column consisting of 10\u00d7 Taq buffer, 0.5 U Taq polymerase, 0.6 mM dNTP  type population, which is designed to handle F1 outbreeding population data containing various genotype configurations. Pairwise analyses were performed and markers were sorted in linkage groups at a minimum LOD score of 4.0. The \u201clocus genotype frequency\u201d function calculated chi-square values for each marker to test for the expected Mendelian segregation ratio. Markers significantly deviating from the expected ratio (p<0.05) were eliminated. The linkage distances were estimated for each LG assuming the Kosambi mapping function. Linkage groups with genetic markers on individual maps were merged to create an integrated map using the \u201cJoin-combine groups for map integration\u201d command.The estimated genome length (Ge) of the consensus female and male genomes was estimated using two different methods. First, Genome estimation size 1 (Ge1) was calculated by adding 2 s to the length of each genetic linkage group to account for the chromosome ends, where s represents the average spacing of the genetic linkage map. Genome estimation size 2 (Ge2) was calculated by multiplying the length of each genetic linkage group by (m+1)/(m\u22121), where m is the number of loci in each genetic linkage group. The estimated genome size (Ge) for each sex was taken as the average of the two estimates. The observed genome length was taken as the length of the framework map (Gof) and the total length (Goa) considering all the markers on the framework map, including the triplets and doublets. The map coverage ratios, Gof and Goa, were calculated as Gof/Ge and Goa/Ge, respectively.QTL analysis was performed with WinQTLCart2.5 software using the composite interval mapping (CIM) method (model 6). Unlinked might act as an additional environmental effect that reduces the significance of the estimated marker-trait association. Therefore, CIM includes neighboring markers and uses the remaining background markers as cofactors in order to remove the effects of multiple QTL. While the CIM analysis was conducted separately for each map, the background markers used in these analyses were derived from both maps. Five background markers were employed in CIM analysis. A stringent LOD score threshold \u22652.5 was set to identify the putative presence of QTL related to growth traits.Table S1Characterization of microsatellite markers genotyped in Japanese flounder mapping family.(DOCX)Click here for additional data file."}
+{"text": "PLoS Genetics show that the ability of Greatwall/Scant kinase to generate an inhibitor of Protein Phosphatase 2A (PP2A) underpins an antagonistic interplay between Greatwall and Polo in DrosophilaCommitment to mitosis is driven by activation of the Cdk1-Cyclin B protein kinase complex known as Mitosis Promoting Factor (MPF). MPF activation promotes downstream protein kinases that control the formation and function of the mitotic spindle. These kinases include members of the NIMA, Greatwall/Scant, Polo, and Aurora kinase families. Each kinase often phosphorylates multiple targets. Sophisticated dependency relationships enable a single kinase to promote distinct events at the same or successive stages of mitosis. Understanding mitosis means cataloguing each target for each kinase and deciphering the interplay between the ensuing pathways. Two papers in this issue of Drosophila genetics. \u201cPolo\u201d describes the circular profile of chromosomes associated with the monopolar spindles in polo mutants Drosophila Polo is considered to be analogous to mammalian Plk1 The mitotic kinases Polo, Aurora, and Greatwall were identified through greatwall mutants fail to correctly condense their chromosomes, leading to the naming of the kinase Greatwall as a protector of chromosome integrity Xenopus egg extracts demonstrate that Greatwall activity is critical to drive mitotic commitment Xenopus Greatwall converts them into potent PP2A-B55\u03b4 inhibitors DrosophilaXenopus kinase, and yeast phospho-Endos subsequently binds components of a ribosome-associated protein complex to control mRNA stability Drosophila endosulfine (endos) mutants is consistent with altered translation in this system Two further functions have been ascribed to Greatwall kinases: the modulation of RNA stability during G0 in budding yeast and, as discussed below, the antagonism of Polo kinase activity in Scant) that failed to complement the 1polo mutant with respect to embryonic viability Scant is a dominant, hyper-activating allele of gwl (denoted scantgwl) 1polo/+ scantgwl/+ embryos. Increasing the scantpolo:gwl ratio by duplication of polo+ suppressed this phenotype, while reducing this ratio by using a Polo inhibitor enhanced it polo+ duplications restore fertility to 1polo/+ scantgwl/+ females scantgwl mutant can be modulated by altering the dose of +polo.The antagonistic relationship between Polo and Greatwall was revealed by a second-site mutant  that participates in Endos control Scant into Xenopus Greatwall increases its interphase activity and promotes premature commitment to mitosis Rangone et al. demonstrate the ability of polo-compromised and Scant flies twins deletion, they employed a genetic analysis to demonstrate that the Greatwall/Endos/PP2A relationship is conserved in DrosophilaWang et al. derived similar conclusions from a very different starting point by systematically seeking deficiencies that enhanced the fertility defect of scantgwl identified mutations in endos, i.e., mutations that increased PP2A activity. In contrast, the search for enhancers of the Scant phenotype identified PP2A and twins mutations that reduced it. More broadly speaking, the biochemical dissection of Xenopus extracts and genetic dissection of Drosophila come to remarkably consistent conclusions: the Greatwall/Endos/PP2A switch. The meiotic progression defects of twins and endos mutants and greatwall over-expressors suggest that these parallels extend to the control of the maintenance of the meiotic state in DrosophilaThese two independent screens used opposite approaches (enhancers versus suppressors) to study Polo and Greatwall and found opposing components of the same regulatory network. The search for suppressors of scantgwl by the same deletions that enhance polo in the Wang et al. study polo mutants so sensitive to a reduction in PP2A levels?\u201d The answer may be linked to the centrosome retention phenotype. Defective centrosome attachment is a common occurrence during the syncitial divisions of mitotic mutants, suggesting that it may simply be indicative of compromised spindle function . However, the role this phenotype played in unlocking the Polo Greatwall/Endos/PP2A relationship suggests that the possibility of a functional link merits consideration.The enhancement of the fertility defect of The nuclear envelope remains largely intact throughout syncitial divisions of the early embryo. Limited fenestration at the spindle poles, beginning at pro-metaphase, enables the microtubules emanating from the centrosomes to capture kinetochores and form the central spindle Reducing Polo activity can cause one of the two centrosomes to disassociate from the nuclear envelope around the time of fenestration Scant (\u201cScott of the Antarctic\u201d), holds the key: 1polo/+ scantgwl/+ mutants have greater problems in retaining the association between the centrosome and the nuclear envelope  than do 1polo/+ +/+ mutants. Scant was named after British explorer Captain Scott, who set off on an unsuccessful mission to the South Pole.So how does the Greatwall/Endos/PP2A pathway impact centrosome retention? The name of the dominant Greatwall mutation, scantgwl is a hyperactive mutation, the enhancement of the detachment phenotype of polo mutants might indicate that PP2A assists Polo in promoting prophase attachment. However, this appears not to be the case, as PP2A mutants have no defect in prophase attachment As polo-dependent prophase centrosome loss by re-capturing the errant centrosome on their anaphase spindles polo and PP2A.Are the two attachment phenotypes (Polo-driven association in prophase and PP2A-driven association during mitotic exit) connected (Alternatively, Rangone et al. provide plausible arguments for a functional link between the Polo and PP2A-driven dephosphorylation to promote centrosome attachment after mitotic exit. There are precedents in which Polo recruitment and subsequent function is driven by dephosphorylation or the dephosphorylated state. The structural component of the anaphase mid-zone, PRC1, is unable to recruit Polo until a Cdk phosphorylation site is dephosphorylated in anaphase Clearly, greater insight into the molecular basis of centrosome attachment is required before we can resolve these possibilities.Drosophila genetics to reveal the interplay between signalling networks. It is no accident that \u201ccell cycle speak\u201d has accumulated abstract names such as \u201cPolo\u201d, \u201cAurora\u201d, \u201cScant\u201d, and \u201cGreatwall\u201d at the heart of its everyday vocabulary. Drosophila genetics remains at the forefront of our attempts to piece together multiple regulator target relationships into a holistic view of the networks that constitute mitotic control. The focus and simplicity of the Scant screens in particular suggest that many insights into the Greatwall/Polo/PP2A axis will continue to emerge from this approach. In the immediate future, the attenuation of the phospho-Endos inhibitory signal is a particularly pressing objective for the field.An overriding message from these studies is the power of"}
+{"text": "PSMD9) lies in the NIDDM2 region and is implicated in diabetes in mice. PSMD9 mutations rarely cause T2D and common variants are linked to both late-onset T2D and maturity-onset-diabetes of the young (MODY3). In this study, we aimed at determining whether PSMD9 is linked to macrovascular pathology of T2D.Coronary artery disease (CAD) and stroke share a major linkage at the chromosome 12q24 locus. The same chromosome region entails at least a major risk gene for type 2 diabetes (T2D) within NIDDM2, the non-insulin-dependent-diabetes 2 locus. The gene of Proteasome Modulator 9  and performed a non-parametric linkage study to test for linkage for coronary artery disease, stroke/TIA, macro-vasculopathy and macrovascular pathology of T2D. We performed 1,000 replicates to test the power of our significant results. Our results show a consistent significant LOD score in linkage with all the above-mentioned phenotypes. Our 1000 simulation analyses, performed for each single test, confirm that the results are not due to random chance.In our 200 T2D families from Italy, we characterized the clinical phenotype of macrovascular pathology by defining the subjects for presence or absence of CAD, stroke and/or transitory ischemic attacks (TIA), plaques of the large arterial vessels (macro-vasculopathy) and arterial angioplasty performance. We then screened 200 T2D siblings/families for PSMD9 IVS3+nt460A/G, +nt437C/T and exon E197G A/G SNPs are linked to CAD, stroke/TIA and macrovascular pathology of T2D in Italians.In summary, the  Type 2 diabetes (T2D) has long-term complications, both microvascular and macrovascular. The microvascular complications entail the diabetic retinopathy, diabetic neuropathy and diabetic nephropathy. The macrovascular complications are represented by coronary artery disease (CAD), stroke, transitory ischemic attacks (TIA), and atherosclerotic plaques in major arterial vessels. The history of arterial angioplasty in a patient also implicates the presence of macro-vasculopathy. Given the high impact of macrovascular pathology on life quality and life expectancy, it is quite important to identify risk genes contributing to such polygenic and complex phenotype.PSMD9 is located.Recently, a study reported a significant linkage to coronary artery disease (CAD) , myocardPSMD9 unique mutations rarely cause T2D [PSMD9 common single nucleotide polymorphism (SNPs) to late-onset T2D, via recessive/additive model [ause T2D . We idenve model , as wellve model . The PSMve model .IVS3+nt460A/G, +nt437C/T and exon E197G A/G T2D risk (SNPs) of PSMD9 cause evidence for linkage with other T2D-associated phenotypes such as CAD, stroke/TIA, vasculopathy and macrovascular pathology of T2D in Italians.The aim of the present study was to determine whether the The subjects were all recruited from Rome, Italy following the Helsinki declaration guidelines. Subjects gave written informed consent and the Penn State College of Medicine Ethical Committee approved the study.We previously recruited 200 Italian T2D affected siblings and families with both T2D affected and unaffected members . We exclWe characterized the family subjects with T2D and without T2D for presence/absence of coronary arterial disease , cerebrovascular events (stroke/transitory ischemic attacks), vasculopathy (plaque(s) on carotid arteries/lower extremities arteries, and/or arterial angioplasty), and macrovascular pathology (vasculopathy and/or CAD and/or cerebrovascular events). Phenotypes of macrovascular pathology are described as unknown if diagnosis is unclear or data are lacking; subjects are described as affected if they carry the phenotype under study and as unaffected if they do not have the phenotype. In our 200 Italian families, for each phenotype, data are less than 100%.PSMD9 IVS3+nt460/+nt437/E197G SNPs in all family members. We directly sequenced the PCR products, status post-purification via EXOSAP-IT on an automated ABI 3730 Sequencer.We sequenced the PSMD9 SNPs with all described phenotypes. The three SNPs studied are in linkage disequilibrium as previously shown [IVS3+nt460A/G, +nt437C/T and exon E197G A/G SNPs. Each phenotype was tested independently using for the above-mentioned three SNPs the same map distance . Merlin calculated the linkage score for each of these SNPs in linkage disequilibrium as a single locus, as it reported only one LOD score per statistical test, which implies that Merlin considered the SNPs located on a single locus.We tested in the 200 Italian families for linkage of the ly shown . The mapAll results are reported as LOD scores calculated by Merlin. For each test performed for each specific phenotype, to exclude false positive results, we calculated the empirical P-value in 1,000 replicates of simulations by using the gene dropping method: this analysis replaces real data with simulated data, while maintaining the pedigree structure, allele frequencies and recombination fraction. These datasets are generated under the null hypothesis of no linkage. The empirical P-value for each phenotype-specific test was calculated based on the P-value corresponding to the single LOD score given by Merlin for the three SNPs analysis at one locus and using the same map distance of the real analysis. Further, the outcome of the simulations was calculated taking in consideration how many times in 1,000 replicates there was a P-value equal to or smaller than the P-value of the real analysis.The LOD scores of the non-parametric linkage analysis, performed for the various phenotypes, are reported, jointly for the three tested SNPs, with their corresponding P-value as well as with the results of the 1000 simulations (Table PSMD9 IVS3+nt460A/G, +nt437C/T and exon E197G A/G SNPs studied are in linkage with CAD, cerebrovascular events, vasculopathy, and macrovascular pathology. The simulation analyses rule out false results for the linkage to CAD, stroke and macrovascular pathology at the significance level of at least \u03b1 = 0.005, however the significance level of the vasculopathy linkage is only at \u03b1 = 0.053. Thus, PSMD9 or any of its variants contribute to linkage of the reported phenotypes.Our study shows that the Of note, the potential contribution of intronic variants to complex disorders such as atherosclerosis and/or major cardiovascular events is widely accepted.However, we think it is important to take in consideration that the linkage signal detected for the macrovascular pathology phenotypes in our study may indeed reflect the underlying linkage for T2D . In factPSMD9 gene has the potential of having pleiotropic effects. Recently, we published the contribution of the same gene to T2D-nephropathy [hropathy , which ihropathy .PSMD9 variants, at least partially. This locus in fact was reported as linked to macrovascular pathology in other populations [Our results should be replicated in different T2D ethnic groups and are of relevance to the T2D families as well as to the families with macrovascular pathology without T2D carrying linkage in the chromosome 12q24 locus, which may be explained by the ulations . In addiLifetime expectancy is highly affected by the presence of atherosclerosis and macrovascular pathology, thus the impact of the identification of a risk gene to macrovascular pathology is inestimable.The authors declare that they have no competing interests.CG conceived and designed the study, collected the clinical information, performed the statistical analysis and drafted the manuscript."}
+{"text": "MAS is quite challenging in conifers because of their large, complex, and poorly-characterized genomes. Our goal was to construct a high-density linkage map to facilitate the identification of markers that are tightly linked to a major recessive male-sterile gene (C. japonica using expressed sequence-derived co-dominant single nucleotide polymorphism (SNP) markers, most of which were genotyped using the GoldenGate genotyping assay. A total of 1261 markers were assigned to 11 linkage groups with an observed map length of 1405.2 cM and a mean distance between two adjacent markers of 1.1 cM; the number of linkage groups matched the basic chromosome number in C. japonica. Using this map, we located ms1 on the 9th linkage group and constructed a partial linkage map around the ms1 locus. This enabled us to identify a marker (hrmSNP970_sf) that is closely linked to the ms1 gene, being separated from it by only 0.5 cM.We constructed a high-density saturated genetic linkage map for ms1 gene on the 9th linkage group and constructed a partial linkage map around the ms1 locus. The map distance between the ms1 gene and the tightly linked marker was only 0.5 cM. The identification of markers that are tightly linked to the ms1 gene will facilitate the early selection of male-sterile trees, which should expedite C. japonica breeding programs aimed at alleviating pollinosis problems without harming productivity.Using the high-density map, we located the  Pinus lambertiana   . Of the Needle tissue was collected from the parents and progeny of both the YI and TO-S pedigrees. Genomic DNA was extracted from individual needles using a modification of the CTAB method . For usehttp://www.ffpri.affrc.go.jp/labs/cjgenome/).For the YI pedigree, six kinds of genetic markers were used to construct the linkage map: cleaved amplified polymorphic sequences (CAPS) markers, restriction fragment length polymorphism (RFLP) markers, microsatellite  markers, EST-derived microsatellite (EST-SSR) markers, amplicon length polymorphism (ALP) markers and single nucleotide polymorphisms (SNP) merkers. The segregation data for 121 CAPSs, 117 RFLPs, 34 SSRs, 1 ALP and 5 SNP markers were obtained in the previous study . In thisms1 gene, 17 male-sterile progenies and the parents of TO-S pedigree were also genotyped using this assay, since the gSNP markers found to be monomorphic in the YI pedigree were excluded from linkage mapping in the TO-S pedigree. The GoldenGate assay employs highly multiplexed allele-specific extension methods and universal PCR amplification reactions. The PCR products, which were fluorescently labeled by the incorporation of 5'-labeled primers P1 (Cy3) and P2 (Cy5), were hybridized to capture probes on the beads in the array. The ratio of the fluorescent signals from 2 allele-specific ligation products was used to determine the sample's genotype. Signal intensity data processing, clustering and genotype calling were performed using the genotyping module in the BeadStudio software (Illumina). Genotyping was conducted exclusively on the basis of SNPs with an Illumina GenTrain score in excess of 0.25; the GenTrain score provides a measure of the reliability of SNP detection based on the distribution of genotypic classes. For each analyzed SNP, individual genotypes with an Illumina GenCall score below 0.25 were excluded; the GenCall score provides a measure of the reliability of an individual SNP call relative to the distribution of genotypic classes.A large set of SNP markers was genotyped using the GoldenGate assay; collectively, this set is referred to as \"gSNPs\". For the YI pedigree, multiplexed genotyping of the gSNP markers was carried out using the 1536-plex GoldenGate array, in accordance with the manufacturer's protocol. A detailed description of the procedures employed and results obtained in the course of discovering these SNPs can be found elsewhere . A totalHigh Resolution Melting (HRM) analysis was also used to obtain SNP genotyping data for linkage map construction; the SNPs identified in this way are henceforth referred to as \"hrmSNPs\". The development procedure and analyzed condition was reported elsewhere . The linArabidopsis [Brassica [Oryza [Nicotiana [Petunia [C. japonica EST sequences using TBLASTN . The following thermal profile was used: 3 min denaturation at 94\u00b0C, followed by 30 cycles of 45 s denaturation at 94\u00b0C, 30 s annealing at 55-64\u00b0C, and 30 s extension at 72\u00b0C, with a final extension step of 72\u00b0C for 10 min. Amplification products were separated by electrophoresis in 2% (w/v) agarose gels run in 1 \u00d7 TAE buffer. The gels were then stained with ethidium bromide and visualized under UV light. Each PCR fragment was sequenced using the BigDye Terminator kit (Applied Biosystems) and ABI Prism 3100 DNA sequencer (Applied Biosystems) to identify SNPs in the parents of the YI pedigree (YI96 and YI38). Primers for both the forward and the backward direction were used. SNP markers associated with genes having significant similarities to genes from other species that are known to be important in male gametophyte development and/or male-sterility were collectively referred to as ssSNPs, while SNP markers associated with genes exhibiting differential expression between male-fertile and male-infertile individuals were collectively referred to as meaSNPs. The ssSNP and meaSNP markers were genotyped by sequencing. For the mapped meaSNP markers, proteins with high similarities to their original ESTs were identified from the NCBI RefSeq database using BLASTX  in reaction mixtures with a total volume of 15 \u03bcL containing 20 mmol/L Tris-HCl (pH 8.0), 50 mmol/L KCl, 2 mmol/L MgClLinkage analyses were conducted for all hrmSNP  were excluded from further linkage analysis. All linkage analyses were performed using the JoinMap v3.0 software with the parameter CP (cross-pollination) [Chi-squared tests were performed for each locus to assess its deviation from the expected Mendelian segregation ratio. Loci exhibiting extreme segregation distortion (ination) . During ination) . For theination) .oG) for the linkage map was calculated as the sum of the sizes of the linkage groups. The expected genome length, eG, was estimated using method 4 of Chakravarti et al. (1991) [oC, is the ratio of the observed and the estimated genome lengths, i.e. oG/eG. The expected genome coverage, eC, was calculated using the equation of Bishop et al. (1983) [The observed genome length (. (1991) , in whic. (1983) :e Gis the estimated genome length.Here, R is the haploid number of chromosomes, N is the number of positioned loci, X is the maximum observed map distance between two adjacent assigned markers in cM at or above a minimum LOD threshold value of 8.0, and x markers will fall within a given interval isIf the markers were randomly distributed and the genome is divided in N intervals, the number of markers per interval would follow a Poisson distribution with a mean of \u03bc. To determine whether the markers were randomly distributed, all linkage groups were divided into 1, 2, 3, 4, 5, 10 and 15 cM intervals. The number of intervals that contained markers were counted and the average number of markers per interval (\u03bc) was calculated. If the average number of random occurrences per interval is \u03bc, then the probability that We compared the actual distribution of markers to that expected for a Poisson distribution using the chi-squared test as described by Kang et al. (2010) .ms1 gene, a total of 19 microsatellite markers on the linkage map . A reaction mixture with a total volume of 8 \u03bcL was used, consisting of 1 \u00d7 Multiplex PCR master mix (Qiagen), fluorescently-labeled forward primers (0.2 \u03bcM), reverse primers (0.2 \u03bcM), and 5 ng of genomic DNA. The following thermal profile was used: 15 min at 94\u00b0C, then 32 cycles of 30 sec at 94\u00b0C, 90 sec at 55-62\u00b0C, 60 sec at 72\u00b0C, followed by 30 min at 72\u00b0C; the results were analyzed using a 3100 genetic analyzer (Applied Biosystems). The independence of the segregation of the SSR markers and the ms1 gene was investigated using chi-square tests to identify markers linked to the ms1 gene.Microsatellite markers were used to identify the linkage group on which the target gene is located because of their high polymorphism and straightforward analysis. Thus, to identify the linkage group containing the ms1 gene was located in the 9th linkage group, which is hereafter referred to as \"LG9.\" The markers in LG9 that exhibited polymorphism in the parents of the TO-S pedigree were therefore used to construct a partial linkage map around the ms1 locus. In addition, gSNP markers that exhibited polymorphism in the parents of the TO-S pedigree and whose segregation with the ms1 gene deviated significantly from that expected in the absence of linkage  in 17 male-sterile progenies were also used in the construction of the partial linkage map.Linkage analysis using EST-SSR markers indicated that the The markers of LG-9 in the TO-S pedigree except two microsatellite markers and four CAPS markers were genotyped on a BioMark 48.48 Dynamic Array (Fluidigm) using KASPar assays. Primer pairs suitable for the KASPar assays were designed on the basis of the sequences of the relevant markers ; see Additional file ms1 gene was expected in this case.Linkage analyses for the TO-S pedigree were performed using the same conditions as were used for the YI pedigree. Loci whose segregation patterns deviated significantly from Mendelian ratios were not excluded from the further linkage analysis in the TO-S pedigree because distortion of loci linked to the On the basis of the sequence similarity results, 238 primer pairs were designed for various candidate genes; of these, 141 generated PCR products that could be separated and identified after electrophoresis on a 2% agarose gel. Of these obtained STSs, 36 were polymorphic in the parents of the YI pedigree  from the expected Mendelian ratios and were therefore excluded from further linkage analysis; among the excluded markers were 22 gSNPs, 8 hrmSNPs, 20 CAPSs, 13 RFLPs, 6 SSRs, 3 ssSNPs and 2 meaSNPs  were successfully genotyped, of which 795 exhibited clear segregation within the YI pedigree. In addition to these gSNP markers, 144 CAPSs, 135 RFLPs, 41 microsatellites, 16 EST-SSRs, 173 hrmSNPs  and 1430.6 cM (eG), respectively; the observed and estimated map coverages were estimated to be 98.2% (oC) and 100.0% (eC), respectively. The maximum observed map distance between two adjacent assigned markers in cM (X) was 17.3 cM. Significant deviations from the Poisson distribution of markers were observed for marker intervals of 1 cM, 2 cM, 3 cM, 4 cM, 5 cM, 10 cM and 15 cM (P < 0.001), indicating that the 1262 markers used to construct the linkage map for the YI pedigree were not randomly distributed .Of the 1290 markers that exhibited no segregation distortion, 1279 could be assigned to specific linkage groups; in total, 1262 markers were mapped, including 761 gSNPs, 159 hrmSNPs, 121 CAPSs, 117 RFLPs, 34 SSRs, 33 ssSNPs, 15 meaSNPs, 16 EST-SSRs, 5 SNPs and 1 ALP . The YI map indicated that most of the ssSNP and meaSNP markers considered were associated with other linkage groups. The partial linkage map around the ms1 locus was constructed for the TO-S pedigree is shown on the TreeGenes database .A partial linkage map around the s Figure . Three mms1 gene was hrmSNP970_sf, which was separated from it by only 0.5 cM in the TO-S pedigree.The marker most tightly linked to the Picea glauca [P. mariana [Pinus taeda [C. japonica (84.9%), although the evaluation criteria used in this work differed slightly from those used in previous studies. These values are slightly lower than that obtained in crops, e.g. 91.3% in barley [The success rate for SNP genotyping (i.e. the SNP conversion rate) with the GoldenGate assay was 81.6% in a glauca , 82.0% i mariana  and 66.9us taeda ; these vn barley , 89.0% in barley  and 92.0n barley . The lown barley . NonetheC. japonica featuring 1262 markers. The observed map length (oG) was 1405.2 cM, and the average marker interval was only 1.1 cM. A total of 11 distinct linkage groups were identified, which corresponds to the haploid number of chromosomes in C. japonica. The number of mapped markers in C. japonica was higher and the average interval between markers was smaller than those reported for other conifers . The observed and expected genome coverages were 98.2 (oC) and 100.0% (eC), respectively. It thus appears that the C. japonica linkage map developed in this work is almost saturated.The genome length in P. mariana [C. japonica genome was non-random. This suggests that there are marker-rich and marker-poor regions in the C. japonica linkage map. If markers were distributed equally over the genome, they would be expected to concentrate in regions of suppressed recombination such as centromeric regions, in which the map distance between markers becomes shorter than their physical separation, as reported for barley [C. japonica linkage map created in this work might thus reflect the distribution of genes in this species, since most of the mapped markers were based on ESTs. To fill in the gaps, it would be desirable to add data on markers for non-coding regions such as genomic microsatellite markers or random genetic markers such as AFLPs.As was shown to be the case in  mariana , chi-squr barley  and maizr barley . Feuiller barley  suggestems1 gene. It is possible that this is because the genes associated with male gametophyte development and male sterility in other species are not closely related to the ms1 gene in C. japonica. Alternatively, the putative homologs detected in this study might not be orthologous to those involved in male gametophyte development in other species; instead, they may be paralogous, with similar domains. While one would expect that genes involved in male gametogenesis would exhibit differential expression in male-sterile and male-fertile individuals, and that this difference would be detectable by analysing the microarray expression data, it is possible that the difference may be statistically insignificant owing to the limitations of the methodology. Future studies in this area should aim to address these issues. In addition to the problems related to the identification of a specific sequence corresponding to ms1, it should be noted that 217 (80.4%) of the 270 candidate genes could not be located on the linkage map due to a lack of polymorphism in the parents of the YI pedigree or because the sequence data was very complex. The efficiency of the mapping could potentially be improved to address these issues by designing primers that span exon/intron junctions. Once suitable candidate genes have been identified, they should be exploited in studies on the TO-S pedigree.It seems unlikely that any of the ssSNP and meaSNP markers identified in this work correspond to the ms1, we were able to determine that it is located on LG9 and to construct a partial linkage map around the ms1 locus , the male-sterility or -fertility of 96.6% of the 205 individuals in the TO-S pedigree could be accurately determined. In theory, this will facilitate the selection of individuals that are heterozygous for the ms1 gene without needing to create control crosses. The identification of these two adjacent markers will increase the viability of using MAS in the TO-S pedigree. This will make it possible to perform early selection of germinated seedlings, which could be useful in that it would reduce the expenditure of time, labour, and space on the growing of seedlings.Although we were unable to assign a specific sequence for s Figure . To our eG; 1430.6 cM), the average physical distance per cM would be roughly 7.5 Mb. This physical distance per cM in C. japonica suggests that the closest markers we identified may be around 3.8 Mb from ms1 locus. As such, genome walking would be impractical with current methods, but it would be feasible to isolate the ms1 gene by BAC walking if markers lying within 0.1 cM of the target could be identified. We have constructed a BAC library for C. japonica that covers 4 times as much of the genome as the marker libraries employed in this work and relates to a mapping population with a large number of individuals (unpublished data). The construction of a more dense linkage map around the ms1 gene will greatly facilitate its isolation or that of more tightly-linked markers.While the two markers will be very useful for TO-S pedigree, it will be necessary to identify the target gene itself to do MAS in other pedigrees. To this end, candidate gene approaches may become more useful as the amount of information on the species increases and techniques improve. An alternative approach for identifying target genes is genome walking using a BAC library. The likelihood of isolating a gene using genome walking depends on the physical distance (per cM) in the target species. On the basis of the estimated genome length calculated from the recombination rates observed in this study , indicating that the linkage map developed in this work is almost saturated. The distribution of the mapped loci on the linkage map for the YI pedigree was not random, as demonstrated by a chi-squared test .We have constructed a high-density linkage map for C. japonica is known to be controlled by a major recessive gene (ms1). We mapped the ms1 gene to the 9th linkage group and constructed a partial linkage map around the ms1 locus using information from the dense map constructed in this work. A total of 42 markers were located on this partial linkage map. A marker, hrmSNP970_sf that is tightly linked to the ms1 gene was identified; the two are separated by only 0.5 cM. The markers linked to ms1 identified in this work will facilitate the early selection of male-sterile trees, which should prove useful in C. japonica breeding programs.Genetic male-sterility in C. japonica; KS: providing the funding for the screening and genotyping of the candidate gene; YT: supervising the project, providing the funding for genotyping using the SNP array. All authors read and approved the final manuscript.YM: preparation of manuscript, construction of linkage map, experimental work and genotyping for EST-SSR, HRM and SNP markers; TUI: SNP discovery, assistance with HRM analysis; KU: SNP discovery; NF: Screening of the candidate gene; MS: development of the TO-S mapping population; SU: design of primers for KASPar assays; AM: assistance with sequencing; NT: provision of segregation data for RFLP, CAPS, SSR and ALP markers; HT: discovery of male-sterile tree in Candidate markers developed from genes with significant similarity to genes related to male gametophyte development and male sterility in other plant species.Click here for fileCandidate markers developed from genes differently expressed between male-fertile and male-sterile individuals.Click here for filePrimer information of hrmSNP markers. The method used for marker development has previously been reported by Ujino-Ihara et al. (2010).Click here for filems1) in C. japonicaLinkage association between microsatellite markers and a male-sterile gene (.Click here for filePrimer information for BioMark 48.48 Dynamic Array (Fluidigm) using KASPar assays.Click here for fileResults of marker distribution analysis in each marker interval.Click here for file"}
+{"text": "Amphilophus spp.) have been described so far and have formed repeated adaptive radiations in several Nicaraguan crater lakes. Here we apply double-digest restriction-site associated DNA sequencing to obtain a high-density linkage map of an interspecific cross between the benthic Amphilophus astorquii and the limnetic Amphilophus zaliosus, which are sympatric species endemic to Crater Lake Apoyo, Nicaragua. A total of 755 RAD markers were genotyped in 343 F2 hybrids. The map resolved 25 linkage groups and spans a total distance of 1427 cM with an average marker spacing distance of 1.95 cM, almost matching the total number of chromosomes (n = 24) in these species. Regions of segregation distortion were identified in five linkage groups. Based on the pedigree of parents to F2 offspring, we calculated a genome-wide mutation rate of 6.6 \u00d7 10\u22128 mutations per nucleotide per generation. This genetic map will facilitate the mapping of ecomorphologically relevant adaptive traits in the repeated phenotypes that evolved within the Midas cichlid lineage and, as the first linkage map of a Neotropical cichlid, facilitate comparative genomic analyses between African cichlids, Neotropical cichlids and other teleost fishes.Cichlid fishes are an excellent model system for studying speciation and the formation of adaptive radiations because of their tremendous species richness and astonishing phenotypic diversity. Most research has focused on African rift lake fishes, although Neotropical cichlid species display much variability as well. Almost one dozen species of the Midas cichlid species complex ( Cichlid fishes are a well-known evolutionary model system for studying the mechanisms of speciation and the formation of adaptive radiations. African Rift Lake cichlids in particular display an astonishing variability in phenotypes and comprise an enormous number of species, which makes Cichlidae one of the most species-rich families in vertebrates . Becausee.g., Because of their geographic distribution, which includes the two Nicaraguan Great Lakes and the independent colonizations of at least eight crater lakes of Nicaragua, Midas cichlid communities comprise repeatedly evolved populations at different evolutionary stages of local adaptation and speciation . The twoin situ in the form of sympatric species by ecological speciation  whether the same type of genes are involved in the process of speciation . Behavioral and morphological characters that have been proposed to be responsible for the elevated speciation rate of cichlids include parental care, sexual selection, functional design, and phenotypic plasticity and polymorphisms  within or between individuals. Here, we apply a newly developed double-digest RADSeq (ddRADSeq) protocol  and a wild-caught female A. astorquii  were reared separately in the laboratory until sexual maturity and then crossed to obtain F1 hybrid progeny. The offspring were raised to sexual maturity in a community tank to allow full-sib pairing. When a pair formed, they were moved to a separate tank and after spawning naturally their eggs were harvested and reared in another tank. F2 offspring were killed when they reached approximately 2 cm total length to get enough tissue for DNA extraction. The F1 pair that had the greatest number of F2 offspring was chosen for construction of the linkage map.A wild-caught male 1 hybrid pair and 347 F2 individuals using the QIAGEN DNeasy Blood & Tissue Kit following the manufacturer\u2019s instructions including RNase A treatment. Then, 1 \u03bcg of DNA template from each individual was double-digested using the restriction enzymes PstI-HF (20 units/reaction) and MspI (20 units/reaction) in one combined reaction for 3 hr at 37\u00b0. Subsequently, each fragmented sample was purified using the QIAGEN MinElute Reaction Cleanup Kit, eluted in 20 \u03bcL of Elution Buffer (EB), and quantified. Fragments were then ligated to P1 adapters that bind to PstI-HF created restriction sites and P2 adapters binding to overhangs generated by MspI . In each reaction, \u223c0.4 \u03bcg of DNA, 1 \u03bcL of P1 adapter (10 \u03bcM), 1 \u03bcL of P2 adapter (10 \u03bcM), 1 \u03bcL of T4 ligase , 4 \u03bcL of 10\u00d7 T4 ligation buffer, and double-distilled water were combined to a total volume of 40 \u03bcL. The ligation was processed on a polymerase chain reaction (PCR) machine using the following conditions: 37\u00b0 for 30 min, 65\u00b0 for 10 min, followed by a decrease in temperature by 1.3\u00b0 per minute until reaching 20\u00b0.Genomic DNA was extracted from the two parents (\u201cP generation\u201d), the F1 DNA revealed that the pilot analysis gel size range of 250\u2212500 bp resulted in sequencing too many fragments and therefore a range of \u223c330\u2212400 bp was chosen for subsequent libraries. Manual size selection from agarose gel electrophoresis was performed on the library containing the parental and F1 samples and samples were then cleaned using the QIAGEN MinElute Gel Purification Kit and eluted in 10 \u03bcL of EB. Fifty F2 individuals were pooled per library and size selected using Pippin Prep technology . Seven PCRs  were performed in replicate on size-selected fragments  \u00d7 10, 72\u00b0 (300 sec). Gel electrophoresis was performed on pooled products to remove remaining oligo dimers and other contaminants. Fragments ranging from 330 to 400 bp were excised from a gel after electrophoresis and cleaned using the QIAGEN MinElute Gel Extraction Kit and eluted in 10 \u03bcL of EB. The eight libraries  were diluted to 1 nM and sequenced single end to 150 bp length with the Illumina TruSeq SBS Kit v5 in eight lanes on an Illumina Genome Analyzer IIx (GeCKo: Genomics Center University of Konstanz) over two consecutive runs, each containing a single PhiX control lane.After we ligated each individual\u2019s DNA to the adapters, samples were pooled and size-selected from an agarose gel. A preliminary Illumina sequencing run containing the parental and Fi.e., RADSeq tags) was performed using Stacks  within individuals, followed by building a catalog of loci present in the two parents and then mapping all stacks from the offspring to the parental catalog and finally creating the offspring genotypes for those loci. Multiple SNPs in one RADSeq stack were treated as a single polymorphic marker . The parameters were optimized and set as follows: the minimum amount of identical reads to create a stack in each parent and progeny was set to three  the number of mismatches allowed between loci from the parents was set to three (\u2212n 3), and highly repetitive RAD tags were removed or disjointed (\u2212t), other parameters were set to default.Bioinformatic processing of the reads  containing alleles from the F1 pair that could be matched with confidence to each parental unit and did not deviate from Mendelian segregation ratios. Markers under segregation distortion (P = 0.05\u22120.001) were added step by step from the less significant to the most and their effect on the map was checked. Marker groupings were calculated using an independent logarithm of odds (LOD) score of 6.0, but the number of calculated linkage groups did not change between LOD scores of 6.0 to a more stringent LOD score of 10.0. Each linkage group was calculated using the regression mapping algorithm and Kosambi\u2019s mapping function , stickleback , and medaka  using blastn searches and an e-value cut-off of 10\u221215 for tilapia  and 10\u221210 for stickleback and medaka. Tilapia was used as a representative of the African cichlid lineage, whereas stickleback and medaka were used as phylogenetically close teleost outgroups to the cichlids. If a marker mapped to more than one locus it was either excluded or, if the difference in the bit score between best hit and second-best hit was more than 20, the best hit was retained. Markers that mapped to unlinked scaffolds were excluded from the synteny analyses. Synteny was calculated in percentage of markers mapping to a single orthologous linkage group in contrast to those mapping to different linkage groups. Markers that solely map to a single linkage group, i.e., if no other markers mapped to that same linkage group, were excluded.All RAD markers used in the linkage map were indexed with the respective linkage group and position and compared with the genomes of the fishes tilapia  loci and checked for SNPs only present in the progeny. The coverage for a locus to be included in the analysis was set to at least 8\u00d7 . In addition, an empirical distribution of allele-to-allele coverage ratio was estimated from all heterozygous RAD markers in the F2 generation. New mutation candidates in the progeny were checked against this distribution, and only those mutations that had an allele ratio within the 95% confidence interval of the loci present in the empirically estimated distribution were retained. This step yielded loci with a ratio lower than 0.5 or greater than 2.0 to be discarded and was performed to exclude candidate loci with odd allele-to-allele ratios . For example, a locus with a mutation candidate and 10 reads in total, including seven reads of the original allele and three reads of the allele with the new mutation, would be excluded because the allele-to-allele ratio is lower than 0.5.A. astorquii individual, a SNP frequency of 0.80 SNPs/kb  and for A. zaliosus 0.89 SNPs/kb  were detected (Table S2). Of 118,213 loci that were scored in both parents, 12,429 were polymorphic with one to three SNPs, with a total of 15,035 SNPs and a frequency of 1.2 SNPs/kb. Of those loci that were polymorphic, 18.5% were fixed between parents. A total of 38,203 loci were detected in the parents and were present in at least 90 of the 343 F2 individuals. The number of loci shared between parents and F2 progeny was smaller than that between parents and F1 because the F2 progeny gel size range was narrower and therefore included fewer fragments. A total of 3,184 loci that were shared between parents, F1 and F2s were polymorphic and retained for further analyses. Of these loci, 755 were informative  and could serve for the construction of the linkage map .A total of 254 million reads were used, with an average of 31.8 million reads per library (SD: 3.1 million reads). On average, approximately 588,000 reads were obtained per individual  with 15\u00d7 coverage per read (SD: 5.1\u00d7). Within the parental kage map . Because2 individuals genotyped with ddRADSeq, four were excluded because of low coverage . From linkage mapping analysis, 25 linkage groups were identified with a total size of 1427 cM and an average marker spacing distance of 1.95 cM (Table S4). Linkage groups were between 51.5 and 77.1 cM in length except for the two small linkage groups LG24 and LG25 with 2.5 and 2.0 cM, respectively. With the exception of two small linkage groups with only two and three linked markers, all linkage groups consisted of at least 10 markers . On average, a typical linkage group comprised 30 markers (Table S4). A total of 17 markers  were completely linked (no recombination was observed between them). The maximum distance between two markers on a single linkage group was 30.7 cM on LG20.Of the 347 FP-value between 0.05 and 0.001 (Table S3). Markers that were distorted significantly with P < 0.0001 were not considered because ratios that are too skewed might create inaccurate distances and false linkages (Table S3).Almost 10% of all markers (74 of 755 markers) deviated from Mendelian segregation ratios with a Table S5) or small linkage group sizes (due to insufficient data in tilapia genomic resources) in tilapia. In addition to the linkage groups that share homologous markers (conserved synteny), the respective position of these markers (linkage order) is mostly the same, suggesting retained syntenic relationships among those loci . In general, however, it is clear that overall levels of synteny between these two cichlid lineages are quite highly conserved, with 86% of all markers mapping to a single orthologous linkage group when those linkage groups containing two or more markers are considered.The vast majority of markers present in any given particular Midas cichlid linkage group also map to a single orthologous linkage group in tilapia , a basal African cichlid that last shared a common ancestor at least 60\u221290 million years ago . Four liose loci . HoweverAs expected, fewer homologous markers were found for stickleback (44 markers) and medaka (38 markers) compared with tilapia and the Midas cichlid . The sti1 progeny and five new mutations in the F2 progeny were found. The same mutation was observed in two different F2 individuals and was therefore only counted as one mutation. The F1 mutation rate was calculated to 7.4 \u00d7 10\u22128 and the F2 5.8 \u00d7 10\u22128, leading to an average mutation rate of 6.6 \u00d7 10\u22128 mutations per nucleotide per generation.Two new mutations in the Fe.g., Here, we report the first linkage map of a Neotropical cichlid species. It will serve as important addition to the genomic resources for cichlid fishes and facilitate in searching for genes that contribute to ecological and phenotypic diversity in Midas cichlids in particular and cichlids more generally. With a total of 25 linkage groups, the present linkage map closely matches the number of chromosomes (n = 24) in the Midas cichlid  will be lost. Accordingly, while maximizing the number of loci, more missing data are accepted, which might result in incorrect distances in linkage map construction and in QTL analyses  from expected Mendelian segregation ratio. Single markers deviating from the expected ratio might result from genotyping errors, though blocks of deviating linked markers as on LGs1, 6, 15, 23, and 24 probably have a biological basis and might be caused by segregation distortion genes , including several interchromosomal rearrangements that occurred after their split . Although the data for genomic comparisons between the Midas cichlid and the noncichlid fish species stickleback and medaka are limited because of the large phylogenetic distance of more than 100 million years since these lineages last shared a common ancestor (u) estimation of 6.6 \u00d7 10\u22128 per site per generation in Midas cichlids indicates a somewhat elevated mutation rate compared to estimates from other vertebrates . A mutaper year , which, ee e.g., . It mighCichlid fishes represent wonderful examples for phenotypic diversity and convergence, rapid speciation, and adaptive radiation ("}
+{"text": "The zinc finger transcription factor Snail1 regulates epithelial to mesenchymal transition, repressing epithelial markers and activating mesenchymal genes. Snail1 is an extremely labile protein degraded by the cytoplasmic ubiquitin-ligases \u03b2-TrCP1/FBXW1 and Ppa/FBXL14. Using a short hairpin RNA screening, we have identified FBXL5 as a novel Snail1 ubiquitin ligase. FBXL5 is located in the nucleus where it interacts with Snail1 promoting its polyubiquitination and affecting Snail1 protein stability and function by impairing DNA binding. Snail1 downregulation by FBXL5 is prevented by Lats2, a protein kinase that phosphorylates Snail1 precluding its nuclear export but not its polyubiquitination. Actually, although polyubiquitination by FBXL5 takes place in the nucleus, Snail1 is degraded in the cytosol. Finally, FBXL5 is highly sensitive to stress conditions and is downregulated by iron depletion and \u03b3-irradiation, explaining Snail1 stabilization in these conditions. These results characterize a novel nuclear ubiquitin ligase controlling Snail1 protein stability and provide the molecular basis for understanding how radiotherapy upregulates the epithelial to mesenchymal transition-inducer Snail1. Expre(GSK-3\u03b2) . Snail1 and Zeb2 . Contrarand Zeb2 . Both liand Zeb2 . Mdm2, aand Zeb2 ,9.O-glycosylation  for proteasome degradation ,21. We hCell lines used in this study have been previously described ,23. WhenThe pcDNA3-Snail1-HA and GFP-Snail1 constructs  have been previously described . The pcDShort hairpin RNAs (shRNAs) against all the described F-box ubiquitin ligases were purchased from Sigma (MISSION\u00ae shRNA plasmids) and used to produce lentiviral particles . TransduEscherichia coli. pFastBac baculovirus vectors coding for HA-Cullin1, 6\u00d7His-Skp1, Roc/Rbx1, 3\u00d7Flag-FBXL5, 6\u00d7His-Snail1-HA and GST-Snail1-HA were used to generate recombinant bacmids with the Bac-to-Bac\u00ae system (Invitrogen). Isolated bacmids were transfected in Sf9 cells using the Cell Fectin II reagent (Invitrogen) to generate high-titer baculovirus. Infection of Sf9 cells was performed for 2\u20133 days and cell pellets were snap-frozen and stored at \u221280\u00b0C. Purification of the SCFFBXL5 or SCFFBXL14 complexes was done after infection of 6xHis-Skp1, HA-Cullin1, Rbx1 and 3xFlag-FBXL5 or FBXL14-HA baculovirus at ratio 1:2:1:1, respectively. Lysis and purification was done essentially as previously described , the protocol was previously described , 10 \u03bcg ubiquitin, 40 ng E1 , 200 ng E3  in 20 \u03bcl reaction buffer containing 10 mM HEPES, pH 7.5, 10 mM KCl, 100 mM NaCl, 1.5 mM MgCl2, 1 mM DTT and 25 mM ATP. After 1\u20132.5 h at 30\u00b0C, reactions were stopped with 5\u00d7 Laemmli buffer and boiled at 95\u00b0C for 5 min. For in vivo ubiquitination assays using anti-FK2 (Millipore) antibody, cells were transfected and treated with 100 \u03bcM FAC and 10 \u03bcM MG132 for 4 h. The cytoplasmic fraction was washed out and the pellet resuspended in RIPA buffer  and the protocol continued as previously described  as baits. Samples were eluted with 30 \u00b5l 2\u00d7 Laemmli buffer, boiled for 5 min at 95\u00b0C and analysed by western blot.HEK293T cells were transfected with the indicated plasmids and pull-down was done as previously described . For pulRNA was extracted and retro-transcribed as described before . AnalyseInfection of pBabe-Snail1-HA or 6xMyc-FBXL5 was carried out using HEK293 Gag-pol cells .32P-labelled probe corresponded to E-box 1 in the E-cadherin promoter (positions \u221264 to \u221292) with the following sequence: 5\u2032-GGCTGAGGGTTCACCTGCCGCCACAGCC-3. The mutated E-box 1 used was 5\u2032-GGCTGAGGGTTAACCTACCGCCACAGCC-3\u2032 (mutated bases are underlined).Electrophoretic mobility shift assay (EMSA) was performed as described before . The 32Pn = 7). Infection of shRNAs against eight F-box proteins  upregulated Snail1 protein levels (at least by 2-fold) compared with parental cells or cells infected with a control shRNA . We repeated the same screening in MCF7 cells, a breast cancer cell line that expresses low Snail1 levels and presents an epithelial phenotype. Only depletion of FBXL5 . FBXL5-depletion promoted the acquisition of a more mesenchymal phenotype that was reversed by transfection of an siRNA against SNAIL1, suggesting that Snail1 protein stabilization is responsible for the phenotypic change in these cells . Therefore, taking into consideration the consistent and remarkable effect observed on Snail1 protein stability after FBXL5 depletion in several cell lines we focused our attention on this ubiquitin ligase.To identify new F-box proteins that degrade Snail1, we infected SW620 colon cancer cells with a shRNA library targeting 53 human F-box proteins. We chose SW620 cells, as they express moderate Snail1 levels . After iof FBXL5 A caused of FBXL5 B. In botof FBXL5 C. SNAIL1of FBXL5 C. To disSupplementary Figure S2A) and its sensitivity to FBXL5 shRNA (see later in the text); and also a rabbit FBXL5 pAb capable of immunoprecipitating FBXL5. Snail1 was detected in complexes immunoprecipitated with the FBXL5 antibody and not with an irrelevant antibody . Surprisingly, subcellular fractionation of cells revealed that endogenous FBXL5 presents a strong nuclear localization in both MCF7 and RWP-1 cells, unlike the other Snail1-E3 ubiquitin ligases, which have been localized in the cytosol , further validating the screening and antibody specificity. Presence of FBXL5 in the nucleus was also confirmed by confocal immunofluorescence in RWP-1 and MCF7 cells: both the endogenous and ectopic FBXL5 proteins were detected in the nucleus . Endogenous FBXL5 immunofluorescent detection was also decreased by sh FBXL5 . Ectopic FBXL5 was localized both in the nucleus and the cytosol in basal conditions and after iron supplementation with FAC . Interestingly, the nucleus export inhibitor LMB caused the nuclear accumulation of FBXL5  suggesting that FBXL5 is being actively exported from the nucleus to the cytosol. Finally, we detected a strong interaction between ectopic Snail1-HA and Myc-FBXL5 in the nuclear fraction of HEK293T cells in the presence of the proteasome inhibitor MG132  destabilized FBXL5 protein and strongly upregulated both endogenous and exogenous Snail1 in RWP-1 cells F. ReintrSupplementary Figure S3A). Finally, incubation of RWP1 cells with LMB, which blocks Snail1 nuclear export , further supporting the conclusion that this modification takes place in the nucleus.Because FBXL5 is localized in the nucleus, we investigated whether it is capable of ubiquitinating Snail1 in this subcellular compartment. As shown in r export , did notin vitro. For this we purified Snail1 and the SCFFBXL5 complex from Sf9 insect cells infected with different baculoviruses corresponding to Flag-FBXL5, His-Skp1, HA-Cullin1 and Rbx1 . Purified SCFFBXL5 ubiquitinated Snail1 only when E1 and E2 enzymes, ubiquitin and ATP were added to the reaction  was as effective as wild-type (wt) ubiquitin for in vitro polyubiquitination of Snail1 . Interestingly, we have previously identified Lys146 as one of the residues in Snail1 required for FBXL14- and \u03b2-TrCP1-mediated degradation . We confirmed the ubiquitination of the CT lysine by in vivo ubiquitination assays, although Snail1CT was modified to a lower extent than the full-length protein . Presence of SCFFBXL5 in the EMSA did not substantially decrease the association of Snail1 with the DNA, suggesting that the Snail1 affinity for DNA is much greater than for the ubiquitin ligase.The interference in DNA binding was also verified promoter . Snail1 mponents D. Ubiquimponents E. This bSupplementary Figure S5A). This Snail1T203 mutant was resistant to FBXL5 degradation . Moreover, in co-immunoprecipitation assays carried out with extracts from cells where degradation was prevented with MG132, we observed that Snail1 and FBXL5 associated to a similar extent in the presence or absence of Lats2  while it was ubiquitinated by FBXL5 with a similar efficiency as the wt protein .Lats2 stabilization of Snail1 has been associated to Snail1 retention in the nucleus . It is palf-life E, indicaalf-life ] was resSnail1 stability increases after several types of cellular stress such as DNA damage or hypoxia . We chec\u03b2-TrCP1, that acts in the cytoplasm, dependent on previous GSK-3\u03b2 phosphorylation and negatively regulated by the Wnt pathway , mainly to ZnF2, although ZnF1 and ZnF3 are also necessary to achieve a full binding capacity. This Snail1 CT domain is well conserved among the different orthologues . Accordiin vitro ubiquitination data showing that the SCFFBXL5 complex modifies Lys85 and Lys146, both placed in the NT, as well as Lys234, which is located in the CT. However, Lys234 seems to be modified to a lower extent and may not participate in degradation. On the other hand, Lys146 has been shown to be involved in both \u03b2-TrCP1 and FBXL14 degradation ; The authors also acknowledge support from ISCIII/FEDER [RD06/0020/0040] and Generalitat de Catalunya [2009SGR867]; a predoctoral fellowship from ISCIII (to R.V.-C.). Funding for open access charge: Ministerio de Ciencia y Tecnolog\u00eda [SAF2010-16089].Conflict of interest statement. None declared."}
+{"text": "Congenital heart defects (CHD) is the most common cause of death from a congenital structure abnormality in newborns and is often associated with fetal loss. There are many types of CHD. Human genetic studies have identified genes that are responsible for the inheritance of a particular type of CHD and for some types of CHD previously thought to be sporadic. However, occasionally different members of the same family might have anatomically distinct defects \u2014 for instance, one member with atrial septal defect, one with tetralogy of Fallot, and one with ventricular septal defect. Our objective is to identify susceptibility loci for CHD in families affected by distinct defects. The occurrence of these apparently discordant clinical phenotypes within one family might hint at a genetic framework common to most types of CHD.Pempirical\u2009=\u20090.0004) and at chromosome 18 .We performed a genome-wide linkage analysis using MOD score analysis in families with diverse CHD. Significant linkage was obtained in two regions, at chromosome 15 (15q26.3, ELS, SNRPA1, and PCSK6 on 15q26.3, and TCF4 on 18q21.2. The new loci reported here have not previously been described in connection with CHD. Although further studies in other cohorts are needed to confirm these findings, the results presented here together with recent insight into how the heart normally develops will improve the understanding of CHD.In these two novel regions four candidate genes are located: S Congenital heart defects (CHD) refer to abnormalities in the heart structure or function that arise at the fetal stages and affect approximately 1% of newborns [, TGFB-BMP/TGFBR, VEGF/FLT1-FLK1, NODAL/ACVRA-ACVRB and RTK/RAS), signal transduction , and transcription factors that determine the expression of cardio-specific genes . Mutations that show a greater penetrance and that therefore approximate a monogenic inheritance are those affecting transcription factors or genes transcribed by them [Normal heart development involves many regulatory pathways including receptor-ligand interactions  in South Indian cases born to consanguineous parents. However, the linkage finding was not robust in a genetic association follow-up study in a general United States population. Despite the successes over the past few years, much remains unknown about the genetics of CHD. It is believed that much of the missing heritability is likely to be due to rare variants with larger effect sizes as well as epigenetic effects. Their systematic characterization is beyond the means of studies that currently rely on linkage disequilibrium (LD) patterns such as genome-wide association analysis. In such situations taking family information into consideration using a direct mapping approach complemented by segregation techniques will be a useful strategy. For complex diseases linkage analysis is a powerful method for detecting gene effects [However, occasionally, different members of one family are observed to suffer from anatomically distinct defects \u2014 for example, one member with ASD, one with TOF, and one with VSD. These apparently discordant clinical phenotypes arising within one family are difficult to rationalize. Benson et al. observedor et al. reported effects ,19. EspeIn this study we performed a detailed genome-wide linkage analysis using MOD scores and 6 families affected by different CHD to test for a common genetic background among different types of heart defects.Pempirical\u2009=\u2009(0.0005-0.0006), where Pempirical refers to the P value obtained after simulations were performed. The associated parametric model is a recessive mode of inheritance with incomplete penetrance with a probability of 0.27 to be affected for individuals carrying two copies of the disease allele. Incomplete penetrance refers to the failure of individuals who carry the high risk genotype to exhibit the trait. This phenomenon is often observed in complex diseases. Besides different ways of gene action or degrees of expression, incomplete penetrance can arise from further genetic or environmental factors. The second significant region was at chromosome 15  comprising 27 SNPs, all of them with a MOD score of 3.5 and Pempirical\u2009=\u2009(0.0004-0.0005). The best-fitting model points to a recessive mode of inheritance, with a probability of 0.98 to be affected for individuals carrying two copies of the disease allele. It should be noted that the estimate of the disease allele frequency obtained by a MOD-score analysis has the largest variance of all trait-model parameters. Furthermore, in some cases, the estimated disease allele frequency will be markedly higher than the true value. This is due to the fact that specifying a higher disease allele frequency can compensate for a general model misspecification and hence lead to robustness in a multipoint analysis [The genome-wide results for MOD scores are plotted in Figure\u00a0analysis .P value\u2009\u2264\u20090.01). Using this criterion a total number of 304 SNPs  and 211 SNPs  were included in the association analysis for 18q21.2-18q21.3 and 15q26.3, respectively. A total number of 34 SNPs resulted to be significant at a type I error rate of 0.05 on chromosome 18. Nevertheless, none of them was significant after using the Bonferroni correction to adjust for multiple testing. On the other hand, on chromosome 15 a total number of 39 SNPs were found to be nominally significant and only one (rs3784481 at 99749\u00a0kb) was significant after Bonferroni correction .To corroborate these findings, family-based association analysis was performed in these two significant regions with SNPs displaying a MOD >2.5  analysis of individual pedigrees under the best fitted model for the two linked regions. As shown in Table\u00a0SELS, SNRPA1, and PCSK6). In addition, one gene is located in the linkage region identified on chromosome 18 (TCF4).When looking in detail at the region around the significant SNPs on chromosome 15, three genes are found very closely to each other . The action of snRNPs is essential to the removal of introns from pre-mRNA, a critical aspect of post-transcriptional modification of RNA. To date no association has been reported between SNRPA1 and heart defects. However, post-transcriptional modification is one of the multiple mechanisms that regulate the level and activity of transcription factors which are responsible for regulating formation and function of the heart. Perturbations of transcription factor expression and regulation disrupt normal heart structure and function [The function .PCSK6 gene belongs to the subtilisin-like proproteinconvertase family. The members of this family are proproteinconvertases that process latent precursor proteins into their biologically active products. This encoded protein is a calcium-dependent serine endoprotease that can cleave precursor protein at their paired basic amino acid processing sites. Some of its substrates are transforming growth factor beta related proteins, proalbumin, and von Willebrand factor. In 1998, a study reported abnormalities of von Willebrand factor in adults with CHD [PCSK9 gene, is a key regulator of plasma low density lipoprotein cholesterol and has emerged as a promising target for prevention and treatment of coronary heart diseases. A recent genome-wide association study further bolstered the importance of PCSK9 by establishing a link between a nearby SNP and early onset myocardial infarction [The protein encoded by the with CHD . Interesfarction .Pempirical\u2009=\u20090.0005) and the second highest peak at 15q26.3  using MOD-score analysis. In addition, at 15q26.3 a significant association was obtained for a SNP  using family-based association analysis. Imprinting analysis of the data in the present study did not confirm the hypothesis of a possibly imprinted locus. When looking at these two linkage regions in detail, four genes are located there, three very close to each other at 15q26.3 and one at 18q21.2.We have conducted a whole genome linkage analysis for CHD using MOD score analysis. We used a total number of 177,568 SNPs providing a good coverage of the genome in a sample of 6 families of Spanish origin with members affected with anatomically distinct CHD: ASD, VSD, TOF, TGV, and DORV. The aim of this study was the identification of chromosomal regions that are implicated in a possible common genetic framework shared by distinct types of CHD. Two interesting linkage regions were found, on chromosome 15 and 18. The highest linkage peak was obtained at position 18q21.2 . Clearly, recruiting more families with CHD will help to verify this linkage evidence as well as to identify new linkage regions.SELS, SNRPA1 and PCSK6) and one gene at 18q21.2 (TCF4). Only one of these genes, SELS, was previously found to be implicated in cardiovascular disorders. The other three genes reported in this study may be considered as novel candidate loci for CHD.We report in this study two genetic regions linked to CHD predisposition. There are three candidate genes located in the 15q26.3 region very close to each other  and 7 trios (an affected proband and both parents) of Spanish origin were recruited at the University Hospital Son Espases (HUSE) of the Balearic Islands and at the Puerta del Hierro University Hospital in Madrid. Pedigree structures are illustrated in Figure\u00a0All patients had a single tube of blood drawn at the time of enrollment, which was collected centrally for subsequent DNA extraction. Genotyping reactions of 52 individuals were performed at the Spanish genotyping center (CeGen) facilities in Barcelona using the Illumina  Human660W-Quad chip, following the manufacturer\u2019s instructions.Because genotyping errors can lead to incorrect inferences about gene flow in pedigrees and may reduce the effectiveness of linkage analysis, strict quality controls (QC) were performed in our data. First of all, the software Graphical Representation of Relationship Errors (GRR) was used to identify errors in the structure of the pedigrees and to eliminate duplicate samples, monozygotic twins, and unrelated subjects . In one 2 values from haplotype frequencies estimated via the expectation maximization (EM) algorithm of Excoffier and Slatkin [2\u2009>\u20090.20), one of the two SNPs was removed from the analysis. With the resulting data set a genome-wide linkage analysis was performed on a total number of 6 pedigrees with sizes varying from 4 to 13 members. After QC, a total number of 177,568 SNPs was analyzed, with 174,475 from the autosomes, 3078 from the X-chromosome, and 15 SNPs from the pseudoautosomal regions (PAR1 and PAR2).In linkage, when analyzing SNPs in pedigrees where some parental genotypes are missing it is necessary to model for marker-marker LD prior to the analysis. Ignoring such LD can result in severe biases in linkage results . That is Slatkin . In our In parametric linkage analysis (LOD scores) the trait-model parameters, i.e., the penetrances and disease allele frequency, must be specified prior to the analysis. It is well known that the power to detect linkage decreases when the specified model is not sufficiently close to the true one. Consequently, we calculated multipoint MOD scores, i.e., LOD scores that were maximized over trait-model parameters using the program GENEHUNTER-MODSCORE (GH-MOD v3.0) . This prIt has also been observed that the CHD risk for children of mothers with a CHD is consistently higher than for those of fathers with a CHD ,40. One P values need to be calculated. We used 5000 replicates, each one generated with Monte-Carlo simulations of marker alleles under the assumption of no linkage and Hardy-Weinberg equilibrium, using the same pedigree structure, affection status, marker spacing, and allele frequencies as in the real data set. Replicates were analyzed like the original data. The empirical P value was obtained as the proportion of replicates showing a MOD score equal to or higher than the one observed in the real data set.A MOD-score analysis leads to an inflation of the type I error when compared to LOD scores calculated under a single parametric model. Therefore, in the context of MOD-score analysis, significance criteria for LOD scores cannot be applied without correction. To assess significance for a MOD score empirical We computed MOD and MOD-imprinting for all autosomes. The MOD score has not been implemented so far for the X-chromosome. Therefore, we performed LOD-score analysis assuming different genetic models for the X-chromosome, ranging from completely dominant to completely recessive modes of inheritance. In addition, the PAR1 and PAR2 were also investigated. Genetic markers in the PARs follow the same inheritance pattern as autosomal markers, becoming progressively more sex-linked  as they approach the pseudoautosomal boundary. When using multipoint linkage analysis in the PARs, it is crucial to use sex-specific maps since there exists a marked difference in recombination frequencies between males and females . We usedP values obtained by the family-based association analysis were adjusted using Bonferroni correction  to account for multiple testing. We used an overall type I error rate of 0.05.Finally, only in those regions where the most significant linkage signals were obtained, to corroborate and narrow the significant regions, family-based association analysis together with the trio families was performed using the LAMP software . LAMP usThe authors declare that they have no competing interests.AF carried out all statistical analyses, interpretation of the results and wrote the manuscript. CB carried out all the simulation analyses, prepared the figures and edited the manuscript. EP carried out the SNP-array DNA preparation and partial analysis. FGA, MADLF, JT, LAP and PGP characterized clinically heart defects of patients. JR characterized clinical dysmorphology and evaluated inheritance. KS critically revised the manuscript and all statistical content. DHS provided samples and revised the manuscript critically for important molecular content. All authors gave final approval of the version to be published."}
+{"text": "Five lod scores greater than 3.0 were observed assuming heterogeneity. The greatest were among families with mean age of diagnosis less than 50 years at 4q21.1  and among all families at 12q24.32 . Among families with four or more affected individuals and among clinic-based families, a common peak was observed at 15q22.31 . Analysis of families with only two affected individuals yielded a peak at 8q13.2 . These previously unreported linkage peaks demonstrate the continued utility of family-based data in complex traits and suggest that new CRC risk alleles remain to be elucidated.A substantial proportion of familial colorectal cancer (CRC) is not a consequence of known susceptibility loci, such as mismatch repair (MMR) genes, supporting the existence of additional loci. To identify novel CRC loci, we conducted a genome-wide linkage scan in 356 white families with no evidence of defective MMR -high tumors, or no evidence of linkage to MMR genes). Families were ascertained via the Colon Cancer Family Registry multi-site NCI-supported consortium (Colon CFR), the City of Hope Comprehensive Cancer Center, and Memorial University of Newfoundland. A total of 1,612 individuals (average 5.0 per family including 2.2 affected) were genotyped using genome-wide single nucleotide polymorphism linkage arrays; parametric and non-parametric linkage analysis used MERLIN in  MLH1, MSH2, MSH6 and PMS2Colorectal cancer (CRC) is the third most common cancer and the third leading cause of cancer death in the United States. Approximately 141,210 new cases and 49,380 deaths from CRC were expected in the United States in 2011 DUSP10) MYNN) EIF3H) BMP4) CDH1) SMAD7) RHPN2), 20p12.3 (rs961253) LAMA5) To identify novel loci, case-control association studies and family-based linkage analyses serve as complementary approaches. At least fifteen, common low-penetrance risk alleles have emerged from genome-wide association studies (GWAS) including at 1q41  CRC families to date and suggests novel regions with evidence of high-penetrance loci.All participants gave written informed consent. Ontario Cancer Research Ethics Board, University of Southern California Institutional Review Board, University of Melbourne Institutional Review Board, University of Hawaii Institutional Review Board, Mayo Clinic Institutional Review Board, Fred Hutchinson Cancer Research Center Institutional Review Board, Memorial University of Newfoundland Institutional Review Board and City of Hope Institutional Review Board approved protocols.A total of 578 linkage-informative families were identified having at least two affected individuals diagnosed with invasive CRC in sibling, half-sibling, cousin, grand-parental, or avuncular pairs The majority of families (N\u200a=\u200a480) were from the Colon Cancer Family Registry multi-site NCI-supported consortium (Colon CFR) ascertained between 1997 and 2007 by Cancer Care Ontario , a University of Southern California Consortium , a University of Melbourne Consortium , the University of Hawaii , the Mayo Clinic , and the Fred Hutchinson Cancer Research Center  Clinic-based families from a City of Hope consortium (N\u200a=\u200a59) were recruited between 1998 and 2005 at the City of Hope , Tufts University , the University of Pittsburgh , Northwestern University , the University of Wisconsin , Vanderbilt University , the University of South Florida/Moffitt Cancer Center , Maine Medical Center , and Rose Medical Center . White CRC cases older than 18 years of age, who had at least one living sibling diagnosed with CRC, were enrolled. Blood samples, pathology reports, and a brief questionnaire focused on ethnicity and family history were collected on all cases.Population-based and clinic-based families (N\u200a=\u200a39) from Newfoundland and Labrador, Canada were obtained at Memorial University of Newfoundland as previously described We genotyped all available affected individuals within each family, as well as key unaffected individuals, including siblings, children, and spouses of deceased affected individuals; parents of affected siblings; grandparents of affected cousins; and other individuals useful for estimation of phase 2>0.10)  in order to minimize false-positive linkage findings (Hardy-Weinberg equilibrium (HWE) testing relied on mean p-values from exact testing of 100 random samples of one individual per pedigree. SNPs were excluded with unknown genetic position (n\u200a=\u200a147) (build 36.3), call rate <95% , minor allele frequency (MAF) <1% (n\u200a=\u200a377), HWE p-value<0.001 (n\u200a=\u200a17), duplicate concordance <95% (n\u200a=\u200a10), or Mendelian error in >2% of families (n\u200a=\u200a4). We also excluded SNPs to reduce LD . Thus, 356 white families with no evidence of MMR deficiency were included in the analysis.MMR proficiency was evaluated using MSI testing, immunohistochemical (IHC) analysis, and LOD scores at known MMR loci. MSI testing of Colon CFR and Newfoundland families was performed on multiple family members using paired normal and tumor DNA isolated from formalin-fixed, paraffin-embedded (FFPE) material all statistics a priori based on presumed genetically relevant characteristics. Thus, family groups were based on mean age at diagnosis , ascertainment scheme , and number of affected individuals . Likelihood ratio testing evaluated heterogeneity of linkage across the independent subsets of each subgroup factor .Multipoint parametric and nonparametric linkage analyses used MERLIN version 1.1.2 st degree relative with CRC, respectively) and 997 controls from population-based collections of the Colon CFR who were genotyped using the Illumina 1M/1M Duo SNP array, as described previously In key regions identified by linkage analysis, we also performed association testing among an additional 1,136 cases . The mean age at diagnosis was 59.7 years  and 56.2 years  among population- and clinic-based families, respectively. The majority of families had two affected members (56%) and an older (>50 years) mean age at diagnosis (84%). MSI data were available on 224 families (209 MSS and 15 MSI-L), and IHC data were available on 255 families and showed no evidence of MMR deficiency . Both MSNAAA encoding N-acylethanolamine acid amidase. The linkage region, defined as a 1-HLOD support interval, spanned 16.0 cM (8.7 Mb). This peak was not seen in older mean age at diagnosis families  region. Three suggestive regions in analysis of all families (HLOD>2.0) were seen on chromosomes 4, 15, and 17  at 12q24.32 with a maximum dominant HLOD of 3.60  and an estimated 48% of families linked . The pea, and 17 ; Table 3MEGF11 which encodes multiple EGF-like-domains 11.Additional linkage peaks with HLODs just over 3.0 were observed on chromosome 15q22.31  among 67 families with four or more affected individuals and among 88 clinic-based families . Among fLOC100129096, \u03b1\u200a=\u200a0.51, NPL\u200a=\u200a0.08). Linkage assuming a recessive mode of inheritance is consistent with an affected sibling pair family structure. This region was not highlighted in analysis of larger families (MSI2 which encodes musashi homolog 2 (Drosophila). Additional linkage results are provided in An additional HLOD over 3.0 was observed in recessive analysis of 200 families with only two affected family members , Table 2families , althougfamilies ; the pea\u22125; family history positive OR 1.82, p\u200a=\u200a1.0\u00d710\u22124)  and controls (N\u200a=\u200a997). In 4q21.21, which showed evidence of linkage in younger age at diagnosis families, the linkage SNP rs10518142 showed no evidence of association; however, rs12643573, which is 2 cM downstream, showed some evidence of association  . At rs14NAAA. NAAA encodes an N-acylethanolamine-hydrolyzing enzyme and is shown to be expressed in variety of human tissues including colon NAAA are members of the chemokine family that are clustered in 4q12-21 region. The CXC chemokines modulate tumor behavior by regulation of angiogenesis, activation of a tumor-specific immune response, and direct stimulation of tumor proliferation in an autocrine or paracrine fashion Results of this genome-wide linkage scan provide strong evidence for four previously- unreported CRC susceptibility loci. Notably, we identified a region at 4q21.1 among families with younger mean age at diagnosis (dominant HLOD\u200a=\u200a4.51) and estimated that 84% of these families were linked. The 1-HLOD-support interval of this region, 16 cM (139 cM\u2013155 cM) spanning 8.7 Mb, contains multiple known genes including TMEM132C, SLC15A4, GLT1D1, and TMEM132D), four hypothetical genes , and one microRNA (MIR3612). The four known genes in this region are conserved in dog, mouse, and chicken and, in some cases, zebrafish and Arabidopsis. One of these transmembrane proteins (TMEM132D) is known to be expressed in mature oligodendrocytes GLT1D1 (glycosyltransferase 1 domain containing 1) in humans. Members of the SLC15 (solute carrier family 15) family are electrogenic transporters of short-chain peptides into a variety of cells MEGF11 and RAB11A. Very little is known about MEGF11RAB11A is a RAS oncogene family member expressed in tumor cell lines and suggested to be involved in membrane trafficking SULF1 in this region has been suggested to modulate signaling by heparin-binding growth factors, and downregulation represents a novel mechanism by which cancer cells can enhance growth factor signaling Among all families, evidence for linkage was seen at 12q24.32 (HLOD\u200a=\u200a3.60) with an estimated 48% of families linked to this locus . This region contains four known genes  Self-reported ethnicity and (B) Genetically-inferred ethnicity which shows a circle surrounding the samples analyzed for linkage.(DOCX)Click here for additional data file.Figure S2Genome-wide Linkage Scans of White pMMR Family Groups with HLOD<3.0. Genome-wide linkage scans of three white pMMR family groups with HLODS<3.0. The blue line represents HLODs under the dominant model and the red line represents the HLODs under the recessive model. (A) Family mean age at diagnosis \u226550 years (N\u200a=\u200a298). (B) Families with 3 affected members (N\u200a=\u200a89). (C) Population-based families (N\u200a=\u200a189). (D) Families with unknown ascertainment (N\u200a=\u200a79).(DOCX)Click here for additional data file.Figure S3Regional Association Plot from Population-based Colorectal Cancer Case Control Analysis in 4q21.1. Plot shows the 1-HLOD interval surrounding rs10518142, the peak linkage SNP among families with younger mean age at diagnosis. The x-axis indicates genomic position. The y-axis indicates \u2212log10 association p-values for genotyped SNPs (solid circles) adjusted for age, gender, study site, and four principal components representing ancestry. The most significantly associated SNP is a indicated by a purple diamond. Other than rs10518142 which is indicated by a yellow circle, the colored points indicate the strength of LD with the SNP most associated with CRC risk (purple diamond). Also shown are the SNP build 36 coordinates in kilobases (kb) and a subset of the known genes in the region (below x-axis).(DOCX)Click here for additional data file.Table S1SNP Exclusions and Number of Analyzed SNPs.(DOCX)Click here for additional data file.Table S2Genetic Models Assumed for Parametric Analyses.(DOCX)Click here for additional data file.Table S3Comparison to Prior Colorectal Cancer Linkage Studies.(DOCX)Click here for additional data file."}
+{"text": "P<9\u00d710\u22128) for hypertension. One candidate gene, PDC, was replicated, with rs3817586 on 1q31.1 attaining P\u200a=\u200a2.5\u00d710\u22124 and 2.9\u00d710\u22125 in the within-family tests for DBP and MAP, respectively. We also identified regions of significant linkage for systolic and diastolic blood pressure on chromosomes 2q22 and 5p13, respectively. Further family-based association analysis of the linkage peak on chromosome 5 yielded a significant association  for DBP. This is the first combined linkage and association study of hypertension and its related quantitative traits with Chinese ancestry. The associations reported here account for the action of common variants whereas the discovery of linkage regions may point to novel targets for rare variant screening.Hypertension is caused by the interaction of environmental and genetic factors. The condition which is very common, with about 18% of the adult Hong Kong Chinese population and over 50% of older individuals affected, is responsible for considerable morbidity and mortality. To identify genes influencing hypertension and blood pressure, we conducted a combined linkage and association study using over 500,000 single nucleotide polymorphisms (SNPs) genotyped in 328 individuals comprising 111 hypertensive probands and their siblings. Using a family-based association test, we found an association with SNPs on chromosome 5q31.1 (rs6596140;  Hypertension, which affects nearly 27% of the population worldwide The high heritability (30\u201360%) of blood pressure has prompted extensive efforts to dissect its genetic basis in silico comparison (CHARGE consortium), identified association with SBP or DBP in eight regions. GWAS on young-onset hypertension STK39.Genome-wide association studies (GWAS), using hundreds of thousands of single nucleotide polymorphism (SNP) markers, are the current method of choice for dissecting the genetic basis of complex disease and can provide a potentially more powerful method of identifying the causal variants that underlie susceptibility to common disease, including hypertension, as compared to linkage analysis. From 2007 to 2009, there have been several large-scale GWAS of hypertension. One of them The present study used sibships recruited in Hong Kong, where one member of the sibship is hypertensive and at least one additional member is normotensive, with as many members of the sibship included in the study as available. We employed the Illumina HumanHap610-Quad Array for genotyping all phenotyped sibship members. This unique use of a family-based design allows testing for both linkage and association for loci influencing hypertension and its related quantitative traits. Here, we present the extensive analyses performed using this high-density SNP data and identify independent and novel genome-wide significant results by both linkage and association analyses.This study was approved by the Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster, reference UW06-177 T/1202. Consent was obtained from all participants involved in this study with a Participant Information Statement and Consent Form, which was approved by the ethics committee.The hypertensive subjects were identified in the hypertension and general outpatient clinics of the Prince of Wales Hospital in Hong Kong and referred to the Clinical Pharmacology Studies Unit (CPSU). Hypertensive probands were ascertained, along with as many of their siblings who agreed to participate. Treatment for hypertension was withdrawn to enable the determination of off-treatment blood pressure (BP). Following withdrawal of antihypertensive medication, subjects were monitored weekly to check if their BP did not exceed the upper exclusion limit and to confirm that they were not at risk from severe hypertension. After the four to eight week washout period Genotyping was performed by deCODE Genetics, Inc, using the Illumina HumanHap 610-Quad BeadChip technology, which enables whole-genome genotyping of 620,901 single nucleotide polymorphisms (SNPs). Image intensities were extracted using Illumina's BeadScan software. Data for the BeadChip were self-normalised using information contained within the array. Allele calling was carried out using Illumina's Genotyping Module version 3.3.7 in BeadStudio version 3.1.3.0.The first stage of data-cleaning examined the genetic relationships by checking the genome-wide identity by descent (IBD) sharing for all pairs of individuals in the sample after pruning markers in LD (r2>0.25). Based on the pair-wise IBD estimation, 4 individuals distributed in 4 families were recognized to be half-siblings to the probands and recoded as such, and 6 individuals were excluded because of sample contamination or unknown familial relationships. Then, inbreeding coefficients were calculated, which found five individuals with either strong positive or negative inbreeding coefficient estimates, indicating that these individuals have more or fewer homozygous genotypes than one would expect by chance, reflecting potential sample contamination. Gender status was also checked using the X chromosome data, which detected 10 problematic samples for whom the reported sex did not match the estimated sex and were removed since these discrepancies could not be reconciled with our records. After cleaning, 315 individuals within 111 families remained.p<0.0001) were excluded from analysis. 503,984 SNPs passed quality control (QC) and were used for analysis. The total GCR in remaining individuals was 99.94%, with all chips having a call rate >98%.SNP genotyping quality was evaluated by examining the genotyping call rate (GCR), the minor allele frequency (MAF), and testing for Hardy-Weinberg Equilibrium (HWE). 8400 SNPs with missing rate >5%, 92169 SNPs with MAF<0.01, and 1686 markers which violated HWE , a regression-based method suitable for selected samples r2 equal to 0.25 The presence of linkage disequilibrium (LD) among markers violates an underlying assumption of linkage in multipoint linkage approaches. Appropriate handling of marker LD can avoid such false positive evidence We also employed QTDT p-values against those expected under the null hypothesis are presented in P\u226410\u22125, with one SNP (rs6596140) attaining P<10\u22127 (FSTL4) (>70 kb away)  and corresponding quantile-quantile (QQ) plot of observed g P<10\u22127 . The lockb away) . FSTL4, P\u226410\u22125 for DBP, 1 for SBP and 9 for MAP. For the total association test, we observed 6 independent signals with P\u226410\u22125 for DBP, 1 for SBP and 3 for MAP. Results of the most significant SNPs are presented for the three quantitative traits in p-values against those expected under the null hypothesis suggest an excess of associations with DBP and MAP in within-family tests, as compared to a null distribution of no associations, but no distinct deviations for other tests , this is not too surprising. The observation that each SNP shows stronger association with one trait or another could reflect sampling variation or true differences in the underlying biological basis of the blood pressure traits.Nine of the SNPs listed in r2\u200a=\u200a0.84) were associated with hypertension status, with P<9\u00d710\u22128 and <6\u00d710\u22126, respectively. Five other markers  had moderate P values around .002 to .0004. The other two markers  showed no association.For the nine SNPs that were significant in continuous trait analysis, two markers  were selected instead. In total, this involved examining 101 SNPs. One SNP in PDC on 1q31.1 was significantly associated after Bonferroni correction for multiple testing of 101 SNPs, with rs3817586  attaining P\u200a=\u200a2.5\u00d710\u22124 and 2.9\u00d710\u22125 in the within-family tests for DBP and MAP, respectively. Nonetheless, when examining the QQ plot of these tests  for the replicated 101 SNPs from previous studies are presented in We also looked for association signals at a subset of our SNPs where there was previous evidence of involvement in hypertension, from both genome-wide association studies se tests , there w\u22125) around 38 cM . The nearest gene is GDNF,, glial cell derived neurotrophic factor isoform, which encodes a highly conserved neurotrophic factor and has been found to be associated with the development of schizophrenia AGXT2 which encodes the mitochondrial alanine-glyoxylate aminotransferase. Baker et al. AGXT2 and their findings suggest that human hepatocyte mitochondria possess AGXT2 activity. For the SBP phenotype, the highest peak was found on chromosome 2  at around 144 cM (130\u2013145 cM), encompassing ARHGAP15, or RHO GTPase-activating protein 15. RHO GTPases regulate diverse biologic processes, and their activity is regulated by RHO GTPase-activating proteins (GAPs), such as ARHGAP15ARHGAP15 showed specificity toward RAC1 in vitro, suggesting that ARHGAP15 is a regulator of RAC1. Suggestive linkage was found for MAP on chromosome 2  at around 157 cM and chromosome 5  at around 146 cM. The peak on chromosome 2 encompasses one interesting gene, GPD2, which encodes protein to localize to the inner mitochondrial membrane, and also contributes to the genetic liability of type 2 diabetes PPP2R2B, which encodes a brain-specific regulatory subunit B of protein phosphatase 2. Although the precise role of the subunit encoded by PPP2R2B remains to be determined, protein phosphatase 2A (PP2A) has been implicated in a number of cellular functions Linkage plots from the multipoint analyses using MERLIN-REGRESS are shown in DRD2) and Angiotensinogen (AGT), with variants contributing an increased risk of hypertension in Chinese subjects. Howerer, none of these regions show strong linkage results in our study.In comparison of the linkage results with other studies in Chinese populations, we found three regions with previous evidence of linkage for hypertension . A linkaP value under the linkage peak was for rs1605685 (p<7\u00d710\u22125) for DBP, which was significant after Bonferroni correction for the 808 SNPs under the peak, but no gene has been characterized around this SNP in the literature.We also performed the simultaneous analysis of both linkage and association for the three quantitative traits. In effect, this allows partialing out the effects of association from the test of linkage. Combined plots for the two chromosomes with strongest linkage peaks (chromosomes 2 and 5) of the tests of within-family association, tests of linkage (when not modeling association), and the combined tests of linkage and association, are presented in We tested 503,984 SNPs for association with hypertension status and BP traits. Our study is the first report of a combined genome-wide linkage and association scan for these traits in families with Chinese ancestry. The two traits are correlated and heritable, and SBP shows stronger increases with age, with DBP starting to plateau and in some individuals fall at ages above 60\u201365 years P values for quantitative traits. The region of association . Our association analysis has identified a significant variant (rs6596140) for hypertension and also detected effects of relatively common alleles with modest ociation  is limitThere is limited overlap between the regions of strongest linkage and association. However, further assessment of SNP association under the linkage peak on chromosome 5 did yield a significant variant, rs1605685, after correcting for multiple testing of the 808 SNPs under the peak.Both linkage and association analysis provide useful, but different forms of information in identifying genetic contributions to complex traits. Linkage analyses are used with family data to find broad genomic regions that contain putative disease loci, while association analyses can identify much smaller regions, either a causal variant or one which is in linkage disequilibrium with such a disease-causing locus. While association can generally detect smaller effects than linkage, association is limited to detecting variants that are either directly assayed or are in strong LD with typed SNP. These are more likely to be common variants, which generally have small effects. In contrast, linkage in families can detect rare variants specific to a subset of families and these rare variants are likely to have larger effects. An advantage of family data in association analyses is to provide a perfect control for population stratification. Our combined linkage and association design brings the best of both worlds to the problem at hand. If significant linkage is detected in the presence of significant association, it suggests that the putative locus is not the functional gene, but rather is a locus in disequilibrium with a trait locus. In contrast, if the linkage signal disappears at a point of significant association, this is suggestive that the associated SNP is, in fact, causal. In addition, all our subjects are Hong Kong Han Chinese, a relatively homogeneous group with regard to genetic background and environmental risk factors. Nevertheless, our findings are limited by relatively small sample size. Furthermore, as prior studies have reported ethnic differences in frequencies of alleles and effects of genes involved in blood pressure traits Figure S1Whole genome association scan and QQ plots for dichotomous hypertension.(TIF)Click here for additional data file.Figure S2Plots of whole genome association scan results for three quantitative traits using within-family tests. SNPs from each chromosome are represented by a different color and ordered by physical location.(TIF)Click here for additional data file.Figure S3Plots of whole genome association scan results for three quantitative traits using the total association test. SNPs from each chromosome are represented by a different color and ordered by physical location.(TIF)Click here for additional data file.Figure S4QQ plots of P-values observed vs expected under the null hypothesis, for the three quantitative traits , obtained from tests of within-family and total association.(TIF)Click here for additional data file.Figure S5QQ plots for previous reported SNPs by eight genome-wide scan studies as well as candidate gene studies in our association analysis of within-family and total tests for DBP, SBP and MAP.(TIF)Click here for additional data file.Figure S6Results of the genome-wide linkage analysis are illustrated for DBP, SBP and MAP, respectively. The multipoint LOD scores are shown on the y-axis plotted against the chromosomal position on the x-axis.(TIF)Click here for additional data file.Figure S7Three regions  with evidence of linkage are shown in the results of linkage analysis as illustrated for DBP, SBP and MAP, respectively. The multipoint LOD scores are shown on the y-axis plotted against the chromosomal position on the x-axis.(TIF)Click here for additional data file.Figure S8QQ plots of the Merlin-Regress peak findings in within-family association analysis for DBP.(TIF)Click here for additional data file.Table S1Strongest associations obtained for dichotomous hypertensive/normotensive disease status, sorted by P value.(PDF)Click here for additional data file.Table S2Strongest associations obtained from both within and total tests for DBP, SBP and MAP, sorted by P value.(PDF)Click here for additional data file.Table S3Relationship of SNPs at 9 significant loci to three blood pressure traits.(PDF)Click here for additional data file.Table S4Family-based association results for all three quantitative traits  for the 101 SNPs previously reported.(XLSX)Click here for additional data file."}
+{"text": "Genome wide linkage studies (GWLS) have provided evidence for loci controlling visceral leishmaniasis on Chromosomes 1p22, 6q27, 22q12 in Sudan and 6q27, 9p21, 17q11-q21 in Brazil. Genome wide studies from the major focus of disease in India have not previously been reported.P\u22640.05) for linkage on Chromosomes 2, 5, 6, 7, 8, 10, 11, 20 and X, with peaks at 6p25.3-p24.3 and 8p23.1-p21.3 contributed to largely by 31 Hindu families and at Xq21.1-q26.1 by 27 Muslim families. Refined mapping confirmed linkage across all primary scan families at 2q12.2-q14.1 and 11q13.2-q23.3, but only 11q13.2-q23.3 replicated . Linkage at 6p25.3-p24.3 and 8p23.1-p21.3, and at Xq21.1-q26.1, was confirmed by refined mapping for primary Hindu and Muslim families, respectively, but only Xq21.1-q26.1 replicated across all Muslim families . STRUCTURE and SMARTPCA did not identify population genetic substructure related to religious group. Classification and regression tree, and spatial interpolation, analyses confirm geographical heterogeneity for linkages at 6p25.3-p24.3, 8p23.1-p21.3 and Xq21.1-q26.1, with specific clusters of families contributing LOD scores of 2.13 (P\u200a=\u200a0.0009), 1.75 (P\u200a=\u200a0.002) and 1.84 (P\u200a=\u200a0.001), respectively.We undertook a GWLS in India in which a primary \u223c10 cM (515 microsatellites) scan was carried out in 58 multicase pedigrees  and replication sought in 79 pedigrees . The primary scan provided evidence (\u22652 adjacent markers allele-sharing LOD\u22650.59; nominal GWLS has identified novel loci that show geographical heterogeneity in their influence on susceptibility to VL in India. Leishmania donovani species complex . Ninety percent of the (under)-estimated 500,000 annual new cases occur in India/Bangladesh/Nepal, Brazil, and Sudan (WHO statistics on leishmaniasis http://www.who.int/topics/leishmaniasis/en/). Although only 10\u201320% of infected individuals develop disease, clinical VL is fatal if left untreated. The remaining 80\u201390% of human infections is sub-clinical or asymptomatic, usually associated with strong cell-mediated immunity to Leishmania antigen Visceral leishmaniasis (VL) is a parasitic disease primarily caused by the 2\u200a=\u200a34) of disease in further siblings of affected sibling pairs Genetic susceptibility to VL was initially demonstrated in mice P\u22642\u00d710\u22125; following Lander and Kruglyak P\u200a=\u200a3\u00d710\u22125) in a village near Gedaref in Eastern Sudan occupied by the Aringa ethnic group. Miller et al. P\u200a=\u200a1.72\u00d710\u22127) and 6q27  for two villages, located \u223c40 kilometers (km) apart and 100 km south of Gedaref, that are occupied by members of the related Masalit ethnic group. It was proposed that these major differences over a small geographical range were due to founder effect and consanguinity in recently immigrant populations. GWLS conducted in Brazil also provide positive evidence  for loci on Chromosomes 6q27, 7q11.22, and 17q11.2-q21.3 Family-based genome-wide linkage studies (GWLS) have been used to identify novel regions of the genome linked to disease susceptibility. Bucheton and coworkers India accounts for up to 50% of VL globally, with adverse effects linked to socio-economic status of its population L. donovani sensu strictu (zymodeme MON-2) was confirmed as the causative agent of VL in the study region, in accordance with other reports on clinical isolates from kala-azar patients in the state of Bihar In India, anthroponotic clinical VL is also known as kala-azar. Our study sample comprises Indian multicase families with clinical VL derived from villages located within a radius of \u223c60 km from the city of Muzaffarpur covering the districts of Muzaffarpur, Vaishali, and Sitamarhi. Global positioning system (GPS) coordinates for each family were collected to facilitate spatial analysis. Further epidemiological and demographic details relating to the study site are described elsewhere in vitro culture) using splenic aspirates Families with at least two siblings affected with clinical VL were ascertained from medical records held in the Kala-Azar Medical Research Centre (KAMRC) in Muzaffarpur, India. Diagnosis of VL was based on presence of typical clinical features of VL i.e. fever with rigors and chills, splenomegaly, weight loss and pancytopenia followed by demonstration of parasites by parasitological methods  , comprisGenotyping of the primary genome scan families was performed by deCODE genetics  for 515 polymorphic markers covering all 22 autosomes and the X chromosome. The average interspacing between markers was 10cM according to the deCODE Haldane genetic positions. The overall rate of missing genotypes was 9%. We also genotyped 17 additional polymorphic markers (Applied Biosystems) to produce a 2-3cM refined map of the primary scan positive regions. The 79 replication families were genotyped for positive markers from the original deCODE panels, as well as the 17 additional markers, as indicated. The integrity of all families was assessed using the PedCheck v1.1 software et al. manuscript in preparation), and data files outputted in formats appropriate for analyses in SPLINK http://www-gene.cimr.cam.ac.uk/clayton/software/stata/) implemented within STATA v8.0. Markers that deviated from HWE (P<9.5\u00d710\u22125) were excluded from analysis. Non-parametric linkage analysis was performed in ALLEGRO pairs scoring function was used to compare allele-sharing identical-by-descent (IBD) across all affected relative pairs within the extended pedigrees, including the singleton families within larger pedigrees xLOD. All LOD scores reported are multipoint. Nominal P-values (i.e. without correction for multiple testing) are reported throughout. Information content for markers was estimated in ALLEGRO.Allele data for all microsatellites were entered in the Genetic Information Environment (GenIE) database, developed in house . The separate Hindu and Muslim family sets had >89% and >94% power to detect a major gene at an allele-sharing LOD score value of 3 (P\u200a=\u200a1\u00d710\u22124) and 2.07 (P\u200a=\u200a1\u00d710\u22123), respectively. The power within the additional families to detect a major gene at an allele-sharing LOD score of 3 (P\u200a=\u200a1\u00d710\u22124) was 63% in the Muslim families, and 100% in the Hindu families and across all additional families.Simulations (100) performed within ALLEGRO using data for a typical set of six linked polymorphic microsatellite markers  showed that the primary genome scan family set  across bP0, P1, and P2 of two individuals sharing 0, 1, and 2 alleles identical-by-descent over the 515 microsatellite markers successfully genotyped in the primary genome scan families. Unrelated individuals should have probabilities 1, 0, and 0, respectively. Full sibs have P0 sharing probabilities of 0.25; half sibs plus first cousin 0.375; grandparent-child, avuncular or half-sib 0.50; double first cousins 0.5625; first cousins or half-avuncular 0.75; half first cousins 0.875; and second cousins 0.9375. We therefore considered any relationship between parents in the families with P0 sharing probabilities <0.95 to be indicative of a consanguineal marriage, with predicted relationships as outlined in The Pedigree RElationship Statistical Test (PREST) program Admixture/Correlated frequencies, (ii) No admixture/Correlated frequencies and (iii) Linkage/Correlated frequencies. The correct value of K is determined by the estimated posterior probabilities, for which values closer to zero indicate a better fitted model. All models were run ten times using 50,000 iterations for the burning period and 100,000 Monte Carlo Markov Chain repetitions.The population structure of the primary scan families was inferred using the model-based clustering software STRUCTURE n alleles were recorded as n-1 biallelic loci Population structure was also examined by carrying out principal component analysis using the SMARTPCA within EIGENSOFT http://members.rediff.com/gisindia/zone_num.jpg).GPS data for latitude and longitude for all CART and spatial analyses were converted to the universal transverse mercator (UTM) coordinates system. Following this system, each family is located at a point defined in terms of UTM northing meters (m) (Y-axis in http://cran.r-project.org/web/packages/rpart/index.html) in R j \u200a=\u200a 1,\u2026, J of a tree separates families i into two branches determined by a splitting node Sj defined by a splitting rule Rj of a particular variable Vj. For example in chromosome 6 marker D6S244 (presented below in 1(i), UTM north for family i, on S1(i): V1(i) <2886078, where 2886078 is the splitting rule Rj of this branch. The left branch contains cases where the decision rule evaluates to be true, that is i: Sj(i) \u200a=\u200a T (i.e. families located UTM north <2886078), and the right branch contains the remaining families i: Sj(i) \u200a=\u200a F (i.e. families >2886078). We denote nj as the number of families at node j. Using the RPART algorithm, variables are selected to maximize the difference between the left and right branches of the regression tree, according to the Gini or Information criterion http://cran.r-project.org/web/packages/rpart/index.html). Cross-validation is applied in RPART to evaluate the cross-validation prediction error of the tree, and choose the tree size where this first achieves a distinct minimum. The results of the trees were then mapped spatially using ArcGIS software.To determine whether geographical location and religion were influencing linkage results, we first carried out classification and regression tree (CART) analysis implemented using the Recursive Partitioning algorithm in the RPART package In a second and independent approach to visualizing the spatial distribution of LOD scores for markers at the peaks of linkage on Chromosomes 6 and 8 we used spatial interpolation within ArcGIS to attribute values for LOD scores at regular grid locations based on measurements taken within the same area P\u22640.05 equating to allele-sharing LOD\u22650.59. These criteria were chosen because in a true disease-susceptibility locus, adjacent markers should be correlated and share positive LOD scores. Thus observing nearby markers with low P-values by chance should be unlikely A 10cM primary GWLS of 515 polymorphic markers was carried out in 58 pedigrees (74 nuclear families). Regions of linkage were defined as having more than one marker with nominal Indian populations are ethnically and genetically diverse P\u22640.02) in analysis of all families and/or the religion-specific analysis were taken forward in refined mapping and replication studies.Only those chromosome regions that achieved LOD\u22651 , while the Chromosome 11q14.2 peak was retained and enhanced at D11S1780 . For Chromosomes 2 (7 markers) and 11 (7 markers), microsatellites that were positive for linkage in the primary scan, as well as the 7 additional microsatellites added for the refined mapping, were genotyped in the 79 additional multicase families. No evidence for linkage at Chromosome 2q12.2-q14.1  was observed in the additional families. Positive evidence for replication at Chromosome 11q13.2-q23.3 was observed with the peak of linkage at D11S4090  \u223c22cM distal to the peak observed at D11S1780 in the primary genome scan families. The combined analysis of primary and additional families for Chromosome 11 confirmed the peak of linkage at D11S4090  on Chromosome 11q23.1.In the refined mapping study, 7 additional markers in positive regions of linkage on Chromosomes 2  and 11  were genotyped in all 58 primary genome scan families . FollowiP\u200a=\u200a0.021), while the Chromosome 8p23.1 peak was refined to the closely adjacent D8S550 . For Chromosomes 6 (5 markers) and 8 (5 markers) microsatellites that were positive for linkage in the primary scan, as well as the 4 additional microsatellites added for the refined mapping, were genotyped in 52 of the additional 79 multicase families that were Hindu. No evidence for linkage at Chromosome 6p25.3-p24.3  or at Chromosome 8p23.1-p21.3  was found in these additional Hindu families.For Chromosomes 6 and 8, grid tightening by genotyping of 4 additional markers was only undertaken in the 31 Hindu primary genome scan families . FollowiP\u200a=\u200a0.007). Six microsatellites that were positive for linkage in the primary scan, as well as the 6 additional microsatellites added for the refined mapping, were genotyped in 26 of the additional 79 multicase families that were Muslim. Borderline evidence for linkage at DXS8055  was found in these additional Muslim families. The combined analysis of primary and additional Muslim families retained the peak of linkage at DXS8055  on Chromosome Xq23.For Chromosome X, grid tightening by genotyping of 6 additional markers was only undertaken in the 27 Muslim primary genome scan families . The ChrFor refined mapping we corrected st versus 2nd (st versus 3rd (not shown) or 2nd and 3rd . Hence, there was geographical heterogeneity even amongst the original 31 Hindu families that contributed to the primary linkage peak observed at Chromosome 6p25.3-p24.3. Splitting functions at all other parts of the D6S244 tree were based on UTM northings/eastings rather than religious group. For the adjacent marker D6S1617 . Following the primary binary decision down the left hand side of the tree  reveals a second cluster of 12 families living west of the UTM easting 339,381 m that also contribute a total LOD score 0.634  for which there is no further splitting function based on religious group. Essentially similar results were obtained for the adjacent marker D8S520  for susceptibility loci for clinical VL in India on Chromosomes 2, 5, 6, 7, 8, 10, 11, 20 and X. Linkage peaks at 2q12.2-q14.1 and 11q13.2-q23.3 were supported by refined mapping across all primary genome scan families, but only 11q13.2-q23.3 replicated in a second set of families. Linkage peaks at 6p25.3-p24.3 and 8p23.1-p21.3 that appeared to be specific to Hindu families, and at Xq21.1-q26.1 putatively specific to Muslim families, were likewise supported by refined mapping in primary genome scan families, but only Xq21.1-q26.1 was replicated across all Muslim families. As with previous GWLS studies in Brazil per se is not the genetic reason for linkage peaks that appeared at first to be Hindu- or Muslim-specific. These results expand on previous findings by Gutala et al (2006) showing greater correlations between genetics and geography as opposed to religion when comparing populations from North and South India. Other variables related to the complexity of Indian populations, such as caste, socio-economic status, and environmental risk factors related to VL infection Results presented here provide preliminary evidence  or strain of the parasite might also determine different genetic control. Ability to map these loci has been limited by employment to date Leishmania infection has been demonstrated in humans and mice. For example, the interleukin (IL)-1 cytokine family gene cluster which plays a prominent role in pro-inflammatory responses to infection lies close to the peak of linkage on Chromosome 2q12.2-q14.1. Lipopolysaccharide (LPS)-induced IL-1\u03b2 production is inhibited in human peripheral blood monocytes (PBMCs) infected with L. donovaniLeishmania lipophosphoglycan (LPG) mediates its suppressive effect on IL-1\u03b2 transcription via a unique promoter sequence Leishmania infection, IL-18 has been shown to act as an important stimulator of Th1 cytokine responses IRF-4-deficient C57BL/6 mice infected with L. majorIL-13RA1) and -2 (IL-13RA2), and the NF-\u03baB repressing factor (NKRF) and activating protein (NKAP) are also under the extended peak at Xq21.1-q26.1. Expression of the IL-13 alpha-2 receptor is required for activation of the TGF-\u03b21 promoter in macrophages Leishmania infection L. major infection 1NF-\u03baB2NF-\u03baBThe fact that different linkage peaks are identified within and between the major global foci on VL in India, Sudan and Brazil is not necessarily surprising, since the complex nature of the disease means that we expect many loci to contribute small effects to disease susceptibility. The species  provides a spatial representation of the CART tree for D8S516 presented in ; (b) shows the spatial interpolation of LOD scores for D8S516 independently derived in ArcGIS. Axes show UTM eastings (X axis) and northings (Y axis) in metres.(TIF)Click here for additional data file.Figure S2CART trees for markers at the peaks of linkage on Chromosome X: (a) DXS6799; (b) DXS8020; (c) DXS8055; (d) DXS1001; and (e) DXS8059. UTM \u200a=\u200a Universal Transverse Mercator; E\u200a=\u200a easting, N\u200a=\u200a northing, in meters. M\u200a=\u200a Muslim. N (n) is the number of families contributing to the cluster on each branch of the tree; the number directly above is the average LOD score for these families; the number directly beneath is the summed LOD score for these families.(TIF)Click here for additional data file.Figure S3CART trees for markers at the peaks of linkage on Chromosomes 2 and 1: (a) D2S293; and (b) D11S1780. UTM \u200a=\u200a Universal Transverse Mercator; E\u200a=\u200a easting, N\u200a=\u200a northing, in meters. M\u200a=\u200a Muslim. N (n) is the number of families contributing to the cluster on each branch of the tree; the number directly above is the average LOD score for these families; the number directly beneath is the summed LOD score for these families.(TIF)Click here for additional data file."}
+{"text": "However, linkage analysis for lipid traits using QTL-ALL analysis revealed promising linkage signals with p\u22640.005 for total cholesterol, LDL cholesterol, and HDL cholesterol at chromosomes 5p15, 9q21, 10p11, 10q21, and 22q13. The most significant signal (p\u200a=\u200a0.0011) occurred at 10q21.2 for HDL cholesterol. We also observed linkage signals for total cholesterol at 22q13.32 (p\u200a=\u200a0.0016) and 5p15.33 (p\u200a=\u200a0.0031) and for LDL cholesterol at 10p11.23 (p\u200a=\u200a0.0045). Interestingly, some of linkage regions identified in this Sikh population coincide with plausible candidate genes reported in recent genome-wide association and meta-analysis studies for lipid traits. Our study provides the first evidence of linkage for loci associated with quantitative lipid traits at four chromosomal regions in this Asian Indian population from Punjab. More detailed examination of these regions with more informative genotyping, sequencing, and functional studies should lead to rapid detection of novel targets of therapeutic importance.In this investigation, we have carried out an autosomal genome-wide linkage analysis to map genes associated with type 2 diabetes (T2D) and five quantitative traits of blood lipids including total cholesterol, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, very low-density lipoprotein (VLDL) cholesterol, and triglycerides in a unique family-based cohort from the Sikh Diabetes Study (SDS). A total of 870 individuals  from 321 families were successfully genotyped using 398 polymorphic microsatellite markers with an average spacing of 9.26 cM on the autosomes. Results of non-parametric multipoint linkage analysis using S According to Global Burden of Disease Study predictions, India, China and USA will be the top three leading countries for the prevalence of diabetes Type 2 diabetes (T2D) is a major public health problem of 21Elevated serum lipid levels are important risk factors for the development of cardiovascular disease (CVD). The genetic basis of several monogenic forms of lipid disorders has been determined, including familial lipoprotein lipase (LPL) deficiency, apoC-II deficiency, defective apoB, familial hypercholesterolemia, and familial triglyceridemia Recent genome-wide association studies (GWAS) performed for many complex traits are revolutionizing the dissection of genetic determinants of several complex traits including T2D and serum lipids. Although these studies are adding to the list of reliably associated common loci controlling T2D and blood lipids and even other complex traits, these loci explain only a small portion of the heritable component associated with these complex diseases. Clearly, additional loci that can explain a large proportion of the variation await discovery.Asian Indians, one quarter of the global population, have unusually high CVD mortality and very high prevalence of insulin resistance and T2D This study was carried out on an endogamous community of Khatri Sikhs living in Northern Indian states of Punjab, Haryana, and New Delhi. The Khatri population was chosen because of its relatively higher prevalence of diabetes as compared to other Sikh castes. Khatri Sikhs are more affluent and live in cities and are traders by profession. In general, Sikhs do not smoke for religious and cultural reasons and about 50% of the study participants are life-long vegetarians. A total of 1,115 individuals from 338 families were extensively phenotyped >95%), were used for linkage analysis of T2D. These 321 diabetic families comprised 275 affected sibling pairs, 59 affected cousin pairs, 127 affected parent-child pairs, 1 affected grand parent-child pair, and 61 affected avuncular pairs. We collected an average of 6.5 participants per family with family size ranging from 3 to 105 members. The average number of generations per family was 2.5. Of these 321 families, 316 families containing 846 individuals  were used in the linkage analysis of blood lipids .A total of 557 families were investigated and 236 families were excluded because they did not meet the eligibility criteria for the study. A total of 321 families containing 870 individuals , who were successfully genotyped  searching medical records for indications of symptoms of diabetes or measures of blood glucose levels, (b) use of diabetic medication, and (c) measuring fasting glucose levels following the guidelines of American Diabetes Association 2], and waist-to-hip ratio (WHR) was calculated as the ratio of abdomen or waist circumference to hip circumference. Despite having comparable BMI (27.5\u00b14.0 T2D cases vs. 27.3\u00b14.7 controls), patients had a pronounced abdominal adiposity as reflected by their significantly higher WHR  than controls. Interestingly, WHR in Khatri Sikh men (BMI 26\u201327 kg/m2) was higher than obese Mexican American men (BMI>32 kg/m2); Sikhs (0.97\u00b10.05) vs. Mexican Americans (0.95\u00b10.06) Body mass index (BMI) was calculated as  were quantified using standard enzymatic methods . Fasting serum insulin was measured by radio-immuno assay . All quantitative parameters were determined by following manufacturer's instructions using a Hitachi 902 auto-analyzer .http://www.marshmed.org/genetics). A total of 870  samples were used in linkage analysis of T2D and 846  samples were used in linkage analysis of lipid levels after excluding those with call rate <95%, relationship errors, gender errors, and those with missing phenotypes.DNA was extracted from buffy coats using QiaAmp blood kits  or by the salting out procedure A variety of statistical software was used to complete this study. To set up the files for analysis, we extensively used the statistical software R (version 2.0.1). Data cleaning was performed following several steps. To check for inconsistencies in the self-reported family structures, we carried out relationship testing using PREST 2 sex, BMI, dietary and lifestyle factors , socio-economic status (education and job-grade) as covariates. To select significant covariate, both stepwise regression and backward elimination were used in genetic models. Significant covariates considered for selection in the model were age, age2, sex, job grade, level of alcohol consumption. Additionally, analysis was performed including and excluding BMI in the model despite its elimination in stepwise regression. Univariate analysis was performed to obtain summary statistics for each trait (online supplementary Table S2). A classical multiple linear regression model: 2, these variables were mean centered. Since most of the traits were right skewed, they were transformed using the Box-Cox transformation method  associated with T2D in these families.As shown in online online . Regression models were then fitted for the transformed traits. In the variable selection step, in most cases forward stepwise-regression and backward elimination agreed with each other. 2, and socio-economic status (job grade) were significant predictors of serum LDL cholesterol levels . All the estimated coefficients are presented in online Table S1. High leverage points and Cook's distance were calculated to detect influential observations and poorly fitted observations. After removing the maximum Cook's distance points, there was no significance change in the model. Calculated jackknife statistics was also within the acceptance region. Residuals of each trait were calculated and these residuals were used for the final QTL-ALL analysis.Univariate analysis of the lipid traits showed some individuals with very high or very low outlier values, which were removed from the analysis. As needed a Box-Cox transformation was used to make the error distribution of the data more normal online . Because obesity is a major risk factor for CVD and T2D risk, and affects lipid levels, we also tested linkage signals including and excluding BMI. Our results did not change after including BMI in the model.QTL-ALL analysis, using the Score.Max statistics, was performed for the five quantitative traits. An overview of the linkage results for the significant signals associated with serum lipid associated traits is given in online . Note that the non-parametric method for linkage (used in our study) only considers allele sharing between affected individuals, therefore, the ambiguous phenotype of unaffected members is unlikely to have led to the failure to detect linkage in this large sample. These results reaffirm the highly complex nature of T2D phenotype. Essentially, our study failed to identify genes associated with T2D even when a homogenous population was used to control genetic heterogeneity associated with T2D phenotype and a sample collected from one geographic location was used to reduce environmental heterogeneity. These finding suggest that the genes responsible for T2D in Sikhs have small effects, as seen in other ethnic groups, and are difficult to detect using linkage analysis. It can be argued that in comparison to random-mating population, higher identity by descent (IBD) sharing in this inbred population might have reduced the power of detecting significant linkage. In this scenario, one would expect to see increased average IBD leading to false positive indications of linkage. On the contrary, we found the opposite with no substantial increase in IBD among affected individuals and thus no linkage. At the same time, we believe that our linkage data may still contain considerably useful information that could enable the discrimination of causal variant from a near-by variant that is merely in linkage disequilibrium (LD) TCF7L2, PPARG, KCNJ11, FTO and KCNQ1) associated with T2D in this population Our study represents the first large scale genome-wide effort to identify chromosomal regions with putative loci affecting T2D and lipid traits in a unique community of Asian Sikhs from Northern India. This diabetic cohort from a genetically homogenous subgroup was collected with the initial goal of identifying T2D predisposing genes. However, the results of our non-parametric linkage scan did not identify any chromosomal region to be significantly linked to T2D . Classical multiple linear regression models were used to adjust for environmental effects on the serum lipid traits. In view of strong environmental component associated with T2D and lipid metabolism, we have carefully analyzed the environmental factors, particularly the unique life style factors such as diet, physical activity, obesity, job status, socio-economic status, gender, and medication that could potentially influence these traits. As explained in the Results section, the significant covariates with potential to modify linkage effect were identified and included in the analysis model. The strongest evidence of linkage (p\u200a=\u200a0.0011) for HDL cholesterol was detected on chromosome 10q21.1\u201321.2. Suggestive evidence of linkage to ApoA-I was observed on chromosome 10q21.1 in the Quebec Family Study (QFS) PCDH15) gene (10q21.1) has been associated with multiple lipid traits in Finnish and Dutch multigenerational dyslipidemic families SCL16A9) that also maps to chromosome 10q21.2 KIAA1462 (10p11.23) that encodes a yet uncharacterized protein. However, a recently published GWAS showed an unambiguous evidence for association of rs3739998 (p\u200a=\u200a7.2\u00d710\u22128) within this gene with CVD and myocardial infarction in German MI Family cohort (GerMIFS) III (KORA) PCDH15 and SCL16A9 and KIAA1462 genes are also associated with multiple lipid traits including HDL cholesterol, LDL cholesterol, and triglycerides  in our provisional results of lipid GWAS being performed on the population originated from the same Asian Indian community (unpublished results).The other aim of this investigation was to identify genomic regions affecting lipid-related phenotypes in this cohort. We performed QTL-ALL analysis on this non-randomly ascertained dataset, which revealed several suggestive linkage signals associated with serum lipid levels A linkage peak for total serum cholesterol (p\u200a=\u200a0.0031) was detected near marker D5S2488 at the proximal region of chromosome 5p15.33. This region was previously linked to LDL cholesterol in the NHLBI Family Heart Study PPAR\u03b1, which is a ligand-activated nuclear transcription factor and controls extracellular and intracellular lipid metabolism, and also inhibits progression of atherosclerotic lesions CELSR1 (located at 22q11\u201313) is associated with ischemic stroke in recent Japanese GWAS CELSR2 on chromosome 1p13 (homologous to CELSR1) is associated with LDL cholesterol and myocardial infarction in a meta-analysis study by Myocardial Infraction Genetics Consortium The linkage signal at chromosome 22q13.32 near marker TCTA015M (p\u200a=\u200a0.0016), detected for total cholesterol was linked with familial hypercholesterolemia in a Utah study KIAA1462, PCDH15, PPAR\u03b1, SLC16A9, and CELSR1) implicated with lipid traits in recent GWAS and meta-analysis studies and also some of these regions overlap with prior linkage studies Our study does not represent a common replication attempt to identify lipid loci in an independent population. Rather, this investigation has been carefully carried out in this unique family-based cohort using a conservative statistical approach applying score-based statistics to map quantitative lipid traits in a non-randomly ascertained dataset. Exceeding our expectations, this study has identified linkage regions, primarily HDL cholesterol (10q21.1\u201321.2) and total cholesterol (22q13.32) that were previously reported for lipid traits or CVD. The most interesting part of this study is that some of these linkage signals also harbor important candidate loci  suggests that this region may contain novel gene(s) influencing serum HDL cholesterol levels and other lipid traits. Further denser and more informative genotyping in each of these regions would be important to discover functional loci influencing blood lipids.Figure S1Genome-wide non-parametric linkage scans for type 2 diabetes using 321 diabetic pedigrees and 398 microsatellite markers (9.26 cM). Individual plot shows linkage signals (Kong and Cox LOD score) on Y axis and microsatellite markers on X axis. None of the chromosome regions revealed any signal associated with T2D in these pedigrees.(TIF)Click here for additional data file.Figure S2Plot of Box-Cox coefficient lambda and the distribution of five quantitative traits including total cholesterol, triglycerides, HDL cholesterol, LDL cholesterol, and VLDL cholesterol before and after transformation.(TIF)Click here for additional data file.Figure S3Genome-wide autosomal linkage scan for five blood lipid phenotypes. Individual plot shows allele sharing LOD  on Y axis and chromosome distance (cM) on X axis.(TIF)Click here for additional data file.Table S1Linear regression model for quantitative traits.(DOC)Click here for additional data file."}
+{"text": "We conducted a genome-wide linkage study to identify loci influencing mammographic breast density (MD).Mammographic breast density is a highly heritable  genotyping was performed on 3,952 individuals using the Illumina Infinium 6K linkage panel.Using a variance components method, genome-wide linkage analysis was performed using quantitative traits obtained by adjusting MD measurements for known covariates. Our primary trait was formed by fitting a linear model to the square root of the percentage of the breast area that was dense (PMD), adjusting for age at mammogram, number of live births, menopausal status, weight, height, weight squared, and menopausal hormone therapy. The maximum logarithm of odds (LOD) score from the genome-wide scan was on chromosome 7p14.1-p13  for covariate-adjusted PMD, with a 1-LOD interval spanning 8.6 cM. A similar signal was seen for the covariate adjusted area of the breast that was dense (DA) phenotype. Simulations showed that the complete sample had adequate power to detect LOD scores of 3 or 3.5 for a locus accounting for 20% of phenotypic variance. A modest peak initially seen on chromosome 7q32.3-q34 increased in strength when only the 513 families with at least two sisters below 50 years of age were included in the analysis . In a subgroup analysis, we also found a LOD score of 3.3 for DA phenotype on chromosome 12.11.22-q13.11 , overlapping a region identified in a previous study.The suggestive peaks and the larger linkage signal seen in the subset of pedigrees with younger participants highlight regions of interest for further study to identify genes that determine MD, with the goal of understanding mammographic density and its involvement in susceptibility to breast cancer. Mammographic density (MD), adjusted for age and body mass index (BMI) is a strong risk factor for breast cancer. The radiographic appearance of the breast on mammography varies among women and reflects differences in breast tissue composition and X-ray attenuation characteristics . Fat is Twin studies have shown that, after adjustment for other factors such as age, parity, menopausal status, body weight and hormonal use, about 60% of the variance in PMD is explained by genetic factors . Both thFamilies consisting of at least two sisters were assembled from several family registries and cohorts: families from Ontario, Australia and Northern California enrolled in the Breast Cancer Family Registry (BCFR) , dizygouMammograms were obtained from at least two sisters in each family. If a woman had breast cancer, the selected mammogram (from the contra breast) had been taken at or before the diagnosis of breast cancer. We sought that the ages at mammogram for two sisters should be within 5 years of each other to minimize the requirement for age-adjustment within a sibship. This was achieved in more than 95% of the families. The cranio-caudal view in each mammogram was digitized and sent to Toronto where all images were measured by one reader (NFB) using a computer-assisted thresholding method. Using Cumulus software, thresholds were set that defined the edge of the breast and outlined the areas of dense tissue. The pixels within these thresholds defined, respectively, the total area of the breast and the area of dense tissue (DA), from which percent density (PMD) was calculated. The non-dense area (NDA) of the mammogram was also calculated from these measurements . This meImages were measured in batches of approximately 100 images at a time. Within each batch, 10 images were duplicated, placed in random order within the set, and read twice in a blind fashion. Some images were also re-read in different batches, in order to assess reliability of the PMD measurements both within and between batches. The correlation between the two reads in the same batch for PMD was estimated to be 0.90  or QIAamp DNA Blood Maxi Kit (Qiagen) according to the supplier's instructions. DNAs were quantified with Quant-iT PicoGreen dsDNA Reagent  using fluorimetry measured with a SpectraMax Gemini EM instrument (Molecular Devices).http://www.cidr.jhmi.edu using the Illumina Infinium II Human Linkage-12 panel. Out of 6,090 SNPs, 413 were removed due to poor clustering, leaving 5,677 SNPs with an overall genotype missing rate of 0.073%. After examining Mendelian errors and estimating relationships [Genotyping for linkage analysis was performed by the Center for Inherited Disease Research (CIDR) ionships -13, 49 pionships  were setGenotypes were missing in less than 20 individuals for 97.4% of the markers; and the poorest marker failed in 81 of 3,952 individuals. Ten or fewer marker genotypes were missing in 91% of the genotyped individuals. Allele frequencies were estimated from all genotyped individuals. Only 1.9% of the markers had minor allele frequency (MAF) less than 0.10, and 76% of the markers had MAF over 0.3; average heterozygosity across SNPs was 43.7%. Multipoint marker informativity remained very stable across the genome, ranging from 0.6 to 0.8.Although self-reported race/ethnicity was available, we estimated population structure by using Eigenstrat  on one ihttp://www.ncbi.nlm.nih.gov/. The Rutgers linkage map was obtained from Rutgers Map Version 2 http://compgen.rutgers.edu/[Physical marker locations were obtained from NCBI Build 36 gers.edu/. For mar\u00ae SNP Genotyping Assays  as recommended using a 96-well format. End-point fluorescence was measured with the plate reader component of the 7900HT Real Time PCR System (Applied Biosystems) and aided by Taqman\u00ae Genotyper software for allele discrimination with call rates > 98%. A portion of the samples (4%) was run in duplicate and corresponded to individuals used in the linkage study to assure quality control and permit assessment across genotyping platforms. The concordances of replicate genotypes and cross platform genotypes were > 99%.Genotyping of individual single-nucleotide polymorphisms (SNPs) to attempt validation of association was performed with allele-specific fluorescent probes in Taqmanth percentile. Trait distributions were unaffected by breast cancer status as mammograms were measured only from the opposite breast, prior to or at a diagnosis. Linear models and generalized additive models were used to model the relationships between the MD scores and known covariates, and the residuals were used in the linkage analysis. Family relationships were not taken into account when fitting the linear models. In general, the expected relationships between MD and covariates were seen. We also estimated heritability in Merlin [Linkage analysis was performed using the Merlin (version 1.1.2)  variancen Merlin  and in Sn Merlin , for then Merlin . To estiAlthough this was primarily a linkage study, we calculated evidence for association using the orthogonal test for quantitative traits implemented in QTDT  at 5,677There were 1,616 pedigrees assembled for this study from existing collections, including 4,526 individuals with DNA. Data verification and cleaning included checking for monozygotic twin status , gender consistency, whether the mammogram was readable, whether relevant covariate data were available, and whether reported family relationships were consistent with the genotyping. Pedigrees were adjusted when data genotyping supported alternative relationships. A total of 1,415 families were retained for analysis containing 6,638 individuals, of whom 4,993 were women  with analysis information . Almost half the families (47%) were recruited from Australia . Tests for Hardy-Weinberg equilibrium (HWE)  were perLogarithm of odds (LOD) score linkage plots for PMD, DA and NDA are shown in Figure We then performed several different analyses of ancillary phenotypes and subgroups to investigate whether the evidence for linkage was stronger under different assumptions.Weight and height are partially determined by genetic factors. We considered the possibility that by including weight and height in the models for calculating residuals, we may have minimized genetic effects due to pleiotropy. Linkage was re-estimated using residuals to models containing all predictors except weight and height; no additional linkage peaks were identified. Conversely, we also considered the possibility that we were not adequately capturing the relationships between PMD and weight, height, and age at mammogram in our linear models. We therefore fitted generalized additive models  for thesP = 0.12) highlighting this chromosomal region.It has been often argued that the influence of genes, relative to the environment, will be larger for early onset features or diseases. Further, it has been noted in at least one study that three SNPs that had been reported to be associated with breast cancer were marginally associated with MD, but only when women with younger ages  were considered . We examrs2061192 and rs10785424, 1-LOD drop interval from 56.0 cM - 64.0 cM, Figure MD decreases with age and is influenced by many factors that alter endogenous sex hormone levels, such as the number of pregnancies, hormone therapy use, and menopausal status. Although in our primary analysis, we had analyzed residuals to linear models adjusted for these factors, we hypothesized that the magnitude of the potential effect of a locus on MD measurements could vary as a function of these reproductive and hormonal variables. To explore this hypothesis, we used our linear models with covariates to obtain a predicted PMD value for each individual, and then we calculated the smallest difference in predicted PMD among all pairs of sisters in each family. We then divided the families into two equally-sized groups based on this predicted PMD difference. Hence, in one group, age, menstrual and reproductive values led to predictions of more similar PMD for the sisters, whereas in the other group, larger PMD differences were predicted by the covariate model; this concept is illustrated in Additional file rs1029482 rising to 3.29   occurred on chromosome 7 near our linkage peak. To pursue this further, sliding two- and three-marker haplotypes were tested for association using FBAT [rs1486155 and allele A at rs723149 showed a stronger association at P = 6.9 \u00d7 10-7. There was weak linkage disequilibrium between these two markers . The minor allele frequency at rs723149 varies across populations http://hapmap.ncbi.nlm.nih.gov, however our test of association using QTDT examines within-family patterns of allele transmission and is robust to population stratification. Furthermore, similar results were obtained when we analyzed the subset of Caucasian families (not shown).The smallest ociation  are showing FBAT  around trs723149 by using PLINK [rs1486155 and rs723149. This covariate was then included in the linkage analysis, but was found to have minimal impact. The LOD score fell by 0.21 for PMD_res, from 2.69 to 2.48.In order to see whether the linkage signal on chromosome 7p was explained by this association, we imputed haplotypes near ng PLINK , and crers723149 of additional sets of women with MD measurements and covariate information was available. These included a non-overlapping set of 235 women from Ontario and Australia [\u00ae SNP Genotyping Assay specifically for rs723149. No evidence for association with PMD was observed in these 1,024 women , P = 0.35, for association with the square root of PMD adjusted for age, age squared, weight, weight squared, height, height squared, parity, and menopausal status).Genotype information at ustralia  selectedPhenotype definition is critical in linkage analysis. The extent of MD can be measured by the percent of the breast that is dense or by the total area that is dense. DA is highly correlated with PMD, but has a skewed distribution. Nevertheless, after transformation and adjustment for covariates, repeated reads of the same mammogram showed very high correlations for DA and for PMD. Therefore, we examined the evidence for linkage to both these phenotypes, and found evidence for suggestive linkage at multiple locations. Furthermore, since MD is known to vary through life, genes that appear to influence MD in mid-life could reflect early influences on MD at the time the breast forms in adolescence, or through subsequent changes or rates of change with increasing age, parity and the menopause, or through influences on both formation and age change in MD . Our stuGiven the number of study participants and available pedigree information, we had estimated that we would have 80% power to obtain a LOD score in the range of 3 with a locus that could explain 25% of the heritability of PMD. Despite the large collection of families and careful consideration of mammographic density parameters, our primary linkage analysis did not yield LOD scores that exceeded desired thresholds set by genome-wide gene-dropping simulations. With different modeling conditions, three loci on chromosomes 7p14.1-p13, 12p11.22-q13.11, and 7q32.3-q34, showed LOD scores that approach or exceed 3. However, given that these results were obtained after multiple analyses, these signals should be considered as 'suggestive'.Chromosome 7p: With the development of a model for covariate effects that was inspired by Pike and colleagues [rs1949880 and rs2054789 corresponds to a 7.2 Mb region containing 72 genes, including small clusters of snoRNA and piRNA genes toward its proximal boundary. Of interest at the proximal boundary, are the insulin-like growth factor binding protein genes, IGFBP1 and IGFBP3, both of which have been hypothesized to be involved in mammographic density (and in breast cancer and other cancers) and have been considered in previous association studies examining MD phenotypes [RALA) with its implicated role in signalling and growth.lleagues , evidencenotypes -35. Of tenotypes ,3 with ienotypes . As linkChromosome 7q: When we examined families containing younger sisters (two sisters with mammograms under age 50 years), the peak on chromosome 7q became stronger. This peak with a 1-LOD drop interval bounded by rs4728251 and rs1476640, corresponds to 9.6 Mb containing 69 genes. Phenotypes at younger ages are expected to display a stronger genetic component [2 varied by age in our previous twin studies [RAS oncogene family, RAB19.omponent  but ther studies ,37, and Chromosome 12: On chromosome 12, a maximum LOD score of 3.3 was seen in families where the sisters would be expected to have dissimilar DA, after adjustment for factors affecting sex hormone levels. Such predicted differences could be due to: differences in the numbers of pregnancies; ages at menopause or menarche; weight; or height. Linkage in this context would identify families where phenotypes are more similar than expected, and this could imply a larger potential genetic effect. The 1-LOD interval of our linkage signal, bounded by rs1909160 and rs1978161 corresponds to a large physical distance of 16.7 Mb encompassing the centromere and contains 80 genes. Genes of potential interest within this region include the vitamin D receptor (VDR) and collagen type II (COL2A1) given their association to breast biology.We did not detect any significant evidence for linkage on chromosome 5 as was reported in a previous study of 89 multi-generation pedigrees , which, BRCA1/2 high risk families, more recently with families of confined ancestries  Senior Research Fellow and a Victorian Breast Cancer Research Consortium Group Leader. JLH is an Australian Fellow of the NHMRC and a Victorian Breast Cancer Research Consortium Group Leader.Supplementary Figures and Tables. Supplementary Table 1. Recruitment of families for linkage with details of Ontario studies. Supplementary Table 2. Family characteristics. Supplementary Table 3. Breast cancer rates among women with complete data, by study type. Supplementary Table 4. Coefficients (standard errors) for covariates in multi-variable linear models predicting DA and NDA, all three sites together. Supplementary Figure 1. Participant recruitment by site. Supplementary Figure 2. Reliability of mammographic density scoring. Supplementary Figure 3. Data cleaning for genetic analysis. Supplementary Figure 4. Principal component analysis of study participants. Supplementary Figure 5. QQ plot of tests of Hardy-Weinberg equilibrium. Supplementary Figure 6. Illustration of the division of families into two groups as a function of predicted mammographic density. Supplementary Figure 7. QQ plot of association tests for the residuals from a linear model for the square root of PMD.Click here for file"}
+{"text": "There was an error in the placement in Figures 1 and 2. The figures were switched. The captions are placed correctly."}
+{"text": "Enterococcus faecalis growth.The anatomical complexity of dental root canals represents a major limitation for a successful endodontic treatment, due to the impossibility of complete instrumentation. Therefore, irrigations are required to facilitate removal of microorganisms. The aim of the study was to test the antimicrobial activity of different endodontic irrigants against E faecalis strain ATCC 29212 was used for culture tests. Statistical analysis was performed using non-parametric tests.Forty-one extracted single-rooted teeth were included in the present study. After content removal and autoclaving, they were divided into eight groups among which two were used as positive and negative controls. The remaining six groups were instrumented and irrigated with solutions containing 17% EDTA, sterile saline solution, and different concentrations of NaOCl. In addition, chlorhexidine 2% was also used in three groups. Microbiological evaluation was performed after 30 minutes, and 24 hours, respectively. E faecalis growth comparisons between successive dilutions within the same group. Use of chlorhexidine 2% did not influence the results.NaOCl 6% recorded statistically significant higher antibacterial effect than NaOCl 2.5% (p<0.05). With this regard, no significant differences were recorded between the effects of NaOCl 6% and NaOCl 5.25%. The same outcome was obtained in E faecalis was improved by use of higher concentrations of NaOCl (5.25% and 6%).The antimicrobial activity of endodontic irrigants against"}
+{"text": "The increased feasibility of whole-genome (or whole-exome) sequencing has led to renewed interest in using family data to find disease mutations. For clinical phenotypes that lend themselves to study in large families, this approach can be particularly effective, because it may be possible to obtain strong evidence of a causal mutation segregating in a single pedigree even under conditions of extreme locus and/or allelic heterogeneity at the population level. In this paper, we extend our capacity to carry out positional mapping in large pedigrees, using a combination of linkage analysis and within-pedigree linkage trait-variant disequilibrium analysis to fine map down to the level of individual sequence variants. To do this, we develop a novel hybrid approach to the linkage portion, combining the non-stochastic approach to integration over the trait model implemented in the software package Kelvin, with Markov chain Monte Carlo-based approximation of the marker likelihood using blocked Gibbs sampling as implemented in the McSample program in the JPSGCS package. We illustrate both the positional mapping template, as well as the efficacy of the hybrid algorithm, in application to a single large pedigree with phenotypes simulated under a two-locus trait model. The increased feasibility of whole-genome (or whole-exome) sequencing has led to renewed interest in using family data to find disease mutations. For clinical phenotypes that lend themselves to study in large families, this approach can be particularly effective, because it may be possible to obtain strong evidence of a causal mutation segregating in a single pedigree even under conditions of extreme locus and/or allelic heterogeneity at the population level.The template for this type of \u201csingle large pedigree\u201d design is straightforward. Linkage analysis can be used to narrow the region of interest to a relatively small locus. From there, linkage disequilibrium  analysis can be used for fine-mapping within the linked locus. This step can be based on all sequence variants within the region -chip data in remaining family members). That is, rather than relying solely on bioinformatic filtering approaches to reduce the set of all observed sequence variants down to a manageable number, the set of candidate sequence variants is obtained by (i) restricting the region of interest based on co-segregation with the phenotype, and then within that region, further restricting the set of interesting variants to specific individual mutations co-segregating with the phenotype. Of course, in the presence of appreciable LD among mutations, further filtering and follow-up experiments may be needed to resolve which among a set of correlated mutations is the functional one.One challenge to this approach is that linkage analysis of large pedigrees is itself not trivial. As is well-known, the Elston\u2013Stewart (ES) algorithm  can handOne widely used approach to circumventing the computational complexity of large pedigree calculations is to use statistical methods that avoid calculation of the full pedigree likelihood, such as variance-components  present background on Kelvin, the software package in which the PPL framework is implemented, and (ii) on McSample, which implements the underlying MCMC techniques used here. We restrict attention to background directly relevant to this paper  model (One distinguishing feature of this framework is how it handles the trait parameter space. An underlying likelihood in a vector of trait parameters is used. The base models are a dichotomous trait (DT) model parameterized in terms of a disease allele frequency, three genotypic penetrances, and the admixture parameter \u03b1 of T) model .Whatever specific model is used, Kelvin handles the unknown parameters of the model by integrating over them for a kind of model-averaging. [Independent uniform priors are assumed for each (bounded) parameter, with an ordering constraint imposed on the penetrances (DT) or genotypic means (QT); see Due to the nature of the underlying trait models, which are formulated based on genetic considerations without regard to computational convenience, analytic solutions to the resulting multi-dimensional integrals are not possible. Instead, Kelvin carries out the integration over the trait parameters using a modified version of DCUHRE , a sub-rhttp://kelvin.mathmed.org/ and Kelvin documentation is accessible on the same site. Help with access, installation, and use can be requested by emailing http://kelvin@nationwidechildrens.org.Kelvin source code is available for download at http://balance.med.utah.edu/wiki/index.php/Download). The sampling is done using blocked Gibbs updates of two types: ones involving all the inheritance states associated with a locus, and ones involving inheritance states associated with sets of individuals as described by McSample is a program for sampling the inheritance states in a pedigree of relatives from the conditional distribution given the structure of the pedigree, observed genotypes and/or phenotypes for individuals in the pedigree, and a model for the founder haplotypes. It is written in Java and is part of the Java Programs for Statistical Genetics and Computational Statistics (JPSGCS) package available from Alun Thomas  Trait calculations per position are also done based on the ES algorithm regardless of pedigree complexity . Thus thFigure 1). The client is the driver for the generation of trait vectors, deciding which trait vectors are needed for the likelihood evaluation at each position, as described in detail in In order to decouple the adaptive trait\u2013model integration process from the likelihood calculations, we use the software engineering trick of employing a client\u2013server architecture together with a database to facilitate the operations  The integration process is fast and efficient, requiring very little in terms of computing resources, and for this reason only a few client processes are required. By contrast, the likelihood calculations are highly computationally intensive. Thus the more servers, the faster the overall speed of the analysis. Here the database serves not only as a bookkeeping device, but also as the single server interface to a large pool of server processes.The client\u2013server architecture supports considerable flexibility in overall Kelvin functionality. It allows us to dynamically add and delete servers as needed. It also allows us to dedicate each server to one pedigree, with the amount of memory and number of cores tailored to the complexity of the pedigree, for efficient use of a distributed computing resource. The client is also by design indifferent as to how the underlying marker likelihood is calculated, i.e., the mechanism used to request and retrieve likelihoods is the same regardless of what approach was used to generate the likelihood. This allows us in principle to mix and match approaches to the marker data, e.g., using the LG algorithm for pedigrees small enough for LG to handle while simultaneously employing MCMC for larger pedigrees, all within the same data set.Figure 2); all but 10 individuals were genotyped. We used actual genotypes for 664,278 SNPs (after comprehensive cleaning) from the Illumina Human OmniExpress 12 V1.0. However, we simulated a new phenotype  by selecting two SNPs , with population frequencies  matching our generating model as specified below; these SNPs were selected additionally for entering the pedigree through the top-most founders and segregating to the next generation at least four times to ensure they would be at least moderately informative in this pedigree.To illustrate the use of this new hybrid MCMC\u2013Kelvin approach, we selected a single large pedigree from an ongoing study of real human data. The pedigree has 48 individuals spanning four generations  disease model based on genotypes at this pair of SNPs. The generating model stipulated disease gene frequency of 1% (locus 1) and 20% (locus 2), and a fully penetrant dominant\u2013dominant (DD) model. This model was selected from a set of 2L models considered in Our overall approach to analyzing the pedigree is as follows:1.We thinned the marker map following standard procedures to eliminate marker\u2013marker LD, after filtering out markers with minor allele frequencies lower than 25%, and applied the new hybrid MCMC\u2013Kelvin method to perform genome-wide linkage analysis. For purposes of this analysis the locus 1 and 2 SNPs were omitted from the marker set analyzed. We based the analysis on 2,000 MCMC iterations combined from 10 independent sampling processes (with different seeds), each with a 1,000-sample burn-in and 200 iterations/sampling run.  Linkage calculations were made every 2 cM under Kelvin\u2019s standard single-locus (SL) DT model.2.We applied the PPLD to fine map under the (primary) linkage peak obtained in the first step, now utilizing all of the available SNPs (including those trimmed out during the first step and the locus 1 and 2 SNPs). While we did not have whole-genome sequence available for this pedigree, if such data were available, then this step would be applied to each variant in turn under the peak(s).3.We repeated step 1, this time conditioning on genotypes at the most highly associated SNP from step 2, under a 2L model. Specifically, we assigned each individual to a LC based on the individual\u2019s SNP genotype. Kelvin then integrates over the trait parameters separately within LC as described above, which allows for dependence of penetrances on LC. We rescanned the genome under this model in order to look for possible modifier loci interacting with the gene under the primary linkage peak. We also carried out conditional 2L-PPLD analyses to see if we could fine map under a secondary linkage peak down to the level of the individual modifier SNP .In addition to these analyses, we also used the simulated pedigree to assess variability of the MCMC portion of the calculations. First, we repeated the entire MCMC process as described above five times, and examined variability of the results across these five runs. Second, we ran a single, much longer sampling process  for which convergence was almost certainly achieved, then compared our results as described in step 1 with the final 5,000 iterations from the tail (post-convergence) end of this run. Finally, we considered variability across individual runs of 200 iterations with a 1,000-sample burn-in, that is, the length of runs that were averaged over in step 1 above.In this section we (i) show results of the analysis of the single large pedigree. We then (ii) consider the accuracy of the MCMC component of the analysis.Figure 3A shows the initial linkage scan. A peak on chromosome 4 clearly stands out above background noise, and we considered this to be our primary linkage finding. The PPL is elevated across a broad region of the chromosome . However, the strongest evidence of linkage spans a relatively short region at approximately 175\u2013181 cM.Table 1). Two SNPs (rs6851302 and rs654089) clearly stand out from the rest, with PPLD = 0.43 in both cases. These two are in complete LD with one another (R2 = 1) and in fact they share the same genotypes across this pedigree; rs6851302 and rs11934037 also show some LD (R2=0.28). Note that even had we restricted fine-mapping to just the best supported region (175\u2013181 cM), we would have successfully found this LD peak. Also for reference purposes, had we selected all 15,531 SNPs from all regions across the entire genome with PPL \u2265 10%, only one additional SNP would have given PPLD \u2265 5% .For purposes of fine-mapping, we considered any positions on this chromosome with PPL \u2265 10%. The resulting (non-contiguous) region contained 9,433 SNPs from the full original marker set. Forty-nine percent of the analyzed SNPs within the linked regions gave evidence against LD (PPLD < 0.0004), while only six SNPs (0.064%) showed PPLD \u2265 5% . There are no large 2L peaks . However, using the difference between the 2L and SL-PPLs as an indication of how much the data \u201cprefer\u201d the 2L model over the SL model , the largest positive difference occurs on chromosome 6 at 112 cM . The doubling of the PPL under the epistasis model suggested a possible modifier gene location. We determined the width of the linkage peak by visual inspection as covering approximately 100\u2013114 cM , and ran conditional 2L-PPLD analyses on all 3,120 SNPs in this region.We then conditioned on rs6851302 in order to rescan the genome for evidence of modifier loci.  Figure 5A shows the SL-PPLD under the linkage region on chromosome 4, and Figure 5B shows the 2L-PPLD \u2013 SL-PPLD across the linkage region on chromosome 6; again a single region is elevated in the 2L analysis, with the highest positive change in the PPLD occurring at rs1145787 . While these numbers are very small, they are still considerably higher than the prior probability of LD, and viewed in terms of 2L\u2013SL differences, rs1145787 is clearly salient.In summary, SL linkage analysis in this single pedigree enabled us to narrow the primary genomic region of interest to 6 cM on chromosome 4, while fine-mapping based on LD within this region detected the true causal variant (locus 1) within this region along with one other variant in complete LD with the causal one. The modifier locus was not salient in the initial linkage scan, however, 2L analysis conditioning on genotype at locus 1 led to discovery of the true modifier variant. While both the PPL and the PPLD at this locus were relatively small, they were easily detected based on the amount of increase of the 2L signals relative to the original SL signals.Table 2). The disease allele frequency is estimated quite accurately by both PPL and PPLD analyses at locus 1; while at locus 2, the 2L-PPLD in particular returns an estimate reasonably close to the generating model.  following the theory developed in Figure 6A, repeating the entire MCMC sampling process five times produced very similar, albeit not identical, PPL profiles across chromosome 4. The marker log likelihood for chromosome 4 from the single long MCMC run still showed some upward convergence up to about 14,000 iterations, at which point it remained essentially flat. Comparing the final (post-convergence) 5,000 iterations with the original results  again supported the accuracy of the original analysis in terms of the PPLs themselves. Again, however, the results are not identical. Figure 6C shows PPLs based on each of the component shorter sampling runs  considered independently. There is considerable variation, particularly at positions further away from the true casual SNP. This strongly suggests, not surprisingly, that shorter runs of this length are not individually sufficient.As seen in However, averaging across this set of shorter runs did enable us to achieve accurate results. Compared to a single, extremely long run, this is also a highly cost-effective approach insofar as it enables us to distribute the MCMC iterations to run concurrently on separate processors. On our hardware, the pooled-iteration process (using 10 servers with 2.5 GHz CPUs and 8 G memory) required 4 h, 40 min to complete chromosome 4, while the single long run (using one server) required 3 days, 5.5 h. Additional simulation studies are needed to further compare averaging across shorter sampling runs with use of single long sampling runs, especially across different pedigree structures with different patterns of missing data.We have illustrated an approach to gene discovery based on a single, highly informative family. This approach involves narrowing the genomic region(s) of interest using linkage analysis, followed by fine-mapping based on targeted LD (association) analysis in the same family. We have additionally illustrated how not just primary but even modifier genes can in principle be detected within a single pedigree.Of course, we \u201ccheated\u201d by including the two causal SNPs in our association panel. In general, we might expect to have data from a standard SNP chip available on most family members for purposes of linkage and association mapping, together with sequence on a subset of individuals. In this case, the association analyses could be conducted on every observed sequence variant in the regions of interest, ensuring that the true mutation would be included (assuming that the relevant disease-causing element is a SNP). Of course to do this, the sequence variants would need to be measured in many family members, but at least in principle this could be done in part through imputation of sequence using sequencing in a subset of individuals combined with SNP data on the remaining individuals.We chose our 2L generating model to be moderately mappable at the primary locus but with a modifier locus that was much harder to find. Of course in practice, realistic models may present more difficult challenges at all component loci, and this illustration is in no way to be construed as an estimate of any kind of power to find the genes. However, one salient feature of this approach is that it is not dependent on bioinformatic \u201cfiltering\u201d approaches to prioritizing sequence variants as likely candidates. Instead, following the now classical reverse genetic paradigm, we rely entirely on positional mapping even at the variant-selection stage. Again, in practice this is likely to still leave a number of variants as candidates, since highly correlated variants under a peak may still be difficult to resolve statistically. Nevertheless, the number of such variants likely to be left on the list of candidates is greatly reduced by focusing on the linked and associated regions.p-values, is required for this. However, Kelvin outputs posterior marginal distributions, which can be used to sequentially update results across data sets without the need to actually pool the data themselves across sites.As noted above, the PPL framework is designed to measure strength of evidence, and not to test hypothesis or serve as a decision making algorithm. Thus at no point in the discussion did we consider \u201csignificance levels\u201d or decide whether the evidence was \u201cstrong enough\u201d to declare success. Rather, we relied on the accuracy of the framework overall as an evidence measurement technique, and simply followed up on the strongest evidence wherever that occurred. In this particular case, doing so led us to find both genes and both causal SNPs, without any \u201cfalse positive\u201d results. In practice, of course, difficult decisions would need to be made before, e.g., expending substantial resources following up functionally on the locus 2 SNP, given the very low PPLD. Nevertheless, had we set very stringent significance criteria from the outset and refused to follow-up on the strongest evidence regardless of the absolute numbers involved, we would have missed the modifier locus entirely. We note too that in consortium settings, Kelvin\u2019s use of Bayesian sequential updating to accumulate evidence across data sets provides an alternative to traditional meta-analysis. Access to primary data, and not just summary measures such as The study design utilized here presented us with one salient computational challenge: how to compute the (parametric) likelihood for so large a pedigree. For this purpose we engineered a hybrid version of Kelvin using MCMC for the marker data and a non-stochastic method for integration over the trait parameters. This method proved to be quite accurate and computationally feasible, at least for data of this type. Of course the method can also be applied to sets of large pedigrees, and as noted, combined with ES- or LG-based analyses of smaller pedigrees or pedigrees with sparser marker maps for greater computational efficiency when analyzing data sets with variable family sizes.Further studies in additional pedigree structures are needed to make specific recommendations regarding burn-in lengths and number of iterations needed to maximize the chances of accurate results for the MCMC portion of the calculation. In this regard, our new method is no better and no worse than McSample itself. However, we have some reason to think that the PPL and PPLD themselves may be relatively robust to some level of sampling variability in the underlying marker likelihood, possibly in part because integration over the trait model protects against modest amounts of imprecision at the marker level. This remains a subject for further investigation.In this particular application, however, 2,000 samples derived from pooling the results of 10 independent sampling processes, each with 200 iterations following a 1,000-sample burn-in, appears to have been highly accurate. Still, this approach remains out of reach for genome-wide analysis on a typical desktop machine, requiring instead a distributed cluster environment to make real-time completion of results feasible. As high performance computing environments become more common for purposes of whole-genome sequence analysis and other \u201c-omics\u201d applications, we hope that this will become less of an impediment to analyses of the sort proposed here. Given the costs of data collection, we would argue that the additional computational demands are worth while if the methods are effective. The most definitive demonstration that they were effective in the current application is in the final results: successful mapping of two interacting disease loci down to the level of the individual causal variants.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "Philomachus pugnax) genome was constructed based on segregation analysis of 58 microsatellite loci from 381 captive-bred individuals spanning fourteen breeding years and comprising 64 families. Twenty-eight of the markers were resolved into seven linkage groups and five single marker loci, homologous to known chicken  and zebra finch (Taeniopygia guttata) chromosomes. Linkage groups range from 10.1 to 488.7 cM in length and covered a total map distance of 641.6 cM, corresponding to an estimated 30\u201335% coverage of the ruff genome, with a mean spacing of 22.9 cM between loci. Through comparative mapping, we are able to assign linkage groups Ppu1, Ppu2, Ppu6, Ppu7, Ppu10, Ppu13, and PpuZ to chromosomes and identify several intrachromosomal rearrangements between the homologs of chicken, zebra finch, and ruff microsatellite loci. This is the first linkage map created in the ruff and is a major step toward providing genomic resources for this enigmatic species. It will provide an essential framework for mapping of phenotypically and behaviorally important loci in the ruff.A linkage map of the ruff ( Philomachus pugnax) exhibit three different and distinct permanent alternative male reproductive morphs, with correlated differences in territorial lekking behavior, body size, and the presence or coloration of ornamental breeding plumage. All populations include: (1) dark-plumed territorial \u201cIndependents,\u201d (2) white-plumed nonterritorial \u201cSatellites,\u201d and (3) small female mimics called \u201cFaeders\u201d  determined parentage prior to this study.http://www.ensembl.org), following approaches described elsewhere  using CERVUS v.3.0. The resulting parentage assignments were compared with the previous pedigree, held by DBL and SBM, for inconsistencies. Grandparent\u2013Parent\u2013Offspring genotypic inconsistences arising from incorrect parentage assignment or microsatellite genotyping errors were detected through a three-generation Pedigree Program  and either resolved by rechecking the parentage and past genotyping records held by DBL and SBM, reviewing raw allele peaks on GENEMAPPER v.4.0 or, in any remaining cases of uncertainty, rescored as untyped.a priori knowledge .The mean marker spacing was calculated by dividing the total length of the map by the number of intervals. Average intramarker spacing for each linkage group was calculated by dividing the length of each linkage group by the total number of intervals on that linkage group. Linkage map coverage was calculated by summing the difference in base-pair position in chicken of the first and last interval on each linkage group, and dividing by the total base-pair length of the chicken genome  could not be assigned predicted chromosomal locations in either genome based on sequence similarity.Based on comparative mapping methods of microsatellite loci homologous to the ruff, chicken, and zebra finch, homologs of 55 of the 58 typed microsatellite loci were assigned predicted chromosomal locations in the chicken genome and 53 were assigned locations in the zebra finch Table . Five ruThe first-generation linkage map of the ruff consisted of 23 microsatellite markers resolved into 7 linkage groups  homologous to chicken and zebra finch chromosomes. Each linkage group was numbered according to the homologous chicken and zebra finch chromosome number . However, additional sequence is covered by the ruff map if the five chromosomes with single markers and the sequence immediately beyond the first and last markers on each linkage group are included. Assuming the ruff has a similar genome size to the chicken (http://www.genomesize.com/), it may be estimated that our map covers 30\u201335% of the ruff genome. The proportion of the total genetic  length of the ruff genome covered by the map is harder to assess, as the microchromosomes are mostly unmapped. Although microchromosomes are physically short and contribute little to the physical genome size, they each have an obligate crossing-over event during meiosis, which contributes 50 cM to the total map length , chromosome 2 (loci Ppu018 and Ppu022) and chromosome 7  genome, comparative analyses between the turkey, zebra finch (Taeniopygia guttata), and chicken  have identified a large number of intrachromosomal rearrangements, reflective of avian genome evolution (Skinner and Griffin Despite the highly conserved synteny generally believed to exist among avian genomes (Griffin et al. In summary, the map of seven linkage groups and length 641.6 cM covers an estimated 30\u201335% coverage of the ruff genome. It is the first linkage map of any shorebird species and will be of utility, even at this low density, as previous studies with approximately 30% map coverage have met with some success in the mapping of phenotypic loci (Miwa et al."}
+{"text": "Genetic linkage maps are important tools in breeding programmes and quantitative trait analyses. Traditional molecular markers used for genotyping are limited in throughput and efficiency. The advent of next-generation sequencing technologies has facilitated progeny genotyping and genetic linkage map construction in the major grains. However, the applicability of the approach remains untested in the fungal system.Lentinula edodes, is a basidiomycetous fungus that represents one of the most popular cultivated edible mushrooms. Here, we developed a rapid genotyping method based on low-coverage (~0.5 to 1.5-fold) whole-genome resequencing. We used the approach to genotype 20 single-spore isolates derived from L. edodes strain L54 and constructed the first high-density sequence-based genetic linkage map of L. edodes. The accuracy of the proposed genotyping method was verified experimentally with results from mating compatibility tests and PCR-single-strand conformation polymorphism on a few known genes. The linkage map spanned a total genetic distance of 637.1 cM and contained 13 linkage groups. Two hundred sequence-based markers were placed on the map, with an average marker spacing of 3.4 cM. The accuracy of the map was confirmed by comparing with previous maps the locations of known genes such as matA and matB.Shiitake mushroom, We used the shiitake mushroom as an example to provide a proof-of-principle that low-coverage resequencing could allow rapid genotyping of basidiospore-derived progenies, which could in turn facilitate the construction of high-density genetic linkage maps of basidiomycetous fungi for quantitative trait analyses and improvement of genome assembly. Genetic linkage maps are maps that show the relative positions and distances between markers or genes along the chromosomes as determined by their recombination frequency. Such maps are important in breeding programmes as they facilitate discovery of quantitative trait loci (QTL), association analysis as well as map-based localization and cloning of genes encoding agronomically essential phenotypes. However, the usefulness of a genetic linkage map depends on the choice and number of polymorphic markers used . TraditiLentinula edodes, is a basidiomycetous fungus that represents one of the most popular cultivated edible mushrooms. It also plays important roles in the pharmacological [L. edodes follows a typical basidiomycete life cycle, in which two monokaryotic mycelia with compatible mating types first fuse to form a dikaryon, the mycelia of which then aggregate to form a primordium under appropriate environmental conditions, which finally mature into a fruiting body. Currently available genetic linkage maps of L. edodes were constructed mainly using the fingerprinting type of markers such as randomly amplified polymorphic DNA (RAPD) [L. edodes is currently unavailable.Shiitake mushroom, ological  and biotological . L. edodA (RAPD) -10 or amA (RAPD) ,12 that L. edodes monokaryon L54A by Roche 454 GS FLX-Titanium and ABI SOLiD sequencing at over 20-fold coverage [L. edodes strain L54 and constructed, for the first time, a high-density sequence-based genetic linkage map of L. edodes. We used this as an example to provide a proof-of-principle that low-coverage resequencing could allow rapid genotyping of basidiospore-derived progenies for construction of high-density genetic linkage maps of basidiomycetous fungi.Previously, we sequenced the draft genome of coverage . The avaL. edodes monokaryon L54A we sequenced previously contained 767 scaffolds after de novo genome assembly [The draft genome of assembly . The totThe genetic linkage map spanned a genetic distance of 637.1 cM, and was composed of 13 linkage groups >20 cM in length  in all the 14 SSIs examined. Mating compatibility tests also confirmed all but one of the genotypes of the mating-type genes matA and matB. Therefore, the overall accuracy of the proposed genotyping approach was over 98%.The accuracy of the proposed genotyping method was verified experimentally  (Data not shown). This probably represents a limitation of the current genotyping method, which is based on simple majority voting. The issue may be more prevalent in large scaffolds such as S499 here. As such, it is recommended to examine large scaffolds in more detail if genotypes of individual genes are to be investigated. However, as the overall estimated accuracy of the proposed approach was over 98%, we expect limited effects of the issue on the linkage relationships revealed between the scaffold markers.To verify the accuracy of the proposed genotyping approach, we compared the genotypes of four known genes called using the current method with those determined experimentally using either mating compatibility tests or PCR-SSCP  on LG2  on LG2. The matB locus was on LGII in Kwan and Xu [matB and hyd1 on the same linkage group reported previously [matB, hyd1 and mfbB on the same linkage group reported in Miyazaki et al. [To examine the accuracy of the current linkage map, we compared with previous maps the locations of some known genes on the linkage groups. The wan & Xu , LGII inwan & Xu ,12 and Lwan & Xu ,10, all  on S499 0.6 cM onge group -10,12. Tn and Xu , LGV in n and Xu ,12 and LL. edodes that comprised 301 markers and spanned ~900 cM  of LG5 here, and in linkage with genes encoding P450 monooxygenase and oxidoreductase, which were on S48 on the same location of the same linkage group  and grown for 1\u20132 months at 25\u00b0C in the dark. The mycelia were collected by filtration, frozen and homogenized in liquid nitrogen. Genomic DNA was extracted from the mycelial fragments using the DNeasy Plant Mini Kit (Qiagen).Twenty basidiospores were randomly isolated from fresh-fruiting bodies of the Shotgun sequencing of the 20 SSIs was performed in multiplex with multiplex identifiers (MIDs) on a GS FLX-Titanium sequencer (454 Life Sciences Corporation) in 1.5 plates run. Genotyping was performed by mapping the sequencing reads of each SSI to the parental L54A reference genome using the GS Reference Mapper version 2.6 (454 Life Sciences Corporation) with default parameters. Identical mapped reads were assigned to genotype \u201cA\u201d whereas reads with high-quality SNPs were regarded as genotype \u201cB\u201d. Here, each scaffold sequence (genotype A or B) served as a genetic marker for linkage map construction. The genotype profile of markers for each SSI was generated using simple majority voting. Briefly, on a certain scaffold, if a particular SSI contained more mapped reads with genotype \u201cA\u201d than \u201cB\u201d, then the marker was genotyped as \u201cA\u201d, and vice versa. A \u201cX\u201d was assigned in cases where there were an equal number of \u201cA\u201d and \u201cB\u201d, and a \u201c-\u201cwas assigned when no reads were mapped on the scaffold. The genotyping process was automated using in-house developed Perl scripts. A flowchart of the procedure was given in Figure\u00a0\u03c72 test. Markers with a segregation ratio of 1:1 (P < 0.05) were clustered into linkage groups when the two-point LOD scored \u2265 3. Map distances between markers were calculated based on the Kosambi\u2019s mapping function. The maximum distance between markers in a single linkage group allowed was 25 centiMorgan (cM). Orientation of markers within individual linkage groups was confirmed by feeding smaller marker subsets to MAPMAKER/EXP V3.0 [The genetic linkage map was built using MSTMap . GenotypEXP V3.0 . The genEXP V3.0 .matA, matB, priA, and hyd1 were determined experimentally in 14 randomly selected SSIs .The data set supporting the results of this article is available in the NCBI Sequence Read Archive under the accession number SRP026195 (AFLP: Amplified fragment length polymorphism; cM: centiMorgan; MID: Multiplex identifier; NGS: Next-generation sequencing; PDA: Potato dextrose agar; PDB: Potato dextrose broth; QTL: Quantitative trait loci; RAPD: Randomly amplified polymorphic DNA; RIL: Recombinant inbred line; SNP: Single-nucleotide polymorphism; SSCP: Single-strand conformation polymorphism; SSI: Single-spore isolate.The authors declare that they have no competing interests.CHA performed genotyping and genetic linkage map construction and helped draft the manuscript. MKC participated in genetic linkage map construction and wrote the manuscript. MCW was responsible for SSI cultivation and DNA extraction. AKKC carried out the mating compatibility tests and PCR-SSCP analysis. PTWL performed genome sequencing. HSK conceived and supervised the study. All authors read and approved the final manuscript.L. edodes L54A strain is 40.2 Mb.Whole-genome resequencing throughput of the SSIs, Description. The genome size of the reference Click here for fileVerification of the genotyping results. Description: Genotypes before the slashes were determined experimentally whereas those after the slashes were determined by the proposed genotyping approach. The genotype in parentheses could be corrected after manual curation.Click here for fileExample of genes on the linkage map markers. Description: Examples of genes located on each of the linkage map markers.Click here for fileL. edodes genetic linkage maps. Description: Comparison of characteristics of major L. edodes genetic linkage maps.Comparison of major Click here for filePCR primers and protocol for PCR-SSCP. Description: Detailed protocol for PCR-SSCP.Click here for file"}
+{"text": "Despite many years of research, most of the genetic factors contributing to myopia development remain unknown. Genetic studies have pointed to a strong inherited component, but although many candidate regions have been implicated, few genes have been positively identified.We have previously reported 2 genomewide linkage scans in a population of 63 highly aggregated Ashkenazi Jewish families that identified a locus on chromosome 22. Here we used ordered subset analysis (OSA), conditioned on non-parametric linkage to chromosome 22 to detect other chromosomal regions which had evidence of linkage to myopia in subsets of the families, but not the overall sample.Strong evidence of linkage to a 19-cM linkage interval with a peak OSA nonparametric allele-sharing logarithm-of-odds (LOD) score of 3.14 on 20p12-q11.1  was identified in a subset of 20 families that also exhibited strong evidence of linkage to chromosome 22. One other locus also presented with suggestive LOD scores >2.0 on chromosome 11p14-q14 and one locus on chromosome 6q22-q24 had an OSA LOD score=1.76 .The chromosome 6 and 20 loci are entirely novel and appear linked in a subset of families whose myopia is known to be linked to chromosome 22. The chromosome 11 locus overlaps with the known Myopia-7  locus. Using ordered subset analysis allows us to find additional loci linked to myopia in subsets of families, and underlines the complex genetic heterogeneity of myopia even in highly aggregated families and genetically isolated populations such as the Ashkenazi Jews. Myopia is a leading cause of visual impairment worldwide, affecting approximately 1 in 4 US adults . Myopia Many genetic linkage studies of myopia, predominantly focusing on highly penetrant, severe manifestations in highly ascertained families have identified several regions across the genome that were linked to myopia including 2q37.1 , 4q22-q2We have previously reported genomewide linkage studies of ocular refraction phenotypes performed in Ashkenazi Jewish families ,32,36. TFamily recruitment and selection criteria have been reported elsewhere and are summarized here . In brieSixty-three multiplex Ashkenazi Jewish families were included in the study. Eligibility for family participation in the study required an index case whose spherical equivalent refraction was \u22121.00 Diopters (D) or lower in both eyes (as long as there was \u22121.00 D or lower in each meridian if astigmatism was present) and had no history of a systemic or ocular disease that might predispose to myopia, including premature birth. Cycloplegic refractions were used for index cases under 50 years of age while manifest refractions were used for those above age 50. The same classification scheme was used to determine affection status for all individuals in the pedigrees, and subjects who did not meet this standard were regarded as unaffected. If a subject was reported to have been myopic but this diagnosis could not be confirmed with either medical records, measurement of the prescription of a pair of eyeglasses, or current physical examination, the individual was treated as being of \u201cunknown\u201d phenotype.Because of the normal developmental changes in refraction during childhood and the potential for misclassification, a more stringent approach to classification of affected versus unaffected subjects was used for the groups of individuals aged 6\u201310 years and 11\u201320 years. All individuals with a \u22121.00 D or lower spherical equivalent were considered affected, as above, regardless of age. However, subjects in the group of individuals aged 6\u201310 years with a +2.00 spherical equivalent refraction or higher in both eyes were classified as unaffected, since they are not likely to develop myopia. Individuals in this age group with a spherical equivalent between +2.00 and \u22121.00 were classified as \u201cunknown.\u201d Individuals in the group of subjects aged 11\u201320 years with +1.50 spherical equivalent or higher in both eyes were classified as unaffected. Any individual with a spherical equivalent of between +1.50 and \u22121.00 in this age group was placed in the \u201cunknown\u201d class. This conservative approach balances the power loss that results from our lack of a good segregation-analysis model of age-dependent penetrance and the concomitant confusion about appropriate genotype probabilities for young unaffected subjects, with the power loss resulting from the classification of normal children as \u201cunknown.\u201dCooperative Human Linkage Center marker set, version 9 . The final 19 families were genotyped at a later date also by CIDR using 402 markers from the modified Cooperative Human Linkage Center version 9. All genotyping was performed blind to clinical status.High\u2013molecular-weight DNA was isolated from buffy coats with a kit . Samples were stored in a DNA repository under a unique code. Altogether 481 DNA samples from 63 families including 220 affected males and 141 affected females were genotyped at the Center for Inherited Disease Research . The first 44 families were genotyped by CIDR using automated fluorescent microsatellite analysis. PCR products were sized on an ABI 3700 sequencer . The marker set used was a modification of the Since the two genome-wide linkage scans were performed at different times, we felt it was problematic to attempt to combine them by reconciling the genotypes at the same loci in the two data sets. Instead, for each microsatellite marker, we created two dummy markers with a genetic distance of 0 between them. The first data set had real genotypes at dummy marker 1 and missing data at dummy marker 2. Individuals in the second data set were coded with missing data at dummy marker 1 and their real genotypes at dummy marker 2.GENEHUNTER-PLUS software [These analyses have been described elsewhere and are summarized here ,32. Multsoftware ,45 to obTo address genetic heterogeneity we used linkage to 22q12 as a covariate and performed Ordered Subset Analysis (OSA) . Non-parGENEHUNTER-PLUS for chromosome 22 at the position of the known linkage peak were used as the covariate in the non-parametric OSA. The distribution of individual family NPL scores can be seen in Individual family NPL scores calculated by Also found by ordering families by descending maximum non-parametric linkage to chromosome 22 was a locus with a maximum OSA LOD score of 2.36 obtained at D11S1344 (68.72 cM) in a subset of 45 families with the strongest evidence of nonparametric linkage to chromosome 22 with NPL score ranging from 12.2 to \u22120.3. This was an increase of 1.68 in the OSA LOD compared with the unconditional test using all families and this increase was nominally significant by permutation testing (p=0.035). The 1-LOD-unit support interval spans 48cM, from 42 to 90 cM . This suThe third locus, at 6q22-q24, was also found by ordering families by descending maximum non-parametric linkage to chromosome 22. This locus had a maximum OSA LOD score of 1.76 at D6S1009 (142.57 cM), an increase of 1.65 in the OSA LOD compared to the unconditional test using all families and this increase was significant by permutation testing (p=0.02) after correction for multiple tests. This subset contained 36 families with the strongest evidence of nonparametric linkage to chromosome 22. The 1-LOD-unit support interval was 20 cM, from 129 to 149 cM. .This sub609256); and another locus on 20p12-p11.We have identified three regions with suggestive evidence of linkage to myopia in subsets of families already linked to chromosome 22: a locus on 6q22-q24 which seems to be entirely novel; a locus on 11p14-q14 which, although large, does overlap with Myopia-7  [PAX6 polymorphisms and myopia is mixed [The locus on chromosome 11, although broad, overlaps MYP7, a locus previously reported in a population of UK twins and of particular interest as it contains the known eye gene, paired box gene 6 (PAX6) . So far is mixed -58, and is mixed , is comiis mixed  but not is mixed . Again, PEX7) gene, mutations in which can cause ocular phenotypes as part of the severe systemic syndromes Refsum disease [The chromosome 6 locus is also novel and is the only one of these three loci to withstand correction for multiple testing. The region contains few genes, but D6S1009 is within 100 kb of the peroxisome biogenesis factor 7  or targeted sequencing of the region in selected individuals would be useful approaches to try and narrow down the variants responsible for the signal. Given the current advances in sequencing technology, whole genome sequencing of appropriate individuals from these families may be more cost effective than targeted sequencing or custom genotyping.Using ordered subset analysis allowed us to find additional loci linked to myopia in subsets of Ashkenazi Jewish families, and underlines the complex genetic heterogeneity of myopia even in highly aggregated families and genetically isolated populations. It is also of note that when these data were analyzed as the refractive error quantitative trait, linkage to the 22q12 locus was not significant and instead a locus on 1p36 was strongly significant . This em"}
+{"text": "Diabetic nephropathy (DN) affects about 30% of patients with type 1 diabetes (T1D) and contributes to serious morbidity and mortality. So far only the 3q21\u2013q25 region has repeatedly been indicated as a susceptibility region for DN. The aim of this study was to search for new DN susceptibility loci in Finnish, Danish and French T1D families.pairs LOD score 3.58). Nominal or suggestive evidence of linkage to this locus was also detected when the three populations were analyzed separately. Suggestive evidence of linkage was found to six additional loci in the Finnish and French sample sets.We performed a genome-wide linkage study using 384 microsatellite markers. A total of 175 T1D families were studied, of which 94 originated from Finland, 46 from Denmark and 35 from France. The whole sample set consisted of 556 individuals including 42 sib-pairs concordant and 84 sib-pairs discordant for DN. Two-point and multi-point non-parametric linkage analyses were performed using the Analyze package and the MERLIN software. A novel DN locus on 22q11 was identified in the joint analysis of the Finnish, Danish and French families by genome-wide multipoint non-parametric linkage analysis using the Kong and Cox linear model (NPLThis study identified a novel DN locus at chromosome 22q11 with significant evidence of linkage to DN. Our results suggest that this locus may be of importance in European populations. In addition, this study supports previously indicated DN loci on 3q21\u2013q25 and 19q13. Diabetic nephropathy (DN) is a major complication in patients with type 1 diabetes (T1D) and contributes to serious morbidity and mortality. It affects about one-third of the patients The incidence of DN reaches its peak after 20 years of diabetes The chromosome region 3q21\u2013q25 has been linked to DN in patients with T1D in several genetic studies The aim of this study was to search for susceptibility loci for DN in Finnish, Danish and French T1D families using a genome-wide linkage strategy. We found a new susceptibility locus and were able to replicate two previously identified loci.All participants provided written, informed consent. Approvals to conduct the research were obtained from the Helsinki University Central Hospital Ethics Committee, Finland, the Ethical Committee of Copenhagen, Denmark, and the Angers University Ethics Committee, France. The study was in accordance with the principle of the Helsinki Declaration.Altogether 175 Finnish, Danish and French families with T1D were studied. The families were mainly nuclear families including one or both parents and siblings concordant or discordant for DN or solely sib-pairs concordant or discordant for DN. In addition, sib-pairs with T1D and normal renal status were included. Patients with DN were identified among those with age of onset of T1D at 35 years or less and who had had T1D for at least 10 years. The distribution of siblings per family and the number of parents and other relatives studied are presented in The Finnish families were recruited by the Finnish Diabetic Nephropathy (FinnDiane) Study Group In Denmark, a nationwide survey of the Danish Registry of Diagnosis was used to identify patients diagnosed with T1D and DN. After verification of patient records, a letter of invitation was sent to all patients with a diabetic sibling. A total of 46 families with at least two siblings with T1D were included in the study .The French and Belgian families (from now on indicated as French families) were collected on the occasion of the Genesis (Genetic Nephropathy Sib pair Study) France-Belgium study The diagnosis of T1D was based on age at onset less than 35 years and permanent insulin treatment initiated within one year of the diagnosis. Normal renal status, i.e. normoalbuminuria (DN\u2212), was defined as an albumin excretion rate (AER) <20 \u00b5g/min or <30 mg/24 h in two out of three consecutive overnight or 24-hour urine collections. Incipient nephropathy  was defined as an AER 20\u2013200 \u00b5g/min or 30\u2013300 mg/24 h and overt nephropathy  as an AER >200 \u00b5g/min or >300 mg/24 h. The patients on dialysis or with a kidney transplant due to diabetic nephropathy were also considered to have nephropathy (DN+). Retinopathy was defined as the presence or absence of retinal laser treatment. Cardiovascular hard end-points were defined as a history of myocardial infarction, coronary revascularization procedure, stroke or amputation. The clinical characteristics of the subjects with T1D in each sample are presented in Genotyping was performed at the Finnish Genome Center. The procedure was conducted using the MegaBACE  or the ABI genotyping systems  and the LMS-MD10 microsatellite marker set (Applied Biosystems) as previously described HLA DQA1-DQB1 haplotypes and DRB1*04 subtypes of DQB1*0302 positive haplotypes were screened in the Finnish families using the PCR-based lanthanide-labelled oligonucleotide hybridization and time-resolved fluorometry detection as described earlier pairs was used to analyze affected pairs (DN+/DN+). In order to use the information from unaffected individuals as well, a NPL quantitative trait linkage statistic (NPLqtl) was applied as described previously Initially, two-point non-parametric affected sib-pair (ASP) (DN+/DN+) linkage analysis was performed genome-wide. This analysis, as well as the transmission-disequilibrium-test (TDT), were performed using the ANALYZE program package implemented in the AUTOGSCAN software tool We estimated the significance thresholds for ASP analysis of 400 markers using the formulae presented by Feingold et al. pairs are shown in We first wanted to identify regions linked to DN in each population sample separately utilizing the concordant DN+/DN+ sib-pairs. Summary of the best ASP and NPL results is presented in In the Finnish study sample no chromosomal region showed significant evidence of linkage to DN in the ASP analysis. Suggestive evidence of linkage was, however, found between DN and four chromosomal regions: 6p21 (ASP LOD score 2.31), 16p12 (ASP LOD score 1.82), 17q25.3 , and 22q11 (ASP LOD score 2.09) . Nominalpairs analysis. The test indicated suggestive evidence of linkage between DN and 6p21 (NPLpairs LOD score 2.10), and 16p12 (NPLpair LOD score 2.09).In order to pool information from multiple markers we also performed a multipoint, nonparametric NPLpairs analysis indicated several loci also supporting linkage to 22q11.In the Danish sample, no chromosomal region showed significant or suggestive evidence of linkage to DN in the ASP analysis. Nominal evidence of linkage was found to six chromosomal regions . Multipopairs analysis supported evidence of linkage to most of these regions and also indicated linkage to 7q36 and 16q12  and 9p21 . Nominal evidence of linkage was found to five additional chromosomal regions including 22q . Resultsnd 16q12 .pairs analysis was applied, the linkage result at 22q11 improved showing significant evidence of linkage between D22S420 and DN  providing validation that our patient population is comprised of T1D patients \u22127 for DN. However, this was mainly driven by the major known T1D risk haplotype DRB1*0401_DQB1*0302, p-value for this allele was 2.6\u00b710\u22125 for DN. All DN+/DN+ pairs are by definition also concordant for T1D.We decided to study the 6p21 region further and genotyped the Finnish patients for the common HLA DR-DQ haplotypes. We also analyzed the Finnish sample set for T1D, and found significant evidence of linkage to the HLA-region on 6p21  or suggestive  evidence of linkage in the ASP analysis and for the two loci previously indicated in DN (3q21\u2013q25 and 19q13). IBD estimation indicated true linkage between DN and 22q11, 3q24, 9p21, 16p12 and 19q13 .qtl Z-score statistic. In the Finnish sample suggestive evidence of linkage between DN and loci on 5p15-p14 and 15q15 was detected (qtl LOD scores 2.06 at 46 cM and 1.94 at 37 cM). The linkage peak at 15q15 (NPLqtl LOD score 1.74 at 43 cM) was surrounded by two markers also indicating linkage , we used a non-parametric quantitative trait linkage NPLdetected . Two conIn the Danish sample nominal evidence of linkage was detected to 5q33.2 and 22q11 .qtl LOD score 2.19) was detected  even after these markers were included in the analysis. The results are detailed in Since the joint analysis of the Finnish, Danish and French families showed significant evidence of linkage to 22q11, we genotyped four additional markers on 22q11\u2013q12 in all three study samples to increase the linkage information across the implicated region. The highest linkage peak was found at 3 cM . When the 13 markers were included in the analysis, the highest peak was found at 152.3 cM .Since the 3q21\u2013q25 region has been suggested as a DN susceptibility region in several previous studies pairs LOD score 3.58) between DN and 22q11 in the joint analysis of the Finnish, Danish and French T1D families. When analyzed separately, the samples showed either suggestive or nominal evidence of linkage between DN and 22q11. The IBD pattern in all three samples indicated that concordant sib-pairs share two alleles more often than expected while discordant pairs do not share alleles more often than expected. For example, when viewing marker D22S420 in the Finnish sample 45% of the DN+/DN+ pairs share two alleles while 27% of the DN+/DN\u2212 pairs share two alleles. This difference indicates that our finding is most likely due to DN and not to T1D. Similar effect was observed in the joint sample where the corresponding values were 38% and 20%. After fine-mapping the region around the 22q11 locus the best linkage signal was detected at 3 cM (D22S420) and this locus does not overlap with any previously reported DN locus detected in patients with T1D. On the other hand, a locus approximately 20 Mb apart from our susceptibility region has in the FIND Study recently been linked to urinary albumin\u2236creatinine ratio in Mexican-Americans with diabetes MYH9  and APOL1 (Apolipoprotein L1) genes associated in African-Americans with diabetic or nondiabetic nephropathy The present study was conducted in three different population samples, the Finnish, the Danish, and the French, among which T1D is most common in the Finns IL-17RA encodes interleukin 17 receptor precursor protein, a ubiquitous type I membrane glycoprotein that binds with low affinity to interleukin 17A. Interleukin 17A and its receptor play a pathogenic role in many inflammatory and autoimmune diseases such as rheumatoid arthritis SLC25A18  nuclear gene encoding mitochondrial protein (GC2) is involved in the transport of glutamate across the inner mitochondrial membrane The 22q11 region contains over 60 protein coding genes. Two of these are especially relevant candidates for DN: The 22q11\u2013q12 region also contains low-copy repeats and is known for its susceptibility to genomic rearrangements due to non-allelic homologous recombination. Various syndromes including the velo-cardia-facial/diGeorge syndrome, the cat-eye syndrome, schizophrenia, and glial tumors of the brain In addition to the 22q11 locus, our study further confirms the previously reported susceptibility loci for DN in T1D on 3q21\u2013q25 and 19q13. Including our report, 3q21\u20133q25 has now been indicated as a susceptibility region for DN in four different populations: the American Caucasians FRMD3 (FERM domain containing 3) gene have been associated with DN in Northern American populations with T1D AUH gene (AU RNA binding protein/enoyl-Coenzyme A) on the other hand, has been implicated in T2DM-ESRD in African Americans In addition to these three susceptibility loci, we identified in each population other possible DN loci. In the French sample two susceptibility regions were found on chromosome 9. The susceptibility region on 9p21.3 has in previous studies shown to harbor two major disease susceptibility loci: one for T2D qtl analysis that is able to retrieve information also from unaffected individuals thus increasing the number of the families in the analysis. We also looked at the IBD sharing patterns on several loci. The results of this analysis support the loci on 22q11, 3q24, 9p21, 16p12, and 19q13 as true DN loci. Linkage to the 6p21 HLA-region, on the other hand, is probably due to T1D since 61% of the DN+/DN+ pairs and 55% of the DN+/DN\u2212 pairs share both alleles.The strength of our study design is the use of both concordant and discordant sib-pairs as well as other relatives. This is evident especially when we view the Finnish sample and the loci at 22q11, 3q24 and 5p14\u2013p15. The locus at 22q11 was detected in the analysis of the concordant sib-pairs. The two other loci surfaced in the NPLA limitation in our study is the sample size. The power to detect susceptibility loci with moderate effects is relatively low, especially when each sample is studied alone. However, all the samples are part of thoroughly characterized and prospectively followed cohorts that have generated a large amount of clinically useful information and contributions to our understanding of the pathogenesis of DN.The two GWAS performed to date have detected DN susceptibility regions on chromosomes 9q In summary, we found significant evidence of linkage between a novel locus on 22q11 and DN. This may indicate that the locus is of importance in the European populations. Our results further support the previous linkage findings between DN and chromosomes 3q21\u2013q25 and 19q13 and implicate several other possible loci in the pathogenesis of DN in T1D.Table S1Summary of the non-parametric linkage results for diabetic nephropathy on the regions showing LOD scores 1.00\u20131.73.(DOC)Click here for additional data file.Table S2List of participating centers and people involved.(XLS)Click here for additional data file."}
+{"text": "Linkage analysis has the potential to localize disease genes of interest, but the choice of which subjects to select for follow-up sequencing after identifying a linkage peak might influence the ability to find a disease gene. We compare nine different strategies for selection of subjects for follow-up sequencing using sequence data from the Genetic Analysis Workshop 17. We found that our more selective strategies, which included methods to identify case subjects more likely to be affected by genetic causes, out-performed sequencing all case and control subjects in linked pedigrees and required sequencing fewer individuals. We found that using genotype data from population control subjects had a higher benefit-cost ratio than sequencing control subjects selected as being the opposite extreme of the case subjects. We conclude that choosing case subjects for sequencing based on more selective strategies can be reliable and cost-effective. The well-established genome-wide linkage analysis method has the potential to focus the search for and identification of genetic loci responsible for a disease or trait. Although linkage analysis has been successfully used to find high-penetrant rare loci for a number of different diseases, with the advent of high-density marker sets, the challenges associated with linkage analysis have limited its use somewhat. These challenges include 1) the difficulty and cost of ascertaining large high-risk pedigrees, (2) shifts in general thought about whether common complex diseases are caused by common variants  or, most recently, by multiple rare variants [ the diffIn this study we focus on the third challenge, namely, the inability to identify a disease gene under a linkage peak. It is possible that the choice of subjects selected for follow-up sequencing after finding linkage evidence influences detection of underlying genetic loci. Because sequencing costs are still prohibitively expensive, particularly for large chromosomal regions, the number of subjects selected for follow-up sequencing may be limited. Here, we explore different strategies for selecting subjects for follow-up sequencing to identify the most reliable and cost-effective means for choosing appropriate sequencing candidates.We use the mini-exome sequence data available for Genetic Analysis Workshop 17 (GAW17). Eight extended pedigrees are simulated based on gene dropping from the pedigree founders\u2019 single-nucleotide polymorphism (SNP) genotypes, which were obtained from sequence alignment files provided by the 1000 Genomes Project pilot3 study. The original files include exonic genotype data for 3,205 genes and 24,487 SNPs. Eight multilineal pedigrees are provided and include 697 individuals, all of whom are genotyped. For each of the 697 individuals, we are also provided with information on affection status, age, sex, smoking status, and three quantitative trait variables called Q1, Q2, and Q4. According to the answers provided by the GAW17 data simulators, disease risk is influenced directly by age, smoking status, Q1, Q2, and Q4 and indirectly by sex through Q4. There are 200 replicates of the phenotype data variables; genotype data are constant over the 200 replicates.r2 = 0.16). Heterozygosity was calculated as 1 \u2212 (p2 + q2), where p is the major allele frequency and q is the minor allele frequency. We calculated linkage disequilibrium between all pairs of markers in pedigree founders using PLINK [We split the pedigrees into 23 unilineal pedigrees with the intent to include as many individuals in a pedigree as possible. Because multiple genes were known to contribute to the disease phenotype, we split pedigrees to reduce intrafamilial genetic heterogeneity. No duplication of individuals was allowed; parents of some individuals were set to zero to break links. After splitting, our data set for each replicate contained 648 individuals. We determined a linkage subset of markers on the basis of marker heterozygosity (minimum heterozygosity = 0.1) and linkage disequilibrium  using a cut point of 0.5. By graphing the predicted probabilities, we divided case subjects and control subjects into four quadrants based on case and control status and predictive probability (<0.5 or \u22650.5). Liability classes for the linkage analysis were divided into these four categories. Control subjects with low predictive probability  and case subjects with high predictive probability  were assigned a weaker penetrance function. Control subjects with high predictive probability , which we call high-covariate control subjects, and case subjects with low predictive probability , which we call low-covariate case subjects, were assigned a more powerful penetrance function.We performed a parametric model-based analysis using general dominant and recessive models. For the dominant model, we used a minor allele frequency of 0.01; for carriers of 0, 1, or 2 copies of the rare allele, the weaker penetrance function was set to be 0.2, 0.6, 0.6, respectively, and the more powerful penetrance function was set to be 0.0005, 0.5, 0.5, respectively. For the recessive model, we used a minor allele frequency of 0.01; for carriers of 0, 1, or 2 copies of the rare allele, the weaker penetrance function was set to be 0.2, 0.2, 0.6, respectively, and the more powerful penetrance function was set to be 0.0005, 0.0005, 0.5, respectively.p-value of 0.05, not accounting for multiple testing.Linkage analysis was performed using the multipoint Markov chain Monte Carlo linkage method MCLINK , which aAfter identification of linkage evidence and pedigrees linked to a region, we performed association testing for only the subjects in the linked pedigrees, using Genie , a softwWe compared nine different strategies for selection of case subjects for sequencing in the eight linked pedigrees  had individual LOD scores greater than 0.588. Variation existed for C17S4578, but not C17S4581, across the eight pedigrees. Association testing proceeded for C17S4578 using individuals from the eight linked pedigrees.We identified two regions that met the criteria for suggestive evidence of linkage, and using the GAW17 answers, we identified one of them as harboring a disease gene. The linkage peak identified was on chromosome 17 between C17S3663 and C17S5325 under a dominant model; our peak HLOD score was 2.44 at marker C17S5244. From the answers, we identified a disease gene, In Table In general, we observed that the overall benefit-cost ratio was higher under both a dominant and a recessive model using the 202 founders as the control population, because of the increased power obtained using a larger control group and the additional benefit of not incurring a cost to genotype the control specimens. However, the effect size for both the dominant and recessive models tended to be higher when the opposite extreme control subjects were used as the comparison group  from regression analysis. For this GAW17 analysis, the data simulators provided us with the covariate risk factors for the disease. It was our intent to find subjects who had the disease but whose disease was more likely attributable to genetic causes. This strategy can be applied to many complex diseases. Although all risk factors may not be known for a particular disease, major risk factors are likely to be known and could be incorporated into both the linkage model and the subsequent selection strategy for identification of sequencing candidates. We also saw good success with selection of the youngest haplotype-carrier case subject or the youngest case subject per pedigree. Use of early-onset case subjects as a surrogate for the low-covariate strategy may be useful when major risk factors for a disease are not known.In this study, we also explored two types of control groups: founders from the original pedigrees, which represented population controls, and control subjects selected as being the opposite extreme of the case subjects. We found that the benefit-cost ratio was higher, in general, across both dominant and recessive models for the population control group than for the model in which control subjects were selected to be the opposite extreme of case subjects. The assumption that population control data can be obtained in the future without cost is reasonable, because genotype data can be obtained freely now for various cohorts . However, it should be noted that if a disease is common in the population, results using publicly available sequence data will be conservative.We identified two linkage peaks that had suggestive evidence of linkage, only one of which contained a variant of interest. Suggestive evidence is defined as a LOD score expected once per chance per genome scan . Hence fIn conclusion, choosing case subjects for sequencing based on being a haplotype carrier and having low-covariate status can be reliable and cost-effective.The authors declare that there are no competing interests.KAB: Designed the study, performed the statistical analyses and drafted the manuscript. JF: Assisted with data analysis, attended the GAW17 meeting and assisted with manuscript preparation. LCA: Conceived of the study and helped to draft the manuscript. All authors read and approved the final manuscript."}
+{"text": "Estimated glomerular filtration rate (eGFR), a measure of kidney function, is heritable, suggesting that genes influence renal function. Genes that influence eGFR have been identified through genome-wide association studies. However, family-based linkage approaches may identify loci that explain a larger proportion of the heritability. This study used genome-wide linkage and association scans to identify quantitative trait loci (QTL) that influence eGFR.Genome-wide linkage and sparse association scans of eGFR were performed in families ascertained by probands with advanced diabetic nephropathy (DN) from the multi-ethnic Family Investigation of Nephropathy and Diabetes (FIND) study. This study included 954 African Americans (AA), 781 American Indians (AI), 614 European Americans (EA) and 1,611 Mexican Americans (MA). A total of 3,960 FIND participants were genotyped for 6,000 single nucleotide polymorphisms (SNPs) using the Illumina Linkage IVb panel. GFR was estimated by the Modification of Diet in Renal Disease (MDRD) formula.P\u200a=\u200a4.4\u00d710\u22125) in MA and chromosome 15q12  in EA. In all subjects, the strongest linkage signal for eGFR was detected on chromosome 10p12 (P\u200a=\u200a5.5\u00d710\u22124) at 44 cM near marker rs1339048. A subsequent association scan in both ancestry-specific groups and the entire population identified several SNPs significantly associated with eGFR across the genome.The non-parametric linkage analysis, accounting for the effects of diabetes duration and BMI, identified the strongest evidence for linkage of eGFR on chromosome 20q11 (log of the odds [LOD]\u200a=\u200a3.34; The present study describes the localization of QTL influencing eGFR on 20q11 in MA, 15q21 in EA and 10p12 in the combined ethnic groups participating in the FIND study. Identification of causal genes/variants influencing eGFR, within these linkage and association loci, will open new avenues for functional analyses and development of novel diagnostic markers for DN. Diabetes mellitus is responsible for approximately 50% of cases of incident end-stage renal disease (ESRD) in the United States and other Western societies, with projections of up to 70% of ESRD in 2015 Epidemiological studies demonstrate that eGFR is a complex trait, whose level in a given individual reflects contributions from genes whose expression is modulated by a hyperglycemic environment. Genome-wide linkage and association analyses have been used to localize susceptibility genes influencing eGFR. Several prior genome-wide linkage scans, including our previous genome scan in a subset of the same study subjects, identified positional candidate genes potentially influencing eGFR based on implicated chromosomal regions In an attempt to identify and characterize susceptibility genes influencing kidney disease in diabetes, we chose the family-based genome-wide linkage scan approach that can identify genetic regions where there are multiple susceptibility variants or other complex mechanisms that may in aggregate explain a larger proportion of the heritability than the single polymorphisms typically identified in GWAS. A genome-wide linkage screen was performed for eGFR based on 6,000 single nucleotide polymorphisms (SNPs) from Hispanic American (HA), African American (AA), European American (EA), and American Indian (AI) participants in the Family Investigation of Nephropathy and Diabetes (FIND). The FIND study was established to provide genome-wide coverage for localization of genes with pathogenically significant effects on risk of progressive DN and related traits, such as eGFR.The FIND study protocol and patient recruitment procedures have been reported eGFR was estimated using the Modification of Diet in Renal Disease (MDRD) equation : eGFR (ml/min per 1.73 m2)\u200a=\u200a186\u00d7(plasma creatinine)\u22121.154\u00d7(age)\u22120.203\u00d7\u00d7(1.210 if AA). For patients with ESRD (N\u200a=\u200a1275) receiving dialysis treatments or kidney transplants, eGFR was imputed at 5.0 ml/min/1.73 m2 because (1) eGFR is meaningless with respect to the participant's true kidney function under these circumstances; and (2) imputing at zero, an extreme value, would give the data from ESRD cases undue influence relative to those of the non-ESRD cases. A total of 3960 subjects, comprising 3547 sib pairs, were included in the analysis . Allele frequencies were estimated separately in the four ethnic groups using the maximum likelihood method implemented in the program FREQ. Mendelian inconsistencies were identified with the MARKERINFO program and inconsistent genotypes were coded as missing. Errors in relationship specification were identified with the program RELTEST. When necessary, a second relationship testing program, RELPAIR version 2.0.1, was enlisted to resolve potential errors involving complex relationships. Multipoint identity by descent (IBD) allele sharing probabilities were estimated by the method of maximum likelihood, using all available information in the pedigree as implemented in the program GENIBD. Multipoint IBD-sharing estimates are robust to misspecification of population allele frequencies, as may occur with admixed samples, because most of the parental information is inferred when the available information is high p values reported by SIBPAL to LOD scores using the one-sided chi-squared distribution with one degree of freedom , appropriate for a one-sided test. In principle, the sib pairs who are identical by descent (IBD) at a marker locus will be phenotypically similar for traits influenced by a nearby linked gene. Evidence for linkage of eGFR was assessed with and without incorporating covariate effects of diabetes duration and body mass index (BMI), entered in the regression model as the sibpair sum. Non-parametric multipoint linkage analysis was carried out separately in each ethnic group, and P values were combined across ethnicities according to Fisher's method P values were obtained for the major linkage peaks using the \u201csimulation\u201d option in SIBPAL, which performs a permutation test. Association analysis was conducted as described previously Genetic analyses were performed using the S.A.G.E.  software package, version 5.3  \u22650.5, MAF (specific to ethnic group) \u22650.05, and P\u200a=\u200a5.5\u00d710\u22124) at 44 cM near rs1339048 (\u22123), flanked by SNPs rs221972 and rs735264  in MA participants (P\u200a=\u200a1.5\u00d710\u22124) and suggestive evidence for linkage was found near rs913375 on 10p14 , primarily contributed by EA (\u22124) in the combined data, that was primarily driven by AI (\u22124), which was driven by AA, AI and MA. Another SNP (rs1345561) associated with eGFR (P\u200a=\u200a6.4\u00d710\u22124) in the combined data was located approximately 16 Mb from the eGFR linkage marker rs1339048 on 10p12 (\u22124), rs580839 on 15q14 (P\u200a=\u200a4.81\u00d710\u22125), rs666478 on 9p21 (P\u200a=\u200a1.46\u00d710\u22124) and rs2928972 on 18q21.2 (P\u200a=\u200a1.30\u00d710\u22124) in AA, AI, EA, and MA, respectively .Following the linkage scans, coarse association analyses between eGFR and SNPs that passed quality control was performed, using the approach implemented in ASSOC. ed by EA . We alsoen by AI . Our asson 10p12 . This ason 10p12 . Severalectively . These rEstimated GFR provides an accurate index of the degree of renal dysfunction and plays a prominent role in the staging of chronic kidney disease \u22124). It is interesting to note that the linkage of eGFR on 10p12 was contributed by three  of the four ethnic groups participating in this study, indicating that the 10p12 region may potentially harbor genes influencing GFR in the FIND participants (CACNB2), ARL5B ADP-ribosylation factor-like 5B (ARL5), and nebulette (NEBL), were previously associated with eGFR-related traits such as blood pressure and hypertension CACNB2 were also associated with sudden cardiac arrest.The most significant linkage to eGFR in ethnicity-combined data was found near rs1339048 on 10p12.31  gene on 9p21. TEK is a cell-surface receptor for angiopoietin (ANGPT) 1, 2, and 4. Through TEK-dependent signaling, ANGPT regulates endothelial cell survival, proliferation, migration, adhesion and cell spreading, and controls vascular permeability and quiescence. Mutations in TEK were previously associated with autosomal dominant forms of venous malformations TEK on cyclin-dependent kinase inhibitor (CDKN) 2A, 2B genes have been previously associated with type 2 diabetes mellitus Of the SNPs most strongly associated with eGFR in population-specific analyses, rs666478 is located within an intronic region of the tyrosine kinase receptor (PDIA6) gene on 2p25. PDIA6 belongs to a thioredoxin superfamily oxidoreductase from the endoplasmic reticulum that acts as a redox signaling adaptor protein, adjusting reactive oxygen species intermediates to specific signals and redox signals to cell homeostasis PDIA6 and regulating renal function needs to be examined, genetic variants located about 7 Mb upstream of PDIA6 on the SRY (sex determining region Y)-box 11 (SOX11) gene were previously associated with T2DM and CKD in Europeans Population-specific association analysis identified several SNPs (rs1686430 and rs1734449) that are associated with GFR only in the AA group. They were located 100 kb apart within an intronic region of the protein disulfide isomerase family A, member 6 (\u22128) for statistical significance on account of the large number of statistical tests involved, linkage studies with less stringent P values are powerful because the number of effectively independent comparisons is much smaller. Conventionally p<0.0001 (LOD>3) has been considered significant linkage, while p<0.001 (LOD>2) has been considered suggestive Utilizing a relatively dense set of 6,000 SNPs as a linkage panel as opposed to the conventional use of a set of about 400 microsatellite markers, the present study reveals quantitative trait loci influencing eGFR to 20q11 in MA, 15q21 in EA and 10p12 in the combined ethnic groups from the FIND study. Several suggestive linkage peaks were also identified in population-specific and population-combined linkage scans in this multi-ethnic cohort. In contrast to GWAS that requires a very stringent p-values (e.g., P<5\u00d710As expected for a complex trait, multiple linkage peaks for eGFR were observed. Although the functional relevance of the linkage findings remains to be established and replicated, genetic regions suggestively linked with eGFR in population-specific and population-combined studies suggest that multiple loci are involved in regulating eGFR in diabetes. Disappointingly, there was no significant overlap with loci linked with renal function-related traits in other studies 2; whereas the CKD-EPI equation appears more accurate for those with eGFR values between 60 and 90 ml/min per 1.73 m2. Although all analyses adjusted for diabetes duration and BMI, other potentially relevant confounding variables such as degree of blood pressure control and cardiovascular disease risk factors were unavailable.While this large study in a severely affected study sample had several advantages, potential limitations are that eGFR was estimated using a single random blood sample for serum creatinine concentration and employed the modified MDRD equation. This equation performs best for eGFR <60 ml/min per 1.73 mIn conclusion, several loci influencing eGFR were identified in the multi-ethnic FIND cohort. Linkage and association results emanating from this multi-ethnic study represent a first step towards improving our knowledge of the mechanisms underlying genetic susceptibility to renal function in diabetes. Furthermore, the results of linkage and association analyses reported in this study will help interpret future genome-wide association/whole-genome sequencing data that should accelerate the identification of causal genes for variation in kidney function in patients with diabetes. Defining the genetic architecture responsible for eGFR loss in individuals of different ethnicities may help develop ethnicity-specific intervention programs and services specifically targeted toward this devastating complication of diabetes. With existing high-throughput genome technologies and novel statistical methodologies, we envision promising new therapies to prevent loss of eGFR, a strong and independent risk factor for cardiovascular morbidity and mortality in patients with diabetes."}
+{"text": "We report analyses of a Brazilian study of early onset schizophrenia (BEOS) families. We genotyped 22 members of 4 families on a linkage SNP array and report here non-parametric linkage analyses using MERLIN\u00ae software. We found suggestive evidence for linkage on two chromosomal regions, 13q32 and 11p15.4. A LOD score of 2.71 was observed at 13q32 with a one LOD interval extending from 60.63\u201392.35 cM. From simulations, this LOD score gave a genome-wide empirical corrected p\u200a=\u200a0.33, after accounting for all markers tested. Similarly 11p15.4 showed the same maximum LOD of 2.71 and a narrower one LOD interval of 4\u201314 cM. Of these, 13q32 has been reported to be linked to schizophrenia by multiple different studies. Thus, our study provides additional supporting evidence for an aetiological role of variants at 13q32 in schizophrenia. Schizophrenia is a chronic, highly disabling disease that has a point prevalence estimate of \u223c0.5% general population, which usually leads to persistent functional impairment with considerable morbidity and economic cost Linkage analysis is a standard approach for identifying the location of genes that cause genetic diseases Several reasons may explain these results, including locus and phenotypic heterogeneity, inadequate sample size, differences in ascertainment, marker sets, ancestry and statistical methods In the present study we perform a pilot linkage study in Brazilian families with early-onset schizophrenia in Sao Paulo, Brazil. We report here the results of non-parametric linkage analyses in 4 small multiplex families.This study was approved by Research Ethics Committee of UNIFESP [CEP No. 1737/06]. Written informed consent was obtained from all participants recruited or caregivers, on the behalf of minors/children, and, clinical and laboratory investigations were strictly conducted according to the principles expressed in the Declaration of Helsinki. All subjects invited accepted to enroll this study.22 subjects from 4 families, including 11 patients with schizophrenia and 1 with schizoaffective disorder were ascertained, yielding 8 affected sib-pairs and 6 other affected relative-pairs. All patients were being treated at the same specialized outpatient clinic in Sao Paulo, Brazil. Eligibility criteria were: one or more affected members with age of onset <18 years, and to have two or more members diagnosed with schizophrenia, excluding monozygotic twins. Exclusion criteria were comorbid mood disorders, mental retardation, and inability in providing informed consent.The Structured Diagnostic Interview (SCID) was performed by trained psychiatrists to provide DSM-IV psychiatric diagnosis. Individuals under 18 years old were assessed with the Kiddie\u2013Sads-Present and Lifetime Version The mean age of the whole sample was 41.4 (sd\u200a=\u200a16.2); 43.5 (sd\u200a=\u200a18.8) for non-affected individuals, and 39.7 (sd\u200a=\u200a14.29) for patients. This difference was non-significant . For the whole sample, 54.5% are male and 45.5% are female; there was no significant difference in gender proportion between affected and non-affected individuals . 7 out of 12 affected individuals presented onset age \u226418 years old, with a mean age of 15.57 (sd\u200a=\u200a2.1). The 5 other affected subjects had a mean age of onset of 24.20 (sd\u200a=\u200a4.6). A brief description of each family and the respective pedigree diagrams are provided in the figures below \u20134:Whole blood was collected into tubes containing 0.1% EDTA and genomic DNA isolation was performed using the Gentra\u2122 Puregene\u2122 Kit , according to the manufacturer\u2019s protocol. DNA concentration was measured using a spectrophotometer  and each sample was accurately quantified in triplicate and diluted to 100\u00b10.5 ng/\u00b5L. Genotyping was performed using standard methods at laboratories at the MRC SGDP Centre, KCL. Briefly, Affymetrix 10 k arrays were typed in all samples and genotypes were called as per the manufacturer\u2019s protocol.All phenotypic information from interviews and questionnaires was coded and samples were assigned a number with removal of any personal identifying information. The program MERLIN We found a suggestive linkage with a LOD score of 2.71 at 13q32 at 13q31/32 . The peaIn our study we found suggestive evidence for linkage on chromosomal regions 13q32 and 11p15.4 under the criteria of Lander and Kruglyak TH) and the dopamine receptor D4 (DRD4) (both at 11p15). However, the evidence for linkage in the region is weak. A review of the linkage results in the region concluded that they show a lack of replication but suggested that modest interest was maintained by possible signals scattered across a 30cM interval Chromosome 11 includes several plausible candidate genes for major psychiatric disorders, especially psychosis, including the tyrosine hydroxylase (ZIC2, SLC15A1, and FGF14. Goes et al. This relative lack of previous evidence for chromosome 11p15 contrasted with the strong previous findings available for 13q32. Thus, although we should not disregard the linkage with 11p15.4, our primary finding is an additional evidence for a potential role for 13q32 region in a small Brazilian sample of families with early-onset schizophrenia affected individuals. The allele-sharing model tested is compatible with allelic homogeneity within the families. This region encompasses several genes, some of them already investigated in schizophrenia but with predominantly negative results like The major limitation of our study was the small sample size. However, families with early-onset schizophrenia were ascertained in an attempt to focus on a more severe and homogenous form of the illness. Likewise, although we find replication evidence for 13q32 and schizophrenia, the most recent meta-analysis Further analyses of family based early onset schizophrenia cohorts and large scale sequencing efforts may shed further light on the specific genetic factors influencing risk for schizophrenia in this region."}
+{"text": "Bacterial non-necrotizing erysipelas and cellulitis are often recurring, diffusely spreading infections of the skin and subcutaneous tissues caused most commonly by streptococci. Host genetic factors influence infection susceptibility but no extensive studies on the genetic determinants of human erysipelas exist.We performed genome-wide linkage with the 10,000 variant Human Mapping Array (HMA10K) array on 52 Finnish families with multiple erysipelas cases followed by microsatellite fine mapping of suggestive linkage peaks. A scan with the HMA250K array was subsequently performed with a subset of cases and controls.all) 3.84, p\u200a=\u200a0.026), which is syntenic to a quantitative trait locus for susceptibility to group A streptococci infections on chromosome 2 in mouse. Sequencing of candidate genes in the 9q34 region did not conclusively associate any to erysipelas/cellulitis susceptibility. Suggestive linkage  was found at three loci: 3q22-24, 21q22, and 22q13. A subsequent denser genome scan with the HMA250K array supported the 3q22 locus, in which several SNPs in the promoter of AGTR1 (Angiotensin II receptor type I) suggestively associated with erysipelas/cellulitis susceptibility.Significant linkage was found at 9q34 (nonparametric multipoint linkage score (NPLSpecific host genetic factors may cause erysipelas/cellulitis susceptibility in humans. Streptococcus pyogenes) and G  \u03b2-hemolytic streptococci are the predominant causative agents of cellulitis/erysipelas but infections may also be caused by group B and C streptococci and Staphylococcus aureusBacterial non-necrotizing erysipelas and cellulitis are often recurring, diffuse, and spreading infections of the skin and subcutaneous tissues, which manifest with local erythema, pain, and warmth usually accompanied by fever, leukocytosis, lymphangitis, and lymphadenitis Mycobacterium leprae, Mycobacterium tuberculosis, Streptococcus pneumoniae, Neisseria meningitidis, Schistosoma mansoni, Leishmania donovani, Epstein-Barr virus, and human papilloma virus Plasmodium vivax, human immunodeficiency virus-1, meningococcal disease, and norovirus Streptococcus pneumoniae infection in mice (syntenic to human 19q13.1-13.3); and chromosome 2, including genes of the interleukin 1 alpha, and prostaglandin E synthetase pathways (syntenic to human 2q14 and 9q33-34) Mendelian and polygenic host genetic factors are known to influence susceptibility to infection by bacteria, parasites, and viruses including We have used erysipelas/cellulitis (hereafter referred to as erysipelas) as a marker infection to identify families with two or more family members suffering from erysipelas and suggesting a possibly increased susceptibility to streptococcal infections. To identify putative susceptibility loci we performed a whole-genome genetic linkage scan and identified suggestive loci on chromosomes 9q34, 3q22-24, 21q22, and 22q13.This study was approved by the Ethical Review Board of Pirkanmaa Hospital District, Tampere, Finland. Written informed consent was obtained from all study participants. All clinical investigations have been conducted according to the principles expressed in the Declaration of Helsinki.We recruited individuals with recurrent erysipelas infections for which preventive monthly intramuscular benzathine penicillin injections are reimbursed in Finland. We contacted all 960 individuals reimbursed for benzathine penicillin through the National Health Insurance Institution in the year 2000. Of these, 50% (483) gave consent to participate and 25% had a first-degree relative with a history of erysipelas. We then collected blood samples from 204 recurrent erysipelas patients and 124 relatives from 52 pedigrees with two or more family members suffering from erysipelas. The diagnosis of erysipelas was verified from hospital records for all patients except for six who self-reported to have had erysipelas but no hospital records were available for verification.An acute erysipelas cohort of 90 patients with acute erysipelas and 90 population controls matched for age and sex was also recruited. An infectious disease specialist recruited the patients from Tampere University Hospital and Hatanp\u00e4\u00e4 City Hospital, Tampere, Finland when they were hospitalized for erysipelas. The cohort is described in detail elsewhere Samples from twenty affected individuals from six most representative families  were genall scores was estimated by simulating data 100 times with MERLIN and extracting the highest NPLall score from each simulation. The minimum NPLall score for suggestive linkage was 2.1 (occurring once at random in a genome scan) and the threshold for significant linkage 4.77 (occurring with a 5% probability in a genome scan). Non-parametric linkage analysis was repeated using Caucasian allele frequency estimates obtained from Affymetrix.MERLIN (Multipoint Engine for Rapid Likelihood Inference) software The NPL results were verified with 31 microsatellites surrounding the suggested linkage peaks at 3q22-24, 9q34, 21q22, and 22q13 at approximately 2 cM (0.85\u20132.2 Mb) intervals . We genoall scores for configuration 0 was estimated by simulating data 1000 times with MERLIN and extracting the highest NPLall score from each simulation. Minimum NPLall score for significant linkage was 2.49, p\u200a=\u200a0.05. For fine mapping of 9q34, linkage analysis was done using four configurations: 0; unconfirmed affected individuals were analyzed as unknown, and 2; they were analyzed as affected. In configurations 0_186 and 2_186, analysis was identical except that allele 186 was called for marker D9S65.MERLIN was used for multipoint NPL analysis as described above. Allele frequencies were estimated from all individuals, non-affected individuals were assigned affection status unknown, and Mendelian inconsistent genotypes were removed prior to analysis. Linkage analysis was done using two configurations: 0; the unconfirmed affected individuals were analyzed as unknown, and 2; they were analyzed as affected. The genome-wide significance of NPLWe selected 15 affected patients and 15 unaffected controls for additional genomic screening with the Affymetrix GeneChip Human Mapping 250KSty Array to search for possible allele or haplotype associations assuming a strong genetic effect. Twelve patients were from the families 1, 2, 4, 5, 8, 9, 12, 14, 22, 32, 37, and 38, and their genetically independent family members served as controls. Three patients and three controls were from the acute cohort. Genotypes were called with BRLMM using Affymetrix default parameters. Analysis focused on the defined linkage peaks: 3q22-24 (D3S1306-D3S1299), 9q34 (D9S290-D9S1863), 21q22 (D21S1898-D21S1920), and 22q31 (D22S1159-D22S1141) . To evalAGTR1  were sequenced in six probands from the families  showing linkage to the 3q22 area. All PCR reactions were performed in 5 \u00b5l volumes containing 20 ng of DNA, with standard reagent concentrations and temperature profiles. Sequencing was performed using dye-terminator chemistry and automated sequencers . Primer sequences are available on request.Altogether five candidate genes in the 9q34 linkage peak were chosen based on their biological relevance in immunity or infections . After Pall score of 3.84 and a suggestive genome-wide p-value of 0.24. Results were identical when the analysis was repeated with Caucasian allele frequency estimates from Affymetrix, except that the chromosome 3 peak marker moved from 3p24 (rs1994987) to 3p22 (rs2167176) with an NPLall score of 2.64 and a genome-wide P-value 0.94. Generally, different families contributed strongest to the most significant peaks: 9q34 ; 3q22-q24 ; 21q22 ; and 22q13 .We found seven suggestive linkage peaks on chromosomes 9q34, 3q22-24, 21q22, 22q13, 3p24, 10q25, and 11q24, in descending order of genomic significance, on the Affymetrix HMA10K Array . The strall of 2.77 and p\u200a=\u200a0.026 for D9S159    . Only suas 2.49) . The hig34\u20131.41) .all 2.9) was observed at D9S65 (132190620 bp) if allele 186 was called, otherwise it shifted to marker D9S64 (134380110 bp) . NPL plots for the four configurations were essentially unchanged  is expressed in proliferating fetal fibroblasts and the developing dermal layer, with lower expression in adult skin. An increase in expression of this gene during fetal but not adult wound healing suggests a role in controlling mammalian dermal regeneration and prevention of scar formation LAMC3  gene belongs to the family of laminins, which are extracellular matrix glycoproteins and the major noncollagenous constituent of basement membranes. They have been implicated in a wide variety of biological processes including intracellular invasion by several bacterial pathogens such as GAS strains FIBCD1 (Fibrinogen C domain-containing 1) is a transmembrane endocytic receptor that binds acetylated structures via a highly conserved fibrinogen-related domain (FReD). Ficolins also have FReDs and they play an important role in innate immunity ABL1  is a proto-oncogene which encodes a cytoplasmic and nuclear protein tyrosine kinase implicated in the processes of cell differentiation, cell division, cell adhesion, and stress response. ABL tyrosine kinases are related to the cell penetration of Shigellae and their signaling is required T-cell development and mature T-cell function Altogether, 59 annotated protein-coding genes are located within the chromosome 9q34 linkage peak (D9S290 to D9S1199) . The fivPTGES (prostaglandin E synthase) is induced by proinflammatory cytokine interleukin 1 beta (IL1B) and synthesizes prostaglandin E2 (PGE2), a key regulator of inflammation by modulating the regulation and activity of T cells and the development and activity of B cells, and by enhancing the production of cytokines and antibodies PTGES is associated with inflammatory diseases, fever, and pain associated with inflammation, and the deletion of Ptges leads to an impaired febrile response in mice PTGES as well as the coding region, but found no specific variants, mutations or indels implicating it directly in erysipelas susceptibility.AGTR1) between SNPs rs9862062 (148359724 bp) and rs4681157 (148412408 bp) showing nominal association , 9q34 (D9S290 to D9S1863), 21q22 (D21S1898 to D21S1920), and 22q23 (D22S1159 to D22S1141). The 3q22 locus was the most significant with several SNPs in the promoter region of the Angiotensin II type receptor 1  in the 3\u2032UTR. The A allele of rs5186 has been associated with increased serum levels of high-sensitivity C-reactive protein and inflammation, and the CC genotype is putatively correlated with hypertension AGTR1.AGTR1 promoter area SNPs (rs9862062 and rs718424) that showed association to erysipelas in Haploview analysis, and genotyped them in the family material and in the acute erysipelas cohort by direct sequencing. The reference G-allele of rs9862062 was suggestively associated in the combined family (probands and marry-ins) and acute erysipelas cohort  and the reference T-allele of rs718424 showed suggestive association with a p-value of 0.017.We chose two Il1a, Il1rn (both located on 2q14 in humans), Ptges (located on 9q34 the linkage peak identified here), and Ptges2   . IncreasAGTR1 on 3q22. AGTR1 is a G-protein-coupled receptor that mediates the major cardiovascular effects of angiotensin II, a potent vasopressor hormone involved in the development of hypertension, atherosclerosis, and insulin resistance. Angiotensin II is the end product of the renin-angiotensin system (RAS), where renin stimulates the production of angiotensin I from angiotensinogen, which is then converted to angiotensin II by angiotensin converting enzyme (ACE). The activation of the RAS correlates with organ injury and mortality in clinical sepsis, possibly by contributing to the enhanced microvascular tone AGTR1, it increases the expression of cytokines, chemokines, growth factors, and adhesion molecules Higher density analysis with the Affymetrix HMA250K Array revealed the nominal association to erysipelas of several SNPs in the promoter region of ACE and other angiotensinogen genes have been associated with susceptibility to inflammatory diseases such as SLE and psoriasis with frequent tonsillitis ACE, AGTR1, and Angiotensin receptor associated protein (AGTRAP) showed significant association with increased 28-day mortality to septic shock for the GG genotype of AGTRAP rs11121816. AGTRAP interacts specifically with the C-terminal tail of AGTR1, and negatively regulates the receptor leading to a functional desensitization to angiotensin II. Pharmacological blockage of the RAS is used to treat hypertension, diabetic nephropathy, and congestive heart failure, but antigen II receptor blockers (ARBs) and ACE inhibitors also suppress proinflammatory cytokines and reduce oxidative stress. Prior usage of ARBs has been shown to reduce mortality in patients hospitalized for sepsis Polymorphisms in both AGTR1 and especially the C allele of rs5186 (+1166A>C) have been associated with hypertension and the A allele of rs5186 has been associated with higher serum levels of high-sensitivity C-reactive protein (CRP) and inflammation AGTR1 and PTGES are involved in the same pathway, as AGTR1 induces the production of COX, which coverts arachidonic acid into Prostaglandin H2 that in turn is converted by PTGES into Prostaglandin E2.Polymorphisms in We found evidence for host genetic factors influencing susceptibility to bacterial non-necrotizing erysipelas/cellulitis, but did not find a common susceptibility factor in all families. We did not find linkage or association with the HLA region previously linked with GAS infection severity in humans Figure S1NPL plots for the fine mapping of the chromosome 9q34 linkage peak with 22 microsatellite markers. The NPL plots for the four configurations were essentially identical. MERLIN was used for multipoint NPL analyses using four configurations. (A) In configuration 0, unconfirmed affected individuals were analyzed as unknown, and (B) in configuration 2, they were analyzed as affected. In configurations (C) 0_186 and (D) 2_186, analysis was identical to configurations 0 and 2, respectively, except that allele 186 was called for marker D9S65.(TIF)Click here for additional data file.Table S1all scores for the 9q34 linkage region.Family-wise NPL Families showing significant linkage are shaded dark grey. Families showing suggestive linkage are shaded light grey.(DOCX)Click here for additional data file.Table S2SNPs found in the family probands in AGTR1.(DOCX)Click here for additional data file."}
+{"text": "Few finance theories consider the influence of \u201cskewness\u201d (or large and asymmetric but unlikely outcomes) on financial choice. We investigated the impact of skewed gambles on subjects' neural activity, self-reported affective responses, and subsequent preferences using functional magnetic resonance imaging (FMRI). Neurally, skewed gambles elicited more anterior insula activation than symmetric gambles equated for expected value and variance, and positively skewed gambles also specifically elicited more nucleus accumbens (NAcc) activation than negatively skewed gambles. Affectively, positively skewed gambles elicited more positive arousal and negatively skewed gambles elicited more negative arousal than symmetric gambles equated for expected value and variance. Subjects also preferred positively skewed gambles more, but negatively skewed gambles less than symmetric gambles of equal expected value. Individual differences in both NAcc activity and positive arousal predicted preferences for positively skewed gambles. These findings support an anticipatory affect account in which statistical properties of gambles\u2014including skewness\u2014can influence neural activity, affective responses, and ultimately, choice. Winning the lottery and contracting a life-threatening illness are both life-changing but unlikely outcomes. Most people will never experience either event, yet the profits of casinos and insurance companies indicate that individuals are willing to pay a high premium for the potential to win big or to cheat death. Although traditional economic theories have little to say about how skewed  outcomes motivate choice, skewed outcomes may influence not only individual fortunes but also movements of the market expected value of an outcome could be calculated by multiplying its magnitude with its probability. Thus, expected value can be statistically approximated with the mean  of repeated gamble outcomes. Current financial models also imply that while individuals are attracted to expected value, they are instead repelled by risk. These mean-variance models thus approximate risk as the mathematical variance  of repeated gamble outcomes An improved understanding of individuals' responses to skewness can inform financial theory as well as practice. Traditional economic models assume that people seek to maximize value. For instance, Blaise Pascal initially proposed that the anticipatory affect model, statistics can influence how individuals feel about a financial option, which can then increase or decrease their willingness to choose that option From a psychological standpoint, statistical properties of gambles may influence choice by altering affective states Accumulating evidence from neuroimaging studies indicates that expected value elicits NAcc activity F\u200a=\u200a10.78, p<0.001, such that all higher variance  gambles elicited more positive arousal than Low-Variance gambles (p<0.001), and Positive-Skew gambles elicited more positive arousal than High-Variance (p<0.05) and Negative-Skew gambles (p<0.05). Negative arousal also varied, F\u200a=\u200a14.97, p<0.001, such that all higher variance gambles  elicited more negative arousal than Low-Variance gambles (p<0.001), and Negative-Skew gambles elicited more negative arousal than High-Variance (p<0.001) and Positive-Skew gambles (p<0.05). As with negative arousal, subjects' perceived risk varied, F \u200a=\u200a12.07, p<0.001, such that all higher variance gambles  were considered riskier than Low-Variance gambles (p<0.001), and Negative-Skew gambles were considered riskier than both Positive-Skew (p<0.05) and High-Variance gambles (p<0.01).Different gambles elicited different levels of self-reported positive arousal, negative arousal, and perceived risk . With reF\u200a=\u200a11.17, p<0.001, such that subjects preferred High-Variance to Low-Variance gambles (p<0.01), and preferred all other gambles to Negative-Skew gambles (p<0.01). Nonparametric analyses of rankings yielded similar results. Comparison of High-Variance versus Low-Variance gamble rankings using a Wilcoxon Signed-Ranks test confirmed that subjects preferred High-Variance to Low-Variance gambles . Contrasts of Positive-Skew and Negative-Skew gamble preferences revealed that subjects preferred Positive-Skew to Negative-Skew gambles  and High-Variance to Negative-Skew gambles . Subjects were indifferent between the two highest-ranked gambles .Different gambles also elicited different preference rankings, p<0.05)  threshold , which was further verified with timecourse analyses. Beyond group differences, individual difference analyses also indicated a link between NAcc activation and preference for Positive-Skew gambles (described below).Whole-brain analyses revealed that anticipatory activation  in predicted regions differed in response to variance and skewness (at a whole-brain corrected threshold of p<0.05) . High vap<0.05) . Among sF\u200a=\u200a12.03, df\u200a=\u200a3, p<0.001) and NAcc  activation during anticipation. Post-hoc t-tests (one-tailed) confirmed significant pairwise differences, such that both Positive- and Negative-Skew gambles elicited more anterior insula activation than High-Variance  and Low-Variance gambles ; while Positive-Skew gambles elicited more NAcc activation than Low-Variance (p<0.01), High-Variance (p<0.05), and Negative-Skew gambles (p<0.05).Activation timecourses were extracted from predefined anatomical NAcc and anterior insula volumes of interest (VOI) to verify neural responses to the gambles indicated by the whole-brain analyses . RepeateR2\u200a=\u200a0.27, p<0.05) and positive arousal  separately predicted preference. Combining both measures significantly improved the explanatory power of the model to account for individual differences in preference for Positive-Skew gambles . Specifically, including neural (NAcc) activation explained additional variance over and above reported positive arousal . Anterior insula activation and reported negative arousal, however, were not significant predictors of preference for Positive-Skew gambles in this model .Consistent with previous findings indicating that NAcc activation correlates with positive arousal The present study examined how financial skewness influences neural activity, affect, and choice. Although previous behavioral research has explored preferences for skewness Expected value and mean-variance approaches do not predict preference for variance and positive skewness. Further, prospect theory These behavioral findings demonstrate that skewness influences choice in a way not accounted for by normative economic or financial models. Previous neuroimaging studies of financial risk-taking have not systematically investigated skewness. The current findings represent a logical but novel extension of a growing neuroimaging literature examining the influence of statistical moments on choice To systematically manipulate skewness , we used mixed gambles with an expected value of zero. Since investigators have observed differences in the attractiveness of gambles with positive and negative expected values Together, these findings provide initial neural and behavioral evidence for an independent influence of skewness on financial preferences. At both group and individual levels of analysis, skewness elicited affect and neural activity that predicts future choice. Contrary to normative models that specify that higher-order moments  should influence decisions less than lower-order moments , these findings suggest that skewness has a disproportionately large impact on reported affective experience, neural activity, and choice. Eventually, choice may be best modeled instead by a flexible multi-attribute framework that incorporates the affective impact of gambles Because people are willing to pay a premium for positively skewed financial investments and receive a premium for shouldering negatively skewed investments Nineteen healthy native English-speaking adults  participated in the study. Subjects had no history of neurological or psychiatric disorders. Written informed consent was obtained from all subjects, under a protocol approved by the Institutional Review Board of the Stanford University School of Medicine. In addition to the 19 subjects included in the analysis, two were excluded for excessive head motion  and two more were excluded for not complying with experimental instructions. In addition to a flat fee of $40.00 for two hours of participation and a $25.00 endowment, subjects' payments were determined by the outcomes during the gambling task as well the outcome of their chosen gamble at the end of the experiment (M\u200a=\u200a$66.00\u00b1 SD $3.00).Subjects received spoken and written instructions and completed a brief training session prior to the first experimental run in the scanner. In the gambling task, subjects played a series of repeated gambles that counted for real money. On each trial, subjects viewed the gamble (2 s), followed by a spinning wheel (2 s), and the outcome highlighted in yellow (2 s). They then pressed a button to verify the gamble outcome (2 s), and observed the cumulative total for the run (2 s). Presentation of all gambles and response prompts were counterbalanced on both right and left sides of the screen. To ensure a high response rate, failures to respond within 2 seconds resulted in an automatic loss of $0.10 in addition to the gamble outcome for that trial , consistent with methods of previous studies t-tests to verify significant differences between gamble types, using SPSS 16.0.After completing the gambling task in the scanner, subjects rated their affective responses as they anticipated each of the gamble outcomes. Previous studies have demonstrated high concordance and reliability between online anticipatory and cued retrospective affective ratings Images were acquired with a 1.5T General Electric MRI scanner  and a standard quadrature head coil. Twenty-four contiguous axial 4-mm-thick slices (in-plane resolution 3.75\u00d73.75 mm) extended axially from the mid-pons to the top of the skull. Functional scans were acquired with a T2*-sensitive spiral in/out pulse sequence  Analyses of neural data utilized AFNI software  p<0.001 with a cluster of 16 contiguous 2.00 mm cubic voxels  Analyses proceeded through three stages: whole-brain localization, volume of interest (VOI) time course verification, and individual difference regressions. Localization analyses utilized a multiple regression model that included independent  regressors modeling anticipation of gamble outcomes: (i) high versus low variance, (ii) skewed versus symmetric, and (iii) positive versus negative skew. The model also included regressors of noninterest indexing residual motion (n\u200a=\u200a6) and anticipation versus the rest of the trial . Maps of contrast coefficients for regressors of interest were coregistered with structural maps, spatially normalized by manually warping to Talaraich space, spatially smoothed to minimize effects of anatomic variability (FWHM\u200a=\u200a4 mm), and collectively submitted to a one-sample t-test against the null hypothesis of no activation in order to test for group differences while controlling for random effects. The threshold for statistically significant foci activation in group maps was set at t-tests to verify differences between types of gambles.For activation time course analyses, VOIs were specified as 8 mm diameter spheres centered on previously identified foci  and confirmed by localization analyses. Activation time courses were spatially averaged within each VOI and then divided by the average activation over the course of the entire experiment to derive measures of percent signal change. To separately examine the influence of each gamble on activation in each region, VOI time courses were averaged into four conditions representing each type of gamble . VOI peak activations during the anticipatory period (lagged by 6 s to match the hemodynamic peak) were submitted to repeated-measures ANOVA. VOI peak activations from regions showing significant main effects were then submitted to post-hoc pairwise For individual differences analyses, timecourse data during anticipation of Positive-Skew gambles were extracted from the NAcc and anterior insula for each individual. Regression analyses examined whether individual differences in peak neural activation during anticipation as well as cue-induced affect predicted subsequent preference for Positive-Skew gambles.Table S1Regressors of interest Z-scores and Talaraich coordinates for peak activation foci. Variance contrast compared high variance versus low variance gambles (High-Variance + Positive-Skew + Negative-Skew > Low-Variance). Skewness contrast compared skewed versus symmetric gambles of equal variance (Positive-Skew + Negative-Skew > High-Variance). Positive Skewness contrast compared positively skewed versus negatively skewed gambles (Positive-Skew > Negative-Skew). Regions surpassed threshold of Z>3.28 .(DOC)Click here for additional data file."}
+{"text": "Aristichthys nobilis, 2N\u200a=\u200a48), which belongs to Cyprinidae. A total of 218 microsatellites were selected across 24 linkage groups (LGs) of a recently well-defined genetic linkage map for bighead carp, with 151 being heterozygous in at least one of six dams in diploid meiogynogenetic families. After tests for Mendelian segregation in two diploid control families, 103 microsatellites were used for G-C distance calculation in 383 gynogens. The second division segregation frequency (y) was computed through half-tetrad analyses, and the values ranged from 0 to 0.97 (mean 0.40). High G-C recombination frequencies (over 0.667) were observed in 18 (17.5%) of the loci examined, which revealed a low level of chiasma interferences compared with other fishes studied previously. Distribution of G-C distances across LGs ranged from 0 cM to 48.5 cM (mean 20 cM) under the assumption of complete interference. All 24 centromeres were localized according to their closest-related microsatellites at 95% confident intervals. The average distance between centromeres and their closest-linked markers was 6.1 cM with 15 out of 24 LGs having a distance below 5 cM. Based on the centromere positions in this study, we proposed a formula of 24 m/sm+24 t/st chromosomes with 92 arms for bighead carp, which was mostly in accordance with a previously reported karyotype for bighead carp (24 m/sm+24 st). These results of centromere localization provide a basic framework and important resources for genetics and comparative genomics studies in bighead carp and its closely-related cyprinid species.Gene-centromere (G-C) mapping provides insights into structural and behavioural properties of chromosomes. In this study, G-C mapping using microsatellite markers and meiogynogenetic (meiotic gynogenetic) families were performed in bighead carp ( Genetic mapping provides a framework for studies of quantitative trait loci (QTL) identification y) The approach of half-tetrad analysis is the basis for G-C mapping, only if two of the four chromatids from a single meiosis were recovered, half-tetrad analysis could be performed Oncorhynchus mykissDenio rerioMisgurnus anguillicaudatusAnguilla japonicaPseudosciaena croceaScophthalmus maximusCynoglossus semilaevisClarias macrocephalusHaliotis discus hannaiMost G-C mapping studies in aquatic animals were based on allozyme markers around a decade ago Aristichthys nobilis) is one of the most important aquaculture fish in China and has been introduced into many other countries for plankton control and human consumption Bighead carp , and followed the experimental basic principles. A slight fin tissue from the parents and control families was sheared under MS222 anesthesia, progenies of the experimental families were sacrificed with anhydrous ethanol, and all efforts were made to minimize suffering.Cyprinus carpio) sperm which was four times diluted by Hank\u2019s solution, and then immersed into 4\u00b0C water bath immediately to inhibit the release of the second polar body. For the bighead carp control families, eggs from a dam were fertilized with sperm from a sire to produce normal diploid progenies. Fertilized eggs were hatched in circulating water with a temperature of approximately 25\u00b0C. Gynogens were raised in laboratory tanks and fed with hatched Artemia cysts until sampling, while control families  were raised in muddy ponds. At the age of one month after hatching, fingerlings of each meiogynogenetic family were sampled and preserved in anhydrous ethanol at 4\u00b0C. Fin tissues were sampled for control families G and H at the ages of 3 years and 1 year old, respectively. Fin clips from each parental fish were also sampled. Genomic DNA was extracted from alcohol-preserved fin tissues and fingerlings following a standard phenol-chloroform protocol Parental females and males of bighead carp were selected from broodstocks of the Zhangdu Lake Fish Farm  to generate experimental families. Totally, six gynogenetic families (A\u2013F) and two normal diploid control families  were produced by artificial propagation during 2008\u20132011. Gynogenetic families were obtained through a previous method 2\u03c7) test (p<0.05). Microsatellites in accordance with the Mendelian segregations were applied to perform analyses of M-C distances and centromere positioning. Some of these microsatellite markers had trans-species ability to amplify common carp-specific alleles, therefore, they were used to verify the success rates of meiogynogenesis for six experimental families of bighead carp.A set of microsatellite markers were chosen from each of the 24 LGs of a recently well-defined genetic linkage map for bighead carp Taq polymerase , 0.4 \u00b5L of forward and reverse primer mixture (2.5 \u00b5mol/L), 20\u201350 ng of template DNA and 9.4 \u00b5L of sterile water. A 96 well thermal cycler  was used to perform PCR amplifications using the following program: 94\u00b0C denaturing for 5 min, followed by 35 cycles of 94\u00b0C for 35 s, optimal annealing temperature , 1 U of perature  for 35 sy). Because the y value was defined as the proportion of heterozygous recombinant genotypes in meiotic gynogens for each locus y in this study could be expressed with this formula: y\u200a=\u200aNe/(Ne+No), where Ne is the number of heterozygotes  and No is the number of the two homozygotes  in a mapping family. If a marker was informative in two or more families and showed unbiased y values among these families, an average was taken as the y value for this marker. Differences of G-C recombination frequencies among families were tested by contingency 2\u03c7 test (p<0.05). Homozygosity induced by one generation of gynogenesis, which is defined as fixation index (F), was calculated by F\u200a=\u200a1\u2212yAs we found no any gynogens that had heterozygous genotypes in all microsatellites used in this study, therefore, parental tetratypes would not play a role in the calculation of G-C recombination rate  were calculated in three different mapping methods: i) complete interference, where x\u200a=\u200a100(y/2), assuming that one recombination exchange precludes additional crossovers x\u200a=\u200a[ln (1+y)\u2212ln (1\u2212y)]\u00d7100/4 x\u200a=\u200a\u2212[ln (1\u2212y)]\u00d7100/2, assuming no chiasma interference G-C distances (The correlation of distances between markers in this study and corresponding distances in the linkage map A consensus genetic linkage map for bighead carp y/N \u00b11.96{[(y/N)(1\u2212y/N)]/N}1/2, where y is the number of heterozygous progenies for the indicated locus, and N is twice the number of progenies y in the second term of the above formula is set equal to 1 The relative position of each marker to the centromere was estimated by considering the minimum number of multiple recombination events, under the hypothesis of complete interference. The 95% confidence interval for a probable centromere region was estimated according to the formula As examples, the patterns and frequencies of crossovers, and the values of chiasma interference in chromosomes were estimated in selected LGs of the bighead carp genetic map, following the methods described previously Of the 218 markers, 151 were heterozygous in at least one of the six dams of gynogenetic families, and these markers were amplified in alternative control families to verify their segregation patterns. To obtain more reliable segregation data in meiogynogenetic families, those markers with possible null alleles were eliminated for recombination analysis, no matter they were in accordance with Mendelian expectations or not. Of the genotypic ratios for 151 markers, 103 were in accordance with Mendelian expectations at 5% level after sequential Bonferroni correction for multiple tests , and theCommon carp-specific alleles were observed in a total of 13 progenies from families A, B, E and F , while n2\u03c7 test (p<0.05) (data not shown).The number of heterozygous microsatellites segregated in gynogenetic families A to F were 39, 15, 47, 42, 21 and 37 respectively, and G-C distances were estimated initially based on these loci . The ratArsd298 in LG10, HysdE11798-1 in LG4, Hysd942-1 in LG20, Arsd542 in LG15 and Arsd276 in LG12) to 0.97 (HysdE4406-1 in LG12) with an average of 0.40, corresponding to a fixation index (F) of 0.60 after one generation of gynogenesis. Low G-C recombination frequencies (y<0.1) were detected in 18 markers , whereas 18 markers  showed high recombination frequencies over 0.667, a value expected for independent segregation between a given locus and its centromere under the assumption of zero interference. These results indicated the existence of interference after a single chiasma formation in some chromosomes of bighead carp.The overall heterozygote frequencies ranged from 0 (y/2), 50% interference (Kosambi function) and zero interference  ranged from 0 to 48.5 cM, from 0 to 104.6 cM and from 0 to 175.3 cM, respectively . Regression between G-C distance and linkage distance, forced through the origin, had a slope of 0.771; but if those distances longer than 30 cM were excluded, then calculated slope was 0.931, which was much closer to 1 . Of thosComparisons of marker positions in LGs of the linkage map We examined half-tetrad genotypes in details for three cases , i.e. LG16 in family A, LG18 in family C and LG12 in family D, to evaluate the distribution patterns of crossovers between markers . In LG16Arsd700 and distal marker Hysd660-1 in this LG theoretically produced nine genotypic combinations in family C \u00d7(1\u201342/48)\u00d748\u200a=\u200a3.125. Thus, the coefficient of coincidence is 2/3.125\u200a=\u200a0.64, corresponding to an interference value of 0.36.The interference value between two markers based on the DCO was estimated in LG18 as an example. The proximal marker family C . Two DCOApostichopus japonicus) only 2.3% microsatellites deviated from the Mendelian ratios in the M-C study using 24-h larvae A. japonicusOstrea edulisCrassostrea gigasSegregation distortions have been observed in many aquatic organisms, especially in marine fish and shellfish Significant differences in the proportions of segregation distortions in bighead carp were most probably caused by stages of sampling. The ages for progenies of the control families in this study was 3 years old for family G and 1 year old for family H, respectively, while the progenies for preparation of the genetic linkage map We found that the ratio between two non-recombinant homozygous genotypes significantly deviated from the expected Mendelian ratio of 1\u22361 in 19 cases involving 18 of the 103 loci and 5 mapping families. Surprisingly, families A and F occupied 15 of these 19 cases with 10 in family A and 5 in family F, respectively. As suggested by previous studies, one of the two homozygous genotypes may link to recessive lethal or deleterious genes causing a significant segregation distortion in diploid meiogynogenetic families F), calculated by 1-y, is an evaluation for the extent of homozygosity F in this study (0.60) was similar to that estimated in our previous study for bighead carp (0.523) F obtained in this study is 2.4 times of the inbreeding coefficient after one generation of sib-mating (F\u200a=\u200a0.25), indicating that meiogynogenesis could provide an effective means for rapid inbreeding in bighead carp.The fixation index (y) in bighead carp gynogenesis ranged from 0 to 0.97 with an average of 0.40, which is similar to that (0.477) in our previous study y above 0.667, which is lower than ratios detected previously in bighead carp (25.76%) The second division frequencies  to distal (telomeric) regions of bighead carp chromosomes. The distribution pattern of G-C distances in bighead carp was similar to that of Pacific abalone Significant correlation of G-C distances and genetic linkage map distances indicated that high interference of crossovers may exist in bighead carp genome, as suggested by previous studies that this correlation is a reflection of complete or nearly complete interference of crossovers in the recombination Arsd700 and Hysd660-1 in LG18 was 0.36, a medium value when compared with Pacific abalone (0.18) Chiasma interference is common in fish, which may be due to mechanical difficulties of double cross-over in fish with relatively small size of chromosomes Hippoglossus hippoglossusThe identification of centromere positions is a perfection for genetic linkage maps, and is also an initial step towards understanding the composition and structure of the centromeric region as well as the whole genome. Mainly due to the lack of well-defined genetic linkage maps using co-dominant markers, centromeres have been located only in very limited aquatic species so far. Zebrafish is the first fish in which all 25 centromeres were localized on genetic linkage maps The closest distance between marker and centromere is very important in the estimation of centromere regions, the closer of the distance the more accurate of the centromere regions Based on the results of our M-C mapping, 24 bighead carp LGs can be divided into two types, with a proposed karyotype formula of 24 m/sm+24 t/st chromosomes for diploid genome. This is in coincidence with a previous formula of 24 m/sm+24 st proposed by Almeida-Toledo et al. (1995) Four of the five previous karyotypes had 96 chromosome arms for diploid bighead carp (48 for haploid) Since genes (microsatellites) can be mapped in relation to their centromeres, G-C mapping allows us to compare gene orders between bighead carp and other fishes, which can provide insight into the mechanism of chromosomal rearrangements G-C recombination frequencies of 0\u20130.97 (mean 0.40) were obtained for bighead carp based on 103 microsatellites through half-tetrad analysis. The patterns and proportions of chiasma interferences were different among LGs, and the rates of both recombination and chiasma interference in bighead carp were lower than those reported in other fishes. Under the assumption of complete interference, all 24 centromeres were localized onto their respective LGs of our second generation genetic map for bighead carp with 95% confident intervals. Based on centromere positions in this study, we proposed a karyotypic formula of 24 m/sm+24 t/st for bighead carp chromosomes. The results of this M-C mapping study successfully integrated the centromere map and genetic linkage map in bighead carp, which provide valuable information for consolidation of genetic map and physical map in future. This study would be also helpful for studies on genome structure, chromosome evolution, and positional cloning for genes of interest in this aquaculture species.Figure S1Regression of inter-marker distances between G-C map in this study and the genetic linkage map of bighead carp, for all 77 marker-pairs formed by 103 microsatellites. The solid line is regression line for all marker-pairs and the dotted one is for those with genetic distances shorter than 30 cM on the genetic linkage map. Slopes of the two lines are marked.(TIF)Click here for additional data file.Table S1Summary information for microsatellite markers used in this study.(XLS)Click here for additional data file.Table S2Genotypic distribution of 103 microsatellites in two control families (G and H) of bighead carp.(XLS)Click here for additional data file.Table S3Microsatellite\u2013centromere (M-C) recombination rates (second meiosis segregation frequency) and M-C map distances based on 103 microsatellites segregating in six meiogynogenetic families of bighead carp.(XLS)Click here for additional data file."}
+{"text": "First, the number of progeny inheriting a dominant sulfadiazine resistance marker linked to p5cs2 was determined. Second, the number of p5cs2/p5cs2 embryos was determined. A ratio of resistant to susceptible plantlets close to 50%, and the absence of aborted embryos were consistent with the hypothesis that the male gametophyte carrying both p5cs1 and p5cs2 alleles is rarely transmitted to the offspring. In addition, in reciprocal crosses with wild type, about 50% of the p5cs2 mutant alleles were transmitted to the sporophytic generation when p5cs1 p5cs2/P5CS2 was used as a female, while less than 1% of the p5cs2 alleles could be transmitted to the outcrossed progeny when p5cs1 p5cs2/P5CS2 was used as a male. Morphological and functional analysis of mutant pollen revealed a population of small, degenerated, and unviable pollen grains, indicating that the mutant homozygous for p5cs1 and heterozygous for p5cs2 is impaired in pollen development, and suggesting a role for proline in male gametophyte development. Consistent with these findings, we found that pollen from p5cs1 homozygous mutants, display defects similar to, but less pronounced than pollen from p5cs1 p5cs2/P5CS2 mutants. Finally, we show that pollen from p5cs1 p5cs2/P5CS2 plants contains less proline than wild type and that exogenous proline supplied from the beginning of another development can partially complement both morphological and functional pollen defects.To confirm the fertility defects of pollen from P5CS1 and P5CS2 is severely compromised, and indicate that proline is required for pollen development and transmission.Our data show that the development of the male gametophyte carrying mutations in both  Arabidopsis reproductive tissues up to 26% of the total amino acid pool, while in vegetative tissues represents only 1-3%. Among floral organs, different authors[In addition to its role as proteinogenic amino acid, and as a molecule involved in responses to a number of biotic and abiotic stresses, proline has been implicated in plant development, particularly flowering and reproduction-3. The f authors-11 point authors.It is not clear, to date, the reason for such a massive proline accumulation in pollen. Because pollen grains undergo a process of natural dehydration, a role of compatible osmolyte capable of protecting cellular structures from denaturation, has been proposed by some authors,12,13, wAtProT1 (AT2G39890), a gene encoding an amino acid carrier recently shown to mediate proline uptake in plants, is highly expressed in mature pollen[Irrespective of its function, proline may accumulate in pollen due to an increased transport from external sources, or to an increased ratio between synthesis and degradation of endogenous proline, or because of a combination of the two, but no conclusive evidence has been produced, as yet, to distinguish among these alternative models. Long distance transport of proline through phloem vessels has been documented,18 and se pollen, transpoe pollen, raising1-pyrroline-5-carboxylate ynthetase (P5CS), and \u03941-pyrroline-5-carboxylate reductase (P5CR). The existence of an alternative route for proline synthesis, converting ornithine to proline by the action of \u03b4-ornithine-amino-transferase  and P5CR, has been hypothesized by some authors[P5CS1 (At2g39800) and P5CS2 (At3g55610)[P5CR (At5g14800).In higher plants proline synthesis proceeds from glutamate that is converted to proline in a two-step pathway catalyzed by the enzymes \u0394 authors,21. Howe authors and glut3g55610), while nP5CS1  and P5CS2 , providing hints for assigning gene functions. P5CS1 is responsible for abiotic stress-induced proline accumulation, as homozygous p5cs1 mutants do not accumulate proline upon stress induction and are hypersensitive to environmental stresses[P5CS2 is necessary for embryo development, as homozygous p5cs2 mutants are embryo lethal and the p5cs2 mutant allele can be propagated only in p5cs2/P5CS2 heterozygous mutants[p5cs1 and heterozygous for p5cs2, is more delayed than that of the single p5cs1 mutant[T-DNA insertional mutants have been characterized,3,24 forp5cs2 mutation was rarely transmitted to the offspring when the proline-deficient p5cs1 p5cs2/P5CS2 was used as a pollen donor suggesting yet another role for proline in affecting male fertility. This prompted the analysis presented in this work, aimed to evaluate the role of endogenous proline in pollen development and fertility. We show here that the development of the male gametophyte carrying mutations in both P5CS1 and P5CS2 is severely compromised, indicating a role for proline in pollen function and development.In the course of a genetic screen designed to identify the floral pathway(s) proline interacts with in Arabidopsis , we found that the p5cs1 p5cs2/P5CS2, used as male, and Arabidopsis flowering time mutants, used as females, aimed to understand the flowering pathway proline interacts with , the p5cs1 mutant allele was always transmitted to the outcrossed progeny, while the transmission frequency of the p5cs2 mutant allele was exceedingly low (in average 0.8 \u00b1 0.1%). Since no obvious gametophytic defects have been ever noticed neither on p5cs1 nor on p5cs2 single mutants, this result suggests that a male fertility defect may be linked to pollen grains bearing mutations in both P5CS1 and P5CS2 genes.In crosses between p5cs1 p5cs2/P5CS2 mutants, the segregation of the sulfadiazine gene \u2013 a dominant resistance marker associated to the T-DNA insertion on P5CS2 \u2013 was analyzed in a seed population from selfed p5cs1 p5cs2/P5CS2 plants. Since the homozygous p5cs2 single mutant is embryo lethal[p5cs2-linked sulfadiazine resistance in the selfed population would approximate a 2:1 ratio of resistant over susceptible plants. If, on the other hand, the segregation ratio for the p5cs2 mutant allele should approximate a 1:1 ratio, a fertility defect for the gametophyte carrying both p5cs1 and p5cs2 mutations would be confirmed. To clarify this point, 831 seeds, from p5cs1 p5cs2/P5CS2 plants, were planted on sulfadiazine plates in six independent experiments. As shown in Figurep5cs2 mutation segregated in a 1:1 resistant:susceptible ratio, as about 50%  plantlets were sulfadiazine resistant  gametophyte carrying both p5cs1 and p5cs2 alleles is rarely transmitted to the offspring.To further confirm the gametophytic defect of the p5cs1 and p5cs2 mutant alleles, reciprocal backcrosses were made between p5cs1 p5cs2/P5CS2 and wild type plants. All seeds produced by the outcrossed siliques were collected and germinated on sulfadiazine-containing media, to follow the transmission of the p5cs2 mutant allele , producing a p5cs1 p5cs2/P5CS2 genotype. In contrast, transmission of the p5cs2 allele in reciprocal crosses occurred in 41 out of 92 cases, suggesting that mutations in both P5CS1 and P5CS2 have no effects on the female gametophyte.To confirm genetically the male sterility of pollen grains carrying both p5cs1 p5cs2/P5CS2 \u00d7 female wt, and female p5cs1 p5cs2/P5CS2 \u00d7 male wt) and analyzed by PCR for the presence of the T-DNA insertion on P5CS2 , by using a primer pair specific for P5CS1, indicating that the chosen plants derived from an outcrossing event, and excluding the possibility of an unintended contamination from self-pollinated parental genotypes. Overall, the reciprocal crosses with wild type plants provide genetic and molecular evidence that p5cs1 p5cs2/P5CS2 is impaired in male fertilization.To confirm these data at molecular level, 24 individuals were randomly chosen from each outcrossed progeny , including both large and small pollen grains, was reduced by ~ 50% (22 \u00b1 4% compared to 54 \u00b1 2.9% of control) compared to wild type  should fail to germinate, while all the large and wild type-looking pollens (p5cs1 P5CS2 genotype) should elongate a pollen tube. To verify this point, the percentage of pollen germination was calculated separately for large and small pollen grains, either as number of large germinated pollen grains out of total large pollen grains, or as small germinated pollen grains out of total small pollen grains , as shown in Figurep5cs1 p5cs2 genotype might occasionally have degenerated and looking small, while some pollen of p5cs1 p5cs2 genotype may be not, or not completely, degenerated and yet looking large. To clarify this point, pools of either large or small pollen grains were analyzed by PCR for the presence or absence of T-DNA insertions in P5CS1 and P5CS2. As shown in FigureP5CS1, only the T-DNA insertion on P5CS1 could be amplified  of the Bates method[p5cs1 p5cs2/P5CS2 plants. The low content of proline measured in pollen from mutant plants (15 pg/pollen), compared to that found in wild type pollen (48 pg/pollen), provides a direct correlation between proline deficiency and pollen defects. Because in a pollen population from p5cs1 p5cs2/P5CS2 plants, approx 50% of the pollen grains look aberrant, a ~ 50% reduction of proline content is expected. However, a proline reduction exceeding 70% was detected in pollen from mutant plants. This result suggests that the observed proline reduction cannot be accounted for only by the abortion of the small misshaped pollen, but that a reduction in proline content must take place, to some extent, also in the large wild type-looking pollen grains. In addition, 10 \u03bcM L-proline was supplemented in vitro to mature pollen from p5cs1 p5cs2/P5CS2 plants grown on germination medium, or in planta to developing anthers. As shown in Figurein vitro  responsible for proline transport in plants, have been isolated and characterized but none of them revealed alterations, compared to wild type, neither in proline content nor in pollen germination efficiency[However, since pollen from ficiency. Althougficiency or AtLHTficiency, may comficiency,34, and ficiency, who detP5CS in pollen[P5CS gene as observed by different authors[P5CS1/2 genes could be expressed only in particular stages of pollen development, and still accumulate enough P5CS enzyme to satisfy overall proline demand for pollen maturation. Temporal discrepancies between transcript and protein levels have been reported in pollen also for other genes, such as AtSUC1 (AT1G71880), whose transcript level is high at tricellular stage and low in mature pollen[AtSTP9 (AT1G50310), whose gene product can only be detected by immunofluorescence microscopy after the onset of germination[P5CS1, P5CS2, and P5CR are detected in pollen by microarray analysis[p5cs1 p5cs2/P5CS2. These contrasting pieces of evidence can be reconciled if ornithine pathway does not contribute to proline synthesis. Incidentally, this evidence supports the finding of Funck et al.[The question whether proline may be synthesized directly in pollen grains has been the object of controversial discussions, because some authors could detect low,32 or non pollen, while on pollen. However authors,19,24,32e pollen and AtSTmination. In addianalysis,34, direk et al. who demop5cs1 p5cs2/P5CS2, and with the absence of obvious defects in female gametopytes, always embedded in sporophytic cells.Overall, proline accumulation in pollen may rely essentially on endogenous proline synthesis, although is yet to be understood whether proline derives uniquely from endogenous synthesis inside the male gametophyte or also from proline synthesized in nearby sporophytic cells and transported or diffused inside pollen grains. A likely hypothesis is that, as pollen lose desmosomal connections to surrounding sporophytic cells, becomes dependent on endogenous proline synthesis, consistent with the late appearance (stage 11) of visible aberrations in developing pollen from P5CR, emb-2772-1 and emb-2772-2[p5cs2 mutants, halting embryo development at a preglobular stage. In sharp contrast, however, no gametopytic defects have been associated, so far, to these mutants.Intriguingly, two Arabidopsis mutants bearing T-DNA insertions on mb-2772-2 exhibit P5CR and P5CS2, two genes coding for proline synthesis enzymes belonging to the same pathway, may lead to similar defects in embryo development, it is puzzling that, contrary to p5cs2, emb-2772 exhibits no gametophytic defects. In Arabidopsis a number of mutants have been described by Muralla et al.[While it is not surprising that lesions in a et al. with defTo explain this apparent paradox, the authors propose that gene products derived from transcription of wild type alleles in heterozygous sporocytes may compensate the deficiency of the mutant gametophytes, and that embryo lethality results when these products are eventually depleted. Likewise, we may speculate that, contrary to P5CS2, the P5CR transcript and/or protein, synthesized in heterozygous sporocytes, is stable enough to sustain pollen but not embryo development.p5cs1 p5cs2 genotype, may be caused by the irreversible damages on cellular membranes caused by the process of dehydration in absence of the protective action of proline.The data presented here suggest that proline is required for pollen development, but gives no indication on the role of proline in pollen development. We know from histological analysis Figure that a fOnce pollen has reached full maturation, accumulated proline is catabolized and serves as source of energy - to fuel the rapid and energy-demanding elongation of the pollen tube,39 - andp5cs1 p5cs2/P5CS2 plants non-metabolizable compatible osmolyte, such as glycine betaine. Equally interesting it will be to dissect the role of proline synthesized in the haploid male gametophyte from that synthesized in diploid sporophytic tissues of the anther.In the future it will be interesting to address this issue by uncoupling these two putative functions, for example targeting in developing pollen grains from p5cs1 and heterozygous for p5cs2, defective in proline synthesis, the development of the male gametophyte with mutations in both P5CS1 and P5CS2 is severely compromised, and provide genetic evidence that proline is needed for pollen development and fertility.We show here that in mutants homozygous for Arabidopsis thaliana from Columbia-0 (Col-0) ecotype used in this work were grown in a growth chamber at 24/21\u00b0C with light intensity of 300-\u03bcE\u00b7m-2\u00b7s-1 under 16 h light and 8 h dark per day. Arabidopsis homozygous for p5cs1 (SALK_063517), originally obtained from the SALK collection, are knockout insertional mutants described in[p5cs2 (GABI_452G01), originally obtained from the GABI-Kat collection, are insertional mutants described in[p5cs2/P5CS2 is embryo lethal in homozygous state and must be propagated in heterozygous state. Arabidopsis homozygous for p5cs1 and heterozygous for p5cs2 (p5cs1 p5cs2/P5CS2), have been characterized and described elsewhere[p5cs1 p5cs2/P5CS2 plant were stratified for three days at 4\u00b0C, surface-sterilized, and germinated on MS1/2 plates supplemented with 12 \u03bcg/ml sulfadiazine. Segregation ratios were calculated by scoring the number of resistant over susceptible plantlets, and confirmed by PCR analysis of random samples. using primers 5\u2019-CAAGCAATGGTGGAAGAGTAAA-3\u2019 and 5\u2019- CGGGGCTCAAGAAAAATCC -3\u2019 for the sulfadiazine resistance gene. For embryo analysis, siliques derived from self-fertilized wild types, p5cs2/P5CS2 mutants, or p5cs1 p5cs2/P5CS2 mutants, were dissected and analyzed under a Zeiss Stevi SV 6 light stereomicroscope . Digital images were acquired with a Jenoptik ProgRes\u00ae C3 digital camera . All the analyses have been repeated at least four times. Statistical significance was inferred from percentage data by using \u03c72 analysis.Wild-type and mutant p5cs1 p5cs2/P5CS2 and flowering time mutants the F1 generation was allowed to self-fertilize and the presence of the p5cs2 mutant allele was assessed from the F2 generation, by sulfadiazine selection or by PCR genotyping of the sulfadiazine resistance gene. To confirm the data and rule out any possible interference of the flowering time mutant genotypes in the transmission of the p5cs2 mutation, reciprocal crosses between p5cs1 p5cs2/P5CS2 and wild type were performed. Transmission of the T-DNA insertion on P5CS2 gene was assessed, either by sulfadiazine selection of outcrossed seeds germinated on sulfadiazine-containing solid medium, or by PCR genotyping of sulfadiazine resistance gene on plantlets grown without selection Statistical significance was inferred from percentage data by using \u03c72 analysis.In crosses between For orcein staining, pollen was collected by dabbing mature flowers, from four weeks old plants, on a microscope slide. After a brief incubation in 1% acetic orcein, the pollen grains were rinsed in 50% acetic acid and examined under a Leitz Laborlux D light microscope  equipped with a Jenoptik ProgRes\u00ae C3 digital camera . For histological analysis, floral buds, of different developmental stages, were embedded in Technovit 7100 (Kulzer), and 3-mm cross-sections were stained with 1% Toluidine blue as described in and analP5CS2, 5\u2019-GGAGCAGAATGGTTTTCTCG-3\u2019 and 5\u2019-TGGAAAACAGCAGCACTGTC - 3\u2019 for the gene P5CS2, 5\u2019-CTGTTGGGGGTAAACTCATTG-3\u2019 and 5 -GCGTGGACCGCTTGCTGCAACT-3\u2019 for the T-DNA insertion on P5CS1, 5\u2019-CTGTTGGGGGTAAACTCATTG-3\u2019 and 5\u2019-CTCTGCAACTTCGTGATCCTC-3\u2019 for the gene P5CS1.Pollen grains were separated by size  under a Zeiss Stevi SV 6 light stereomicroscope  and pools of about 500 pollen grains were prepared and frozen at \u221220\u00b0C. Pollen DNA was extracted from these samples with a modified CTAB (cetyl trimethylammonium bromide) protocol according to. Becausein vitro germination variability, pollen from wild type plants was always included nearby pollen from p5cs1 p5cs2/P5CS2 plants on the same microscope slide.Mature pollens from stage 13 flowers were col2 analysis.For pollen germination analysis, the slides were examined under a Leitz Laborlux D microscope  and digital pictures of randomly chosen fields were acquired with a Jenoptik ProgRes\u00ae C3 digital camera . To determine pollen germination efficiency, the number of germinated and non-germinated pollens was scored from five, randomly chosen, fields per replica in four independent experiments. Statistical significance was inferred from percentage data by using \u03c7In vitro complementation was attempted by spotting mature pollens in microscope slides coated with germination medium supplemented with 10 \u03bcM L-proline. In vivo complementation was performed by daily spraying early developing inflorescences with 10 \u03bcM proline. Mature pollens from stage 13 proline-treated flowers were collected and analyzed as described above.Proline measurements were modified from as folloThe authors declare that they have no competing interests.MT wrote and supervised the work, performed reciprocal crosses, orcein stains and Alexander\u2019s stains, RM carried out segregation and silique analyses, DAPI stains, germination assays and proline complementation assays, CL carried out histological analyses, MB performed molecular analyses, PC gave financial support and revised the manuscript. All authors read and approved the final manuscript.Figure S1. p5cs1 p5cs2/P5CS2 anther stained with Alexander\u2019s stain. Close up of a p5cs1 p5cs2/P5CS2 anther stained with Alexander\u2019s stain. The small and misshaped pollen grains are clearly visible, within a p5cs1 p5cs2/P5CS2 anther, as non-stained pollen pollen grains alongside wild type-like, red stained, pollen grains. Bar = 50 \u03bcm.Click here for fileFigure S2. In vitro germination assays of pollen from p5cs1 single mutants. To evaluate possible defects in pollen viability of a mutant bearing a single mutation in P5CS1 gene, pollen from a homozygous knockout p5cs1 mutant was incubated in vitro on germination medium and scored for germination. (A) Percentage of germinated versus total pollens (germinated + non germinated), including both large and small pollen grains (right column), compared to wild type (left column). (B) Percentage of large (middle column), small (right column), and wild type pollen (left column) versus large (middle column), small (right column) and wild type (left column) total germinated pollen. Values in (A) and (B) represent the means of four independent experiments \u00b1 SE.Click here for file"}
+{"text": "Bipolar disorder is a common, heritable mental illness characterized by recurrent episodes of mania and depression. Despite considerable effort to elucidate the genetic underpinnings of bipolar disorder, causative genetic risk factors remain elusive. We conducted a comprehensive genomic analysis of bipolar disorder in a large Old Order Amish pedigree. Microsatellite genotypes and high-density SNP-array genotypes of 388 family members were combined with whole genome sequence data for 50 of these subjects, comprising 18 parent-child trios. This study design permitted evaluation of candidate variants within the context of haplotype structure by resolving the phase in sequenced parent-child trios and by imputation of variants into multiple unsequenced siblings. Non-parametric and parametric linkage analysis of the entire pedigree as well as on smaller clusters of families identified several nominally significant linkage peaks, each of which included dozens of predicted deleterious variants. Close inspection of exonic and regulatory variants in genes under the linkage peaks using family-based association tests revealed additional credible candidate genes for functional studies and further replication in population-based cohorts. However, despite the in-depth genomic characterization of this unique, large and multigenerational pedigree from a genetic isolate, there was no convergence of evidence implicating a particular set of risk loci or common pathways. The striking haplotype and locus heterogeneity we observed has profound implications for the design of studies of bipolar and other related disorders. Bipolar disorder is a common, heritable mental illness characterized by recurrent episodes of mania and depression. Despite considerable efforts genetic studies have yet to reveal the precise genetic underpinnings of the disorder. In this study we have analyzed a large extended pedigree of Old Order Amish that segregates bipolar disorder. Our study design integrates both dense genotype and whole-genome sequence data. In a combined linkage and association analysis we identify five chromosomal regions with nominally significant or suggestive evidence for linkage, several of which constitute replication of earlier linkage findings for bipolar disorder in non-Amish families. Association analysis of genetic variants in each of the linkage regions yielded a number of plausible candidate genes for bipolar disorder. The striking genetic heterogeneity we observed in this genetic isolate has profound implications for the study of bipolar disorder in the general population. CACNA1C  and ANK3 (ankyrin 3) implicating these as potential susceptibility genes for bipolar disorder CACNA1C and identified new susceptibility alleles in ODZ4Bipolar affective disorder is a life-long mental illness characterized by recurrent episodes of depression and mania or hypomania, with a typical age of onset in young adulthood. Twin and family studies have shown that bipolar disorder has a strong genetic component with heritability estimated to be in the range of 80% Genetic studies of isolated populations have led to the identification of genetic variants underlying a number of Mendelian disorders, particularly those caused by autosomal recessive mutations The Old Order Amish are a genetic isolate of European ancestry currently residing in several states in North America, with a concentration in Pennsylvania and Ohio HRAS1 and INS loci on chromosome 11 The original genetic linkage study of a large extended Old Order Amish pedigree with bipolar affective disorder reported positive findings with DNA markers around the To identify the genetic basis of bipolar disorder in the Amish, our research returned to the expanded multigenerational Old Order Amish pedigree available as the Amish Study of Major Affective Disorders at the Coriell Institute for Medical Research and applied contemporary genomic and statistical methodology by integrating genotype and whole-genome sequence data. This investigation combines linkage analyses on multiallelic microsatellite genotypes for the large pedigree (n\u200a=\u200a497) with the analysis of Illumina Omni 2.5M SNP genotypes (n\u200a=\u200a388) and whole-genome sequences of 50 family members. Our findings reveal multiple linkage regions that each harbor a considerable number of sequence variants, supporting initially reported locus heterogeneity. Dissection of exonic and intronic variants that reside in these linkage peaks has identified credible candidate genes that will be further examined in large-scale population-based studies. Our results underscore the complexity of the genetic etiology of bipolar disorder in this genetic isolate and have important implications for the study of bipolar disorder in the general population.The Amish Study of Major Affective Disorders includes biomaterials and clinical data for a large extended family (497 family members) . The AmiIn order to address genetic and phenotypic heterogeneity, analysis was restricted to 49 nuclear families with at least one BPI subject. Among the 364 selected subjects were 92 individuals with Major Affective Disorder including bipolar disorder I (BPI) (n\u200a=\u200a62), bipolar disorder II (BPII) (n\u200a=\u200a15) and Major Depressive Disorder, recurrent form (MDD-R) (n\u200a=\u200a15); 52 individuals with minor psychiatric diagnoses (classified as \u201cunknown\u201d phenotype) and 220 \u201cwell\u201d subjects. Three diagnostic hierarchies were defined for the analysis: the first and most stringent classification included only BPI individuals as affected (BPI phenotype), the second included BPI and BPII individuals as affected (narrow phenotype) and the third included BPI, BPII and MDD-R as affected (bipolar spectrum disorder or BPS).2) of bipolar susceptibility in the Amish pedigree using the Sequential Oligogenic Linkage Analysis Routines (SOLAR) To quantify the fraction of phenotypic variance accounted for by genetic causes, we estimated the narrow sense heritability  in the 1000 Genomes Project data, the EVS database and a set of 54 HapMap WGS made publicly available by CGI. Of the resulting list of 514 variants predicted to be \u201cdamaging/deleterious\u201d by both Polyphen2 GRM7) gene on chromosome 3 (TTLL2) and t-complex 10 homolog (TCP10) on chromosome 6 , we performed family based genome-wide association analysis on 388 samples genotyped with Illumina Omni 2.5 Million SNP arrays. We applied two methods for association analysis: (a) EMMAX mosome 3  top and mosome 6  bottom. mosome 6 . However13207753 .\u22124) association was observed  and 18p11 . The analysis of subpedigrees yielded nominally significant linkage peaks on 2p25  and 4p16.3  observed within subpedigree 310/410 as well as 16p13 (expLOD 3.15 at D16S3127) within sub-pedigree 310 (D18S453) maps less than 1 Mb distal to the translocation breakpoint [t] segregating with schizophrenia in a seven member family Linkage analysis of all 49 nuclear families and the sub-pedigrees detected suggestive linkage (LOD>2.2) on six and eleven chromosomes respectively . Within gree 310  and 4. SD4S3360-D4S2936-D4S412) covering the highest observed linkage peak (HLOD 3.96). Here among the four contributing haplotypes , a single haplotype, 5-3-2, was shared by 3 out of 4 linked nuclear families. The 5-3-2 haplotype was present in 9/10 affected individuals within the contributing families. There was significant haplotype association of the 5-3-2 haplotype (FBAT p\u200a=\u200a0.0267) as well as for the global omnibus test  in affected individuals within linked families  and 4p16.3 (in POLN and EVC genes). However a significant number of variants under the linkage peaks are either novel or rare  or moderately frequent (e.g ERCC4 and MLL5). Using the current level of resolution, it is difficult to evaluate whether these variants contribute to nominal association signals within the five regions detected by the analysis of SNP array data using EMMAX and FBAT  and the neighboring distal-less homeobox 5 and 6 genes (DLX5 and DLX6) (P\u200a=\u200a0.008 and P\u200a=\u200a0.003 respectively). The most strongly associated gene under the 16p13 peak was the ATP-binding cassette, sub-family C, member 6 (ABCC6) gene (P\u200a=\u200a0.00055). The 2p25 region harbors the integrin beta 1 binding protein 1 (ITGB1BP1) gene, which is involved in cell adhesion. The class of cell adhesion molecules has been implicated to play a role in bipolar disorder in multiple gene set enrichment analyses studies We also analyzed sequence variation on linkage-driving haplotypes by focusing on exonic missense variants and SNPs associated with the expression level of genes under linkage peaks  but rather several non-coding and exonic SNPs. Due to the long haplotype blocks observed in founder populations, a linkage signal may reflect a combined effect of multiple causal alleles within the same chromosomal region as commonly described in the dissection of behavioral traits in model organisms KCNH7 (Arg394His), potassium voltage-gated channel, subfamily H, member 7, is of considerable interest. Voltage-gated ion channels have previously been implicated in a number of psychiatric disorders, including bipolar disorder, due to their known involvement in neuronal excitability and synaptic transmission KCNH7 locus with bipolar disorder was also reported at rs6736615, an intronic variant, in a recent case-control study of 400 Taiwanese subjects ADAM18, INSL6, IFT81) that play roles in spermatogenesis and fertilization. To determine whether mutations in these genes may underlie bipolar disorder and/or other complex traits, it will be necessary to explore additional Amish families with mental illness and comorbid medical conditions.Using WGS we identified a number of non-synonymous, likely deleterious variants that are rare in the 1000 Genomes Project dataset (2%) but present in 10\u201330% of the BP subjects and their first-degree relatives. Such differences in allele frequency, resulting from a population bottleneck and expansion from a relatively small number of founders, have been previously described in several isolated populations , involved in hormone-dependant transcriptional regulation. Among several genes identified in the second publication ODZ2 . A rare variant in this gene and in a paralogous gene ODZ4, with an intronic SNP (rs12576775) associated with bipolar disorder at a genome-wide level Our analysis of previously published genome-wide significant GWAS loci did not show any convergence with the results from our family-based GWAS. Recently, two publications employed whole exome sequencing to identify rare variants associated with bipolar disorder using a family-based design Isolated founder populations provide a unique opportunity to evaluate the genetic architecture of human disease Leveraging family-based design with the population-based studies of BP-associated quantitative traits in a founder population will be critical for effective gene and human-genetics driven drug discovery. Although candidates detected by our comprehensive approach were not significant after the correction for multiple testing, we provide supportive evidence and a biological rational for further investigating these variants in large cohorts, especially when more genome and exome data become available.th Edition (DSM-IV) for uniform clinical criteria http://ccr.coriell.org/Sections/Collections/NIGMS/AmishStudy.aspx?PgId=600). Collection of blood/tissue samples followed diagnostic consensus, using two informed consent forms: a) one with annual Univ. Miami IRB approval defining (with language appropriate for Old Order Amish) how their cells would be preserved for medical research on Major Affective Disorders; and, b) the Informed Consent Form required by the Institute for Medical Research (CIMR), later Coriell - National Institute for General Medical Sciences (NIGMS) Human Genetic Cell Repository (HGCR). Since completion of the Amish Study BPI cell collection at CIMR (JAE curator), the NIGMS-HGCR consent form has undergone revisions. The CIMR/NIGMS Informed Consent Forms makes no reference to specific diseases, types of genetic analysis that will be performed in future studies . Analysis of whole-genome re-sequencing data from consented individuals in this pedigree was also approved by the IRB of the Weill Cornell Medical College and the Perelman School of Medicine at the University of Pennsylvania.The genetic-epidemiologic study of bipolar disorder among the Old Order Amish, in settlements throughout Pennsylvania, was documented extensively, including ascertainment protocols and diagnostic methods Based on genealogy the whole pedigree is subdivided into four large subpedigrees  G\u200a=\u200a where each node in V represents a nuclear family in the pedigree. Edges in E correspond to parent-child relationships connecting the nuclear families. If there is an individual who is a child in one family and the parent in another, we connect the two families by a directed edge. The nuclear family graph also protects the privacy of individuals by allowing the visualization of the relatedness between families without revealing family structure .To visualize the high-level structure of the pedigree and the degree of relatedness of nuclear families we constructed the nuclear family graph . The nuccgatools software  made available by CGI. The listvar tool was used to generate a master list of the 11.1M variants present in the 50 Amish samples. The testvar tool was used to determine presence and absence of each variant within the 50 Amish WGS. Only variants with high variant call scores (\u201cVQHIGH\u201d tag in the data files) were included. Variants that were observed in a single WGS were excluded.Whole genome sequencing (WGS) was performed by Complete Genomics Inc.  using a sequence-by-ligation method http://evs.gs.washington.edu/EVS/). For novel variants in our Amish WGS the allele frequencies in the Complete Genomics Diversity Panel (http://www.completegenomics.com/public-data/), a set of WGS from 54 unrelated HapMap samples, was used as a technical control to guard against platform specific false positive calls. Predictions of functional impact of exonic missense variants using Polyphen2 In each sample sequencing depths was averaged within 100 Kb windows across the genome. Median sequence coverage over all windows across the 50 samples was \u223c50\u00d7. Within each sample averaged sequencing depth was >40\u00d7 for \u223c70% of the genome and >10\u00d7 \u223c98% in all 50 sequenced individuals . Coveraghttp://pngu.mgh.harvard.edu/~purcell/plink/). The initial dataset contained 2,379,855 SNPs and 394 samples. The following filters were applied in sequence; the numbers of markers or samples excluded is given in parentheses: a) exclude SNPs with missing rate >0.5 , b) exclude samples with missing rate >0.02 (6), c) exclude SNPs with missing rate >0.02 , d) exclude SNPs with MAF<0.02 , e) exclude SNPs with informative missingness p<1e-6 (0), f) exclude SNPs with Hardy-Weinberg equilibrium p<1-e6 (0), g) exclude individuals with >5% Mendel errors (0) and h) exclude SNPs with >1% Mendel errors (1334). After quality control we retained 1,309,937 SNPs and 388 samples. A single sample with WGS was removed by quality control filtering. For the remaining 49 samples with both WGS and SNP array genotypes, we evaluated concordance using the cgatools snpdiff program (http://cgatools.sourceforge.net/). We found an average concordance of 0.996 (SD 9.1e-05) in the called variants, suggesting a very high consistency of the WGS and SNP array datasets.Genotyping of 394 samples from the extended Amish pedigree using Illumina Omni 2.5 M SNP arrays was performed at the Center for Applied Genomics . We performed rigorous quality control of the raw genotype calls by applying a series of filters on both markers and samples using PLINK  EMMAX st degree relatives, [0.0884\u20130.177] for 2nd degree relative, [0.0442\u20130.0884] for 3rd degree relatives and <0.0442 for unrelated subjects. Observed ranges of kinship coefficients for known family relationships in the extended pedigree match well to these expectations  We performed phasing and imputation of variants identified by WGS into the Omni 2.5 M SNP genotypes using the Genotype Imputation Given Inheritance (GIGI) software Parametric and non-parametric linkage analyses were performed using the MERLIN software version 1.1.2 To test for gene-wise burden under an additive model we applied a family-based test of additive burden (rareFBAT) 2). Age and sex were used as covariates.The heritability of bipolar disorder was estimated using the Sequential Oligogenic Linkage Analysis Routines (SOLAR) Sequence variants that significantly impact gene expression in the human brain Click here for additional data file.Figure S2Weighted sequence coverage for the whole genome (blue) and exome (red) of WGS of 50 Amish samples.(PNG)Click here for additional data file.Figure S3Pairwise kinship coefficient matrices for 388 samples with Omni2.5 SNP genotypes. A) Kinship coefficients based on known pedigree relationships. B) Estimated kinships coefficients based on 1.3M SNP genotypes.(TIF)Click here for additional data file.Figure S4Quantile-Quantile plots of genome-wide association analysis. A) FBAT analysis and B) EMMAX analysis.(TIF)Click here for additional data file.Figure S5Manhattan plots of genome-wide association analysis of 388 samples from the extended Amish pedigree. A) Family-based association analysis using FBAT, B) Mixed model case-control analysis with correction for relatedness using EMMAX.(TIF)Click here for additional data file.Figure S6GRM7 (top) and TTLL2-TCP10 loci (bottom) (plots generated with LocusZoom).FBAT (left) and EMMAX (right) association results at the (TIF)Click here for additional data file.Figure S7GRM7 (left) and TTLL2-TCP10 (right) loci.LocusZoom plots of case-control association for 2,836 BP cases and 2,744 controls (GAIN) at the (PNG)Click here for additional data file.Figure S8SNP-based and gene-wise burden association tests in the linkage regions. For each of the five identified linkage regions (A) 2p25, B) 4p16.3, C) 7q21, D) 16p13 and E) 18p11) association results with EMMAX and FBAT using Omni2.5 SNP array data (top and middle panel) are shown. The bottom panels display the results of gene-wise burden tests of exonic and regulatory variants using the rareFBAT test based on imputed WGS and Omni2.5 genotype data. The x-axis shows the chromosomal position in Mb, the y-axis the -log for the respective method. Each data point corresponds to a single SNP in the EMMAX and FBAT panels and a combined burden test for exonic and regulatory variants in a single gene in the rare FBAT panels.(TIF)Click here for additional data file.Figure S9Example of an Ingenuity Pathway Analysis (IPA) of the top 100 genes from table S1. No significant enrichment for a specific pathway, biological function or tissue-specific gene expression was observed.(TIF)Click here for additional data file.Figure S10Normalized coverage (i.e. coverage relative to the genome average) for different GC content percentiles. GC content was calculated for bins of 501 bp across the genome. It can be seen that the normalized coverage only drops off at the very extremes of the GC content distribution in all 50 samples.(PNG)Click here for additional data file.Figure S11Evaluation of imputation performance for different thresholds on the genotype posterior probability. Concordance between imputed and SNP array genotypes (red) and call rate of the imputed genotypes (blue) are shown. The plot shows averages over A) all samples in the dataset, B) families in the neighborhood of WGS samples and C) first degree relatives of subjects with WGS.(TIF)Click here for additional data file.Table S1Top 50 most conserved rare exonic variants by GERP conservation score.(XLSX)Click here for additional data file.Table S2514 Amish-specific putative damaging exonic missense variants. Exonic missense variants, identified in 50 subjects with WGS, were filtered by <2% allele frequency in 1000 Genomes, EVS and 54 HapMap WGS. Functional impact was assessed by consensus of Polyphen2 and SIFT.(XLSX)Click here for additional data file.Table S3Top SNPs from genome-wide association analysis with FBAT and EMMAX association analysis of 1.3M SNPs for 388 subjects from the extended pedigree. Only SNPs with p<\u200a=\u200a5e-4 are shown.(DOCX)Click here for additional data file.Table S4Result from rareFBAT burden test analysis of 62 candidate regions for BP and schizophrenia identified by recent GWAS studies. The table shows chromosomal region, number of Omin2.5 SNP included, rareFBAT P-value and (if applicable) the previously reported associated SNP.(XLS)Click here for additional data file.Table S5ANK3, ODZ4 and NEK4-ITIH1-ITIH3-ITIH4 region as well as 24 genes reported in two recent whole-exome sequencing studies.Exonic missense variants in genome-wide significant BP association hits in the (XLS)Click here for additional data file.Table S6A) Linkage results from the analysis of 49 nuclear families from the extended pedigree and B) linkage results from the analysis of defined subpedigrees. The table lists the chromosomal region, top LOD marker, length of peak region for a 2 LOD confidence interval, phenotype under which maximum linkage was observed, linear non-parametric LOD score (NPL LOD), exponential non-parametric LOD score (NPL expLOD), parametric LOD score, parametric HLOD score and the alpha value denoting the fraction of linked families in the HLOD computations.(DOC)Click here for additional data file.Table S7Number of linked families and contributing haplotypes for five identified linkage regions.(DOC)Click here for additional data file.Table S8D4S3360, D4S2936, D4S412) association test results for linked families in the 4p16 linkage region under an additive model. Haplotypes identified by allele sharing in affected siblings are shown in bold. Significant haplotype association (p\u200a=\u200a0.0267) is observed for the 5-3-2 haplotype. The global FBAT haplotype association test also was significant with p\u200a=\u200a0.0291.FBAT haplotype ((DOC)Click here for additional data file.Table S9Genes within the five linkage regions with nominally significant burden test association (P<0.05). The table shows the peak region, Gene, number of exonic and regulatory SNPs included in the burden test and rareFBAT P-values for the Null of hypotheses of \u201cNo linkage, no association\u201d and \u201clinkage and no association\u201d.(DOCX)Click here for additional data file.Table S10Result from rareFBAT burden test analysis of 47 genes under the 2p25 linkage peak. Exonic missense and eSNP variants were included in the test for each gene.(XLS)Click here for additional data file.Table S11Result from rareFBAT burden test analysis of 83 genes under the 4p16.3 linkage peak. Exonic missense and eSNP variants were included in the test for each gene.(XLS)Click here for additional data file.Table S12Result from rareFBAT burden test analysis of 249 genes under the 7q21 linkage peak. Exonic missense and eSNP variants were included in the test for each gene.(XLS)Click here for additional data file.Table S13Result from rareFBAT burden test analysis of 98 genes under the 16p13 linkage peak. Exonic missense and eSNP variants were included in the test for each gene.(XLS)Click here for additional data file.Table S14Result from rareFBAT burden test analysis of 66 genes under the 18p11 linkage peak. Exonic missense and eSNP variants were included in the test for each gene.(XLS)Click here for additional data file.Table S15Numbers of families and subjects with WGS data for the entire pedigree and seven more homogeneous subpedigrees chosen based on family relationship and imputation quality.(DOC)Click here for additional data file."}
+{"text": "Brassica napus L.) to obtain a high density genetic map for this species and to study the linkage disequilibrium pattern.High density genetic maps built with SNP markers that are polymorphic in various genetic backgrounds are very useful for studying the genetics of agronomical traits as well as genome organization and evolution. Simultaneous dense SNP genotyping of segregating populations and variety collections was applied to oilseed rape  and along the linkage groups. Overall, polymorphism level was higher and LD decayed faster in spring than in \u201c00\u201d winter oilseed rape types but this was shown to vary greatly along the linkage groups.We developed an integrated genetic map for oilseed rape by high throughput SNP genotyping of four segregating doubled haploid populations. A very high level of collinearity was observed between the four individual maps and a large number of markers (>59%) was common to more than two maps. The precise integrated map comprises 5764 SNP and 1603 PCR markers. With a total genetic length of 2250\u00a0cM, the integrated map contains a density of 3.27 markers (2.56 SNP) per cM. Genotyping of these mapped SNP markers in oilseed rape collections allowed polymorphism level and linkage disequilibrium (LD) to be studied across the different collections (winter Brassica napus sequence assembly and genome organization analyses.Our study provides a valuable resource for further genetic studies using linkage or association mapping, for marker assisted breeding and for  LD heatmaps were built for each linkage group with the R package LDheatmap implemented in R software  based on their collinearity with Click here for fileDot-plots obtained for each linkage group between the integrated map and all four individual TNDH, DYDH, AADH and AMDH maps. The marker order on the vertical axis is from the four individual maps and the marker order on the horizontal axis is from the integrated map. Cumulative genetic distance in cM is indicated on each axis. (PDF 471 kb)Click here for fileList of the 222 SNPs that were assigned to different linkage groups (LG) according to the mapping populations.Click here for fileList and characteristics of the 1536 public SNPs : Position on the integrated map , Context sequence and SNP position, Arabidopsis and B. rapa hits.Click here for fileDistribution of PIC values along the linkage groups in the fodder rape (FO), spring (SOSR) and winter (WOSR) oilseed rape and the different seed quality subgroups  within WOSR. PIC was averaged across a sliding window of 10\u00a0cM with a step of one cM.Click here for file2 as a function of genetic distance (in cM) between pairs of SNPs on each linkage group in the whole, spring, winter and \u201c00\u201d winter oilseed rape collections. Red curves show the non-linear regressions trend line of r2 versus genetic distance.Plots of rClick here for fileLinkage disequilibrium heatmaps for each linkage group in the whole, spring (SOSR), winter (WOSR) and \u201c00\u201d WOSR collections. The putative position of the centromeres is indicated by a black arrow.Click here for fileList of accessions used for the diversity analysis and LD study.Click here for file"}
+{"text": "We applied a new weighted pairwise shared genomic segment (pSGS) analysis for susceptibility gene localization to high-density genomewide SNP data in three extended high-risk breast cancer pedigrees.NBS1, BRCA1 and RAD51L1.Using this method, four genomewide suggestive regions were identified on chromosomes 2, 4, 7 and 8, and a borderline suggestive region on chromosome 14. Seven additional regions with at least nominal evidence were observed. Of particular note among these total twelve regions were three regions that were identified in two pedigrees each; chromosomes 4, 7 and 14. Follow-up two-pedigree pSGS analyses further indicated excessive genomic sharing across the pedigrees in all three regions, suggesting that the underlying susceptibility alleles in those regions may be shared in common. In general, the pSGS regions identified were quite large (average 32.2 Mb), however, the range was wide (0.3 \u2013 88.2 Mb). Several of the regions identified overlapped with loci and genes that have been previously implicated in breast cancer risk, including Our analyses have provided several loci of interest to pursue in these high-risk pedigrees and illustrate the utility of the weighted pSGS method and extended pedigrees for gene mapping in complex diseases. A focused sequencing effort across these loci in the sharing individuals is the natural next step to further map the critical underlying susceptibility variants in these regions. Breast cancer MIM #114480) is the most prevalent cancer among women in developed countries 14480 is . HoweverBRCA1 or BRCA2. Regions of excessive sharing in the cases of these pedigrees have good potential for harboring breast cancer susceptibility variants.We performed genomewide pSGS analysis in three Utah high-risk breast cancer pedigrees selected as unlikely to be due to BRCA1 and BRCA2. Each met the following criteria [BRCA1 or BRCA2 mutations, and (2) the pedigree had no significant linkage to the BRCA1 or BRCA2 regions. Hence, a-priori these pedigrees have a low probability of segregating mutations in BRCA1 or BRCA2.Using existing mutation screening and microsatellite linkage data, pedigrees were selected to have low probability of being due to mutations in the genes criteria : (1) theThe three extended, high-risk Utah pedigrees studied are shown in Figure Informed consent was obtained from all participants in this study. This study is approved by the Institutional Review Board at University of Utah.http://www.illumina.com). Principal components analysis was carried out on the set of all self-declared Caucasian samples in iControlDB with genotype data available for the 550K Illumina SNP array. We pruned the 550K Illumina SNPs to a set with r2<0.5 and used smartpca[In SGS approaches, control samples are required to estimate genomewide LD structure that is used in the empirical assessment of significance. The primary set of controls used was ascertained locally via the UPDB resource . These control individuals were known to be cancer-free and were self-declared Caucasian. These 224 \u2018local controls\u2019 comprised 117 males and 107 females. To ensure robustness of our findings, for regions of interest identified from the genomewide pSGS analysis based on the LD model estimated from the local controls, we also assessed significance based on a set of genomically-matched controls. This second set of controls comprised individuals selected from the Illumina Genotype Control Database (iControlDB) ( smartpca to extrap < 10-5), those with a missing rate greater than 5%, a minor allele frequency (MAF) of less than 1%, or significantly different from Hardy-Weinberg Equilibrium (p < 10-4) were removed. This resulted in a total of 516,475 SNPs genomewide included in the SGS analyses.SNPs from the Illumina 550K array were used. SNPs with a significantly different missing data rate between cases and controls  we also performed SGS and multipoint linkage analysis as secondary analyses, for comparison.Si to be the number of cases sharing at least one allele IBS at SNP i.Thomas et al.  proposedN is the total number of cases in a pedigree, and N11i,\u2009N22i are the counts of cases homozygous 11 and 22, respectively. Missing genotypes are treated as heterozygotes.where Ri (t) to indicate the number of consecutive SNPs (which includes the ith SNP) with IBS sharing among at least t cases , where t is usually the total number of individuals whose genotypes are in comparison (t = N). We recently introduced a new SGS test statistic, the weighted mean pairwise Shared Genomic Segment (pSGS) statistic [N cases, and denote djk as the number of meioses between cases j and k, and Rijk(2) as the run length shared by the pair of cases j,k at locus i. The test statistic for the pSGS is defined as follows:We use tatistic . It combhttp://balance.med.utah.edu/wiki/index.php/Access_programs_by_name).The significance is assessed empirically based on expected sharing under a model that includes LD as described in Thomas . Our metO(nm), given n individuals with m genotyped markers [We used FitGMLD to obtain a LD model based on the 224 local control samples using default parameters . This pr markers .p-values for each locus using Monte Carlo procedures, by comparing the observed lengths to expected lengths under the null. Sharing under the null was achieved using a gene-dropping procedure assuming random mating, Mendelian inheritance and a genetic map for recombination. Founder haplotypes in the pedigree were generated using the estimated LD model. These were segregated through the known pedigree structure using random Mendelian inheritance to generate genotypes for each descendant in the pedigree. Recombinant events were based on an established genetic map [We estimated nominal etic map . SimulatThe simulation procedure was implemented using a parallel Java program to improve computational efficiency.th ranked p-value across all 1,000 genomes  to determine the level for the significant threshold; and the 1,000th ranked p-value  to determine the suggestive threshold.Genomewide thresholds provide a correction for the multiplicity of tests performed across the genome. For SGS methods, the multiple testing corresponds to the number of SGS segments across the genome, and this depends on the pedigree structure (number of meioses between the studied cases) and the sharing statistic considered (pSGS or SGS). Hence, we estimated genomewide thresholds empirically for each pedigree for both pSGS and SGS. A genomewide significant threshold was defined as the level of significance that would be achieved at a rate of 0.05 times per genome under the null . A genomewide suggestive threshold was defined as the level of significance achieved at a rate of 1 per genome under the null (\u03bc=1.0). To estimate these thresholds we generated 1,000 null genome configurations for each pedigree , performed SGS and pSGS, identified the shared segments and their respective p-values . For each pedigree and statistic, the p-values for all segments across all 1,000 genomes were ranked. We identified the 502 of 0.16 over a sliding 500 kb window in the public available HapMap CEU data, and exceeded an individual call rate of 98% of genotyped subjects. We used an established genetic map [We also performed multipoint linkage analysis on each pedigree. In order to eliminate inflation of linkage statistics due to LD, a pruned set of SNPs n=26,177) were used for the linkage analysis. This set of \u201cLD-free\u201d SNPs had a minimum spacing of 0.1 cM, a minimum heterozygosity of 0.3 (to maintain good information content), and a maximum r7 were us-3 and p=3.0\u00d710-4, respectively, and the genomewide suggestive and significant thresholds for SGS were p=1.3\u00d710-4 and p<1.0\u00d710-6. For pedigree 2, no results surpassed the nominal threshold therefore empirical genomewide thresholds were not determined. For pedigree 3, the empirical genomewide suggestive and significance thresholds for pSGS were p=5.0\u00d710-3 and p=2.5\u00d710-4, respectively, and the suggestive and significant thresholds for SGS were estimated as p=3.8\u00d710-5 and p<1.0\u00d710-6. Genomewide thresholds for suggestive and significant linkage signals have been previously established to be LODs of 1.86 (p=1.7\u00d710-3) and 3.30 (p=4.9\u00d710-5), respectively [For all three pedigrees, nominal evidence was considered to be p\u22640.05. For pedigree 1, the empirical genomewide suggestive and significant thresholds for pSGS were p=6.5\u00d710ectively .N cases sharing) and the multipoint LOD score from linkage analysis. Table Figure Four genomewide suggestive pSGS regions were identified, with an additional two borderline. One of these regions was also genomewide suggestive in the SGS analysis, and one was genomewide suggestive using a dominant linkage analysis Table . Three oN-1 cases), although the range of the number of cases sharing was wide; generally spanning the total range possible. The size of the shared regions also varied quite widely; for the six regions of interest 14.5 \u2013 88.2 Mb  will share segments of size 25 cM on average. Under the alternate hypothesis that a disease locus exists, the length follows a Gamma distribution with mean 2/d. In the cousin-pair example, the average segment length surrounding a shared disease locus would be 50 cM. In our pedigrees, breast cancer pairs ranged from siblings (d=2), to pairs separated by 11 meioses  [BRCA1/2 and sporadic breast cancer tumors [KIT and VEGFR2 found in triple negative breast cancer, an aggressive and difficult to treat form of the disease [RAD51L1, which contains one of the two most significant associations reported in a multi-stage genomewide association study of 9,770 cases and 10,799 controls [AHR gene that has been associated with breast cancer risk [IL6[ZEB2 that is involved in RAS pathway that has been proposed as involved in clinical breast cancer progression [POLB[NBS1 (NBN) [EBAG9 has been suggested to be involved in early stage breast cancer [We investigated three extended Utah high-risk breast cancer pedigrees using weighted pSGS analysis to identify regions of excessive sharing that could potentially harbor breast cancer susceptibility loci. Five regions of interest were identified on chromosomes 2, 4, 7, 8 and 14. Three of these regions  showed evidence for excessive sharing in two pedigrees (pedigrees 1 and 3), with chromosome 4 being perhaps of particular interest because the region gained significance in the two-pedigree analysis. All five of these regions have either been identified previously in genomewide searches or candidate susceptibility genes reside in them. Our region on chromosome 4 is supported by evidence from two previous genomewide linkage studies of families not attributable to LOD=1.8) . This loLOD=1.8) . In addir tumors ,22. Furt disease . Our regcontrols . Our regcer risk ,26, and risk [IL6, which crisk [IL6. Our 88.gression . There agression , and earion [POLB and NBS1S1 (NBN) ,33 have t cancer .ELAC2, which was previously proposed as a susceptibility gene for prostate cancer using Utah high-risk pedigrees [BRCA2[BRCA1. It is perhaps surprising that a region containing BRCA1 would arise, given our aim to screen out families with known BRCA mutations. In agreement with our selection criteria, we show no linkage at this locus (LOD=0.03). Nonetheless, it is possible that BRCA1 remains a potential factor for risk in this pedigree.Seven nominal regions were also identified in our analyses. Notably two of these regions are on chromosome 17. One of the chromosome 17 regions in pedigree 1 (10.8-19.9 Mb) contains the candidate gene, edigrees . Genes tes [BRCA2. The secWe selected weighted pairwise SGS as our primary analysis specifically because the original SGS method will lose power quickly with intra-familial heterogeneity, and breast cancer is known to be a complex and very heterogeneous disease. In line with this assumption, only one of our genomewide suggestive pSGS regions also showed genomewide suggestive evidence using the original SGS algorithm. Furthermore, while the number of sharers across our regions of interest remained high, the range was wide and often reduced to the minimum possible number sharing. Hence, it appears that the pairwise algorithm may have been successful at providing more robustness to noise from heterogeneity, in addition to any residual genotyping error. One of our most significant pSGS regions (chromosomes 2) also showed genomewide suggestive evidence using multipoint linkage analysis with a dominant model.An advantage of a pedigree design for gene identification is that a small number of cases and a well-delimited region can be easily defined and increases the efficiency of downstream experiments. Sequencing multiple cases selected for their high likelihood of sharing the underlying susceptibility variant provides an additional and powerful filter that can be used to parse findings from sequencing efforts. Hence, follow-up regionally-focused sequencing of the most compelling of these regions is a cost-effective and logical next step to identify the critical underlying risk variant at these loci.Our pSGS analyses have highlighted several regions that have the potential to harbor susceptibility variants for breast cancer, some of which confirm loci previously proposed by others. Three of our most significant regions  were observed in two pedigrees and show evidence for shared risk variants across those pedigrees. Arguably, these three regions in pedigrees 1 and 3 are particularly good candidates to pursue using regionally-focused sequencing to identify novel breast cancer risk variants. In addition, and more broadly, this study has illustrated the potential utility of our new weighted pSGS method and extended pedigrees for gene mapping in complex diseases.IBD: Identity-By-Descent; IBS: Identity-By-State; iControlDB: Illumina Genotype Control Database; LD: Linkage Disequilibrium; MAF: Minor Allele Frequency; MCMC: Markov chain Monte Carlo; pSGS: pairwise Shared Genomic Segment; SGS: Shared Genomic Segment; SNP: Single Nucleotide Polymorphism; UCR: Utah Cancer Registry; UPDB: Utah Population Database.The authors declare that they have no competing interests.ZC performed the statistical analyses, drafted the manuscript and participated in algorithm and parallelization software development. AT participated in algorithm development, study design and statistical oversight. CT performed statistical analyses. JF contributed to data coordination and processing. LACA conceived of the study. NJC had oversight for study design, algorithm development, statistical analyses and manuscript writing. All authors read and approved the final manuscript."}
+{"text": "A novel method for haplotype phasing in families after joint estimation of recombination fraction and linkage disequilibrium is developed. Results from Monte Carlo computer simulations show that the newly developed E.M. algorithm is accurate if true recombination fraction is 0 even for single families of relatively small sizes. Estimates of recombination fraction and linkage disequilibrium were 0.00 (SD 0.00) and 0.19 (SD 0.03) for simulated recombination fraction and linkage disequilibrium of 0.00 and 0.20, respectively. A genome fragmentation phasing strategy was developed and used for phasing haplotypes in a sire and 36 progeny using the 50 k Illumina BeadChip by: a) estimation of the recombination fraction and LD in consecutive SNPs using family information, b) linkage analyses between fragments, c) phasing of haplotypes in parents and progeny and in following generations. Homozygous SNPs in progeny allowed determination of paternal fragment inheritance, and deduction of SNP sequence information of haplotypes from dams. The strategy also allowed detection of genotyping errors. A total of 613 recombination events were detected after linkage analysis was carried out between fragments. Hot and cold spots were identified at the individual (sire level). SNPs for which the sire and calf were heterozygotes became informative (over 90%) after the phasing of haplotypes. Average of regions of identity between half-sibs when comparing its maternal inherited haplotypes (with at least 20 SNP) in common was 0.11 with a maximum of 0.29 and a minimum of 0.05. A Monte-Carlo simulation of BTA1 with the same linkage disequilibrium structure and genetic linkage as the cattle family yielded a 99.98 and 99.94% of correct phases for informative SNPs in sire and calves, respectively. Using initial values of the haplotype frequencies and iterating over equation\u00a0A2 will converge to ML estimates of haplotype frequencies. Linkage disequilibrium is estimated by where If the linkage phase of the sire is Tm/tM then the E.M. equations are:The probability of phase TM/tm is:LTM/tm and LTm/tM are the likelihoods of phase (TM/tm) and phase (Tm/tM) under a model estimating linkage disequilibrium (\u03b4), allele frequencies for one of the alleles at markers T/t and M/m , and the recombination fraction (c).where The likelihood equation to estimate LD across half-sib families is:Li is the likelihood for the i-th half-sib family conditional to genotype marker information (nG) and nf is the number of families. Note that depending on the sire genotype, allele frequencies for T and M (double homozygote) or M (homo-heterozygote) do not need to be estimated. The E.M. algorithm can be applied to multiple families by iterating on the four haplotype frequencies and recombination fraction:where where equations for haplotype frequencies for each single family varies depending on the sire genotype (see  for a fuSNP: Single Nucleotide Polymorphism; LD: Linkage disequilibrium; ROI: regions of identity; GFPS: Genome fragmentation phasing strategy.The authors declare that they have no competing interests.LGR contributed to the developed the idea and carried out the mathematical modeling for the joint estimation of LD and linkage disequilibrium. He drafted the first version of the manuscript. AMH participated in the design of the study, contributed with preparation of DNA samples and writing of several parts of the manuscript. DT carried out the tissue sampling, and contributed to the writing of the manuscript. WMR contributed to the development of the idea, helped to carry out tissue sampling and contributed to the writing of the manuscript. All authors read and approved the final manuscript."}
+{"text": "Dianthus caryophyllus L.) is an important ornamental flower worldwide. We previously reported a random amplified polymorphic DNA (RAPD)-based genetic linkage map derived from Dianthus capitatus ssp. andrezejowskianus and a simple sequence repeat (SSR)-based genetic linkage map constructed using data from intraspecific F2 populations; however, the number of markers was insufficient, and so the number of linkage groups (LGs) did not coincide with the number of chromosomes (x\u2009=\u200915). Therefore, we aimed to produce a high-density genetic map to improve its usefulness for breeding purposes and genetic research.Genetic linkage maps are important tools for many genetic applications including mapping of quantitative trait loci (QTLs), identifying DNA markers for fingerprinting, and map-based gene cloning. Carnation  could be mapped to 17 linkage groups (LGs) covering 969.6 cM. Comparison of five minor LGs covering less than 50 cM with LGs in our previous RAPD-based genetic map suggested that four LGs could be integrated into two LGs by anchoring common SSR loci. Consequently, the number of LGs corresponded to the number of chromosomes (x\u2009=\u200915). We added 192 new SSRs, eight RAPD, and two sequence-tagged site loci to refine the RAPD-based genetic linkage map, which comprised 15 LGs consisting of 348 loci covering 978.3 cM. The two maps had 125 SSR loci in common, and most of the positions of markers were conserved between them. We identified 635 loci in carnation using the two linkage maps. We also mapped QTLs for two traits  and a phenotypic locus for flower-type by analyzing previously reported genotype and phenotype data.Dianthus, including carnation, and will be useful for mapping QTLs associated with various traits, and for improving carnation breeding programs.The improved genetic linkage maps and SSR markers developed in this study will serve as reference genetic linkage maps for members of the genus  Genetic linkage maps are valuable resources that provide a framework for many genomic analyses. They are an important tool for many genetic applications, including mapping of quantitative trait loci (QTLs), comparative mapping, identifying DNA markers for fingerprinting, analyses of population genetics and phylogenetics, genome sequence assembly, relating physical and recombination distances along the genome, and map-based cloning of genes. The wide applications of linkage maps and their importance in genetic research has led to numerous linkage mapping projects being undertaken in plants -3.Dianthus caryophyllus L.), in the Caryophyllaceae, is one of the major floricultural crops in Japan and around the world. Hundreds of cultivars are grown around the world and many new varieties are bred and registered every year ) in our previous study [Cap1 (LOD\u2009=\u20096.1) and Cap2 (LOD =5.3) were mapped onto LG NP_4 and LG NP_10, respectively. We also mapped a phenotypic locus for flower-type. We identified a single flower-type locus in a previous study [D. capitatus ssp. andrezejowskianus was mapped onto LG NP_15. In addition to the four co-segregating RAPD markers identified previously, four SSR markers  were identified in this study.We mapped QTLs onto the two maps by analyzing data reported previously ,15,21. Ton tests ,23. A neus study . Cbw1 aneviously ,24. Therus study , was notus study . The sinHigh-density linkage maps are useful for locating genes of interest for marker-assisted selection and to identify QTLs. In this study, we revised previously constructed linkage maps to add new markers and increase their resolution. In total, 412 single-locus SSR markers are mapped, 234 of which are new markers. We have mapped a large number of SSR markers in the linkage map of carnation, which is not a model crop and had few available SSRs. Recently, NGS has been used to develop several types of genetic markers such as restriction site associated DNA (RAD) and single nucleotide polymorphism (SNP) markers, including SSR markers -29. One n\u2009=\u200911) on the 85P map, but the corresponding LG on the NP map, NP_6, had no distorted loci  than in the 85P map . Most of the distorted loci were located on LG 85P_ 6 , was nous study  to compous study ,41) usinus study . MoreoveWe identified a total of 635 loci including 488 SSR in carnation using the two linkage maps. We believe that these linkage maps will serve as reference genetic linkage maps for carnation and that many of the mapped SSR markers will be useful to compare these maps with others constructed using data from different mapping populations. Currently, we are producing another mapping population and will construct a new linkage map. Moreover, whole genome sequencing by the NGS system is underway.Dianthus, including carnation, which will be useful for mapping QTLs associated with various traits, and for improving elite carnation cultivars in breeding programs.We improved two carnation linkage maps and mapped a large number of SSR loci. The genetic linkage map derived from a cross between the CBW-resistant line 85\u201311 and susceptible \u2018Pretty Favvare\u2019 comprising 412 SSR loci covering 969.6 cM, with an average distance of 2.4 cM between loci. The genetic linkage map derived from a cross between CBW-resistant \u2018Carnation Nou No.1\u2019 and susceptible \u2018Pretty Favvare\u2019 comprised 201 SSR, 145 RAPD, and two STS loci covering 978.3 cM, with an average 2.8 cM distance between loci. These two refined maps were anchored with 125 SSRs loci. We identified 635 loci in carnation using the two linkage maps. The number of LGs was coincident with the haploid chromosome number of carnation (x\u2009=\u200915). Our genetic linkage maps, combined with the SSR markers developed in this study, represent reference genetic linkage maps for members of the genus 2 progenies of the line 85\u201311 and \u2018Pretty Favvare\u2019 for SSR-based map construction. To develop useful SSR markers for carnation cultivars derived from closely related crosses, we improved this mapping population to construct a higher density linkage map. The standard-type carnation line 85\u201311 is highly resistant to CBW and the spray-type carnation cultivar \u2018Pretty Favvare\u2019 is susceptible to CBW. Line 85\u201311 was selected from the third-generation lines in our breeding program for improving flower vase life, and showed low ethylene production in whole flowers during senescence [2 progenies used in our previous study [We used 91 Fnescence . Of the us study , one proD. caryophyllus \u2018Super Gold\u2019 and the highly resistant wild species, D. capitatus ssp. andrzejowskianus[D. caryophyllus, with \u2018Carnation Nou No.1\u2019. Therefore, the segregating progenies represent a backcross (BC1) population. The marker loci derived from D. capitatus ssp. andrzejowskianus were genotyped for map construction in this study.To compare the LGs with those in our RAPD-based map , 94 lineowskianus. The segTo improve the 85\u201311 map, we screened for polymorphisms between parents using 1,013 \u201cGS\u201d and 897 \u201cNES\u201d primer pairs. We also rescreened for polymorphisms using 1,530 \u201cCES\u201d primer pairs using fluorescent fragment analysis instead of electrophoresis on polyacrylamide gels. Polymorphic markers between the parents were used for mapping in the progeny. To improve the NP map, we screened for polymorphisms between parents using the SSR primer pairs used in Yagi et al. , includiThe PCR conditions and the subsequent analyses were as described by Yagi et al. . The reaRAPD analysis was performed according to the methods of Yagi et al. . We testP\u2009<\u20090.05 and P\u2009<\u20090.01 were plotted as asterisks on the graphs. Construction of the NP map revealed that LG A1 in Yagi et al. [We constructed the genetic linkage map using JoinMap ver. 4.1 . Marker data were assigned to the LGs using a minimum LOD score of 7.0. The linkage groups were displayed using version 2.2 of MapChart . We usedi et al.  was diviAll SSR loci located on LGs with a small number of loci on the 85P map  were transferred onto the NP map. More than five SSR loci in each LG on the 85P map were tested on the NP map.Genotype data and phenotype data from previous studies ,15,21 weThe authors declare that they have no competing interests.MY participated in the design of the experiments, developed and genotyped the SSR, RAPD, and STS markers, constructed the maps, interpreted the data, and wrote the paper. TY participated designing the experiments and revised the manuscript. SI, ST, and HH developed and screened SSR markers from Sanger-sequenced ESTs and developed SSR markers from RNA-seq. KT collected RNA-seq data and grew the plants with MY and TO. TO participated in designing the experiments, grew the plants, and revised the manuscript. HY grew the plants and participated in the discussion of the results and helped to revise the manuscript. All the authors read and approved the manuscript.List of new SSR markers mapped onto 85P map.Click here for fileList of new SSR markers only mapped onto NP map.Click here for file"}
+{"text": "Geographical isolation has generated a distinct difference between Atlantic salmon of European and North American Atlantic origin. The European Atlantic salmon generally has 29 pairs of chromosomes and 74 chromosome arms whereas it has been reported that the North American Atlantic salmon has 27 chromosome pairs and an NF of 72. In order to predict the major chromosomal rearrangements causing these differences, we constructed a dense linkage map for Atlantic salmon of North American origin and compared it with the well-developed map for European Atlantic salmon.in situ hybridization (FISH) analysis. The proposed rearrangements, which define the differences in the karyotypes of the North American Atlantic salmon relative to the European Atlantic salmon, include the translocation of the p arm of ssa01 to ssa23 and polymorphic fusions: ssa26 with ssa28, and ssa08 with ssa29.The presented male and female genetic maps for the North American subspecies of Atlantic salmon, contains 3,662 SNPs located on 27 linkage groups. The total lengths of the female and male linkage maps were 2,153\u2009cM and 968\u2009cM respectively, with males characteristically showing recombination only at the telomeres. We compared these maps with recently published SNP maps from European Atlantic salmon, and predicted three chromosomal reorganization events that we then tested using fluorescence This study identified major chromosomal differences between European and North American Atlantic salmon. However, while gross structural differences were significant, the order of genetic markers at the fine-resolution scale was remarkably conserved. This is a good indication that information from the International Cooperation to Sequence the Atlantic salmon Genome, which is sequencing a European Atlantic salmon, can be transferred to Atlantic salmon from North America. The com\u2013100 MYA . A conse\u2013100 MYA  compared\u2013100 MYA  and a re\u2013100 MYA . Chromos\u2013100 MYA .Salmo salar) represents an important model for studying the mechanisms under-lying vertebrate chromosome re-arrangements and re-diploidization because of the recent genome duplication and varying modal chromosomal numbers in populations. The European Atlantic salmon metapopulation extends from Northern Europe to Spain, while the North American Atlantic salmon metapopulation is found in the Northeastern USA and Canada [The Atlantic salmon . There are at least three possible reasons for this. First, the majority of the markers included on the SNP-array were developed based on sequence data generated predominantly from European Atlantic salmon, which could introduce ascertainment bias when the SNP-array is used to genotype salmon of North American origin. Second, the majority of the mapping families were from an aquaculture stock that was imported to New South Wales from the River Phillip in Nova Scotia in the 1960s and subsequently introduced to Tasmania in the 1980s . Since tThe linkage maps constructed for European and North American Atlantic salmon have 3,055 SNPs in common, which allowed us to perform a comprehensive comparison of chromosomal structures between Atlantic salmon from either side of the Atlantic Ocean. This comparison suggests the creation of three composite chromosomes, including a novel metacentric chromosome, in North American Atlantic salmon differing from the European karyotype Figures\u2009, 3 and 4The first proposed rearrangement involves the European chromosomes ssa01 and ssa23. We suggest that a chromosomal translocation event joined the p-arm of ssa01 to ssa23  and ssa29 (acrocentric). Although linkage results are conclusive regarding fusion of these two chromosomes in North American Atlantic salmon, it is not easy to deduce the architecture of this chromosome. We hypothesise that ssa29 combined through a tandem fusion with the q-arm of ssa08, preserving the centromere position of ssa8 Figure\u2009. This isThe proposed chromosomal rearrangements in the North American Atlantic salmon relative to the European Atlantic salmon described above provide an explanation for the reported differences in karyotype between the two sub-species. However, our results from FISH-analysis also indicated a heterogeneous state of the two fusion chromosomes, suggesting that there are polymorphic variations in the North American karyotype that need to be studied in more detail.et al. [et al.[Lubieniecki et al.  represen. [et al. came fro. [et al.. TherefoThe Atlantic salmon genome seems to be remarkable in its tolerance to chromosomal rearrangements and plasticity. The ability of two individuals with different numbers of chromosomes to mate successfully may relate to the presence of pseudolinkage and residual tetrasomy in male salmonids ,31,32 thOur comparison of the dense SNP linkage maps for Atlantic salmon derived from either side of the Atlantic Ocean identified major chromosomal differences between European and North American Atlantic salmon, which were confirmed by FISH analysis. Despite this, marker order is well conserved and a large proportion of the SNPs generated from European Atlantic salmon work are informative in the North American population. Thus, while gross structural differences are significant, the DNA sequence at the fine-resolution scale is remarkably conserved. This is a good indication that information from the International Cooperation to Sequence the Atlantic Salmon Genome , which iThe mapping panel consisted of two distinct sample groups. The first group comprised 160 parents and 1669 offspring from a Tasmanian aquaculture program, which was established from salmon originating from the River Philip, Nova Scotia in the 1960s. The second group comprised 38 parents and 107 offspring from an aquaculture program in Atlantic Canada, which were founded using salmon from the Saint John River, New Brunswick (Cooke Aquaculture Ltd). Within the 97 families from both groups, offspring represented a mixture of half-sibs and full-sibs. Where information from one parent was missing, the family was entered as half-sib family. For all samples, high molecular weight genomic DNA was isolated from fin-clips using commercially available kits. DNA was quantified, normalized and quality checked before genotyping.All 1974 samples were genotyped using a custom Atlantic salmon iSelect SNP-array manufactured by Illumina . This particular array is a newer version of the salmon array described by Bourett et al. , and wasBecause this is a non-standard array, a pre-existing cluster file was not available; therefore, automatic clustering was used to produce general SNP and sample statistics. However, the partially tetraploid nature of the Atlantic salmon genome means that some markers did not show standard diploid distribution and required manual cluster adjustment. For this purpose additional samples (480) from diverse geographic locations were included. This helped to ensure that all alleles were represented for each SNP and improved our confidence when re-clustering. Once the best possible clustering had been done, genotypes were exported to a database. Individuals and families were then examined for pedigree errors, and this resulted in a final set of 1,974 salmon being used for the linkage map construction.Modified versions of the CRIMAP 2.4 software  were utiEuropean Atlantic salmon  weighing 2\u20135\u2009kg were anaesthetized using MS222. The fish skin was cleaned with 70% EtOH and Kimwipes. Up to 2\u2009mL of blood was aseptically drawn from the caudal vein of the fish using a sterile syringe inserted near the anal fin. The blood was collected into a Vacutainer tube containing heparin, gently mixed and transported at 4\u00b0C to the Davidson lab at Simon Fraser University.A similar procedure was used to obtain blood from Atlantic salmon from the Aqua Bounty Canada transgenic broodstock in Prince Edward Island. The blood was collected early in the morning in Prince Edward Island and shipped at 4\u00b0C to the Davidson lab at Simon Fraser, arriving within 24\u2009h.The original transgenic Atlantic salmon was produced at Memorial University, St. John\u2019s, Newfoundland , and theThe heparinized blood was thoroughly mixed with 5\u2009mL of media L-15 (Gibco) in a 15\u2009mL sterile plastic tube and placed on ice for 5\u2009minutes. The diluted blood was then centrifuged at 1,200\u2009rpm for 5\u2009minutes at room temperature. After centrifugation, the buffy coat (containing lymphocytes) above the red blood cells was floated in plasma by a gentle stirring with a 1\u2009mL pipette. The lymphocyte-enriched plasma was then collected in a new 15\u2009mL sterile plastic tube. The plasma was centrifuged at 1,500\u2009rpm for 5\u2009minutes, and the resulting cell pellet was suspended in 5\u2009mL of complete media L-15 containing 10% fetal bovine serum (FBS), 60\u2009\u03bcg/mL of kanamycin sulfate, 1 x antibiotic-antimycotic solution , 25\u2009\u03bcM of 2-mercaptoethanol and 18\u2009\u03bcg/mL of phytohemagglutinin (PHA-W) and 100\u2009\u03bcg/mL of lipopolysaccharide (LPS). The cells were cultured at 18\u00b0C in a culture tube slanting to an angle of about 30\u00b0 with gentle daily mixing for 6\u2009days. About 90\u2009minutes before cell harvest, the lymphocyte culture was supplemented with 500\u2009ng/mL colcemid. The cells were collected by centrifugation at 1,500\u2009rpm for 5\u2009minutes, and the supernatant was discarded. The cell pellet was suspended in 2\u2009mL of 0.075\u2009M KCl hypotonic solution for 20\u2009minutes at 20\u00b0C. The hypotonic solution was slowly added to a volume of 2\u2009mL. Then, 2\u2009mL of fresh Carnoy\u2019s fixative (3 methanol: 1 acetic acid) was added slowly. After centrifugation at 1,500\u2009rpm for 5\u2009minutes, the supernatant was discarded. The fixed cells were gently suspended in 3\u2009mL of Carnoy\u2019s fixative. The fixation step was repeated two more times, and then the cells were suspended 1\u20132\u2009mL of Carnoy\u2019s fixative. A microscope slide was exposed to hot water vapor at 73.5\u00b0C for 30\u2009seconds.The cell suspension was immediately dropped on to the slide. After the slide surface became \u201cgrainy\u201d, the slide was immediately exposed again to the hot water vapor at 73.5\u00b0C for 30\u2009seconds. The slide was then quickly dried on a hot surface, which provided good chromosome spreading.FISH analyses were carried out using a modification of the procedure described in Li et al. . BAC DNAHybridization of the fluorescent-labeled DNA probe with chromosome spreads was performed as suggested by the manufacture (Vysis) with minor modifications. Five \u03bcL of the nick translation reaction mixture of the BAC containing one microsatellite marker and 5 \u03bcL of the nick translation reaction mixture of the BAC containing another microsatellite marker, which were used to predict the chromosomal locations of the BACs  were mixThe authors declare that they have no competing interests.SB: Participated in the linkage analysis, interpreted data and drafted the manuscript. JL: Carried out the FISH-analysis. MPK : Was responsible for genotyping, SNP filtering and assisted in finalizing the manuscript. EGB: Provided family material and assisted in finalizing the manuscript. SD: Provided family material and assisted in finalizing the manuscript. WSD: Was responsible for the FISH-analysis, helped draft and finalize the manuscript. SL: Conceived the study, provided financial support, was responsible for the linkage analysis and helped draft and finalize the manuscript. All authors read and approved the final manuscript.Where possible and appropriate information regarding animals and research methods has been provided according to the ARRIVE guidelines.This work has received funding from the Norwegian Research Council (NFR) , the CSIRO Food Futures Flagship Program, and the Natural Sciences and Engineering Research Council of Canada (NSERC) Strategic Grants Program. Linkage map for North American Atlantic salmon. The columns in the table are ID of the marker, North American Atlantic salmon chromosomes number, order of markers within each linkage group, female linkage map in cM, male linkage map in cM, number of meioses, dbSNP accession number and North American specific SNPs (marked with x). (TXT 203 kb)click here for file"}
+{"text": "Studying population isolates with large, complex pedigrees has many advantages for discovering genetic susceptibility loci; however, statistical analyses can be computationally challenging. Allelic association tests need to be corrected for relatedness among study participants, and linkage analyses require subdividing and simplifying the pedigree structures. We have extended GenomeSIMLA to simulate SNP data in complex pedigree structures based on an Amish pedigree to generate the same structure and distribution of sampled individuals. We evaluated type 1 error rates when no disease SNP was simulated and power when disease SNPs with recessive, additive, and dominant modes of inheritance and odds ratios of 1.1, 1.5, 2.0, and 5.0 were simulated. We generated subpedigrees with a maximum bit-size of 24 using PedCut and performed two-point and multipoint linkage using Merlin. We also ran MQLS on the subpedigrees and unified pedigree. We saw no inflation of type 1 error when running MQLS on either the whole pedigrees or the sub-pedigrees, and we saw low type 1 error for two-point and multipoint linkage. Power was reduced when running MQLS on the subpedigrees versus the whole pedigree, and power was low for two-point and multipoint linkage analyses of the subpedigrees. These data suggest that MQLS has appropriate type 1 error rates in our Amish pedigree structure, and while type 1 error does not seem to be affected when dividing the pedigree prior to linkage analysis, power to detect linkage is diminished when the pedigree is divided. Complex pedigrees from isolated populations have gained popularity for genetic studies due to their pedigree size, genetic homogeneity, and environmental homogeneity Pedigree size and complexity also present problems when running linkage analyses because even the best available linkage programs, such as Allegro GenomeSIMLA We extended the software package GenomeSIMLA to generate complex pedigree structures based on a template pedigree. Once a population of chromosomes has been created, a collection of founders is drawn and are mated based on the pedigree structure to produce all generations of the pedigree. Affection status is assigned by applying a penetrance function with the option of only assigning known phenotype and genotype data to the same individuals with known phenotype and genotype data in the template pedigree, maintaining a more realistic distribution of genotyped affected and unaffected individuals in the pedigree. We simulated a null disease model into 1000 pedigree replicates, each with 124 autosomal SNPs with a spacing of 0.062 centimorgans and no linkage disequilibrium between them, using our recently published 4998-member Amish pedigree with almost identical affection status  For studies of power, we modified the null simulation, forcing one of the 124 SNPs to have either a dominant, recessive, or additive effect with odds ratios of 1.1, 1.5, 2.0, or 5.0 for this locus, resulting in 12 total disease models. The minor allele frequency for the \u2018disease\u2019 SNP was held constant at 0.2, consistent with the GWAS hypothesis of at least one common variant increasing risk of a common disease. One thousand replicates were simulated for each disease model.We ran MQLS (software version 1.2) to test for association and used option \u20181\u2019 to include all individuals, cases, controls, and individuals with unknown phenotype in the analyses. More recent versions (starting at version 1.5) of MQLS include a more robust variance estimator To generate sub-pedigrees within a bit-size limit of 24, we ran PedCut We also ran MQLS on the sub-pedigrees to compare those results to running MQLS on the unmanipulated large simulated pedigrees. Prior to running MQLS, we re-calculated kinship coefficients using the sub-pedigree structures rather than the entire pedigree structure to model some of the effect of losing the entire pedigree structure that might occur when using association to follow-up linkage analysis in these sub-pedigrees. We determined type 1 error rates and power as before.All computations were performed using either the Center for Human Genetics Research (CHGR) computational cluster or the Advanced Computing Center for Research and Education (ACCRE) cluster at Vanderbilt University. Scripts and pedigree structures are available upon request.In 1000 runs of MQLS, each with the entire 4998-member pedigree and 124 null SNPs, we see average type 1 error rates of 5.06%, 1.02%, 0.56%, and 0.13% associated with p-values less than 0.05, 0.01, 0.005, and 0.001, respectively. Therefore, we do not see an inflated type 1 error rate when running MQLS in our pedigree structure.Evaluating power, we find, as expected, that we have the least power to detect association when the underlying disease model is recessive and the most power to detect association when the underlying disease model is additive. For dominant and additive models we have >90% power to detect association at p\u22640.05 when the simulated odds ratio is at least 2.0, but power drops significantly at an odds ratio of 1.5. With a very strong effect of OR\u200a=\u200a5, we have very high power to detect association even as low as a p-value of 5.0E-8 (such as would be needed for Bonferroni-corrected GWAS). Under the recessive models power was only >80% using a p-value threshold of 0.05 for an odds ratio of 5.0 .Using the same sets of pedigrees, but dividing them into subpedigrees using PedCut, the type 1 error rates when running MQLS hardly changed from the MQLS analysis using whole pedigrees. The type 1 error rates were 5.16%, 1.06%, 0.51%, and 0.11% for the same p-value thresholds.On the other hand, evaluating power when subdividing the pedigree before running MQLS we do see a loss of power. Power is only >80% for dominant and additive models at an odds ratio of 5.0 .Averaging across 1000 replicates of two-point parametric linkage analysis using sub-pedigrees with a bit-size \u226424, we see low type 1 error rates, which were nearly the same when running dominant and recessive models. The type 1 error rate using a critical value of HLOD of 3 under the dominant model was only 0.01% and under the recessive model was only 0.02%. Nonparametric analyses had no type 1 error at LOD threshold of 2 and 3 .According to our simulations, we had >80% power to detect a two-point HLOD \u22651.0 with a simulated additive model with OR\u200a=\u200a5.0 when a dominant model is assumed during linkage analysis. All other circumstances had <80% power; however, with the simulated dominant model with OR\u200a=\u200a5, Merlin was able to detect the disease SNP almost 80% of the time at or above an HLOD of 1 when a dominant model was assumed. Even when a recessive model was assumed two-point linkage analysis was not powerful for any of the simulated recessive scenarios. Parametric analyses were more powerful than nonparametric analyses .When running multipoint analysis on the same sets of sub-pedigrees we see both higher type 1 error and higher power for most circumstances except for a simulated dominant model with OR\u200a=\u200a5. For multipoint analyses we see higher type 1 error and power for nonparametric analyses than for parametric analyses  and 6. FPedigrees from population isolates provide rich datasets for genetic analyses; however, the size and complexity of the pedigrees contribute to ambiguity when running analyses and interpreting results. We have used this approach to discover novel susceptibility loci for complex diseases, such as Alzheimer disease and Parkinson\u2019s disease, by studying the Amish communities of Ohio and Indiana Simulations of pedigrees as large and as complex as an Amish pedigree and other population isolates to assess the type 1 error rate and power of MQLS have not been previously published, so we sought to fill this void. We did not see an inflated type 1 error rate in our simulated pedigrees. Therefore, MQLS is an appropriate method for analyzing pedigrees as large and as complex as the Amish. MQLS has sufficient power to detect a strong effect of OR\u200a=\u200a5 when the mode of inheritance is recessive, dominant, and additive and a more moderate effect of OR\u200a=\u200a2 when the mode of inheritance model is dominant or additive. While these are large effect sizes compared to those typical of complex diseases, in a homogeneous founder population a larger effect size is more likely.Linkage analyses for a pedigree of this size and complexity require pedigree splitting, but the effect of using PedCut to subdivide the pedigrees on the type 1 error and power of linkage analysis is not known. Using a bit-size limit of 24 for sub-pedigree size , we saw a low type 1 error rate associated with an HLOD of 3.0 for both two-point and multipoint linkage (lower for two-point). An HLOD of \u223c3 has traditionally been a \u2018significant\u2019 HLOD score, and the low type 1 error rate in this instance all allows us to confidently use this threshold when evaluating linkage results from the Amish sub-pedigrees. These approaches, however, were not powerful when we analyzed simulated 1-locus disease models in pedigrees with this number of variously related individuals.Unfortunately, we cannot analyze the entire 4,998 member pedigree for linkage to compare the type 1 error and power to analyses of sub-pedigrees for linkage. We can, however, compare the type 1 error of association analysis using MQLS on the entire pedigree versus using MQLS on the sub-pedigrees. Splitting the simulated pedigrees did not affect the type 1 error when running MQLS. This result does not guarantee that splitting a pedigree will not lead to any spurious positive results, since other studies suggest otherwise (14). We do see a loss of power due to splitting the pedigrees because many pedigree connections are disrupted. In a previous simulation study McArdle et al saw that type 1 error increased but power was not affected when ignoring family structure while performing association analysis. Their conclusion was based on testing relatively simple pedigrees compared to singletons, which was a common approach at that time Through these simulations we see that MQLS has acceptable type 1 error rates even when using an extremely complex pedigree structure. Type 1 error rates are also acceptable when splitting pedigrees prior to linkage analysis, consistent with a related study (13). Unfortunately, but not surprisingly, significant power is lost when pedigrees are divided. Development of new methods or extensions of current methods to use more pedigree information to perform multipoint linkage analyses or implementation of alternative methods such as identifying identical by descent (IBD) shared segments"}
+{"text": "We report two approaches for linkage analysis of data consisting of replicate phenotypes. The first approach is specifically designed for the unusual (in human data) replicate structure of the Genetic Analysis Workshop 17 pedigree data. The second approach consists of a standard linkage analysis that, although not specifically tailored to data consisting of replicate genotypes, was envisioned as providing a sounding board against which our novel approach could be assessed. Both approaches are applied to the analysis of three quantitative phenotypes  in two sets of African families. All analyses were carried out blind to the generating model . Using both methods, we found numerous significant linkage signals for Q1, although population colocalization was absent for most of these signals. The linkage analysis of Q2 and Q4 failed to reveal any strong linkage signals. The data consist of information on 3,205 genes containing 24,487 single-nucleotide polymorphisms (SNPs) on 697 unrelated individuals drawn from 7 population samples. The family data consist of 8 extended pedigrees , also with a total of 697 individuals. The pedigrees were seeded with 202 founders, who were described as having been drawn at random from the 697 unrelated individuals. Two hundred replicates were provided for both the unrelated individuals data set and the family data set. A noteworthy feature of these data is that the SNP genotypes from replicate to replicate are identical. In other words, for any given individual over all replicates, each person is represented as though he or she is one of 200 monozygotic twins.The simulated mini-exome data for Genetic Analysis Workshop 17 were derived from the pilot3 study of the 1000 Genomes Project  of the 24,487 SNPs have a frequency of 1/(2N); that is, only one copy of the variant exists in the entire set of unrelated individuals, and fully 87.2% have a minor allele frequency less than 5.0%. Under usual circumstances, this collection of SNPs would have such a low polymorphic information content as to render ordinary linkage analysis powerless to identify genes involved in the quantitative phenotypes of interest. Fortunately for the family data set, the data simulators provided fully informative identity-by-descent (IBD) matrices for each pair of individuals at each gene. These IBD scores were given as 0, 0.5, and 1, denoting the sharing of 0, 1, or both genes identical by descent. Both methodological approaches reported here used these IBD matrices exclusively for the linkage analyses.Because our analyses were blind to the generating model, we began our investigation by obtaining some descriptive statistics. We started with the genotypes of the We note that in the process of simulation, recombination could occur only once per chromosome per meiosis and that this recombination could not occur within a gene. This allows us to refer to IBD at a particular gene. In real data, we would not have perfect information, although with extensive genotyping and sequencing we would be able to approach this. Recombination within a gene is also possible, although it is less likely than in the larger intergenic regions. Furthermore, a multipoint analysis is unnecessary because the single point information is completely informative.Before performing the linkage analyses, we began by making sure that the data had the properties we expected. We believe that this is an important but often neglected step for both real and simulated data. While attempting to identify the population origin of the 202 founders of the 8 pedigrees, we discovered to our surprise that 4,239 SNPs had been altered, although these alterations did not prevent the unambiguous identity and population origin of the founders. We were even more surprised to discover that the founders were not a random sample from the unrelated individuals data set. Instead, all but three of the founders were drawn from the two African samples. Moreover, three of the eight pedigrees were established by approximately equal numbers of founders from the Yoruba and the Luhya population samples. The remaining five pedigrees were primarily founded by one ethnic group or the other. Under ordinary circumstances when using genetic markers for a linkage analysis, it is more cumbersome to analyze families composed of individuals drawn from various ethnic groups because differences in allele frequencies need to be taken into account  pedigree 2 , pedigree 3, and pedigree 4 ; and (B) pedigrees 5 and 6. Individuals in kindred group A are all sampled from the Luhya population, a Bantu ethnic group living primarily in Kenya. It is unclear whether the Luhya sample from the 1000 Genomes Project was randomly sampled from all of the 16 tribes that make up the Luhya ethnic group or from a smaller subset. Individuals in kindred group B are all Yoruba, a large heterogeneous group of about 30 million people from West Africa, predominantly Nigeria.The unusual structure of the data replicates (varying covariates but a constant genetic structure) led us to generate a novel quantitative phenotype from the data as our target of analysis. For a typical variance components linkage analysis, we control for covariates by using the residual after linear regression as a quantitative phenotype. In the linear regression, we model the trait as:\u03b1 is the global mean value of the trait, \u03b2 is a vector representing the linear effect sizes of the covariates (x), \u03b3 is a vector representing the linear effect sizes of true (but unknown) causative genetic variants (g), and \u03b5 represents the sum of all other factors. Typically, we analyze:where \u03b1 and \u03b2, are computed from the data and G represents the sum of the genetic contributions to the phenotype.as the quantitative phenotype, where i given by:For our analysis, we treated the observations of an individual across multiple data replicates as repeated measures. To do this, we used the framework of a generalized linear mixed model (GLMM) as implemented in PROC GLIMMIX in SAS 9.2 [In this way, each unique genetic observation is counted only once in the analysis, but full information from the repeated measures is used.p-values were combined over multiple replicates using Fisher\u2019s method [For the standard linkage analysis (which does not take into account the repeated measures feature of these data), we used SOLAR , and thes method . SmokingWe carried out an Eigenstrat analysis  using alp < 10\u22124 from either method) are reported in Table p-value from the individual replicates, the range of p-values, and the p-value obtained by combining the data from the families across the 50 replicates . The novel repeated measures analysis (performed using the GLMM) is an analysis based on only the individuals from a single replicate (N = 400), but it uses the information from 50 repeated measures of each trait to provide a more accurate estimate of the total genetic liability for each individual.We applied our two approaches to the first 50 replicates for which SOLAR obtained a convergent solution. The significant results .It would be na\u00efve to expect any single method to pinpoint all the genetic factors that were simulated, especially for private polymorphisms . For instance, Hill et al.  recentlyPLAT and a modest false positive for Q4 on chromosome 20 at LOC643659. Similarly, the GLMM identified modest true positives for Q2 at SREBF1 on chromosome 17 and at PLAT and a modest false positive for Q4 on chromosome 5 at RNF145.The linkage results for Q2 and Q4 (not shown in the figures) did not produce any compelling evidence for the involvement of a well-demarked chromosomal region for these phenotypes. Fisher\u2019s method identified a modest true positive for Q2 on chromosome 8 at R2 is 0.787 with a range of 0.759\u20130.811.) We concluded that Q4 was primarily environmentally determined but that the generating model did include unlinked heritable factors.Other analyses by our group  indicatep-values of approximately 10\u22124, which normally would not be considered strong evidence of linkage. The other two signals (on chromosomes 8 and 17), however, would be considered persuasive evidence of linkage if the families were independent.Although we correctly located most of the simulated signals for Q1, we also found a number of false-positive signals (Table CDCA2 on chromosome 8) to determine whether there was a reasonable explanation for this strong false-positive finding obtained in the SOLAR analysis. Briefly, we found that this false-positive signal resulted from a chance deviation from random segregation between the A SNP at C8S775 in CDCA2  and the C SNP at C13S434 in FLT1 . Eighteen informative meioses involving these two SNPs were observed in the Yoruba kindred (pedigree 5) responsible for this false-positive signal , it is not persuasive evidence in a linkage setting. However, when 50 marker-identical families are pooled, this minor nonrandom segregation is telescoped into 750NR:150R, which is a highly significant deviation from the 450NR:450R expected under the hypothesis of no linkage. We believe this is the explanation of the false-positive signal at CDCA2.We selected the best localized of the two stronger signals and conducted a post hoc analysis of the false-positive signal (i.e., the signal centered over Finally, we note that the present situation does not reflect any situation observed in human genetics. However, these analyses are appropriate for plant and animal genetics. In particular, homozygous inbred strains and their F1 hybrids offer investigators the ability to observe the characteristics of hundreds of genetically identical organisms in a variety of contexts. Furthermore, the analyses of monozygotic twin data and longitudinal phenotype data are also amenable to analysis with generalized linear models.The authors declare that there are no competing interests.AH participated in the study design, carried out the analyses, and helped draft the manuscript. RC participated in the study design and helped draft the manuscript. BS participated in the study design and helped draft the manuscript. All authors read and approved the final manuscript."}
+{"text": "In the last years GWA studies have successfully identified common SNPs associated with complex diseases. However, most of the variants found this way account for only a small portion of the trait variance. This fact leads researchers to focus on rare-variant mapping with large scale sequencing, which can be facilitated by using linkage information. The question arises why linkage analysis often fails to identify genes when analyzing complex diseases. Using simulations we have investigated the power of parametric and nonparametric linkage statistics , to detect the effect of genes responsible for complex diseases using different pedigree structures.As expected, a small number of pedigrees with less than three affected individuals has low power to map disease genes with modest effect. Interestingly, the power decreases when unaffected individuals are included in the analysis, irrespective of the true mode of inheritance. Furthermore, we found that the best performing statistic depends not only on the type of pedigrees but also on the true mode of inheritance.When applied in a sensible way linkage is an appropriate and robust technique to map genes for complex disease. Unlike association analysis, linkage analysis is not hampered by allelic heterogeneity. So, why does linkage analysis often fail with complex diseases? Evidently, when using an insufficient number of small pedigrees, one might miss a true genetic linkage when actually a real effect exists. Furthermore, we show that the test statistic has an important effect on the power to detect linkage as well. Therefore, a linkage analysis might fail if an inadequate test statistic is employed. We provide recommendations regarding the most favorable test statistics, in terms of power, for a given mode of inheritance and type of pedigrees under study, in order to reduce the probability to miss a true linkage. Linkage analysis has been a very popular method for detecting genes of major effect. It has been used since the '80s with either sibling pairs or large multiplex pedigrees. In complex diseases, where the mode of inheritance is characterized by factors such as reduced penetrances, presence of phenocopies, and heterogeneity, it has been argued that a better approach to identify variants involved in such diseases is genome wide association analysis (GWAS). In the last years, GWAS have grown in scale and complexity, with studies looking at over a million genetic markers in samples with many thousand individuals. These studies have proved to be successful in identifying common single nucleotide polymorphisms (SNPs) and common risk alleles that contribute to complex diseases. Nevertheless, it is believed that many genetic and epigenetic factors are likely to contribute to common complex diseases, including multiple rare SNPs, i.e., those that occur in less than 5% of the world's population. In factGenome wide linkage studies are performed with parametric or nonparametric methods. Parametric analysis provides the most powerful method when the mode of inheritance (MOI) is known. The most used parametric statistic is the LOD score. HoweverSamples of different pedigree structures and sizes were considered, the same structures as by Mattheisen et al.. Five peHO using MERLIN software[HO, MERLIN generates each replicate under the assumption of marker segregation independent of the trait, using identical pedigree structure, affection status, marker spacing, allele frequencies and patterns of missing data as given in the input file.In order to investigate the influence of the pedigree structure on the distribution of parametric and non-parametric linkage scores, 100,000 replicates of 500 pedigrees were generated under software for each0\u2009=\u20090.04, f1\u2009=\u20090.20, f2\u2009=\u20090.20, p\u2009=\u20090.05), additive , and recessive mode , where fi stands for the penetrance of the genotype with i copies of the disease allele and p stands for the disease allele frequency. The genetic parameters were obtained using the R package POWERPKG[0). Phenocopies represent individuals who are affected not owing to genetic predisposition at the locus under study but due to unspecified environmental factors or genes at another location[H1 respecting the pattern and the allele frequencies for the phenotypes and genotypes of the input files, but introduces linkage between the phenotype and the marker depending on the specified MOI.Three scenarios reflecting a dominant, an additive and a recessive MOI were studied to evaluate the power of detecting linkage in complex genetic diseases. For each scenario, 5,000 replicates of 500 pedigrees were generated for each of the pedigree structures shown in Figure POWERPKG for affelocation. MERLIN To simplify the discussion, we restrict the analysis to a single genetic position. The simulated data were analyzed using parametric and nonparametric methods. The following statistics were used:0\u2009=\u20090.003, f1\u2009=\u20090.5, f2\u2009=\u20090.5), additive model , and recessive model  where fi stands for the penetrance of the genotype with i copies of the disease allele. Since a high disease allele frequency (p) can compensate for misspecified penetrance parameters when analyzing complex traits[p was fixed at 0.25 for all three models. In the following, the term MOI refers to the parametric model used in data simulation and AMOI refers to the parametric model used in the analysis of the simulated data. We have deliberately chosen different parameters in the MOI and in the AMOI, corresponding to the real life situation of studying complex diseases in which the correct parameter values are not known beforehand. Hence, there is some degree of model misspecification even when MOI and AMOI are the same . Misspecification of the penetrance does not generally have a strong effect on the two-point LOD score (only on the estimate of the recombination fraction), as long as dominance is specified correctly[The LOD is computed as the ratio between the likelihood of obtaining the test data if the two loci are indeed linked, versus the likelihood of observing the same data purely by chance. Here, MLINK was usedx traits, p was forrectly,17.The MOD is based on maximizing the maximum LOD with respect to the trait-model parameters . MOD scores were computed with GENEHUNTER-MODSCORE,18 with The NPL focuses on the sharing of disease status as well as the sharing of alleles by relatives. Its extension, KC-LOD, is maximized over a parameter \u03b4 representing the degree of allele sharing among affected individuals. Indeed, when the IBD information is complete, there is a one-to-one correspondence between the NPL score and the KC-LOD score, which means that the tests based on the two statistics are formally equivalent. In our H0 was analyzed using the parametric and nonparametric linkage statistics mentioned above. The scores corresponding to empirical P-values of 0.0017 \u201csuggestive evidence for linkage\u201d [\u201cclassical LOD-3 criterion\u201d [\u201csignificant evidence for linkage\u201d [To study the effect of pedigree structure on the distribution of the linkage statistics, each replicate generated under linkage\u201d , 0.0001 iterion\u201d  and 0.00linkage\u201d  were calH1 was analyzed using the parametric and nonparametric linkage statistics mentioned above. The values for each statistic were ordered from highest to lowest over the 5,000 replicates for a given model and for a given structure. Observed power levels P(Z) were determined as a function of the score Z used as a critical value for each test statistic T, as follows: P(Z)\u2009\u2261\u2009(number of replicates yielding T\u2009\u2265\u2009Z) /N, where N represents the number of replicates performed .To study the power of the different statistics to detect linkage, each replicate generated under 0 for the corresponding test statistic. The NPL score is constructed on the basis of a score statistic[0. Instead of using the critical values obtained in this study, we decided to rely on the ones provided by Mattheisen et al.[0 , and 630,000 analyses were performed under H1 .In order to compare the different linkage statistics regarding their power, critical values were obtained using the theoretical distribution under Htatistic,22. Theotatistic. The LODtatistic-25, for n et al. for the A graphical overview of the results is shown in FigureThe observed power of NPL, KC-LOD, MOD and LOD scores for each pedigree structure is shown in Additional fileThe NPL score is computed using affected pairs only. For that reason, very similar results are expected for ASP, DST and DSQ as well as for A3G and D3G. In fact, the minor differences arise because the disease locus genotypes are conditioned on the phenotypes of the ascertained individuals. A very poor power (8.4%-42.0%) is obtained with ASP, DST or DSQ. The power is increased (99.5%-99.9%) when adding an affected sib (AST), and it is even better (99.6%-100%) when A3G and D3G structures are used. The best power is obtained using ASQ (100%). At the level of significant evidence for linkage, a bad performance of the NPL is acquired when using pedigrees with less than three affected sibs, especially when the MOI is recessive.The KC-LOD is also computed using affecteds only. For that reason, very similar results are observed for ASP, DST and DSQ as well as for A3G and D3G. An effect of pedigree structure is also obtained with the same trend as for the NPL score, although at the level of significant evidence for linkage, a better power is obtained with the KC-LOD than for the NPL statistic , the MOD score provides a less powerful test with DST and DSQ than with ASP.When the true MOI is dominant and the AMOI is also dominant, then AST, ASQ, A3G and D3G structures are performing well. Surprisingly, the power slightly increases when the AMOI is misspecified as being additive. When the true MOI is additive then the additive AMOI performs best, as one would expect, with the dominant or recessive AMOI having only slightly decreased power. Similarly, for a recessive MOI, the best power is obtained if the AMOI is recessive as well. Overall, when the true MOI is recessive the power ranges are much lower than if a dominant or additive MOI underlies the trait. At the level of significant evidence for linkage a very similar power is obtained as the one obtained using the KC-LOD score.As expected, the results of our simulations show that the power to detect linkage is low, at least with samples of 500 pedigrees, when using ASP, DST or DSQ to map complex diseases. Considering a type I error of 0.000049 .We undertook this simulation analysis to answer two questions. First, what is the impact of the pedigree structure on the null distribution of NPL, KC-LOD, LOD and MOD score statistics? Second, what is the power of the NPL, KC-LOD, LOD and MOD score statistics when a complex disease is present? Results show that an effect of the pedigree structure under no linkage is seen only on the MOD score. The critical MOD score values for suggestive evidence for linkage are markedly increased for DST and DSQ compared to ASP , unaffected sibs carry only very limited information with regard to their unobserved trait-locus genotype. The aforementioned explanation particularly applies to a MOD score analysis, but the finding that including unaffected individuals leads to a reduced power to detect linkage in the case of reduced penetrance holds for all parametric and nonparametric linkage statistics investigated in this simulation study. We want to emphasize that simply not genotyping an unaffected sibbling will not correct this deficiency, as it is an issue with the type of pedigree from which siblings are ascertained rather than the analysis method or who in the family is genotyped.Clearly, it can be expected that larger pedigrees provide more linkage information than small ones. This is in fact what we principally have observed in our simulation study. Nevertheless, a decrease of power is observed in all statistics when adding one or two unaffected sibs to ASP, resulting in DST and DSQ, respectively. For the nonparametric statistics (NPL and KC-LOD), this can be explained by the fact that only the affected phenotype is used to calculate the statistic, and there are on average less mutant alleles segregating in DST or DSQ than in ASP because the disease-locus genotypes are conditioned on the trait phenotypes of the ascertained individuals. In the case of the parametric LOD score which makes use of both, the affected and unaffected phenotype, this finding is less evident. It could be explained by the fact that trait-model parameters need to be specified prior to the analysis and a misspecification of these parameters has a more aggravating effect with larger sibships. Still tThe question arises which test statistic should be used for a given type of sample, in order to obtain a power as high as possible. Additional file0 performed in this study). Apparently, in the case of a mixture of pedigrees with varying numbers of affected and unaffected children, it is worthwhile to jointly explore all dimensions of the parameter space corresponding to a MOD score analysis with each of the different pedigree types, such that the higher score outweighs the increased critical value that is required to declare linkage. Anyway, this result applies only to the specific mix sample considered in this analysis; if the mix sample changes the power values will also change. Another important difference compared to real data sets is that large pedigrees containing multiple affected members are usually rare for complex traits, especially those with late onset. This is due to relatively small recurrence risks for complex diseases. Small pedigrees, such as nuclear families, e.g. ASP, are relatively common and easier to collect. In these situations, an alternative would be to increment the number of pedigrees to be analyzed. To give at least a hint regarding the number of ASP needed to achieve the same power as with AST, we did simulations for the KC-LOD and obtained that one would need approximately 1500 ASP to reach the same power as with 500 AST.We have to notice that all our results are based on a highly informative marker. It is expected that with a reduced information content the power would decrease. Indeed, there are other differences between real data sets and our simulation settings. For example, in most cases data sets consist of pedigrees of different structure. To give an idea regarding the power in this situation, we conducted the analysis with a mixture of pedigrees   rather than multipoint analysis, because the former requires significantly less computation time, which is especially important in a large-scale simulation analysis. Furthermore, the conclusions for multipoint analysis do not seem to differ substantially from those for two-point analysis. In factAnother concern in this study could be that the decreasing cost of SNP genotyping has made it much harder to justify the use of microsatellite genotyping in linkage studies. SNPs are likely to be the genotyping method applied to samples currently being collected. Still, our results should also be valid in the presence of SNP data. In a study of linkage analysis with imprinting, ASP and ASQ were simulated. The study considered first 40 SNPs (MAF 0.15 and 0.32\u2009cM apart to each other) and then one microsatellite marker . Results showed no substantial difference in the power to detect linkage between the analysis with SNPs and the analysis with the microsatellite (manuscript in preparation). When using SNPs, one should only be cautious about the closely spaced SNP markers and to model LD, or exclude SNPs in LD before using linkage analysis.Generally it is difficult to realistically simulate the MOI of complex diseases. Here we have focused on three realistic models with reduced penetrance as well as phenocopies, allowing for other genetic and/or environmental factors. We believe that these set of model parameters, together with their moderate genotype-relative risks, reflect a realistic scenario.Linkage analysis is not hampered by allelic heterogeneity, and so it genuinely achieves the combination of the peak signals for many single variants obtained in a certain genetic region. We have demonstrated how important is the impact of pedigree structure on the power to detect linkage when diseases with a complex MOI are considered. The pedigree structure has a more severe effect than a model misspecification in the case of two-point parametric LOD score analysis. Based on these results, it is important to use moderate to large pedigree structures with at least three affected members, unless a very large number of ASPs is available for study, and to be cautious when modeling unaffected individuals with complex diseases. When using small pedigrees, one might miss the true genetic linkage when actually a real effect of linkage exists. We believe that missing a true location is an error at least as severe as the false conclusion of linkage. Furthermore, we have shown that the test statistic has an important effect on the power to detect linkage. Here, according to our simulation results we have provided guidelines to researchers regarding which test statistic should be used when studying a certain pedigree structure. By this means, it is ensured that the collected pedigree data are exploited in the best possible way. After all, such extensive gene-mapping studies should not fail to detect linkage due to a suboptimal test statistic used for the analysis.The authors declare that they have no competing interests.AF made substantial contributions to conception and design, carried out all the analyses and interpretation of data. KS made substantial contributions to conception and design, revised the manuscript critically for important intellectual content; and gave final approval of the version to be published. Both authors read and approved the final manuscript.Table S1. Power (%) to achieve a given Z value or higher, for each statistic and for each pedigree structure.Click here for fileTable S2. Statistics to be used given the sample structure.Click here for file"}
+{"text": "Based on Life Cycle Assessment (LCA) and Eco-indicator 99 method, a LCA model was applied to conduct environmental impact and end-of-life treatment policy analysis for secondary batteries. This model evaluated the cycle, recycle and waste treatment stages of secondary batteries. Nickel-Metal Hydride (Ni-MH) batteries and Lithium ion (Li-ion) batteries were chosen as the typical secondary batteries in this study. Through this research, the following results were found: (1) A basic number of cycles should be defined. A minimum cycle number of 200 would result in an obvious decline of environmental loads for both battery types. Batteries with high energy density and long life expectancy have small environmental loads. Products and technology that help increase energy density and life expectancy should be encouraged. (2) Secondary batteries should be sorted out from municipal garbage. Meanwhile, different types of discarded batteries should be treated separately under policies and regulations. (3) The incineration rate has obvious impact on the Eco-indicator points of Nickel-Metal Hydride (Ni-MH) batteries. The influence of recycle rate on Lithium ion (Li-ion) batteries is more obvious. These findings indicate that recycling is the most promising direction for reducing secondary batteries\u2019 environmental loads. The model proposed here can be used to evaluate environmental loads of other secondary batteries and it can be useful for proposing policies and countermeasures to reduce the environmental impact of secondary batteries. Secondary batteries, also known as rechargeable batteries, are a group of batteries that can be used after discharge by charging the active substances. As they can be used repeatedly, the secondary batteries\u2019 lives are longer than those of primary batteries. As a result, the use of secondary batteries has the potential advantages of conserving resources and reducing waste. Moreover, given their high capacity and high energy density, secondary batteries are widely used throughout the World ,2.However, these environmentally friendly features do not imply that secondary batteries have no impact on the ecological environment. Different types of secondary batteries have different potential effects on resources, ecosystems and human health during their production, use, disposal, or recycle stages. For example, the production and disposal phases of heavy metals, which secondary batteries contain, can be hazardous to the environment ,4,5 due To date, the environmental effects of secondary batteries, their resource pressures, their potential risks and hazards have not attracted enough or sufficient attention. More attention should be paid to the environmental characteristics of secondary batteries as the ecological impacts of electronic products come into focus . EnvironVarious tools have been developed to evaluate the environmental impacts of different systems ,10,11,12et al. performed a LCA study of two lithium-ion batteries to optimize the design of lithium-ion batteries for plug-in hybrid electric vehicles [et al. assessed the energy and environmental impacts of sodium/nickel chloride batteries [To reveal the environmental impact of secondary batteries, Matheys compared the environmental indicators of five electric vehicle batteries using the LCA method . Zackrisvehicles . The LCAvehicles . Longo eatteries . In resoatteries . Slack [atteries  focused atteries  studied et al. performed a critical analysis of natural resource savings in lithium ion battery recycling [The existing research has built a good foundation for the further study of the secondary batteries\u2019 environmental impact. The extant research considers limited aspects of the relation between environmental impact and the end-of-life treatment. Dewulfa ecycling . HoweverThe increasing public concern about the environment has resulted in stricter regulations worldwide on spent portable batteries related to the adequate destination of hazardous residues . The envThe analysis was carried out following the ISO 14040  and ISO The goal and scope and inventory of a LCA study depend on the intended use of the study. The LCA model was built on a Li-ion battery and a Ni-MH battery based on a theoretical design. Material needs were based on laboratory tests and literature references. The main goal of this LCA study was the assessment of the environmental impact of different secondary batteries made from laboratories, as well as the comparison and analysis of the impact factors from different batteries. Furthermore, it highlights the environmental points within end-of-life treatment analysis.Functional Unit. The assessment objects of this study were secondary batteries. Two types of experimental batteries were selected: (1) A type of Li-ion battery  and (2) a type of Ni-MH battery . During the batteries\u2019 production processes, the types and quantities of their raw materials were analysed and recorded. The functional unit was defined as a battery whose specific capacity is 1,000 mAh/g under sustained charge cycles. All figures for environmental impact were related to one battery under the functional unit conditions.System boundaries. The system boundaries were based on the general rules of ISO 14040 and 14044. This study focused on secondary batteries. The product system includes production, use and recycling. Metals and other materials will be recycled as raw materials. The battery charger and other hardware were outside the system boundary. Only the battery itself and its casing were considered. Life cycle inventory. The life cycle inventory of the components and materials included the LiNil/3Col/3Mnl/3O2 + 1% Fe3O4 ternary cathode material Li-ion battery and the LaMg12+200% Ni alloy anode material Ni-MH battery. The first discharge-specific capacities of the LiNil/3Col/3Mnl/3O2 + 1% Fe3O4 ternary cathode material Li-ion battery and the LaMg12 + 200% Ni alloy anode material Ni-MH battery, both produced in the laboratory, were 170.9 mAh/g and 932.8 mAh/g, respectively. The types and mass of the main raw materials and energy consumption are shown in Environmental impact assessment. In line with the recommendations in Eco-indicator 99 system [9 system , three mThe Eco-indicator methodology that is used to calculate the standard values conforms well to the ISO 14042 standard on Life Cycle Impact Assessment (LCIA), although some details will perhaps deviate. The LCA method was selected to comprehensively evaluate the environmental impact of secondary batteries. In the Life Cycle Impact Assessment process of LCA, the Eco-indicator 99 system was chosen. The Eco-indicator 99 LCIA method is based on the principles of environmental damage. The term \u201cdamage\u201d includes 11 aspects, which can be grouped according to three main types of damage . Standar6/PC-DMC were ignored after considering the lack of data in the Ecoinvent database for the relevant components. Acetylene black was mainly composed of carbon, and its environmental impact is mainly caused by carbon. The environmental impact of carbonyl nickel was represented by nickel sediment.Material synthesis and the assembly of batteries were implemented in the Beijing Institute of Technology under quality assurance conditions. The raw material masses and the charge-discharge data were measured in the lab. Energy consumption and environmental emission were collected from databases and data from the scientific literature . The resFor the Eco-indicator comparison of the two selected batteries, it was necessary to determine a unified functional unit and standardized inventory data. Quality standardisation of the raw materials was then performed in accordance with the standard. In this paper, the functional unit is a battery whose specific capacity is 1,000 mAh/g. The cycles are the times a consumer charges and discharges a battery in the use phase. The requirements of the use step impact the charge-discharge cycles. Each battery requires a material independently from the use step. The needs are related to the choice of the functional unit for two different batteries. Depending on the relationship between the charge-discharge cycles and the specific capacity, the material needs can be related with the use step. And this would be reflected in the life cycle inventory. kN:iC can be calculated by fitting the formula obtained through curve fitting of the cycle performance.Based on the condition of battery tests in the laboratory, the total capacity of batteries can be calculated by Equation (1) in which the cycle was sm) under standard specific capacity  can then be calculated by Equation (2):m is the battery mass under test, and sm is the result of quality standardisation of raw materials for the functional unit. Through this calculation method, a life cycle inventory of the raw materials was assembled. To facilitate the comparison of results, the mass of raw material needs  were obtained. For discarded batteries, incineration and recycle were chosen to be the end-of-life treatment measures. The cycle performance of the selected batteries is shown in The results were obtained in accordance with the method described in Three-dimensional Eco-indicator slices of the two selected batteries are shown in The influence of cycles and incineration rate on the Eco-indicator distribution of two selected batteries at a certain recycle rate (50%) is shown in In summary, to reduce the environmental impact of selected batteries, the charge-discharge cycle number during the use stage should be at least 200. Battery incineration has a greater impact on the Eco-indicator point of the Ni-MH battery, while its impact on the Li-ion battery is small. Battery recycling can reduce the battery\u2019s environmental impact, particularly for Ni-MH batteries, and the incineration has little effect on reducing the environmental impact of Ni-MH batteries.\u22124 Pt to 2.0 \u00d7 10\u22124 Pt when the charge-discharge cycle number ranges from 0 to 1,000. For the Li-ion battery, the Eco-indicator point ranges from 0.1 \u00d7 10\u22124 Pt to 1.0 \u00d7 10\u22124 Pt when the charge-discharge cycle number ranges from 0 to 1,000. According to the results, if the battery has a short use phase, the environmental impact will increase. If secondary batteries are under unsteady use conditions, they always have a short use phase and short charge-discharge cycles. Since the cycle numbers are related with energy consumption, the uncertainty of energy consumption in battery use phase has larger impacts on the Li-ion battery. Improving the cycle performance of the Li-ion battery can efficiently reduce the environmental impact. Based on the laboratory battery test conditions, the charge-discharge cycle numbers of the batteries and the masses of raw materials were the input data of the LCA. According to the quality standardisation of the raw materials\u2019 masses, the cycle number of the batteries was assumed to be normally distributed under 95% confidence interval. The distributions presented the impact of uncertainty data. For the Ni-MH battery, the Eco-indicator point ranges from 0.5 \u00d7 10In this paper, an assessment framework was applied to conduct an environmental impact and end-of-life treatment analysis for secondary batteries based on the Eco-indicator 99 system. This applied model comprehensively evaluated the cycle, recycle and waste  stages of secondary batteries. The analysis of the end-of-life treatment is helpful to improve some extant policies concerning secondary batteries. Two types of secondary batteries were studied by means of life cycle assessment. The results show that: (1) The Eco-indicator points of the two selected batteries decrease rapidly with the increase of the cycle number up to 200, but the attenuation is small after 200; (2) the incineration rate has greater impact on the Eco-indicator point of the Ni-MH battery than of the Li-ion battery, so policies and regulations concerning the disposal of the two kinds of batteries should be different; (3) the Eco-indicator points of the two types of batteries decay with the increase of the recycle rate, which means a strict recycle policy should be set; and (4) the influence of the recycle rate on the Ni-MH battery is more obvious, while the influence on the Li-ion battery can be clearly observed when the recycle rate is 40%\u201350%.Due to the assessment of environmental loads about the selected secondary batteries, several policy implications should be considered for reducing environmental impact of secondary batteries. First, policy-makers should encourage the use of higher energy density batteries with better chemical characteristics. Additional cycles in a longer service life will result in lower environmental loads. To reduce the environmental impact of selected batteries, the charge-discharge cycle number during the use stage should be at least 200. In other words, according to the assessment, a policy should be proposed to require a minimum of 200 cycles for secondary batteries. Second, secondary batteries should be sorted out from refuse because their contents and properties are different from those of common household garbage. For instance, most batteries contain heavy metals, such as cobalt and nickel, as well as some organic pollutants. Environmental risks will increase if secondary batteries are buried or combusted with mixed municipal waste. Third, end-of-life treatment policies for secondary batteries should be carefully selected. For example, incineration measures should be put in place for Ni-MH batteries, and battery recycle can significantly reduce the environmental impact of Ni-MH batteries. Additionally, the recycle of Li-ion batteries may result in higher efficiency and lower environmental loads. In summary, the mechanism analysis tools proposed in this paper can also be used to evaluate the environmental impact of other secondary batteries, and the policies proposed in this paper may be useful in setting measures to reduce the environmental impact of these batteries."}
+{"text": "The images for Figures 4 and 5 were reversed. The legends of these figures are correct."}
+{"text": "Temperate fruit and nut trees require adequate winter chill to produce economically viable yields. Global warming has the potential to reduce available winter chill and greatly impact crop yields.We estimated winter chill for two past (1975 and 2000) and 18 future scenarios . For 4,293 weather stations around the world and GCM projections, Safe Winter Chill (SWC), the amount of winter chill that is exceeded in 90% of all years, was estimated for all scenarios using the \u201cDynamic Model\u201d and interpolated globally. We found that SWC ranged between 0 and about 170 Chill Portions (CP) for all climate scenarios, but that the global distribution varied across scenarios. Warm regions are likely to experience severe reductions in available winter chill, potentially threatening production there. In contrast, SWC in most temperate growing regions is likely to remain relatively unchanged, and cold regions may even see an increase in SWC. Climate change impacts on SWC differed quantitatively among GCMs and GHG scenarios, with the highest GHG leading to losses up to 40 CP in warm regions, compared to 20 CP for the lowest GHG.The extent of projected changes in winter chill in many major growing regions of fruits and nuts indicates that growers of these commodities will likely experience problems in the future. Mitigation of climate change through reductions in greenhouse gas emissions can help reduce the impacts, however, adaption to changes will have to occur. To better prepare for likely impacts of climate change, efforts should be undertaken to breed tree cultivars for lower chilling requirements, to develop tools to cope with insufficient winter chill, and to better understand the temperature responses of tree crops. Commercially successful cultivation of many fruit and nut trees requires the fulfillment of a winter chilling requirement, which is specific for every tree cultivar th century, winter chill was found to have declined in California and Oman, and this process was expected to continue in the future. The differences between these studies indicate that different growing regions may be differentially impacted, but to date, no estimates are available at a global scale to indicate which regions will maintain adequate winter chill for temperate fruits and nuts in the future. This study aims to fill this knowledge gap and to provide important information needed to evaluate the future viability of fruit and nut growing regions around the world.Climate change is likely to affect future winter chill and could have a major impact on the US$ 93 billion global fruit and nut industry . Based on this analysis we calculated the safe winter chill (SWC) metric st century; A1B - emissions leveling off at mid 21st century; and A2 - continually increasing rate of greenhouse gas emissions). Projections were made for the middle (2040\u20132059) and end (2080\u20132099) of the 21st century. Idealized daily temperature curves were used for converting daily to hourly weather records and allow calculation of winter chill. For each scenario, 101 years of weather records were generated and the 10% quantile of the resulting distribution of annual winter chill interpreted as Safe Winter Chill. For each scenario, SWC from all stations was spatially interpolated, and winter chill for all scenarios was extracted from the resulting layers for 24 important growing regions around the world.Daily weather records for all weather stations were obtained from the National Climatic Data Center of the United States In all climate scenarios, estimates of Safe Winter Chill ranged from 0 CP in tropical and very cold regions to about 170 CP in maritime temperate climates of Northwestern Europe \u20134. WhiTrends in site-specific projections for different growing regions varied substantially  and the B1 scenario . For each weather station, emissions scenario and GCM, mean monthly anomalies of minimum and maximum temperatures and precipitation relative to the calibration period were obtained for two periods of time: 2040\u201359 and 2080\u20132099, representing conditions at mid and end 21st century. For several weather stations, mostly along coast lines, no GCM projection data were available. These stations were excluded, bringing the total number of weather stations down to 4293.Using the same method, we evaluated the weather record for 18 future scenarios, based on projections by three Global Climate Models , UKMO-HadCM3 and CSIRO-Mk3.0) that had been statistically downscaled to a 0.5 degree resolution using the CRU TS 2.0 data set to calibrate the downscaling . Using the Climate Wizard tool , the 10% quantile of the distribution int). Subtracting this grid from the original dataset of mean annual temperatures produced an estimate of the temperature variation that was not accounted for in the original interpolation of chill portion values (Tdiff). For the correction, the effect of temperature on chill portion numbers was then estimated by a 5th order polynomial regression between mean annual temperatures at all weather stations and the amount of safe winter chill calculated for them . A simple interpolation based on only the available weather stations would not be a very accurate representation of winter chill around the world, because temperatures at many locations may differ substantially from those of the closest weather station, which is often far away. We therefore used a high-resolution temperature dataset obtained from the WorldClim database for them . The resFrom the surfaces of safe winter chill, site-specific results were extracted for 24 point locations representing important growing regions around the world. Comparing safe winter chill estimates for different combinations of GCM, GHG emissions scenario and point in time provides an impression of the agreement between scenarios, on a case-study basis. We also evaluated differences between scenarios based on the entire distribution over all relevant grid cells . Because differences between models in site specific estimates can be both negative and positive, we evaluated the mean absolute difference among all grid cells. The results are indicative of the agreement between models."}
+{"text": "Plasmodium through their heparan sulphate chains, suggesting that genetic variations in genes involved in heparan sulphate biosynthesis may influence parasitaemia. Interestingly, Hs3st3a1 and Hs3st3b1 encoding enzymes involved in the biosynthesis of heparan sulphate are located within a chromosomal region linked to Plasmodium chabaudi parasitaemia in mice. This suggests that HS3ST3A1 and HS3ST3B1 may influence P. falciparum parasitaemia in humans.There is accumulating evidence that host heparan sulphate proteoglycans play an important role in the life cycle of HS3ST3A1 and HS3ST3B1 were identified in 270 individuals belonging to 44 pedigrees and living in Burkina Faso. Linkage and association between parasitaemia and the polymorphisms were assessed with MERLIN and FBAT. A genetic interaction analysis was also conducted based on the PGMDR approach.Polymorphisms within P. falciparum parasitaemia and the chromosomal region containing HS3ST3A1 and HS3ST3B1 was detected on the basis of the 20 SNPs identified. In addition, rs28470223 located within the promoter of HS3ST3A1 was associated with P. falciparum parasitaemia, whereas the PGMDR analysis revealed a genetic interaction between HS3ST3A1 and HS3ST3B1. Seventy-three significant multi-locus models were identified after correcting for multiple tests; 37 significant multi-locus models included rs28470223, whereas 38 multi-locus models contained at least one mis-sense mutation within HS3ST3B1.Linkage between HS3ST3A1 and HS3ST3B1 are associated with P. falciparum parasitaemia. This suggests that those variants alter both the function of heparan sulphate proteoglycans and P. falciparum parasitaemia.Genetic variants of  Plasmodium falciparum malaria is transmitted to humans through the bite of infected female Anopheles mosquitoes. Sporozoites that have been injected into the skin migrate from the site of injection, and reach the liver, where they invade hepatocytes and change into merozoites; merozoites penetrate and replicate inside red blood cells. Heparan sulphate proteoglycans (HSPGs) may play an important role in the biology of Plasmodium through their carbohydrate chains (heparan sulphate) in both the mammalian host and the vector. Anopheles heparan sulphate has been shown to bind circumsporozoite protein (CSP), suggesting a role for the carbohydrate chains within Anopheles salivary glands for infection and transmission of the parasite [Plasmodium berghei for invasion, whereas the parasite migrates through cells with low sulphated HSPGs in skin and endothelium [P. falciparum merozoite antigen (EBA-140) has been shown to bind to red blood cells in a heparan sulphate-manner, whereas soluble heparan sulphate and heparin inhibit the merozoite invasion into red blood cells [parasite . CSP alsparasite . Highly othelium . In addiod cells ,4. Finalod cells .HS3ST3A1 and HS3ST3B1, and the only significant sequence homology between these proteins occurs in the sulphotransferase domains. Nevertheless, HS3ST3A1 and HS3ST3B1, which encode the 3-O sulphotransferases 3-OST-3A1 and 3-OST-3B1 respectively, are 700 kb apart in the same chromosomal region, and show a high sequence identity [HS3ST3A1 that encodes 3-OST-3A1 has been associated with mother-to-child transmission of human immunodeficiency virus (HIV) through a genome-wide association study [These observations suggest that the outcome of malaria infection may be influenced by variations in the biosynthesis of heparan sulphate, owing to genetic variations within genes encoding the enzymes involved. These include O-sulphotransferases, which catalyze 2-O, 6-O, or 3-O sulphation . The O-sidentity . 3-OST-3identity . Recentlon study , whereason study . This suP. falciparum malaria resistance or susceptibility [Plasmodium chabaudi parasitaemia (Char1-10) or cerebral malaria [Char3) and 11 (Char8), which show extensive conservation of synteny with human chromosomes 6p21.3 and 5q31-q33, respectively [Hs3st3a1 and Hs3st3b1, which encode 3-O sulphotransferases in mice [HS3ST3A1 and HS3ST3B1 may influence parasitaemia in humans. This hypothesis was further supported by a linkage study based on microsatellite markers in humans . This prompted us to screen HS3ST3A1 and HS3ST3B1 in a population living in an endemic area in Burkina Faso to identify polymorphisms, to evaluate their linkage and association with parasitaemia, and gene-gene interactive effects by using the pedigree-based generalized multifactor dimensionality reduction (PGMDR) method [There is a growing body of evidence for human genetic factors controlling the outcome of infection. Familial aggregation and segregation analyses showed the existence of a genetic component of phenotypes related to tibility ,15. Sevetibility . Linkagetibility -19. The tibility ,20,21. Htibility ,23. In c malaria ,24,25. Nectively ,27. The  in mice ,27, sugg) method .The study subjects live in a rural area, Logoforousso, a village to the south-west of Bobo-Dioulasso (Burkina Faso). The population and the area of parasite exposure have been extensively described . VolunteP. falciparum parasitaemia measurements were considered in this study. The mean number of asymptomatic P. falciparum parasitaemia measurements per subject was 14.9 + 8.1 (range one to 28). The parasitaemia was defined as the number of parasitized erythrocytes observed per microlitre in thin blood films. The analysis was conducted on a logarithmic transformation of parasitaemia adjusted for seasonal transmission and for age that showed a significant effect on parasitaemia (P < 10-4). The standardized residual was the phenotype used for linkage and association analyses.Parasitaemia was measured as described . During HS3ST3A1 coding sequence and that of HS3ST3B1, particularly between exon 2 sequences [Each subject underwent a venipuncture, and DNA was extracted from mononuclear cells separated by Ficoll-Hypaque density gradient as described . DNA conequences . TherefoThe compatibility with Mendelian inheritance of marker alleles was checked with the FBAT and MERLIN programs ,32. All 2 = (p11p22-p12p21)2/p1p2q1q2, where p11, p22, p12, and p21 were two-locus haplotype frequencies, and p1, p2, q1, and q2 were allele frequencies. r2 is the standard c2 statistic divided by the number of chromosomes in the sample. It ranges from 0 to 1. When r2 is 1, SNPs are in complete LD.Allele frequencies were calculated by gene counting and deviation from Hardy-Weinberg equilibrium was tested using a Chi-2-test with 1 degree of freedom. Haplotypes were generated based on family genotypic data with MERLIN. Pair-wise LD (Linkage Disequilibrium) was calculated with Haploview and graphical overview of linkage disequilibrium (GOLD) ,34. LD bP value based on a normal approximation. Association in the presence of linkage was assessed using the orthogonal model released in the QTDT program [P values were calculated using the likelihood-ratio criterion.Multipoint linkage analyses were performed for sibship and half-sibship data using the software package the MERLIN Package . The reg program . P valueP value were calculated for the training set. The testing set was used to estimate the prediction accuracy of the model, which is the ratio of correct classifications to the total number of instances classified. The non-parametric sign test was used to evaluate the significance of the prediction accuracy.Genetic interactions were analysed by using the pedigree-based generalized multifactor dimensionality reduction method (PGMDR) . The PGMThe false discovery rate (FDR) procedure was performed to account for the multiple tests performed ; an FDR HS3ST3A1 and HS3ST3B1  within HS3ST3A1; there was also a highly significant linkage disequilibrium between rs2072243 and rs2072242  within HS3ST3B1.Twenty polymorphisms were identified in the promoter, the exons, the intron-exon junctions, and the 3' untranslated region of HS3ST3A1 and HS3ST3B1 after applying an FDR of 10% (Table P = 0.009). A linkage was also detected based on the analysis of the 10 SNPs with the highest Minor Allele Frequency (MAF) after applying a FDR of 5% (Table P = 0.005).A regression-based procedure was applied for multipoint linkage analysis using MERLIN. Based on the analysis of the 20 SNPs, parasitaemia was genetically linked to the region containing P = 0.005), whereas the other SNPs were not associated with parasitaemia (P > 0.09). The allele C was negatively associated with parasitaemia , whereas the allele T was positively associated with parasitaemia . This result remained significant after applying an FDR of 5%, and confirmed the linkage signal detected with MERLIN. Furthermore, the linkage was taken into account to test the association between each SNP and parasitaemia by using QTDT; an association in the presence of linkage was detected for rs28470223 (P <0.003) after applying an FDR of 5%.Combined linkage and association between each SNP and parasitaemia was further evaluated. FBAT showed evidence of linkage and association between rs28470223 and parasitaemia  and the cross-validation analysis with the testing set  after applying a FDR of 5%. The analysis revealed 73 significant multi-locus models, which could be consistently cross-validated  rs3744337, rs3744335, rs28470223, rs78863672, rs2072243, rs2072242, and rs115229628 that are located within the 5'UTR region of either HS3ST3A1 or HS3ST3B1: ii) rs3785655 and rs7379332 that are within the 3'UTR region of HS3ST3B1; iii) rs9906855, rs62057033, rs61732181, rs61744056, rs62636622 and rs55688668 that are synonymous mutations of either HS3ST3A1 or HS3ST3B1; and iv) rs62636623, rs62056073, rs61729712, and rs9906590 that are mis-sense mutations within HS3ST3B1. Interestingly, rs28470223 was in 37 of the 73 significant multi-locus models, and 38 multi-locus models contained at least one mis-sense mutation.The epistatic effect of HS3ST3A1 and HS3ST3B1), were considered as candidate genes.This is apparently the first study to investigate the association between a phenotype related to malaria susceptibility and genes involved in HS biosynthesis. Two genes 700 kb apart, which encode 3-O sulphotransferases involved in the synthesis of HS  the binding of P. falciparum antigen on host cells and/or ii) the pro-inflammatory response.In all, the results suggest that several SNPs within P. falciparum antigen on host cells and the parasite invasion rate. Highly sulphated HS has been shown to promote a productive invasion of cells by P. berghei sporozoites, whereas sporozoites migrate through cells harbouring low sulphated HS [P. falciparum antigen on the human erythrocyte surface or the merozoite invasion rate [Together with previous reports -4,41, thhated HS . Since thated HS , and altion rate , the resion rate ,43, and HS3ST3A1 and HS3ST3B1 might also alter the immune response, and more specifically, the inflammatory response. Indeed, pro-inflammatory cytokine and chemokine binding to HS that depends on the sulphation profile of HS controls both the tissue targeting and the local accumulation of cytokines and chemokines [CCR5 (chemokine (C-C motif) receptor 5) up-regulation was associated with the up-regulation of HS3ST3A1 and HS3ST3B1 in humans infected by HIV; the authors suggested either that interaction between HS and CCR5 causes up-regulation, or that the promoters of CCR5, HS3ST3A1 and HS3ST3B1 share a cis-regulatory motif binding the same transcription factor [CCR5 and HS3ST3A1 have been associated with HIV infection [Genetic variations in emokines ,45. Inten factor . Since Cnfection ,47, thisHS3ST3A1 and HS3ST3B1 are linked to P. falciparum parasitaemia, that rs28470223 within the promoter of HS3ST3A1 is associated with P. falciparum parasitaemia, and that interactions between HS3ST3A1 and HS3ST3B1 polymorphisms alter P. falciparum parasitaemia. The results also indicate that rs28470223 and four mis-sense mutations within HS3ST3B1 strongly contribute to genetic interactions. This study also suggests that other genes involved in HS biosynthesis may affect malaria resistance. In this way, NDST1 that plays a major role in HS biosynthesis is located within the chromosome 5q31-q33, which is linked to P. falciparum parasitaemia [This study shows that sitaemia .CCR5: Chemokine (C-C motif) receptor 5; CSP: Circumsporozoite protein; GRCh37: Genome Reference Consortium Human genome build 37; HIV: Human immunodeficiency virus; HS: Heparan sulphate; HSV-1: Herpes simplex virus 1; HSPG: Heparan sulphate proteoglycan; LD: Linkage Disequilibrium; MAF: Minor Allele Frequency; iRBC: infected red blood cells; PGMDR: Pedigree-based generalized multifactor dimensionality reductionThe authors declare that they have no competing interests.AA carried out the molecular genetic studies, participated in the sequence alignment and performed the statistical analysis. SG participated in the molecular genetic studies and sequence alignment. SA participated in the statistical analysis. FF participated in the design of the study, and revised the results and the manuscript. PR performed the design of the study, supervised the experiments and the statistical analyses, and wrote the manuscript. All authors read and approved the final manuscript.HS3ST3A1 and HS3ST3B1Primer pairs and annealing temperatures used to amplify .Click here for fileThe best multi-locus models identified with PGMDR. All the P values were significant after applying a FDR of 5%.Click here for file"}
+{"text": "As schizophrenia is genetically and phenotypically heterogeneous, targeting genetically informative phenotypes may help identify greater linkage signals. The aim of the study is to evaluate the genetic linkage evidence for schizophrenia in subsets of families with earlier age at onset or greater neurocognitive deficits.Patients with schizophrenia  and their first-degree relatives  from 557 families with schizophrenia were recruited from six data collection field research centers throughout Taiwan. Subjects completed a face-to-face semi-structured interview, the Continuous Performance Test (CPT), the Wisconsin Card Sorting Test, and were genotyped with 386 microsatellite markers across the genome.A maximum nonparametric logarithm of odds (LOD) score of 4.17 at 2q22.1 was found in 295 families ranked by increasing age at onset, which had significant increases in the maximum LOD score compared with those obtained in initial linkage analyses using all available families. Based on this subset, a further subsetting by false alarm rate on the undegraded and degraded CPT obtained further increase in the nested subset-based LOD on 2q22.1, with a score of 7.36 in 228 families and 7.71 in 243 families, respectively.We found possible evidence of linkage on chromosome 2q22.1 in families of schizophrenia patients with more CPT false alarm rates nested within the families with younger age at onset. These results highlight the importance of incorporating genetically informative phenotypes in unraveling the complex genetics of schizophrenia. Family and twin studies have indicated that schizophrenia has a strong genetic basis Cumulative evidence suggests that age at onset is one potential component phenotype for schizophrenia. Compared with schizophrenia patients with late onset, those with early age at onset have been associated with more severe cognitive impairments and poorer outcomes A number of candidate gene studies have explored the relations of certain genetic variants to schizophrenia by subgrouping patients using these genetically informative phenotypes of schizophrenia, including age at onset This study aimed to evaluate the genetic linkage evidence for empirically derived subtypes of schizophrenia by applying ordered subset linkage analyses in a large sample of families of siblings co-affected with schizophrenia, which consisted mainly of a single ethnic group Participants of this study included patients with schizophrenia and their first-degree relatives recruited from six data collection research center throughout the nation in the Taiwan Schizophrenia Linkage Study between 1998 and 2002. The study design, ascertainment process, and sample characteristics have been described in more detail elsewhere The DIGS includes a psychosis section that inquires about the age at onset of the first psychotic episode that could not be attributed to medical illness, medications, or substance abuse. When the information with regard to onset of the first psychotic episode on the interview was unclear, research psychiatrists would review the medical history and determine the age at onset.Probands and their relatives completed both the CPT and the WCST. The degraded and undegraded version of CPT -\u20181-9\u2019 procedure has been described in detail elsewhere For the WCST, we used a computerized version The adjusted z scores of the CPT indices were derived by means of standardizing the raw scores with adjustments for sex, age, and education against a community sample of 345 individuals http://www.cidr.jhmi.edu). A series of procedures for quality control was adopted for the genotyping, such as duplicate assays with a discordancy rate of 0.06%, and checking for non-Mendelian inheritance and pedigree inconsistencies with an overall rate of 0.39% in genotype and family errors http://zork.wustl.edu/nimh.The original genotyping was conducted by the Center for Inherited Disease Research, with 386 markers spaced at an average of 9-cM intervals, following the center's standard genotyping procedures , and three WCST indices . Meanwhile, the remaining indices were ranked using the descending order: four CPT indices  and five WCST indices .P value of \u2264 0.05 was considered significant, suggesting that a particular subset of individuals in the sample is more strongly linked to the specified chromosomal region than the whole sample. The procedures are summarized in the left part of The statistical significance of the difference between the greatest LOD scores in a subset of families identified by the OSA and the LOD scores at the same position in the full set of families was evaluated via 1,000 permutations. Under the null hypothesis that the ranking of the covariate is independent of the family's LOD scores on the target chromosome, the families were randomly permuted with respect to the covariate ranking and a chromosome-wide p value for each chromosome was yielded. A P value of <0.05 in permutation was considered significant.Because OSA-derived subsets of families might have tied covariates, we attempted to introduce another subphenotype to further dissect the families , and thus the threshold of a corrected genome-wide significance level was set as 0.0029 . For the nested OSA part, there were 33 covariates examined (17 covariates in the OSA and 16 indices on the CPT and the WCST), and the threshold of a corrected genome-wide significance level was set as 0.0015 .After the permutation on the composition of a subset of families, which provides chromosome-wide significance level, we further evaluated its genome-wide significance by simulating the genotype data of the families and then estimating the empirical significance level with correction for multiple phenotypes, as illustrated in the right part of ad hoc correction procedure was also performed, which was calculated as LOD(corrected) \u200a=\u200a 3.6 + log10 (numbers of tests) Second, according to the Lander and Kruglyak threshold The family covariate values between the subset of families that resulted in the greatest linkage signals and the remaining families were then compared. A linear mixed effect model with family treated as a random effect was applied using the Proc MIXED of the SAS software version 9.1  to adjust for within-family correlation.P<0.0001), 0.14 to 0.31 for CPT indices (P ranging from 0.004 to 0.0001), and 0.12 to 0.24 for WCST indices (P ranging from 0.04 to 0.0001).The distributions of individual covariates, which were averaged over the affected siblings of each family, are listed in P \u2264 0.05) in a subset-based maximum LOD score from the initial LOD scores of the whole sample. The results of the OSA identified distinct chromosomal regions implicated for schizophrenia with different informative phenotypes: 1) 2q22.1 for younger age at onset; 2) 7q32.2, 8q13.1, and 9q34 for greater undegraded CPT-related deficits; 3) 7q32.3 for greater degraded CPT-related deficits; and 4) 8q13.1 and 8q21.1 for greater WCST-related deficits. For the five loci with significant increases in subset-based LOD scores, the percentage of linked families in each subset ranged from 10% to 53% of the whole sample. When the families within the subset were compared with the remaining families for the corresponding subphenotype, there were indeed significant differences between the two groups in the covariates, with earlier age at onset or more neurocognitive deficits in the subset of families. Of those covariate/locus combinations with a significantly increased LOD score, only \u201cage at onset/2q22.1\u201d reached genome-wide significance based on simulations after an ad hoc correction for multiple informative phenotypes, whereas no locus reached the Lander and Kruglyak genome-wide significance threshold after an ad hoc correction for multiple informative phenotypes.P\u200a=\u200a0.004) in 81% of 282 families  and 7.71  in 87% of 281 families , respectively. Of note, both the LOD score of 7.36  and of 7.71  reached genome-wide significance, regardless of which method was used in the significance evaluation, i.e., simulated genotype data or the adjusted Lander and Kruglyak threshold after accounting for multiple phenotypes. More severe CPT or WCST deficits were indeed found in the families of the nested subset than those in the remaining families, as summarized in Among the loci identified in The distributions of the genome-wide LOD scores of the nested OSAs that were based on age at onset and CPT false alarm rates over 22 autosomal chromosomes are illustrated in This study found that subsets of families of patients with schizophrenia characterized by younger age at onset or neurocognitive deficits exhibited increased linkage signals and reached genome-wide significance on 2q22.1, which was nearby a suggestive locus (a LOD score of 1.16 on 2q14.1) in the initial genome-wide scan for the whole sample Our nested OSA approach demonstrates an exploratory way to incorporate various schizophrenia-related characteristics in deciphering the genetic linkage signal to schizophrenia for families with tied covariates . Our results revealed that in the onset-based subset of families having maximal linkage signals, families with tied covariates might in fact have linkage scores in the opposite directions. Accordingly, a further subsetting by one CPT or WCST index might partition the families into more homogeneous subsets and then result in more increase in the LOD scores. Unlike other statistical methods for detecting linkage by dissecting heterogeneity, which require a prior stratification of the data An important feature of this study is that we used age at onset and several facets of the CPT and WCST as covariates to reduce phenotypic and genetic heterogeneity of schizophrenia. These are important features as they clearly impact the outcome and severity of the illness. In addition, cognitive deficit is a core symptom for schizophrenia Unlike previous linkage studies for psychiatric disorders that used selective indices of neurocognitive tasks, either as a composite profile of neurocognitive performance P values were derived from simulations. The current results of particular subset-based NPL-Z scores attaining genome-wide significance might be very conservative because a Bonferroni-type correction of the P value was applied for the correlated indices among the CPT or WCST. It should be pointed out that it is very difficult to assess the statistical significance of our subset-based linkage signals due to the complexity inherent in the many possible sources of multiple testing. Accordingly, the results reported here might be better treated as exploratory and independent replication is warranted to reassure the robustness of the findings.However, our approach might suffer from the issue of multiple testing. To fully address this, several strategies were used in this study to help interpret the significance of the OSA results. First, we used permutation tests to estimate the statistical significance of the change in LOD score from the subsets, which controlled for inflation in the false-positive rate induced by examining multiple family subsets for a given covariate. Second, two simulation-based strategies were applied to estimate the genome-wide significance level of subset-based LOD scores, with one based on the simulated genotype data of this sample and the other one based on adjusting the Lander-Kruglyak threshold that was also obtained by simulations. Such simulations can help control for the inflation in the false-positive rate over multiple chromosomes and markers since genome-wide significance was estimated by the proportion of times that a particular LOD score was reached anywhere in the simulated genome. Finally, we used a Bonferroni correction to adjust for multiple trait-related covariates after empirical genome-wide The most striking linkage signal found in this study was obtained from a nested OSA on chromosome 2q22.1 (D2S4422 at 147.4 cM). Intriguingly, a rank-based meta-analysis showed evidence of linkage in this region This study is limited in several aspects. First, our samples consisted of adults with Han Chinese ancestry only, and hence the results may not extrapolate to populations of other ethnicities where allele frequencies may vary. Second, despite our use of different strategies to deal with multiple testing, our linkage findings may not rule out the possibilities of false positive or false negative because genome-wide statistical significance of OSA-derived subset results remains a difficult issue. Third, the age at onset of schizophrenia in this study was determined mainly on the initiation of first psychotic symptoms. Although the age at first functional impairment may be closer to the true onset of the disease, there is an overall agreement that the onset of the first psychotic episode is a better marker for the onset of schizophrenia because it has a higher specificity than negative or nonspecific symptoms In summary, we obtained possible evidence of linkage on chromosome 2q22.1 in families of schizophrenia patients with more CPT false alarm rates nested within the families with younger age at onset. Our results suggest the presence of etiologic heterogeneity in schizophrenia and highlight the importance of unraveling the complex genetics of schizophrenia by incorporating genetically informative phenotypes."}
+{"text": "Gadus morhua) and in the analysis of traits such as temperature tolerance, growth characteristics and sexual maturation. We used an Illumina GoldenGate panel and the KASPar SNP genotyping system to analyse SNPs in three Atlantic cod families, one of which was polymorphic at the Hb \u03b21 locus, and to generate a genetic linkage map integrating Pan I and multiple Hb loci.Haemoglobin (Hb) and pantophysin (Pan I) markers have been used intensively in population studies of Atlantic cod  have been mapped on linkage group (LG) 2 while the other five  were placed on LG18. Pan I was mapped on LG 1 using a newly developed KASPar assay for a SNP variable only in Pan IThe genetic linkage map presented here incorporates the marker Pan I, together with multiple Hb loci, and integrates genetic linkage data produced by two different research groups. This represents a useful resource to further explore if Pan I and Hbs or other genes underlie quantitative trait loci (QTL) for temperature sensitivity/tolerance or other phenotypes. Gadus morhua) represents one of the most valuable commercial resources for international fisheries [The Atlantic cod  matches the number of haploid chromosomes reported for the Atlantic cod by Fan and Fox [We used a curated Illumina GoldenGate panel containing 1536 SNPs , includi\u00ae4 . The gen\u00ae4 . The 23 al. 2010 ; a 1:1 c and Fox . The com and Fox ; therefo and Fox ,26. Alle and Fox ,20 suggeA and Pan IB described at the Pan locus can be determined by assessing the polymorphism present at a DraI site located in intron 4 [A homozygotes  with thet al. . To iden [et al. ) and Moe [et al.  we perfo [et al.  using 12is group . Using t [et al.  might re [et al. ,12 where [et al. ,14 sequeThe genetic linkage map presented here, that includes the marker PanI and multiple Hb loci, represents a useful resource for studying genotype-phenotype relationships, for QTL studies, as well as for population studies. Our data indicate that Hb genes are located on two different linkage groups while Pan I locus was mapped on a third linkage group. Further studies are needed to elucidate which of these genes/linkage groups will correlate with phenotypic traits. The Hb \u03b21 gene, which has been linked to variation in haemoglobin oxygen binding capacity and water temperature preference ,28  are indicated on the left of each linkage group, with SNP identifiers on the right. PanI and Hb loci are in bold, italicized and highlighted in red while SNPs common to Moen et al. 2009 are in bold and highlighted in red.Click here for fileet al. 2009Linkage group correspondence between the genetic linkage groups presented in this study and Moen . This file contains the data related to the linkage group correspondence between the genetic linkage groups presented in this study and Moen et al. 2009.Click here for file"}
+{"text": "Oncorhynchus clarkii) following the introduction of rainbow trout (O. mykiss). Neither the evolutionary consequences nor conservation implications of rainbow trout introgression in cutthroat trout is well understood. Therefore, we generated a genetic linkage map for rainbow-Yellowstone cutthroat trout (O. clarkii bouvieri) hybrids to evaluate genome processes that may help explain how introgression affects hybrid genome evolution.Introgressive hybridization is an important evolutionary process that can lead to the creation of novel genome structures and thus potentially new genetic variation for selection to act upon. On the other hand, hybridization with introduced species can threaten native species, such as cutthroat trout  contained confirmed chromosome rearrangements between rainbow and Yellowstone cutthroat trout indicating that rearrangements may suppress recombination. The frequency of allelic and genotypic segregation distortion varied among parents and families, suggesting few incompatibilities exist between rainbow and Yellowstone cutthroat trout genomes.The hybrid map closely aligned with the rainbow trout map (a cutthroat trout map does not exist), sharing all but one linkage group. This linkage group (RYHyb20) represented a fusion between an acrocentric (Omy28) and a metacentric chromosome (Omy20) in rainbow trout. Additional mapping in Yellowstone cutthroat trout indicated the two rainbow trout homologues were fused in the Yellowstone genome. Variation in the number of hybrid linkage groups (28 or 29) likely depended on a Robertsonian rearrangement polymorphism within the rainbow trout stock. Comparison between the female-merged FChromosome rearrangements suppressed recombination in the hybrids. This result supports several previous findings demonstrating that recombination suppression restricts gene flow between chromosomes that differ by arrangement. Conservation of synteny and map order between the hybrid and rainbow trout maps and minimal segregation distortion in the hybrids suggest rainbow and Yellowstone cutthroat trout genomes freely introgress across chromosomes with similar arrangement. Taken together, these results suggest that rearrangements impede introgression. Recombination suppression across rearrangements could enable large portions of non-recombined chromosomes to persist within admixed populations. The widespread occurrence of hybridization has been a catalyst for intensive study in evolutionary biology and has provided rich opportunities for investigating genome evolution, adaptation, speciation, reproductive isolation, and hybrid fitness-3. HybriGenetic linkage maps provide a means for investigating genome evolution and function, and have contributed to an improved understanding of hybridization and introgression-10. TheyGenome maps for salmonid fishes show signatures of two significant events: genome duplication and chromosome rearrangements-21. The Oncorhynchus mykiss) and cutthroat trout (O. clarkii) are two salmonid species that inhabit western North America. Rainbow and cutthroat trout are sister species and shared a common ancestor approximately 3 million years ago[Rainbow trout . Rainbow trout (RBT) have 58\u201364 diploid chromosomes depending on the chromosome race[2 hybrids between RBT and YCT and developed a F1 hybrid linkage map to investigate the genomic consequences of introgression. The objectives for constructing the F1 hybrid linkage map were to 1) determine if linkage groups were conserved between the F1 map and existing RBT maps, 2) determine if introgression suppressed recombination, and 3) estimate the prevalence of segregation distortion in the F1 hybrid map. Our hybrid linkage map has application to conservation and management of indigenous cutthroat trout subspecies because it localizes species-specific markers to linkage groups and identifies genomic regions where recombination is suppressed, both of which may assist resource managers in determining accurate estimates of RBT admixture throughout the native cutthroat trout range. It will also have application for identifying QTL associated with species-specific traits. Finally, it can be used as a baseline for future comparative mapping studies in rainbow-cutthroat trout hybrids.Here, we present the first hybrid genetic linkage map between two introgressing salmonid species, rainbow trout and Yellowstone cutthroat trout   and RYHyb28 (RBT sex chromosome homologue) , and one species-specific insertion/deletion (indel) were mapped in YCT-RBT F1 hybrids, indicating that a major portion of RYHyb20 was inherited as a single, non-recombining block of markers. Additional mapping in a male YCT (48 progeny) indicated that the Omy20 and Omy28 homologues were fused in YCT.We found evidence for a fusion between two RBT chromosomes in the hybrids. Rainbow trout chromosomes Omy20 (a metacentric chromosome in RBT) and Omy28 (an acrocentric chromosome in RBT), were fused into a single linkage group, RYHyb20, in both sexes Figure\u00a0. Several\u0398\u2009=\u20090.46, LOD\u2009=\u20090.03) confirmed that the RYHyb25 and RYHyb29 homologues are not fused in YCT. We found a similar recombination estimate between the same loci in Male2 , but not in Male 1 .Linkage groups RYHyb25 and RYHyb29  differed in arrangement among parents. The distal mapping loci OMM1301 (on RYHyb25) and Ogo2UW/ii (on RYHyb29) were not linked in Female 1 and Male 2, indicating two independent linkage groups ) to specific linkage groups  that have been previously identified within RBT. We alsoWe found eight pseudolinkage groups exclusive to both male maps . Pseudolinkage was represented in males by statistical linkage between markers that mapped to independent linkage groups in the two female maps. All cases of pseudolinkage were between chromosomes identified as homeologous within RBT.P\u2009<\u20090.001, G-test) and 5.65 , respectively , with the following exceptions. Female and male recombination rates were not different across RYHyb24, and the female to male recombination ratios could not be estimated across RYHyb11, RYHyb14, and RYHyb23 because male pairwise recombination values were zero for all corresponding female pairwise comparisons. The recombination rate was not different between mapping parents of the same sex across the genome.Females within both families had a significantly higher recombination rate across the genome than males. The female to male recombination ratio in Family 1 and Family 2 was 6.92 , appeared elevated across the four loci closest to the telomere in the hybrid map (2.44). However, the recombination distance appeared suppressed in the hybrid map between the two markers closest to the centromere. Recombination suppression near the centromere was likely associated with the fusion to RYHyb20(Omy20). Linkage group RYHyb25_29 had a relative hybrid:RBT recombination distance of 0.59; however, we were unable to include the majority of the q-arm in the comparison. We observed five non-recombining loci in the hybrid map, whereas the same five loci mapped over 40\u00a0cM in the RBT. In contrast, the single-armed linkage groups RYHyb25 and RYHyb29 had relative hybrid:RBT recombination distance of 0.82 and 1.12, respectively. The reduced relative map distance in RYHyb25_29 compared to RYHyb25 and RYHyb29 suggests that the fusion between the two chromosome arms caused a reduction in the recombination rate across the centromere. Linkage group RYHYb15 had a relative hybrid:RBT recombination distance of 0.66. Although the hybrid map had a reduced recombination distance across the centromere compared to the RBT map, the recombination distance was greater in the hybrid map than in the RBT map near telomeres on both chromosome arms. Linkage group RYHyb14 had a relative hybrid:RBT recombination distance of 0.59; however, we were unable to include the majority of the q-arm in the comparison.We found five instances of suppressed and one instance of elevated recombination distance across large numbers of loci within several linkage groups in the female hybrid map relative to the female RBT map  contained different chromosome arrangements between YCT and RBT. Segregation distortion was generally limited and distortion patterns varied among parents and families, suggesting that few incompatibilities exist between RBT and YCT genomes. Taken together, these results suggest that RBT and YCT genomes freely introgress, with the exception that differences in chromosome rearrangements between the species could impede introgression across large portions of specific linkage groups.O. tshawytscha), as the Omy20 homologue in Chinook is an acrocentric chromosome and the segment homologous to Omy20q appears inverted compared to RBT . The second rearrangement would have been a Robertsonian type involving centric fusion between the acrocentric Omy20 homologue and Omy28, conserving chromosome arm number. The three remaining differences may be explained by comparing acrocentric chromosome numbers between RBT (seven in the 60 chromosome race) and YCT (twelve). The seven RBT acrocentric chromosomes have been identified as such in the hybrid map, suggesting that they have an acrocentric YCT homologue. The remaining five YCT acrocentric chromosomes likely have homologs with RBT metacentric chromosomes. The third and fourth rearrangement differences probably represent centric fusion/fission events between two RBT metacentric and four of the five remaining YCT acrocentric chromosomes, which would not change the chromosome arm number. The fifth rearrangement difference may have involved a fission event within a RBT metacentric chromosome arm, generating the fifth acrocentric and a submetacentric chromosome in YCT. Fission within a RBT metacentric arm would gain one YCT chromosome arm and restore the 52 haploid chromosome arm number, countering the loss of a chromosome arm by the pericentric inversion.The hybrid map is a product of differences in chromosome rearrangements between RBT and YCT, as well as mixed ancestry of the RBT source stock. Using data from karyotypes,28 and t1 hybrids that were Robertsonian heterozygotes  comprised of 28 linkage groups, containing the RBT metacentric fusion Omy25_Omy29 and the YCT acrocentric homologues to Omy25 and Omy29, as well as Robertsonian acrocentric homozygotes  comprised of 29 linkage groups, containing RBT acrocentric chromosomes Omy25 and Omy28 and their YCT homologues  is likely due to mixed ancestry of the source RBT stock. Linkage analysis in YCT indicated that the Omy25 and Omy29 homologues each represent acrocentric linkage groups in YCT, but these chromosomes are known to be a Robertsonian polymorphism in RBT and 29 i,35. The Conclusions based on recombination differences between the hybrid and RBT maps have limitations, as rates can vary between related species, as well1 hybrids. First, conservation of synteny and marker order between the hybrid and RBT maps[1 hybrids. Second, because RBT and YCT contain the same number of chromosome arms, Robertsonian type rearrangements are considered to have played a significant role in generating the chromosome number differences between the species[1 hybrids, which would not necessarily cause malsegregation and unbalanced gametes. For example, the rate of nondisjunction was not different in individuals that were heterozygous for Robertsonian rearrangements compared to homozygotes in pink salmon (O. gorbuscha)[Mus musculus domesticus)[Sorex araneus)[Chromosome rearrangements can generate recombination suppression in heterokaryotypes  through unbalanced gametes which results in non-recombinants being the only viable gametes, or by rRBT maps,33 sugge species. Meioticrbuscha), house mesticus), and Euraraneus), suggest1 hybrids across several loci spanning chromosome fusion and fission differences between RBT and YCT; the Robertsonian fusion/fission within RYHyb25_29 and RYHyb20. This suggests that chromosome rearrangements did indeed suppress recombination. Rearrangements generate extensive linkage disequilibrium in heterokarotypic hybrids[1 hybrids could indicate the presence of rearrangements. We suggest that three other metacentric hybrid linkage groups, RYHyb11, RYHyb14, and RYHyb15, contained chromosomes that differed by arrangement between RBT and YCT. These linkage groups might represent Robertsonian rearrangements because recombination was suppressed across putative centromeres which could indicate that F1 hybrids were heterozygous for centric fusions/fissions. However, we found marker order differences within the suppressed regions in each of these three linkage groups compared to RBT linkage maps[Recombination was restricted in the F hybrids and supp hybrids,30,43. Tage maps,33, whicSignificant differences in recombination rates between several hybrid and RBT linkage groups could indicate the presence of inter-specific, genic incompatibilities. However, our broad-scale analysis hinders inference across smaller genomic scales where incompatibilities have been reported-46. NeveThe recombination suppression pattern across the Robertsonian rearrangement RYHyb25_29 and the presumed Robertsonian rearrangement RYHyb15 differed from patterns reported between chromosome races in the house mouse,48 and cDrosophila pseudoobscura and D. persimilis[Helianthus petiolaris and H. annuus[S. araneus and S. antinorii[Our results suggest that chromosome rearrangement is the main genomic obstacle for gene exchange between RBT and YCT. Rearrangements have been observed to reduce gene flow between several species pairs, including ersimilis, HeliantH. annuus, and S. antinorii,50. Rearantinorii,51. As aantinorii,51. Indeantinorii,32. The antinorii,49,50,52antinorii.Given the hybrid linkage map results, we predict that, within introgressed populations, inter-specific recombination will be restricted in particular genomic regions where chromosome arrangement differs between YCT and RBT. This prediction is supported by studies that have reported reduced gene flow across chromosome rearrangements within stable hybrid zones,11,50,52P\u2009<\u20090.05, within hybrid maps across a variety of taxa, including interspecific crosses between Mimulus guttatus and M. nastutus[Lepomis cyanellus and L. megalotis[Nasonia giraulti and N. vitripennis[Salvelinus alpinus[M. guttatus[Ceratodon purpureus[Coregonus clupeaformis[Tigriopus californicus[et al.[The low level of segregation distortion observed in the RBT-YCT hybrid maps was unexpected. Several studies have reported high levels of segregation distortion, greater than 15% of loci at  nastutus, Lepomismegalotis, and Nastripennis and betws alpinus, M. gutt guttatus, Ceratodpurpureus, Coregonpeaformis, and Tigifornicus. Divergeifornicus-63. We wus[et al.. NeverthAlthough segregation distortion was limited, we found a few noteworthy cases. A variety of mechanisms may cause distortion,64,65. US. alpinus) and brook charr [Salmo trutta) and Atlantic salmon  because their sex chromosomes lack homology[Conservation of synteny with the RBT sex chromosome, Omy1, suggests that RYHyb28 is the sex-linkage group. Indeed, sex has been established as mapping to homologs in YCT and RBT. Conservtinalis), introgrtinalis),74. The homology, yet thehomology. Howeverhomology and intrhomology. NeverthOur results are consistent with a growing number of studies demonstrating that chromosome rearrangements reduce gene flow by suppressing recombination,49,50,521 hybrids provides an initial framework for investigating hybridization and is likely relevant to other cutthroat trout subspecies that introgress with RBT, and, therefore, may serve as a general model of genomic introgression. In addition, our study defines a set of genome-wide species markers that can be applied to conservation and management of indigenous YCT.The genetic linkage map established herein for YCT-RBT F1 YCT-RBT parents were generated by crossing female YCT collected from Henry\u2019s Lake Fish Hatchery and Fish Management Station, Idaho Department of Fish and Game (IDFG), with male RBT (Kamloops stock) from Hayspur Hatchery (IDFG). Mature F1 hybrids were collected at Henry\u2019s Lake in March of 2004 and used to generate two F2 hybrid full-sib crosses . Because a YCT genetic map does not exist, we also constructed a YCT cross (N\u2009=\u200948 mapping progeny) so that we could clarify linkage anomalies observed between the F1 hybrid and published RBT maps[1 hybrid and YCT parents by screening 12 species-specific markers that differentiate RBT and YCT , 1.5-2.5\u00a0mM MgCl2, 200\u00a0\u03bcM each dNTP, 1.5 pmol of each primer, and 0.5 units Taq polymerase . PCR products were visualized on 2-4% agarose gels stained in ethidium bromide.In addition, 14 insertion/deletion and four RFLP species-diagnostic primers , between adjacent markers to estimate map distances. Male and female maps were compared by generating sex-specific and sex-merged maps using LINKMFEX. The total number of linkage groups in the F1 hybrid map was determined from sex-merged maps. Linkage maps were graphically represented using the program MAPCHART[1 hybrid parents, and thus these markers were not ordered within the map. However, we did assign each of these markers to a specific linkage group.Linkage maps were established using LINKMFEX v2.3 software package with an MAPCHART. MarkersWe estimated the average recombination ratio across the genome and across each linkage group between parents within each family and between parents of the same sex using LINKMFEX. Significant differences in genome-wide and linkage group-wide recombination rates were identified by summing G-test values and degrees of freedom across each comparison.et al.[et al.[1 map. The consensus female RBT map was generated using only markers in common with the female-merged F1 hybrid map  or areas of high recombination (telomeric regions). Kosambi map distances from Rexroad et al.[\u0398 map distances by applying the formula \u03b8\u2009=\u20090.5(ek4\u2009\u2212\u20091)/(ek4\u2009+\u20091)[1 map and the consensus female RBT map across linkage groups against the null hypothesis of no difference in recombination rate. Significant differences in the recombination rate were identified as described above.To determine if introgression suppressed recombination rates, we generated a consensus female RBT map from Guyomard et al. and Rexrl.[et al. using read et al. were cone4k\u2009+\u20091), where k1 parent chromosome phases as either YCT or RBT using LINKMFEX. Locus-specific allele frequencies were tested for deviation from 1:1 Mendelian expectations by summing G-test values and degrees of freedom within each 25\u00a0cM interval.We tested for allelic distortion using a sliding window analysis, because loci exhibiting segregation distortion often cluster together within the genome. We esta2 hybrid progeny as YCT homozygote (YCT/YCT), RBT homozygote (RBT/RBT), or heterozygote (YCT/RBT). Significant deviation from the expected 1:2:1 Mendelian genotypic proportions was determined for each locus by applying chi square tests followed by multiple comparison corrections using a false discovery rate (B-Y FDR)[For testing genotypic segregation distortion, we used the most likely allele phases obtained from LINKMFEX in the sliding window analysis to assign locus-specific genotypes in the FB-Y FDR) across aThe authors declare that they have no competing interests.COO performed genotyping of microsatellite and indel markers, carried out the statistical analyses, and drafted the manuscript. VLP and JCG performed SNP genotyping. LH and KN helped interpret the data and draft the manuscript. COO, LH, and KN conceptualized the study. All authors read and approved the final manuscript.C. O. Ostberg: U.S. Geological Survey, Western Fisheries Research Center, 6505 NE 65th Street, Seattle, WA 98115 and School of Aquatic and Fishery Sciences, University of Washington, 1122 NE Boat Street, Box 355020, Seattle, WA 98105, USA, costberg@usgs.gov L. Hauser: School of Aquatic and Fishery Sciences, University of Washington, 1122 NE Boat Street, Box 355020, Seattle, WA 98105, USA, lhauser@uw.edu V. L. Pritchard: Department of Biological Sciences, University of Turku, 20014 Turku, Finland, victorialpritchard@gmail.com J. C. Garza: Southwest Fisheries Science Center, National Marine Fisheries Service and University of California, Santa Cruz, 110 Shaffer Road, Santa Cruz, CA 95060, USA, carlos.garza@noaa.gov K. A. Naish: School of Aquatic and Fishery Sciences, University of Washington, 1122 NE Boat Street, Box 355020, Seattle, WA 98105, USA, knaish@uw.edu.This file contains numbered worksheets that provide information on mapping loci and linkage maps in Excel file format. The worksheets includes mapping loci and references (worksheet 1), parent-specific linkage maps (worksheets 2\u20135), female- and male-merged linkage maps (worksheets 6 and 7), sex-merged linkage map (worksheet 8), species diagnostic markers localized to hybrid linkage groups (worksheet 9), and F1 and RBT consensus maps used to investigate recombination suppression.Click here for fileThis PDF file includes figures representing parent-specific F1 hybrid linkage maps and inferred Yellowstone cutthroat trout (YCT) allele frequencies for each locus.Click here for fileThis PDF file includes figures representing female-, male-, and sex-merged F1 hybrid linkage maps.Click here for fileThis PDF file includes figures that compare map distances, in centiMorgans, across the same markers in the female-merged F1 hybrid linkage map (X-axis) and the female consensus rainbow trout map (Y-axis) for each linkage group.Click here for fileThis PDF file includes figures representing inferred genotypic frequency distributions for Yellowstone cutthroat trout homozygotes, rainbow trout homozygotes, and heterozygotes at each locus in Family 1, Family 2, and loci scored in both families combined across each hybrid linkage group.Click here for file"}
+{"text": "Family-based study designs are again becoming popular as new next-generation sequencing technologies make whole-exome and whole-genome sequencing projects economically and temporally feasible. Here we evaluate the statistical properties of linkage analyses and family-based tests of association for the Genetic Analysis Workshop 17 mini-exome sequence data. Based on our results, the linkage methods using relative pairs or nuclear families had low power, with the best results coming from variance components linkage analysis in nuclear families and Elston-Stewart model-based linkage analysis in extended pedigrees. For family-based tests of association, both ASSOC and ROMP performed well for genes with large effects, but ROMP had the advantage of not requiring parental genotypes in the analysis. For the linkage analyses we conclude that genome-wide significance levels appear to control type I error well but that \u201csuggestive\u201d significance levels do not. Methods that make use of the extended pedigrees are well powered to detect major loci segregating in the families even when there is substantial genetic heterogeneity and the trait is mainly polygenic. However, large numbers of such pedigrees will be necessary to detect all major loci. The family-based tests of association found the same major loci as the linkage analyses and detected low-frequency loci with moderate effect sizes, but control of type I error was not as stringent. Family studies have been an integral part of genetic research since the 1950s. This useful and powerful study design fell out of favor with the advent of genome-wide association studies (GWAS), which focus on unrelated, population-based study designs. However, advances in the field of next-generation sequencing techniques have resurrected family-based designs as an alternative to the population-based approach. In this study, we evaluate methods from the linkage analysis and family-based association analysis era and apply these methods to the analysis of the Genetic Analysis Workshop 17 (GAW17) mini-exome sequence data . After aIn the simulation process, the 202 founders of the eight extended pedigrees were a random sample from the 697 individuals in the simulated unrelated data set, so only a subset of the total trait-generating SVs were present in the family data, with rare variants being particularly underrepresented . The simPairwise identity-by-descent (IBD) sharing values were provided by GAW17 for a fully informative marker at the location of each gene in the mini-exome data. Because the actual genotypes of this set of highly informative linkage markers were not provided, we were not able to analyze the fully informative marker data using the standard linkage programs used for the SVs. Instead, we used a modified Haseman-Elston regression to analyze relative pairs. We selected sib pairs and grandparent-grandchild pairs based on phenotype data from replicate 1. For the qualitative trait, we selected all the discordant sib pairs and a subset of concordant affected and concordant unaffected sib pairs. For the quantitative traits we selected all possible sib pairs. We analyzed all possible grandparent-grandchild pairs. In replicate 1, we performed standard logistic regression of the qualitative trait  and standard linear regression of the squared difference in phenotype values of the members of each pair for the quantitative traits, using the provided IBD sharing values for the pairs as the independent variables.Because of the large size of the pedigrees, the Lander-Green algorithm implemented in Merlin could not analyze the entire pedigrees. Pedigrees were broken down into nuclear families with Sib-Pair . We analDD/Dd and dd, respectively. For Q1, the model was frequency of D = 0.01, mean DD = 1, mean Dd = 0, mean dd = \u22121, common variance = 1. The number of causal loci that exhibited GWS evidence of linkage (LOD \u2265 3.3) and suggestive linkage (LOD \u2265 1.9) are reported. Time and computer program constraints did not allow for multipoint analysis on replicate 1 or for single-point analysis to be performed on all replicates.The Elston-Stewart algorithm in FastLink ,5 is ablGenotypes for 13,784 nonmonomorphic sequence variants were used as given (uncollapsed), coded as the number of minor alleles, and also with rare variants collapsed by a method based on the work of Li and Leal  and Morrp-values were obtained by performing a Bonferroni correction based on the number of SV markers analyzed per collapsing method .We used ROMPrev version0.2 , to estiWe used ASSOC  in the eVEGFA and VEGFC were the only nonmonomorphic causal variants with a major effect (generating model \u03b2 > 1) on Q1. BCHE contained the only nonmonomorphic (but rare) SV with a major effect on Q2. There were no nonmonomorphic SVs with major effects on the qualitative trait except for the three SVs acting through the effects of Q1 and Q2 on the qualitative trait.The family data did not include all possible causal and noncausal variants because of selection resulting from random sampling of the founders . In the For replicate 1 for Q1 and Q2 using sib pairs and grandparent-grandchild pairs, we observed many peaks greater than the 2.2 threshold for suggestive linkage for sib pairs  or the 1p-values less than the 1 \u00d7 10\u22123 threshold for suggestive linkage, but only one signal was due to a causal variant. For Q2, no SVs were significant at p = 1 \u00d7 10\u22123. In Q4, one SV was significant at p < 1 \u00d7 10\u22123. Again, no GWS results were observed  had a major locus effect (\u03b2 > 1) on Q1 in these families. In replicate 1, using single-point Elson-Stewart linkage analyses in complete pedigrees, both SVs were detected at genome-wide significance . However, for both rare and common variants with smaller effect sizes on Q1, no suggestive or GWS linkage signals were detectable in replicate 1 in these eight large families. No SVs that contributed to the qualitative trait were detected at genome-wide significance   for the two major loci for Q1 that had variants segregating in the families. Although time constraints did not allow this analysis to be performed on all replicates, comparison of this result to the total lack of power in sib pairs, grandparent-grandchild pairs, and nuclear families in replicate 1 suggests that the well-known improvement in linkage power when using extended pedigrees holds true for analysis of SVs with major gene effects, even in the presence of extreme genetic heterogeneity and polygenic effects. In all linkage methods except VC analysis, false-positive rates were well controlled at GWS levels but not at suggestive levels, another classic result. More large pedigrees would be needed to find the major loci not represented in these families as a result of locus heterogeneity and to detect moderate effect loci, but these results show that linkage can be powerful to detect oligogenic effects on variance of a quantitative trait even when the causal variant is rare in the population.Family-based tests of association were powerful only for detecting association of genes that contained SVs of large effect in this small number of families. However, they did detect some SVs of moderate effect in a small percentage of replicates. Larger sample sizes should improve power. An important observation is that one GWS type I error was found in 50% of replicates, suggesting that associations that are only occasionally replicated may still be false positives. ROMP and ASSOC had comparable results. ROMP has the advantage that it does not require genotyping of the parents, which would greatly reduce sequencing costs. However, ROMP had more GWS false positives because it treats related trios as independent. The type I error rate in ROMP is a known problem that can be addressed easily with permutation testing because of the speed of the method.One of the approaches suggested for the analysis of high-throughput sequencing data in families is to use extended pedigrees and classic linkage analysis methods for rare variant sequence analysis. This appears to be useful for detecting major loci and for controlling type I error. However, certain aspects of these methods are problematic. First, for most current multipoint linkage methods, intermarker linkage disequilibrium must be removed by dropping single-nucleotide polymorphisms, if all the founders in the family are not genotyped. Second, the combination of a large number of markers with large pedigree sizes is cumbersome. Two-point linkage using the Elston-Stewart algorithm performed well, but most implementations do not easily allow analysis of large numbers of markers and this algorithm is not computationally feasible for more than two or three markers in multipoint linkage. Large extended pedigrees are most powerful for detecting major loci, but the Lander-Green algorithm, which is able to handle large numbers of markers in multipoint linkage, does not scale well for large pedigree sizes. In real data, the large size of pedigrees with a full exome- or genome-wide set of variants would represent a significant analytical challenge. Variance components methods on extended pedigrees using SOLAR or methods that calculate approximations to Elston-Stewart likelihoods, such as SimWalk2, are computationally challenging but may be feasible for sequence data in extended pedigrees given powerful computing resources. More method development is clearly needed.Overall, the ASSOC and ROMP association methods appear promising. Family-based association tests, which can leverage both family structure and linkage disequilibrium to their advantage, were able to find the same loci as the linkage analyses. Again, those variants with the largest effect sizes were the ones detected at GWS levels. Reducing the significance threshold inflated the false-positive rate without increasing the number of real signals found. Our results underline the importance of correcting for multiple testing and replicating significant results for control of family-wise error.Family-based association study designs offer several advantages over population-based designs: better control of population stratification, enrichment of rare variants, and the ability to discriminate relevant variants that segregate with the trait. The downside to these designs is that many analysis methods such as ASSOC, although powerful, require sequencing of many family members, which increases the cost of analysis. Some family-based association methods can reduce sequencing costs by using only parent-offspring trios or distantly related affected pairs. For continuous traits, ROMP is a powerful method that reduces the number of individuals who need to be sequenced, because it requires only phenotype information on the parents.For traits such as those simulated for GAW17, which exhibit extreme genetic heterogeneity and mostly polygenic effects on variation, a much larger sample would be required to detect more of the causal SVs using either linkage or family-based association methods. The GAW17 simulation assumed that exome sequencing was performed on 697 individuals in 8 extended pedigrees. At current whole-exome sequencing prices, this would be an expensive study. However, sequencing costs are falling rapidly. It is not unreasonable to believe that within the next few years the costs of a whole-exome sequence will fall below $1,000 and may approach $500 per person, making large family studies feasible. Sequencing a few distant relatives, followed by genotyping of all shared rare variants, is an alternative strategy that can take advantage of the strengths of family-based sequencing studies while controlling costs.The authors declare that there are no competing interests.All authors contributed to the design of the study. CLS did the HWE tests and estimated allele frequencies in the families, performed the Haseman-Elston regression (assisted by TG and DL), split the pedigrees into nuclear pedigrees, performed Lander-Green linkage analyses. RW performed the variance-components linkage analysis. JEBW performed the Elston-Stewart linkage analyses. CMJ carried out the ASSOC family based association analyses. MK performed the ROMP family based association analyses. AFW and CMJ planned the family based association analyses. HS collapsed the rare variants for the association analyses. JC produced the residuals of the traits adjusted for the covariates. CLS and CMJ drafted the paper, all authors edited the drafts and read and approved the final manuscript."}
+{"text": "Family history is a major risk factor for colorectal cancer and many families segregate the disease as a seemingly monogenic trait. A minority of familial colorectal cancer could be explained by known monogenic genes and genetic loci. Familial polyposis and Lynch syndrome are two syndromes where the predisposing genes are known but numerous families have been tested without finding the predisposing gene. We performed a genome wide linkage analysis in 121 colorectal families with an increased risk of colorectal cancer. The families were ascertained from the department of clinical genetics at the Karolinska University Hospital in Stockholm, Sweden and were considered negative for Familial Polyposis and Lynch syndrome. In total 600 subjects were genotyped using single nucleotide polymorphism array chips. Parametric- and non-parametric linkage analyses were computed using MERLIN in all and subsets of families. No statistically significant result was seen, however, there were suggestive positive HLODs above two in parametric linkage analysis. This was observed in a recessive model for high-risk families, at locus 9q31.1  and for moderate-risk families, at locus Xp22.33 . Using families with early-onset, recessive analysis suggested one locus on 4p16.3  and one on 17p13.2 . No NPL score above two was seen for any of the families. Our linkage study provided additional support for the previously suggested region on chromosome 9 and suggested additional loci to be involved in colorectal cancer risk. Sequencing of genes in the regions will be done in future studies. Colorectal cancer (CRC) is increasing in incidence and is ranked as the second and third most common cancer type in the western world and Sweden respectively. CRC has a lifetime risk of 5% and affects men and women equally. One major risk factor is a family history of the disease and 20-25% of all CRC cases have a close relative with the same disease . Known sThe study was undertaken in agreement with the Swedish legislation of ethical permission and according to the decision in the Stockholm regional ethical committee (2008/125-31.2). All participants gave written informed consent to participate in the study.The families were ascertained through the clinical genetics department at the Karolinska University Hospital in Stockholm, Sweden between 1990 and 2005. FAP was excluded using medical records from affected individuals and Lynch syndrome was excluded using our current clinical protocol . FamilieGenomic DNA was extracted from peripheral blood using standard procedures. Genotyping was done separately in two different sets of family material. Genotyping of 548 patients with 6090 markers was performed by the Illumina Infinium assay ,29 usingPedcheck  was usedIndividuals with colorectal cancer or a polyp with high degree dysplasia were coded as affected. Family members with unclear status were coded as unknown. Families were ascertained assuming a dominant trait and spouses were therefore coded as unaffected. Four different analyses were performed using different sets of families and patients; all families, all families with at least three cases (high-risk), all families with CRC among sibs (moderate-risk) and families with a mean age below 50 (early-onset) to be comparable with the results from the recent linkage study by Cicek et al.All 121 families, including all subjects from both genotyping sessions, were used for linkage analysis. Thus, two marker files were merged and 7256 markers were used in the analysis. Merlin by default allows a maximum of 24 bits for each family, why four large families had to be split. The families were split so that each sub-family used one common ancestor and fitted into the limit as defined while running the program. The original 121 families were analysed as 126 (one family had to be split into three). 2 = 0.1 has been used to avoid that false positive results inflate the statistics [2>0.1 was accounted for, by MERLIN organizing the markers into clusters. MERLIN makes use of the population haplotype frequencies to assume LD within each cluster. To maintain uniformity in our study subsets, the same clusters were continuously used in all analysis. Since presence of linkage disequilibrium (LD) may inflate multipoint linkage statistics, a threshold of ratistics . LD amonA total of 600 individuals from 121 families were successfully genotyped. The analyses were conducted for all and each of three different subgroups; high-risk, moderate-risk and early-onset families . There wFor high-risk families  one locus on chromosome 9q31.1, showed an HLOD above two assuming recessive inheritance and an estimated 66% of families linked . MaximumFor moderate-risk families  one locus on the tip on chromosome Xp had LOD and HLOD above two with maximum 2.2 and 2.5 respectively, for the marker rs2306737 in recessive analysis . LOD scoFinally, for the group of families with early-onset (only 8 families) two loci showed positive LOD scores and HLODs above two in recessive analysis . One wasNo NPL score above two was seen for any of the families. HLODs above 2 are presented in We used SNP genotyping to perform a linkage analysis in 121 CRC families and did not find any overall statistically significant results with a LOD or HLOD over 3. A few large families were split to be able to use MERLIN, and lost a bit of its power in the analysis. The effect of this was of little importance. However, we did find LODs and HLODs above 2, suggestive of linkage. A previous linkage study used 356 families and showed one locus with HLOD > 3 (12q) and 4 with HLOD > 2 , all in dominant analysis . We founIn our substudy of large, high-risk families (more than three affecteds), a locus on chromosome 9q31 was found. The same region has been suggested before, although in dominant models, and was here again identified by us using linkage analysis in a recessive model. This locus was previously suggested by one sib-pair study and one study using linkage in many kindred and also previously by us in one large family with rectal cancer and adenomas -20. ThosFor moderate-risk families, with affected sibs only, one region on the tip on chromosome X was suggested in recessive model (max HLOD=2.2) and similar to the chromosome 9 locus there was also a positive but lower LOD using a dominant model (0.7). This region is almost 6 Mb, has not been suggested before and contains numerous candidate genes. The Cicek study included 200 moderate risk families and also using recessive model they identified one locus with an HLOD>3 (8q) and 4 loci with an HLOD>2 . We could not find support for any of those loci. Finally we observed using a recessive model two loci with high HLODs (4p and 17p) for the early-onset families. However, this group consisted of only 8 families. Family 8 was also among the high-risk families linked to chromosome 9 locus and for this size of family it is expected that a whole genome study should generate linkage to many regions and thus most will be false positives. We could not replicate any of the loci from Cicek et al. using dominant or recessive model .A previous study focusing on adenoma and colorectal adenoma and carcinoma found linkage to chromosomal regions on chromosomes 18q21 and 2p22 in 69 families while a sub-analysis of 55 families with cancer only showed linkage to chromosome 3q21-24,35. TherSlightly to our surprise we could see also positive parametric LOD-scores in this study compared to our previous ones ,23 whereIn conclusion, our linkage study provided additional support for the region on chromosome 9, some evidence for new loci to be involved in colorectal cancer risk and no support for other previously reported loci. This suggested heterogeneity if familial CRC should influence the design of future association and linkage studies."}
+{"text": "The aim of this study was to map the disease locus of autosomal dominant cataracts (ADC) in a Chinese family.A four-generation family with multiple individuals affected by ADC was investigated. Genomic DNA was collected from 22 family members. A gene-scan at known candidate ADC loci was performed. To achieve fine-mapping we genotyped fourteen STR markers at the critical region of 19q.The two-point logarithm of odds (LOD) score was calculated using Linkage Software Package Version 5.1 for linkage analysis. The haplotype was constructed using Cyrillic software.max=4.25) was obtained at the D19S877 (\u03b8=0). Haplotype analysis also confirmed the locus and further narrowed it down to a critical interval from the marker D19S924 to the 19qter.Ten members of this Chinese family were affected by nuclear cataracts. Initially,  linkage analysis revealed a significant LOD score of 3.82 at the STR marker D19S418. Subsequently, after refine-marker analysis, a maximum LOD score (ZWe have successfully mapped an ADC locus to 19q13-qter. Previous studies have identified three cataract loci on 19q; however, we found no overlap between the locus of this study and any of the previously identified loci. We therefore suggest that the 19q13-qter locus in this family is a new locus for ADC. Cataract is a disease associated with opacity of the ocular lens. Along with the cornea, the lens is the organ that transmits and refracts light. Also, the lens is the only tissue in mammals capable of accurately focusing light onto the retina through a process called accommodation . TherefoThe phenotype (or spectrum of morphology) for cataracts is variable and complex. However, according to the location of the opacification the cataract can be categorized as embryonic nucleus, fetal nucleus, cortex, or pole. Of these types, opacification affecting the nucleus is the most common .According to the age of onset, cataracts can be categorized as: 1) congenital cataract:  onset age is less than one year; (2) juvenile: onset-age is from 1 to 10 years; 3) presenile cataract: onset-age is above 10 and up to to 45 years; (4) age-related cataract: above 45 years. Of these, about 25 percent of the congenital and juvenile cataracts are believed to be inherited [ congenit presenil110700) in 1963  [Inherited cataracts, transmitted with the inheritance of autosomal dominant (AD), autosomal recessive (AR) and X-linked (XL) genes , are a gin 1963  ; the DufLIM2 (AR) [Here, we report on a Chinese family affected by inherited, isolated nuclear cataracts. In our study, we present evidence linking the family\u2019s disorder to chromosome 19q. Refined analysis of STR-linkage and haplotype construction have further confirmed the autosomal dominant cataracts locus and narrowed it to 19q13-qter. Previous studies have identified three isolated cataract loci on 19q; these include the 19q12-q13.1  gene, between markers D19S928 and D19S425) , the 19qA four-generation family with inherited isolated cataract was investigated . Twenty-Marshfield Clinic Human Genetics linkage map) at 19q were synthesized with fluorescein-labeled 5\u2032. For selected markers, multiplex PCRs were performed in a 5.0 \u03bcl reaction mixture containing 25 ng of genomic DNA, 1\u00d7PCR buffer, 100 umol of each dNTP, 3.0 mmol MgCl2, 80 pmol each of forward and reverse primers and 0.2 U of AmpliTaq Gold DNA polymerase. The mixture, including reaction products 1.0\u00a0\u03bcl, Liz Size Standard-500 0.2 \u03bcl and Hi-Di formylamine 9 \u03bcl, was denatured, electrophoresed and visualized on a 3130 Genetic Analyzer (Applied Biosystems). The allele size was analyzed using Genescan analysis software V3.7 and Genotyper software V3.7 (Applied Biosystems).Initially, a part-genome scan was performed with highly polymorphic STR markers  at the known ADC candidate loci, including 1p36, 1p32, 1q21-q25, 2p24, 2p12, 2q33-q35, 3q21-q22, 3q26-qter, 8q13.3, 10q25, 11q23-q24, 12q12-q14, 13q11\u201313, 15q21-q22, 16q22-q23, 17p13, 17q11-q12, 17q24, 19q13, 20p12.2-p11.23, 21q, and 22q. Subsequently, fourteen refining primers of short tandem repeat (STR) markers . The disease was an assumed autosomal dominant trait with 98% penetrance. Marker allele frequencies were set at 1/n, where n is the number of alleles observed. We assumed gene frequencies of 0.0001 and no gender-based difference in rates of recombination. Haplotype construction was then performed to define the borders of the cosegregating region using the Cyrillic program.A four-generation Chinese family  was invefamily to an interval of 4.9Mb, from the D19S924 to 19qter.Twenty-two members of the family, including ten affected individuals and twelve unaffected individuals, were genotyped with microsatellite markers at the following 22 ADC loci: 1p36, 1p32, 1q21-q25, 2p24, 2p12, 2q33-q35, 3q21-q22, 3q26-qter, 8q13.3, 10q25, 11q23-q24, 12q12-q14, 13q11\u201313, 15q21-q22, 16q22-q23, 17p13, 17q11-q12, 17q24, 19q13, 20p12.2-p11.23, 21q and 22q. Using two-point linkage analysis, we excluded all the loci mentioned above (data not shown) except for 19q. In the 19q locus a significant logarithm of odds (LOD) score was initially found at the marker D19S418 . To confirm this finding, we subsequently synthesized fourteen pairs of refining primers (some of the STRs are low-informative). The following genotyping and linkage analysis of the refining markers showed a maximum LOD score of 4.25 and 3.99 at markers D19S877 and D19S544 (\u03b8=0), respectively . EnsuingIn this study, we report on a Chinese family affected by inherited autosomal dominant nuclear cataract. We extracted gDNA samples from 22 family members and performed linkage analysis of the known-candidate ADC loci using the STR markers. The results excluded all known loci except for 19q. A maximum LOD SCORE of 4.25  was obtaLIM2\u2014an identified ARC gene [LIM2 gene. Therefore, the chromosome 19q  is perhaps a hotspot for  cataract pathogenesis. To make the 19q cataract loci clear, we have illustrated them  locus is a new ADC locus. Further study is needed to identify the disease-causing gene and provide new insights into the molecular mechanisms of the cataractogenesis occurring in this family."}
+{"text": "The need to integrate information from multiple linkage maps is a long-standing problem in genetics. One way to visualize the complex ordinal relationships is with a directed graph, where each vertex in the graph is a bin of markers. When there are no ordering conflicts between the linkage maps, the result is a directed acyclic graph, or DAG, which can then be linearized to produce a consensus map.New algorithms for the simplification and linearization of consensus graphs have been implemented as a package for the R computing environment called DAGGER. The simplified consensus graphs produced by DAGGER exactly capture the ordinal relationships present in a series of linkage maps. Using either linear or quadratic programming, DAGGER generates a consensus map with minimum error relative to the linkage maps while remaining ordinally consistent with them. Both linearization methods produce consensus maps that are compressed relative to the mean of the linkage maps. After rescaling, however, the consensus maps had higher accuracy (and higher marker density) than the individual linkage maps in genetic simulations. When applied to four barley linkage maps genotyped at nearly 3000 SNP markers, DAGGER produced a consensus map with improved fine structure compared to the existing barley consensus SNP map. The root-mean-squared error between the linkage maps and the DAGGER map was 0.82 cM per marker interval compared to 2.28 cM for the existing consensus map. Examination of the barley hardness locus at the 5HS telomere, for which there is a physical map, confirmed that the DAGGER output was more accurate for fine structure analysis.The R package DAGGER is an effective, freely available resource for integrating the information from a set of consistent linkage maps. The need to integrate information from multiple linkage maps into a consensus map is a long-standing problem in genetics ,2. ConseWenzl et al.  differenv to vertex w in the graph if and only if bin v comes before bin w in one of the linkage maps.It was Yap et al.  who firsWu et al.  have capClose et al.  used MerThe discovery that the simplified consensus graphs produced by MergeMap are not ordinally equivalent to the linkage maps prompted the development of new algorithms for the simplification and linearization of consensus graphs. These algorithms have been implemented as a package for the R computing environment  called DM that is to be integrated with graph G. Let v Sbe the set of vertices in G that contain one or more markers in bin v of M. For each vertex w in vS, if all of the markers in w are contained in v, then w remains intact. If only some of the markers in w are in v, then w is split into two vertices: w1 contains the common markers between v and w, and w2 contains the remaining markers in w. All of the edges directed in and out of w are replicated for w1 and w2. Vertex w1 also receives new edges directed in and out of it according to the immediately proximal and distal bins in M. Any markers in v that were not present in G are added as a new vertex with appropriate edges. When completed, the consensus graph contains a directed edge for every map interval between adjacent bins in the linkage maps . Although depicted in the consensus graph Figure , the zerv and w are strongly connected if there is a path from v to w and from w to v. In the context of markers, that would mean the markers in v mapped before those in w, which in turn mapped before those in v; this represents an inconsistency between the linkage maps.DAGGER checks for ordering conflicts between the linkage maps by identifying the strongly connected components of the consensus graph. Two vertices R G(formed by reversing the edges in G), and then on G itself. The traversal of R Ggenerates a topologically sorted list of the vertices. By conducting the depth-first search of G in this order, the algorithm identifies the strongly connected components of G.Identifying the strongly connected components of a directed graph is a standard exercise in computer science . DAGGER G for visualization with the Graphviz dot software )L1 norm (mean absolute error) or L2 norm (root-mean-squared [RMS] error). With only one linkage map the average RMS error was 4.7 (SE 0.2) cM, while with eight linkage maps the average RMS error was 2.4 (SE 0.1) cM for the QP method. Using either norm, the QP maps had consistently less error than the LP maps .Table The original impetus for developing DAGGER came from analyzing the results of Close et al. , who usehinb/hina/gsp from the distal end.Figure hinb/hina/gsp markers in the DAGGER graph are that marker 3_0984(hinb) is distal to markers 3_0975(gsp), 3_0979(hina), and 3_0977(gsp). Table None of the ordinal information in the DAGGER graph violates this physical order. The only relationships among the hinb/hina/gsp markers. MergeMap shows the markers 3_0976(gsp), 3_0978(hina), 3_0980(hina), and 2_0226(hina) as mapping distal to both hinb markers, and the marker 3_0975(gsp) is shown distal to 3_0979(hina). The order of these markers is indeterminate from the linkage maps, which is how they are portrayed by DAGGER. The unphysical relationships in the simplified graph from MergeMap arise because of the way markers are binned. MergeMap will potentially bin markers if they are binned in at least one of the linkage maps and if their order is indeterminate  makes a full column rank matrix \u00c3 . With this addition, the combined adjacency matrix \u00c3 on B) is also full column rank, and the quadratic form is positive definite DAGGER uses the R packages quadprog  and Rglpg linkage maps, 2g parents with a fixed average level of sequence identity were randomly generated and paired to create g doubled haploid populations. For each population, gamete formation was simulated for 200 individuals assuming no crossover interference. Linkage maps were constructed using Haldane's mapping function and a LOD-score weighted-least-squares approach [To simulate approach . The addStatistical analysis was conducted using PROC MIXED in SAS 9.2 , with each simulation as \"subject\" to properly model the covariance structure (LP and QP consensus maps were generated for each simulation).When the four linkage maps published by Close et al.  were usede novo results generated by submitting the four linkage maps to MergeMap Online [de novo MergeMap output. The results in this manuscript are for the de novo MergeMap output.Comparisons were made to both the published results of Close et al.  as well p Online  on 23 Fep Online  was 2.23AH014393.1[The physical map in Figure H014393.1. The posH014393.1, with thH014393.1. The conH014393.1.DAG: directed acyclic graph; LP: linear program(ming); QP: quadratic program(ming); RMS: root-mean-squared; SNP: single nucleotide polymorphism; SE: standard error.Figure S1. Consensus graph for chromosome 1H.Click here for fileFigure S2. Consensus graph for chromosome 2H.Click here for fileFigure S3. Consensus graph for chromosome 3H.Click here for fileFigure S4. Consensus graph for chromosome 4H.Click here for fileFigure S5. Consensus graph for chromosome 5H.Click here for fileFigure S6. Consensus graph for chromosome 6H.Click here for fileFigure S7. Consensus graph for chromosome 7H.Click here for fileTable S1. Consensus map for the entire genome.Click here for file"}
+{"text": "To describe the phenotype of a family with an autosomal dominant macular dystrophy and identify the chromosomal location of the gene that causes this phenotype.Twelve members of a three-generation family underwent routine clinical examination, including fundus photography. Four of the patients underwent extended examination with Goldmann perimetry, full-field electroretinogram, dark adaptation, and color vision testing, and two patients underwent optical coherence tomography and fundus autofluorescence examination. DNA samples were obtained from 12 family members and 3 spouses and genotyped at the known North Carolina Macular Dystrophy (NCMD) locus on chromosome 6q (MCDR1: OMIM 136550) using short tandem repeat polymorphisms. DNA samples were subsequently examined with a genome-wide scan of single nucleotide polymorphisms and the genotypes that were produced were studied with linkage and haplotype analyses.The 10 affected family members had clinical findings of macular lesions that are typical for NCMD. The small drusen-like yellowish lesions of mild NCMD were hyperautofluorescent. Hyperpigmented foveal lesions were surrounded by a zone of confluent hyperautofluorescence. Linkage analysis of short tandem repeat polymorphism genetic markers excluded the NCMD locus on chromosome 6. However, analysis of single nucleotide polymorphism genotypes from a genome-wide scan showed that NCMD in our pedigree is linked to a region on chromosome 5p that overlaps the previously mapped macular dystrophy (MCDR3) locus with a maximum log of the odds (LOD) score of 2.69 at a recombination fraction of 0.00 .We report the first pedigree with NCMD from Scandinavia, and the first confirmation that a gene for this condition is located on chromosome 5p13-p15. The bright elements or lesions typical of NCMD differed from drusen in that no sign of accumulation of material between the retinal pigment epithelium and Bruch\u2019s membrane was seen. While the present study has found indications that the elements are located in the outermost layers of the retina, their precise location remains to be identified directly. North Carolina macular dystrophy (NCMD) is an autosomal dominant dystrophy that was first reported as hereditary macular degeneration and aminoaciduria by Lefler, Wadworth, and Sidbury . ExtensiThe main characteristics of the disease consist of: symmetric, tiny drusen-like, yellowish deposits confined to the foveal region with little or no impact on visual acuity (Grade 1) ; conflue136550) on chromosome 6q16 [608850) [Linkage studies of NCMD pedigrees mapped a disease-causing gene to a locus  . Further[608850) . Lastly,[608850) . Here, wThe study adhered to the Declaration of Helsinki and was approved by the local ethics committee. All patients gave their written consent after being informed about the purpose and implications of the investigation.Two patients from the same family were independently referred to the National Eye Clinic for the Visually Impaired (NEC) for diagnostic examination. The findings initiated a supplementary examination of additional family members. All patients underwent a standard examination, including a review of their visual symptoms; assessment of refraction, Snellen visual acuity, ocular alignment and motility; slit lamp examination; ophthalmoscopy; and color fundus photography of macular lesions. Some patients underwent supplementary examination with Goldmann perimetry (object size IV4e and I4e); color vision screening (Ishihara\u2019s 38 pseudoisochromatic plates and Lanthony tritan plates); and full-field ERG , according to the recommendations by the International Society for Clinical Electrophysiology and Vision . In addiSty1 array of the GeneChip Human Mapping 500K Array Set; Affymetrix, Santa Clara, CA), which interrogate 238,304 single nucleotide polymorphisms (SNPs). Sample processing and labeling were performed using the manufacturer\u2019s instructions. The arrays were hybridized, washed, and scanned in the University of Iowa DNA core facility. Array images were processed with GeneChip DNA Analysis software (Affymetrix). Microarray data were analyzed and multipoint nonparametric linkage scores were calculated using the EasyLinkage software package [MLINK and LODSCORE programs, as implemented in the FASTLINK (v2.3) version of the LINKAGE software package [max for D51987 was 2.69 when the \u201caffected\u201d allele frequency was arbitrarily set to 50%, suggesting that linkage is present despite assumptions about allele frequencies.Pedigree members were first genotyped with short tandem repeat polymorphism (STRP) genetic markers flanking the MCDR1 locus. Genotyping with STRP genetic markers was conducted using standard methods, as previously described . Twelve  package . Pairwis package -28. PeneMembers of a three-generation pedigree  receivedThe proband, a 13-year-old school girl , III-3, The proband\u2019s paternal uncle , II-6 haThe proband (III-3) and her paternal uncle (II-6) were also examined with fundus autofluorescence imaging using confocal scanning laser ophthalmoscopy and optical coherence tomography OCT, . The brirs13154455. The linkage to 5p was confirmed by genotyping all 10 affected members of the pedigree with seven STRP markers that span the region  was ruled out using a panel of STRP markers (data not shown). Next, a genome-wide scan for linkage was conducted using microarrays of SNP genetic markers on 9 of the 10 affected family members. Analysis of the SNP genotypes identified a region of chromosome 5p with a maximum nonparametric linkage score of 9.88 (p=0.0001). The affected family members shared an allele of 1081 consecutive SNPs that span 13.96 Mbp between the telomere and e region . The maxThe linked region for our pedigree partially overlaps with the previously described NCMD locus, MCDR3 , and comThe NCMD phenotype is characterized by diverse macular lesions: tiny, drusen-like material in the macular region, foveal pigment epithelial atrophy, or bilateral coloboma-like scars with hyperpigmentations. The lesions are either congenital or appear in early infancy, and the visual acuity in the best eye is often astonishingly good compared with the extent of the macular affection. Among our patients, only one  had gradWhile in most pedigrees NCMD is linked to the chromosome 6q locus MCDR1 , the macMinor phenotypic differences between NCMD that maps to MCDR1 and that which maps to MCDR3 have been reported. Color vision defects were detected in members of the original MCDR3 pedigree, while pedigrees linked to MCDR1 have normal color vision. Progression of disease has been reported in a single member of the original MCDR3 pedigree, but its rare occurrence does not seem to differentiate between pedigrees linked to the MCDR1 and MCDR3 loci ,19. OverThe pathogenesis of NCMD is poorly understood. Histopathological studies have provided some insight. Light microscopy of the eyes from a 72-year-old woman with grade 2 NCMD showed complete loss of photoreceptor cells and the underlying RPE, Bruch\u2019s membrane attenuation, and marked atrophy of the choriocapillaris ,30. The Structural elements of the retina that appear bright in infrared fundus photographs should show up on infrared OCT, but in the case of NCMD elements, the lateral resolution of the instrument may be insufficient to resolve the individual elements. The lack of abnormal hyperreflectivity in the neurosensory retina in the young patient we investigated by spectral-domain OCT suggests that the bright elements could be located in the RPE and that their presence could be masked by the high reflectivity of the normal RPE. The bumpy, irregular contour of the RPE noted on several of the OCT scans also suggests that the photoreceptor outer segment/RPE- complex is the primary target of disease in NCMD. This would be compatible with the eventual development of geographic outer retinal atrophy in the later stages of the disease.Decaying tissue of the outer retina is often seen outside the rim of a chorioretinal scar such as the central hyperpigmented scar in patient II-6. Such bright, hyperautofluorescent, amorphous material may be a nonspecific product of cell death.In this report, we present a second NCMD family linked to chromosome 5p that confirms the previous discovery of the MCDR3 locus. Combining the linkage studies of our family and the original MCDR3 produced a new critical region of 12.75 Mbp that contains a gene that causes NCMD. These results represent important steps toward the discovery of genes that are capable of causing NCMD, which will provide novel insights into the pathogenesis of this condition."}
+{"text": "Alternatively, one can study the genetics of intermediate traits that are known risk factors for CVD, which can be measured quantitatively. Using the latter strategy, we have measured 21 cardiovascular-related biomarkers in an extended multigenerational pedigree, the CARRIAGE family . These biomarkers belong to inflammatory and immune, connective tissue, lipid, and hemostasis pathways. Of these, 18 met our quality control standards. Using the pedigree and biomarker data, we have estimated the broad sense heritability (H2) of each biomarker (ranging from 0.09\u20130.56). A genome-wide panel of 6,015 SNPs was used subsequently to map these biomarkers as quantitative traits. Four showed noteworthy evidence for linkage in multipoint analysis (LOD score \u2265 2.6): paraoxonase , the chemokine RANTES (22q13.33), matrix metalloproteinase 3 , and granulocyte colony stimulating factor . Identifying the causal variation underlying each linkage score will help to unravel the genetic architecture of these quantitative traits and, by extension, the genetic architecture of cardiovascular risk.Given the importance of cardiovascular disease (CVD) to public health and the demonstrated heritability of both disease status and its related risk factors, identifying the genetic variation underlying these susceptibilities is a critical step in understanding the pathogenesis of CVD and informing prevention and treatment strategies. Although one can look for genetic variation underlying susceptibility to CVD  Cardiovascular disease (CVD) is the leading cause of death, accounting for over 500,000 deaths per year in the United States and greater than seven million deaths worldwide. It has been estimated that over 82 million Americans are afflicted with at least one form of CVD and over 16 million Americans are afflicted with clinically significant CVD    , which was recently identified in a genome-wide association study (GWAS) for ulcerative colitis [IL17RE, it is very likely that it binds a cytokine and has an immunologic role and thus could affect levels of RANTES. This locus is linked to several traits including pulse pressure [The chemotactic cytokine RANTES recruits T-cells, eosinophils, and basophils to sites of inflammation and is therefore a likely participant in CVD through the contribution of inflammation to atherosclerotic plaque formation and response to plaque rupture. The region around the LOD score peak for RANTES (22q13) contains the gene  colitis . The ligpressure , bone mipressure , serum cpressure , rheumatpressure , schizoppressure , height pressure , and brepressure .SCARF1) and a highly conserved intracellular signaling protein (YWHAE). In addition, this region is linked to ventricular hypertrophy [Matrix metalloproteinase (MMP3) plasma concentrations are linked to increased risk of plaque rupture and, thus, to myocardial infarction . In our ertrophy , childhoertrophy , and rheertrophy , among oertrophy , schizopertrophy , readingertrophy , pulse pertrophy , and earertrophy . The thiertrophy  and stroertrophy .RUNX1T1. Although there are no data specifically suggesting this, one might postulate regulatory networks in hematopoiesis wherein alterations in RUNX1T1 function or expression impact GCSF levels. Other studies have reported linkage at 8q22 for hypertension [GCSF is a growth factor and a cytokine which, in addition to its obvious role in promoting the growth of granulocytes, also promotes the growth of stem cells and their release from the bone marrow and has been implicated in the response of vascular endothelial cells to oxidative stress . We obsertension , dihydrortension , and Tourtension , althougThe significant heritabilities of these CVD biomarkers are not necessarily surprising, as they are potential predictors or risk factors of CVD, and CVD itself has a relatively strong genetic component (i.e. heritability of 0.38-0.57 ). A signin cis and possibly allelic variation in the PON genes themselves, as has been previously observed [in trans could contribute in some significant way to variability in paraoxonase levels. The role of regulation in trans is underscored by the fact that, of the remaining LOD scores \u2265 2.6 , none were coincident with the physical loci encoding the biomarker. Thus, just by examining the locations of our LOD scores relative to the loci directly encoding the biomarker in question, we were able to unravel some of the genetic architecture of the trait. Interestingly, we did not find overlap between the only interesting QTL for hsCRP in our study  with other published genomic regions linked to and/or associated with hsCRP levels  on chromosome 7q. In our study, we had linkage to this region on 7q, suggesting regulation observed . Howeverls ; CRP lels ; this m2 > 0.4) between any of the SNPs in our most significant linkage peaks (data not shown). The genome scans themselves were conducted under the assumption that the biomarkers were not correlated and that each genome scan was a set of independent tests. However, the level of inter-marker correlations between biomarkers could invalidate that assumption and imposes a multiple testing burden that could influence the significance interpretation of our LOD scores. Identifying all MLODs greater than or equal to two as \u201cinteresting\u201d resulted in 15 loci, translating to only 3.7% of the MLODS across all biomarkers and the 22 autosomes. Even so, any of the 15 highlighted results could be type I errors. However, our intent was to highlight the regions providing the most evidence for linkage in our study. Finally, there is no single method that allows for maximal power while using all measurements of the quantitative trait, including extreme outliers. In the current study, we have elected to remove extreme values at either range of the quantitative measure, thus creating distributions that are closer to normality. In so doing, it is possible that some biologically meaningful information has been lost. Other analytic methods (such as the lodadj option in SOLAR) would allow those values to be included, but could also introduce compromises in power, particularly when the overall data does correspond well to a normal distribution.There are limitations to the current approach. Namely, the study was conducted in a population with a specific ancestry, primarily African and Native American and therefore the genetic loci detected in our genome screen may not be applicable to other ethnicities and populations. However, in some ways this \u2018limitation\u2019 is also a great strength, as African Americans are an understudied population at high risk for CVD. Furthermore, it is not known to what extent a primarily African American sample would be expected to share causal variation underlying biomarker levels with another ethnic group. In addition, it is possible that the presence of linkage disequilibrium (LD) between SNPs can inflate LOD scores and, as our population is of mixed African American descent, there exists the potential for the significant LD inherent in recently admixed populations. However, the SNPs selected for the genome scan were designed to limit LD between markers and there was no significant LD , subsequent evaluation of those genetic markers for association with CVD, and validation in further cohorts are necessary. The identification of the putative causal variants underlying the linkage results for these four biomarkers will not only advance our understanding of cardiovascular risk but hopefully serve as a model for the study of other complex diseases via the genetic dissection of intermediate traits.Table S1Percent of samples measured as below lower limits of quantification for a given biomarker assay.(DOCX)Click here for additional data file.Table S2Displayed are the maximum two-point LOD score at theta equal zero (MaxLOD) for each biomarker by chromosome, with the name of the probe (RS number), the centimorgan (cM) position of the probe, and the gene annotation for the SNP (or the closest gene for intergenic SNPs).(DOCX)Click here for additional data file.Figure S1Chromosome linkage plots for the most significant multipoint linkage peaks.(DOCX)Click here for additional data file.Figure S2Genome-wide autosomal multipoint linkage results for each biomarker .(DOCX)Click here for additional data file."}
+{"text": "Network analysis of cases and controls sharing haplotypes on chromosome 19 further strengthened the association as there are more large networks of cases sharing haplotypes than controls. This linkage region includes a cluster of zinc finger genes of unknown function. Analysis of genome wide transcriptome data suggests that genes in this zinc finger cluster may be involved in very early developmental regulation of the CNS. Our study also indicates that BEAGLE fastIBD allowed identification of rare variants in large unrelated population with moderate computational intensity. Even with the development of whole-genome sequencing, IBD mapping still may be a promising way to narrow down the region of interest for sequencing priority.Genome-wide association studies (GWAS) have identified around 60 common variants associated with multiple sclerosis (MS), but these loci only explain a fraction of the heritability of MS. Some missing heritability may be caused by rare variants that have been suggested to play an important role in the aetiology of complex diseases such as MS. However current genetic and statistical methods for detecting rare variants are expensive and time consuming. \u2018Population-based linkage analysis\u2019 (PBLA) or so called identity-by-descent (IBD) mapping is a novel way to detect rare variants in extant GWAS datasets. We employed BEAGLE fastIBD to search for rare MS variants utilising IBD mapping in a large GWAS dataset of 3,543 cases and 5,898 controls. We identified a genome-wide significant linkage signal on chromosome 19 (LOD\u200a=\u200a4.65; p\u200a=\u200a1.9\u00d710 HLA-DRB1*15:01 allele in the major histocompatibility complex (MHC), which was first detected in the 1970's Multiple sclerosis (MS) is a complex neurological disease of the central nervous system (CNS) triggered by environmental and genetic factors. There is considerable evidence for a significant genetic component to MS susceptibility, such as a higher concordance rate in monozygotic twins (24%\u201330%) than dizygotic twins (3%\u20135%) Despite this success, the variants identified by GWAS to date only explain 18\u201324% of the heritability of MS IFIH1 conferring protection to type I diabetes Standard analyses of GWAS data are not designed to detect associations with low frequency variants (MAF\u22645%), and other strategies are required. One approach is to re-sequence loci containing common susceptibility variants identified from GWAS studies. This strategy was used to detect rare variants in n like n log n instead of 2n.Several methods of IBD mapping have been published: these include PLINK \u22126; LOD\u200a=\u200a4.65) using thresholds based on IBD segment length greater than 3 cM and the probability p-value less than 10\u22129 (3cM_1e-9). This locus was deemed genome-wide significant according to the recently established genome-wide significance thresholds set for IBD mapping To detect MS rare variants, we here use BEAGLE fastIBD to perform an IBD analysis on several large MS GWAS datasets comprised 3543 cases and 5898 controls. We identified a region of high significance on chromosome 19q13.43, with a genome-wide significant localisation signal  \u22127) were discarded, as were samples with call rates less than 0.98. Duplicates and close relatives were also removed. This data cleaning was performed using PLINK.Conservative quality control measures were imposed both on the individual datasets before merging, and in the combined dataset after merging: SNPs with call rates less than 0.95 or in Hardy-Weinberg disequilibrium  was conducted by EIGENSTRAT http://faculty.washington.edu/browning/beagle/). In brief, genotypes for the merged, cleaned dataset were converted to BEAGLE format by using the linkage2Beagle.jar utility program. We then used the BEAGLE method for phasing the data and identifying IBD segments simultaneously, using the \u2018fastibdthreshold\u2019 option. This procedure was run 10 times for each chromosome starting with different seeds of the random number generator.The fastIBD analyses were conducted using BEAGLE (yi), case-control pairs (ui) and control-control pairs (vi) estimated to share haplotypes IBD at each SNP.The output of these calculations was a series of \u201cputative\u201d IBD segments shared between pairs of individuals. Each segment comes with the following information attached: ids for the pair of individuals, first and last SNPs in the IBD segment, length of the segment in centimorgans, and probability of the two individuals both carrying the segment if it was not IBD. We filtered these segments using various maximum probabilities and minimum segment lengths, as recommended in the BEAGLE manual. Results from the 10 runs were combined by taking the union of IBD segments detected in each run. From the final list of segments, we wrote a Perl script to count numbers of case-case pairs (yi in case-case pairs (\u201ccase pairs\u201d) as a function of IBD sharing in xi \u200a=\u200a ui + vi in case-control pairs and control-control pairs combined (\u201ccontrol pairs\u201d).We focused on the detection of loci where groups of cases have inherited rare susceptibility alleles IBD. To do this, we modelled IBD sharing yi as a function of the xi: linear regression, negative binomial regression and Poisson regression. Models were fitted using R SR_commands S1).We tried various methods to model the i with more IBD sharing in cases than expected, residuals zi from the fitted models should be large and positive. To present residuals on a scale more familiar to geneticists, we converted them to LOD scores using the formula LODi \u200a=\u200a zi2/(2*loge(10).At SNPs http://cran.rproject.org/web/packages/network/index.html).At the SNPs with the highest LOD scores, we calculated the proportions of case pairs sharing IBD in various populations, and plotted networks of case and control pairs sharing IBD with each other using the R network package . In summary, following cleaning there were 3,243 cases and 5,725 controls with 274,735 autosomal SNPs in the final analysis  were excluded. All datasets overlapped well after the removal of outliers . A strong linkage signal was observed in the HLA region (LOD\u200a=\u200a3.58), while the strongest signal in non-HLA region was on chromosome 19 (LOD\u200a=\u200a4.65), which reached genome-wide significance according to the recent established genome-wide significance threshold set for IBD mapping We detected IBD with the threshold of IBD segment greater than 3 cM and the haplotype probability p-value less than 10yi versus control-pair sharing xi as each of the 274,735 SNPs i. Using different colours to represent SNPs on different chromosomes, an outlier group of SNPs with relatively high case pair sharing on one chromosome stands out in green.Figure S2, S3, S4), we found that the Poisson model provided the best fit for these data. \u22126. As expected, a strong signal also was observed in the HLA region  and corresponds to a cluster of zinc finger genes at 19q13.4, many of which have arisen by gene duplication. None of the genes in this region have been previously identified in published GWAS or associated with MS or autoimmune diseases.\u22123) equivalent to a p value threshold of 0.01 and a fold change minimum 1.5. Those probe sets that passed this threshold were plotted on the UCSC browser screen view The genes in this region were examined to identify candidate genes with putative roles, which could, impact on susceptibility to MS. Published microarray expression data ZNF274 is a DNA binding protein involved in regulation of H3K9me3 methylation at the 3\u2032 end of some ZNF genes by recruitment of the histone methyltransferase SETDB1, and the corepressor TRIM28 (KAP1) KAP1, SETDB1 and ZNF274 binding in K562 cells KAP1 and SETDB1 at the 3\u2032 end of the gene. A small number of genes are also bound at the 3\u2032 end of the transcript by ZNF274. We also observed a pattern in the level of H3K9me3 methylation, with two maximum levels at about position 62,850,000 and 63,400,000 and trailing off at position 63,250,00. This bimodal pattern also occurs in the KAP1 and SETDB1 binding data and is even more apparent when viewing a wider view of the region. This position, marked in the figure by vertical black line, also corresponds with the position of rs159870 (chr19: 63239261) and there is break in synteny with rodent genomes in this zone.Although little is known about the majority of genes in this region, \u22125; We next examined patterns of IBD sharing at the SNP with the highest LOD score on chromosome 19 (rs159872). We compared the proportion of IBD case pairs in different populations to determine whether there are particular populations that contribute to more IBD case pairs at this locus; and found the Tasmanian population has the highest proportion of IBD case pairs. When compared with all other combined Australian populations, the Tasmanian population significantly contributed more IBD case pairs at this locus (p\u200a=\u200a0.004); and was significant when compared with all other combined non-Tasmanian populations (p\u200a=\u200a5.44\u00d710\u22126 (LOD\u200a=\u200a4.65). In classical linkage analysis in small families, individuals are closely related and the segments of IBD tend to be fairly long (>10 cM) which are easier to detect and less independent than IBD mapping, the generally-accepted threshold for genome-wide significance is p\u200a=\u200a2.0\u00d710\u22125\u22128\u22128 and 2.0\u00d710\u22125. Recently, researchers demonstrated that the genome-wide significance thresholds for IBD mapping depend on the IBD segment size detected or IBD generations \u22126, while the segment size of 3.2 cM corresponds to 15 generations and the genome-wide significance threshold is 4.0\u00d710\u22126\u22126 and 2.0\u00d710\u22126. As such, the linkage signal on chromosome 19, with a p-value of 1.9\u00d710\u22126, was determined to be genome-wide significant.We have applied BEAGLE fastIBD for the detection of rare MS variants utilising a large-scale GWAS dataset. We identified a high linkage signal on chromosome 19 with a p-value of 1.9\u00d710http://genome.ucsc.edu/). Seven genes  in this region belong to the Kr\u00fcppel family of ZNF genes. Only a few ZNF genes in this region have known vertebrate homologues and it includes a number of primate specific KRAB-ZNF genes Most genes in this linkage region are zinc finger (ZNF) proteins of which 32 genes have been suggested to be transcriptional regulators Detailed analysis of genes in this region did not reveal any direct links with MS. However examination of their expression profiles in published data revealed a shared early developmental CNS specific expression profile with 22 genes in this region being members of the expression module M20 described in ZNF274, located within the linkage region, is involved in gene silencing through recruitment of the histone methytransferase complex TRIM28 (KAP1)/SETDB1 to the 3\u2032 end of specific ZNF genes ZNF274 also interacts with p75NTR and is predicted to play a role in programmed cell death during development ZNF549, ZNF324, ZNF548, ZNF264, ZNF671, ZSCAN1 and ZSCAN18 are members of cluster A ZNF304 is implicated in lymphocyte activation ZNF274 has very high expression in activated eosinophils compared with other immune cell types Epigenetic mechanisms such as histone modification and DNA methylation are responsible for silencing many specific transcription factors including zinc finger genes, and the 3\u2032 end of many ZNF genes are specifically covered by H3K9me3 http://developinghumanbrain.org/).Together these findings suggest that many of the genes in this cluster may be involved in early differentiation of neuronal cells and potentially the silencing of genes required for myelination. Expression of ZNF genes is commonly detected in foetal brain and they are predicted to be involved in development of the nervous system, a KRAB zinc finger cluster on chromosome 8 has also been proposed to be involved in regulation of CNS development Thus this may be an example of a gene cluster of KRAB -ZNF genes exhibiting coordinated expression regulation, indicating the presence of a genomic regulatory block (GRB). Such regions are usually transcription factors controlled by highly conserved noncoding regions. Although the identification of GRBs remains difficult the evidence that we have collated is suggestive of two genomic regulatory blocks within the linkage region, interrupted at the position of SNP rs159870 where there is an absence of H3K9me3 methylation and a break in synteny  with the highest LOD score on chromosome 19, we hypothesise that there are some difference between cases and controls sharing haplotypes in the linkage region among different populations. We found the Tasmanian MS population has the highest proportion of case IBD sharing, significantly higher than non-Tasmanian combined populations as well as other non-Tasmanian combined Australian populations. While Tasmania has the highest prevalence of MS in Australia, it is generally agreed that this is primarily driven by environmental effects related to, sunlight and/or vitamin D Even though Beagle fastIBD is several orders of magnitude faster than Beagle IBD, IBD analysis remains moderately computationally intensive on a dataset of this size . For instance, on chromosome 2 with 22,607 SNPs, the computation time for each run was approximately 4.6 hours with memory requirement of 3.3 GB on 2 cores of a SGI Altix ICE 8200 HPC cluster computer node.BRCA1 and BRCA2 gene all increase risk of breast cancer IFIH1 gene all protect against type I diabetes However, we also found IBD analysis limitations: it is only suited to discover rare variants if all variants act in the same direction in one gene. For example, the identified rare variants in The optimal method to detect rare disease-causing variants is whole genome sequencing of thousands of samples. When this becomes affordable, there will remain a role for IBD analysis to prioritize regions for follow-up analysis and minimize the massive multiple testing burden. Just as linkage analysis is now used to identify regions for follow-up in whole genome sequencing and exome sequencing of Mendelian disease families, and linkage analysis can be used to weight regions for GWA analysis In summary, we have applied IBD analysis to a large complex disease GWA dataset and identified a linkage signal with genome-wide significance, although it. While our most significant result is of equivocal significance, and lies in a region that is hard to validate via sequencing, we believe IBD analysis has considerable potential, particularly to help interpret whole-genome sequencing data in complex trait studies.Figure S1Principal components analysis for the dataset. Most individuals in the dataset are of predominantly northern European ancestry (right hand side), but some have southern European ancestry (left hand side) .(TIF)Click here for additional data file.Figure S2Fitting Poisson model for the IBD data. All the four real lines in these four modules fit well with the default lines, suggesting Poisson model is appropriate for this data. The residuals of the green region are higher than others.(TIF)Click here for additional data file.Figure S3Fitting negative binomial model for the IBD data. All the four real lines in these four modules fit not well with the default lines, suggesting negative binomial model is not suitable for this IBD data.(TIF)Click here for additional data file.Figure S4Fitting linear model for the IBD data. All the four real lines in these four modules fit not well with the default lines, suggesting linear model is not suitable for this IBD data.(TIF)Click here for additional data file.SR_commands S11) Fitting and testing model for IBD data.2) Plot of residuals from the Poisson model converted to LOD scores.3) Network analysis.(PDF)Click here for additional data file."}
+{"text": "Microtus ochrogaster) is an emerging rodent model for investigating the genetics, evolution and molecular mechanisms of social behavior. Though a karyotype for the prairie vole has been reported and low-resolution comparative cytogenetic analyses have been done in this species, other basic genetic resources for this species, such as a genetic linkage map, are lacking.The prairie vole (Here we report the construction of a genome-wide linkage map of the prairie vole. The linkage map consists of 406 markers that are spaced on average every 7 Mb and span an estimated ~90% of the genome. The sex average length of the linkage map is 1707 cM, which, like other Muroid rodent linkage maps, is on the lower end of the length distribution of linkage maps reported to date for placental mammals. Linkage groups were assigned to 19 out of the 26 prairie vole autosomes as well as the X chromosome. Comparative analyses of the prairie vole linkage map based on the location of 387 Type I markers identified 61 large blocks of synteny with the mouse genome. In addition, the results of the comparative analyses revealed a potential elevated rate of inversions in the prairie vole lineage compared to the laboratory mouse and rat.Microtus.A genetic linkage map of the prairie vole has been constructed and represents the fourth genome-wide high-resolution linkage map reported for Muroid rodents and the first for a member of the Arvicolinae sub-family. This resource will advance studies designed to dissect the genetic basis of a variety of social behaviors and other traits in the prairie vole as well as our understanding of genome evolution in the genus  Mus musculus) and rat (Rattus norvegicus), are currently available [Genomes evolve by a number of molecular mechanisms including chromosomal rearrangements . The genvailable ,4, intervailable ).Microtus is comprised of 62 species of voles and is one of the most, if not the most, speciose Muroid genus [Microtus is particularly interesting to study with respect to the process of genome evolution because the rate of speciation in this genus is estimated to be 20-fold higher than the average mammalian lineage [Microtus genus differ from one another, the likely number and type of large-scale chromosomal rearrangements that have led to those observed differences, as well as the reconstruction of a proposed ancestral Microtus genome [Microtus genomes and that of the mouse [Microtus genus has not reported.The genus id genus . Microtu lineage  and beca lineage ,9. Compas genome -15. In ahe mouse ,15. HoweMicrotus ochrogaster) is an emerging model for studying the genetic and molecular bases of social behavior and how it evolves [Microtus genomes.The North American prairie vole ( evolves . Breedin evolves ) and a k evolves . However evolves ,18, no g evolves . With thA total of 624 SNPs were genotyped on a panel of 353 prairie voles. After applying quality control measures and other filters to the genotyping results, the final data set used for the linkage mapping included the genotypes of 431 SNPs from 285 individuals . Most (n = 392) of the prairie vole loci tagged by the filtered set of SNPs could be assigned an orthologous position in the mouse genome, i.e., Type I markers (e.g. ). The avThirty-five linkage groups that included a total of 406 SNPs were identified Figure . PrairieThe physical linkage, and to a lesser extent the order of genes tend to be conserved between mammalian genomes (e.g. ). Of the2 test, p < 0.012). This difference is primarily due to an estimated higher rate of inversions in the prairie vole versus the mouse/rat lineages , should be able to address this question further and better resolve when shifts in the rate of genome evolution may have occurred.The rate of chromosomal rearrangements varies across the mammalian phylogeny . From ouevolving ,9, it isMicrotus genus, identified derived characteristics of the prairie vole genome [Microtus genomes other than the prairie vole by cross-species chromosome painting has led to the reconstruction of a putative ancestral Microtus karyotype [Microtus genomes with respect to our higher-resolution comparative mapping results is of potential value. For example, by using synteny maps with the laboratory mouse reported here for the prairie vole and previously for other voles as a common point of reference [Microtus autosomes 2, 5, 6, 7, 12, 13, 14, 19, 21, 23 and 24 proposed in [Microtus chromosome, or vice versa, it does suggest that at the macro-level the prairie vole genome differs from the ancestral Microtus karyotype by the aforementioned pair of fission events, and presumably at least two fusion events which would be needed to maintain the diploid karyotype of 2n = 54 present in both the prairie vole and ancestral state. However, it is important to note that future improvement of the vole linkage map may alter some of the comparisons we report here. Future comparative mapping studies of other Microtus genomes that can now be undertaken by leveraging the genomic resources being developed for the prairie vole will provide further insights into the rates and mechanisms by which genomes within this genus have evolved.Comparative analyses of G-banded karyotypes from a number of Arvicolid rodents, including the prairie vole and other members of the e genome . More rearyotype . While weference , we predMicrotus. Further, it will provide a beneficial resource for furthering our understanding of the genetic basis of social behaviors and other traits while giving insight into how these traits evolve.The genetic linkage map of the prairie vole will provide an important resource towards our understanding of genome evolution in the genus SRX018685, SRX018510, SRX018516, SRX018515, SRX018511, SRX018514, SRX018513, SRX018512). In the case of the BAC-end re-sequencing, loci-specific M13F and M13R tailed primers were designed with PRIMER3 [\u00ae (Beckman Coulter) platform. Transcriptome sequencing data generated with the Roche 454 sequencing platform [http://repeatmasker.org/), or predicted intron-exon boundary defined based on alignment of the prairie vole transcripts to the mouse genome.SNPs were discovered by either re-sequencing loci corresponding to previously reported bacterial artificial chromosome (BAC)-end sequences from the prairie vole CHORI-232 library , or usin PRIMER3  and used PRIMER3  were manplatform  was scanThe chromosome locations for the mouse orthologs of the 2773 prairie vole transcripts in which at least one SNP was identified were used as a proxy to select a uniformly-spaced and genome-wide set of 384 SNPs for genotyping on the GoldenGate (Illumina) platform. The names, sequences, and other accessory information for each genotyped locus are described in Additional File Guide for Care and Use of Laboratory Animals published by the National Research Council.A total of 353 prairie voles from our local colony were genotyped. After excluding individuals with questionable parentage, i.e. children for which genotypes at > 3% of the makers were inconsistent with the parental genotypes, two pedigrees were selected for constructing the linkage map: a 3-4 generation pedigree derived from interbreeding descendents of three founder breeding pairs , were monomorphic (n = 109), or associated with an inferred genotype error rate in excess of 7% (n = 37) were excluded from the linkage analyses. Inconsistent genotypes, which represented 0.5% (n = 638) of the genotype calls for the remaining 438 markers, were re-coded as no data. The final data set used for the linkage analyses included 122,071 genotype calls and 2,759 missing data points.A modified version of CRI-MAP , v2.503 An inherent methodological limitation of the likelihood-based method used by CRI-MAP is that it does not necessarily explore all possible marker orders, and thus the marker order found to have the best likelihood score starting from a given framework map may not represent the true optimal order of the markers. To address that limitation, when the linkage groups included more than 2 loci that mapped to the same orthologous mouse chromosome and the order of the markers was not the same as that in mouse we also calculated the likelihood score of each group of markers assuming an order equivalent to that of the mouse. For prairie vole chromsomes 6, 10, 15 and 21, the marker order predicted by the orthologous positions in the mouse yielded a map with a better likelihood score than the synteny na\u00efve method, in which case the mouse order was used as starting point to improve the ordering of the prairie vole markers with the FLIPS option. Additionally, several markers were uniquely placed to the same position on the map Pair-wise LOD scores for these loci are indicated in Additional File The prairie vole-mouse comparative cytogenetic map described in  was intehttp://genome.ucsc.edu/hgLiftOver. GRIMM [Loci in the prairie vole linkage map with a known orthologous position in the mouse genome (Additional File r. GRIMM  was usedr. GRIMM  was thenJKD performed the re-sequencing of the BES for SNP discovery. LAM, LJY, and JWT conceived and oversaw the project. LAM and JWT performed the analyses and wrote the manuscript. All authors approved the final version of the manuscript.Supplementary Table 1. Marker information. Locus name refers to either the BAC-end locus or the orthologos mouse gene name. SNP names were asssigned similarly where names beginning in \"FI\" correspond to vole BAC-end loci names and names beginning in \"NM\" correspond to the accession number of the orthologos mouse gene.Click here for fileSupplementary Table 2. Integration of the prairie vole cytogenetic and linkage maps. Markers that did not have an orthologus position in the mouse genome were omitted from this table. Concordant markers were those in which the cytogenetic and genetic linkage maps agreed with respect to blocks of prairie vole-mouse synteny.Click here for fileFamilyPedigree1. Pedigree of large multi-generational family used for constructing the linkage map.Click here for fileFamilyPedigree2. Pedigree of small nuclear family used for constructing the linkage map.Click here for fileSupplementary Table 3. Pair-wise LOD scores for markers that were uniquely placed to the same position on the map.Click here for file"}
+{"text": "Chiari Type I Malformation (CMI) is characterized by displacement of the cerebellar tonsils below the base of the skull, resulting in significant neurologic morbidity. Although multiple lines of evidence support a genetic contribution to disease, no genes have been identified. We therefore conducted the largest whole genome linkage screen to date using 367 individuals from 66 families with at least two individuals presenting with nonsyndromic CMI with or without syringomyelia. Initial findings across all 66 families showed minimal evidence for linkage due to suspected genetic heterogeneity. In order to improve power to localize susceptibility genes, stratified linkage analyses were performed using clinical criteria to differentiate families based on etiologic factors. Families were stratified on the presence or absence of clinical features associated with connective tissue disorders (CTDs) since CMI and CTDs frequently co-occur and it has been proposed that CMI patients with CTDs represent a distinct class of patients with a different underlying disease mechanism. Stratified linkage analyses resulted in a marked increase in evidence of linkage to multiple genomic regions consistent with reduced genetic heterogeneity. Of particular interest were two regions  identified within the subset of \u201cCTD-negative\u201d families, both of which harbor growth differentiation factors  implicated in the development of Klippel-Feil syndrome (KFS). Interestingly, roughly 3\u20135% of CMI patients are diagnosed with KFS. In order to investigate the possibility that CMI and KFS are allelic, GDF3 and GDF6 were sequenced leading to the identification of a previously known KFS missense mutation and potential regulatory variants in GDF6. This study has demonstrated the value of reducing genetic heterogeneity by clinical stratification implicating several convincing biological candidates and further supporting the hypothesis that multiple, distinct mechanisms are responsible for CMI. Chiari Type I Malformation (CMI) is characterized by displacement of the cerebellar tonsils below the base of the skull and occurs with an estimated prevalence of less than one percent in the United States Although multiple mechanisms have been proposed for cerebellar tonsillar herniation, including cranial constriction, cranial settling, spinal cord tethering, intracranial hypertension, and intraspinal hypotension While no disease gene has been identified for CMI to date, several lines of evidence support a genetic contribution to disease in at least a subset of nonsyndromic cases. These include twin studies One major challenge is the variability of clinical presentation within the CMI patient population. This clinical heterogeneity presents as differences with respect to the pattern and severity of symptoms, response to surgery, presence of associated conditions, age of onset, and the extent of tonsillar herniation. As CMI is thought to be influenced by multiple genetic and environmental factors, this clinical heterogeneity likely reflects in part an underlying genetic heterogeneity. While this can have substantial implications during the design stage of a genetic study, the selection of families that are genetically homogeneous is not straight forward. One approach is to stratify families using clinical features that may identify groups of families that share similar genetic risk factors. In other words, reducing phenotypic variability may lead to a reduction in genetic variability. Although the pool of candidate clinical features to use for stratification can be quite large, previous clinical associations observed with the disorder provide some insight into which features to select.To address these issues, we performed the largest whole genome linkage screen to date using 367 individuals from 66 nonsyndromic CMI multiplex families. Based on the limited evidence for linkage using the complete collection of families, we performed a stratified whole genome linkage analysis using the presence or absence of CTD related conditions and successfully identified putative CMI susceptibility genes in the genetically more homogeneous strata.All participating family members provided written informed consent for this study. If participants were minors, written consent was obtained from a parent or legal guardian for participants younger than 18 years of age. Participants between 12 and 17 years of age were asked to provide written assent. Written informed consent and assent, when applicable, were obtained by approved clinical staff. Consent forms were either discussed in person or were mailed and then discussed over the telephone. All participant interactions were logged in Progeny 8 , our clinical data collection software program. The original signed consent is maintained by the study and a copy was provided to participants. The consent form, procedure described above, and this study were specifically approved by the institutional review board of Duke University Medical Center.Participants were ascertained across the United States primarily through self-referral in response to advertisements on the web (e.g. Duke Center for Human Genetics and GeneTests), mailings and/or presentations to patient support groups and physician referral. Families were enrolled in the current study if at least two sampled individuals were diagnosed with CMI with or without syringomyelia. Exclusion criteria included the following: 1) families with a positive family history of a known genetic syndrome , 2) family history of spina bifida or tethered cord syndrome, and 3) individuals thought to have a secondary form of CMI, such as occurring due to a brain tumor. Although syndromic families formally diagnosed with hereditary CTDs were excluded from our genetic screen, many family members exhibited conditions such as hypermobility, mitral valve prolapse and scoliosis which are often associated with CTDs as described in further detail below. Blood samples were collected from affected individuals and all available connecting family members, regardless of affection status. Additionally, study participants completed a family and medical history telephone interview, responded to a detailed clinical questionnaire, and submitted release forms for medical records and pre-surgical MRIs. When possible, a diagnosis of CMI was determined based on MRI measurements in which affection status was defined as cerebellar tonsillar herniation of 3 mm or more for both tonsils or herniation of 5 mm or more for either tonsil . A small amount of DNA (0.3 \u00b5g) was run on a 0.8% agarose gel in order to assess quality and each sample was quantified using the Nanodrop . In total, 436 individuals from 75 families were genotyped using Illumina Human610-Quad BeadChips  per the manufacturer\u2019s instructions and chips were scanned using the Illumina iScan system . Due to the duration of ascertainment for this study, genotyping was performed in two separate batches . In addition to samples from study participants, replicate samples were included across sample plates and checked for mismatches. Specifically, two CMI family  and two Centre d\u2019Etude du Polymorphism Humain (CEPH)  samples were included across three 96-well sample plates per batch in an alternating pattern.Quality control (QC) procedures were performed to ensure high quality data were used for analysis. Initial quality assessment was performed separately for each batch using the Illumina GenomeStudio genotyping module . Single nucleotide polymorphism (SNP) data (N\u200a=\u200a585497 combined across batches 1 and 2) quality were further assessed using PLINK v1.07 replicates\u200a=\u200a1,000). Additional model parameters used for the simulations included: disease MAF of 0.001, marker MAF of 0.30, and an affecteds-only, low penetrance function .Power for the whole genome linkage study was determined using SIMLINK all scoring function was used which assesses IBD sharing across subsets of affected individuals All linkage analyses were performed using MERLIN 1.1.2 and MINX (MERLIN in X) 2 threshold of 0.16 For both the parametric and nonparametric linkage analysis, two-point and multipoint analyses were performed. In order to maintain the correct type I error rate when conducting a multipoint analysis in families when one or both parents are missing, the option, \u201c\u2013rsq\u201d, in MERLIN was implemented which allows for the modeling of inter-marker linkage disequilibrium (LD) between SNPs Prior to whole genome linkage analysis, MERLIN\u2019s error detection option was used to identify possible genotyping errors, such as unlikely double recombinants Families (N\u200a=\u200a66) were stratified based on medical record documentation or self-reported family history of any of the following CTD related conditions: hypermobility (N\u200a=\u200a4), kyphosis (N\u200a=\u200a2), aneurysm (N\u200a=\u200a11), mitral valve prolapse (N\u200a=\u200a9), pectus excavatum (N\u200a=\u200a1), scoliosis (N\u200a=\u200a15), orthostatic hypotension (N\u200a=\u200a1), supraventricular tachycardia (N\u200a=\u200a2), heart valve disease (N\u200a=\u200a12), and/or heart murmur (N\u200a=\u200a6). In total, 34 families were grouped as \u201cCTD-positive\u201d and the remaining 32 families were \u201cCTD-negative\u201d. CTD-positive families had a significant history for one (47.1%), two (32.4%), three (14.7%), or five (5.9%) of the CTD-related conditions described above.total\u200a=\u200a1000).A series of permutation tests were performed using custom scripts in order to determine genome-wide and chromosome-wide empirically derived significance levels for the stratified analyses conditional on the prior evidence for linkage. This was used to assess the relationship between the increased evidence for linkage and clinical criteria used to stratify families. For both the parametric (two-point and multipoint) and nonparametric  analyses the following was performed: 1) The dataset was randomly split in half creating two datasets each containing 33 families, 2) Linkage analyses were conducted using MERLIN 1.1.2 and MINX for the X chromosome de novo sequencing was based on results from the CTD stratified whole genome linkage analysis described below. All affected individuals from any of the 66 linkage families that showed a positive family specific LOD score for the peak marker on chromosome 8 (rs2446871) or chromosome 12 (rs10505755) were selected for Sanger sequencing of growth differentiation factors, GDF6 and GDF3. In total, 96 affected individuals from 39 families and 75 affected individuals from 28 families were initially screened for mutations in GDF6 and GDF3, respectively. Seventeen GDF6 primer sets were designed to cover the exons (including intron-exon boundaries), 5\u2032 and 3\u2032 untranslated regions (UTR), as well as three intronic regions with high conservation . Three GDF3 primer sets were designed to cover exons (including intron-exon boundaries) and 5\u2032 and 3\u2032 UTRs. Primer sequences, PCR conditions and kits are described in detail , 7544 (1.3%) and 6835 (1.2%) SNPs were excluded from batches 1 and 2, respectively, due to call rates <98%, presence on chromosomes 24\u201326, high replicate error rate, as well as Illumina specific quality metrics including AB T Mean, AB R Mean, cluster separation, among others. Within each batch, replicate reproducibility rates exceeded 99.999% and all samples, except for one of the CEPH samples in batch 2, had a call rate >99%. Additional SNPs were excluded with Mendelian errors in >4% families (N\u200a=\u200a220), MAF <0.05 (N\u200a=\u200a66355), HWE p<0.001 (N\u200a=\u200a275), identical physical location , no genetic distance available from deCODE (N\u200a=\u200a948), call present in only batch 1 (N\u200a=\u200a2445), call present in only batch 2 (N\u200a=\u200a2991), and identical genetic position . Genotypes for all SNPs showing non-Mendelian inheritance were set as missing for the entire family. A total of 221343 SNPs remained after filtering and were used to construct the two-point linkage map. From those remaining SNPs, 12056 were selected for use in the multipoint linkage analysis using criteria such as genetic distance in order to create an evenly spaced map and high MAF estimates resulting in increased marker heterozygosity (Mean distance (cM) between SNPs: 0.31\u00b10.008; Mean MAF: 0.42\u00b10.09). In addition to SNP exclusions, three individuals were excluded due to large genomic duplications and/or regions of loss of heterozygosity detected from log R ratio and B allele frequency plots in Illumina GenomeStudio. This ultimately resulted in a total loss of 14 individuals due to two families that were no longer useful for linkage analysis. After additional sample exclusions were applied, 367 individuals from 66 families remained for analysis. Detailed sample exclusions are provided, along with the multidimensional scaling analysis used to identify sample outliers .SIMLINK Following data quality assessment, both two-point and multipoint parametric and nonparametric linkage analyses were conducted. Initial findings across all 66 families showed minimal evidence for linkage, with no multipoint maximum LOD scores exceeding 2 although several two-point LOD scores exceeded 3 across the various models  and chromosome-wide (CW) empirical p-values were obtained for both multipoint and two-point analyses under the three linkage models. Although no marker met GW significance, the peak marker for 8q21.3\u2013q22.1 had a GW empirical p-value of 0.07 with a highly significant CW empirical p-value of 0.008. Additionally, several markers from the two-point and multipoint analyses had CW empirical p-values less than 0.05 as shown in Sanger sequencing was performed on all affected individuals from families with a positive LOD score for the linkage peak marker in the chromosome 8 or 12 linkage regions. The primary focus was on the most significant multipoint linkage peak found on chromosome 8 within the CTD-negative group of families . The 1 LOD down supporting interval contained 49 candidate RefSeq genes . Of those, one of particular interest was Growth differentiation factor 6 (GDF6) which is a member of the bone morphogenetic protein (BMP) sub-family and has been previously associated with a wide range of phenotypes including ocular, such as microphthalmia and coloboma, as well as skeletal, such as Klippel-Feil syndrome (KFS) which is characterized by fusion of any two of the seven cervical vertebrae In total, 22 SNPs, 2 insertions, and 1 deletion were found in GDF6 and 3 SNPs were found in GDF3 . Of thesIn addition, the two intronic variants  that are shared across all affected family members are located within potential regulatory regions . The novIn order to gain a better understanding of the genetic architecture of CMI, we conducted a whole genome linkage screen using a collection of 66 nonsyndromic families with at least two sampled individuals presenting with CMI with or without syringomyelia. It was hypothesized that the limited evidence for linkage across all 66 families collectively was due to genetic heterogeneity and may be associated with the phenotypic variability observed. Based on the co-occurrence of CMI and CTDs, families were stratified by CTD related conditions in order to identify phenotypically and potentially genetically more homogeneous groups of families for linkage analysis. Stratified analyses identified multiple genomic regions showing increased evidence for linkage consistent with reduced genetic heterogeneity across families as a result of the CTD related stratification criteria. Furthermore, several plausible disease genes were identified as discussed in detail below.Prior to describing our most significant results, it is important to relate our findings to the only other whole genome linkage screen conducted to date which implicated regions on chromosomes 9 and 15 in-vitro. Asai-Coakwell and colleagues evaluated changes to bone morphogenetic protein (BMP) signaling by co-transfecting an expression construct with the A249E mutation and a Sex determining region Y-box 9 (SOX-9)-responsive reporter gene into primary limb mesenchymal cells and assessed SOX-9 reporter activity While we presented linkage results within the subsets of both CTD-positive and CTD-negative families, the focus of the current paper has been on the CTD-negative families as these are thought to represent more \u201cclassical\u201d CMI due to cranial constriction and also resulted in the identification of the only genomic region with a maximum LOD score exceeding 3. The most significant of our findings implicated the growth differentiation factors, GDF6 and GDF3, both of which had been previously implicated in KFS Although there is evidence for a functional effect, the expression of A249E is complex with previous evidence of pleiotropy , variable expressivity , and reduced penetrance Additional variants of interest from our study include two intronic GDF6 variants, rs140757891 and a novel SNP, g.406+2780C>T. ChIP-seq data from a small number of cell lines indicate that both variants are located within predicted targets of SUZ12, a polycomb protein involved in epigenetic silencing of developmental genes. Interestingly, haploinsufficient SUZ12 mice exhibit cerebellar herniation, as well as spina bifida, an enlarged brainstem, and occipital cortical changes While encouraged by our findings, we acknowledge several limitations of this study. First, because we enforced strict eligibility criteria (exclusion of syndromic cases) and required families to have multiple affected individuals, the total number of families eligible for the study was low and likely contributed to reduced power. However, despite the relatively small sample size, the number of families examined was almost three times as large as the collection of families used in the only other whole genome linkage screen published to date de novo variant detection. Other candidate genes, such as low density lipoprotein receptor-related protein 6 (LRP6) present within the chromosome 12 candidate interval could also be investigated as LRP6, when specifically deleted from early mesenchyme, causes a slight delay in mouse skull ossification Future work will include functional follow-up of variants of interest as well as sequencing GDF3 and GDF6 in a larger cohort of sporadic and familial CMI cases. Furthermore, the distant regulatory elements previously identified for GDF6 in vitro, and the fact that it has been identified in multiple skeletal and ocular disease cohorts, and 5) Identified two potential regulatory variants  shared across all affected individuals in the families they were identified in and located within predicted regulatory regions for SUZ12 which itself is an excellent candidate gene for CMI. Further investigation of GDF3 and GDF6, other plausible biological candidates such as SUZ12, NF1, and LRP6, as well as the genetic relationship between CMI and KFS is warranted.The current study demonstrates the utility of using clinical stratification to reduce genetic heterogeneity in CMI by identifying genomic regions showing increased evidence for linkage with maximum LOD scores exceeding 2 and even 3, as well as having implicated credible candidate genes in CMI susceptibility. Although further work is necessary to confirm the involvement of these genes and individual sequence variants in the development of CMI, this work makes several important contributions to the field of CMI research: 1) We conducted the largest whole genome linkage screen to date providing multiple candidate intervals for future investigation and replication, 2) Our results suggest a relationship between CTD related conditions and genetic etiology which is consistent with the hypothesis that CMI with CTDs versus CMI without CTDs occur through different mechanisms , 3) Multiple biological candidates were implicated from the analysis, including the only two GDFs currently known to be associated with KFS suggesting a shared genetic etiology between CMI and KFS. This is consistent with the fact that KFS is known to co-occur with CMI and share a common cellular etiology, 4) Identified a known KFS missense mutation in two of our families that is not necessary for disease but likely contributes to the phenotype due to its rare frequency in the general population, known functional effect Figure S1Multidimensional scaling (MDS) analysis. This was performed using a pruned marker dataset and only one representative individual from each family. The red and black colors correspond to different clusters (PLINK's pairwise population concordance test: \u2013ppc 1e-4). Individuals shown in red represent families that are self-reported Caucasian, Hispanic. MDS plot was created in R 2.15.0.(TIFF)Click here for additional data file.Figure S2Segregation of select variants in three CMI pedigrees. Family 9772 (A), Family 9496 (B), and Family 9432 (C). Alleles \u201cA\u201d and \u201ca\u201d represent a novel SNP at Chr8\u223697154593, Alleles \u201cB\u201d and \u201cb\u201d represent a novel SNP at Chr8\u223697157811, alleles \u201cC\u201d and \u201cc\u201d represent RS140757891, and alleles \u201cD\u201d and \u201cd\u201d represent a novel SNP at Chr8\u223697169735. Individual 108 from family 9496 has previously had brain surgery and a shunt; no additional information is known. Symbols shaded in black indicate a diagnosis of CMI with or without syringomyelia and small diamonds represent miscarriages. Lower case letters shown in red indicate the variant allele. Genotype calls are based on bidirectional sequencing. Progeny 8  was used to construct the pedigrees.(TIFF)Click here for additional data file.Table S1PCR primer sequences and conditions.(DOC)Click here for additional data file.Table S2PCR primer kits and thermocycler conditions.(DOC)Click here for additional data file.Table S3Quality control of sample data.(DOC)Click here for additional data file.Table S4Most significant two-point and multipoint LOD scores.(DOC)Click here for additional data file.Table S5GDF6 and GDF3 identified sequence variants.(DOC)Click here for additional data file."}
+{"text": "PSMD9) lies in the 12q24 locus and is in linkage with MODY3, type 2 diabetes (T2D), microvascular and macrovascular pathology, carpal tunnel syndrome, and hypercholesterolemia in Italian families.Chromosome 12q24 was recently associated with hypertension. Proteasome Modulator 9  IVS3+nt460 A > G, IVS3+nt437 T > C and E197G A > G with elevated blood pressure/hypertension. The non-parametric linkage analysis was performed for this qualitative phenotype by using the Merlin software; the Lod score and correspondent P-value were calculated. Parametric linkage analysis was also performed. For the significant linkage score, 1000 replicates were run to calculate the empirical P-value.We characterized the Italian T2D families' members for presence and/or absence of elevated blood pressure (\u2265 130/80) and/or hypertension. The phenotypes were described as unknown in all cases in which the diagnosis was either unclear or the data were not available for the subject studied. We tested in the 200 Italians families for the presence of the linkage of the PSMD9 gene SNPs studied are in linkage with elevated blood pressure/hypertension in our Italian families.The PSMD9 gene and/or any variant in linkage disequilibrium with the SNPs studied contribute to the linkage to hypertension within our family dataset. This is the first report of PSMD9 linkage to hypertension within the 12q24 locus.We conclude that the  PSMD9), a coactivator of insulin gene transcription, which is highly expressed in pancreatic islets [PSMD9 overexpression cause beta-cell dysfunction and contribute to type 2 diabetes (T2D) [PSMD9 may rarely cause T2D by unique mutations [PSMD9 A/T/G haplotype to late-onset T2D, with the strongest evidence under the recessive model [PSMD9 T2D risk single nucleotide polymorphisms (SNPs) with T2D-nephropathy [PSMD9 within the locus 12q24, and the evidence of linkage with microcirculation within the same locus [PSMD9 is a candidate gene for hypertension. Further, as PSMD9 is linked to T2D, it should be screened to identify any inheritance contributing factors to the T2D-associated phenotypes, of which hypertension is a major player.A recent study has shown that changes in retinal vascular caliber are linked to the chromosome 12q24 locus in a large Caucasian population [utations . We idenve model  in Italihropathy , T2D-neuhropathy , retinophropathy , carpal hropathy , and machropathy . Given tPSMD9 IVS3+nt460, IVS3+nt437, E197G T2D risk SNPs for linkage with elevated blood pressure and/or hypertension in our 200 Italian T2D families.In the present study, we aimed at testing the the subjects were all recruited from center Italy following the Helsinki declaration guidelines. Subjects gave written informed consent. The Penn State College of Medicine Ethical Committee approved the study.We recruited 200 Italian T2D affected siblings and extended families, including also unaffected members. The families originate from the center of Italy. The members are at least three generations Italians. Identical twins were excluded. These T2D families have been helpful in the whole or as unrelated T2D cases in identifying or excluding diabetes risk genes and/or variants in previous studies -24. We cIVS3 PSMD9 region containing the +nt460 A > G and +nt437 C > T SNPs and the exon 5 coding region containing the E197G A > G SNP with specific primers in the affected and unaffected family members. We directly sequenced the PCR products, status post-purification via EXOSAP-IT, on an automated ABI 3730 Sequencer.Via PCR, we amplified the PSMD9 SNPs with elevated blood pressure/hypertension. Both non-parametric and parametric linkage analysis for the qualitative phenotype were performed for the three SNPs via Merlin software [PSMD9 SNPs IVS3+nt460, IVS3+nt437, E197G are in strong linkage disequilibrium (LD) [We tested in the 200 Italian families for linkage of the software . Allele software . Merlin For each test performed, to exclude the presence of any false positive in our results, we calculated how many times similar P-values were expected by chance in 1,000 replicates of simulations by using the gene dropping method: this analysis replaces real data with simulated data, while maintaining the pedigree structure, allele frequencies and recombination fraction. These datasets are generated under the null hypothesis of no linkage.PSMD9 SNP IVS3+nt460 is slightly more significant than the other two SNPs. Under the parametric model analysis, the most significant is the dominant model. The simulation analyses of 1,000 replicates have excluded false positives and establish the validity of the real data.The results of the non-parametric and parametric linkage analysis performed for the qualitative phenotype hypertension are reported in table PSMD9 IVS3 +nt460 A > G and +nt437 C > T and exon 5 E197G A > G SNPs studied and/or any variants in LD with them are in linkage with elevated blood pressure/hypertension in our 200 Italian families. The PSMD9 SNPs studied contribute to the linkage of the reported phenotype. It is possible that the recessive and additive models appear less significant than the dominant model, as the disease penetrance value given for the heterozygous state under both models, and for the homozygous state under the additive model, is inferior to 1. Thus, some of the power of the data may be lost. On the other hand, recessive and additive models are still significant as the variants in homozygous state are likely inherited by the affected family subjects more often than expected by the null hypothesis of no linkage and thus contribute to the linkage signal. The limitation of our study is that the linkage may capture the signal from the potential co-inheritance of genetic variants linked to another phenotype and to the underlying T2D that is commonly shared with hypertension; however, as T2D patients have high prevalence of hypertension, virtually all linkage studies performed in T2D may as well mask a linkage with hypertension. In fact, when we statistically analyze only a single phenotype, we cannot protect the results from any potentially known and unknown associated factor with the phenotype under study and from the co-inheritance of genetic variants related to that factor underlying the linkage signal.Our analysis show that the The strength of our study resides in the significant data both at the level of non-parametric analysis, which is not vulnerable to miscalculation of allele frequencies and penetrance values, as well as at the level of the parametric analysis, which may suffer from not perfect estimates of allele frequencies and penetrance values.However, the best statistical power test is the simulation empirical P-value, and in this study all empirical P-values have excluded the possibility of false positives in our data analysis.These findings have not yet been confirmed in other ethnicities.PSMD9 SNPs IVS3+nt460, IVS3+nt437, E197G are significantly linked to elevated blood pressure/hypertension in the Italian T2D family dataset.The PSMD9: Proteasome Modulator 9; SNP: single nucleotide polymorphism; T2D: type 2 diabetes; MODY3: maturity-onset diabetes of the young 3; IVS: intervening sequence (intron)The authors declare that they have no competing interests.CG conceived and designed the study, collected the clinical information, performed the statistical analysis and drafted the manuscript. CG also read and approved the final manuscript."}
+{"text": "Efforts to uncover the risk genotypes associated with the familial nature of autism spectrum disorder (ASD) have had limited success. The study of extended pedigrees, incorporating additional ASD-related phenotypes into linkage analysis, offers an alternative approach to the search for inherited ASD susceptibility variants that complements traditional methods used to study the genetics of ASD.We examined evidence for linkage in 19 extended pedigrees ascertained through ASD cases spread across at least two (and in most cases three) nuclear families. Both compound phenotypes  and more narrowly defined components of these phenotypes, e.g., social and repetitive behavior, pragmatic language, and anxiety, were examined. The overarching goal was to maximize the aggregate information available on the maximum number of individuals and to disaggregate syndromic phenotypes in order to examine the genetic underpinnings of more narrowly defined aspects of ASD behavior.Results reveal substantial between-family locus heterogeneity and support the importance of previously reported ASD loci in inherited, familial, forms of ASD. Additional loci, not seen in the ASD analyses, show evidence for linkage to the broad autism phenotype (BAP). BAP peaks are well supported by multiple subphenotypes  showing linkage to regions overlapping with the compound BAP phenotype. Whereas 'repetitive behavior\u2019, showing the strongest evidence for linkage , appears to be linked to novel regions not detected with other compound or narrow phenotypes examined in this study.These results provide support for the presence of key features underlying the complexity of the genetic architecture of ASD: substantial between-family locus heterogeneity, that the BAP appears to correspond to sets of subclinical features segregating with ASD within pedigrees, and that different features of the ASD phenotype segregate independently of one another. These findings support the additional study of larger, even more individually informative pedigrees, together with measurement of multiple, behavioral- and biomarker-based phenotypes, in both affected and non-affected individuals, to elucidate the complex genetics of familial ASD. Autism spectrum disorder (ASD), a neurodevelopmental condition associated with lifelong disability, has now become an urgent public health challenge. Research into the genetics of ASD is motivated by the very real possibility that genetic testing can play a role in improving the diagnostic process and in providing targets for the development of new drugs . HoweverEarly family studies based on retrospective data found a 2\u20139% recurrence of ASD among siblings of affected probands  observedHowever, studies attempting to uncover the risk genotype associated with the familial nature of the disorder have been largely unsuccessful. A review of linkage studies using mostly affected sibling pairs has indicated many 'significant\u2019 linkage peaks with some replication among linkage signals . Fine made novo copy number variants (CNVs) contribute, at least in part, to the etiology of sporadic  ASD [de novo structural variants [In startling contrast, there is substantial evidence that a multitude of rare ial) ASD ,20. To qial) ASD ), have uvariants ,22. Everde novo CNVs than to inherited ones in spite of the fact that ASD is a familial disorder and that rare inherited CNVs have also been consistently reported in ASD. Several pedigrees showing inherited CNVs or point mutations in a number of key CNS genes or regions  have also been published [The field has been more likely to assign a causal role to ublished -27. In mublished  (such diThe study of extended pedigrees (defined here as including relatives outside of the nuclear family identified by an ASD proband) offers an opportunity to study the inherited nature of ASD more thoroughly. Such pedigrees may have enough information to be able to follow the segregation of a genetic variant through a single family to see if it is associated with phenotype status. Several groups have employed the extended pedigree strategy, ranging from a search for shared CNVs across cousin pairs to studying a single, extensive Finnish pedigree from 20 nuclear families -32. One Incorporating additional phenotypes into linkage analysis may be a robust way to uncover inherited ASD susceptibility variants. Consider a genetic mutation that increases the risk of ASD and which can also produce a sub-clinical phenotype in mutation carriers. Misclassifying carriers as 'unaffected\u2019 artificially reduces penetrance estimates for linkage analysis, while misclassifying non-carriers as 'affected\u2019 introduces apparent phenocopies; either of these errors will tend to depress linkage signals. Thus, in a set of pedigrees segregating this mutation, the more accurately the sub-clinical phenotype is measured, the stronger the linkage evidence should be. In other words, a clinical definition that correctly captures the segregation pattern of the mutation will, all other things being equal, result in stronger linkage evidence. Similarly, a quantitative trait (QT) that correctly segregates with the mutation will produce stronger linkage evidence, in general, than one that does not. Here, we utilize clinical data on several ASD-related phenotypes in conjunction with linkage analysis of extended pedigrees to explore genotype-phenotype relationships in ASD.We recruited extended pedigrees with at least three ASD cases spread across at least two nuclear families . All families were either known to the authors through previous studies or identified through advertising. In all, 19 families were available for this study, 6 recruited in Canada (CA) and 13 in the US. The CA pedigrees had an average of 24 genotyped individuals and 25 phenotyped individuals, while the US pedigrees had an average of 16 genotyped individuals and 18 phenotyped individuals. To minimize etiologic heterogeneity, families were excluded from the study if there was evidence of the following co-occurring medical conditions thought to be etiologically-related to autism in one of the index probands with autism: tuberous sclerosis, neurofibromatosis, phenylketonuria, Fragile X screening, or significant CNS injury. We did not exclude individuals with a chromosome abnormality in order to determine whether that abnormality might also be inherited and play a role in susceptibility. All individuals were of northern European heritage. All data collection took place under Institutional Review Board approval and the research was conducted in accordance with the World Medical Association Declaration of Helsinki . Writtenth ed) (DSM-IV) pervasive developmental disorder; and to ii) characterize all pedigree members on phenotypes of interest. For the latter goal of characterizing pedigree members, the strategy employed was to assess for both compound phenotypes  and more narrowly defined components of these phenotypes including social and repetitive behavior, pragmatic language, and anxiety. The overarching goal in taking this multi-tiered approach was to maximize the aggregate information available on the maximum number of affected individuals  as well as to disaggregate these global phenotypes to look at the genetic underpinnings of individual behavioral aspects of the broad construct of autistic behavior.Our overarching clinical strategy included clinical assessments performed to i) index eligible extended pedigrees, by identifying at least three related individuals with a Diagnostic and Statistical Manual  ,40 and MWhen MPAS-R and MPRS consensus ratings were not available, as for all CA pedigrees and some US individuals, the BAP Questionnaire (BAP-Q)  was usedThe US (but not CA) families were additionally assessed on seven secondary phenotypes. The secondary US phenotypes address various aspects of autism and include anxiety (ANX), repetitive/ritualistic behavior (RRB), social functioning (SOC), and language abnormalities, i.e., social communication (PRS), core language ability (CLF), non-word repetition (NWR), and rapid naming (RAP). The CL, NWR, and RAP phenotypes were considered exploratory as there is less support for these constructs showing familiality in the literature, and were added to enrich the overall assessment for communication phenotype information, one of three major criteria for autism in the DSM IV.ANX, RRB, and SOC are quantitative variables. ANX is based on the anxiety (N1) facet t-score of the NEO Personality Inventory Revised , given tth Edition below 84 (1 SD below the mean) [The remaining variables are dichotomous. PRS is based on ASD status and the social score of the MPRS, used above to diagnose BAP; 61% of those assessed with MPRS were positive on PRS and 24% of non-ASD cases were positive on PRS. All ASD positive individuals were considered as affected for PRS. The pedigrees have between 6 and 10 PRS cases. CLF affection indicates a standard core language score from the Clinical Evaluation of Language Fundamentals 4he mean) . Half ofhe mean) : NWR cashe mean) . For NWRp value <1x10-10 and MSs below 1x10-4 were dropped. After thinning of the SNP map to remove marker-marker linkage disequilibrium (R2 >0.20), we based linkage analyses on a combined map comprising 10,364 SNPs + 1,078 MSs. The genetic map was based on the Build 36 hg18 (Build 37 hg19 used for X only) Rutgers Combined Linkage-Physical Map (http://compgen.rutgers.edu/RutgersMap) [Three hundred and twenty-two individuals  were genotyped using a dense microsatellite (MS) marker set (avg. intermarker distance 4 cM) obtained from DeCode; 280 individuals  were also genotyped using the Illumina OMNI 2.5 M chip. In preparation for linkage analysis, both MS and SNP data were used to verify family structure; founders were assessed for relatedness (no relatedness found), and extended relationships were confirmed. All genotypes were cleaned for marker missingness , sample missingness , and excess Mendel errors both by marker and individual; SNPs with a Hardy-Weinberg gersMap)  , and the penetrance vector fi, representing the probability that an individual with genotype i develops disease, for i \u2013 1..3. The QT model replaces the penetrances with a vector of three genotypic means and three genotypic variances. Note that normality is assumed at the genotypic level, but not at the population level, and there is no inflation of scores under violations of normality [Three different trait models were employed, depending on the type of trait being analyzed: dichotomous trait , quantitative trait , and QT threshold . The DT model is parameterized in terms of \u03b1 , the trait parameters are integrated across all pedigrees as a set at each locus. This is appropriate under the expectation that at each locus, the trait model is essentially the same across pedigrees. Under 'sequential\u2019 (PPLSEQ), trait parameters are integrated over separately for each pedigree at each locus, and the marginal evidence for or against linkage itself is accumulated across pedigrees using Bayesian sequential updating. Sequential updating is appropriate under the expectation that each pedigree may implicate different loci and/or the same loci but under different trait models . When there is relative genetic homogeneity, pooling will yield larger signals at linked loci; when there is extensive heterogeneity, however, sequential will yield larger signals at linked loci and also smaller signals at unlinked ones [The PPL has two basic approaches to the accumulation of evidence, which we employ here to consider evidence across pedigrees. Under 'pooled\u2019  to any given locus. Based on empirical data  we set \u03c0Additional distinctive features of the statistical framework are related to the fact that the PPL is a measure of statistical evidence, not a decision-making procedure. There are, therefore, no 'significance levels\u2019 associated with it  and it is not interpreted in terms of associated error probabilities ,63. By tThe results are presented in four sections: i) we consider the comparison between pooled and sequentially updated results for both ASD and BAP, in order to gauge the extent of between-family heterogeneity, and then present primary ii) ASD and iii) BAP results, followed by iv) results for the secondary phenotypes.POOL \u22650.20, but 4 under sequential ; while for BAP the corresponding numbers were 1 (PPLPOOL = 0.41) and 5 (with max PPL = 0.46 at that same position). Across the board, where PPL >10%, PPLSEQ > PPLPOOL . At the same time, the proportion of the genome showing evidence against linkage (PPL <2%) was 74% and 79% for ASD pooled and sequential, respectively, and 66% and 70% for BAP pooled and sequential, respectively. Thus, for both phenotypes, a greater proportion of the genome showed evidence against linkage under sequential analysis, and at these locations, the evidence against linkage tended to be stronger. This pattern of results is consistent with appreciable genetic heterogeneity across pedigrees. In what follows, we therefore rely on sequential as the primary data analytic approach to the accumulation of evidence going forward, dropping the 'SEQ\u2019 subscript.Figure\u00a0Four loci show PPL >20% , with additional loci on chromosomes 2, 8, 9, and possibly 12 standing out above the background as well . Apart from 2q37.2-q37.3 (which is best supported by BAP), only 2 loci are strongly supported by 2 or more of these phenotypes . At the remaining loci, only one phenotype clearly supports linkage; particularly notable in this regard are 9p21.3 and 15q26.3, which are supported by ANX with both PRS and SOC giving evidence against linkage. Thus, in general, there does not seem to be a clear pattern of correlation across these phenotypes. On the contrary, while it is difficult to draw definitive conclusions based on these pedigrees alone, there is some suggestion that the three phenotypes might be picking up different underlying genetic loci. Focusing on the 5 loci with PPL >30% for either ANX, PRS, or SOC (in order to minimize noise due to permutation over the phenotypes): 8q24.13 is supported by SOC with ANX and PRS neutral; 9q21.31-q22.31 is supported by SOC as well as PRS and probably ANX; 13q11-q12.3 is supported by ANX as well as PRS but not SOC; 15q26.3 is supported by ANX but neither PRS or SOC; and 19p12-q12 is supported by SOC but not by ANX, with PRS neutral.Finally, a striking conclusion emerges regarding RRB. RRB itself gives the two highest PPLs seen for any phenotypes : PPL = 62% at 6p25.2-24.3, and PPL = 69% at 19p13.3. Each of these loci is at best very slightly supported by one additional phenotype, with the remaining phenotypes either neutral or giving evidence against linkage. Particularly notable is that even ASD and BAP do not support these loci  produced lower PPLs at all loci with PPL >20% compared to allowing for different genetic loci and models across pedigrees (PPLSEQ) . Moreover, as previously mentioned, support was found for some previously implicated ASD loci in one individual pedigree but not others. This pattern is consistent with results from the CNV analyses published over the last decade that have shown very little sharing of loci across different families. This highlights the need for locus- and gene-discovery methods that are robust to locus heterogeneity, and should inform our interpretation of negative as well as positive findings going forward.Secondly, in aggregate, our results strongly support the presence of substantial between-pedigree locus heterogeneity for both ASD and BAP analyses. Assuming common loci across pedigrees .Finally, the phenotypes considered here are almost certainly still just proxies for more biologically defined underlying features. This complicates interpretation of results across phenotypes. For example, perhaps the most tantalizing result is the apparent independent segregation of RRB even from ASD within these ASD-multiplex families. This seems contradictory, unless ASD as diagnosed via the Autism Diagnostic Interview and Autism Diagnostic Observation Schedule (ADI and ADOS) is a measure for some aspects of autism but not all. It is worth noting here that the measurement of repetitive behaviors in the RBS-R is not equivalent to what is used for the ASD diagnosis itself; 'repetitive behaviors\u2019 do not constitute a unitary construct, and more finely grained or biologically direct phenotypes that are likely to bring us closer to the complex mechanisms occurring in ASD, would help clarify the ASD-RRB relationship from a genetic point of view .Overall, our primary conclusions speak to key features of the complexity of the genetic architecture of ASD: i) there appears to be substantial between-family locus heterogeneity; ii) in keeping with previous epidemiologic findings, the BAP does appear to correspond at least roughly to sets of subclinical features segregating with ASD within pedigrees, so that equating non-ASD relatives with non-carriers of ASD genes is not correct in general; and iii) different features of the ASD phenotype appear to segregate independently of one another within these pedigrees, in support of the multiple hit model articulated by Eichler and others -73. If tANX: Anxiety; ASD: Autism spectrum disorder; BAP: Broad autism phenotype; BAP-Q: BAP Questionnaire; CLF: Core language ability; CNV: Copy number variants; MPAS-R: Modified personality assessment schedule revised; MPRS: Modified pragmatic rating scale; MS: Microsatellite; NWR: Non-word repetition; PPL: Posterior probability of linkage; PRS: Pragmatic rating scale; QT: Quantitative trait; RAP: Rapid naming; RRB: Repetitive/ritualistic behavior; SOC: Social functioning.The authors declare that they have no competing interests.JP, VJV and PS conceived of the study, participated in design and coordination, and performed data interpretation and manuscript preparation; BF, M W-S, IO, MP and AT participated in the design of the study, oversaw data collection, and aided manuscript preparation; YH was involved in development and application of computer programs used for the analyses; KW performed data analysis. All authors read and approved the final version of the manuscript."}
+{"text": "Measurement of thyroglobulin (Tg) protein in the washout of the needle used for fine needle aspiration biopsy cytology (FNAB-C) has been shown to increase the sensitivity of FNAB-C in identifying cervical lymph node (CLN) metastasis from well-differentiated thyroid cancer (TC). In this study, we evaluated whether routine measurement of Tg protein (FNAB-Tgp), Tg mRNA (FNAB-Tgm) and calcitonin (CT) mRNA (FNAB-CTm) in the FNAB washout of CLN increases the accuracy of FNAB-C in the diagnosis of suspicious metastatic CLN.In this prospective study 35 CLN from 28 patients were examined. Histology showed metastatic papillary TC (PTC) in 26 CLN, metastatic medullary TC (MTC) in 3 CLN, metastatic anaplastic TC (ATC) in 3 CLN and 3 metastatic CLN from extra-thyroidal cancers.The overall accuracy of FNAB-C was 84.4%, reaching 95.7% when the analysis was restricted to PTC. Both FNAB-Tgp and FNAB-Tgm compared favorably with FNAB-C and shown diagnostic performances not statistically different from that of FNAB-C. However, FNAB-Tgp and FNAB-Tgm/FNAB-CTm were found useful in cases in which cytology results were inadequate or provided diagnosis inconsistent with patient's clinical parameters.We demonstrated that FNAB-C, Tg/CT mRNA and Tg protein determination in the fine-needle washout showed similar accuracy in the diagnosis of metastatic CLN from TC. The results of this study suggest that samples for Tg protein and Tg/CT mRNA measurements from CLN suspicious for metastatic TC should be collected, but their measurements should be restricted to cases in which FNAB-C provides uninformative or inconsistent diagnosis with respect to patient's clinical parameters. Thyroid cancer represents the most frequent endocrine neoplasia, accounting for 1.7%  of all new malignant diseases and 0.5%  of deaths related to cancer worldwide . The accIn the present work we evaluated, in 35 consecutive CLN for which the histological diagnosis was available, if routine measurement of thyroglobulin (Tg) protein and Tg and calcitonin (CT) mRNA in the washout of the needle used for FNAB increases the sensitivity of FNAB-C in identifying cervical lymph node (CLN) metastasis from either DTC or MTC. The results obtained suggest that while samples for Tg protein and Tg/CT mRNA measurements from CLN suspicious for metastatic thyroid cancer should be always collected, their measurements should be restricted to cases in which FNAB-C gives uninformative or inconsistent diagnosis with respect to patient\u2019s biochemical and/or clinical parameters.The case study included 35 cervical lymph nodes (CLN) with definitive diagnosis from 28 consecutive patients referred, from September 2004 to October 2012, to the outpatients\u2019 clinic of Endocrinology and Thyroid Diseases of the Policlinico Umberto I general hospital of Rome . The study was approved by the ethical committee of the Policlinico Umberto I hospital in agreement to the declaration of Helsinki and all patients gave written informed consent. Patients, 7 males and 21 females with a median age of 39.5 yr (range 20-72 yr), with single or multiple suspicious CLN underwent fine-needle aspiration biopsy (FNAB) followed by cytological (FNAB-C) evaluation, Tgp measurement, and Tgm and CTm assessment in the fine needle washout. Among them, 9 patients previously underwent total thyroidectomy with histological diagnosis of papillary thyroid cancer (see Table Thyroid ultrasonography (US) was performed on the cervical area using the Aplio XV  system equipped with a linear transducer (PLT-805AT). The scanning was performed on patients\u2019 neck hyperextended in supine position. US parameters suggestive of malignant lymph node infiltration included: rounded shape and long-to-short axis ratio inferior to 1.5; irregular echogenicity; presence of microcalcifications; irregular vascularity, i.e. peripheral or mixed  vascularity. Following US, patients selected for the FNAB were instructed not to take aspirin or any other anticoagulant in the 5 days prior to biopsy. A 25 -gauge needle, attached to 20 ml plastic syringe, was used to aspirate nodes under US assistance. Two to three separate aspiration from each CLN were made. All aspirates were smeared directly on glass slides for cytological examination as described below. The needle was then washed with 1 ml of phosphate buffered saline (PBS) by multiple pumping actions and the suspension was centrifuged at 1200 rpm for 5 min. The supernatant was collected and frozen to -20\u00b0C until analyzed for Tg protein level (FNAB-Tgp), while the pellet was quickly frozen in liquid nitrogen and stored at -80\u00b0C.Following FNAB the needle\u2019s material was expelled onto glass slides and smeared with a second slide to spread the material across the surface. The slides were then either air-dried or wet-fixed using the Bio-Fix . Air-dried slides were stained with a May Grunwald-Giemsa solution, while the wet-fixed slides were stained with the Papanicolaou solution. For the histological diagnosis, soon after lymphadenectomy the tissue was placed in 10% buffered formalin and paraffin embedded. Four \u03bcm thick sections were then prepared and stained with hematoxylin and eosin . Both cyTgp levels in the FNAB washout were measured using the immunoluminometric assay (ILMA) Tg-PluS . The analytical and functional sensitivity of the assay was, respectively, 0.02 ng/ml and 0.15 ng/ml. Tg values below the functional sensitivity were considered undetectable . Samples2+, specific Tg, CT and beta-2-microglobulin (B2M) primers 0.5 \u03bcM, 2-3 \u03bcl of cDNA, and molecular biology-grade water to 50 \u03bcl. In particular, the sequences of the above primers were as follows: Tg, forward CTCTGGAAAGATTCTGACATGG (exon 28-29), reverse CTCTGGAAAGATTCTGACATGG (exon 30-31) (amplicon 243 bp); CT, forward CCTTCCTGGCTCTCAGCATC (exon 2), reverse GAGTTTAGTTGGCATTCTGG (exon 4) (amplicon 407); B2M, forward CAGCAGAGAATGGAAAGTC (exon 2), reverse CATGCTGCTTACATGTCTCG (exon 3) (amplicon 269 bp). After 2 min of initial denaturation at 94\u00b0C, 40 cycles have been run as follows: 94\u00b0C for 20 sec, 56-60\u00b0C for 10 sec, 65\u00b0C for 50 sec. Positive controls, represented by normal human thyroid tissue for Tg mRNA and the MTC derived cell line TT for CT mRNA, have been included each time. The obtained amplicons have been visualized by agarose gel electrophoresis, and their specificities have been checked by automated DNA sequencing .The above mentioned pellets have been resuspended in 500 \u03bcl of Isol-RNA lysis reagent  and total RNA extracted as previously described . In partSensitivity, specificity, diagnostic accuracy, positive and negative predictive value of each diagnostic test were calculated. Differences in sensitivity, specificity, negative and positive predictive values and accuracy among the different tests were evaluated by the Fisher exact test. Cohen\u2019s k coefficient was used to estimate the agreement between various diagnostic tests. Statistical calculations were performed using Stata version 8.0 . The results were considered statistically significant when the two-tailed p value was <0.05.As reported in Table As reported in Figure Tg and CT mRNA levels were measured in 32 out of the 35 CLN with definitive diagnosis. In 2 cases samples were judged inadequate because of the absence of \u03b22-microglobulin mRNA. As shown in Tables The CT mRNA analysis provided correct results in all 30 samples (3 true positive and 27 true negative).The comparison of the diagnostic parameters of FNAB-C, FNAB-Tgp and FNAB-Tgm, alone or in combination, was performed excluding from the case series 3 CLN harboring metastasis from MTC and 3 CLN affected by extrathyroidal cancers. Specificity, sensitivity, PPV, NPV and accuracy are reported in Table As it may be noticed, the diagnostic performances were comparable and not significantly different among FNAB-Tgp, FNAB-Tgm and FNAB-C. In addition, the combined use of FNAB-Tgp\u2009+\u2009FNAB-Tgm or FNAB-C\u2009+\u2009FNAB-Tgp\u2009+\u2009FNAB-Tgm did not improve significantly the diagnostic value of FNAB-C alone Table .When the analysis was restricted to the 26 CLN harboring metastatic PTC FNAB-C and FNAB-Tgp showed, respectively, 95.7 and 100% accuracy and, in the 20 CLN in which both diagnoses were available, a 95% overall agreement between the 2 tests was observed Table . On the Regarding the 3 CLN harboring metastatic ATC (Table FNAB-Tgp provided a correct diagnosis in all 3 CLN with uninformative cytology. Also FNAB-Tgm provided correct diagnosis in 2 of the 3 CLN with inadequate cytology, being inadequate as well as in the third case.Cytological evaluation of FNAB represents the gold standard technique for the diagnosis of CLN suspected to harbor metastatic disease from thyroid cancer as well as from other primary tumors. The technique accuracy, highly dependent on the experience and ability of the cytopathologist, has been reported to vary from 73% to 94% ,18,22,25The results showed that, on 32 CLN harboring metastases from papillary and anaplastic thyroid carcinomas, FNAB-C had 88.5% accuracy, that is within the range reported in other studies ,18,22,25A major diagnostic value of FNAB-Tgp and FNAB-Tgm was observed in cases of uninformative FNAB-C, given that in all 3 CLN characterized by inadequate cytology FNAB-Tgp provided the correct diagnosis. In these cases, FNAB-Tgm also provided the correct diagnosis in 2 of them, being inadequate in one. Other instances in which the combined use of FNAB-Tgp, FNAB-Tgm and FNAB-CTm is valuable include those cases in which FNAB-C diagnosis is not consistent with patient\u2019s clinical and biochemical parameters. This was the case (Table FNAB-C remains the gold standard technique for the diagnosis of suspicious CLN due to its ability to efficiently diagnose metastasis not only from thyroid cancers, but also from other primary tumors. If confirmed on larger case studies, the results of this study suggest that samples for Tg/CT mRNA and Tg protein measurements from CLN suspicious for metastatic thyroid cancer should be always collected Figure , but theThe authors declare that they have no competing interests.EB, SS, PM, EDeA and SU conceived the study. EB, CDG, AA, LG, GC and EDA run the experimental procedures and organized data collection. SU and CDV performed the statistical analysis. SS, PM, EDeA and SU drafted the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6890/13/7/prepub"}
+{"text": "Many exome sequencing studies of Mendelian disorders fail to optimally exploit family information. Classical genetic linkage analysis is an effective method for eliminating a large fraction of the candidate causal variants discovered, even in small families that lack a unique linkage peak. We demonstrate that accurate genetic linkage mapping can be performed using SNP genotypes extracted from exome data, removing the need for separate array-based genotyping. We provide software to facilitate such analyses. Whole exome sequencing (WES) has recently become a popular strategy for discovering potential causal variants in individuals with inherited Mendelian disorders, providing a cost- effective, fast-track approach to variant discovery. However, a typical human genome differs from the reference genome at over 10,000 potentially functional sites -4 and reOther WES studies have not performed formal linkage analysis, but have nonetheless considered inheritance information, such as searching for large regions of homozygosity shared by affected family members using genotypes obtained from genotyping arrays -18 or exClassical linkage analysis using the multipoint Lander-Green algorithm , which iWe investigated whether linkage analysis using the Lander-Green algorithm could be performed using genotypes inferred from WES data, removing the need for the array-based genotyping step . We infeWe anticipated two potential disadvantages to this approach. Firstly, exome capture only targets exonic SNPs, resulting in gaps in marker coverage outside of exons. Secondly, genotypes obtained using massively parallel sequencing (MPS) technologies such as WES tend to have a higher error rate than those obtained from genotyping arrays . The useWe compared the results of linkage analysis using array-based and exome genotypes for three families with different neurological disorders showing Mendelian inheritance Figure . We sequAllele frequencies and genetic map positions were available for 3,269,163 HapMap Phase II SNPs that could be translated to UCSC hg19 physical coordinates. The Illumina TruSeq platform used for exome capture targeted 61,647 of these SNPs (1.89%). After discarding indels and SNPs whose alleles did not match the HapMap annotations, a median 56,931 (92.3%) of targeted SNPs were covered by at least five high-quality reads  and 731,306 genotypes for M-3 and M-4 (Illumina OmniExpress BeadChip). Table t = 0.5 (see below), 77% (346 of 450) of discordant SNPs were ambiguous SNPs, while ambiguous SNPs composed just 2.7% of all SNPs . Such SNPs are prone to strand annotation errors, as the two alleles are the same on both strands of the SNP. We therefore discarded ambiguous SNPs, which left 29,459 to 52,892 SNPs available for comparison  comprised a high proportion of SNPs with discordant genotypes, despite being a small proportion of SNPs overall. For example, for A-7 at coverage \u2265 5 and ow), 77% 46 of 450t, upon concordance of genotyping array and WES genotypes , while the male T-1 had one such call out of 635 genotypes (0.16%). The same SNPs were not called as heterozygous by the genotyping arrays. No heterozygous \u00d7 chromosome calls were observed at the default value of t = 0.001.We note that Prior to performing linkage analysis on exome and array SNP genotypes, we selected one SNP per 0.3 cM to ensure linkage equilibrium while retaining a set of SNPs dense enough to effectively infer inheritance. The resulting subsets of WES genotypes Table  containeDespite this difference, there was good agreement between LOD scores achieved at linkage peaks using the different sets of genotypes Figure , Table 5If our genetic model is correct, then variants lying outside of linkage peaks cannot be the causal mutation and can be discarded, thus reducing the number of candidate disease-causing variants. Table Linkage analysis is of great potential benefit to WES studies that aim to discover genetic variants resulting in Mendelian disorders. As variants outside of linkage peaks can be eliminated, it reduces the number of identified variants that need to be investigated further. Linkage analysis of WES genotypes provides information regarding the location of the disease locus to be extracted from WES data even if the causal variant is not captured, suggesting regions of interest that may be targeted in follow-up studies. However, many such studies are being published that employ less sophisticated substitutes for linkage analysis or do not consider inheritance information at all. Anecdotal evidence suggests that a substantial proportion of MPS studies of individuals with Mendelian disorders fail to identify a causal variant, though an exact number is not known due to publication bias.We describe how to extract HapMap Phase II SNP genotypes from massively parallel sequencing data, providing software to facilitate this process and generate files ready to be analyzed by popular linkage programs. Our method allows linkage analysis to be performed without requiring genotyping arrays. The flexibility of linkage analysis means that our method can be applied to any disease model and a variety of sampling schemes, unlike existing methods of considering inheritance information for WES data. Linkage analysis incorporates population allele frequencies and genetic map positions, which allows superior identification of statistically unusual sharing of haplotypes between affected individuals in a family.We demonstrate linkage using WES genotypes for three small nuclear families - a dominant family from which two exomes were sequenced and two consanguineous families from which a single exome was sequenced. As these families are not very powerful for linkage analysis, multiple linkage peaks with relatively low LOD scores were identified. Nonetheless, discarding variants outside of the linkage peaks eliminated between 81.2% and 99.43% of all nonsynonymous exonic variants detected in these families. The number of variants remaining could be reduced further by applying standard strategies, such as discarding known SNPs with minor allele frequencies above a certain threshold. Our work demonstrates the value of considering inheritance information, even in very small families that may consist, at the extreme, of a single inbred individual. As the price of exome sequencing falls, it will become feasible to sequence more individuals from each family, resulting in fewer linkage peaks with higher LOD scores.Exome capture using current technologies yields large numbers of useful SNPs for linkage mapping. Over half of all SNPs covered by five or more reads were not targeted by the exome capture platform. Approximately 78% of these captured untargeted SNPs lay within 200 bp of a targeted feature. This reflects the fact that fragment lengths typically exceed probe lengths, resulting in flanking sequences at both ends of a probe or bait being captured and sequenced. The serendipitous result is that a substantial number of non-exonic SNPs become available, which can and should be used for linkage analysis.t = 0.2 for inferring genotypes at known SNPs [t = 0.001. Our results highlight the need to tailor this parameter to the specific application, either genotyping or rare variant detection. Although we anticipated WES genotypes being less accurate than array genotypes, all four samples achieved a high concordance of 99.7% for SNPs covered by five or more reads at t = 0.5We found that setting the prior probability of heterozygosity to 0.5 during genotype inference resulted in the best concordance between WES and array genotypes. The authors of the MAQ SNP model recommend using own SNPs , while tWe found that LOD scores obtained from WES genotypes agreed well with those obtained from array genotypes from the same individual(s) at the location of linkage peaks, with the median difference in LOD score zero to two or three decimal places for all three families. This was despite the fact that the array-based genotype sets used for analysis contained more markers and had higher average heterozygosities than the corresponding WES genotype sets, reflecting the fact that genotyping arrays are designed to interrogate SNPs with relatively high minor allele frequencies that are relatively evenly spaced throughout the genome. By contrast, genotypes extracted from WES data tend to be clustered around exons, resulting in fewer and less heterozygous markers after pruning to achieve linkage equilibrium. We conclude that if available, array-based genotypes from a high resolution SNP array are preferable to WES genotypes; but if not, linkage analysis of WES genotypes produces acceptable results.Once WGS is more economical, we will be able to perform linkage analysis using genotypes extracted from WGS data, which will obviate the problem of gaps in SNP coverage outside of exons. The software tools we provide can accommodate WGS genotypes without requiring modification. In the future, initiatives such as the 1000 Genomes Project [The classic Lander-Green algorithm requires markers to be in linkage equilibrium . ModelinWe have integrated the relatively new field of MPS in families with classical linkage analysis. Where feasible, we strongly advocate the use of linkage mapping in combination with MPS studies that aim to discover variants causing Mendelian disorders. This approach does not require purpose-built HMMs, but can utilize existing software implementations of the Lander-Green algorithm. Where genotyping array genotypes are not available, we recommend utilizing MPS data to their full capacity by using MPS genotypes to perform linkage analysis. This will reduce the number of candidate disease-causing variants that need to be evaluated further. Should the causal variant not be identified by a WES study, linkage analysis will highlight regions of the genome where targeted resequencing is most likely to identify this variant.Written informed consent was provided by the four participants or their parents. Ethics approval was provided by the Royal Children's Hospital Research Ethics Committee (HREC reference number 28097) in Melbourne. Genomic DNA was extracted from participants' blood samples using the Nucleon\u2122 BACC Genomic DNA Extraction Kit .All four individuals were genotyped using Illumina Infinium HumanHap610W-Quad BeadChip  or OmniExpress  genotyping arrays . These arrays interrogate 598,821 and 731,306 SNPs respectively, with 342,956 markers in common. Genotype calls were generated using version 6.3.0 of the GenCall algorithm implemented in Illumina BeadStudio. A GenCall score cutoff  of 0.15 was used.Target DNA for the four individuals was captured using Illumina TruSeq, which is designed to capture a target region of 62,085,286 bp (2.00% of the genome), and sequenced using an Illumina HiSeq machine . Individual T-1 was sequenced using one-quarter of a flow cell lane while the other three individuals were sequenced using one-eighth of a lane. Paired-end reads of 110 bp were generated.Reads were aligned to UCSC hg19 using Novoalign version 2.07.05 . QualitySNP genotypes were inferred from WES data using the samtools mpileup and bcftools view commands from release 916 of the SAMtools package , which iThese genotypes were formatted for linkage analysis using a modified version of the Perl script linkdatagen.pl  with an The two Perl scripts used to extract genotypes from VCF files and format them for linkage analysis are freely available on our website , as is tMultipoint parametric linkage analysis using WES and array genotypes was performed using MERLIN . A popult = 0.001. Variants were annotated by ANNOVAR [SAMtools mpileup/bcftools was also used to detect variants from the reference sequence with the default setting of  ANNOVAR  using thbp: base pair; HMM: hidden Markov model; MPS: massively parallel sequencing; SNP: single nucleotide polymorphism; VCF: variant call format; WES: whole exome sequencing; WGS: whole genome sequencing.KRS conceived of the study and performed all analyses described in the article. MB provided guidance and ideas. CJB wrote software tools. MSH, AES, and RJHS performed whole exome sequencing. MSH performed array-based SNP genotyping. RJHS, RJL, HN, GM and DJA collected families and clinical data. PJL contributed reagents and materials. KRS and MB drafted the initial article. All authors discussed the results and commented on the manuscript.Supplementary tables.Click here for file"}
+{"text": "VEGFC and one common variant on chromosome 4q conferred the greatest risk for the dichotomous trait. We identified two rare variants on chromosomes 4q (VEGFC) and 6p (VEGFA) that explain 12.4% of the total phenotypic variance of trait Q1. We also identified four variants (including one at VNN3) on chromosome 6q that are able to drop the linkage LOD from 3.7 to 1.0. These results suggest that the use of classical linkage and association methods in large families can provide a useful approach to identifying variants that are responsible for diseases and complex traits in families.Compared to genome-wide association analysis, linkage analysis is less influenced by allelic heterogeneity. The use of linkage information in large families should provide a great opportunity to identify less frequent variants. We perform a linkage scan for both dichotomous and quantitative traits in eight extended families. For the dichotomous trait, we identified one linkage region on chromosome 4q. For quantitative traits, we identified two regions on chromosomes 4q and 6p for Q1 and one region on chromosome 6q for Q2. To identify variants that contribute to these linkage signals, we performed standard association analysis in genomic regions of interest. We also screened less frequent variants in the linkage region based on the risk ratio and phenotypic distribution among carriers. Two rare variants at  Common variants have been successfully identified for many diseases and complex traits through the use of genome-wide association studies (GWAS). Although many of the findings from GWAS have been replicated in different populations, current association results have yet to explain many existing linkage regions of interest. Furthermore, GWAS have limited power to identify rarer variants, in contrast to linkage analysis, which is not sensitive to allelic heterogeneity . Thus thIn this paper, we present our analysis of the Genetic Analysis Workshop 17 (GAW17) family data  , in thiij\u03c0 denote the proportion of alleles shared IBD between relatives i and j at a locus. ij\u03c0 can be estimated as ij\u03c0 is twice the kinship coefficient ijf . For highly informative markers, the mean and variance of ij\u03d5 and var(ij\u03c0) , respectively, under the null hypothesis of no linkage. It is straightforward to consider the Z statistic:For the dichotomous trait, we use an affected relative pair nonparametric linkage (NPL) scan. The NPL method assumes that, in the genomic region in linkage with the trait, affected relative pairs are expected to share more alleles identical by descent (IBD) than average. Let ormation . The expi, j) and  index all affected relative pairs. The covariance of the estimated IBD sharing can be well approximated with the technique used in the regression-based quantitative trait linkage method [i,j\u03a3k,l Cov, which is a function of the pedigree structure . Becausfor each affected relative pair (parent-offspring pairs are excluded for the lack of variation). Because the covariate Age is a strong risk factor for the dichotomous trait (a 10-year increase in age doubles the risk of being affected), it is crucial to take into account the effect of aging in the NPL analysis. We include only affected relative pairs with an age difference no larger than 16 years in the NPL analysis. Although this threshold is somewhat arbitrary, we subsequently applied other threshold values to ensure that our results were not too sensitive to this value. Note that our linkage results are not inflated by potential linkage disequilibrium (LD) between adjacent markers, because the IBD statistics are known in our simulated data. In practice, IBD statistics need to be estimated, and the LD needs to be properly modeled in the linkage analysis when genotype data for the parents of affected sib pairs are not complete [To examine the association of SNPs with simulated phenotypes, we apply the standard transmission disequilibrium test (TDT)  and the ij\u03c0, kl\u03c0) in the test statistic can be conveniently calculated as a function of the pedigree structure without using the SNP data). Covariates in the linear regression of the quantitative traits include Age, Smoking status, and the first principal component from a multidimensional scaling (MDS) structure analysis [For the quantitative traits, we implement a score-based robust linkage analysis . Althouganalysis  in whichWe examine the identified linkage region using the variance component score test as implemented in the GDT software package (through parameter fastAssoc) . Althougp = 2 \u00d7 10\u221216), with a 10-year increase in age doubling the risk of being affected. Smoking is the second largest risk factor. Sex is not significantly associated with the affection status. Our NPL scan on 22 autosomes with adjustment for Age revealed one significant linkage region on chromosome 4q with maximum LOD = 3.1 at position 170.34 Mb and a one-LOD support interval between 142.8 Mb and 177.9 Mb. The linkage evidence is derived primarily from the largest family . Family 7 alone provides the maximum LOD (4.2) in a region between 153.57 Mb and 177.90 Mb.For the dichotomous trait, Age is the most statistically significant risk factor . However, this result is sensitive to the age threshold. When Age is not incorporated into the analysis, the evidence supporting linkage dropped to LOD = 1.7; thus we restricted our subsequent analyses to the region on chromosome 4q.p < 0.001). Rather than comparing allele frequency differences between affected and unaffected individuals, we computed the odds of being affected among carriers of rare and infrequent variants. We estimated allele frequencies based on all 202 founders, representing the general population. In the linkage region, four SNPs had odds greater than or equal to 1: C4S4373 at 167.01 Mb , C4S4915 at 176.14 Mb , C4S4916 at 176.14 Mb , and C4S4935 at 177.85 Mb . C4S4373 and C4S4916 (9 Mb apart) are in complete LD  so only one of these two SNPs needed to be further studied. In addition, C4S4915 and C4S4916 (101 bp apart) are in strong LD. All C4S4916 variant carriers were also C4S4915 variant carriers, whereas three of the C4S4915 carriers were not C4S4916 carriers. All C4S4916 and C4S4935 carriers were private to family 7 and were not present in other families.To localize the variants that contribute to the evidence supporting linkage, we first performed TDT and GDT analyses. No significant association was found , indicating that all carriers inherited the rare variant from a common founder in family 7. When we focused only on family 7, by screening all SNPs, we also found two common variants with a large affected/unaffected ratio: C4S5105 and C4S5108. These two SNPs are 240 bp apart and in high LD; we selected C4S5108 because it provides a slightly higher significance. Together, these three variants are able to explain 36 out of 50 affected individuals in family 7. We summarize the three variants in Table ADAM29), 6.80 , and 1.77 (C4S5108). The LD block between C4S4373 and C4S4916 (9 Mb apart) together with the other two variants can explain the large interval of support in the linkage analyses.We performed a logistic regression analysis with the three SNPs included in the model Table . We usedp-values were not substantially improved using the collapsed alleles.In addition to the SNP-by-SNP association analysis, the rare variants with larger effect identified through our screening procedure were collapsed within genes before the association analysis. We assumed that the collapsed genotype of an individual was a heterozygote if the individual was a carrier of any of the rare variants (a homozygote with two copies of the rare variant is rather rare). However, the GDT h2 = 0.615 (Q1), h2 = 0.432 (Q2), and h2 = 0.697 (Q4). A bivariate analysis identified a modest genetic correlation between Q1 and Q2 (Gr = 0.255), suggesting that these two traits may share genes in common. There was no evidence of a common genetic basis between Q4 and the other two quantitative traits.The MDS structure analysis  identifiThe robust quantitative trait linkage analysis identified two linkage regions for Q1 and one linkage region for Q2. The first region for Q1 is on chromosome 4q, overlapping with the linkage region for the dichotomous trait. The maximum linkage support is LOD = 14.8 at 167.1 Mb, with a wide region of support (88.0\u2013186.1 Mb with LOD > 3). The second region supporting linkage for Q1 is on chromosome 6p  with a large support region (LOD > 3 from 0 to 80.0 Mb). The region supporting linkage for Q2 is on chromosome 6q at position 143.6 Mb, with a maximum LOD of 3.7.VEGFC, MAF = 0.002, p = 2.3 \u00d7 10\u221215) and 6p . A conditional linkage analysis adjusting for these two SNPs resulted in a maximum LOD of 0 in these two regions, with no other significant associations. Table Our association scan identified rare variants on chromosomes 4p , the two variants explained 26.7% of the total phenotypic variance (the estimated heritability was reduced from 0.814 to 0.547). We screened the rare variants according to the phenotypic distribution of the carriers, but we did not identify any other rare variants that contributed independently to the variation beyond that from C4S4935 and C6S2981. On chromosome 6p, besides C6S2981 with a large effect on Q1, other potential rare variants include C6S752 at 25.83 Mb, C6S2245 at 31.71 Mb, and C6S2432 at 32.92 Mb; however, all C6S752 carriers  are also C6S2981 carriers, and all C6S2432 carriers are also C6S752 carriers. Thus a LD block exists in the region between 25.83 Mb and 43.85 Mb. This LD block could explain why the SNP with the strongest effect is at 43.85 Mb even though the linkage peak is at 26 Mb.The estimated heritability was reduced with the two rare variants included, from VNN3), C6S6047 at 144.8 Mb , C6S6659 at 155.5 Mb , and C6S6839 at 155.6 Mb . When the four SNPs were adjusted in the linkage analysis, the LOD was reduced from 3.7 to 1.0. Although the four variants were able to explain most of the linkage in the region, they had small effects on Q2: They explained only 4.3% of total phenotypic variance, and none of the variants were significantly associated with Q2.Q2 exhibits significant linkage on chromosome 6q (with a maximum LOD = 3.7 at 143.6 Mb). By screening the phenotypic distribution among the carriers, we identified two rare variants, one less frequent variant, and one common variant that partly explained the evidence for linkage in this region: C6S5449 at 133.1 Mb  and one common variant on chromosome 4q for the dichotomous trait, two rare variants on chromosomes 4q (at VEGFC) and 6p (at VEGFA) that explain 12.4% (or 26.7% in the largest family) of the phenotypic variance of trait Q1, and rare variants at VNN3 on chromosome 6q that explain the linkage to trait Q2. No linkage regions were identified for trait Q4. The variant at VEGFC on chromosome 4q underlies both the dichotomous trait and the quantitative trait Q1. Compared to the true model that was used to simulate the GAW17 family data [Our linkage and association analysis identified two rare variants . Bioinformatic annotation  should be incorporated as well.The variants identified in our analysis are rare in the general population  and would be difficult to identify in a population-based study. These variants have a much higher frequency in some families, and our work shows that the use of linkage and association in large families provides a powerful way to identify variants that are responsible for diseases and complex traits.The authors declare that there are no competing interests.WC designed and carried out the statistical analysis, and drafted the manuscript. AM participated in the design of the analysis and drafted the manuscript. SSR helped to draft the manuscript. All authors read and approved the final manuscript."}
+{"text": "Diuretic agents are widely used on the treatment of water retention related diseases, among which acetazolamide (AZA) acts originally as a carbonic anhydrase (CA) inhibitor. Aquaporin-1 (AQP1) being located in renal proximal tubules is required for urine concentration. Previously our lab has reported AZA putatively modulated AQP1. Aim of this study is to testify our hypothesis that regulating AQP1 may mediate diuretic effect of AZA.in vivo study, we utilized Sprague Dawley rats, as well as AQP1 knock-out (AQP1\u2212/\u2212) mice to examine urine volume, and human kidney-2 (HK-2) cell line was used for in vitro mechanism study. In our present study we found that AZA decreased CAs activity initially but the activity gradually recovered. Contrarily, diuretic effect was consistently significant. AQP1 protein expression was significantly decreased on day 7 and 14. By utilizing AQP1\u2212/\u2212 mice, we found diuretic effect of AZA was cancelled on day 14, while urine volume continuously increased in wild-type mice. Surface plasmon resonance (SPR) results indicated AQP1 was physiologically bound by myosin heavy chain (MHC), immunoprecipitation and immunofluorescence results confirmed this protein interaction. In vitro study results proved AZA facilitated AQP1 translocation onto cell membrane by promoting interaction with MHC, dependent on ERK/ myosin light chain kinase (MLCK) pathway activation. MHC inhibitor BDM and ERK inhibitor U0126 both abolished above effect of AZA. Eventually AZA induced AQP1 ubiquitination, while proteasome inhibitor MG132 reversed AZA's down-regulating effect upon AQP1.For Our results identified AZA exerted diuretic effect through an innovative mechanism by regulating AQP1 and verified its inhibitory mechanism was via promoting MHC-dependent translocation onto cell membrane and then ubiquitin mediated degradation, implicating a novel mechanism and target for diuretic agent discovering. Aquaporin-1 (AQP1) was the first water channel to be identified 3 re-absorption and acid secretion in the process of urine formation. AZA exerts its diuretic role by inhibiting both cytoplasm form CAII and membrane-bound form CAIV located in renal proximal tubular epithelial cells, which catalyze the equilibration between carbon dioxide and carbonic acid and mediate re-absorption of HCO3\u2212. Thus, after CAs activity is inhibited by AZA, HCO3\u2212 re-absorption is suppressed, resulting in increase of HCO3\u2212 excretion. CAs inhibition also decreases the production of H+ and reduces the H+-Na+ exchange, resulting in suppression of Na+ re-absorption and H2O re-absorption in proximal tubules. Eventually CAs inhibition by AZA induces a mild diuretic effect. As a diuretic agent, AZA is clinically used to treat edema due to congestive heart failure and drug-induced water retention. However, the rapid development of tolerance has limited its application. Previous study in our lab Acetazolamide (AZA) is a potent inhibitor of carbonic anhydrases (CAs), which catalyze the equilibration of carbon dioxide and carbonic acid and plays a key role in NaHCOThe purpose of the present study is to determine whether the diuretic effect of AZA is partially mediated by modulating AQP1. The mechanism of AQP1 reduction after administration of AZA was also discussed. Our data suggested that AZA promoted interactions between AQP1 and myosin heavy chain (MHC). Consequently more AQP1 was carried to cell membrane and then ubiquitination of AQP1 followed by the degradation through proteasome occurred. Therefore, decreasing AQP1 protein expression was proved to be a novel diuretic mechanism of AZA.Firstly, we found single administration of AZA caused a remarkable increase in urine volume of rats in the first 8 hours. Then urine volume was maintained on a high plateau until administration for 3 days and sustained to increase on day 7 and day 14 . To exam3 as clinically used. NaHCO3 was commonly used to improve metabolic acidosis induced by AZA. The combination of AZA and NaHCO3 significantly reduced carbonic anhydrase activity within 8 hours as well. Moreover, in this combination condition recovery of carbonic anhydrase activity was inhibited. On day 14 of administration, CAs activity only recovered to 62.65% of control. The expression of carbonic anhydrase II and IV was not increased either . AZA induced diuretic effects in both AQP1\u2212/\u2212 mice and wild-type mice after 8 hours of administration. However, after 14 days of administration, AZA only increased urine outputs in wild-type mice, but could not exert a diuretic role in AQP1\u2212/\u2212 mice contrarily. AQP1 deficiency blunted the diuretic effect of AZA on day 14.Furthermore, to determine the role of AQP1 in diuretic effect induced by AZA, AQP1in vitro tool to discover how this inhibition occurred, and in pre-experiments, we found AZA incubation for 24 hours could significantly reduce AQP1 protein level in HK-2 cells (data not shown). At first, we focused on whether AZA could affect AQP1 mRNA level because transcriptional regulation is a fundamental manner of protein expression change. We used RT-PCR amplification of AQP1 mRNA prepared from HK-2 cells to examine AQP1 mRNA content. Unfortunately, it did not change from 5 minutes to 24 hours in a medium contained 3\u00d710\u22126 mol/L AZA  cells as an ol/L AZA .\u22126 mol/L AZA, and then cell membrane and cytoplasm were separated. Besides of transcriptional regulation, the other mechanism about AQP1 expression change is ubiquitin-proteasome mediated degradation It has been shown that AQP1 degradation through ubiquitin-proteasome system mediated inhibitory effect of AZA, and additionally we noticed that AQP1 ubiquitination only occurred on cell membrane. Considering that previous work has proved translocation between cytoplasm and membrane was an important way of AQP1 content regulation Above results led us to a further question: How AZA promoted AQP1 translocation onto cell membrane? Since it has been reported microtubules belonging to motor proteins in cells may participate in translocation of AQP1 Next we examined whether AZA could affect the co-localization of AQP1 and MHC in HK-2 cells, we concluded that the increased merged fluorescence intensity indicated the co-localization was enhanced when cells were treated with AZA . However\u22122 mol/L, AQP1 translocation was abolished ) and MLC as well as their activate forms, which may take part in regulation of AQP1 and MHC, were examined by western blot analysis. The results  signaling pathway plays an important role in AQP1 content regulation  results  showed t results . The act results . MLCK acERK/MLCK/MLC pathway activation orderly after AZA treatment gave us the clue that this signaling pathway eventually activated MHC to interact with AQP1, then promoting translocation onto cell membrane and reduction by degradation. To validate the role of this ERK-dependent pathway, we used U0126 specifically inhibiting ERK and found it prevented AQP1 from being increased on cell membrane after AZA incubation (data not shown) and finally reversed the AQP1 reduction induced by AZA . Moreove2++Aquaporins are a family of homologous intrinsic membrane proteins that are responsible for the water permeability of membranes in a variety of cell types \u2212/\u2212 mice validated the critical role of AQP1 in the long-term diuretic process. As is reported, AQP1 deficiency produced remarkable diuretic effect before administration. In AQP1\u2212/\u2212 mice, because of absence of AQP1, AZA's diuretic effect was abolished when CAs activity totally recovered on day 14 after dosing. By contrast, for wild type mice, when AQP1 was present, AZA produced notable diuretic effect on day 14 after administration despite of recovering of CAs activity. On day 14, if AQP1 was not the only or main target of AZA, AZA would increase urine volume in AQP1 knockout mice through other mechanisms despite absence of AQP1. However, on Day 14 we demonstrated AZA could not increase urine volume in knockout mice compared to wild type mice  group was significantly higher than that in NaHCO3 (\u2212) group on day 14 of administration, because now the diuretic effect come from adding of both AQP1 and CAs inhibition, which indicates that inhibiting AQP1 or CAs by AZA are two independent events in kidney both leading to diuresis. To further exclude the impact of NaHCO3 in our present study, we added a group of rats receiving only 30 mg/kg/day of NaHCO3, and urine volume, CAs activity and expression, as well as AQP1 protein expression were measured. We found single use of NaHCO3 could slightly elevate rats' urine volume compared with control group, but only on Day 7 there was statistical significance  since SPR has been considered as a powerful and reliable tool to discover novel protein interaction Action of myosin heavy chain needs regulation and binding of myosin light chain, which can be phosphorylated by myosin light chain kinase. In our experiments, we found that upstream of MLCK-signal molecule ERK-has been activated by AZA. Therefore, we propose that AZA induces phosphorylation of MLCK via activation of ERK pathway, and then promotes interaction between MHC and AQP1 to increase the translocation of AQP1. As predicted, ERK pathway inhibitor U0126 can reverse the effect of AZA on AQP1. In addition, like U0126, MLCK non-specific wortmannin also abolished down-regulating effect of AZA upon AQP1. Besides, U0126 or wortmannin alone could not affect expression of AQP1 in our present study (data not shown). Therefore, we consider ERK-MLCK pathway as an important mediator in suppression effect of AZA upon AQP1. However, there is one limitation that that wortmannin is a non-specific inhibitor of MLCK in vitro study, we have examined the level of cAMP after administration of AZA, however, the change of cAMP level is not consistent with trafficking of AQP1 (data not shown). In another study, researchers have demonstrated that ultraviolet radiation (UVB) and H2O2 induced AQP1 down-regulation which was mediated by ERK activation, however, the mechanism was not mentioned in this article 2+-dependent manner A lot of signaling pathways have been proved to take part in trafficking and content regulation of aquaporins. Due to AQP2 translocation, cAMP-protein kinase A (PKA) pathway has a crucial role; however, other signaling cascades also induce AQP2 translocation, such as phosphoinositide 3-kinase (PI3K) pathway Eventually we focused on the fate of AQP1 which has been transported onto cell membrane after administration of AZA. Previous work of our lab has proved that AZA binds directly to AQP1 and inhibits water permeability In conclusion, the present study provides evidence that AZA has different diuretic mechanisms at different time points. At beginning, AZA inhibits carbonate anhydrases activity. Then CAs activity recovers and the diuretic mechanism of AZA is caused by reducing AQP1 protein expression. The reduction of AQP1 is due to increase of translocation onto the cell membrane dependent on ERK/MLCK pathway and interaction with MHC, and then degradation through uibiquitin-proteasome system. Our study implicates an innovative mechanism for action of diuretics AZA and also provides a novel sight for diuretic agent discovery.AZA, MG132, U0126, Wortmannin and BDM were from Sigma\u2013Aldrich Corporation . Biacore3000 CM5 series sensor chips were purchased from Biacore AB . Other biochemical reagents were from Beijing Chemical Plant .This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of China Association for Laboratory Animal Science. All animal care and experimental protocols were approved by the Animal Care Committee of Peking University Health Science Center. All sacrifice was performed under pentobarbitone anesthesia, and every effort was made to minimize suffering.\u2212/\u2212 and wild-type mice were gifts from Professor Yang Baoxue in Peking University. Rats were divided into 18 groups randomly. Five to seven rats for each group were treated with AZA at 40 mg/kg/day or vehicle for 8 h, 1, 3, 7, 14 days with or without combination with 30 mg/kg/day NaHCO3. Rats were maintained on axenic laboratory chow with free access to water. All rats were kept in metabolic mages in a Specific Pathogen Free (SPF)-level laboratory. After administering drugs or vehicle 8 hours, urine volumes of rats were measured and recorded. AQP1\u2212/\u2212 and wild-type mice were randomly divided into 3 groups respectively. Six mice for each group were treated with vehicle or AZA at 60 mg/kg/day for 8 h and 14 days. Animals were kept in metabolic mages in a SPF-level laboratory and free access to water. Urine volumes of mice were measured pre-treatment and after administering vehicle or drugs.Male rats of Sprague Dawley strain, initially weighting 160 g, were obtained from the Animal Center of Peking University Health Science Center and AQP1An endpoint colorimetric microtechnique 2 at 37\u00b0C. Cells passaged into 0.5\u20131\u00d7105 cells per 60 mm cell culture dish. The cells were incubated with drugs or vehicle after growing to 70% to 80% confluence, and then cells were harvested and extracted by RIPA lysis buffer containing PMSF (phenylmethylsulfonyl fluoride) and protease inhibitor cocktail . Next, lysates were centrifuged at 12,000 g for 10min at 4\u00b0C. The supernatants were collected and stored at \u221280\u00b0C. Protein concentration was determined by BCA assay .HK-2 cells were purchased from Cell Culture Centre, Institute of Basic Medical Science Chinese Academy of Medical Sciences . Briefly, HK-2 cells were cultured in DMEM/F12 medium supplemented with antibiotics (penicillin and streptomycin) and 10% fetal bovine serum  in a humid atmosphere incubator with 5% CO4HCO3, 2 mmol/L CaCl2. The bound proteins were eluted with 2 \u00b5L of 0.1% TFA and deposited to a vial in a sample rack for the following analysis.AQP1 protein was isolated and purified from the rat red blood cells. The purified AQP1 was injected onto the CM5 chip. Binding activity of AQP1 immobilized on the CM5 chip was assayed using AQP1 antibody at an increasing concentration. After immobilization of AQP1, rat kidney extraction was diluted in five-fold of HBS-EP buffer. The insoluble residue was pelleted by centrifugation and discarded. The supernatant was diluted ten-fold in the same buffer (in the following this preparation is referred to as kidney extract), and 200 \u00b5L kidney extract was injected at a flow rate of 20 \u00b5L/min. HBS-EP buffer was used as the running buffer in this experiment. The capture and recovery of the AQP1 binding proteins was conducted according to the RECOVERY program encoded in the instrument. The flow system was then washed with 50 mmol/L NaOH and the flow cells were rinsed with 50 mmol/L NHThe masses of the peptides obtained above were analyzed by MALDI-TOF/MS (Matrix-Assisted Laser Desorption/Ionization-Time of Flight/Mass Spectrometry) on an AXIMA-CFR Plus MALDI-TOF mass spectrometer (Shimadzu Biotech). The peptide masses were submitted to the MSDB database via the Mascot database search engine (Matrix Science) for protein identification.Rats were injected with pentobarbitone (100 mg/kg) i.p. to induce anesthesia. The kidney was removed and washed three times in ice cold PBS (pH 7.4), and then cortex portion was homogenized in lysis buffer. HK-2 cells were lysised in the RIPA buffer on ice. The homogenate was then centrifuged at 12,000 g for 10 min at 4\u00b0C. The protein concentration of the supernatant was determined using the BCA assay. 0.5 mg/ml protein was added in 1 \u03bcg/mL IgG and 20 \u03bcl/ml protein A-Agarose . After centrifugation 1,000 g for 5min at 4\u00b0C, the supernatant was added in 10 \u03bcl/ml AQP1 or MHC antibody at 4\u00b0C for 1 hour. Then 20 \u03bcl/ml protein A- Agarose was added to the lysate and mixed at 4\u00b0C overnight. After centrifugation 1,000 g for 5min at 4\u00b0C, the supernatant was removed. Wash the pellet 4 times and take a part of the protein for protein concentration determination. The others were re-suspended with loading buffer. The sample was boiled for 5min, and then centrifuged at 4\u00b0C, at 1,000 g for 5min. Finally, obtained samples were analyzed by Western blot analysis.Samples (60 \u03bcg) were separated by 12% SDS-PAGE. Upon completion of the electrophoresis, the proteins were transferred to PVDF membrane. Non-specific binding sites were blocked by pre-incubating the membrane in 5% skimmed milk in Tris-buffered saline Tween . After PVDF Membranes were incubated overnight at 4\u00b0C with specific antibodies: anti-p-ERK (Tyr204), anti-ERK1/2, anti-p-p38 (Thr180/Tyr182), anti-p38, anti-p-JNK (Thr183/Tyr185), anti-JNK, anti-carbonic anhydrase II, anti-carbonic anhydrase IV, anti-MHC and anti-AQP1  and washed three times with TBS-T buffer, the membranes were incubated with alkaline phosphatase-conjugated goat anti-rabbit or goat anti-mouse IgG . Then the western blot bands were scanned, and bands intensity was analyzed by Bio-Rad Quantity One software for quantification.18 and ReverAidTM M-MuLV reverse transcriptase  were used to generate the first strand cDNA mix. The mixture were incubated for 1 hour at 42\u00b0C, then the reverse transcriptase was inactivated by heating to 95\u00b0C for 5min and the contents were cooled to 4\u00b0C before allocating for polymerase chain reaction (PCR). Sequence specific primers for human AQP1 (5\u2032-AGATCAGCATCTTCCGTG-3\u2032 and 5\u2032-AGTTGTGTGTGATCACCG-3\u2032) -3\u20325\u2032-AACGGATTTGGTCGTATTG and -3\u20325\u2032-GCTCCTGGAAGATGGTGAT) were employed in the PCR system. Amplification was performed in a thermal cycler  using the following program: initial melt at 94\u00b0C for 3 minutes, 55\u00b0C for 1minute, 31 cycles at 95\u00b0C for 45 seconds, 55\u00b0C for 40 seconds, and 72\u00b0C for 45 seconds, followed by a final extension at 94\u00b0C for 1minute and 60\u00b0C for 10 minutes and storage at 4\u00b0C. The products were analyzed using 1% agarose gels with DNA ladders.HK-2 cells were harvested and total mRNA was prepared by a commercial available kit . The RNA was quantified spectrophotometrically, by measuring absorbance at 260 nm (A260), and its purity was determined by the ratio of A260/A280. 5 \u03bcg each of total RNA together with the oligo (dT) HK-2 cells were grown on poly-lysine treated glass slide. Firstly, Wash 3 times with PBS before staining. After fixation by 4% paraformaldehyde at room temperature for 20min, cells were penetrated by 0.5% Triton X-100 at room temperature for 20min. Then wash 3 times with PBS. Next cells were incubated by a normal corresponding serum working solution at room temperature for 30min. Primary antibodies for AQP1 and MHC were incubated at 4\u00b0C overnight, subsequently the appropriate secondary antibody was incubated at 37\u00b0C for 1 hour. Nuclear was stained by Hoechst 33342 of 1 min. Lastly Wash 3 times with PBS. The images were recorded by confocal microscope .The myosin light chain kinase (MLCK) ELISA kit was purchased from Beijing ZhongHaoShiDai Biotechnology Center . Briefly, 100ul sample was added into one well. Cover microfilter plates and react at 37\u00b0C for 90 minutes. After being washed twice, biotin antibody working solution was added to wells. Then react at 37\u00b0C for 60 minutes. After washing 3 times, ABC working solutions were added to wells. Then react at 37\u00b0C for 30 minutes. Following washing for 5 times, TMB color working solution was added to wells at 37\u00b0C in dark. Then add TMB stop solution to wells. OD values of 450 nm were examined by ELISA Reader .P<0.05 was considered to be statistically significant.All results are expressed as means \u00b1 S.E.M. For multiple comparisons, the statistical analysis was performed by using one-way ANOVA followed by the Newman-Keuls multiple comparison tests. Figure S13 alone on rats urine volume, carbonic anhydrases activity and expression, as well as aquaporin-1 expression.Impact of NaHCO(DOC)Click here for additional data file.Figure S2Effect of MLCK inhibitor wortmannin on AQP1 expression.(DOC)Click here for additional data file.Figure S3Time-course effect of acetazolamide on AQP1 protein expression on the cell membrane and cytoplasm.(DOC)Click here for additional data file.Table S13.Blood pH values in Rats administrated with acetazolamide combined with or without NaHCO(DOC)Click here for additional data file."}
+{"text": "Generally, SNPs are abundant in the genome; however, they display low power in linkage analysis because of their limited heterozygosity. Haplotype markers, on the other hand, which are composed of many SNPs, greatly increase heterozygosity and have superiority in linkage statistics.Here we developed Haplo2Ped to automatically transform SNP data into haplotype markers and then to compute the logarithm (base 10) of odds (LOD) scores of regional haplotypes that are homozygous within the disease co-segregation haploid group. The results are reported as a hypertext file and a 3D figure to help users to obtain the candidate linkage regions. The hypertext file contains parameters of the disease linked regions, candidate genes, and their links to public databases. The 3D figure clearly displays the linkage signals in each chromosome. We tested Haplo2Ped in a simulated SNP dataset and also applied it to data from a real study. It successfully and accurately located the causative genomic regions. Comparison of Haplo2Ped with other existing software for linkage analysis further indicated the high effectiveness of this software.http://bighapmap.big.ac.cn/software.html.Haplo2Ped uses haplotype fragments as mapping markers in whole genome linkage analysis. The advantages of Haplo2Ped over other existing software include straightforward output files, increased accuracy and superior ability to deal with pedigrees showing incomplete penetrance. Haplo2Ped is freely available at:  Linkage analysis plays an important role in mapping disease-causing genes. Compared to other methods, such as association research, not only are very limited samples needed in linkage study, but also the high disease homogeneity among pedigree members increases the possibility of locating causative genes ,2. FurthAlong with the achievement of high-throughput SNP genotyping, using whole genome SNP data for linkage analysis has been shown to be an efficient strategy ,6. HowevSoftware packages have been developed to carry out multi-point analysis. The traditional linkage methods employed two basic algorithms: the Elston-Steward algorithm, used in Allegro, and the Lander-Green algorithm, used in Merlin. SNPLINK, a Perl Script that performs full genome linkage analysis, uses both these algorithms. However, the application of these two algorithms is limited, either by the number of markers or by the size of the pedigrees. Another program, SNP4Linkage, is based on allele sharing determination and is better adapted to high-density SNP genotyping data. Nevertheless, it still lacks a tool for considering haplotype fragments as genomic markers for linkage research -10. TherHaplo2Ped is an effective tool using haplotypes as markers for linkage analysis. It is well-suited for genome-wide linkage mapping with high density SNP data. It provides a user-friendly interface to select input files and set parameters to perform the analysis Figure . The RunFirstly, we consider the example of a dominant disease model. In a trio, if the child and his father are both affected, the father's transmitted haploid will be selected as an aHap. Conversely, if the child is healthy, the affected father's untransmitted haploid will be deemed as an aHap. When we cannot be sure of the child's affected status (the child is too young to show symptoms or it is a disease with incomplete penetrance), then the affected father's two chromatids would be selected and treated with the rule that at least one of them is an aHap.Once the set of aHaps is determined from all the trios, the haplotype sharing analysis is performed. A window-length and step-size are set to scan these aHaps to determine disease candidate segment(s) generated from recombination events Figure . For theFor a disease with incomplete penetrance, we cannot determine whether the asymptomatic healthy child is really disease free or not. As referred to above, we treat both the transmitted and un-transmitted haploids of their affected parent as paired aHaps. The two assumed aHaps are then compared to the assured aHaps. A true disease co-segregation haplotype fragment should be found in at least one of the two assumed aHaps. Regarding determination of a candidate region by window sliding, although paired aHaps are not as informative as the assured aHaps, they may still contribute to shortening the linked regions and identifying whether or not the child carries the disease targeted haplotype.Using the above method to analyze a disease caused by fragment deletion may result in two linked regions separated by a homozygous region caused by the deletion. For large deletions (> 500 Kb), such a result may lead to confusion or an incorrect conclusion. Therefore, Haplo2Ped provides a LOH (loss of heterozygous) test to detect large fragment deletions.We used the pedigree data generated from Illumina 370 K CNV-Quad chip as an example for analysis. The raw dataset came from a family with RP (retinitis pigmentosa) disease. To test Haplo2Ped, we made certain changes in the raw data to generate simulated disease targets. The pedigree structure is shown in Figure The genotype data of the simulated pedigree was then submitted to Haplo2Ped for linkage mapping. Figure To compare the efficiency of Haplo2Ped with other existing software, we submitted the same simulated data to Merlin, SNPLink, and SNP4Linkage. The output results are listed in Table SNPLINK did not report LOD scores in the final output files although it showed good accuracy on four regions co-segregating with the disease. Furthermore, SNPLINK missed some regions on the left edge of two expected regions on chromosomes 1 and 13. The results from SNP4Linkage were the same as SNPLINK. There were no false positive regions detected by these two programs.In a real study of a digital-anomaly family, we applied Haplo2Ped to SNP genotype data from 13 family members for the linkage analysis by haplotype, and successfully located the linkage region. Further study determined the mutation of the causative gene [To evaluate the false positive rate of Haplo2Ped, we simulated genotype data sets for thirty pedigrees with one causal mutation each using an in-house developed Perl script (packaged with Haplo2Ped). Each data set was analyzed by both Haplo2Ped and Merlin. The false positive regions reported by Merlin were significantly more than those reported by Haplo2Ped . Two or more disease founders would result in more than one type of disease haplotype for the family, which could lead to either loss of linkage signals, or generate false positives. Haplo2Ped analysis is based on deduced parental haplotypes; therefore, in cases where one parent is missing in a nuclear family, it is still applicable for linkage study.Our simulation analysis showed that Haplo2Ped was consistently accurate in pinpointing the regions co-segregating with the disease. It did not miss any expected regions, while other software reported biased results, especially on the left edge of certain regions. Given the limited recombination events accumulated in a pedigree, both the disease-causing mutation and the neighboring SNPs in a shared haplotype co-segregate with the disease phenotype. However, when a disease-causing haplotype is transmitted to offspring, recombination occurs at random sites of this haplotype, indicating that the disease-causing mutation also probably locates at the edge of our assumed regions. If any expected regions are missed, the risk of not locating the final mutation is increased.A gain of LOD score using haplotypes as markers in our tool demonstrated an advantage over Merlin, which is based on classical maximum-likelihood methods. Employing haplotypes with high heterozygosity as markers avoided the false positive results generated by Merlin, which is subject to the low heterozygosity of individual SNPs. Furthermore, the LOD score of the SNPs reported by Merlin in the assumed regions usually varies over a wide range. Many SNPs even show lower LOD scores than those in the unlinked regions. This adds to the difficulty of locating the linked regions. Thus, we suggest that our method of combining the heterozygosity of multi-SNPs and the breakpoints of recombination (borders of co-segregating haplotype) better reflects the stable strength of a linkage region compared to a method that only uses the heterozygosity of individual SNPs.Another advantage of Haplo2Ped is its capability of dealing with the diseases that exhibit incomplete penetrance, a model of which is not included in software such as SNPLINK. Using simulated data with incomplete penetrance, although Merlin reported expected linkage regions similar to those of Haplo2Ped  detection for pedigrees in which fragment deletion causes the disease. We propose that haplotype fragments could be powerful genomic markers in linkage analysis.Software name: Haplo2Pedhttp://bighapmap.big.ac.cn/software.htmlSoftware home page: Operating system(s): Windows or LinuxProgramming language: Matlab platformOther requirements: NoLicense: NoThe authors declare that they have no competing interests.FC and CZ conceived the study. FC and XZ wrote the paper. FC implemented the program. XZ carried out comparison analysis between different software. YZ and CZ improved the manuscript. CL conducted the microarray experiments. All authors read and approved the final manuscript.The pedigree simulation process. The pedigree simulation process was carried out based on a real genotype data set of a RP (retinitis pigmentosa) family. The family members' affected status was reset and six disease co-segregating haplotypes were simulated in chromosomes 1, 5, 9, 13, 17, and 21.Click here for fileSoftware comparisons using real data. Linkage regions detected by three different softwares are from data of a real study (Table S1). The pedigree analyzed is a three-generation Han Chinese family with complex digital anomalies.Click here for fileEvaluation of false positive rate of Haplo2Ped with completely simulated genotype data. Comparison of false positive rate between Haplo2Ped and Merlin based on simulated genotype data of thirty different pedigree structures.Click here for fileSoftware comparison with simulated pedigree in incomplete penetrance. The detected regions reported by Haplo2Ped and Merlin using simulated pedigree in incomplete penetrance.Click here for file"}
+{"text": "Ovis aries) to construct a linkage map for bighorn sheep, Ovis canadensis. We then assessed variability in genomic structure and recombination rates between bighorn sheep populations and sheep species.The construction of genetic linkage maps in free-living populations is a promising tool for the study of evolution. However, such maps are rare because it is difficult to develop both wild pedigrees and corresponding sets of molecular markers that are sufficiently large. We took advantage of two long-term field studies of pedigreed individuals and genomic resources originally developed for domestic sheep , resulting in an autosomal female map of 3166 cM and an autosomal male map of 2831 cM. Despite differing genome-wide patterns of heterochiasmy between the sheep species, sexual dimorphism in recombination rates was correlated between orthologous intervals.Ovis species. However, domestication may have played a role in altering patterns of heterochiasmy. Finally, we found that interval-specific patterns of sexual dimorphism were preserved among closely related Ovis species, possibly due to the conserved position of these intervals relative to the centromeres and telomeres. This study exemplifies how transferring genomic resources from domesticated species to close wild relative can benefit evolutionary ecologists while providing insights into the evolution of genomic structure and recombination rates of domesticated species.We have developed a first-generation bighorn sheep linkage map that will facilitate future studies of the genetic architecture of trait variation in this species. While domestication has been hypothesized to be responsible for the elevated mean recombination rate observed in domestic sheep, our results suggest that it is a characteristic of  The construction of genetic linkage maps in model organisms and domesticated species enables studies of the genetic architecture of trait variation and genome evolution. However, such resources for free-living populations of non-model species are still rare because it is difficult to acquire large enough pedigrees and associated sets of molecular markers ,2. The uOvis canadensis), a mountain ungulate inhabiting western North America )We constructed a first-generation bighorn sheep linkage map using DNA from two wild pedigreed population and genomic resources originally developed for domestic sheep. Since bighorn sheep and domestic sheep genomes are very similar, future efforts to increase marker density in specific chromosomal regions should be relatively straightforward. This could be achieved using bighorn sheep single nucleotide polymorphism (SNP) markers recently discovered using the OvineSNP50 Beadchip , additioThe main reason for developing genomic resources in bighorn sheep is to allow studies of complex trait genetic architecture and evolution under natural settings. In the NBR population, genomic resources will enable investigations into the genetic basis of fitness, inbreeding depression and genetic rescue . In RM, Ovis species while the unusual male-biased heterochiasmy might have been a consequence of domestication. Finally, we have demonstrated that interval-specific patterns of sexual dimorphism could be conserved among closely related species, possibly due to the position of these intervals relative to centromeres and telomeres.While resources developed for domestic sheep are obviously highly useful to bighorn sheep research, genomic research in bighorn sheep can also yield valuable information through comparisons with domestic sheep in return. For example, we have demonstrated how linkage mapping in bighorn sheep can be used to infer ancestral marker order in domestic sheep. Also, by comparing the domestic sheep map with the map of a close wild relative, we were able to determine that the elevated recombination rates observed in domestic sheep were likely a characteristic of The NBR population was established by transplanting four rams and eight potentially pregnant ewes from Banff National Park  in 1922 . The popThe RM population is native to a small isolated mountain range located about 50 km east of the Canadian Rockies in Alberta, Canada . ImmigraIn both populations, parentage was originally determined with ~30 microsatellite loci using the 95% confidence threshold in Cervus . For theIn addition to markers used for the initial pedigree reconstruction, microsatellites putatively distributed throughout the genome of our focal species were identified using the domestic sheep IMF map version 4.7 ,51. MarkWe constructed population-specific linkage maps as well as an integrated map where populations were treated as independent families using CRI-MAP . The sam10 likelihoods. The relative sex-averaged length of different maps was compared by summing the length of intervals that were present in both maps  of Lovich and Gibbons . This inSince the length of an interval partly depends on the subset of markers included in a linkage analysis, we assessed the validity of comparing intervals between maps constructed using different number of markers (the domestic sheep IMF map 4.7 contains about 1400 markers). To accomplish this, we repeated cross-species analyses using information derived from additional linkage maps based solely on markers mapped in both species. Results and conclusions were essentially the same as for previous analyses and are therefore not presented. These maps are available in Additional file All research protocols were approved by the University of Alberta Animal Use and Care Committee, affiliated with the Canadian Council for Animal Care (Certificate 610901).JP designed the study, conducted laboratory work, performed data analysis and wrote the manuscript. JTH designed and supervised fieldwork at NBR and performed paternity analyses. CSD implemented laboratory methods and performed laboratory work. JMM participated in laboratory work and data analysis. JFM provided marker information and constructed a domestic sheep linkage map composed solely of markers mapped in bighorn sheep. DWC planned and supervised the study. All co-authors read and commented on draft versions of the manuscript. All authors read and approved the final manuscript.List of markers, map position and variability. Excel document displaying List of markers, map position and variability.Click here for fileComparison of bighorn sheep population-specific maps. PDF displaying comparison of bighorn sheep population-specific maps.Click here for fileComparison of intervals present in both population-specific bighorn sheep maps. XLS file displaying comparison of intervals present in both population-specific bighorn sheep maps.Click here for fileComparison of intervals present in bighorn sheep and domestic sheep maps. XLS file displaying comparison of intervals present in bighorn sheep and domestic sheep maps.Click here for fileBighorn sheep mapping pedigrees. PDF file displaying bighorn sheep mapping pedigreesClick here for fileBighorn sheep and domestic sheep linkage maps based on shared markers only. XLS bighorn sheep and domestic sheep linkage maps based on shared markers onlyClick here for file"}
+{"text": "Background: Recurrent spontaneous abortion (RSA) is one of the most common health complications with a strong genetic component. Several genetic disorders were identified as etiological factors of hereditary X linked RSA. However, more genetic factors remain to be identified.Objective: In this study we performed linkage analysis on a large X linked RSA pedigree to find a novel susceptibility locus for RSA. Materials and Methods: A linkage scan using 11 microsatellites was performed in 27 members of a large pedigree of hereditary X-linked RSA. Two point parametric Linkage was performed using Superlink v 1.6 program.Results: Evidence of linkage was observed to markers at Xq23, DXS7133 and at Xq22.1 DXS101, with LOD score of 3.12 and 1.60, respectively.Conclusion: Identified locus in this study may carry a responsible gene in RSA. Narrowing down of this region may leads to identification of this gene.This article extracted from M.Sc. thesis. (Sahar Shekouhi) Spontaneous abortion is the fate of approximately 15% of all clinically recognized pregnancies in the general population . Recurrent spontaneous abortion (RSA) is defined as three or more consecutive pregnancy losses prior to 20-22 week of gestation . RSA is RSA patients often occur as isolated case in families; however almost rarely occur as a hereditary complication in pedigrees. Unknown single-gene or polygenic disorders, involved in fundamental cellular and reproductive processes, can cause idiopathic recurrent euploid losses. Theoretically single gene abnormalities, along with balanced chromosomal abnormalities, can be inherited by offspring and leads to RSA as a Mendelian disorder in pedigree members, hereditary RSA -15. In other hand, hereditary RSA is a genetically heterogeneous phenomenon and occurs either as autosomal dominant, autosomal recessive or X linked disorder. Up to now several single gene disorders for example hydrops fetalis result from alpha thalassemia and thrombophilic factors, have been identified to cause or increase the risk of hereditary RSA , 17. HowLinkage mapping study of hereditary RSA pedigrees, can lead to the identification of genetic factors responsible for such complications. For assessment of genetic linkage, linkage analysis was performed. The present study is the first one to investigate linkage to known RSA susceptibility locus in Mashhad population which is characterized by its heterogenous genetic background. Regarding the non-random X inactivation in women with repetitive miscarriage, it has been concluded that probably there are several loci on the chromosome X which can lead to occurrence of hereditary RSA , 19.So far, several X linked disorders such as Rett syndrome, incontinentia pigmenti and some kinds of hemophilia type-A are identified as the causes of hereditary RSA pedigrees , 17. HowIn this experiment, we have analyzed a large pedigree of hereditary RSA and, identified a region which may be involved in hereditary RSA by using linkage.PatientsThis experiment was a linkage analysis study. A large family with a five generation pedigree and 9 RSA patients was assessed in this experiment. Proband was a 30 year old woman having 5 consecutive spontaneous abortions without any live offspring. Various studies investigating recurrent abortion causing disorders, including anatomical abnormalities, immunological, hormonal, Thrombophilic and cytogenetic abnormalities did not show any abnormality. In addition, 8 more affected and 7 non affected women along with 11 healthy men were genotyped in this family. DNA genotypingAfter completing the informed consent, 5 to 10 ml of brachial venous blood was collected in EDTA tubes. DNA was extracted from lymphocytes by a standard salting out method. 11 short tandem repeats (STRs) loci  which scattered through the X chromosome with an average spacing of 10 cM were selected for linkage analysis. 2, 0.2 \u03bcM of each primer and 1 unit of Taq HotStart DNA polymerase. PCR reaction was performed in 2720 ABI thermal cycler which is programmed for 35 cycles of denaturation at 94oC for 40s, 64oC for 40s and 72oC for 2min; an additional step of denaturation at 95oC for 10min preceded the first cycle and another step of extension at 72oC for 7 min was extended at the end of reaction. PCR products were resolved using 10% Polyacrylamide Gel Electrophoresis (PAGE). According to total number of alleles in pedigrees each allele was numbered from 1  to 6  and each patient woman receives a tow number code for each STR locus. Since men are hemi-zygote for X linked genes they receive unicharacter code. Microsatellites were genotyped by using of PCR amplification  using Superlink v1.6 program. Pedigree was analysed as recessive model and LOD scores were obtained at 0.05 recombination increment steps until to recombination value of 0.45. Penetrance of disease and disease allele frequency were given 99% and 0.001, respectively. STR alleles were set to codominant and Map order and genetic inter-STRs distances were taken from the Marshfield STRP map.Pedigree is shown in Genotype of pedigree members is shown in In other hand STR between qTelomer and DXS7133  does not show any evidence of linkage .Recurrent pregnancy loss can be caused by several factors involved in fundamental events during human reproduction. Anatomical, immunological, hormonal and infectious factors along with known genetic factors are involved in 50% of cases, however remaining 50% of abortions has not any known reason and named as idiopathic abortion. In recent decade increasing number of evidence indicate that genetic disorders may be responsible in major part of idiopathic abortions. Although RSA often occur as isolated case, however, sometimes hereditary RSA found with apparent Mendelian inheritance mode. These RSA pedigrees have important role in identification of new RSA etiologic loci. Linkage analysis can be performed on these pedigrees to candidate loci as genetic factors of these RSA cases. Moreover, in some experiments on X linked RSA pedigrees some loci such as Incontinentia pigmenti, Rett syndrome and hemophilia were identified to be linked with RSA , 17. HowBetween RSA pedigrees, X linked RSA pedigrees has interesting characteristics which encourage linkage analysis. As possible locus located on X chromosome, all of analyzed polymorphic markers can be confined on this chromosome. As a result, the number of needed markers decrease in compare with autosomal pedigrees and performing linkage become very easy. Furthermore, it has been suggested that a significant portion of isolated RSA cases may have X chromosome disorders. These disorders lead to reduction of fitness in cell which has defected X chromosome as active one. This leads to deviation of 50:50 ratio of cells inactivating either X chromosome, a phenomena known as Skewed X inactivation.Several studies suggest that Skewed X inactivation, defined as 90% inactivation of one parental allele, has association with increasing of recurrent abortion , 18, 19.http://research.-marshfieldclinic.org/ genetics/ Map_Markers/ maps/IndexMap-Frames.html). In this work, we present the genotyping results of 27 members  of an X linked RSA pedigree from a village located near Mashhad, Iran. Loci DXS7132, DXS6800, DXS6789, DXS7133, HPRTB, GATA31E08, DXS7423, DXS101, DXS6807, DXS143, and DXS7130 have been investigated , 21. Allet al and linkage for X-linked dominant genodermatosis incontinentia pigmenti (In this study we report mapping of a novel region on X chromosome Xq23) in relation to X linked recurrent abortion following linkage analysis. Our result is in agreement with previous reports of linkage for autism loci; particularly those reported by Kilpinen pigmenti , 23. Thi in relat"}
+{"text": "To date, genome-wide association studies have yielded discoveries of common variants that partly explain familial aggregation of diseases and traits. Researchers are now turning their attention to less common variants because the price of sequencing has dropped drastically. However, because sequencing of the whole genome in large samples is costly, great care must be taken to prioritize which samples and which genomic regions are selected for sequencing. We are interested in identifying genomic regions for deep sequencing using large multiplex families collected as part of earlier linkage studies. We incorporate linkage analysis into our search for Q1-associated alleles. Overall, we found that power was low for both whole-exome and linkage-guided sequencing analysis. By restricting sequencing to regions with high LOD peaks, we found fewer associated single-nucleotide polymorphisms than by using whole-exome sequencing. However, incorporating linkage analysis enabled us to detect more than half of the associated susceptibility loci (52%) that would have been identified by whole-exome sequencing while examining only 2.5% of the exome. This result suggests that incorporating linkage results from large multiplex families might greatly increase the efficiency of sequencing to detect trait-associated alleles in complex disease. Linkage studies have fallen out of favor in recent years as genome-wide association has become the new paradigm for gene discovery. However, genome-wide association itself is perhaps reaching its limit, because the price of sequencing has decreased and is likely to drop much further. At this point, the cost of whole-genome sequencing is still high enough that great care must be taken to select which samples or genomic regions to sequence. Much of this sequencing will not include newly collected samples but will use samples from existing studies, either of the case-control or pedigree variety. We are interested in the potential of large multiplex families , obtained as part of linkage studies, to guide subsequent sequencing efforts. This analysis could be done either by identifying highly informative individuals to sequence, by directing the analysis to gain greater power, or by prioritizing certain regions for deep sequencing rather than taking a genome-wide approach.In this paper, we explore the utility of linkage analysis of large pedigrees to prioritize certain genomic areas for sequencing. This method can be viewed as an extreme case of guiding an analysis for greater power . Of courIn this study, we compute the variance component logarithm of odds (LOD) scores for Q1 and Q4 for all 200 replicates provided in the Genetic Analysis Workshop 17 (GAW17) data set. The median heritability for the 200 simulation replicates is 58% for Q1 and 63% for Q4. We then examine the power of the 17 truly associated Q1 SNPs by controlling the type I error inferred from the association results with 218 unassociated SNPs for Q4 because the simulation model does not include any truly associated SNPs for Q4. This allows us to compare power and type I error rates for two sequencing strategies: (1) whole-exome sequencing followed by association tests on all SNPs detected from the whole exomes and (2) targeted sequencing of exomes under linkage peaks followed by family-based association tests using polymorphisms in these linked regions.For each of the 200 GAW17 simulation replicates, we used the 697 individuals from 8 families for linkage and association analyses. We did not split the large multiplex families into small families. We performed genome-wide variance components linkage analysis . We incoFor the association analysis, we computed residuals from a linear model that included Age, Sex, and Smoking status for traits Q1 and Q4 and used the residuals in subsequent association analyses. From the simulation model, we selected 17 SNPs that were truly associated with Q1. A set of 218 SNPs, including 201 SNPs that were not associated with Q1 and the true Q1 SNPs (17 SNPs), was tested for association with Q4. Each SNP was coded as 0, 1, or 2 with respect to the number of minor alleles and was used as a covariate in the RELPAL program in S.A.G.E. (version 6.0) [ik\u03a5 is the trait value of individual i in pedigree k, ikX is the design vector for fixed effects for individual i , B is the coefficient vector of fixed effects, ikZ is the design vector for within-pedigree random effects, b is the coefficient vector for pedigree-specific covariates and polygenetic effects, and the ike are individual-specific random effects assumed to be independently and identically distributed [where tributed . SignifiWe evaluated the power for association using Q1 and type I error using Q4. Because we found unexplained genotype correlation across chromosomes, the unassociated trait Q4 was an appropriate choice to calculate the type I error. To address the problem of multiple testing, we applied two adjustments for significance thresholds using the Sidak correction:N is the number of statistical tests. First, an adjustment was based on the total number of SNPs  analyzed in the whole exome. The second adjustment was based on the number of SNPs under the 1.5-LOD support interval for regions with a LOD score greater than 3.3 in each of the 200 replicates. We applied the significance threshold for linkage signal at a LOD of 3.3 for a conservative genome-wide significance level [p-value threshold ranging from 0.05 to 2.81 \u00d7 10\u22126). P-values outside the LOD support area are set to 1 and are therefore never considered significant. That is, true Q1 risk alleles that are not under a support interval with a peak LOD score greater than 3.3 are not carried forward for association analysis in the linkage-guided strategy and thus are considered false negatives.where VEGFA and C4S4935 in VEGFC. After correcting for the number of SNPs tested in the genome-wide approach, we found that the power to detect both of these SNPs was greater than 99%. The power to detect the truly associated loci was greater than the nominal \u03b1 level for only three of the remaining SNPs, all of them located in the FLT1 gene  for each replicate. The number of SNPs within 1.5-LOD support intervals from the most significant linkage peaks varied substantially by replicate. Significant linkage loci were observed at all but 12 of the Q1 replicates but only at 7 of the Q4 replicates. On average, 611.7 SNPs per replicate were under linkage peaks, which represent only about 2.5%  of the exome. The percentage of the genome included in the linkage peaks varied across replicates but was never larger than 7% of the whole exome. Therefore a great reduction in sequencing cost could be achieved by restricting sequencing to areas under linkage peaks. The linkage analysis of Q4 indicates that a much smaller percentage of the exome would be sequenced for unassociated traits, with only seven replicates requiring any sequencing at all, and that none of the regions overlapped in different replicates. The average proportion of the genome sequenced for unassociated traits if sequencing were restricted to linked regions would be 11.4/24,487, or 0.04%, which suggests a low false-positive rate.We examined the power for the true Q1 susceptibility loci using linkage results to guide our association analyses (Table Seven out of 43,600 SNPs showed significant association for Q4. However, among 218 unassociated SNPs only 10 were under the linkage region, and none of these were significant. This implies that the linkage-based sequencing produced zero false positives out of 43,600 tests.We examined 17 causal SNPs for Q1 and 218 unassociated SNPs for Q4. We then examined these SNPs using two sequencing paradigms: whole-exome and linkage-guided sequencing. Association results with the whole-exome sequencing approach with appropriate corrections accounting for multiple testing revealed that overall power to detect association with small effect sizes, regardless of SNP minor allele frequency, was quite low. Only two SNPs were detected with a power greater than 80%.For the second approach, we first performed genome-wide robust variance components linkage analyses for Q1 and Q4 using the supplied IBD sharing. Then, we identified SNPs linked to traits in each replicate, defined as being within a 1.5-LOD support interval of a LOD score greater than 3.3. Finally, we recomputed the power to detect each of the Q1 SNPs under a linkage-guided sequencing paradigm, using a less stringent multiple testing penalty that accounted only for SNPs falling under linkage peaks. Using the linkage results, we detected association with the two easily detected SNPs about 70% of the time. Comparing 90% with 70% of the power to detect only 2 of the 17 susceptibility loci might seem low, but it is important to keep in mind that power to detect the other Q1 loci is also low under a whole-exome paradigm. By using the linkage-guided approach to reduce the amount of sequencing, we found that restricting sequencing under the linkage peaks would have detected more than 52% of the loci found by whole-exome sequencing despite the fact that only 2.5% as much of the genome would have to be sequenced. This statistic seems better if we restrict our attention to two loci that could be detected with high power, where restricting sequencing under linkage peaks would have detected association approximately 70% of the time. This demonstrates that sequencing under linkage peaks can be an efficient strategy for examining large multiplex families in terms of the number of true associations obtained per base pair sequenced.Our method is only the first step in an evaluation of the utility of linkage information in association analysis. It would also be important to evaluate the difference between analyses of the full sample and analyses that sequenced only families that appeared to be linked. When we examined the significance of SNPs by family, it was clear that for most SNPs the evidence for association emerged from a single family or a small group of families. Performing pedigree-specific LOD score analysis may enable a further reduction in the number of base pairs to be sequenced without compromising the power to detect mutations associated with the traits of interest. One limitation of our study is that we did not account for population substructure in our current association analyses. Further analysis would be necessary to evaluate whether or not the substructure confounds the reported findings.The authors declare that there are no competing interests.SHC carried out all association analyses and power calculation, and participated in drafting the manuscript. CL carried out the linkage analyses and provided assistance with the power calculation, and participated in drafting the manuscript. JD conceived of the study, participated in the design of the study, and participated in drafting the manuscript. MWL conceived of the study, participated in its design and statistical analyses, and drafted the manuscript. GJ conceived of the study, participated in its design and statistical analyses, and helped to draft the manuscript. All authors read and approved the final manuscript."}
+{"text": "For each population, a genome with four linkage groups (100 cM) was generated. The linkage groups possessed 51, 21, 11 and 6 marks, respectively, and a corresponding distance of 2, 5, 10 and 20 cM between adjacent marks, thereby causing various degrees of saturation. For very saturated groups, with an adjacent distance between marks of 2 cM and in greater number, i.e., 51, the method based upon stochastic simulation by simulated annealing presented orders with distances equivalent to or lower than rapid chain delineation. Otherwise, the two methods were commensurate through presenting the same SARF distance.The efficiency of simulated annealing algorithms and rapid chain delineation in establishing the best linkage order, when constructing genetic maps, was evaluated. Linkage refers to the phenomenon by which two or more genes, or even more molecular markers, can be present in the same chromosome or linkage group. In order to evaluate the capacity of algorithms, four F Genetic mapping favors breeding activities, by associating one or more marks to those genes of economic interest and/or control quantitative characteristics (QTL), with a reasonable chance of use in assisted selection, hence the extreme importance of the precise construction of genetic maps in the successful introduction of strategies in breeding programs.One of the most important stages in the construction of linkage maps is the ordering of the genetic markers within each linkage group  were simulated. Genomes were generated for each population, with four linkage groups, each 100 cM in size. There were 51, 21, 11 and 6 marks in each linkage group, with distances of 2, 5, 10 and 20 cM, respectively, between adjacent marks, thus causing various degrees of saturation. The groups were composed of:In order to create a real situation and compare the efficiency of the methods, four Fm1), marker 2 (m2), ..., marker 51 (m51), with intervals between adjacent marks of 2 cM;\u2022 First linkage group: marker 1 (m52), marker 53 (m53),..., marker 72 (m72), with intervals between adjacent marks of 5 cM;\u2022 Second linkage group: marker 52 (m73), marker 74 (m74),..., marker 83 (m83), with intervals between adjacent marks of 10 cM;\u2022 Third linkage group: marker 73 (m84), marker 85 (m85),..., marker 89 (m89), with intervals between adjacent marks of 20 cM.\u2022 Fourth linkage group: marker 84  for computing application was used in obtaining the above populations.I = {1, ..., k} be a set of indices and M = {mi: i \u2208 I} a set of markers indexed by i. Consider that Dij represents the distance between the marker mi and the marker mj and define \u039b as a set of all the possible permutations of the elements of the M set. An M element will be denoted by i, ..., \u03c3k) is a permutation of the elements of set I. A permutation xm \u2208 \u039b can be understood as an order to by-pass all the markers. The problem is to find an order that minimizes the distance necessary to by-pass all the markers only once, without the need of returning to the origin.The problem of mark ordering by performing the analogies necessary for solving the traveling salesman problem, can be described in the following way: let f(xm) be the function that associates SARF, or the total distance covered, to each order xm \u2208 \u039b, or, in other words, xm \u2208 \u039b order that minimizes f(xm). Simulated annealing and rapid chain delineation algorithms were used for obtaining a numeric approximation for the solution of this problem.Let Simulated annealing is a small modification in the famous MCMC algorithm of Metropolis-Hastings , therebyx and that the energy of this state is H(x). A candidate state y of energy H(y) is generated by applying slight perturbation to state x. The following probability is used in the decision-rule for accepting the candidate state:Suppose that the current state of the solid is T indicating temperature. If cooling is slow, the solid reaches thermic balance at each temperature. From the point of view \u2018simulation', this means generating several transactions at a certain temperature T , or, in other words, the elements H(x);\u2022 The function ym of distance given by y of energy H(y);\u2022 A candidate order c > 0 is equivalent to the temperature.\u2022 A control parameter c0 the initial control parameter and L0 the initial number of iterations used for an equal value of c0. Simulated annealing can thus be described in the following manner:Let n = 0, c0 and L0;1) Choose i vary from 1 to Ln2) Make ym in the neighborhood of xm and generate a random variable X ~ U;\u2022 Generate f(ym) \u2264 f(xm), then xm \u2190 ym;\u2022 If f(ym) > f(xm) and xm \u2190 ym;\u2022 If \u2022 End of operation;n \u2190 n + 1cn and Ln, and return to step 2 until the \u2018stop' criterion, where Ln is the number of chain transactions in each temperature (cn).Define The rapid chain delineation algorithm  constitumi, mj) the estimate of recombination fractions between pairs is the lowest. These markers will start the chain;1) Verify for which pairs of markers (mk) presenting the lowest estimate of recombination fractions with one of the terminal markers. Place the two together accordingly;2) Verify which is the unmapped marker (3) Repeat the procedure until all the markers are added to the chain;4) Then, attempt successive inversions in double and triple marks, in order to minimize SARF (the sum of adjacent recombination fractions).One hundred repetitions were carried out with the stochastic simulation algorithm, simulated annealing, and the results compared to those provided by the rapid chain delineation method. The criterion used for reaching this solution was minimum SARF.The results obtained with GQMOL software, which finds the solution for the problem through the rapid chain delineation method, are presented in Figures was defined asym in the set of possible orders. The algorithm was implemented in the R version 2.7.1 programming language . An Intel Core 2 Duo T5750 2.0 GHz processor was used with a 3 Gb RAM memory, Windows XP SP2.During the application of the algorithm, it was defined to uniformly choose an order nth algorithm iteration, denoted by cn, was calculated based upon the expressionThe parameter of control in the m is the number of iterations of the algorithm and A is a constant chosen in a convenient form, described as follows:where A is undertaken in such a way that the simulated annealing algorithm escapes from the minimum places of interest function (SARF) to reach the global minimum. Therefore, constant A must be chosen in such a way that all the initial orders are accepted. In the present case, 2 was considered as the value of this constant.The choice of One hundred repetitions were carried out, with a comparison of the best result from simulated annealing to that from the rapid chain delineation method.m3, m2, m4, m5, ..., m14, m15, m17, m16, m18, m19, ..., m32, m34, m33, m1, m35, m36, ..., m49, m50, m51, with a total SARF distance of 129,90 cM, thus being of smaller size than the 135,00 cM from rapid chain delineation  and double-haploid. Nevertheless, on comparison, algorithmic performance in simulated annealing was superior to that in rapid chain delineation, even with sufficiently large populations.According to m51, m50, ..., m19, m1, m18, m17, ..., m3, m2. The total distance was 112,30 cM, thus shorter than that arising from the other method evaluated (SARF) of 115,60 cM. The numeric order appears in The analysis of a population of 1000 individuals revealed that the order established by the rapid chain delineation method was identical to that from a population of 200 individuals, thus corroborating the results by The total distances for these orders are 104,10, 113,90 and 97,80 cM, for the second, third and fourth linkage groups, respectively. The evolution of the total distances of algorithmic iteration in the linkage groups was analyzed .In all the cases studied, execution of simulated annealing took less than 131 s, at the most . As rapiFigures i.e. the lowest SARF value, the former proved to be more efficient. Such a superior performance can also be explained by the number of markers, for, as the algorithm in question is stochastic, the higher the number of markers, the more efficient the method when compared to rapid chain delineation, ultimately leading to the possibility of analyzing a higher number of possible orders, as occurred here. As to the other linkage groups, with lower saturation levels and consequently less markers, results were similar with the two methods.It is obvious from the data that, in the case of the most saturated linkage groups, namely those with shorter distances between adjacent marks, viz., 2 cM, achievements through simulated annealing were similar or better than those by rapid chain delineation in less than 50% of the repetitions. Nevertheless, on considering the criterion used for constructing linkage maps, Furthermore, the number of individuals constituting the population has no effect on results when using the algorithm, since recombination frequencies, previously calculated for each pair of markers, are fundamental when ordering. So, the number of individuals exerts an influence only on the precision of estimates, but not on the ordering, thereby possibly leading to the construction of imprecise linkage maps. According to e.g. 51, the method based on stochastic simulation, viz., simulated annealing, presented orders with distances (SARF) equal to or shorter than rapid chain delineation in less than 50% of the repetitions. Nevertheless, the former method appears to be more interesting than the latter in these cases, as the criterion used for constructing linkage maps is to take into consideration the order of markers with lower SARF values. In the other cases, the two methods were alike, presenting the same SARF distances. Furthermore, it was noted that the number of individuals in the population does not affect ordering, although it does affect the estimates of recombination frequencies. The average time taken for simulated annealing execution did not exceed 112 s, thus not an obstacle for implementation.In the present study, simulated annealing and rapid chain delineation algorithms were compared when establishing the best linkage order in the construction of genetic maps, in populations of different sizes and saturation levels. It was observed that, for very saturated linkage groups, with an adjacent distance between marks of 2 cM, and a higher number of marks, The data from the present work demonstrate the relevance of the method used for ordering markers in the construction of genetic maps. Therefore, future studies should be carried out, in order to evaluate all the methods encountered in the literature, and thus facilitate their use according to the situation."}
+{"text": "Figures 1-3 are incorrect. The correct versions of Figures 1-3 can be viewed here: and"}
+{"text": "Previous studies reported that a subpopulation of mouse and rat trigeminal neurons express water channel aquaporin-1 (AQP1). In this study we make a comparative investigation of AQP1 localization in the human and mouse trigeminal systems. Immunohistochemistry and immunofluorescence results showed that AQP1 was localized to the cytoplasm and cell membrane of some medium and small-sized trigeminal neurons. Additionally, AQP1 was found in numerous peripheral trigeminal axons of humans and mice. In the central trigeminal root and brain stem, AQP1 was specifically expressed in astrocytes of humans, but was restricted to nerve fibers within the central trigeminal root and spinal trigeminal tract and nucleus in mice. Furthermore, AQP1 positive nerve fibers were present in the mucosal and submucosal layers of human and mouse oral tissues, but not in the muscular and subcutaneous layers. Fluorogold retrograde tracing demonstrated that AQP1 positive trigeminal neurons innervate the mucosa but not skin of cheek. These results reveal there are similarities and differences in the cellular localization of AQP1 between the human and mouse trigeminal systems. Selective expression of AQP1 in the trigeminal neurons innervating the oral mucosa indicates an involvement of AQP1 in oral sensory transduction. Aquaporins (AQPs) function as water selective channels providing a major route for osmotically driven water transport through cell membranes AQP1 is also expressed in the mammalian nervous system In the present study, we examined the cellular localization of AQP1 in the human and mouse trigeminal nerve systems by immunohistochemistry and immunofluorescence staining. Additionally, using fluorogold (FG) retrograde tracing combined with immunostaining, we identified a subpopulation of AQP1 positive trigeminal neurons that innervate the oral mucosa. These morphological evidences will contribute to the further exploration of the role of AQP1 in sensory functions and diseases associated with the trigeminal nerve.Immunoreactivity for AQP1 was localized to the cytoplasm and cell membrane of medium and small-sized trigeminal neurons in humans and mice, as demonstrated by the immunohistochemistry  and doubThe quantitative data showed that AQP1-immunoreactive neurons composed 39.74% in humans and 37.94% in mice of the total trigeminal neurons. Moreover, the percentages of small-sized and medium-sized trigeminal neurons immunoreactive for AQP1 were 63.65% and 43.7% in humans, and 53.28% and 22.47% in mice, respectively . The larAll satellite cells were positive for AQP1 in human trigeminal ganglia \u2013B, but oThere were numerous AQP1 positive nerve fibers in the peripheral trigeminal branches in humans  and miceAs shown in Our results revealed that AQP1 immunoreactivity, aside from endothelial cells of capillaries and microvessels, was localized to nerve terminals within the mucosa and nerve fibers within the submucosa of tongue , cheek , palate In order to confirm the selective innervation of AQP1-positive nerve fibers to the oral mucosa rather than skin or muscle, we performed the double immunofluorescence for AQP1 and \u03b2-tubulin III on the human and mouse check sections. As expected, a high colocalization of AQP1 and \u03b2-tubulin III was detected in a proportion of nerve fibers within the human and mouse cheek submucosa . In contFG retrograde tracing, combined with immunofluorescence, was performed to confirm that AQP1 labeled nerve fibers in the submucosa were originated from the trigeminal sensory neurons . A subseIn this study we compared the expression and distribution of AQP1 throughout the human and mouse trigeminal systems. The main results are summarized in in vitro and in vivoPublished data support the existence of species specificity of AQP1 localization in the mammalian nervous system, although the basis for these differences remains unclear. For example, earlier studies show that mouse and rat astrocytes express AQP4 but not AQP1 The present results revealed several selective expression patterns of AQP1 in the human and mouse trigeminal systems. First, AQP1 is expressed in a subpopulation of small and medium sized trigeminal neurons, but not large sized neurons. Second, AQP1-positive nerve fibers are present in oral mucosa and submucosa, but not subcutaneous or intramuscular regions. Third, a subpopulation of small to medium-sized trigeminal neurons innervating mouse cheek mucosa, but not the skin, express AQP1. Finally, in the mouse brain stem, AQP1 positive axons are only observed in caudal part of the trigeminal root, the spinal trigeminal tract and nucleus.Supporting the present results, selective expression of AQP1 in the nervous system is also found in the recent literature. For example, in the sheep duodenum, nearly 30% of submucosal neurons are immunoreactive for AQP1, whereas none of myenteric neurons are AQP1-positive It is well known that trigeminal neurons are responsible for transmission of orofacial somatosensory information. The selective expression of AQP1 in a subpopulation of small- and medium-sized trigeminal neurons innervating the oral mucosa indicates its potential role in oral somatosensory transduction. Several groups have addressed the roles of AQP1 in pain transmission in primary afferent neurons. An early study reported that AQP1 gene null mice exhibit mild impairment in nociceptive response after thermal (tail-flick) or chemical (capsaicin) stimuli There is still a lack of direct evidence for involvement of AQP1 in pain transmission of the trigeminal system. Borsani and colleagues (2009) investigated the expression of AQP1 and AQP2 in the trigeminal ganglia of mice, using an animal model of perioral acute inflammatory pain Apart from somatosensory transduction, we propose that expression of AQP1 in the trigeminal system may contribute to water sensation in the oral cavity. The moisture extent of the oral mucosa can be sensed by the central nervous system In summary, our results demonstrate the similarities and differences in the cellular localization of AQP1 between the human and mouse trigeminal systems. We also provide the morphological evidence that AQP1 positive trigeminal neurons innervate the oral mucosa. These findings suggest that AQP1 may mediate trigeminal sensory transmission, but requires further study.Three cadavers without neurological or psychiatric illnesses, aged 36\u201370 years, were obtained via informed donation for the medical education and research of Nanjing Medical University with corresponding written consents prepared by the donors and their families. The utilization of human tissues was approved by the Ethics Committee of Nanjing Medical University.Sixteen three-month old male ICR mice were purchased from the Animal Breeding Facility of the Nanjing Medical University and housed under conditions of controlled illumination (12\u223612 h light/dark cycle), humidity (30\u201350%), and temperature (18\u201322\u00b0C). Food and water were available at all times, except during the experiments. All procedures were approved by the Animal Welfare Committee of Nanjing Medical University. Every effort was made to reduce the number of animals and their suffering during the experiments.For FG retrograde tracing experiments, mice were deeply anesthetized intraperitoneally using 3.5% chloral hydrate (350 mg/kg). One microlitre of 1% FG  in distilled water was injected into the cheek submucosa or subcutaneous layer (n\u200a=\u200a5 in each group) via a 5-\u00b5l Hamilton microsyringe. After recovery from anesthesia, mice were returned to their home cages and sacrificed 3 days later.The cadavers were perfused through the internal carotid artery with 10,000 ml of a 10% formalin solution within 10 hours of death. The mice were deeply anesthetized with 2% sodium pentobarbital , then transcardially perfused with 0.9% saline followed by 4% freshly-prepared paraformaldehyde in phosphate buffer . Bilateral trigeminal ganglia of the two species were dissected and removed. The brain stem and oral tissues including the lip, soft and hard palates, cheek skin and mucosa, and tongue were also collected and each of them was cut into half. Human tissues were post-fixed in 4% paraformaldehyde/PB at 4\u00b0C for 3 days, and mouse tissues were post-fixed overnight.One group of human and mouse tissues were cryoprotected overnight in 30% sucrose in 0.1 M PB, embedded in optimal cutting temperature compound , and processed for frozen sections at 20\u201330 \u00b5m thickness using a cryostat . The other group was dehydrated in graded ethanol, embedded in paraffin and cut at a thickness of 6 \u00b5m using a paraffin slicing machine (Leica RM2135). Trigeminal ganglion samples were fixed in a horizontal position and cut into serial longitudinal sections. Samples of the mandibular division of the human trigeminal nerve, mouse and human oral tissues and brain stem were cut into transverse sections. All sections were mounted on gelatin-coated slides. In addition, the cheek and trigeminal ganglion tissues of FG injected mice were processed for frozen sections.Paraffin sections were deparaffinized with xylene and ethanol, followed by antigen retrieval with citric acid buffer (pH 6.0). After quenching endogenous peroxidase with 3% hydrogen peroxide for 10 min, the sections were incubated with blocking solution  for 1 hour at room temperature and overnight with the rabbit polyclonal anti-AQP1  or anti- glutamine synthetase (GS) antibody  at 4\u00b0C. After repeated washings in PBS, the sections were incubated with biotin-conjugated goat anti-rabbit IgG (1\u2236500) followed by horseradish peroxidase-conjugated avidin . The positive reactions were visualized with 3,3\u2032-diaminobenzidine tetrahydrochloride (DAB). Some sections were counter-stained with hematoxylin.For double immunofluorescence staining, slide-mounted frozen sections were preincubated in 0.01 M PBS containing 0.2% Triton X-100 and 0.25% bovine serum albumin for 1 hour at room temperature. The sections were incubated overnight with a mixture of mouse monoclonal anti-\u03b2-tubulin III , or -GFAP antibody  and polyclonal rabbit anti-AQP1 antibody  overnight at 4\u00b0C. After rinsing, sections were incubated for 1 hour at room temperature in a mixture of fluorescein isothiocyanate-conjugated donkey anti-mouse IgG  and rhodamine isothiocyanate -conjugated donkey anti-rabbit IgG . The sections were then washed and cover-slipped with PBS/glycerol buffer.For FG combined with AQP1 immunofluorescence staining, sections were preincubated in 0.25% bovine serum albumin PB for 1 hour at room temperature, and then incubated overnight with the polyclonal rabbit anti-AQP1 antibody  at 4\u00b0C. After rinsing, sections were incubated with rhodamine isothiocyanate-conjugated donkey anti-rabbit IgG , then washed and cover-slipped with buffered PBS/glycerol.To confirm the antibody specificity, we conducted immunostaining for AQP1 on human and mouse tissues where AQP1 protein has been described. In agreement with the current literature In addition, the primary antibody for AQP1 was pre-absorbed with the corresponding antigen  did not stain any specific immunoreaction in the above human and mouse tissues \u2013F. NegatThe light or fluorescence micrographs were captured by Leica DM4000B digital microscope equipped with image capturing software . The cytoarchitecture of human and mouse brain stem sections with hematoxylin counterstaining was identified according to The Human Central Nervous System: A Synopsis and Atlas 2 in humans and <300 \u00b5m2 in mice), medium-sized neurons (600\u20132000 \u00b5m2 in humans and 300\u2013600 \u00b5m2 in mice) and large-sized neurons (>2000 \u00b5m2 in humans and >600 \u00b5m2 in mice). The percentages of AQP1-positive neurons with respect to the total number of neurons for each class were determined. Data are expressed as means \u00b1 standard derivation of the mean.For quantitative analysis of AQP1 expression, paraffin sections of the human and mouse trigeminal ganglia, immunostained with AQP1 and counter-stained with hematoxylin, were captured in sequence at 400\u00d7magnification. Four sections containing the maximum plane of the trigeminal ganglia per specimen were selected for quantitative analysis. The cell number and cross-sectional areas of AQP1-positive and -negative trigeminal neurons with a clearly visible nucleolus were measured throughout the entire ganglion regions using an Image-Pro Plus 6.0 Analysis System . According to the previous studies Figure S1AQP1 staining human and mouse trigeminal sections. (A\u2013B) Human satellite cells expressing AQP1 (arrowheads) are observed around either AQP1-negative neurons (stars) or AQP1-positive neurons (triangle). (C\u2013D) In contrast, mouse AQP1-positive satellite cells (arrowheads) are only localized to AQP1-positive trigeminal neurons (triangle). No AQP1 immunoreactive signals are observed around AQP1-negative trigeminal neurons (stars). (E\u2013F) After neutralizing rabbit-ant-AQP1 antibody by the C-terminal peptide, no immunostaining was present at the human (E) and mouse (F) trigeminal ganlia. Scale bars\u200a=\u200a200 \u00b5m in A, 50 \u00b5m in B, 100 \u00b5m in C, 25 \u00b5m in D, 75 \u00b5m in E and F.(TIF)Click here for additional data file.Figure S2Immunolocalization of AQP1 in human and mouse choroid plexus and mouse renal tissues. (A\u2013C) AQP1 immunoreactivity was selectively and densely expressed at the apical surface of human (A) and mouse (B) choroid epithelium and the apical and basolateral membranes of mouse renal proximal tubules (C). (D\u2013F) Rabbit-ant-AQP1 antibody pre-incubated with the C-terminal peptide caused no immunostaining on the human (D) and mouse (E) choroid epithelium, and mouse renal tissues (F). Scale bars\u200a=\u200a100 \u00b5m.(TIF)Click here for additional data file."}
+{"text": "Trifolium repens L.) is an allotetraploid species (2n = 4X = 32) that is widely distributed in temperate regions and cultivated as a forage legume. In this study, we developed expressed sequence tag (EST)\u2013derived simple sequence repeat (SSR) markers, constructed linkage maps, and performed comparative mapping with other legume species. A total of 7982 ESTs that could be assembled into 5400 contigs and 2582 singletons were generated. Using the EST sequences that were obtained, 1973 primer pairs to amplify EST-derived SSR markers were designed and used for linkage analysis of 188 F1 progenies, which were generated by a cross between two Japanese plants, \u2018273-7\u2019 and \u2018T17-349,\u2019 with previously published SSR markers. An integrated linkage map was constructed by combining parental-specific maps, which consisted of 1743 SSR loci on 16 homeologous linkage groups with a total length of 2511 cM. The primer sequences of the developed EST-SSR markers and their map positions are available on http://clovergarden.jp/. Linkage disequilibrium (LD) was observed on 9 of 16 linkage groups of a parental-specific map. The genome structures were compared among white clover, red clover (T. pratense L.), Medicago truncatula, and Lotus japonicus. Macrosynteny was observed across the four legume species. Surprisingly, the comparative genome structure between white clover and M. truncatula had a higher degree of conservation than that of the two clover species.White clover ( Trifolium repens L.) is widely distributed in temperate regions of the world and is cultivated as a forage legume mainly in grazing pasture. It propagates both by seeds and stolons, and it grows well in a wide range of soil and environmental conditions , random amplified polymorphic DNA (RAPD), and amplified fragment length polymorphism (AFLP) markers (R locus) and the self-incompatibility locus (S locus)  mapping has been progressing mainly for marker-assisted selection (MAS) in white clover. Significant QTLs were identified for seed production . Of the tribe Trifolieae, the genus Trifolium is the largest and contains approximately 255 species (T. pretense L.) and alfalfa (Medicago sativa) with 167 and 37 commonly mapped markers, respectively  (M. truncatula. This result indicated the predominant colinearity between most of the homeologous groups (HG) of white clover and chromosomes of M. truncatula, except for F and H in white clover and chromosomes 2 and 6 in M. truncatula. Before this report, linkage groups of the white clover map were named using letters (A\u2013H). M. truncatula.Comparative genetic mapping is an effective strategy for sharing genetic and genomic information between model species and those with more complex genome structures . White cM. truncatula and Lotus japonicus. The resulting EST-SSR markers, integrated linkage map, and observed macrosynteny produced by this work will be valuable resources for genetic mapping, QTL analysis, and molecular breeding of white clover in the future.To accelerate the advance of molecular genetics in white clover, we performed EST-SSR marker development and constructed an integrated high-density linkage map. For broadening the knowledge across legume species, the genome structure of white clover was compared with that of red clover and two model legumes, An integrated genetic linkage map was constructed using a full-sib mapping population of 188 individuals derived from a cross between \u2018273-7\u2019 as a female parent and \u2018T17-349\u2019 as a male parent. \u2018273-7\u2019 is a wild accession in the Hokkaido region in Japan, with characteristics of early flowering, large leaves, a red leaf mark, and a sparse stolon network. \u2018T17-349\u2019 was derived from \u2018Hokkai 1,\u2019 a breeding line of the National Agricultural Research Center for Hokkaido Region (Japan). \u2018Hokkai 1\u2019 was bred by a maternal line selection method, which consisted of 10 maternal lines generated in eight countries . \u2018T17-349\u2019 was selected for its special characteristics of micro-leaves, late flowering, a white leaf mark, and a dense stolon network.Eco RI-Xho I site of a pBluescript II SK(\u2011) plasmid vector (Stratagene) and introduced into an E. coli ElectroTen-Blue strain (Stratagene) by electroporation. For generation of ESTs, plasmid DNA was amplified from the colonies using TempliPhi  and was subjected to sequencing using the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems). The reaction mixtures were run on the automated DNA sequencer ABI PRISM 3730 (Applied Biosystems).Total RNAs were extracted from 68 g of seedlings of a Swedish white clover variety \u2018Sonja\u2019 using the Plant RNA Purification Reagent (Invitrogen). The seeds were sown on petri dishes containing moistened filter papers, and whole seedlings were used for RNA extraction when primary leaves were fully developed. Purification of polyadenylated RNA and conversion to cDNA were performed as described previously . The EST contigs were classified into KOG categories according to the results of BLASTX searches against amino acid sequences in the KOG set (http://www.ncbi.nlm.nih.gov/COG/) , tri-nucleotide (NNN), and tetra-nucleotide (NNNN) repeats, were identified from the non-redundant white clover ESTs using the SSRIT (Simple Sequence Repeat Identification Tool) program , so that1 progeny. The names of markers and their sources are listed in Table S1.DNA was extracted from young leaves of white clover using DNeasy Plant Mini Kit (Qiagen). A total of 4619 primer pairs of SSR markers, including 1973 SSR markers developed in this study (hereafter WCS markers), 2518 red clover SSR markers (RCS markers) developed by 2, 0.08 U of BIOTAQ DNA polymerase (Bioline), 0.8 mM dNTPs, and 0.4 \u00b5M of each primer. A modified touchdown PCR protocol was followed as described by 1 progenies were selected and used for segregation analysis of a mapping population of 188 progenies.PCR amplification was performed in 5-\u00b5l reaction volumes using 0.6 ng of genomic DNA in 1X PCR buffer (Bioline), 3 mM MgClSegregation data obtained from a mapping population of 188 progeny were analyzed by a combination of a color map method . GeneralFirst, segregated marker loci were categorized into two parental-specific data sets by comparison of the sizes of polymorphic bands of parents and progenies. The segregation data were re-scored using the \u2018HAP1\u2019 population type codes employed in the JoinMap analysis. Next, the segregated marker loci in each parental-specific data set were roughly classified into 16 linkage groups using the color map method. Then, the robustness of the data sets for each linkage group was confirmed by the Grouping module of JoinMap using a logarithm of odds (LOD) threshold of 10. Finally, homeologous linkage groups within each parental-specific data set and corresponding linkage groups between the two parental-specific data sets were assumed by comparison of the names of bi-parental and multiple polymorphic marker loci. The locus orders in each parental-specific map were calculated by a Regression Mapping module of JoinMap. Each parental-specific data set was handled as a \u2018HAP1\u2019 population type, and the following parameters were used for the calculation: Kosambi\u2019s mapping function, LOD \u2265 1.0, REC frequency \u2264 0.4, goodness-of-fit jump threshold for removal of loci = 5.0, number of added loci after which to perform a ripple = 1, and third round = yes.Figure S1). Therefore, we concluded that the loci belonging to multiple linkage groups were not correctly integrated into the largest linkage group. Meanwhile, most of the 16 linkage groups of the \u2018273-7\u2019-specific data showed one-on-one colinearity to the eight chromosomes in M. truncatula by comparative analysis. Therefore, the marker loci consisting of the largest linkage group of the \u2018T17-349\u2019-specific data were carefully disassembled into multiple linkage groups according to the classified groups of the corresponding markers of the \u2018273-7\u2019-specific data and chromosomes of M. truncatula, as well as their segregation pattern by color mapping. Finally, the reclassified linkage groups were confirmed again based on their robustness under the Grouping module of the JoinMap program.After this procedure, the linkage map derived from the \u2018273-7\u2019-specific data were considered reasonable, whereas an error persisted in the \u2018T17-349\u2019-specific data. This error was because one of the 16 linkage groups of the \u2018T17-349\u2019-specific data consisted of an extremely larger number of marker loci  than the other 15 groups, and corresponding marker loci classified to the largest linkage group were found on most of the linkage groups of the \u2018273-7\u2019-specific data. In addition, graphical genotypes of the largest linkage group showed mosaic patterns .Syntenic regions between the genomes of white clover, red clover, and two model legumes, L. japonicas, and M. truncatula) and the A. thaliana genome, 7082 non-redundant ESTs had significant similarity  to the registered sequences, whereas the remaining 900 ESTs were not previously identified as sequences that show significant similarity on the published genomes for the four reference species (Table S3).A total of 15,214 cDNA clones were sequenced from their 5\u2032 ends, and a total of 10,290,123 qualified bases, of which the average GC content was 42.8%, were obtained. To identify the number of independent EST species, clustering of the EST sequences was performed using the PHRAP program. As a result, 7982 potential non-redundant EST sequences were generated, including 5400 contigs and 2582 singletons. When these non-redundant EST sequences were searched for similarity against proteome databases of three legume genomes  by BLASTX and classified into KOG categories of assigned orthologous A total of 1266 di-, tri-, and tetra-nucleotide SSRs that were \u226515 bp were identified in the non-redundant EST sequences. Provided that the total size of the non-redundant EST sequences is 6.0 Mbp, the frequency of occurrence of the SSRs in transcribed regions of the white clover genome was estimated to be one SSR in every 4.7 kb. Di-, tri-, and tetra-nucleotide SSRs accounted for 19.0%, 69.8%, and 11.1% of the identified SSRs, respectively . The SSRhttp://clovergarden.jp/ and in Table S1.In terms of the repeat motifs among the 1973 generated WCS markers, 1589 (81%) were tri-nucleotide repeats, whereas 139 (7%) and 245 (12%) were di- and tetra-nucleotide repeats, respectively. Three types of di-nucleotide repeats were observed, with poly (AG)n the most frequently observed, constituting 67% of the di-nucleotide repeats . Among te.g. WCS0403a and WCS0403b). Of the 1797 loci, 424, 789, and 584 showed bi-parental, \u2018273-7\u2019-specific, and \u2018T17-349\u2019-specific polymorphisms, respectively.Polymorphisms of the white clover mapping population were examined for a total of 4619 SSR markers, including 1973 white clover SSR markers (WCS), 2518 red clover SSR markers (RCS), and 128 other SSR markers on a previously published white clover linkage map . As a reTable S2). The length of each linkage group ranged from 97.6 cM (LG6a) to 186.5 cM (LG1b). The mean locus density and segregation distortion were 2.19 cM and 35.7%, ranging from 1.43 cM\u00b7locus-1 (LG8a) to 4.07 cM\u00b7locus-1 (LG6a) and from 19.5% (LG3a) to 66.0% (LG2b), respectively. Most of the linkage groups, except LG6a and LG6b, showed colinearity to eight chromosomes of M. truncatula (data not shown). Therefore, the linkage group numbers were designated based on the corresponding M. truncatula chromosome number. The numbers of loci showing significant similarity with genome sequences on the corresponding M. truncatula chromosomes were 22 , 32 , 17 , 20 , 37 , 54 , 52 , 32 , 37 , 33 , 1 , 6 , 18 , 36 , 43 , and 35 . Homeologous linkage groups were distinguished by randomly attached lower case letters (e.g. \u2018a\u2019 and \u2018b\u2019).Of the 1173 loci showing polymorphisms on \u2018273-7\u2019, 1059 were mapped onto 16 linkage groups, representing a total map length of 2322.9 cM  than the other 15 linkage groups (data not shown). All of the loci of the largest linkage groups were successfully ordered by the JoinMap software, but the result appeared to be incorrect because graphical genotypes of mapping populations showed mosaic patterns . Moreover, the corresponding loci of the linkage map were mapped on most of linkage groups of the \u2018273-7\u2019-specific map, and no colinearity was found between the linkage group and chromosomes of M. truncatula. Therefore, we concluded that the largest linkage group was consistent with loci that were originally generated from multiple chromosomes and showing similar segregation patterns. For this reason, the loci consisting of the largest linkage group of the \u2018T17-349\u2019-specific data were disassembled to multiple linkage groups according to the homeologous linkage groups of the corresponding markers of the \u2018273-7\u2019-specific map and chromosomes of M. truncatula, along with their segregation pattern by color mapping. As a result, the \u2018T17-349\u2019-specific map was constructed with 863 loci of 16 linkage groups, with a total length of 2450.3 cM (Table S2). The graphical genotypes of the individuals showed less inconsistency than the previous map . By comparison of the marker positions between the two parental-specific maps, the linkage groups of the \u2018T17-349\u2019-specific map were numbered according to the names of the \u2018273-7\u2019-specific map. The numbers of loci showing significant similarity with genome sequences on the corresponding M. truncatula chromosomes were 25 , 14 , 14 , 29 , 27 , 37 , 33 , 44 , 36 , 35 , 3 , 2 , 32 , 24 , 36 , and 11 . The length and locus density of each linkage group of the \u2018T17-349\u2019-specific map ranged from 89.6 (LG8b) to 252.3 cM (LG7a) and from 1.71 cM\u00b7locus-1 (LG3b) to 7.77 cM\u00b7locus-1 (LG6b), respectively. Segregation distortion (P < 0.05) of each linkage group tended to be higher than that of \u2018273-7\u2019 and ranged from 35.9% (LG5a) to 75.8% (LG4a), with 53.3% as a mean.As briefly described in Table S2). The length of each linkage group varied from 122.9 (LG2a) to 207.8 cM (LG2b). The locus density and segregation distortion were 1.44 cM\u00b7locus-1 and 45.6%, ranging from 0.98 cM\u00b7locus-1 (LG3b) to 2.12 cM\u00b7locus-1 (LG2b) and from 28.2% (LG3a) to 62.0% (LG1b), respectively.An integrated linkage map was constructed by combining the segregation data of the \u2018273-7\u2019- and \u2018T17-349\u2019-specific maps. Most of the linkage groups were successfully integrated; however, LG6a was not integrated because only two bi-parental loci were commonly mapped on the two parental-specific maps. Therefore, the number of mapped loci and the length of LG6a were calculated as follows. Number of loci =  \u2013 ; length = average length of the parental-specific maps. As a result, 1743 loci generated from 1189 SSR markers were mapped onto 16 linkage groups, which totaled 2511.3 cM in length (Table S4). Both parental-specific and bi-parental markers mapped randomly onto most regions of the integrated linkage groups, except for several distal regions   . The larTable S2). Multiple loci were classified as Type I and Type II (Table S2). Type I was defined as multiple loci identified by observation of multiple bands on 10% polyacrylamide gels. Type II was defined as those generated from single bi-parental segregation data and located on different linkage groups of the parental-specific maps. The total number of multiple loci was 962 , of which the numbers of Type I and Type II were 186 and 836, respectively (60 loci overlapped). Of the 962 multiple loci, 324 and 409 were mapped onto the same or homeologous linkage groups, respectively, whereas 229 multiple loci were mapped onto other linkage groups (Table S4). The multiple loci were identified across all linkage groups  and WCS0080b(LG7b) of the \u2018273-7\u2019-specific map. Of the regions showing significant LD across linkage groups of the \u2018T17-349\u2019-specific map, four regions had extremely high LD compared with other regions, including 75\u201380 cM of LG1a, 55\u201359 cM of LG3b, 143\u2013156 cM of LG5b, and 54\u201363 cM of LG7b.The genome-wide LD was estimated for the marker loci mapped onto parental-specific linkage maps . High r2M. truncatula sequences (Table S5). The total number of mapped loci and the genetic length of the map were 1714 and 833.9 cM, respectively. Each of the two homeologous linkage groups of the white clover integrated map were integrated to a single linkage group to simplify the comparison of white and red clover genomes.To investigate genome synteny between white clover and red clover, 2071 SSR markers developed from white clover and equences  were exaOf the 1743 loci mapped onto the white clover integrated linkage map, 951 showed sequence similarities to the ESTs or genome sequences of markers mapped onto the integrated linkage map of red clover. As shown in M. truncatula, and L. japonicus, respectively, and 725 were common to two genomes. By considering the genes with highest similarity score as putative orthologs, the map locations of the white clover markers and the corresponding genes of the other legumes were compared. As shown in Figure S4, the alignment of homologous sequence pairs along each linkage group revealed an obvious syntenic relationship. Syntenic relationships seemed to be highest against M. truncatula (Mt), in which the syntenic relationships spanned whole chromosome between wc HG1\u2013Mt chr1, wc HG2\u2013Mt chr2, wc HG3\u2013Mt chr3, wc HG5\u2013Mt chr5, and wc HG7\u2013Mt chr7. A segmental syntenic blocks were observed between wc HG1\u2013Mt chr7; wc HG4\u2013Mt chr4 and 8; and wc HG8\u2013Mt chr4 and 8. No synteny blocks were observed between white clover HGs and Mt chr6. A segmental level of syntenic relationships were detected against L. japonicus (Lj) as follows: wc HG1\u2013Lj chr5; wc HG2\u2013Lj chr6; wc HG3\u2013Lj chr1; wc HG4\u2013Lj chr3 and 4; wc HG5\u2013Lj chr2; wc HG7\u2013Lj chr1; and wc HG8\u2013Lj chr3 and 4.Of the 1096 sequences that corresponded to mapped SSR markers, 784 and 807 showed sequence similarities to the genes of Arabidopsis, L. japonicas, and rice. In addition, the distribution of the identified SSR motifs in the accumulated white clover EST sequences was basically similar to the distribution found in the genomes of other legume species, such as L. japonicas . In addition, the patterns of graphical genotypes of those loci were not inconsistent with other markers . Therefore, these data indicate that both the graphical genotypes and the mapping position of loci were adequate. We considered this the most unique result in this study, because linkage analysis has been generally performed under a prerequisite assumption of the absence of linkage between chromosomes. Therefore, it was impossible to identify LD across linkage groups without the assistance of reference information. In this study, we used two reference sources for identification of LD across white clover linkage groups, including another parental-specific map (\u2018273-7\u2019) and a predominant macrosynteny between M. truncatula. This work is an example of the application of comparative genomics to reveal unique molecular genetics phenomena of species that have a complex genome structure. The LD across linkage groups was observed only in the \u2018T17-349\u2019-specific map, which suggested that it was a specific behavior of haplotype combination. Based on the experimental procedures and results of this study, it is not possible to specify the biological factors that affect the phenomenon. However, the result suggested that systematic selection was occurring to promote (or avoid) specific combinations of haplotype blocks across chromosomes. Further analysis, such as an investigation of the segregation of haplotypes of BC1F1 populations derived from a cross between multiple F1 and \u2018T17-349,\u2019 would be required to determine the biological factors that affect the phenomenon.In parallel, we discovered the existence of LD across linkage groups on the \u2018T17-349\u2019-specific map. Although the segregation pattern of polymorphic bands in an F1 mapping population (data not shown). To our knowledge, a total of 168 primer sequences, including 90 SSR and 78 SNP markers, are published for DNA markers generated from the white clover genome or EST sequences  O. In their study, progenitor-specific SNPs were identified on stress tolerance\u2013related genes. We consider that SNPs on amplicons of mapped EST-SSR markers also can be used for identification of the progenitor of genomes. By application of the approach reported by M. truncatula, and they identified predominant synteny between the two species, except HG2(F) and HG6(H) in white clover and chr2 and chr6 in M. trancatula. Although only 14 markers were commonly mapped between the two studies, most of our results agree with M. truncatula than between white and red clover. Because the ancestor chromosome number in the genus Trifolium is supposed to be 2n = 16, our results suggest that the genome structures of white clover and M. truncatula have not drastically diverged after genus Trifolium and Medicago diverged (Trifoliastum, which consists of species with a basic chromosome number of 8, whereas red clover belongs to the section Trifolium, which consists of species with basic chromosome numbers of 5,6,7,8, and 24 (M. truncatula did not show significant macrosynteny between two clover species. M. truncatula was different from other chromosomes: it is the smallest and has multiple heterochromatic sites and retroviral elements scattered throughout its arm. The unique structure of the chromosome might be conserved in white clover HG6 and cause the difficulty of mapping EST-SSR loci.Significant synteny blocks were identified between white clover and red clover. Although the identified synteny blocks in this study showed more complex patterns, the results are consistent with those identified by Along with recent remarkable advances of technologies in genome analysis, knowledge of the genetics and genomics of plant species has rapidly progressed. However, there are still many hurdles in genetic and genomic analysis in polyploid and outcrossing species because of the difficulty in distinguishing between heterozygous, paralogous, and homeologous sequences. There are still many unresolved issues in the genetic analysis of polyploidy and outcrossing species, such as QTL identification, gene expression, and inbreeding depression. We anticipate that the EST-SSR markers and linkage maps developed in this study will accelerate the progress of genetics in white clover and other polyploid and outcrossing species."}
+{"text": "Our publication in PLOS ONE focuses on the development of a high-density genetic linkage and QTL mapping for half-smooth tongue sole. This work is closely related to a publication by our group in Current Zoology titled \"Construction of a microsatellite-based genetic linkage map for half-smooth tongue sole Cynoglossus semilaevi\", which reported a mid-density microsatellite genetic linkage map for the same species:Song WT, Miao GD, Zhao YW, Niu YZ, Pang RY, et al. (2013) Construction of a microsatellite-based genetic linkage map of half-smooth tongue sole (Cynoglossus semilaevis). Current Zoology 59(1): 99-108.The article in Current Zoology should have been cited and discussed in the PLOS ONE article and we apologize for this oversight. However, the two papers contain different results. In our paper in PLOS One, we constructed a high-density microsatellite genetic linkage map in half-smooth tongue sole with 1,009 markers with a high resolution . In addition, 159 sex-linked microsatellite markers were identified; five sex-linked microsatellite markers were confirmed for their association with sex in a large number of individuals selected from different families; and four growt h-related QTLs were mapped. In contrast, in our publication in Current Zoology, we constructed mid-density genetic linkage maps for female and male half-smooth tongue sole with only 480 and 417 markers, respectively; only two sex-linked AFLP markers were mapped; and no QTLs were mapped.Because the two projects both deal with linkage mapping analysis, there is similarity in the text of the Introduction and Discussion between the two articles, the authors apologize for this."}
+{"text": "In previous analyses, we identified a region of chromosome 19 as harboring a susceptibility locus for chronic otitis media with effusion and/or recurrent otitis media (COME/ROM). Our aim was to further localize the linkage signal and ultimately identify the causative variant or variants. We followed up our previous linkage scan with dense SNP genotyping across in a 5 Mb region. A total of 607 individuals from 139 families, including 159 affected sib pairs and 62 second-degree affected relative pairs, were genotyped at 1,091 SNPs. We carried out a nonparametric linkage analysis, modeling marker-to-marker linkage disequilibrium.-5) at position 63.4 Mb, with a LOD-1 support interval between 61.6 Mb and 63.8 Mb, providing significant evidence of linkage between this region and COME/ROM. The support interval contains over 90 known genes, including several genes involved in the inflammasome protein complex, a key regulator of the innate immune response to harmful exogenous or endogenous stimuli. Parametric linkage analysis suggests that for a sib of an affected individual, the recurrence risk of COME/ROM due to this linkage region is twice the recurrence risk in the population. We examined potential associations between the SNPs genotyped in this region and COME/ROM, however none provided evidence for association.The maximum log of the odds (LOD) score increased to 3.75 (P = 1.6 \u00d7 10This study has refined the 19q region of linkage with COME/ROM, and association results suggest that the linkage signal may be due to rare variants. Otitis media (OM), or inflammation of the middle ear, represents a leading reason for physician visits by children and a major component of the pediatric healthcare burden . A signiGenetic factors play an important role in COME/ROM susceptibility. COME/ROM aggregates in families -7, and fet al. identified linkage peaks on chromosome 17q12, 10q22, 7q33, 6p25, and 4p15 [To date, two linkage studies for COME/ROM have been published but the loci identified in the two studies did not overlap. The first genome-wide linkage scan, conducted by our group , identifet al.  and their families were recruited for the study, which has been described previously ,14,16. AWe selected 1,536 SNPs for genotyping, including 1,492 chromosome 19 SNPs chosen from a combination of tagging, nonsynonymous and synonymous coding SNPs, physical coverage, and putative copy number variation (CNV) interrogation. Forty-four Ancestry Informative Markers (AIMs) spaced across the genome were included to verify major ethnic group membership and to detect European stratification in the families. Genotyping was carried out by the Center for Inherited Disease Research (CIDR) using Illumina's GoldenGate assay .After removal of SNPs on the basis of poor genotype quality, missing data, excessive replicate and/or Mendelian errors -20, or mr2 > 0.5 and r2 > 0.2. We also explored other strategies to take into account the potential impact of LD on the validity of our linkage findings. We analyzed a subset of 90 families  in which all affected individuals have complete parental data. This subset of data is expected to be free of the LD effect on linkage [The Kong and Cox linear nonparametric linkage (NPL) method  as imple linkage .The Linkage and Association Modeling in Pedigrees (LAMP) method  was usedQLS [QLS method also incorporates association evidence across families and has been shown to be more powerful in many settings than standard methods. The prevalence of otitis media is specified at 0.1 in MQLS and only 137 Caucasian families (the reported ethnicity has been verified with AIMs) are included in the MQLS analysis. We also imputed the HapMap [In order to investigate whether the support for linkage on chromosome 19q can be explained by a common genetic variant in the region, we applied three family-based association tests, the Transmission/Disequilibrium Test (TDT) , the GenQLS . The TDTQLS . The GDTe HapMap  CEU SNPse HapMap .-5) at 110.5 cM , when the marker-marker LD was modeled at either r2 > 0.5 or r2 > 0.2. This result supported significant evidence for linkage between the 19q region and COME/ROM  with complete parental data were included in the linkage analysis, the maximum LOD score was 2.85. Note the other 35% of data contains additional linkage evidence that can be estimated as shown above . Since t-5. For a sibling of an affected individual, the recurrence risk of COME/ROM attributable to the 19q locus is twice as high as the recurrence risk in the population.The maximum LOD score based upon parametric linkage analysis was 5.42 at 107.5 cM, corresponding to P = 1.6 \u00d7 10-5. Since the number of independent SNPs should be less than 1,091 because of the LD between SNPs, we estimated the number of independent SNPs by pruning pairwise LD between SNPs. There are 216 SNPs that are in approximate linkage disequilibrium with each other (r2 < 0.1), and a Bonferroni correction based on 216 SNPs gives a nominal significance level of P < 0.00023. None of the observed associations reach either level of significance.No statistically significant (P < 0.0003) association between any SNP and COME/ROM was identified Figure . Using aQLS analyses yielded significant evidence of association (P < 0.001). The strongest association detected by TDT reflects the inflation of the statistics due to linkage in the region, rather than by association at a single variant [The five strongest associations identified through the TDT family-based association analyses  all fall in the LOD-1 support interval. In contrast, none of the family-based GDT or M variant . Using GRare variants in this region of 19q are much less represented than common variants in our SNP panel. Among all 1,091 SNPs, there are only 81 SNPs with a minor allele frequency less than 5%. The lack of significant association is consistent with evidence that linkage in the 19q region is not due to common genetic variants.2 > 0.2 modeled). The dense markers, as well as a larger sample size, have therefore helped improve the LOD score and localize the region of linkage. Additionally, parametric linkage analysis of our data suggests that for a sibling of an affected individual, the recurrence risk of COME/ROM that is due to this linkage region is twice as high as the recurrence risk in the population. Our analysis provides evidence that a COME/ROM susceptibility locus can be found within this region.We extended our previously reported linkage analysis on chromosome 19q with dense SNP genotyping in the critical 5 Mb region. Analyses of these additional data confirmed significant evidence supporting linkage in this region to COME/ROM, increasing the LOD score from 2.61  to 3.75 ZNF71, an endothelial zinc finger gene induced by TNF-\u03b1 [ZNF8, which represses BMP and FGF\u00df pathways important during development {Jiao, 2002 #32}, and ZNF304, which has been found to activate lymphocytes [NLRP13, NLRP5 and NLRP8. Inflammasomes are key regulators of the innate immune response to harmful exogenous or endogenous stimuli [CHMP2A) is part of the chromatin-modifying protein/charged multivesicular body protein (CHMP) involved in surface receptor degradation and formation of endocytic bodies [The LOD-1 support interval represents the telomeric region of chromosome 19. This region is high in gene content, containing over 90 known genes. Many of the genes in this region are zinc finger and zinc finger-related genes. Potential candidates for COME/ROM susceptibility include by TNF-\u03b1 , ZNF8, w stimuli ,34 Alpha stimuli . Chromatc bodies . ClearlyIn summary, we confirmed linkage of COME/ROM to chromosome 19q in a family based population with dense genotyping of a previously identified 5 Mb region. The lack of significant association with common variants in this region suggests that the observed significant linkage may be due to rare variants. Numerous studies have provided evidence that rare variant are involved in the etiology of complex traits . Further examination of the 19q region in COME/ROM susceptibility by next-generation sequencing of the region may be required in order to detect rare variants that may have novel and functionally significant effects. These studies are currently underway to determine the COME/ROM susceptibility gene in this region.WMC, MMS, KAD and EKA drafted the manuscript. SSR and KAD conceived of the family-based study, and KAD oversaw recruitment. MMS, JCM, SSR, and KAD participated in the design of the linkage follow-up study. JCM was responsible for SNP selection. MMS oversaw and coordinated molecular genetic analyses. JCM and XH conducted quality control procedures on genotype data. WMC and FC performed the statistical analysis. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2350/12/124/prepub"}
+{"text": "Natural \u03b2-glucans extracted from plants and fungi have been used in clinical therapies since the late 20th century. However, the heterogeneity of natural \u03b2-glucans limits their clinical applicability. We have synthesized \u03b2-glu6, which is an analog of the lentinan basic unit, \u03b2-(1\u21926)-branched \u03b2-(1\u21923) glucohexaose, that contains an \u03b1-(1\u21923)-linked bond. We have demonstrated the stimulatory effect of this molecule on the immune response, but the mechanisms by which \u03b2-glu6 activates innate immunity have not been elucidated. In this study, murine macrophages and human PBMCs were used to evaluate the immunomodulatory effects of \u03b2-glu6. We showed that \u03b2-glu6 activated ERK and c-Raf phosphorylation but suppressed the AKT signaling pathway in murine macrophages. Additionally, \u03b2-glu6 enhanced the secretion of large levels of cytokines and chemokines, including CD54, IL-1\u03b1, IL-1\u03b2, IL-16, IL-17, IL-23, IFN-\u03b3, CCL1, CCL3, CCL4, CCL12, CXCL10, tissue inhibitor of metalloproteinase-1 (TIMP-1) and G-CSF in murine macrophages as well as IL-6, CCL2, CCL3, CCL5, CXCL1 and macrophage migration inhibitory factor (MIF) in human PBMCs. In summary, it demonstrates the immunomodulatory activity of \u03b2-glu6 in innate immunity.  C. albicans uptake and endocytosis  \u03b2-D-Glcp-(1\u21923)-\u03b1-D-Glcp-(1\u21923)-[\u03b2-D-Glcp-(1\u21926)-]D-Glcp) \u21926-branch. HoweverCompared with other immune cells, macrophages are long lived and produce high levels of cytokines and chemokines upon stimulation to recruit immune cells to the site of infection . After aMany signaling pathways are involved in cell activation, differentiation and cytokine secretion of macrophages. The Ras-Raf-MEK-ERK and PI3K-Akt signaling pathways in macrophages are the most commonly studied intracellular transduction cascades. In the Ras-Raf-MEK-ERK pathway, activated Ras  activates RAF kinase, which phosphorylates and directly leads to MEK (MEK1 and MEK2) activation; then, MEK phosphorylates and activates ERK . ERK plaIn this study, we found that \u03b2-glu6 activated ERK and c-Raf phosphorylation but suppressed the AKT signaling pathway. Antibodies against TLR2, TLR4 and Dectin-1 inhibited the \u03b2-glu6-induced alterations in the Ras-Raf-MEK-ERK and PI3K-Akt signaling pathways in murine macrophages. Moreover, we examined the cytokine and chemokine profiles in \u03b2-glu6-stimulated murine macrophages and human PBMCs and proved the immunomodulatory activity of \u03b2-glu6 in innate immunity. Beta-glu6 was synthesized by Fanzuo Kong, Jun Ning and Jianxin Gu. The details of synthesis are described in a United States Patent  and a Chinese Patent (patent number: ZL02813563.6). The structure of \u03b2-glu6 was identified with NMR, MS and HPLC. The purity of \u03b2-glu6 is more than 98% , \u0394\u0394Ct= (CtGene-Ct GAPDH) treatment-(CtGene-CtGAPDH) control.(DOC)Click here for additional data file."}
+{"text": "Linkage analysis is the first step in the search for a disease gene. Linkage studies have facilitated the identification of several hundred human genes that can harbor mutations leading to a disease phenotype. In this paper, we study a very important case, where the sampled individuals are closely related, but the pedigree is not given. This situation happens very often when the individuals share a common ancestor 6 or more generations ago. To our knowledge, no algorithm can give good results for this case.To solve this problem, we first developed some heuristic algorithms for haplotype inference without any given pedigree. We propose a model using the parsimony principle that can be viewed as an extension of the model first proposed by Dan Gusfield. Our heuristic algorithm uses Clark\u2019s inference rule to infer haplotype segments.We ran our program both on the simulated data and a set of real data from the phase II HapMap database. Experiments show that our program performs well. The recall value is from 90% to 99% in various cases. This implies that the program can report more than 90% of the true mutation regions. The value of precision varies from 29% to 90%. When the precision is 29%, the size of the reported regions is three times that of the true mutation region. This is still very useful for narrowing down the range of the disease gene location. Our program can complete the computation for all the tested cases, where there are about 110,000 SNPs on a chromosome, within 20 seconds. Linkage analysis is the first step in the search for a disease gene. The aim is to find the rough location (a region in which the disease gene is) of the gene in the chromosome. Linkage studies have facilitated the identification of several hundred human genes that can harbor mutations leading to a disease phenotype . The priLinkage analysis has been extensively studied in recent years. Almost all the existing methods are for families with clearly given pedigrees. The pedigree information helps a lot in the design of computational methods. Early approaches to linkage analysis were based on sparse microsatellite markers. With the new development of microarray techniques, high-density SNP genotype data can be used for large-scale and cost-effective linkage analysis ,3. With There are two categories of existing approaches to linkage analysis, the probabilistic approaches and the deterministic approaches. In probabilistic approaches, recombinant rates are estimated in a way to maximize the likelihood of the observed data -7. Softwet al.. After the extension process, our program reports all the mutation regions obtained in the algorithm. The complete algorithm to find the mutation regions on the whole chromosome is shown as Algorithm 2.The extension process stops when we find conflicts in both directions. The extended region obtained from  and  in Table Our program may work for the situation where the input individuals are from multiple families. Our algorithm tries to find regions shared by all the diseased individuals. Thus, as long as the diseased individuals from multiple families share the same (or similar) haplotype segment on the true mutation region, our program should be able to find such region. Even if the haplotype segments from different families on the true mutation region are slightly different, the program should be able to report several smaller regions with a few missing SNPs in the middle. Again, it is possible for users to figure out the whole true mutation by ignoring the few missing SNPs in the middle.We developed a software package for linkage analysis where the input individuals are closely related, but the pedigree is not known. We propose a model using the parsimony principle that can be viewed as an extension of the model first proposed by Dan Gusfield ,21). Our. Our21])Project name: MRDProject homepage:http://www.cs.cityu.edu.hk/~wenjuacui/software/mutationRegion/index.html. The source code is also available.Operating system(s): Platform independentProgramming language: JavaOther requirements: Java 1.6.0 or higherLicense: NoneAny restrictions to use by non-academics: NoneThe authors declare that they have no competing interestsLW proposed the topic and ideas for algorithms, WC implemented the program, and both authors devised and developed the method and prepared the manuscript. Both authors read and approved the final manuscript.Supplementary Material: This file includes several figures and additional experimental results mentioned in the paper. It contains the different set of input individuals on Pedigree 2-4 in the paper, the pedigrees containing 6 and 7 generations and 2,3,4,5 diseased individuals in the latest generation respectively. The tables show the results on the input of the above figures.Click here for file"}
+{"text": "A genetic component to the etiology of leprosy is well recognized but the mechanism of inheritance and the genes involved are yet to be fully established.A genome-wide single nucleotide polymorphism (SNP) based linkage analysis was carried out using 23 pedigrees, each with 3 to 7 family members affected by leprosy. Multipoint parametric and non-parametric linkage analyses were performed using MERLIN 1.1.1.Genome-wide significant evidence for linkage was identified on chromosome 2p14 with a heterogeneity logarithm of odds (HLOD) score of 3.51 (rs1106577) under a recessive model of inheritance, while suggestive evidence was identified on chr.4q22 , chr. 8q24  and chr.16q24 . Our study also provided moderate evidence for a linkage locus on chromosome 6q24\u201326 by non-parametric linkage analysis , overlapping a previously reported linkage region on chromosome 6q25\u201326.A genome-wide linkage analysis has identified a new linkage locus on chromosome 2p14 for leprosy in Pedigrees from China. Mycobacterium leprae. It affects the skin and peripheral nerves and can cause irreversible impairment of nerve function and consequent chronic disabilities HLA-DRPARK2/PACRGLTATLRsCCDC122, C13orf31, NOD2, TNFSF15, HLA-DR, and RIPK2 and a trend toward an association with a SNP in LRRK2. Five of these genes encode proteins involved in the innate immune response Leprosy is a chronic infectious disease caused by A total of 82 patients and 16 unaffected individuals from 23 multi-case leprosy families were genotyped in the present study. After quality control filtering,the linkage analysis was carried out using 5525 autosomal SNPs. The most noticeable results of the genome-wide linkage analysis were summarized in To evaluate the empirical significance of our linkage results, we conducted a simulation analysis to evaluate the significance of our results using the criteria proposed by Lander and Kruglyak Linkage was also identified on chr.4q22.2\u201322.3 , chr.8q24.3, chr.16q24.1\u2013q24.2  with the full penetrance, and chr.6q24\u201326 . When the penetrance was varied in the parametric analysis, the maximum HLOD score of these regions changed slightly. According to our simulation results, these linkage results are only suggestive. The linked regions identified by parametric analysis were also supported by nonparametric analysis . The locIn this study, we performed a genome-wide linkage analysis using a high-density whole-genome linkage array with the median distance between SNP markers of 441 kb and identified a novel susceptibility locus for leprosy on chromosome 2p14 under a recessive model of inheritance. Suggestive evidence of susceptibility loci were found on chromosome 4q22 and 16q24 under a dominant model, chromosome 8q24 under a recessive model and chromosome 6q24\u201326 by non-parametric analysis. Not all of our pedigrees showed linkage in each of these chromosome regions and this suggests potential genetic heterogeneity among different leprosy families. Our results suggest the presence of multiple genetic variants predisposing to leprosy under different modes of inheritance. The linked regions were supported by parametric (either under dominant or recessive models) as well as non-parametric linkage. Parametric linkage analysis is more powerful than non-parametric methods for detecting linkage, with differences in power determined by the true underlying model and linkage information content. Although there is uncertainty about the true penetrance of leprosy, varying the disease penetrance has little impact on our linkage results, suggesting that the linkage results are stable and no depending on the penetrance. This is expected, because all the linkage analyses were performed by treating all the family members without disease phenotype as \u2018unknown\u2019.PARK2 and PCARG genes on this locus have been identified to be associated with leprosy susceptibility in two ethnically distinct populations Vietnamese and Brazilian Previous linkage studies using microsatellite markers have identified several linkage loci on chromosome 10p13 There seems to be little overlap between the regions/loci identified in the present linkage study of leprosy families and the ones revealed by our previous GWAS of unrelated leprosy cases and controls. The discordant results are not surprising, since the loci identified by the current linkage and the previous GWAS analyses may be different. The current analysis is more likely to identify variants with relatively strong genetic effects (high penetrance) and thus causing familial aggregation where multiple family members were affected with the disease. Such linkage loci may harbor relative rare variant, potentially showing allelic heterogeneity across families, which would require a direct re-sequencing analysis to uncover. In contrast, the GWAS analysis is more likely to identify common genetic variants with lower penetrance whose genetic effect are too moderate to cause familial aggregation of the disease and thus be detected by linkage analysis with the current sample size. Linkage and association analyses are therefore complement and both needed to reveal the full spectrum of genetic risk variants for leprosy.There are several limitations to our study. First, the size of the sample is modest. Replication of our results in independent samples  will be essential. Second, we concentrated our efforts in large leprosy pedigrees with a possible stronger genetic component. Thus, these results might have overestimated the magnitude of the effect of these loci in general population. Third, while it is possible that MB and PB forms of leprosy have some different predisposing genetic factors, it is not feasible to conduct subgroup analysis in this study due to the sample size. Our study of all the pedigrees together may help to identify genetic factors that are shared by MB and PB. Notwithstanding these limitations, our study provides strong genetic evidence of a novel susceptibility locus for leprosy on chromosome 2p13.3\u201314 and suggested several other regions of potential interest.CLEC4F, CD207, ATP6V1B1, PPP3R1, KIAA1048, ANXA4 and AAK1. These results may help guide further studies on leprosy. The analysis of additional leprosy pedigrees, in addition to fine-mapping and/or resequencing to identify susceptibility genes and functional variation within the linkage regions will further validate these findings. Elucidation of the genetic factors that influence susceptibility to leprosy may provide new insight into the prevention and control of the disease.There are a number of genes of potential interest within 2p14 region that are involved in innate immune response, particularly in endocytosis process, including A collection of 23 multiplex families with 3 to 7 family members affected with leprosy was enrolled from Shandong, Jiangsu and Yunnan provinces, including 13 families of Chinese Han, 5 of Miaozu, 2 of Yizu, 1 of Daizu, 1 of a mixed Han and Yizu and 1 of a mixed Han and Baizu. The diagnosis of leprosy was based on medical records stored in local leprosy control institutions and clinical assessments at the time of blood taken . Demographic characteristics, clinical subtypes and age at onset of the disease were also collected from medical records. The classification of the patients was based on clinical and histological criteria EDTA anticoagulated venous blood samples were collected from all the participants. Genomic DNA was extracted from peripheral blood lymphocytes by standard procedures using Flexi Gene DNA kits . Genomic DNA samples were diluted to working concentrations of 50 ng/\u00b5l for genotyping analysis. DNA samples were surveyed for quality both by a Nanodrop Spectrophotometer (ND-1000) and the electrophoresis assay. Approximately 200 ng of genomic DNA was used for genotyping analysis. Briefly, each sample was whole-genome amplified, fragmented, precipitated and resuspended in appropriate hybridization buffer. Denatured samples were hybridized on prepared Illumina Linkage-12 Human DNA Analysis Kit . After hybridization, the BeadChip oligonucleotides were extended by a single labeled base, which was detected by fluorescence imaging with an Illumina Bead Array Reader. Normalized bead intensity data obtained for each sample were loaded into the Illumina BeadStudio 3.3 software, which converted fluorescence intensities into SNP genotypes.The genome-wide linkage analysis was performed by using a total of 6090 SNP markers, having average 0.58 cM genetic map spacing and average 441 kb physical map spacing. The patterns of disease transmission did not support an X-linked mode of inheritance in the leprosy pedigrees, the X-chromosome was not analyzed. SNPs with a call rate less than 90% or with cluster plots that did not show clear separation of the three genotype clusters were excluded. A total of 5525 autosomal SNPs were retained in the linkage analysis. The average minor allele frequency (MAF) of the SNPs was 0.276.Multipoint parametric and non-parametric linkage analyses were performed via the program of MERLIN version 1.1.1 Simulations were performed to assess the statistical significance of the observed results using the program MERLIN with 1000 replicates. Datasets were simulated according to the null hypothesis of no linkage across the whole genome with the same family structures, marker map, allele frequencies and patterns of missing data as what have been used in our linkage analysis. Both parametric and non-parametric analyses were performed for each replicate with the same parameters as in the linkage analysis. The significance of linkage were defined using the rates of chance occurrence as proposed by Lander and Kruglyak's Table S1Result of linkage analysis on chromosome 2p14.(DOC)Click here for additional data file.Table S2The results of simulation.(DOC)Click here for additional data file.Supporting Information S1Pedigree structures of the families in the study.(DOC)Click here for additional data file."}
+{"text": "After publication of our article , we becap-value of < 0.05.All probe sets were mapped using standard interval mapping methods at 1 cM intervals (~2 Mb) along all autosomes and the X chromosome. This procedure generates estimates of linkage between variation in transcript expression levels and chromosomal location. The entire set of values can be used to construct a set of QTL maps for all chromosomes  in which position is plotted on the x-axis and the strength of linkage--the likelihood ratio statistic (LRS) or log of the odds ratio (LOD)-is plotted on the y-axis. An LRS of 18 or higher is significant at a genome-wide"}
+{"text": "For decades, research efforts have tried to uncover the underlying genetic basis of human susceptibility to a variety of diseases. Linkage studies have resulted in highly replicated findings and helped identify quantitative trait loci (QTL) for many complex traits; however identification of specific alleles accounting for linkage remains elusive. The purpose of this study was to determine whether with a sufficient number of variants a linkage signal can be fully explained.We used comprehensive fine-mapping using a dense set of single nucleotide polymorphisms (SNPs) across the entire quantitative trait locus (QTL) on human chromosome 7q36 linked to plasma triglyceride levels. Analyses included measured genotype and combined linkage association analyses.. However, no single genetic variant was sufficient to account for the linkage. On the other hand, multiple variants capturing the variation in these five genes did account for the linkage at this locus. Permutation analyses suggested that this reduction in LOD score was unlikely to have occurred by chance (p\u200a=\u200a0.008).Screening this linkage region, we found an over representation of nominally significant associations in five genes With recent findings, it has become clear that most complex traits are influenced by a large number of genetic variants each contributing only a small percentage to the overall phenotype. We found that with a sufficient number of variants, the linkage can be fully explained. The results from this analysis suggest that perhaps the failure to identify causal variants for linkage peaks may be due to multiple variants under the linkage peak with small individual effect, rather than a single variant of large effect. For decades, research efforts have tried to uncover the underlying genetic basis of human susceptibility to a variety of diseases. While our efforts untangling the basis of monogenic disorders have been highly successful Linkage studies have resulted in highly replicated findings and helped identify quantitative trait loci (QTL) for many complex traits; however identification of specific alleles accounting for the linkage peak has remained elusive Thus, the purpose of this study was to determine whether with a sufficient number of variants  a linkage signal can be fully explained. To accomplish this goal, we analyzed a quantitative trait locus (QTL) on human chromosome 7q36 linked to plasma triglyceride levels (LOD\u200a=\u200a3.7) The Metabolic Risk Complications of Obesity Genes (MRC-OB) project was established in 1994 to identify the genetic determinants of the metabolic syndrome and its metabolic abnormalities All protocols have been approved by the Institutional Review Board of the Medical College of Wisconsin. Details on the cohort used in this study are included in As previously reported , 2,209 individuals from 507 families were ascertained for basic anthropomorphic phenotypes, plasma lipid measures, and fasting glucose and insulin levels. A genome-wide linkage scan of these families identified a quantitative trait locus (QTL) on human chromosome 7q36 linked to plasma triglyceride levels (LOD\u200a=\u200a3.7). The locus spans approximately 5 Mb of genomic sequence, and contains 18 known genes.We selected 1,048 tagSNPs spanning the entire 5 Mb region using the tagger function of Haploview ln) transformed. Data were re-examined and observations exceeding 4 standard deviation units were removed as outliers, as described previously As triglyceride levels are continuously distributed, the data were first examined for deviations from normality. Raw triglyceride levels exhibited an increased number of high values, so the data were natural log . For the sets of SNPs exhibiting substantial LD, SNPs were eliminated on the basis of missing data, such that SNPs with the lowest percentage of missing data were retained in the analysis. Importantly, previous evidence of association was not considered in the reduction of the SNP sets. The selected SNPs were included in a model where each SNP was modeled additively. Importantly, as our goal was not to determine the significance of any specific SNP in the multi-SNP model, all SNPs were forced to be included in the model but significance testing was not performed. Other covariates included age, age by sex, sex, age squared, age squared by sex, asthma, menopause status, birth control use, smoking status, lipid lowering medications, and diabetes status, as these were the covariates included in the initial linkage analysis when the original QTL was identified.To dissect the genetic effects of these cluster regions and to assess the magnitude of effect on TG levels and the originally observed linkage, we selected all SNPs in these cluster regions that were used in the single SNP screen (n\u200a=\u200a324), including SNPs regions that showed no evidence of single SNP association. To minimize redundant information, SNPs were eliminated from consideration if they exhibited substantial LD with other SNPs in the analysis  The results for the association analysis of individual SNPs with plasma TG levels are illustrated in When examining the effect on the LOD score of each of the 109 SNPs which exhibited nominal p-values we found that no one SNP explained the linkage peak (LOD score dropped below 0.05). Indeed, the median value for the proportion LOD decline was 0.03 (interquartile range: 0.002\u20130.05). Further, only 15 SNPs exhibited LOD drops greater than 0.10. Taken together these data suggest that any one SNP in this region is insufficient to explain the linkage peak.2<0.7). When all 160 SNPs were included in the analysis model, 684 individuals had complete genotype information for all SNPs and thus entered the analysis. The covariates (including the SNPs) explained 36% of the variance in triglycerides and there was no evidence of any additional genetic effect for the locus on 7q36 . When restricting the analysis to the same 684 individuals but not including the SNPs as covariates, the remaining covariates explained only 5% of the variation in triglycerides and there was strong evidence of a genetic effect .For further analysis, we selected all SNPs (n\u200a=\u200a324) surrounding these five genes identified in the single SNP screen. Of these variants, 160 SNPs had low pairwise linkage disequilibrium measures . this linkage peak can be explained completely.With recent findings, it has become clear that most complex traits are influenced by a large number of genetic variants each contributing only a small percentage to the overall phenotype. Thus the question remains for complex traits whether a linkage peak can be explained in entirety with a sufficient number of variants. Using a family based cohort which exhibited linkage for plasma triglycerides to human chromosome 7q36, we have demonstrated that a single genetic variant is insufficient to account for the linkage peak. Further, we demonstrated that using multiple variants capturing the variation in five genes In this study, we coupled information from linkage and association to clarify the genetic basis of variation in plasma triglyceride concentrations. A major benefit of using this combined approach is that these methods provide independent information Using a comprehensive set of 1,048 tagSNPs identified from the publicly available data of the International HapMap Consortium While these genes are plausible, no single SNP explained a significant fraction of the linkage, suggesting that multiple variants of small individual effect account for the QTL. While the identification of causal variants following an initial linkage study has worked well for Mendelian disorders, the failure to identify a single variant that accounts for a linkage peak for a complex trait consistent with previous fine-mapping studies On the other hand, accounting for many variants irrespective of the level of association can account for the linkage peak. One hundred sixty variants (15.3% of the 1048 SNPs tested) randomly selected from these five genes completely account for the linkage on chromosome 7q36. However, as the purpose of this paper was to determine if capturing the variation present in these five genes would be sufficient to explain the linkage, we did not seek to identify the minimum number of variants required to explain the linkage or the specific variants. Given the linkage disequilibrium in this region, it is plausible that the causal variants are not in the 160 variants selected. Future studies will need to examine this locus in more detail to identify the specific variants responsible for the linkage.These results suggest that a single genetic variant is not likely to be the cause of a linkage signal for a complex trait. Indeed, even in the simple Mendelian inherited diseases such as cystic fibrosis In this study, we demonstrate that a single SNP or gene is insufficient to explain the linkage of serum triglycerides on 7q36. Indeed, while we observe a cluster of associated SNPs in five genes, any single SNP or gene fails to completely account for the linkage peak. On the other hand, when we select SNPs across all five genes, evidence of linkage disappears, suggesting that multiple variants in these genes significantly alter plasma triglyceride levels. Our findings suggest that the failure to identify causal variants for linkage peaks may be due to multiple variants under the linkage peak with small individual effect, rather than a single variant of large effect."}
+{"text": "Figures 2 and Figures 3 are switched."}
+{"text": "Here, we aimed to identify familial and sex-differential risk loci in the largest available, uniformly ascertained, densely genotyped sample of multiplex ASD families from the Autism Genetics Resource Exchange (AGRE), and to compare results with earlier findings from AGRE.Autism spectrum disorders (ASDs) are male-biased and genetically heterogeneous. While sequencing of sporadic cases has identified From a total sample of 1,008 multiplex families, we performed genome-wide, non-parametric linkage analysis in a discovery sample of 847 families, and separately on subsets of families with only male, affected children  or with at least one female, affected child . Loci showing evidence for suggestive linkage (logarithm of odds \u22652.2) in this discovery sample, or in previous AGRE samples, were re-evaluated in an extension study utilizing all 1,008 available families. For regions with genome-wide significant linkage signal in the discovery stage, those families not included in the corresponding discovery sample were then evaluated for independent replication of linkage. Association testing of common single nucleotide polymorphisms (SNPs) was also performed within suggestive linkage regions.P\u2009<\u20090.01), while loci at 6q27 and 8q13.2 showed suggestive linkage in our extended sample. Suggestive sex-differential linkage was observed at 1p31.3 (MO), 8p21.2 (FC), and 8p12 (FC) in our discovery sample, and the MO signal at 1p31.3 was supported in our expanded sample. No sex-differential signals met replication criteria, and no common SNPs were significantly associated with ASD within any identified linkage regions.We observed an independent replication of previously observed linkage at chromosome 20p13 (With few exceptions, analyses of subsets of families from the AGRE cohort identify different risk loci, consistent with extreme locus heterogeneity in ASD. Large samples appear to yield more consistent results, and sex-stratified analyses facilitate the identification of sex-differential risk loci, suggesting that linkage analyses in large cohorts are useful for identifying heritable risk loci. Additional work, such as targeted re-sequencing, is needed to identify the specific variants within these loci that are responsible for increasing ASD risk. This seSeparate non-parametric, genome-wide linkage analyses for ASD affection status in the stage 1 MO (n\u2009=\u2009487 families) and FC (n\u2009=\u2009314 families) subgroups identified five loci with LOD scores \u22652.2 (Table\u00a0MO.St1\u2009=\u20092.98 at rs7521242 (92.905\u00a0cM); a 2-LOD interval from this peak SNP spans 10.0\u00a0Mb, 13.7\u00a0cM, and 48 RefSeq genes. Analysis of this locus in the MO subgroup from the combined sample identified a smaller, but still suggestive, peak LODMO.Com\u2009=\u20092.55 also at rs7521242 , which is expressed in the central nervous system and plays a significant role in glial cell fate determination and in normal development of the corpus callosum  to regulate cellular differentiation during development , and the minor allele T was found to be under-transmitted to affected offspring from ALL families (odds ratio\u2009=\u20090.4143). In contrast, the strongest corrected association signal occurred in the FC peak on chromosome 8p21.2 at rs78485638 with a corrected P-value of 0.052 .The strongest unadjusted association signal within any linkage region occurred in the ALL peak on chromosome 4q13.1 at rs115667468 with a To identify and support genomic loci likely to contain variants contributing to ASD risk in multiplex families from the AGRE collection, we performed linkage analyses in all and sex-stratified subsets of multiplex families followed by targeted association testing in 1,521 families  from the AGRE cohort. The strongest linkage signal that we identified was on chromosome 20p13, which exceeded a LOD score of 3.0 in our combined sample of 1,008 multiplex families. At 20p13, we also replicated in an independent sample from the same AGRE cohort a previous report of significant linkage at this locus . AnalyseAll.Com of 3.02, and it also meets the criterion for an independent replication of genome-wide significant linkage [NRSN2 [GenBank:NM_024958] (0.3-LOD drop from peak), a gene expressed throughout the cerebral cortex, thalamus, hypothalamus, and in Purkinje cells [CSNK2A1 [GenBank:NG_011970] (0.5-LOD drop from peak), which encodes a protein involved in the regulation of circadian rhythms , whose gene product has transcription factor activity and has been implicated in central nervous system development [de novo mutations [STC1 [GenBank:NG_029711] (1.1-LOD drop from peak), which encodes a glycoprotein regulated by calcium that may act to protect neurons from ischemia and hypoxia [NEFM [GenBank:NG_008388] and NEFL [GenBank:NG_008492] (both 1.1-LOD drop from peak) whose products likely function in transport to neuronal projections [GNRH1 [GenBank:NG_016457] (0.7-LOD drop from peak) is also located within this linkage region, and mutations in this gene are likely to affect gonadal function [NRG1 [GenBank:NG_012005] (0-LOD drop from peak), a known schizophrenia risk gene [The significant MO and FC signals identified here implicate regions containing promising candidate genes that warrant further exploration by targeted re-sequencing . Theelopment ,56. Rareelopment , as welljections . Potentifunction , perhapsisk gene ,75.A similar stratification approach for the identification of male-specific and female-affecting ASD risk loci has been successfully applied previously by Stone and colleagues  to an eaUnder the assumption of genetic heterogeneity, for complex traits such as ASD, it is possible that the various loci identified by analyses of different family sets flag true sites for ASD risk in a proportion of the families tested. To pursue this, linked loci will need to be investigated more closely to identify the precise variants that effectively increase ASD risk. This has so far proven challenging, as association testing of densely mapped common SNPs within linkage peaks has failed to definitively identify risk variants, both in the present study and in previous work .Although our analyses are likely underpowered to identify regionally significant associations with common variants of small effect size, it is alternatively possible that rare variants, not explicitly tested here, contribute to the heritable component of ASD risk. For example, rare variants, shared between siblings but private to each nuclear family, may cluster in the same gene or set of genes. Since rare variants are less likely to be tagged by common SNPs, they should be more readily localized by allele-agnostic linkage analyses than association testing. As in gene discovery studies of sporadic ASD cases, sequencing of functional genomic features in linked regions will be necessary to identify rare variants and evaluate their role in familial ASD risk. In either case, larger family-based cohorts will be needed to improve power.We conclude that the use of linkage analyses in multiplex family cohorts has complementary utility to genome-wide association studies for the investigation of the familial, inherited contribution to ASD risk. This is especially the case in the context of rare variants in human disease . AddAGRE: Autism Genetics Resource Exchange; ALL: all families; ASD: autism spectrum disorders; CNV: copy number variant; FC: female-containing; IL: interleukin; LOD: logarithm of odds; MO: male-only; SNP: single nucleotide polymorphism; SNV: single nucleotide variant; TDT: transmission disequilibrium test.The authors declare that they have no competing interests.DMW carried out the linkage and association studies and drafted the manuscript. JKL performed preprocessing and quality control on genotype data and provided assistance with genetic analyses. RL carried out the genotype imputation for all samples. RMC advised on the design and interpretation of the study and helped to draft the manuscript. DHG conceived of the study, participated in its design and interpretation, and helped to draft the manuscript. All authors read and approved the final manuscript.Genotyped families and cases from Autism Genetics Resource Exchange (AGRE). *Additional members from families partially genotyped at earlier stage.Click here for fileP-value from TDT) across 2-LOD interval from peak LOD; bottom: RefSeq gene alignment in 2-LOD interval.Linkage observed at suggestive threshold in both stage 1 and combined samples. Regions of suggestive linkage (logarithm of odds (LOD) >2.2) observed in both the full sample (ALL) from stage 1 and combined stage analyses. A) 6q27, B) 8q13.2-3; for each, top: linkage across full chromosome from all stage 1 family groups; middle: linkage from stage 1 (solid line) and combined samples (dashed line) and association signal from transmission disequilibrium test (TDT) in all combined stage families  score by chromosomal band, single nucleotide polymorphism (SNP) or microsatellite, hg19 base pair coordinate(s), and genetic position (cM) is reported on the left side of the table. The left and right boundaries by SNP, hg19 base pair coordinate, and genetic position of a 2-LOD drop interval from the peak marker are reported in the center and right sections of the table, respectively.Click here for file"}
+{"text": "P\u2003<\u20030.02 for linkage in both groups of families is 270\u2003kb from EMX2. In three sibships, we found recessive linkage to KHDRBS3, previously reported in a Somali family. In another family we discovered sex-reversal associated with VUR, implicating PRKX, for which there was weak support for dominant linkage in the overall data set. Several other candidate genes are suggested by our linkage or association results, and four of our linkage peaks are within copy-number variants recently found to be associated with renal hypodysplasia. Undoubtedly there are many genes related to VUR. Our study gives support to some loci suggested by earlier studies as well as suggesting new ones, and provides numerous indications for further investigations.Primary vesicoureteric reflux (VUR), the retrograde flow of urine from the bladder toward the kidneys, results from a developmental anomaly of the vesicoureteric valve mechanism, and is often associated with other urinary tract anomalies. It is the most common urological problem in children, with an estimated prevalence of 1\u20132%, and is a major cause of hypertension in childhood and of renal failure in childhood or adult life. We present the results of a genetic linkage and association scan using 900,000 markers. Our linkage results show a large number of suggestive linkage peaks, with different results in two groups of families, suggesting that VUR is even more genetically heterogeneous than previously imagined. The only marker achieving  Primary vesicoureteric reflux (VUR), the retrograde flow of urine from the bladder toward the upper urinary tract, is the most common urological anomaly in children, and urinary tract infections (UTIs) and renal damage (known as reflux nephropathy) are common in VUR patients  duct. Reciprocal signaling between the bud and the metanephric mesenchyme results in the growth of the ureteric bud to form the ureter and its branching to form the collecting ducts, and organization of the metanephric mesenchyme to form the kidney. Apoptosis occurs in the part of the mesonephric duct between the newly developed ureter and the urogenital sinus. The free end of the developing ureter inserts into the bladder wall and forms the vesicoureteric valve.The ureterovesical junction (UVJ) acts as a valve and closes during micturation or when the bladder contracts. The UVJ is structurally and functionally adapted to allow the intermittent passage of urine and prevent the reflux of urine into the ureter. The main defect in patients with VUR is believed to involve the malformation of the UVJ, in part due to shortening of the submucosal ureteric segment due to congenital lateral ectopia of the ureteric orifice. The precise position at which the ureteric bud grows out from the mesonephric duct is critical not only to the position and angle at which the ureter is inserted into the bladder, but to the degree of renal dysplasia (due to the ureter growing into mesenchyme that is less able to form kidney) and if the budding is bifid, a duplex kidney will form  [Weber et\u2003al. ROBO2 [Lu et\u2003al. SIX2 [Weber et\u2003al. BMP4 [Weber et\u2003al. SOX17 [Gimelli et\u2003al. TNXB [Gbadegesin et\u2003al. The term CAKUT was coined  that could be used for at least one of the three analyses, linkage, transmission disequilibrium test (TDT), and case\u2013control association, of which 500 were from VUR patients . Seventy-one patients had at least one additional urinogenital tract anomaly, 40 of them having at least one duplex kidney. Numbers of samples used for each analysis are given in sections below.A DNA sample collection from peripheral blood samples from healthy members of the Irish population was established as the Irish Blood Transfusion Service \u2013 Trinity College Dublin (IBTS-TCD) BioBank. These samples had already been genotyped using the Affymetrix  Genome-Wide Human SNP Array 6.0 before our patients and families were genotyped  and agarose gel electrophoresis. A few samples that were of low concentration or partially degraded  were sent for whole-genome amplification by Qiagen REPLI-g Services . All samples were then diluted to a standard concentration, plated, and sent to Atlas Biolabs GmbH  who rechecked DNA quality by gel analysis and then genotyped the samples on the Affymetrix Genome-Wide Human SNP Array 6.0.Genotypes were \u201ccalled\u201d (determined from the raw fluorescence data) from the Affymetrix CEL files using Affymetrix Genotyping Console (version 4.1.1.834). CEL files with a contrast QC below 0.4 were removed from analysis, and those remaining were called using the Birdseed V2 algorithm and a two-step workflow. The initial round of genotype calling (which was used to remove poor samples prior to the second genotyping round) was performed using the Birdseed V2 algorithm with VUR and BioBank samples in a single batch; samples with a call rate below 95% or an autosomal SNP heterozygosity rate more than 3 SD from the mean were removed from further analysis. The second round of genotype calling was performed with VUR and BioBank samples in separate batches; no further removal of CEL files was necessary. Genotype data was generated at 834,482 SNPs across the genome. Markers that produced a genotype in at least 95% of the batch of CEL files were exported to PLINK format for use in further analysis.http://www.r-project.org). Samples with genotype call rates below 97% and average heterozygosities outside the range 0.30\u20130.32  were excluded.The next stage of quality control was performed using PLINK  were created and used to check relationships, sample duplications, and ethnicities. Genome-wide identity-by-descent (IBD) sharing was calculated (using the \u201c\u2013Z-genome\u201d command in PLINK). All unexpected findings were checked by microsatellite analysis of the DNA using the PowerPlex\u00ae 16 System  followed by rechecking patient information where necessary. Sample identities/plate maps were then corrected, and the genotype sets subjected to a further round of quality control. This revealed that several pairs of nuclear families, separately ascertained and recruited into the study, were related. These relationships were checked by contacting the families, and the relationships coded into the sample information. Multidimensional scaling of the samples together with 210 unrelated Phase II HapMap  indicated that this thinned set of SNPs (n\u2003=\u20037051) provided adequate linkage information. The data set was then \u201cwiped\u201d by processing with the MERLIN  and nonparametric (model-free) linkage analysis across the genome. A total of 199 families  were used in the linkage analysis, of which 118  had been used in our previous genome scan  and were also checked for consistency of allele frequencies and LD patterns in the region with the known patterns from CEU HapMap data. SNPs deemed to be unreliable on the basis of these checks were removed.In our previous genome scan  some new family members have been added, (b) some samples used last time were not plated, or failed quality control, and (c) some families independently ascertained have been found to be related, and so this time have been recognized as extended families. Also, this time a different set of markers has been used, and precise details of the parameter settings used may also be different. Thus, the results from the old families presented here should not be expected to be exactly the same as previously published, though there is a good correspondence.Figures\u2003KHDRBS3. This gene is in the very center of our own linkage peak  and for the old and new groups of families .Abreu et\u2003al.  suggest P\u2003<\u200310\u22125) along with results from the previous U.K./Slovenian GWAS analysis  reach genome-wide levels of significance. Table\u2003Having added many more families to the study since our previous genome scan, we had hoped that linkage might improve in some of our linked regions, giving us increased confidence and tighter mapping. Instead, the new families showed linkage at different positions, with only a little, or even no linkage in the regions highlighted in the old families, resulting in modification of the larger peaks in size and position, loss of all of the smaller peaks, and addition of some new peaks in the combined set. Overall, the picture is of a large number of regions of increased allele-sharing by affected family members, with some reaching higher scores than others, but none very high, and it seems that VUR may be even more genetically heterogeneous than we previously supposed, and this makes the results more difficult to interpret.As our families are predominantly small nuclear families, every family inevitably contributes regions of allele-sharing where VUR genes do not lie. In the nonparametric analysis, as in single-locus LOD analysis, families can contribute negative linkage scores in positions where alleles are not shared, so in regions in which there is in fact no VUR gene, apparent linkage due to chance allele-sharing in some families may be canceled out by lack of any sharing in others. However, as VUR is heterogeneous, some canceling will also occur at loci where there are VUR genes due to lack of allele sharing at those loci by families with mutations at different loci. Thus, in nonparametric analysis of a multifamily cohort for a highly heterogeneous condition, chance can obscure real linkages as well as creating false linkage peaks of similar size.In parametric linkage analysis, calculation of LOD on the basis of a single dominant gene can result in negative scores across the whole genome if different families actually have mutations at several different loci. This was the case in our original genome scan and again in this one. The HLOD method takes account of heterogeneity, and there are no negative scores, making it possible to achieve positive scores at positions where several sibships share alleles, but it is evident from our own data that the results of combining families that do and do not exhibit sharing at a particular locus are entirely unpredictable. We observe that when there is an HLOD linkage peak in one group of families and no linkage in the other group, the result in all families combined may be that the peak appears again exactly the same size, or half the size or completely disappears, and if there is a small peak in each group, the result in all families combined may also be small, or may be increased. Thus, chance can also have a considerable effect upon parametric linkage results in highly heterogeneous conditions.As greater numbers give greater statistical power, it seems only sensible to concentrate, for the purposes of further investigation, upon regions in which linkage peaks are seen in the full set of families. These consist of a small number of relatively large (but not highly significant) peaks, and a larger number of very small peaks Fig.\u2003. It seemP\u2003<\u20030.02, but two new peaks have been added on 19q and 22q.In the nonparametric linkage analysis, the main peaks from our previous study  have become only slightly larger or slightly smaller in the combined data, as well as shifting slightly in position, and all the smaller peaks have fallen below recognition level of In the dominant HLOD analysis, the same pattern occurs, but there is a slightly different collection of most prominent peaks. The 10q peak is larger than the 2q one, instead of the other way round, as in the nonparametric analysis, but they are again the largest two. However, the peak on 1q, though present in both old and new families, never reaches an HLOD score of 1.2, even in the combined result. The peak on 6q becomes smaller, and the one on 7q, seen in the old families, is reduced to 0.82 by the addition of the new families, but there is a peak on 3q in the old families which is slightly increased in the combined data, with a small contribution from the new families. As in the nonparametric analysis, there are peaks of linkage in the new families that did not appear in the old families, but none of these survives >1.2 in the combined results, while the peak on 22q seen in the nonparametric analysis, reaches only 0.34 in the all families HLOD dominant analysis.P\u2003=\u20030.024, yet it reaches HLOD 1.72 in exactly the same position. This may be due partly to differences in the ways that the different statistical methods work, but also to the particular values chosen for parameters such as allele frequency and penetrance for the HLOD analyses.The very small amount of recessive linkage does not account for much of the difference between the results of the nonparametric and dominant HLOD analyses, either in magnitude or position. For instance, in the combined data the linkage on 1q reaches 1.78 with a p value of 0.002 in the nonparametric analysis (and is on 1q24.1), but only reaches 0.74 HLOD (and that 3.3\u2003Mb away on 1q23.3), while on 3q12.2 nonparametric linkage only reaches 0.86 with FGFR2. It is also only about 600\u2003kb from rs1368532, the peak dominant marker (HLOD 2.44) on 10q in the joint U.K.-Slovenian data , is not close to any known urinary-tract developmental genes and linkage has not been found by any other group studying VUR. The same applies to our nonparametric linkage regions on 1q24.1, 4p16, and 7q36.2. However, since our first genome scan, in which the 2q37 and 7q36 linkage peaks were both found, a patient with duplex kidney and VUR  has been found to have an unbalanced 2;7-translocation with a terminal 9.52\u2003Mb gain in chromosomal band 2q37.1-q37.3 and a terminal 5.65\u2003Mb loss in 7q36.2-q36.3  X-linked dominant VUR has recently been reported  Though some of our linkage peaks corresponded with linkage peaks of other studies, we did not replicate many of other results and found various results not found in other studies. (3) The combined conclusion of points 1 and 2 is that VUR is extremely genetically heterogeneous. However, it is also no doubt true that some of the apparent linkage peaks in our own and other studies will later prove to be artifacts, but at this stage it cannot be determined which ones these will be. (4) Each of our linkage analyses detected some linkage in places where linkage was not found by the other analyses. (5) Some of our linkage peaks were close to genes known to be involved in urinary tract development, and some were close to plausible new candidates, but in other peaks, including our highest linkage, on 2q, there is no obvious candidate.In summary (1) We found almost no correspondence between the linkage patterns in the two groups of families (\u201cold\u201d and \u201cnew\u201d) from the same population, with just a single SNP, close to RSRC1, which produces a protein that takes part in RNA splicing, something that cannot be ruled out from relevance to VUR as another RNA-splicing protein KHDRBS3 appears to be involved in VUR, but adjacent to RSRC1 is a gene far more likely to be involved in VUR, and it is SHOX2. Shox2 regulates Bmp4 expression in the developing mouse heart  so it is highly likely that SHOX2 also regulates BMP4 in the developing urinary tract, and therefore could be involved in VUR. The gene lies between the third and fourth SNPs listed in Table\u2003or there is a particular mutation which is common in Ireland on one haplotype bearing these SNPs while in the U.K. and Slovenia mutations on other haplotypes are as common and balance the ratio so that no association is seen, or the difference is due to the fact that the U.K./Slovenian collaboration deliberately excluded families in which the index case or an affected sibling had additional structural anomalies of the urinary tract, whereas we intentionally included them.Although none of the results from our genome-wide association analysis are highly compelling, some are worthy of further discussion. Firstly, the result of 23 adjacent suggestively significant SNPs on 3p in the TDT analysis is very interesting. Most of these markers are in the gene ELAC2. GUDMAP shows that it is highly expressed in the ureteric bud, and is also expressed in the metanephric mesenchyme. The protein produced by this gene interacts with SMAD2, though so far only SMAD4 has been implicated in urinary tract development . The frizzled proteins act as receptors in Wnt signaling . It has also been found to be highly enriched in the ureteric bud tip compared with the ureteric stalk, and to be one of the most highly induced targets of GDNF signaling in the mouse ureteric bud (embryonic day 11.5) . Whereas, apparently significant SNPs whose adjacent SNPs showed no association at all were rejected, and not reported, as the results most likely resulted from incorrect genotyping , these SNPs, with some elevation of probability of neighboring SNPs, represent the lowest level of possible real association. Some of them are within 0.5\u2003Mb of possible candidate genes , functional studies are necessary, as one cannot just assume that the normal allele on the other chromosome will not be sufficient, or that it will be inhibited by the product of the mutated allele. For variants in noncoding DNA, where we expect the majority of mutations to be, it can be difficult even to guess which variants are worth investigating. The ENCODE project Maher  has idenThe first step after sequencing and identifying variants, is to \u201cfilter\u201d the variants by reference to the SNP database (dbSNP) and other variant databases, by checking the evolutionary conservation of the bases concerned, and by using all the pathogenicity predictors available, including location within likely regulatory elements defined by ENCODE. A systems biology approach might also help. Pathway software might give clues about the affected pathways in VUR by finding interactions between unsuspected candidate genes close to our association results and within our linkage regions, and visualize them in a molecular network. However, many genes still have very little annotation and they would not be included. Then from the shortlist of variants in highly conserved positions that are absent from or rare in variant databases, the next step is to check whether each variant segregates with VUR in the family or families in which it is found. Then, for those that do, the next step is to screen control DNA samples from the same population, and for this we have Irish BioBank controls from blood donors. This will eliminate any variants that are just as common in the general Irish population as they are in VUR patients. After that the remaining variants have to be tested by making the mutations in human cells in culture and observing the effects on gene expression, and after that, the variants that remain promising have to be tested by making transgenic mice and observing whether they develop VUR or CAKUT. We have collaborators ready to do the cell and mouse studies when we have selected promising variants.As mentioned above, the genotyping for this study revealed that a number of our independently ascertained families are genetically related, and these provide one avenue for study. We have already performed whole-genome sequencing on members of one of these extended families, and screened the variants, including using the SNP genotyping data from the genome scan to identify the genomic regions linked to VUR in that particular family, and are currently carrying out BioBank control screens of the shortlisted variants prior to functional studies. Subsequently, we hope to be able to do the same with DNA from our other extended families. We also intend to sequence the genome right across the major linkage peaks in all our patients, and hope that by this we may be able to discover new relevant regulatory elements by clustering of rare novel conserved variants from different families at particular restricted sites. Then functional studies will again be needed. We also intend to sequence across the 8q recessive linkage region in our three families that show linkage to that, and similarly screen variants found there. As will have been learnt from the linkage and association sections above, there are various other interesting findings which we also hope to pursue in due course.The observations that VUR is clearly inherited with high incidence in first degree relatives, and yet linkage studies using large numbers of small families achieve few if any results reaching conventional significance levels, and that studies of smaller numbers of larger families produce conflicting results, can all be explained by there being many genes involved in urinary tract development, disruption of the activity of any one of which may cause VUR and/or CAKUT, so that in large cohorts of families, only a small proportion of them are likely to have mutations at any one locus, and small groups of families will sample different small numbers of loci. Likewise, a candidate gene linkage study is likely to detect very low linkage, even if the gene studied is involved in VUR, because most of the families being tested will be linked to other loci. Also, candidate exon sequencing will often find few mutations (a) because most of the patients have mutations at other loci and (b) because many of the mutations are probably not in the exons, and may be at some distance away from the gene. Most of the conserved DNA in the genome is noncoding, and though it is easy to determine the pathogenicity of a coding mutation, concentrating upon coding sequences inevitably means that most mutations will not be found.With small linkage peaks in studies such as ours, there is inevitably a low signal-to-noise ratio, such that some genuine linkages may be missed while some spurious peaks may appear, and the accuracy of the position of a linkage peak in relation to the true site of mutations is also low, but some of the findings from this study are so striking that they seem likely to be true, and provide numerous leads for further study.It will not be easy to identify functional mutations in noncoding sequences and to demonstrate that they do indeed regulate the genes concerned and do lead to VUR and/or CAKUT, but the recent publications from the ENCODE project should at least help us to identify the best candidate regulatory elements. This, combined with the availability of high-throughput DNA sequencing technologies, should allow the causative genetic changes to be identified.We concluded from the heterogeneity uncovered by our first genome scan (Kelly et\u2003al."}
+{"text": "Aux/IAA genes present in Arabidopsis have not been functionally characterized to date. IAA8 appears to have a distinct function from the other Aux/IAA genes, due to its unique transcriptional response to auxin and the stability of its encoded protein. In this study, we characterized the function of Arabidopsis IAA8 in various developmental processes governed by auxin and in the transcriptional regulation of the auxin response. Transgenic plants expressing estrogen-inducible IAA8 (XVE::IAA8) exhibited significantly fewer lateral roots than the wild type, and an IAA8 loss-of-function mutant exhibited significantly more. Ectopic overexpression of IAA8 resulted in abnormal gravitropism. The strong induction of early auxin-responsive marker genes by auxin treatment was delayed by IAA8 overexpression. GFP-fusion analysis revealed that IAA8 localized not only to the nucleus, but, in contrast to other Aux/IAAs, also to the cytosol. Furthermore, we demonstrated that IAA8 interacts with TIR1, in an auxin-dependent fashion, and with ARF proteins, both in yeast and in planta. Taken together, our results show that IAA8 is involved in lateral root formation, and that this process is regulated through the interaction with the TIR1 auxin receptor and ARF transcription factors in the nucleus.The expression of auxin-responsive genes is regulated by the TIR1/AFB auxin receptor-dependent degradation of Aux/IAA transcriptional repressors, which interact with auxin-responsive factors (ARFs). Most of the 29  TIR1/AFB) TIR1/AFB-mediated ubiquitination following the perception of auxin molecules promotes ARF-mediated transcription. Therefore, the dynamic changes in Aux/IAA abundance, which are controlled by cellular auxin levels, determine the expression levels of auxin-responsive genes.Auxin was the first phytohormone to be discovered and is well-known for its regulatory role in various developmental processes, such as growth and differentiation, and in the cellular responses to light, phosphate starvation, and pathogens Aux/IAA genes, which were initially identified as a large family of early auxin-response genes TIR1 complex and is involved in the instability of Aux/IAA proteins Arabidopsis genome encodes 29 Aux/IAA proteins Aux/IAA genes have been conducted by molecular and biochemical methods and by characterization of mainly gain-of-function mutants. Analysis of proteolytic degradation using transiently expressed Aux/IAA-luciferase fusions suggested that significant primary sequence and structural variations in the receptor recognition motif within domain II affect the stability and longevity of Aux/IAA proteins treated with auxin Aux/IAA genes have little effect on the phenotype Arabidopsis Aux/IAAs regulate various developmental processes that are governed by auxin molecules, including gravitropism, lateral root formation, and root and stem elongation Arabidopsis Aux/IAA genes have been clarified, most of the 29 Aux/IAA genes have not been functionally characterized to date.IAA8 appears to have a distinct function from other Aux/IAA genes; unlike other Aux/IAA genes, the expression of IAA8 is not altered by auxin treatment in various tissues, and IAA8 is more stable than the well-characterized Aux/IAA proteins, IAA1 and IAA17 IAA8 is expressed in the developmental vasculature of the shoot apex, hypocotyl, and root tip, and is developmentally regulated Arabidopsis IAA8 in the transcriptional regulation of the auxin response as well as on the control of root development. We have also analyzed the subcellular localization of IAA8 and carried out a comprehensive interaction analysis of IAA8 with its functional partners, the TIR1 auxin receptor and ARF transcription factors. We found that IAA8 regulates lateral root formation and operates as a transcriptional repressor of the auxin response. Interestingly, although IAA8 physically interacts with both TIR1, via an auxin molecule, and ARF proteins, as do other Aux/IAA proteins, its subcellular distribution is distinct.IAA8 in Arabidopsis, we generated transgenic plants that overexpress IAA8. Initially, we used the Cauliflower Mosaic Virus (CaMV) 35S promoter for the overexpression; however, strong overexpression of IAA8 in any transgenic plants could not be confirmed (data not shown). Therefore, the estradiol-inducible promoter XVE in the pER8 vector IAA8 (XVE::IAA8). The expression of XVE::IAA8 transgenes upon induction with estradiol was examined in over 20 transgenic lines by semi-quantitative reverse transcriptase-PCR (RT-PCR) analysis. Of XVE::IAA8 plants examined, IAA8 expression was induced above endogenous levels only in lines #8 and #11 following estradiol treatment . The absence of visible developmental defects in a loss-of-function mutant of IAA8 is consistent with findings of a previous report These Aux/IAA genes are involved in auxin-related root development, such as primary root elongation, lateral root formation, and grapitropism iaa8-1 and XVE::IAA8 plants. On normal MS medium, the phenotype of all of the genotypes examined was unaffected  was altered (iaa8-1 as compared with Col-0 (XVE::IAA8 plants (line #11), which displayed a strong abnormal root phenotype  XVE::IAA8 plants, especially in line #11, which show drastic changes in root phenotypes  large T antigen NLS Aux/IAA proteins generally possess a nuclear-localization sequence (NLS) and are localized to the nucleus epressor , and alsin planta, we further conducted bimolecular fluorescence complementation (BiFC) assays using a transient expression system in Arabidopsis mesophyll protoplasts as described above. The N terminal of IAA8 was fused to the N-terminal half of YFP (nYFP) to yield nYFP-IAA8, and the N terminal of TIR1 was fused to the C-terminal half of YFP (cYFP) to yield cYFP-TIR1. The nYFP-IAA7 and nYFP-IAA17 fusions were used as positive controls and nYFP-GUS was used as a negative control. In the negative control experiment using nYFP-GUS and cYFP-TIR1, no YFP fluorescence was observed. Similarly, protoplasts expressing both nYFP-IAAs and cYFP-TIR1 did not show any fluorescent signal in the absence of IAA , which contains domain III and IV, of the ARF activators, ARF5, ARF7, and ARF19 . Since gain-of-function mutations in several Aux/IAA genes alter auxin-related root development IAA8 plants. Although primary root lengths were normal for all of the genotypes examined , there was a significant difference in the number of lateral roots and in the gravitropic response  Aux/IAA genes. In dicots, including Arabidopsis, lateral root initiation depends on the activation of pericycle cells by auxin IAA8 promoter-driven GUS expression is found in the vascular tissue of the root tip, which appears to correspond to pericycle cells IAA8 is not up-regulated by auxin In contrast, the number of lateral roots was altered in e latter . Thus, IIAA8 overexpression  and are localized to the nucleus Arabidopsis mesophyll protoplasts. Auxin-dependent interactions between the TIR1 auxin receptor and IAA8 as well as the well-characterized Aux/IAA proteins, IAA7 and IAA17, were observed both in yeast and in planta  was used as wild type. Seeds of iaa8-1XVE::IAA8 construct, which consists of IAA8 driven by an estrogen-inducible promoter, XVE, in the pER8 vector Arabidopsis IAA8 cDNA fragment was amplified by PCR with gene-specific primer sets (see Table S1) and cloned into pDONR207 (Invitrogen), using the Gateway BP recombination method (Invitrogen). After verifying the nucleotide sequence of the PCR fragment by sequencing, the IAA8 cDNA fragment was transferred into the modified pER8 estradiol-inducible plant transformation vector bar gene, for selection of transgenic plants, using LR clonase (Invitrogen), according to the manufacturer's instruction (Invitrogen). The resultant vector was introduced into Agrobacterium tumefaciens strain GV3101 (pMP90) by electroporation. The transformed A. tumefaciens was used for floral dip transformation of Arabidopisis Col-0 as described in To create the etal.All seeds were surface sterilized in 50% commercial bleach and 0.01% Silwet L-77 (Toray DowCorning), subjected to cold treatment, and then vertically grown on half strength Murashige and Skoog (MS) medium supplemented with 1% sucrose under the same conditions as for growth in soil. To monitor root growth and lateral root formation, seedlings were grown for 14 days. The length of primary roots and the number of lateral roots were determined for at least 20 seedlings in each genotype. The gravitropism assays were carried out according to Park The roots of two-week-old plants grown on half strength MS medium as described above were immersed in 10 \u03bcM \u03b2-estradiol for 24 h (estrogen treatment) and then transferred to 1 \u03bcM indole-3-acetic acid (IAA) solution (auxin treatment). Total RNA was isolated using Total RNA Extraction Kit Mini (Plant) (RBC Bioscience). First-strand cDNA was synthesized from 1 \u03bcg of total RNA treated with DNase I (TAKARA BIO) using the PrimeScript II 1st strand cDNA Synthesis Kit (TAKARA BIO). PCR amplification was then performed using GoTaq Green Master Mix (Promega). The gene-specific primers and PCR cycles for each gene are listed in Table S1.IAA7 and IAA17 cDNAs and the C-terminal domain (CTD) fragments of ARF3, ARF4, ARF5, ARF7, ARF11, ARF16, and ARF19 cDNAs were amplified by PCR using plasmids harboring full-length Aux/IAA and ARF cDNAs from RIKEN Arabidopsis cDNA Encyclopedia DNABook IAA8 cDNA fragment was transferred into bait plasmid vector pEG202gw, and IAA7, IAA17, and ARFCTD fragments were cloned into prey plasmid vector pJG4-5gw TIR1 (GC105370) was obtained from ABRC and used for transferring fragments into pEG202gw, as described above. A yeast two-hybrid assay using the LexA-based two-hybrid system was basically carried out as described in MAT\u03b1 ura3 trp1 his3 3LexAop-LEU2) harboring pJK103 [2LexAop-lacZ]) reporter plasmid was carried out with the Frozen-EZ Yeast Transformation II Kit (Zymo Research). The strength of interaction between IAA8 and each ARFCTD was evaluated by measuring \u03b2-gal activity using the \u03bf-Nitrophenyl-\u03b2-D-galactopyranoside (ONPG) method, according to the Yeast Handbook (Clontech). To test for interactions between TIR1 and Aux/IAAs, transformants with both TIR1 in pEG202 and Aux/IAAs in pJG4-5 were transferred onto SD/-Ura/-His/-Trp/-Leu with 100 \u03bcM indole-3-acetic acid (IAA). All plates were incubated for 2\u20133 days at 30\u00b0C.The fragments of To confirm the expression levels of IAA8 and ARFCTD proteins co-expressed in yeast, the extracted total protein from each transformant was subjected to SDS-PAGE and then transferred onto Protoran nitrocellulose membrane  for Western blot analysis. Rabbit anti-LexA (BioAcademia) and rat anti-HA  antibodies were used for the detection of LexA DNA-binding domain fusion of IAA8 and activation domain with HA tag fusion of each ARFCTD, respectively. Luminata Crescendo Western HRP substrate (Millipore) was used for chemiluminescent detection.IAA7-, IAA8- and IAA17-GFP fusions driven by the CaMV 35S promoter, all IAA cDNA fragments made as described above were transferred into p2GWF7 ARF3, ARF4, ARF5, ARF7, ARF11, ARF16, and ARF19 were amplified by PCR using plasmids in the RIKEN Arabidopsis cDNA Encyclopedia DNABook nYFP-Aux/IAAs, ARFs-nYFP, cYFP-TIR1, and IAA8-cYFP fusions driven by the CaMV 35S promoter were constructed by transferring Aux/IAA, ARF, TIR1, and IAA8 full-length cDNAs into nYFP/pUGW0, nYFP/pUGW2, cYFP/pUGW0, and cYFP/pUGW2 For the construction of Arabidopisis Col-0 plants grown on soil were prepared and transformed using the PEG-calcium transfection method Mesophyll protoplasts from the leaves of four-week-old Figure S1The primary root length of Col-0, XVE::IAA8 transgenic plants, and the iaa8-1 mutant. All seedlings were vertically grown for two weeks on 1/2 MS medium containing 10 \u03bcM \u03b2-estradiol (+\u03b2-estradiol) or 0.1% ethanol (\u2212\u03b2-estradiol) and then primary root length was measured. The averages of at least 20 seedlings \u00b1 S.D. are shown.(EPS)Click here for additional data file.Figure S2The phenotype of wild-type (Col-0) and IAA8 loss-of-function mutant (iaa8-1) seedlings. All seedlings were vertically grown for two weeks on 1/2 MS medium containing 10 \u03bcM \u03b2-estradiol (+\u03b2-estradiol). Bar \u200a=\u200a1 cm.(EPS)Click here for additional data file.Figure S3Western blot analysis of IAA8 and ARF proteins co-expressed in yeast. BD-IAA8 and AD-ARFCTD expressed in each yeast transformant as shown in (EPS)Click here for additional data file.Table S1Nucleotide sequences of primers used in this study.(XLSX)Click here for additional data file."}
+{"text": "A bulk heterojunction of porous silicon and eumelanin, where the columnar pores of porous silicon are filled with eumelanin, is proposed as a new organic-inorganic hybrid material for photovoltaic applications. The addition of eumelanin, whose absorption in the near infrared region is significantly higher than porous silicon, should greatly enhance the light absorption capabilities of the empty porous silicon matrix, which are very low in the low energy side of the visible spectral range (from about 600\u2009nm downwards). The experimental results show that indeed the photocarrier collection efficiency at longer wavelengths in eumelanin-impregnated samples is clearly higher with respect to empty porous silicon matrices. The relevance of solar power in the renewable energy field is constantly increasing due to its ready availability and to the fact that the available amount exceeds by several orders of magnitude the needs of the human race. The search for new materials with better performances than standard Si-based solar cells is also constantly increasing. Organic materials  emerged Among the adopted strategies for new materials, interface geometry often plays a major role in the collection of photogenerated carriers, and bulk heterojunctions ,9-12 - in-type porous silicon (PSi) and eumelanin, a natural pigment featuring relatively high electrical conductivity 43]. In dv/v) and fractionated by preparative TLC (CHCl3/MeOH 98:2) to identify the constituents by comparison with synthetic standards with known structure.The residue was acetylated with acetic anhydride-pyridine 95:5  and more than a factor of ten larger than that of PSi whose absolute value becomes less than 103\u2009cm\u22121. This is where the effect of eumelanin may be expected to generate a more significant difference in the photoconductive behavior of empty and impregnated porous layers. It is important to note that from [\u22121 for thin films and 8,000\u2009cm\u22121 for thick films.The data shown in Figure\u2009hat from , the eum2) is, with and without eumelanin, our results conclusively show that even a thin oxide layer on the pore's wall is sufficient to severely limit the layers' photosensitivity.The photoconductive properties of porous Si samples were studied with and without eumelanin for unoxidized layers. A few samples were oxidized at several oxidation levels. This test has been done because, although the partial oxidation process may reduce the PSi conductivity, the oxidation-induced modification of the eumelanin adhesion to the pore walls could, in principle, more than counterbalance the effect. In all cases, however, all the partially oxidized samples showed no measurable photosensitivity. Whatever the impinging light intensity value . The observed variability did not appear to depend on the eumelanin immobilization procedure.Imax\u2009=\u20090.8\u2009\u03bcA for the two empty PSi layers and Imax\u2009=\u20090.9\u2009\u03bcA for the impregnated sample labeled impregnated PSi 1) for three of the samples shown, while for one of the impregnated samples, the maximum absolute photocurrent is significantly lower .To better evaluate the effect of the eumelanin insertion on the photoconductive properties of the samples and to discriminate possible contact issues from the samples' behavior, we measured the wavelength dependence of the light response of impregnated and empty porous Si samples using low-pass optical filters in front of the samples. By this approach, using a given filter the light arriving onto the samples' surface will only have wavelengths longer than the cutoff filter's. This means that the photocurrent measured with each filter will be given by all photocarriers generated by the interaction of the samples with the residual spectral range, that is, the part whose photon energy is lower than that characteristic of the cutoff filter. Consequently, if the carrier photogeneration occurs in the whole source spectrum, the plot of the photocurrent as a function of the cutoff wavelengths is expected to be a monotone curve which is increasing when moving towards shorter wavelengths. The results obtained on typical samples in our experiments are shown in Figure\u2009To separate the contact-related issues from the photogeneration behavior of the samples, the photocurrent intensity and the wavelength dependent photogeneration behavior must be separated. If the behavior of the samples would prove to be the same with respect to the maximum recorded photocurrent, it would be possible to attribute the intensity differences from sample to sample essentially to contact-related issues and not to the hybrid material building procedure adopted. Accordingly, we show in Figure\u2009The striking feature of Figure\u2009From the results shown in Figures\u2009The latter effect is even more significant if we keep in mind the very low intensity of the impinging light in the 700 to 850\u2009nm range: for the impregnated samples, 40\u2009% of the total photocurrent is obtained with a very limited part of the total spectrum; while to obtain the same amount of photocurrent with the empty PSi samples, we need to increase the wavelength range up to 550\u2009nm, including most of the available lamp spectral range.These results show how the impregnation of the PSi matrix with eumelanin significantly increases the capability of the layer to efficiently photogenerate carriers from light especially when approaching the infrared region.We have shown that the photovoltaic properties of PSi may be significantly improved by impregnation with eumelanin. In particular, we showed that introducing the pigment in the porous Si matrix leads to a significantly more efficient photocurrent generation in the lower energy part of the experimental wavelength range explored with respect to empty porous Si layers.This result not only contributes to expand the scope of heterojunctions in developing a new hybrid material but also provides the first evidence of the eumelanin capability to efficiently collect the photon energy. In a given substrate, eumelanin seems to act as a kind of \u2018antenna\u2019, modifying the range of the useful wavelength range for photoconversion application.Although further experimental and theoretical studies are needed to improve the electrical contacts reproducibility and to reach a deeper understanding of the observed behavior in view of future development and applications, the proof of principle device presented here opens a new window into the evolving panorama of eumelanin-based devices and contributes to the development of bioinspired and biocompatible optoelectronic devices.The authors declare that they have no competing interests.GM conceived of the study, participated in its design and coordination, prepared part of the samples and performed the reflectivity measurements and analysis. LM performed the photoconductivity measurements and prepared part of the samples. SS participated in the design of the study. AP participated in the design of the study, carried out the eumelanin preparation and performed the PSi impregnation process with eumelanin. All authors contributed to the data analysis. All authors read and approved the final manuscript."}
+{"text": "Similar results were obtained in the multi-factorial generalized estimating equation analysis. Using different statistical approaches, in this study, we have confirmed that the single nucleotide polymorphism rs3737787 is related to triglyceride and apolipoprotein E levels in type 2 diabetes mellitus families.Upstream transcription factor 1 (USF1) is capable of controlling various members in glucose and lipid metabolism pathways. Much evidence suggests that the rs3737787 polymorphism in the USF1 gene may lead to alteration of lipid metabolism. The objective of this study was to test the association between rs3737787 and type 2 diabetes mellitus (T2DM) and its related lipid metabolic traits in the Chinese population. A total of 287 eligible T2DM families were chosen in Beijing. A set of questionnaires was administered to obtain information on demographic characteristics. Physical measurements were recorded. DNA was extracted from blood samples and genotyped using PCR-RFLP method. Statistical analyses including linkage analysis and family-based association test were performed to detect the relationship between rs3737787 and T2DM related traits. In the non-parametric linkage analysis, it was observed that rs3737787 is potentially linked with triglyceride and apolipoprotein E levels, where the logarithm of the odds scores were 0.87 ( Dyslipidemia is the main cardiovascular risk factor of type 2 diabetes mellitus (T2DM) patients, and may potentially lead to some severe complications, including many common cardiovascular and cerebrovascular diseases. Lipid metabolism of T2DM is modulated by several factors, in which control of blood glucose and insulin resistance are the most important, while insulin resistance is the central link resulting dyslipidemia. Insulin resistance results in increased serum concentrations of very low density lipoprotein (VLDL) and triglyceride (TG) and decreased rates of their clearance. It was found that dyslipidemia is a major modifiable risk factor for atherosclerosis , which iUSF1 gene is located at human chromosome 1q22-q23, which is the most consistently replicated locus showing linkage with T2DM in different populations (Upstream transcription factor 1 (USF1) is a ubiquitously expressed transcription factor, and is a member of the basic helix-loop-helix leucine zipper family. USF1 has been shown to modulate the expression of various crucial genes related to glucose and lipid metabolism, such as glucokinase, fatty acid synthase, and acetyl-CoA carboxylase gene, by specifically binding to E-box motifs in these genes. The important role of USF1 in regulating and coordinating various members in glucose and lipid metabolism pathways leads it to be regarded as a significant candidate gene for some metabolic abnormalities such as T2DM, dyslipidemia, and metabolic syndrome. ulations .USF1 was initially reported to be linked and associated with familial combined hyperlipidemia (FCHL) in Finnish families and in other populations  of pulation . Naukkarpressure . NonetheThis study was conducted in Fangshan District in suburban Southwest Beijing, China. The population in Fangshan District is stable and unlikely to migrate. In addition, the genetic background of the population tends to be unique and the living environments are similar among individuals. Thus, the population is Fangshan District is suitable for genetic epidemiological studies, and has been used as the study population by our research group for years \u201318. The were diagnosed with T2DM according to World Health Organization (WHO) criteria  andwere patients in Fangshan District First Hospital or lived in the communities served by the hospital.There was no evidence of acute infection, trauma or other situation of stress.They did not have mitochondrial mutation related diabetes which was detected using polymerase chain reaction with restriction fragment length polymorphism (PCR-RFLP) to detect the tRNA 3243A/G mutation.They did not have maturity onset diabetes of the young (MODY) which was determined by pedigree analysis and age of onset of diabetes.Eligible probands were identified using the following criteria:had at least one affected sibling and one living parent.All the parents and the siblings agreed to participate in the study with informed consent anddid not have severe disabilities that affected their ability to participate in the study.Eligible families were chosen from the families of eligible probands using the following explicit inclusion criteria:Families that had lived in distant regions and families without informed consent were excluded from the study.After a family was enrolled, a questionnaire was administered by trained interviewers to each member of the family to obtain information on demographic characteristics, previous medical history, family history, and life habits . Physical examination data including height, weight, waist, and hip circumference were recorded by trained doctors using standardized processes. Blood samples were obtained via venipuncture by skilled nurses. DNA was extracted using phenol-chloroform approach, according to the standard protocol .Taq polymerase (2 units/\u03bcL \u00d7 0.5 \u03bcL) in a final volume of 25\u03bcL. After the reaction mixture is preincubated for 5 minutes at 94\u00b0C, 35 rounds of amplification were performed, consisting of denaturation at 94\u00b0C for 30 seconds, annealing at 54\u00b0C for 30 seconds, extension at 72\u00b0C for 30 seconds, and a final extension at 72\u00b0C for 5 minutes. Polymerase chain reaction products are subjected to digestion by HpyCH4IV overnight at 37\u00b0C. The products are electrophoresed on a 2.5% agarose gel (120V for 1 hour) with ethidium bromide staining and visualized under ultraviolet light . Homozygous wild-type (CC) individuals show 96- and 33-base pair (bp) fragment, while heterozygous individual show three bands at 129, 96, and 33 bp, respectively. Homozygous rare-allele individuals, which cannot be digested by HpyCH4IV, show only 129-bp band.For the rs3737787 polymorphism, the sequences of the forward and reverse primers used in the polymerase chain reaction are 5\u2032-GGCCTGCAGTGGTGTGAAA-3\u2032 and 5\u2032-TCCAGTATCCAGCATGGAGACA-3\u2032 (synthesized by Shanghai GeneCore BioTechnologies Co. Ltd.). Genomic DNA (100ng) is added to a buffer with deoxyribonucleoside triphosphates (10 mM \u00d7 0.25 \u03bcL), forward and reverse primers , and p value less than 0.05 was considered statistically significant (two-tailed).Most of the statistical analyses were conducted using SPSS software  unless it was necessary to use other specified software. A We used SIB-PAIR 1.00b to perform Mendelian error checking and genotype imputation in the family data and MERLIN 1.1.2 to perform variance component linkage analyses for blood lipid traits. The standard non-parametric linkage analysis used by MERLIN employs the Kong and Cox linear model to evaluate the evidence for linkage. This model is designed to discover small increases in allele sharing spread across a large amount of families, which is usually expected in a complex disease such as T2DM. The method of variance component first used for quantitative trait loci (QTL) mapping of complex diseases in the 1990s, and has become a common method of QTL linkage analysis . This meWith SAS macro program, we used the family based association test (FBAT) method to test for the association between the SNP and the traits under additive genetic model. The FBAT method was developed by Rabinowitz and Laird based on transmission/disequilibrium test (TDT), which adopted score test to compute the statistic and then to conduct the statistical inference. While conserving the characteristic of TDT that avoids the effects of population stratification, FBAT also is suitable for various types of pedigrees and genetic models, thus, it is a popular method in genetic association studies.We also use generalized estimating equations (GEE) to assess the relationship between the polymorphism and the related traits. GEE is a unification of two major approaches for linkage analysis of quantitative traits: variance components and Haseman-Elston regression. This method considers environmental and other covariates while evaluating the relationship .p=0.175)  and 1.96 (p=0.001), respectively. No other linkage relationships with quantitative traits were observed. We also detected a possible linkage between the polymorphism and T2DM related binary traits; however, all the LODs were less than 1.0, i.e. the potential linkage relationships were not statistically significant  of 0.87 , we found that the TT genotype of rs3737787 was statistically associated with triglyceride and apolipoprotein E Table .Many patients with type 2 diabetes mellitus die of atherosclerosis and its complications, while abnormalities in plasma lipoproteins and derangements in lipid metabolism rank as the most firmly established and best understood risk factors for atherosclerosis . The abnUSF1 gene in northern Chinese population. The results were in accordance with other studies in similar populations  show differential expression of downstream genes in fat biopsies (ABCA1), angiotensinogen (AGT), and apolipoprotein E (APOE), differed significantly in their expression between the two haplotype groups of USF1 (defined by SNPs in LD with rs3737787 and rs2073658). They also indicated that the most downregulated gene in the risk individuals was APOE, which is expressed in at risk individuals at only half of the levels of the non-risk ones. However, rs3737787 in the 3\u2032-untranslated region (UTR) and rs2073658 in intron7 are in complete linkage disequilibrium (LD) . While we know that 3\u2032-UTR can mediate the translational control of corresponding genes , which was first discovered to be a surface protein on human platelets, and has been found to be associated with central obesity and systolic blood pressure in the Chinese population (JAM1.Usually, for complicated diseases such as T2DM or dyslipidemia, the associated polymorphisms do not lead to obvious defects in their translated proteins. They often tend to lie in the presumptive regulatory regions, such as promoters or enhancers in introns , 31. Minbiopsies . Three gng genes , it can pulation . ConsideUSF1 gene and blood lipid profile in Chinese type 2 diabetes mellitus families. However, these findings need to be confirmed in other population and with larger sample sizes, together with a functional analysis of the gene with such polymorphism.In conclusion, our study shows a relationship between an important polymorphism of"}
+{"text": "Reading disability (RD) is a common neurodevelopmental disorder with genetic basis established in families segregating \u201cpure\u201d dyslexia. RD commonly occurs in neurodevelopmental disorders including Rolandic Epilepsy (RE), a complex genetic disorder. We performed genomewide linkage analysis of RD in RE families, testing the hypotheses that RD in RE families is genetically heterogenenous to pure dyslexia, and shares genetic influences with other sub-phenotypes of RE.SEMA3C gene at the 7q21 linkage locus in members of one multiplex RE/RD pedigree and the DISC1 gene in affected pedigrees at the 1q42 locus.We initially performed genome-wide linkage analysis using 1000 STR markers in 38 US families ascertained through a RE proband; most of these families were multiplex for RD. We analyzed the data by two-point and multipoint parametric LOD score methods. We then confirmed the linkage evidence in a second US dataset of 20 RE families. We also resequenced the In the discovery dataset there was suggestive evidence of linkage for RD to chromosome 7q21  and at 1q42 . Much of the linkage evidence at 7q21 derived from families of French-Canadian origin, whereas the linkage evidence at 1q42 was well distributed across all the families. There was little evidence for linkage at known dyslexia loci. Combining the discovery and confirmation datasets increased the evidence at 1q42 , but decreased evidence at 7q21 , possibly because the replication sample did not have French Canadian representation.Reading disability in rolandic epilepsy has a genetic basis and may be influenced by loci at 1q42 and, in some populations, at 7q21; there is little evidence of a role for known DYX loci discovered in \u201cpure\u201d dyslexia pedigrees. 1q42 and 7q21 are candidate novel dyslexia loci. DYX1-DYX9) mapped Reading disability (RD) is one of the most common developmental conditions in childhood with a prevalence ranging from 5\u201312% ELP4 gene at 11p13 Epilepsy is often associated with neurodevelopmental disorders The aim of this study was to map loci for RD in RE families and determine whether there is overlap with \u201cpure\u201d RD loci identified in non-comorbid samples. We asked two questions: (i) does reading disability in RE map to loci different from those so far reported in genetic studies of dyslexia in neurologically normal samples The IRBs of New York State Psychiatric Institute and all collaborating centers approved the study. All subjects gave written informed consent.We employed a two-stage linkage design, the first stage was a genome-wide screen of 38 RE families; the second stage was a follow-up of linkage peaks and reported dyslexia loci in 20 later ascertained RE families. Where an obvious candidate existed, a gene under the linkage peak was resequenced.Rolandic Epilepsy probands and their families were recruited from pediatric neurology centers in the US for a genetic linkage study in 2005\u20132007. Ascertainment was through the proband, with no requirement for other family members to be affected with epilepsy.Cases were enrolled if they met stringent eligibility criteria for RE, consisting of typical orofacial seizures, age of onset between 3\u201312 years, no previous epilepsy type, normal global developmental milestones, normal neurological examination, EEG with centrotemporal sharp waves and normal background, and neuroimaging that excluded an alternative structural, inflammatory or metabolic cause for the seizures. Board-certified experts in epileptology, neurophysiology, and neuroimaging centrally reviewed all of the probands\u2019 charts, EEGs, and neuroimaging for eligibility prior to recruitment (see Acknowledgements). Questionable cases were discussed with an independent expert child neurologist specializing in epilepsy. Cases were not required to be comorbid with any neuropsychiatric disorder, and proband RD and SSD affectedness was unknown at time of ascertainment.The first stage genome-wide linkage screen included 38 two or three generation RE families. In the second stage, we included an additional 20 two or three generation RE families. The number of genotyped individuals with RE, RD or both are listed for each family in A pediatric-trained physician (TC or DKP) interviewed the case families. Both parents were interviewed, either together or separately, and the proband and siblings were also interviewed when age appropriate. The investigator administered a 125-item questionnaire covering perinatal, developmental, medical, educational details, family history and detailed seizure semiology and treatment history. A pediatric neurologist, pediatric neuropsychologist, adult neuropsychologist, and pediatric speech pathologist jointly developed the questionnaire. Questions that were answered positively were followed up in detail by clinical interview to establish ICD-10 diagnoses and to distinguish from global learning disability. The questionnaire included 13 items addressing speech articulation disorder F80.0. A similar batch of questions was used in a high-risk study of phonological disorder . The questionnaire also contained nine items addressing the ICD-10 definitions of reading disorder F81.0. RD was thus identified by significant impairment in the development of reading skills not solely accounted for by mental age, sensory problems, mother tongue, or inadequate schooling. Operationally, we asked about difficulties in learning to read in the first year or two of elementary school, reading remediation, and repeating a grade. We also excluded, by clinical interview, hearing impairment, social and educational deprivation, and other factors that were inconsistent with the ICD-10 diagnosis of RD. We checked available school and psychologist\u2019s reports for confirmation, and all were consistent with our findings.Wechsler Abbreviated Scale of Intelligence; academic achievement including spelling: Woodcock-Johnson III Gray Oral Reading Tests 4Test of Word Reading EfficiencyClinical Evaluation of Language Fundamentals, 4th EditionBoston Naming Test, 2nd EditionA subset of 11 probands and 10 siblings underwent comprehensive neuropsychological evaluation, the details of which are reported elsewhere Blood or saliva samples  for DNA extraction were collected from probands and all potentially informative available family members. DNA was extracted using standard protocols (15). Individuals were classified as affected or not affected for Reading Disability according to clinical evaluation using operational ICD-10 definitions of reading disorder (F81.0) (who.int/classifications/en). In the first stage, a total of 194 individuals were genotyped at deCODE Genetics, Iceland using the deCODE 1000 marker single tandem repeat (STR) set, which has an average genome-wide resolution of 4 cM. In the second stage, a total of 145 individuals were typed for markers in the same STR set on chromosomes 1\u20133, 5\u20137, 11, 12 and 15\u201317. Twenty additional new markers were typed only in the replication sample. Amplified fragments were typed using ABI 3700 and ABI 3730 DNA analyzers with CEPH family DNA used as standards. Alleles were called automatically and checked for Mendelian consistency and Hardy-Weinberg equilibrium.or CTS; and RD or SSD.We analyzed the data by two-point and multipoint LOD score calculations using the Maximized Maximum LOD Score (MMLS) approach SEMA3C gene in a large 17-member pedigree, 10 affected with RD, of French-Canadian origin included in the original linkage analysis. Genomic DNA from 13 individuals was available for screening. Sanger sequencing was also used to screen the DISC1 gene in 20 affected families with RE and RD. We designed primers to amplify all of the exons and alternatively spliced exons, as well as the splice sites, promoter and 5\u2032 and 3\u2032 UTRs. We sequenced DNA amplified by PCR using the ABI 3130 DNA Analyzer and interpreted sequence using the Invitrogen Vector NTI Advance Suite. See We used bi-directional Sanger sequencing for mutation screening of the In two-point analysis of RD in the initial set of families, we observed a LOD score of 3.05 at marker D7S660 in chromosomal band 7q21. The LOD score maximized under a dominant mode of inheritance, with 60% penetrance, at a recombination fraction of 0.01. We also observed LOD scores above 2.0 on chromosomes 1, 2 and 6 .or SSD, and also for the affectedness definition RD or CTS . In a combined analysis of both datasets, the evidence for linkage of RD at 1q42 increased to LOD 3.55 at the same marker as in the original dataset, D1S1540, maximizing under a dominant, 70% penetrance trait model. In multipoint linkage analysis the maximum HLOD rose to 4.70 at D1S2833. However, at chromosome 7, the maximum LOD score in the second dataset was at D7S2409 , which was 10 cM distant from the linkage peak observed at D7S660 in the original dataset. In combined analysis, the evidence for linkage at D7S660 dropped to 2.28 .Exploring the linkage data by family, sixteen families provided positive LOD scores at the 7q21 region, with the majority of the linkage evidence coming from eight French-Canadian families and three Hispanic families. Interestingly, there were no French-Canadian families in our replication dataset. One particular French-Canadian pedigree,  with 17 SEMA3C gene, which resides only 50,000 bp from the start of the microsatellite. Not only is this the closest gene to our linkage peak, but SEMA3C is an attractive candidate for further investigation. Semaphorins act as axonal growth cone guidance molecules, especially important in the developing fetal brain, and thus fit well with the theme emerging in the genetics of dyslexia and other neurodevelopmental disorders SEMA3C from all 13 individuals in the pedigree of French-Canadian origin  which is associated with many psychiatric and neurodevelopmental conditions DISC1 in affected members of 20 families that were linked to the 1q42 locus.The 1q42 locus contains approximately 25 genes in the 1-LOD interval around the linkage peak, and deletions at this locus have previously been associated with rolandic seizures and abnormal EEG DISC1 coding mutations, microdeletions or microduplications, are causative for RD in RE families.We found 6 coding SNPs in the sequenced regions; however, none of them were novel, and all occurred at a frequency expected in a Caucasian population, indicating it is unlikely that DYX loci discovered in relatively \u201cpure\u201d dyslexia samples may not be representative of genetic influences on RD found in samples comorbid for epilepsy. This provides further evidence for the genetic basis of comorbidity in common forms of epilepsy, following our report of pleiotropy between speech sound disorder and centrotemporal EEG spikes We have discovered evidence for possible novel dyslexia loci at 7q21 and 1q42 in families of subjects with rolandic epilepsy, a common type of epilepsy in which speech and language impairments strongly co-aggregate Approximately 55% of RE patients are co-morbid with RD DISC1. DISC1 has been linked and associated with schizophrenia and bipolar disorder in the general population DISC1 mutations have been associated with deficits in sustained attention and memory in schizophrenia patients DISC1 from sequencing affected family members in this study, suggesting an alternative susceptibility gene, an intronic mutation, or other structural variations.The most interesting candidate gene at the 1q42 locus is SEMA3C in one large multiplex French-Canadian pedigree suggests the need to consider other variation in or around this locus.Our linkage results pointed to the possibility of a second, genetically heterogeneous RD locus enriched in French-Canadian pedigrees at 7q21. A chromosomal deletion at this locus (7q11.23q21.2) has previously been associated with microcephaly, ADHD, and epilepsy with bilateral centrotemporal EEG spikes The genetic basis of dyslexia and speech sound disorder is firmly established in the neurodevelopmental genetics field, yet the occurrence of these common comorbidities among children with epilepsy has not previously been attributed to a genetic etiology. We recently showed pleiotropy at the 11p13 locus for both centrotemporal EEG spikes and speech dyspraxia Pleiotropy has been demonstrated between SSD and RD in SSD families Table S1Breakdown by pedigree of number affected with Rolandic Epilepsy, RE, Reading Disability, RD, or both, in original group 1:n and replication group 2:n.(DOC)Click here for additional data file.Table S2Forward and reverse primer sequences used in the PCR and subsequent Sanger sequencing of a. SEMA3C and b. DISC1; nomenclature from Ensembl.(DOC)Click here for additional data file."}
+{"text": "We apply a multiphase strategy for pedigree-based genetic analysis of systolic blood pressure data collected in a longitudinal study of large Mexican American pedigrees. In the first phase, we conduct variance-components linkage analysis to identify regions that may harbor quantitative trait loci. In the second phase, we carry out pedigree-based association analysis in a selected region with common and low-frequency variants from genome-wide association studies and whole genome sequencing data. Using sequencing data, we compare approaches to pedigree analysis in a 10 megabase candidate region on chromosome 3 harboring a gene previously identified by a consortium for blood pressure genome-wide association studies. We observe that, as expected, the measured genotype analysis tends to provide larger signals than the quantitative transmission disequilibrium test. We also observe that while linkage signals are contributed by common variants, strong associations are found mainly at rare variants. Multiphase analysis can improve computational efficiency and reduce the multiple testing burden. In pedigree-based studies, discovery of genomic regions harboring genetic determinants of quantitative traits such as systolic blood pressure (SBP) has conventionally been conducted using linkage analysis based on identity-by-descent allele sharing. In the genome-wide association studies (GWAS) era of cost-effective high-throughput genotyping technology, the mapping of the genetic basis of complex traits/diseases in human populations has been population-based in unrelated individuals, and largely case-control or cross-sectional in design. With the advent of next-generation sequencing technology, investigators are able to examine each single base pair (bp) and test for association with a trait, but the massive amount of variant information available for analysis can be overwhelming. With the development of techniques for pedigree-based imputation from sequence data on selected pedigree members, pedigree-based analysis of whole genome sequencing data is feasible.MECOM, 3q26) identified in a pathway influencing blood pressure and cardiovascular disease risk by the International Consortium for Blood Pressure Genome-Wide Association Studies (ICBP-GWAS) [MECOM genomic region.We demonstrate that multiphase analysis in pedigrees can be an efficient strategy for identifying genetic variants underlying a quantitative trait, in which region discovery by linkage analysis of GWAS single-nucleotide polymorphism (SNP) markers with high minor allele frequency (MAF) is followed by region refinement with densely distributed GWAS SNPs and/or fine mapping with sequence variants in identified regions. Using a summary phenotype derived from longitudinal measurements of SBP together with GWAS and whole genome sequencing genotype data from the San Antonio Family Studies (SAFS) as provided by Genetic Analysis Workshop 18 (GAW18), we report pedigree-based linkage and association analysis conducted to identify genetic variants underlying SBP. Our multiphase analyses are carried out in 3 steps, as illustrated by the workflow in Figure From a total of 1389 participants in 20 pedigrees, 932 have SBP measurements at 1 or more study exams for up to 4 exams. Characteristics recorded include sex, year of exam, age at each exam, current use of antihypertensive medications, and current tobacco smoking. GWAS genotypes were assayed in a total of 959 individuals, with a total of 65,519 GWAS SNPs on chromosome 3 available for analysis. Among these individuals, 464 were also sequenced at an average 60 \u00d7 coverage, resulting in 1,215,399 sequence variants on chromosome 3. For the remaining 495 individuals, the missing genotypes at the sequence variants were imputed using a novel population-based imputation approach [Y be the observed SBP and \u0176 be the fitted SBP from the censored model given exam-specific covariates and pedigree-specific random effects. For an individual receiving medication, let R, was then used as an adjusted phenotype for each individual in subsequent stages of linkage and association analysis.Antihypertensive medication complicates the analysis of SBP, because patients prescribed medication tend to have elevated underlying SBP values. Based on a novel extension developed by Konigorski et al , we treaki et al . We compn members is partitioned into a locus-specific variance component To detect regions with potential loci for SBP, we applied the variance-component linkage method for pedigree-based analysis . In an an. To examine the influence of GWAS SNP density on linkage analysis, we sampled 3 sets of SNPs. Initially, a total of 988 SNP markers was randomly sampled from chromosome 3 GWAS SNPs with MAF \u22655%. To allay concerns about adequacy of SNP density, in the second and third samplings, we randomly sampled 1620 and 2999 SNPs, respectively, excluding previously sampled SNPs and using the same MAF criteria. We first performed quantitative genetic analysis to create a suitable null model for each selected marker [where the elements of the structuring matrix for the locus-specific variance, \u03a0, are proportions representing the identity-by-descent (IBD) sharing of alleles for each relative pair at this locus; the structuring matrix for the additive genetic variance component, 2\u03a6, is twice the kinship coefficient matrix; and the matrix for the variance resulting from unshared environmental effects is specified by the identity matrix Id marker . ApplyinR, is modeled as a linear combination of fixed effects  and random effects . The genotype scores are decomposed into between-family (b) and within-family (w) components, resulting in fixed-effect model \u03b2b = \u03b2w. The QTDT approach estimates both \u03b2b and \u03b2w, and tests whether the within-family parameter \u03b2w is significantly different from 0. QTDT reflects the correlation between SNP genotype and phenotype within families and is robust to population stratification effects [In a candidate region on chromosome 3 identified with some evidence for linkage in the sampled GWAS data and previously reported in GWAS meta-analysis , we comp effects , which c effects .With the first set of 988 GWAS SNPs, evidence for linkage with SBP on chromosome 3 using combined pedigree data was mainly found in 4 regions: 5 to 12 Mb, 47 to 59 Mb, 89 to 115 Mb, and 165 to 175 Mb . Among the 58,651 variants in this region, 20,211 are common (MAF \u22655%), 10,508 are low-frequency (1% to 5% MAF), and 27,932 are rare (MAF <1%). We observed that, as expected, the MG association analysis tended to provide larger signals than the QTDT approach . In family-based association analysis of sequence variants, however, we observed stronger association signals at rare variants compared to common variants. As is typical in fine-mapping studies, we examined association with sequence variants under linkage peaks obtained from a chromosome-wide scan. Depending on the inherent power in a study, it may be advisable to establish a fairly liberal criterion for identification of linkage regions. Although the linkage strategy we used reduces the multiple testing burden in phase 2, it may miss regions of interest that would have been detected by a GWAS association analysis. For purposes of comparison, albeit in a single data set, we examined the results from a complete, dense GWAS scan of chromosome 3 that used mixed models to account for the pedigree structure [p <10\u22125; the chromosome-wide maxima near 150 Mb agreed quite well. Our linkage scan also identified regions at other locations, including those near 10, 27, and 100 Mb, that would have required more liberal GWAS criteria for identification.The main purpose of the proposed multiphase design is to first identify interesting genomic regions for a complex quantitative trait, and then to fine-map those regions in follow-up studies, reducing both the number of tests for association conducted at null variants and the computational processing time. With randomly sampled common GWAS SNP data for large Mexican American pedigrees from SAFS, we identified 4 linkage regions for SBP on chromosome 3. Especially for 2-point linkage, high-density SNP analysis is desirable. In linkage analysis in an identified region, we observed higher LOD scores using imputed sequence data compared to GWAS SNP data, particularly for common variants (Figure tructure . We obseThe authors declare that they have no competing interests.ZC designed the overall study, conducted the sequence variant analyses, and drafted the manuscript. KRT conducted the chromosome-wide linkage scan using sampled GWAS markers. SBB conceived the study, participated in its design and conduct, and helped to revise the manuscript. All authors read and approved the final manuscript."}
+{"text": "Pinctada fucata (P. fucata), is one of the marine bivalves that is predominantly cultured for pearl production. To obtain more genetic information for breeding purposes, we constructed a high-density linkage map of P. fucata and identified quantitative trait loci (QTL) for growth-related traits. One F1 family, which included the two parents, 48 largest progeny and 50 smallest progeny, was sampled to construct a linkage map using restriction site-associated DNA sequencing (RAD-Seq). With low coverage data, 1956.53 million clean reads and 86,342 candidate RAD loci were generated. A total of 1373 segregating SNPs were used to construct a sex-average linkage map. This spanned 1091.81 centimorgans (cM), with 14 linkage groups and an average marker interval of 1.41 cM. The genetic linkage map coverage, Coa, was 97.24%. Thirty-nine QTL-peak loci, for seven growth-related traits, were identified using the single-marker analysis, nonparametric mapping Kruskal-Wallis (KW) test. Parameters included three for shell height, six for shell length, five for shell width, four for hinge length, 11 for total weight, eight for soft tissue weight and two for shell weight. The QTL peak loci for shell height, shell length and shell weight were all located in linkage group 6. The genotype frequencies of most QTL peak loci showed significant differences between the large subpopulation and the small subpopulation (P<0.05). These results highlight the effectiveness of RAD-Seq as a tool for generation of QTL-targeted and genome-wide marker data in the non-model animal, P. fucata, and its possible utility in marker-assisted selection (MAS).The pearl oyster,  Pinctada fucata (P. fucata), is a marine bivalve found in China and Japan, where it is predominantly cultured for pearl production. In China, the regions where it is cultured are mainly distributed along three coastal provinces  of the South China Sea. Artificial breeding may no longer be commercially sustainable due to heavy mortality and the decline of pearl quality observed in established breeding programs P. fucata. Solutions to genetic degeneration include the use of conventional breeding methods, selection, crossbreeding and hybridization to improve the quality of aquaculture strains P. fucata, breeding with the aim of high quality pearl production, for example the selection of pearl color, weight P. fucata is marker-assisted selection (MAS). Molecular genetic polymorphisms can be integrated with artificial selection of phenotypes by applying MAS to achieve substantial increases in the efficiency of artificial selection Crassostrea gigasArgopecten irradiansPinctada maximaPinctada martensiiCrassostrea gigas (disease resistance) Argopecten irradians (growth) Nodipecten subnodosus (orange shell color) P. fucata. Therefore, it is important to identify new SNPs and QTLs that are associated with growth-related traits, which may contribute to the improvement of pearl production in P. fucata.The pearl oyster, Gasterosteus aculeatus. Markers at the Eda locus, linked to plate loss, were identified in linkage group (LG) IV GnathopogonP. fucata for large-scale SNP discovery.The application of restriction fragment length polymorphism, randomly amplified polymorphic DNA, amplified fragment length polymorphism, simple sequence repeats, SNPs and expressed sequence tags has changed the approach to genetic research in the field of aquaculture and has allowed the construction of genetic linkage maps Methods that are frequently used for QTL mapping include the Kruskal\u2013Wallis (KW) non-parametric test, interval mapping (IM) and composite interval mapping P. fucata genome using the RAD-seq method, with particular focus on high density genetic linkage map construction and growth-related QTL detection. In this analysis, we built a high-density sex-average linkage map of P. fucata using 1373 SNPs identified from RAD-seq. We also identified 39 QTL-peak loci for growth-related traits and found that the genotype frequencies were significantly different between the large and small subpopulations (P<0.05). These identified QTLs may be valuable resources for MAS in P. fucata.The aim of this study was to identify a large number of SNP markers from the P. fucata from sample sites because they were not collected from protected areas of land. The pearl oyster P. fucata, is not an endangered or protected species.All animal work has been conducted according to relevant national and international guidelines. No specific permissions are required to work with invertebrates in China. Similarly, no specific permissions were required for the collection of P. fucata was carried out at the Marine Biology Research Station, Daya Bay, Chinese Academy of Sciences, Shenzhen, China. A female and male adult P. fucata were obtained from a wild population in Dapeng Bay , Shenzhen, China. These were used as founders of an F1 intercross. The hinge length (HL) of 1047 6-month-old F1 progeny of the family was measured. Seventy four of the largest (HL >33 mm) and 74 of the smallest oysters (HL <19.7 mm) were sampled. Each oyster was numbered and shell height (SH), shell length (SL), shell width (SW), HL, total weight (Wt), soft tissue weight (Wf), and shell weight (Ws) were measured. The adductor muscle or the whole soft tissue was also sampled and preserved in 90% ethanol for DNA extraction. A total of 100 individuals, which included the two parents, the 48 largest individuals (defined as the large subpopulation) and the 50 smallest individuals  were then used for linkage and QTL mapping.The breeding program of 260/280, and DNA quality was analyzed using agarose gel electrophoresis. The RAD library construction procedure closely followed the methodology described by Baird et alEcoRI . The reactions were heat inactivated at 65\u00b0C for 20 min. Modified Illumina adapters (P1 adapters) that contained barcode sequences were added to the samples, along with 1 \u00b5L of 100 mM rATP (Promega), 1 \u00b5L 10\u00d7 EcoRI buffer, 0.5 \u00b5L (1000 U) T4 DNA Ligase (New England Biolabs) and 5 \u00b5L H2O. The reactions were incubated at room temperature for 20 min and then heat inactivated at 65\u00b0C for 20 min. The ligation products were then combined in appropriate multiplex pools. For each library pool, digested DNA was sheared to an average length of 500 bp. Each of the 22 library samples were then separated by electrophoresis through a 1.3% agarose gel and fragments in the 300\u2013700 bp range were isolated using a MinElute Gel Extraction kit (Qiagen). The Quick Blunting Kit (New England Biolabs) was used to polish the ends of the DNA. The samples were re-purified and 15 U of Exo-Klenow (Enzymatics) were used to generate adenine overhangs on the 3\u2032 end of the DNA, at 37\u00b0C. After subsequent purification, 1 \u00b5L of 10 \u00b5M P2 adapters  were ligated to the DNA products. Samples were re-purified and eluted in 50 \u00b5L. Five microliters of this product were used in PCR amplification with 50 \u00b5L of Phusion Master Mix (New England Biolabs), 5 \u00b5L of 10 \u00b5M modified Solexa Amplification primer mix (Illumina) and 40 \u00b5L of H2O. Phusion PCR settings followed product guidelines for a total of 16\u201318 cycles. Samples were gel purified, to excise the DNA fragments (300\u2013700 bp), and dissolved in Elution Buffer. After quality and quantity tests on the PCR products, the obtained RAD libraries were sequenced on an Illumina Hiseq 2000 platform. For each parent, sequencing data size was set to obtain twice as much of the genome. For each progeny, sequencing data size was set to obtain as much as half of the genome. Raw Illumina reads were filtered to eliminate adapter sequences. The reads with a low quality (Q\u22645 E) base number of greater than half of the total number of nucleotides and reads that did not show the enzyme cleaved sequence, \u201cAATTC\u201d, in the first five bases were eliminated. Sequences were sorted to individuals in accordance with the barcode sequences. They were then used for clustering analysis (a minimum stack depth of two) and genotyping by the software stacks Genomic DNA of the two parents and the 98 F1 progeny was extracted from the preserved samples using E.Z.N.A mollusc DNA Kit , in accordance with the manufacturer\u2019s instructions. The concentration of extracted DNA was estimated with a spectrophotometer, using OD2 test, P<0.05) were excluded. All of the marker information was input into JoinMap 4.0 The SNP markers were categorized into \u201clm\u00d7ll\u201d, \u201cnn\u00d7np\u201d and \u201chk\u00d7hk\u201d types, which represent heterozygosity in the maternal, paternal and both parents, respectively. Markers that were genotyped in less than 85% of progeny and markers that showed significantly distorted segregation  and a degree of correlation between loci and traits that was equal to or greater than four asterisks were used to identify QTL-peak loci Pearson correlation coefficients were used to determine the linear correlation between pairs of growth-related traits, using software SPSS version 16.0 . The mean size of growth-related traits was compared in the large and small subpopulation using the Student\u2019s t-test in SPSS version 16.0. The significance level was set to 0.05. The QTL mapping was performed using MapQTL 5 software P. fucata. Hence, the RAD-seq analysis was employed for DNA polymorphism detection and genotyping. The Illumina Hiseq 2000 sequencing yielded a total of 84.36 gigabases (Gb) of clean bases from the 100 samples. The average number of clean bases per individual was 0.84 Gb. Sample 9 was the individual with least number of clean bases (0.45 Gb) and sample 2 was the individual with maximum number of clean bases (1.93 Gb) (Table S1). A total of 1956.53 million clean reads was generated and 86,342 candidate RAD loci were identified. After 84,336 candidate RAD loci were discarded , 2006 \u201clm\u00d7ll\u201d, \u201cnn\u00d7np\u201d and \u201chk\u00d7hk\u201d type of SNP markers, which were scored in enough individuals, were retained for further analysis. Amongst the 2006 markers, 625 showed deviation from Mendelian segregation (P<0.05). A total of 1381 SNP markers, which included 562 \u201clm\u00d7ll\u201d type markers, 611 \u201cnn\u00d7np\u201d type markers and 208 \u201chk\u00d7hk\u201d type markers, were retained for linkage map construction.There are few known SNP markers available for the linkage mapping of , Figure S1).A total of 562 \u201clm\u00d7ll\u201d type SNP markers were used for the female linkage map construction. After four similarity markers were discarded, 558 markers were located on the female linkage map. Fourteen linkage groups were identified and the number of loci per linkage group varied from nine to 94, with a mean of 39.8. The female linkage map spanned 1024.31 cM, with an average spacing of 3.19 cM and a Coa of 93.51% .For the construction of the male linkage map, 611 \u201cnn\u00d7np\u201d type SNP markers were used and four similarity markers were excluded. The remaining 607 markers were all located on the linkage map. Seventeen linkage groups were identified, which included two triplets and one doublet. The number of loci per linkage group varied from two to 158, with a mean of 35.7. The male linkage map covered 928.00 cM, with an average spacing of 4.03 cM and a Coa of 90.67% (Table S2) were located on the linkage map. Fourteen linkage groups were formed and the number of markers per linkage group varied from 18 to 265, with a mean of 98. The sex-average linkage map covered 1091.81 cM, with an average spacing of 1.41 cM. The length of the linkage groups ranged from 63.22 cM to 115.79 cM, with an average of 77.98 cM.  of the female map and 23 markers of LG 3 (24 markers) of the male map. The SNP markers in LG 3 of the sex-average linkage map also presented a similar order of markers as in the female and male linkage maps.P<0.01), and the mean value of each growth-related trait in the large subpopulation was significantly larger than that in the small subpopulation (P<0.0001)  were 10.913, 12.579, 11.082, 10.885, 7.898, 8.115 and 11.303, respectively. A total of 39 QTL-peak loci for growth-related traits were identified, which included three for SH, six for SL, five for SW, four for HL, 11 for Wt, eight for Wf and two for Ws  between the large subpopulation and the small subpopulation  showed significant differences  P. fucata and can be used as the basis for a better understanding of genetic structure in shellfish species.The sex-average linkage map has fourteen linkage groups, which is consistent with the number of haploid chromosomes of P. fucata was 1122.7 cM. This is shorter than the expected genome length of a consensus map for Pinctada martensii  Chlamys farreri  P. fucata, in this study, and Pinctada maximaP. fucata and this research has provided foundation materials for the utility of RAD-seq in non-model animals.The estimated genome length of the integrated linkage map for Ostrea edulis and Crassostrea gigasP. fucata may be the same as that observed in Crassostrea gigas, in which Mendelian segregation occurs during early developmental stages but is distorted during later development. These distortions are largely attributable to selection against recessive deleterious mutations of fitness genes that are closely linked to the markers P<0.05) were used for the linkage map construction of P. fucata.In this study, 2006 SNPs were scored in enough individuals and belongs to \u201clm\u00d7ll\u201d, \u201cnn\u00d7np\u201d and \u201chk\u00d7hk\u201d type of SNP markers. Amongst the 2006 markers, 625 showed deviation from Mendelian segregation. One of the problems for linkage mapping is marker-segregation distortion, which has also been observed in P. fucata. The use of this method will provide high significance thresholds when the phenotype distribution is skewed P. fucata family with 1047 progeny were produced, and individuals at the extremes of the phenotype distribution  were selected for QTL mapping. The growth-related traits of the selected individuals deviated from a normal distribution, which meant that the permutation test of the IM method was not justified and the LOD value of significance thresholds was too large. The use of the IM method in this analysis failed to identify any regions that reached genome-wide significance. The KW nonparametric test for QTL detection does not assume a normal distribution of quantitative traits Nodipecten subnodosus) Rubus idaeus) Pinctada fucata when a reference genome is available.The IM method from the MapQTL5 software P. fucata are of particular interest to researchers due to their correlations with pearl production. In this study, 39 QTL-peak loci for growth-related traits were identified and most of the QTL-peak loci were distributed on LG 6. Many QTL-peak loci, such as locus 423886, locus 288691 and locus 99925, were also found to be consensus QTLs for growth-related traits. The consensus QTLs may correspond to distinct, closely linked QTLs or one QTL that acts upon several quantitative characters involved in the same metabolic pathway P. fucata highlight their further application for MAS.Although QTL analyses have been carried out for economically important traits in over 20 aquaculture species, most QTLs were only mapped in large spaces between markers P<0.0001) and the genotype frequencies of most QTL-peak loci showed significant differences (P<0.05) between the two subpopulations. For example, the genotype frequencies at locus 423886 and locus 466758 showed significant differences between the two subpopulations. The frequency of the \u201cnp\u201d genotype of locus 423886, plus \u201chk\u201d and \u201ckk\u201d of locus 466758, were higher in the large subpopulation than in the small subpopulation. That may be caused by the accumulation of genotype frequency at marker-trait association loci during the selection of extreme individuals. These identified marker-trait association loci may be valuable resources for molecular selection breeding in P. fucata.Marker-trait association analysis is an alternative approach to QTL mapping that we used to identify QTLs for growth P. fucata. The RAD-seq method was successfully utilized for the identification of a large number of SNPs and the construction of a high-density genetic linkage map of P. fucata. The choice of extreme phenotype individuals from a large full-sib P. fucata family as the mapping population was able to identify QTLs by the KW non-parametric test. Many QTL-peak loci were found to be consensus loci for growth-related traits and were located at similar regions of the linkage groups. Significant differences in genotype frequency, for most of the QTL-peak loci, were also observed between the large and the small subpopulations. The choice of extreme phenotype individuals from a large full-sib P. fucata family as the mapping population could yield QTLs that concentrate distribution across the linkage groups. The location of QTLs at similar regions of the linkage groups and QTLs that act upon several quantitative characters highlight their further application for MAS in P. fucata.The identification of high growth-related traits that may contribute to the improvement of pearl production is the main goal of genetic breeding programs of Figure S1The female linkage map for Pinctada fucata. The map is composed of 14 linkage groups, with 558 markers, and spans 1024.3 cM. The map distances (in cM) are indicated on the left of the chromosomes and the names of the SNP markers are shown on the right.(DOC)Click here for additional data file.Figure S2The male linkage map for Pinctada fucata. The map is composed of 17 linkage groups, with 607 markers, and spans 928.0 cM. The map distances (in cM) are indicated on the left of the chromosomes and the names of the SNP markers are shown on the right.(DOC)Click here for additional data file.Table S1Statistics of sample information. Detailed statistics on 100 samples used as the mapping family. Q20-rate means the percentage of bases with a quality value \u226520 and Q30-rate means the percentage of bases with quality value \u226530.(DOC)Click here for additional data file.Table S2Markers used for linkage and QTL mapping. The information of 1373 SNP markers includes marker name, SNP position, major allele, minor allele and the consensus sequences those contained the SNPs.(XLS)Click here for additional data file."}
+{"text": "The authors and editors retract this publication following an investigation into concerns around the data presented in the figures that were brought to the editors\u2019 attention. The text below has been agreed by the editors and all authors, except for G. Corte (deceased).There were concerns raised about all of the figures. Serious issues were detected in figures 6, 7 and supplementary figures S1 and S3, including duplicated gel bands within and between these figures.An institutional inquiry has been undertaken at the IRCCS AOU San Martino-IST in Genova (formerly IST) where Dr Gherzi undertook this research and a number of the figure anomalies have been verified by the institutional committee. Dr Gherzi would like to note specifically that his co-authors  were not involved in the preparation of these figures. An explanation of inadvertent error was given for some of the issues identified, while for two issues, a satisfactory explanation could not be provided. Dr Gherzi assumes full sole responsibility for the errors leading to these anomalies and apologizes.The authors maintain that the conclusions of the published paper remain valid. However, the detected errors in the published figures undermine editorial confidence in the results presented in the study. Dr Gherzi would like to note that he has sent replicate autoradiograms for each experiment to the institutional inquiring committee. However, the editors, having seen this data, could not confidently validate what was provided. Given the serious concerns that remain about the validity of some of the published data presented in this paper, the authors and editors are issuing a retraction. All authors have agreed to the retraction of this paper  and deeply regret any inconvenience this publication has caused for others."}
+{"text": "The accessibility of high-throughput genotyping technologies has contributed greatly to the development of genomic resources in non-model organisms. High-density genotyping arrays have only recently been developed for some economically important species such as conifers. The potential for using genomic technologies in association mapping and breeding depends largely on the genome wide patterns of diversity and linkage disequilibrium in current breeding populations. This study aims to deepen our knowledge regarding these issues in maritime pine, the first species used for reforestation in south western Europe.He) and long distance linkage disequilibrium (LD) along the chromosomes. We found no particular pattern in the empirical variogram of He across the 12 linkage groups and, as expected for an outcrossing species with large effective population size, we observed an almost complete lack of long distance LD.Using a new map merging algorithm, we first established a 1,712 cM composite linkage map  by bringing together three already available genetic maps. Using rigorous statistical testing based on kernel density estimation and resampling we identified cold and hot spots of recombination. In parallel, 186 unrelated trees of a mass-selected population were genotyped using a 12k-SNP array. A total of 2,600 informative SNPs allowed to describe historical recombination, genetic diversity and genetic structure of this recently domesticated breeding pool that forms the basis of much of the current and future breeding of this species. We observe very low levels of population genetic structure and find no evidence that artificial selection has caused a reduction in genetic diversity. By combining these two pieces of information, we provided the map position of 1,671 SNPs corresponding to 1,192 different loci. This made it possible to analyze the spatial pattern of genetic diversity (These results are a stepping stone for the development of strategies for studies in population genomics, association mapping and genomic prediction in this economical and ecologically important forest tree species. They  forests . Genetic forests  and anim forests  have all forests .2) and population recombination rate (r2 = 1/(4Nec +1)) has obvious consequences for genomic prediction, because both the training population size and marker density vary with Ne, the effective population size [Knowledge about linkage disequilibrium (LD) measured by the squared correlation between two loci is important for applications of molecular markers in association mapping and genomic prediction. The decay of LD over physical and genetic distance determines the resolution and density of the markers required for association mapping ,11. A foion size ,17.Cryptomeria japonica genome, suggesting that recombination rate may vary according to the nature  of DNA, as shown in angiosperms [Pinus taeda[Previous studies of short-distance  LD in conifers, including maritime pine ,19, haveiosperms  and gymniosperms . In the iosperms  discovernus taeda reportedThe main objective of this paper was to describe LD pattern, level and structure of genetic diversity across the maritime pine genome. The result may provide baseline information for future genetic studies  in this economically important conifer. To this end, we first establish a high-density genetic linkage map by merging three existing SNP-based maps  using maWe used the following strategy to integrate the three linkage maps, G2F, G2M and F2, into a single composite map. First, intermediate composite maps were established for G2F-F2 and G2M-F2 because there were few markers common to the G2F and G2M maps suitable for anchoring , whereas 198 SNPs were common to F2 and G2F maps and 240 SNPs were common to F2 and G2M maps (see ). We theK parameter) for intermediate composite maps, but that RMSEs were similar for the two programs after the final step of map merging , for all LGs, between the composite maps constructed with LPmerge and MergeMap. Finally, correlations between marker positions on parental maps  and on the final composite map constructed with LPmerge were high , indicating that the positions of the markers on the composite map were consistent with those on the corresponding source maps.We compared the results generated by LPmerge and MergeMap methods, by carrying out Wilcoxon signed rank tests on two metrics: the linkage group length of the composite map, and the root mean square error (RMSE) calculated from the difference in map position (in cM), between each component map and the resulting composite map. Three hypotheses were tested: i) the map lengths obtained for the intermediate  composite maps do not differ significantly between LPmerge and MergeMap; ii) The difference in RMSE between component (or intermediate composite) maps and the resulting intermediate  composite map does not differ significantly between LPmerge and MergeMap, and iii) the RMSE for each component (or intermediate composite) map does not differ significantly from the intermediate  composite map constructed with LPmerge, and similarly for MergeMap. MergeMap systematically yielded longer maps than LPmerge, for both intermediate and final composite maps  was performed on the composite map, to determine whether genes were evenly distributed between maritime pine chromosomes. With twice as many markers than in our first investigation [P\u2009=\u20090.65). In terms of the distribution of markers on individual chromosomes, we found that a density of at least 10 markers per bandwidth (P\u2009=\u20093.3\u2009\u00d7\u200910-63) was required for the identification of a recombination cold spot, whereas a density of at most three markers (P\u2009=\u20093.2\u2009\u00d7\u200910-63) characterized a hot spot for recombination. Given these upper and lower limits, and considering the stringent confidence interval defined for kernel density function, we identified 13 significant clusters of markers (in 8 LGs), corresponding to recombination cold spots  and is also provided in Additional file The mean call rate  was 92% for the FGB population. Two poorly performing samples were identified by plotting the sample call rate against the 10%\u2009GeneCall score. Three pairs of trees were found to display identical genotypic information for the 2,600 SNPs and were therefore considered mislabeled in the tree archive  and, probably, less polymorphic, due to the stringent cutoffs used: i) MAF\u226533% and coverage\u226510\u00d7, to prevent the selection of SNPs present at such low frequencies that they are likely to be the product of sequencing error, ii) ADT score \u2265 0.75, to minimize the variability of the flanking region surrounding the targeted SNP, thereby increasing the likelihood of a successful Illumina Infinium assay. In addition, the MAF spectrum is likely to be shifted upward, with an underrepresentation of rare alleles not captured due to the small size of the sample used to prepare the cDNA libraries. The possibility of such a bias should be borne in mind when making further evolutionary inferences concerning the demographic and selective history of maritime pine populations.Significant departure from Hardy-Weinberg equilibrium was detected for 12 SNPs from the 2,600 polymorphic markers in the FGB population . After Bonferroni correction for multiple tests  none of these SNPs yielded a value significantly different from the expected value. We can therefore consider that the percentages of each of the three SNP genotypes remained constant in what can be considered a large population, with random mating, without mutation, migration or natural selection. The minor allele frequency (MAF) distribution of these 2,600 SNPs is shown in Figure\u00a02 = 0.17, p\u2009=\u20090.007) between genetic PC1 and the major axis of geographic variation (mostly latitude), with some evidence of PC2 being associated with the second axis (mostly longitude)    calculated for the 2,600 SNPs was 0.391 (SD\u2009=\u20090.127), while that for the 1,421 SNPs corresponding to mapped contigs was 0.434, (SD = 0.067). These are very high estimates given the biallelic nature of these markers . We used the mapped markers to determine whether genetic diversity was equally distributed between the LGs  between He values was observed. Tukey\u2019s HSD test showed that LGs could be classified into three groups, with lower , medium  and higher  levels of diversity.The mean value of Nei\u2019s diversity index , the observed pattern of short-distance LD (not shown) was not consistent with trends typically observed in conifers [At least two SNPs were available in 248 EST-contigs for investigation of the pattern of physical LD. We considered SNPs with a MAF>5%, resulting in the retention of 714 pairs for the analysis. However, given the biased procedure used to select SNPs conifers  based on2) showed that LD decreased rapidly over very short genetic distances for all chromosomes  were not due to inaccurate map position resulting from the construction of the composite linkage map, we directly checked the map position of these pairs in the components maps. From these 380 pairs, 238 originated from the same component map, while 142 were from different component maps. From these 238 pairs, the genetic distance in the component map was equal to 0 cM for 102 pairs and comprised between 0 and 1 cM for 66 pairs, indicating that their position in the r2 plot was probably unreliable and therefore could not be used to infer long distance LD. An extreme case is provided for two outliers markers (F51TW9001DHGV3 and CT583376) in LG3 placed 23 cM apart in the composite map, while they completely co-segregated in the component map (G2M). Thus, only 70 pairs (i.e. 238\u2013102\u201366) were left to construct the distribution of long distance  LD. As rare allele frequency can influence LD, this distribution . Each inter-chromosomal LD value was tested against the upper bound of this null distribution (significant if r2 > 0.32 at the 5% level). Given the number of tests performed, Bonferroni correction was applied to this upper bound  LD was examined for the first time in this species, over the 12 chromosomes, on the basis of SNP markers localized on the composite linkage map and their genotypic profiles in an unstructured population. The distribution of the squared correlation coefficient for allelic frequencies  markers ,41. The et al. were thel.[et al. subsequel.[et al.,51. Endel.[et al. discoverl.[et al., DAGGER,l.[et al.. By usin801 loci , 1,898cMnus taeda. We thennus taeda,53.e) for a population resulting from mass selection in natural forests, with an estimated selection intensity of about 1.5 \u00d7 10-5[We presented a genome-wide map of genetic diversity . Such patterns would have been indicative of decreases in diversity associated with loci underlying the variation of the target traits. However, given the rapid decay of LD in this species (within a few hundred bp on average), the marker density used was probably too low to capture any localized decline in heterozygosity, if any occurred around selected loci.These results contrast with the large reduction of genetic variability observed for the selected traits  between The set of 2,600 SNP markers developed in this study will be used to assess genetic diversity in subsequent generations of the maritime pine breeding program. The maintenance of genetic diversity is not only essential to guarantee the adaptation of future improved varieties to ongoing climatic change [2 were obtained only for physically linked polymorphisms, i.e. SNPs belonging to the same gene. No significant LD was found over larger distances. These results are consistent with population genetics theory for such an undomesticated, outcrossing species, and can be attributed principally to the large effective sizes of the unstructured populations found in most conifers . Samplele et al. for PinuPinus taeda[Our findings suggest that the initial mass selection used to form the base population of the maritime pine breeding program was not only successful in terms of the initiation of a program to develop improved varieties , but alsnus taeda,64. Thennus taeda,66, whilGiven the lack of LD in this population and lack of associations between markers and phenotypes, predictions based on SNP markers for selection would likely have very low reliability. In several simulation studies on domestic animal and trees, LD showed a significant effect on reliability of predictions from genomic prediction models ,16. For We established a 1,712 cM linkage map of maritime pine with 1,838 SNP markers using for the first time a new map merging algorithm that integrates linkage maps from separate populations without any recourse to original genotypic data. We found clear cold spots of recombination consistent with the centromeric and telomeric regions of metacentric chromosomes . We thenAt the dawn of a new paradigm in forest tree breeding -71, nameet al.[The two mapping populations (G2 and F2) for which SNP-based linkage maps were merged in this study were described in : G2 desiet al. construcet al., to seleet al... In total, 9,279 SNPs  were individually inspected with Genome Studio software, using a GenCall score cutoff of 0.15 (according to Illumina\u2019s recommendations) to detect failed SNPs. Loci for which two or three clusters (depending on the type of marker segregation) were identified without ambiguity were considered to be polymorphic markers. SNP clusters were modified manually, to refine cluster positions, when necessary. SNPs and surrounding sequences were submitted to dbSNP , as described in . In totahttp://cran.r-project.org/web/packages/LPmerge/, and MergeMap (http://www.mergemap.org/), which has been used in several barley mapping projects [K was varied from 1 to 8, and the composite map with the lowest RMSE was selected. For both software packages, as few markers were common to G2F and G2M, we first generated two intermediate composite maps (\u201cF2+G2F\u201d and \u201cF2+G2M\u201d). We then merged intermediate maps into a final composite map. The merging of the three maps in a single step yielded the same marker order in the composite map , but we present the two-step procedure here because this approach made it possible to compare LPmerge and MergeMap on three datasets , making it possible to draw more general conclusions.We compared two different software packages to generate a composite map from three existing SNP-based linkage maps  of mariprojects ,51. To c2 tests (P<0.05). The expected number of genes for each LG was obtained by multiplying the ratio \u201csize of LG/total genome length\u201d by the total number of mapped markers. We also analyzed the distribution of markers along the chromosomes, by using a kernel density estimation to calculate optimal window size (bandwidth) for dividing the genome into blocks, in which we counted the number of genes. Kernel density estimation is a non-parametric technique for density estimation, in which a known density function (here a Gaussian function) is averaged across the observed data points to create a smooth approximation. The smoothness of the density approximation depends on the bandwidth. In our case, we used a fixed and robust bandwidth estimator [et al.[2-test) the observed distribution of the number of markers per bandwidth with that expected under a Poisson distribution. A lower bound threshold, defining a cold spot of recombination (i.e. a cluster of markers on the linkage map) was determined when the observed number of markers was greater than the expected value, while the results of the chi2-test were significant. Similarly, to define a hot spot of recombination, an upper bound threshold was determined when the observed number of markers was lower than expected, while the results of the chi2-test were significant. Finally, we compared the position of the confidence interval of the kernel density estimator with these lower and upper bounds, to identify significant hot and cold spots, respectively.We investigated whether the mapped genes were evenly distributed between linkage groups (LG), by comparing the observed and expected numbers of genes per linkage group in chistimator , based or [et al.. Bandwidr [et al., we estir [et al.,78. For K), from 1 to 10. The correlated allele frequency model with admixture was used, with burn-in and run-length periods of 2.5x105 iterations. We used the mean likelihood L(K) and Evanno\u2019s delta K criterion [K could be identified, as expected when discrete populations are present in the data.Genetic structure and cryptic relatedness within the FGB population were assessed in three ways. First, we assessed the patterns of pairwise relatedness, calculated from the genotype matrix as described in . Second,riterion  values oe, Nei\u2019s index of genetic diversity [http://genepop.curtin.edu.au/. Genetic diversity parameters were finally retrieved from the output of GenePop, using a PerlScript. As these three parameters were highly correlated, we considered only He.A SNP diversity map was superimposed on the composite linkage map. We used the FGB population to test departure from Hardy-Weinberg equilibrium and to estimate three genetic diversity parameters for each SNP: minor allele frequency (MAF), observed heterozygosity (Ho) and expected heterozygosity  and the location at which it is measured . The covariance calculated is equal to half the variance of the differences in the value of a metric (Z) between all pairs of points (i and j) separated by a given distance (h). This approach is often referred to as semivariance analysis in geostatistical studies  between a variable of interest  and Nh is the number of paired data (SNP markers) at a distance of h (1 to 10 cM) or less. We calculated variance with a robust estimator, to avoid the influence of outliers, as described in [e values associated with each SNP marker were randomized with respect to chromosomal position. One thousand permuted data sets were generated and the probability of finding a value higher than the observed value for a distance class was calculated from the distribution of the permuted data. We then determined whether diversity was equally distributed between LGs . A simple one-way ANOVA was performed, followed by a Tukey\u2019s HSD test for multiple comparisons of means. This test compares the difference between the He values of each pair of LGs, with appropriate adjustment for multiple testing.where ribed in ,91. We f2[LD between pairs of loci was estimated by the squared allele frequency correlation r2, based o2. LD was 2 values were plotted against the genetic distance between the two loci (starting at 0 cM for SNPs from the same contig). We then built a null model to test for the presence of inter-chromosomal LD, by retaining only genetically linked pairs (i.e. corresponding to two different contigs) with critical values of r2 > 0.1 [We investigated the distribution of intra-chromosomal LD over physical and genetic distances. For the estimation of short-distance  LD, SNPs from the same contig (discarded for linkage map construction) were reintroduced into the LD analysis and placed at the same map position as the marker initially selected for linkage map generation. Pairwise rr2 > 0.1 .2, based on pairs of SNP belonging to the same contig, with MAF>5%. Of the 4,911 contigs studied, 248 contained two or more SNPs and were retained for the intragene LD analysis. The extent of LD was estimated by nonlinear regression analysis on the basis of intragene r2 values [2 between pairs of adjacent sites (E(r2)) were estimated with the formula:At the intragene level, LD was estimated by the squared allele frequency correlation r2 values . The exper where Ne is the effective population size and r is the recombination rate per site and per generation) and n is the sample size. We carried out nonlinear regression (nls function) with R software x, replacing C with C \u00d7 distance (in bp) between pairs of sites, to fit this formula to our data.which is valid under drift recombination equilibrium and low mutation rate and can be adjusted for sample size . In thisSupporting data are available as additional files.The authors declare that they have no competing interests.EC, JE and CP constructed the composite linkage map; EC, LB and CP sampled trees from the Aquitaine breeding population and analyzed the SNP data; JBL analyzed the pan-genomic distribution of recombination and genetic diversity, EM, JV, MB and CP carried out the population genetic analysis, IL and FE performed the bioinformatic analyses, FI brought its expertise in terms of implementation of genomic selection in pine breeding. CP wrote the manuscript with input from all authors, obtained funding for the research, conceived, designed and coordinated the study. All authors read and approved the final manuscript.Alignments of the composite linkage maps obtained with LPmerge (LPM on the left) and MergeMap (MM on the right) software.Click here for fileComparison between LPmerge and MergeMap for composite map construction. The first table provides the two metrics for statistical testing, i.e. linkage group length of the composite map and root mean square error (RMSE), whereas the second table provides the result of the test. Two intermediate composite maps (G2F_F2 and G2M_F2) were constructed before the production of the final composite map (G2F_F2-G2M_F2).Click here for filea) Pairwise kinship relationships between 192 individuals of the FGB population, showing 3 pairs of trees with identical genotypic information over the 2,600 SNPs, which were therefore considered to be mislabeled in the tree archive, b) Pairwise kinship relationships between the 186 individuals of the FGB population, i.e. excluding the three abovementioned pairs.Click here for fileList of SNP markers with dbSNP accession numbers, corresponding contig ID in PineContig_v2, genetic parameters in the first-generation breeding population, and linkage group assignment on the component maps.Click here for filea) Plot of genetic PC1 and PC2 and their relationship to the two geographic components, b) biplot of PCA against geographic coordinates, c) relationship between the first genetic and geographic PC (averaged per location), d) relationship between the second genetic and geographic PC (averaged per location), e) genetic distance  as a function of geographic distance.Click here for fileClustering of the 186 G0 trees of the FGB population using the Structure software. Distribution of Evanno\u2019s delta K values (A) and example of barplots obtained with numbers of groups K varying from 2 to 5 (A).Click here for fileDistribution of genetic diversity  along the 12 linkage groups of the maritime pine composite map. Blue: one SNP in the contig, He value for the SNP; red: two SNPs in the same contig, He value for the second SNP; Green: three SNPs in the same contig, He value for the third SNP; Purple: four SNPs in the same contig, He value for the fourth SNP.Click here for filePlot of linkage disequilibrium, measured as the squared correlation coefficient of allele frequencies (r2), against genetic map distance (cM) between all marker pairs in each of the 12 linkage groups (LG) of the maritime pine genome. r2 was determined with the GGT 2.0 program, from the polymorphism data for 186 unrelated trees of the Aquitaine population. The 0.1 critical level of r2 was determined after Robbins et al. (2011). J Exp Bot, 62:1831\u20131845.Click here for fileDistribution of long distance intra-chromosomal linkage disequilibrium (LD) as estimated by r2. This distribution was used as a null model to test the significance of inter-chromosomal LD potentially due epistatic selection.Click here for fileList of 65 pairs of markers with MAF>20% and associated linkage disequilibrium values (r2).Click here for fileEstimates of effective population sizes.Click here for fileDescription of the three component maps from Chancerel et al. (2013).Click here for fileGeographic origin of the G0 trees.Click here for fileGenotyping dataset .Click here for fileBandwidth values (cM) obtained by kernel density analysis for the composite linkage map obtained with LPmerge.Click here for file"}
+{"text": "Recently Turkey has witnessed an influx of foreign immigrants. This can have an effect in the outcome of health care. We aimed to investigate the knowledge, attitude and behaviors about health among foreign students that have come to Turkey from different countries for collegiate education in order to show at what level these factors play a role in shaping health ,2.In this survey, 154 international students, 44 girls 108 boys, from 44 different countries studying in 11 different faculties in Erciyes University whose mean age is 21,57\u00b12,88 were evaluated by a questionnaire consisting of five scoops; students use of alcohol and cigarette, knowledge of the use of alcohol and cigarette, eating habits, belief of and attitudes pertaining to healthy living in their countries . QuantitIrrespective of country and faculty 70,86% were aware of the negative effects of alcohol and smoking on health.54,30% of the society is engaged in exercises whereas 29,80% agreed that exercising is not practised. 46% are aware of the dangers of obesity whereas 28.66% view it as a sign of wealth. On the importance of breastfeeding, 75% agreed that breastfeeding is widely practised. 30% agree that in their country some people resist immunization due to religous beliefs. 77% agreed to rituals being widely practised for treatment of diseases. 53,70% agreed that evil eye and witchcraft can be the cause of some diseases. 23% agreed to female genital mutilation (FGM) being practised. 35% agreed that there is stigmatisation of diseases like AIDS, leprosy. 61,58% agreed to the direct buying of drugs from chemists without doctor consultation. 68,4% agreed to the use of herbal treatment. 64,5% agreed to the use of family planning methods.Despite translocation individuals tend to hold unto their beliefs, attitudes and practises as they try to acculturate . As phys"}
+{"text": "This paper describes a method for spreadsheet computations of Fresnel integrals to six significant figures, based on successive improvements of known rational approximations which are accurate to only three figures. Outside the range of validity of the improved approximations, known series expansions are used to obtain the Fresnel integrals to six figures. This author required them to six significant figures for a project in optics which employed spreadsheet software to compute and plot three-dimensional diffraction patterns. It was found that software and algorithms for computing the Fresnel integrals in Fortran and C are readily available  + b(x), where a(x) and b(x) are linear functions of x and \u03b1(x) is chosen so that the oscillations of \u03b5(x) are imitated. By a judicious choice of parameters it was arranged that the second residuals, \u03b5(x) \u2212 \u0394(x) also exhibited a sinusoidal behvior so that the same procedure could be repeated. As the techniques used were empirical, based on trial and error rather than mathematical rigor, no further details will be given. The final results are:The improvement of the rational approximations of  in Ref. . It was f(x) and g(x) given by these equations into C(x) to \u00b1 3.5 \u00d7 10\u22126 for 0.6 \u2264 x \u2264 3.1.The digits appearing in these expressions are significant within the uncertainties stated in C(x) and S(x) were computed for 0.6 < \u00d7 < 3 from x \u2264 0.3, and x \u2265 3, the the Taylor series of The results of Sec. 2 were entered into a spreadsheet program and"}
+{"text": "Labeo rohita (Hamilton), were produced in 2010. Aeromonas hydrophila is a bacterial pathogen causing aeromoniasis in rohu, and is a major problem for carp production worldwide. There is a need to better understand the genetic mechanisms affecting resistance to this disease, and to develop tools that can be used with selective breeding to improve resistance. Here we use a 6\u00a0K SNP array to genotype 21 full-sibling families of L. rohita that were experimentally challenged intra-peritoneally with a virulent strain of A. hydrophila to scan the genome for quantitative trait loci associated with disease resistance.Production of carp dominates world aquaculture. More than 1.1 million tonnes of rohu carp, A. hydrophila. The linkage map consisted of 25 linkage groups, corresponding to the number of haploid chromosomes in L. rohita. Male and female linkage maps were similar in terms of order, coverage  and average interval distances . Forty-one percent of the SNPs were annotated with gene identity using BLAST (cut off E-score of 0.001). Twenty-one SNPs mapping to ten linkage groups showed significant associations with the traits hours of survival and dead or alive (P <0.05 after Bonferroni correction). Of the SNPs showing significant or suggestive associations with the traits, several were homologous to genes of known immune function or were in close linkage to such genes. Genes of interest included heat shock proteins , mucin (5b precursor and 2), lectin (receptor and CD22), tributyltin-binding protein, major histocompatibility loci (I and II), complement protein component c7-1, perforin 1, ubiquitin , proteasome subunit, T-cell antigen receptor and lymphocyte specific protein tyrosine kinase.In all, 3193 SNPs were found to be informative and were used to create a linkage map and to scan for QTL affecting resistance to L. rohita to A. hydrophila.A panel of markers has been identified that will be validated for use with both genomic and marker-assisted selection to improve resistance of  Labeo rohita Hamilton) accounting for around 1.2 million tonnes in 2010 . First we determined which markers and individuals should be excluded from the GWA analysis using the check.marker function in GenABEL. This function was used to exclude individuals or markers with call rate <95%, markers with minor allele frequency <0.24%, individuals with high autosomal heterozygosity (FDR <1%) and individuals with identity by state \u22650.95. Genomic kingship was computed between all pairs of individuals. We performed a pedigree based association analysis where the pedigree is a confounder (where the heritable trait is more similar between close relatives and therefore some degree of association is expected between any genetic marker and any heritable trait). The effect of the confounding pedigree is expected to inflate the resulting null distribution of the chi square test statistic by a constant, lambda. Lambda is a function of the traits heritability and pedigree structure (expressed as a kinship matrix). Two fast tests for genome wide association were applied, Family-based Score Test for Association . A mixed polygenic model of inheritance was applied in order to study association in our genetically homogeneous families where the hours of survival or dead or alive traits (y) were modelled as:A genome-wide association study (GWAS) was performed using GenABEL  and Gen  using tG describes the polygenetic effect , f describes fixed effects  and e describes the random residual effects. The joint distribution of residuals in the pedigree was modelled using a multivariate normal distribution with variance-covariance matrix proportional to the identity matrix. A genomic kingship matrix, generated by calculating the average identity-by-state between individuals in the pedigree (ibs in GenABEL), was used as the relationship matrix for FASTA and GRAMMAS. Both FASTA and GRAMMAS exploit maximum likelihood estimates of the intercept from the polygenic model. Two hundred permutations were used to estimate genome wide significance for the FASTA and GRAMMAS test (downward bias in the estimate of SNP effects are expected).where \u03bc is the intercept, http://pngu.mgh.harvard.edu/purcell/plink/[hours of survival and dead or alive respectively. In the case of QFAM, the module used a permutation procedure to correct for family structure. Association testing was performed within families. Ten thousand permutations per SNP  were applied producing a point-wise estimate of each individual SNPs empirical significance.The QFAM and ASSOC analysis modules in PLINK ll/plink/ were useP\u2009<\u20090.001, P <0.01 and P <0.05 levels after Bonferroni correction based on the number of linkage groups (which was 25 for L. rohita) were noted for all tests.GWAS associations with significance at A. hydrophila resistant versus susceptible rohu individuals was checked for some SNP containing contigs using sequence coverage data and methods collected and described in [A. hydrophila. Unchallenged individuals from the 15 highest and 10 lowest ranking families were selected to create the first generation of the resistant and susceptible lines respectively. RNA pools were prepared from liver, intestine, muscle, kidney, spleen and brain tissue samples from 10 resistant and 10 susceptible line fish for comparison. Quantile-based rank normalisation was used to correct for differences between sequencer flow cells or RNA pools [MA scale plots where M\u2009=\u2009log2R-log2S and A\u2009=\u20090.5*(log2R\u2009+\u2009log2S), R and S being average coverage depth for each contig in the resistant and susceptible line pools respectively.Differential transcript expression in selected ribed in . In brieNA pools . Data weA. hydrophila (Ah 15) was isolated from a field outbreak that occurred on a farm at Puri, Odisha, India . Forty eight rohu juveniles (weighing 80\u2013100\u00a0g) were challenged intra-peritoneally with live A. hydrophila at a dose of 1.5 x 106\u00a0cfu/g of body weight. Tissue samples from liver, gill and spleen were collected from infected fish at different time periods viz., 1, 3, 6, 12, 24, 48, 72\u00a0hours, 7 and 15\u00a0days post-infection, along with three non-infected controls, in triplicate after euthanasia with a heavy dose of MS222. The tissue samples were kept in RNAlater  and stored at -20\u00b0C until RNA extraction. Total RNA was extracted using TRI reagent . A total of 1\u00a0\u03bcg of RNA was treated with DNase I . First strand cDNA was synthesized using M-MLV reverse transcriptase  as per the manufacturer\u2019s instructions. qPCR was performed using FastStart Essential DNA Green Master  in a Light Cycler 96  using perforin specific primers (forward- 5\u2019 GACGCCTACCACAACCT 3\u2019 and reverse 5\u2019 TTTGCCCTCCTAACTGG 3\u2019) designed in Primer Premier Version 5  from the sequence of contig 113696_50. Briefly, 1\u00a0\u03bcl of synthesized cDNA was used as a template in a total reaction mixture of 10\u00a0\u03bcl containing 5\u00a0\u03bcl of 2X Light cycler SYBR green I mix, 0.5\u00a0\u03bcl of primer pairs (5 pmole) and 3\u00a0\u03bcl of H2O provided in the kit. The qPCR programme consisted of pre-denaturation at 95\u00b0C for 10\u00a0min and 45\u00a0cycles of amplification at 95\u00b0C for 10sec, 55\u00b0C for 10sec, and 72\u00b0C for 20sec. All reactions were performed simultaneously for each gene with \u03b2-actin [Rohu juveniles from the 2008 year class were collected and kept in 700\u00a0L ferro-cement tanks for acclimatization prior to the experiment. A virulent strain of -\u0394\u0394Cq. Mean fold differences and standard errors were calculated. Differences between the temporal mean values of one gene in an organ was analysed using one-way ANOVA followed by Duncan\u2019s multiple range tests, with values P\u2009<\u20090.05 considered as significantly different.The quantification cycle (Cq) values were calculated using a Light Cycler 96 SW 1.1 and the data was exported. N-fold differential expression was calculated using the comparative Cq method . The Cq http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE27994). Other supporting data  is included in the additional files section.All assembled transcriptome reads are available through GEO Series accession number GSE27994  are shown to the right of each linkage group while position (in Kosambi cM relative to the upper marker in the group) is shown to the left.Click here for fileSequence and annotation of contigs containing mapped SNPs (MS-excel file). LG, linkage group. cM, position on linkage group in centimorgans. GeneID, closest SNP homology from BLAST. snpA and snpB, alternative alleles for SNP. SequenomNot, contig sequence showing alternative SNP allele in square brackets.Click here for fileCorrespondence of annotated SNPs mapped in L. rohita to peptides and chromosome regions in the zebra fish (Danio rerio) genome (MS-excel file).Click here for fileA. hydrophila within L. rohita families A-U.Frequency of hour\u2019s survival after challenge with Click here for file"}
+{"text": "Dear Editor,Orthomyxoviridae family and is the causative agent of seasonal and pandemic flu. There are three types of influenza virus: A, B and C. Influenza A viruses possess the greatest host-range and reassortment ability and all known influenza pandemics have been caused by type A. Influenza A viruses contain 8 negative-strand RNA segments, encoding 14 proteins, including 3 major surface proteins, hemagglutinin (HA), neuraminidase (NA) and the M2 proton channel. Due to their accessibility on the surface of the virus, HA, NA and M2 have been the primary targets for the design of anti-influenza therapeutics allyl-Neu5Ac2en  and selective inhibition of the growth of N1-containing viruses compared with an N2-containing virus in plaque reduction assays allyl-Neu5Ac2en does indeed work against 09N1 allyl-Neu5Ac2en into both forms. We found that 3-(p-tolyl)allyl-Neu5Ac2en bound both proteins in a similar conformation, but distinct from the canonical group 1 members N8 and N5. In contrast, 3-(p-tolyl)allyl-Neu5Ac2en could not be soaked into the group 2 member, N2, but rather the inhibitor was found to bind the second binding site allyl-Neu5Ac2en.Previously we reported that the 2009 pandemic H1N1 influenza virus (pH1N1) NA (09N1) does not have a 150-cavity and therefore it was unclear whether or not group 1 specific inhibitors which access the 150-cavity could be effective against it allyl-Neu5Ac2en were solved by molecular replacement using Phaser from the CCP4 program suite.The protein crystals were grown according to our previous reports allyl-Neu5Ac2en with 09N1 and 09N1-I149V were solved at 1.7 \u00c5 and 2.0 \u00c5, respectively. In both complex structures, the atypical group 1 150-loops are forced half-open into a similar disordered conformation with electron density lacking for residues T148 and I149 allyl-Neu5Ac2en was solved at 1.8 \u00c5 and the complex structure clearly showed the characteristics observed earlier for the N8 complex allyl-Neu5Ac2en did not result in typical NA inhibitor complex structures, with the inhibitor bound in the active site. Rather, for N3, 3-(p-tolyl)allyl-Neu5Ac2en was bound to the second sialic acid binding site between two adjacent NA molecules allyl-Neu5Ac2en exhibits specificity for group 1 NA. This explains further why poorer inhibition of N2 by this group-1-targeted inhibitor was generally observed allyl-Neu5Ac2en, which is based upon Neu5Ac2en, has an advantage over other group 1 inhibitors which are based upon the oseltamivir structure allyl-Neu5Ac2en to both 09 pH1N1 and 09 pH1N1-I149V viruses in cell-based plaque reduction assay. Recombinant viruses were generated by reverse genetics using previously described methods  was used as a fluorescent NA substrate according to previously reported methods allyl-Neu5Ac2en for virus is in micro molar range , which needs to be improved for a clinically-therapeutic inhibitor. Therefore, with this work we provide further evidence of the binding modes that are possible for the novel class of NA inhibitor represented by 3-(p-tolyl)allyl-Neu5Ac2en, which will contribute to further development of this inhibitor class in the future.To further the biological relevance of our data, we evaluated the inhibition efficacy of 3-"}
+{"text": "Oncorhynchus, and with the genus Salmo, to determine the phylogenetic relationship between chromosome arrangements and the retention of undifferentiated duplicated regions. A 6596.7 cM female coho salmon map, comprising 30 linkage groups with 7415 and 1266 nonduplicated and duplicated loci, respectively, revealed uneven distribution of duplicated loci along and between chromosome arms. These duplicated regions were conserved across syntenic arms across Oncorhynchus species and were identified in metacentric chromosomes likely formed ancestrally to the divergence of Oncorhynchus from Salmo. These findings support previous studies in which observed pairings involved at least one metacentric chromosome. Re-diploidization in salmon may have been prevented or retarded by the formation of metacentric chromosomes after the whole genome duplication event and may explain lineage-specific innovations in salmon species if functional genes are found in these regions.Whole genome duplication has been implicated in evolutionary innovation and rapid diversification. In salmonid fishes, however, whole genome duplication significantly pre-dates major transitions across the family, and re-diploidization has been a gradual process between genomes that have remained essentially collinear. Nevertheless, pairs of duplicated chromosome arms have diverged at different rates from each other, suggesting that the retention of duplicated regions through occasional pairing between homeologous chromosomes may have played an evolutionary role across species pairs. Extensive chromosomal arm rearrangements have been a key mechanism involved in re-dipliodization of the salmonid genome; therefore, we investigated their influence on degree of differentiation between homeologs across salmon species. We derived a linkage map for coho salmon and performed comparative mapping across syntenic arms within the genus  Whole genome duplication (WGD) is a mutational mechanism that can serve as a primary driver of evolutionary novelty . ChangesThe stabilization of the duplicated genome through diploidization can be achieved by rearrangements , gene loss, and sequence deletion and divergence . These pSalmonid fishes are descended from a whole genome duplication event in an autotetraploid ancestor , distincThe role of the WGD event in salmonid trait innovation and diversification is unclear. Recent evidence based on molecular clock estimates suggest that duplication is unlinked to a major transition in life history, anadromy , and preSalmo, Salvelinus, and Oncorhynchus) has occurred through Robertsonian rearrangements of whole chromosome arms  and rainbow trout (O. mykiss) has revealed evidence for the retention of at least eight metacentric chromosomes and four acrocentric chromosomes that are ancestral to species divergence within the genus Oncorhynchus  is a species whose genome has not been extensively described to date. A low-density linkage map of coho salmon has been constructed using microsatellites  sequencing  the Washington Department of Fish and Wildlife\u2019s (WDFW) Wallace River Hatchery ; (2) the Domsea broodstock population, which originated in 1973 and 1974 from Wallace River Hatchery; (3) Bingham Creek , a tributary to the Satsop River in the Southwest Washington Coast/Lower Columbia ESU; and (4) Chehalis River located in British Columbia, Canada .An initial framework map was constructed using two haploid crosses (haploid family 1 and 2) comprising 64 and 62 individuals, respectively. These types of crosses have the advantage of identifying duplicated loci, because these loci will appear as heterozygotes in the offspring if they are polymorphic, while nonduplicated loci will be homozygous. Haploid families were created at the University of Washington hatchery facility , following the protocol of Sex-specific maps were created using two F3 outbred diploid crosses and one outbred diploid cross. Specifically, F3 diploid crosses were created from a cultured line originally derived from an outbred cross between two populations in Washington State . F0 maleSbfI, and a 6-nucleotide barcode was added to each sample for individual identification following protocols described by Genomic DNA from the sampled individuals was extracted using the DNeasy extraction kit  following the manufacturer\u2019s procedures. The DNA was digested with Genetic sex was determined in the two diploid families (diploid family 1 and 2) using a Y-linked growth hormone pseudogene (GH5 and GH6)  using the try.seq and map functions implemented in ONEMAP. The data from all diploid male parents were then combined to calculate an integrated male map using MERGEMAP.Ordering markers in the diploid male map was computationally difficult, potentially due to reduced recombination and occasional tetrasomic inheritance in males . TherefoThe reference database for coho salmon containing duplicated and nonduplicated RAD loci was aligned to the 54,937 filtered RAD loci identified in Chinook salmon  or both paralogs were polymorphic (BPP). Duplicated loci with both paralogs polymorphic (BPP) were used to infer homeologous linkage groups because both paralogs could be mapped. The positions of duplicated loci were subsequently examined on the integrated haploid map to determine whether there was a bias in the distribution of these loci across linkage groups. A kernel smoothing approach using a sliding window of 2 cM was used to determine whether there was a regional bias in distribution of these loci for each linkage group following methods described by Supporting Information, File S1.A reference database comprising a unique set of RAD loci was created for the purpose of sequence alignment and identification of polymorphisms across individuals. A total of 70,037 loci were sequenced with a depth greater than 5\u00d7 per individual in at least 496 individuals. These loci formed the temporary reference database of RAD loci, and they were retained for further screening. Sequence alignment using BOWTIE showed that 4075 loci did not align uniquely to themselves and likely corresponded with repeat regions; therefore, these loci were removed from the temporary database. After performing the BLAST search of the temporary reference database against itself, 2085 loci did not return a match or the best match score was not the locus itself. It was possible that these loci contained repeat sequences; therefore, these loci were also removed from the temporary reference database. Sequences from the haploid individuals were aligned to the reference database using BOWTIE; 3706 loci from the haploid individuals aligned to several other loci, and these loci were thus black-listed and removed from the reference database. Additionally, 7235 loci were identified as polymorphic duplicated loci in the haploid families. The final reference database comprising 52,936 nonduplicated loci and 7235 duplicated loci, as well as the 9866 loci that were removed by screening, are given in File S2).An initial framework map was constructed with two haploid families. Haploid family 1 and 2 had 3976 and 4048 biallelic polymorphic RAD loci, respectively, comprising a total of 6652 unique RAD loci. Among these loci, a mixture of duplicated and nonduplicated loci  were successfully assigned to 30 linkage groups with a LOD score of 5.0 to 7.0. The total map length for the haploid family 1 and 2 was 3040.1 cM and 3185.5 cM, respectively. The integrated haploid map had 5377 nonduplicated markers and 1266 duplicated markers with a total map length of 3602.6 cM (File S2).Linkage analyses were conducted in the diploid families following the construction of the integrated haploid map. Diploid family 1, 2, and 3 had 1360, 1176, and 1931 biallelic nonduplicated loci that were polymorphic in each female parent, respectively. Among these loci, a set of loci  were successfully assigned to 30 linkage groups with a LOD score of 4.0 to 8.0. The total map length for the diploid family 1, 2, and 3 was 3714.1 cM, 3068.9 cM, and 5047.2 cM, respectively. Although the diploid family 3 had the largest total map length, it also had the highest number of markers mapped. Because more recombination events are captured with more markers , it is nFile S2). The number of markers in common between the integrated male and female maps varied for each linkage group, ranging from 25 to 106 markers per linkage group (File S3). The male map had a total map length of 4141.76 cM (File S3).The male meioses were mapped using linkage analyses in the diploid families. Among the 8681 loci placed on the integrated haploid/diploid female map, diploid family 1, 2, and 3 had 846, 814, and 879 polymorphic loci in common for each male parent. Among these loci, a set of loci , as well as the Y-linked growth hormone pseudogene and sex-determining gene, sdY, were successfully assigned to 30 linkage groups with a LOD score of 4.0 to 7.0. Both the growth hormone pseudogene and sdY mapped to the telomeric region of the linkage group, Co30. All linkage groups were successfully merged, except for Co22, which was split into two linkage groups (Co22_1 and Co22_2) (File S4). Although telomeres were not mapped in males due to a lack of duplicated markers, many male linkage groups were expanded in size toward the terminal regions relative to the female, as seen by the increased distance in these regions reflecting more recombination events. Such patterns were particularly prominent for several linkage groups . Although qualitative, there was also evidence of suppressed recombination around the region containing the centromere in the male map compared with female integrated map for all linkage groups. The male map had reduced distance in these regions compared to the female map.The comparison between the male and female linkage groups reflected different recombination patterns between the sexes ; File S4Discussion.We mapped 664 RAD loci in coho salmon that had been previously placed on the Chinook salmon map, which permitted the identification of homologous chromosomal arms between the two species . On the Comparative mapping with Chinook salmon permitted inference of the structure of coho linkage groups. Twenty linkage groups in coho salmon corresponded to putative bi-armed metacentric chromosomes, and 10 linkage groups corresponded to putative uni-armed acrocentric chromosomes . These iComparative mapping between the Chinook and coho salmon maps also provided information on chromosomal arrangements that are shared between the two species. Eighteen chromosomes are conserved between the species ; specifiFive metacentric linkage groups in coho salmon (Co10\u2013Co14) consist of one acrocentric chromosome and one arm from a metacentric chromosome in Chinook salmon. Four metacentric linkage groups in coho salmon (Co15\u2013Co18) comprise arms that are found in two separate metacentric chromosome pairs in Chinook salmon. Two metacentric linkage groups  comprise two acrocentric chromosome pairs in Chinook salmon. Finally, one acrocentric linkage group (Co30) corresponds to an arm that is a part of a metacentric chromosome pair in Chinook salmon.File S5), suggesting that marker orders were conserved for all chromosomes or chromosomal arms. Such analyses provide additional evidence for the occurrence of centrometric inversion in Omy20 in rainbow trout after divergence between rainbow trout and Chinook/coho salmon, and this chromosomal inversion may be exclusive to rainbow trout ; 87.0% of the duplicated loci were located on 16 linkage groups  were placed on this map. These loci were not distributed evenly among the linkage groups  and seven metacentric chromosomes across the genus Oncorhynchus (Co01\u2013Co07)  than the consensus female map (6596.7 cM) constructed with two haploid families and three diploid families. There are three main reasons that might explain the reduced map size in the male compared with the female map. First, the difference could simply be a function of more markers being placed on the female map , because map size tends to increase when more markers are added . Second,Accurate identification of chromosome structure in coho salmon relied on aligning homologous chromosome arms with Chinook salmon. In addition, regions containing the centromere were inferred through comparative mapping between coho and Chinook salmon as no gynogenetic diploid families were used for this study to identify the exact location of the centromere. Homology of Co07a and Co12b with Chinook salmon chromosome arms Ots11p and Ots34 was not completely resolved. These arms are homeologous to each other within both species, and markers on Ots34 mapped to both Co07a and Co12b in coho salmon. We inferred that Co07a was homologous to Ots11p, as the arm Co07a is part of a metacentric chromosome that is conserved in Chinook salmon (Ots11) and rainbow trout (Omy19) . In factThe coho salmon linkage map constructed in this study has 8681 markers, spanning all predicted 30 linkage groups. This coverage is comparable with recently published maps across a number of salmon species , and numMale salmonids are the heterogametic sex . In thisOur results showed that suppressed recombination around the region containing the centromere in males was widely apparent, whereas higher recombination was observed in telomeric regions for some male linkage groups relative to female. The results are in agreement with a number of studies performed on other salmonid species ; althougComparative mapping provided insights into the process of chromosomal evolution occurring after the whole genome duplication event, and this is the first study that characterizes chromosomal evolution between coho and Chinook salmon. Nine metacentric and nine acrocentric chromosomes appear to be conserved between these two species. Among these conserved chromosomes, one metacentric (Co08 in coho and Ots12 in Chinook) and five acrocentric chromosomes are unique to coho and Chinook salmon, suggesting that the structure of these linkage groups is ancestral to the divergence of these species relative to rainbow trout and Atlantic salmon. The remaining linkage groups are not conserved, reflecting extensive chromosomal rearrangements since coho and Chinook salmon diverged.Oncorhynchus (Oncorhynchus species (Co26\u2013Co29). Similarly, eight metacentric chromosomes are conserved among all three species . The results support the hypothesis of Oncorhynchus. There is one interesting extension to these earlier observations. One metacentric chromosome in Chinook salmon, Ots08, sometimes occurs as a metacentric chromosome (Omy25p and q) or as two acrocentric arms (Omy25 and Omy 29) in rainbow trout .One metacentric and one acrocentric chromosome, Co09 and Co26, respectively, appear to be conserved across coho salmon, Chinook salmon, rainbow trout, and Atlantic salmon . Such findings suggest that homeologies may be preferentially retained between larger metacentric chromosomes (Salmo and Oncorhynchus. We speculate that this process also differed to some extent following the divergence of the two genera. Although the exact distribution of duplicated markers along chromosome arms in Atlantic salmon has not yet been described (Although our results confirm that the divergence rates of homeologs following the whole genome duplication event have not been uniform (escribed .The implications of our findings for species divergence within the subfamily Salmoninae will become clearer once we gain a greater understanding of the role of duplicated regions in evolution. If the duplicated regions we detected have genes that permit greater flexibility for adaptation by providing the opportunity to acquire additional or novel functions , then reSalmo and Oncorhynchus. We have also identified linkage groups that recently have been or may be involved in ongoing homeologous pairing in coho salmon. Such pairings were conserved with related Pacific salmon species. Ancestral metacentric chromosomes appear to retain recently diverged duplicated regions and may be involved in ongoing homeologous pairings; such results indicate that diploidization may have been prevented or retarded in these ancestral metacentric chromosomes following the whole genome duplication event. The resources developed here will facilitate genome-wide studies in coho salmon, such as genome scans, QTL mapping, and genome-wide association studies (Here, we developed an extensive set of genomic resources for coho salmon: a reference database of unique RAD loci, two types of consensus female linkage maps, and a consensus male linkage map. A dense female map constructed in this study permitted alignment of linkage groups in this species with that of Chinook salmon, enabling interspecies comparisons with related salmon species. Syntenic relationships across multiple salmonid species identified in this study provided strong evidence for chromosomal rearrangements and conservation of metacentric and acrocentric chromosomes following the divergence between  studies , as well"}
+{"text": "More than 500 plant species have demonstrated the ability to absorb metals or metalloids from soils and concentrate them at extremely high levels in leaf tissues . Likewise, traits such as pest or pathogen resistance are often not observable until plants are exposed, and one would not define these resistances as not existing simply because a population does not happen to encounter specific pests or pathogens where it grows. In fact, several crop pest resistance traits have been derived from wild relatives that grow where the pest has never been present . A naturalistic definition also ignores evolutionary history, as it excludes hyperaccumulators that historically occupied soils with high metal concentrations but now do not (for whatever reasons), as well as all lineages retaining hyperaccumulation ability as an ancestral trait or exaptation, or that have evolved hyperaccumulation outside of the context of metalliferous soils by drift or a byproduct of other processes. Even worse, by assuming that hyperaccumulation ability could only evolve in response to metal-rich environments, current naturalistic definitions approach adaptationism , and the utility of understanding the genetic and physiological basis of hyperaccumulation for technological purposes, a working definition for metal hyperaccumulation should be compatible with these pursuits , but ideally plants should be grown to reproduction and assessed for production of viable offspring.For single-level experiments, metal concentrations in amended soils should be appropriately low enough to rule out passive hyperaccumulation but high enough to allow the detection of active hyperaccumulation. The use of soil amendments at multiple levels is more informative.Chelate-induced phytoextraction with mobilizing agents such as EDTA should not be used when screening for hyperaccumulation or tolerance, as such conditions artificially facilitate plant metal accumulation while buffering plants against toxicity, thus poorly reflecting both hyperaccumulation capacity and metal tolerance.We advocate the study of hyperaccumulation and tolerance in greenhouse studies by proposing the following guidelines:In addition to these guidelines, it is helpful to appreciate that the manifestation of both hyperaccumulation and tolerance are environment-dependent, and evaluations in a single environment  are unlikely to capture the complex nature of these traits and their interaction. Greenhouse studies applying continuous gradients of metal concentrations via amended soils can be used to determine the extent of the dependence of hyperaccumulation on soil metal concentrations while simultaneously assessing metal tolerance. In this way, the traits of tolerance and hyperaccumulation can be more fully expressed as concentration-response curves. Phylogenetically explicit studies can further reveal whether tolerance and hyperaccumulation evolve together or sequentially, and provide a controlled framework for assessing the myriad adaptive hypotheses that have been put forward to explain this fascinating phenomenon.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "Introduction: The purpose of this in vitro study was to assess the antifungal activity of final canal rinse with either three concentrations of sodium hypochlorite (NaOCl) , two concentrations of chlorhexidine (CHX) (2% and 0.2%), MTAD, Tetraclean, Hypoclean and Chlor-Xtra on Candida albicans  in a human tooth model. Methods and Materials: Two hundred and thirty five extracted human maxillary central and lateral incisors were used in this study. Teeth were randomly divided into nine test groups (n=25) and positive and a negative control groups (n=5). After cleaning and shaping, teeth were contaminated with C. albicans and incubated for 72 h. The irrigation solution in nine experimental groups included: 6% NaOCl, 2.6% NaOCl, 0.5% NaOCl, 2% CHX, 0.2% CHX, MTAD, Tetraclean, Hypoclean and Chlor-Xtra. After culturing on Sabouraud 4% dextrose agar, colony-forming units (CFU) were counted. Results: 6% NaOCl, 2% CHX and Chlor-Xtra were equally effective (P>0.05) and significantly superior to MTAD and Tetraclean (P<0.05). In addition, the effectiveness of Tetraclean and MTAD was significantly less than Hypoclean, NaOCl at all concentrations (6% 2.6% and 0.5%), MTAD and 0.2% CHX (P<0.05). Furthermore, Tetraclean was significantly more effective than MTAD (P<0.05). Conclusion: Antifungal activity of 6% NaOCl, Chlor-Xtra and 2% CHX was significantly greater than 2.6% NaOCl, 0.5% NaOCl, MTAD, 0.2% CHX and Tetraclean. It has been demonstrated that 0.5% NaOCl kills . It has act time . In order to overcome the high surface tension of NaOCl, two NaOCl-based root canal irrigants have been introduced. Chlor-Xtra is a mixture of 6% NaOCl with powerful wetting agents and proprietary surface modifiers. Alkylating agents have also been added to increase the electrical capacity of the solution. It is sHypoclean  is another NaOCl-based solution which is composed of 5.25% NaOCl and two detergents 13]. Studies on Hypoclean are very limited. Its 28-day substantivity has been demonstrated . Studies.Chlorhexidine (CHX) is a cationic bisguanide with a wide antimicrobial spectrum which is effective against both Gram-positive and Gram-negative bacteria as well as fungi . There are some antibiotic-based root canal irrigation solutions in endodontics, as well. Biopure MTAD  is a mixture of a tetracycline (doxycycline), an acid (citric acid), and a detergent (Tween 80)  . It can in vitro antifungal activity of different concentrations of NaOCl , CHX (0.2% and 2%), MTAD, Hypoclean, Chlor-Xtra and Tetraclean. There is only one study on the antifungal effect of Tetraclean . Furtheret al.  effectiveness of 2% CHX and 6% NaOCl against C. albicans was equal and was superior to 17% EDTA and MTAD. A study using a dentine tube model demonstrated that presence of smear layer lead to more resistance of 5% NaOCl . Anotheralbicans , which i  demonstrated that residual antibacterial activity of Hypoclean was significantly less than Tetraclean. There are a number of studies on the antibacterial efficacy of Tetraclean. Neglia et al.  indicateter 48 h . A study species . Althougi et al.  showed ti et al.  revealedi et al.  showed t [et al.  evaluateAnother issue is the volume of the used root canal irrigant which was approximately similar for all samples. The treatment time chosen for final rinsing in this study (5 min) is based on another study . The effect of 6% NaOCl, Chlor-Xtra and 2% CHX on C. albicans was significantly greater than 2.6% NaOCl, 0.5% NaOCl, MTAD, 0.2% CHX and Tetraclean."}
+{"text": "Suberin is a lipophilic macromolecule found in specialized plant cell walls, wherever insulation or protection toward the surroundings is needed. Suberized cells form the periderm, the tissue that envelops secondary stems as part of the bark, and develop as the sealing tissue after wounding or leaf abscission. Suberin is a complex polyester built from poly-functional long-chain fatty acids (suberin acids) and glycerol. The suberin acids composition of a number of plant tissues and species is now established, but how the polyester macromolecule is assembled within the suberized cell walls is not known. In the last years contributions from several areas have however significantly enriched our understanding of suberin. The primary structure of the polyester, i.e., how the suberin acids and glycerol are sequentially linked was revealed, together with the stereochemistry of the mid-chain functional groups some suberin acids have; solid-state NMR studies showed the presence of methylene chains spatially separated and with different molecular mobility; biophysical studies showed the membrane behavior of suberin acids derivatives, allowing new insights on structure-properties relationships; and a number of candidate genes were conclusively related to suberin biosynthesis. The comprehension of suberin as a macromolecule will be essential to understand its vital protective roles in plants and how they will deal with eventual environmental changes. Suberin is also expected to be a source for high-performing bio-based chemicals, taking advantage of the structural uniqueness of their constituent suberin acids. When plants emerged from the sea and start evolving in the more aggressive land environment, they faced a number of survival challenges, including control of water loss, insulation against climatic variability, and protection against abiotic aggressions . Polyaromatics, which can be analytically determined as lignin, are found associated  to suberin, and in a lesser extent to cutin , a tree found in the west Mediterranean and neighboring Atlantic regions, from which commercial cork is harvested. Cork have a large set of properties, making it a technological performing material, used worldwide in a high number of industrial applications, for some of them without proper substitute  tubers are a typical example. When potatoes are harvested, the suberization process keeps evolving in the periderm cells for a few days, making the \u201cmature\u201d potato skin , the pioneering work of Sitte (mostly based in cork) postulated in a comprehensive manner the ultrastructure of suberized cell walls. Besides confirming the overall structure proposed by van Wisselingh, the main observation of Sitte was that the secondary suberin wall is thinly lamellate Sitte, . Two typThe presence of this mostly organized poly-lamellate structure, besides tissue identity and location, has been used to define suberized cells  or more correctly, as the mass-loss after suberin depolymerization , and the ones with saturated chains, starting at C16 (skipping the C18) and going up to C28. Other types of fatty acids are also found in some suberins, including \u03b1-hydroxyacids, \u03b1,\u03c9-alkanediols, and C16 \u03c9-dihydroxyacids   Figure . The steQ. suber cork and potato periderm suberins , all \u03c9-hydroxyacids which were found as monomers, were also found esterified though their \u03c9-hydroxyl group to ferulic acid as oligomer species  Pereira, ; in natiBesides the insoluble polyaromatics, when the suberin polyester is depolymerized by any ester breaking technique, small amounts of phenolics are co-solubilized together with the main aliphatic suberin monomers. Values from 1 to 10% of all suberin depolymerized materials have been found . A model for the suberin polyaromatics as a poly(ferulic acid) structure, built from ferulic acid units linked by condensation and ether linkages similar to lignin, was developed: after feeding 13C labeled phenylalanine in suberizing wound potato periderm, signal enhancement was found mostly in carbons assignable to hydroxycinnamates , also A key question to be answered is how we define suberin. The original term \u201csub\u00e9rine\u201d (in French) is attributed to the famed XIX century chemist Chevreul, who used it to define all the non-soluble materials specific of cork Gilson, . Later, in vitro: synthesized glycerol\u20141,16-hexadecanedioic acid\u2014glycerol formed stable lipid vesicles in aqueous media; these vesicles became fluid only at relatively high temperatures (57\u00b0C), and their membrane thickness could be estimated close to the length of the C16 chain when stretched  groups, one more mobile, and the other, presumably close to ester groups , much more rigid in molecular motion  macromolecule. Glycerol units linked in succession to \u03b1,\u03c9-diacids will be the core backbone of the suberin macromolecule  show up commonly in suberin oligomers as ferulates. This means that \u03c9-hydroxyacids, besides being part of the acylglycerol suberin matrix, linked through their acidic end group to glycerol, can act at the same time as connectors to the neighboring polyaromatics, through esterification to ferulic acid; the latter can be part of, or be linked to the periphery of those polyaromatics Figure . We alsoin vitro of a specific inhibitor of fatty acid elongases (EPTC), led to a suberin with shorter long-chain monomers; besides, the suberized cell walls after this treatment showed less, and in some cases thinner, light lamellae; the conclusion was that the glycerol-suberin acids structure was necessary for the lamellae formation, and that the long-chain monomers would be perpendicular to the light lamellae plane  acid was the starting point for the mid-chain 9,10 modified suberin monomers: the terminal C-18 carbon is first \u03c9-hydroxylated forming the C18:1\u03c9-hydroxyacid, and further oxidized to the corresponding C18:1\u03b1,\u03c9-diacid; the epoxidation at the C-9/C-10 carbons, and its further hydration, give rise respectively to the C18 9,10 epoxide and C189,10-diol monomers. Straight chain suberin acids followed the known pathway starting at acetyl and malonyl CoA, down to the C16 saturated chain fatty acid, followed by its successive elongation, together with its eventual end chain oxidation to \u03c9-hydroxyacids and \u03b1,\u03c9-diacids  monomers started in the 1970s  or to C22\u2013C24 fatty acids (CYP86B1), were found associated with the suberization process; the epoxidation in vitro of the C-9 double bond of the C18:1(oleic) acid by the CYP94A1 oxygenase, gave rise to dominantly  and much less  stereoisomers, both corresponding to the cis configuration found in suberin epoxyacids discussed above. The silencing of the CYP83A33 gene in potato, which promotes the \u03c9-hydroxylation step in its periderm suberin, led to a highly modified polyester structure and the distortion of the lamellar arrangement in the cell walls  oxygenases were proved to play a crucial role in the end- and mid-chain oxidation steps, starting from mono-functional fatty acids, and leading to \u03c9-hydroxyacids and \u03b1,\u03c9-diacids, either with saturated chains or with secondary oxygenated groups  of glycerol  positions  were found in suberizing tissues, and their role in the suberin biosynthesis proved after their silencing in mutant plants  to ferulic acid. A glycerol-3-phosphate acyltransferase (GPAT5) was found to direct the esterification of \u03c9-hydroxyacids and \u03b1,\u03c9-diacids at the 18 mid-chain modified ones; besides, they carry the properties derived from the long hydrocarbon chains, such as molecular flexibility and hydrophobicity. The preparation of technical polymers with designed properties, starting from these bi- or polyfunctional suberin monomers, is one of the more promising areas  trees harvesting (outer bark) (Pinto et al., The chemical uniqueness of suberin long-chain \u03b1,\u03c9-bifunctional monomers, or the polyester macromolecule as a whole, makes them of high industrial interest. A key point is their bi- or poly-functionality: each suberin acid have at least two reactive groups in the \u03b1,\u03c9-positions, or more in the case of the CThe efforts to obtain isolated suberin acids in industrial scale date back to the 1940s when a company named \u201cSuber\u201d was installed in France, offering \u201cSub\u00e9ryl,\u201d a product obtained from suberin after cork saponification (Guillemonat, The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "The consensus map spanned a total length of 1443.84 cM, and consisted of 5,019 SNP markers derived from RAD tag sequencing and 118 publicly available SSR markers distributed in 17 linkage groups, corresponding to the haploid chromosome number of sunflower. The maximum interval between markers in the consensus map is 12.37 cM and the average distance is 0.28 cM between adjacent markers. Despite a few short-distance inversions in marker order, the consensus map showed high levels of collinearity among individual maps with an average Spearman's rank correlation coefficient of 0.972 across the genome. The order of the SSR markers on the consensus map was also in agreement with the order of the individual map and with previously published sunflower maps. Three individual and one consensus maps revealed the uneven distribution of markers across the genome. Additionally, we performed fine mapping and marker validation of the rust resistance gene 12R, providing closely linked SNP markers for marker-assisted selection of this gene in sunflower breeding programs. This high resolution consensus map will serve as a valuable tool to the sunflower community for studying marker-trait association of important agronomic traits, marker assisted breeding, map-based gene cloning, and comparative mapping.A high-resolution genetic map of sunflower was constructed by integrating SNP data from three F Helianthus annuus L) is a member of the Asteraceae family, and is the fourth most economically important annual crop grown worldwide for edible oil http://sunflower.uga.edu/cmap/), the limited number of markers makes it difficult to conduct fine-scale linkage mapping and map-based cloning. Association mapping and genomic selection are dependent on a large number of polymorphic markers. These analyses are only successful if thousands of markers are available, because of the low level of linkage disequilibrium (LD) present in germplasm resources of sunflower Sunflower (Single nucleotide polymorphisms (SNP) are the most common type of genetic variation 12R, in the constructed consensus map.Construction of a consensus map from multiple linkage maps offers the opportunity to map a larger number of markers than would be possible in any individual bi-parental map and also tends to eliminate many large marker gaps. Statistical software has been developed to pool segregation data from individual populations and compute loci orders and genetic map distances based on mean recombination frequencies and combined LOD scores 2 progeny derived from a cross between HA 89 and RHA 464. HA 89 (PI 599773) is an oilseed maintainer line and RHA 464 (PI 655015) is an oilseed restorer sunflower germplasm which is known to possess resistance genes for both downy mildew and rust diseases R-gene) R12 to linkage group (LG) 11 of sunflower using simple sequence repeat (SSR) markers 2 progeny derived from a cross between a proprietary confection B line  and RHA 464. The third mapping population (Pop3) consisted of 142 F2 progeny derived from a cross between CR 29  and RHA 468 (PI 667184). CR 29 is a proprietary confection restorer line and RHA 468 is an oilseed restorer line. To graphically explain relationships among the parent lines, Jaccard's genetic similarity coefficient Three mapping populations were used to develop SNP genetic maps in sunflower . Five pa12R specific markers. They include 238 inbred lines and 63 germplasm lines released by USDA, and 247 plant introduction (PI) lines originally collected from 32 countries, which together represent a diverse germplasm pool of cultivated sunflower  was used to assess goodness-of-fit to the expected segregation ratio for each marker using the \u2018locus genotype frequencies\u2019 feature of JoinMap 4.1. Markers that showed significant segregation distortion from the expected 1\u22362\u22361 (co-dominant) or 3\u22361 (dominant) ratios were excluded from map construction. Markers were assigned to linkage groups applying the independence LOD (logarithm of the odds) parameter with LOD threshold values ranging from 2.0 to 12.0. We used the \u2018similarity of loci\u2019 command of JoinMap to identify perfectly identical markers  which are supposed to be mapped at exactly the same position on the linkage group. In order to reduce the burden of calculation effort, only one marker was kept of the \u2018similar loci\u2019 for linkage mapping analysis. Linkage analysis and marker order were carried out using the regression mapping algorithm. Recombination fractions were converted to map distances in centimorgans (cM) using the Kosambi mapping function Linkage maps were constructed independently for each mapping population using the same procedures and parameters in each case. All the SNP markers and the majority of the SSR markers used for linkage mapping were co-dominant. The Chi-square test  from marker positions in consensus and individual genetic maps. Significance tests were conducted in R version 2.13.1 x is the actual marker count in each interval, \u03bb is the average number of markers per interval and e is the base of the natural logarithm. Analyses were conducted with the R statistical package All linkage groups were divided into 1, 2, 5, and 10 cM intervals and the observed marker frequency distribution of each interval was calculated. The observed marker frequencies per centiMorgan (cM) unit interval were compared to that of expected frequencies generated from a Poisson distribution using a Chi-square test 12R with SSR markers. Briefly, urediniospores of North America (NA) race 336 were used to inoculate F2 plants, along with the two parental lines HA 89 (susceptible parent) and RHA 464 (resistant parent). Twenty seedlings of each of the F2-derived F3 families were also phenotyped with the same pathogen race to distinguish between F2 plants that were homozygous or heterozygous for the resistance gene. Rust infection types and severity (pustule coverage) were scored 10\u201312 days after inoculation as described by Qi et al. 12R.Rust phenotypic data of Pop1 were obtained from Gong et al. Three segregating populations derived from five parental lines were used in this study . RHA 464The three mapping populations, Pop1, Pop2, and Pop3, were used to produce three separate high-density genetic maps containing 2,286, 3,236, and 2,123 markers in each map, respectively .2 individuals from a cross between HA 89 and RHA 464. Two individuals were discarded because of too much missing data, leaving a total of 139 F2 individuals for the final linkage analysis. Pop1 was first genotyped with a total of 220 polymorphic SSR markers. Of these, 118 informative and co-dominant SSRs were integrated with SNP markers to construct a high-density linkage map of Pop1. The number of SSR markers for each LG varied and ranged from one on LG14 to 13 on LG10 (The construction of the Pop1 linkage map started with 141 F on LG10 . All butP<0.05) from the expected Mendelian ratio, and were discarded from mapping analysis, leaving a total of 2,286 segregating markers   showed significant distortion (168 SNP) . The mar168 SNP) . Ninety 168 SNP) .2 individuals of a cross between a proprietary confection B-line and RHA 464. Filtration of the SNP genotype data yielded 3,464 good quality SNP marker data for linkage analysis in Pop2. A total of 228 markers (6.6%) showed significant (P<0.05) distortion from the expected 1\u22362\u22361, which were removed, yielding a final genetic map of 3,236 SNP markers assembled into 17 LGs. The 17 LGs were identified on the basis of the common SNP markers located on each chromosome relative to the linkage map of Pop1. The Pop2 linkage map covers a total length of 1,370.97 cM with an average density of one marker in every 0.42 cM  showed significant (P<0.05) distortion from the expected Mendelian ratio and were removed from the linkage analysis. The remaining SNP markers were placed onto 17 sunflower linkage groups except for 6 markers, which could not be suitably added in any linkage group, resulting in a final map consisting of 2,123 SNP markers . Genome wide marker distribution in 1-cM intervals shows a clear clustering of markers in certain genomic regions, indicating that the markers were not randomly distributed along the entire length of the sunflower linkage groups . The ave12R, present in the inbred line RHA 464, was previously mapped to LG11 with flanking SSR markers, CRT275 and ZVG53, in an interval of 10.6 cM 2 population (Pop1 in the present study) was used to saturate the 12R region with SNP markers. Rust phenotypic data of Pop1 were integrated with SNP marker data, and seven linked SNP markers were identified, five on one side , and two on the other side , defining an interval less than 2.3 cM surrounding the previously mapped 12R gene in LG11 , while the Pop1 map  was somewhat shorter than the rest of the maps. Comparisons of linkage groups among individual maps revealed that the upper ends of LGs 4 (\u223c42 cM), 13 (\u223c29 cM), 14 (\u223c33 cM), 15 (\u223c28 cM) and 17 (\u223c50 cM) and the lower end of LG12 (\u223c36 cM) in the Pop1 map showed no marker coverage, though the same regions in the other two maps possessed many mapped loci Click here for additional data file.Table S2List of two-hundred forty seven sunflower plant introduction (PI) lines used for marker validation.(XLSX)Click here for additional data file.Table S3SNP sequences for the 10,000 targeted loci.(XLSX)Click here for additional data file.Table S4SNP and SSR marker positions in Pop1, Pop2, Pop3, and consensus genetic linkage maps.(XLSX)Click here for additional data file.Table S5Spearman's rank correlation coefficients between marker positions in the consensus map and individual population maps in each linkage group of sunflower.(DOCX)Click here for additional data file.Figure S1A three-way Venn diagram illustrating all unique, two-way and three-way sets of shared SNP markers mapped in three component populations. The mapping populations are abbreviated as in the text: Pop 1\u200a=\u200aHA 89\u00d7RHA 464; Pop 2\u200a=\u200aB-Line\u00d7RHA 464; Pop 3\u200a=\u200aCR29\u00d7RHA 468.(TIF)Click here for additional data file.Figure S2Integrated genetic linkage map of sunflower. The map shows the linkage groups 1, 2, and 3 developed from three F2 mapping populations. Markers in bold font are SSR markers.(TIF)Click here for additional data file.Figure S3Integrated genetic linkage map of sunflower. The map shows the linkage groups 4, 5, and 6 developed from three F2 mapping populations. Markers in bold font are SSR markers.(TIF)Click here for additional data file.Figure S4Integrated genetic linkage map of sunflower. The map shows the linkage groups 7, 8, and 9 developed from three F2 mapping populations. Markers in bold font are SSR markers.(TIF)Click here for additional data file.Figure S5Integrated genetic linkage map of sunflower. The map shows the linkage groups 10, 11, and 12 developed from three F2 mapping populations. Markers in bold font are SSR markers.(TIF)Click here for additional data file.Figure S6Integrated genetic linkage map of sunflower. The map shows the linkage groups 13, 14, and 15 developed from three F2 mapping populations. Markers in bold font are SSR markers.(TIF)Click here for additional data file.Figure S7Integrated genetic linkage map of sunflower. The map shows the linkage groups 16 and 17 developed from three F2 mapping populations. Markers in bold font are SSR markers.(TIF)Click here for additional data file."}
+{"text": "Linkage of aged care and hospitalisation data provides valuable information on patterns of health service utilisation among aged care service recipients. Many aged care datasets in Australia contain a Statistical Linkage Key (SLK-581) instead of full personal identifiers. We linked hospital and death records using a full probabilistic strategy, the SLK-581, and three combined strategies; and compared results for each strategy.Linkage of Admitted Patient Data for 2000\u201301 to 2008\u201309 and Registry of Births, Deaths and Marriages death registration data for 2008\u201309 for New South Wales, Australia, was carried out using probabilistic methods and compared to links created using four strategies incorporating a SLK-581. The Basic SLK-581 strategy used the SLK-581 alone. The Most Recent SLK-581, Most Frequent SLK-581, and Any Match SLK-581 strategies leveraged probabilistic links between hospital records drawn from the Centre for Health Record Linkage Master Linkage Key. Rates of hospitalisations among people who died were calculated for each strategy and a range of health conditions.Compared to full probabilistic linkage, the basic SLK-581 strategy produced substantial rates of missed links that increased over the study period and produced underestimates of hospitalisation rates that varied by health condition. The Most Recent SLK-581, Most Frequent SLK-581, and Any Match SLK-581 strategies resulted in substantially lower rates of underestimation than the Basic SLK-581. The Any Match SLK-581 strategy gave results closest to full probabilistic linkage.Hospitalisation rates prior to death are substantially underestimated by linkage using a SLK-581 alone. Linkage rates can be increased by combining deterministic methods with probabilistically created links across hospital records. Linkage of data collected from hospital and aged care services has been used to assess aged care service utilisation and mortThe Home and Community Care (HACC) Program, an Australian Government initiative, provides essential community care and support services to enable people who are frail or disabled and at risk of long-term residential care to remain in the community. During 2009\u201310 there were over 244 302 HACC clients in New South Wales (NSW), with 80% being over 65\u00a0years of age[nd, 3rd and 5th letters of family names, the 2nd and 3rd letters of given names, date of birth in the form DDMMYYYY and a single character for sex. Dummy characters are used for insufficient and missing data so the SLK-581 always has a length of 14 characters[Personally identifying information such as name, address, date of birth and gender are required to perform high quality probabilistic record linkage. However, the identifiable information contained in the HACC MDS is in the form of a statistical linkage key (SLK), the SLK-581. The SLK-581 is a concatenation of 14 characters consisting of the 2The Australian Institute of Health and Welfare (AIHW) has shown that the likelihood of common SLK-581 linkage keys for different individuals in large aged care data sets is very low. In a national database of nursing home residents 0.6% of 440 000 people had a non-unique SLK-581. VariousNew South Wales (NSW) has the largest population of all Australian States and Territories, comprising 7.2 million people . We linkEthical approval was obtained from the NSW Population and Health Services Research Ethics Committee.n\u2009=\u200919 874 083) were used for this study.The APD represents a census of all admitted patient services in NSW. It covers demographic and episode related data for every inpatient that is separated  from any public, private, repatriation hospital, private day procedure centre or public nursing home in NSW. The APD contains demographic data items, administrative items and coded information. Principal diagnosis and co-morbidities on the APD are coded according to the ICD-10-AM. APD recn\u2009=\u200947 110) were used for this study.Deaths occurring in NSW must be notified to the NSW Registry of Births, Deaths and Marriages (RBDM) under the Births, Deaths and Marriages Registration Act 1995. NSW death registration data comprise records of persons who died in NSW, regardless of their place of residence. RBDM death records for the period 1 July 2008 to 30 June 2009  Master LFor the entire linked dataset the CHeReL reported the linkage quality as less than 5/1000 missed links and 3/1000 false positive links. Where duplicate RBDM records were found, one record was randomly selected and retained for all five linkage strategies, giving 46 949 records for analysis. The full probabilistic linkage strategy resulted in 44 804 RBDM records, each representing one person, linked to 598 828 APD records; 2145 (4.6%) RBDM records did not link to an APD record.Linkage of APD to RBDM records was carried out using the SLK-581 only. This strategy resulted in 41 633 RBDM records, each representing one person, linked to 432 026 APD records; 5316 (11.3%) RBDM records did not link to an APD record.Each individual identified in the full probabilistic model (Strategy 1) was assigned the SLK-581 from their most recent hospital separation. In the case of incomplete personal identifiers, the most recent complete SLK-581 was used. Where the individual had no records with a complete SLK-581 the most recent incomplete SLK-581 was used. This strategy resulted in 41 081 RBDM records, each representing one person, linked to 555 243 APD records; 5868 (12.5%) RBDM records did not link to an APD record.Each individual identified in the full probabilistic model (Strategy 1) was assigned the complete SLK-581 that occurred most frequently in their hospital separations. Where multiple versions of the SLK-581 occurred with equal greatest frequency, the SLK-581 from the most recent hospital separation was used. Where the individual had no complete SLK-581 the most frequently occurring incomplete SLK-581 was used. This strategy resulted in 41 044 RBDM records, each representing one person, linked to 556 723 APD records; 5905 (12.6%) RBDM records did not link to an APD record.This involved a two stage linkage process. First, each RBDM death record was linked with the APD using the SLK-581. Second, APD records that linked to an RBDM record were drawn from the CHeReL Master Linkage Key using the method described for the full probabilistic model. This strategy resulted in 41 633 RBDM records, each representing one person, linked to 571 923 APD records; 5316 (11.3%) RBDM records did not link to an APD record.A descriptive analysis of the quality of the SLK-581 in the APD and RBDM death registration data was carried out. The proportion of records with missing data and possibly poor quality data[The proportion of persons with at least one missed link or at least one false positive link were examined for strategies 2 to 5 using strategy 1 (Full Probabilistic Model) as the standard. As one individual could have more than one linked hospital record using Strategy 1, it was possible for an individual to simultaneously have both false positive and missed links according to strategies 2 to 5. The trend in the proportion of persons who died with either at least one missed link or at least one false positive link to the APD was examined by year of death.We examined the proportion of persons who died in NSW between 1 July 2008 and 30 June 2009 inclusive that were hospitalised prior to death by linkage strategy and year of death. Mean and median number of hospitalisations and 95% confidence intervals were calculated.For each linkage strategy, the number of hospitalisations among persons who died in NSW during 2008\u201309 was examined for a range of health priority areas  and ambulatory care sensitive conditions. Health Of the 19.9 million APD records, 12.9 million (65.1%) related to public hospital admissions and 6.9 million (34.9%) to private hospital admissions  obtained from the linkage are returned to each data custodian, who in turn add the PPN to the source dataset and provide the health information and PPN to the investigators for analysis. This process separates the linkage  from the analysis (which does not) and provides a robust mechanism for preserving individual privacy.It would be possible to increase linkage rates by improving the quality of reporting of personal identifiers, such as name, on the source datasets. It is likely that the addition of further links from other population-based datasets would further improve the performance of a combined strategy.Our study measured the extent to which deterministic links created using an SLK-581 can be improved by leveraging links created using full probabilistic techniques and linked population based data currently available in NSW. The results of this study indicate that a combined approach of deterministic linkage using an SLK-581 and probabilistic linkage of APD records provides more accurate results than using the SLK-581 alone. While use of the SLK-581 is unique to Australia, other deterministic linkage approaches using limited personal identifiers are possible such as linkage using the first two letters of the first name and surname, sex and date of birth. A combined approach using both probabilistic and deterministic linkage methods could be used in other settings where the datasets to be linked include full personal identifiers on some datasets and limited personal identifiers on others.Where datasets contain high proportions of missing name information, deterministic linkage using a SLK-581 results in a substantial number of missed links associated with a low rate of false positive links. By leveraging existing links in a population-based linked dataset that has been created using a full probabilistic approach, the number of missed links can be substantially reduced, and the associated under-reporting of measures of population health and health service utilisation mitigated, while maintaining a low rate of false positive links.The authors declare that they have no competing interests.KI conceptualised the study. RI, TH and KL carried out the statistical analysis. All authors were involved in study design and manuscript preparation. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6947/14/85/prepubICD-10-AM codes for selecting health priority areas and ambulatory care sensitive conditions (ACSC) associated with hospitalisation.Click here for file"}
+{"text": "MiR156 and miR529 are two combinatorial regulators of squamosa promoter binding protein-like (SBP-box) genes. Previous studies have clarified the evolutionary dynamics of their targets; however, there have been no reports on the evolutionary patterns of two miRNA regulators themselves to date. In this study, we investigated the evolutionary differences between these two miRNA families in extant land plants. Our work found that miR529 precursor, especially of its mature miRNA sequence, has a higher evolutionary rate. Such accelerating evolution of miR529 has significantly effects on its structural stability, and sequence conservation against existence of itself. By contrast, miR156 evolves more rapidly in loop region of the stable secondary structure, which may contribute to its functional diversity. Moreover, miR156 and miR529 genes have distinct rates of loss after identical duplication events. MiR529 genes have a higher average loss rate and asymmetric loss rate in duplicated gene pairs, indicating preferred miR529 gene losses become another predominant mode of inactivation, that are implicated in the contraction of this family. On the contrary, duplicated miR156 genes have a low loss rate, and could serve as another new source for functional diversity. Taken together, these results provide better insight into understanding the evolutionary divergence of miR156 and miR529 family in miRNA combinational regulation network.A complete picture of the evolution of miRNA combinatorial regulation requires the synthesis of information on all miRNAs and their targets.  APETALA2 (AP2), and MADS-box gene families, have been shown to be regulated by distinct miRNAs in plants [MicroRNAs (miRNAs) are small, non-coding RNA molecules that regulate gene expression by binding to target mRNA transcripts, leading to either translational repression or mRNA degradation. A growing body of evidence indicates that, in plants, a single miRNA can target and regulate multiple transcripts and conversely, the same genes can be targeted by a number of different miRNAs \u20133. With n plants ,3. HowevMiR156 and miR529 have been demonstrated to cooperatively target squamosa promoter binding protein-like (SBP-box) genes [miR156 and miR529 is still a mystery in plants until now. However, we found that miR156 and miR529 are significantly different in three aspects. First, analysis of miRNA deep sequencing expression profile from rice has shown that miR156 genes are ubiquitously expressed in the root, shoot and other tissues at the seedling stage, whereas miR529 genes are only detected in the panicle throughout development [Brachypodium distachyon, suggesting that ancient miR156 and miR529 genes have formed conserved expression patterns over long evolutionary periods [miR156 is ubiquitous throughout land plants and its regulatory circuit is extremely well conserved throughout plant evolution [miR529 is only present in bryophytes, lycopods, and monocots, and displays limited taxonomic distributions [miR529 is at least 400 million years old and may have been lost from all core eudicots and that the evolutionary forces driving the loss of this regulatory system are intriguing. Finally, miR529 is decreased in monocots compared to moss, whereas miR156 is increased in number [miR156 family and sharp contraction of the miR529 family during plant evolution. The aforementioned differences between miR156 and miR529: expression pattern, taxonomic distribution, and the number of members in the miRNA family hinted that these two miRNA families may have undergone different evolutionary pathways.x) genes . They sh periods ,8. Seconvolution . By contibutions ,12. ThesmiR156 and miR529 families from moss to flowering plants. By comparing their evolutionary rate, thermodynamic stability, sequence conservation, and rate of gene loss after gene duplication, we will reveal the differences of evolutionary dynamics between miR529 and miR156. And, the correlations between the different evolutionary dynamics of these two miRNA families and their observed differences in extant land plants will be also discussed.The aim of this study was to explore the evolutionary differences between miR156 sequences are registered in miRBase database (release 21) [miR529 exists in several land plant organisms, but some miR529 genes had been obtained by similarity search and had not been validated with sufficiently experimental evidence prior to their addition to miRBase. To eliminate potential inaccuracies, these miR529 sequences were excluded from our analyses. Finally, a total of 11 miR529 members were selected from four plant species: Physcomitrella patens, Oryza sativa subsp. japonica, Brachypodium distachyon, and Zea mays. All precursor and mature sequences of miR156 and miR529 were downloaded from miRBase (release 21) [miR156 and miR529 genes used in this study is summarized in Although over 300 ease 21) , only 43miR156 and miR529 were separately aligned using ClustalX [The precursor sequences of ClustalX  and adjuClustalX ,15. PrevClustalX ,17. TherClustalX : mature miR156 and miR529 precursor structure using default settings. Moreover, we eliminated the influence of the length of the miRNA precursor sequence by normalizing MFE as previously described [RNAfold  was usedescribed . In thismiR156 and miR529 was estimated via the online software Clustal Omega  with the default setting. The same method was also applied to calculate sequence identify of mature alignments of miR156 and miR529.The sequence identity of precursor alignments of miR156 and miR529 gene duplications because it has no less than two members in each miRNA family and a good genome annotation. Segmental duplications of miR156 and miR529 genes in rice genomes were retrieved from the Plant Genomic Duplication Database (PGDD) dataset [miR156 and miR529 gene as guides to identify the duplicated block in which a miR156 or miR529 gene resides. The syntenic relationship between each miRNA gene pair in the duplicated block was visualized using MicroSyn [Tandem duplication and segmental duplication significantly contribute to gene family expansion. Thus, we mainly focused on these two patterns of gene expansion in this study. Tandem duplications are characterized as multiple members of a gene family occurring within the same or neighboring intergenic regions and segmental duplications are defined as segments of DNA with near-identical sequence that map to two or more genomic locations. Of the four organisms analyzed in this study, we chose rice as the representation for analyzing  dataset . In this dataset . We therMicroSyn .\u20139 substitutions/synonymous site/year [The rates of synonymous substitution (Ks) of duplicated genes are expected to be similar over time . Therefoite/year .The rate of gene loss was estimated according to Wang\u2019s method . The twoWhere, S1 is the number of single-copy genes in copy 1; N2 is the number of the extant genes in copy 2.miR529 genes might have higher evolutionary rate than broadly expressed miR156 genes. To test this hypothesis, we first measured the evolutionary rates of the precursor miRNA (pre-miRNA) between the two miRNA families using MEGA with the Kimura 2-parameter model [miR529 precursor sequences was higher than that of miR156 precursor sequences; however the differences were not statistically significant (p>0.05). Subsequently, we measured the independent evolutionary rates for the four parts of the precursor miRNA. In general, mature miRNA, as the functional part of the precursor miRNA, was well conserved between distant lineages and fewer mutations were found within its sequence, while the loop was the most variable region of the precursor sequence. As may be expected, the evolutionary rate was highest in the loop-containing region in both miR156 and miR529 families (miR529 family members than in the miR156 family members (p<0.01). The evolutionary rates in the stem and miRNA* regions were comparable between both miRNA families. Unexpectedly, the evolutionary rate of miR529 sequences was two times higher than that of miR156 sequences in the mature miRNA region (p<0.01), which is contrary to our findings in the loop region. Collectively, these data suggest that miR529 has higher evolutionary rate in precursor, especially in mature sequences, whereas an accelerating evolution of miR156 precursor occurred in its loop region.It has been reported that ubiquitously expressed genes evolve more slowly than tissue-specific genes, which suggests that the extent to which genes are expressed is critical for their evolutionary rates in multicellular organisms . Therefoer model ,15. The families . InteresmiR529 shows higher evolutionary rate than miR156 in its precursor sequence; however the difference is small   . After n(p<0.01) .miR156 and miR529. As shown in miR156 was higher than that of miR529 (p<0.01). When only the mature sequences were considered, the difference of sequence identity between miR156 and miR529 was considerably dramatic. Pairwise sequence identity was over 95% and 77% for mature miR156 and miR529, respectively . This observation agreed with previous reports suggesting that the miR156 family mainly arose from large scale segmental duplication [miR529 family, two miR529 family members (miR529a and miR529b) reside within the duplicated block 50, indicating that miR529 genes also originated from segmental duplication. In addition, two duplicated gene pairs (miR156b/miR156c and miR156h /miR156j) of the miR156 family are located within the same gene and have been characterized as tandem duplications. However, the tandem duplication cannot be inferred because the two miR529 members in rice were characterized as segmental duplicated pairs. As a result, we investigated whether tandem duplication was the mechanism by which multiple members of the miR529 family arose in moss, though its genome is not well annotated. Our results indicate that three miR529 gene pairs of seven genes were located in neighboring regions and the distance between each miRNA pair was less than 100 nt, which satisfies the criteria for tandem duplication (data not shown). Consequently, we can infer that similar duplication mechanisms, including segmental duplication and tandem duplication are at work in the evolution of the miR529 family, as is the case with the miR156 family.Previous studies suggest that tandem and segmental duplications dominated the expansion of the 6 family ,29. As amiR529 resides are almost at the same level as the Ks values calculated for the other four blocks. This means that the segmental duplication events involving miR156 and miR529 genes occurred approximately 70 million years (Myr) ago, which is consistent with the time at which genome duplication events took place in rice [miR156 and miR529 genes produced in the same genome duplication.In addition, we used synonymous silent substitutions per site (Ks) as a proxy for time to estimate the approximate time of occurrence of the segmental duplication events. As can be seen from  in rice ,30. ThermiR156b and miR156c are tandem duplications, yet there is only one corresponding miRNA, miR156l, residing within duplicated block 19. To help estimate the loss rate of the miR156 and miR529 genes, we tailed the number of flanking protein-coding genes of one miRNA copy that does not have its counterparts in the other duplicated copy within the same block. Then, the rates of gene loss were compared between blocks and between two copies of the same block. As seen in miR529 genes resided had a higher average rate of loss than other four duplicated blocks  in which miR156 genes are located (miR529 duplicated pairs have greatly asymmetric rates of gene loss. One copy (copy 2) showed the highest percentage (70%) of gene loss, while the other copy had only 28% of gene loss. By contrast, the majority of miR156 duplicated gene pairs showed very little difference of the gene loss rate between the two copies  [Our results suggest that narrowly expressed fference . TherefoLncRNAs) , indicatmiR529 have a lower evolutionary rate than those of miR156 and the opposite trend was observed in the mature miRNA sequences , and the asymmetric rate of gene loss makes up the important evolutionary forces that cause the contraction of this miRNA family. An extreme phenomenon is that all the miR529 members are extinct in core eudicots. Our previous work demonstrated stronger purifying selection against mutations within the binding sites of miR529 targets [miR529 targets also are cooperatively controlled by miR156. If the mutations occurred on the binding site of miR529 targets, they would be bound to disrupt ligand binding between miR156 and this same target. Therefore, the economical and balanced way to change miR529 combinatorial regulation is to change miR529 itself. Indeed, the retention of miR529 binding sites in some members of SBP-box genes but no miR529 candidate in core eudicots gave it a full interpretation . It is S1 FigError bars indicate the standard error of the mean.(TIF)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file."}
+{"text": "The aim of the study was to evaluate the effects of micro-electric current on sodium hypochlorite\u2019s (NaOCl\u2019s) tissue-dissolution abilities, compared with other activation methods, including sonic, ultrasonic, pipetting, and temperature.n\u2009=\u2009154) with standard sizes and weights were prepared and divided into two temperature groups: room temperature and 45\u00a0\u00b0C. Each temperature group was divided into seven sub-groups by activation methods: D\u2009=\u2009distilled water (\u2212control); NaOCl\u2009=\u20095.25\u00a0% passive NaOCl (+ control); P\u2009=\u20095.25\u00a0% NaOCl with pipetting; SA\u2009=\u20095.25\u00a0% NaOCl with sonic activation; UA\u2009=\u20095.25\u00a0% NaOCl with ultrasonic activation; E-NaOCl\u2009=\u20095.25\u00a0% NaOCl with micro-electric current; and E-NaOCl\u2009+\u2009P\u2009=\u20095.25\u00a0% NaOCl with micro-electric current and pipetting. Specimens were weighed before and after treatment. Average, standard deviation, minimum, maximum, and median were calculated for each group. Resulting data were analyzed statistically using multi-way ANOVA and Tukey HSD tests. The level of the alpha-type error was set at\u2009<\u20090.05.Bovine muscle tissues (p\u2009<\u20090.05), and the UA, SA, and P groups dissolved significantly higher amounts of tissue than did the positive control or E-NaOCl groups (p\u2009<\u20090.05). At 45\u00a0\u00b0C, there was no significant difference between the SA and E-NaOCl groups (p\u2009>\u20090.05), and the E-NaOCl\u2009+\u2009P group dissolved a higher amount of tissue than any other group (p\u2009<\u20090.05).At room temperature, the E-NaOCl\u2009+\u2009P group dissolved the highest amount of tissue (Using NaOCl with micro-electric current can improve the tissue-dissolving ability of the solution. In addition, this method can be combined with additional techniques, such as heating and/or pipetting, to achieve a synergistic effect of NaOCl on tissue dissolution. Successful root-canal treatment depends on removing micro-organisms, which cause infection of pulp tissue, and dentin debris from the root canal , 2. IrriNaOCl has a dynamic balance that tends to change direction continuously, as the formula below shows .2O \u2194 NaOH\u2009+\u2009HOCl \u2194 Na+ + OH\u2212\u2009+\u2009H+ + OCl\u2212.NaOCl\u2009+\u2009HExternal factors that change this dynamic balance also change NaOCl\u2019s efficiency. Although NaOCl has many properties, activation techniques as an external factor affect the dynamic balance of NaOCl use, increasing its tissue dissolution ability, based on activation with sonic or ultrasonic devices, and heating the solution , 9, 10. Recently, we demonstrated that micro-electrical activated NaOCl increased the tissue dissolution capacity of the solution .The null hypothesis was that the micro-electrical energy can increase the tissue dissolution efficiency of sodium hypochlorite solution as well as conventional activation methods such as heating, pippeting, sonic & ultrasonic energy.The purpose of this in vitro study was to compare micro-electric current activation with other methods, such as sonic, ultrasonic, and heat activation on the dissolution ability of NaOCl.This in vitro study conducted on bovine muscle model optained from a public butcher. Therefore authors stated that ethical approval from commitee of human or animal researches was not necessary.Wizard\u2122  NaOCl solution, with a concentration of 5.25\u00a0% chlorine was determined by the iodine/titration method. Prior to the experiments, the NaOCl concentration was kept at +4\u00a0\u00b0C.Bovine muscle tissue was used for these tissue-dissolution experiments. Muscle tissue was kept at \u221216\u00a0\u00b0C and in a 100\u00a0% humid medium. To standardize size and weight, samples were collected with a biopsy punch with a 5-mm diameter  from a 2-mm tissue piece cut from muscle tissue. Prior to testing, samples were weighed with a digital precision scale  and put in a 10-mL NaOCl solution. Before treatment with NaOCl, mean weight of tissue samples was 38\u2009\u00b1\u20091\u00a0mg.In accordance with a study by Stojicic et al., the experiments were conducted at room temperature (25\u00a0\u00b0C) and at 45\u00a0\u00b0C . ExperimSpecimens were divided into two groups according to temperature. Then, each temperature groups was sub-divided into 7 groups by activation methods. These 14 groups contained 154 tissue samples, 11 in each group (Table\u00a0The experiments tested 3 current-activation methods: ultrasonic (UA), sonic (SA), and pipetting (P). In the ultrasonic experiments, the stainless steel size #25 ultrasonic tip  was operated at moderate speed in the solution. The EndoActivator\u2122 using polymer tip no. 25/04 was run at 10,000\u00a0cpm in the solution. Tips used in ultrasonic and sonic activation were submerged up to 10\u00a0mm in the NaOCl solution and operated at a distance of approximately 5\u00a0mm from the tissue. For pipetting, in accordance with Stojicic et al. [In addition, 2 micro-electric methods were used in the experiments: single micro-electric energy (E-NaOCl) and micro-electric energy with pipetting (E-NaOCl\u2009+\u2009P). A potentiometer  was calibrated to supply 10\u00a0mA to the NaOCl . At room temperature, the UA, SA, and P groups dissolved significantly higher amounts of tissue than did the E-NaOCl group (P\u2009<\u20090.001 for both comparisons), and the E-NaOCl\u2009+\u2009P group dissolved the highest amount of tissue (p\u2009<\u20090.05).All activation groups at both temperatures dissolved significantly higher amounts of tissue than did the negative control groups (P\u2009<\u20090.05). At 45\u00a0\u00b0C, the UA and P groups dissolved significantly higher amounts of tissue than did the E-NaOCl group (P\u2009<\u20090.001 for both comparisons). The E-NaOCl\u2009+\u2009P group dissolved higher amounts of tissue than did any other group, including those groups at room temperature (P\u2009<\u20090.05).As Table\u00a0Many studies have been conducted on the tissue-dissolving abilities of NaOCl. These studies have demonstrated that NaOCl\u2019s dissolution effect changes as its concentration, pH, surface tension, and temperature change. In addition, agitation methods increase NaOCl\u2019s dissolution effect , 15, 16.Lumley et al. determined 100\u00a0\u03bcm and less to be the distance limit for creating cavitation during ultrasonic action . In the p\u2009<\u20090.05). We also obtained positive combination results on NaOCl activated with micro-electric current, heat, and agitation methods on NaOCl\u2019s tissue-dissolving ability. This can be explained by the finding that when a micro electric current is activated NaOCl, the dynamic balance of the solution may change.We reported, for the first time, that micro electrically activated 5.25\u00a0% NaOCl has better results than 5.25\u00a0% NaOCl without any activation on tissue dissolution efficiency . Micro eP\u2009<\u20090.0001). Some previous studies have demonstrated that ultrasonic-activated NaOCl cleaned root canals successfully [The present study found no significant difference between sonic, ultrasonic, and pipetting activation at room temperature. These results conform to those found by Stojicic et al. . Conventessfully \u201325. Howeessfully \u201328. The In previous studies, electrolysed water was used as a canal-washing solution , 30. In Within the limitations of the present study combined use of micro-electric energy, heat, and agitation had a positive, synergistic effect on sodium hypochlorite\u2019s tissue-dissolving ability. However, further studies should be conducted on the micro-electric energy to better understand this technique in practice."}
+{"text": "The authors wish to retract this publication due to errors, brought to their attention by a reader, which were made in preparing figures. The figures in question are 2D, 6B and 6G in which the actin panels have been duplicated, Fig 5C and 5D where too much contrast or brightness was used, Fig 7C in which the second panel from the top has been scaled incorrectly and finally 3B and 8B which were assembled inaccurately.All of these published figures were prepared and assembled prior to publication by the first author Dr. Apiruck Watthansurorot. Given the errors noted above, these figures cannot be considered to reliably represent the data collected. Although replicates are available for the figures, the authors are retracting this publication, due to the possibility of misrepresentation of the data. All of the authors believe that it is necessary to confirm that the conclusions drawn are corrected for publication in the future.PLOS Genetics for these errors.All of the authors have agreed to this retraction. The authors apologize to the readers and editors of"}
+{"text": "However, genetic factors associated with lung function in individuals with exceptional longevity have not been identified.Reduced forced expiratory volume in 1\u00a0second , smoking status, pack-years, and field center.We conducted a genome wide association study (GWAS) to identify novel genetic variants associated with lung function in the Long Life Family Study (LLFS) . Replication was performed using data from the CHARGE/SpiroMeta consortia. The association between SNPs and FEVCYP2U1 gene to be associated with FEV1 and a novel SNP (rs889574) associated with FEV1/FVC, none of which were replicated in the CHARGE/SpiroMeta consortia. Using linkage analysis, we identified a novel linkage peak in chromosome 2 at 219\u00a0cM for FEV1/FVC (LOD: 3.29) and confirmed a previously reported linkage peak in chromosome 6 at 28\u00a0cM (LOD: 3.33) for FEV1.We identified nine SNPs in strong linkage disequilibrium in the 1.Future studies need to identify the rare genetic variants underlying the linkage peak in chromosome 6 for FEVThe online version of this article (doi:10.1186/s12931-014-0134-x) contains supplementary material, which is available to authorized users. Pulmonary function measures such as forced expiratory volume in one second FEV, forced  history -9. The Dortality . Due to ortality . We haveortality  suggestiortality . SeveralFEV1/FVC -16. Howeth FEV1) -19 in ge in FEV1 . Further in FEV1  and a pr in FEV1  suggest The LLFS study design has also been described in detail previously . BrieflyAfter excluding 15% of the participants due to presence of non-European ancestry (n\u2009=\u20096), low quality spirometry (defined as 2 or more acceptable spirometry maneuvers with reproducibility within 250\u00a0mL) (n\u2009=\u2009295), self-reported pulmonary fibrosis (n\u2009=\u200911) obtained during an in-person interview, history of lung volume reduction surgery (n\u2009=\u200914), or missing genotypes (n\u2009=\u2009344), a total of 3,889 participants were included in the present analysis.The examinations were conducted in the home setting with portable equipment by centrally trained and certified research assistants using a standardized protocol. Lung function was measured with a portable spirometer  following American Thoracic Society guidelines . Calibrap <1E-06, and with missing genotypes were excluded. Ancestry PCs produced from unrelated subjects were expanded, within EIGENSTRAT framework, to all members of LLFS. Genotype imputations were performed based on the cosmopolitan phased haplotypes of 1000 Human Genome  using MACH and MINIMACH [2\u2009>\u20090.3 for imputed SNP filters were applied to the hybrid dataset for analysis, the number of SNPs for analysis is reduced to 6,522,421 , of which 1,204,935 SNPs were genotyped and 5,317,486 SNPs were imputed.The Human Omni chip 2.5 v1 , was used to genotype all the LLFS participants at the Center for Inherited Disease Research (CIDR). Ancestry principal components (PCs), to control for population structure, were produced with EIGENSTRAT  on 1,515MINIMACH ,25 and a1 and FEV1/FVC) were identical to the models used by the CHARGE/SpiroMeta consortia [2, sex, height, field center and ancestry PCs (PC1-20) in addition to the kinship matrix. The adjusted phenotypic residuals from these models (FEV1 and FEV1/FVC) were inverse normal transformed to normally distributed z-scores. These transformed residuals were then used as the phenotype for association testing under an additive genetic model, separately for ever smokers and never smokers. The associations between individual SNPs the FEV1 (milliliters) and FEV1/FVC (percent) were analyzed using a linear mixed effects model with kinship structure [The statistical models used to test the association between the GWAS SNPs and lung function , pack-years, and field center. The SOLAR package was also used to estimate heritability and empirical p of LOD.To calculate Identity by Descent (IBD) for the linkage analyses, the ZAPLO program was used to estimate haplotypes of SNPs in small regions  . The deC2 (standard deviation: 4.79 Kg/m2). There were 2,203 (57%) never smokers, 1,403 (40%) former smokers and 283 (3%) current smokers. The average number of cigarettes smoked among former smokers was 20.26 pack years (standard deviation: 22.07 pack years) while the average number of cigarettes smoked among current smokers was 28.25 pack years (standard deviation:19.03 pack years). There were 89 participants (2.3%) with self-reported history of chronic obstructive pulmonary disease (COPD) and 339 (8.7%) participants with self-reported history of asthma, 123 (3.1%) participants with a self-reported history of congestive heart failure and 11 (0.28%) participants with a self reported history of lung cancer. As shown in Table\u00a01  and FEV1/FVC  as compared to the CHARGE/SpiroMeta consortia  male participants and 2155 (55%) female participants with an average age of 68.6\u00a0years (standard deviation: 15.2\u00a0years) and an average BMI of 27.13\u00a0kg/m1 and FEV1/FVC are shown in Additional file 1 and FEV1/FVC are shown in Additional file 1 (p\u2009<\u20095E-06)  . In addition, 7 imputed SNPs in the CYP2U1 gene and 1 imputed SNP in the PHACTR2 gene also showed a borderline association with FEV1 (p\u2009<\u20099.2E-07). However, none of these SNPs were associated with FEV1 in the CHARGE/SpiroMeta consortia or in the overall meta-analysis. Five of the 9 previously identified GWAS SNPs (p\u2009<\u20091E-07) for FEV1 were nominally associated with FEV1 in LLFS (p\u2009<\u20090.05)  in the LLFS GWAS, (Table\u00a01/FVC in the LLFS (p\u2009<\u20090.05)  at the q terminus of chromosome 6   over a larger range (10\u00a0cM \u2013 50\u00a0cM) further attenuated the linkage peak (LOD = 2.60) but did not completely explain the linkage peak    ) as the present study [1. While we found a modest attenuation of the FEV1 linkage peak after adjustment for common GWAS SNPs under the linkage peak that were nominally associated with FEV1 (p\u2009<\u20090.001), they did not completely explain the linkage peak. In contrast, the novel linkage peak identified in chromosome 2 at 219\u00a0cM (~372\u00a0kb) for FEV1/FVC was almost completely explained, by adjustment for the common GWAS SNPs under the linkage peak that were nominally associated with FEV1/FVC (p\u2009<\u20090.001). These findings suggest that common variants alone are insufficient to explain some linkage peaks such as the linkage peak in chromosome 6 for FEV1. As shown in other diseases, the inability to identify association under linkage peaks could in part be attributable to the fact that only common variants are examined under the linkage peak whereas the linkage signal could be caused by multiple rare variants with higher penetrance [1 needs to be evaluated in future studies. The linkage peak identified in chromosome 6 at 28\u00a0cM (~280\u00a0kb) for FEV1 does not contain any known genes. However, there are several DNase I hypersensitivity sites and putative transcription factor binding sites (9H3K27Ac marks) that have been identified in cells from pulmonary epithelium and blood vessels derived from the pulmonary artery within this linkage peak (EnCode data) suggesting that regulatory elements in this region may play an important role in determining lung function. The linkage peak for FEV1/FVC in chromosome 2 at 219\u00a0cM (~373\u00a0kb) contains the genomic region that codes for the DIRC3 gene, a non coding RNA that is involved in the pathogenesis of familial renal cancers (EnCode data). Though DIRC3 is expressed in pulmonary tissue its role in determining lung function has not been evaluated. Furthermore, this region also contains DNase I hypersensitivity sites or putative transcription factor binding sites (9H3K27Ac marks) in cells derived from the pulmonary epithelium or vasculature. Thus the linkage peaks identified in this study on chromosomes 2 and 6 may indicate previously unidentified regulatory pathways that may influence longevity through their effect on lung function.Two previous reports identified a linkage peak for FEVnt study ,33. As cnetrance . Hence, 1 and FEV1/FVC, none of these findings could be replicated in the CHARGE/SpiroMeta consortia. However, only 38% of SNPs associated with FEV1 or FEV1/FVC in LLFS were available for replication in the CHARGE/SpiroMeta consortia. Thus, the remaining SNPs and insertion/deletions polymorphisms associated with FEV1 and FEV1/FVC in LLFS but not genotyped/imputed in the CHARGE/SpiroMeta consortia will need to be evaluated in future studies. Since the LLFS study participants were not randomly selected to represent the general population but were specifically selected for their family history of exceptional longevity, it is possible that there may be some unique genotypes associated with lung function that may not be replicated in studies that are more representative of the general population. One previous study showed that elderly male offspring (range: 65\u201389 years) with long lived parents (age at death of at least on parent >80\u00a0years) had FEV1 that was 330\u00a0ml larger than FEV1 for male offspring with short lived parents even after controlling for smoking [1 and FEV1/FVC identified in previous meta-analysis (Additional file Though this study identified a few novel GWAS SNPs that were associated with FEV smoking . The fin smoking  might in1, this study also identified a novel linkage peak in chromosome 2 for FEV1/FVC. Repeated measurements of lung function in this study population along with targeted resequencing under the observed linkage peaks in future studies may help clarify the role of genetic variants in determining preserved lung function among exceptionally long lived individuals.The family-based cohort design of the LLFS with extensive genotype information and detailed lung function measurements makes this study a valuable resource to identify genetic determinants of lung function. In addition to confirming some of the previously identified GWAS SNPs and a previously identified linkage peak in chromosome 6 for FEV"}
+{"text": "The construction of high-density linkage maps appears more feasible since the advent of next-generation sequencing (NGS), which eases SNP discovery and high-throughput genotyping of large population. However, the marker number explosion and genotyping errors from NGS data challenge the computational efficiency and linkage map quality of linkage study methods. Here we report the HighMap method for constructing high-density linkage maps from NGS data. HighMap employs an iterative ordering and error correction strategy based on a k-nearest neighbor algorithm and a Monte Carlo multipoint maximum likelihood algorithm. Simulation study shows HighMap can create a linkage map with three times as many markers as ordering-only methods while offering more accurate marker orders and stable genetic distances. Using HighMap, we constructed a common carp linkage map with 10,004 markers. The singleton rate was less than one-ninth of that generated by JoinMap4.1. Its total map distance was 5,908 cM, consistent with reports on low-density maps. HighMap is an efficient method for constructing high-density, high-quality linkage maps from high-throughput population NGS data. It will facilitate genome assembling, comparative genomic analysis, and QTL studies. HighMap is available at  Linkage maps, especially high-density ones, play an important role in the study of genetics and genomics. Application of high-density linkage maps has greatly facilitated discovery of functional genes Great efforts have been made to study algorithms for constructing high-density and high-quality linkage map Several practical strategies have been used to tackle the difficulties in constructing high-density linkage map in species such as sunflower Here, by using an iterative ordering and error correction strategy, we present an efficient method that simplifies and enhances the construction of high-density, high-quality linkage map from high-throughput population NGS data (HighMap). Our studies reveal that HighMap has excellent performance of high-density linkage map construction. HighMap provides an important tool for understanding genetics and genomics.All experimental procedures were conducted in conformity with institutional guidelines for the care and use of laboratory animals in Centre for Applied Aquatic Genomics of the Chinese Academy of Fishery Sciences. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Centre for Applied Aquatic Genomics at Chinese Academy of Fishery Sciences (2011AA1004020012).k-nearest neighbor algorithm to correct genotyping errors and impute genotyping missing SARF) as objective function and adopted Blocked Gibbs sampler after trying different Gibbs sampling methods and different objective functions in simulated annealing. HighMap consists of four modules, designed for linkage grouping, marker ordering, error genotyping correction and map evaluation, respectively . JoinMap4.1 was incapable of grouping large data of markers. To compare ordering performance of HighMap with that of JoinMap4.1, we used HighMap rather than JoinMap4.1 to cluster the marker data when we constructed the linkage map using JoinMap4.1.HighMap performance was further confirmed using the sequencing data of a real full-sib family of common carp which consisted of 211 offsprings Depth and quality of sequencing reads fluctuate randomly across genomes due to sampling randomness. To ensure genotype quality, reads with low depth of sequencing should be discarded in the process of genotype calling Comparative analysis revealed that HighMap permitted the utilization of more markers than JoinMap4.1 . HighMapHighMap contains an error correction algorithm, which can impute missing observations and eliminate erroneous genotyping from mapping data in the mapping process. Data showed that the algorithm is efficient. Take the dataset of 700 markers as an example. We introduced 10.25% of erroneous data and 12.86% of missing observations . After eA singleton is a single locus in one offspring which is different in parental origin from both its direct neighboring loci To assess the performance of HighMap in marker order accuracy and map distance stability, simulation data set was generated from the full-sib family consisting of 200 offsprings with 200 markers, which contained different missing observations or different genotyping errors. Results showed that the Spearman correlation coefficient between the true and calculated marker order based on HighMap decreased less obviously than that of JoinMap4.1 as the marker error rate increased. The differences of the correlation coefficient between HighMap and JoinMap4.1 were more pronounced when error rate exceeded 20% . This reMap distance expansion is mainly caused by genotyping errors and the map distance reflects the quality of a linkage map. In the presence of genotyping errors, it may be necessary to make a balance between controlling the expansion of map distance and ensuring validity of the marker order. We gave the priority to address issue of marker order accuracy for it is more important than the map distanceComputational efficiency is a concern in linkage mapping. Both grouping and ordering are important in the construction of high-density linkage map. JoinMap still suffers from the limit of marker number in the linkage grouping To test HighMap performances on real data, we generated a high-density linkage map of common carp based on a full-sib family NGS data. The integrated map was comprised of 10,004 markers with an average of 11 fold sequencing depths. Among them, 19% were the data with sequencing depths of less than 5 fold. The segregation patterns for these markers are shown in In this study, we intended to develop a method that can efficiently utilize NGS data and ease the construction of high-density and high-quality linkage map. The challenges of such an effort are associated with the marker density explosion and potential genotyping errors, which involve sequencing depth and sequence heterozygosity. The higher the heterozygosity is, the more the genotyping is prone to error. As was shown in the simulation study, the error rate reached up to 34.1% for markers with ab\u00d7cd segregation pattern when markers were sequenced at one fold depth, for markers with ef\u00d7eg segregation pattern it arrived at 21.3%, and for markers with hk\u00d7hk or nn\u00d7np or lm\u00d7ll segregation pattern, the error rate stood at 17.4% . To addrLinkage maps are widely used in marker-assisted selection, quantitative trait loci mapping, and comparative genome analysis. They are also necessary to anchor scaffolds on chromosomes during genome assembly. Due to the limitation of marker density and population size of linkage maps, there left many scaffolds unanchored or unordered in genome assembly recently published. For example, the cacao genome had only 67% and 50% of assembled sequences anchored and ordered, respectively In summary, we offer a computationally efficient method for linkage mapping using population NGS data. The development of HighMap should propel the application of NGS in linkage mapping. It is a lasting task to make full use of NGS data at lower cost and to construct high-density linkage maps. Great efforts are guaranteed to further improve the potentials of NGS data utilization in the linkage studies.Figure S1Simulation data sets containing both the missing and erroneous markers. Missing and erroneous rates increased simultaneously as markers increased from 100 to 1,000.(TIF)Click here for additional data file.Figure S2Changes in linkage map quality as missing observation increased. The X-axis indicates missing observation. The Y-axis indicates Spearman rank correlation coefficient between estimated map marker order and true marker location for A, B and C, singleton rates for D, E and F, estimated genetic map distances for G, H and I, respectively. \u201cIntegrated\u201d, \u201cFemale\u201d, and \u201cMale\u201d indicate integrated, female, or male linkage maps, respectively.(TIF)Click here for additional data file.Figure S3Computational speed of HighMap. Running time was reported as number of 100 seconds. JoinMap4.0 is computationally demanding when marker data contained more than 200 markers.(TIF)Click here for additional data file.Figure S4Heat maps of pair-wise recombination of the common carp. Yellow color represents tight linkage; red represents weak linkage; blue represents no linkage.(TIF)Click here for additional data file.Figure S5Haplotype maps of the family of common carp consisting of 211 offsprings. Each two columns represent the genotype of an individual. Rows correspond to genetic markers. Green and blue boxes indicate one chromatid from parents; gray boxes indicate missing data.(JPG)Click here for additional data file.Figure S6HighMap indicates the Spearman correlation coefficient between marker order of linkage map estimated by HighMap and genome sequences of zebra fish. rJoinMap4.1 indicates the Spearman correlation coefficient between the marker order of linkage map estimated by JoinMap4.1and the genome sequences of zebra fish.The difference between the correlation coefficient of HighMap and JoinMap4.1. r(JPG)Click here for additional data file.Table S1Segregation patterns of common carp linkage map.(DOC)Click here for additional data file.Table S2Singleton rate of common carp linkage map estimated by HighMap and JoinMap4.1.(DOC)Click here for additional data file.Table S3Genetic distance of common carp linkage map estimated by HighMap and JoinMap4.1.(DOC)Click here for additional data file."}
+{"text": "Fusarium oxysporum, an important replant disease pathogen in Pseudostellaria heterophylla rhizospheric soil. Moreover, HPLC was used to identify phenolic acids in root exudates then it was further to explore the effects of the phenolic acid allelochemicals on the growth of F. oxysporum f.sp. heterophylla. The amount of F. oxysporum increased significantly in P. heterophylla rhizosphere soil under a consecutive replant system as monitored through qPCR analysis. Furthermore, the growth of F. oxysporum f.sp. heterophylla mycelium was enhanced by root exudates with a maximum increase of 23.8%. In addition, the number of spores increased to a maximum of 12.5-fold. Some phenolic acids promoted the growth of F. oxysporum f.sp. heterophylla mycelium and spore production. Our study revealed that phenolic acids in the root secretion of P. heterophylla increased long with its development, which was closely related to changes in rhizospheric microorganisms. The population of pathogenic microorganisms such as F. oxysporum in the rhizosphere soil of P. heterophylla also sharply increased. Our results on plant-microbe communication will help to better clarify the cause of problems associated with P. heterophylla under consecutive monoculture treatment.In this study, quantitative real-time PCR (qPCR) was used to determine the amount of  P. heterophylla root tubers are commonly used to treat chronic diseases associated with the lung and as a spleen tonicP. heterophylla under consecutive monoculture.PP. heterophylla, but also in many other greenhouse crops, fruits, forages, horticultural and medicinal plants. Several groups of chemicals have been implicated for autotoxicity, including terpenoids, phenolics, steroids, alkaloids and cyanogenic glycosides789Three major causes have been described for consecutive monoculture problems in previous studies: imbalance of soil nutrients, autotoxicity of root exudates and shifts in the microbial community345p-coumaric acid in cucumber root exudates significantly affects soil microbial communities in the rhizosphere and promoted the growth of soil-borne pathogensHowever, many other studies suggested that the autotoxicity of root exudates could shape rhizosphere microbiology by deterring or attracting specific microbial species1113P. heterophylla monoculture system. We hypothesize that some allelopathic compounds may have an indirect detrimental impact on plants, and predispose roots to infection by pathogenic fungi through certain biochemical and physiological interactions. Additionally, root allelochemicals in the rhizosphere have an ecological role in plant-microbe-soil interactionsBased on our field observations, severe fungal diseases, especially root rot and Fusarium wilt, are commonly found in the Fusarium oxysporium has numerous specialized forms, and is considered one of the most important fungal pathogens of crops that causes root rot and wilt diseaseP. heterophylla. Previous studiesP. heterophylla growth and development. Nevertheless, how the specific pathogenic microorganism such as F. oxysporum varies in consecutive monoculture system and how the root exudates affect the soil-borne fungi under in vitro conditions still remain unknown. Therefore, the aim of the study is to quantify the population of the specific pathogen in rhizosphere soils of P. heterophylla with different years of monoculture using qPCR, and consequently to further assess the stimulatory effect of allelochemicals produced by host on the growth of soil-borne fungi and its relationships with consecutive monoculture problems. This paper provides a new insight into understanding the chemoecological process of host\u2013pathogen interactions in rhizosphere, and attempts to explain the increased incidence of root rot and Fusarium wilt disease in the monocultured P. heterophylla.2 = 0.993) (Amplification efficiency E = 10\u22121/slope = 1.898) for our qPCR analysis. The result of qPCR analysis showed a significant increase in the amount of pathogenic F. oxysporum in the rhizosphere of P. heterophylla as the monoculture years increased , the growth rate reached a plateau at 0.67\u2005\u03bcg/mL concentration. Therefore, a single phenolic acid may promote the mycelium growth of F. oxysporum f.sp. heterophylla within a specific concentration range. This result is consistent with the effect of total root exudates on the mycelium growth of F. oxysporum f.sp. heterophylla as observed above. However, for phenolic acids mixture, the promoting rate on mycelium growth increased as the concentration increased, implying that the synergistic effects of phenolic acids might be involved in the mixture.The mycelial growth patterns of m growth . Among tF. oxysporum f.sp. heterophylla spore production at concentrations from 0 to 54\u2005\u03bcg/mL (F. oxysporum f.sp. heterophylla at a range of concentrations. Vanillin showed the greatest enhancement (357%), followed by p-hydroxybenzoic acid (314%) and mix-7 (300%).Phenolic acids showed a significant dose-response effect on the capacity of 54\u2005\u03bcg/mL . TherefoF. oxysporum f.sp. heterophylla increased dramatically as compared with control ; vanillin, y = 13050x \u2013 14191 (R2 = 0.9991); ferulic acid, y = 12194x + 38266 (R2 = 0.9998).Based on the previous results, standard curves for ferulic acid, F. oxysporum f.sp. heterophylla. We observed different patterns in the usage of the three compounds by F. oxysporum f.sp. heterophylla as compared to the control. After 22\u2005h, p-hydroxybenzoic acid and vanillin were completely consumed and after 26\u2005h ferulic acid was completely consumed in the fermentation media  method to study the rhizospheric microbial composition under consecutive monoculture system of P. heterophylla and we found that the poor growth of P. heterophylla under consecutive monoculture system resulted from an altered microbial community F. oxysporum in the rhizosphere of P. heterophylla significantly increased under a monoculture system, to a level fivefold higher than that of the control  in a consecutive monoculture system.The population of Pseudostellaria heterophylla Demonstration District of Agroecological Institute, Fujian Agriculture and Forestry University, Zherong , Fujian, is known as a geo-authentic production zone, i.e., an optimal production area for P. heterophylla. The rhizosphere soil samples of P. heterophylla were collected from both newly planted and replanted fields at the harvesting time, and the adjacent uncultivated field soil sample was collected as a control. A total of five random plots (15\u2005m2) for each rhizosphere soil sample were used in our trial tests.In this study, the trial field located at the Fusarium oxysporum f.sp. heterophylla was isolated from infected P. heterophylla plants grown in the Pseudostellaria heterophylla Demonstration District, the Agroecological Institute, Fujian Agriculture and Forestry University. The separation frequency was 88.5%, and it only infected P. heterophylla plants but was not able to infect the seedlings of other crops . We isolated the pathogenic fungus from the infected parts of P. heterophylla plants again and verified it to be exactly the same as the original strain (Fusarium oxysporum) based on morphological observation, infection characteristic and ITS sequence analysis. It has been preserved in the Agroecological Institute, Fujian Agriculture and Forestry University, P.R. China.The sequencing information of the pathogenic strains was searched using BLAST and homologous comparison. Specific primers YD1/YD2  were desF. oxysporum, F. graminearum, F. solani, Aspergillus flavus, Bacillus amyloliquefaciens, and the different-year monoculture soils was extracted using the cetyl trimethyl ammonium bromide (CTAB) method, and used to test the specificity of the primer sets YD1/YD2 and for subsequent PCR analysis2O. Cycle conditions were 5\u2005min at 95\u00b0C, 35 cycles of 30\u2005s at 95\u00b0C, 30\u2005s at 62.5\u00b0C and 20\u2005s at 72\u00b0C, followed by 10\u2005min of extension at 72\u00b0C and a final hold at 4\u00b0C. PCR products were analyzed on an agarose gel.DNA from 2O). The parameters were as follows: 95\u00b0C for 5\u2005min (denaturation and hot-start activation), followed by 40 cycles of 95\u00b0C for 10\u2005s and 62.5\u00b0C for 20\u2005s. After the qPCR ran, melting curve analysis was performed to verify the specificity of the amplified product under the following conditions: 95\u00b0C for 15\u2005s, 60\u00b0C for 15\u2005s, followed by an increase to 95\u00b0C over 10\u2005min and a hold at 95\u00b0C for 15\u2005s.The Foh106 fragment was gel-purified, cloned into the PeasyTM-T4 Zero Cloning Kit , and sequenced. The sequenced DNA was reamplified using YD1/YD2 from the plasmid and purified using a TIAN pure Mini Plasmid Kit (TIANGEN BIOTECH (BEIJING) CO., LTD). The concentration of the target DNA (Foh106) was determined using a spectrophotometer , and was then diluted to 5, 4, 3, 2, 1, 0.1, 0.01, and 0.001\u2005ng/\u03bcL. qPCR was monitored on a Mastercycler ep realplex (Germany). Standard curve plotting and melting-curve analysis was performed following the qPCR amplification protocol3 lux and the relative humidity was 50% in the growth chamber. All mediums were then collected after the tissue culture plantlets were incubated for one month and two months, respectively. Blank culture medium was used as a control. A total of 200\u2005mL of methyl alcohol (chromatography) was added to each group mediums. After ultrasonic processing and incubating for several minutes, the extracting solution was centrifuged and concentrated using rotary evaporation at 40\u00b0C until the final volume was 5\u2005mL, and then dried with N2, weighed and stored at \u221280\u00b0C. Thereafter, half of the exudates were dissolved with sterile distilled water to 100\u2005\u03bcg/mL26F. oxysporum f.sp. heterophylla was inoculated onto a 9\u2005cm diameter petri dish and cultivated at 28\u00b0C for 5\u20137 days. F. oxysporum f.sp. heterophylla growth diameter was measured when the control hyphal diameter covered two thirds of the culture dish.Batches of uniform seedlings with healthy roots at the 4-leaf-stage were transferred into sterile robust seedling medium . Each group included three replicates in 30\u2005mL of MS medium and three tissue culture plantlets. The tissue culture plantlets were grown at 25\u00b0C under bacteria-free condition. The illumination time was from 6:00 to 18:00, the light intensity was 3.97 \u00b1 0.15 \u00d7 10F. oxysporum f.sp. heterophylla spores were flooded with sterilized water and the conidia suspensions were filtered through three layers of gauze to separate conidia from myceliumUnder sterile conditions, P. heterophylla were filtered through a 0.22\u2005\u00b5m filter prior to the analysis of the root exudation profiles. The analysis was conducted using an HPLC system  with an ODS - C18 column . The root exudation profiles were determined under the following HPLC conditions, by which eight standard phenolic compounds  could be completely separated. Under these conditions, the mobile phase consisted of methanol (A) and 1% phosphoric acid (B) with a gradient elution of 100\u201350% A plus 0\u201350% B at a rate of 0.8\u2005mL/min for 12\u2005min, 50% A plus 50% B at a rate of 0.8\u2005mL/min for 20\u2005min, 50\u201330% A plus 50\u201370% B at a rate of 0.8\u2005mL/min for 30\u2005min, 30% A plus 70% B at a rate of 0.8\u2005mL/min for 32\u2005min, and 30\u201350% A plus 70\u201350% B at a rate of 0.8\u2005mL/min. The UV detector wavelength was set at 280\u2005nm and the column temperature was maintained at 40\u00b0C. The standard compounds were chromatographed alone and in mixtures. Retention times for the standard compounds and the major peaks in the extracts were recorded. The phenolic compounds from each fraction were identified based on their retention times and the addition of standards to the samplesRoot exudates (100\u2005\u03bcg/mL) of the monocultured p-hydroxybenzoic acid, vanillin, vanillic acid, syringic acid, salicylic acid and cinnamic acid were made in the same ratio as the results by HPLC analysis and diluted to the same concentrations as single phenolic acid dissolved in the basal medium . After 7\u2005h, 0.1% HgCl2 was added to all the experimental groups to stop spore germination. Then the spore germination rate was calculated. Each treatment had three replicates.Based on our preliminary study, seven standard compounds identified in root exudates were selected and made as mother solution, then diluted to 54, 18, 6, 2 and 0.67\u2005\u03bcg/mL in each medium alone, at the same time, the mixtures with ferulic acid, F. oxysporum f.sp. heterophylla (3 \u00d7 106\u2005microconidia/mL) and 64\u2005\u03bcg/mL of each standard phenolic acid  were added to modified Czapek's medium (with sucrose removed). Basal medium was used as the control. F. oxysporum f.sp. heterophylla was then cultivated at 28\u00b0C with 150\u2005rpm rotation. Each group comprised three replicates. The concentration of each phenolic acid was quantified in the liquid fermentation media using HPLC at 4\u20137\u2005h intervals. Standard compounds of the three concerned phenolic acids  were calibrated with 5, 1, 0.5, and 0.1\u2005\u03bcg/mL standard solutions for HPLC chromatography. The standard compounds were injected alone and in mixtures. Retention times for the standard compounds and the major peaks in the medium were recorded. The phenolic compounds from each fraction were identified based on their retention times and the addition of standards to the samples. The HPLC conditions were as following: The column was ODS - C18 , with a mobile phase of methanol and 1% phosphoric acid aqueous solution (30: 70). The UV detector wavelength was set at 280\u2005nm at a rate of 1\u2005mL/min, and the column temperature was maintained at 40\u00b0C.A total of 100\u2005\u03bcL of Analysis of variance was performed using DPS7.05. The differences in the means were determined by calculation of the least significant difference (LSD) at the 5% level.W.X.L. designed the experiments, and Y.P.Z., Z.F.L. and L.X.C. executed the experiments and wrote the manuscript. W.X.L. improved the manuscript. Y.Q.Y., L.K.W., S.A., Z.X.Z. and C.X.F. helped to polish the language. All authors reviewed the manuscript.Dataset 1"}
+{"text": "The objective of this study was to investigate depth perception in astronauts during and after spaceflight by studying their sensitivity to reversible perspective figures in which two-dimensional images could elicit two possible depth representations. Other ambiguous figures that did not give rise to a perception of illusory depth were used as controls. Six astronauts and 14 subjects were tested in the laboratory during three sessions for evaluating the variability of their responses in normal gravity. The six astronauts were then tested during four sessions while on board the International Space Station for 5\u20136 months. They were finally tested immediately after return to Earth and up to one week later. The reaction time decreased throughout the sessions, thus indicating a learning effect. However, the time to first percept reversal and the number of reversals were not different in orbit and after the flight compared to before the flight. On Earth, when watching depth-ambiguous perspective figures, all subjects reported seeing one three-dimensional interpretation more often than the other, i.e. a ratio of about 70\u201330%. In weightlessness this asymmetry gradually disappeared and after 3 months in orbit both interpretations were seen for the same duration. These results indicate that the perception of \u201cillusory\u201d depth is altered in astronauts during spaceflight. This increased depth ambiguity is attributed to the lack of the gravitational reference and the eye-ground elevation for interpreting perspective depth cues. Depth perception is the visual ability to perceive the world in three dimensions and the distance of an object. In a recent investigation we found that the perception of distance and depth of objects was altered in astronauts after several months spent in weightlessness . One intThe objective of this study was to further investigate if the perception of depth was altered in astronauts during and immediately after spaceflight by using reversible perspective figures known to generate ambiguous illusions of depth. Perceiving depth in these figures is a form of illusion because the images are actually two-dimensional. The best known of these ambiguous figures is the Necker cube  that canIllusions that result from the difference between the objective and the subjective features of the environment are a common feature of visual perception. Consequently, illusions are a valuable tool for exploring the adaption of visual perception to unusual representation of the environments . Based oPrevious experiments have compared the perception of reversible figures in subjects upright and tilted supine or on their side, and concluded that the direction of the gravitational vertical influenced their responses , 8. ExpeThis experiment was undertaken with the understanding and written consent of each subject. The test procedures were approved by and in compliance with the standards of the NASA Johnson Space Center Institutional Review Board for human testing and were performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki.Six astronauts  and 14 control subjects , ranging in age from 22\u201353 years (mean 35.5 years) participated in this experiment. All subjects were tested during three sessions on the ground. For the control subjects, the mean interval between the first and the second session was 49 days, and the mean interval between the second and third session was 28 days. The astronauts were tested 10 times in total. They were first tested at approximately launch minus (L-) 220 days, L-160 days, and L-80 days. They were then tested on board the International Space Station during missions lasting 124\u2013187 days (mean 152 days). The in-flight sessions took place on flight days (FD) 5\u201314 (mean FD08), FD20-36 (mean FD26), FD63-102 (mean FD72), and FD106-174 (mean FD134). Finally, they were tested again one day after return to Earth (R+1 day) and at R+5 and R+9 days (\u00b1 1 day depending on subject availability).Two sets of figures were used in this study: the first set induced a depth ambiguity, such as a cube seen from above or from underneath; the second set generated an anthropomorphic ambiguity, such as a man\u2019s head or a begging woman. Because the perception of these images temporarily changes back and forth even as one continues to look at the same stimulus figure, these images are referred to as reversible (or ambiguous) figures.interpretation of perspective, i.e. a mental construction of perceived depth, that can be reversed. Perceiving depth in these figures is a form of illusion because the figures themselves are actually flat.The figures that induced depth ambiguity were two-dimensional (2D) geometrical figures that elicited the perception of the same three-dimensional (3D) object in two different perspectives. These reversible perspective figures included the well-known Necker cube  and threFour other figures were used: the Mach book, the Ames trapezoidal window, the moving plaid , and theThe figures that induced anthropomorphic ambiguity were black or white silhouettes that elicited the reversibility of the meaningful content of what was seen: a man\u2019s face or an old woman begging with her arm extended (based on ), a SparBecause it has been suggested that eye movements to different fixation points could cause reversals  a red doOn the ground, subjects were tested when sitting at a desk. In orbit, the subjects were tested while free-floating to eliminate orientation-related cues . The figSubjects were informed about the two alternative perceptions beforehand. Since we were interested in studying perceptual reversals, we ensured that the subjects knew what the alternatives were. However, we also told the subjects that it was okay not to see the reversal, so that they did not assume that reversal was expected.The subjects held a mini trackball finger mouse with two push buttons. The sample rate of the trackball was 40 Hz. To accurately record both the intervals of perceptual reversal and the perceived interpretation, subjects were asked to press the button on the finger mouse corresponding to the interpretation they perceived. Prior to each recording, subjects were shown the figure and a text message indicating the two different ways in which it could be seen  and the corresponding clicking instructions . Then the ambiguous figure was presented continuously for 1 minute. Subjects were asked to identify which interpretation they saw first, and then to repeatedly indicate when they saw the respective alternate interpretation by pressing down either of the two buttons of the finger mouse.For each figure, the measurements included: (a) the reaction time, defined as the temporal period from stimulus onset to the subject\u2019s first perceptual response; (b) the time to first reversal, defined as the temporal period from the subject\u2019s first percept to the first reversal; (c) the number of reversals; and (d) the percentage of time for seeing each interpretation.The measurements obtained in the two trials were averaged for each session and each subject. One-way ANOVAs were conducted to compare these measurements across the three sessions performed on the ground between the 14 control subjects and the six astronauts. This test was to verify that the responses of our small number of astronauts were within the range of a larger population. Within subjects (repeated measures) ANOVAs were then conducted to compare the effect of session days (10 sessions) and figures  on the various response parameters. When a significant effect of days was seen, paired samples t-tests were used to make post hoc comparisons between the first session and the subsequent sessions.p = 0.07]. A repeated-measures ANOVA in the control subjects\u2019 data yielded a significant difference in reaction time across the 3 test sessions  and the 6 figures  but no interaction between the two  and the 6 figures  and no interaction between the two  and the perspective figures . However, the time to first reversal was longer for the perspective figures than for the silhouettes for both the control subjects  and the astronauts . The time to first reversal averaged across all 20 subjects was 11.7 s (SD 9.1 s) for the perspectives figures and 5.1 s (SD 3.1 s) for the silhouettes. For both perspective figures and silhouettes, no significant difference was found across pre-flight sessions and figures for both the astronauts and the control subjects  = 0.02, subjects .p = 0.588] and the figures . No significant difference in the time to first reversal was observed for the silhouettes either, both across session days  and figures .For the perspective figures, a two-way ANOVA in the astronauts\u2019 data yielded no significant difference in the time to first reversal across the 10 sessions , but was significantly different for the perspective figures . The number of reversals was smaller for the perspective figures than for the silhouettes for both the control subjects  and the astronauts . The number of reversals per minute averaged across all 20 subjects was 8.7 s (SD 5.4 s) for the perspectives figures and 20.5 s (SD 15.4 s) for the silhouettes. For the perspective figures, no significant difference in the number of reversals was found across pre-flight sessions and figures for both the astronauts and the control subjects  = 0.66, subjects . Howeverp = 0.392] and the figures . However, a significant difference in the number of reversals was observed for the silhouettes across session days , but not across figures . The mean number of reversals increased from L-220 to FD08 and stabilized thereafter, but with a larger variability across subjects  = 1.06, subjects .Because previous studies have reported an inverse correlation between the time to first reversal and the number of reversals , 20, we p = 0.115] and the perspective figures . However, there was a clear difference in the duration for seeing each interpretation between the silhouettes and the perspective figures for both the control subjects  and the astronauts . When averaged across all 20 subjects the mean percentage of time for seeing the old woman and the golfer in the silhouettes was 50.5% (SD 11.9), i.e. close to chance. However, there was a 67%-33% asymmetry for seeing the perspective figures. The interpretations corresponding to the low inserts in 2 = 0.74). Repeated measures ANOVA yielded no significant difference in the duration for seeing each interpretation across the 3 pre-flight sessions and the 6 figures for the control subjects, but a significant difference across the perspective figures for the astronauts   = 2.52, = 0.004] .p < 0.001] and the figures , but no interaction . The pre-flight asymmetry for seeing the perspective figures (67\u201333%) gradually decreased during the flight until reaching 52\u201348% on FD172, and re-increased after the flight   = 0.52, p = 0.471]. The first interpretations of the figures that were seen on each trial did not change significantly across the 10 sessions, for both the perspective figures  and the silhouettes .For the perspective figures, a two-way ANOVA in the astronauts\u2019 responses yielded a significant difference in the percentage of time for seeing each interpretation across the 10 sessions [F  = 4.55,  < 0.05) . No signOur results indicate that the reaction time progressively decreased with the repetition of the test sessions. The number of percept reversals of the anthropomorphic silhouettes progressively increased with the repetition of test sessions, but the number of reversals of the depth perspective figures was not altered by spaceflight. There was, however, a higher probability for seeing one of the two possible 3D interpretations in normal gravity. For example, when looking at the Necker cube on Earth, subjects saw a cube resting on a surface (i.e. as though viewed from above) more often that a cube hanging from the ceiling (i.e. as though viewed from below). Our results showed that this asymmetry progressively decreased in weightlessness as the flight progressed, and then returned to the preflight level after return to Earth.Visual illusions are classically considered as a systematic mismatch between the basic response of the sensory organs to a stimulus , and the percept this object gives rise to. In the ambiguous figures, information is insufficient to result in a single interpretation, so these figures can be perceived as two different persons or two objects with different orientation .The bi-stability of ambiguous figures is commonly thought to be the result of perceptual processes involving \u201cbottom-up\u201d messages (carrying sensory information) and \u201ctop-down messages\u201d (carrying prior expectation). The percepts are the result of combining both types of messages . The decillusory depth was affected in weightlessness. Weightlessness is characterized by changes in sensory information from gravity-sensing receptors , as well as alterations in the mental representation of three-dimensional space [The question asked in this experiment was whether the perception of al space . Actual al space . The meaUnder terrestrial conditions, depth corresponds to the distance relative to the observer, in the direction of the line of sight; width, i.e. the line joining the two eyes, corresponds to the horizon; and height corresponds to the direction of gravity. In a drawing, vertical lines represent either the direction of the terrestrial gravitational vertical or the direction of depth: the farther away an object is, the higher it is represented on the page. Thus, there is therefore confusion between up-down and fore-aft. Such confusion is reflected in illusions of distortions, such as geometrical illusions, and in illusions of ambiguities, such as figure/ground illusions and ambiguous figures.Geometrical (distortion) illusion figures are essentially skeleton perspective drawings suggesting depth. When the illusion is about judgment of parallelism or alignment, it is minimal for horizontal and vertical orientations and maximal for oblique orientations (\u00b145\u00b0). In judgments of size, we are also less accurate for the oblique directions. For example, the oblique lines in the Muller-Lyer and the Ponzo illusions and the vertical line in the horizontal-vertical illusion all give the same projection as the receding lines, for example, of a railway . These iGibson  interpreThe \u201cvisual cliff\u201d studies have shown that the capacity to perceive depth is present from the beginning of an organism\u2019s life . HoweverAnthropological studies have also shown that geometrical illusions were more prevalent in people living in the constructed environment of cities than those dwelling in more natural environments . AlthougIn a normal gravity environment, we are more likely to see a cube resting on a table than hanging from the ceiling; hence the longer duration for seeing a cube from above when looking at the Necker cube. Another well-known example of knowledge built into our visual system is the assumption that scenes are illuminated from above . An elegGeometrical illusions were found to generate less distortion in astronauts during spaceflight . The numGround-based experiments have also shown a decreased susceptibility to geometrical illusions in vestibular patients . PatientIt was also observed that blind people who recovered sight later in life  and patiAnother pictorial source of depth information that arises from perspective projection is the height of objects in the picture-plane relative to the horizon. Because humans are bipedal creatures standing upright in normal gravity, the objects located in the lower part of our visual field (i.e. at our feet) are perceived to be closer to us than those in the higher part of our visual field (i.e. at the ceiling or in the sky) . It can After adaptation to weightlessness, free-floating astronauts have become accustomed to seeing the visual environment from various viewpoints. The objects in the lower part of their visual field are not necessarily the closest. Also, their feet are not always in contact with the \u201cfloor\u201d and they do not maintain an upright posture . ConsequTheorists like Gibson have argued that the core of our perception of the third dimension is our interpretation of planar surfaces such as the ground rather than our interpretation of objects separated from one another in empty space. Of all the cues we know about, only perspective could provide direct information about planes and their orientation with respect to us. However, the contribution of perspective is not a necessary factor, since the objects look three-dimensional even when linear perspective is eliminated .During the vast majority of everyday life situations on Earth, we resolve the ambiguity of the environmental stimuli through sensory information and prior knowledge. After adaptation to weightlessness, astronauts develop larger depth perception instability, manifested by an equal probability for seeing each 3D interpretation after three months in space. This perceptual instability is presumably due to impairment in sensory information related to the spatial orientation relative to the vertical and in top-down processes implicitly driven by prior normal gravity knowledge. This model may also account for the cognitive deficits observed in what the astronauts call the \u201cspace fog\u201d syndrome, which is characterized by difficulties in correctly allocating attention and filtering out irrelevant information [These observations confirm that depth is not so much the direct result of certain specifiable stimulus cues as it is a mental construction. This mental construction would obey some rules related to our experience of living in a gravitational environment. The vertical reference, the eye-ground elevation, and the size-constancy scaling are presumably some of the rules that the visual system utilizes to interpret perspective cues to indicate distance and depth."}
+{"text": "Proprioception plays an important role in the complex mechanism of joint control. Contemporary sport activities impose extremely high physical demands on athletes. Winter sports are played in areas with excessively low temperatures. Moreover, many athletes are subjected to treatments that involve local lowering of the body temperature before, during, and after physical activity. This work reviews the current knowledge regarding the influence of local cryotherapy on the proprioception system. The reviewed literature identified several tests that evaluate different aspects of proprioception. There is no universally agreed protocol, or clear set of criteria for test conditions. The outcomes of different tests and assessments of cryotherapy procedures using different cold modalities are poorly correlated. In general, the published results on the mechanism of cryotherapy effects on proprioception are not uniquely conclusive and are frequently contradictory. Additional high-quality research is required to explicitly answer the following questions: (1) whether local cryotherapy influences all aspects of proprioception; (2) whether the current methods of evaluation are adequate for the exploration of the relationship between cryotherapy and proprioception; and (3) whether the application of local cryotherapy is safe for athletes regarding proprioception. The review clearly showed that there is no comprehensive model relating cryotherapy and proprioception. Prop sense)\u201d \u20136. Joint sense)\u201d \u20139. Kines sense)\u201d , and, foNeuromuscular control is based on subconscious information from mechanoreceptors and processes within the central nervous system that allow control movement through coordinated muscle activity. It results from a complex interaction between the nervous system and the musculoskeletal system through feedback and feedforward mechanism control . RiemannTo avoid the misuse of the components of proprioception and evaluate the correct variables during investigations into proprioception, these definitions are reviewed in this paper. Joint position sense determines the ability of a subject to perceive a required (desired) joint angle \u201318. KineThe following three major testing procedures for the proprioception system are described in the literature, depending on which submodality is tested: (1) the reproduction of positioning, commonly called joint position sense (JPS), (2) the threshold of the detection of passive motion (TTDPM), and (3) the force (re)production sense (FR). In the JPS test, the joint is moved (actively or passively) towards the earlier requested (reference) angle. After a few seconds, the joint is returned to the original position. Following this movement, the subject should reproduce the perceived angle with the identical or contralateral knee or, in some cases, demonstrate the perceived angle on the joint model , 27, 28.Contemporary sports activities impose extremely high physical demands on athletes. Many of them are subjected to treatments that involve local changes in body temperature to obtain therapeutic effects. Cryotherapy consists of the lowering of tissue temperature by withdrawing heat from the body to achieve an analgesic effect . SuperfiThough it is relatively simple to use cryotherapy by decreasing the tissue temperature, much controversy and confusion remain regarding the clinical practice, particularly in the literature on the influence of cryotherapy on the proprioception system. This controversy was reported in the most recent review of Costello and Donnelly . This spThe purpose of this review is to answer questions regarding (1) the effects of locally applied cryotherapy on each aspect of proprioception system, (2) the appropriateness of today's methods of proprioception assessment, and (3) the safety of local cryotherapy for athletes when proprioception is considerate. The first two questions are of fundamental importance for the proprioception studies, while the third is more specific and narrow.The primary objective of this paper was to review the literature in the context of the relationship between cryotherapy and the proprioception system. The effects of local cooling on functional performance and other factors influencing proprioception, including aging, fatigue, warm-up, regular physical activity, and exercise, are found in comprehensive review papers by Ribeiro and Oliveira  and BleaThe field of human kinetics was selected for a search of original research contributions. In addition, the fields of medicine and sports medicine were examined. A search was conducted using the following databases: PubMed, SPORTDiscus, Scopus, EBSCOHost, ScienceDirect, and Web of Science. Keywords including cryotherapy, cooling, cold packs, ice, gel packs, proprioception, joint position sense, kinesthesia, sense of force, proprioception system, and balance were used in various configurations. The search period regarding the relationship between proprioception and cryotherapy was observed from January 1990 to December 2013.The criteria for the study selection were as follows: the publication language was English; the subjects were young adults, humans, and healthy; tests were conducted within at least five minutes after the application of cryotherapy; and cryotherapy was applied locally. The outcome measures were all aspects of proprioception, including balance, joint position sense, kinesthesia, and force sense. Studies regarding the effects of local cooling on maximal functional performance were excluded from this review.The summary and details of the methods and procedures used in the reviewed publication are listed in P < 0.05 and compared the posttreatment results with the baseline or a control group.The average time of cryotherapy application was up to 20.3 \u00b1 5.3 minutes and depended on the cooling medium. Ice (mean time 21.4 \u00b1 3.8\u2009min) or cold water immersion  was used. One team cooled the tissue as long as required for the muscle temperature to decrease to 3\u00b0C. Seven teams used water immersion, and seven used ice compresses. One group used commercial cold packs, one group used an icing system, and one group used an ice blanket consisting of sacks filled with crushed ice (P = 0.958). Uchio et al. [P = 0.003). Additionally, Surenkok et al. [P < 0.005). These authors unanimously suggested that caution should be exercised if cryotherapy is used immediately after physical activity. Hopper et al. [P = 0.049) after cold-water immersion (4\u00b0C) of the ankle joint, concluded that these results should not be deemed clinically significant.Joint position sense (JPS) was the most frequently investigated aspect of the proprioception system, with eleven of the eligible studies focusing on JPS. The review of this literature revealed that cryotherapy had a negative effect on the JPS in four studies , 55, 56.o et al.  demonstrr et al. , who obtNone of the remaining seven papers reported any significant changes in the JPS . The results of those studies showed that there is no evidence of an increased risk of injury following a return to exercise after cryotherapy. Though no differences were found for active JPS after cryotherapy application, Wassinger et al.  concludeBased on the above conflicting evidence, it is very difficult to categorically state whether cryotherapy impairs the joint position sense. Some authors , 55, 58 In the literature, there are a number of papers regarding kinesthesia \u201323. NoneIn a review of the literature, two studies investigated the influence of cryotherapy on the force sense as one of proprioception aspects , 29. RubThe studies concluded that the perception of force signal is not affected by local cooling, though the studies used different cryotherapy procedures  and targRelatively few papers have been published regarding the influence of local cryotherapy on balance sense , 59, 60.Twelve studies reported no adverse effects on proprioception , 57\u201360. Our discussion of the reviewed papers focused on addressing the three questions stated in the introduction. Specifically, we tried to determine the following: (1) whether locally applied cryotherapy affects all aspects of the proprioception system; (2) whether the current methods of proprioception evaluation are adequate for exploring the relationship between cryotherapy and the proprioception system; and (3) the safety of the use of cryotherapy by athletes in cases in which proprioception is considered.P < 0.05). With theprogression of skin temperature lowering to 10\u00b0C, the pain threshold and pain tolerance increased (P < 0.05). In contrast, several investigators [The critical review of the literature clearly shows contradicting evidence on the influence of local cryotherapy on proprioception. A number of researchers , 55, 56 tigators , 57\u201360 ftigators , 58. Coltigators . Water itigators . The invtigators , 54. HarOther aspects of the proprioception system such as balance and force sense have been investigated. These characteristics of proprioception appear to be the least influenced by cryotherapy. Douglas et al.  obtainedThis review found that only five studies , 55, 56 Twelve of the seventeen studies indicate that there is no evidence for a deleterious effect by cryotherapy on the proprioception system including JPS, force sense, and balance.McCloskey  postulatMany studies report that laboratory investigations on proprioception seldom reflect the actual load demands during functional movement (during sports or daily activities). Costello and Donnelly  proposedAdditionally, during the local application of cryotherapy, including treatment of one part of the joint or muscle , proprioception could be partially compensated for by other receptors such as ligament receptors or flexor muscle receptors. During an investigation using ice water immersion as a cold modality , all ofOf the seven reviewed studies in which the investigators used cold/ice water immersion, only two obtained a significant influence on proprioception , 52, andThe investigation of proprioception allows an estimation in the subjects of the actual level of the neuromuscular system functionality, the participation of the neuromuscular system in joint stabilization, the magnitude of damage to the neuromuscular system functioning, and the physiotherapy progression and its efficiency. The following key factors should be considered in proprioception investigations and the analysis and interpretation of the data to obtain reliable and validated databases: the precision of the measuring device (instrument) and measurement error; the aspects of proprioception; the method of measuring ; the quantity of the measurements; the methods of averaging; the investigated variables ; the units of variable ; the elimination of external factors that influence proprioception ; the investigated joint; the starting position; the gravitational effects ; the movement direction; and the movement velocity. Considering the above factors, the results of the reviewed papers should be interpreted with care.The reviewed literature identified several tests that evaluate different aspects of proprioception. There is no standardized protocol or clear set of criteria for those test conditions. The outcomes of the tests and the cryotherapy procedures using different cold modalities are poorly correlated.This review clearly showed that there is no comprehensive model relating cryotherapy and proprioception, and the results from the proprioception assessment methods provide a limited view of the manner in which cryotherapy and proprioception are related.The most frequently investigated aspect of proprioception was JPS, which is perceived to be one of the more functional tests for proprioception evaluation. In the published literature, there are no publications concerning the relationship between kinesthesia and cryotherapy. Two other aspects of proprioception, balance, and force sense appeared to be unaffected by cryotherapy.The reviewed literature does not provide conclusive evidence regarding the safety of local cryotherapy use for athletes or the evaluation of proprioception.The following general conclusion was determined in the study: a novel, standardized protocol, better evidence, and better quality studies regarding the relationship between proprioception and local cryotherapy should be conducted on a broader range of samples to answer unequivocally the major areas of investigation of this study.propose a priority list of proprioception aspects that should be evaluated when the individual subject condition is to be considered (differentiation between athletes and patients);establish a standardized protocol for proprioception evaluation. The outcomes of tests for the aspects of proprioception poorly correlate with each other. The proposed procedure should attract greater attention to proprioception evaluation in other parts of the body, including the elbow, wrist, hip, and spine. The proposed procedure should include rotational and sectional body movement when testing proprioception;design and build a new and comprehensive instrument for measuring the aspects of proprioception , which will be sensitive and reliable and will limit the risk of bias;specify the required duration and spatial extent of local cryotherapy to achieve a specific penetration depth of treatment in cases in which intramuscular and joint cooling studies are conducted;extend the proprioception investigation to a broader range of initial conditions regarding the conditions of the subjects, for example, large groups of subjects, clinical groups, elite athletes, and children;conduct proprioception investigations at various times in the rehabilitation process;establish a multifaceted training regime of the proprioception system;assess the possibility of introducing a placebo effect into the investigation of proprioception.Future research should concentrate on establishing a functional relationship between the proprioceptive system and local/entire body cryotherapy. To achieve this objective, the following actions are needed:"}
+{"text": "Coronary artery disease (CAD) is the leading cause of death worldwide. Recent genome-wide association studies (GWAS) identified >50 common variants associated with CAD or its complication myocardial infarction (MI), but collectively they account for <20% of heritability, generating a phenomena of \u201cmissing heritability\u201d. Rare variants with large effects may account for a large portion of missing heritability. Genome-wide linkage studies of large families and follow-up fine mapping and deep sequencing are particularly effective in identifying rare variants with large effects. Here we show results from a genome-wide linkage scan for CAD in multiplex GeneQuest families with early onset CAD and MI. Whole genome genotyping was carried out with 408 markers that span the human genome by every 10 cM and linkage analyses were performed using the affected relative pair analysis implemented in GENEHUNTER. Affected only nonparametric linkage (NPL) analysis identified two novel CAD loci with highly significant evidence of linkage on chromosome 3p25.1 (peak NPL \u200a=\u200a5.49) and 3q29 (NPL \u200a=\u200a6.84). We also identified four loci with suggestive linkage on 9q22.33, 9q34.11, 17p12, and 21q22.3 (NPL \u200a=\u200a3.18\u20134.07). These results identify novel loci for CAD and provide a framework for fine mapping and deep sequencing to identify new susceptibility genes and novel variants associated with risk of CAD. Long-term prospective clinical and epidemiological studies have identified several major risk factors for development of CAD, including smoking history, older age, male gender, high fat diet, personal history of angina pectoris, family history of myocardial infarction (MI), obesity, diabetes mellitus, high blood pressure, increased plasma total and low-density lipoprotein cholesterol, increased plasma triglycerides, and decreased plasma high-density lipoprotein cholesterol Coronary artery disease (CAD) is the leading cause of death globally, and accounts for >13% of all deaths  using family samples and genome-wide association studies (GWAS) using population samples. There are advantages and disadvantages for both. GWAS are more powerful than GWLS to detect common alleles at a locus, but less powerful if the phenotypes of interest are due to the segregation of low-frequency or rare alleles The major disadvantage of GWAS is that its discovering power is limited to common variants with relatively low risk (odds ratio or OR of 1.2 on average) In this study, we report results from a genome-wide linkage scan from a well-characterized U.S. population with 428 nuclear families with early onset CAD and myocardial infarction (MI) . Multiple new susceptibility genetic loci were identified for CAD, which provide new genetic bases of CAD and a framework to identify underlying susceptibility genes at each locus.The study subjects have been recruited in the United States through the Center for Cardiovascular Genetics and Department of Cardiovascular Medicine at the Cleveland Clinic The GeneQuest study was the first large-scale prospective collection of families with early-onset CAD and MI for identifying the underlying genes. The GeneQuest cohort consists of 1,613 subjects from 428 multiplex families with early-onset CAD and MI. Each family has at least two affected sibs, and the majority of families also have one unaffected sibling. The details, including diagnostic criteria and ascertainment of the patients and their families have been described elsewhere Patients with hypercholesterolemia, insulin-dependent diabetes, childhood hypertension, and congenital heart disease were excluded from this study.Whole blood samples were drawn from each participant, and genomic DNA was isolated from the blood by use of the commercial Puregene Kits (Gentra).Genotyping of microsatellite STR markers was performed by the National Heart, Lung, and Blood Institute (NHLBI) Mammalian Genotyping Service (directed by Dr. James L. Weber), Center for Medical Genetics at Marshfield Clinic by use of the Screening Set 11 consisting of 408 markers that span the entire human genome at every 10 cM P value of 7.4\u00d710\u22124 or LOD score of 2.2), 4.08  and 4.99 , respectively.Before statistical analysis of genome-wide genotyping data, obvious pedigree errors, data errors, genotyping errors, and locus-order errors that commonly occur with a large-scale linkage analysis were corrected as described previously http://www-genome.wi.mit.edu/ftp/distribution/software/genehunter/). The genomic regions with a peak two-point or multiple point NPL score that meets the suggestive linkage threshold (i.e. NPL \u22653.18) are listed in Genome-wide NPL analysis of affected sibling pairs in the GeneQuest families was conducted using GENEHUNTER  and four loci with suggestive linkage  \u20133. ThesePPARG gene encoding peroxisome proliferator-activated receptor gamma is located within the CAD locus, and is a short distance of 697 kb from D3S2403. A variant in PPARG  was found to be associated with type 2 diabetes PPARG was associated with HDL-cholesterol levels PPARG and CAD was controversial because many studies reported inconsistent results. Meta-analysis showed that the H161T variant in PPARG was associated with risk of CAD under a fixed-effect model, but the association became non-significant under a random-effect model; the P12A variant was not associated with CAD PPARG is the gene responsible for CAD at the 3p25.2 locus. Moreover, the nearest gene from the CAD marker D3S2403 is IQSEC1, which encodes a protein with an IQ motif and a Sec domain. Interestingly, marker MFD427-AAA with the peak NPL score for the 3q29 CAD locus is located within the IQCG gene encoding another protein with an IQ motif. Therefore, IQSEC1 and IQCG became candidate genes for CAD for the 3p25.2 and 3q29 loci for CAD, respectively.Using genotyping data from the same GeneQuest families, we previously identified a novel linkage for HDL-cholesterol, which spanned a 65 cM region, showed a bi-phasic pattern, and peaked at markers MFD433 and GATA131D09 on chromosome 3p25\u201326 P\u200a=\u200a0.0001) IQCG gene, the 3q29 CAD locus harbors several other possible candidate genes for CAD. The LMLN gene encodes an M8 family of metallopeptidase associated with lipid droplets and playing a role in lipid storage, generation of ROS, cell migration and invasion CEP19 gene encodes a 19 kDa centrosomal protein involved in obesity, glucose intolerance and insulin resistance The 3q29 locus for CAD was supported by several other studies showing positive but not significant linkage. For instance, meta-analysis of four genome-wide linkage studies for CAD indicated that the genetic region of 3q26-27 might contain susceptibility genes for CAD (The linkages on chromosome 17 are of particular interest. A suggestive linkage for MI was previously reported on chromosome 17 at a position of 50.7 cM in a cohort with 739 affected sibpairs (LOD \u200a=\u200a2.85). The entire putative MI linkage spanned from marker D17S921 at 17q11.2 (40 cM) to D17S787 at 17q21 (79 cM) ABO gene identified as a risk variant for CAD by GWAS is located 4.20 Mb from marker AGAT125 at the 9q34.11 region showing peak linkage to CAD in this study. It is important to note that in other CAD loci identified by GWLS in this study, GWAS did not identify any risk variants for CAD. There are several potential explanations. First, GWLS signals may represent variants or susceptibility genes for familial CAD, but not for sporadic CAD as used in GWAS. Second, GWLS signals may represent low frequency or rare variants with large effects on risk of CAD, which were generally ignored by GWAS. Considering the fact that larger meta-GWAS may not be easily practical for CAD now, re-focus on family-based studies may be the priority in the short future to identify new risk variants for CAD in our opinion.We compared our linkage results to GWAS data reported to date. Interestingly, we found that SNP rs579459 in the The identification of new CAD linkage loci might have been facilitated by our strict criteria for pedigree recruitment, which focused on white families enriched with well-diagnosed early-onset CAD, and pre-empted common risk factors, such as hypercholesterolemia and insulin-dependent diabetes. Furthermore, compared with other reported genome-wide linkage scans for CAD and MI, our patient population has the youngest age at onset, <45 years in males and <50 years in females, which is expected to significantly increase the genetic component involved in CAD. Family history was another factor for us to recruit families, with aim to identify inheritable CAD cases and to exclude environmental ones. Seeking for families with strong and multi-generational CAD history remains the most accessible way of increasing the inherited component for the recruited familiar cohort Despite a great success in identifying new genetic loci for CAD in this study, several technical limitations associated with non-parametric linkage analysis should be well-recognized. First, the families were ascertained through probands with a strong family history of CAD, and by the unique selection strategy, which focused on white families enriched with well-diagnosed early-onset CAD. Such family recruitment may be subjected to some selection biases and some parameter estimates for CAD risk may not straightforwardly be extended to the general population. Therefore, a precaution is clearly needed for interpreting the risk estimate when translated into clinical practice. Second, due to very nature of non-parametric linkage analysis, our analysis did not model person-specific covariates to remove the effects of potentially confounding factors. Although some measures such as pre-empting common risk factors like hypercholesterolemia and insulin-dependent diabetes were taken during the family recruitment, some sporadic or environmental cases might be included in the analysis, causing identity-by-decent values between affected sib pairs downward-estimated, particular when gene-environment interactions are present. Third, due to limited sample sizes, we did not perform a stratified analysis by different subtypes of CAD and hence only genomic regions linked to general definition of CAD were identified. Fourth, this study is limited by inadequate marker density provided by microsatellite markers, fine mapping and analyses of genetic variants by use of high-density SNP markers or new-generation DNA sequencing technologies might be a better design in our future studies. Finally, there is discrepancy between our previous report of linkage to chromosome 1p34-36 In conclusion, the present study has identified two novel genetic loci for CAD that passed the threshold of highly significant and four genetic loci with suggestive evidence of linkage. Future fine mapping and deep sequencing should facilitate the identification of an underlying major (or minor) gene for this complex disorder at each locus. Identification of the CAD gene(s) should uncover the biological pathways and molecular mechanisms for the pathogenesis of CAD."}
+{"text": "Emergent properties of global political culture were examined using data from the World History Survey (WHS) involving 6,902 university students in 37 countries evaluating 40 figures from world history. Multidimensional scaling and factor analysis techniques found only limited forms of universality in evaluations across Western, Catholic/Orthodox, Muslim, and Asian country clusters. The highest consensus across cultures involved scientific innovators, with Einstein having the most positive evaluation overall. Peaceful humanitarians like Mother Theresa and Gandhi followed. There was much less cross-cultural consistency in the evaluation of negative figures, led by Hitler, Osama bin Laden, and Saddam Hussein. After more traditional empirical methods  failed to identify meaningful cross-cultural patterns, Latent Profile Analysis (LPA) was used to identify four global representational profiles: Secular and Religious Idealists were overwhelmingly prevalent in Christian countries, and Political Realists were common in Muslim and Asian countries. We discuss possible consequences and interpretations of these different representational profiles. As processes of globalization connect the world , an impoHero) . Am. Am7]. AHero) . These cHero)  across nHero) , 14]..Hero) . T. Tcultur\u201d (p. 2) , who see\u201d (p. 2) , 20]. S. Scultur\u201d (p. 2) , 21]..culturaldemocratic cultures and regimes, which seems an imbalanced proposition for a century where, for example, the rise of non-democratic China has been so salient. Indeed, Fuchs  us us36] usIf not otherwise mentioned, we used SPSS 18 for all statistical analyses. th and 21st century figures known for their roles in dictatorships, terrorism, mass murder, and/or driving their countries into unjust wars. They were followed by Mao Zedong, Vladimir Lenin, Genghis Khan, Saladin and The Qin Emperor. The last two had evaluations close to the neutral point of 4.04 and so are not evaluated as \u201cvillains\u201d by our samples. Clearly time has dimmed the ferocious reputation of Genghis Khan, whose rating also approached the midpoint.The 10 most positively evaluated historical figures across all countries were (in descending order): Albert Einstein, Mother Theresa, Mahatma Gandhi, Martin Luther King, Isaac Newton, Jesus Christ, Nelson Mandela, Thomas Edison, Abraham Lincoln and Buddha. These are scientists, humanitarians, and religious figures with one exception of a political leader (Abraham Lincoln). It seems reasonable to argue that maybe Abraham Lincoln was perceived as a humanitarian, since it is known that he abolished slavery in the United States. The five most negatively evaluated historical figures across all countries were (from the bottom): Adolf Hitler, Osama bin Laden, Saddam Hussein, George W. Bush and Joseph Stalin. These are 20Intraclass correlation coefficients (ICC)  were calrd, 4th, and 5th highest ICCs in the inventory. The average ICC for the bottom eight  was. 18 compared to. 10 for the top eight. There was thus more agreement across cultures about the \u201cgreatest heroes\u201d of world history compared to its \u201cworst villains\u201d. In Among the top 10 most admired figures in world history, there was more consensus across countries about Buddha, Newton, Einstein, Mother Theresa and Mandela; in descending order, there was less consensus about Edison, Gandhi, Christ, Martin Luther King, and the least consensus about Lincoln. The pattern suggests that scientists are most consensually evaluated as \u201cheroes\u201d across cultures, together with religious and humanitarian figures who are unambiguously associated with peace. At the bottom, there was less consensus about the evaluation of historical figures, with Saddam Hussein having the highest ICC of any figure, and Osama bin Laden, Stalin, and Mao having the 3We attempted next to map the figures into a meaningful two-dimensional space using a multidimensional scaling procedure (MDS) with Proxscal and subsequent generalized procrustes analysis (GPA) . MDS plaThe cluster analysis thus converged on four clusters of countries: a Catholic-Orthodox  cluster , a mainly Western cluster , a Muslim cluster , and an Asian cluster .Having a reduced set of coherent groups in the form of four country clusters, we repeated the MDS procedure with a subsequent GPA with the following results. The rotated centroid configuration accounted for 69% of the squared distances for two dimensions of evaluations of figures. This was not a satisfactory improvement. The two-dimensional spaces for historical figures were relatively similar only for the first two clusters consisting of predominantly Christian populations. As shown in Interpretation for The horizontal axis of the Asian cluster MDS is not straightforward either, as can be seen in constructs groups of individuals using an inductive procedure that analyzes the configuration of their responses.Since our attempts to find universal dimensions of meaning somewhat failed, we decided to introduce a new type of statistical analysis that to our knowledge has not heretofore been employed in dealing with large scale cross-cultural data. It does not presume country as a unit of analysis but rather In the next step, we employed a LPA for the evaluation of 28 historical figures using Mplus . We usedLPA is suited for identifying distinct groups or types of people who may have quite a different set of beliefs or evaluations of different sets of historical events or figures in world history from other distinct types or groups of people. These different types may be overlooked when examining the overall mean levels of responses to a set of attitude or opinion items. Our analysis differentiates people in our data set into latent profiles based on similarities and differences in their overall pattern of responses across a range of continuous indicators\u2014as noted previously, different groups appear to rate controversial figures like Stalin or Mao very differently, and it cannot be assumed that country or culture is the primary determinant of these variances .We examined a range of different solutions, with models ranging from one to five latent profiles. We examined the Sample-size adjusted Bayesian Information Criterion (SABIC) and entropy as indicators of model fit. The SABIC can be used to compare a series of models that differ in the number of parameters. Entropy values range from 0 to 1.0, where a high value indicates a lower classification error. Entropy values close to 1.0  indicate that there is a clear separation of classes, or in other words that the model clearly separates the data into distinct profiles. SABIC and entropy statistics for the different solutions were as follows: single-class solution SABIC = 649585.54; two-class solution SABIC = 638239.86, Entropy = .822; three-class solution SABIC = 631105.97, Entropy = .870; four-class solution (preferred) SABIC = 627066.55, Entropy = .823; five-class solution SABIC = 624197.31, Entropy = .814.The SABIC and entropy statistics indicated that a four profile solution provided a reasonably parsimonious model of the latent profiles (or discrete categories of people) underlying the observed data. This was further supported by inspecting each solution qualitatively, which indicated that the fifth and subsequent solutions simply provided incrementally finer splits in the profiles identified in the four-class solution without showing any distinctly new patterns . Likewise, a three-class solution did not provide a fine-grained enough solution and failed to differentiate between the religious and secular idealist profiles identified in the four-class solution.representational profiles  or orth cenThird, and perhaps most importantly respondents regarded figures associated with the rise of human rights and resistance to oppression with a relatively high degree of positive consensus. That is, there was more consensus about the positive valence of particular historical figures compared to the historical figures evaluated as negative in world history. While the genocidal Hitler was undisputed as the worst rated figure in world history, his ICC  was much higher than that for the benevolent figures of Albert Einstein and Mother Theresa. Even more controversy surrounded Osama bin Laden and Saddam Hussein, who were rated close to the midpoint in our small (n = 4 countries) and non-representative (mostly Asian countries) sample of Muslim nations but negatively everywhere else.Our findings thus seem to suggest that where evaluations of negative figures in particular are concerned, there may be highly in-group favoring discourses that provide means-ends justifications for rating figures like Mao, Stalin, and Lenin favorably and Osama and Saddam towards the midpoint. That is, according to ingroup accounts, Mao, Stalin and Lenin may have \u201cmade mistakes\u201d, but they also made hard choices required for nation-building, whereas Osama and Saddam may be viewed as \u201cfighters against Western imperialism\u201d. We suspect that these means-ends justifications of less favored figures are culture-specific, and may counter the homogenization of global political culture among young educated people even though there is substantial agreement about its \u201cheroes\u201d and preferred values. From our MDS analyses, it appears that some Asian and Muslim countries may have alternative systems for mapping the meaning of figures in world history, supporting Sahlins\u2019  contentiOur analyses of representational profiles revealed a gap between Western and Catholic/Orthodox clusters versus Muslim and Asian countries clusters in prevalence mappings. The Western clusters and Catholic/Orthodox clusters consisted almost entirely (88%) of \u201cHistorical Idealists\u201d, whereas the Muslim cluster had a plurality of \u201cPolitical Realists\u201d (39%) and \u201cHistorical Indifferents\u201d (30%). The \u201cHistorical Indifference\u201d found in Muslim countries may be a product of a rejection of Eurocentrism in the selection of historical figures (including relatively few Muslims). The Asian country cluster was more of a hybrid, containing a plurality of \u201cSecular Idealists\u201d (34%) and \u201cPolitical Realists\u201d (30%).ensemble representations of historical figures strongly influenced by Western liberal democratic ideals. History is a crucial symbolic resource ..ensembleOur four Muslim countries provide a powerful counterpoint against over-generalizing this pattern, made all the more poignant by the fact that one of the four samples was Tunisia, the tinderbox that started the so-called Arab Spring. In a global representational framework, it is incumbent upon the citizens of Western countries to understand that the overwhelming support for \u201cHistorical Idealism\u201d prevalent in their countries is not universal, and cannot be expected to be transplanted without growing pains to Muslim countries. Rather, we should anticipate a long trajectory of democratization in these countries rather than an unbroken chain of immediate successes, as localized and marginalized formations are very important even in the analysis of cosmopolitanism . Becauseand a respect for the top-down, authoritarian traditions that governed Asia prior to recent times in many places . T. Tand a lization .We are at a juncture in time where the world is becoming fully connected economically . Underst"}
+{"text": "Vigna vexillata (L.) A. Rich. (tuber cowpea) is an underutilized crop for consuming its tuber and mature seeds. Wild form of V. vexillata is a pan-tropical perennial herbaceous plant which has been used by local people as a food. Wild V. vexillata has also been considered as useful gene(s) source for V. unguiculata (cowpea), since it was reported to have various resistance gene(s) for insects and diseases of cowpea. To exploit the potential of V. vexillata, an SSR-based linkage map of V. vexillata was developed. A total of 874 SSR markers successfully amplified single DNA fragment in V. vexillata among 1,336 SSR markers developed from Vigna angularis (azuki bean), V. unguiculata and Phaseolus vulgaris (common bean). An F2 population of 300 plants derived from a cross between salt resistant (V1) and susceptible (V5) accessions was used for mapping. A genetic linkage map was constructed using 82 polymorphic SSR markers loci, which could be assigned to 11 linkage groups spanning 511.5 cM in length with a mean distance of 7.2 cM between adjacent markers. To develop higher density molecular linkage map and to confirm SSR markers position in a linkage map, RAD markers were developed and a combined SSR and RAD markers linkage map of V. vexillata was constructed. A total of 559 (84 SSR and 475 RAD) markers loci could be assigned to 11 linkage groups spanning 973.9 cM in length with a mean distance of 1.8 cM between adjacent markers. Linkage and genetic position of all SSR markers in an SSR linkage map were confirmed. When an SSR genetic linkage map of V. vexillata was compared with those of V. radiata and V. unguiculata, it was suggested that the structure of V. vexillata chromosome was considerably differentiated. This map is the first SSR and RAD marker-based V. vexillata linkage map which can be used for the mapping of useful traits. Vigna vexillata (L.) A. Rich. is a perennial herb belonging to the genus Vigna and considered to be closely related to cowpea (Vigna unguiculata (L.) Walp.), the most important food legume in Africa , [, [Vigna  Ohashi: ), black  Hepper: ), mungbeWilczek: ), rice b Ohashi: ) and yariculata: ) were recompared .V. vexillata consist of the 11 linkage groups which correspond to the number of chromosomes of this promising crops for the future [Recently, two sequencing-based linkage maps of azuki bean and mungbean ,37) weree future .V. vexillata (1) using SSR markers developed from related Vigna and Phaseolus crops, (2) using RAD markers developed by de novo RAD-sequencing and (3) to compare constructed linkage map with those of two most important Vigna crops, V. radiata and V. unguiculata.Hence, the objectives of this study were to construct linkage map of 2 mapping population was developed from a cross between wild V. vexillata accession V1  and V5 . V1 is a salt tolerant accession collected from Paramaribo, Suriname while V5 is a salt sensitive accession collected at a site 35km E of Santa Marta, Columbia. The V1 was used as a male parent and V5 was a female parent in the cross to produced F1 seeds. The F1 plant was self-pollinated to produce 300 F2 plants which were grown in a greenhouse of NIAS from May 2013.An F2 plants were extracted from fresh leaf tissue using the CTAB method [Total genomic DNA of the parents and FB method  with a sA total of 1336 SSR markers consisting of 329 SSR markers of azuki bean , 480 ESTr) of 0.25. Kosambi mapping function [V. radiata), yardlong bean (V. unguiculata) and V. vexillata were compared. In case more than 2 SSR markers of a mungbean linkage group  were mapped with several SSR markers of another mungbean linkage group , it is estimated that a translocation is occurred.An SSR genetic linkage map was constructed with JoinMap ver. 4.0 . The calfunction  was usedfunction . Double function . Based oRAD-seq analysis was performed based on the protocol of Matsumura et al. with minor modifications .Adapter-1 for BamHI-digested DNA site was prepared by annealing the two synthesized oligonucleotides 5\u2019-biotin-GTACAGGTTCAGAGTTCTACAGTCCGACGATCXXXXXX-3\u2032 and 5\u2032-GATCXXXXXXGATCGTCGGACTGTAGAACTCTGAACCTGT-3 . Adapter 2 for NlaIII-digested DNA site was prepared by annealing of the two complementary oligonucleotides 5\u2032-amino-CAAGCAGAAGACGGCATACGACATG-3\u2032 and 5\u2032-TCGTATGCCGTCTTCTGCTTG-3\u2032.2 individuals were simultaneously digested with BamHI-HF and NlaIII and purified. Adaptor-1 and adaptor-2 were ligated to the digested DNA samples and purified. Adaptors ligated biotinylated DNA samples were collected using streptavidin coated magnetic beads . Adaptor ligated DNA on the beads was amplified by PCR using Phusion High-Fidelity DNA polymerase (Thermo Fisher Scientific) and the adapter primers. Size of the PCR amplified fragments were checked by electrophoresis in agarose gel. The 96 purified PCR products were pooled and sequenced using the Illumina HiSeq2000 system. The sequencing primer was 5\u2032-CGACAGGTTCAGAGTTCTACAGTCCGACGATC.DNA library for RAD sequencing was constructed as follows. Each genomic DNA samples (100\u2013300 ng) of parents and 286 F2 individuals based on the six-base variable index sequences. RAD-tags with the sequence reads of less than 2 sequence mismatch were grouped as a stack. Stacks of each parent with less than 3 sequence mismatch were estimated as the stacks derived from a homologous locus. A list of RAD-tag sequences and their count was constructed for each sample. For the genotyping of F2 individuals, stacks which have more than 20 RAD-tags (minimum stack depth of 20) were used as potential RAD-markers.Extraction of RAD-tag sequence and bi-allelic RAD-marker detection were conducted using a software, Stacks ver. 1.12 . Sequenc2 individuals, which show identical F2 individuals genotypes, and/or which show significant segregation distortion  were not used. Suitable values of recombination fractions and LOD scores were estimated using the est.rf command. After these steps, initial linkage group construction was carried out using the formLinkageGroups command with a maximum recombination fraction of 0.25 and LOD threshold of 15. Robustness of linkage groups was checked using plot.rf command. Marker order within a linkage group was estimated using a program TMAP [Linkage group construction was conducted by a software R/qtl ver. 1.36.6 . Potentiram TMAP . Some RAAmong 1,336 SSR markers screened, 874 SSR markers (65.4% on average) amplified single DNA fragment . The perV. vexillata genome at an average marker distance of 7.2 cM  covering a total length of 510.5 cM of the f 7.2 cM , Table 1f 7.2 cM . The lenf 7.2 cM .2 individuals was 412,372, 1,138,645 and 450,040,150, respectively  and Fectively . The aveLinkage analysis identified 11 linkage groups (LG1\u2013LG11) containing a total of 475 RAD markers loci and 84 SSR markers loci after the removal of unlinked markers , Fig 2. V. radiata (left) and V. unguiculata (middle) were highly conserved. Most of the common markers were mapped on the same linkage group with the same order. One reciprocal translocation was found between LG4 and LG7. On the other hand, 3 translocations were detected in V. vexillata compared with V. radiata  was estimated to be conserved with those of V. radiata and V. unguiculata. Other 3 LGs of V. vexillata  were composed of the fragments of 2 LGs of V. radiata or V. unguiculata.Comparative linkage maps based on the common SSR markers were developed . Linkage radiata , Table 2V. vexillata. In addition, this is the first genetic linkage map consisting of 11 linkage groups that correspond to the haploid chromosome number of V. vexillata (n = 11). Former V. vexillata linkage map was constructed by 70 RAPD, 47 AFLP and one SSR marker, and was consisted of 14 linkage groups [The genetic linkage map developed in this study is the first SSR and RAD markers-based linkage map of Vigna and Phaseolus showed good amplification and a total of 874 markers were selected as potentially useful markers for V. vexillata , we could construct a high-density linkage map with 11 linkage groups with 475 RAD-markers (SNPs based of RAD-tag sequence) (V. radiata) and an azuki bean (V. angularis) linkage maps were constructed by SNPs using GBS method (genotyping-by-sequencing) [By the application of RAD-seq analysis to the genetically close parental accessions of equence) . Recentluencing) ,36. HoweSince the genetic linkage map based on the RAD and SSR markers could confirm the linkage group accuracy of SSR-based linkage map, comparative genomic analysis were performed using common SSR markers.V. vexillata chromosomes, while genomic structure of V. radiata and V. unguiculata was basically conserved with only one translocation. The remarkable chromosomal re-arrangements in V. vexillata was surprising because molecular phylogenetic analysis based on RFLP and the sequences of nuclear ribosomal DNA (rDNA) regions suggested that V. vexillata was more closely aligned with V. unguiculata compared with V. radiata [V. vexillata group (subgenus Plectrotropis) formed an evolutionary intermediate group between V. unguiculata group (subgenus Vigna) and V. radiata group (subgenus Ceratotrois) [The present study suggested several translocations occurred in  radiata ,57,58. Itotrois) ,59.V. marina, V. radiata and V. unguiculata linkage maps [V. marina and V. radiata, which belong to the different subgenera, i.e., subgenus Vigna and Ceratotropis, respectively. On the contrary, one reciprocal translocation between LG4 and 7 was found between V. marina and V. unguiculata, which belong to the same subgenus Vigna. However, since the number of common SSR markers used for developing the comparative linkage map was not sufficient in these studies, it should be confirmed by the comparison of higher density molecular maps or more directly by cytogenetic analysis. Gomathinayagam et al. [V. vexillata x V. unguiculata and reported high frequency of univalent formation, hence suggested that the genomes of the two species are structurally differentiated.Similar phenomenon was reported in the comparative analyses among age maps . Structum et al.  observedV. vexillata suggested in the present study is commonly seen in V. vexillata or only occurred in some local accessions (e.g. American accessions). In case of azuki bean (V. angularis), we also suggested reciprocal translocation between LG4 and LG6 based on the comparison of SSR-based linkage maps developed for 2 different mapping populations [In addition, it is unknown that the chromosomal re-arrangement of ulations ,30. Receulations . In thatVigna vexillata is a promising food crop which could be grown under harsh environmental conditions [macrosperma) recognized having large seeds, non-dehiscent pods and bushy plant type [V. vexillata var. macroperma showed earlier maturity with higher seed yield than Bali cultivated accessions [macrosperma) with African and Austronesian wild V. vexillata accesions, it was revealed that var. macrosperma and the wild accessions can be considered to belong to the same gene pool with strong genetic compatibility [1 plants with a cultivated Bali accession having vigorous growth, but it showed complete F1 sterility. However, backcrossing both to wild and Bali cultivated parents could produce BC1 plants, suggesting the possibility of backcross breeding of improving these domesticated forms [V. vexillata could be used to incorporate adaptation gene(s) into these domesticated forms to grow under both biotic and abiotic stress environments.nditions . There anditions . There iant type ,64. V. vtibility . In conted forms . As was V. vexillata in the present study could be used as basic genome mapping resources.In the breeding procedure, especially using wild germplasm, it is essential to map the position of useful traits and use marker assisted selection in the segregating population . The SSRS1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file."}
+{"text": "Wikipedia is a huge global repository of human knowledge that can be leveraged to investigate interwinements between cultures. With this aim, we apply methods of Markov chains and Google matrix for the analysis of the hyperlink networks of 24 Wikipedia language editions, and rank all their articles by PageRank, 2DRank and CheiRank algorithms. Using automatic extraction of people names, we obtain the top 100 historical figures, for each edition and for each algorithm. We investigate their spatial, temporal, and gender distributions in dependence of their cultural origins. Our study demonstrates not only the existence of skewness with local figures, mainly recognized only in their own cultures, but also the existence of global historical figures appearing in a large number of editions. By determining the birth time and place of these persons, we perform an analysis of the evolution of such figures through 35 centuries of human history for each language, thus recovering interactions and entanglement of cultures over time. We also obtain the distributions of historical figures over world countries, highlighting geographical aspects of cross-cultural links. Considering historical figures who appear in multiple editions as interactions between cultures, we construct a network of cultures and identify the most influential cultures according to this network. The influence of digital media on collective opinions, social relationships, and information dynamics is growing significantly with the advances of information technology. On the other hand, understanding how collective opinions are reflected in digital media has crucial importance. Among such a medium, Wikipedia, the open, free, and online encyclopedia, has crucial importance since it is not only the largest global knowledge repository but also the biggest collaborative knowledge platform on the Web. Thanks to its huge size, broad coverage and ease of use, Wikipedia is currently one of the most widely used knowledge references. However, since its beginning, there have been constant concerns about the reliability of Wikipedia because of its openness. Although professional scholars may not be affected by a possible skewness or bias of Wikipedia, students and the public can be affected significantly , 2. ExteWikipedia is available in different language editions; 287 language editions are currently active. This indicates that the same topic can be described in hundreds of articles written by different language user groups. Since language is one of the primary elements of culture , collectCultural bias or differences across Wikipedia editions have been investigated in previous research \u201317. A spWe note that the analysis of persons\u2019 importance via Wikipedia becomes more and more popular. This is well visible from the appearance of new recent studies for the English Wikipedia  and for Here we investigate interactions and skewness of cultures with a broader perspective, using global ranking of articles about persons in 24 Wikipedia language editions. According to Wikipedia  these 24On the basis of this data set we analyze spatial, temporal, and gender skewness in Wikipedia by analyzing birth place, birth date, and gender of the top ranked historical figures in Wikipedia. We identified overall Western, modern, and male skewness of important historical figures across Wikipedia editions, a tendency towards local preference , and the existence of global historical figures who are highly ranked in most of Wikipedia editions. We also constructed networks of cultures based on cross-cultural historical figures to represent interactions between cultures according to Wikipedia.To obtain a unified ranking of historical figures for all 24 Wikipedia editions, we introduce an average ranking which gives us the top 100 persons of human history. To assess the alignment of our ranking with previous work by historians, we compare it with the Hart\u2019s list of the top 100 people who, according to him, most influenced human history . We noteAij. If there is a link (one or more) from node (article) j to node (article) i then Aij = 1, otherwise, Aij = 0. The out-degree kout(j) is the number of links from node j to other nodes and the in-degree kin(j) is the number of links to node j from other nodes. The links between articles are considered only inside a given Wikipedia edition, there are no links counted between editions. Thus each language edition is analyzed independently from others by the Google matrix methods described below. The transcriptions of names from English to the other 23 selected languages are harvested from WikiData (http://dumps.wikimedia.org/wikidatawiki) and not directly from the text of articles.In this research, we consider each Wikipedia edition as a network of articles. Each article corresponds to a node of the network and hyperlinks between articles correspond to links of the network. For a given network, we can define an adjacency matrix To rank the articles of a Wikipedia edition, we use two ranking algorithms based on the articles network structure. Detailed descriptions of these algorithms and their use for Wikipedia editions are given in , 28, 29.Sij of Markov transitions by normalizing the sum of the elements in each column of A to unity  and replacing columns with zero elements by elements 1/N with N being the matrix size. Then the Google matrix is given by the relation Gij = \u03b1Sij + (1 \u2212 \u03b1)/N, where \u03b1 is the damping factor [\u03b1 = 0.85. It is known that the variation of \u03b1 in a range 0.5 \u2264 \u03b1 < 0.95 does not significantly affect the probability distribution of ranks discussed below P of a node i at iteration t in a network with N nodes is given byPageRank is a widely used algorithm to rank nodes in a directed network. It was originally introduced for Google web search engine to rank web pages of the World Wide Web based on the idea of academic citations . CurrentP(i) of P is the PageRank of node i. More detailed information about the PageRank algorithm is described in [P(i), we obtain the PageRank ranking index K(i). In qualitative terms, the PageRank probability of a node is proportional to the number of incoming links weighted according to their own probability. A random network surfer spends on a given node a time given on average by the PageRank probability.The stationary state ribed in . OrderinP* of a node at iteration time t is given byIn a directed network, outgoing links can be as important as ingoing links. In this sense, as a complementary to PageRank, the CheiRank algorithm is defined and used in , 28, 36.P*(i) of P* as the CheiRank probability of node i with \u03b1 = 0.85. High CheiRank nodes in the network have large out-degree. Ordering all nodes by their decreasing probability P*(i), we obtain the CheiRank ranking index K*(i). The PageRank probability of an article is proportional to the number of incoming links, while the CheiRank probability of an article is proportional to the number of outgoing links. Thus a top PageRank article is important since other articles refer to it, while a top CheiRank article is highly connected because it refers to other articles.Same as the case of PageRank, we consider the stationary state i which is Ki-th ranked by PageRank and K*i ranked by CheiRank. Then we can assign a secondary ranking j has lower 2DRank and vice versa. A detailed illustration and description of this algorithm is given in [PageRank and CheiRank algorithms focus only on in-degree and out-degree of nodes, respectively. The 2DRank algorithm considers both types of information simultaneously to rank nodes with a balanced point of view in a directed network. Briefly speaking, nodes with both high PageRank and CheiRank get high 2DRank ranking. Consider a node given in .We note that the studies reported in  show thaWe consider 24 different language editions of Wikipedia: English (EN), Dutch (NL), German (DE), French (FR), Spanish (ES), Italian (IT), Portuguese (PT), Greek (EL), Danish (DA), Swedish (SV), Polish (PL), Hungarian (HU), Russian (RU), Hebrew (HE), Turkish (TR), Arabic (AR), Persian (FA), Hindi (HI), Malaysian (MS), Thai (TH), Vietnamese (VI), Chinese (ZH), Korean (KO), and Japanese (JA). The Wikipedia data were collected in middle February 2013. The overview summary of each Wikipedia is represented in We understand that our selection of Wikipedia editions does not represent a complete view of all the 287 languages of Wikipedia editions. However, this selection covers most of the largest language editions and allows us to perform quantitative and statistical analysis of important historical figures. Among the 20 largest editions  we have not considered the following editions: Waray-Waray, Cebuano, Ukrainian, Catalan, Bokmal-Riksmal, and Finish.First we ranked all the articles in a given Wikipedia edition by PageRank and 2DRank algorithms, and selected biographical articles about historical figures. To identify biographical articles, we considered all articles belonging to \u201cCategory:living people\u201d, or to \u201cCategory:Deaths by year\u201d or \u201cCategory:Birth by year\u201d or their subcategories in the English Wikipedia. In this way, we obtained a list of about 1.1 million biographical articles. We identified birth place, birth date, and gender of each selected historical figure based on DBpedia  or a manWe attribute each of the 100 historical figures to a birth place at the country level , to a birth date in year, to a gender, and to a cultural group. Historical figures are assigned to the countries currently at the locations where they were born. The cultural group of historical figures is assigned by the most spoken language of their birth place at the current country level. For example, if someone was born in \u201cConstantinople\u201d in the ancient Roman era, since the place is now Istanbul, Turkey, we assign her/his birth place as \u201cTurkey\u201d and since Turkish is the most spoken language in Turkey, we assign this person to the Turkish cultural group. If the birth country does not belong to any of the 24 cultures (languages) which we consider, we assign WR (world) as the culture of this person. We would like to point out that although a culture can not be defined only by language, we think that language is a suitable first-approximation of culture. All lists of top 100 historical figures with their birth place, birth date, gender, and cultural group for each Wikipedia edition and for each ranking algorithm are represented in . A part Jesus, Aristotle, or Napoleon appear in multiple Wikipedia editions, we have 1045 unique top PageRank historical figures and 1616 unique top 2Drank historical figures.To apply PageRank and 2DRank methods, we consider each edition as the network of articles of the given edition connected by hyper-links among the articles . The errors appear due to transcription changes of names or missing cases in our name-database based on English Wikipedia. For Western languages the number of errors is presumably reduced since transcription remains close to English. Based on the manual inspection for the Korean and the Russian Wikipedia, we expect that the errors of our automatic recovery of the top people from the whole articles ordered by PageRank and 2DRank are on a level of two percent.We also note that our study is in compliance with Wikipedia\u2019s Terms and Conditions.Above we described the methods used for the extraction of the top 100 persons in the ranking list of each edition. Below we present the obtained results describing the spatial, temporal and gender distributions of top ranked historical figures. We also determine the global and local persons and obtain the network of cultures based on the ranking of persons from a given language by other language editions of Wikipedia.The birth places of historical figures are attributed to the country containing their geographical location of birth according to the present geographical territories of all world countries. The list of countries appeared for the top 100 persons in all editions is given in The obtained spatial distribution of historical figures of Wikipedia over countries is shown in Western (Europe and USA) skewed patterns are observed in both top PageRank historical figures  ranking algorithm as shown in Regional skewness, the preferences of Wikipedia editions for historical figures who were born in geographically or culturally related countries, is also observed. For example, 18 (5) of the top 100 PageRank historical figures in the Korean (Japanese) Wikipedia were born in China. Also 9 of the top 100 PageRank historical figures in the Persian Wikipedia were born in Saudi Arabia. The distribution of top persons from each Wikipedia edition over world countries is shown in To observe patterns in a better way at low numbers of historical figures, we normalized each column of In the case of the top 2DRank historical figures shown in In short, we observed that most of the top historical figures in Wikipedia were born in Western countries, but also that each edition shows its own preference to the historical figures born in countries which are closely related to the corresponding language edition.Socrates, Plato, and Herodotus), Roman politicians  and Christianity leaders ). We also observe that the Arabic and the Persian Wikipedia have more historical figures than Western language Wikipedia editions from AD 6th century to AD 12th century. For the case of 2DRank in The analysis of the temporal distribution of top historical figures is done based on their birth dates. As shown in Augustine the Hippo and Justinian I in common. Similar distributions obtained from 2DRank are shown in The distributions of the top PageRank historical figures over the 24 Wikipedia editions for each century are shown in The data of ML, C and NL, C for a given language edition L and a given century C. Here ML, C is the number of historical figures born in all countries being attributed to a given language L, and NL, C is the total number of historical figures for a given century C and a given language edition L. For example, among the 21 top PageRank historical figures from the English Wikipedia, who were born in AD 20th century, two historical figures (Pope John Paul II and Pope Benedict XVI) were not born in English speaking countries. Thus in this case NEN, 20 = 21 and MEN, 20 = 19. rL, C = ML, C/NL, C for each edition and each century. In ancient times (i.e. before AD 5th century), most historical figures for each Wikipedia edition are not born in the same language region except for the Greek, Italian, Hebrew, and Chinese Wikipedia. However, after AD 5th century, the ratio of same language historical figures is rising. Thus, in AD 20th century, most Wikipedia editions have significant numbers of historical figures born in countries speaking the corresponding language. For PageRank persons and AD 20th century, we find that the English edition has the largest fraction of its own language, followed by Arabic and Persian editions while other editions have significantly large connections with other cultures. For the English edition this is related to a significant number of USA presidents appearing in the top 100 list . Fo. FoML, From the gender distributions of historical figures, we observe a strong male-skewed pattern across many Wikipedia editions regardless of the ranking algorithm. On average, 5.2(10.1) female historical figures are observed among the 100 top PageRank (2DRank) persons for each Wikipedia edition. Above we analyzed how top historical figures in Wikipedia are distributed in terms of space, time, and gender. Now we identify how these top historical figures are distributed in each Wikipedia edition and which are global historical figures. According to previous research , there aP, A of a historical figure P is given byFollowing , the ranRP, E, A is the ranking of a historical figure P in Wikipedia edition E by ranking algorithm A. According to this definition, a historical figure who appears more often in the lists of top historical figures for the given 24 Wikipedia editions or has higher ranking in the lists gets a higher ranking score. Carl Linnaeus is the 1st global historical figure by PageRank followed by Jesus, Aristotle. Adolf Hitler is the 1st global historical figure by 2DRank followed by Michael Jackson, Madonna (entertainer). On the other hand, the lists of the top 10 local historical figures ordered by our ranking score for each language are represented in supporting Tables S1\u2013S25 and [Here \u2013S25 and .Carl Linnaeus is related to the fact that he laid the foundations for the modern biological naming scheme so that plenty of articles about animals, insects and plants point to the Wikipedia article about him, which strongly increases the PageRank probability. This happens for all 24 languages where Carl Linnaeus always appears on high positions since articles about animals and plants are an important fraction of Wikipedia. Even if in a given language the top persons are often politicians , these politicians have mainly local importance and are not highly ranked in other languages . As a result when the global contribution is counted over all 24 languages Carl Linnaeus appears on the top PageRank position.The reason for a somewhat unexpected PageRank leader NA \u2265 18) and are highly ranked (\u27e8K\u27e9 \u2264 50) for each Wikipedia such as Carl Linnaeus, Plato, Jesus, and Napoleon (Right-Top of the NA < 18) but are highly ranked (\u27e8K\u27e9 \u2264 50) in the Wikipedia editions in which they appear, such as Tycho Brahe, Sejong the Great, and Sun Yat-sen (Left-Top of the NA < 18) and who are not highly ranked (\u27e8K\u27e9 > 50). Here NA is the number of appearances in different Wikipedia editions for a given person and \u27e8K\u27e9 is the average ranking of the given persons across Wikipedia editions for each ranking algorithm. In the case of 2DRank historical figures, due to the absence of global historical figures, most of them belong to two types of local historical figures .Our analysis suggests that there might be three groups of historical figures. P, A we determine also the top global female historical figures, presented in Following ranking of persons via \u0398The comparison of our 100 global historical figures with the top 100 from Hart\u2019s list  gives anWe also compared the distributions of our global top 100 persons of PageRank and 2DRank with the distribution of Hart\u2019s top 100 over centuries and over 24 languages with the additional WR category . We find that these 3 distributions have very similar shapes. Thus the largest number of persons appears in centuries AD 18th, 19th, 20th for the 3 distributions. Among languages, the main peaks for the 3 distributions appear for EN, DE, IT, EL, AR, ZH. The deviations from Hart\u2019s distribution are larger for the 2DRank list. Thus the comparison of distributions over centuries and languages shows that the PageRank list has not only a strong overlap with the Hart list in the number of persons but that they also have very similar statistical distributions of the top 100 persons over centuries and languages.The overlap of the top 100 global persons found here with the previous study  gives 54It is interesting to note that for the top 100 PageRank universities from the English Wikipedia edition the overlap with Shanghai top 100 list of universities is on a even higher level of 75 percent .Napoleon III (and even not Napoleon I) [Peter the Great, that has much more historical grounds. In a similar way for FR the results of [Adolf Hitler, that is rather strange for the French culture, while we find a natural result Napoleon.Finally, we note that the ranking of historical figures using the whole PageRank (or 2DRank) list of all Wikipedia articles of a given edition provides a more stable approach compared to the network of biographical articles used in . Indeed,oleon I) , who hassults of  give at A appears in another edition B, then we can consider this as a \u2018cultural\u2019 influence from culture A to B. Here we consider each language as a proxy for a cultural group and assign each historical figure to one of these cultural groups based on the most spoken language of her/his birth place at the country level. For example, Adolf Hitler was born in modern Austria and since German language is the most spoken language in Austria, he is considered as a German historical figure in our analysis. This method may lead to some misguiding results due to discrepancy between territories of country and cultures, e.g. Jesus was born in the modern State of Palestine (Bethlehem), which is an Arabic speaking country. Thus Jesus is from the Arabic culture in our analysis while usually one would say that he belongs to the Hebrew culture. Other similar examples we find are: Charlemagne (Belgium\u2014Dutch), Immanuel Kant , Moses (Egypt\u2014Arabic), Catherine the Great .We consider the selected top persons from each Wikipedia edition as important historical figures recognized by people who speak the language of that Wikipedia edition. Therefore, if a top person from a language edition In total there are such 36 cases from the global PageRank list of 1045 names (these 36 names are given in SI). However, in our knowledge, the birth place is the best way to assign a given historical figure to a certain cultural background computationally and systematically and with the data we have available. In total we have only about 3.4 percent of cases which can be discussed and where a native speaking language can be a better indicator of belonging to a given culture. For the global 2DRank list of 1616 names we identified 53 similar cases where an attribution to a culture via a native language or a birth place could be discussed (about 3.3 percent). These 53 names are given in SI. About half of such cases are linked with birth places in ancient Russian Empire where people from Belarus, Litvania and Ukraine moved to RU, IL, PL, WR. However, the percentage of such cases is small and the corresponding errors also remain small.A to culture B is given by the number of historical figures belonging to culture B (e.g. French) appearing in the list of top 100 historical figures for a given culture A (e.g. English). The persons in a given edition, belonging to the language of the edition, are not taken into account since they do not create links between cultures. In Based on the above assumption and following the approach developed in , we consFor example, there are 5 French historical figures among the top 100 PageRank historical figures of the English Wikipedia, so we can assign weight 5 to the link from English to French. Gij for each network is constructed following the standard rules described in [K and the CheiRank index K* that order all cultures according to decreasing PageRank and CheiRank probabilities  and German is on  in the case of PageRank historical figures  and German is on .To identify which cultures (or language groups) are more influential than others, we calculated PageRank and CheiRank of the constructed networks of cultures by considering link weights. Briefly speaking, a culture has high PageRank (CheiRank) if it has many ingoing (outgoing) links from (to) other cultures see . The dis figures  and top  figures . Here K  figures . In the K = 1 for 9 editions [K = 4 and 3 in It is important to note that there is a significant difference compared to the previous study : there, editions  to K = 4K, K*) changes significantly in time. Indeed, if we take into account only persons born before the 19th century then the ranking is modified with EN going to 4th  and French, Russian and Arabic (for 2DRank persons). For historical figures before the 19th century, we find respectively Arabic, Turkish and Greek (for PageRank) and Arabic, Greek and Hebrew (for 2DRank). The high position of Turkish is due to its close links both with Greek culture in ancient times and with Arabic culture in more recent times. We see also that with time the positions of Greek in 2DRank improves due to a global improved ranking of Western cultures closely connected with Greece.By investigating birth place, birth date, and gender of important historical figures determined by the network structure of Wikipedia, we identified spatial, temporal, and gender skewness in Wikipedia. Our analysis shows that the most important historical figures across Wikipedia language editions were born in Western countries after the 17th century, and are male. Also, each Wikipedia edition highlights local figures so that most of its own historical figures are born in the countries which use the language of the edition. The emergence of such pronounced accent to local figures seems to be natural since there are more links and interactions within one culture. This is also visible from to the fact that in many editions the main country for the given language is at the first PageRank position among all articles (e.g. Russia in RU edition) . DespiteIt is very difficult to describe history in an objective way and due to that it was argued that history is \u201can unending dialogue between the past and present\u201d . In a siWe use a computational and data mining approach, based on rank vectors of the Google matrix of Wikipedia, to perform a statistical analysis of interactions and entanglement of cultures. We find that this approach can be used for selecting the most influential historical figures through an analysis of collectively generated links between articles on Wikipedia. Our results are coherent with studies conducted by historians , with anInfluence of digital media on information dissemination and social collective opinions among the public is growing fast. Our research across Wikipedia language editions suggests a rigorous mathematical way, based on Markov chains and Google matrix, for the identification of important historical figures and for the analysis of interactions of cultures at different historical periods and in different world regions. We think that a further extension of this approach to a larger number of Wikipedia editions will provide a more detailed and balanced analysis of interactions of world cultures.S1 Filehttp://dumps.wikimedia.org/. All the raw data necessary to replicate the findings and conclusion of this study are within the paper, supporting information files and this Wikimedia web site.For a reader convenience the lists of all 100 ranked names for all 24 Wikipedia editions and corresponding network link data for each edition are also given at  in addit(PDF)Click here for additional data file."}
+{"text": "Autoimmune Addison\u2019s disease (AAD) is a rare, highly heritable autoimmune endocrinopathy. It is possible that there may be some highly penetrant variants which confer disease susceptibility that have yet to be discovered.DNA samples from 23 multiplex AAD pedigrees from the UK and Norway  were genotyped on the Affymetrix SNP 6.0 array. Linkage analysis was performed using Merlin. EMMAX was used to carry out a genome-wide association analysis comparing the familial AAD cases to 2706 UK WTCCC controls. To explore some of the linkage findings further, a replication study was performed by genotyping 64 SNPs in two of the four linked regions (chromosomes 7 and 18), on the Sequenom iPlex platform in three European AAD case-control cohorts . The data were analysed using a meta-analysis approach.-7). A meta-analysis of the replication study data demonstrated that three chromosome 18 SNPs were associated with AAD, including a non-synonymous variant in the NFATC1 gene.In a parametric analysis, applying a rare dominant model, loci on chromosomes 7, 9 and 18 had LOD scores >2.8. In a non-parametric analysis, a locus corresponding to the HLA region on chromosome 6, known to be associated with AAD, had a LOD score >3.0. In the genome-wide association analysis, a SNP cluster on chromosome 2 and a pair of SNPs on chromosome 6 were associated with AAD (P <5x10NFATC1 gene is worthy of future investigation, however each of the regions identified require further, systematic analysis.This linkage study has implicated a number of novel chromosomal regions in the pathogenesis of AAD in multiplex AAD families and adds further support to the role of HLA in AAD. The genome-wide association analysis has also identified a region of interest on chromosome 2. A replication study has demonstrated that the  MHC [MICA [CIITA [CTLA4 [PTPN22 [CYP27B1 [NLRP-1 [STAT4 [GATA3 [CD274 [Autoimmune Addison\u2019s disease (AAD) is, a rare endocrine condition with a prevalence in Caucasian European adults of up to 140 per million . Its rarMHC , MICA [4HC [MICA , CIITA [A [CIITA ,7, CTLA4A [CTLA4 ,9, PTPN2 [PTPN22 ,11,12, C[CYP27B1 , NLRP-1 1 [STAT4 , GATA3 [4 [GATA3  and CD273 [CD274  loci. Th3 [CD274 . However3 [CD274 . FinallyEthical approval for this work was obtained in each participating country as follows: Stockholm, Sweden\u2014Regionala etikprovningsnamnden i Stockholm Dnr 2008/296-31/2; Bergen, Norway\u2014Regional Ethics Committee West; Newcastle, UK\u2014Leeds (East) Research Ethics Committee, 2005 (REC reference number 05/Q1206/144). Informed, written consent was sought from each study participant at all centres with the exception of the Norwegian controls. These samples were gathered through the national blood donor scheme. All blood donors are informed of ongoing research through written information and are given the opportunity to opt out should they wish to do so. All Norwegian control samples collected in this way are anonymised at source.23 Caucasian multiplex AAD families from the UK and Norway, comprising 117 individuals, were included in the final analysis. None had autoimmune polyendocrinopathy syndrome type 1 features . Details of these families are included in For the genome-wide association study, genotype data from 2706 controls  from the WTCCC publicly available UK 1958 Birth Cohort was used.For the replication study, DNA was available from unrelated AAD case-control cohorts from the UK , Norway  and Sweden .All single nucleotide polymorphism (SNP) locations refer to HapMap data release number 28, on NCBI B36 assembly .Genotyping for the linkage study and genome-wide association study was undertaken on the Affymetrix SNP 6.0 array, according to manufacturer\u2019s instructions, in two batches. The first batch was genotyped at Almac Diagnostics  and the second at AROS Applied Biotechnology . To ensure genotyping fidelity, one sample was genotyped twice, once in each batch and the results were found to be comparable. Called genotype results were returned for each genotyped individual by the provider. Following the linkage and association analyses, results were analysed. A replication study was then planned to further investigate two of the linked regions. For the replication study, 64 HapMap tag SNPs were selected and genotyped at CIGMR, Manchester, on the Sequenom iPlex platform as per the manufacturer\u2019s instructions in UK, Norwegian and Swedish AAD case-control cohorts  measures taken for each aspect of the study are shown in Following data QC, 14,771 informative SNPs (approximately 4 SNPs per centimorgan (cM)) were selected for linkage analysis in Merlin . The inf-7 was chosen to account for multiple testing.To compare genetic variation in the AAD families with that seen in a population-based control sample, a dense, quality controlled marker map  was used for a genome-wide association analysis using EMMAX software . EMMAX . On chromosome 9, a linkage peak was observed between 36.0 and 40.4 cM (17486802\u201319751149 bp), with a maximum LOD and HLOD score within this peak of 2.90 at marker SNP_A-1996138 . On chromosome 7, a maximum LOD and HLOD score of 2.88 was seen at marker SNP_A-4232044  at position 70082089 bp within a linkage peak spanning 82.4\u201386.2 cM (70020160\u201373809454 bp). A second, smaller linkage peak between 69.8 and 71.7 cM (47565504\u201349876993 bp) was also seen on chromosome 7. Within this peak, a maximum HLOD of 2.09 at marker SNP_A-2279338  was observed (estimated proportion of linked families (\u03b1) 0.66). At this locus, the maximum LOD score was 1.28. Applying this model, no other locus had a LOD/HLOD score of greater than 2.0.Applying a rare dominant model, three loci on chromosomes 7, 9 and 18 had LOD/HLOD scores of greater than 2.5 . The maxSNP_A-1923640, rs2072633 at 31919578 bp). At this locus, the linear LOD score was 3.13. In this analysis, no other loci had LOD scores greater than 2.0. A full list of genes in the linked regions can be found in In a non-parametric analysis, which effectively excludes the parent-offspring pairs (18 kindreds informative), one locus on chromosome 6 had a LOD score of greater than 3.0 . Here, a\u20137) (2 >0.8) were not similarly associated (data not shown). However, association was noted with a cluster of five intergenic SNPs in tight LD (r2 >0.8), spanning 228 kilobases (kb) on chromosome 2, with maximal association at rs10495950 (P 9.7 x 10\u20138). In addition to these 5 SNPs reaching genome-wide significance, a further 5 SNPs in LD with these (r2 >0.6) were associated with AAD, although to a lesser degree, not reaching genome-wide significance. In addition, a pair of SNPs on chromosome 6  were also associated with AAD. Again, an additional 7 SNPs in LD with these (r2 >0.6) were also associated, but the degree of association did not reach genome-wide levels of significance.In the genome-wide association analysis, 17 SNPs were associated at a level of genome-wide significance (P<5 x 10\u20137) . The majNFATC1). The linkage peak on chromosome 7 was then also chosen for further investigation over the linkage peak on chromosome 9, as the region of interest is very gene-rich.As the linkage peaks and the associated regions were large and numerous, an initial replication study to look at just two regions of interest was undertaken, with plans to look at the other regions of interest in the future. Chromosome 6, which contained both linked and associated loci, was eliminated from further analysis because of the proximity of the findings to the MHC. MHC is already known to be strongly associated with AAD and most other autoimmune conditions and more novel findings were sought. The linkage peak on chromosome 18 was selected as the first area for more detailed analysis, as this had the highest LOD score and the region of linkage was close to a plausible candidate gene, nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1  and 18 (75241668\u201377639585 bp) were selected and genotyped on the Sequenom iPlex platform in UK, Norwegian and Swedish AAD case-control cohorts (primer sequences available on request).2 <0.2), all on chromosome 18, were associated with AAD (P<0.05). Maximal association was seen at rs7236339 where the minor A allele was present in 20.4% of the cases across the three national cohorts compared to 24.1% of the controls . Nominal association was also noted at rs754093 where the minor G allele was present in 47.1% of cases compared to 43.7% of controls  and at rs8091998 where the minor T allele was present in 20.2% of cases compared to 23.0% of controls . The genotype frequencies from the national cohorts and meta-analysis results are shown in This is the first linkage study in AAD and takes advantage of a unique resource of carefully phenotyped multiplex AAD families from both the UK and Norway. Importantly, in a non-parametric analysis, a linkage peak with a linear LOD score of 3.13 was seen on chromosome 6, corresponding to the HLA region, a known susceptibility locus for AAD. This finding demonstrates that the non-parametric analysis within the linkage study was sufficiently powerful to detect a true AAD locus with an odds ratio previously estimated to be between 3 and 15 ,27. ApplAssessing the pedigrees included in this analysis, no unifying mode of disease inheritance was apparent across all families. In any linkage analysis, this apparent heterogeneity can be allowed for either by using a non-parametric approach, or by using a parametric approach with a \u201cbest fit\u201d model and assessing the proportion of linked families, represented by \u03b1. The non-parametric approach, which assesses sharing of alleles that are identical by descent (IBD) and therefore by necessity must exclude affected parent-offspring pairs, loses some power as a result of the reduced number of kindreds suitable for the analysis. In contrast, the parametric approach allows all pedigrees to be included, thus increasing study power, and the calculation of the HLOD allows for genetic heterogeneity between the kindreds. At the linkage peaks on chromosomes 7, 9 and 18, all families were contributing to the results and therefore the observed LOD and HLOD scores were the same.CTLA4 and PTPN22. At loci where the previously reported associations are modest, this may be because parts of this study, in particular the genome-wide association study, are underpowered. However, it is perhaps not surprising given that the hypotheses underlying linkage and association analyses are different. Linkage analysis could be positive if distinct rare variants in a single gene were conferring disease susceptibility in several families, however these variants would not be replicated by association analysis due to their rarity, as association analysis relies on the common disease, common variant (CDCV) hypothesis [vice versa. However, the finding of linkage with the HLA region suggests that the multiplex AAD families do at least share some susceptibility loci with sporadic cases.Aside from linkage to, and association with, the MHC region of chromosome 6, the linkage peaks and clusters of associated SNPs detected in the linkage and genome-wide studies presented here do not correspond to known, widely replicated AAD susceptibility loci such as pothesis . It is aHLA region (rs1150753 is located in the Tenascin B gene while rs2187668 is found in the HLA-DQA1 gene) and again validates the approach. In addition, a cluster of five SNPs spanning 230 kb on chromosome 2 were also associated. These SNPs are located between the FBXO11 (F-box only protein 11) and FOXN2 (Forkhead box N2) genes, both of which are expressed in adrenal cortex. The FBXO11 protein product contains arginine methylation domains [FOXN2 gene encodes a forkhead domain DNA binding protein. However, this is a region of extended LD, which includes other genes expressed in adrenal cortex including mutS homolog 6 (MSH6), a DNA repair gene [(PPP1R21), a gene involved in the regulation of cell signalling and DNA repair [(STON1), involved in molecule trafficking [FBXO11 gene is the most likely candidate in AAD; however the true association could lie with any of these genes, or with a variant in a non-coding regulatory element. This region therefore requires further investigation.A genome-wide association analysis, comparing the 50 individuals with AAD from the pedigrees to 2706 UK 1958 Birth Cohort controls, revealed two clusters of associated SNPs on chromosomes 6 and 2 meeting genome-wide levels of significance. The associated SNPs on chromosome 6 correspond to the  domains  and reguair gene  protein A repair  and stonfficking . Based oOne significant limitation of the genome-wide association study is that a publicly available UK cohort of controls was used as a comparator, however just over half of the cases from the multiplex families were from Norway. Using the unaffected multiplex family relatives as controls would have been one way of controlling for this bias, however the small size of the cohort meant that this analysis would be greatly underpowered. The UK WTCCC controls were selected for this study as the data are freely available, the demographics of the control population are well described and the data have undergone strict quality control measures and are therefore known to be robust. A similar Norwegian control cohort that could be used for such a comparison is not freely available. Furthermore, if the 50 familial AAD cases were to be divided into individual UK and Norwegian cohorts for separate analyses, the study would lose significant power. Moreover, we have recently demonstrated that the UK and Norwegian populations are genetically similar  and therrs754093, encodes a non-synonymous variant (pCys751Gly) in exon 9 of the NFATC1  gene. This amino acid residue is conserved across many species including primates and rodents. SIFT analysis, which uses an algorithm to predict whether an amino acid change will disrupt a protein, predicts that this is a deleterious mutation [NFATC1 is a plausible candidate gene for AAD as it is expressed in adrenal cortex and encodes a transcription factor which plays a central role in gene transcription during the immune response [A replication study, looking at 64 SNPs in the linkage peaks of chromosomes 7 and 18, was conducted in AAD case-control cohorts from the UK, Norway and Sweden . To gain maximum study power, a meta-analysis was performed across the 3 national cohorts, using a random effects model to allow for any heterogeneity. Nominal association was observed with three independent SNPs on chromosome 18 and AAD. One of these, mutation . NFATC1 response . The twoUnfortunately, linkage analysis has low resolution and the linkage peaks generated in the study of the multiplex families were large and numerous. We selected two linkage peaks for further study and selected these on the basis that the linked regions were gene-rich and contained some plausible candidates for AAD which had not previously been investigated. In the replication study, despite focussing our efforts on only two of the four linkage peaks, we were still unable to account for all variation in the regions of interest on chromosome 7 and 18. Therefore, a significantly associated locus may have been overlooked. Further systematic exploration of the loci of interest generated in both the linkage analysis and the genome-wide association study is now required. To do this, whole exome sequencing of family members is underway.NFATC1 gene is a promising locus and is certainly worthy of future investigation.This linkage study has implicated a number of novel chromosomal regions in the pathogenesis of AAD in multiplex AAD families. However, these regions are large; neither the genome-wide association analysis nor the regional case-control association results have provided substantive evidence to confirm a new AAD susceptibility gene. Nevertheless, the S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file."}
+{"text": "Gadus morhua, in previously identified hybrid zones in the North Atlantic. Here, previously identified clinal loci were mapped to the cod genome with most (\u223c70%) occurring in or associated with (<5 kb) coding regions representing a diverse array of possible functions and pathways. Despite the observation that clinal loci were distributed across three linkage groups, elevated ILD was observed among all groups of clinal loci and strongest in comparisons involving a region of low recombination along linkage group 7. Evidence of ILD supports a hypothesis of divergence hitchhiking transitioning to genome hitchhiking consistent with reproductive isolation. This hypothesis is supported by Bayesian characterization of hybrid classes present and we find evidence of common F1 hybrids in several regions consistent with frequent interbreeding, yet little evidence of F2 or backcrossed individuals. This work suggests that significant barriers to hybridization and introgression exist among these co-occurring groups of cod either through strong selection against hybrid individuals, or genetic incompatibility and intrinsic barriers to hybridization. In either case, the presence of strong clinal trends, and little gene flow despite extensive hybridization supports a hypothesis of reproductive isolation and cryptic speciation in Atlantic cod. Further work is required to test the degree and nature of reproductive isolation in this species.Hybrid zones provide unprecedented opportunity for the study of the evolution of reproductive isolation, and the extent of hybridization across individuals and genomes can illuminate the degree of isolation. We examine patterns of interchromosomal linkage disequilibrium (ILD) and the presence of hybridization in Atlantic cod,  Understanding the complex contributions of ecological factors and genomic architecture to the formation of reproductive isolation is central to an understanding of speciation Recent studies continue to highlight the importance of genomic architecture to speciation and adaptation Gadus morhua, is a commercially exploited demersal marine fish found throughout the North Atlantic characterized by high dispersal potential, large fecundities, and large population sizes G. morhua) in conjunction with linkage mapping to demonstrate the utility of genomic islands of adaptive divergence Atlantic cod, The goal of the present paper is to examine the nature of the trends observed previously e.g.,  and in pet al. (2013) and explored the presence of LD and hybridization in the wild. These data represent cod sampled (N\u200a=\u200a466) from throughout the North Atlantic in 1996\u20132007, and approximately 20 individuals were sampled from each location . We accepted only E values >1.0e\u221240 with sequence similarity >96% as a hit to the correct position in the Atlantic cod genome. In the instance that a SNP did not map to a protein coding region of the genome, the distance (bases) to the nearest flanking genes on either side were calculated. SNPs within annotated genes and SNPs within a 5 Kb distance from an annotated gene were given a HGNC (Human Genome Nomenclature Committee) ID. We also used these annotations from the cod genome scaffold to obtain gene ontology and functionality information from the Uniprot (www.uniprot.org), or GeneCards (www.genecards.org) databases.Here we focus on linkage groups containing clinal (latitude) SNP outliers . Of SNPs previously identified as outliers, most could not be annotated using BLAST FST was calculated for each locus again in these three linkage groups using ARLEQUIN FST were examined in conjunction with map position using the linkage map In previous work, outlier tests and spatial analysis Two Bayesian approaches were used to infer the presence and frequency of various hybrid classes in the wild using only the outlier loci. Bayesian clustering was performed using STRUCTURE v.2.2.4 In addition to STRUCTURE, NEWHYBRIDS To evaluate the ability of each approach to correctly identify the various hybrid classes, individuals of each class were simulated using HYBRIDLAB 1.0 Outlier loci see . In LG7,To identify genes containing or associated with (<5 kb) these SNPs, SNP sequences were mapped to cod genome scaffold FST). The largest region of consistent high divergence and D\u2019 was near the middle of LG7. This region displayed some of the highest LD values and most consistent in comparison to the other LG\u2019s. A uniform block of approximately 4 cM near the centre of LG7 was strongly associated with an approximately 3 cM section of LG2  were characterized by pure N-type, with S-type and hybrid individuals dominating to the south . On bothHybridization along hybrid zones or between ecological species can provide rare opportunity for the study of the evolution of reproductive isolation e.g., . The degThe parallel nature of environmental gradients in the North Atlantic provide replicated environmental and latitudinal gradients and a model for the identification of genes associated with ecological influences Understanding the nature of these clinal loci such as whether or not they implicate similar physiological pathways or functions is critical to the interpretation of these clines. Previous attempts to explore the gene associations of the key SNPs that distinguish the N and S types using BLAST had very limited success, producing annotations of only a few sequences DrosophilaPlasmodium falciparumThe study raises the question of how the non-random association of genes on three different chromosomes can exist despite apparent high rates of interbreeding in some locations. The evidence of extensive LD among these outliers and islands of divergence on different chromosomes is consistent with either a lack of interbreeding, the suppression of recombination, or low hybrid fitness. A lack of successful interbreeding could explain the association among islands of divergence, but the apparent common occurrence of F1 individuals suggests this is not the case. However, as F2 or backcrossed individuals were rare, it is difficult to rule out intrinsic barriers to gene flow. In theory, transmission ratio distortion e.g.,  could paThe absence of most hybrid classes supports a hypothesis of reproductive isolation and limited gene flow, although several possible explanations exist for this apparent lack of hybrid classes in southern locations where the N and S forms co-occur. First, it is possible that this analysis lacked sufficient power to detect some hybrid classes. However, the simulation and analysis of hybrid classes supports the hypothesis that these individuals would have been correctly identified if present. A second possible explanation is that the presence of Bateson-Dobzhansky-Muller incompatibilities among islands of divergence may explain the observed linkage among chromosomes and the lack of some hybrid classes in the wild. The Bateson\u2013Dobzhansky\u2013Muller model predicts that postzygotic isolation evolves due to the accumulation of incompatible epistatic interactions, but few studies have quantified the relationship between genetic architecture and patterns of reproductive divergence In addition to the lack of some hybrid classes, the high proportion of individuals classified as F1\u2019s (>50%) in some samples was noteworthy. The number of individuals classified as F1\u2019s was highest at the mid latitudes examined (i.e. Georges Bank) and declined to the south in the east and west, with only a single F1 individual being present in the sample from Ireland. Despite the high proportion of F1\u2019s in some samples, the overall proportion among all samples was low (\u223c15%) suggesting that some samples or regions may have been biased towards hybridization or the collection of hybrids e.g., . AdmitteAnopheles mosquitoes where two sympatric forms have been described Regardless of which scenario is valid in this case, observation here of genetically discrete N- and S-types despite interbreeding suggests reproductive isolation and an evolutionarily significant division. There is also some evidence that the forms possess divergent phenotypes e.g.,  and occuDisentangling the contributions of ecological factors and genomic architecture to the formation of reproductive isolation is central to an understanding of speciation File S1Supporting Information file that includes Table S1, Figure S1, and Figure S2. Table S1. SNP associated genes and map position from SNP sequence alignment to cod genome using BLAT. FST between N and S types from either side of the North Atlantic  with linkage map distance across (A) LG2, (B) LG7 and (C) LG12.(PDF)Click here for additional data file."}
+{"text": "Fragaria vesca) reference genome and to disentangle the subgenomes of the wild octoploid progenitors of cultivated strawberry, Fragaria virginiana and Fragaria chiloensis. Our novel approach, POLiMAPS (Phylogenetics Of Linkage-Map-Anchored Polyploid Subgenomes), leverages sequence reads to associate informative interhomeolog phylogenetic markers with linkage groups and reference genome positions. In contrast to a widely accepted model, we find that one of the four subgenomes originates with the diploid cytoplasm donor F. vesca, one with the diploid Fragaria iinumae, and two with an unknown ancestor close to F. iinumae. Extensive unidirectional introgression has converted F. iinumae-like subgenomes to be more F. vesca-like, but never the reverse, due either to homoploid hybridization in the F. iinumae-like diploid ancestors or else strong selection spreading F. vesca-like sequence among subgenomes through homeologous exchange. In addition, divergence between homeologous chromosomes has been substantially augmented by interchromosomal rearrangements. Our phylogenetic approach reveals novel aspects of the complicated web of genetic exchanges that occur during polyploid evolution and suggests a path forward for unraveling other agriculturally and ecologically important polyploid genomes.Whole-genome duplications are radical evolutionary events that have driven speciation and adaptation in many taxa. Higher-order polyploids have complex histories often including interspecific hybridization and dynamic genomic changes. This chromosomal reshuffling is poorly understood for most polyploid species, despite their evolutionary and agricultural importance, due to the challenge of distinguishing homologous sequences from each other. Here, we use dense linkage maps generated with targeted sequence capture to improve the diploid strawberry ( Our justification for this new assembly is 2-fold. First, the high density of segregating polymorphisms in our linkage maps relative to the F. vesca ssp. vesca \u00d7 Fragaria bucharica linkage map used in the original genome assembly   (hereafter Fvb-s for \u201cselfed\u201d\u2014both ovule and pollen donor). Following our previously described methodology (\u22124); otherwise, genotypes were considered missing. We retained putative polymorphisms for linkage mapping if they showed missing genotypes in fewer than eight offspring, did not show greater than 85% of offspring with the same genotype, and had informative parental genotypes consistent with segregation in the offspring.We revised the assembly of the assembly  providesid clade . We previd clade , referrehodology , we extrhodology . Our prohodology  to map ohodology . We retaWe used OneMap  to generPrunus persica (peach) genome .We compared our Fvb-s map with our previous Fvb-m and Fvb-p maps, and tested whether the position of any polymorphism was inconsistent among maps. We then used these maps to generate a new genome assembly that placed all polymorphisms in linear order based on their linkage map positions. To do so, we assumed that scaffolds were assembled correctly, and therefore we rearranged the order of whole scaffolds rather than breaking scaffolds into sections, unless we had multiple polymorphisms on the same scaffold mapping to different locations. We defaulted to the scaffold order and orientation in FvH4 unless we had conflicting evidence from our linkage maps. We used the ) genome  to guidesed BLAT  to identsupplementary tables S1 and S2, Supplementary Material online): One for F. virginiana ssp. virginiana  using a modified CTAB (cetyltrimethylammonium bromide) procedure . In addition, we sequenced the complete genomes of the maternal parent in both octoploid crosses, following our previous methodology for low coverage whole-genome sequencing of Fragaria  markers (rocedure . TargeteFragaria .https://github.com/listonlab/POLiMAPS). Our goal was to identify variants occurring with approximately one-eighth frequency , which segregated in a Mendelian fashion . Fixed differences between homeologs can be distinguished from true SNPs because they will not show Mendelian segregation. We coded genotypes as heterozygous if the rare variant occurred at least twice and at \u22652.5% frequency. We coded genotypes as homozygous if there was no polymorphism or if the rare variant occurred a single time and at less than 1.25% frequency, which we considered to be a potential sequencing error. Ambiguous genotypes that did not meet these criteria or which had less than 32\u00d7 coverage were coded as missing. To filter for Mendelian segregation, we only retained sites in which the two genotypes were each observed in at least eight progeny. We retained markers for linkage mapping (LG SNPs) only if they had missing data for no more than a single progeny. We converted remaining genotypes to OneMap format and used OneMap to generate linkage maps as with Fvb-s. Due to the complexity of octoploid linkage groups, we did not attempt to manually add LG SNPs with larger numbers of missing genotypes, as we did with the diploid Fvb-s. For both octoploid species, we used a LOD of 5 to assign LG SNPs to linkage groups, and LG SNPs were assigned to the most likely position within the linkage group based on their LOD score. Some linkage groups were then manually joined based on visual inspection of segregation patterns, Fvb position and phylogenetic position. \u201cMinor\u201d linkage groups with fewer than five LG SNPs are reported but excluded from subsequent analysis. For all linkage groups, we assigned a haploid chromosome number (1\u20137) corresponding to the Fvb pseudochromosome with the largest number of LG SNPs in the linkage group. Each \u201cmajor\u201d linkage group of \u22655 LG SNPs was then named for the species, haploid chromosome number , subgenome , and parent ; for example, \u201cFvirg-IV-Av-p\u201d is a linkage group from F. virginiana, with most of its LG SNPs coming from Fvb4 . We used these reads to infer the sequence of the subgenome in the vicinity of the LG SNP (Perl script available at https://github.com/listonlab/POLiMAPS). In cases where a linkage group had an LG SNP less than a read length away from a variant differentiating other linkage groups and/or diploid taxa, we used this adjacent variant as a phylogenetic marker. We did not use the LG SNPs themselves in phylogenetic analysis because they are variable within the linkage group and they were chosen such that the minor variant only occurred in a single subgenome, so we did not expect any meaningful phylogenetic signal from the LG SNPs.For all LG SNPs in the octoploid linkage groups, we identified all original octoploid Illumina reads that mapped to Fvb and contained the minor variant A. We useF. iinumae, Fragaria mandshurica, and Fragaria viridis  for use in phylogenetic analysis, following our previous methodology for low coverage whole-genome sequencing of Fragaria (Fragaria nipponica (http://www.ncbi.nlm.nih.gov/assembly/GCA_000512025) and F. bucharica (http://www.ncbi.nlm.nih.gov/assembly/GCA_000511995) and used BLAT with default parameters to match contigs to Fvb .We sequenced whole genomes of Fragaria  . First, the site would be supportive of the phylogeny if the subgenome shares a base with its sister ingroup species (either F. vesca or F. iinumae), whereas the other two species share a different base. Second, the site would show \u201cintrogression-like homoplasy\u201d if the subgenome shares a base with the wrong ingroup species, whereas its sister species shares a different base with the outgroup, consistent with introgression of DNA between the two ingroup lineages. Third, the site would show \u201coutgroup homoplasy\u201d if the subgenome shares a base with the outgroup, whereas F. vesca and F. iinumae share a different base. In the absence of true introgression, homoplasy is only due to factors such as independent mutations, incomplete lineage sorting, and sequencing errors, and thus introgression-like homoplasy should occur at the same prevalence as outgroup homoplasy. Therefore, we tested for an excess of introgression-like homoplasy in order to detect introgression , and then we identified markers with an Fvb position that did not match this primary chromosome. We tested whether any translocations matched FvH4 but not Fvb, which could indicate errors in our Fvb assembly. We tested whether rearrangements were significantly clustered in their reference genome position by generating 10,000 simulated data sets in which LG SNPs showing rearrangements were randomly distributed among all targeted regions with LG SNPs. As with introgression (above), we tested whether the ratio of octoploid:diploid normalized depth differed between targeted regions with or without interchromosomal rearrangements.In order to identify interchromosome rearrangements, we first incorporated data from a recent linkage map of ananassa  along wiananassa  and we pananassa  on our FFragaria chromosomes with two linkage groups for different sections of FvH4 pseudochromosome 4, which were manually joined for a final Fvb-s linkage map of seven linkage groups spanning 326 cM.In the targeted capture sequence data from the 41 Fvb-s progeny, 3.244 Mb had a mean per-individual depth \u226520\u00d7 . Within these high-coverage sites, 1,825 polymorphic sites passed our quality thresholds and were used in linkage mapping. Our initial Fvb-s linkage map consisted of eight linkage groups corresponding to the seven F. vesca ssp. bracteata maps, we observed 44 interchromosome translocations, 40 intrachromosome translocations, 39 inversions, and 18 placements of unmapped FvH4_0 scaffolds . Our new assembly, here designated Fvb, contains 208.9 Mb of scaffold sequence, with 207.0 Mb assembled into seven pseudochromosomes (Fvb1\u2013Fvb7) and 1.9 Mb remaining unassembled on a false chromosome Fvb0. With 10-kb gaps separating all scaffolds, the complete size of Fvb is 211.7 Mb . We observed 164 distinct stretches of contiguous Prunus orthologs in FvH4, but only 116 such stretches in Fvb , and phylogenetic position (see below). Our final count was thus 28 major linkage groups ( \u2265 33 LG SNPs each) for both parents, representing the 28 haploid chromosomes, plus 4 maternal and 5 paternal minor linkage groups ( \u2264 3 LG SNPs each) (In the targeted capture reads from the two Ps each) .Fig. 4.F. chiloensis parents and their 42 progeny, 1,632 kb had a mean per-individual depth \u226532\u00d7 , whereas 705 kb had a mean per-individual depth \u226580\u00d7 . A total of 2,542 LG SNPs passed our quality thresholds and were used in linkage mapping, including 1,100 maternal LG SNPs and 1,442 paternal LG SNPs. As with F. virginiana ssp. virginiana, an additional 780 LG SNPs polymorphic in both parents were used to pair corresponding linkage groups. Using a LOD threshold of 5 initially yielded 39 maternal linkage groups and 36 paternal linkage groups. Splitting at gaps greater than 33 cM resulted in 39 maternal linkage groups and 40 paternal linkage groups. As with F. virginiana ssp. virginiana, some of these linkage groups were then manually joined, resulting in 28 major linkage groups ( \u226517 LG SNPs each) for both parents, representing the 28 haploid chromosomes, plus 2 maternal and 2 paternal minor linkage groups ( \u2264 4 LG SNPs each) (In the targeted capture reads from the two Ps each) .F. vesca, F. iinumae, and one of the diploid outgroup species . These markers represented 160\u2013421 regions of 1 kb in Fvb (A), so at most sites the majority of taxa had missing data . Nevertheless, phylogenetic signal was sufficient to resolve trees with high bootstrap support  for the major clades at all seven chromosomes (http://purl.org/phylo/treebase/phylows/study/TB2:S15849). In all seven trees, one of the four octoploid subgenomes groups with F. vesca, which we designated subgenome Av. Chromosomes from the other three subgenomes consistently form a clade with F. iinumae. The subgenome sister to F. iinumae we designated subgenome Bi, and the two basal to the F. iinumae/Bi clade we designated subgenomes B1 and B2, with B1 being the subgenome showing greatest divergence from F. iinumae. Because this divergence criterion is weak relative to the topology-based criteria, this distinction may not be evolutionarily meaningful across haploid chromosomes of subgenomes B1 versus B2.We obtained 540\u20131,517 markers per haploid chromosome for phylogenetic analysis, 317\u2013902 of which were phylogenetically informative with respect to the relationship between an octoploid subgenome, b in Fvb . For anyb in Fvb A, so at F. vesca than to F. iinumae, or where subgenome Av is closer to F. iinumae than to F. vesca (B). Introgression homoplasy was 1.7 times as common as outgroup homoplasy .We observed phylogenetic support at 54\u2013198 regions per haploid chromosome  for subgenome Av, 44\u2013178 regions  for subgenome Bi, 43\u2013120 regions  for subgenome B1, and 46\u2013113 regions  for subgenome B2 ; fig. 6.F. vesca B. IntrogF. iinumae-like subgenome  evolving to be more similar to F. vesca, with no instances of introgression making subgenome Av more similar to F. iinumae . More specifically, 54% of introgression clusters involve subgenome B1 acquiring F. vesca-like sequence. Because we defined subgenome B1 as having greater divergence from F. iinumae, introgression may be confounded with the distinction between B1 and B2. However, the average amount of introgression between B1 and B2  is higher than both Bi  and Av (no introgression). In the low-coverage whole-genome data, the mean ratio of F. vesca-like reads to F. iinumae-like reads is significantly higher within clusters (1.37) than in the rest of the genome (0.98) , and nontargeted regions (possibly duplicated throughout Fragaria).We were able to assign 777 SSR markers from t-test, t = 3.14, P < 0.01), suggesting copy number variation caused by duplication of these regions in some lineages since the common Fragaria ancestor (B).To minimize variance in coverage and standardize expected distribution patterns, we restricted further analysis to the 105 rearrangements that occur within 1 kb of one of 76 targeted regions. There are 20 targeted regions with two or more of these rearrangements, and each of the 105 of them occurs on a targeted region with 0.74 other rearrangements on average. However, if 105 rearrangements were randomly distributed among all 2,598 targeted regions with LG SNPs in the octoploids, only two regions with multiple rearrangements are expected (95% CI: 1\u20133), and the average rearrangement would share a targeted region with only 0.04 others (95% CI: 0.00\u20130.10), a highly significant difference indicating nonrandom clustering. The ratio of mean normalized depth per targeted region in the octoploids versus Fvb-s was higher and more variable at targeted regions showing rearrangements  relative to other targeted regions with LG SNPs in the octoploids  group with F. iinumae , which then hybridized with an unknown F. iinumae-like autotetraploid (B1B1B2B2) to form the octoploid ancestor of F. chiloensis and F. virginiana. Our results otherwise support the relationships among Fragaria species suggested by previous studies of nuclear . For our analyses we assumed, based on previous work  groups with  iinumae . These dbgenomes . A parsi nuclear  and plas nuclear  sequenceous work , that otegregate . Specifiegregate . Althougegregate .F. virginiana ssp. virginiana and F. chiloensis have a Mendelian sex locus mapping to different ends of different chromosomes in homeologous group VI , suggesting either independent origins or translocation . The \u201cuse\u201d of different autosomes for sex chromosomes has been seen among species in the same genera in other taxa  to be more F. vesca-like, whereas none of them converts subgenome Av to be more F. iinumae-like, a highly nonrandom pattern. Second, instances of introgression homoplasy are much more common than outgroup homoplasy, even though both should occur at a similar rate if they are only due to mutation, sequencing errors, or incomplete linage sorting  and one F. vesca individual, natural selection may not have played a role. On the other hand, if such homoploid hybrids were rare, it is unlikely that all three F. iinumae-like subgenomes would have hybrid ancestry unless this somehow facilitated polyploid hybridization and the subsequent success of the octoploids. If introgression took place among subgenomes after polyploidization, the unidirectional pattern was likely to have been driven by some consistent evolutionary process, presumably strong selection. Although homeologous exchange can occur preferentially in one direction for mechanistic rather than adaptive reasons . In other words, many of these observations may represent a \u201ccopy-and-paste\u201d rather than a \u201ccut-and-paste\u201d mechanism, consistent with the activity of Class I or Class II, Subclass 2 transposable elements (Fragaria or just in F. vesca ssp. bracteata (nontargeted and low octoploid:diploid depth ratio), most rearrangements occur in subgenomes B1, B2, or Bi, reflecting their greater phylogenetic distance from Fvb. Thus, many of the differences between subgenomes are likely to be interchromosome rearrangements that occurred in the diploid ancestors as they diverged from each other. However, those that do occur in subgenome Av must have happened in the relatively short time since this subgenome diverged from its diploid F. vesca-like ancestor, suggesting that postpolyploidization rearrangements may also be important, consistent with the common observation of increased transposition in interspecific hybrids . Fourth, Fvb shows much higher synteny with the Prunus genome than FvH4 does  is F. vesca ssp. bracteata ."}
+{"text": "The linkage scan found a single significant linkage peak on chromosome 12. Using an independent cohort of unrelated nonagenarians, we carried out a fine-scale association mapping of the region suggestive of linkage and identified three sites associated with healthy aging. These healthy-aging sites (HASs) are located in intergenic regions at 12q13\u201314. HAS-1 has been previously associated with multiple diseases, and an enhancer was recently mapped and experimentally validated within the site. HAS-2 is a previously uncharacterized site possessing genomic features suggestive of enhancer activity. HAS-3 contains features associated with Polycomb repression. The HASs also contain variants associated with exceptional longevity, based on a separate analysis. Our results provide insight into functional genomic networks involving non-coding regulatory elements that are involved in healthy aging and longevity.We have completed a genome-wide linkage scan for healthy aging using data collected from a family study, followed by fine-mapping by association in a separate population, the first such attempt reported. The family cohort consisted of parents of age 90 or above and their children ranging in age from 50 to 80. As a quantitative measure of healthy aging, we used a frailty index, called FI Aging can be defined as the occurrence of changes over time that adversely affect the vitality and functions, increasing the mortality rate . The onsOne quantitative indicator of the genetic basis of a complex trait is heritability, which is a measure of the extent of genetic control of the trait. The heritability of human longevity ranges from 0.15 to 0.35 , 7, whicEfforts to find such genetic elements have been made using genetic epidemiological methods . A major34, composed of 34 common health and function variables [34 increases exponentially with age, indicating declining health and function ability. The rates of increase differ significantly between offspring of long-lived parents (\u2265 90 years old) and offspring of short-lived parents (< 76 years old at death), indicating that FI34 is associated with parental longevity. The genetic basis of FI34 is substantiated by a narrow-sense heritability estimate of 0.39. Using FI34, we found elevated levels of resting metabolic rate (RMR) linked to declining health in nonagenarians [34 as a means of identifying additional physiological factors involved in healthy aging.For a quantitative measure of heathy aging, we developed the frailty index FIariables . FI34 inenarians . This reWe have completed genome-wide linkage scanning followed by fine-scale association mapping and found three sites associated with healthy aging at 12q13\u201314. These HASs are located in intergenic regions. Functional annotation of the HASs indicates that they possess genomic features indicative of enhancer or silencer activity. Our results indicate that healthy aging, and longevity as well, can be controlled by non-coding regulatory elements.npl) analysis in the MERLIN package was carried out on the data from the HAFS offspring only, the most significant linkage peak was found at 77cM  on chromosome 12 . When the inferred parental data were incorporated in the npl analysis, the linkage peak on chromosome 12 became more significant with a higher LOD score . Dense SNP markers can result in inflation of linkage when significant linkage disequilibrium (LD) between adjacent markers exists (see METHODS). However, linkage analysis software assumes marker-marker linkage equilibrium. Therefore, it is important to take into account potential marker-marker LD in linkage analysis. We obtained similar linkage results with different models of LD between SNP markers . The dependent variable was the FI34 score and the independent variable was the genotype of the SNP marker in the additive mode. Age and sex were included as covariates. This way, we tested whether the risk of healthy aging or unhealthy aging among the oldest-old increases additively as the number of copies of the minor allele increases, after adjustment for age and sex. Three groups of SNPs stood out in this analysis , SNPs in HAS-2 (the lowest P = 4.2 \u00d7 10\u22123), and SNPs in HAS-3 (the lowest P = 1.3 \u00d7 10\u22122). Employing the very conservative Bonferroni correction for multiple comparisons to maintain a family-wise error rate of 0.05, no SNP exceeded the adjusted significance level of 1.52 \u00d7 10\u22124  and applied logistic regression to the same SNPs with age and sex as covariates. This time, SNPs in HAS-2 and HAS-3 remained conspicuous, whereas HAS-1 became less prominent  was very close to the significance cutoff set by the Bonferroni adjustment  is well defined and characterized, with substantial familial clustering and heritability. Fourth, we leveraged the linkage analysis to establish association of gene variants with the phenotype in a separate cohort.By employing linkage analysis followed by association mapping, we mapped genomic sites on chromosome 12 that are linked to healthy aging, which in consequence uncovered multiple, novel genetic markers of longevity. Our approach deserves several comments. The genome-wide linkage scan is unbiased in that it doesn't need any prior knowledge. Secondly, having found a significant linkage, we were able to focus on the linkage region with a lowered power barrier. Thirdly, our phenotype  phenotypes for the first time, healthy aging and longevity. Somewhat different outcomes were observed for association of SNPs depending on the phenotype. The allelic association tests of longevity involved both young controls and nonagenarian cases, whereas the regression tests of healthy aging involved the oldest-old cases only. Furthermore, the phenotype of healthy aging is not exactly the same as the phenotype of longevity. Although FI earlier , FI34 prAll the functionally annotated SNPs that we examined in the healthy-aging associated sites (HAS) are non-coding variants. In particular, HAS-2 and -3 are located in intergenic regions barren of any known mRNA-encoding loci. Thus, our foremost task was to assign functionality to the SNPs that could be causative for the healthy-aging association. Many post genome-wide association studies of human pathological traits have found non-coding variants capable of modulating gene expression by affecting transcriptional enhancer or silencer activity .P values ranging from 9.96 \u00d7 10\u22126 to 1.79 \u00d7 10\u22127. Although these eQTL associations are considered not significant in this particular cell type (the study-wise threshold of significance was set at P < 5.78 \u00d7 10\u221212), these findings suggest that these healthy-aging associated sites may physically interact with other genomic sites to exert their regulatory effects over the course of healthy aging.SNP rs10877013 in HAS-1, located within a putative binding site for the CCAAT/enhancer binding protein (C/EBP), affects enhancer activity of DNA fragments containing the SNP in an allele- and orientation-dependent manner . Data frAVPR1A and DPY19L2, whose bivalent promoters are surrounded by Polycomb-group (PcG) protein-repressed sites , recruited in sibships. Ethnicity was self-declared. The Louisiana Healthy Aging Study (LHAS) was also described elsewhere [N = 869), ranging in age from 20 to over 100 years old. Ethnic affiliation was genetically-inferred using Structure analysis (0.8 assignment probability) [The Healthy Aging Family Study (HAFS) and demographic characteristics of its participants were described elsewhere . The parlsewhere . Its parability) , 39. OnlP < 0.01 using unrelated subjects) and SNPs with minor allele frequencies below 1% were eliminated using PLINK [e.g., claimed full-sib instead of actual half-sib), and an incorrect report of ethnicity. Any genotype errors in the corrected pedigrees were removed by another application of PEDSTATS. The final dataset contains 5, 533 SNPs for 392 subjects, including 98 dummy subjects created for missing parents (only one parent was available in most of the families).Following genomic DNA extraction and quantification, genotypes of 5,913 biallelic SNPs for 324 subjects were generated, using the Illumina Infinium Linkage 24 set. Genotype data were imported into GenomeStudio Data Analysis Software and analyzed using the Genotyping Module. The starting dataset contained genotype data for 5, 913 SNPs from a total of 320 subjects. The project threshold  was set at the default value of 0.25, and genotypes of samples with call rates \u2265 0.9 were exported and further analyzed. SNPs not in Hardy-Weinberg proportions  [We inferred healthy aging status of each parent from offspring data as follows. In selective breeding of animals or plants, response (R) is proportional to selection (S) with the constant of proportionality being the narrow-sense heritability (h2) , 45:R=hoff) is equal to h2 times the average phenotypic value of parents (Ppar):In other words, the average phenotypic value of offspring (Pa):Narrow-sense heritability is the proportion of phenotypic variance (V) accounted for by the additive genetic variance (VWith a is the covariance (Cov) between parents and offspring (slope between independent variable x and dependent variable y). Thus, equation Vpar is the regression coefficient. Thus, equation Cov/V2 is equal to regression coefficient , parents were divided into two age groups   , and parnpl module of MERLIN-1.1.2 with \u2013pairs \u2013npl command line options [pair statistic to test for allele sharing among pairs of affected individuals, whereas the \u2013npl option gives the Whittemore and Halpern NPL all statistic to test for allele sharing among all affected individuals [all statistics were better than the pair statistics , and the linkage statistics and plots presented in this paper are from the all statistics. The npl module of MERLIN requires binary traits. The HAFS subjects were divided into four age groups: three offspring age groups  and two parent age groups   for healthy aging; otherwise, the subject is coded \u20181\u2019 (no). We obtained similar results from using the lower limit of the 90% CI  was included  . The dive 90% CI . With thas lower . The linincluded .2 exceeds 0.16 (or 0.4), and the grid option is to have MERLIN carry out analysis at a 2-cM (or 5-cM) interval along the chromosome. Boyden and Kunkel [2 value of 0.16.The LD modeling in MERLIN is based on haplotype frequency estimation within clusters of markers , 49. We d Kunkel  and Edwad Kunkel  used the34 value for each age group was calculated in the same way as the cutoff values for the npl linkage analysis  for healthy aging; otherwise, the subject was coded \u20181\u2019 (no). All statistical analyses to test SNP associations were performed in PLINK. The additive mode of inheritance for each SNP assumed an increasing effect of its genotype with the increasing dose of the minor allele. The linear regression of raw FI34 scores on additive effects of SNPs was performed by issuing \u2013linear command line option along with \u2013covar and \u2013sex options, which were to adjust for age (in a separate covariate file) and sex. Similarly, the logistic regression of dichotomized FI34 on additive effects of SNPs was carried out by using \u2013logistic. The association for longevity between cases and controls was carried out by using the standard \u2013assoc option, which generated asymptotic P values based on 1-df \u03c77;2 statistics. We also performed Fisher's Exact test using \u2013fisher option and permutation by \u2013perm option. The PLINK outputs were directly fed into Haploview (v4.2) [We conducted a case-control association analysis using the LHAS sample. Following quality control measures as described above, we obtained genotype data for 453 SNPs in 312 subjects (136 controls of age from 31 to 59 plus 176 cases of age \u2265 90). The 176 cases were divided into two age groups (90 to 94 and 95 to 103), and the cutoff FIanalysis . The samw (v4.2)  to consthttp://www.well.ox.ac.uk). Imputation was carried out as directed by the user guide, using reference files downloaded from https://mathgen.stats.ox.ac.uk/impute/impute_v2.html#reference. The output files were converted to PLINK files for association tests.IMPUTE2 (v2.3.0)  was used"}
+{"text": "OSBPL10--a disease susceptibility gene for dyslipidemia--may also influence systolic blood pressure (SBP). In particular, our NGLA dense single-nucleotide polymorphism (SNP) analysis identified a 2.5-megabase (Mb) region that strongly cosegregates with low SBP (maximum posterior probability of linkage [PPL] = 68%). Furthermore, using the posterior probability of linkage disequilibrium (PPLD), we fine-mapped this region and identified 12 SBP-associated variants (PPLD ranging between 4% and 14%) that comprise a rare, 4-site haplotype. This haplotype extends into the candidate gene, OSBPL10 (oxysterol-binding protein-like 10). In contrast to our NGLA methods, a commonly used filter-based approach identified 23 variants with little evidence for spatial clustering around any particular gene or region of interest.To realize the full potential of next-generation sequencing, it is important to consider multiple sources of genetic information, including inheritance, association, and bioinformatics. To illustrate the promise of such an approach, we applied our next-generation linkage and association (NGLA) methods to the sequence data of a large 57-member Mexican American family with hypertension. Our results show that  The Genetic Analysis Workshop 18 (GAW18) distributed phenotype, genotype, and sequence data on 20 large Mexican American families. For 772 individuals who were not taking medication for hypertension, SBP was measured repeatedly with a maximum of 4 observations per person. Eighty-four individuals were actively taking hypertension medication, and the phenotypes of 530 individuals were unobserved. For individuals who were not taking medication for hypertension, we used SBP at the first available time point as our quantitative phenotype and regarded treated individuals  as affected. All analyses were performed on the unadjusted phenotype data without correction for covariates, and, as recommended by the GAW18 Program Committee, we restricted our attention to chromosome 3.p values less than 0.0001 were removed.We analyzed the phenotypic data under a quantitative trait threshold model using the program KELVIN , which ir2 between adjacent SNPs of 0.20. The genetic map was obtained from the Rutger's published map [To maximize the chance of detecting linkage, we analyzed all 20 families across a relatively sparse, but highly informative subsample of the 65,519 genome-wide association study (GWAS) SNPs. In particular, the selected subsample contained 719 SNPs with an average heterozygosity of 43% and a maximum pairwise To identify SBP susceptibility genes , we selected the pedigree with the largest PPL on chromosome 3 and applied our NGLA methods to the sequence data of a 2.5 Mb region covering the peak. These methods include (but are not limited to) a bayesian approach to linkage analysis , dense SNP linkage analysis , generalized association analysis with both family-based and population-based components , and standard techniques in bioinformatics. As such, we extracted all sequence variants in the region, and computed the PPLD for the 9263 sequence variants observed in this family. To compare our methods to a commonly used filter-based approach (FBA) we:1. Filtered out all common variants  from the 1,215,400 total variants of chromosome 3.n = 3) over the age of 31 years with low SBP.2. Selected distant relatives (3. Filtered out variants that were not shared by the distant relatives in (2).4. Used a phastCons 44-way threshold of 220  to priorWe performed the FBA analysis based on low SBP because the estimated trait model from the linkage analysis indicated that the locus would be protective against high SBP.Of the 20 large Mexican American families, we focused on Ped15, which is the family with the strongest evidence for linkage  of our results. Third, we confined our analyses to a 4-cM region of chromosome 3, which means that candidate genes in other regions  cannot be excluded. As such, candidate genes in these regions deserve further investigation. Also, to the extent possible, we examined the sensitivity of our methods to MCMC error and to different phenotype definitions. In each case, our conclusions remained qualitatively the same.OSBPL10. Furthermore, the 4-site rare haplotype that we identified, was not seen in any of the unlinked families, but was seen in one of the other linked families. Although the commonly used FBA identified OSBPL10, it missed the strongest PPLD association: 3_31907631 (rs7624739), and it found several variants in apparently unlinked regions of chromosome 3 (PPL <2%).Our NGLA approach to the analysis of sequence data led us to a manageable number of intronic (and intergenic) variants that lie in an excellent candidate gene: p value; detects linkage (PPL) and association (PPLD); and, in this case, permits the simultaneous use of both quantitative (SBP) and dichotomous phenotypic information . Our approach also gives researchers the opportunity to make efficient use of all of the available dense SNP data, and permits the use any existing linkage analysis method that can handle multipoint analysis of dense SNP data in large pedigrees . TThe authors declare that they have no competing interests.VJV and DAG designed the overall study, WCLS and YH conducted statistical analyses and drafted the manuscript. All authors read and approved the final manuscript."}
+{"text": "Vaccinium macrocarpon Ait.), by generating large numbers of markers for genomic studies such as genetic mapping.The application of genotyping by sequencing (GBS) approaches, combined with data imputation methodologies, is narrowing the genetic knowledge gap between major and understudied, minor crops. GBS is an excellent tool to characterize the genomic structure of recently domesticated (~200\u00a0years) and understudied species, such as cranberry  in a cranberry pseudo-testcross population wherein 5477 SNPs and 211 short sequence repeats (SSRs) were used to construct a high density linkage map in cranberry of which a total of 4849 markers were mapped. Recombination frequency, linkage disequilibrium (LD), and segregation distortion at the genomic level in the parental and integrated linkage maps were characterized for first time in cranberry. SSR markers, used as the backbone in the map, revealed high collinearity with previously published linkage maps. The 4849 point map consisted of twelve linkage groups spanning 1112\u00a0cM, which anchored 2381 nuclear scaffolds accounting for ~13\u00a0Mb of the estimated 470\u00a0Mb cranberry genome. Bin mapping identified 592 and 672 unique bins in the parentals and a total of 1676 unique marker positions in the integrated map. Synteny analyses comparing the order of anchored cranberry scaffolds to their homologous positions in kiwifruit, grape, and coffee genomes provided initial evidence of homology between cranberry and closely related species.GBS data was used to rapidly saturate the cranberry genome with markers in a pseudo-testcross population. Collinearity between the present saturated genetic map and previous cranberry SSR maps suggests that the SNP locations represent accurate marker order and chromosome structure of the cranberry genome. SNPs greatly improved current marker genome coverage, which allowed for genome-wide structure investigations such as segregation distortion, recombination, linkage disequilibrium, and synteny analyses. In the future, GBS can be used to accelerate cranberry molecular breeding through QTL mapping and genome-wide association studies (GWAS).The online version of this article (doi:10.1186/s12864-016-2802-3) contains supplementary material, which is available to authorized users. The advent of next generation sequencing (NGS) technologies, coupled with reduced representation genome sequencing strategies, such as genotyping by sequencing (GBS), can generate vast quantities of single nucleotide polymorphism (SNP) markers in minor crop species lacking extensive genomic resources . SNPs, thttp://www.biotech.cornell.edu/) with or without reference genomes for the creation of high density linkage maps 95 and GH1x35 .The current study was initiated to generate a large SNP dataset using GBS in order to: 1) develop a saturated cranberry linkage map, 2) characterize genome-wide recombination, linkage disequilibrium, and segregation distortion, 3) anchor available cranberry genomic scaffolds and putative coding DNA sequences (CDS)  for candEcoT221 digested DNA from cranberry parental plants (repeated 8 times each) and progeny (n\u2009=\u2009362) were sequenced yielding 3,000,842,566 total reads and 7,213,626 tags after merging. P1 accounted for 12,189,203 reads, whereas P2 accounted for 16,185,112 reads, each of the 362 siblings accounted for 8,348,193 reads on average. The samples were divided into four 96-well plates, and a linear model of the form y\u2009=\u2009X\u03b2\u2009+\u2009\u03b5 found significant differences between the number of reads per plate and per column, mainly due to variation in initial DNA concentrations and quality of the sample. A similar model was fitted to detect differences in missing data due to library preparation (plates) and samples  resulting from sequencing errors and/or other unknown biological mechanisms were discarded prior to map construction and only loci with mild levels of distortion p-values\u2009>\u20090.001 were included. Therefore, 5,477 segregating SNPs were selected for further analysis in addition to 211 SSR markers previously reported by Schlautman et al. 95 x GH1x35. The integrated linkage map was constructed using a total of 4648 SNP and 201 SSR biparental and uniparental markers (Table\u00a0n\u2009=\u2009x\u2009=\u200912 95 (P1) and GH1x35 (P2), selected due to their phenotypic differences for agronomic traits of interest. Both parental and progeny clones are maintained by the Valley Corporation in Tomah, WI. Total genomic DNA from 0.1\u00a0g of leaf tissue was extracted from fresh leaves of single uprights for each of the accessions using a modified CTAB protocol [Genetic analyses were performed using a full-sib segregating population of 362 cranberry progeny from a cross of two elite cranberry selections, protocol .Experion\u00ae  traces showing fragment distribution sizes of libraries made with ApeKI (4-base cutter), EcoT22I (6-base cutter), and PstI (6-base cutter), commonly used to reduce genome complexity according to Elshire et al. , were coApeKI cut the genome most frequently and the highest amount of SNPs would probably be identified in libraries made with this library. Nevertheless, sequence coverage per SNP locus, will likely be lower compared to libraries prepared with the 4-base cutter enzymes. Past studies in other crops suggest that fewer overall SNP calls will be made with the EcoT22I library compared to the PstI library; however, tighter fragment size distribution observed in the Experion\u00ae traces of the EcoT22I library suggested that sequence coverage would likely be higher for EcoT22I.EcoT22I, which cuts the site 5\u2032-ATGCA\u2193T-3\u2032//3\u2032-T\u2191ACGTA-5\u2032, was selected for reducing genome complexity in this study based on optimization results in cranberry , and such markers were removed from the study.R scripts were used to remove SNP and SSR markers with excessive missing data (>20\u00a0%) to avoid the loss of accuracy during the imputation process , and mar1 and P2 and initially ran in JoinMap 4.1\u00ae by removing all identical loci. Data was extracted and phased to be used as a DH population type and transformed to ASMap format for DH populations [colorize option to view graphical genotypes and the genotyping probabilities tab sheet in JoinMap 4.1\u00ae, markers causing discrepancies were manually removed.The imputed marker data was separated into 2 unique sets of uniparental configurations (ABxAA and AAxAB) to create starting parental maps for Pulations . The MSTRecombination matrices were calculated and plotted to validate the most parsimonious marker order found by the MST algorithm. Graphical presentations of parental maps were prepared using MapChart v2.2 to inspect collinearity between the current integrated map and the previous SSR map developed from Schlautman et al. .GO) was calculated by adding individual lengths of all linkage groups. The expected length of each linkage group was estimated according to method four of Chakravarti et al. [m\u2009+\u20091)/(m-1) where m is the number of mapped makers in the linkage group. The expected genome length (GE) was then estimated summing the estimated linkage group lengths. Observed genome coverage (GCO) was calculated as the ratio of GO and GE [.Genome length  [A and a at locus one and B and b at locus two with allele frequencies \u03c0A, \u03c0a, \u03c0B, and \u03c0b respectively and haplotype frequencies \u03c0AB, \u03c0Ab, \u03c0aB, and \u03c0ab, the difference between the observed and expected haplotype frequencies is D\u2009=\u2009\u03c0AB\u2009\u2212\u00a0\u03c0A\u03c0B. LD plots were obtained to visually assess the extent of r2 within the cranberry linkage groups and genome, and to determine if the calculated LD correlated with the marker positions in the high density linkage map. To assess the genome-wide rate of LD decay, marker positional information from all 12 linkage groups was combined into a single data frame, and r2 was computed for bins of 5\u00a0cM across to assess LD decay for each parental bin map.Linkage disequilibrium in the cranberry genome was analyzed using the two uniparental datasets generated for parental linkage bin mapping. The markers were sorted according to their positions in the parental linkage maps (recombination based). Linkage disequilibrium (r2) , definedChi-square analyses of segregation distortion implemented in JoinMap 4.1\u00ae  were perUsing the position information generated by the high density map we order the scaffolds containing such markers, and such sequences were merged to create pseudo-molecules. Using R capabilities, we generated additional files containing the marker, position in cM, scaffold origin, sequence, and genes annotated for such scaffold to be used in the synteny analysis , coffee , kiwifrun CIRCOS .1 cross, cross of two highly heterozygous parents; GBS, genotyping by sequencing; LDA, linear discriminant analysis; LG, linkage group; LOD, Logarithm (base 10) of odds; MST, minimum spanning tree; PCR, Polymerase chain reaction; QTL, quantitative trait loci; SNPs, single nucleotide polymorphisms; SSRs, simple sequence repeats; V. macrocarpon, Vaccinium macrocarpon Ait.CDS, coding DNA sequence; cM, centiMorgan; CP, cross pollination; DH, double haploid; F"}
+{"text": "EAC) and its premalignant precursor, Barrett's esophagus (BE). We aim to demonstrate the utility of linkage analysis to identify the genomic regions that might contain the genetic variants that predispose individuals to this complex trait (BE and EAC).Familial aggregation and segregation analysis studies have provided evidence of a genetic basis for esophageal adenocarcinoma (BE/EAC pedigrees, and performed both model\u2010based and model\u2010free linkage analyses, using S.A.G.E. and other software. Segregation models were fitted, from the data on both the 42 pedigrees and the 1000 pedigrees, to determine parameters for performing model\u2010based linkage analysis. Model\u2010based and model\u2010free linkage analyses were conducted in two sets of pedigrees: the 42 pedigrees and a subset of 18 pedigrees with female affected members that are expected to be more genetically homogeneous. Genome\u2010wide associations were also tested in these families.We genotyped 144 individuals in 42 multiplex pedigrees chosen from 1000 singly ascertained Linkage analyses on the 42 pedigrees identified several regions consistently suggestive of linkage by different linkage analysis methods on chromosomes 2q31, 12q23, and 4p14. A linkage on 15q26 is the only consistent linkage region identified in the 18 female\u2010affected pedigrees, in which the linkage signal is higher than in the 42 pedigrees. Other tentative linkage signals are also reported.BE/EAC pedigrees identified linkage regions on chromosomes 2, 4, 12, and 15, with some reported associations located within our linkage peaks. Our linkage results can help prioritize association tests to delineate the genetic determinants underlying susceptibility to BE and EAC.Our linkage study of  National statistics estimate 18,140 new cases of esophageal cancer, the majority adenocarcinomas, in 2014 , ASCC1 (10q), and CTHRC1 (8q). Our initial studies of FBE determined that families with three or more affected members develop esophageal cancer at an earlier age compared to families with only one or two affected members, suggesting a genetic basis for this complex trait  have been published, but they are in relatively small to moderate\u2010sized samples. Hu et\u00a0al.  found liThe multi\u2010center methodology for approaching probands and accruing FBE pedigrees has been previously described  . Fourteen relative pairs that were identified as unrelated and two full sib pairs identified as half sibs were accordingly corrected. There were 395 SNPs that have Mendelian inconsistencies and they were automatically excluded from the analyses. After relationship correction, the genotyped pedigrees include 78 affected and 66 unaffected individuals, comprising 111 sib pairs, 10 half sib pairs, 18 avuncular pairs, and nine cousin pairs.We thus obtained a set of data with 1000 singly ascertained Barrett's esophagus pedigrees comprising 10,594 individuals that was used to estimate the genetic mode of inheritance of FBE. The dataset used here is a corrected and expanded version of the data that were analyzed by Sun et\u00a0al. . ClinicaTo find appropriate models for model\u2010based linkage analysis, we fitted segregation models using the program SEGREG in S.A.G.E. 6.3. This was done on both the 1000 singly ascertained pedigrees with 10,594 individuals and the 42 linkage informative pedigrees with 1132 individuals. We fitted two types of statistical segregation models: the finite polygenic mixed model (FPMM) \u00a0\u2265\u00a00.2 and the intervals between any two consecutive SNPs at least 0.2\u00a0cM. The single marker model\u2010based linkage analysis was performed for all the SNPs.P\u00a0<\u00a00.05, empirical P\u2010values were evaluated by permutation, the number of permutations determined for the P\u2010values to be within 20% of their true values with 95% confidence, up to 100,000 permutations. Because the permutation test currently in SIBPAL can lead to inflated significance of very small P\u2010values, especially in larger sibships  is used here whenever it was found to be larger than the asymptotic P\u2010value.Successively using the programs FREQ, GENIBD, and SIBPAL in the S.A.G.E. program package, allele frequencies and sibpair identity by descent (IBD) were estimated for all the SNPs and single marker model\u2010free linkage analysis was performed. By using the W4 option in SIBPAL, the optimally weighted average of the squared sibpair trait sum and squared sibpair trait difference  less than 0.01 by a likelihood ratio test is reported.Association tests were also performed for each SNP in the linkage panel, separately using the 42 pedigrees and the 18 female\u2010affected pedigrees. In order to account for familial correlations, this analysis was performed using the program ASSOC in S.A.G.E. The association model in ASSOC can include both a polygenic variance component and a common sibship variance. However, both these two variance components converged to 0 on testing 95% of the SNPs, and only one of them could be estimated for the remaining 5% of the SNPs. In testing the association, each SNP was coded in three ways \u2013 dominant, recessive, or additive. Sex and founder status were included as covariates of FBE . For each SNP, the minimum association On fitting the FPMM model with 1 polygenic locus, we found that \u2013 on the basis of Akaike's A information criterion (AIC) \u2013 using the 1000 pedigrees or the 42 linkage pedigrees, the best\u2010fitting model was found to be a dominant model ; on fitting the MLM model, using either dataset the best\u2010fitting model was a recessive model   or suggestive linkage lod\u00a0>\u00a02) regions by single\u2010marker or multipoint linkage analysis Fig.\u00a0A. Using  regions Under the recessive model, a wide, consistent linkage region was identified by both single\u2010marker and multipoint linkage analyses on chromosome 2q31 (174\u2013190\u00a0cM), within which three SNPs have lod\u00a0>\u00a02 by single\u2010marker analysis (Table\u00a0The linkage region on chromosome 4 (48\u201359\u00a0cM) was identified by multipoint linkage under the dominant model (lod\u00a0=\u00a03.2), and a SNP with single\u2010marker linkage was identified in this region under the recessive model (lod\u00a0=\u00a02.0). Another region on this chromosome, at 73.2\u00a0cM, was identified by single marker linkage under the recessive model. A region on chromosome 8 at 99\u2013125\u00a0cM was identified by model\u2010based multipoint linkage analyses under the dominant model (lod\u00a0=\u00a03.8 at 121\u00a0cM).P value\u00a0<\u00a00.0012 (or \u2212log10(P)\u00a0>\u00a02.92), which corresponds to lod\u00a0>\u00a02 (Table\u00a0S2). However, this SNP identified by model\u2010free linkage is not consistent with any other linkage analyses we performed.Single\u2010marker model\u2010free linkage in the 42 pedigrees identified one SNP having permutation In the 18 female\u2010affected pedigrees, the only region that was consistently identified to have suggestive linkage by two analyses is on chromosome 15q26, identified by both the single\u2010marker and multipoint linkage analyses under the recessive model, with lods of 2.45 and 2.98, respectively, at 128.8\u00a0cM Table\u00a0. This reWe also performed multipoint model\u2010based linkage using MERLIN , 4 (a SNP at 72.8\u00a0cM had P\u00a0=\u00a00.008), and 8 (a SNP with P\u00a0=\u00a00.002) . However, association tests with the 42 pedigrees identified 4 SNPs that showed some possible evidence of association in the linkage regions on chromosomes 2  was identified by multiple linkage analyses , and two SNPs in this region also showed suggestive association. The linkage region on chromosome 12q23 (110\u2013120\u00a0cM) was identified by multipoint linkage under both dominant and recessive models. The region on 4p14 (48\u201359\u00a0cM) was identified by multipoint linkage under the dominant model, but with a single SNP linkage under the recessive model. Furthermore, chromosome 4q (72\u201373\u00a0cM) and 8q22 (99\u2013125\u00a0cM) were identified in the 42 pedigrees by one of the linkage analyses and showed some evidence of association.In the 18 female\u2010affected pedigrees, the only region identified by two or more linkage analyses was on chromosome 15q26 under the recessive model. This region was also identified in the 42 pedigrees, but the linkage was stronger in the subset of 18 pedigrees.In this study, we performed extensive linkage analyses on Barrett's esophagus (BE) and its associated adenocarcinomas on 42 multiplex pedigrees and a subset of them, 18 female\u2010affected pedigrees. The best fitting inhertiance models were dominant or recessive, depending on the penetrance model we used and whether it included a polygenic component. By model\u2010based linkage analyses under the dominant or recessive models, regions on chromosomes 2q, 4p, and 12q were identified to have consistent linkage by at least two linkage analyses in the 42 pedigrees, and a narrow region on chromosome 15 was identified by model\u2010based linkage analyses under the recessive model in the 18 female\u2010affected pedigrees. Some other regions or SNPs on chromosome 4q and 8q were also identified by linkage and association analyses. All these linkage regions or positions are candidates for further study or verification and potential genetic testing in BE/EAC families.It could be that the 42 pedigrees are not genetically different from the 18 female affected pedigrees and only appear to be so by chance. However, the proportion of linked pedigrees at the two linkage regions on chromosome 2 and chromosome 8 estimated by MERLIN suggests that, for the chromosome 2 linkage region, the 18 female\u2010affected pedigrees are more genetically heterogeneous than the whole set of 42 pedigrees. On the other hand, for the linkage on chromosome 15, the 18 female\u2010affected pedigrees are more genetically homogeneous than the 42 pedigrees.In the previous segregation analysis, we found that BE was transmitted in a dominant mode by fitting the FPMM model  to be associated with BE, EAC, or reflux esophagitis. The associated SNPs in two of the genes, IGF1 and IGF1R, are near the linkage peaks we have identified. The associated SNP rs6214 in IGF1 that was reported by McElholm et\u00a0al. is located in our linkage region on chromosome 12q23, 685\u00a0kb from the peak SNP rs3205421; but in another association study of BE, in a cohort of 1852 cases and 5172 controls, rs6214 did not reach genome\u2010wide significance  that regulates the KIAA1715 expressed in esophagus mucosa, and the peak SNP rs3205421 in the chromosome 12 region is a significant cis\u2010eQTL (P\u00a0=\u00a02\u00a0\u00d7\u00a010\u221213) that regulates the GNPTAB gene expressed in esophagus muscularis . These reported functional effects further support our findings.There are some reported associations located in the linkage regions that we have identified. McElholm et\u00a0al.  studied f et\u00a0al.  reportedon Table\u00a0 reportedon Table\u00a0. In addion Table\u00a0, we founIn this study, we genotyped all individuals with blood samples available. This is not an ideal study with a sufficiently large sample size to come up with results that can stand on their own. Nevertheless, the corroborative results already found in recent genome\u2010wide association studies demonstrate that thorough linkage analyses, even on non\u2010ideal data, can help focus the search for causal genes. On the reasonable assumption that it is genetically more homogeneous, we specifically studied linkage in the subset of 18 female\u2010affected pedigrees. It is always possible that females have some gender\u2010specific protective mechanisms from developing Barrett's esophagus, but the definitive reason for such a gender difference is unclear. An ideal linkage study would include more covariates, such as BMI and gastroesophageal reflux symptoms, easily done with the software we used. If, for example, evidence for linkage changes when BMI is included as a covariate, a genome\u2010wide association study of this obesity\u2010related disease would locate the risk variants by addressing the impact of BMI on the association, if we assume that 5\u00a0\u00d7\u00a010s Galwey  to deterThe rapidly rising incidence of EAC over the past four decades is undoubtedly related to an uncharacterized environmental factor. We propose that this change in the environmental factor is interacting with an underlying complex genetic susceptibility, which is contributing to this rising incidence of BE and EAC, and the genes involved will be easier to find if the linkage results we report here are taken into account.None declared.Data S1. Description of the permutation test.Figure\u00a0S1. Minimum P values of association at each SNP on testing additive, dominant, and recessive effects respectively by the likelihood ratio test using 42 pedigrees, adjusting for sex and founder status.Figure\u00a0S2. Minimum P values of association at each SNP on testing additive, dominant, and recessive effects, respectively, by the likelihood ratio test using 18 female\u2010affected pedigrees, adjusting for sex and founder status.Table\u00a0S1. Penetrance functions of the four segregation models in Table\u00a0Table\u00a0S2. Summary of linkage  and association analyses (ASSOC) using all 42 pedigrees at the positions with suggestive linkage or association by any of the analyses.Table\u00a0S3. Summary of linkage  and association analyses (ASSOC) using 18 female affected pedigrees at the positions with suggestive linkage or association by any of the analyses.Click here for additional data file."}
+{"text": "Two-point linkage analyses of whole genome sequence data are a promising approach to identify rare variants that segregate with complex diseases in large pedigrees because, in theory, the causal variants have been genotyped. We used whole genome sequence data and simulated traits provided by Genetic Analysis Workshop 18 to evaluate the proportion of false-positive findings in a binary trait using classic two-point parametric linkage analysis. False-positive genome-wide significant log of odds (LOD) scores were identified in more than 80% of 50 replicates for a binary phenotype generated by dichotomizing a quantitative trait that was simulated with a polygenic component (that was not based on any of the provided whole genome sequence genotypes). In contrast, when the trait was truly nongenetic (created by randomly assigning affected-unaffected status), the number of false-positive results was well controlled. These results suggest that when using two-point linkage analyses on whole genome sequence data, one should carefully examine regions yielding significant two-point LOD scores with multipoint analysis and that a more stringent significance threshold may be needed. The development of low-cost, high-throughput sequencing technologies has reignited the interest in family-based study designs for finding causal genes in rare and common diseases. Linkage analysis was designed for the investigation of rare variants of large effect and is robust to allelic heterogeneity. Most researchers are familiar with using multipoint methods for assessing linkage, which leverage cosegregation of a marker-haplotype with the trait in a family as a proxy for the location of causal variants. Whole exome sequencing (WES) and whole genome sequencing (WGS) of several or all individuals in a pedigree has now become feasible. These data provide information about genetic variability at a very high resolution. Multipoint linkage analyses of such dense sets of markers are time-consuming, do not scale well to millions of markers, and intermarker linkage disequilibrium (LD) can cause inflated type I error rates when pedigree founders are not genotyped . A powerGenotype data for all 8,348,674 sequenced and imputed single nucleotide variants (SNVs) on the odd-numbered chromosomes were analyzed. There were 959 individuals with genotypes; 430 individuals without genotype data were added to provide complete pedigree information for linkage analysis, and one member was removed from each of two monozygotic twin pairs. There were 413 founders: 108 had genotypes and phenotypes, 9 had genotypes but no phenotypes, and 296 had neither. Because our goal was to estimate the false-positive rate under the null hypothesis of no linkage, we used two phenotypes that were simulated independently of any genotyped SNVs in the GAW18 data set. We requested the details of the simulation, and therefore knew the generating model of the simulated traits. The first analyzed phenotype was a binary trait based on the provided quantitative phenotype Q1 that was simulated by the GAW18 data providers to allow estimation of false-positives. Q1 was simulated as a normally distributed quantitative trait correlated among family members with heritability = 0.68, but was not influenced by any of the genotyped SNVs [Several pedigrees contained loops that cause problems for likelihood estimation in the linkage analysis program we used. Because our goal was to estimate false-positive rates under the null , we chose very simple strategies for breaking these loops to produce simple pedigrees. Different approaches to break loops were applied depending on their complexity. Removing the unaffected individual T2DG2501049 in pedigree 25 broke a loop caused by a woman with children from two brothers. The simple loops in pedigrees 2 and 3 were removed by duplicating an unaffected individual (T2DG0200031 and T2DG0300138) with both parents and children in the pedigree. Duplicated individuals had the same genotype and phenotype data but modified family relationships. Pedigrees 6 and 7 were excluded because their more complicated loops could not have been broken with the simple approaches used for the other pedigrees. Thus we had a set of simple pedigrees that allowed analysis of false-positive rates of two-point parametric linkage. In analyses of real data, use of a program that can analyze large pedigrees with loops intact would have optimal power.http://cran.r-project.org/web/packages/paramlink/index.html). A dominant model was chosen because no, or only a very low number of, rare allele homozygotes are observed for rare SNVs and because we were seeking to evaluate type I error rate rather than power. In a real analysis, one could either use a model from segregation analysis or could utilize dominant, recessive, and additive models with reduced penetrance [Allele frequencies of all SNVs were estimated with PLINK , using fnetrance  and adjunetrance -13. Penenetrance , with a netrance . The GWSResults are presented separately for the two different unlinked phenotypes.For the binary trait based on the provided quantitative null trait Q1, 43 of 50 replicates showed at least one GWS LOD score, with values ranging from 3.3001 to 5.685 , only two out of the 50 replicates had GWS results. In one replicate, one SNV on chromosome 1 had a combined LOD score of 3.336 that was driven by a LOD score of 3.334 in family 3. The MAF was estimated as 0.0085, and all seven affecteds in family 3 were heterozygous for the rare allele. Only three of 53 unaffecteds in this family were heterozygotes. In another replicate, 19 SNVs in a 4.6 Mb region on chromosome 3 had combined LOD scores between 3.308 and 3.751, with family 5 having the largest family-specific LOD scores, ranging from 2.128 to 2.578. These SNVs were in two blocks with nearly complete LD, and estimated MAF was 0.389 on average. Again, all affecteds, or all but one affected, were heterozygous or homozygous for the rare allele in family 5.Linkage analysis fell out of favor in the genome-wide association studies era, but the power of families can be well leveraged in sequencing studies. The advent of large-scale WES and WGS studies has rekindled interest in family-based analysis designs ,14. FamiIn this study we were interested in type 1 error rates of two-point linkage analysis of a binary trait under the null hypothesis of no linkage. GAW18 provided only one quantitative trait simulated under the null hypothesis of no linkage  so we chose to dichotomize this trait; we also simulated our own qualitative trait that had no genetic effects whatsoever. As our purpose was evaluation of false-positive rates for traits under the null, issues of power do not apply, and this approach is reasonable. However, we do not recommend dichotomization of a quantitative trait in real data because linkage analysis is generally more powerful for a quantitative trait compared to a binary dichotomization of that trait. Incorporating covariates into the linkage analysis can also improve power in a real study especially for established risk factors.Our analysis revealed very different results for the two null phenotypes. We observed a large number of false-positive results for the polygenic trait, although it was simulated without using any of the provided genotypes and was unlinked to the provided chromosomes. Most of the false-positive results observed for this trait involved SNVs with relatively common minor alleles, and only 12 of the 2398 significant SNVs across all 50 replicates had arbitrary MAF of 0.0001. Although incorrect specification of both the trait model and the marker allele frequencies can inflate type I error rates, estimation of marker allele frequencies from the data (as we did here) has been shown to control false-positive rates even when the trait model is misspecified, so model misspecification is not likely the cause of the type 1 error rate inflation observed here. Differences in pedigree structure and model misspecification also appeared to have little effect on false-positive rates of two-point LOD score linkage of complex traits in a study of a single marker with 8 equifrequent alleles . The simIn contrast, the number of false-positive results based on the classic LOD score threshold of 3.3 is well controlled if affected-unaffected status is randomly assigned , even when more than eight million SNVs are analyzed by two-point parametric linkage analysis.Both two-point and multipoint linkage analysis can detect linkage to a region containing a causal variant even if the causal variant was not genotyped. Even though we only evaluated LOD scores at \u03b8 = 0, in a real analysis, one should also obtain the LOD score at the maximum likelihood estimate of \u03b8 to detect SNVs very close to a causal variant. One must be careful interpreting a two-point analysis because the most significant two-point LOD may not indicate the actual causal variant, but simply the closest variant to the untyped causal variant as, for example, when the causal variant is not analyzed because it is in a region with low coverage or was filtered out because of bad quality. One should examine the strongest linkage region for variants dropped because of low quality and possibly resequence the most strongly linked region.This study of false-positive rates in WGS data simulated under the null hypothesis of no linkage to any chromosomes provided in the data set suggests that a higher LOD score threshold may be required when analyzing a complex trait with nonzero heritability. However, more extensive simulations are required before such new thresholds can be proposed. Reestimating allele frequencies and performing multipoint linkage in regions showing significant two-point scores may help to resolve this problem. Single significant SNVs with no supporting linkage to nearby variants may be eliminated by the multipoint analysis. This remains an area of future research.The authors declare that they have no competing interests.CLS, SS, and JEBW designed the study; CLS, SS, and CDC conducted analyses. CLS and SS drafted the manuscript. All authors read and approved the manuscript."}
+{"text": "SNAI1 gene located on human chromosome 20q13.2, Snail1 is composed of 264 amino acids and usually acts as a transcriptional repressor. Phosphorylation and nuclear localization of Snail1, governed by PI3K and Wnt signaling pathways crosstalk, are critical in Snail1\u2019s regulation. Snail1 has a pivotal role in the regulation of epithelial-mesenchymal transition (EMT), the process by which epithelial cells acquire a migratory, mesenchymal phenotype, as a result of its repression of E-cadherin. Snail1-induced EMT involves the loss of E-cadherin and claudins with concomitant upregulation of vimentin and fibronectin, among other biomarkers. While essential to normal developmental processes such as gastrulation, EMT is associated with metastasis, the cancer stem cell phenotype, and the regulation of chemo and immune resistance in cancer. Snail1 expression is a common sign of poor prognosis in metastatic cancer, and tumors with elevated Snail1 expression are disproportionately difficult to eradicate by current therapeutic treatments. The significance of Snail1 as a prognostic indicator, its involvement in the regulation of EMT and metastasis, and its roles in both drug and immune resistance point out that Snail1 is an attractive target for tumor growth inhibition and a target for sensitization to cytotoxic drugs.Snail1 is the founding member of the Snail superfamily of zinc-finger transcription factors, which also includes Snail2 (Slug) and Snail3 (Smuc). The superfamily is involved in cell differentiation and survival, two processes central in cancer research. Encoded by the  The Snail superfamily of transcription factors includes Snail1, Slug, and Scratch proteins, all of which share a SNAG domain and at least four functional zinc fingers ,3]. The. The3]. Drosophila melanogaster in 1984, Snail1 also has well-documented homologs in Xenopus, C. elegans, mice, chicks, and humans . Sna. SnaDrosEpithelial-to-mesenchymal transition (EMT) is the process by which epithelial cells lose their apical polarity and adopt a mesenchymal phenotype, thereby, increasing migratory properties, invasiveness and apoptotic resistance. The expression of mesenchymal markers, like vimentin and fibronectin, replaces that of the usual epithelial markers, including E-cadherin, cytokeratins and Mucin-1 . EMT is The Notch intracellular domain, LOXL2, NF-\u03baB, HIF-1\u03b1, IKK\u03b1, SMAD, HMGA2, Egr-1, PARP-1, STAT3, MTA3, and Gli1 all interact directly with the Snail1 promoter to regulate Snail1 at the transcriptional level \u201329]. Hy. Hy29]. In addition, ILK promotes PARP-1 binding, and STAT3 binds as a final result of an IL-6/JAK/STAT pathway ,24]. In. In24]. YY1 and Snail1 itself are two special instances of transcriptional Snail1 regulation. YY1 binds to the 3\u2019 enhancer, rather than the promoter, and knockdown of YY1 has been shown to decrease Snail1 expression . FurtherExperiments conducted to elucidate the relationship between p53, a tumor suppressor protein, and Snail1 have shown that p53 acts via miR-34a, -34b, and -34c to repress Snail1 at a 3\u2019 untranslated region (UTR). Consequently, when p53 is repressed, the repression of Snail1 is lifted, and the expression of Snail1 rises .107, S111, S115, S119), results in Snail1\u2019s being exported to the cytoplasm. The second instance of phosphorylation  leads to its ubiquitination by \u03b2-Trcp, which recognizes the destruction motif D95SGxxS100 and ubiquitinates Lys98, 137, and 146. Consequential proteasomal degradation follows . Th. ThCDH1,cadherin . Total Ecadherin . Decreascadherin .Four Snail1 complexes have been identified as mechanisms of E-cadherin repression. (1) Snail1 interacts with G9a, which concurrently recruits DNA methyltransferases (DNMTs) to the E-cadherin promoter. Snail1\u2019s zinc fingers are thought to interact with the G9a ankyrin repeats, SET domain, or both. The complex has been shown to increase H3K9me2 and decrease H3K9 acetylation . (2) TheRaf kinase inhibitor protein (RKIP), a member of the phosphatidylethanolamine-binding protein (PEBP) group, suppresses metastasis by inhibiting the Raf-MEK-ERK and NF-\u03baB pathways \u201365]. In. In65]. PTEN promoter, which contains two E-boxes, and represses PTEN . A . A PTEN poptosis . Akt, oppoptosis . Throughpoptosis .Occludin, an integral membrane protein crucial to the integrity of tight junctions, was first identified in 1993. The transmembrane protein has four hydrophobic domains within its 522 amino acid sequence and a molecular weight of 65 kDa ,74]. Th. Th74]. The claudin family contains more than twenty members, all of which are integral proteins spanning the membrane four times. Family members range from 20-27 kDa, but they all share PDZ binding motifs, which allow them to interact with ZO-1, ZO-2, and MUPP-1, among others . ClaudinSpecifically, claudin-1, -3, -4, and -7 are all susceptible to repression by Snail1. The promoter sequence of each of these proteins contains multiple E-box binding motifs: claudin-1 has two E-boxes, claudin-3 has six, claudin-4 has 8, and claudin-7 has eight. As such, Snail1 can completely inhibit their transcription . The desMUC1, is an epithelial marker expressed at the apical surface of epithelial cells in the reproductive tract, digestive tract, lungs, kidney, liver, eyes, and other tissues . Ad. AdMUC1, tissues . Mucin-1 tissues . O-linke tissues . In epit tissues ,80]. Sp. SpMUC1, tissues . PerhapsThe 2872 bp promoter facilitates much of Mucin-1\u2019s regulation, and it notably includes five sites for YY1 binding . Snail1 ZEB-1, like Snail1, is a zinc-finger transcription factor that assists in the induction of EMT. Using E-boxes and co-repressors such as CtBP and BRG1, ZEB-1 represses E-cadherin and Mucin-1 ,84]. Ho. Ho84]. The presence of Snail1 increases both RNA and protein levels of ZEB-1 during EMT. Snail1 expression in MDCK clones causes a 2.5-fold increase in ZEB-1 promoter activity compared to control cells. The abilities of Snail1 and ZEB-1 to repress E-cadherin are additive, and the two transcription factors work together to achieve a complete EMT .Vimentin is 57 kDa intermediate filament generally restricted to mesenchymal cells . VimentiFibronectin is a glycoprotein involved in cell adhesion, differentiation, and migration ,93]. A . A 93]. CK18 gene, which is located on chromosome 12q13. Cytokeratin 18 is an intermediate filament protein involved in cell structure, cell signaling, and the cell cycle . C. CCK18 gll cycle . Snail1 ll cycle . Unlike ll cycle .Matrix metalloproteinases (MMP) cleave extracellular matrix substrates and, thereby, alter cell-matrix adhesions . MMP-2 aSnail1\u2019s presence increases the mRNA levels of both MMP-2 and -9 . One sugLymphoid enhancer-binding factor 1 (LEF-1) is a T-cell factor commonly detected in tumors ,120]. T. T120]. Snail1 is expressed in many types of cancer. Snail1 overexpression usually correlates with increased migration, invasion, and metastasis. An inverse relationship with E-cadherin is expected, and Snail1 consequently corresponds with poor differentiation as well. Frequently, more advanced malignancies and poor prognosis also accompany elevated Snail1 expression Table\u00a0.Invasive breast carcinomas, including infiltrating ductal (IDC) and infiltrating lobular carcinomas (ILC), spread to surrounding breast tissues, lymph nodes and the pleural cavity. Assigned histological grades, with three being the highest, correlate with prognosis . Breast Snail1 is not present in normal breast epithelium, nor is it present in ILCs (n = 21). Of 17 patients, Snail1 was expressed in 47% of IDCs, and its expression correlated with lymph node metastases and high histologic grades . E-cadheSnail1 mRNA and protein levels are inversely correlated with E-cadherin in hepatocellular carcinoma (HCC) . Snail1 Overall, Snail1 expression is lower in ovarian carcinoma than in breast carcinoma, though its expression is still associated with distant metastases . ExpressE-cadherin expression is drastically reduced in gastric carcinoma, and Snail1 expression levels once again share an inverse relationship with E-cadherin expression levels . Snail1 Oral squamous carcinoma is another case of E-cadherin/Snail1 expression inversion, and the higher the Snail1 expression, the more invasive the cancer. E-cadherin positive cells maintain their cuboidal shape while E-cadherin negative cells turn spindle-shaped. This is a typical sign of EMT, and it shows Snail1\u2019s repression of E-cadherin .Pancreatic carcinoma tissues show significantly reduced E-cadherin levels and relatively high Snail1 expression . In one Colorectal cancer (CRC) begins in gland cells that line the colon and rectum, and it is one of the most commonly newly diagnosed cancers and a leading cause of cancer-related deaths . Snail1 Though the expression level of Snail1 is lower in bladder carcinoma than in other types of cancer, its presence still has a significant impact on the cancer\u2019s progression. In one study, only 16% of the 120 tested tissues expressed Snail1, indicating that Slug and Twist, whose expression levels were 63% and 44% respectively, play larger roles. However, Snail1 expression increased in node-positive compared to node-negative tumors, and Snail1\u2019s presence lowered the three-year progression free survival rate to only 15% . Since SIn melanoma, there is increased Snail1 mRNA and low E-cadherin in the presence of Snail1 expression. By contrast, no Snail1 mRNA was detected in primary melanocytes . Snail1 CDH1 gene, which encodes E-cadherin, seem to be more influential than the presence of Snail1 . S. Shigh wIn a similar study of non-small cell lung cancer, Wang et al. compared ciplatin-resistant A549 cells with their A549 counterparts . A549/CDPoor differentiation, sphere-forming capacity, self-renewal, and typical markers such as ALDH and CD44, among other properties, characterize the stem-like phenotype . ClearlySnail1 drives EMT primarily through the direct repression of E-cadherin . Other tZhu et al. have examined the relationship between the expression of the Response Gene to Complement-32 (RGC-32) and TGF-\u03b2-mediated EMT . RGC-32 Snail1 is a target of NF-\u03baB activity and its expression and role in EMT are well recognized. Since NF-\u03baB is activated by many signals, clearly, such signals will also regulate Snail1 among other target gene products. Tsubaki et al. have reported that various solid tumors express the Receptor Activator of Nuclear Factor-\u03baB (RANK) and it is activated by RANK-ligand resulting in the promotion of tumor cell growth, migration, metastasis, and anchorage independence in breast cancer cells . In addiHuang et al. investigated the expression level of Notch1 in lung adenocarcinoma and its relationship to metastasis . They foThe relationship between the expression of cyclooxegnase-2 (Cox-2) and the downregulation of E-cadherin and its relationship to the EMT phenotype was reported by Fujii et al. . These iReducing Snail1 expression has proven Snail1\u2019s involvement in tumor resistance to many chemotherapeutic drugs and immunotherapies. In melanoma, Snail1 knockdown, as a result of siRNA treatment, stops both tumor metastasis and immunosuppression. Tumor-specific T cell responses also intensify as a result of this knockdown . SimilarSnail1 also factors into resistance because of its involvement in survival pathways. Snail1\u2019s activation of MAPK and PI3K survival pathways leads to resistance to serum depletion and TNF-\u03b1 . The repBeclin-1 sensitized the EMT cells to CTL killing. Hence, tumor cells\u2019 resistance to CTL is mediated by EMT-induced activation of autophagy-dependent mechanisms ..Beclin-1Few chemical inhibitors target Snail1 directly. However, Snail1-induced EMT has been successfully abrogated by a select few chemical inhibitors. LSD and HDAC inhibitors, as well as drugs targeting Snail1/p53 and Snail1/E-cadherin interactions, have shown efficacy Figure\u00a0, Table\u00a04TP53 gene, by binding directly and inducing exocytosis . P. P187]. Snail1\u2019s roles in metastasis, recurrence, and resistance make it a novel and pleiotropic target in cancer, and improving our understanding of Snail1 could thus provide new ways of approaching the treatment of metastatic cancer.Akt: Protein kinase BALDH: Aldehyde dehydrogenase\u03b2-Trcp: Beta-transducin repeat-containing proteinCDDP: CisplatinCo-REST: REST corepressor 1CSN2: COP9 signalosome 2CtBP: C-terminal binding proteinEGF: Epidermal growth factorEgr-1: Early growth response factor-1ER: Estrogen receptorERK: Extracellular signal-regulated kinaseFGF: Fibroblast growth factorFIGO: International Federation of Gynecology and ObstetricsGSK-3\u03b2: Glycogen synthase kinase-3 betaHB-EGF: Heparin-binding EGF-like growth factorHDAC: Histone deacetylaseHGF: Hepatocyte growth factorHIF-1\u03b1: Hypoxia-inducible factor 1-alphaHMGA2: High-mobility group A2HRE2: Hypoxia response element-2IKK\u03b1: I\u03baB kinase alphaILK: Integrin-linked kinaseIL-6: interleukin 6JAK: Janus kinaseLEF-1: Lymphoid enhancer-binding factor 1LOXL2: Lysyl oxidase-like 2LSD1: Lysine-specific demethylase 1MAPK: Mitogen-activated protein kinaseMDM2: Mouse double minute 2 homologMEK: MAPK kinaseMTA3: Metastasis-associated protein 3MUPP-1: Multi-PDZ domain protein 1NF-\u03baB: Nuclear factor kappa-BNuRD: Nucleosome remodeling deacetylaseOct-4: Octamer-binding transcription factor 4PAK1: p21-activated kinase 1PARP-1: Poly(ADP-ribose) polymerase-1PI3K: Phosphatidylinositol 3-kinasePRMT5: Protein arginine methyltransferase 5RANKL: Receptor activator of nuclear factor kappa-B ligandSTAT3: Signal transducer and activator of transcription 3Sox-2: Sex determining region Y-box 2TGF-\u03b21: Transforming growth factor beta 1TNF\u03b1: Tumor necrosis factor alphaTRAIL: TNF-related apoptosis-inducing ligandYY1: Yin Yang 1ZEB1/2: Zinc finger E-box-binding homeobox 1/2ZO-1/2: Zonula occludens protein 1/2The authors declare that they have no competing interests.SK was responsible for reviewing the literature, summarizing data and preparing a draft of the manuscript. BB conceptualized and developed an outline for the manuscript as well as edited the manuscript for publication. Both authors read and approved the final manuscript."}
+{"text": "The authors would like to make the following clarifications regarding the manuscript figures, as well as provide a corrected version of Figure S1, provided here as The authors would like to clarify that some of the figures in this article contain juxtaposed northern blot panels depicting distinct biological comparisons that have not been separated by vertical splice lines between the juxtaposed panels. Although splice lines were not indicated in those published figures, black lines or brackets were superimposed above panels to identify the distinct sets of gel lanes. The lack of splice lines does not affect any of the conclusions drawn from the experiments.YRA1, C-672, N-372, N-311, N-712, and C-943 northern blots in Figure 3 were included in the more extensive deletion series blots of Figures S1, S2, S3, S6 and S7 and the blot for the C-672/R-CDE synonymously named allele is included in both Figures 3 and 4 .The authors would also like to clarify some intentional duplications in the figures, which were done for purposes of comparison. Panels depicting the N400 panel in Figures S1 and S6, once again for purposes of comparison. The Figure S1 panel should have been associated with the corresponding SCR1 blot shown in Figure 5F. Figure S1 has now been corrected accordingly and is provided here as The authors acknowledge the intentional duplication of the Figure 5F S1 Figyra1 alleles containing deletions from the 3\u2032-end of the YRA1 intron was constructed and the steady-state levels of transcripts encoded by each of these alleles in wild-type (1), upf1\u0394 (2), edc3\u0394 (3), and upf1\u0394edc3\u0394 (4) cells were determined by northern blotting. Blots were hybridized with probes complementary to the YRA1 or SCR1 transcripts, with the latter serving as a loading control. The positions of YRA1 pre-mRNAs encoded by the endogenous and all the exogenous YRA1 alleles are marked by a triangle and by diamonds, respectively. A schematic diagram of the analyzed yra1 alleles is shown above the northern blot, with the relative position of each deletion indicated. Pre-mRNAs encoded by each of the YRA1 mutant alleles cannot be spliced to produce mRNAs, as the 3\u2032 splicing signals were deleted from these pre-mRNAs. The transcripts are divided into three groups by broken lines based on their distinct decay phenotypes manifested in the northern blots.A panel of (TIFF)Click here for additional data file."}
+{"text": "The aim of a genome-wide association study (GWAS) is to isolate DNA markers for variants affecting phenotypes of interest. This is constrained by the fact that the number of markers often far exceeds the number of samples. Compressed sensing (CS) is a body of theory regarding signal recovery when the number of predictor variables  exceeds the sample size. Its applicability to GWAS has not been investigated.h2 = 1), there is a sharp phase transition from poor performance to complete selection as the sample size is increased. For heritability below one, complete selection still occurs, but the transition is smoothed. We find for h2 \u223c 0.5 that a sample size of approximately thirty times the number of markers with nonzero coefficients is sufficient for full selection. This boundary is only weakly dependent on the number of genotyped markers.Using CS theory, we show that all markers with nonzero coefficients can be identified (selected) using an efficient algorithm, provided that they are sufficiently few in number (sparse) relative to sample size. For heritability equal to one (Practical measures of signal recovery are robust to linkage disequilibrium between a true causal variant and markers residing in the same genomic region.\u2009Given a limited sample size, it is possible to discover a phase transition by increasing the penalization; in this case a subset of the support may be recovered. Applying this approach to the GWAS analysis of height, we show that 70-100% of the selected markers are strongly correlated with height-associated markers identified by the GIANT Consortium. Mock dad/100094/.ARIC: Atherosclerosis risk in community; CS: Compressed sensing; FDR: False discovery rate; FPR: False positive rate; GENEVA: Gene environment association studies; GIANT: Genetic investigation of anthropometric traits; GS: Genomic selection; GWAS: Genome-wide association study; LD: Linkage disequilibrium; LE: Linkage equilibrium; MAF: Minor allele frequency; MR: Marginal regression; NE: Normalized error; OLS: Ordinary least squares; PPV: Positive predictive value; SNP: Single-nucleotide polymorphism.The authors declare that they have no competing interests.L1 -penalized regression codes to PLINK 2 for use in the height analysis. All authors read and approved the final manuscript.SV performed the numerical experiments and analyzed the data. SV, JJL, SDHH, and Chow contributed to the conception of the study, drafted the article, and endorsed the final version for submission. Chang ported the MATLAB"}
+{"text": "Balance control improvement is one of the most important goals in sports and exercise. Better balance is strongly positively associated with enhanced athletic performance and negatively associated with lower limb sports injuries. Proprioception plays an essential role in balance control, and ankle proprioception is arguably the most important. This paper reviews ankle proprioception and explores synergies with balance control, specifically in a sporting context. Central processing of ankle proprioceptive information, along with other sensory information, enables integration for balance control. When assessing ankle proprioception, the most generalizable findings arise from methods that are ecologically valid, allow proprioceptive signals to be integrated with general vision in the central nervous system, and reflect the signal-in-noise nature of central processing. Ankle proprioceptive intervention concepts driven by such a central processing theory are further proposed and discussed for the improvement of balance control in sport. In many sports, superior balance ability is necessary to achieve the highest competitive level and avoid lower limb injuries \u20133. To coAnkle proprioception can be altered by general  and sporBalance ability and ankle proprioception are both related to competition level in a range of sports. A systematic review on balance ability and athletic performance found that static balance ability of rifle shooters and archers was associated with their shooting accuracy, and dynamic balance ability of ice hockey players displayed a significant relationship with maximum skating speed . In addiSimilarly, ankle proprioception and sports performance are related. Han et al.  measuredThus, although visual  and vestBoth balance control and ankle proprioception are negatively associated with ankle injuries , 35. TheSimilar reports of the relationship between ankle proprioception and ankle injury risk are also noted in the literature. For example, a longitudinal study found ankle proprioception could predict ankle injuries in college basketball players . In addiAnkle injuries often lead to disruption of muscles and tendons with associated damage to inherent mechanoreceptors , 41, whiThese findings suggest that ankle proprioception is closely related to balance control in sport injuries, and balance ability may be significantly affected by impaired ankle proprioception after injuries.Sensory noise and sensory information reweighting are two possible mechanisms for the optimal use of sensory information in balance control , 6, 9. BIn addition, the observation of bilateral deficits in both ankle proprioception and balance control after ankle injury , 47 favoIf central processing of proprioceptive information links ankle proprioception and balance control, then this has implications for ankle proprioceptive assessment. It suggests that the most appropriate measurement technologies are those that are relevant to normal function and that encompass ecologically valid components related to balance function . This isProprioception can be assessed using different technologies/methodologies , 28, 54.The three different technologies used for testing ankle proprioception are presented in In a method designed to increase ecological validity, an upright, weight-bearing stance TTDPM option has been used, along with the AMEDA apparatus , for ankIn addition to ecological validity, assessment of ankle proprioception should acknowledge and incorporate the signal-noise nature of central processing \u201368. It hWaddington and Adams  applied In this way, the AMEDA technique both fulfills ecological validity and captures data in such a way as to address the signal-noise nature of central processing of proprioceptive information relevant to balance function. Although this method has not yet been used to determine the precise association between ankle proprioception measured with an AMEDA and balance ability in athletes, Guo et al.  has asse passive or active interventions to improve ankle proprioception and balance control, particularly after ankle injury, have been extensively reported in the literature [Both ankle proprioception and balance control are essential in sports , 18, 40,terature , 11. RegA number of studies have explored effects of passive interventions, such as taping, bracing, compressing, or sport shoe insoles, on ankle proprioception , 74\u201378, In contrast, there is some evidence that the use of insoles, another passive intervention, has a positive effect on ankle proprioception in soccer players , 76. It Various active exercise interventions, delivered in a task-specific paradigm, have been found to be effective for the improvement of ankle proprioception. It has been proposed that this occurs through neural mechanisms such as neural learning and neural plasticity . Neural  per se but rather trains the CNS to shift the weighting of sources of proprioceptive signals to improve balance. If this is the case, yachting and figure-skating athletes whose daily activities involve performing motor tasks on an unstable surface would benefit from exercise on a similar surface [In contrast, some neural changes may require weeks, months, or even years of practice. Several weeks of wobble-board training , 85, 86, surface . What is per se when conducted on each leg and by optimizing ankle proprioceptive information reweighting for balance control in sport. Future research is needed to elucidate the CNS process associated with active interventions.Apart from training surfaces, another issue associated with active ankle proprioceptive training is whether the training should focus on the injured side or should involve both sides after sports injury. Given a significant and positive correlation found between performance of both ankles in healthy and injured participants , 46, 61,Although developing better proprioception and balance control through training is a common goal for athletes and there is mounting evidence suggesting that active interventions such as wobble board training aid in doing this, there may also be a significant genetic component to proprioceptive ability and balance control. This is likely to be more evident in elite athletes, who are striving to be the best of the best, where training levels are already extensive. In studies by Han et al. , 30, it Proprioception plays an essential role in balance control, and ankle proprioception is arguably the most important aspect of this. Central processing of ankle proprioceptive information, along with other sensory information, enables integration for postural and balance control. When assessing ankle proprioception for generalization to applied situations, the method used should have ecological validity and allow proprioceptive signals to be integrated in the central nervous system, in order to reflect the signal-noise nature of central processing in sports activities. In addition, ankle proprioceptive interventions, passive or active, should therefore be predicated on discriminating signal from noise in central processing, to attain optimal outcomes."}
+{"text": "We reported previously that root elongation in Arabidopsis is promoted by exogenous proline, raising the possibility that this amino acid may modulate root growth.CYCLINB1;1  gene.To evaluate this hypothesis we used a combination of genetic, pharmacological and molecular analyses, and showed that proline specifically affects root growth by modulating the size of the root meristem. The effects of proline on meristem size are parallel to, and independent from, hormonal pathways, and do not involve the expression of genes controlling cell differentiation at the transition zone. On the contrary, proline appears to control cell division in early stages of postembryonic root development, as shown by the expression of the G2/M-specific The overall data suggest that proline can modulate the size of root meristematic zone in Arabidopsis likely controlling cell division and, in turn, the ratio between cell division and cell differentiation.The online version of this article (doi:10.1186/s12870-015-0637-8) contains supplementary material, which is available to authorized users. P5CS1 and P5CS2, while a single gene, P5CR, encodes the second committed enzyme of proline synthesis in plants [Thanks to its unique cyclic structure and physical-chemical properties, proline is of paramount importance in plants, both as building block for protein synthesis and as a compatible osmolyte accumulating during, and protecting from, environmental stress. It is synthesized in the cytosol from glutamate in a two-step pathway catalyzed by \u03b4-pyrroline-5-carboxylate synthetase (P5CS), and \u03b4-pyrroline-5-carboxylate reductase (P5CR). The first enzyme of this pathway, catalyzing the rate-limiting step of proline synthesis in higher plants, is encoded in Arabidopsis by two paralog genes In the last years it has been increasingly evident that the amino acid proline, in addition to its role in protein synthesis and stress response, plays a key role in plant development, particularly in developmental processes related to reproduction , such asP5CS2  are embryo lethal in homozygosis, and can be propagated only as heterozygotes, unless complemented by exogenous proline [p5cs1 and heterozygous for p5cs2 (p5cs1 p5cs2/P5CS2) are late flowering [Agrobacterium rhizogenes [rolA, rolB, rolC and rolD, are responsible of hairy root insurgence and elongation. This latter, by insertional mutagenesis [rolD, later on recognized as a proline-producing ornithine cyclodeaminase gene [Accordingly, Arabidopsis mutants carrying a knock out T-DNA insertion in lowering . A more lowering . Intriguizogenes . Virulenagenesis , has beease gene , 13.Arabidopsis thaliana. The simplicity of its cellular organization, the possibility to be grown on agar plates under well-defined conditions, and the wealth of genetic and molecular resources available for Arabidopsis, have greatly contributed to build a solid picture of the molecular mechanisms behind growth and development of the Arabidopsis root [SHY2 [.In recent years our understanding of the genetic and molecular mechanisms underlying root growth and development has tremendously improved thanks to the exploiting of the model species sis root . The dimot [SHY2 .SHY2 is induced by the cytokinin-responsive transcription factors ARR1 and ARR12, and regulates the size of the root meristem by downregulating the PIN-FORMED (PIN) genes that encode auxin efflux facilitators. In addition to plant hormones, however, novel effectors have been recently proposed to affect root meristem size in Arabidopsis [p5cs1 p5cs2/P5CS2, compared to wild type. Here we show that proline can modulate the size of root meristematic zone in Arabidopsis by controlling cell division and, in turn, by modulating the ratio between cell division and cell differentiation.According to the current model , SHY2 isbidopsis \u201318 and op5cs1 p5cs2/P5CS2 [p\u2009<\u20090.01). As shown in Fig.\u00a0p5cs2/P5CS2 and homozygous p5cs1 parental lines, compared to partial double mutant p5cs1 p5cs2/P5CS2 and wild type lines. The proline content of the parental lines turned out to be intermediate between p5cs1 p5cs2/P5CS2 and wild type lines, with measured values of 0.15\u2009\u00b1\u20090.05 \u03bcmoles/g of proline for homozygous p5cs1 roots, and 0.11\u2009\u00b1\u20090.02 \u03bcmoles/g of proline for heterozygous p5cs1/P5CS2 roots. In spite of the reduction in proline content, roots from homozygous p5cs1 mutants appeared indistinguishable from wild type roots, while roots from heterozygous p5cs1/P5CS2 looked slightly shorter (not shown) suggesting that the levels of endogenous proline present in these mutants are reduced, relative to wild type, but still sufficient (p5cs1) or nearly sufficient (p5cs2/P5CS2) to sustain normal root growth. Overall these data confirm the positive correlation between proline content and root growth. Clearly mutations on P5CS2 have a stronger effect on proline accumulation and root growth, compared to mutations on P5CS1. However both genes seem to contribute to the overall proline content in roots, as indicated by the lower proline level and by the shorter roots exhibited by the p5cs1 p5cs2/P5CS2 partial double mutant, compared to parental lines. In Arabidopsis, the maintenance of the root meristematic zone and, consequently, of the root growth is ensured by the balance between the rate of cell division in the root meristematic zone and the rate of cell differentiation in the TZ [p5cs1 p5cs2/P5CS2 mutant and in wild type, the size of the root meristematic zone expressed as number of cortex cells spanning from the quiescent center (QC) to the first elongated cell in the TZ. As shown in Fig.\u00a0p5cs1 p5cs2/P5CS2 are accounted for by smaller meristems that stop growing at 3 dag with an average number of cells of 16.4\u2009\u00b1\u20090.47 . To confirm the effect of proline on the size of the root meristematic zone, we scored the number of meristem cells in wild-type roots, grown either in presence or in absence of proline  increased meristem size from 1 to 10 dag, with an average number of 37.3\u2009\u00b1\u20090.54 cells, as compared to 28.5\u2009\u00b1\u20090.42 of the untreated controls , indol-3-acetic acid (IAA) or cytokinin. As described previously [p5cs1 p5cs2/P5CS2 appears significantly larger than untreated controls (p\u2009<0.001), but significantly smaller than hormone-treated wild types  - a G2/M phase-specific cyclin gene regarded as a reliable marker of cell cycle progression [p5cs1, p5cs2/P5CS2, either treated or non-treated with exogenous proline, as compared as to a wild-type root meristem. Since CYCB1;1 is expressed only in meristem cells, unlike the other genes analyzed in this work, we used the meristem-specific ROOT CLAVATA HOMOLOG 1 (RCH1) [CYCB1;1 expression to root meristems of different sizes. As shown in Fig.\u00a0CYCB1;1 is downregulated at 3 dag, but not at 5 dag, when the level of expression of this gene, relative to RCH1, becomes similar in the proline-deficient mutant and in the wild type. Supplementation of 10 \u03bcM exogenous proline to p5cs1, p5cs2/P5CS2 roots, however, restored the levels of CYCB1;1 expression to wild-type levels, confirming the effect of proline on the expression of CYCB1;1.To assess whether proline can modulate the size of the root meristematic zone by controlling cell division, we analyzed by RT-PCR (not shown) and qRT-PCR Fig.\u00a0, the expgression  - in p5c1 (RCH1)  as a refCYCB1::GUS construct in p5cs1, p5cs2/P5CS2, and analyzed the activity of CYCB1::GUS in a p5cs1, p5cs2/P5CS2 , the lower ratio of GUS spots to root meristem cells found in early stages of meristem growth of p5cs1 p5cs2/P5CS2, CYCB1;1::GUS roots, relative to wild-type CYCB1;1::GUS roots, indicates that proline-deficient meristems are growing at a slower pace than wild-type ones.Since we never saw differences in germination rates between p5cs1 p5cs2/P5CS2 grew slower than root meristem cells of wild types, with a rate of cell division at 3 dag of 0.018\u2009\u00b1\u20090.001 cells cells\u22121 h\u22121, compared to 0.022\u2009\u00b1\u20090.001 cells cells\u22121 h\u22121. At 5 dag the rate of cell division in the mutant root meristem dropped down to 0.008\u2009\u00b1\u20090.001 cells cells\u22121 h\u22121, in sharp contrast to the wild type root that showed a cell division rate of 0.012\u2009\u00b1\u20090.001 cells cells\u22121 h\u22121. The overall data indicate that in the root meristem of p5cs1 p5cs2/P5CS2, because of a slower cell division rate, the balance between cell division and cell differentiation is reached at 3 dag, when the root meristem of p5cs1 p5cs2/P5CS2 gets its final dimension.To further confirm this indication, we determined the rate of cell division in mutant and wild-type roots by calculating, with a modification of the Beemster and Baskin method , the varCYCB1 is upregulated and cell division prevails over cell differentiation, consequently the meristem enlarges. However, since both blue-stained spots and meristem cells increase, the ratio between GUS-expressing and meristem cells remains unchanged in the proline-treated wild type. Our data are compatible with a model in which proline affects the ratio between cell division and cell differentiation, modulating, in turn, the size of the root meristematic zone. In the proline-deficient mutant, SHY2-mediated differentiation activity is normal, but cell division at early stages of meristem growth is hampered as CYCB1 is downregulated. As a consequence, the p5cs1 p5cs2/P5CS2 root meristem grows less in the first days after germination, and results in a smaller meristem than wild type, as the balance between cell division and cell differentiation is reached earlier (3 dag) than in the wild type (5 dag). To test this model we analyzed the effect of proline on meristem size upon variation of the expression of SHY2. We examined the root meristem size in a loss-of-function shy2-31 mutant line [p5cs1 p5cs2/P5CS2, and in a shy2-2 gain-of-function mutant line [. Because of the absence of SHY2, the root meristematic zone of shy2-31 null mutants never reaches a balance between cell division and cell differentiation and become much larger than in wild types.Upon proline treatment ant line  crossed ant line  treated p5cs1 p5cs2/P5CS2 shy2-31 was found to be much larger than the root meristem of p5cs1 p5cs2/P5CS2 and nearly as large as the root meristem of shy2-31 . Following spontaneous cyclization, GSA is converted in \u03b4-pyrroline-5-carboxylate (P5C) that is further reduced to proline by the enzyme P5CR. Despite high sequence similarity and partially overlapping transcription pattern, these two paralog genes seem to play different, non-redundant functions in stress regulation and embryogenesis, because p5cs1 homozygous mutants are hypersensitive to stress conditions [p5cs2 homozygous mutants are embryo lethal [P5CS1 and P5CS2 may have partially redundant functions. Indeed, while p5cs1 homozygous mutants have roots indistinguishable from wild type and p5cs2/P5CS2 heterozygous mutants have roots only slightly shorter (not shown), roots from p5cs1 p5cs2/P5CS2 partial double mutants are always much shorter than parental mutants . Intriguingly, asparagine and methionine were found to increase while glutamate was found to decrease root meristem size, raising the question whether or not these amino acids may share a common mechanism with proline. The inhibitory effect of exogenous glutamate on root growth seems quite different from the effect of proline. Unlike other amino acids that inhibit root growth at high concentration, including proline, glutamate is effective only at low concentrations and leads to inhibition of the primary root but also to proliferation of secondary roots [+-ATPase [The cyclic amino acid proline has been implicated in root elongation ever since the discovery of ongation , and encongation . Consistver time . In Arabnditions , while po lethal . In rootnts Fig.\u00a0 to c. Hewth Fig.\u00a0 or on CYry roots . Its mecry roots  and probry roots . Methionry roots  and may +-ATPase . It is tp5cs1 p5cs2/P5CS2 compared to wild types, as judged by germination rates, rosette leaf diameters , rosette leaf area  and fresh weight . The root architecture is somehow less branched compared to wild type, suggesting that proline may also affect secondary root development. However the effects of proline on secondary roots appear late in development and we don\u2019t know, at present, whether these effects are direct or indirect. In addition p5cs1 p5cs2/P5CS2 partial double mutants exhibit also a slight delay in flowering [p5cs2 mutants described by Funck et al. [p5cs1 p5cs2/P5CS2, the two latter mutants carry homozygous mutations in P5CS2, which have been associated to embryo lethality and severe morphological defects [Apart from the short-root phenotype, no major defects are seen in the general plant growth of k et al.  in MaizePIN genes, which, in turn, are controlled by the levels of SHY2. In a regulatory circuitry, the crosstalk between auxin, cytokinin and gibberellin finely tunes the expression of SHY2 in the root to fix the position of the root transition zone and determine the boundaries of the root meristematic zone. Some authors [CYCB1;1 expression in secondary roots of Arabidopsis, while M\u00e4h\u00f6nen et al. [PLETHORA (PLT) genes define the location of developmental zones of the primary root, and affect the expression of cell cycle regulator genes, including CYCB1;1.Plant hormones play pivotal roles in plant growth and development and four of them, auxin, cytokinin, gibberellins and brassinosteroids are essential for plant growth, with auxin, cytokinin and gibberellins mainly involved in cell division, and brassinosteroids in cell elongation . Since p authors , 36, how authors , for exaPIN1  are not altered in a p5cs1 p5cs2/P5CS2 background, corroborating the notion that proline does not interfere with auxin signaling. In addition, proline supplementation can partially rescue the small meristem size of the gain-of-function shy2-2 mutant   is the op5cs1 p5cs2/P5CS2 roots by following the expression of the G2/M phase-specific CYCB1;1 cyclin gene. Our data clearly show a positive correlation between proline and CYCB1;1 expression and, in turn, cell division activity, although, at present, we don\u2019t know the molecular mechanism by which proline can modulate the rate of cell division of the primary root. A hint to explain the effects of proline on cell cycle is given by Wang et al. [p5cs1 p5cs2/P5CS2 mutants may be a limiting factor both for protein synthesis and cell cycle progression. However, from the analysis of protein accumulation in roots and shoots that found no significant difference between mutant and wild-type lines  ecotype were mostly used in this work, with the exception of shy2-31, which was in Landsberg erecta (Ler). Since the effects of proline on root length and root meristem size have been preliminary tested either in Col-0 or in Ler, and have been found to be indistinguishable between the two ecotypes (not shown), we decided to use Col-0 as wild-type control in all the experiments.Wild-type and mutants p5cs1 p5cs2/P5CS2, arr1-4, arr1-3, gai-t6 rga-24, pSHY2::GUS, and pCYCB1;1::GUS were previously described [A. thaliana seeds were plated on one-half MS  supplemented with sulfadiazine \u2013 to counter select P5CS2/P5CS2 from the p5cs2/P5CS2 heterozygous population - and grown in vertical position. In all genetic crosses using p5cs1 p5cs2/P5CS2, this mutant was used as a female, because of the male sterility of the p5cs1 p5cs2 pollen grains [escribed , 18, 19.Proline content was measured according to Bates , using Lp5cs1 p5cs2/P5CS2, this partial double mutant was always used as a female. The F1 generation was allowed to self fertilize and the presence of the p5cs2 mutant allele was assessed from the F2 generation, by sulfadiazine selection or by PCR genotyping of the sulfadiazine resistance gene.In crosses using http://rsb.info.nih.gov/ij). For each experiment, at least 90 plants, coming from three independent experiments, were analyzed. For meristem size analysis, roots were cleared with a 8:3:1 mixture of chloral hydrate:water:glycerol, mounted on a glass slide, and observed, under Nomarski optics, with an Axio Imager.A2 (Zeiss) light microscopy. The size of root meristems was performed by counting the number of cortex cells in a file extending from the QC to the first elongated cell in the TZ, as described [Root length was measured with IMAGE J software  supplemented with sulfadiazine and grown in vertical position. At daily intervals, roots were cleared with a 8:3:1 mixture of chloral hydrate:water:glycerol, observed under Nomarski optics with an Axio Imager.A2 (Zeiss) light microscopy and acquired with a DC500 digital camera . For every time point the average number of cortex cells (N) from a file extending from the QC to the TZ was determined. Next the number of dividing cells per root meristem was calculated by subtracting the number of cells from adjacent time points (N2-N1). Finally the rate of cell division, for every time point, expressed as cells cells\u22121 h\u22121, was calculated by dividing the resulting scores by N1*24 (N2-N1/N1* t), and averaging the results from x data points. The difference in cell division rates between p5cs1 p5cs2/P5CS2 and wild-type root meristem cells was tested for statistical significance with a student\u2019s test a produced a significance level of **p\u2009<\u20090.05, at 3 dag, and ***p\u2009<\u20090.01, at 5 dag.Cell division rates in root meristems were determined with a modification of the kinematic method described by Beemster and Baskin . A. thalArabidopsis DNA was extracted with a modified CTAB method, according to Stewart and Via [p5cs1 and p5cs2 were already described [ARR1, ARR12 and ACT8 were as follows: ARR1-FW: 5\u2032-GAGATGGCATTGTCTCTGCTC-3\u2032; ARR1-RV: 5\u2032-GATCAAACCCATT CAATGTCG-3\u2032; ARR12-FW: 5\u2032-CGGTACAATATGCGGATTTTGATTCGGTAT-3\u2032; ARR12-RV: 5\u2032-TCACCATTATTATTACTCCCACGGTTCTTA-3\u2032; ACT-FW: 5\u2032 -ATG AAGATTAAGGTC GTGGCA-3\u2032; ACT-RV: 5\u2032-TCCGAGTTTGAAGAGGCTAC-3\u2032. PCR conditions were: 3\u2032 at 94\u00a0\u00b0C followed by 30 cycles of 30\u2033 at 94\u00a0\u00b0C, 30\u2033 at 60\u00a0\u00b0C and 1\u2032 at 72\u00a0\u00b0C. All primers used in this work were designed using Primer3 PLUS (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi/). Real-time qRT-PCR analyses were carried out with a Bio-Rad iCycler iQ (Bio-Rad). Amplifications were monitored using the SYBR Green fluorescent stain. The presence of a single PCR product was verified by dissociation analysis in all amplifications. The comparative threshold cycle (\u0394\u0394CT) method was used to calculate the relative amount of gene expression, normalized using the CT values derived for either RCH1  or ACT . All the analyses were performed in triplicate on three independent samples. qRT-PCR Primers for SHY2, ARR1, ARR12, CYCB1;1, RCH1 and PIN1 were as follows: qSHY2-FW: 5\u2032-AGATGGTGATTGGATGCTCA-3\u2032; qSHY2-RV: 5\u2032-GCCTAA ACCTTTGGCTTCTG-3\u2032; qARR1-FW: 5\u2032-TGGTACAGCACCATCAGGTT-3\u2032; qARR1-RV: 5\u2032-TGCTGCATC CGTAGCCACTC-3\u2032; qARR12-FW: 5\u2032-CTCTTCGACTCACCC TCCTC-3\u2032; qARR12-RV: 5\u2032-CACATTGTTCCATTCCAAGG-3\u2032; qCYCB1 -FW: 5\u2032-TGGTAGCTGCTTCTGCA ATC-3\u2032; qCYCB1-RV: 5\u2032-AGCTTTGCACAGTCCATGAG-3\u2032; qRCH1-FW: 5\u2032-AGAGAACGT GCCAAAGATGA-3\u2032; qRCH1-RV: 5\u2032-CGCAGAGAAA CTCGTGCTAC-3\u2032; qPIN1-FW: 5\u2032-GGTGGTGGTCGGAACTCTAA-3\u2032; qPIN1-RV: 5\u2032-TAGCAGGACCACCGTCT TCT-3\u2032.Molecular techniques were performed according to standard protocols. Total RNA for RT-PCR was extracted from roots using RNeasy Plant Mini Kit (Quiagen) according to manufacturer\u2019s instructions. Reverse transcription was performed from 1\u03bcg of total RNA using the Superscript II\u2122 kit (Invitrogen) as recommended by the manufacturer. For genomic PCR  and Via . Primersp5cs1 p5cs2/P5CS2 plants were collected. Roots were crushed in Laemmli buffer  under liquid nitrogen and subsequently treated at 65\u00a0\u00b0C for 15 min. Equal volumes of each sample were loaded in a 30\u00a0% Sodium Dodecyl Sulphate PolyAcrylamide Gel (SDS-PAGE) for protein analysis. Protein detection was performed by Silver Staining according to Bio-Rad protocol (Silver Stain Plus\u2122 Cat. N\u00b0 #161-0449).For protein analysis 5 mg (fresh weight) of root or shoot apexes from either wild type or All the supporting data are included as additional files."}
+{"text": "Fragaria \u00d7ananassa Duch.) is an allo-octoploid considered difficult to disentangle genetically due to its four relatively similar sub-genomic chromosome sets. This has been alleviated by the recent release of the strawberry IStraw90 whole genome genotyping array. However, array resolution relies on the genotypes used in the array construction and may be of limited general use. SNP detection based on reduced genomic sequencing approaches has the potential of providing better coverage in cases where the studied genotypes are only distantly related from the SNP array\u2019s construction foundation. Here we have used double digest restriction-associated DNA sequencing (ddRAD) to identify SNPs in a 145 seedling F1 hybrid population raised from the cross between the cultivars Sonata (\u2640) and Babette (\u2642). A linkage map containing 907 markers which spanned 1,581.5 cM across 31 linkage groups representing the 28 chromosomes of the species. Comparing the physical span of the SNP markers with the F. vesca genome sequence, the linkage groups resolved covered 79% of the estimated 830 Mb of the F. \u00d7ananassa genome. Here, we have developed the first linkage map for F. \u00d7ananassa using ddRAD and show that this technique and other related techniques are useful tools for linkage map development and downstream genetic studies in the octoploid strawberry.The cultivated strawberry ( Fragaria is an important soft fruit genus, primarily due to the cultivation of the genetically complex garden strawberry . In 2012, the world production of strawberries exceeded 5 million tons and the crop was valued in excess of US$10 billion [n = 8x = 56). Very recent studies of the sub-genome structure of the species have determined that at least three diploid donors have contributed to the extant genome composition. The genome contains one sub-genome which displays similarity to Fragaria iinumae, two additional sub-genomes that are not clearly distinguishable, but that are clearly segregating disomically, and a single sub-genome with a strong similarity to F. vesca [F. iinumae and undefined sub-genomes existed initially as a hexaploid, which subsequently hybridized at a much later stage with an F. vesca-like diploid to form the extant octoploid genome configuration [F. \u00d7ananassa has thus been revised as A-A, b-b, X-X, X-X, where the A genome is F. vesca-like, the b genome is F. iinumae-like, and the two X genomes are of unknown origin but are delimited X-X, X-X to reinforce that they segregate independently in a disomic fashion, and may or may not be derived from the same ancestral progenitor.The cultivated strawberry is a genetically complex allo-octoploid  \u20138. Howevil et al  describeF. vesca [a priori knowledge of the genomes of the organism under investigation is required, and are thus not dependent on previously identified SNPs being present in the genome of the study organism. They are also much cheaper per sample to assay than the use of arrays, but have the disadvantage of returning fragmented datasets containing high percentages of missing data. However, this might be resolved by imputation strategies to reduce the noise in the data used for map construction [The IStraw90 array should prove to be a powerful tool for genetics studies in the cultivated strawberry, however, the cost per sample of the array has implications for its applicability as a broad genotyping tool, both in very large experiments where the total cost of its implementation would be prohibitive, as well as in exploratory or pilot studies where initial proof of concept funding might be limited. In these scenarios, other high-throughput genotyping techniques may be more cost-effective for SNP genotyping and linkage map construction. Such approaches are based on genomic DNA enrichment using restriction digestion and adaptor ligation, followed by second-generation sequencing of multiplexed libraries. These techniques, along with others such as the direct re-sequencing of a sub-set of progeny individuals , allow tF. vesca , red rasF. vesca , and appF. vesca , while RF. vesca , has beeF. vesca  and aubeF. vesca  amongst truction .F. \u00d7ananassa. Whilst it has recently been reported that GBS libraries have been constructed for the cultivated strawberry [F. \u00d7ananassa. In this investigation, we tested the efficacy of a modified RAD-tag protocol using double digestion with two restriction enzymes .Tennessen et al  employedrawberry , to date  for lin1 hybrid seedlings was raised from the cross between the Dutch cultivar Sonata (\u2640) and the Norwegian cultivar Babette (\u2642). The F1 seed were germinated in mist chambers before being transplanted to flats and subsequently to larger pots. Young leaf tissue from one representative of each of the 145 F1 progeny plants and the two parents was flash frozen in liquid nitrogen and lyophilized for DNA extraction. DNA was extracted using DNeasy Plant Mini Kit (Qiagen) according to the manufacturer\u2019s protocol. The resulting DNA was quantified using a Qubit Fluorometer  and the quality was assessed subjectively by agarose gel electrophoresis.An experimental population comprising 145 FEcoRI and MspI  before being purified using AMPure XP Beads . Paired combinations of double-stranded adapters were used to uniquely tag samples which, after cleaning to remove un-ligated adapter, were quantified and pooled in equimolar amounts; one pool included 96 samples, another included 49 while a third included just the parental genotypes. All three pools were subjected to size selection using a Pippin Prep  and fragments were separated using a 2% agarose cartridge to capture a narrow distribution around 400bp. Sequencing was performed using an Illumina MiSeq  and V2 sequencing kit chemistry (2\u00d7251 nt).Individual, high-molecular weight DNA samples, were prepared for sequencing according to the ddRAD protocol of Peterson et al . BrieflySNPs were detected using Stacks (v1.18) software . Briefly1 segregating population using the genotypes of the parental lines to assign segregation. Data were filtered for all markers containing more than 50% missing values and a chi-squared analysis was performed to determine segregation distortion at the 5% level of significance (Chi-squared = 3.841 (1 d.f.), 5.991 (2 d.f.) or 7.815 (3 d.f.)). Initially, only robust markers for which no significant segregation distortion was observed at the 5% level were used for linkage mapping using JoinMap 4.1 . An initial linkage map was constructed using the Maximum Likelihood mapping function and assessed for spurious linkages or inflated genetic distances, with individual genotypes being converted to missing values where necessary or loci being removed completely where they caused conflicts in the data. Following scrutiny of the Maximum Likelihood data, linkage mapping was performed with the initial marker set using regression mapping and v1.0 linkage groups were produced. Additional data were then added to the dataset for markers exhibiting significant segregation distortion. Marker placement was determined using regression mapping with a minimum logarithm of odds (LOD) score threshold of 3.0, a recombination fraction threshold of 0.35, ripple value of 1.0, jump threshold of 3.0 and a triplet threshold of 5.0, and mapping distances were calculated using the Kosambi mapping function to produce a v2.0 linkage map. Data from the v2.0 map were compared to the v1.0 linkage maps, and any markers causing shifts in the placement of the initial robust markers were removed from the linkage groups, following which, marker ordering was recalculated to produce the linkage map presented. Due to the level of missing values in the dataset, marker bins were calculated from map positions with no decimal places. The sequence tags associated with each SNP were used as queries for a BLAST analysis against the v2.0 (Fvb) genome sequence of F. vesca \u2018Hawaii 4\u2019 [Data produced by Stacks were coded as an Fawaii 4\u2019 ,23. Linkawaii 4\u2019 . PhysicaA total of 66,318 stacks were detected from analysis of the forward reads, 20% of these  contained one or more SNPs. To maximize data completeness and minimize false positive SNPs, stacks were only included if they were detected in both parents and \u2265 100 offspring (\u2265 69%) and had \u2264 5 SNPs within the 240 nt sequence. This resulted in a list of 1,098 SNP-containing stacks that were used for mapping.F. \u00d7ananassa genome , whilst a further 306 exhibited significant segregation distortion. The remaining 733 were used to construct the initial v1.0 linkage maps. Following scrutiny of the resultant linkage groups, the markers exhibiting significant segregation distortion were added to the dataset and 1,039 markers were analyzed for linkage and marker ordering. The final linkage map produced contained a total of 902 sequence characterized SNP markers in 650 mapping bins spanning 1,581.5 cM across 31 linkage group fragments that corresponded to the full complement of 28 chromosomes of the a genome . The mapFvb genome sequence, the linkage groups resolved covered 656.2 Mb (79%) of the estimated 830 Mb of the F. \u00d7ananassa genome size. Fvb genome sequence on each linkage group  pseudomolecules indicating that the map positions of the ddRAD markers was reliable. This, along with the total length of the linkage map, and the estimated proportion of the F. \u00d7ananassa genome it covered (79%) indicates that the map represents the majority of the \u2018Sonata\u2019 and \u2018Babette\u2019 genomes, and will provide a useful resource for future studies of segregating traits of interest in the progeny. As in other F. \u00d7ananassa linkage maps, the proportion of markers heterozygous in each of the parental genotypes varied between linkage groups, but no clear patterns were observed that would permit us to speculate on the effects of breeding and selection as in previous studies [The S\u00d7B linkage map has a total genetic distance over the 31 linkage groups of 1,581.5 cM, which is comparable to other maps produced using regression mapping. The SNP-based \u2018Darselect\u2019 \u00d7 \u2018Monterrey\u2019 Fnt et al  1,400.1 F. \u00d7ananassa using ddRAD, a technique exploiting the power of short read sequencing technology and reduced representation genome coverage to call sequence variation in the progeny of a segregating mapping population. Whilst the number of markers we were able to score using this approach was less than when using the IStraw90 whole genome genotyping array, the map produced covered the genomes of the two parental cultivars adequately, spanning some 79% of the total estimated genome size, and placement of markers was robust and reliable, evidenced through a good correlation between the genetic positions of the markers mapped and their physical positions on the Fvb genome sequence. Our investigation provides clear evidence that ddRAD, and by extension, other related techniques such as RADseq and GBS, are useful tools for linkage map development in cultivated strawberry, and permit the rapid development of good quality linkage maps for downstream genetic studies of segregating traits.We have developed the first linkage map for S1 Table(XLSX)Click here for additional data file."}
+{"text": "While the importance of record linkage is widely recognised, few studies have attempted to quantify how linkage errors may have impacted on their own findings and outcomes. Even where authors of linkage studies have attempted to estimate sensitivity and specificity based on subjects with known status, the effects of false negatives and positives on event rates and estimates of effect are not often described.We present quantification of the effect of sensitivity and specificity of the linkage process on event rates and incidence, as well as the resultant effect on relative risks. Formulae to estimate the true number of events and estimated relative risk adjusted for given linkage sensitivity and specificity are then derived and applied to data from a prisoner mortality study. The implications of false positive and false negative matches are also discussed.Comparisons of the effect of sensitivity and specificity on incidence and relative risks indicate that it is more important for linkages to be highly specific than sensitive, particularly if true incidence rates are low. We would recommend that, where possible, some quantitative estimates of the sensitivity and specificity of the linkage process be performed, allowing the effect of these quantities on observed results to be assessed. Record linkage is the task of bringing together information from two or more different sources that pertain to the same individual. Increasingly it is used to determine outcomes, particularly cancer and mortality, in large cohort studies or registry based populations Probabilistic linkage assigns weights to potentially matched records, based on the contribution from each partial identifier It has long been recognised that misclassification, and as a result linkage errors, can lead to biased results The purpose of this paper is to provide further assistance to researchers who are attempting to appraise the possible impact of errors in the linkage process. The problem of missing linkage is viewed as misclassification of outcome and with this in mind we present a simple quantification of the effect of sensitivity and specificity on incidence and event rates as well as the resultant effect on relative risks. Furthermore, we present a derived formula that allows the \u201ctrue\u201d number of events in a linkage study to be estimated from the observed number of events based on the sensitivity and specificity of the data linkage and illustrate its use in a linkage study sensitivity (SE) is the probability of detecting an event via linkage if an event has truly occurred and is equal to TP/(TP+FN). The specificity (SP) is the probability of not detecting an event if the event is truly absent and is equal to TN/(TN+FP). N is the number of individuals in the population and equals TP+FP+TN +FN. By substituting the nomenclature above into the right hand side of formula one,Cancelling the TP+FN in the first part of the equation and simplifying throughout results inMultiplying the TN+FP the last part of the equation results inMisclassification as a result of poor sensitivity results in an under estimation of the number of events, while poor specificity results in an over estimation of events. EN is the number of individuals in the exposed population, EO is the number of events in the exposed population, NEN is the number of individuals in the non-exposed population and NEO is the number of events in the non-exposed population.Linkage studies are often used to examine the effect of certain risk factors on a specific event. The impact of sensitivity and specificity on incidence carries through to the estimation of effect size such as relative risks (RR) or standardised ratios and can result in significant bias in these estimators. This effect is illustrated by applying So the true relative risk is given by:However if the number of events can only be determined with a sensitivity of 0.90 and specificity of 0.95 then by applying And hence The observed RR 1.46 is a considerable under estimate of the true RR\u200a=\u200a2.Additionally, if the specificity of event incidence is 100%, then the relative risk will not be biased irrespective of sensitivity. To examine this further, consider our application of Sensitivity terms will cancel in the equation and thus the equation for observed relative risk simplifies to the equation for the true relative riskSo far, we have described the effect of sensitivity and specificity on the observed number of events. However, it is more common in linkage studies that the observed number of events is known and the aim is to determine how much the observed number of events is biased from the true number of events. If the sensitivity and specificity for a linkage is known, the true number of events, The observed number of events is described as:The observed number of non-events is described as:Multiplying Solving these two simultaneous equations for the true number of events, Rearrange the right hand side of the equation to get:Simplifying the left hand side of the equation to get:Multiplying both sides of the equation by Simplifying this equation you get:If linkage study estimates of sensitivity and specificity are available, then these estimates can be used with Adjusted measures of effect can be derived from linkage studies which are able to estimate sensitivity and specificity either via internal or external validation. This is illustrated by an empirical example from the population-based studies of prisoners in New South Wales E is the expected number of deaths based on mortality rates in the comparator population.The sensitivity and specificity of linkage was estimated to be 88.4% and 99.7% respectively. For the study cohort, a total of 5,137 of 85,203 inmates were found to have died according to data linkage. This compared to an expected 1,323 events, giving an SMR  of 3.9. After adjusting for the sensitivity and specificity obtained in the sub-study using The relative risk can be similarly adjusted. Among male prisoners, the RR for death in those with psychiatric hospital admission was determined. In those admitted to psychiatric hospitals and not admitted, respectively, the following were reported: observed deaths , expected deaths  and populations  to give a RR of 2.03. After applying these data to Results of epidemiological studies using outcomes determined by linked datasets are affected by errors in linkage. To date, methods to quantitatively assess the effect of misclassification on observed study events in linkage studies have not been described. This study develops and tests a simple formula for adjusting observed events and relative risk by known estimates of sensitivity and specificity. This formula and the conceptual framework behind it are analogous to the methods used to adjust for misclassification when calculating odds ratios and hazard ratios in general epidemiological studies If the estimates of sensitivity and specificity are not valid, then it is possible for the formula to give nonsense adjusted estimates, for example, negative values if specificity is estimated to be low. The formula is also only a simple, approximate adjustment. It only adjusts the observed number of events, and does not adjust estimates of person-years at risk that would also be affected. However, for events that are relatively uncommon, the person-years at risk would be altered only minimally and are probably not an important component of uncertainty.et al. reminds us that there are many non-random factors which impact on linkage producing a range of less unpredictable biases A further limitation is that our method does not allow for uncertainty in the estimates of sensitivity and specificity. We would recommend that the formula is used as a quantitative assessment of sensitivity and specificity of the linkage process, but that unadjusted results are presented as the main study findings. Our analyses have assumed that errors in linkage are random or non-differential and demonstrate how random error will bias outcomes towards null findings. A review by Bohensky Our results have important implications for study design. Comparisons of the effect of sensitivity and specificity on incidence indicate that it is much more important for linkages to be highly specific than sensitive, particularly if true incidence rates are low. Typically, linkage studies commonly use probabilistic linkage methods and these methods are primarily designed to improve the sensitivity of the linkage process. However, the trade-off between sensitivity and specificity means any improvement in sensitivity must be at some cost in terms of poorer specificity. Despite this trade-off, our results suggest that linkage methods that maximise specificity will lead to the most robust study results, particularly for events that are rare. Other approaches which have accounted for the impact of linkage error on statistical inference include work done by Scheuren and Winkler As a result of our deliberations we would recommend that analyses which only consider exact linkage matches, an approach that would probably result in close to 100% specificity but at a possibly much lower sensitivity, should routinely be included as sensitivity analyses. Furthermore, in all linkage studies we would recommend that some quantitative estimates of the sensitivity and specificity of the linkage process be performed if possible, allowing the effect of these quantities on observed results to be assessed."}
+{"text": "Background and aims. Increasing the temperature of sodium hypochlorite (NaOCl) enhances its dissolutionand antibacterial properties. However, the high resistance of multi-species biofilms couldrestrict the effect of the solution regardless of its temperature, enabling the long-termrecovery of the surviving bacteria. The aim of this study was to investigate if theincrease of temperature of NaOCl improves its antibacterial and dissolution ability onoral biofilms and if the post-treatment remaining bacteria were capable of growing in anutrient-rich medium.Materials and methods. Forty dentin blocks were infectedintra-orally for 48 hours. Then, the specimens were treated with 1% and 2.5% NaOCl at roomtemperature (22\u00baC) and body temperature (37\u00baC) for 5 and 20 min. The percentage of livecells and the biovolume were measured pre- (control) and post-treatment and after thebiofilm revitalization. Four confocal \u2018stacks\u2019 were chosen from random areas of eachsample. Statistical analysis was performed using Kruskal-Wallis and Dunn tests.Statistical significance was defined at P <0.05.Results. All the NaOCl groups were effective in dissolvingthe biofilm at any temperature, concentration and contact time without statisticaldifferences among them (P >0.05). The 1%-NaOCl for 5min was not able to significantlykill the bacteria, regardless of its temperature and contact time (P >0.05).Conclusion. The temperature variation of the NaOCl was notrelevant in killing or dissolving bacterial biofilms. Twenty-four hours of reactivationdid not appear to be enough time to induce a significant bacterial growth. Among all existing endodontic irrigants,NaOCl is the solution most recommended in endodontic therapy, due to its great tissuedissolution capacity and antibacterial effect.2 In order to eliminate biofilms as far as possible from the root canal system duringendodontic treatment, mechanical instrumentation and the applications of chemical solutionsare used,3 This condition is aggravated as the solutionconcentration is increased. So, considering the caustic potential of NaOCl, it would not beadvisable to use the solution in a high concentration. As an attempt to resolve thislimitation, increasing the temperature of the NaOCl in low concentrations was proposedbecause it seems to enhance the dissolution and antibacterial properties of the irrigant.Furthermore, the toxicity of preheated NaOCl in a low concentration should be lower than thesame solution in a high concentration at room temperature because the properties of bothshould be similar in the root canal when the solution achieves the bodytemperature.4 Although, there is almosta general consensus that increasing the NaOCl temperature improves its antibacterial anddissolution properties, there is little information in the endodontic literature on thissubject.5 In addition, although NaOCl is biologically acceptable when it is confined within thecanal, it is highly caustic when it is extravasated beyond the apical region.9Nonetheless, eight of them focusedon evaluating either dissolution,10 orantibacterial effects,12 and only one analyzed both effects.4 In addition, all the publications mentioned above were carriedout in laboratory conditions using human or bovine pulps, bovine muscle, rat dermalconnective tissue and collagen matrices to study the NaOCl dissolution ability; anduni-species biofilms to study the antibacterial capacity of NaOCl. Therefore, the authorsconsider that analyzing the effect of NaOCl in different temperatures on poly-microbialbiofilms formed in situ offers a significant approximation with thein vivo biofilms, such as those formed in a necrotic root canal. At the time of conducting this research, it was possible to find nine studies on theantibacterial and dissolution effects of pre-heated NaOCl in PubMed search.13 These adaptivemechanisms alter the bacterial metabolism from biosynthesis and reproduction towardobtaining energy for its existing biological functions at that moment.14 Thus, when the nutrient supply is favorableagain, the metabolic functions and cell division of the bacteria are resumed untilexhaustion of the nutrients, then and once again; the bacteria begin their \u201chibernationperiod.\u201d15 On the other hand, the supply of nutrients to the bacteria in the oral cavity is one ofthe most abundant in comparison with other ecosystems because the host consumes food.However, in microenvironments such as root canals, the bacteria develop efficient metabolicadaptive mechanisms in order to survive under conditions of stress, nutrient scarcity ornutrient deprivation.insitu infected dentin, and like wise test if the post-treatment biofilm wascapable of growing again in a nutrient-rich medium. The aim of this study was to investigate if the concentration, exposure time andpreheating of NaOCl improved its antibacterial effect and dissolution ability on  The study protocol was approved by the Institutional Human Ethical Committee (Protocol166/2011). The irrigant solutions used in this study were 1% and 2.5% NaOCl . The temperatures tested were 22\u00baC (room temperature) and37\u00baC (body temperature), and the exposure times were 5 and 20 min. To heat the NaOCl,disposable syringes containing 1 mL of solution were placed 30 min before being used in anincubator at 37\u00b0C. A new syringe was used every 5 min. The laboratory temperature wascontrolled using a mercury barometer. The temperature of the solutions was controlled usinga pH meter with a temperature sensor .16Samples were autoclaved for 30 min at 121\u00baC  and then treated with 2.5% NaOCl for 15 min and 17% EDTA for3 min. An in situ model was modified to induce the dentininfection.2 Bovine dentin blocks of 5\u00d75\u00d73 mm were obtained from freshly extracted bovine teethaccording to a methodology previously published by the authors.16 The opposite side to theinfected dentinal surface was marked with nail varnish to facilitate the identification ofthe biofilm side. The dentin blocks with enamel or irregular surfaces were discarded. The samples were fixedin the cavities of Hawley\u2019s orthodontic device using sticky wax . Five dentin blocks were used for each experimental procedure and fourconfocal \u2018stacks\u2019 were analyzed from random areas of each sample, totaling 40 samplesthroughout the experiment; this means that 480 images were recorded . The same samples were utilized to perform theseprocedures because the Live/Dead dye did not interfere with the cellularviability.\u00a05 mL\u00a0BHIbroth\u00a0and\u00a0incubated\u00a0at 37\u00b0C for 24hours to standardized the bacterial growth.The subject maintained recommended oral hygienepractices. A healthy single volunteer used the Hawley\u2019s retainer for 48 hours. After this time, thesamples were transferred to\u00a0test tubes containing17Thepre-NaOCl treatment samples were used as a control group. The images of the pre-treatedsamples were obtained and recorded using a Confocal Laser Scanning Microscope . Four random areas of each block were scanned using a \u00d740 oillens, 1.5 \u03bcm step-size, and a format of 512 \u00d7 512 pixels. The area of each image represented275 \u00d7 275 \u03bcm. The rank of laser penetration into biofilm was 30-80 \u03bcm. The biofilm wasmeasured from the outer biofilm layer to the dentinal surface. When the dentinal infection period was finalized, the samples were rinsed with distilledwater to remove the non-adherent cells. Next, the biofilm was stained with Live/DeadBacLight Bacterial Viability ; inaccordance with Stojicic et al. The samples were then immersed in 24-well tissue culture plates, containing 2mL of theexperimental solutions for 5 min (n=5) and 20 min (n=5). The solution of 20-min groups wererefreshed every 5 min to simulate the clinical conditions (the old solution was sucked andthe fresh solution was added). After the contact time tests, the samples were immersed in 2mL of 5% sodium thiosulphate for 10 min to counter the NaOCl residual effect. After that,the samples were stained again and the post-treatment biofilms were analyzed in the CLSM.The dentin blocks were then irrigated with distilled water and incubated in 5 mL of BHI at37\u00baC for 24 hours. After the incubation period, the samples were stained and analyzed againto verify the bacterial reactivation. Representative images of the pre- and post-treatmentsamples and revitalized biofilm can be observed in 3/\u03bcm2),18 and the percentage of viable cells werecalculated using the Bioimage_L program.19 The total biovolume  was utilized as the analyticalsoftware. Statistical analysis was performed using D\u2019Agostino-Pearson omnibus normality testto verify the normal distribution of the data. The Kruskal-Wallis and Dunn tests (P <0.05) were used for multiples comparisons because the data did not pass the normalitytest.\u201375% percentiles of the total biovolume and the live cells percentageafter treatment with NaOCl at 22\u00baC and 37\u00baC are shown in  All the experimental solutions were able to decrease the ratio of the total biovolume(P<0.05) without statistical differences among the experimental groups (P>0.05). Nostatistical differences were found between the percentage of live cells of the controlgroup and 1%-NaOCl-5 min at 22\u00baC (P=0.09) and heated at 37\u00baC (P=0.08).The medians and the25 The total biovolume and percentage of live cells of the revitalized biofilm aftertreatment with 1% NaOCl for 5 min at room temperature showed no statistically significantdifferences in comparison with the control (P=0.08). Although the 1%-NaOCl at roomtemperature was able to significantly dissolve the organic matter (P=0.01), the remainingbiofilm showed high percentages of bacterial viability  to eliminate Enterococcus faecalis.Similar results were observed in the present study, where the antimicrobial ability of NaOClwas more dependent on its concentration and contact time than its temperature. It isimportant to note that the 1%-NaOCl-5 min groups, regardless of their temperature, showed asimilar percentage of live cells compared to the control group. The synergistic effects thatpromote bacterial biovolume, the resistance of bacteria to antimicrobial agents, and theirinvasion in multispecies biofilms,36 couldbe the main causes of the differences among the present study and other publishedinvestigations. Anyhow, more studies on the effect of NaOCl on polimicrobial biofilms formedin situ are necessary. Although the literature provides some insight into chemical stability and antimicrobialability of NaOCl preparations, the findings appear to be somewhat contradictory. Previouspublications analyzed the antimicrobial effect of the NaOCl temperature variation onuni-species biofilm37 In line with this statement, Chavez de Paz et al13 showed that Streptococcusanginosus biofilm cells were able to recover 78% of the dehydrogenase activityand 61% of the esterase activity and its biomass mm\u22122 increased around 35% after72 hours of reactivation. In addition, Shen et al38 using the Live/Dead technique, found that the revitalized biofilm took4 weeks to reach a 75% bacterial growth, similar to the 3 weeks biofilms with nutrientsupplementation. Considering these findings, it is possible that 24 hours is an insufficienttime for the biofilm to change its stress metabolism to a rich nutrientmetabolism.Consequently, the bacteria in the biofilm remained in a stationary phase. Regarding the bacterial revitalization, statistical differences were not found in thepresent study among the NaOCl room temperature groups and their respective reactivations.The same situation was repeated in the NaOCl-37\u00baC groups. This would seem contradictorygiven that in all groups varying percentages of viable cells were found, but the slow growthrate of the bacteria within a biofilm is not necessarily due to the nutrient limitation.Infact, this slowed-down bacterial reproduction could be a response to stress conditions. Thisresponse to stress causes physiological changes that protect the bacteria from the effect oftemperature and pH changes and many chemical agents. Based on the aforementioned, the authors concluded that an increase of the NaOCltemperature did not enhance its dissolution or antibacterial ability when it was tested onmixed-species biofilms. Heating the NaOCl at 37\u00baC did not improve its dissolution ability; its antimicrobialeffect is more dependent on the concentration and contact time than the temperature. Thebiofilm treated with NaOCl is not capable of achieving significant levels of biomass andcell viability after 24 hours of exposure to nutrient-rich conditions, except when thesolution is used in low concentrations and contact times. S\u00e3o Paulo Research Foundation (FAPESP 2011/08184-8). The authors declare that they have nocompeting interests."}
+{"text": "We conduct pedigree-based linkage and association analyses of simulated systolic blood pressure data in the nonascertained large Mexican American pedigrees provided by Genetic Analysis Workshop 18, focusing on observed sequence variants in MAP4 on chromosome 3. Because pedigrees are large and sequence data have been completed by imputation, it is feasible to conduct analysis for each pedigree separately as well as for all pedigrees combined. We are interested in quantifying and explaining between-pedigree heterogeneity in linkage and association signals. To this end, we first examine minor allele frequency differences between pedigrees. In some of the pedigrees, rare and low-frequency variants occur at a higher prevalence than in all pedigrees combined. In simulation replicate 1, we conduct variance-components linkage and association analysis of all 894 MAP4 variants to compare analytic approaches in single pedigree and combined analysis. In all 200 replicates, we similarly examine the 15 causal variants in MAP4 known under the generating model. We illustrate how random allele frequency variation among pedigrees leads to heterogeneity in pedigree-specific linkage and association signals. Whole genome sequencing holds out the promise of being able to more effectively map the effects of genetic variants on complex traits, and thereby identify the causal variants involved in disease expression . Even whIn analysis of the original Genetic Analysis Workshop pedigree data , we obsWe analyzed the imputed \"best guess\" sequence genotype data for a total of 959 study participants in the 20 pedigrees, as provided, including 894 MAP4-designated sequence variants encompassing the chromosome 3 region from 47.892183 to 48.130741 megabases (Mb). Under the generating model for the phenotype simulations, the sequence data were the same for each replicate, and the same set of 15 variants was defined as causal, so only the randomly generated phenotypes varied from replicate to replicate. We estimated the minor allele frequency (MAF) at each segregating locus within a pedigree. As described in Chen et al [We applied the genetic analysis software SOLAR for variance-components models to assess linkage and association in pedigrees . With sig) are decomposed into between-family (b) and within-family (w) components in a fixed-effect model E(phenotype) = \u03bc + \u03b2bb + \u03b2ww. The MG approach estimates regression coefficients with the constraint \u03b2b = \u03b2w. The QTDT approach estimates both \u03b2b and \u03b2w and tests whether the within-family parameter \u03b2w is significantly different from zero. The parameter \u03b2w reflects the within-pedigree correlation between phenotype and the allelic transmission score w = (g-b) which is the deviation between the observed and expected genotype. It is, therefore, robust to stratification effects [In the QTDT method, the mean phenotype is modelled as a linear combination of fixed effects  and random effects . The genotype scores  regional profile plots. For processing of all 200 replicates, we considered only the 15 MAP4 causal variants specified in the simulation model. Given the small size of MAP4 relative to a typical linkage region, and the limitations of single-point IBD estimation for linkage analysis, we took the maximum of the 2-point LOD as a regional measure of linkage. We constructed box plots of the LOD score and \u2212log10 to examine variation across replicates by pedigree and differences in power among the causal loci.In replicate 1, for each of 894 loci with sequence variants we computed the LOD score, and the asymptotic MG and QTDT p-values for all pedigrees combined and for each pedigree separately, and constructed LOD score and \u2212log10 and as expected, rare variants (MAF <1%) were most prevalent. Overall MAF values for the 15 \"causal\" loci in MAP4 ranged from 0.5% to 37.8%: 3 common variants (>5% MAF) were observed in all 20 pedigrees, 3 low-frequency variants (1% to 5% MAF) appeared in 7 or more pedigrees, with 9 rare variants (<1% MAF) in 1 to 5 pedigrees. In any one pedigree, only 3 to 8 of 15 variants were observed. When a rare variant was observed, the corresponding pedigree-specific MAF was substantially higher, with nearly all being >1% for causal variants.p values, whereas pedigree 10, in contrast, had small LOD scores but more significant MG p values. The combined pedigree maxLOD of 2.26 and minimum MG asymptotic p value of 1.3 \u00d7 10\u221214 occurred at causal locus 10 corresponding to a low-frequency variant (3.2%) with a large effect size and the highest SBP %variance explained within MAP4. At this locus . At the other extreme, pedigree-specific MG and QTDT analysis failed in pedigrees 4 and 14 which had very low MAF and negligible LOD scores.In replicate 1, there was marked variation in the combined-pedigree 2-point LOD scores across the MAP4 region  and the within-family score for an individual is the deviation of their observed genotype from the expected, which is taken as a measure of allelic transmission . Like otp values may be inaccurate, so, as illustrated in Table Under the fixed effects part of the QTDT model, the between-family and within-family scores are approximately orthogonal so the between and within regression coefficients are approximately independent , but onlp values across all 200 replicates demonstrates considerable heterogeneity among pedigrees  and low-frequency  variants were reasonably well-detected by the MG test. Analysis of loci 5 and 8 gave nearly identical results, although only locus 5 had a nonzero effect size under the generating model, reflecting an indirect effect of an underlying haplotype. Loci 7 and 10 were similarly subject to complete linkage disequilibrium within pedigrees; each had independent effects, but these were not distinguishable; the locus 10 regression coefficients  included in the phenotype-generating model, segregation of the variant was observed in 5 or fewer of the 20 pedigrees, with mean MAF >1% in the subset. The low-frequency variant responsible for the largest SBP %variation explained (locus 10), which segregated in 12 pedigrees, exhibited the best power for association with good agreement between MG and QTDT signals. Pedigrees with some evidence for linkage at this locus were enriched for the low-frequency variant , and it followed that these pedigrees were also more informative in the within-family QTDT assessing transmission disequilibrium, and contributed to smaller standard errors in the MG regression analysis. As we understand it, the Genetic Analysis Workshop 18 (GAW18) pedigrees comprise a sample of large Mexican American families, not ascertained on the phenotype. Therefore differences in MAF between pedigrees arise from random differences in the genotypes of the founder individuals for each pedigree. Because the genetic model used to generate the phenotype data in the simulated data sets is the same in each pedigree, variation in MAF among the pedigrees is a major source of heterogeneity in the pedigree-specific linkage and association tests.The authors declare that they have no competing interests.SBB and ZC designed the overall study and drafted the manuscript. ZC, JP, and KRT conducted statistical analyses. All authors read and approved the final manuscript."}
+{"text": "We were recently made aware that one of the figures  in"}
+{"text": "Triticum aestivum L.). In the present study, a recombinant inbred line (RIL) population derived from a Gaocheng 8901/Zhoumai 16 cross grown in three environments was used to identify quantitative trait loci (QTLs) for dough rheological and starch pasting properties evaluated by Mixograph, Rapid Visco-Analyzer (RVA), and Mixolab parameters using the wheat 90 and 660 K single nucleotide polymorphism (SNP) chip assays. A high-density linkage map constructed with 46,961 polymorphic SNP markers from the wheat 90 and 660 K SNP assays spanned a total length of 4121 cM, with an average chromosome length of 196.2 cM and marker density of 0.09 cM/marker; 6596 new SNP markers were anchored to the bread wheat linkage map, with 1046 and 5550 markers from the 90 and 660 K SNP assays, respectively. Composite interval mapping identified 119 additive QTLs on 20 chromosomes except 4D; among them, 15 accounted for more than 10% of the phenotypic variation across two or three environments. Twelve QTLs for Mixograph parameters, 17 for RVA parameters and 55 for Mixolab parameters were new. Eleven QTL clusters were identified. The closely linked SNP markers can be used in marker-assisted wheat breeding in combination with the Kompetitive Allele Specific PCR (KASP) technique for improvement of processing quality in bread wheat.Dough rheological and starch pasting properties play an important role in determining processing quality in bread wheat ( Triticum aestivum L.). Gluten protein consists of glutenin and gliadin, responsible for dough rheological properties. Glutenin is divided into high- and low-molecular-weight glutenin subunits (HMW-GSs and LMW-GSs); these are encoded by Glu-1 and Glu-3 loci on chromosomes 1A, 1B, and 1D, respectively  construct a high-density linkage map with the 90 and 660 K SNP assays, (2) provide a comprehensive insight into genetic loci for dough rheological and starch pasting properties using a genome-wide QTL mapping approach, and (3) identify SNP markers closely linked to QTLs associated with quality traits for MAS in wheat breeding.2:6 RILs for QTL analysis were developed from a cross between Gaocheng 8901 and Zhoumai 16 by single seed descent. Gaocheng 8901 was a strong gluten variety with alleles 1, 7+8, 5+10, Glu-A3g, Glu-B3d, Wx-A1a, Wx-B1a, Wx-D1a, Pina-D1b, Pinb-D1a, and Pinb-2v2, whereas Zhoumai 16 has weak gluten with alleles Null, 7+9, 2+12, Glu-A3c, 1B.1R, Wx-A1a, Wx-B1a, Wx-D1a, Pina-D1a, Pinb-D1b, and Pinb-2v3. The compositions of all alleles were confirmed in parents by molecular markers . Kernel moisture and protein were determined with a near infrared transmittance (NIT) analyzer . Samples were tempered overnight to 14, 15, and 16% moisture contents for soft (SKCS hardness index (HI) < 40), medium , and hard (HI > 60) types, respectively. All samples were milled using a Brabender Quadrumat Junior Mill . Flour extraction rates were about 60%.3 to eliminate the effect of \u03b1-amylase activity in flour on starch pasting properties.A 10-g Mixograph  was used to assess dough mixing characteristics and MPT, MPV, MPW, and MTxW were measured according to AACC  method 5A Mixolab  was used to determine dough mixing and pasting properties of wheat flour simultaneously during dough mixing. About 50-g of flour was put into the Mixolab bowl and an appropriate amount of water was added to ensure that the torque of the dough was in the 1.1 \u00b1 0.07 Nm range. Processing was divided into five stages based on the \u201cChopin 12heat\u201d protocol as follows: establishing equilibrium at 30\u00b0C for 8 min, then heating to 90\u00b0C at a rate of 4\u00b0C/min for 15 min, holding at 90\u00b0C for 12 min, cooling to 50\u00b0C at a rate of 4\u00b0C/min for 10 min, and finally holding at 50\u00b0C for 5 min. The mixing speed was kept constant at 80 rpm. The parameters water absorption (WA), development time (DT), stability time (ST), C1 (the torque of maximum point in the first mixing stage), C2-C5 (the torque of end points in the corresponding mixing stages) were recorded during the procedure.http://www.capitalbio.com). Genotypic clusters involving each SNP were confirmed using the polyploidy version of GenomeStudio software .A 90 K iSelect SNP array containing 81,587 markers and a 660 K SNP array with 630,517 markers were used to genotype all 176 RILs and 59 randomly selected RILs, respectively, at CapitalBio Corporation . However, many SNPs were mapped at the same loci or within 0.01 cM among them. Therefore, BIN-Mapping function from IciMapping 4.0  was used to construct a skeleton map containing 2375 markers for subsequent QTL analysis. Secondly, among 90 and 660 K SNP analyses of 59 random RILs, 12,205 and 109,209, respectively, were polymorphic between the parents. Of those, 4809 and 20,498, respectively, had more than 10% missing data, and 2081 and 31,371, respectively, were not anchored to the linkage map. Thus, 5315 and 57,340 from two arrays, respectively, were used to construct the linkage map (defined as Map 2) with Joinmap 4.0 software; this was used to enrich the markers in several QTL regions with larger marker intervals from Map 1. Thirdly, Maps 1 and 2 were integrated into a high-density linkage map with MergeMap Online .SNPs of poor quality, or with more than 10% of missing data, or segregation distortion of more than 0.35 were removed. Three procedures were followed in constructing the high-density linkage map. Firstly, among 81,587 SNPs (90 K) used in screening all 176 RILs, 12,205 were polymorphic between the parents. Of those, 2080 had more than 10% missing data, and 828 were not anchored to the linkage map. Finally, 9297 SNPs were used to construct a linkage map (defined as Map 1) using Joinmap 4.0 software . QTL analysis was carried out with QTL Cartographer 2.5 using composite interval mapping  based on Map 1. Logarithm of odds (LOD) scores ranged from 1.8 to 2.6 for all traits tested according to 2000 permutations tests at a probability of 0.01. Therefore, a LOD score of 2.6 was used for declaring significant QTL. The QTL \u00d7 Environment (QE) interaction was analyzed by IciMapping 4.0 using the multi-environment trials (MET) , with a LOD threshold based on 1000 permutation tests at a probability of 0.01. For the 90 K iSelect SNP genotyping assay, candidate genes were confirmed following Zhai et al. (http://plants.ensembl.org/Triticum_aestivum/Info/Index) to determine SNP markers corresponding to the original genes, and these gene sequences were used as queries to blast the NCBI database (http://www.ncbi.nlm.nih.gov/) to identify putative gene functions. BLAST hits were filtered to an e-value threshold of 10\u22125 with an identity higher than 75%. Collinearity analysis was conducted by Ensembl Plants database (http://plants.ensembl.org/index.html) and blast hits were filtered with an e-value threshold of 10\u221216.The short and long chromosome arms of each linkage group were confirmed according to the wheat 90 and 660 K consensus SNP maps. The linkage map was constructed using MapChart 2.2  and correlation were performed by SAS 9.0 . ANOVA was conducted using the PROC MIXED procedure, where environments were treated as fixed effects, and lines, line \u00d7 environment interactions and replicates nested in environments were all treated as random effects. The broad-sense heritabilities . MTxW was significantly correlated with Mixolab parameters ST, C2, C3, C4, and C5.The significant and positive correlations were found among MPT, DT, ST, and C2 Table . MPV andThe high-density linkage map comprised 8067 (90 K) and 38,894 (660 K) SNP markers, and spanned a total length of 4121 cM involving all 21 chromosomes, with an average chromosome length of 196.2 cM, ranging from 78.0 cM (3D) to 387.5 cM (5B) Tables , S3. TheA total of 119 additive QTLs were identified for 17 quality parameters using the 90 K iSelect SNP genotyping assay linkage map; 15 stable QTLs explained more than 10% of the phenotypic variation across environments Table . In addiQMPT.caas.1DL, tightly linked to GENE-0511_484 with a genetic distance of 0.02 cM, explained the highest phenotypic variation ranging from 37.6 to 52.6% across environments. The QTLs on 1AL and 1BL tightly linked to Glu-A1 and H20 with genetic distances of 1.03 and 0.05 cM, respectively, had major effect on MPV and MPW. QMTxW.caas.1DL, QMTxW.caas.2BS, and QMTxW.caas.4B were identified across three environments, explaining 5.9\u201312.0% of the phenotypic variation. Alleles with increasing effects for the QTLs influencing Mixograph parameters located on chromosomes 1AL, 1BL, and 1DL were from Gaocheng 8901.MPT, MPV, MPW and MTxW were controlled by 10, 11, 8, and 6 QTLs, respectively Table . QMPT.caQPV.caas.6BL.2 tightly linked to RAC875_c50348_93 at a genetic distance of 5.00 cM showed the highest contribution. The major QTV.caas.1BL was 0.02 cM from Kukri_c7770_176 and explained up to 17.9% of the phenotypic variation. QBD.caas.7DL closely linked to Excalibur_c4508_1007 was detected in all environments, accounting for 10.4\u201314.0% of the phenotypic variation. QFV.caas.5BL.2 was 0.01 cM from BS00031339_51, explaining up to 13.6% of the phenotypic variation. QPTI.caas.2AL and QPTI.caas.4AS tightly linked to Excalibur_c6710_2192 and RAC875_rep_c106151_123 at genetic distances of 0.00 and 0.03 cM, respectively, were identified in all environments. Several QTLs  were strongly affected by QE interaction.PV, TV, BD, FV, SB, and PTI were associated with 5, 5, 2, 5, 3, and 9 QTLs, respectively Table . QPV.caaHa-Excalibur_c49805_63 on chromosome 5DS was associated with WA in all environments. The stable QTL tightly linked to Glu-D1, was the most important locus for ST and C2. QC3.caas.1DS was 0.04 cM from TA003135-0494, explaining 7.4\u201317.3% of the phenotypic variation. The largest phenotypic variations for C4 and C5 were contributed by QC4.caas.5BL.1 and QC5.caas.6DS closely linked to BS00031339_51 and RAC875_rep_c85994_258 at genetic distances of 0.02 and 1.07 cM, respectively. Alleles increasing DT and ST on chromosome 1DL came from Gaocheng 8901 and exhibited strong interaction with environments.WA, DT, ST, C2, C3, C4, and C5 were conditioned by 13, 8, 9, 5, 8, 6, and 6 QTLs, respectively , 5AS, 5BL, 6BL, and 7DL based on Map 1 , ST (r = 0.85), and C2 (r = 0.60) were found in the present study. FV also exhibited significant correlations with C3 (r = 0.31), C4 (r = 0.44), and C5 (r = 0.38). These results suggested that wheat quality can be effectively assessed by Mixolab parameters. However, it needs about 50-g flour in one Mixolab test, whereas only 10 and 3.5-g of flour were needed in one Mixograph and RVA analysis, respectively. As a result, Mixograph and RVA are more suitable for analyzing early generational material in wheat quality breeding while Mixolab could be used in bakery factories.Mixograph and RVA are most commonly used to evaluate the dough rheological characteristics and starch pasting properties in wheat breeding, respectively. Recently, a new device Mixolab has been introduced to assess dough rheological characteristics, starch pasting properties and flour enzyme activity in one test sample, reducing labor requirements. The instrument also provide information on effects of different ingredients  and QTL clusters  were significantly increased by the high-density linkage map  and MTxW(QMTxW.caas.2BS). Zhang et al.  with Pina-D1a/Pinb-D1b/Pinb-2v3. Grain hardness mainly affected by Ha locus on chromosome 5DS exhibited significant influence on milling quality. It has been reported that hard wheat has much more the amount of damaged starch than soft wheat . Therefore, QTL for WA on chromosome 5DS was contributed by both Pina and Pinb genes at Ha locus, in agreement with Ma et al. (Gaocheng 8901 (HI = 66) with alleles a et al. .QMPT.caas.5AL positioned at 87 cM on chromosome 5AL was not previously reported. A stable QTL QMPT.caas.7DL at 93 cM on chromosome 7DL is different from one reported by Tsilo et al.  to identify SNP markers corresponding to the original genes, and then the genetic map constructed in the present study was inspected for the presence of the same markers. Kukri_rep_c101946_496 derived from an isoamylase 2 gene, was mapped on chromosome 1AL at a distance of 2.45 cM from the LOD contour peak marker for QSB.caas.1AL. Wsnp_Ex_rep_c66900_65313836 derived from an isoamylase 3 gene, was mapped on chromosome 5AL at a distance of 9.50 cM from the LOD contour peak marker of QFV.caas.5AL , FV(QFV.caas.5BL.2), SB (QSB.caas.5BL), C3 (QC3.caas.5BL.2), C4 (QC4.caas.5BL.1), and C5 (QC5.caas.5BL) may represent the region affecting WE-AX.Arabinoxylans are separated into water-extractable (WE-AX) and water-unextractable (WU-AX) based on the solubility. It is widely accepted that WE-AX showed positive influence on dough properties because it could construct networks with protein and starch by hydrogen bonding  ,    showed the best conservation of gene order among these species, suggesting that these regions may facilitate further fine mapping and discovery of candidate genes.With rapid development of next-generation sequencing, genomic sequence of many species such as rice (QMPT. caas.1BL, QMPT.caas.1DL, QMPV.caas.1AL, QMPV.caas.1BL, QMPV.caas.5AL, QMPV.caas.6BL, QMTxW.caas.4B, QPV.caas.6BL.2, QSB.caas.5BL, QPTI.caas.2AL, QPTI.caas.4AS, and QTL clusters can be used in MAS for improvement of wheat processing quality.The present study indicates that MPT, MPV, PV, SB, and PTI are good parameters for the evaluation of processing quality in wheat breeding due to higher broad-sense heritabilities. A high-density linkage map was constructed in the Gaocheng 8901/Zhoumai 16 population with the 90 and 660 K SNP arrays; it provided a powerful tool to identify QTLs/genes for important quality traits and candidate genes. Eleven QTL clusters for dough strength and starch pasting properties were discovered. The SNP markers tightly linked to We declare that these experiments comply with the ethical standards in China.HJ carried out the experimental and wrote the paper. WW, JL, YZ, and JY participated in field trials. SZ contributed to flour milling. ZL, XX, and ZH designed the experiment and assisted in writing the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "Astyanax mexicanus, is a unique model system consisting of cave-adapted and surface-dwelling morphotypes that diverged >1 million years (My) ago. This remarkable natural experiment has enabled powerful genetic analyses of cave adaptation. Here, we describe the application of next-generation sequencing technology to the creation of a high-density linkage map. Our map comprises more than 2200 markers populating 25 linkage groups constructed from genotypic data generated from a single genotyping-by-sequencing project. We leveraged emergent genomic and transcriptomic resources to anchor hundreds of anonymous Astyanax markers to the genome of the zebrafish (Danio rerio), the most closely related model organism to our study species. This facilitated the identification of 784 distinct connections between our linkage map and the Danio rerio genome, highlighting several regions of conserved genomic architecture between the two species despite \u223c150 My of divergence. Using a Mendelian cave-associated trait as a proof-of-principle, we successfully recovered the genomic position of the albinism locus near the gene Oca2. Further, our map successfully informed the positions of unplaced Astyanax genomic scaffolds within particular linkage groups. This ability to identify the relative location, orientation, and linear order of unaligned genomic scaffolds will facilitate ongoing efforts to improve on the current early draft and assemble future versions of the Astyanax physical genome. Moreover, this improved linkage map will enable higher-resolution genetic analyses and catalyze the discovery of the genetic basis for cave-associated phenotypes.The Mexican tetra,  Anoptichthys (\u201cbony fish without eyes\u201d)  . Breedint eyes\u201d) , b. Bothresearch  focusingresearch , developresearch , physiolOca2) (brown phenotype (Mc1r)  phenotypes evolved through heritable genetic changes. These studies centered on both Mendelian and complex phenotypes, including eye regression , and int1 \u00d7 Pach\u00f3n cave backcross pedigree using markers generated from random amplified polymorphic DNA (RAPD) fingerprinting  technology. This technology enables accurate and high-throughput collection of massive amounts of sequence data  bred from a male surface fish and female cavefish from the Pach\u00f3n cave. In addition, surface (n = 4), Pach\u00f3n cave (n = 4), and surface \u00d7 Pach\u00f3n F1 hybrid (n = 4) specimens were used to evaluate and code GBS markers for use with JoinMap software , but were not included in linkage mapping calculations. Parental specimens belonged to laboratory populations originally sourced from the El Abra region of northeastern Mexico and all fish used were generously provided to our laboratory by Dr. Richard Borowsky (New York University). All live fish used in this study were maintained as previously described (see 2 population (n = 129) was individually reared in a 1-liter tank. All phenotypic data from the \u201cAsty12\u201d F2 population (n = 41) were obtained from paraformaldehyde-preserved specimens.Linkage mapping and QTL studies were performed using genotypic and phenotypic data obtained from two separate F1 and F2 hybrid Astyanax mexicanus specimens using the DNeasy Blood and Tissue Kit (Qiagen) as previously described . Samples were processed by the Institute for Genomic Diversity (Cornell University), where genomic libraries were constructed and GBS was performed as described elsewhere  surface or cave individuals had the identical nucleotide at a particular locus, then the genotype was assigned to the consensus parental population. Likewise, a true \u201chybrid\u201d genotype was assigned if three or more F1 individuals harbored the same heterozygous condition  at a given locus. Those genotypes with an ambiguous morphotypic origin were denoted \u201cNA.\u201dGenotypes for each of 7956 GBS markers (each consisting of a single SNP in a 64-bp-long sequence fragment) were screened in cave and surface  forms to assign the morphotypic origin of each allele. Fi.e., both the surface and cave genotypes were scored \u201cNA\u201d) or if the assigned surface and cave genotypes were identical; 6006 genomic markers were deemed suitable and prepared for linkage map calculation using the \u201ccross-pollination\u201d (CP) segregation coding used in JoinMap. At this stage, 107 markers were found to be uninformative  and discarded from further analysis. We screened the remaining set (n = 5899) to identify markers failing to conform to predicted genotypic ratios ; 2896 markers demonstrated a \u03c72 value more than 50, implying significant departure from the predicted genotype ratio and were discarded from further analysis. Our final GBS marker set included 3003 markers evaluated in 170 F2 individuals.Markers were then screened for suitability in linkage calculations. Markers were deemed unsuitable and discarded from further analysis if neither parental genotype could be assigned . Our workflow used program default settings, with the following exceptions: the maximum grouping independence LOD value was set to 50.0; linkage groups were calculated using regression mapping; and linkage mapping was performed using the Kosambi method . LinkageAstyanax mexicanus has karyotypic number of 25; The first round of mapping produced 28 linkage groups comprising a total map length of 2956 cM. At this stage, one linkage group  failed to assemble into a consolidated group and was therefore eliminated from further analysis. The remaining individual linkage groups ranged in length from 27.25 to 187.46 cM, containing between 10 and 225 markers with an average intermarker distance between 0.51 and 6.40 cM. After this initial round of mapping, we further screened existing linkages to target the most optimal 25 groups  was eliminated. A densely populated group with a high independence LOD  was split and 12 linkage groups (103.982 \u2264 n \u2264 142.783 cM) were trimmed.Astyanax mexicanus and the zebrafish genome and used to map albinism as a proof of concept. Albinism was scored as a binary phenotype wherein presence of melanin (0) or absence of melanin (1) was assigned to each of the members of our experimental F2 pedigrees. All QTL analyses of albinism were conducted using R/qtl .At present, physical genome resources for 0 My ago . Despitee.g., three different Danio rerio chromosomes). In cases where a single marker sequence aligned multiple times with the same target, raw results were filtered by e-value, retaining the lowest e-value alignment for each marker-target pairing. There are two 64-bp sequences for each GBS marker, differing only in that each contains one of the two alleles for the imbedded SNP. Because both of these sequences were included when BLAST searches using the 64-bp marker sequences were conducted, this filtering step also served to collapse these results into a single set of results, retaining the better of the two alignments for each marker-target pairing.These and all subsequent searches were performed using a BLASTN script run on the Ohio Supercomputing Cluster (OSC). All quality control defaults, including an expect value  cutoff of 10, were maintained. The script permitted the return of alignments between a given 64-bp marker sequence and regions of up to three distinct targets  for a given marker sequence agreed with the target reported for one or more other markers on the same linkage group that returned only a single robust hit, then the top hit for the marker in question was considered \u201csupported\u201d and retained. If the top hit was not supported in this fashion but a different BLAST result was, then the latter \u201cnot top hit, supported\u201d result was retained instead. If none of the results returned for a marker sequence were supported, then the top hit was retained, despite the lack of support. In rare cases, there was no way to resolve which result should be retained. Results for these \u201cunresolved\u201d markers were discarded.In some instances, a single queried sequence returned alignments with multiple targets. These instances were resolved by sorting results to determine the \u201ctop hit,\u201d which was defined as having the lowest e-value and highest percent identity  to a particular target sequence. If the target of the top hit . The collective sequence data for the Astyanax genome (GenBank Assembly ID GCA_000372685) were downloaded from Ensembl, along with the transcript sequences for 23,042 predicted genes. BLAST searches were used to determine putative locations for the 64-bp sequences of the 2235 GBS markers comprising our final linkage map in the Astyanax genomic and transcriptomic data sets. After initial searches were performed as described, \u223c2000-bp stretches of genomic sequence harboring our 64-bp GBS marker sequences were aligned with the Danio genome. Similarly, full sequences for predicted Astyanax transcripts to which our GBS markers aligned were queried against a Danio cDNA database downloaded from Ensembl. Both data sets were then filtered (as described), yielding a single \u201cbest\u201d Danio alignment for each informative query. This process enabled us to leverage draft genomic and transcriptomic data to augment the amount of sequence information associated with our 64-bp GBS markers and to identify homologous genomic positions in a well-characterized model system.When using BLAST searches to align our 64-bp markers directly to the Astyanax and Danio rerio genomes.After BLAST searches using the direct, genomic, and transcriptomic alignment methods were completed, the filtered results for all three were combined. When multiple methods returned results for the same marker, a single result was chosen and retained using the same filtering process applied to single data sets (above). The Circos program  for each data set were aligned with Astyanax genome scaffolds using the same BLAST and filtering protocols used for our own data (above). Both previous studies included markers located in candidate genes. The locations of Astyanax orthologs of these candidate genes were identified using Ensembl.Previous maps published by http://dx.doi.org/10.5061/dryad.6s718).GBS marker sequences and genotyping data are available from the Dryad Digital Repository  comprising 2235 markers spanning 2110.7 cM, with an average intermarker distance of 1.052 cM (Table S1). The strategy we used enables application of powerful, cost-effective, next-generation sequencing technology to facilitate genetic studies in emerging or nonmodel systems.Here, we present a dense linkage map for Astyanax genomic and transcriptomic resources, followed by searches of the homologous sequences in Danio (Astyanax genomic (26.5%) or transcriptomic (13.3%) sequences as an intermediary  comprising ancient syntenic blocks shared with each of 25 zebrafish chromosomes . However, other Danio chromosomes appear scattered across several linkage groups, without a consensus representation for any particular group .Our analysis of synteny between e groups . CertainDanio chromosomes within our GBS marker set. We examined this possibility by assessing the number of syntenic links between our GBS-based linkage map and each Danio chromosome. We would anticipate that longer chromosomes would naturally harbor more syntenic links. Values were therefore expressed as a ratio of syntenic links per megabase (mean = 0.59 GBS markers/Mb). Although the mean value for chromosomes that were not strongly represented on any particular linkage group in our map  was lower than that for chromosomes demonstrating strong synteny with a particular linkage group , there was not a significant difference between the two groups . This leads us to conclude that, although representation of particular chromosomes in our data set may be a contributing factor, it is unlikely that this is the primary cause of the differences in chromosomal representation patterns observed.We believe these findings most likely reflect genomic rearrangements that have occurred since the divergence of these two species. However, this finding could also be attributed to low representation of particular Astyanax GBS markers  may include paralogous genes or otherwise ambiguous results that could lead to erroneous links between a linkage group and a Danio chromosome. Although we cannot rule out this possibility, we feel our strategy prioritized the \u201coptimal\u201d BLAST result among multiple hits for a single marker leading to alignments that agree with nearby unambiguous results  of final calls were unsupported by the results for other markers belonging to the same linkage group (Table S1). Given that chromosomal arrangements have occurred over the \u223c150 My since divergence, we feel our systematic approach best identifies paralogous genes and other potential sources of ambiguity.Alternatively, BLAST results for  results . As a reMaterials and Methods); however, we relied on a relatively small number of cave, surface, and F1 hybrid individuals (n = 4 each) to identify parental allelic origin. Similarly, the relatively small number of meiotic events represented by the 170 F2 individuals may have resulted in linkage map inaccuracies  of the 2235 GBS markers present in our map. These markers were localized to positions spread across 598 different Astyanax genome scaffolds. Our 25 Astyanax linkage groups contain markers representing between 12 (linkage groups 8 and 22) and 55 (linkage group 3) genome scaffolds each, with a map-wide average of 27.64 scaffolds/linkage group. Individual genome scaffolds contained between 1 and 31 GBS markers appearing in our final map, with an average of 3.50 markers per scaffold. GBS markers located on the same genomic scaffold colocalized to a single linkage group 87.3% of the time. This suggests that our recombination mapping successfully recapitulated the true genomic positions of the markers used to construct our map.Positional locations in the current draft of the Astyanax and Danio. Metrics such as the number of linkage groups, total map length, number of markers, and marker density are commonly used to compare linkage maps within species. Both our GBS-based map and the RAD-seq and microsatellite-based map published by Astyanax mexicanus karyotype number of 25. The microsatellite-based map presented by Danio  per Danio rerio chromosome  and Danio chromosome = 6.92, minimum = 1, maximum = 20). Additionally, although instances of synteny strongly represented in previous maps were also identified in this analysis, our map demonstrated increased representation of certain Danio chromosomes poorly represented in previous maps. For example, Danio rerio chromosome 11; however, we identified 36 links between our map and chromosome 11. Similarly, Danio chromosomes 17 and 19 are each represented once in the map of Our map contains a total of 784 links between our linkage groups and the romosome . This reAstyanax genomic scaffolds and each linkage map. We examined the five strongest syntenic links between single linkage groups in our GBS-based map and single Danio chromosomes and then identified analogous connections between those chromosomes and specific linkage groups in the maps presented by Our linkage map uses an entirely different marker set than those used in previous maps. Therefore, it was not possible to make direct comparisons with the linkage groups across prior studies. However, we could indirectly compare maps by examining connections between Astyanax genomic scaffolds harboring markers associating each linkage group with a particular Danio chromosome were then compared  GBS marker sequences. As expected, based on the significant divergence between these species, we recovered varying levels of synteny between portions of our Astyanax linkage groups and regions of the Danio genome. As a proof of concept, we successfully mapped a strong QTL associated with albinism and demonstrated significant conserved genomic architecture in the regions surrounding the gene Oca2, between Astyanax and Danio. We successfully anchored emerging Astyanax genomic information to our GBS-based linkage map, identifying the putative location of thousands of anonymous GBS marker sequences within unplaced Astyanax genome scaffolds. This strategy revealed significant colinearity between genomic scaffolds and our linkage map, and it demonstrated the utility of high-density, GBS-based linkage maps to inform and improve nascent genomic resources. Multiple comparisons with previously published maps suggest that our GBS-based map offers a higher level of resolution and a greater number of connections between Astyanax and Danio genomes. We hope that this resource and technology will accelerate the search and identification of genes mediating cave-associated traits in Astyanax, facilitate the genomic assembly for this system, and prove useful to other natural model systems of evolutionary and biomedical relevance.We constructed a high-density linkage map for"}
+{"text": "The technical challenges associated with national data linkage, and the extent of cross-border population movements, are explored as part of a pioneering research project. The project involved linking state-based hospital admission records and death registrations across Australia for a national study of hospital related deaths.The project linked over 44 million morbidity and mortality records from four Australian states between 1st July 1999 and 31st December 2009 using probabilistic methods. The accuracy of the linkage was measured through a comparison with jurisdictional keys sourced from individual states. The extent of cross-border population movement between these states was also assessed.Data matching identified almost twelve million individuals across the four Australian states. The percentage of individuals from one state with records found in another ranged from 3-5\u00a0%. Using jurisdictional keys to measure linkage quality, results indicate a high matching efficiency (F measure 97 to 99\u00a0%), with linkage processing taking only a matter of days.The results demonstrate the feasibility and accuracy of undertaking cross jurisdictional linkage for national research. The benefits are substantial, particularly in relation to capturing the full complement of records in patient pathways as a result of cross-border population movements.The project identified a sizeable \u2018mobile\u2019 population with hospital records in more than one state. Research studies that focus on a single jurisdiction will under-enumerate the extent of hospital usage by individuals in the population. It is important that researchers understand and are aware of the impact of this missing hospital activity on their studies.The project highlights the need for an efficient and accurate data linkage system to support national research across Australia. Administrative datasets are a powerful resource enabling health researchers to answer epidemiological questions that require long-term follow up on large samples of the population . Access To allow researchers to gain a picture of an individual\u2019s health over time, data linkage techniques are utilised to identify which administrative records from multiple datasets belong to the same person. This process allows the researcher to answer questions about the health of individuals over time, rather than solely about discrete health events .Data linkage has several advantages over other study methods. It is far less intrusive and costly than collecting the same information by other means, such as through large-scale surveys. It allows entire populations to be studied, reducing common problems with follow-up encountered in survey based research designs . Its shoIn the absence of a unique identifier, data linkage is carried out using demographic information such as name, date of birth and address. As these identifiers can change and be in error (or contain missing information), probabilistic statistical methods are used to ensure the highest quality of linked data .Two types of errors impact linkage quality: false positives, where two records are designated as a match when they should not be, and false negatives, where two records are designated as a non-match when they should not be. The rate of these two errors, measured through precision  and recall (sensitivity) statistics, determines overall linkage quality .Ensuring high linkage quality is difficult and typically requires manual efforts. Organisations involved in routine, large-scale data linkage frequently employ a system of manual review of created links to monitor and maintain linkage quality , 8. ThisData linkage facilities exist in many parts of the world including Australia, the UK and Canada , 9\u201312. AFrom 2009, there has been significant additional government investment in expanding the data linkage research infrastructure in Australia . The creSeveral \u2018Proof of Concept\u2019 (POC) collaboration projects were initiated by the PHRN to demonstrate the feasibility of moving large datasets across the country, linking these to a high quality in a short period of time, and using the subsequent linked data to answer research questions of national importance .The first of these POC collaborations linked hospital admissions records with death data across several states, focusing on deaths occurring in hospital or within 30\u00a0days of hospitalisation. The project was the first of its kind in Australia.The purpose of this paper is twofold. Firstly, to highlight the technical achievements associated with undertaking data linkage for this first POC collaboration.The paper intends to show that national linkage of \u2018big data\u2019 can be carried out efficiently and accurately. As well as scalable linkage services, an effective national linkage infrastructure needs to deliver high quality linkage results. Current methods for ensuring high linkage quality rely heavily on manual processes, which are not feasible on large datasets. For national linkage to be viable, high linkage quality must be achieved and maintained through automated methods alone.The second aim of the paper is to demonstrate the importance and impact of cross-jurisdictional linkage. The study will capture population movement at individual or person-based level through linkage of disparate datasets, enabling researchers to assess the full extent of health service utilisation across state borders. The effect of more complete patient pathways on research outcomes has not been previously documented and is not well understood. With reliable estimates of cross-border population flows and service utilisation, researchers can gain a better picture of the need for national linkage studies over state-based linkages projects.The data for the POC collaboration included up to ten years of state-based hospital admissions and mortality records from four Australian states between 1st July 1999 and 31st December 2009: Western Australia (WA), New South Wales (NSW), South Australia (SA) and Queensland (QLD) .As a cross jurisdictional project, which involved data files with large number of records, it was not feasible to compare all possible record pairs to establish links. Instead a series of blocks were employed which aimed to reduce the number of comparisons without having an impact on linkage quality (i.e. reduce comparisons without missing \u2018True Positive\u2019 links). To assess the efficiency and quality of the blocks we calculated two complexity metrics, the reduction ratio and pairs completeness score .The reduction ratio provided an assessment of the decrease in comparisons as a result of the blocking strategy. This was calculated as the ratio of actual blocked comparisons to the total possible comparisons and measured the efficiency of the strategy without measuring the impact on linkage quality.within their own jurisdiction.The percentage of \u2018true pairs\u2019 blocked or pairs completeness metric measured the number of true positive pairs compared in the blocking strategy as a percentage of all possible true positive pairs identified using the jurisdictional linkage keys for WA and NSW records. Records from these states were used as they have been linked and extensively reviewed There is an obvious balance between the reduction ratio and percentage of \u2018true pairs\u2019 blocked. If the comparisons are reduced for efficiency it can have an impact on linkage quality and increasing comparisons to maximise quality can significantly impact the time required to process the linkage. The blocking strategy is therefore the reference point for all additional linkage quality estimates .Over 44 million records across morbidity and mortality collections were linked within and between each jurisdiction. The linkage strategy produced a series of records pairs each with a matching score which were used to identify records belonging to an individual across all data sources. The linkage strategy was evaluated in terms of blocking efficiency and linkage quality.Using the blocking strategy outlined, approximately 142 billion comparisons were performed during the linkage process. These matching assessments made up only 0.014\u00a0% of all possible record pairs from the full comparison space. The blocking process was similar within each jurisdiction, with the state-based reduction ratio ranging between 0.99973 and 0.99987. Table\u00a0Linkage results were compared against those produced by state-based linkage units in WA and NSW . The jurisdictional links from these states were used as a gold standard and allowed an evaluation of linkage quality against each individual state .The accuracy results for all linkages were exceptionally high with over 99.76\u00a0% of all \u2018true pairs\u2019 made available for comparison through blocking i.e. a very small number of pairs identified by WA and NSW jurisdictional linkage keys were lost as a result of the blocking strategy Table\u00a0. This prIn WA, over 99.9\u00a0% of the morbidity pairs identified as links were found to be correct, and 98.1\u00a0% of all possible within-jurisdiction morbidity links were found. This resulted in a maximum F-measure quality score of 0.99 where 1.000 would indicate a perfect linkage . Records from public hospitals showed much higher results (F-Measure\u2009=\u20090.995) indicating that the lack of demographic information accounted for the drop in linkage quality Table\u00a0.The final results of the linkage across the various jurisdictions are summarised in Table\u00a0The number of individuals with a single hospital record varied across the four jurisdictions with Western Australia (WA) having the smallest proportion (35\u00a0%) and South Australia\u00a0(SA) having the highest (52\u00a0%). Similarly, the average group size  varied between 6.2 and 5.2 in WA and SA respectively. It should be noted that the South Australian figures do not include private hospital records which may influence the proportion of singleton groups in that state.Cross-border population movements and hospital usage statistics over the study period are summarised in Table\u00a0The linkage described here was part of a large POC collaboration that tested the efficiency and accuracy of newly established national data linkage infrastructure in Australia.The accuracy and efficiency of the linkage was shown to be high with a large number of \u2018blocked\u2019 pairs comparisons removed from the matching process with very little impact on the linkage quality. Using validated linkage information from WA and NSW, little discrepancy was found between the created links and those found by jurisdictional linkage units in those states. The existence of some discrepancies can be attributed to the additional quality work carried out by those jurisdictional linkage units. Jurisdictional linkage units in Australia typically employ extensive manual review of created links, along with stringent regular manual quality checks. Further errors are identified through feedback following the use of the linked data in research projects. Some of the difference in results could also be attributed to the limited number of identifiers supplied for cross-jurisdictional linkage. Linkage quality depends heavily upon the quality of the underlying dataset. NSW data, with one third of names missing, had the lowest overall linkage quality using our linkage strategy .These quality comparisons rely on the use of jurisdictional linkages as the gold standard. These links from WA and NSW have been validated by researchers who have used them widely. In addition, significant expertise has been developed by these organisations which have a long history of linkage. Having access to two entire sets of extensively checked links allowed us to gain a very accurate estimate of our quality. Few previous investigations into linkage quality have had such a reliable and large gold standard with which to test their results. Typical measures of linkage quality have used samples of links to gain an estimate of quality, often able only to estimate the number of incorrect links created, with the number of links missed essentially unknown , or haveLinking hospital records across four states over a ten year time span showed that, on average, between 3\u00a0% and 5\u00a0% of patients within one state had hospital record in another state. The results further showed that between 4\u00a0% and 7\u00a0% of hospital records occurring in a state can be attributed to an individual who also has records in another state.These findings suggest that research studies examining patient pathways may underestimate the total number of event records belonging to individuals if they do not factor in cross-border hospital admissions. In studies involving hospital admissions events from a single state, it is important that researchers are aware of the incomplete nature of information and the impact this may have on research outcomes. The size and impact of this underestimation will depend on several factors such as the selection of study cohort and the study period, with longer study periods being more susceptible to population movement into and out of the jurisdiction.It has been shown that data linkage quality can have an overall impact on research outcomes, potentially biasing results . HoweverThese results show the feasibility of large scale data linkage infrastructure, producing high quality results through efficient linkage processes. Overall, data linkage quality in large scale linkage remains very high, despite the lack of stringent manual quality review procedures, which would be extremely costly on datasets of this size. Importantly, this type of linkage identifies cross-border population movement, enabling researchers to fully describe patient pathways.The national linkage infrastructure has been successfully used to join together records from multiple administrative datasets which belong to the same person. The infrastructure has been developed to be flexible and scalable, addressing the traditional challenges and limitations of efficiently linking national data. With an increasingly \u2018mobile\u2019 population with life event records in different states, this \u201ccross-jurisdictional\u201d linkage service will have positive benefits on Australian health research."}
+{"text": "Crassostrea gigas, a widely cultivated marine bivalve mollusc, is becoming a genetically and genomically enabled model for highly fecund marine metazoans with complex life-histories. A genome sequence is available for the Pacific oyster, as are first-generation, low-density, linkage and gene-centromere maps mostly constructed from microsatellite DNA markers. Here, higher density, second-generation, linkage maps are constructed from more than 1100 coding (exonic) single-nucleotide polymorphisms (SNPs), as well as 66 previously mapped microsatellite DNA markers, all typed in five families of Pacific oysters . The map comprises 10 linkage groups, as expected, has an average total length of 588 cM, an average marker-spacing of 1.0 cM, and covers 86% of a genome estimated to be 616 cM. All but seven of the mapped SNPs map to 618 genome scaffolds; 260 scaffolds contain two or more mapped SNPs, but for 100 of these scaffolds (38.5%), the contained SNPs map to different linkage groups, suggesting widespread errors in scaffold assemblies. The 100 misassembled scaffolds are significantly longer than those that map to a single linkage group. On the genetic maps, marker orders and intermarker distances vary across families and mapping methods, owing to an abundance of markers segregating from only one parent, to widespread distortions of segregation ratios caused by early mortality, as previously observed for oysters, and to genotyping errors. Maps made from framework markers provide stronger support for marker orders and reasonable map lengths and are used to produce a consensus high-density linkage map containing 656 markers.The Pacific oyster  Crassostrea gigas is one of the most widely cultivated marine species, having been introduced for this purpose from Asia to all continents but Antarctica  and maximum likelihood (ML) mapping methods, we identify three factors behind uncertainties in marker orders and inflated map distances: (i) markers segregating from only one parent, (ii) markers linked to chromosomal regions impacted by strong, early viability selection, and (iii) genotyping errors. In the end, a single consensus map, constructed from the most reliable set of individual framework maps, is achieved. SNP linkage information has an unexpected bearing on the current genome assembly, insofar as it implies that a large proportion of genome scaffolds may be incorrectly assembled.2 families, 2 \u00d7 10 (n = 90) and 51 \u00d7 35 (n = 108), each derived from an intercross of full-sib F1 hybrids, which, in turn, were produced by crosses of partially inbred lines . The adult libraries contributed 126,331 (90.7%) of the 139,335 EST sequences obtained from these libraries. Additional sequence was generated from a putative, nonredundant subset of 11,904 cDNA clones, which were pooled, their inserts fragmented,and sequenced by Illumina Genome Analyzer methods (SRA accession SRR2119182). Further sequence information came from two other larval cDNA libraries, CFPP and CFPS, which were made, respectively, from 6-d- and 18-d-old larvae of a reciprocal hybrid between inbred lines 35 and 51; these libraries were sequenced by 454 GS FLX Titanium methods (GenBank accessions SRX032364 and SRX032365). Sequences from all libraries were assembled, using miraEST (http://bio-bwa.sourceforge.net/). The three SAM output files were combined, using the pileup command in SAMtools (http://www.htslib.org/) to find 126,392 sites with potential variants (whether true SNPs or read errors). Candidate SNPs were sorted according to the amount of supporting sequence evidence and whether they were segregating within or between the two inbred lines; each alternative allele had to be supported by two or more sequences. Additionally, the unigene contigs were mapped on a draft version of the oyster genome, so that intron-exon boundaries could be identified. The list of high-quality SNPs was further refined to remove candidates located within 50 nucleotides of an intron-exon boundary, so that a 100-nt GoldenGate assay probe centered on the SNP site  were compared with the transcriptome assembly, using BLAST  and the best hit for each EST was aligned with the EST sequence using CLUSTAL. Alignments of cDNA sequences from the two inbred lines 51 and 35 were combined to generate a SAM file for all ESTs. Separately, reads from two additional Illumina RNA-Seq libraries (SRP061799) were compared with the assembled transcriptome, using BWA .From the list of potential probes, we chose the top 1536 SNPs for the final GoldenGate bead array . Markers missing data for more than 10% of individuals in a family were excluded. Initial groupings of markers were based on independence log of the odds (LOD) tests in steps from a LOD of 1.0  to a LOD of 10.0 . The independence LOD is less likely than the linkage LOD test to find spurious linkage, when segregation distortion, such as that observed for oysters  and three rounds of optimization per sample. Default parameters for map-order optimization were used except that chain length was increased from 10,000 to 20,000. Parameters for multipoint estimation of recombination frequency were generally increased  to ensure convergence.We attempted to resolve discrepancies in map orders and marker distances between RG and ML maps by focusing, first, on biparentally segregating framework markers and then on a set of well-spaced anchor loci. When suitable congruence between RG and ML maps for anchor loci was achieved, we applied the resulting order as a fixed order to the mapping of framework markers and all markers. Maps of framework markers were used to construct consensus maps and to compare pairwise recombination rates between male and female parents.s, the average spacing of markers, as the sum of the lengths of the 10 linkage maps within each family divided by the total number of mapped intervals . Genome length was then estimated as the sum of linkage group lengths plus 20s, to account for intervals beyond the most distal markers on both ends of the 10 linkage groups (following m + 1)/(m \u2212 1), where m is the number of markers per linkage group  and each SNP location was identified using a custom script. Loci were named  according to the genome scaffold to which they aligned (3 in the example), followed by the nucleotide position within that scaffold ; the position is preceded by the base in the reference genome (G) and followed by the substitution observed in that strand (A).The SNP-containing sequences were aligned with the reference genome using BWA-MEM , but the two F2 families have many more hk \u00d7 hk mating types, reflecting derivation from a cross of inbred grandparents . The two F2 families differ significantly from each other, however, with 51 \u00d7 35 having a much greater relative proportion of hk \u00d7 hk mating types than 2 \u00d7 10 ; this difference is consistent with the initial selection of SNPs fixed for different alleles in the 51 vs. 35 inbred parent lines. Microsatellite DNA markers account for all of the ab \u00d7 cd mating types and most of the ef \u00d7 eg mating types. Some SNPs fall into the ef \u00d7 eg category, owing to inference of a shared null allele . In keeping with their inbred history, the two F2 families differ noticeably from the three, random-bred G0 families, in having almost 100 fewer markers mapped (526 vs. 623) and many fewer ab \u00d7 cd mating types , tegories  reflectsG-tests of independent segregation. Summing across all five mapping families, a total 1085 coding SNPs are grouped, together with enough previously mapped microsatellite DNA markers, 66 in total, to establish consistency with previous linkage maps  are mapped in two or more families , lendingTable S6). Of the 618 scaffolds with mapped SNPs, 358 contain one SNP, and 260 contain two or more SNPs . The median length of the 100 scaffolds that map to two or more linkage groups, 740 kbp, is significantly longer than the median length of the 160 scaffolds that map to a single linkage group, 403 kbp (n = 118), whereas the average distance between adjacent SNPs that map to the same linkage group is 58.2 kbp . Numbers of ambiguous bases (coded as N in the published genome), between adjacent SNPs on a scaffold that either do or do not map to the same linkage group, are also significantly different  can have one breakpoint  or two breakpoints (ABA or BAB); of the 27 scaffolds in this category  and none has an order requiring two breakpoints (AABA) or three breakpoints (ABAB). Indeed, across the 60 informative combinations in i.e., cases in which the number of SNPs > number of LG assignments), only two cases require more than the minimum number of breakpoints.Finally, we consider the number of \u201cbreakpoints\u201d necessary to account for the order with which same-scaffold SNPs are mapped to linkage groups. For example, a scaffold with three SNPs mapping to two linkage groups , default thresholds for linkage had to be relaxed to permit all markers to be added. For 44 of 50 maps, linkage of all markers could only be achieved by forcing remaining markers onto a third-round RG map, sometimes resulting in large contributions to total \u03c72 or negative distances to existing mapped markers; on average, 10.6 makers were forced onto the 44 linkage groups, with a range from one to 42 markers. Maximum values for the nearest-neighbor fit statistic range from a low of 0.2 cM, for LG 2 in 2 \u00d7 10, to 621.3 cM, for LG 10 in the same family; indeed, 36 of 50 fit statistics exceed the estimated lengths of their linkage groups .Provisional linkage maps made by the RG method generally provide poor fits to observed recombination rates among markers (e groups . Maximumnp \u00d7 nn or ll \u00d7 lm in r2) of RG marker rank order on ML marker rank order. These r2 values range from 0.01 to 1.0, with 24 of 50 exceeding 0.75 but 8 falling below 0.1 . The mean proportion of distorted markers on a linkage group, 0.31, does not vary among families but does vary among linkage groups , ranging from a low of 0.034 on LG 1 to highs of 0.56 on LG 10 and 0.61 on LG 3. There is no significant relationship between proportions of markers distorted and either map lengths or maximum nearest-neighbor fit statistics across linkage groups; but certain distorted markers clearly do contribute to poor fit and inflated map lengths (examples given below).Distortion of Mendelian segregation ratios is widespread over these maps, as observed in previous studies .Having observed effects of markers segregating from only one parent on map orders and lengths, we proceed with linkage maps of framework markers segregating from both parents , to illustrate factors influencing map lengths and marker orders. In the first example, LG 1 for family 51 \u00d7 35, segregation ratios conform to Mendelian expectations, so selection is not a factor. However, markers segregating from only one parent cause moderately large nearest-neighbor fits  and, at the termini of both parental maps, expansions of map lengths from about 70 cM to more than 200 cM. Still, agreement in the rank order of 66 markers between the RG and ML maps is fairly good, with only a few outliers . Single-parent segregations also contribute to regions of poor fit and length expansion. Regression of RG on ML marker orders is quite poor for all 79 markers  and is 2.4\u00d7 as long as the comparable RG map .To build a consensus linkage map for the Pacific oyster, we used individual family ML framework maps, with the exceptions described in Crassostrea gigas, along with 66 microsatellite DNA markers to establish coherence with first-generation maps. On average, the map has a spacing of 1 cM and covers 86% of a genome that is estimated, here, to be \u223c616 cM in total length. This observed map length corresponds well with both a cytological estimate of map length in the eastern oyster Crassostrea virginica  applies to the oyster.More than 1100, coding SNPs are placed on a second-generation genetic linkage map for the Pacific oyster 2vs. wild-caught) and an ascertainment bias stemming from the initial identification of SNPs fixed for different alleles in the inbred parent lines of the 51 \u00d7 35 F2 family.In contrast to what et al. . For this reason, only framework maps are merged into consensus linkage maps.In the course of this study, three factors emerged as evident causes of discrepancies in map orders and lengths between RG and ML methods and among families. The first, mentioned above and illustrated by two examples . The average relative fitness of heterozygotes compared to the most abundant homozygotes at each marker across this ML map is 0.75, reaching a nadir of \u223c0.3, where one homozygote disappears entirely. Here, the gross asymmetry and partial dominance of viability selection is clearly associated with inflation of ML map distances and incongruence of RG and ML marker orders. Thus, the number, location, and phase of loci under viability selection produce idiosyncratic effects on map lengths and marker orders. Detailed analyses of the location and effects of viability mutations in the G0 families are reported elsewhere .Mode of selection appears to make a difference, because massively distorted but well-behaved LGs occur only in the Ffamilies . For exae.g., Distortion of Mendelian segregation ratios is commonly observed in mapping studies, especially in plants and marine molluscs, although the phenomenon may generally be underreported, because distorted markers often are discarded from data sets before linkage analysis. Distortion of segregation ratios can arise in interspecific or intersubspecific crosses, because of Dobzhansky-Muller incompatibilities e.g., . Viabilivs. zygotic), by the dominance of viability genes, and by the dominance of markers  populations .The consensus linkage map made with MergeMap ("}
+{"text": "Salmo salar). This paper describes the development and characterisation of a high density SNP linkage map based on SbfI RAD-Seq SNP markers from two Atlantic salmon reference families.Genetic linkage maps are useful tools for mapping quantitative trait loci (QTL) influencing variation in traits of interest in a population. Genotyping-by-sequencing approaches such as Restriction-site Associated DNA sequencing (RAD-Seq) now enable the rapid discovery and genotyping of genome-wide SNP markers suitable for the development of dense SNP linkage maps, including in non-model organisms such as Atlantic salmon (Approximately 6,000 SNPs were assigned to 29 linkage groups, utilising markers from known genomic locations as anchors. Linkage maps were then constructed for the four mapping parents separately. Overall map lengths were comparable between male and female parents, but the distribution of the SNPs showed sex-specific patterns with a greater degree of clustering of sire-segregating SNPs to single chromosome regions. The maps were integrated with the Atlantic salmon draft reference genome contigs, allowing the unique assignment of ~4,000 contigs to a linkage group. 112 genome contigs mapped to two or more linkage groups, highlighting regions of putative homeology within the salmon genome. A comparative genomics analysis with the stickleback reference genome identified putative genes closely linked to approximately half of the ordered SNPs and demonstrated blocks of orthology between the Atlantic salmon and stickleback genomes. A subset of 47 RAD-Seq SNPs were successfully validated using a high-throughput genotyping assay, with a correspondence of 97% between the two assays.This Atlantic salmon RAD-Seq linkage map is a resource for salmonid genomics research as genotyping-by-sequencing becomes increasingly common. This is aided by the integration of the SbfI RAD-Seq SNPs with existing reference maps and the draft reference genome, as well as the identification of putative genes proximal to the SNPs. Differences in the distribution of recombination events between the sexes is evident, and regions of homeology have been identified which are reflective of the recent salmonid whole genome duplication. The dramatic increase in the production of farmed fish in the past few decades has resulted in aquaculture species becoming of huge economic importance, promising a sustainable resource of high quality protein and long-chain fatty acids. Salmonids, in particular Atlantic salmon, are amongst the most important aquaculture species. In 2010, approximately 1.5 million tonnes of Atlantic salmon were produced from farms worldwide, corresponding to a value of just over $7.8 billion [Salmonidae originate from a common ancestor whose genome underwent a duplication event around 25 - 100 million years ago (MYA) . Extant http://www.ncbi.nlm.nih.gov/Traces/wgs/?val=AGKD01). However, the recent genome duplication and frequent long repeat regions are hampering the genome assembly [The genomic resources for Atlantic salmon are amongst the most extensive of all aquaculture species ,13, and assembly . The linassembly -11,22. Massembly -25). Theassembly ,10,22), assembly -11.While dense SNP genotyping platforms are still in development for salmonid species, next generation sequencing (NGS) technologies are making the SNP discovery and genotyping process much more feasible, efficient, and cost-effective ,27. SeveThe main aim of this study was to construct a high density SNP linkage map of the Atlantic salmon genome using SNP markers derived from a RAD-Seq analysis using the SbfI restriction enzyme. Additional aims, building on this linkage map, were to (i) investigate the differences in recombination rate and distribution between male and female maps; (ii) integrate the new RAD-Seq linkage map with existing linkage/physical maps and the draft Atlantic salmon reference genome; (iii) identify putative genes proximal to the SNPs in the linkage map using comparative genomics; and (iv) investigate the salmonid genome duplication by comparisons to rainbow trout and stickleback linkage groups.The Atlantic salmon samples used in this study were from the two SalMap reference families, for which dense sex-specific microsatellite linkage maps exist . FouFollowing the merging of reads into RAD loci across individuals, 75,688 distinct RAD loci were detected  which is indicative of 37,844 SbfI cleavage sites in the Atlantic salmon genome. This number is comparable to a previous study by our group  in familca. 1 million reads per individual  is required to ensure high levels of high confidence genotype coverage. A further 4 individuals were removed from further analysis due to a high Mendelian error rate (> 200 errors). Post-filtering, the number of SNPs retained for the construction of the linkage map was 8,257, and the total number of individuals remaining was 77 .A total of 28,415 putative SNPs were discovered, of which 11,103 were detected in the RAD loci and 17,312 were detected in the PE contigs. Stringent filtering criteria  . On average, a 97% correspondence between KASP and RAD-Seq genotypes was found . This allowed the assignment of 4,367 Atlantic salmon reference genome contigs (corresponding to 57\u00a0Mb of sequence) to at least one linkage group (Table\u00a0Flanking sequences for the mapped RAD-Seq SNPs (RAD loci and PE contigs) were aligned to the Atlantic salmon draft reference genome sequence contigs  gene sequences using tblastx. The stickleback was chosen because it is one of the most closely-related species to Atlantic salmon for which there is a near-complete annotated reference genome sequence. Significant sequence similarity  between mapped RAD contigs and stickleback genes was observed for approximately 17% of the mapped RAD contigs  was screened for sequence similarity to all known three-spined stickleback  between the Atlantic salmon and stickleback genomes, the linkage group positions of the genes associated with the mapped RAD contigs in the stickleback reference genome were recorded. For each of the salmon linkage groups, the stickleback linkage groups to which the mapped RAD contigs most frequently aligned to was identified . Regions of conserved synteny were identified for 26 of the 29 Atlantic salmon linkage groups. However, no clear pattern of orthology with a stickleback linkage group was observed for Atlantic salmon linkage groups 5, 19 and 22  . While we did not observe more than 25% of markers segregating as a single unit on linkage maps , an improved reliability of marker order may be obtained by analysing additional families with larger numbers of offspring to increase the number of informative meioses.The number of SNPs initially assigned to each linkage group using the CRI-MAP software and anchor marker information in this study was compared to the SNP linkage map constructed by Lien et al  and a stThe length of genomic DNA sequenced at each RAD locus, including the RAD locus itself and the PE contig, is approximately 500\u00a0bp. Therefore, multiple SNPs are frequently observed at a single locus and recombination between these SNPs is unlikely. As such, these SNPs are expected to map to the same position on the linkage map. To test this, we analysed the positions of SNPs from RAD loci and PE contigs which originate from the same restriction cut site within the genome. We identified 26 restriction cut sites with mapped SNPs from both the RAD locus and PE contig. In ~60% of cases, SNPs from the RAD locus and associated PE contig mapped to the same cM position on the map. Where this did not occur, PE SNPs were found to be positioned at the terminal ends of linkage groups. Given the lower read coverage for the PE contig due to the nature of the RAD-Seq protocol, SNPs derived from the PE contig may have a higher genotyping error rate than those from the RAD locus. A common feature of linkage mapping software is the positioning of markers with higher error rates at the ends of linkage groups, which could explain the instances where RAD loci and PE contig derived SNPs did not co-localise on linkage maps.The large difference in recombination rate between Atlantic salmon males and females, and the distribution of the recombination events along the chromosome, have been a subject of much discussion in the literature -11,22. Wca. 50%. There is a GC bias in the SbfI recognition sequence (5\u2032 CCTGCA/GG 3\u2032 and 3\u2032 GG/ACGTCC 5\u2032) which may result in a higher-than-expected frequency of SbfI cut sites within gene-rich regions of the genome. This bias in SbfI cut sites in potentially gene-rich regions of the genome has been observed in other SbfI RAD-Seq studies [Approximately 17% of the SNPs had flanking sequence data which gave a significant alignment with an annotated gene in the stickleback genome. Including the additional step of aligning the mapped SNP flanking sequences to the salmon reference genome contigs increased the proportion of mapped SNPs associated with a putative gene to  studies . In totaviz. 29, equal to the number of linkage groups). Most salmon linkage groups were assigned to at least one stickleback group , with three salmon linkage groups  remaining unassigned, possibly due to the lower number of gene-associated markers on these linkage groups. Twelve stickleback linkage groups aligned to more than one Atlantic salmon linkage group, with one (stickleback linkage group 20) aligning to three Atlantic salmon linkage groups , which is fewer than Atlantic salmon  the mapped RAD-Seq SNPs. Homeologous regions within the Atlantic salmon genome and the putative orthologues of the salmon linkage groups in the stickleback and rainbow trout genome were identified and confirmed, providing support for a salmonid specific genome duplication. RAD-Seq is an increasingly popular tool for QTL mapping and population genomics, and this new map will provide a useful framework for future genomics studies.https://genomics.ed.ac.uk/). Raw sequences are available from the ENA (http://www.ebi.ac.uk/ena/), accession number PRJEB4502.The two SalMap families Br5 and Br6)  used in  and Br6 http://picard.sourceforge.net/) and excluded. Overall, 482,547 consensus RAD contigs were generated, of which 366,219 were from the RAD loci and 116,328 were from the PE contig. 2% of RAD loci had more than one PE contig associated with it. SNPs were called using samtools v0.1.18 [The process for generating SNP genotype data for the individuals in the RAD-Seq experiment was as follows. Firstly, raw reads were \u2018demultiplexed\u2019 and assigned to individual samples according to their nucleotide barcode using RADpools v1.2.1 , resultihttp://www.rqtl.org/), (ii) putative PSVs (inferred by excess heterozygosity)  and (iii) apparent Mendelian errors  was carried out. Given parental genotypes, a Mendelian error was defined as a highly improbable offspring genotype at a given SNP. Removing SNPs with genotypes in fewer than half of the individuals left 13,637 SNPs. Removing individuals with poor genotyping coverage (genotyped at fewer than 25% of the SNPs) resulted in the removal of 15 individuals  was calculated according to the Haldane mapping function. Marker genotypes containing errors can appear as recombination events and result in erroneous positioning of SNPs some distance removed from other markers at the ends of linkage groups. Therefore, maps for each parent and each linkage group were investigated manually, and SNPs with a gap of greater than 30\u00a0cM in male maps and 20\u00a0cM in female maps from the neighbouring SNP at the ends of the linkage groups were removed. Maps were drawn using the MapChart software  . Therefore, the male map was split into 18 intervals of 5\u00a0cM. The female map was then split into 18 marker intervals of equal size 18 x ~13\u00a0cM) and the percentage of SNPs mapping to each interval for both sexes was calculated. For each linkage group and each sex across both families, the five intervals with the highest percentage of markers were identified, and the averages of these percentages are given in Figure\u00a0\u00a0cM and thttp://web.uvic.ca/grasp/). Masked sequences were then aligned with the Atlantic salmon reference genome  using Blastn with an E-value threshold of 1e-30 for RAD loci and 1e-80 for PE contigs. RAD loci or PE contigs aligning to multiple (> 2) reference genome contigs were excluded.In order to assign Atlantic salmon reference genome contigs to linkage groups, the mapped RAD contigs were first repeat-masked using the RepeatMasker software using the \u201cSalmon Raw Repeat DB v1.6\u201d database ). The identification of SNPs within or close to putative genes was performed in two stages. In the first stage, the mapped RAD contigs were screened for sequence similarity to stickleback (Gasterosterus aculeatus) genes using tblastx . tblastx was chosen as it is more sensitive to protein homologies between distantly related species using sequence data . Only the two most significant alignments were retained in an attempt to avoid spurious alignment to multiple genes from different stickleback linkage groups (for example due to similarity of genes from the same gene family).All known three-spined stickleback gene nucleotide sequences were downloaded from Ensembl BioMart , sinAs described above, for each linkage group and each individual separately, RAD loci and PE contigs associated with mapped SNPs were aligned against the stickleback gene sequences. RAD loci and PE contigs were then grouped into a single RAD locus in order to be counted only once  and the total number of RAD loci showing significant alignment to a gene on a particular stickleback linkage group was counted. For each Atlantic salmon linkage group, a single synteny relationship was assigned only if the number of significant alignments for a particular stickleback linkage group was twice (or more) than the number of significant alignments for any other stickleback linkage group, and this relationship was seen in all mapping parents. The only exceptions to this was in cases where two stickleback linkage groups showed identical numbers of RAD loci alignments, then both were assigned to that Atlantic salmon linkage group.http://www.lgcgenomics.com/genotyping/kasp-genotyping-reagents/), a subset of RAD-Seq SNPs with flanking sequence  repository, [PRJEB4502, The authors declare that they have no competing interests.Conceived and designed the experiments: RDH, SCB. Prepared RAD libraries: NL. Managed sequencing of RAD libraries: KG. Analysed data: SG, TC. Wrote the paper: SG, RDH. All authors read and approved the final manuscript.This file contains Supplementary Tables A1-A4. Table A1 \u2013 Library structure and read depth for the paired-end RAD-sequencing libraries. Table A2 \u2013 Markers used as anchors in CRI-MAP for assignment of RAD-derived SNPs to linkage groups, and their corresponding Atlantic salmon linkage groups/chromosomes. Table A3 \u2013 Map length (cM) for each mapping parent and the comparison between the sexes. Table A4 \u2013 Homeologous Atlantic salmon linkage groups with the stickleback and rainbow trout linkage groups and proto-Acinopterygian linkage groups which they have in common.Click here for filePutative PSVs: Variants removed from analysis due to excess heterozygosity. Contains the following information for each PSV identified: ID of the SNP identified as a PSV (column 1); RAD consensus ID (column 2) and RAD consensus sequence (column 6) from which the SNP originates; position of the SNP identified as a PSV in the RAD consensus sequence (bps) (column 3); SNP alleles (columns 4 and 5).Click here for fileSNPs used for RAD-Seq validation. Contains ID and sequence information for the 47 SNPs validated by KASP technology. For each SNP, the percentage concordance between the genotypes obtained from RAD-Seq and those obtained from the KASP technology for the two SalMap families is given in column 2.Click here for fileLinkage maps. Annotated with alignment information to stickleback genes and Atlantic salmon genome contigs. Map distances are given in centiMorgans (cM). Each parent\u2019s linkage map is given in separate sheets of the file.Click here for file4,367 Atlantic salmon reference genome contigs and the Atlantic salmon linkage groups to which they map.Click here for file112 reference genome contigs which mapped to more than one linkage group and the Atlantic salmon linkage groups to which they align.Click here for file"}
+{"text": "Brassica napus L.). However, the genetic basis of both traits is poorly understood. The main objectives of this study were to dissect the genetic basis of SW and SL in rapeseed through the preliminary mapping of quantitative trait locus (QTL) by linkage analysis and fine mapping of the target major QTL by regional association analysis.Seed weight (SW) and silique length (SL) are important determinants of the yield potential in rapeseed  and uq.A09-3 , were detected in all four environments and showed the opposite additive-effect direction. These QTLs were validated and fine mapped  by regional association analysis with a panel of 576 inbred lines, which has a relatively rapid linkage disequilibrium decay (0.3\u00a0Mb) in the target QTL region.Preliminary linkage mapping identified thirteen and nine consensus QTLs for SW and SL, respectively. These QTLs explained 0.7-67.1% and 2.1-54.4% of the phenotypic variance for SW and SL, respectively. Of these QTLs, three pairs of SW and SL QTLs were co-localized and integrated into three unique QTLs. In addition, the significance level and genetic effect of the three co-localized QTLs for both SW and SL showed great variation before and after the conditional analysis. Moreover, the allelic effects of the three QTLs for SW were highly consistent with those for SL. Two of the three co-localized QTLs, A few QTLs with major effects and several QTLs with moderate effects might contribute to the natural variation of SW and SL in rapeseed. The meta-, conditional and allelic effect analyses suggested that pleiotropy, rather than tight linkage, was the genetic basis of the three pairs of co-localized of SW and SL QTLs. Regional association analysis was an effective and highly efficient strategy for the direct fine mapping of target major QTL identified by preliminary linkage mapping. Linkage and association analyses are two complementary strategies for the genetic dissection of complex quantitative traits. Compared with each other, linkage mapping has relatively high power and a low false positive rate, whereas association mapping has relatively high resolution ,2. LinkaBoth the seed weight (SW) and silique length (SL) are important determinants of yield potential in rapeseed and are good targets for selection in breeding ,17 due tIn particular, neither of the QTLs for SW and SL has been fine mapped. Following preliminary linkage mapping, the classical/traditional fine mapping strategy is based on the recombinant individuals screened from a large-scale NIL (near isogenic lines)-segregating population, which requires several rounds of successive backcrossing and self-crossing (cost of at least two years) and the genotyping of thousands of individuals ,31. ThusIn the current study, we used regional association mapping to validate and fine map two major SW and SL QTLs on the A09 linkage group of rapeseed that were identified by the preliminary linkage mapping. In detail, the main objectives of this study were as follows: (1) preliminary mapping of the QTLs for SW and SL using linkage analysis; (2) validation and fine mapping of the target major QTLs using regional association analysis; and (3) determination of the genetic basis of the co-localization of SW and SL QTLs using meta-, conditional and allelic effect analyses.m (main raceme thousand seed weight) was higher than SWb (raceme branch thousand seed weight) by approximately 10% for both the parents and all of the populations in all environments, which was in agreement with a previous report [The two parents, Zhongshuang11 and No. 73290, differed significantly for SL but not SW in all the investigated environments  and SL, respectively), which was generally consistent with previous studies [The analysis of variance indicated that the genotypic, environmental and genotype\u2009\u00d7\u2009environment effects were all extremely significant for both SW and SL  showed distorted segregation. The biased loci were distributed unevenly: most of them were located on A01, A04, A06, A08, A09, C04 and C08 linkage groups, and the loci biased to the same parent tended to cluster together, which is a common phenomenon in B. napus[A framework of the genetic linkage map containing 529 loci . The meta-analysis integrated 48 overlapping identified QTLs into 10 repeatable consensus QTLs on the A01, A03, A04, A07, A08, A09 and C02 linkage groups , cqSW.A08 and cqSW.A09-3 were detected in three environments , and only one consensus QTL, cqSW.A09-1, was consistently detected in all four environments (mean R2\u2009=\u200920.1%).A total of 51 SW identified QTLs (25 significant QTLs and 26 overlapping suggestive QTLs) were detected . The meta-analysis integrated 12 overlapping identified QTLs into three repeatable consensus QTLs on the A09 and C02 linkage groups (Table\u00a0cqSL.A09-2 was detected in two environments (mean R2\u2009=\u200913.2%), cqSL.C01 was detected in three environments (mean R2\u2009=\u20094.9%), and only cqSL.A09-1 was detected in all four environments (mean R2\u2009=\u200919.0%).A total of 18 SL identified QTLs (14 significant QTLs and four overlapping suggestive QTLs) were detected  and uq.A09-3 (flanking 13.2\u00a0cM region) were located on the A09 linkage group, with opposite additive-effect directions for both SW and SL.The consensus QTLs for SW and SL were subjected to meta-analysis again, which resulted in 19 unique QTLs Table\u00a0. Of thesm) was conditioned by SL (SWm|SL), none of the three loci  remained significant for SW in all experiments; when SL was conditioned by SW (SL|SWm), these loci were not significant for SL in half of the experiments. These results strongly suggested that pleiotropy, rather than tight linkage was likely to be the genetic cause of the three unique QTLs for both SW and SL, and that SW was possibly contributed by SL for these loci.To determine the genetic basis of three unique QTLs for both SW and SL (pleiotropy or tight linkage), conditional QTL analysis was performed Table\u00a0. When SWuq.A09-1 and uq.A09-3) were identified by the alignment between the primer sequences of tightly linked SSR markers (BrSF6-2562 and BrSF0358) and the genomic sequences of B. napus (unpublished data) and B. rapa[B. rapa and B. napus[The corresponding genomic regions of two major unique QTLs . The extent of the LD decay was evaluated using linked markers (markers from the same chromosomes). The LD decay decreased within 1.40\u00a0Mb over the whole genome and within 1.19\u00a0Mb on the A09 linkage group. In particular, the extent of the LD decay for the target QTL region  and SL  and was very near to BrSF6-2562, the nearest marker for uq.A09-1. Within the second peak, the marker BrSF6-1572 showed the strongest association signal for both SW  and SL  and was near to BrSF0358, the nearest marker for uq.A09-3.Considering the population structure  to BrSF6-2562 (at 31.19\u00a0Mb), indicating a resolution of approximately 1\u00a0cM (0.51\u00a0Mb). Another LD block around the marker BrSF6-1572 extended roughly from BrSF6-1390 (at 29.02\u00a0Mb) to BrSF0358 (at 30.28\u00a0Mb), indicating a resolution of approximately 2.5\u00a0cM (1.26\u00a0Mb).To determine the resolution of this association study, the extent of the LD around the best associated SSR markers (BrGMS0025 and BrSF6-1572) was investigated. As expected, this region was divided into two LD blocks . Eight aR2 of the association markers showed great variation before and after the conditional analysis using both methods (Table\u00a0To determine the genetic basis (pleiotropy or tight linkage) of the common association markers for SW and SL, conditional analysis was performed using two methods. The first method used the conditional phenotypic values, while the second method used one trait as a covariate for the other, to perform the association analysis. The results showed that the p value and ds Table\u00a0. Taking uq.A09-1, uq.A09-3 and uq.C02-1), their allelic effects for SW were highly accordant with those for SL in both the linkage and association populations. For example, the corresponding phenotypic values of the three major haplotypes  of the marker BrSF6-1572 (nearest to uq.A09-3) for SW and SL were 4.42\u00a0g and 65.39\u00a0mm, 4.11\u00a0g and 62.51\u00a0mm, and 3.97\u00a0g and 60.41\u00a0mm, respectively. This finding increased the likelihood that pleiotropy rather than tight linkage was the underlying genetic basis for the three pairs of co-localized SW and SL QTLs.The allelic effects of the co-localized SW and SL QTLs in the linkage and association populations were estimated using the phenotypic values of the different genotypes for the nearest marker . Our results suggested that this strategy is effective for direct fine mapping after preliminary linkage analysis. Compared with the traditional/classical NIL-based fine mapping approach , this stm), branch raceme (SWb) [w) [m, SWb and SWw were all measured for both the genetic and QTL analyses. Strikingly, SWm showed an extremely high correlation with both SWb (mean r\u2009=\u20090.93) and SWw (mean r\u2009=\u20090.96), and most of the QTLs identified for SWm, SWb and SWw were consistent. However, SWm is more easily measured than SWb and SWw. We therefore suggested the measurement of SWm rather than SWb and SWw in futher studies.In previous genetic and QTL mapping studies, seed weight was usually measured separately from the main raceme (SWme (SWb)  and wholSWb) [w) ,27,29. IcqSW.A07 and cqSW.A09-3) and three , respectively, have also been confirmed by the previous studies. The currently identified consensus QTLs, cqSW.A07, cqSL.C01 and cqSL.C02-3, likely corresponded with TSWA7a, sl11 and qSL.N12, respectively, which were detected in one of the previous studies and are located around the common markers BRMS036 [cqSW.A09-3 and cqSL.A09-2, were very close (<1\u00a0Mb) to the cqSWA9 and cqSLA9 QTLs, respectively, which were identified in a previous study [In the previous linkage QTL mapping studies, approximately 120 and 30 QTLs have been identified for SW ,27-29,37 BRMS036  and CB10us study . In addius study ,28,37, Mus study ,27,29, CB. napus (AACC) was derived from chromosome doubling after the recent (~0.01 million years ago) natural hybridization between its two diploid ancestors, B. rapa (AA) and Brassica oleracea (CC) [B. rapa and B. napus showed co-linearity [B. napus linkage map and the B. rapa physical map of the QTL interval of uq.A09-1 and uq.A09-3  in B. rapa.The allotetraploid cea (CC) . The preinearity ,39, someinearity , insertiinearity ,41. In t3 Figure\u00a0, which wB. napus association population was 1.4\u00a0Mb, which corresponds with approximately 2.8\u00a0cM [The estimated genome-wide LD decay of the current y 2.8\u00a0cM ,43 and wy 2.8\u00a0cM . The esty 2.8\u00a0cM . However6 lines with extremely large (SW\u2009>\u20096.0\u00a0g) and small (SW\u2009<\u20093.0\u00a0g) seeds were in high accordance with the silique length of the corresponding lines . Thus, the variations in SW might be primarily affected by those in the SL in the current linkage population, which is in accordance to the abovementioned conditional analysis for the three co-localized QTLs for both SW and SL. This finding is understandable because long siliques enable an increased photosynthesis area and assimilation, thereby providing the basis for the increase in the SW, and implicating maternal control of the underlying gene (s) [From the linkage and association analyses, a total of three co-localized SW and SL QTLs were identified, with the same additive-effect direction, which agreed with the significantly and moderately positive correlations in both populations. In fact, the co-localization of the SW and SL QTLs was also commonly observed in other previous studies ,21,27,29gene (s) -49. TherIn the present study, we proposed a regional association mapping strategy to directly fine map the target QTLs identified in preliminary linkage mapping. Compared with the traditional/classical NIL-based fine mapping strategies, our approach has many advantages, for example, it is time-saving, labor-saving and cost-effective. Using this strategy, the confidence intervals of the two major QTLs for both SW and SL on the A09 linkage group were successfully narrowed to a large extent, demonstrating the effectiveness of our strategy. Interestingly, the meta-, conditional and allelic effect analyses all suggest that pleiotropy, rather than tight linkage, was the genetic basis of the three unique QTLs for both SW and SL. Furthermore, the variations in SL are more likely to be the cause of the variation in SW, not vice versa. These results provide a solid basis for candidate gene screening and further gene cloning. In addition, several SW and/or SL QTLs identified by the current linkage mapping appeared to be \u201crepeatable\u201d in previous studies and could be the potential targets for marker-assisted selection in rapeseed breeding.2, F2:3 and F2:4 individuals/lines that were derived from two sequenced rapeseed cultivars, Zhongshuang11  and No. 73290 . The association population consisted of a panel of 576 rapeseed inbred lines  from Oct. 2008 to May 2009 (code W09F2). The F2:3 lines were planted in Wuhan from Oct. 2009 to May 2010 (code W10F2:3) and Oct. 2010 to May 2011 (code W11F2:3) and in Xining  from April to Aug. 2011 (code X11F2:3). The F2:4 lines were planted in Xining from April to Aug. 2011 (code X11F2:4). The association population was planted in Wuhan from Oct. 2011 to May 2012 (code W12AP).Location-year combinations were treated as environments, and environment-population combinations were treated as experiments. The experiments were performed in two contrasting environments (semi-winter and spring rapeseed area). Details of the climate conditions during the growing season are described in Additional file 2 individuals). Each block contained two rows with 15 plants per row with spacing of 33.3\u2009\u00d7\u200916.7\u00a0cm. The seeds were sown by hand, and the field management followed standard agriculture practice. In each block, 10 representative individuals from the middle of each row were harvested by hand at maturity.Both the linkage (including both parents) and association populations were arranged in a randomized complete block design with three replications (except Fm), raceme branch thousand seed weight (SWb) and whole-plant thousand seed weight (SWw) were each evaluated. For the F2 individuals, only SWm was measured. For the F2:3 and F2:4 lines, SWm, SWb and SWw were measured. The SL was measured based on 10 well-developed siliques (not including the beak) from the main raceme. For the association population, SWm and SL were measured using the same method described above.For the linkage populations, the seeds from the main raceme and branch raceme were threshed separately. The SW was measured based on 1000 fully developed seeds. The main raceme thousand seed weight (SWh2\u2009=\u2009\u03c32g / (\u03c32g\u2009+\u2009\u03c32ge / n\u2009+\u2009\u03c32e / nr), where \u03c32g was the genetic variance, \u03c32ge was the interaction variance of the genotype with environment, \u03c32e was the error variance, n was the number of environments and r was the number of replications. The estimation of \u03c32g, \u03c32ge and \u03c32e were obtained from the SAS ANOVA procedure. Pearson\u2019s correlation coefficients were calculated using the SAS CORR procedure based on environment means.The broad-sense heritability was calculated as 2 individuals. Three groups of markers from different sources were used for polymorphism screening between the two parents. The first group, mainly consisted of SSR and STS (sequence tagged site) markers, were selected from database of publish molecular markers in Brassica (http://www.brassica.info/resource/markers/ssr-exchange.php) and published papers [Brassica crops  by our lab [Genomic DNA was extracted from leaf tissues of the two parents (Zhongshuang11 and No. 73290) and its derived 184\u00a0Fd papers ,35,50-60 our lab . The thi our lab .http://www.kyazma.nl/index.php/mc.JoinMap) with the threshold for goodness-of-fit of \u2266 5, a recombination frequency of\u2009<\u20090.4 and minimum logarithm of odds (LOD) score of 2.0. All genetic distances were expressed in centimorgans (cM) as derived by the Kosambi function [The genetic linkage map was constructed using the JoinMap 4.0 software (function . The seghttp://statgen.ncsu.edu/qtlcart/WQTLCart.htm). A forward-backward stepwise regression following model 6 was performed to choose the co-factors (chosen with Pin\u2009=\u20090.05 and Pout\u2009=\u20090.05) before the QTL detection. The control marker numbers, window size and walking speed were set to 5, 10\u00a0cM and 1\u00a0cM, respectively. A default genetic distance of 5\u00a0cM was used to define a QTL in a specific experiment. The experience-wise LOD threshold was determined by a permutation test of 1000 repetitions [The linkage mapping of QTLs was performed separately for SW and SL using the composite interval mapping program  of the Wetitions . LOD scoetitions ,65 and nMeta-analysis was used to estimate the number and positions of the meta-QTLs underlying the QTLs of the same or related traits, which were repeatedly detected in different environments and located on the same chromosomal region . The comEach identified QTL was designated with the initial letter \u201cq\u201d, followed by the name of the trait abbreviation (SW/SL) and linkage group. Each consensus QTL was designated with the initial letters \u201ccq\u201d, followed by the name of the trait abbreviation and linkage group. Each unique QTL was designated with the initial letters \u201cuq\u201d, followed by the name of the linkage group. Arabic numerals were added if more than one QTL was located on the same linkage group.(T1/T2) were obtained by the mixed model approach for the conditional analysis of quantitative traits [http://ibi.zju.edu.cn/software/qga/index.htm), where T1|T2 indicates that trait 1 is conditioned by trait 2. Then, the conditional mapping of the QTLs was conducted according to the conditional phenotypic values using the same method as the unconditional QTLs mentioned above.To dissect the genetic basis (pleiotropy or tight linkage) of the co-localization of the SW and SL QTLs, conditional analysis was performed. The conditional phenotypic values y e traits  using QGR2 value of LD and the corresponding significance level  were calculated using the TASSEL 3.0 standalone software (http://www.maizegenetics.net/index.php?option=com_content&task=view&id=89&Itemid=119) with a permutation test of 1000 repetitions. Rare alleles with an allele frequency of\u2009<\u20090.05 were treated as missing data [R2 value among unlinked loci pairs [R2 values among the linked SSR pairs vs. the physical distance (Mb) between those markers were generated. The LD decay was calculated as previously described [The ing data . The locci pairs ,70. Lociescribed .Both the Q matrix and K matrix were calculated using allelic data from 93 SSR markers  method  incorporSW: Seed weight; SWm: Main raceme thousand seed weight; SWb: Raceme branch thousand seed weight; SWw: Whole-plant thousand seed weight; SL: silique length; QTL: Quantitative trait locus; NIL: Near isogenic lines; MAGIC: Multi-parent advanced generation inter-crosses; NAM: Nested association mapping; LD: Linkage disequilibrium; GWA: Genome-wide association; SSR: Single sequence repeat; STS: Sequence tagged site; SNP: Single nucleotide polymorphism; InDel: Insertion /Deletion; cM: Centimorgans; MLM: Mixed linear model.The authors declare no competing financial interests.Conceived and designed the experiments: JQS, HZW; performed the experiments: NL, JQS; analyzed the data: NL, JQS; contributed reagents/materials/analysis tools: HZW, GHL, XFW; wrote the manuscript: NL JQS. All authors read and approved the final manuscript.Table S2. ANOVA and broad-sense heritability (h2) of seed weight and silique length. Table S3. Correlation coefficients of seed weight in different tissues in the same experiment. Table S4. The constructed genetic linkage map for the F2 population. Table S5. Identified QTLs for seed weight and silique length. Table S6. Molecular markers (within the target QTLs regions) used for association mapping. Table S7. Markers used for population structure analysis. Table S8. Genotype of the 17 SSR loci in association population. Table S9. Family relatedness for the association population. Table S10. Effect estimates of the three co-localized SW and SL QTLs in the linkage population.Descriptive statistics of seed weight (g) and silique length (mm) in the linkage and association populations. Click here for fileDetails of the climate conditions, including monthly mean temperature, monthly maximum temperature, monthly minimum temperature, monthly sunshine and monthly rainfall during the growing season.Click here for file"}
+{"text": "The ability to identify regions of the genome inherited with a dominant trait in one or more families has become increasingly valuable with the wide availability of high throughput sequencing technology. While a number of methods exist for mapping of homozygous variants segregating with recessive traits in consanguineous families, dominant conditions are conventionally analysed by linkage analysis, which requires computationally demanding haplotype reconstruction from marker genotypes and, even using advanced parallel approximation implementations, can take substantial time, particularly for large pedigrees. In addition, linkage analysis lacks sensitivity in the presence of phenocopies . Combinatorial Conflicting Homozygosity (CCH) analysis uses high density biallelic single nucleotide polymorphism (SNP) marker genotypes to identify genetic loci within which consecutive markers are not homozygous for different alleles. This allows inference of identical by descent (IBD) inheritance of a haplotype among a set or subsets of related or unrelated individuals.A single genome-wide conflicting homozygosity analysis takes <3\u00a0seconds and parallelisation permits multiple combinations of subsets of individuals to be analysed quickly. Analysis of unrelated individuals demonstrated that in the absence of IBD inheritance, runs of no CH exceeding 4\u00a0cM are not observed. At this threshold, CCH is >97% sensitive and specific for IBD regions within a pedigree exceeding this length and was able to identify the locus responsible for a dominantly inherited kidney disease in a Turkish Cypriot family in which six out 17 affected individuals were phenocopies. It also revealed shared ancestry at the disease-linked locus among affected individuals from two different Cypriot populations.http://sourceforge.net/projects/cchsnp/.CCH does not require computationally demanding haplotype reconstruction and can detect regions of shared inheritance of a haplotype among subsets of related or unrelated individuals directly from SNP genotype data. In contrast to parametric linkage allowing for phenocopies, CCH directly provides the exact number and identity of individuals sharing each locus. CCH can also identify regions of shared ancestry among ostensibly unrelated individuals who share a trait. CCH is implemented in Python and is freely available (as source code) from The online version of this article (doi:10.1186/s12864-015-1360-4) contains supplementary material, which is available to authorized users. Genetic analysis of multiply affected pedigrees has seen a resurgence of interest in the post-GWAS era ,2. Typica priori this rate is unknown and analyses are sensitive to model misspecification [A second problem in the analysis of extended pedigrees is that if some individuals are phenocopies  this causes erroneous exclusion of the causative locus, since phenocopies lack the true disease-linked haplotype. Although parametric linkage analyses can be performed under the hypothesis of a specified phenocopy rate, fication  so analyfication . Even utfication . Furthernot the result of IBD inheritance becomes vanishingly small. This exclusion approach contrasts with homozygosity mapping  in which identical homozygous genotypes define the subset of the genome included in analysis. We demonstrate how CCH can be used to identify regions of the genome where a single haplotype is shared by some, but not all, members of a family and that this information can be used to identify individuals harboring a heterozygous disease-linked variant in a family with multiple phenocopies, and compare CCH with traditional parametric linkage analysis. Unlike pedigree-based linkage methods used to analyse dominant traits, this approach is equally applicable to related and unrelated individuals or combinations thereof.We have developed a tool called Combinatorial Conflicting Homozygosity (CCH) that addresses these challenges. Rather than calculating the likely flow of haplotypes through a family, CCH uses dense SNP genotypes to identify individual markers that cannot have been inherited identical-by-descent (IBD) among a set of individuals from a recent common ancestor. This allows identification of regions of the genome that contain consecutive markers in which IBD inheritance is not excluded. We show that above a threshold size (of 4\u00a0cM in the example here) the probability that such regions are The underlying algorithm of CCH is based on the simple, previously described principle that inheritance of at least one shared haplotype among two or more individuals is excluded by the occurrence of SNPs homozygous for different alleles, i.e. identical-by-state for zero alleles (IBS0) ,11  of approximately 300,000 SNPs taken from the HapMap Project . Groups -6). SNPs demonstrating Mendelian errors were removed. A cohort of 17 unrelated population-matched controls genotyped on the same array were used to prune for LD at r2\u2009<\u20090.2. From these, the SNP with the highest heterozygosity within the family and lowest frequency in the controls within 0.5\u00a0cM windows were selected. This yielded 5,344 informative SNPs for linkage. Affected-only parametric linkage analyses was undertaken with SwiftLink [All research involving human participants was performed with written informed consent and was approved by the ethics committee of Lefkosa Burhan Nalbanto\u011flu State Hospital. All participants provided informed consent for their involvement in the research in accordance with the Declaration of Helsinki and for the publication of the study results. Individuals were genotyped on the HumanCytoSNP 12v2 array  according to the manufacturers\u2019 instructions. Genotype data have been deposited in the NCBI Gene Expression Omnibus (GEO)  and are wiftLink . As SwifSNPs with low minor allele frequency (MAF) are likely to be identical by state purely by chance, so we sought to determine whether CCH might indicate apparent regions of IBD inheritance because of low marker diversity. However, since it only takes a single instance of CH to break up a haplotype, we hypothesised that CCH analysis would be relatively robust to this effect: very large numbers of consecutive markers showing no CH would be required to mimic the very large  haplotypes inherited within a family. Similarly, local linkage disequilibrium (LD) between nearby markers mean that blocks of alleles tend to be inherited together even in the absence of IBD inheritance from a recent common ancestor. In the Caucasian population, fewer than 10% of SNPs separated by 160 kbp  show significant evidence of LD, and where the recombination rate (i.e. the number of cM per megabase-pair) is greater, the span of LD tends to be reduced . We ther6 of these genome-wide CH analyses, when subsets comprising 4 or more unrelated individuals (whatever the group size) were included, no runs comprising >100 SNPs and extending >4\u00a0cM in length were observed. Since CH cannot occur among a set of individuals at a locus where a haplotype is shared  much longer runs of no CH are expected to occur in regions of the genome where there is IBD inheritance of at least one haplotype from a recent common ancestor. The length of runs arising from IBD inheritance is dependent on the size of the shared haplotype, which decreases as a function of the number of meioses separating the individuals, starting at an expected length of ~34\u00a0cM  for a pair of siblings. When related individuals  of no CH observed in these analyses were distributed exponentially Figure\u00a0 about a The statistical test we use to infer IBD inheritance at a given locus is the likelihood that the observed length of no CH at that locus occurred under the null hypothesis .-7 and >7\u00a0cM was <10-12. For combinatorial CH analyses, these likelihoods should be corrected for the multiple independent analyses performed . Since, in a group of individuals, any IBD locus could harbor the allele responsible for a shared inherited trait, the size of the locus  does not indicate the likelihood of the allele of interest being located there. Rather, it represents the likelihood that the locus is inherited IBD by all those individuals. This is equivalent in linkage terms to identifying loci which cosegregate perfectly with a trait and therefore exhibit the maximum observed LOD score within a pedigree, with the magnitude of the maximum LOD score depending on the certainty with which haplotypes can be inferred and the family structure. In genome-wide analyses, these IBD loci  represent the subset of the genome within which a co-segregating allele may lie. When analysing k out n individuals, the number of independent analyses, nCk, is equal to n!/(k!(n-k)!). This number rises exponentially as n and n-k increase, exceeding 108 when n\u2009>\u2009~30 and (n-k)\u2009>\u2009~10. This places a limit on the size of groups amenable to CCH analysis due both to the computing time required for such a large number of analyses, and also the reduction in statistical power due to multiple independent tests being performed. Nonetheless, CCH analysis remains practical even among large groups, where\u2009<\u2009~8 phenocopies are hypothesized.Using the observed underlying exponential distribution of no CH run lengths Figure\u00a0 to compuTo assess the sensitivity and specificity of CCH to accurately determine the maximum number of individuals IBD at a locus within a pedigree, Monte Carlo genome-wide gene-dropping simulations using the pedigree structure shown in Figure\u00a0When searching for a disease-causing variant within a family, we make the assumption that the variant lies within a shared haplotype that is larger than the detection threshold \u2013 double recombination events immediately flanking the variant responsible will therefore prevent its detection by CCH. However, conventional linkage analysis is similarly susceptible to this possibility: linkage is performed either with widely spaced polymorphic markers  or else with a SNP chip using a thinned-out set of perhaps 5\u201310,000 informative biallelic markers (mean spacing ~0.5 cM) where multiple consecutive markers are needed to reconstruct a haplotype with enough certainty to yield a high-magnitude LOD score. This means that double-recombinations flanking small (i.e. sub-cM) regions will be invisible to haplotype reconstruction algorithms and are therefore missed regardless of the approach used. Because recombinations occur with a median spacing of ~75 cM per meiosis, when dealing with a small number (i.e. <20) meioses in a single family, the probability of recombinations occurring so close together at the disease-causing locus is small. Furthermore, because CCH is robust to the existence of phenocopies, a small number of individuals in whom such a double recombination event has occurred will not prevent detection of the disease-linked haplotype in the rest of the family when CCH is employed. In contrast, the whole locus will be excluded if parametric linkage analysis is performed with a non-negligible phenocopy rate.We investigated a Turkish Cypriot family with apparent autosomal dominant kidney disease  among 19 family members Figure\u00a0A. No extA genome-wide parametric linkage analysis using a subset of 5,344 informative SNPs took ~105\u00a0hours of computing time run in parallel 220 fold (ten repeats of each of the 22 chromosomes) and failed to identify any loci significantly co-segregating with kidney disease  in exon 32 of the COL4A3 gene. This variant was absent in unaffected members of the family and the six phenocopies, including the index case. Analyses of smaller subsets identified 11 loci shared by \u226510 and 22 loci shared by \u22659 individuals  shared by 12 individuals. These loci contain no genes associated with kidney disease, so further analyses were performed on all 12,376 possible subsets of 11 out of 17 individuals. Together, these analyses took approximately 7\u00a0hours to complete serially on a desktop computer and indicated two further loci (of 11.4 and 7.75\u00a0cM) shared by 11 individuals  in the Turkish Cypriot population is not known, however one or more of these abnormalities is detectable in >16% of Australian adults . ConsideCOL4A3 locus by linkage at variable phenocopy rates from 0 to 0.45 yielded a maximum LOD score of 1.4 at a phenocopy rate of 0.25  identified numerous loci with LOD scores exceeding zero and a smaller number with LOD scores exceeding one  with 200 fold parallelisation per subset size was finished within 30\u00a0minutes (including queuing), with a total running time of ~41\u00a0hours. The limit on computing time with parallel CCH is simply a product of the number of available cluster nodes.CCH has a number of advantages over tradition linkage approaches: First, CCH directly provides the exact number and identity of individuals sharing each locus . Second, CCH only requires genotype data for the affected individuals under examination without a need for data from unaffected connecting relatives. CCH may thus be particularly useful in the context of pedigrees in which affected subjects are distantly related via individuals for whom precise relationship information and DNA is unavailable. Third, pre-processing of data for CCH is minimal only requiring standard quality control procedures with no need to generate an informative marker subset or to provide population specific allele frequencies. Formatting of the data and executing the software is therefore straightforward. Finally, each genome-wide CH analysis of a subset of individuals is rapid (~2.5\u00a0seconds) and completely independent the analysis of other subsets, thus permitting significant parallelisation. In the example family under study, to perform linkage analysis using SwiftLink running on a four core processor took an average of ~30\u00a0minutes per chromosome. Parallelised 220 fold, each genome-wide linkage took 3.5\u00a0hours (including cluster queuing time) with a total running time of ~110\u00a0hours. Given that CCH can rapidly identify the disease-linked locus in autosomal dominant disease, even in the presence of multiple phenocopies. CCH is also able to detect regions of shared ancestry among ostensibly unrelated individuals using un-phased genotypes. We believe that CCH should be considered in the investigation of inherited diseases where phenocopies are suspected, and may be especially powerful in detecting the location of founder mutations within isolated populations."}
+{"text": "Brassica napus, a highly duplicated amphidiploid species, to assess the proportion of resistance QTL located at duplicated positions.Several major crop species are current or ancient polyploids. To better describe the genetic factors controlling traits of agronomic interest (QTL), it is necessary to understand the structural and functional organisation of these QTL regions in relation to genome duplication. We investigated quantitative resistance to the fungal disease stem canker in B. napus. Overall, 44% of these regions (28/64) are duplicated homoeologous regions. They are located in duplications of six  of the 24 ancestral blocks that constitute the B. napus genome. Overall, these six ancestral blocks have 34 duplicated copies in the B.napus genome. Almost all of the duplicated copies (82% of the 34 regions) harboured resistance associated markers for stem canker resistance, which suggests structural and functional conservation of genetic factors involved in this trait in B. napus.Genome-wide association analysis on a panel of 116 oilseed rape varieties genotyped with 3228 SNP indicated that 321 markers, corresponding to 64 genomic regions, are associated with resistance to stem canker. These genomic regions are relatively equally distributed on the A (53%) and C (47%) genomes of B. napus. Further investigation of the similarity/divergence in sequence and gene content of these duplicated regions will provide insight into the conservation and allelic diversity of the underlying genes.Our study provides information on the involvement of duplicated loci in the control of stem canker resistance in The online version of this article (doi:10.1186/1471-2164-15-498) contains supplementary material, which is available to authorized users. Polyploidy or whole genome duplication (WGD) is an important phenomenon that has occurred during speciation and diversification of most plant species, 2. It iBrassica napus is a suitable model for studying the effects of WGD on genetic factors involved in the control of complex traits. B. napus  is an allotetraploid species formed from the hybridization between B. rapa  and B. oleracea \u2009=\u20091/(1\u2009+\u20094Nec) where c is the recombination rate in morgans and Ne the effective population size. The E[r2] was plotted against the genetic distance between molecular markers (in cM) in order to estimate the extend of LD with R software\u2009=\u20091/(1\u2009+\u20094Nec) as described in Sved et al.[r2 threshold of 0.2 are given for each linkage group, for the A and C genomes and for the whole genome. (XLSX 10 KB)Additional file 1: Table S1: Extent of linkage disequilibrium on each linkage group of d et al.. The effB. napus genome. The linkage disequilibrium decay was measured in the whole panel of 116 winter oilseed rape cultivars. The red curve shows the non-linear regression trend line of r2 versus genetic distance. (PDF 125 KB)Additional file 2: Figure S1: Linkage disequilibrium related to the genetic distance between markers along 10 p values obtained from the K and KP CML models were plotted against the genetic distance (in cM) for each linkage group. The corresponding linkage disequilibrium pattern calculated between pairs of tested markers for association is presented below the Manhattan plot. The more the colour is closer to red, the higher the linkage disequilibrium is. (PDF 1 MB)Additional file 3: Figure S2: The linkage disequilibrium pattern and results for association analysis of resistance to stem canker for each linkage group. Negative logAdditional file 4: Figure S3: Principal Component Analysis of 116 winter oilseed rape cultivars based on 727 SNP markers. The proportion of variance explained by the two principal components (Eigenvector 1 and 2) is indicated in parentheses. Varieties were tagged according to their euric acid and glucosinolate contents, \u201c00\u201d indicates a double low in erucic acid and glucosinolate content, \u201c0+\u201d indicated low erucic acid and high glucosinolate content, \u201c+0\u201d indicates high erucic acid and low glucosinolate content and \u201c++\u201d indicated high erucic acid and high glucosinolate content. The spring type variety \u2018Yudal\u2019 is circled in red on the right of the graph. (PDF 167 KB)Additional file 5: Figure S4: Distribution of the kinship coefficients calculated between 116 winter oilseed rape cultivars. (PDF 171 KB)Additional file 6: Table S2: List of the 321 resistance associated markers. The positions (in cM) of the markers on our linkage map, including information about their assignation to AK block are provided. When possible, context sequences of associated SNP are given. For a set of markers mentioned \u201cprivate\u201d, context sequences are available under request (see \u2018Availability of supporting data\u2019 section). (XLSX 33 KB)B. napus genome. P values given in the table for the K and KP linear models were those not corrected with the FDR test. Information about the co-localisation of resistance-associated markers with previously identify QTL in our laboratory (in a double haploid -DH- and/or a connected -CP- populations) is indicated. (XLSX 20 KB)Additional file 7: Table S3: Position of resistance-associated markers in relation to the duplicated blocks in Brassica napus varieties used for the genome-wide association analysis. *The G2 index is a mean of three replicates. **\u201c00\u201d indicates a double low in erucic acid and glucosinolate content, \u201c0+\u201d indicated low erucic acid and high glucosinolate content, \u201c+0\u201d indicates high erucic acid and low glucosinolate content and \u201c++\u201d indicated high erucic acid and high glucosinolate content. The dash indicate missing data. IHAR: Instytut Hodowli I Aklimatyzacji Roslin, Poznan; INRA: Institut National de la Recherche Agronomique; JD: Jouffray-Drillaud; KWS: KWS Saat AG; Momont: Sarl Adrien Momont & Fils; NK: Syngenta Seeds SAS; NPZ: NPZ Lembke Semences Sarl; RAPS Gbr: Raps GbR Saatzucht Lundsgaard; SW: Sval\u00f6f Weibull AB. (XLSX 15 KB)Additional file 8: Table S4: List of"}
+{"text": "For two marker loci, there are nine scenarios that allow the estimation of female, male, and/or combined recombination frequencies. In a double cross population derived from four inbred lines, five categories of markers are classified and another five scenarios are present for recombination frequency estimation. Theoretical frequencies of identifiable genotypes were given for each scenario, from which the maximum likelihood estimates of one or more of the three recombination frequencies could be estimated. If there was no analytic solution, then Newton-Raphson method was used to acquire a numerical solution. We then proposed to use an algorithm in Traveling Salesman Problem to determine the marker order. Finally, we proposed a procedure to build the two haploids of the female parent and the two haploids of the male parent in clonal F1. Once the four haploids were built, clonal F1 hybrids could be exactly regarded as a double cross population. Efficiency of the proposed methods was demonstrated in simulated clonal F1 populations and one actual maize double cross. Extensive comparisons with software JoinMap4.1, OneMap, and R/qtl show that the methodology proposed in this article can build more accurate linkage maps in less time.In this study, we considered four categories of molecular markers based on the number of distinguishable alleles at the marker locus and the number of distinguishable genotypes in clonal F However, EM algorithm and Markov chains used in recombination frequency estimation and linkage phase determination were time-consuming. In addition, some configurations in the previous studies  gave 18 cross combinations based on the genotypes of the two parents. The first four each generates four genotypes, which can be properly identified in the progenies. They are identical when used in linkage analysis. Redundant configurations complicate the application of those methods in practical populations.1 and phase-unknown double cross. It has been noted that software packages in R software were computationally slow and always failed to construct dense maps  can be made from four inbred lines to extend the genetic diversity in genetic studies and plant breeding. In clonal F1 and double cross populations. Our objectives in this study were: (1) to identify and classify informative markers based on the number of distinguishable alleles and the number of distinguishable genotypes; (2) to derive the theoretical frequencies of identifiable genotypes for each scenario of marker pairs and maximum likelihood estimates of recombination frequencies; (3) to build the female, male, and combined linkage maps; (4) to build the four haploids of the female and male parents based on the estimated recombination frequencies and the combined linkage map; and (5) to demonstrate the advantage of the proposed methods in comparison with other software.The unknown linkage phase and multiple alleles complicate recombination frequency estimation in clonal F1 hybrids of two clonal parents, one used as female and the other used as male  represents the case of fully informative markers. By fully informative, we mean the four genotypes at one locus in progenies can be clearly identified. In other words, the two alleles in any clonal progeny can be traced back to its female and male parents . For cat, and BD . When no1 progenies  represents the case of male polymorphism markers. By male polymorphism markers, we mean they show no polymorphism in the female parent, but they show polymorphism in the male parent. For category II markers, only two genotypes can be observed in the clonal Frogenies . Genotypallele B . When no1 progenies  represents the case of female polymorphism markers. By female polymorphism markers, we mean they show polymorphism in the female parent, but they show no polymorphism in the male parent. For category III markers, only two genotypes can be observed in the clonal Frogenies . Genotype C or D . When no2 population derived from two inbred parents in self-pollinated and cross-pollinated species. For category IV markers, three genotypes can be observed in the clonal F1 progenies, which are coded by AA, AB, and BB, respectively (Category IV (or AB = CD) represents the case of co-dominant markers. By co-dominant markers, we mean they show the same polymorphism pattern in both female and male parents, similar to an Fectively . When noXX.Missing marker types are common in most genetic populations . In the case of genotype A1A2/B1B2, gametes A1A2 and B1B2 are the two noncrossover  types with a frequency of A1B2 and B1A2 are the two crossover  types with a frequency of Fr will be lower than 0.5 if the two loci are linked. In the case of genotype A1B2/B1A2, gametes A1A2 and B1B2 are the two crossover types with a frequency of A1B2 and B1A2 are the two noncrossover types with a frequency of Fr will be more than 0.5 when the two loci are linked. Obviously, whether the estimated Fr is less or more than 0.5 could help to determine the linkage phase and genotype of the female parent. Similarly, whether the estimated Mr is less or more than 0.5 could help to determine the linkage phase and genotype of the male parent.Taking the female parent as an example, the four female gametes are Fr is less than 0.5, then the female parent will be in linkage phase A1A2/B1B2; otherwise, it will be in linkage phase A1B2/B1A2. If estimated Mr is less than 0.5, then the male parent will be in linkage phase C1C2/D1D2; otherwise, it will be in linkage phase C1D2/D1C2.Therefore, linkage phases and genotypes of both parents can be determined by their estimated recombination frequencies, respectively. If estimated A1A2/B1B2 and the male parent has genotype C1C2/D1D2. In phase II, the female parent has genotype A1A2/B1B2 and the male parent has genotype C1D2/D1C2. In phase III, the female parent has genotype A1B2/B1A2 and the male parent has genotype C1C2/D1D2. In phase IV, the female parent has genotype A1B2/B1A2 and the male parent has genotype C1D2/D1C2. The four phases will be used later for some scenarios in estimating the combined recombination frequency r, to make sure the estimated r is less than 0.5, and the estimation will not be affected by the linkage information confounding in one or both parents.Considering the two phases to be determined in both parents together, four potential linkage phases of the two parents can be defined. In phase I, the female parent has genotype A1C1, A1D1, B1C1, and B1D1, and locus 2 has four identifiable genotypes A2C2, A2D2, B2C2, and B2D2. The first row and first column of Table S1 show the four female and male gametes and their frequencies, from which we can easily derive theoretical frequencies of the 16 identifiable genotypes at the two linked loci. For convenience, the 16 genotypes were rearranged in n1, n2, \u2026, and n16. Based on theoretical frequencies and sample sizes in L) and logarithm likelihood (logL) can be constructed in C is a constant independent of the unknown recombination frequencies. The maximum likelihood estimates (MLE) of recombination frequencies can be calculated either by solving the likelihood equation .We begin with the most ideal situation where locus 1 has four identifiable genotypes r in r, when directly calculated from its likelihood function.Define the estimate of the combined recombination frequency A1C1, A1D1, B1C1, and B1D1, and locus 2 has two genotypes X2C2 and X2D2. In scenario 3, locus 1 has four genotypes A1C1, A1D1, B1C1, and B1D1, and locus 2 has two genotypes A2X2 and B2X2. Fr cannot be estimated in scenario 2; Mr cannot be estimated in scenario 3. MLE of Mr in scenario 2 can be calculated from its likelihood functions, given in in is the observed sample size for the ith genotype . Define the estimate of r in r, when directly calculated from its likelihood function.In scenario 2, locus 1 has four genotypes rF in scenario 3 can be calculated from its likelihood function, given in in is the observed sample size of the ith genotype . Define the estimate of r in r.MLE of genotype , ni:j isA1C1, A1D1, B1C1, and B1D1, and locus 2 has three genotypes A2A2, A2B2, and B2B2. Fr and Mr is confounded in half of the genotypes. MLE of Fr and Mr using the other half of the genotypes are given in in is the observed sample sizes of the ith genotype and in:j is the sum of in to jn.In this scenario, locus 1 has four genotypes Fr and Mr in r . Because the theoretical frequencies . Define the estimate of r in In scenario 5, locus 1 has two genotypes Fr in scenario 7 can be calculated from its likelihood functions, given in r in r for scenarios 5 and 7, respectively.MLE of X1C1 and X1D1, and locus 2 has three genotypes A2A2, A2B2, and B2B2. In scenario 8, locus 1 has two genotypes A1X1 and B1X1, and locus 2 has three genotypes A2A2, A2B2, and B2B2. Fr cannot be estimated in scenario 6, and Mr cannot be estimated in scenario 8. MLE of Mr in scenario 6 can be calculated from its likelihood function, given in in is the observed sample size of the ith genotype . For linkage phases II and III, MLE of r can be found from in is the observed sample size of the ith genotype and n is the total sample size .For linkage phases I and IV, Newton-Raphson algorithms to estimate r from the four potential linkage phases, i.e., two loci were linked) and true linkage phase was I , then r should be estimated at approximately 0.5 in all linkage phases . First, one F1 hybrid is made between inbred lines A and B; the other F1 hybrid is made between inbred lines C and D. Then, a double cross is made between the two F1 hybrids; one is used as female and the other one is used as male. When polymorphism markers are screened in the four inbred lines, the four alleles in double cross populations can be clearly assigned. In this case, five marker categories can be differentiated on the number of identifiable alleles in the four original lines and the number of identifiable genotypes in their double cross progenies . Categories I to III are similar to those in clonal F1. Category IV in clonal F1 can be further divided into two categories in double cross, which are denoted as categories IV and V. For category IV (or A = CB = D), allele A is the same as allele C, and allele B is the same as allele D. For category V (or A = DB = C), allele A is the same as allele D, and allele B is the same as allele C.Double cross populations in plants have four inbred lines, A, B, C, and D, as parents that are homozygous at most chromosomal locations . In scenario 11, the six identifiable genotypes are the same as scenario 6 in Mr with 1\u2212Mr (Table S3). In scenario 12, the six identifiable genotypes and their theoretical frequencies are the same as scenario 8 in Table S3). In scenario 13, genotypes and their theoretical frequencies are the same as linkage phases II and III of scenario 9 in Table S4). In scenario 14, genotypes and their theoretical frequencies are the same as linkage phase I of scenario 9 in Table S4). Methods for estimating r are similar to the corresponding scenarios in clonal F1. For convenience, theoretical genotypic frequencies at two loci for scenarios 10 to 14 are given in Table S2, Table S3, and Table S4.In scenario 10, the 12 identifiable genotypes are the same as scenario 4 in H0 is the null hypothesis corresponding to no genetic linkage, AH is the alternative hypothesis corresponding to the linkage relationship between two loci, and r is the combined recombination frequency. The log-likelihood function under the null hypothesis is LOD score can be calculated from the log-likelihoods under the two hypotheses, i.e., The existence of the linkage can be tested by the following two hypotheses.We considered one chromosome with 20 evenly distributed markers in simulation. Recombination frequencies between any two neighboring markers were set at 0.05, equivalent to a genetic distance of 5.27 cM using Haldane mapping function .1 progenies was simulated by the genetics and breeding simulation tool of QuLine . Algorithms for estimating recombination frequencies and building linkage map were implemented in the software called GACD (available from www.isbreeding.net). For comparison, JoinMap4.1, OneMap, and R/qtl were used for linkage map construction in the simulated population. The mapping algorithm in JoinMap4.1 was maximum likelihood mapping with the following parameters: chain length = 1000; initial acceptance probability = 0.25; cooling control parameter = 0.001; and stop after 10000 chins without improvement. Function \u201corder.seq\u201d in OneMap was used for ordering, where the best order was determined in a window size of five markers. The best order in R/qtl was determined by function \u201corderMarker,\u201d where the initial order was established by a greedy algorithm and was refined by rippling. In the simulated population, Haldane mapping function was used to convert recombination frequency (r) to map distance (d) in cM. In the maize population, Kosambi mapping function . The closer to the diagonal, the lower the recombination frequencies would be. For example, recombination frequencies between marker 1 and markers 2, 8, and 19 were 0.05, 0.26, and 0.42, respectively. Recombination frequencies of marker pairs 8 and 9, 8 and 15, and 8 and 20 were 0.05, 0.26, and 0.36 (Table S5), respectively.Theoretical recombination frequencies between the 20 simulated markers were shown in the upper triangular matrix . The closer between two markers, the smaller the estimated distance is. For example, the true recombination frequencies of marker pairs 1 and 2, 1 and 8, and 1 and 19 were 0.05, 0.26, and 0.42 (Table S5). Their estimated distances were 5.3 cM, 29.0 cM, and 160.9 cM (Table S6), respectively. The true recombination frequencies of marker pairs 8 and 9, 8 and 15, and 8 and 20 were 0.05, 0.26, and 0.36 (Table S5). Their estimated distances were 3.1 cM, 37.8 cM, and 88.6 cM (Table S6), respectively. It should be noted that the map length of a chromosome is calculated from lengths of individual ordered intervals, rather than the recombination frequency between the first and the last markers.If combined recombination frequency cannot be estimated, then the corresponding marker distance and LOD score cannot be calculated either. The upper triangular matrix showed the estimated map distance between the 20 markers . Their LOD scores were 43.0, 14.4, and 0.1 (Table S6), respectively. The true recombination frequencies between marker pairs 8 and 9, 8 and 15, and 8 and 20 were 0.05, 0.26, and 0.36 (Table S5). Their LOD scores were 48.5, 10.0, and 1.3 (Table S6), respectively.The lower triangular matrix of Estimates of the combined recombination frequencies were used to order the 20 markers, and the best order with the shortest map length was shown in Table S4 and Table S5) and therefore do not appear on the female map. Marker 3 was located at the beginning and marker 19 located at the end on the female map, which explained the reduced female map length. Markers, 4, 5, 7, 9, and 17 are category III (Table S4 and Table S5); therefore, they do not appear on the male map. However, marker 1 was still located at the beginning and 20 was still located at the end on the male map, which explained the map length similar to the combined one.The female map does not contain markers of category II, and the male map does not contain markers of category III. The order of markers in the female and male maps were the same as that in the combined map, but map distances between markers were estimated by the female and male recombination frequencies, respectively. In the simulated population, lengths of the female and male maps were 81.90 cM and 103.02 cM, respectively . For theFr and Mr between neighboring markers, four haploids of parents at 20 marker loci were determined . Alleles on HapA and HapB were represented by X, which could be either allele A or allele B. Alleles on HapC and HapD were C and D, respectively, which were the same haploids as those of the previous locus. Marker 3 was the first on the female map . In a simulated population, markers 3, 6, 10, 12, and 16 were category IV. Taking marker 3 as an example, alleles on HapA, HapB, HapC, and HapD were A, B, D, and C, respectively. Its category was redefined as category V of double cross . R/qtl can only conduct linkage mapping in phase-known double cross, so marker categories and genotypes after haploid building were imported into R/qtl. Marker orders given by GACD, OneMap, and R/qtl were the same as the predefined order in the simulated model. However, marker order given by JoinMap4.1 was far from the predefined (Table S7). The first and last markers were Marker 12 and Marker 18, respectively. The true map length was 100.13 cM. Length was estimated at 101.79 cM from GACD, 15211.04 cM from JoinMap, 103.83 cM from OneMap, or 104.22 cM from R/qtl. The reason for the extremely large map length from JoinMap was the estimated value of 0.5 of recombination frequency between some neighboring markers in the female or male maps, which was converted to a distance of 10,000.0 cM in JoinMap. For example, recombination frequency between markers 3 and 5 belonging to category V and III was estimated at 0.5 on the female map, corresponding to a distance of 10,000.0 cM on the female map and 5007.99 cM on the combined map. Time spent for building the maps was 8 sec by GACD, 30 sec by JoinMap, 455 sec by OneMap, and 63 sec by R/qtl on a computer with 1.60 GHz CPU and 3.00 GB RAM.General information of combined linkage maps of the simulated population built by GACD, JoinMap4.1, OneMap, and R/qtl were shown  and a simulated clonal F1 population with 200 individuals and 200 markers belonging to category IV . A greater advantage was observed for the marker number 200 in one single chromosome (Table S8). GACD took 0.5 min, JoinMAP took 5 min, OneMAP took 537 min, and R did not output any results. GACD results in the shortest linkage map closest to the true length in the shortest time (Table S8). The reason may be as follows. Previous studies tried to estimate recombination frequency, determine linkage phase, and build linkage map simultaneously. In our study, we first estimate all pair-wise recombination frequencies . Linkage phases were determined from the estimated recombination frequencies . Linkage map was built based on the matrix of all pair-wise recombination frequencies . Finally, the four haploids were built from the completed linkage maps . Separating a complicated genetic question into four clearly defined steps results in more accurate genetic linkage maps in shorter time. In addition, we believe the adoption of the optimization algorithm in solving the Traveling Salesman Problems also contributes to the ordering efficiency.Comparison of different software packages was also conducted in a simulated clonal FFigure S3). The whole genome was 1778.09 cM in length, and the average marker distance was 8.51 cM.In the actual population, the missing marker rate was at 6.49%. Among the 220 markers, 60 markers showed segregation distortion under significance level 0.05. Recombination frequencies of all marker pairs were estimated and then used for linkage map construction. The combined genetic linkage map was constructed by 219 SSR molecular markers using the software GACD. One marker cannot be linked with any other markers and was deleted. The 10 chromosomes had 25, 28, 25, 24, 21, 19, 18, 16, 25, and 18 relatively evenly distributed markers, respectively  had 19, 19, 20, 13, 16, 13, 12, 14, 17, and 15 markers, respectively, with a total of 158 markers. The total female map length was 1796.92 cM. The 10 male chromosomes  had 18, 19, 22, 21, 17, 14, 14, 9, 19, and 15 markers, respectively, with a total of 168 markers. The total male map length was 1599.13 cM.The 10 female chromosomes  markers are more and more often being used in genetic analysis. 1, genotype of the female parent can be either A1B1/A2B2 or A1B2/B1A2; genotype of the male parent can be either C1D1/C2D2 or C1D2/D1C2. In double cross, there are four homozygous inbred lines whose genotypes may be known. Alleles A, B, C, and D at each polymorphism locus can be traced back to the four inbred lines, when the four lines have been genotyped. In this case, genotype of the single cross between lines A and B is A1B1/A2B2; genotype of the single cross between lines C and D is C1D1/C2D2. Therefore, double cross is actually a special case of clonal F1 in which only linkage phase I is applicable .In clonal FA1B1/A2B2 or A1B2/B1A2; genotype of the other single cross can be either C1D1/C2D2 or C1D2/D1C2. In this case, the double cross must be treated as one clonal F1 population for genetic analysis , as is the case for the actual maize population used in this study.In a double cross where polymorphism loci are only screened in the two single crosses, linkage phases become unknown before estimating recombination frequencies. Genotype of one single cross can be either 1 can be determined by linkage analysis, from which four haploids can be built. If the four haploids could be viewed as haploids of the four inbred lines in a double cross, then clonal F1 is equivalent to double cross. In short, there are many similarities between clonal F1 and double cross, although difference does occur . It is important in genetics to know when clonal F1 and double cross are equivalent and when they are not. Previous genetic studies focused on only one of clonal F1 or double cross population. To our understanding, this study is the first that tried to combine the two kinds of populations. Based on the linkage analysis, two haploids of the female parent and two haploids of the male parent can be built, and then the clonal F1 progenies can be viewed as a double cross population derived from four inbred lines. The unified QTL mapping method for the two kinds of populations will be fully investigated in another article . In this case there are two alleles at each locus, and only marker category IV and linkage phases I and IV are applicable. Methods proposed in this study can be readily used to estimate recombination frequency, identify linkage phase, and build the two haploids of the clonal parent. In self-pollinated and cross-pollinated species, an F2 population is the selfing generation of one F1 hybrid between two inbred parents. Linkage phases are known when both inbred parents are genotyped. In this case, the clonal F1 derived from the selfing of one clonal parent can be viewed as an F2 population, after the two parental haploids are built.In practice, clonal F1 hybrid and itself, the F2 population becomes a special case of clonal F1 when linkage phases are unknown, or a special case of double cross when linkage phases are known . In the F2 population, there are two alleles at each locus; therefore, only marker category IV is applicable. Haploids built in clonal F1 and double cross may help to identify and correct markers that are misclassified for the two inbred parents. Moreover, genetic analysis in an F2 population can still be performed by the clonal genetic analysis methods, even when there is no genotypic data on its two parental lines or on its F1 ancestry.If selfing can be viewed as a cross between the F1, it is possible to build the female and male linkage maps to reflect the sex-specific recombination frequencies.More broadly, methodology proposed in this study can be applied in genetic populations derived from any two heterozygotes in animals and plants. For example, in animals, linkage analysis is normally conducted in progenies between one female parent and one male parent, both are highly heterozygous, and they are drawn from a large random-mating population. By using the methodology of clonal F"}
+{"text": "To analyze the accuracy of deterministic and probabilistic record linkage to identify TB duplicate records, as well as the characteristics of discordant pairs. The study analyzed all TB records from 2009 to 2011 in the state of Rio de Janeiro. A deterministic record linkage algorithm was developed using a set of 70 rules, based on the combination of fragments of the key variables with or without modification (Soundex or substring). Each rule was formed by three or more fragments. The probabilistic approach required a cutoff point for the score, above which the links would be automatically classified as belonging to the same individual. The cutoff point was obtained by linkage of the Notifiable Diseases Information System \u2013 Tuberculosis database with itself, subsequent manual review and ROC curves and precision-recall. Sensitivity and specificity for accurate analysis were calculated. Accuracy ranged from 87.2% to 95.2% for sensitivity and 99.8% to 99.9% for specificity for probabilistic and deterministic record linkage, respectively. The occurrence of missing values for the key variables and the low percentage of similarity measure for name and date of birth were mainly responsible for the failure to identify records of the same individual with the techniques used. The two techniques showed a high level of correlation for pair classification. Although deterministic linkage identified more duplicate records than probabilistic linkage, the latter retrieved records not identified by the former. User need and experience should be considered when choosing the best technique to be used. Linkage between databases aims to identify if two or more records relate to the same entity, generally the same individual. This technique is used to identify duplications in a same file or between two or more files whose information must be gathered in a single databaseProbabilistic record linkage uses approximate comparison functions. Different weights are assigned to each field based on their discrimination power and vulnerability to error. Deterministic record linkage uses exact comparison functions and classification based on rules developed from the knowledge of specialists-,The Ministry of Health is responsible for developing, managing and storing data from national health information systems. Although the systems are not automatically interconnected, it is possible to link these databases thanks to the existence of nominal identification variables with high discrimination power, especially when used in combination, which are standardized in the systems. Tuberculosis (TB) cases are compulsorily recorded in a national information system for disease notification (Notifiable Diseases Information System \u2013 Sinan-TB)Few studies compare the accuracy of linkage processes between secondary databasesThe aim of this study was to analyze the accuracy of deterministic and probabilistic record linkage to identify TB duplicate records, as well as the characteristics of discordant pairs.The study used information from the Sinan-TB database from 2009 to 2011 related to the state of Rio de Janeiro, available from the State Health Department. TB notifications terminated due to diagnosis change were excluded.A data preprocessing phase was carried out to correct errors and standardize the content of the key variables  used in each technique. Data transformations included: removal of punctuation marks, accents, repeated blanks and prepositions; conversion of letters to uppercase; removal of numbers from variables intended to be exclusively composed of letters and vice versa; removal of terms indicating lack of information ; replacement of double letters by a single one; standardization of date formats; standardization of address terms .Subsequently, new variables were generated from standardized mother\u2019s name and address formats, with the performance of the following processes: 1) Parsing  and 2) Substringing . Each new variable was also transformed into another containing its Soundex code,The probabilistic approach involves standardizing, blocking and forming links (record pairs to be compared), applying comparison algorithms and generating similarity scores, setting thresholds to classify links into true pairs, non-pairs and doubtful pairs, manually reviewing doubtful pairs and removing duplications,The cutoff score, above which the links were classified as belonging to the same individual,To identify duplicate records, the same blocking strategy was used. The linkage variables were name, mother\u2019s name and date of birth. The first two were compared using Levenshtein distance, and the third by using an exact algorithmA deterministic record linkage algorithm was developed in Stata 12.0, based on 70 rules, to identify the record groups of a same individual and classify them as a pair or non-pair. These rules were based on the key variables, as well as those created in the preprocessing phase. Some rules also used Soundex codes in a concatenated format . In each rule, the content of three or more variables was compared exactly, regardless of the presence of data constraints .The variable name was used in almost all rules, given its high discrimination power. Date of birth was used in some rules with information on day, month and year, while in others one or more of its components were used in combination. The variables city of residence or of notification, sex, date of notification, date of conclusion, notification number, notification unit code, as well as others related to place of residence  were used in the original form. District was the only one of these variables to undergo preprocessing.The rules were laid out sequentially, from those using fewer variables, with greater discrimination power, to those using several variables in combination, but which individually had less discrimination power. Discrimination power is understood as a variable\u2019s capacity to independently discriminate an individual.The inclusion of rules aimed to increase the algorithm\u2019s sensitivity without losing specificity. With each new rule, new groups were found or new records added to existing groups, even though they did not pair with all the records already identified in the group. Rules that generated incorrect groups were modified or ultimately discarded following an extensive manual review.Several data constraints were imposed in many rules. The universal constraint for all rules was that the groups could only be formed with records whose variables had no missing values. In addition, for certain rules which used a combination of variables with lower discrimination power, the content of some of those variables needed to have a minimum length of characters. Similarly, in some rules, the Soundex code had to have a minimum number of blocks (Maria Antonia Santos \u2192 M600-A535-S532 \u2192 3 blocks).Name infrequency was also considered in some rules. To this end, three separate databases were created indicating the frequency of the first, middle and last name in the Brazilian Mortality Information System (SIM) database for 2008-2010. Names appearing in the TB database but absent in SIM were considered rare. Records containing the same rare first, middle or last names were combined into groups, even when some of the other variables used in the rule had incomplete information. However, records with common rare names but with birth dates more than 20 years apart were not considered as belonging to the same group, since they could refer to father and son, for example. This possibility had to be considered, especially in the case of an infectious disease whose main transmission occurs within the household. The definition of rarity ranged from 500 to 1.000 for name frequency, according to the discrimination power of the other variables of the rule.A manual review was carried out of the links classified as pairs by both techniques and of those with discordant classification. The classification of the manual review was considered the gold standard for calculations of sensitivity and specificity.The following indicators were analyzed to profile records with discordant classification between the techniques:Percentage of missing values for the variables name, mother\u2019s name, sex, date of birth and address;Score median of duplicate records identified by probabilistic linkage and not identified by deterministic linkage;Percentage of same group records identified by probabilistic linkage with differences in the variable sex;Similarity measure median for the variables name, mother\u2019s name and date of birth of discordant records .Levenshtein distance (LD) should be analyzed considering the size of the character string, as long strings are more likely to generate higher valuesFor groups with three or more records, the record link used to calculate SM was formed by the record presenting discordant classification between both techniques and the record which, in probabilistic linkage, had the highest score in the group. This alternative was adopted assuming that the record associated with the highest score would tend to be spelled more correctly. Records presenting difference in sex were excluded from the SM analysis, since they were not submitted to probabilistic linkage because they belonged to different logical blocks. Stata 12.0 software was used to analyze the records.The project was approved by the Research Ethics Committee of the Institute of Studies in Collective Health of the Federal University of Rio de Janeiro .The Sinan-TB database contained 43,825 records. In deterministic linkage, 21.3% of the records were classified as pairs, and in probabilistic linkage, 19.5%. Discordant classification appeared in 1,812 records. In total, 527 records were classified as pairs by probabilistic linkage and not by deterministic linkage, and 1,285 records formed pairs by deterministic linkage and not by probabilistic linkage. The subsequent manual review, performed to determine the gold standard, showed no classification change for the group of records classified as pairs by both techniques .In the accuracy analysis, sensitivity and specificity values for deterministic linkage were 95.3% and 99.9%, respectively. For probabilistic linkage the figures were 87.2% and 99.8%, respectively .Of the records classified as pairs by deterministic linkage and non-pairs by probabilistic linkage, none had missing values for the variables name and sex, 5.3% had missing values for mother\u2019s name, 6.3% for date of birth and 0.5% for address. Ten percent of the records presented missing values for at least one of these variables. The score median of records classified as pairs by probabilistic linkage and as non-pairs by deterministic linkage was 24.2, ranging from 20.3 to 32.3. No records were found with missing information for the variables name and sex. For mother\u2019s name, 14.4% of records had missing values; for date of birth, 11.0%; and for address, 3.8%. More than a quarter of the records had missing values for at least one of the analyzed variables.Of the 733 links classified as pairs by deterministic linkage and non-pairs by probabilistic linkage, 15.7% showed differences for the variable sex. Approximately 26.0% had SM lower than 70.0% for the variable name, 26.6% for mother\u2019s name, and 8.3% for date of birth. For links classified as pairs only by probabilistic linkage, 16.6% had SM lower than 70.0% for the variable mother\u2019s name and 10.6% for date of birth. As to the SM median, the biggest difference between the groups was for the variable name (81.6 for pair links by deterministic linkage and 100 for pair links by probabilistic linkage) .,,,,The two techniques showed a high correlation in classifying records as pairs. However, deterministic linkage retrieved 8.8% more records than probabilistic linkage, despite the latter having identified records that were not identified by the former. Although the specificity of both techniques was similar, sensitivity was higher in deterministic linkage, corroborating the findings of other studies using the Sinan database,,,,,The occurrence of variables with missing values and SM lower than or equal to 70.0% for key variables was the main reason for not identifying pairs in both techniques. Additionally, blocking by sex in probabilistic linkage was also responsible for the failure to form certain groups. Removing the variable sex from the initial blocking to set the cutoff point might minimize this problem, but greatly increase the volume of links to be manually reviewed, because the blocking key would become even more sensitive. On the other hand, the manual review would be performed only once and the established cutoff point would be applied to works with the same database. Although there are no missing values for the variable name, the lack of information for the variables mother\u2019s name and date of birth in Sinan-TB is still significantVariables related to address were used as auxiliary criteria in classifying records by deterministic linkage. Even though this variable should be used with caution, since patients might change their address between notifications, it assists in the decision. In probabilistic linkage between different databases, these variables can be used in the manual review to support classification. In this study, we chose not to perform the manual review after implementing the routine to identify duplications. In deterministic linkage, a different treatment was considered for common names in the algorithm. The influence of these strategies in retrieving pairs was not investigated, but they are believed to have influenced classification. The strategy of matching by frequency (information on name infrequency) could be incorporated into the probabilistic linkage software, minimizing the occurrence of false positives in high scores due to the presence of homonyms.The high-score median for records classified as pairs by probabilistic linkage and non-pairs by deterministic linkage indicates that mutually similar key variables were not identified. This attests the need to improve the deterministic routine.,The deterministic algorithm used commercial statistical software whose interface is in English and requires prior knowledge of users of the programming language used by the software. In addition, the adaptation of the algorithm to databases other than Sinan-TB is conditional on knowledge of these databases and programming experience. Transcribing it using free software might facilitate its wider use. Probabilistic linkage used free national software which also requires prior knowledge. However, as the OpenReclink routine to identify duplications allows automatic classification of groups, it is easier to useThe study did not measure time and difficulties in using each technique. However, both the elaboration of the deterministic algorithm and the manual revision to set the cutoff point for probabilistic linkage demanded a considerable amount of time and experience from the researchers.Sinan has specific intrinsic routines to identify and treat duplicate recordsThe definition of which technique to use to identify duplicate records in Sinan-TB depends on the purpose and need of users. As the search for methodological perfection is a premise in research, deterministic linkage may be used initially to retrieve most of the links, followed by probabilistic linkage to retrieve the links that were not found by the former. In routine TB control programs at the three levels of government, removal of duplication is performed several times a year, which is extremely time-consuming for the professionals involved. The use of deterministic linkage is recommended provided the prerequisites for its use are met. Otherwise, probabilistic linkage is indicated, which, if necessary, can be rendered more sensitive with the performance of a manual review of the groups with scores below the set cutoff point, to retrieve new links previously unidentified. As for the construction of new deterministic routines and improvement of existing ones, probabilistic linkage, being generic, should be used as an initial strategy to accumulate knowledge to be incorporated in the development of deterministic linkage. Such accumulation of knowledge is important, since deterministic linkage is less specific and might generate far less accurate results than those obtained by probabilistic linkage. For Grannis et al., although the sensitivity of deterministic linkage may reach 100%, it can be considerably reduced for data with different characteristics of identification , and therefore probabilistic linkage is considered to be more efficientSinan undergoes routine updates to meet the needs of users and of the national surveillance system"}
+{"text": "Penicillium digitatum is one of the most destructive postharvest pathogen of citrus fruits, causing fruit decay and economic loss. The emergence of fungicide-resistant strains made the control of P. digitatum more difficult. While the genome of P. digitatum is available, there has been few reports about its resistant mechanism from the transcriptome perspective and there has been no large-scale functional annotation of the genome using expressed genes derived from transcriptomes.\u00a0P. digitatum\u00a0strain HS-F6 (prochloraz-resistant strain) and HS-E3 (prochloraz-susceptible strain) before and after prochloraz-treatment were extracted and sequenced on an Illumina Hiseq 2000 platform. The transcriptome data of four samples were compared and analyzed using differential expression analysis, novel transcripts prediction and alternative splicing analysis, SNP analysis and quantitative real-time PCR. Total RNA ofP. digitatum. The whole RNA was extracted from a prochloraz-resistant strain (HS-F6) and a prochloraz-susceptible strain (HS-E3) before and after prochloraz-treatment and sequenced by Illumina technology. A total of more than 100 million reads were generated and de novo assembled into 9760 transcripts that contained annotated genes after quality control and sequence assembling. 6625 single nucleotide variations (SNVs) were identified from the sequences aligned against the reference genome. Gene expression profiling analysis was performed upon prochloraz treatment in HS-F6 and HS-E3, and differential expression analysis was used to identify genes related to prochloraz-response and drug-resistance: there are 224 differentially expressed genes in HS-E3 and 1100 differentially expressed genes in HS-F6 after prochloraz-treatment. Moreover, gene expression profile in prochloraz-resistant strain HS-F6 is quite different from that in HS-E3 before prochloraz-treatment, 1520 differential expression genes were identified between the two strains. Gene ontology (GO) term enrichment and KEGG enrichment were then performed to classify the differential expression genes. Among these genes, there are a lot of transporter encoding genes including 14 MFS (Major Facilitator Superfamily) transporters, 8 ABC (ATP-binding cassette transporter) and 3 MATE (multidrug and toxic compound extrusion family) transporters. Meanwhile, the roles of typical MFS, ABC and MATE proteins in prochloraz resistance were investigated using real-time quantitative PCR.We present a large scale analysis about the transcriptome data of P. digitatum demonstrate differences between prochloraz-resistant and prochloraz-susceptible strains with prochloraz-treatment. The differences existed in expressed transcripts, splice isoforms and GO categories, which would contribute to our knowledge on the molecular mechanisms involved in drug resistance of P. digitatum.The sequencing-based transcriptome data of The online version of this article (doi:10.1186/s12864-015-2043-x) contains supplementary material, which is available to authorized users. P. digitatum is the most destructive disease of citrus fruit which cause green mold and responsible for up to 90\u00a0% of total crop losses during postharvest packing, storage, transportation, and marketing .The datasets supporting the results of this article are available in the NCBI SRA repository ["}
+{"text": "However, joint modeling methods are usually computationally intensive; hence they cannot currently accommodate large pedigrees with dense markers. This article proposes a simple method to combine the linkage and association evidence with the aim of improving the detection power of disease susceptibility genes. Our detection power comparisons show that the combined linkage-association  Our detection power comparisons show that the combined linkage-association p values can improve the causal gene detection power remarkably in Genetic Analysis Workshop 18 (GAW18) simulation data.Linkage analysis in family data looks for the genomic region where the disease phenotype of interest and a stretch of genetic markers are cosegregated. As a result of the strong identity-by-descent (IBD) sharing among family members and a limited number of recombination events present in collected pedigrees, the critical regions detected by linkage analyses rarely pinpoint a single gene. However, linkage analysis is immune to the confounding of population stratification suffered by association analyses. Association analyses regress quantitative phenotypes on a marker's genotypes or compare allele frequencies of a single-nucleotide polymorphism (SNP) between cases and controls, and can narrow down the putative disease regions to small regions of high linkage disequilibrium (LD blocks), which are usually much shorter than linked regions. With the advance of next-generation sequencing technology and highly accurate imputation methods, association analyses with dense marker coverage can even potentially locate candidate causal variants (and thus candidate genes) directly. Because the genotype-phenotype correlation information is investigated differently by linkage and family-based association analyses, various efforts have been made to model linkage and association jointly -9. Namind Liptak  method tAll the analyses and comparisons in this report are performed with the disease causal variants known.We adopt the method found in Levy et al  to adjus10 in likelihood between the alternative and the restricted models. A total of 3071 genome-wide association studies (GWAS) array SNPs were randomly selected so that they were not in high LD in unrelated individuals. Multipoint linkage analysis in SOLAR [SOLAR  is a varin SOLAR  was applp values.The multimarker version of family-based association test (FBAT) statistics is a linear combination of single-marker FBAT statistics with the data-driven combination weights . We adopp value by observing that 2*loge(10LOD) is asymptotically distributed as a 0.5:0.5 mixture of a p values for a gene are inverse-normal transformed to Z1 and Z2 respectively. We then adopt the following unweighted Liptak method [p value. When Z1 and Z2 are independent, k is a k-element vector of 1, \u03a6 is a 2 \u00d7 2 identity matrix, and  is a row vector made up of Z1 and Z2 that follows the standard normal distribution asymptotically. When Z1 and Z2 are correlated [j = 1,2) is an N-element column vector of test statistics for test j when the phenotypes are permuted N times. The combined linkage and association p values were calculated using Liptak method with and without correlation correction.In the output from SOLAR, LOD scores were given with respect to genetic distances; the physical boundaries for each gene were mapped to genetic distances, and a gene was assigned the average LOD score of the genetic region to which it is mapped. Next, the linkage LOD score is converted to a at 2*logeLOD is asrrelated , \u03a6 can bThe linkage analysis showed that chromosome 3 had an LOD score >1.5 three and nine times among simulations 1 to 10 for the traits of Q1 and mean SBP, respectively. Most of the linkage regions for the trait of mean SBP were mapped around 55 to 70 cM, whereas for the trait of Q1, the linkage regions were quite scattered, being0 to 30 cM, 125 cM, and 165 to 220 cM for the 3 simulations with LOD scores >1.5. It turned out that chromosome 3 had the strongest linkage signal.p value threshold so that top 50 genes were checked against the simulated disease model. For mean SBP, on average, 49 of 8047 genes had combined p values less than 0.001 among simulations 1 to 10. Only 2 causal genes, MAP4 and FLNB on chromosome 3, were ever among the top 49, so we investigated their detection power. For Q1, on average, there were 9.5 and 9.1 genes out of 8047 with FBAT p values and combined p values smaller than 0.001, corresponding to an empirical false-positive rate of 0.0012 and 0.0011, respectively.FBAT was applied to 8047 genes among 11 chromosomes that have more than 1 nonsynonymous SNP. We mimicked the fast validation strategy in practice, which took top 50 candidates to validate in independent samples. Because we investigated gene-based analyses, we took a p values were slightly different when the correlation between linkage and association p values was corrected, the ranks of these 2 genes (out of 8047) based on the combined p values did not change. Table p values and the combined p values for the traits Q1 and mean SBP.Although the combined p values were viewed to improve the FBAT p values if the rank of the causal gene based on the latter was beyond 49, and the rank based on the former was within 49. There were 5 and 4 improvements for MAP4 and FLNB, respectively . Combined p values improved the detection power for MAP4 from 50% to 100%. For FLNB that explains a much lower percentage of SBP variance (0.29%); FBAT had no detection power. Combined p values improved the power to 40%. Moreover, the type I error was well controlled in our combined p values. These results indicated a promising strategy of combining the linkage and association evidence to improve the true discovery rate/power. Furthermore, our method combines the linkage and association p values in a simple way; thus it is applicable to large pedigrees as long as large pedigrees can be accommodated in the linkage analyses. The option -e in FBAT software forces an estimation of association in the presence of linkage, thus the association signal detected is expected to be independent of the linkage signal. That the combined p values with and without correlation correction were very similar  verified this.Generally speaking, the power for detecting the causal genes was low, except for p values we propose to calculate depend on the strength of both linkage and association signals. Moderate signals in both linkage and association will generate a more significant combined p value than a significant signal in one test but a null signal in the other. To maximize the association power, we analyzed only nonsynonymous SNPs in gene-based association tests, as we know that the nonsynonymous SNPs are enriched with causal variants with relatively large effects from the released disease model. In real sequencing projects, especially whole genome sequencing studies, we may select other functional variants to analyze, such as deleterious or regulatory SNPs, to improve the association power.The combined p values makes some causal genes that have moderate supports in both tests stand out. For example, for simulation 8, chromosome 3 had a LOD score of <1.5. However, the regions to which MAP4 and FLNB were mapped still have moderate linkage evidence, with LOD scores of 0.82 and 0.53, respectively. As a result, the ranks improved from 154  to 6  for MAP4, and from 2372  to 343  for FLNB.In our opinion, the combined test is more powerful because linkage and association analyses investigate different parts of phenotype-genotype correlation, thus providing nonredundant information. Combining these 2 p values do improve the causal gene detection power remarkably. The improved true discovery will render a higher chance for the top genes to be validated.We proposed a simple method to combine the linkage and family-based association evidence that is applicable to large pedigrees. Our results showed that the combined linkage and FBAT The authors declare that they have no competing interests.YL and JL conceived of the study and its design, YL analyzed the data and wrote the manuscript. JNF helped with the long-term mean SBP calculation; HQL and HL processed the genotype data. All authors read and approved the final manuscript."}
+{"text": "CDH7 and CDH19. The locus at 4q22.1 overlaps the previously identified region of musical aptitude, music perception and performance giving further support for this region as a candidate region for broad range of music-related traits. The other regions at 18q21 and 16p12.1-q12.1 are also adjacent to the previously identified loci with musical aptitude. Pathway analysis of the genes suggestively associated with composing suggested an overrepresentation of the cerebellar long-term depression pathway (LTD), which is a cellular model for synaptic plasticity. The LTD also includes cadherins and AMPA receptors, whose component GSG1L was linked to arranging. These results suggest that molecular pathways linked to memory and learning via LTD affect music-related creative behaviour. Musical creativity is a complex phenotype where a common background with musicality and intelligence has been proposed. Here, we implicate genetic regions affecting music-related creative behaviour, which also include genes with neuropsychiatric associations. We also propose a common genetic background for music-related creative behaviour and musical abilities at chromosome 4.Creative activities in music represent a complex cognitive function of the human brain, whose biological basis is largely unknown. In order to elucidate the biological background of creative activities in music we performed genome-wide linkage and linkage disequilibrium (LD) scans in musically experienced individuals characterised for self-reported composing, arranging and non-music related creativity. The participants consisted of 474 individuals from 79 families, and 103 sporadic individuals. We found promising evidence for linkage at 16p12.1-q12.1 for arranging , 4q22.1 for composing  and Xp11.23 for non-music related creativity . Surprisingly, statistically significant evidence for linkage was found for the opposite phenotype of creative activity in music  at 18q21 , which contains cadherin genes like  Creative activities in music represent cognitive functions of the human brain. A creative performance can be defined as a production of work or performance that is both original and appropriate for the situation in which it occurs \u20133. CreatCreative activities in music  can also be considered as a component of musical aptitude . MusicaCompositional processes have been studied, and composition research has been based on various theoretical foundations . HoweveThe biological background of creativity and insight in music have been studied via neuroimaging experiments: using functional magnetic resonance imaging (fMRI) during composing , completDRD2 has been linked to ideational fluency , 4935, 4elopment . Nearby families  suggesti-8; www.qiagen.com/ingenuity), of which 17 genes across ten genomic regions were found in our joint LD and linkage analysis  pathway , a locus that is also associated with schizophrenia [Concerning composing, we also found suggestive evidence for joint linkage and LD at ophrenia .CDH7 and CDH19 genes (LOD 3.3), and the best joint LD and linkage was found at rs11152166 , which has previously been linked to major depressive disorder [Unexpectedly, we found evidence of linkage in the group of participants who did not compose or arrange but have practised music, on chromosome 18q21 (LOD 3.09), exceeding the genome-wide significance threshold. The best two-point linkage within the region was found at 18q22.1 near MAP3K7  at 6q15 (\u22126) within the TSHZ2 intron. Among these suggestively associated markers, rs2225994  is located near a set of markers that have been suggestively associated with cognitive capabilities: cognitive performance [Overall, the best joint linkage and LD for NCNA pinpointed rs1528983 near  at 6q15 . The secformance , 54, socformance  and postformance .SYN1, ELK1, KCND1 and SYP) that affect brain function. Synapsin 1 (SYN1) is involved in synaptic vesicle clustering and recycling, neuronal development and synaptogenesis. Mutations in SYN1 have been associated with epilepsy and autism spectrum disorder [ELK1 regulates genes expressed in the striatum, the reward centre of the human brain [KCND1, a voltage-dependent Kv1.4 channel subunit is expressed in the hippocampus, olfactory bulb, and heart [SYP lead to X-linked intellectual disability implicating its involvement in learning and memory.We did find suggestive evidence for linkage at Xp11.23 (LOD 2.50), for non-music related creativity . The lindisorder . ELK1 rean brain . KCND1, nd heart . SYP is nd heart . Mutatio-7) in intron region of STOX1 were suggested (-7) was suggested. This SNP lies in an intron of RAPGEF4 and near the ZAK gene  and at 6q15 for NCNA . However, we were unable to tag significant SNPs within linked regions.This study gives preliminary evidence for the molecular genetic background of creative activities in music. We found suggestive evidence for linkage at 16p12.1-q12.1 (LOD 2.75) for self-reported arranging and at 4q22.1 (LOD 2.15) for self-reported composing and significant evidence at 18q21 (LOD 3.09) for NCNA, the opposite phenotype of creative activity in music. Joint LD and linkage analysis including both families and sporadic individuals suggested that there are also multiple other genomic locations: the best were at 2p24.3 for arranging . LTD is considered to be a cellular model for synaptic plasticity and memory . Neurone et al. . In musie et al. ; it has e et al. , 20 and e et al. . Recentle et al. . All theGSG1L. The AMPAR complex consists of multiple proteins, and its compositions are brain region-specific [GSG1L containing AMPAR complexes are enriched in the cortex and are also found in the hippocampus, striatum and the thalamus [NRG1 that has previously been shown to be associated with divergent thinking, also regulates cerebellar AMPARs activity-dependently [Interestingly, the AMPAR relates also to arranging, where the best two-point linkage was found at specific . GSG1L cthalamus , 68. Thethalamus , 70. Espthalamus ). Additithalamus . Interesendently . Overallendently  is imporCDH7, CDH19 and CDH20 from the cadherin family. Cadherins are calcium-dependent cell adhesion molecules. The CDH7 gene and many other genes from the cadherin family have been associated with neuropsychiatric disorders like schizophrenia ). Also, the region at 14q21.3 that was suggestively associated with NCNA includes alleles with cognitive and psychiatric implications [RAPGEF4 that is known to affect cognitive abilities. Obviously, the relevance of these cognitive and personality linked genes in creativity related traits should be studied in detail.To our surprise, NCNA yielded significant linkage on 18q21. The region includes numerous genes related to brain functions including s et al.  for reviof finch . There iof finch , 77. Theof finch . It is kof finch . Overallications \u201356. On tications ). Whetheications . We hypoUGT8 gene were significantly associated. More recently, we assigned the linkage to 4q22 with musical aptitude using a Bayesian approach [\u03b1-synuclein gene (SNCA) that was found to be one of the most up-regulated genes after listening to and performing music is also located at 4q22 [UNC5C gene. This gene is a netrin receptor for axon guidance during neural development and may affect neuronal cell death in the adult nervous system [FAM49A for arranging is also interesting as there is evidence for its paralog FAM49B in other music-related phenotypes.To date, several genomic approaches have pinpointed chromosome 4q22-23 as the candidate region for music-related traits , 76, 77.approach . Interess system . In concet al. [According to Salimpoor et al.  a composet al.  and audiet al. . Clearlyet al. . Thus, cet al. .et al. [We found a difference in the prevalence of creative activities in music between genders and age groups. As noticed also by Vinkhuyzen et al. , the oldet al. . Thus, wet al. , 84.et al. [There are some limitations in this study. The phenotype was based on self-reported creativity in music, determined using a questionnaire, as no specific test to define the phenotype is available. There might be biases like dishonesty and diversity of the participants in the questionnaire answers as explained by Ukkola et al. . Geneticet al. , which sAccording to Levitin , musicalS1 FigThe number of the \u201cyes\u201d answers for composing, arranging and other creativity are shown here with overlap between the answers. Over half of the \u201cyes\u201d answers between arranging and composing overlapped. Overall, 320 individuals arranged, composed or considered themselves to be creative.(PNG)Click here for additional data file.S2 FigThe population stratification was studied with principal component analysis using EIGENSOFT 6.0.1. Only unrelated individuals  were used. Plots from the top two principal components are here shown for arranging (A), composing (B), NCNA (C) and other creativity (D).(PNG)Click here for additional data file.S3 FigHistograms of age among active and passive subjects in arranging (A) and composing (B). Frequency of the individuals who compose or arrange drops after the age of 32. This age threshold was used to split the data into two liability classes for genetic analyses.(PDF)Click here for additional data file.S4 FigThe frequencies within each age group are shown here for arranging, composing, NCNA and other creativity separately for males and females. The music-related creative activities are more common among music-experienced males than females in all three age groups .(PDF)Click here for additional data file.S5 FigThe chromosomes are shown on the X-axis and results on the Y-axis. The results are given here as\u2013log; the larger values denote stronger association. The best associations remain suggestive; significant results would rise above 8.(PNG)Click here for additional data file.S6 FigThe joint linkage and LD p-values are given on the Y-axis as negative logarithms (Observed). The linkage disequilibrium between the best associated SNP, rs6531144, and the other SNPs are colour-coded according to their pairwise R-squared values (see the figure legend). The light blue curve shows the background recombination rate (HapMap). Genes in the region are shown at the bottom. From them, FAM49A with unknown function is on the same LD block as the associated SNPs.(PDF)Click here for additional data file.S7 FigThe joint linkage and LD p-values are given on the Y-axis as negative logarithms (Observed). The linkage disequilibrium between the best associated SNP, rs6531144, and the other SNPs are colour-coded according to their pairwise R-squared values (see the figure legend). The light blue curve shows the background recombination rate (HapMap). Genes in the region are shown at the bottom. From them, MAP3K7 is nearest of the associated SNPs.(PDF)Click here for additional data file.S8 FigThe joint linkage and LD p-values are given on the Y-axis as negative logarithms (Observed). The linkage disequilibrium between the best associated SNP, rs1417940, and the other SNPs are colour-coded according to their pairwise R-squared values (see the figure legend). The light blue curve shows the background recombination rate (HapMap). Genes in the region are shown at the bottom.(PDF)Click here for additional data file.S9 FigThe joint linkage and LD p-values are given on the Y-axis as negative logarithms (Observed). The linkage disequilibrium between the best associated SNP, rs4972527, and the other SNPs are colour-coded according to their pairwise R-squared values (see the figure legend). The light blue curve shows the background recombination rate (HapMap). Genes in the region are shown at the bottom.(PDF)Click here for additional data file.S1 TableHere are the translations of the original Finnish questions (available from the authors) considering creativity. The questionnaire was available either through the Internet or on paper.(PDF)Click here for additional data file.S2 TableThe best functions are listed here with related genes that were associated in joint LD and linkage analyses (Genes and # genes). Cancer, especially solid tumour, related functions were overrepresented with all phenotypes, but this is highly likely to be due to cancer being overrepresented in expression databases and thus it was not considered as a truly associated function.(PDF)Click here for additional data file."}
+{"text": "Linkage maps are essential tools for the study of several topics in genome biology. High density linkage maps for the porcine autosomes have been constructed exploiting the high density data provided by the PorcineSNP60 BeadChip. However, a high density SSCX linkage map has not been reported up to date. The aim of the current study was to build an accurate linkage map of SSCX to provide precise estimates of recombination rates along this chromosome and creating a new tool for QTL fine mapping.Sscrofa10.2 annotation. The total length of this chromosome was 84.61\u00a0cM; although the average recombination rate was 0.60\u00a0cM/Mb, both cold and hot recombination regions were identified. A Bayesian probabilistic to genetic groups and revealed that the animals used in the current study for linkage map construction were likely to be carriers of X chromosomes of European origin. Finally, the newly generated linkage map was used to fine-map a QTL at 16\u00a0cM for intramuscular fat content (IMF) measured on longissimus dorsi. The sulfatase isozyme S gene constitutes a functional and positional candidate gene underlying the QTL effect.A female-specific high density linkage map was built for SSCX using The current study presents for the first time a high density linkage map for SSCX and supports the presence of cold and hot recombination intervals along this chromosome. The large cold recombination region in the central segment of the chromosome is not likely to be due to structural differences between X chromosomes of European and Asian origin. In addition, the newly generated linkage map has allowed us to fine-map a QTL on SSCX for fat deposition.The online version of this article (doi:10.1186/s12863-014-0148-x) contains supplementary material, which is available to authorized users. Linkage maps are key tools to genetically map and dissect complex traits, as well as for the study of several topics in genome biology such as the molecular basis of recombination and evolutionary genomics . Interesinactive X specific transcripts (XIST), androgen receptor (AR) and thyroid-binding globulin (TGB), and over 370 QTLs for productive and reproductive related traits have been reported on this chromosome . However, the location of these QTL is not precise, due to the low density of the available linkage map. In spite of its relevance, the highest density linkage map for the porcine X chromosome to date includes only 60 markers [The X chromosome plays an important role in the evolution of human and animals , and expHigh density genetic linkage maps are not only essential for QTL fine-mapping, they are also needed to successfully identify functional and positional candidate genes that may carry causal mutations. Therefore, the aim of the current study was to construct a high density linkage map of the SSCX, obtaining precise estimates of the recombination rate along this chromosome. Furthermore, we have employed the new dense marker linkage map to identify possible QTL for several production and meat quality traits in an experimental Iberian x Landrace cross.Sscrofa10.2 genome assembly [The animals used in the current study belong to three generations of an experimental Iberian x Landrace cross, the so-called IBMAP pedigree . Brieflyassembly  were rethttp://www.animalgenome.org/bioinfo/tools/share/crimap/) was used for linkage map construction. The order given to the SNPs followed the physical order of the Sscrofa10.2 assembly. Note that possible errors in the Xq tail assembly (from 125\u00a0Mb in Sscrofa9 version corresponding to 144\u00a0Mb in Sscrofa10.2 version) have been reported [The linkage map was built employing exclusively the female genotypes and using those high-quality SNPs with a minor allele frequency higher than 0.15   [Correlations between recombination rate and SNP number, GC content (%GC) and gene content were examined. The SNP number, %GC and gene content were calculated along the X chromosome in 1\u00a0Mb non-overlapping windows. The gene content was estimated using the porcine genome annotation biomart)  and the To infer the origins of the chromosome X segregating in the mentioned IBMAP experimental cross, 100 animals with available genotypes from the Porcine60SNP BeadChip [The SNPs contained within the pseudoautosomal (PAR) and non-pseudoautosomal (NPAR) regions of X chromosome were differentiated following Burgos-Paz et al.  criterian\u2009=\u200955) and BC2 (n\u2009=\u200979) generations of the IBMAP experimental cross. The BC1 pigs were not included in the QTL scan as the segregating X chromosomes were exclusively of Landrace origin (F1 boars (\u2642 Iberian x \u2640 Landrace) x Landrace sows). A summary of the phenotypic traits analyzed is shown in Table\u00a0A QTL scan on SSCX was conducted for growth, fatness, intramuscular fat content (IMF) and body conformation traits recorded on 134 fattened pigs from the F3  and generation/cross (three levels), uk is the random polygenic effect of the kth individual, xk is a covariable  and b its respective slope, a is the QTL additive effect; Pak is the probability of the kth individual having an allele of Iberian and eijk is the random residual. The probabilities P were obtained using a modification of the MCMC algorithm described by P\u00e9rez-Enciso et al. [A\u03c32u, A being the numerator relationship matrix.The QTL scan was performed with the following basic model:o et al.  allowingP-values were calculated assuming a \u03c72 distribution of the LRT with the degrees of freedom given by the difference between the number of estimated parameters in the reduced and full models. Note that permutation techniques cannot be used to calculate chromosome-wide P with animal models because the pedigree structure is broken. It is assumed that a nominal 10\u22123P-value is equivalent to a 1% chromosome-wide level [Likelihood ratio tests (LRT) were calculated comparing the full model and a reduced model without the corresponding QTL effect. The nominal de level . All thede level . The conde level Sscrofa10.2 annotation , comprising the centromere, showed almost no recombination events (average recombination rate\u2009=\u20090.05\u00a0cM/Mb). Note that few useful SNPs were available in this interval not only in the selected set for the analyzed population , recombination rate and % GC was 0.20  and recombination rate and gene content was 0.01 . The only chromosomal feature significantly correlated with the recombination rate was to the GC content. The regions displaying higher recombination rates tended to show enriched GC sequences, in agreement with the tendency observed in autosomes [In order to analyze the chromosome features influencing recombination rate on the X chromosome, the correlations between recombination rate and SNP number, GC content (%GC) and gene content were calculated. The correlation between recombination rate and SNP number was \u22120.14 , located at 4.1\u00a0Mb on SSCX and involved in energy metabolism regulation and insulin sensitivity [The confidence interval of the QTL for IMF was 13\u201320\u00a0cM, which corresponds to 3.5-5\u00a0Mb. The QTL falls within the PAR and outside of the cold and hot recombination intervals. This region contains only three protein-coding genes, including an interesting candidate gene for fat deposition, which require further investigation, the sitivity .The present study presents for the first time a high density linkage map for SSCX and supports the existence of cold and hot recombination regions. The large cold recombination region in the central segment of the chromosome is not likely to be due to structural differences between Asian and European X chromosomes as previously hypothesized. The new generated linkage map has allowed us to identify a QTL for IMF content, an important meat quality parameter."}
+{"text": "Lucania parva and L. goodei, that differ in salinity tolerance and still hybridize in their contact zone in Florida. Using SNPs from orthologous EST contigs, we compared synteny between the two species to determine how genomic architecture has shifted with divergence. Karyotyping revealed that L. goodei possesses 24 acrocentric chromosomes (1N) whereas L. parva possesses 23 chromosomes (1N), one of which is a large metacentric chromosome. Likewise, high-density single-nucleotide polymorphism\u2212based linkage maps indicated 24 linkage groups for L. goodei and 23 linkage groups for L. parva. Synteny mapping revealed two linkage groups in L. goodei that were highly syntenic with the largest linkage group in L. parva. Together, this evidence points to the largest linkage group in L. parva being the result of a chromosomal fusion. We further compared synteny between Lucania with the genome of a more distant teleost relative medaka (Oryzias latipes) and found good conservation of synteny at the chromosomal level. Each Lucania LG had a single best match with each medaka chromosome. These results provide the groundwork for future studies on the genetic architecture of reproductive isolation and salinity tolerance in Lucania and other Fundulidae.Linkage maps are important tools in evolutionary genetics and in studies of speciation. We performed a karyotyping study and constructed high-density linkage maps for two closely related killifish species,  Linkage maps allow the exploration of genotype-phenotype relationships through quantitative trait loci mapping  and the rainwater killifish (L. parva) are two closely related species that differ radically in their salinity tolerance  karyotyped both species and (2) produced two high-density single-nucleotide polymorphism (SNP)-based linkage maps. Transcriptome sequencing and high-throughput SNP genotyping were performed in both species to create the linkage maps. These data allowed us to determine (1) the number of chromosomes possessed by each species, (2) whether a fusion/fission event had occurred, and (3) patterns of synteny between the two species. We analyzed synteny at the linkage group (LG) level and at the level of marker order to determine whether large or small-scale genomic rearrangements have occurred during divergence  to ask how Lucania genomic architecture corresponds to that of other teleost fish , then fixed in a 3:1 methanol/glacial acetic acid mixture (30 min). The gills were dabbed on the slides to break the cells and release the chromosomes. Slides were cleared, dried, stained with 5% Giemsa solution , mounted with Permount, and visualized using a compound microscope.Somatic karyotypes were determined for both L. goodei and L. parva from F2 mapping crosses between two geographically isolated populations. The key to comparing the genetic linkage maps between the two species was to use SNP markers from orthologous expressed sequence tags  that were expressed in both species  and a swamp population in the Everglades . For Lucania parva, the populations were Indian River Lagoon, an Atlantic coast population where salinity is typically 35 ppt  and Pecos River, a freshwater inland river in Texas . Between population breeding pairs were established in both hybrid cross directions. Cross designs for the creation of the F1 parents are described for L. goodei in L. parva in 1 hybrid offspring were then raised to adulthood and paired with unrelated F1 hybrid individuals to create F2 offspring. For L. parva, we created and genotyped 161 F2 offspring from 8 F2 families and 16 F1 parents .For each species, crosses were set up between geographically and ecologically divergent populations. For 2 eggs were collected and raised in freshwater with dilute methylene blue (an antifungicide). Fry were fed newly hatched Artemia and raised to 1 month postfertilization. The fry were then killed in MS-222, preserved in ethanol, and stored at \u221280\u00b0. F1 parents and F0 grandparents were preserved in ethanol. F2 family sizes ranged from 15 to 24 fry.FL. goodei populations (Upper Bridge and Everglades) and the two L. parva populations (Indian River and Pecos River). Fish were killed in MS-222. Tissue samples were taken from the gills (1\u22122 arches), dorsal fins, eyes, brain, and the gonads (ovaries or testes). RNA was extracted using a protocol modified from File S1). A pooled sample containing RNA from all tissues was created for each population that contained equal amounts of RNA from all individuals. The RNA was submitted to the Keck Center for Comparative and Functional Genomics at the University of Illinois for creation of cDNA libraries and sequenced using Illumina HiSequation 2000 (see File S1). The two populations from L. goodei were uniquely barcoded and run on a single lane of Illumina HiSeq for 100-bp paired-end sequencing. Indian River L. parva and Pecos River L. parva were run on separate lanes. Average quality scores were above 20 for all cycles. Samples produced from 5 to 10 billion bases of data . The population specific Illumina transcriptome sequences are archived in Genbank (bio-project ID: PRJNA215087).RNA was extracted from five males and five females from the two L. goodei populations across Florida . These sequences were used to construct a reference upon which subsequent assembly was based . Tissue samples were taken from the gills, fins, eyes, brain, and gonads (ovaries or testes). Additional details on the 454 project can be found in In addition, 454 sequencing was done on pooled samples of RNA from five Lucania goodei 454 sequences were used as a reference for the assembly. For the reference, contigs were assembled using Newbler assembler . Assembly parameters were as follows: the minimum contig length was set at 200 bp, the minimum overlap length was 60 bp, and the minimum overlap identity was 95%. A total of 29,838 contigs were generated by the Newbler assembler. Using the L. goodei 454 contigs as a reference anchored the analysis of shorter Illumina sequences and allowed identification of contigs that contained multiple SNPs. The goal was to find contigs containing a SNP that was diagnostic for the two L. goodei populations as well as a SNP that was diagnostic for the two L. parva populations. This method may have missed L. parva contigs that were not expressed in L. goodei, but these contigs are uninformative for the comparison of linkage maps. Illumina sequences were trimmed to 75 bp in length and aligned against the reference using Novoalign . Alignment parameters were as follows: the maximum alignment score acceptable for a best alignment was set at 45; the gap extend penalty was set at 10; and the number of good quality bases for an acceptable read was set at 50.i.e., Mapping and Assembling with Quality) software  between the two species. The between species SNPs were designed for another study. The custom Infinium bead chip held probes for 4545 SNPs. Of these, 1497 were candidate SNPs for L. goodei, 1369 were candidate SNPs for L. parva, and 1679 were candidate between species SNPs. All SNPs were labeled with the ID number of the contig in which they occur. Protein annotations for the linkage map contigs were obtained using blastX searches against teleost reference proteomes  and human complete proteome (Uniprot release 2012_8 available at: ftp://ftp.uniprot.org/pub/databases/uniprot/previous_releases/release-2012_08/uniref/). Only contigs that matched a single protein in each species with a blast score >100 were considered high confidence annotations (Table S1). Proteins that matched multiple contigs located on different LGs were removed from the annotations.Candidate SNPs were submitted to Illumina for initial evaluation for suitability for the Infinium Genotyping Assay. There were three classes of SNPs: www.gentra.com) extraction protocol over 4 days (see File S1). Sample concentration and quality were verified using a Nanodrop spectrophotometer . DNA samples were diluted to 75 \u00b5g/\u00b5L in nuclease-free water and then genotyped at all SNPs using the Lucania Illumina Infinium Bead Chip. Source and probe sequences from the chip are accessible on Dryad (http://datadryad.org/resource/doi:10.5061/dryad.hv75h/9). Bead chips were scanned using the iScan System (Illumina) at the Keck Center for Comparative and Functional Genomics at The University of Illinois. Illumina GenomeStudio (v2011.1) was used for genotype calls. Cluster positioning was performed separately for L. goodei and L. parva SNPs . Population-specific SNP alleles were verified from genotypes of 16 Upper Bridge L. goodei , 17 Everglades L. goodei , 6 Indian River L. parva , and 5 Pecos River L. parva . Genotype data were analyzed separately for each family. For each family, SNPs were removed if either (1) genotypes were homozygous in both parents thereby guaranteeing all offspring were homozygotes, or (2) the genotype was a no-call  for one or both parents. Not all SNPs were fixed between the two L. goodei populations, so we also genotyped the F0 grandparents to clarify phases.DNA was extracted using a modified version of the PureGene  using JoinMap 4.1 (L. goodei) and 3.5 (L. parva) were used and markers significantly out of Hardy-Weinberg equilibrium (P < 0.001) were excluded. The Kosambi mapping function .For each species, individual maps were constructed for each individual Ffunction  was usedfunction  was usedL. parva and L. goodei, SNPs designed from a common contig were considered to be putatively orthologous. Consistent clusters of SNPs from common contigs in both species provided evidence of synteny at the LG level . Within LGs, marker order was compared among orthologous SNPs by performing rank order correlations between species. Lucania goodei and L. parva LGs also were compared with chromosomes from medaka (Oryzias latipes), the most closely related species with a fully sequenced genome [both are in the superorder Acanthopterygii (Lucania linkage maps were blasted against medaka sequences using blastn. If a given contig had highly significant blast hits (bit score > 100) against a single medaka LG, then its approximate position was estimated in the medaka genome. However, if a given contig had multiple, highly significant blast hits on multiple medaka LGs, then the orthologous location in the medaka genome could not be determined. Hence, synteny was examined at the level of LG clustering for the L. goodei and L. parva, L. goodei and medaka, and L. parva and medaka comparisons. Synteny was examined at the level marker order for only the L. goodei and L. parva comparison. False-discovery rate P-values were calculated for rank order correlations using fdrtool in R . Betta splendens was used as a standard.The genome size of both species was estimated using flow cytometry to determine how much recombination was occurring per megabase. Four Lucania species differed in chromosome number. Lucania parva had 23 chromosomes . The number of markers per linkage group (LG) ranged from 18 (LG 20) to 59 (LG 1). The total length of the map was 605 cM with the average LG being 26.3 cM (L. parva was 1.423 pg (range: 1.396-1.450). One centimorgan in L. parva is thus approximately 2.3 Mb.The number of chromosomes corresponded well to the number of LGs that were recovered. For o 23 LGs . Many of 26.3 cM . Marker L. goodei matched the number of chromosomes (1N = 24). Significant linkages were found for 915 SNP markers making up 24 LGs (Table S1). The number of markers per LG ranged from 4 (LG 21) to 66 (LG 2). The linkage map spanned 392 cM with the average LG size being 16.33 cM (L. goodei was 1.349 pg (range: 1.342-1.356). One centimorgan in L. goodei is thus approximately 3.44 Mb.Similarly, the number of LGs recovered for p 24 LGs . Again, 16.33 cM . AverageL. goodei and L. parva, allowing us to unambiguously assign orthologous LGs. Across all LGs, we found 368 markers shared between the linkage maps that were from putatively orthologous ESTs. L. goodei and L. parva. Specifically, 364 (98.91%) markers (those from a common contig and in both linkage maps) showed a pattern of synteny, whereas only 4 markers (1.09%) deviated and clustered differently in the two species .The LGs were highly syntenic between L. goodei were syntenic with the largest LG in L. parva, strongly suggesting that LG 1 represents the metacentric chromosome observed in the L. parva metaphase spread. L. goodei was syntenic with the top portion of LG 1 in L. parva . When rank order correlations were performed on LGs separately , marker order was significantly correlated in 11 of 23 syntenic LGs . Medaka has 24 chromosomes, and all Lucania LGs had a single best match to a medaka chromosome (listed at the bottom of L. goodei and medaka 559 of 585 (95.6%) orthologous markers were syntenic at the LG level. Similarly, L. parva had 532 of 545 (97.6%) orthologous markers that were found to be syntenic with medaka chromosomes. LG 1A in L. goodei corresponded to chromosome 3 in medaka and LG 1B corresponded to medaka chromosome 11.Synteny at the LG level was also well preserved between Lucania parva and L. goodei with high marker density (1.26 markers/cM in L. parva and 2.41 markers/cM in L. goodei). These linkage maps establish genomic resources for Lucania and provide the groundwork for future linkage disequilibrium studies, quantitative trait loci mapping, molecular population genetic studies, and further synteny comparisons with other teleost species. The fact that many of these SNPs came from ESTs whose protein functions are known in other groups allows us to estimate the position of functionally important loci in Lucania (see Table S1). This is the first linkage map for any member of the Fundulidae family, a group that exhibits an extraordinary ability to tolerate and adapt to physiological extremes  is syntenic with two smaller L. goodei chromosomes (LG 1A and 1B). Comparisons of interspecific linkage maps have previously been used in other taxa to identify chromosomal rearrangements between species . First, Fundulus parvipinnis is the closest relative to Lucania  that L. parva\u2019s karyotype of 1N = 23 deviates from the typicial fundulid karyotype of 1N = 24  is unique among related fish species for which linkage maps exist. Guppies (Poecilia reticulata) also possess a fused chromosome, but it has occurred between two chromosomes that are syntenic to medaka 2 and 21 (Oreochromis niloticus) possess two fused chromosomes: one between chromosomes that are syntenic to medaka 2 and 4, and another between chromosomes syntenic to medaka 6 and 12  were collinear (Oncorhynchus) also suggest high preservation of marker order between congeners (Lucania is genuine or simply the result of (1) low number of orthologous markers on some linkage groups, (2) genotyping errors, or (3) map construction errors due to merging maps from multiple families within each species. If genuine, then the low marker-order synteny found in Lucania would be indicative of small-scale rearrangements, which could potentially contribute to the reduction of gene flow and the evolution of reproductive isolation.Our comparisons of synteny at the marker order level revealed significant marker order preservation in only half of the linkage groups between Lucania goodei and L. parva showed that a large-scale genomic rearrangement has occurred between species. This Robertsonian fusion may have aided divergence in these closely related species and helped to maintain species boundaries in zones of contact by suppressing recombination in hybrids. Other fundulid species also differ in karyotype, and our work may give insight into the mechanisms by which these changes occur. These maps will enable the use of numerous genomic techniques to determine how reproductive isolation has evolved in Lucania. The maps will also further a general understanding of the evolution of many other unique traits in this group including vision, color pattern, and salinity tolerance.Our linkage maps for"}
+{"text": "Nonsyndromic orofacial clefts are one of the most common birth defects worldwide. It occurs as a result of genetic or environmental factors. This study investigates the genetic contribution to nonsyndromic cleft lip and/or palate through the analysis of family pedigrees. Candidate genes associated with the condition were identified from large extended families from the Malay population.LPHN2 at 1p31, SATB2 at 2q33.1-q35, PVRL3 at 3q13.3, COL21A1 at 6p12.1, FOXP2 at 7q22.3-q33, FOXG1 and HECTD1 at 14q12 and TOX3 at 16q12.1.A significant nonparametric linkage (NPL) score was detected in family 100. Other suggestive NPL and logarithm of the odds (LOD) scores were attained from families 50, 58, 99 and 100 under autosomal recessive mode. Heterogeneity LOD (HLOD) score\u2009\u2265\u20091 was determined for all families, confirming genetic heterogeneity of the population and indicating that a proportion of families might be linked to each other. Several candidate genes in linkage intervals were determined; We have identified several novel and known candidate genes for nonsyndromic cleft lip and/or palate through genome-wide linkage analysis. Further analysis of the involvement of these genes in the condition will shed light on the disease mechanism. Comprehensive genetic testing of the candidate genes is warranted. Orofacial clefts are congenital malformation that comprises of a large fraction of human birth defects that affects the lip and/or palate. Orofacial clefts are one of the most common human congenital disorder reported in Western countries and second most common birth defects among newborn , 2. It aClefts can be categorized into syndromic and isolated forms, according to whether affected individuals have other physical and developmental anomalies. The focus of this study was on nonsyndromic clefts. Nonsyndromic cleft lip with or without palate (NSCLP) is a malformation disease that does not show any other signs or symptoms of abnormal condition such as abnormal physical appearance or psychological disorder. Nonsyndromic cases for both cleft lip with or without cleft palate are heterogenous with multifactorial etiology, in which both genetic and environmental factors take place . ContribLinkage analysis of multiplex families and association studies using either case\u2013control or family-based designs has become primary methods in identifying potential genes for CLP . The firEven after many years of carrying out investigation, the mode of inheritance of CLP remains controversial and challenging that were probably due to the samples and models employed . LinkageEight large extended families with 91 individuals were included, comprised of \u22653 affected members in each family. The blood withdrawal was undertaken either by appointment at clinic or home visit. All individuals have been referred to the craniofacial specialist. This study was approved by the Research Ethics Committee (Human) of the Universiti Sains Malaysia, Health Campus, Malaysia; Reference No.: USMKK/PPP/JEPeM [258.3.(3)] and written informed consent for blood withdrawal was obtained from the participants or their parent for the patients below 16 years old. Informed consent for publication permission submitted nationally or internationally with regards to any data output, personal information or family background was also obtained from all the participants or their parent for the patients below 16 years old.All families either having cleft lip (CL), cleft lip and palate (CLP) or cleft palate (CP) were all included in this study. Patients were screened by specialists of plastic surgery for any major abnormalities or syndromes. Patients with minor anomaly such as low-set ears, hypertelorism, clinodactyly and single palmar crease were included. Meanwhile, if the patients have a heart disease, any syndromes associated with cleft, or a major or more than two minor defects, they would be excluded from this study.260/A280 ratio of 1.7\u20131.9.Genomic DNA was extracted from 200 \u03bcl of heparinized peripheral blood using QIAamp DNA Mini Kit  following the manufactures instruction. The quality and quantity of DNA samples were quantified using Infinite\u00ae 200 PRO NanoQuant (Tecan). The concentration was measured at absorbance of 260/280 nm. Purity was determined by calculating the ratio of absorbance at 260 nm to absorbance at 280 nm. Pure DNA has an AExtracted DNA from eight large extended families was used for microarray analysis using Illumina Infinium Human Linkage-24 Beadchip. All the samples were genotyped at St George\u2019s Hospital, University of London.On day 1, Standard DNA plate (250 ng/\u03bcl) and blank (0 ng/\u03bcl) DNA were prepared and the quantitated DNA was read with Infinium LIMS Database. After that, DNA samples were denatured and neutralized with 4 \u03bcl 0.1 N NaOH followed by vortex the plate at 1600 rpm for 1 min and centrifuge to 280 xg for 1 min. The plate was incubated overnight in the Illumina Hybridization Oven at 37\u00a0\u00b0C.On Day 2, DNA were fragmented and precipitated. Precipitated DNA then were incubated in Illumina Hybridization Oven for 1 h at 48\u00a0\u00b0C. After that, the resuspended DNA samples were dispensed onto BeadChips and incubated into the 48\u00a0\u00b0C Illumina Hybridization Oven for at least 16 h.On Day 3, BeadChips were prepared for staining process. After washing and submerged in PB1, labeled nucleotides were added to DNA to extend the primers. The primers then were stained using a sandwich-staining protocol followed by disassembled the Flow-Through Chambers and coated the BeadChips for protection. Image of the BeadChip were scanned using iScan Reader. Data from the scanned images were analyzed using Illumina\u2019s Genome Studio Genotyping Module. Markers with a minor allele frequency greater than 5\u00a0% and call rate higher than 95\u00a0% were used in the analyses. The data were converted to \u201c.txt\u201d file using Beadstudio program for further analysis by downstream software programs.Easy Linkage Plus v5.08 software was used to enable linkage analyses for large scale SNP data. It allows multipoint simulation studies as well as checking for Mendelian/non-Mendelian genotyping errors and Hardy-Weinberg equilibrium (HWE) . Gene Hup values and cM values. Plots display details of the used inheritance model, marker map, sex specific or sex-averaged marker positions. Plots were generated as \u2018TOTAL\u2019 plots averaging all families or plus individual family plots [Parametric analysis was carried out under both dominant and recessive mode of inheritance. A disease allele frequency of 0.001 and penetrance vectors of 0.99 for homozygous and heterozygous mutant was used for autosomal dominant. Similar allele frequency and penetrance parameters were used for homozygous mutant for autosomal recessive. Nonparametric analysis was carried out to evaluate allele sharing among affected individuals . The outly plots .The thresholds for genome-wide linkage analysis were defined based on criteria proposed by Lander and Kruglyak . ParametQuality controls have been carried out to detect and eliminate errors in both genotype and pedigree data files. Phenotype error is detected when an affected individual misclassified as an unaffected individual or vice versa, which occurred due to sample mixing, inaccurate family information, misdiagnosis and wrong ID number in pedigree file and genotype file. Quality control is mandatory since phenotype errors can highly affect a value of LOD score.PedCheck is used to identify genotype incompatibilities in linkage analysis. It is essential to eliminate all the Mendelian inconsistencies in the pedigree data due to genotyping error or others. Mendelian errors was checked using PedCheck program version 1.1 . PedChecOn the first level, the nuclear-family algorithm uses the known genotypes to check for inconsistencies between parents and offspring. There is no genotype elimination in this level. At the second level, genotype elimination was performed via extended version of the Lange-Goradia algorithm. It uses the nuclear-family relationships to eliminate invalid genotypes in the pedigree. The third level Critical-Genotype algorithm and fourth level of Odds-Ratio algorithm attempts to identify any critical genotypes in the pedigree. The program will not move forward if detect level-1 errors. Therefore, level-1 errors need to be corrected.In addition to PedCheck program, Merlin was used to detect for genotyping errors. Unlikely genotypes are equivalent to double recombinations in a short chromosomal segment. Unlikely genotypes were corrected by Pedwipe program together with Merlin package.Both Illumina 6K Linkage 24 deCODE Human Genetic Map and AFFY 100k Marshfield Human Sex-Averaged from Gene Hunter-Multipoint Linkage Analysis v2.1r5 were used for linkage analysis.An output of linkage analysis was used to determine linkage interval of selected SNPs using National Center for Biotechnology Information (NCBI) web-based. The data from NCBI were transferred into Gene Distiller database. It is used to select and project genes from within a linkage interval, display gene specific information and sort the genes according to phenotype parameter .Eight large extended families consist of total 91 individuals were included. Size of each family was ranged between 9 and 14 individuals  SNPs were eliminated from the analysis.p value\u2009<\u20090.05. The overall call rate was\u2009>\u200998\u00a0% for all samples. Suggestive and significant linkage intervals were yielded with NPL score\u2009>\u20092 among the families using both genetic map. The output of total families has reached to the level of suggestive linkage for chromosomes 2q33.1-q35, 4q22.1-q25, 7q22.3-q33, and 14q12. However, by analyzing each family, family 100 was the only family that reached a significant linkage level with NPL scores of 3.67\u20133.68, p value\u2009<\u20090.05 (p\u2009=\u20090.02) on chromosome 1, 13 and 22. Meanwhile, other linkage intervals in family 50 and family 58 showed suggestive linkage as shown in Table\u00a0All the subjects were genotyped using Illumina 6K Linkage 24 deCODE Human Genetic Map and the AFFY 100k Marshfield Human Sex-Averaged. All markers were in Hardy-Weinberg equilibrium (HWE) Under the recessive model, suggestive linkage peaks were obtained in family 99 at one linkage interval and in family 100 at six linkage intervals Table\u00a0. The genNo linkage peak was obtained under autosomal dominant with assumed parameters neither using Illumina 6K Linkage 24 deCODE Human Genetic Map nor AFFY 100k Marshfield Human Sex-Averaged. Family 99 was the solo family resulted in a maximum LOD score of 1.51 at rs39552 at position 114.94 cM on 16q23.3-q24.1. Linkage intervals for chromosomes 4q22.3  and 17q22  reached HLOD \u22651 in total families.Both nonparametric and parametric analysis methods were applied to the data with an assumption of the presence of both autosomal dominant and recessive inheritance. As the sample size was not large, both types of analysis were carried out to ensure reliability and accuracy of the results. Parametric analysis with LOD score requires specified mode of transmission of all loci to avoid erroneous results. Other alternative methods of linkage analysis have been developed and are known as nonparametric. Nonparametric analysis is beneficial as it does not require specified values in the transmission model. Generally, based on linkage analysis, the most probable mode of inheritance among families is autosomal recessive. Furthermore, analysis of family pedigrees has found parent-offspring inheritance for NSCLP; 33.3\u00a0% of cases were autosomal dominant and 66.7\u00a0% were autosomal recessive. Autosomal dominant occurs when a disease allele from an affected parent is transmitted to the offspring and the offspring suffers from the disease and tends to occur in every generation of an affected family. Conversely, autosomal recessive inheritance assumes that both parents are unaffected but that both carry the disease allele. It is not likely to occur in every generation of an affected family. The overall analysis through family tree leads to a conclusion of autosomal recessive inheritance which most probably occurred since the normal phenotype could not be distinguished as a carrier or non-affected person.The data set produced from both genetic maps was helpful in exploring larger probability of genes involved in clefting. Some produced data were found overlapped between the two genetic maps but some findings are exclusive to each genetic map. All interested findings were combined for better understanding of linkage distribution. Several linkage intervals attained the suggestive statistical threshold in NPL analysis in total families on locus 2q33.1-q35, 4q22.1-q25, 7q22.3-q33, and 14q12. However, the analysis of each family found that family 50 and family 58 attained several suggestive linkage thresholds while family 100 attained significant linkage of NPL scores on locus 1p31.1-31.3, 2q31.1-q35, 7q22.3-32.2, 8q22.1-q23.3, 11p15.3-p13, 13q22.2-q31.1, 14q12-q21.1 and 22q11.1-q11.21. Similar NPL scores was found in family 100 which means that it has higher probability of mutation harbored in larger linkage intervals since they have been intact throughout generations [Parametric analysis for autosomal recessive that reached LOD score at suggestive level was found in family 99 and family100. Suggestive linkage was attained at locus 3q13.3-q13.33 in family 99 and the rest 1p31.1, 2q31.2-33.3, 8q22.1 and 11p15.1 in family 100. Nonsyndromic clefting is known to be genetically heterogenous, therefore susceptible genes may vary in each family.LPHN2).Linkage interval in the locus of 1p31.1-p31.3 has attained significant NPL and suggestive LOD score in family 100 and suggestive linkage in family 50 with HLOD\u2009=\u20091.73, \u03b1\u2009=\u20090.308 under recessive model. Positive \u03b1 indicates that a subset of families might be linked to this chromosomal region. Previous study has reported that HLOD can provide robust and powerful tool for detection of linkage in the presence of heterogeneity . FindingLPHN2  in 1p31 region in human craniofacial development so far. LPHN2 is a tumor suppressor genes that was found within oral submucous fibroris [Several studies have documented their findings regarding 1p chromosomal region. A novel homozygous intergenic deletion of 237-kb at 1p31 was reported in four affected siblings of consanguineous marriage and 1p31 is an autosomal recessive locus . It was fibroris .SATB2) and Small ubiquitin-like modifier 1 (SUMO1). The role of SATB2 in craniofacial development has been discussed widely. Strong expression of SATB2 was detected during palatal shelves development with maximum expression in the mesenchyme underlying the medial edge epithelia [SATB2 mutation.The linkage interval in 2q31.1-q35 has shown one of the highest significant NPL score in total data set and in family 100 and suggestive NPL score in family 50. This chromosomal region also attained a suggestive LOD score in family 100 and HLOD score of 2.63, \u03b1\u2009=\u20090.124 under autosomal recessive mode of inheritance for total family. Several genes attained between 2q31.1-q35 linkage intervals may play a role in craniofacial development. However, the most common genes associated with craniofacial development were Special AT-rich sequence-binding protein 2  gene related to orofacial cleft. S\u00f6zen et al., [PVRL3 and concluded that no variant mutations found in the selected regions in Caucasian polpulations. However, there is possibility that the variants were not detected by the SSCP/heteroduplex screening method used [COL21A1  that have never reported associated with craniofacial development. COL21A1 is a member of FACIT collagen family (fibril-associated collagens with interrupted helices). Type XXI collagen is localized to tissues containing type I collagen. Therefore, like other members of this collagen family, it serves to maintain the integrity of the extracellular matrix. However, two previous studies have reported haplotype in COL11A2 at 6p21.3 associated with nonsnyndromic cleft [Suggestive linkage of LOD score in family 99 and HLOD \u22651 in total family for 3q13.3-13.33 chromosomal region brought to the interest of this study, with the findings of Poliovirus Receptor-Related 3  was found among 36 multiplex Chinese families [FOXG1) and HECT domain containing E3 ubiquitin protein ligase 1 (HECTD1) gene found within the chromosomal region of 14q12 in family 58 and family 100, which play a role in craniofacial development. TOX High Mobility Group Box Family Member 3 (TOX3) found in the chromosomal region of 16q12.1. Microdeletion in this region has led to a syndromic disease [The 14q12 linkage interval was significant in LOD analysis and suggestive in NPL analysis. Role of 14q in the etiology of oral clefts has been well known and this chromosome contains several oral cleft candidate genes such as  disease . It was  disease .LPHN2 at 1p31, PVRL3 at 3q13.3, COL21A1 at 6p12.1 and TOX3 at 16q12.1 and known genes SATB2 would shed more light on the disease mechanism that has been discovered through genome-wide linkage analysis. A comprehensive investigation of the candidate genes in orofacial clefts is warranted.A high degree of heterogeneity and linkage was detected in family 50, 58, 99 and 100 from the Malay population. Significant NPL score was detected primarily on family 100 thus assuming high gene linkage in this family. The novel findings of The microarray data for the all families are available by the following doi:10.6070/H41C1TWH that was deposited in LabArchives ."}
+{"text": "EVC, EVC2, SLC2A9, NKX3-2, and HMX1. The coding regions of three genes, EVC known for cartilage development and NKX3-2, HMX1 involved in microtia, were selected for sequencing with 5 individuals from the pedigree. Of the 38 identified sequence variants, none segregates along with the disease phenotype. Other genes or DNA sequences of the 10-Mb region warrant for further investigation. In conclusion, we report a susceptibility locus of isolated microtia, and this finding will encourage future studies on the genetic basis of ear deformity.Microtia is a congenital deformity where the external ear is underdeveloped. Genetic investigations have identified many susceptibility genes of microtia-related syndromes. However, no causal genes were reported for isolated microtia, the main form of microtia. We conducted a genome-wide linkage analysis on a 5-generation Chinese pedigree with isolated bilateral microtia. We identified a suggestive linkage locus on 4p15.32\u20134p16.2 with parametric LOD score of 2.70 and nonparametric linkage score (Zmean) of 12.28 . Haplotype reconstruction analysis of the 4p15.32\u20134p16.2 region further confined the linkage signal to a 10-Mb segment located between rs12505562 and rs12649803 . Various human organ developmental genes reside in this 10-Mb susceptibility region, such as  Microtia includes a spectrum of congenital anomalies of the auricle, ranging from mild structural abnormalities to complete loss of the auricle (termed anotia) HOX gene family, SIX gene family, EYA, TBX1, IRF6, CHUK, and GSCHOXA2 gene were identified in syndromic microtia families Microtia has heterogeneous natures in both etiology and pathology. Strong evidence showed the involvement of the genetic and environmental factors, as well as their combined effects in the disease Linkage analysis is a classic and effective way for mapping Mendelian disorders. In recent years, despite the identification of many gene variants involved in diseases through genome-wide association studies, the inability of these variants to account for much of the heritability of common disorders has led to a renewed interest in linkage analysis and other family-based methods Written informed consent forms were obtained from all individuals  for genetic and biological investigations. This study was reviewed and approved by the ethics committee of the Beijing Institute of Genomics, Chinese Academy of Sciences and Plastic Surgery Hospital, Chinese Academy of Medical Science.We recruited a five-generation pedigree with isolated bilateral microtia from Zhejiang, China . All of All 14 DNA samples were extracted using DNA-extraction kits  and genotyped on the Human Omni-Zhonghua chips  according to the manufacturer's specifications. Genotyping module of Genomestudio v3.0  was employed to call the genotype of 900,000 SNPs. The genotypes status was estimated conditionally on the fluorescent signal and standard cluster file provided by Illumina. All DNA samples were successfully genotyped at call rate >99.7% with genotype call threshold  of 0.15. We removed SNPs  that could not be accurately clustered or were located on sex chromosomes, and obtained 873,539 SNPs for subsequent linkage analyses. We used cnvPartition 3.1.6 to detect the copy number variation (CNV) with parameters of log R ratio (logged ratio of observed probe intensity to expected intensity) and B allele frequency . Only CNVs detected with more than 5 probes with confidence scores greater than 35 were considered.2, was greater than 0.5, c) shifted the window 5 SNPs forward and repeated the procedure. A total of 23,139 SNPs were obtained after the sliding window test. Then, the Mendelian consistency was checked on the genotype data of each SNP with Pedcheck v1.00 To prevent false positive linkage signals due to strong linkage disequilibrium (LD) between adjacent SNP markers, we used a sliding-windows method in Plink Power calculation was performed by SLINK (v3.00) with 1000 replicates, assuming the above autosomal-dominant model P value with a correction factor derived from the length of a simulated chromosome over the genome length in centimorgans .To determine the genome-wide significance level of the linkage signal, we performed simulation studies of 5000 replicates generated with the gene-dropping method in MERLIN. The parameters of marker allele frequency, genetic distance, pedigree structure, and missing data pattern were retained in each simulation. For individual chromosome, the proportion of simulations exhibiting equal or greater linkage scores was used to estimate the frequency of the observed linkage signal occurring by chance. Genome-wide significance levels were calculated by multiplying the chromosome-wide EVC, NKX3-2, and HMX1, were amplified. Sequencing reactions using Applied Biosystems Big Dye Terminator chemistry were resolved on ABI Prism 3730XL DNA Analyzers . Phred/Phrap/Polyphred/Consed  software was used to analyze sequence files and to call SNPs. The base-quality value threshold was set to 20 . All polymorphic sites were manually inspected.Candidate gene sequencing was conducted with three affected and two unaffected individuals  from the pedigree. The coding regions of three candidate genes, Our proband (6 years old) was identified as bilateral microtia. A visual malformation of external ear without other accompanying facial malformations was observed in the proband. The upper part of the affect ear was smaller than normal. Her two deformed ears measurements were same and as follows: ear length, 3.9 cm; ear width, 2.1 cm. The structure of crus helicis, cavity of concha, tragus and ear lobe were as normal, but triangular, scaphoid fossae and antihelix were absent, leaving a shell-like appearance with 130 degree of the auriculocephalic angle.Through the proband, we identified a large microtia pedigree, which composed of 51 living individuals with 23  affected. All affected individuals have bilateral microtia in concha-type and the remnant external ear showed similar shell-like appearance. Only slight variation in the degree and symmetry of the affected auriculocephalic angle, auricular triangular, scaphoid fossae, and antihelix structures were observed in the affected individuals. They have no other accompanying facial malformation or hereditary disease, such as hearing loss, craniofacial deformity, and congenital heart disease. The characteristics of the affected individuals are summarized in 2<0.5) with others. Both the parametric and nonparametric analyses indicated the highest linkage scores on chromosome 4p15.32\u20134p16.2 . According to Lander & Kruglyak We performed a genome-wide linkage analysis on this isolated microtia pedigree using a pruned set of SNPs, removal of SNPs generating strong pairwise LD  in 14 genotyped samples . Eight CEVC, EVC2, HMX1, HTRA3, NKX3-2, CC2D2A, CRMP1, DRD5, and SLC2A9. Of the nine genes, EVC, EVC2, NKX3-2, and HMX1, are closely related to facial development. SLC2A9 plays roles in the development and survival of chondrocytes in cartilage matrices.There are 47 known genes in the 10-Mb linkage region. Since microtia is a developmental defect, we simply focused on genes involved in human organ development from the 10-Mb region. With the information of the function and gene ontology, we found nine organ developmental genes, EVC gene was selected for its roles in cartilage development, and the NKX3-2 and HMX1 gene was for their link to syndromic microtia EVC, including 6 non-synonymous mutations and 3 synonymous mutations  approximately take 65% of microtia cases EVC, EVC2, HMX1, HTRA3, NKX3-2, CC2D2A, CRMP1, DRD5, and SLC2A9) were identified in the 10-Mb susceptibility region on 4p15.3\u20134p16.2. CC2D2A, DRD5, and CRMP1 are known for neural development EVC, EVC2, SLC2A9, NKX3-2 and HMX1 genes for this isolated microtia pedigree.Microtia is a congenital abnormity, and genes involved in embryonic development, especially the development of first and second pharyngeal arches EVC or EVC2 can cause disorders with facial abnormality, such as Ellis-van Creveld syndrome or Weyers acrodental dysostosis NKX3-2, expressed in lateral plate mesoderm and embryonic skeleton, plays important roles in ear morphogenesis NKX3-2 from disorders of oculo-auriculo-vertebral spectrum and spondylo-megaepiphyseal-metaphyseal dysplasia  et al observed HMX1 expression in the eyes and ears of mice and human embryos. A 26-bp deletion of HMX1 was identified as causative mutation for oculo-auricular syndrome, an autosomal-recessive disorder with malformation of eyes and ears EVC, NKX3-2, and HMX1 play key roles in facial development, we sequenced the coding regions of them with 5 family members. Although the identified mutations were not segregated well with microtia phenotype, possible causal mutations may still locate in the noncoding regions of these three genes or the other genic regions of the 4p15.3\u20134p16.2 (such as EVC2 and SLC2A9).The above 5 candidate genes are associated with many facial anomalies. Defects in Balikova et al identified a \u223c750-kb amplified CNV region (approximately ranged from chromosome 4:8659910 to 4:9433782) within 4p16 was linked to syndromic microtia In this study, we identified a novel susceptibility locus for isolated microtia. Several genes within the locus exhibit key roles in facial development and are promising for the medical genetic study of the disease. Through detecting genetic variants of these genes, we may uncover causal mutations for isolated microtia. Our results warrant further studies of the 10-Mb region in relation to external ear morphogenesis.Figure S1Copy number variation map of 14 genotyped individuals. The schematic summarizes the distribution of duplications, deletions and multi-allelic loci on each human chromosome.(TIF)Click here for additional data file.Table S1The Mendelian inconsistencies rate within the pedigree.(DOCX)Click here for additional data file.Table S2The Kong and Cox's LOD score at 4p15.32\u20134p16.2.(DOCX)Click here for additional data file.Table S3Identified SNPs in candidate genes within susceptibility locus.(DOC)Click here for additional data file."}
+{"text": "Torreya grandis, naturally distributed in mountains of the southeastern China. The linkage\u2013LD map constructed from various types of molecular markers opens a powerful gateway for studying the history of plant evolution.The relationship between linkage disequilibrium (LD) and recombination fraction can be used to infer the pattern of genetic variation and evolutionary process in humans and other systems. We described a computational framework to construct a linkage\u2013LD map from commonly used biallelic, single-nucleotide polymorphism (SNP) markers for outcrossing plants by which the decline of LD is visualized with genetic distance. The framework was derived from an open-pollinated (OP) design composed of plants randomly sampled from a natural population and seeds from each sampled plant, enabling simultaneous estimation of the LD in the natural population and recombination fraction due to allelic co-segregation during meiosis. We modified the framework to infer evolutionary pasts of natural populations using those marker types that are segregating in a dominant manner, given their role in creating and maintaining population genetic diversity. A sophisticated two-level EM algorithm was implemented to estimate and retrieve the missing information of segregation characterized by dominant-segregating markers such as single methylation polymorphisms. The model was applied to study the relationship between linkage and LD for a non-model outcrossing species, a gymnosperm species,  One of the strategies to resolve this issue is constructing a LD map from which to infer population history by visualizing the decline pattern of LD with genetic distance. This strategy has been widely used in human genetics5 and increasingly recognized in other species.7 Many of these LD maps are constructed from the relationship of pairwise LD with the physical distance of the same marker pair, which do not estimate the frequency of recombination between marker loci.Linkage disequilibrium (LD), a concept to describe the non-random association of alleles at different loci, has been a focus of population genetic studies during the last several decades.1 Therefore, the estimation of the linkage, apart from estimating LD, is an essential step towards constructing a LD map. Wu and Zeng8 pioneered the application of a sampling design to simultaneously estimate these two parameters. By sampling parents randomly from a natural population and the seeds of the sampled parents, this design constructs a two-level hierarchic structure of molecular data, which enables the characterization of how different markers are associated in the original population and how the markers co-transmit their alleles in a Mendelian fashion from the parent to offspring. Lou et al.9 derived a close-form EM algorithm to estimate the LD and recombination rate within a unifying framework. Such a joint linkage\u2013LD analysis has been applied to the genetic mapping of complex traits, leading to the identification of biologically validated quantitative trait loci (QTLs) for drought resistance in maize.10 More recently, this strategy has been modified to accommodate to the estimation of genetic imprinting11 and genetic variance.12 Pikkuhookana and Sillanpaa13 implemented a Bayesian algorithm for parameter estimation from this strategy.The basic principle by which LD is used for historical inference results from its relationship with the recombination rate.8 open-pollinated design to construct a linkage\u2013LD map using biallelic co-dominant markers. To make this framework more useful for a broader area of applications, we extended it to enable the utilization of dominant-segregating markers. Several recent studies have shown that epigenetic variation provides a source for the generation of phenotypic diversity in natural populations15 and also epigenetic marks, such as differential cytosine methylation, may be inherited and have experienced the pressure of natural selection.16 Thus, it has become increasingly important to construct a more comprehensive linkage\u2013LD map by including methylation markers. In epigenetic population studies, there are many ways to score and analyse methylation-sensitive amplification polymorphisms, of which one common approach is to score those fragments that stay unmethylated as 1 and all others methylated as 0. This scoring approach leads to the segregation pattern of the so-called single methylation polymorphism (SMP) markers equivalent to that of dominant genetic markers.17 Lu et al.18 found a possibility of using three-point analysis to enhance the precision and power of linkage detection for dominant markers. Likewise, Li et al.19 developed a three-point analysis to analyse LD among three dominant markers and establish a procedure for testing and estimating multiple disequilibria at different orders. However, the simultaneous estimates of LD and recombination fraction between dominant markers are methodologically challenging, because their genotypes can little explain the information of allelic segregation. We implemented a two-level EM algorithm for joint linkage and LD analysis by modelling and retrieving the unobservable feature of segregating genotypes for dominant-inherited markers. An example was demonstrated to show the utility and usefulness of the model by analysing a real data collected from an OP design of an outcrossing species, Torreya grandis, naturally distributed in mountains of the southeastern China.In this article, we described a general computational framework built on Wu and Zeng's2.2.1.n unrelated maternal individuals and open-pollinated seeds from each sampled plant. This constitutes a two-level hierarchic sampling setting in which both parental plants and their offspring are genotyped by the same set of molecular markers. Consider a pair of biallelic markers A and B, which generate nine joint genotypes, AABB (coded as 1), AABb (coded as 2), \u2026, aabb (coded as 9). Let in denotes the number of mother plants with marker genotype i, and j derived from mother genotype i. Depending on the genotype of a mother, all offspring from her have different numbers of marker genotypes. Table From a natural plant population at Hardy\u2013Weinberg equilibrium (HWE), we randomly sample coded as , AABb  for marker A and Bp and bp (Bp + bp = 1) for marker B, respectively, and D is the LD between the two markers. Under the assumption of HWE, the expected frequency of two-marker genotype i in the parental population (iP) is expressed as the product of the two corresponding haplotype frequencies. Based on the principle of co-transmission of two genes from a parent to its progeny, we derived the expected frequency of two-marker genotype j in the progeny population from mother genotype i  denote the unknown parameters including all haplotype frequencies (arrayed in \u03a9p) and the recombination fraction \u03b8. A unifying log-likelihood that integrates two-level maternal and progeny genotype data can be expressed as\u03a9p that form expected maternal genotype frequencies  or Ab|aB (with probability of AbpaBp), where the vertical lines are used to separate the two underlying haplotypes of a diplotype. For a given parental genotype combination, a certain group of offspring genotypes is produced. For a mother with genotype AaBb, there will be two possible diplotypes, AB|ab and Ab|aB, whose relative frequencies areAB, Ab, aB and ab with frequencies defined as follows:We use es Table  and the AB or ab and Ab or aB.Let Based on the information about genetic segregation in each family, the lower lever likelihood is constructed asBelow, an algorithmic procedure will be described to estimate the parameters that define the likelihood.2.2.2.We implement two EM algorithms to estimate the unknown parameters. The first is implemented to estimate the haplotype frequencies and therefore allelic frequencies and linkage disequilibria by jointly maximizing log-likelihoods (2) and (4). The second is implemented to estimate the recombination fraction that is contained with double heterozygote by maximizing the log-likelihood (4). In the E step of the second EM algorithm, we calculate the probability with which a considered haplotype produced by double heterozygote parent is the recombinant type usingm equals the sum of following terms,In the M step, the estimate of the recombination fraction is obtained byThe E and M steps are iterated between Equations (5) and (6) until convergence.2.2.3.After genetic parameters are estimated, we test whether the two markers are associated and/or linked on the same genomic region. This can use the following hypotheses:H0 and H1 are calculated from which a log-likelihood ratio is calculated. By comparing this test statistic with a \u03c72 threshold with two degrees of freedom, we can accept or reject H0.The likelihoods under the D and \u03b8 separately, showing how the two markers are related. Under the null hypothesis It is also needed to test the significance of 2.3.2.3.1.17 For a dominant marker, the homozygote AA for the dominant allele cannot be distinguished from the heterozygote Aa. Thus, these two genotypes are observed as a single \u2018phenotype\u2019 (A_). For two dominant markers A and B, some cells for the observations and expected genotype frequencies in Tables A/jBj, Aj= A_ (coded as 1) or aa (coded as 0) and Bj= B_ (coded as 1) or bb (coded as 0), in the parental population. Similarly, let Ak/Bk given its parent genotype Aj/Bj  is formulated to jointly estimate haplotype frequencies and recombination fraction by implementing two EM algorithms. The first EM algorithm is used to estimate haplotype frequencies by jointly maximizing the upper and lower level likelihood, whereas the second EM algorithm used to estimate the recombination by maximizing the lower level likelihood. Here, we show how the second EM algorithm is implemented to estimate the recombination fraction. In the E step, we calculate the overall frequencies of the genotype with the progeny cells Table  using.In the M step, we estimate the recombination fraction by2.3.2.We formulate the hypothesis tests for the significance of the LD and linkage. The estimation of allele frequencies of two dominant markers under the null hypothesis of no LD should also be based on the EM algorithm; i.e. in the E step, we calculateA and B by usingIn the M step, we estimate the allele frequencies of markers \u03c72 distribution.The E and M steps between Equations (9) and (10) are iterated until the estimates are stable. The log-likelihood ratio under the null and alternative hypotheses is calculated and compared with a threshold determined from a 3.8 design, we sampled a natural population of Torreya grandis distributed in the southeastern China.20 In spite of the economic value of T. grandis, this species has not been extensively studied in population genetics. Zeng et al.21 constructed a first low-density genetic map for genus Torreya using an open-pollinated progeny derived from half-sib seeds of a landrace T. grandis \u2018Merrillii\u2019, providing basic information for marker genotyping of this species. We sampled 50 unrelated trees randomly from a natural population of T. grandis and 20 progeny for each sampled tree. In total, we obtained (50 + 50 \u00d7 20) = 1,050 trees, which were genotyped by 233 sequence-related amplified polymorphism (SRAP) markers. SRAPs are dominant-segregating markers,22 providing an excellent demonstration for the practical utility of our model. This data set constitutes a two-level hierarchic framework with a high level from the parents and a lower level from the progeny. We analysed each pair of these markers using the dominant model to simultaneously estimate the LD and recombination fraction and further test the significance of these two parameters.We used a real data analysis to demonstrate how the model is used to construct a linkage\u2013LD map. According to Wu and Zeng's23Of 233 \u00d7 232/2 = 27,028 pairs, 5,733 (21.21%) display significant non-random associations, and 2,140 (7.92%) are significantly linked. It was seen that much fewer pairs  are both associated in the original natural population and linked when they co-transmitted during meiosis from the parent to progeny. All this suggests that, for many marker pairs, significant associations are inconsistent with significant linkage. In other words, a pair of unlinked markers may be associated with each other, and also a pair of linked markers may not necessarily have a significant LD. Significant associations of unlinked markers may be due to the impact of some recent evolutionary forces on these markers, whereas the absence of associations between linked markers implies that this particular region of the genome has experienced the random mating of numerous generations.\u03b8 = 0.3 and LOD = 3.0, 233 markers were grouped into 8 linkage groups, but with 73 markers unlinked. Markers in each linkage group were ordered with an objective function of the sum of adjacent recombination fractions. When the optimal order of a linkage group was determined, the map distance between any two adjacent markers was calculated by Haldane's map function. To the end, we obtained a low-density genetic linkage map for T. grandis  are needed to characterize the biological function of these loci and relate this function to possible anthropological selection or climate change towards an in-depth understanding of the evolutionary mechanisms of T. grandis.By plotting pair-wise LD over the genetic distance, we constructed a LD map from which to infer the population history of dis Fig. . In geneT. grandis genome may have experienced a long evolutionary history. Relative to linkage Group 7, linkage Groups 2 and 8 has much more frequent LD between different loci from the same and different linkage groups, implying that some recent pressure of natural selection may have taken place in this part of the genome.From the distribution of all pair-wise LD, it was found that most pairs of markers do not display a large LD value Fig. , conform4.D = 0.15 and 0.02, respectively, in the population. The allele frequencies for the two markers are Ap = 0.6 vs. ap = 0.4 and Bp = 0.5 vs. bp = 0.5, respectively. The two markers are linked with two sizes of then recombination fraction \u03b8 = 0.20 and 0.05. In each design, 1,000 simulation replicates were performed to estimate the means of the MLEs for each parameter and their standard deviations. By collapsing the simulated co-dominant marker genotypes into a dominant setting, we can test how well our model performs to construct a dominant LD map.To examine the statistical properties of the model for constructing the LD map with dominant markers, we performed computer simulation by mimicking a natural population at HWE. We randomly sample a panel of unrelated open-pollinated families . Given a total of 1,000 progeny, the simulation considers three sampling strategies, 1,000 \u00d7 1 , 200 \u00d7 5  and 50 \u00d7 20 . For each strategy, we simulated two co-dominant markers with strong and weak LD, \u03b8 is first dependent on the size of LD, followed by the degree of linkage and the sampling strategy. If LD is near zero, then \u03d5 is close to \u03c91 and \u03c92 will not contain \u03b8. Thus, \u03b8 is not estimable when there is no association between the two markers. To better estimate the linkage, precise estimation of LD is essential.Table \u03b8 can be well estimated. In the package of software, we provide the function of determining such an LD value given a sampling strategy and allele frequencies.An additional scenario of simulation was conducted by collapsing only one of the two co-dominant markers into a dominant status. As expected, this scenario was intermediate in the precision of parameter estimation between those in which both markers are co-dominant and dominant, respectively. We have also performed simulation studies using the same schemes described above, but by quantitatively changing the values of LD within its interval. This simulation allows us to determine the minimum value of LD beyond which 5.1 For this reason, a test of LD about its departure from LE may tell us more stories about the evolutionary history of the population. Just because the use of LD to infer a population's past events is founded on its relationship with the frequency of recombination, a joint estimation of the LD and recombination fraction can provide more precise information about evolutionary inference.8Similar to the HWE, significant departure from linkage equilibrium (LE) indicates that the population studied is undergoing some evolutionary pressure by extensive inbreeding, gene flow, genetic drift, mutation, natural selection, etc. However, unlike HWE, LE cannot be established in one generation of random mating, rather than it needs a number of generations to be reached, because LD declines at a rate that depends on the recombination fraction.8 open-pollinated progeny design to construct a linkage\u2013LD map and particularly showed how this design can accommodate to missing information of dominant-segregating markers such as cytosine methylation markers. DNA methylation, as a covalent base modification of plant nuclear genomes, is thought to be accurately inherited through both mitotic and meiotic cell divisions.14 Also, similarly to spontaneous mutations in DNA, errors in the maintenance of methylation states would violate the equilibrium of natural populations, leading to changes in associations between epialleles at different methylated loci. Thus, by constructing a linkage\u2013LD map using those so-called SMPs, we can infer evolutionary pasts of the natural populations from a different perspective.15In this article, we extended Wu and Zeng's22 are still being used for many under-represented species including forest trees and wildlife species.26 Thus, the dominant model described can widen the usefulness of the open-pollinated design in practical population studies. Simulation studies have determined an appropriate sampling strategy to construct a linkage\u2013LD map using dominant markers. Since the precise estimation of LD is of primary importance to linkage estimation, we recommend using many smaller families over small larger families. In addition, the efficiency of linkage\u2013LD map construction can be enhanced by three-point analysis, which has proved to not only provide more information about the genome structure and organization, but also reduce a possibility of biased estimation of the linkage when LD has a small value.28 This is especially true for dominant markers.Indeed, as a simple and cheap technique, dominant markers, such as SRAP markers,8 open-pollinated design to study the population structure and history of an outcrossing species. Torreya grandis is a gymnosperm tree species with a large size, endemic to the eastern and southeastern China.20 Because of economic and ecological values, this species has been increasingly studied in terms of its evolutionary history and the genetic control of complex traits.21 The results from a joint linkage and LD analysis with dominant markers suggest that this species has experienced a long history of evolution, but some regions of its genome are subject to a certain recent evolutionary forces. This information will provide guidance for better germplasm management of this important woody plant. Advances in understanding the evolutionary history of Torreya can be made by sampling multiple populations in a range of its distributions. This study, along with the previous one based on half-sib seeds from a single tree of T. grandis reporting a linkage map covering a total of 7,139.9 cM in 10 groups,21 was among the first to construct genetic linkage maps for genus. It is important to align the two maps into a single one for a better coverage of the Torreya genome. Also, much more markers that can align those unlinked markers detected from the current and Zeng et al.'s studies are needed to completely cover 11 chromosomes of T. grandis. A complete coverage of markers allows more extensive studies of variation and examination of LD patterns, which will better reveal levels of complexity for this species.Although the original model for joint linkage and LD analysis was proposed more than a decade ago, its practical use has not occurred until recent years when the collection of molecular markers for under-represented species has been feasible. The current study presents one of the first applications of Wu and Zeng's2 A joint linkage and LD analysis can overcome this false-positive discovery.27 Thus, the LD map constructed from genetic and epigenetic markers will provide an important fuel to map key QTLs that affect quantitative traits of economic and environmental importance.7It has been recognized that genetic mapping based on LD analysis helps to fine map complex traits or disease, but this approach may have a high likelihood to detect spurious signals of association, because allelic association can also be due to evolutionary forces rather than physical linkage.Forest Scientific Research in the Public Welfare (201404102); Open Fund for Key Discipline of Forestry of Zhejiang Province, Zhejiang A&F University (AF201308); Key Scientific Project of Selection and Breeding of New Agricultural Varieties of the Department of Science and Technologies of Zhejiang Province (2012C12904-12); and \u2018One-thousand Person Plan\u2019 Award. Funding to pay the Open Access publication charges for this article was provided by Special Fund for Forest Scientific Research in the Public Welfare (201404102).This work is supported by Special Fund for"}
+{"text": "PSMD9) single nucleotide polymorphisms (SNPs) rs74421874 [intervening sequence (IVS) 3+nt460-G>A], rs3825172 (IVS3+nt437-C>T) and rs14259 (E197G-A>G) are linked to: T2D, depression, anxiety, maturity-onset-diabetes-of the young 3/MODY3, obesity, waist circumference, hypertension, hypercholesterolemia, T2D-macrovascular disease, T2D-microvascular disease, T2D-neuropathy, T2D-carpal-tunnel syndrome, T2D-nephropathy, T2D-retinopathy and non-diabetic retinopathy. PSMD9 SNP rs1043307/rs14259 (E197G-A>G) plays a role in anti-depressant therapy response, depression and schizophrenia. We aimed at determining PSMD9 rs74421874/rs3825172/rs14259 SNPs potential linkage to primary insomnia and sleep hours in T2D families. We recruited 200 Italian T2D families phenotyping them for primary insomnia and sleep hours per night. PSMD9-T2D-risk SNPs rs74421874/rs3825172 and rs1043307/rs14259 were tested for linkage with insomnia and sleep hours. Non-parametric-linkage analysis, linkage-disequilibrium-model analysis, single-SNP analysis, cluster-based-parametric analysis, quantitative-trait and variant-component analysis were performed using Merlin software. To validate data, 1000 replicates were executed for the significant non-parametric data. PSMD9 rs74421874 (IVS3+nt460-G>A), rs3825172 (IVS3+nt437-C>T) and rs1043307/rs14259 (E197G-A>G) SNPs are linked to insomnia in our Italian families.Insomnia increases type-2 diabetes (T2D) risk. The 12q24 locus is linked to T2D, depression, bipolar disorder and anxiety. At the 12q24 locus, the Proteasome-Modulator 9 ( Insomnia is associated with increased risk for type-2 diabetes (T2D)6789PSMD9), contributing to T2D through rare variants [rs149556654 SNP (N166S A>G) and S143G A>G variant]PSMD9 is significantly linked via the common single nucleotide polymorphisms (SNPs) rs74421874 (IVS3+nt460 G>A), rs3825172 (IVS3+nt437 C>T) and rs14259 (E197G A>G) to T2D151726PSMD9 SNP rs1043307/rs14259 (E197G A>G) is implicated in anti-depressant therapy responsePSMD9 SNPs rs74421874 (IVS3+nt460 G>A), rs3825172 (IVS3+nt437 C>T) and rs14259 (E197G A>G) are linked to depressionPSMD9 is strongly implicated in depressionPSMD9 may well play a role in insomnia.Within the 12q24 locus654 SNP N6S A>G anrs14259 E7G A>G to IVS3+nt4 C>T and http://www.genecards.org/cgi-bin/carddisp.pl?gene=PSMD9&search=psmd9). Therefore, PSMD9 may have a pathogenic role in inflammation and autoimmunityPSMD9 variants impairing PSMD9 protein sequence and/or protein dosage may alter downstream effects of its functions as well as interaction with key receptors such as the IL6 receptor. As PSMD9 is part of a complex network of transcription and co-activators, reduced or increased PSMD9 gene may lead to different phenotypes, or to an underlying pathological factor such as inflammation contributing to the various phenotypes, and commonly shared by them. Its proven interaction with the IL6 receptor may potentially explain its pleiotropic role in multiple disorders, whose pathogenesis is mediated by inflammation. However, other possible explanations for a shared pathogenesis among neuro-psychiatric disorders exist. For example, adult neurogenesis might be a more plausible pathogenic mechanism for neuro-psychiatric disorders. The proteasome-mediated proteolysis plays a recognized role in neuronal development, synaptic plasticity, and protein quality control. A proteomic study has reported the positive finding of PSMD9 at both the level of neuronal cytosol as well as synapsisPSMD9 gene role in the ubiquitin-proteasome system and its proven protein interaction with dysbindin and dysbindin-related role in neuroplasticity is intriguingPSMD9 is ubiquitously expressed and highly concentrated in eukaryotic cells as part of the 26S proteasome complex, which degrades intracellular proteins into antigenic peptides in the immune response and antigen presentation by MHC class I cells (PSMD9-depression-risk SNPs rs74421874 (IVS3+nt460 G>A), rs3825172 (IVS3+nt437 C>T) and rs14259 (E197G A>G) for linkage with insomnia and sleep hours in the 200 Italian T2D families.In light of these data, our object in the present study was to test the The subjects were all recruited from central Italy following the Helsinki-declaration guidelines. Subjects gave written informed consent. The Penn State Institutional Review Board approved the study.The Italian T2D families with affected sib-pairs were recruited via standardized interview from Rome. They are genetically homogeneous, and all have been Italian for at least three generations; T2D families have a T2D-family history. We excluded families with identical twins and siblings with uncertain identical paternity. The Italian family dataset has provided useful genetic information in T2D and associated phenotypes124445464748495051525354555657585960The Italian T2D families with mainly affected sib-pairs were recruited via standardized interview. The 200 Italian families are mainly constituted by 360 ungenotyped parents of 180 affected sib-pairs  for a total of 380 affected siblings, plus 20 families inclusive of 101 genotyped family members with an average family size of 5.05. The total of genotyped family members is 481, including 245 female subjects and 236 male subjects. The median age was 63. T2D was diagnosed based on the fasting criteria of blood glucose \u2265126\u2009mg/dl on at least two occasions, or a random glucose of \u2265200\u2009mg/dl associated with symptoms of diabetes, exclusion of type 1 diabetes, and a positive family history for non-insulin-dependent diabetes/T2D. T2D was present in 94.10% of the families\u2019 members. Major depression and generalized anxiety disorder were diagnosed based on the subject meeting the DSM-IV criteria during his/her lifetime. Generalized anxiety disorder was present in 52.50% of families and major depression in 29.90% of families.We characterized the Italian families for the presence or absence of insomnia based on the DSM-IV diagnosis criteria for primary insomnia; the subject needed to have had in the past year trouble falling or staying asleep or not feeling rested waking up for at least one month; this had to significantly interfere with the routine activities; also any medical condition , which may cause this similar status, was excluded. If the subjects were negative or positive for the DSM-IV diagnostic criteria for generalized anxiety disorder or major depression that was annotated. Other mental disorders were excluded. Further, an average sleep time in the past year of less than four hours per night was considered insomnia. Of the very few patients with insomnia on sleep medication, we considered only the average sleep hours before starting the medication, independently on when the medication was started. Primary insomnia was present in 42.20% of families.The phenotype of insomnia is described as unknown if data are lacking. We could not collected insomnia data for all of the members in our 200 Italian families.IVS3-PSMD9 region containing the SNPs rs74421874 (IVS3+nt460 G>A), rs3825172 (IVS3+nt437 C>T) and the exon 5-coding region, containing the rs1043307/rs14259 (E197G A>G). We directly sequenced the PCR products, post-purification via EXOSAP-IT, on an automated ABI 3730 Sequencer.Using polymerase chain reaction (PCR) and with specific primers, we amplified in all family members the PSMD9 SNPs rs74421874 (IVS3+nt460 G>A), rs3825172 (IVS3+nt437 C>T) and rs1043307/rs14259 (E197G A>G) for linkage with primary insomnia and average sleep hours per night. Both non-parametric and parametric-linkage analysis for the qualitative phenotype were performed for the three SNPs, using Merlin softwareIn the 200 Italians families we tested the PSMD9 SNPs rs74421874 (IVS3+nt460 G>A), rs3825172 (IVS3+nt437 C>T) and rs1043307/rs14259 (E197G A>G) are in strong linkage disequilibrium (LD)We previously reported that the In order to rule out the effect of depression or anxiety on insomnia, we also performed a likelihood ratio test of each SNP association with insomnia considering anxiety or depression as a covariate, by using a mixed effects logistic regression model taking into account the families as a random effect factor in the model. For the Chi-square statistics, a corresponding p-value less than 0.05 means that the SNP tested has a significant effect on insomnia in the presence of the tested covariate and families as a random effect. The p-value is calculated by performing a likelihood ratio test, where the test statistic is the Chi-square statistic, which has a degree of freedom \u201cDF\u201d depending on the covariate and random effects.The following parameters were used for the parametric-linkage analysis based on the SNPs cluster, thus eliminating the LD inflation of the linkage signal, as it considers the SNPs all in the same locus: cluster haplotype disease-allele frequency 0.25; dominant model with penetrance for homozygous non-risk allele 0.13 , for heterozygous risk allele 1.00, for homozygous risk allele 1.00; recessive model with penetrance for homozygous non-risk allele 0.13, for heterozygous risk allele 0.13, for homozygous risk allele 1.00; additive model with penetrance for homozygous non-risk allele 0.13, for heterozygous risk allele 0.45, and for homozygous risk allele 0.90. Merlin computed the lod score from all the informative families for the insomnia phenotype (n\u2009=\u200928).For each significant non-parametric test performed, we calculated how many times similar p-values were expected by chance in 1,000 replicates of simulations using the gene-dropping method in order to exclude the presence of any false positives in our results. This analysis replaces real data with simulated data, while maintaining the pedigree structure, allele frequencies, and recombination fraction. These datasets were generated under the null hypothesis of no linkage.The quantitative-trait linkage analysis and variance-component analysis for the quantitative trait of average sleep hours per night was performed using MerlinAll results are reported as LOD scores as calculated by Merlin.PSMD9 SNPs rs74421874 (IVS3+nt460 A>G), rs3825172 (IVS3+nt437 C>T) and rs1043307/rs14259 (E197G A>G) are linked to primary insomnia via the non-parametric model .Our analysis shows that the PSMD9 SNPs to primary insomnia by using the LD-based model analysis and by testing each single SNP independently. The simulation analyses of 1,000 replicates exclude false positives and establish the data validity. The result of the non-parametric linkage analysis for primary insomnia are reported in Additionally, we identified a positive non-parametric linkage of the above-mentioned PSMD9 SNP rs74421874 (IVS3+nt460G>A) and SNP rs3825172 (IVS3+nt437C>T), independently, with insomnia using anxiety as covariate , but not using depression as covariate . The Chi-square statistics also shows a significant association of rs1043307/rs14259 (E197G A>G) with insomnia using anxiety as covariate  as well as using depression as covariate , rs3825172 (IVS3+nt437 C>T) and rs1043307/rs14259 (E197G A>G) studied are linked to primary insomnia in the Italian T2D families; it shows a trend towards linkage to sleep hours, with a trait heritability estimated to be circa 11%, and gene-trait heritability circa 25%. The different types of non-parametric-linkage analyses performed determined that the linkage is present independently from the presence of LD among variants; the simulations excluded the possibility that the results are due to random chance.Our study demonstrates that the PSMD9 coding variant rs14259 (E197G A>G) for a significant association with insomnia, considering anxiety or depression as a covariates. The two PSMD9 intronic SNPs rs74421874 (IVS3+nt460 G>A) and rs3825172 (IVS3+nt437 C>T) are significantly associated with insomnia considering anxiety as covariate, but lose significance in the association test with insomnia considering depression as covariate. Thus, the PSMD9 intronic variants rs74421874 (IVS3+nt460 G>A) and rs3825172 (IVS3+nt437 C>T) may provide vulnerability to the missense coding variant rs1043307/rs14259 E197G, which by itself may be the pathogenic variant.The logistic regression model supports the role of the In addition, we identified the recessive model as the most significant for the linkage, as our families\u2019 structure contain sib-pairs, which are the most statistically powerful family dataset to identify recessive disorders.Our study may have also captured the linkage signal due to the presence of T2D; however, this risk is always present if we test for linkage in a complex disorder which is associated with another common disorder. Nevertheless, the variance-component analysis of sleep hours, based on a quantitative trait independent from the T2D qualitative phenotype, shows at least a trend towards linkage of sleep hours.PSMD9 is a ubiquitously expressed gene, which is implicated in anti-depressant therapy response and depression2435As insomnia is a risk factor for T2D, it is reasonable to anticipate that a gene may be linked to both insomnia and T2D. Furthermore, The IL6 receptor has been identified as a novel PSMD9-interacting partner which further supports PSMD9 role in the inflammatory responsePSMD9 is linked to primary insomnia in our T2D Italian families. Specifically, the PSMD9 coding variant rs1043307/rs14259 E197G is significantly associated with insomnia taking into account anxiety or depression as covariate, while the PSMD9 intronic SNPs rs74421874 (IVS3+nt460 G>A) and rs3825172 (IVS3+nt437 C>T) remain significantly associated with insomnia only when taking into account anxiety \u2013and not depression\u2013 as covariate. These findings are new, and should be independently replicated in other populations. These data, if confirmed in other populations, imply that PSMD9-targeted therapies may prevent or treat primary insomnia, depression, and T2D, and/or to manage personalized therapy based on patient genotypes.In summary, How to cite this article: Hao, H. et al. T2D and Depression Risk Gene Proteasome Modulator 9 is Linked to Insomnia. Sci. Rep.5, 12032; doi: 10.1038/srep12032 (2015)."}
+{"text": "The BnaZNF2 genetic map exhibited perfect collinearity with the physical map of B. napus, indicating its high quality. Comparative mapping revealed several genomic rearrangements between B. napus and B. rapa or B. oleracea. A total of eight and 16 QTLs were identified for pod number and seed number per pod, respectively, and of which three and five QTLs are identical to previously identified ones, whereas the other five and 11 are novel. Two new major QTL respectively for pod number and seed number per pod, qPN.A06-1 and qSN.A06-1 (R2\u2009=\u200922.8% and 32.1%), were colocalised with opposite effects, and only qPN.A06-1 was confirmed and narrowed by regional association analysis to 180\u2009kb including only 33 annotated genes. Conditional QTL analysis and subsequent NILs test indicated that tight linkage, rather than pleiotropy, was the genetic causation of their colocalisation. Our study demonstrates potential of this reference genetic population/map for precise QTL mapping and as a base for positional gene cloning in rapeseed.To facilitate the pseudochromosomes assembly and gene cloning in rapeseed, we developed a reference genetic population/map (named BnaZNF Brassica napus L. , is mainly cultivated for the production of edible oil and is the second largest oil crop globally, after soybean. Rapeseed originated from recent (\u22480.01 million years) allopolyploidy between the ancestors of two diploid species, B. rapa  and B. oleracea Rapeseed, Nearly ten linkage QTL mapping studies and one association analysis study involving pod number and/or seed number per pod in rapeseed have been reported24567891011bzhhttp://www.brassica.info/resource/sequencing.php) by our own and several other institutes. Approximately a dozen linkage maps have been developed using different genetic populations of rapeseed (http://www.brassica.info/resource/maps/published-data.php); however, the number of sequence-based markers in these linkage maps was insufficient, and none of these genetic populations were derived from Zhongshuang11. To accurately anchor and orientate the 2730 sequence scaffolds of Zhongshuang11 (http://www.ncbi.nlm.nih.gov/genome/genomes/203), a skeleton linkage map of sequence-based markers must be constructed using a reference population derived from itself. In addition, tens of rapeseed cultivars have also been resequenced (http://www.brassica.info/resource/sequencing.php)Recent innovations in genome sequencing technology and bioinformatics have enabled the sequencing of European winter type rapeseed cultivar Darmor-de novo sequencing) and No.73290 (resequencing) exhibit significant differences in many traits related to agronomy, plant/root architecture and yield (particularly pod number and seed number per pod)B. napus and B. rapa/B. oleracea; (3) mapping and comparison of QTLs for pod number and seed number per pod using the reference skeleton linkage map; (4) validation and fine mapping of the major QTLs by regional association analysis; and (5) dissection of the genetic basis of the colocalised QTLs for pod number and seed number per pod by conditional QTL analysis.Among the sequenced cultivars, Zhongshuang11 , 2592 single nucleotide polymorphism (SNP), 179 sequence tagged site (STS) and 76 Insertion/Deletion (InDel), 961 (10.3%) primer pairs displayed clear polymorphism between the two parental cultivars Zhongshuang11 and No.73290, producing 1038 sequence-based PCR markers in the BnaZNFpulation .All 1038 markers were subjected to Joinmap 4.0 software for linkage analysis, which resulted in a linkage map of 19 linkage groups and 803 markers  ; Table 1Only 40  markers exhibited significant (P\u2009\u2264\u20090.001) segregation distortion, of which 5 and 35 skewed to Zhongshuang11 and No.73290, respectively . InteresB. napusB. rapaB. oleraceaBrassica. Comparative mapping between the BnaZNF2 linkage map and the physical maps of B. napus and B. rapa/B. oleracea was based on the markers aligned to their pseudochromosomes, and the results are displayed using dot-plots  to 0.997 (A03) for the different chromosomes (mean\u2009=\u20090.963)  of B. napus and B. rapa/B. oleracea: translocation in one location in A01, A02 and C01 and in two locations in C05 and C06; inversion in one location in C02 and C06; and reshuffling in A05 and C04  . These r and C04 .The two parents (Zhongshuang11 and No.73290) differed significantly in pod number and seed number per pod in all four investigated environments . The podB. napus L.) could be improved by increasing its component traits, such as pod number and/or seed number per pod.The pod number of the whole plant was significantly positively correlated with those of the main and branch inflorescence with moderate and high coefficients, respectively , suggestAnalysis of variance (ANOVA) revealed that genotype, environment and the genotype \u00d7 environment interaction all have significant effects on both the pod number and seed number per pod of the main and branch inflorescence as well as whole plant . The broA total of 69 QTLs  were detected for pod number and seed number per pod in five experiments . After dqPN.A06-1 and qSN.A06-1) were repeatedly detected in four and all five experiments, respectively, and displayed a large effect (R2\u2009=\u200922.8% and 32.1%); thus, they can be treated as major QTLs  overlapped well  of these QTL could be determined, of which approximately a quarter overlapped . In deta0% of theB. napusB. rapaB. rapaTo further fine-map the two colocalised major QTLs for pod number and seed number per pod, regional association analysis was conducted. The corresponding genomic region of the target major QTL was identified by the alignment of the primer-pairs sequences of the molecular markers within the QTL interval and reference genomic sequences of R2\u2009=\u20098.7%); this signal was very close (13\u2009kb) to the peak marker (BrSF46\u2013167) of qPN.A06-1 identified by linkage analysis  including only 33 annotated genes in the reference genome of B. napus (qPN.A06-1 and qSN.A06-1 was not likely caused by pleiotropy.A large range of phenotypic variation was observed for both pod number (32\u2013106) and seed number per pod (7.6\u201328.0) among the 576 inbred lines of the association population. Based on population structure and family relatedness, regional association analysis was conducted using mixed linear mode (MLM) by TASSEL3.0 software. Interestingly, a significant (P\u2009<\u20090.001) association signal was only observed for pod number and not for seed number per pod . Of the analysis ; Table 5B. napus . BecauseB. napus , it was B. napus . These rqPN.A06-1 and qSN.A06-1 in the same experiments, conditional QTL analysis was performed in both the linkage and association populations  values and R2 of the six associated markers, exhibited only a small variation. These results suggest that tight linkage rather than pleiotropy is more likely the genetic basis of the colocalisation of qPN.A06-1 and qSN.A06-1.To determine the genetic basis (pleiotropy vs. tight-linkage) of the colocalisation of ulations . Regardl2 population from two sequenced rapeseed cultivars was developed. Among the reported genetic populations in rapeseed51920212223et al. unpublished data). Due to the heterozygosity of the current BnaZNF2 population, the corresponding recombinant inbred lines population (named BnaZNRIL) was also developed in our lab. All major QTLs identified from the BnaZNF2 population have been fine-mapped in our laboratory, and the functional validation of the candidate genes is in progress. Therefore, the BnaZNF2/BnaZNRIL population is an ideal reference population for QTL mapping and map-based gene cloning in rapeseed.In the current study, an F2 population (2 linkage map were developed in this study (B. napus (http://www.brassica.info/resource/maps/published-data.php). These favourable characteristics will facilitate the easy transfer of these markers to other genetic populations and the comparison/integration of different linkage maps in B. napus as well as in other Brassica species25To facilitate pseudochromosome assembly of Zhongshuang11, a skeleton linkage map of 803 sequence-based PCR markers was constructed using the BnaZNFpulation . A totalis study , increasB. napus59122728292 linkage map ; therefore, they were unlikely to be caused by an insufficient number of markers. Because its genomic coverage ratio was not low, the short length of the C09 linkage group was obviously due to its low recombination frequency among all 19 linkage groups. For the A08, A10 and C07 linkage groups, their short length is likely due to the low polymorphism between parents because their genomic coverage ratios were very low, while the recombination frequencies were not low. In fact, the A08 and A10 linkage groups are also short in more than half of the published linkage maps of B. napus5912272829The existence of short linkage groups is commonly observed in published linkage maps of kage map . Of thesInterestingly, most of the distorted markers tended to cluster on both ends of the linkage groups and skew to the same parent . This cl2 linkage map and physical map of B. napus were collinear  between these species. These rearrangements should occur very recently, after the formation of B. napus from the progenitors of the two diploid species B. rapa and B. oleracea. This finding is consistent with the reported genomic rearrangements in the resynthesised Brassica polyploids, including B. napus333435The major part of the constructed BnaZNFollinear ; Table 2ollinear  may be dqSNP2) and C01 (qSNP11b) for pod number, three on A01 (qSNA1), C03 (cqSS.N13) and C09 (qSS.C9) for seed number per pod, and one on C01 (qSNP11a/qSS11) for both traits could be considered major QTLs67R2\u2009\u2265\u200910%) or moderate (R2\u2009<\u200910%) effect, and only two each for pod number and seed number per pod should be considered major QTLs. The recent completion of sequencing and assembling of rapeseed genome enabled the first physical map-based comparative QTL analysis in the current study, which undoubtedly increased the accuracy of comparison. Only three QTLs for pod number and five QTLs for seed number per pod identified in the current study had been reported previously  identified in the current study, a regional association strategy was proposed by using the high resolution of the natural populationqPN.A06-1 was successfully narrowed to a much smaller (approximately 1/7) interval of 180 kb including only 33 annotated genes . This is the first study that provides solid evidences to clearly demonstrate a negative correlation between yield component traits in rapeseed as well as other crops. The existence of two tightly linked major QTLs for pod number and seed number per pod also indicates the retention of a large linkage drag during the breeding of the elite rapeseed cultivar Zhongshuang11. Therefore, the strategy described in this paper will be effective in increasing the pod number of Zhongshuang11 without decreasing the seed number per pod via the introgression of qPN.A6 through backcross and marker-assisted selectionResource limitation force an organism to allocate energy to processes in a competitive manner2692) used for the linkage analysis included 184 F2 individuals derived from Zhongshuang11 and No.73290. The natural population used for the association analysis was described in our previous study and comprises 576 global inbred lines, including the two parents of the linkage populationThe reference genetic population . The F2:3 families were planted in Wuhan from Oct. 2009 to May 2010 (code W10F2:3) and Oct. 2010 to May 2011 (code W11F2:3) as well as in Xining from April to Aug. 2011 (code X11F2:3). The F2:4 families were planted in Xining from April to Aug. 2011 (code X11F2:4). The association population was planted in Wuhan from Oct. 2011 to May 2012 (code W12AP). The planting of the F2:3, F2:4 and association populations followed a randomised complete block design with two to three replications. Each block contained two rows, with 33.3\u2009cm between rows and 16.7\u2009cm between individual plants. The seeds were sown by hand, and the field management followed standard agricultural practice. In each block, 10 representative individuals from the middle of each row were harvested by hand at maturity.The FPod number was measured as the number of effective pods on the main inflorescence, branch inflorescence and whole plant, respectively . Seed number per pod was calculated as the number of well-filled seeds on the main inflorescence, branch inflorescence and whole plant, respectively, divided by the corresponding pod number .Brassica . The second group, consisting of SSR markers , was developed by our lab from the sequence scaffolds of B. rapa, B. oleracea and B. napusB. oleracea cultivarsThree groups of molecular markers were first used to screen polymorphism between the two parents of the linkage population, and the single-locus markers were then used to screen the mini-core collection of the association population. The first group, mainly consisting of SSR and STS markers, was downloaded from the public database of http://www.kyazma.nl/index.php/mc.JoinMap) using a threshold for goodness-of-fit of \u2264 5, a recombination frequency of < 0.4 and minimum logarithm of odds (LOD) score of 2.0. All genetic distances were expressed in centimorgans (cM) as derived by the Kosambi function. Double-crossover events were examined, and the original scores were rechecked for potential scoring errors.The genetic linkage map was constructed using the software JoinMap 4.0 (B. napus (http://www.brassica.info/CropStore/maps.php)52022495051Linkage groups were assigned to the corresponding chromosomes  based on the common markers in the reported linkage maps of http://statgen.ncsu.edu/qtlcart/WQTLCart.htm). The walk speed, number of control markers, window size and regression method were set to 1\u2009cM, 5, 10\u2009cM and forward regression, respectively. The default minimum distance between QTLs (5\u2009cM) and minimum LOD from top to valley (1) were used to define a QTL peak in an experiment. The experiment-wise LOD threshold was determined by permutation analysisQTL mapping was conducted using the composite interval mapping (CIM) procedureBecause QTLs detected in different experiments and mapped to the same region of a chromosome might be several estimates of the position of a single QTLR2 between all pairs of markers using the standalone software TASSEL v3.0R2 value among unlinked markersLinkage disequilibrium (LD) was estimated as the correlation coefficient ad hoc statistic \u0394k based on the rate of change in LnP(D) between successive kThe structure of the association population was inferred using the software STRUCTURE v2.3.4The relative kinship of the 576 inbred lines of the association population was calculated using SPAGeDi v1.4 softwareThe regional association analysis was conducted using single-locus SSR markers within the confidence interval of the target major QTL in the association population of 576 inbred lines. Based on the population structure and relative kinship (Q and K matrix), the calculation was performed with a mixed linear model (MLM) incorporated into the TASSEL v3.0 software. The threshold of the significant marker-trait association was set to P\u2009\u2264\u20090.001.http://ibi.zju.edu.cn/software/qga/index.htm), where T1|T2 indicates that trait 1 is conditioned by trait 2.To determine the genetic basis of the colocalisation of QTLs for different traitsh2\u2009=\u2009\u03c3g2/(\u03c3g2\u2009+\u2009\u03c3ge2/n\u2009+\u2009\u03c3e2/nr), where \u03c3g2, \u03c3ge2 and \u03c3e2 are the variances of genotype, genotype by environment and error, respectively, while n and r are the number of environments and replicates, respectively. The genetic correlation was calculated as rG\u2009=\u2009covGxy/(\u03c3Gx2\u2009\u00d7\u2009\u03c3Gy2)1/2, where covGxy is the genotypic covariance and \u03c3Gx2 and \u03c3Gy2 are the variances of the pairwise traits. The significance of each genetic correlation was determined using a t-test of the correlation coefficientsx2-test and Pt-test) of the degree-of-fit and differences, respectively.Broad-sense heritability was calculated as How to cite this article: Shi, J. et al. Linkage and regional association analysis reveal two new tightly-linked major-QTLs for pod number and seed number per pod in rapeseed (Brassica napus L.). Sci. Rep.5, 14481; doi: 10.1038/srep14481 (2015)."}
+{"text": "Melampsora larici-populina causes significant yield reduction and severe economic losses in commercial poplar plantations. After several decades of breeding for qualitative resistance and subsequent breakdown of the released resistance genes, breeders now focus on quantitative resistance, perceived to be more durable. But quantitative resistance also can be challenged by an increase of aggressiveness in the pathogen. Thus, it is of primary importance to better understand the genetic architecture of aggressiveness traits. To this aim, our goal is to build a genetic linkage map for M. larici-populina in order to map quantitative trait loci related to aggressiveness. First, a large progeny of M. larici-populina was generated through selfing of the reference strain 98AG31 (which genome sequence is available) on larch plants, the alternate host of the poplar rust fungus. The progeny's meiotic origin was validated through a segregation analysis of 115 offspring with 14 polymorphic microsatellite markers, of which 12 segregated in the expected 1:2:1 Mendelian ratio. A microsatellite-based linkage disequilibrium analysis allowed us to identify one potential linkage group comprising two scaffolds. The whole genome of a subset of 47 offspring was resequenced using the Illumina HiSeq 2000 technology at a mean sequencing depth of 6X. The reads were mapped onto the reference genome of the parental strain and 144,566 SNPs were identified across the genome. Analysis of distribution and polymorphism of the SNPs along the genome led to the identification of 2580 recombination blocks. A second linkage disequilibrium analysis, using the recombination blocks as markers, allowed us to group 81 scaffolds into 23 potential linkage groups. These preliminary results showed that a high-density linkage map could be constructed by using high-quality SNPs based on low-coverage resequencing of a larger number of M. larici-populina offspring.The poplar rust fungus  Melampsora larici-populina , is the main phytosanitary constraint for commercial poplar cultivation in Europe and other parts of the world  most of the rust fungi are heteroecious, thus the completion of sexual crosses requires two non-related host plants  molecular markers. For a genetic map of sufficient density, the development of a large number of markers and the genotyping of a large number of progeny represent significant costs, both in time and in money. With the advent of next generation sequencing (NGS) techniques, combined with genome-wide marker discovery techniques such as reduced-representation sequencing (RRS), restriction-site-associated DNA sequencing (RAD-seq) or multiplexed shotgun genotyping (MSG), it is now possible to overcome the technical and financial constraints of genetic mapping  to characterize the S1 progeny through a segregation analysis of polymorphic microsatellite loci; (iii) to test for linkage disequilibrium between these loci in the progeny; and (iv) to perform a pilot study of genetic mapping through whole-genome resequencing of a subset of 47 S1 individuals.The purpose of this work was (i) to generate S1 progeny of M. larici-populina is a heteroecious and macrocyclic rust fungus, the completion of the life cycle requires two unrelated host plants, poplar and larch ), where x is the sequencing depth at a given SNP, to the observed proportion of observed heterozygotes in the 98AG31 parental strain, which is necessarily heterozygous in all true SNP positions. Minimum mean square error estimation yielded r = 0.51714, b = 3.9148, and c = 0.98404. This function was assumed to give the probability of observing a heterozygote if the genotype is truly heterozygous. Since, in our setting, true heterozygotes and true homozygotes are equally likely, we can use the same function as the probability that a genotype is truly homozygous if it is observed to be homozygous. If a genotype was observed to be homozygous, we used an arbitrary weighting scheme to take into account both depth and allele frequency evenness when considering genotypes observed to be heterozygous. The score was computed as (M/m-1)/200-1 where M and m are the majority and minority allele absolute frequencies, respectively. In addition, the score of each SNP incorporates the scores of neighboring SNPs. Thus, the score of a given position was actually computed as the sum of the score of the focus SNP and the 15 neighboring SNPs on each side, weighted by the distance .In order to take into account genotype uncertainty when sequencing depth for a single individual is low, we used an A further step of smoothing was performed in order to identify recombination blocks. We assumed that a region of consecutive SNPs with constant heterozygous or homozygous status to be a block that was transmitted without recombination. We defined these blocks as regions of consecutive SNPs for which the score kept the same sign and at least one SNP exhibited an absolute value larger than 0.5. To avoid excessive false positive recombination points, we extended recombination blocks over SNPs with scores of opposite sign provided that they did not exceed |0.2| and up to a SNP with a score of at least |0.2| of the corresponding sign. These blocks represented overlapping regions of the genome with different putative recombination points defined in each individual. We defined a sample-wise set of blocks by dividing the genome in regions based on all putative recombination points. As a result recombination blocks were regions in which no recombination event had taken place.Next, we generated the phased sequence of the two parental haplotypes of all blocks. We retrieved the majority allele of all SNPs for individuals that were classified as homozygous for the region under consideration. This generated the sequence of all putative homozygotes for each non-recombining region. We generated the maximum-likelihood phylogeny of those sequences using PhyML version 20120412  as a measure of linkage disequilibrium. The P-value was not computed if fewer than 10 individuals had non-missing data for both sites, or if there was less than one copy of each homozygous genotype and fewer than two copies of each heterozygous genotype (over the two loci).For all pairs of recombination blocks, we used M. larici-populina S1 progeny, 4-month-old poplar plants were spray-inoculated with M. larici-populina strain 98AG31 in a greenhouse. Uredinia appeared on the abaxial surface of the inoculated poplar leaves 8\u201310 days after inoculation  and the second  verification. Isolates identified as contaminated were deleted from further analyses.The genetic purity of 138 of the offspring was verified twice  through genotyping with a set of 25 polymorphic microsatellite loci specific to P-value = 0.034 and 0.005, respectively), exhibiting an excess of heterozygotes .In order to validate the progeny's meiotic origin, a segregation analysis was performed on 115 genetically pure S1 individuals, using the 14 microsatellite loci (out of the 25), which are heterozygous in the parental strain 98AG31. Twelve out of the 14 microsatellite loci exhibited a Mendelian segregation of 2 (heterozygous): 1 (homozygous 1): 1 (homozygous 2), as expected   Table . Highly M. larici-populina, plus the parental strain 98AG31, was resequenced using the Illumina HiSeq 2000 technology. A total of over 40 billions bp were sequenced, with a relatively uneven repartition of sequencing depth per individual  linkage disequilibrium extends to over a distance of 1 Mbp , of which 61,743 involved markers belonging to the same assembly scaffold. The analysis of the decay of linkage disequilibrium against physical distance shows that highly significant  allowed us to identify signatures of statistical linkage between different scaffolds , the production of F1 and F2 progenies through selfing or outcrossing was easier compared to heteroecious rusts. Nevertheless, early work on genetics of heteroecious cereal rusts also began in the 1940's  and MLP59 (located on scaffold 37). This result can still be explained by chance alone under Mendelian segregation. Applying the Bonferroni correction for multiple testing leads to a significance threshold of 0.0035, so that both loci no longer depart significantly from the expected ratio. Alternatively, a significant excess of heterozygotes may be due to the proximity of these loci to mating type loci. Eleven putative pheromone precursor genes and four pheromone receptor genes, which may be involved in the mating type, were annotated in the M. larici-populina is an obligate biotroph and can be cultivated only in interaction with its host. Analysis of genome-wide data could help analyze putative signatures of selection. In this first study, however, SNPs obtained through whole-genome sequencing were filtered such as all three genotypes were represented, which could have caused a bias when testing for departure from Mendelian ratios, by excluding sites more often in case of distortion of Mendelian segregation.Other forms of selection may cause an excess of heterozygotes, such as over dominant selection. This type of selection is known to occur within host-pathogen interactions  loci known to be physically linked because they are located on the same scaffold and found genetically linked; (ii) loci that are not physically linked but found genetically linked; and (iii) loci that are not found genetically linked despite being physically linked.Q-value < 10\u22124), since they are located on the same scaffold at 569,936 bp distance, which proved the efficiency of this method to confirm physical linkage by genetic linkage tests. In addition, locus MLP77 also was found in significant linkage disequilibrium with locus MLP56 , with a Q-value < 10\u22124, despite located on different scaffolds. Consequently, this pair of loci is genetically linked and the respective scaffolds (scaffold 5 and scaffold 15) should be grouped into a linkage group. Furthermore, since locus MLP56 is located at the end of scaffold 5 and MLP77 is located at first third of scaffold 15, the most likely physical link should be between the end of scaffold 5 and the beginning of scaffold 15. Thus, the microsatellite-based linkage disequilibrium analysis allowed us to build one linkage group for a total length of 4,252,591 bp, which accounts for about 4.2% of the total genome length. However, these two scaffolds were not found in the same linkage group as defined from the SNP-based linkage analysis  and MLP82  were found in significant linkage disequilibrium . Loci MLP94 and MLP55 are located on scaffold 1 at a distance of 2,568,380 bp, which could be far enough to break any genetic linkage. This result is consistent with the SNP-based linkage analysis, which showed that linkage is unlikely to be detected at distances higher than 2 Mbp. However, the situation is different for loci MLP54 and MLP59, which are located on scaffold 8 at a distance of 737,315 bp, and for which no linkage disequilibrium was found despite this relatively small distance. The SNP-based linkage analysis showed that linkage could be still detectable at distances up to 1 Mbp. This loss of linkage disequilibrium could be due to a recombination hotspot in this specific region /MLP55 , and MLP54 /MLP59  pairs. Although each pair of loci is located on a single scaffold, no linkage disequilibrium was detected .In a second step, we aimed to adopt a whole-genome perspective on linkage disequilibrium patterns. We used shifts from homozygous to heterozygous genomic regions detected from whole-genome sequencing data to scale genomic data down to 2580 recombination blocks in which all SNPs were non-recombining. These blocks were then treated as markers, allowing us to integrate data from successive SNPs, and to cope with sequencing errors and missing data that could have had a strong impact due to the relatively small sample size and the unequal sequencing depth among individuals. The approach was however limited to genomic regions that were polymorphic within the parental strain, thereby generating informative segregating markers in the offspring. Thanks to the low level of inbreeding and the relatively high level of polymorphism in the M. larici-populina is not easy to assemble properly, even based on high-depth genome sequencing. It is thus possible that some of the scaffolds are actually chimers.Linkage disequilibrium could be detected between markers located at nearby locations on the same assembly scaffold, but also between markers located on different scaffolds, while many pairs of markers located on the same scaffold exhibited no linkage disequilibrium. We found that, as expected, linkage disequilibrium decays with increasing distance, as shown with microsatellite markers cited earlier, with linkage still detectable at distances up to 1 Mbp. Based on the 2580 SNP-based markers, we detected 23 putative linkage groups, including scaffolds that exhibit signals of linkage disequilibrium with at least one other scaffold of the same group. Interestingly, some of the between-scaffold signatures of linkage disequilibrium obtained with whole-genome sequencing data pointed out potential genome assembly issues. As tentatively evidenced using microsatellite-based analysis, it appears that, at several instances, contiguous genomic regions within a scaffold display discontinuities in genetic linkage. These results point to the fact that most of the largest scaffolds might have to be redefined, i.e., split and rearranged. Due to the large size and high repetitive sequence content (for a fungus), the genome of Noteworthy, the pattern of linkage disequilibrium among these whole-genome markers (23 linkage groups) did not match the observation made using the 14 microsatellite markers. In other words, the linkage group made of scaffold 5 and 15 evidenced by the microsatellite-based linkage analysis is not supported by the SNP-based scaffold merging. This discrepancy can be further explained by a careful look at the pattern of SNP-based linkage along scaffold 15 Figure  and may M. larici-populina genome. The subsequent use of 2480 markers integrating the information of almost 150,000 SNPs detected in 47 S1 individuals suggested 23 linkage groups. Both methods have strengths and weaknesses\u2014microsatellites were typed on more offspring individuals, and SNPs are available at higher density along genomes\u2014but they are complementary. While the SNP-based and microsatellite-based linkage analyses appeared inconsistent at first sight, we demonstrated that these two analyses converged to question current genome assembly. These encouraging results demonstrate that an accurate genetic map could be constructed with a larger number of M. larici-populina S1 offspring, by using the method for constructing ultra-high-density linkage maps with high-quality SNPs based on low-coverage resequencing, as already described for plants  of microsatellite markers allowed us to define one linkage group in the M. larici-populina, such a genetic map will provide complementary data for completing the genome assembly. The assembly of the genome of M. larici-populina has been made difficult by its large size (compared to most fungi) and especially its high content in repetitive sequences (Duplessis et al., M. larici-populina might therefore represent a significant advance.Besides constituting by itself a valuable resource for investigating the architecture of complex traits of Micha\u00ebl Pernaci, St\u00e9phane De Mita, Fabien Halkett, and Pascal Frey designed research; Micha\u00ebl Pernaci, St\u00e9phane De Mita, Axelle Andrieux, J\u00e9r\u00e9my P\u00e9trowski, and Pascal Frey performed research; Micha\u00ebl Pernaci, St\u00e9phane De Mita, Fabien Halkett, and Pascal Frey analyzed data; and Micha\u00ebl Pernaci, St\u00e9phane De Mita, Fabien Halkett, S\u00e9bastien Duplessis, and Pascal Frey wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "JME), the most common genetic epilepsy, remains enigmatic because it is considered one disease instead of several diseases. We ascertained three large multigenerational/multiplex JME pedigrees from Honduras with differing JME subsyndromes, including Childhood Absence Epilepsy evolving to JME , JME with adolescent onset pyknoleptic absence , and classic JME . All phenotypes were validated, including symptomatic persons with various epilepsies, asymptomatic persons with EEG 3.5\u20136.0\u00a0Hz polyspike waves, and asymptomatic persons with normal EEGs. Two\u2010point parametric linkage analyses were performed with 5185 single\u2010nucleotide polymorphisms on individual pedigrees and pooled pedigrees using four diagnostic models based on epilepsy/EEG diagnoses. Haplotype analyses of the entire genome were also performed for each individual. In pedigree 1, haplotyping identified a 34\u00a0cM region in 2q21.2\u2013q31.1 cosegregating with all affected members, an area close to 2q14.3 identified by linkage . In pedigree 2, linkage and haplotyping identified a 44\u00a0cM cosegregating region in 13q13.3\u2013q31.2 . In pedigree 3, haplotyping identified a 6\u00a0cM cosegregating region in 17q12. Possible cosegregation was also identified in 13q14.2 and 1q32 in pedigree 3, although this could not be definitively confirmed due to the presence of uninformative markers in key individuals. Differing chromosome regions identified in specific JME subsyndromes may contain separate JME disease\u2010causing genes, favoring the concept of JME as several distinct diseases. Whole\u2010exome sequencing will likely identify a CAE/JME gene in 2q21.2\u20132q31.1, a JME/pA gene in 13q13.3\u2013q31.2, and a cJME gene in 17q12.Juvenile myoclonic epilepsy ( According to the World Health Organization , \u201cEpilepThe \u201csine qua non\u201d in the diagnosis of JME are the myoclonic seizures or \u201cmyoclonias\u201d. French authors established the link between these myoclonias and epilepsy in the mid nineteenth century. In Aside from the myoclonic seizure phenotype, a more precise definition for the JME syndrome and the awakening morning myoclonias was not provided until two separate groups, one writing in German and the other in Spanish reported cohorts of JME patients. Janz and Christian writing in German in /EFHC1 in 6p12 as disease causing in JME  (Delgado\u2010Escueta BRD2 [HGNC:1103]), Connexin 36 (Cx\u201036) also called GJD2 (Gap Junction protein Delta2) (HGNC:19154), and malic enzyme 2 (ME2) (HGNC:6984) and microdeletions in 15q13.3, 15q11.2, and 16p13.11 have also been identified and may contribute risk to JME  multigeneration families from Honduras ascertained through a JME proband, each exhibiting a different subsyndrome of the disease, namely, Childhood Absence Epilepsy evolving to JME (CAE/JME) in pedigree 1, JME with adolescent onset pyknoleptic absence (JME/pA) in pedigree 2, and classic JME (cJME) in pedigree 3. In each pedigree, at least seven other family members were diagnosed with a form of generalized epilepsy or were clinically asymptomatic but demonstrated EEG diffuse 3.5\u20136.0\u00a0Hz polyspike waves.The study was approved by the Medical Institutional Review Boards for the David Geffen School of Medicine at UCLA, National Institute of Neurology and Neurosurgery in Mexico City, Mexico, and the National Autonomous University of Honduras. Informed consent was obtained for all participants, patients or from parents of epilepsy\u2010affected or unaffected children.The probands and their families were collected by neurologists at the local study sites of the international consortium of Genetic Epilepsy Studies (GENESS). Diagnosis of JME subsyndromes is based on the work of Mart\u00ednez\u2010Ju\u00e1rez et\u00a0al. . DiagnosThe proband of pedigree 1 with CAE/JME presented at 10\u00a0years of age with pyknoleptic (at least daily) absences with 3\u20104\u00a0Hz spike and wave complexes. She developed myoclonic seizures and clonic\u2010tonic\u2010clonic and tonic\u2010clonic seizures with EEG 4\u20136\u00a0Hz polyspike wave complexes during menarche at 12\u00a0years of age. At 32\u00a0years of age, absences occurred at 5 per day while convulsions appeared at 3 to 4 am, 4\u00a0days before menses, during ovulation, or when fatigued or sleep deprived.The proband of pedigree 2 with JME/pA presented at 13\u00a0years of age with myoclonic, and tonic\u2010clonic and clonic\u2010tonic\u2010clonic seizures. Pyknoleptic absences started at 18\u00a0years of age with 3\u20135\u00a0Hz single spike and slow wave complexes.The proband of pedigree 3 with cJME experienced frequent, severe, awakening myoclonic seizures of shoulders and arms, forearms, hands, and infrequently legs, without loss of consciousness at 14\u00a0years of age. Grand mal clonic\u2010tonic\u2010clonic or tonic\u2010clonic seizures were also present  and were interviewed and examined by a GENESS neurologist. Probands of pedigrees 1 and 2 had 24\u2010h ambulatory EEG and video recordings. When asymptomatic relatives had abnormal diffuse 3.5\u20136.0\u00a0Hz polyspike waves, further clinical examinations were performed and video\u2010EEG recordings confirmed the absence of clinical seizures. All members were studied by a  team composed of at least two GENESS neurologists to confirm diagnosis, and VEEG results were read independently and blindly by two investigators. Blood samples were collected from each living and participating member.Members were considered to have the EEG polyspike wave trait when they were clinically asymptomatic but had evidence of the fast variety (3.5\u20136.0\u00a0Hz) polyspike wave or spike wave complexes. The EEG of affected members have also displayed diffuse, bilateral, synchronous 2.0\u20133.0\u00a0Hz single spike and slow wave complexes or more irregular diffuse 4.0\u20136.0\u00a0Hz sharp and slow waves mixed with random spikes along with the fast variety of spike wave complexes  as the sole clinical phenotype, or generalized clonic\u2010tonic\u2010clonic or tonic\u2010clonic grand mal seizures plus absence seizures, or absence seizures/epilepsy only, absence seizures/epilepsy with eyelid myoclonia, or a combination of these generalized epilepsies  following the manufacturer's protocols. At least 25\u00a0\u03bcL of DNA were obtained at a concentration of 50\u2013100\u00a0ng/\u03bcL. Genotyping services were provided by the Center for Inherited Disease Research (CIDR). Using a custom resynthesis of the InfiniumLinkage\u201024 marker panel, genotyping of 5208 SNPs were attempted and 5185 SNPs (99.6%) were released. SNPs were called using GenomeStudio version 2011.1, Genotyping Module 1.9.4, Gentrain version 1.0. The Mendelian consistency rate of these pedigrees was 99.987% and the concordance rate was 99.81%.We developed four diagnostic models for purposes of linkage analysis: Model #1: all family members with any form of clinically symptomatic epilepsy were considered affected; Model #2: all family members with any form of clinically symptomatic epilepsy plus clinically asymptomatic members with polyspike waves evidenced on their EEGs were considered affected; Model #3: only JME affected members were considered affected; and Model #4: all family members with JME plus clinically asymptomatic members with polyspike waves evidenced on their EEGs were considered affected. Linkage analyses were run on each of the individual pedigrees under each diagnostic model. Additionally, linkage analyses were also run on the combination of the three Honduran pedigrees under each diagnostic model.Two\u2010point parametric linkage analysis was completed using Superlink v1.6 .The highest average and maximum ELOD scores occurred when all pedigrees were combined under diagnostic model #2 . In fact, regardless of which diagnostic model was run, the combination of the pedigrees produced a higher expected LOD score than any of the pedigrees could have produced on their own.When individual pedigrees were run, the addition of the clinically asymptomatic members with the EEG trait to the affected pool in pedigrees 1 and 2 consistently produced a higher ELOD score than that produced when only clinically affected members were considered affected . This increase in ELOD score was particularly evident when comparing models #3 and #4 in pedigree 1, as only one family member in this pedigree was diagnosed with JME. The simulations using diagnostic models #3 and #4 produced smaller average and smaller maximum ELOD scores than those run under diagnostic model #1 and #2, particularly in pedigrees 2 and 3 as it counted less members as affected. In each pedigree and the combination of pedigrees, the results from diagnostic model #3 consistently produced the smallest scores, as it counted the fewest members as affected. Pedigree 3 has no clinically asymptomatic members with the EEG polyspike wave trait, therefore, the ELODs for this pedigree were unchanged between diagnostic models #1 and #2 and diagnostic models #3 and #4.Linkage analyses were run under all four diagnostic models for Honduras pedigrees 1, 2, and 3 , as well as for the combination of the pedigrees (Table\u00a0Zmax (\u03b8m\u00a0=\u00a0f\u00a0=\u00a00.00) of 2.59 at rs2272386 on 2p23.2 under diagnostic model #2, which included all the epilepsy affected individuals  and those with the EEG polyspike wave trait. A drop in Zmax occurred when asymptomatics with EEG polyspike waves were not considered affected (diagnostic model #1). Diagnostic model #3 produced a negligible LOD score in this pedigree because only one member has a JME diagnosis. Diagnostic model #4 produced LOD scores higher than those produced by diagnostic model #3 with Zmax (\u03b8m\u00a0=\u00a0f\u00a0=\u00a00.00) of 1.77 at rs1028184 on 2q14.3. This is a result of the addition of the clinically asymptomatic members with the EEG polyspike wave trait to the only person with JME.The highest LOD obtained from all analyses was suggestive for linkage with Zmax\u00a0=\u00a02.26) on 4q34.2 when all forms of epilepsy  and three asymptomatic persons with the EEG polyspike wave trait were included as affected under diagnostic model #2. Aside from 4q34.2, the other locus identified by this model was 13q21.31 with Zmax (\u03b8m\u00a0=\u00a0f\u00a0=\u00a00.00) of 1.90 at rs1335686. Diagnostic model #3 produced LOD scores very similar to those produced under diagnostic models #1 and #4, though none of the same markers were seen among the top results of the three models and all failed to reach scores comparable to diagnostic model #2.The LOD score peaked at rs13132745 (Zmax\u00a0=\u00a02.55), including all epilepsy affected members . Interestingly 13q21.31 was also identified by models #1 and #2 with Zmax (\u03b8m\u00a0=\u00a0f\u00a0=\u00a00.00) of 2.04 at rs1553161. Because no pedigree members were clinically asymptomatic with abnormal EEGs, the results for diagnostic models #1 and #2 are identical, as are the results for models #3 and #4.LOD scores were suggestive for linkage at rs1888952 on chromosome 9p22.3 under diagnostic models #1 and #2 (Zmax reached 2.95 at a second marker in 4q35.2 (rs6553022) and also had markers at 5q21.3 (rs1045706) and 13q31.1 (rs3127540) surpass the level suggestive for linkage. Similar to the individual pedigree analyses, the addition of the clinically asymptomatic members with EEG polyspike waves in the combined pedigree analysis increased LOD scores, crossing the threshold for significance.When the three Honduran pedigrees with JME proband diagnoses were combined, diagnostic model #2 produced LOD scores significant for linkage, and diagnostic model #1 produced LOD scores suggestive for linkage. When all clinically affected members and all clinically asymptomatics with EEG polyspike waves were included as affected under model #2, LOD score peaked at 3.50 at rs3127540 on 13q31.1. Under this analysis, rs1869941 at 4q35.2 and rs1203974 at 16p13.3 also reached the level of significance. When only the epilepsy affected members were considered affected under model #1, Zmax of 1.77. Chromosome loci 2p23.2 and 5q23, which had Zmax of 2.59 and 2.11, respectively, along with all other loci, are eliminated by haplotype analysis.Haplotype analysis of the entire genome of pedigree 1 showed only one cosegregating chromosome region, namely a 34\u00a0cM area in 2q21.2\u201331.1, bordered by rs2872920 and rs17664, that cosegregates with all clinically affected persons and clinically asymptomatic individuals who carry the EEG polyspike wave trait . Since SNP markers were all biallelic, the number of allele combinations that can provide informative meiosis is reduced. However, SNPs do have the advantage that their density and distribution across the genome makes it much easier to impute an optimal haplotype due to the decreased likelihood of a double recombination occurring over a very small region. The segregating region in H3, for example, spanned approximately 55\u00a0cM, covered by 94 SNP markers.\u00a0Of those 94 SNPs, using model #2, only 3 SNPs had LOD scores greater than 1.50 and 17 SNPs had LOD scores greater than 1.00 . In the case of H5, all of the reported regions could only \u201cpotentially\u201d cosegregate, due to the fact that only one \u201cmarried\u2010in\u201d was collected and haplotyped. This increased the space between informative markers where the parental haplotypes could not be definitively assigned.Most importantly, whole\u2010genome haplotype analysis was essential in eliminating chromosome regions that had appeared significant for linkage and then failed to cosegregate in any of the pedigrees, such as 9q22.3 in pedigree 3, as well as 4q35.2 and 16p13.3 in the pooled linkage analysis of the three pedigrees. Linkage analysis combined with haplotype analysis did identify one locus in 2q21.2\u2010q31.1 for a CAE/JME gene and another locus in 13q13.3\u2010q31.2 for a JME/pA gene.Juvenile myoclonic epilepsy remains an enigmatic epilepsy. Despite exhibiting varying seizure phenotypes within family members, differing frontocortical\u2010subcortical networks that underlie EEG polyspike waves, and changing prognoses for seizure remission, JME researchers continue to explain JME as a single disease  in 2q24 (Krepischi et\u00a0al. SLC4A10, or solute carrier family 4, sodium bicarbonate transporter, member 10 in 13q31 (Gurnett et\u00a0al. The large cosegregating region from chromosome 13q13.3\u2013q31.2, suggested by individual linkage and haplotyping results in pedigrees 2 and 3, as well as the pooled pedigree linkage results, contains several chromosome areas that have previously been implicated in various epilepsies. Idiopathic generalized epilepsies have previously been genetically linked to locus 13q31.3 (EPICURE Consortium et\u00a0al. CAMSAP1L1 or calmodulin regulated spectrin\u2010associated protein family, member 2 found in a GWAS of Chinese epilepsy patients (Guo et\u00a0al. SRGAP2 or SLIT\u2010ROBO Rho GTPase activating protein 2 in a single patient with early infantile epileptic encephalopathy and severe psychomotor disability (Saitsu et\u00a0al. Pedigree 3 produced more varying results, with haplotypes definitely cosegregating in 17q12 and possibly cosegregating in 1q32.1 and 13q14.2. The 17q12 locus has been linked with generalized epilepsies and febrile seizures, as well as with migraines comorbid in Rolandic epilepsy (Sir\u00e9n et\u00a0al. There is a wide range of seizure phenotypes across JME families, and even within the same JME family. While our pedigrees were ascertained through a proband with a JME diagnosis, these probands exhibited three distinct subsyndromes of the disease based on the age at occurrence of each seizure phenotype and specific EEG pattern, the presence of myoclonias, generalized clonic\u2010tonic\u2010clonic or tonic\u2010clonic grand mal seizures and absence seizures, and the sequence of their appearance. The family members of these pedigrees exhibited a wide range of epilepsy phenotypes and did so with different patterns based on the proband's JME subsyndrome. Pedigree 1, whose proband had CAE/JME, had the highest number of family members with \u201cabsence epilepsy only\u201d. There were no cases of classic JME without absence seizures in family members of pedigree 1 suggesting this family exhibits more of an absence syndrome. Pedigree 2, whose proband had JME/pA, had cases that were primarily \u201cJME only\u201d or \u201cabsence epilepsy only\u201d in family members suggesting this family has a syndrome with mixtures of absence and JME. Pedigree 3, whose proband had cJME, had family members consisting mostly of JME cases and because none of the family members had \u201cabsence epilepsy only\u201d this pedigree 3 is a true classic JME family. These observations coincide with previous reports that epilepsy diagnoses in family members vary by the proband's diagnosis, and that family member diagnoses are more likely to share epilepsy syndrome and seizure type with the proband (Winawer et\u00a0al. Because differences can be seen in the presentation and frequency of epilepsy phenotypes in family members of specific pedigrees based on the JME subsyndrome, it has been argued that the subsyndromes are actually different forms of epilepsy diseases that should not be analyzed together under the single diagnosis of JME. The three candidate chromosome loci, namely 2q21.2 to 2q31.1 for CAE/JME in pedigree 1, 13q13.3 to 13q31.2 for JME/pA in pedigree 2 and 17q12 for cJME in pedigree 3 support this concept.We conducted linkage analysis using different diagnostic models to simulate varying levels of clinical heterogeneity within and among the pedigrees and assess the effects on the LOD score. Evidently, the highest LOD scores for each pedigree and the combination of pedigrees were produced under the most clinically heterogeneous diagnostic model #2, which included all forms of epilepsy plus the clinically asymptomatic members with EEG polyspike waves. Haplotype analysis of the entire genome for each pedigree was critical for determining which chromosome regions cosegregated and which regions coincided with the chromosome loci identified by linkage analysis. These results argue that clinical phenotypic heterogeneity within a pedigree will not inhibit analysis and can actually increase the LOD score. Despite having a proband diagnosis of JME, these results further support the notion that these pedigrees should not be combined, because we would have expected the cosegregating regions to overlap among the pedigrees and the combined LOD score to be closer to the higher ELOD score.Despite our best efforts, our results are subject to limitations, which, in turn, advise caution in interpretations. First, is the unavailability of DNA due to deceased members and members unwilling to participate which is not uncommon in family\u2010based studies. We lacked DNA for deceased founders in all three pedigrees and for several \u201cmarried\u2010in\u201d individuals, most notably in pedigree 3. This prevented notations of phase known meioses, forcing imputations of haplotypes, and preventing definition of a single candidate region for pedigree 3. Second, is the caution in how our results should be interpreted in the overall context of the complex heredity of epilepsy. Linkage results of complex traits can be unreliable and inexact. Most analyses of the complex hereditary factors in epilepsy have often failed to disentangle heredity from environment. Usually a multifactorial or complex genetic model of polygenes acting on environment is inferred. Here, linkage and haplotype analysis in three large multigeneration kindreds with many matings and a large number of offspring with well defined epilepsy and EEG endophenotypes emphasize the role of locus heterogeneity and differing mutated genes in 13q13.3\u201031.2 for JME with adolescent onset pyknoleptic absence, 2q21.2\u2013q31.1 for CAE evolving to JME and 17q12 for classic JME. Definite understanding of the differing clinical epilepsy and EEG endophenotypes within these JME families will require identification of the many variants within the many specific epilepsy\u2010causing genes and their neurobiological disease mechanisms.None of the authors has any conflicts of interests to disclose.Figure\u00a0S1. Individual genome scans for all three pedigrees for each of the four diagnostic models.Click here for additional data file.Figure\u00a0S2. Haplotype of pedigree 3, chromosome 1q32.1.Click here for additional data file.Figure\u00a0S3. Haplotype of pedigree 3, chromosome 13q14.2.Click here for additional data file.Figure\u00a0S4. 170 SNPs genotyped on chromosome 13 from Family 2.Click here for additional data file.Table\u00a0S1. Simulated ELOD results for Honduran pedigrees.Click here for additional data file.\u00a0Click here for additional data file."}
+{"text": "High-throughput sequencing studies (HTS) have been highly successful in identifying the genetic causes of human disease, particularly those following Mendelian inheritance. Many HTS studies to date have been performed without utilizing available family relationships between samples. Here, we discuss the many merits and occasional pitfalls of using identity by descent information in conjunction with HTS studies. These methods are not only applicable to family studies but are also useful in cohorts of apparently unrelated, \u2018sporadic\u2019 cases and small families underpowered for linkage and allow inference of relationships between individuals. Incorporating familial/pedigree information not only provides powerful filtering options for the extensive variant lists that are usually produced by HTS but also allows valuable quality control checks, insights into the genetic model and the genotypic status of individuals of interest. In particular, these methods are valuable for challenging discovery scenarios in HTS analysis, such as in the study of populations poorly represented in variant databases typically used for filtering, and in the case of poor-quality HTS data. High-throughput sequencing (HTS) has proven to be highly successful at identifying causal variants for many genetic disorders with a variety of underlying genetic etiologies , The Human Gene Mutation Database  and ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/). However, these databases are known to be incomplete, missing known mutations. Additionally, Bell et al.  extending quality control (QC) steps, (2) identifying IBD regions, (3) validating the genetic model, and (4) validating haplotype segregation by making use of IBD information, with or without additional SNP chip data. Using this type of information can lead to the accumulation of evidence in favor of particular variants, determine the correct genetic model, lead to the elimination of irrelevant variants and can also narrow the search space for variants to particular genomic regions.HTS data is highly non-uniform in coverage which is influenced by GC content of the underlying sequence where both very high and very low GC content DNA sequence is captured by fewer HTS reads  due to poor WES data generation, yet we were still able to detect a linkage peak and rapidly identify the causal variant despite the inability to identify the variant in many samples due to low coverage  IBD process. IBD can be calculated as a genome-wide summary, or at specified locations in the genome. In contrast, linkage analysis is a likelihood ratio test statistic that compares the probability of the genotyping data, for possibly more than two individuals, under two different hypotheses. The null hypothesis calculates the probability of the data assuming no linkage between the phenotype (modeled as another genetic marker) and the genotyping data from many genetic markers, and compares it to the alternative hypothesis of linkage between the phenotype and the set of genetic markers. This function of the likelihood ratio is known as the LOD score and can be calculated at any position in the genome.Linkage and IBD analyses are powerful approaches for the identification of genomic regions harboring the causal variants for Mendelian diseases. Thus, both linkage and IBD methods are useful tools for filtering detected variants in cohorts of related individuals with HTS data. To implement this, one performs IBD or linkage analysis as described above, with or without SNP chip data, prior to variant filtering. These regions are then used as an additional filter for variants that remain to be considered. Other authors have designed genetic model-specific hidden Markov models for IBD detection  and small indels only . The identification of moderate-sized indels require additional analysis of the HTS data with specialized algorithms such as Pindel (Ye et al. While rare, it is possible that the assumed genetic model, under which the linkage or IBD analysis has been performed, may not be correct. This would lead to an incorrect filter. This can occur and is of particular concern for consanguineous pedigrees where it is usual to infer an autozygous recessive model. However, the inbreeding may be a coincidence, with the true mutation being most likely an incompletely penetrant dominant mutation or two compound heterozygous recessive mutations. The deeper the inbreeding loop, the less likely it is that an affected individual will still share a segment of an ancestor that is homozygous by descent, as indicated by an increasing LOD score for such regions in affected individuals, as parents are more distantly related. Extensive inbreeding may also make mapping results difficult to interpret, especially in the presence of locus heterogeneity (Markus et al. The trio design for HTS projects is an extremely attractive approach for detecting hypothesized de novo variants in a cohort of sporadic cases where DNA from both parents is available. Without additional evidence for inbreeding in the population of origin, which suggests the possibilities of an autozygously inherited variant, the de novo genetic model is the most likely genetic model for singleton affected individuals and provides a powerful filter for variants. The trio design for de novo analysis is another family-based study design but it does not make use of linkage information, instead using the family data to eliminate inherited variants. Again, however, the success of this approach relies heavily on the hypothesized inheritance model being correct. In Taft et al. , the iniHere, we have shown how IBD sharing analysis can be used in tandem with HTS to ensure data veracity, to identify appropriate genetic models that facilitate genotype filtering and to make inferences about which individuals are likely to share the ancestral susceptibility haplotype according to the best-fitting genetic model at each locus.HTS is increasingly being applied to high morbidity, rare, Mendelian disorders where only sporadic cases are observed due to individuals being unlikely, or unable, to reproduce. These types of cohorts usually lack familial information for linkage or IBD studies available, unless the affected individuals are the product of a consanguineous union, in which case a powerful linkage analysis is possible. Other studies can only avail themselves to small, underpowered pedigrees for linkage, however, there is no longer a requirement for a priori statistically significant linkage, since WES and WGS are genome-wide variant identification methods, with no additional costs involved to examine further regions in the genome. A filter based on using multiple regions identified by either linkage or IBD analysis can still be a powerful method for the reduction of the number of candidate variants.Many of the approaches we have outlined will become even more powerful as WGS supersedes WES and other targeted sequencing approaches. WGS is known to have less bias overall than WES and thus may identify causal variants that have been missed with WES (Bamshad et al. HTS is not fail-safe. True causal variants may fail to be detected due to insufficient coverage or erroneous data (Koboldt et al. The forthcoming improvements in HTS will lead to increased read length. These will improve our capacity to infer CNVs, microsatellites, other structural variations and facilitate de novo assembly and in silico haplotyping, which we have described above to also aid variant filtering. Detection of these types of variants in HTS data will also benefit from the use of family-based information. PennCNV (Wang et al. With the decreasing costs of HTS larger cohorts of affected individuals will be sequenced and some of these individuals will be related, either cryptically or by a known pedigree. Despite the undoubted success of HTS studies, the incorporation of familial information has the potential to enhance our efforts in this fast-moving field."}
+{"text": "Plasmodium falciparum mild malaria and parasitaemia, only two of them being significant at the genome level. The objective of the present study was to identify malaria resistance loci in individuals living in Burkina Faso.Genome-wide studies have mapped several loci controlling A genome scan that involved 314 individuals belonging to 63 families was performed. Markers located within chromosomes 6p21.3 and 17p12 were genotyped in 247 additional individuals belonging to 55 families. The linkage and the association of markers with parasitaemia and mild malaria were assessed by using the maximum-likelihood binomial method extended to quantitative trait linkage and the quantitative trait disequilibrium test, respectively.\u22125), a suggestive linkage of mild malaria to chromosome 19p13.12 , and a suggestive linkage of asymptomatic parasitaemia to chromosomes 6p21.3  and 17p12 . Genome-wide family-based association analysis revealed a significant association between three chromosome 5q31 markers and asymptomatic parasitaemia, whereas there was no association with mild malaria. When taking into account 247 additional individuals, a significant linkage of asymptomatic parasitaemia to chromosome 17p12  was detected.Multipoint linkage analysis showed a significant linkage of mild malaria to chromosome 6p21.3 (LOD score 3.73, P\u2009=\u20091.7 10A new genome-wide significant malaria locus on chromosome 17p12 and a new suggestive locus on chromosome 19p13.12 are reported. Moreover, there was evidence that confirmed the influence of chromosomes 5q31 and 6p21.3 as loci controlling mild malaria or asymptomatic parasitaemia. Plasmodium falciparum who progress to severe malaria. Many studies provide evidence for human genetic factors controlling the outcome of infection by P. falciparum[HBB locus as a major locus in P. falciparum severe malaria [Char3 and Char8 that control Plasmodium chabaudi in mice [Malaria remains a major problem of public health in over one hundred countries worldwide. According to the World Health Organization (WHO), the number of cases in 2010 reached 219 millions (95% CI 154\u2013289 millions), among which over 660 000 (95% CI 490 000\u2013836 000) were fatal [ in mice .The present study reports results from a genome-wide scan and additional further testing of promising regions. The maximum-likelihood binomial method extended to quantitative trait linkage analysis (MLB-QTL) and the quantitative trait disequilibrium test (QTDT) were applied to search for genetic linkage and association in the presence of linkage with mild malaria and asymptomatic parasitaemia.P. falciparum parasitaemia, fever (axillary temperature more than 37.5\u00b0C) and clinical symptoms ; in that case no threshold of parasitaemia was used. In the absence of classical symptoms of malaria, and once others pathologies could not be eliminated, only children (age\u2009<\u200915\u00a0years) with more than 5,000 parasites per \u03bcl and older subjects with more than 2,000 parasites per \u03bcl were considered as having had a malaria attack. Each episode of illness was treated according to the recommendation of the CNRFP  of Burkina Faso. Parasitaemia was checked at the end of the treatment. Subjects who presented at least one mild malaria attack during the survey were considered in the analysis affected, while the others were considered unaffected. Determination of parasitaemia was described in previous studies [P. falciparum asexual forms were retained to determine parasitaemia. Parasitaemia was defined as the number of parasitized erythrocytes observed per \u03bcl in thin blood films.The initial study population consisted of 314 individuals belonging to 63 families living in an urban district of Bobo Dioulasso in Burkina Faso; the mean age of sibs was 13.6\u2009\u00b1\u20096.3\u00a0years (range 1\u201334 years). The additional study subjects live in a rural area, Logoforousso, a village to the south-west of Bobo-Dioulasso (Burkina Faso). The study population comprised 247 subjects from 55 nuclear families; the mean age of the sibs was 10.1\u2009\u00b1\u20094.7 (3\u201329 years). The populations and the areas of parasite exposure have been extensively described . Informe studies . BrieflyP\u2009=\u20090.013). The residual of the logistic regression model and the one of the linear regression model were calculated for mild malaria and parasitaemia, respectively; the residuals were further used in linkage or association analyses. All the sibs (in full) were included in linkage analyses.Mean of adjusted asymptomatic parasitaemia was a logarithmic transformation of the parasitaemia adjusted for seasonal transmission ,12, afteGenome scan was performed by using 400 microsatellite markers , with an average distance of 10\u00a0cM. Multiplex PCR was performed under standard conditions, and the products were analysed by using a ABI377 systems (Applied Biosystems). Additional genotypes that were previously characterized in the initial study population were used for chromosomes 6p21.3  and 5q31-q33  , whereas\u201310). The approach with the assumption on the normal distribution was used for parasitaemia. The MLB statistics was expressed in terms of a MLB-LOD and a one-sided standard normal deviate, denoted ZMLB. According to Kruglyak and Lander recommendations [Nonparametric multipoint linkage analyses were performed with the MLBGH 3.0 program, which uses the general framework of Genehunter program ,16. The ndations , linkageCombined association and linkage analyses of quantitative traits were carried out using the orthogonal model released in the QTDT 2.6.1 program . Varianc\u22125) and on chromosome 19p13 , respectively. The 1-LOD drop area for QTL location on chromosome 6p21.3 was very small, and included only 5 genes: TNF, LTA, LTB, LST1, and NCR3.The genome scan was performed on 314 individuals belonging to 63 families. Figure\u00a0\u22124); the 1-LOD drop area for QTL location was very similar than the one for mild malaria, and contained the same genes. The best LOD score was achieved on chromosome 17p12 at D17S969/D17S799 . The 1-LOD drop area for QTL location on chromosome 17p12 spanned 14\u00a0cM (D17S1875-D17S839), and contained 11 genes: MAP2K4, ARHGAP44, MYOCD, ELAC2, COX10, DNAH9, HS3ST3B1, HS3ST3A1, ZNF18, CDRT15, and SHISA6.Figure\u00a0Combined linkage and association between malaria phenotypes and microsatellite markers were further tested. After applying a FDR of 10%, there was no significant association with mild malaria, whereas there was an association of asymptomatic parasitaemia with D5S642, the IL9 microsatellite, and D5S2017. Strikingly, those genetic markers are located within chromosome 5q31; noticeably, the distance between D5S642 and the IL9 microsatellite was 3.7\u00a0cM, and the one between the IL9 microsatellite and D5S2017 was 6.7\u00a0cM, suggesting the existence of several distinct malaria resistance loci in this region.\u22125) and 17p12 , as shown in Figure\u00a0TNF, LTA, LTB, LST1, and NCR3. The whole 1-LOD drop area for QTL location on chromosome 17p12 spanned 13\u00a0cM (D17S969-D17S839), and contained the 11 genes identified in the primary analysis.Additional individuals n\u2009=\u2009247) that belonged to 55 families were also genotyped for the microsatellite markers in chromosomes 6p21.3 and 17p12, resulting in a total of 118 families with 561 individuals genotyped. The additional genotyping led to higher LOD scores for chromosomes 6p21.3  synthesis. They catalyse the 3\u20130 sulfation of carbohydrates, which is the last step of the synthesis, and which reflects the completion of HS synthesis. The variation in human genes controlling the sulphation of HS may alter the binding of sporozoites to hepatocytes and their development in those cells, as it has been shown in mice infected by P. berghei[Finally, a new locus controlling parasitaemia was identified based on a genome-wide significant LOD score after adding new families in the study. The peak of linkage located within chromosome 17p12 was sharp, and the 1-LOD drop area contained 11 genes both in the initial population and the extended one, the mouse orthologs of which are within nt study ; interes. berghei. Taken tThis is the first report of a malaria resistance locus on chromosome 17p12, whereas it confirms previous loci on chromosomes 6p21.3 and 5q31. Although replication studies are needed, our results support the recent hypothesis that genetic variation within genes involved in HS synthesis strongly influences parasitaemia. This may provide new insights for developing new therapeutic strategies.The authors declare that they have no competing interests.AB carried out the linkage and association analyses. BK and SW genotyped the microsatellite markers, and constructed the genotype database. AA and SG participated in genotyping. FF participated in the design of the study, and revised the results and the manuscript. PR performed the design of the study, supervised the experiments and the statistical analyses, participated in the statistical analyses and the interpretation of data, and wrote the manuscript. All authors read and approved the final manuscript."}
+{"text": "Talent is Overrated , 23-year old Carlsen is ranked first with 2881 pointst-test confirms that Carlsen's rating is statistically different from the next ten grandmasters (M = 2780.6), t(9) = \u221219.38, p < 0.001, mean difference = \u2212100.4; 95% CI . One hundred points is a considerable difference: it is half a standard deviation in skill and means that, against the very best players in the world, Carlsen's probability of winning is 63.7%.A particularly spectacular example is provided by chess grandmaster Magnus Carlsen (Norway), who became world champion in classic chess in November 2013 by beating Viswanathan Anand (India) and who also became world champion in rapid chess  and speed chess  in June 2014. In the June 2014 rating list published by the World Chess Federation (M = 24.6 years), t(9) = 2.83, p < 0.05, mean difference = 6.6 years; 95% CI . However, this result is exactly the opposite of what is predicted by deliberate practice: on average, Carlsen practiced statistically significantly fewer years than the other players.  but not statistically significant (p = 0.55)).To test the monotonic assumption, we collected information from the internet and biographies about the age at which these grandmasters started playing chess and about their current age . The fact that Carlsen dominates the chess world so outrageously, being world champion not only in classic chess but also in rapid chess and in blitz, refutes this hypothesis, central to the theory of deliberate practice.Several objections can be leveled at this analysis. We discuss three of them, and show that they do not invalidate our argument. First, Carlsen's prodigious skill throughout adolescence and early adulthood may not be as remarkable as it first appears, as numerous young players perform better that their older competitors. For example, Howard  has showr = \u2212 0.21) is not statistically significant (p = 0.54), but Carlsen is reliably younger than the other ten top players, t(9) = 3.16, p < 0.05, mean difference = 7.6 years; 95% CI . Nevertheless, the age variable does not explain why Carlson is so clearly better than the four players who are roughly his age.Second, it could be argued that, just like in sport, age plays an important role in chess and youth will give an edge to younger top competitors. It is known that the effects of ageing occur depressingly early with cognitive variables such as reasoning, visualization and processing speed, peak performance being observed in the early to mid-twenties (Salthouse, Third, Carlsen might have engaged in more intense deliberate practice. Although we do not know the details of Carlsen's training, this is unlikely, in particular if we use Ericsson et al.'s  criterioThus, the question arises, in the risk of offending the proponents of deliberate practice: Does Carlsen have a particular talent for chess? The answer to this question is so obvious in the chess world that it is not even posed\u2014Carlsen is known as the \u201cMozart of chess.\u201d Several factors support the hypothesis of talent. Carlsen showed clear signs of intellectual precocity early in his life. At the age of five, he knew \u201cthe area, population, flag, and capital of all the countries of the world,\u201d and memorized similar information for all Norway's 430 municipalities (Agdestein, The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "Plasmodium falciparum. \u25ba Genetic maps and recombination parameters. \u25ba Quantitative trait locus analysis of parasite phenotypes..\u25ba The principles of genetic linkage analysis of experimental genetic crosses in  Plasmodium falciparum. Three experimental genetic crosses have been performed using the human malaria parasite P. falciparum. Linkage analysis of the progeny of these crosses has been used to identify parasite genes important in phenotypes such as drug resistance, parasite growth and virulence, and transmission to mosquitoes. The construction and analysis of genetic maps has been used to characterise recombination rates across the parasite genome and to identify hotspots of recombination.We review the principles of linkage analysis of experimental genetic crosses and their application to  The processes of independent segregation of the 14 chromosomes and crossing over between homologous chromosomes both occur during meiosis in the diploid zygote stage of the parasite , which iThe haploid sporozoites that develop within an oocyst can therefore possess combinations of chromosomes, and genes along those chromosomes, which did not exist in the gametes (known as recombinants). Meiosis produces recombinants only following fertilisation events between genotypically distinct gametes; fertilisation between a male and female gamete from the same parasite genotype (self-fertilisation) would result in a zygote that was totally homozygous at all loci, and chromosomal segregation and crossing over would produce progeny that were identical to the original gametes .In the laboratory, cross- and self-fertilisation occur between gametes in the blood meal of a mosquito feeding on an artificial mixture of gametocytes of two different genotypes, at frequencies determined by random associations between gametes, i.e. there does not appear to be a favouring of self- or cross-fertilisation . A similLaboratory crossing experiments are easier to analyse than those occurring during natural transmission, because the genotypes of the gametocytes are known and defined, and the maximum number of alleles at any locus is two. The recombinant progeny can be cloned and analysed for their inheritance of the two parental alleles . The frePlasmodium falciparum  within the progeny clones in the former \u2013 those existing in the two parents used for the cross \u2013 whereas there could be very many within a population sample in the latter studies. Therefore, loci can be identified by analysing smaller numbers of parasite clones from an experimental cross, and there are less likely to be confounding effects from multiple forms of multiple genes.If variation in a parasite phenotype is controlled by polymorphisms within a single gene, each haploid progeny clone will inherit the allele from either one parent or the other, and the progeny will all exhibit one of two variants of the phenotype \u2013 two parental-like groups \u2013 with no intermediate phenotypes. Any variation in phenotype will be due to environmental differences or to experimental noise. The gene controlling the phenotypic variation of interest can be identified by examining the genomes of the progeny clones, and finding the region(s) of the genome that are shared among the parent and progeny that exhibit one phenotypic state. This is the principle of linkage analysis.In cases where multiple genes contribute to the phenotypic variation, non-parental phenotypes would be observed, which could fall between the two parental phenotypes, or be greater or smaller than either parent. These multigenic or complex traits are analysed using a statistical approach known as quantitative trait locus (QTL) analysis .A linkage analysis study of an experimental genetic cross involves three steps: (i) the phenotype of interest is examined in the progeny of a genetic cross; (ii) a genetic map of each of the progeny clones is generated, based on the inheritance of polymorphic markers, to identify the regions of each chromosome inherited from each of the two parents in the cross; and (iii) the phenotype and genotype data are linked using statistical methods such as maximum likelihood (ML) analysis and Log of Odds (LOD) scores .2.1To perform any linkage analysis, it is necessary to identify polymorphic markers throughout the genome and \u201cmap\u201d their positions and relative genetic distances along each chromosome . The polP. falciparum used genetic maps based on restriction fragment length polymorphism (RFLP) markers , which represents the likelihood of linkage versus no linkage . A LOD sPlasmodium crosses have been analysed for single-locus as well as QTL, which will be discussed in Section 4. Analysis is made simpler because the parasite stages undergoing phenotyping are haploid; for a single-locus study there are no heterozygotes and parasite clones are treated as being fully homozygous at all loci. Analysis of experimental genetic crosses has allowed the identification of parasite loci controlling both simple and complex traits, including resistance to antimalarial drugs such as chloroquine and quinine , the abi3Genetic maps define the relative order of loci along a chromosome in terms of genetic distance, which measures the likelihood of crossover events between those two loci. Crossover events are more likely to occur between markers further apart on a chromosome than between those close together. Crossover events are initiated by the formation of a double strand break , which iTheileria parva  with relatively high frequencies of crossover and gene conversion events . In contromosome , possibleric var . Analysieric var , which ieric var .P. falciparum reduces the number of progeny clones that need to be phenotyped and genotyped to identify QTL to an experimentally manageable number. For example, the Pfmdv1 locus underlying a male gametocyte defect was initially mapped using just 11 progeny clones  was found to be required for resistance  mapped to pfdhfr, while parasite response to dihydroergotamine methanesulfonate , mapped to pfmdr1 . Disruption of the pvmdv-1 gene resulted in a reduction of mature gametocytes and developmental arrest at stage I. Although this was not the exact phenotype seen in the cross, the study identified Pfmdv-1 as a key contributor to gametocyte development.The parasite Dd2 infects mosquitoes poorly, despite producing morphologically normal gametocytes; analysis of 11 progeny from the HB3\u00a0\u00d7\u00a0Dd2 cross indicated that this failure to infect mosquitoes was linked to an 800\u00a0kb locus on chromosome 12 . FurtherQTL analysis has also been carried out to investigate parasite genes contributing to parasite development within the mosquito . QTL analysis of progeny from the 3D7\u00a0\u00d7\u00a0HB3 cross revealed a major locus on chromosome 12 which accounted for 94% of the variation in infection intensity (number of oocysts per mosquito midgut) and 44% of the variation in infection prevalence (proportion of mosquitoes infected).4.4P. falciparum genetic cross revealed that expression levels of many transcripts during the asexual parasite lifecycle were heritable and could be mapped . Analysis of a e mapped . Approxi5Analysis of experimental crosses  remains an important tool in malaria research, allowing unbiased identification of gene(s) affecting a phenotype, rather than a candidate gene approach. Modern genotyping methods and computing power allow more rapid identification of genetic regions controlling a trait and thus have significantly strengthened the power of linkage mapping approaches in recent years. The approach allows the identification of multiple genes contributing to a phenotype, including loci interacting epistatically and additively, and it is possible to estimate the relative contribution of a genetic locus to a phenotype.P. falciparum has allowed the discovery of genetic mechanisms behind a variety of phenotypic traits for which there were no obvious candidate genes. Experimental crosses have advantages over studies using field isolates with diverse genetic backgrounds, for example (i) the limited genetic variation in the progeny (derived from only two parents) avoids possible confounding effects of multiple forms of multiple genes, and (ii) the ability to phenotype laboratory clones repeatedly under controlled conditions can reduce environmental variation .Analysis of experimental genetic crosses of Progeny clones from the three experimental crosses performed to date, coupled with the high density genetic maps now available for each cross, provide a useful community resource for future work to understand the influence of parasite genetic polymorphism on biological phenotypic variation. Further refinement of the genetic linkage maps, including complete sequence information for the progeny clones, will reveal new crossovers, assist in identifying precise points of recombination (chromosome breakpoints) and hot spots of recombination, and will facilitate positional mapping of candidate genes."}
+{"text": "Xenopus laevis and the juvenile turtle Pseudemys scripta. This analysis served to highlight the organization of the hypothalamus in the anamniote/amniotic transition. We have identified supraoptoparaventricular and the suprachiasmatic regions (SCs) in the alar part of the hypothalamus, and tuberal and mammillary regions in the basal hypothalamus. Shared features in the two species are: (1) The supraoptoparaventricular region (SPV) is defined by the expression of Otp and the lack of Nkx2.1/Isl1. It is subdivided into rostral, rich in Otp and Nkx2.2, and caudal, only Otp-positive, portions. (2) The suprachiasmatic area contains catecholaminergic cell groups and lacks Otp, and can be further divided into rostral (rich in Nkx2.1 and Nkx2.2) and a caudal (rich in Isl1 and devoid of Nkx2.1) portions. (3) Expression of Nkx2.1 and Isl1 define the tuberal hypothalamus and only the rostral portion expresses Otp. (4) Its caudal boundary is evident by the lack of Isl1 in the adjacent mammillary region, which expresses Nkx2.1 and Otp. Differences in the anamnio-amniote transition were noted since in the turtle, like in other amniotes, the boundary between the alar hypothalamus and the telencephalic preoptic area shows distinct Nkx2.2 and Otp expressions but not in the amphibian (anamniote), and the alar SPV is defined by the expression of Otp/Pax6, whereas in Xenopus only Otp is expressed.Most studies in mammals and birds have demonstrated common patterns of hypothalamic development highlighted by the combination of developmental regulatory genes (genoarchitecture), supporting the notion of the hypothalamus as a component of the secondary prosencephalon, topologically rostral to the diencephalon. In our comparative analysis we have summarized the data on the expression patterns of different transcription factors and neuroactive substances, used as anatomical markers, in the developing hypothalamus of the amphibian  The hypothalamus is considered the forebrain territory par excellence dedicated to control homeostatic processes, and its neuroanatomical regionalization has been a much debated topic in recent years. The term \u201chypothalamus\u201d was coined during the last century with the beginning of neuroanatomical studies His, ,b, folloTwenty years ago, the first proposal of the prosomeric model pointed out several evidences to discard the columnar paradigm of the forebrain organization, revealing the discrepancy between the traditional anatomical landmarks and the morphogenetic organization of the brain, what eventually led to refute the boundary role of the ventricular sulci  between the Isl1/Nkx2.1 positive PO and the SPV-Otp expressing region  was traditionally included within the hypothalamus until genoarchitectonic studies revealed that this region is derived from the FoxG1-positive telencephalic neuroephitelium  the expression of Otp in the SPV territory has been reported  Nkx2.1 and Nkx2.2 are found in the ventricular zone, in contrast to the caudal portion (SCc) that is devoid of expression , within the Isl1-positive basal territory  of  Figures  and, exp Figures , a trans Figures . Moreove Figures , where i Figures . Thus, i Figures , whereas Figures , and lik Figures .Pseudemys a thin Nkx2.1 positive band , suggesting an implication of Dll4 in the specification of the GABAergic phenotype in the Tub, as occurs in the majority of the histogenetic domains where both markers colocalize  that is Otp/Nkx2.1+/Shh-, and the RMa, where the expression patterns of Shh and Nkx2.1 are inverted, being Nkx2.1-/Shh+ (Figures Xenopus, in the turtle the Nkx2.1 expression is continuous throughout the mammillary band, abutting directly the Pax7 + p3b (Figures Xenopus (Figures In recent years, the regionalization of the this area has been under analysis and the terminology used for its various subdivisions and their actual extent in the basal hypothalamic region have progressively varied with the appearance of the molecular approach (Shimogori et al.,  Figures . In cont Figures . However Figures , within  Figures  a portio Figures . It has  Figures . In addi Figures , coincid Figures . This su Figures  and simi Figures , likely  Figures .Xenopus Lhx7 is expressed in the alar hypothalamus and continues ventrally into the mammillary territory (Moreno et al., The mammillary area is also characterized by the expression of genes of the LIM-HD family, whose combinatorial expression pattern led to propose a new regionalization of this territory in mammals (Shimogori et al., Several studies in fishes have also reported the differential expression of LIM genes, such as Lhx6 and Lhx1/5, in the basal hypothalamic territory (Osorio et al., The lack of Shh expression in RMa has also been reported in the chicken, where the Shh becomes downregulated in the tuberomammillary primordium, but not in the RMa, at a specific point during development (Mart\u00ed et al., Thus, regarding the situation in fishes, some studies have described the presence of Shh in the basal hypothalamus, although so far there are no data about the specific location of Shh expression within this basal hypothalamic territory. It has been reported the presence of two hedgehog genes, expressed in a Sonic Hh-like pattern, in the basal hypothalamus of lamprey (Osorio et al., In terms of chemical specification, the amphibian and reptilian mammillary region is characterized by the presence of a rich catecholaminergic cell population (Smeets et al., Xenopus could suggest the existence of a forerunner of the subthalamic nucleus in anurans (Dom\u00ednguez et al., Regarding the nuclear specification, controversy exists regarding the origin of the different neuronal groups and several data support the contribution of diencephalic areas to the mammillary territory. In mammals, the retromammillary area was considered a caudoventral hypothalamic specification located between the diencephalic tegmentum (in P3; see for review Puelles et al., The organization of the brain undergoes evolutionary/adaptative changes during the anamnio-amniotic transition. The evolutionary leap from amphibians to reptiles involves relevant adaptation changes to conquer a new environment that have clear consequences on brain organization. However, it seems that during the transition from aquatic to terrestrial life the hypothalamus has maintained a major general pattern of organization, but with subtle differences that could be related to the new requirements for adaptation to the new environment. These variations in hypothalamic organization/regionalization highlighted in the present comparative genoarchitectonic analysis appear to have occurred gradually during the anamnio-amniotic transition starting with amphibians, which are the first tetrapods that arose, being anamniotes Table . ConsideIn the evolutionary context Table , our resComparative studies of the hypothalamus across vertebrates encompass many difficulties because the different degree of topographical modification of its parts, due to diverse forces during development that lead to the final different anatomy in each group Figures , 8. The All authors had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. This review is based on previous studies in which the three authors were involved (Moreno et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "Here, we identify nine strains  in which \u0394lepA confers a synthetic growth phenotype. These strains are compromised for gene regulation, ribosome assembly, transport and/or respiration, indicating that LepA contributes to these functions in some way. We also use ribosome profiling to deduce the effects of LepA on translation. We find that loss of LepA alters the average ribosome density (ARD) for hundreds of mRNA coding regions in the cell, substantially reducing ARD in many cases. By contrast, only subtle and codon-specific changes in ribosome distribution along mRNA are seen. These data suggest that LepA contributes mainly to the initiation phase of translation. Consistent with this interpretation, the effect of LepA on ARD is related to the sequence of the Shine\u2013Dalgarno region. Global perturbation of gene expression in the \u0394lepA mutant likely explains most of its phenotypes.LepA is a paralog of EF-G found in all bacteria. Deletion of  Eleven GTPases are universally conserved in bacteria ,2. TheselepB) in Escherichia coli -like state of the ribosome, LepA preferentially forms this state by binding the PRE complex rather than by catalyzing reverse translocation  to rescue stalled ribosomes and otherwise is stored in an inactive form at the membrane  acetyltransferase (cat) gene by the recombineering method  by electroporation, and recombinants were selected on Luria Broth (LB) plates containing 25 \u03bcg/ml Cm. This \u0394lepA::cat mutation was then moved by P1 transduction into BW25113 (The g method . Briefly BW25113  to generTM mutagenesis (Stratagene) and primers 5\u2032-ATCGACACCCCGGGCGCCGTAGACTTCTCCTATG-3\u2032 and 5\u2032-CATAGGAGAAGTCTACGGCGCCCGGGGTGTCGAT-3\u2032. The In-FusionTM (Clontech) method was used to generate pRB35. A DNA fragment corresponding to pLEPA without codons 487\u2013594 of lepA was amplified using primers 5\u2032-TAATACGTCTACACGTACCATGTCGGACGCCTGGAA-3\u2032 and 5\u2032-CGTGTAGACGTATTAGGCAAAGACAACAAATAA-3\u2032. This product was then circularized as recommended by the supplier (Clontech) and transformed into the cloning strain C2992. Plasmid pRB35 was purified from the transformed cells and confirmed by sequencing.To make pRB34, the H81A mutation was introduced into pLEPA  using Qu600) as a function of time. Data were fit to the equation OD600 = a \u00b7 2t/d, where t represents time, d represents the doubling (or generation) time and a represents the OD600 at time t = 0.Cells were grown in LB media at 37\u00b0C. Growth was monitored by measuring the turbidity of the culture (ODE. coli LepA (Prosci Inc.) were used to probe the membranes, and detection was carried out with the secondary antibody ECL system .Total lysate from mid-log phase cells were resolved using 8% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (PAGE), and proteins were transferred to nitrocellulose membranes . Polyclonal antibodies raised against purified Gly3-specific oligonucleotide 5\u2032-[32P\u2032-CTCGCGACCCCGACCTTGGCAAG-3\u2032 as described  strain [KLF2604(pWSK29)] and the complemented (C) strain [KLF2604(pLEPA)] were grown in LB media at 37\u00b0C to mid-log phase (OD600 \u2248 0.5), chilled by pouring over crushed ice and harvested by centrifugation. Pellets were resuspended in 500 \u03bcl lysis buffer  and 400 U/ml Superase inhibitor (Ambion) and lysed in three freeze/thaw cycles. Clarified lysates were loaded onto 10\u201340% sucrose gradients in TMN buffer  and spun in an SW-41 rotor (Beckman) for 2.5 h at 35000 revolutions per minute. Gradients were fractionated using a syringe-pump system (Brandel) with in-line ultraviolet absorbance detector .Cells of the wild-type (WT) strain [BW25113 containing the empty vector pWSK29 ], the \u0394l2 for 1 h at room temperature prior to sucrose gradient sedimentation. Monosomes were collected, RNA was extracted using phenol and CHCl3 and RNA fragments in the 20\u201330 size range were gel purified and used to generate cDNA libraries as described (\u00ae (Life Technologies), fragmented by alkaline hydrolysis, fragments in the 30\u201340 size range were gel purified and cDNA libraries were made as described  in the presence of 5 mM CaClescribed . To prepescribed .Barcoded libraries were pooled into two lanes of an Illumina HiSeq 2500 Sequencer, single-end sequenced with a read length of 50 nucleotides (nts) and demultiplexed. For libraries resulting in fewer than 15 million reads, cDNA for the corresponding samples was re-sequenced, and reads obtained were combined with the original reads. Data are available at the NCBI Sequence Read Archive (study accession number SRP048921).In order to eliminate low quality reads, any nt with phred quality score below 20  was converted to an \u2018N\u2019 and any trailing \u2018N's were removed. 3\u2032 adapters were identified and removed if at least 5 nts of the 3\u2032 adapter sequence matched the sequenced read from a given nt to the end of the read and there were less than 20% mismatches between the candidate read region and the adapter sequence . Finally, post-adapter-removal, any reads shorter than 20 nts or containing more than 5 \u2018N's were discarded.E. coli genome (NCBI Reference Sequence: NC_000913.2) using Bowtie2 v 2.0.0-beta5 , the rRNA reads were computationally identified and removed. We downloaded genomic locations for 5S rRNA from the UCSC Genome Browser's Rfam track . We alsorch Tool  to identFor downstream analysis, each read needed to be associated with a single genomic location. The midpoint of reads was used for total RNA libraries. Ribosome footprints were mapped to the predicted center of the P site of the ribosome. To identify this P site, we created a histogram of 3\u2032-end positions of ribosome-footprint reads (shown to vary in distance from the P site less than the 5\u2032 ends ) as a fuAll statistical tests were performed using the R statistical programming package version 3.1.0 .To validate reproducibility across replicates, raw read coverages across the genome (with +/\u2212 strand coverages combined) were compared from replicate to replicate across WT, mutant (M) and complemented (C) samples for both total RNA and ribosome footprints. The Pearson's correlation coefficient was determined for each of the replicate comparisons .E. coli K12's Genbank RefSeq gene annotation file . For eat-test, corrected for multiple testing utilizing the Benjamini\u2013Hochberg correction  was determined by taking ribosome footprint coverages per gene and dividing by the coverage of the corresponding replicate of total RNA. To test for significance in ARD fold changes across experimental conditions (WT versus M and M versus C), we took the base 2 logarithm of all gene coverages, and performed a Student's rrection .y-intercept to be zero in the non-log, normalized coverage space . A measure of spread, t-test was performed, comparing the differences in spread between WT versus M and M versus C samples.Gene-by-gene correlations were performed comparing ribosome footprints coverages t-test.Read counts from the respective samples were compared for total RNA libraries, comparing total mRNA, rRNA, tRNA and other stable small RNAs to total aligned reads. Significances of LepA effects were tested using Student's E. coli genome mentioned previously. Genes infC and pcnB were left out of the analysis, as they have the non-standard start codon AUU. The percentage of purines and pyrimidines was then determined for each list, in a location-dependent manner, and finally, the respective percentages from the LepA-affected genes were divided by the percentages for all high coverage genes to yield an enrichment/suppression of purine and pyrimidine prevalence compared to background levels. Any locations where the LepA-affected and the high-coverage genes had a percentage for purine or pyrimidine prevalence that were both zero were re-labeled to have an enrichment of 1. A two-tailed binomial test was performed for the pyrimidine frequencies for 8\u201311 nts prior to the start codon.We determined purine and pyrimidine frequencies for each position of the TIR for genes that exhibit altered ARD due to loss of LepA . The nt sequence, aligned with respect to the start codon, was obtained for these genes, as well as all high coverage genes (to be used as a background), utilizing the gene annotations and the Ribosome density (RD) was determined for each gene at every position in the gene by determining:ic is the relative ribosome occupancy at a given nt, i, in the gene, normalized for gene-by-gene coverages . A local coverage comparison across sample types was performed by taking 10 nt windows, shifted by 5 nts at a time, scanning over each gene, comparing the window coverages. Window coverage, per window, is the sum over ic's over every nt in the window. Only windows which showed a complemented average window coverage  were tested for the significance of the coverage differences (WT versus M and M versus C) utilizing a Benjamini\u2013Hochberg  for both WT versus M and M versus C comparisons, consecutive and overlapping windows were combined, then the local coverages were visually examined. Of the 38 regions examined in genes with enhanced coverages in the mutant , we identified 25  to contain a single, clear, complemented peak, and examined the peak-aligned codon frequencies across the windows, normalized to background levels across the prevalence of codons in all high coverage genes. A two-tailed binomial test was performed for GGU, which appeared in greater abundance compared to all other codons at the A site of the peaks. Although the 12 regions with reduced coverage in the mutant  were also examined in a similar manner, no clear complemented peaks were observed.For the 56 windows deemed significant . Reverse translocase activity was tested in various complexes and under various conditions, including those employed in Qin et\u00a0al. . We founet\u00a0al. ,12 and cet\u00a0al. .kcat/KM = 0.11 min\u22121 \u03bcM\u22121) . The rate of translocation in the presence of LepA-trunc is similar to that seen for EF-G\u03944 (0.11 versus 0.48 min\u22121 at 1 \u03bcM factor)  that lacks 50\u201360 residues (\u223c6 kDa) of the C-terminus. A previous crystallographic study of LepA revealed that the last 44 residues of the protein are disordered in the crystal and presumably flexible ( factor) , a varia\u0394lepA in E. coli, using a method described previously -resistance gene . Reduced growth was seen for crosses involving deletions of dksA, molR1, rsgA, tatB, tolR, tonB, rseA, rseB, rseC and recO. Because the latter four genes are closely linked to lepA, we suspected that these were \u2018false positives\u2019, due to the predicted low probability of generating the double-mutant recombinants. Indeed, when these crosses were further analyzed individually, frequencies of colony formation were about 300-fold lower than crosses involving unlinked markers, and the resulting transconjugants exhibited no obvious growth defects relative to the parent strains. Three crosses of the screen, involving deletions of ubiF, ubiG and ubiH, showed larger colonies on the Cm Kn plates, raising the possibility that the growth phenotypes caused by these mutations were being suppressed by \u0394lepA. However, subsequent PCR analysis of these selected recombinants indicated the presence of the WT gene (e.g. +ubiF) in addition to the kan-marked null allele (e.g. \u0394ubiF::kan). Thus, something more complex was occurring in these crosses, presumably involving regional duplications of each ubi locus.We used two different Hfr donor strains\u2014one with the F plasmid integrated near 44 min and oriented to transfer lepA, we used P1 transduction to construct each double mutant de novo. Double mutants harboring \u0394lepA and either \u0394dksA, \u0394molR1, \u0394rsgA, \u0394tatB, \u0394tonB, \u0394tolR, \u0394ubiF, \u0394ubiG or \u0394ubiH showed a reduced growth rate, indicating negative interactions  (rsgA)    ,37, ribo) (rsgA) , respiraG, ubiH) ,40 and/oB, tolR) \u201343, sugg\u0394lepA was combined with \u0394ubiF, \u0394ubiG or \u0394ubiH, even though the original screen predicted positive genetic interactions in these cases. We suspect that the original Hfr-mediated crosses involving \u0394ubiF, \u0394ubiG and \u0394ubiH allowed for chromosomal duplication events, resulting in merodiploid recombinants that outgrew the parental \u0394ubi strains. While the basis of this effect remains unclear, mutations in these ubi genes have been shown to cause partial resistance to aminoglycosides, due to defects in uptake  to express LepA lacking its CTD (\u0394487\u2013594) and the other (pRB34) to express LepA carrying mutation H81A. Histidine 81 is predicted to be critical for GTPase activity, as analogous substitutions of EF-Tu and EF-G cause specific loss of GTPase activity ,47. When\u0394lepAmutant (M) and \u0394lepA(pLEPA) complemented (C) strains, using the ribosome profiling technique .To investigate the role of LepA in translation, we analyzed the WT, echnique ,48. Cellechnique . Polysomechnique . Three iq-values of \u22640.05  showed increased ARD of this magnitude. Displayed in Figure \u0394lepA. The degree to which \u0394lepA globally perturbs gene expression (i.e. the collective processes of transcription and translation) can be seen in plots comparing gene-by-gene coverage of ribosome footprints versus total RNA  for many coding regions (Supplementary Tables S1\u2013S3). Of 1872 genes analyzed , 520 had significantly altered ARD due to loss of LepA (i.e. both WT versus M and C versus M comparisons yield Benjamini\u2013Hochberg corrected of \u22640.05 ). Twenty\u0394lepA(pLEPA) strain. These data suggest that ARD is generally reduced in the mutant, in line with the ribosome profiling data.In preparing lysates for the ribosome profiling analysis, it was evident that the mutant strain had significantly more free subunits and fewer polysomes than the WT strain Figure . This de\u0394lepA increased the fraction of coding mRNA by a factor of 1.2, an effect reversed in presence of pLEPA , and hence we reasoned that read counts should provide a proxy for the relative abundance of various classes of RNA molecules in the cell. Mutation PA Table . A propoPA Table . The oveIn theory, reduced ARD could result from slower initiation or faster elongation. However, the large effects on ARD observed here Table  are diff\u0394lepA on ribosome distribution along mRNA. Plots representing the average ribosome distribution for the 1872 coding regions, aligned with respect to the start codon, revealed lower ribosome occupancy for the first \u223c20 codons in the presence of \u0394lepA::cat . However, this trend was just as evident in the complemented strain and hence cannot be attributed to loss of LepA. Next, we looked for complementable changes in the pattern of ribosome occupancy on a gene-by-gene basis, initially by visually scanning the profiling data on the genome browser. Few differences were seen, the most notable being putative pause sites in ftsW and dppA. While these effects seemed by eye to be complementable, subsequent analysis for each of the corresponding 10 nt windows failed to confirm a significant difference in the mutant versus complemented strains. Finally, we computationally searched for 10 nt windows that exhibited significant differences in local ribosome occupancy . In the WT versus M comparison, 3185 windows exhibited significant differences, whereas in the C versus M comparison this number was much smaller\u2014187. These data are in line with the metagene analysis, which revealed obvious but non-complementable effects of \u0394lepA::cat at the 5\u2032 ends of genes . Forty-three windows showed significantly higher ribosome occupancy in the mutant strain compared to either strain containing LepA, whereas 13 windows showed lower occupancy. Upon combining consecutive windows, the former 43 windows were reduced to 36 regions, in which we identified 25 clear pauses that are highly position specific  and clearly due to loss of LepA. Notably, RD was not reduced after these pause sites, suggesting that pausing in these cases does not decrease the overall rate of protein synthesis. When the corresponding coding sequences were aligned with respect to the pause position, a glycine codon (GGU or GGC) was often seen to occupy the A site of the paused ribosome , although the same cannot be said of GGC. These data provide evidence that LepA prevents or reduces ribosomal pausing at certain GGU codons in the cell.Ribosome occupancy tends to vary locally on a given mRNA, due, for example, to ribosome pausing . To inveGly3 in E. coli . Thus, the observed ribosomal pausing at GGU codons in the absence of LepA is not an indirect consequence of lower Gly-tRNAGly3 concentration.Because GGU and GGC codons are only read by Gly-tRNA E. coli , we used E. coli  to compa\u0394lepA strains . These data indicate that LepA does not generally impact average elongation rate.We directly measured elongation rates for the synthesis of LacZ and another large (\u223c80 kDa) well-resolved protein, in the presence and absence of LepA. A pulse-chase method was employed , albeit E. coli cells, loss of LepA leads to altered ARD for more than 500 coding regions. In many cases the magnitude of this effect is substantial, with changes in ARD of \u22655-fold for dozens of genes. We also find evidence that LepA influences translation elongation, preventing ribosomal pauses at certain GGU codons. However, these latter effects are relatively few, highly localized (i.e. no impact on RD upstream or downstream), and occur in genes that differ from those that exhibit pronounced LepA-dependent ARD changes. Moreover, LepA does not generally impact average elongation rate, based on direct measurements of synthesis rates for two large polypeptides in vivo. Collectively, these observations lead us to conclude that LepA primarily influences initiation, either directly or indirectly. Consistent with this interpretation, we detect a relationship between the TIR sequence and the effect of LepA on ARD. For genes with significantly lower ARD in the mutant (i.e. LepA-dependent), pyrimidines are underrepresented in the SD region; the opposite trend is seen for genes with higher ARD in the absence of LepA.A number of observations from the Weissman group suggest that ARD is largely determined by the rate of initiation. For example, (i) use of the specific inhibitors harringtonine and cycloheximide allowed elongation rates to be measured globally in eukaryal cells, and the results show remarkable uniformity of elongation rates among genes, regardless of functional class or codon usage ; (ii) RDWhile the effect of LepA on translation depends on the particular gene, LepA clearly enhances the translation efficiency (i.e. increases ARD) of many genes. In the mutant strain, genes with \u226510-fold lower ARD outnumber those with \u226510-fold higher ARD by 26 to 1, total polysome levels are reduced by half and the overall amount of mRNA per ribosome in the cell is increased by \u223c16%. The latter observation implies that the actual ARD values for the mutant are across-the-board smaller than reported and hence the number of LepA-dependent genes larger. Together, these findings suggest that LepA generally promotes translation initiation in the cell.rsgA  LepA has no effect on the fidelity of decoding or the frequency of frameshifting in various contexts and conditions  Lenditions .Gly3 are as high in the mutant strain as in either control strain , arguing against the idea that pausing is a consequence of \u2018hungry\u2019 ribosomes awaiting cognate substrate. Although both GGU and GGC are recognized by tRNAGly3, only GGU appears significantly overrepresented in the A site of the paused complexes. This implies that the pausing is codon-specific and entails either slower decoding of GGU or slower translocation of GGU-paired peptidyl-tRNAGly3. Whether LepA acts directly or indirectly to promote one of these events remains an open question.The basis of the observed ribosome pausing at GGU codons remains unclear, as does the mechanism by which LepA prevents such pauses. Levels of Gly-tRNAet\u00a0al., despite an extensive effort to do so . In our hands, LepA exhibits ribosome-dependent GTPase activity but fails to promote reverse translocation. Consistent with our results, Cooperman et\u00a0al. have shown that addition of LepA to POST-state ribosomes induces tRNA movement within the 50S subunit but fails to accelerate codon\u2013anti-codon movement within the 30S subunit ppGpp, regulates transcription of many genes . However, in the absence of DksA, the transcriptional response is compromised, unmasking a \u0394lepA phenotype. Notably, characteristic of growth defects previously attributed to \u0394lepA cells is a lengthened lag phase . The origin of this effect remains unclear. Plasmid-encoded LepA fails to  reverse this effect, and hence it cannot be attributed to the loss of LepA. The mutation entails replacement of the lepA gene with a DNA cassette carrying the cat gene and its upstream promoter element, oriented in the same direction as the lep operon. Insertion of the cat cassette has polar effects on the downstream lepB gene (encoding leader peptidase), which might be responsible (via an unclear mechanism) for the ribosome distribution changes seen . The lepB mRNA level is increased by about 4-fold in the M and C strains, based on read coverage of the 3\u2032 half of gene (pLEPA contains the 5\u2032 part of lepB (cat cassette disrupts translational coupling that normally contributes to lepB translation. Aside from a polar effect on lepB, another possibility is that Cm acetyltransferase is somehow responsible for the observed changes in ribosome distribution, as the cat gene is uniquely absent from the WT strain.Mutation  of lepB , complicE. coli. Whether LepA acts directly or indirectly to facilitate TIR selection remains to be determined. For two reasons we favor the hypothesis that LepA acts during ribosome assembly/maturation and hence indirectly contributes to initiation. First, \u0394lepA confers a synthetic phenotype in the absence of RsgA, a GTPase known to catalyze release of RbfA during late-stage ribosome maturation (A major finding of this work is that LepA contributes predominantly to the initiation phase of protein synthesis in turation ,59. An aturation \u201368. We eturation ,70.Supplementary Data are available at NAR Online."}
+{"text": "The genetic background to bipolar disorder (BPD) has been attributed to different genetic and genomic risk factors. In the present study we hypothesized that inherited copy number variations (CNVs) contribute to susceptibility of BPD. We screened 637 BP-pedigrees from the NIMH Genetic Initiative and gave priority to 46 pedigrees. In this subsample we performed parametric and non-parametric genome-wide linkage analyses using ~21,000 SNP-markers. We developed an algorithm to test for linkage restricted to regions with CNVs that are shared within and across families.PSG) gene, a gene-family not previously implicated in BPD etiology. Two SNPs in the shared CNV are likely transcription factor binding sites and are linked to expression of an F-box binding gene, a key regulator of neuronal pathways suggested to be involved in BPD etiology.For the combined CNV and linkage analysis, one region on 19q13 survived correction for multiple comparisons and replicates a previous BPD risk locus. The shared CNV map to the pregnancy-specific glycoprotein (Our CNV-weighted linkage approach identifies a risk locus for BPD on 19q13 and forms a useful tool to future studies to unravel part of the genetic vulnerability to BPD.The online version of this article (doi:10.1186/s13040-015-0076-y) contains supplementary material, which is available to authorized users. Bipolar disorder (BPD) is a burdensome  and commThe genetic architecture of BPD is however more complicated than previously anticipated . A varieIn this study our hypothesis was that CNVs irrespective of their frequencies predispose to BPD. We further hypothesized that such structural variants are inherited. Under such assumptions, families ascertained for having high genetic liability of BPD constitutes an unprecedented opportunity to find such genomic variations in regions with evidence for linkage. To increase power to find families with linkage to BPD we screened 637 BP-pedigrees, provided by the NIMH Bipolar Genetic Initiative, and selected a subsample presumed to carry a genetic form of BPD. Pedigrees were selected based on family-wise genome-wide linkage analysis or by analyzing candidate genes for presence of stretches of deletions. We selected 46 BP-pedigrees for our present study and conducted two separate forms of analyses. First, we performed parametric and non-parametric genome-wide linkage analyses using a dense SNP-marker map with genetic data filtered for genotypes mapping to CNVs.We next tested the same sample with parametric and non-parametric linkage analyses restricted only to regions with CNVs that are shared among at least two individuals within the same family. To do this we developed an algorithm to sum family-wise linkage scores in regions with CNVs that are shared within and across families.Our results demonstrate that for the linkage part of this study, several signals surpassed threshold of suggestive linkage for both the non-parametric and parametric models and confirm several previously reported linkage regions. For the combined CNV and linkage analysis, one region on 19q13 survived correction for multiple comparisons and confirms a previously reported risk locus for BPD.FBXO30) with a suggestive role in BPD susceptibility.Several plausible candidate genes for BPD reside in 19q13. Moreover, two markers in the identified CNV have been reported as eQTLs for an F-box binding protein  .The criteria we used for selecting BP-pedigrees were a family-wise logarithm of odds (LOD) score\u2009>\u20091.1, or if several families were found to have overlapping family-wise LOD scores\u2009>\u20091.0 in the same genomic region. Pedigrees were also screened for stretches of homozygous genotypes (ROH), possibly indicating deletions, as this type of genetic polymorphism has been shown to have a suggestive role in the etiology of BPD . To do tIn total we selected 46 families for our analyses consisting of 277 individuals with DNA and 97 individuals for whom DNA was not available  were obtained from Rutgers University and Cell Repository  and were genotyped using the Illumina Human 610quad chip at Uppsala University, Sweden. We applied quality control (QC) analysis of individuals, SNP and CNV data consisting of an ordered series of steps to prevent spurious signals that may otherwise mislead statistical inference. Of notice, in order to reduce the presence of erroneous genotypes, SNPs located within CNV-regions were removed. Methods of genotyping and QC analysis are described in Additional file After final QC filter 269 individuals with DNA from 46 pedigrees (44 pedigrees with Caucasian ancestry and 2 pedigrees with African American ancestry) and 20,714 SNP-markers were ready for the linkage analysis. CNV calling with PennCNV and QC analysis, as described in the Additional file All of our CNVs overlapped with variations reported in three publicly available databases in August 2013; Database of Genomic Variants , WelcomeTo unravel a segregating risk locus to BPD in our sample the different levels of heterogeneity, both clinical and genetic, prompted us to categorize individuals in different affection status models (ASMs) and to calculate linkage using multipoint parametric dominant and recessive models that take into account inter-familial heterogeneity using heterogeneity LOD scores (HLOD) and multipoint non-parametric models in MERLIN (v.1.1.2) .ALL) statistics, in order to identify linkage. The Z scores were converted to LOD scores and P values according to Kong and Cox 1997 [For the non-parametric analysis we used the linear model  with an Cox 1997 . For theCox 1997 . The GENCox 1997  was usedFor the linkage part of this study we defined the suggestive linkage level to be that which on average would be exceeded by one linkage peak by chance in a genome-wide scan, and significant linkage to be that which would be expected to exceed once per 20 genome-wide scans as proposed by Lander and Kruglyak 1995 . To defiP value corresponding to suggestive linkage was then calculated based on a family-wise error rate (FWER) of 1-e^{\u22121}\u2009=\u20090.632, this is the probability that a Poisson distributed random variable with an expected value of 1, is positive, and it approximates the probability of at least one significant linkage peak [The threshold for the empirical age peak .As the models are nested, correction for multiple comparisons across the different models would have been too conservative, we corrected only within each model. Additional file In order to find inherited genomic regions conferring risk for BPD, the sum of average family-wise parametric LOD scores or non-parametric Z scores were calculated over regions and families sharing overlapping CNVs. For families with at least two members with overlapping CNVs, the average linkage score in the region was calculated and added to those observed in the same region in other families. Thus, the algorithm generates a CNV-weighted linkage scores for genomic segments representing regions with CNVs that are shared within and across families  linkage analysis met suggestive level on 3p14.1. The three affection status models generated comparable results with ASM3 exhibiting the strongest peak, NPL Z\u2009=\u20093.56 [The multipoint non-parametric , exhibited a significant CNV-weighted linkage score after a 1000-fold simulation analysis including correction for multiple comparisons, with empirical 033 Fig.\u00a0. The sig033 Fig.\u00a0 resides 033 Fig.\u00a0. The posPSGs)  which otherwise has inherited the same haplotype as his siblings from the father for whom we did not have any DNA sample. This indicates either that there is a double crossover in this region or that we missed the detection of a CNV in the child or that the father has two identical haplotypes with the exception of the presence of the CNV.Similarly, in pedigree 29\u20130209 the son (29\u201310643) has inherited the same haplotype as his sister from the father, but no CNV was detected despite being present in both the father and the sister. This could be explained either by a double crossover or by us failing to detect the CNV. In family 26\u20135011 the haplotype with the CNV was only transmitted to one of the affected children. In family 20\u20131049 the CNV was transmitted from the untyped father (20\u201310855) to a child with diagnosis under ASM2 but not to the sibling with diagnosis under ASM1.de novo CNV in pedigree 20\u20130172 for individual 29\u201310511 and deletions in families 11\u2013130 and 11\u2013156 that were not transmitted  in three additional pedigrees  Fig.\u00a0. We furtted Fig.\u00a0 and therOur genome-scan using 20,123 markers resulted in a high resolution mapping to identify narrow regions linked to BPD. We performed QC analyses aiming to reduce influences from markers located in CNV regions and selected informative markers using an independent study population. We estimated thresholds for suggestive linkage through simulation according to a well established criteria of Lander and Krygluak 1995  to obtaiMAGI1, has recently been reported [MAGI1 gene polymorphism was further validated in an independent sample of unrelated bipolar, schizophrenia and schizoaffective disorder cases, and thus, represents an interesting candidate for future studies. The synaptic function gene, MAGI1, has also been proposed as a putative candidate in a linkage and expression analysis by Lopez de Lara 2010 [MAGI1 CNV is the major reason for the observed linkage at 3p14 in our sample since the CNV reported by Karlsson et al. [The most notable linkage was to chromosome 3p14.1. This signal reached suggestive linkage in both the non-parametric and parametric dominant models under ASM3, and is consistent with several previous reports of linkage to BPD \u201332. Severeported  to harbon et al.  was onlyOur suggestive linkage to 1q23 in the parametric recessive analysis is consistent with the model-free linkage analyses reported previously , 35. ThiAlthough chromosome 6 has been a focus for a BPD risk locus , 40, nonThe present study was aimed for a CNV-weighted analysis and apparently several pedigrees in our sample were not optimal for a linkage analysis. Since the number and sizes of pedigrees were small, the number of meioses was limited, leading both to low power and reducing the ability to pinpoint linkage to a small region. Of note, the same BP-pedigrees have been included in previous linkage scans of both parametric and non-parametric models  without P\u2009=\u20090.033). We did not adjust thresholds for significance for the CNV-weighted linkage analyses according to all tested models. Our CNV-weighted linkage score on 19q13 would not be significant if all nine models were accounted for according to Bonferroni\u2019s approach. However, a Bonferroni correction would be too conservative as the models are correlated  genes  but also that 5 of 15 unaffected (i.e. not classified BP-I) individuals were positive for the CNV, that is, they had a deletion (del) or duplication (dup) of a chromosomal region that included the CNV-weighted linkage peak. This indicates that if the CNV is functional in causing risk for BPD it still has incomplete penetrance, whereas some individuals got BPD due to other reasons.In a series of subsequent analyses we aimed to find possible implications of structural variation in the This observation can also be construed as the CNV having no involvement in the etiology of BPD. Although this cannot be entirely ruled out, the counter-argument that nonetheless underscores the involvement of the CNV polymorphism in 19q13 in the etiology of BPD concerns issues of disease classification. In spite of the fact that this polymorphism occurs in individuals without disease classification according to ASM1, they are not classified as never mentally ill. The fact that the CNV deletions and duplications are not entirely overlapping are most likely due our CNV calling algorithm based on SNPs, as described in the Additional file Of interest, our result with a significant signal on 19q13 for BPD susceptibility agrees with previous reports.GSK3A) gene and the glutamate receptor, ionotropic kainate 5 (GRIK5) gene. The GSK3A protein is homologous to GSK3B, a target molecule for lithium treatment [First, without implicating a specific gene, Francks et al.  detectedreatment , which hreatment . Severalreatment . Taken tCEACAM21 gene in the 19q13 region to associate with SZ. The CEACAM genes, or the carcinoembryonic antigen-related cell adhesion (CEA) gene family, have several structural and functional similarities to the PSG genes (OMIM: 109770). Recent results show that the CEA genes are both brain and immune cell expressed (http://genome.ucsc.edu/). Although the exact molecular function of these gene products remains elusive, some studies reveal function related to cell-cell adhesion, innate immune system and signal modulation in various tissues [PSG gene in BPD etiology.Secondly, Alkelai and collaborators  found th tissues \u201348. Thes tissues , 50 suggOther candidates in this linkage peak region include genes related to neurotrophin  and immuThe mechanisms of the clinical manifestations and phenotypic effects of CNVs are well documented  and inclhttp://regulome.stanford.edu/) and screened all available markers in the CNV region for being transcription factor binding sites. Two markers (rs4802370 and rs7252967) are likely transcription factor binding sites and linked to expression of the F-box binding gene (FBXO30). The F-box protein functions as an ubiquitin-ligase and targets the transcription factor NF-kB [NF-kB pathway has been shown to be a key regulator of neuroplasticity, neuronal survival and pro-inflammatory status and thus serves as a one of many etiology correlates to BPD [Based on these observations we made a bioinformatic search Additional file 2:Selection criteria for inclusion of BP-pedigrees. Data obtained from a large number of pedigrees from NIMH Genetic Initiative Wave 1\u20134 was screened to generate informative pedigrees and to reduce any sporadic and environmental form of BPD. Two different analyses were used to select pedigrees. Test for runs of homozygosity (ROH) and analyzing regions with increased parametric family LOD scores under different assumed modes of inheritance. (DOC 62\u00a0kb)Additional file 3:Pedigrees included in linkage and CNV weighted analysis for BPD. To retrieve informative pedigrees from the NIMH Genetic Initiative Wave 1\u20134 sample, 46 pedigrees consisting of 269 genotyped individuals and 97 individuals with no available DNA were selected. Three affection status models were considered (ASM1-3) based on the different bipolar affective disorder sub-types, described in the figure-box. Individuals with a diagnosis of bipolar spectrum disorders which only apply to a certain ASM were coded as \u201cunknown\u201d under the other ASMs. In order to illustrate full details of pedigree composition different subtypes of bipolar spectrum disorders are illustrated. For linkage analyses with different ASMs only genotyped individuals were coded as ASM1-3. All other individuals, irrespective of diagnosis, were coded as \u2018unknown\u2019. (PDF 364\u00a0kb)Additional file 4:Results of the parametric and non-parametric genome-wide linkage analyses under affection status models ASM1-3. Simulation based thresholds for genome-wide significant and suggestive linkage levels are illustrated in red and black dashed lines respectively. A-C: Non-parametric linkage analyses. NPL Z scores are illustrated. D-F: Parametric dominant linkage analyses. Heterogeneity LOD scores (HLOD) are illustrated. G-I: Parametric recessive linkage analyses. Heterogeneity LOD scores (HLOD) are illustrated. (PDF 367\u00a0kb)Additional file 5:Principles for region for which one CNV-weighted linkage score is calculated. LOD scores from two families (family ID: 20\u20131049 and 12\u2013330) for a certain chromosome are depicted. The occurrence of CNVs for individuals within these families is also illustrated. The average linkage score (parametric LOD score or non-parametric Z-score) is calculated over the region with overlapping CNV from at least two individuals in the same family. Note, for those individuals with the presence of a CNV were SNPs zeroed out. In the non-CNV carriers were genotypes intact. The average linkage score from all families with overlapping CNV in at least two individuals is added. A CNV-weighted linkage score is thus generated for a defined region that share overlapping CNVs for more than 1 individual per family. To illustrate the calculation; an example is given with three markers that are located in a region with four overlapping CNVs, at the position 60\u201375\u00a0cM. For these 3 markers, the LOD scores for the two families are: Fam-ID 12\u2013330: 0.9, 0.9 and 0.9, Fam-ID: 20\u20131049: 0.2, 0.4 and 0.1. The CNV-weighted linkage score (the sum of average linkage score) is then calculated as: Fam-ID 12\u2013330: (0.9\u2009+\u20090.9\u2009+\u20090.9)/3\u2009=\u20090.9, Fam-ID: 20\u20131049: (0.2\u2009+\u20090.4\u2009+\u20090.1)/3\u2009=\u20090.23. Then, a total CNV-weighted linkage score\u2009=\u20090.9\u2009+\u20090.23\u2009=\u20091.13. (PDF 179\u00a0kb)Additional file 6:Genomic positions of CNVs identified in 19q13. The table shows the positions of all CNVs that were identified in 19q13 (chr19:48066441\u201348157656) and that harbors a significant CNV-weighted linkage score. All genomic coordinates are according to NCBI36/hg18. (DOC 38\u00a0kb)Additional file 7:Table of the 25 strongest CNV-weighted linkage scores of the parametric dominant ASM1 model. The two strongest signals withstood test for significance using a 1000-fold simulation analysis including a FWER correction. Asterisk (*) denotes a significant value. Start and end positions are shown for the genomic regions that were identified to contain a shared CNV within (and across) family members. The full length of the CNV is also presented for each individual. Affection status is given for each individual, with BP-I\u2009=\u2009bipolar type I, BP-II\u2009=\u2009bipolar type II and OMD\u2009=\u2009other mental disorder. Of note, different ASMs were assumed, and affection status under a certain model implies a non-diseased status under the remaining ones. The copy number variation state is deletion or gain, where CN\u2009=\u20091 refers to 1 copy deletion and CN\u2009=\u20093 refers to 1 copy gain. All genomic coordinates are according to NCBI36/hg18. (DOC 326\u00a0kb)"}
+{"text": "Rana temporaria, and the densest linkage map to date for this species. We found high homology with the published linkage maps of the Eastern and Western lineages but with differences in the order of some markers. Homology was also strong with the genome of the Tibetan frog Nanorana parkeri and we found high synteny with the clawed frog Xenopus tropicalis. We confirmed marked heterochiasmy between sexes and detected nonrecombining regions in several groups of the male linkage map. Contrary to the expectations set by the male heterogamety of the common frog, we did not find male heterozygosity excess in the chromosome previously shown to be linked to sex determination. Finally, we found blocks of loci showing strong transmission ratio distortion. These distorted genomic regions might be related to genetic incompatibilities between the parental populations, and are promising candidates for further investigation into the genetic basis of speciation and adaptation in the common frog.By combining 7077 SNPs and 61 microsatellites, we present the first linkage map for some of the early diverged lineages of the common frog,  Adults were sampled in two localities from the North of Spain . We crossed a male from Candioches [high altitude: 1687 m.a.s.l. (metres above sea level)] and a female from B\u00e1rcena  belonging to two different mtDNA clades . The controlled cross was carried out in the laboratory of the Amphibian Facilities at the University of Oviedo. Female and male abdomens were pressed gently by hand to obtain eggs and sperm without harming the animals. Tadpoles were individualized at Gosner stage 25 and were kept at 12 hr light:12 hr dark photoperiod and 14\u00b0 until they reached Gosner stage 42. Then, they were killed with an overdose of benzocaine and frozen. DNA from the brains of parents and 184 F1 offspring was extracted with the DNeasy Blood and Tissue Kit (QIAGEN). DNA quality was assessed via 0.7% agarose gel electrophoresis and DNA concentration was determined with a Qubit fluorometer. DNA extractions were diluted to 100 ng/\u03bcl for subsequent library preparation.This study is based on a large full-sib family from a single File S1). Briefly, DNA was digested with restriction enzymes PstI, and BamHI and the fragments of each individual were ligated to one of the 94 modified Illumina adapters, which contain barcode sequences, with T4 DNA ligase. Adapter-ligated DNA was combined to two library pools consisting of 92 offspring and the two parents in each pool. After purification, DNA fragments of a certain size (300\u2013600 bp range) were selected in each library using an E-Gel iBase Power System as in Two libraries were prepared according to the slightly modified protocol of R. temporaria, the reads from the presumably heterogametic parent  were used to generate the reference sequence. To that end, identical reads were first collapsed with FASTQ/A Collapser of the FASTX-toolkit  were discarded. Finally, the selected SNPs were used to map the progeny reads. Following Raw Illumina reads with low quality were discarded as well as reads with ambiguous barcode sequences. As the forward and reverse reads did not overlap, forward reads were used for the first part of the analysis. We demultiplexed the sequences and barcodes were trimmed as a result of this process. Low quality ends were discarded resulting in 91 bp reads. Because of the lack of a reference genome for -toolkit . Second,-toolkit  at 90% ossembler  and a sissembler . The dat2X test, p < 0.05). However, to later determine potentially incompatible regions between the two lineages, we retained loci showing relatively mild segregation distortion  for construction of new linkage maps and permutation analysis (see Transmission ratio distortion). Genotyping error was calculated for both sire and dam by comparing replicated genotype calls obtained from two separate sequencing lanes.SNP-calling was performed again with a quality threshold of 100 and a maximum of four SNPs allowed per contig. Parents and progeny were deemed heterozygotes if the MAF was > 0.1 and SNPs were excluded if > 25% of progeny genotypes were missing and > 6% of progeny showed noncompatible genotypes. SNPs showing segregation distortion were not included in the initial linkage maps (File S3), 113 microsatellites were successfully amplified within 19 multiplex reactions following the protocol of the QIAGEN Multiplex PCR Master Mix (2 \u00d7) at half volume (25 \u03bcl). Amplification reactions started with an initial polymerase activation step at 95\u00b0 for 15 min, followed by 40 cycles of: 94\u00b0 for 30 sec, 55\u00b0  or 60\u00b0 (for the rest) for 90 sec, and 72\u00b0 for 60 sec; and finally, an extension stage of 60\u00b0 for 30 min. Microsatellite analysis was performed using an ABI 3100 automatic DNA Sequencer.In addition to the SNPs, microsatellite markers were also included to enable direct comparison with the previous maps (File S3).Genotypes were determined using the software GENEMARKER v 2.4 . Uninformative microsatellites, as well as those with null alleles, were excluded from subsequent analysis  were removed before the linkage analysis.As for the SNPs from the RAD-seq, microsatellites showing Mendelian segregation violations  using the cross type \u201cdoubled haploid\u201d (DH) and Maximum Likelihood algorithm in MSTmap  iteratively followed by an additional round of linkage map reconstruction, until no possible double crossover was found.Markers, both SNPs and microsatellites, were assigned according to their segregation pattern to five categories: n MSTmap . The Kosi.e., < 4 markers) were excluded from further analysis. The order of the markers within each linkage group was determined with the Maximum Likelihood mapping algorithm, which assumes no crossover interference. Therefore, the distance between markers was calculated using the Haldane mapping function. Spatial sampling was used with five thresholds: 0.1, 0.05, 0.03, 0.02, and 0.01. Three map optimization rounds were run in each spatial sample, using the following parameters: chain length was 10,000, cooling control parameter was 0.0001, and the rounds were stopped after 100,000 chains without improvement. Finally, for the multipoint estimation of recombination frequencies, we used a burn-in of 100,000, five cycles of Monte Carlo Expectation Maximization , and six sampling periods for recombination frequency. Stabilization of the recombination frequencies was monitored with the sum of recombination frequencies of adjacent fragments and the mean number of recombination.The average linkage map was created using the option appropriate for an outbred full-sib family  in Joinmap 4.1 . IdenticXenopus tropicalis homology. The expected genome length and observed genome coverage were calculated according to GO) was the sum of the observed length of the linkage groups. The expected length (GE) was the sum of the expected length of the linkage groups, which was calculated multiplying the observed length of each linkage group by the factor (m + 1) / (m \u2212 1), m being the number of markers of the linkage group. The observed genome coverage, understood as the proportion of the genome comprised in our recombination map, was the ratio GO/GE.The name of the linkage groups follows the 2X, p-values from 0.05 to 0.005) in the linkage analysis. None of the markers caused big gaps  of recombination distance. To identify distorted regions, instead of only distorted markers, we used a kernel smoothing and permutation test  and Nanorana parkeri  were used to evaluate the homology and the synteny among amphibian genomes. References from both forward and reverse sire contigs containing SNPs of the linkage map were aligned to a draft genome of R. temporaria (File S2) using Bowtie2 (http://ncbi.nlm.nih.gov) and Swissprot (http://uniprot.org) databases as well as in X. tropicalis and N. parkeri genomes using blast. Homologous sequences of other species were retained if the best hit e-value was five orders of magnitude higher than the second-best hit e-value . File S4 contains sex-specific and average maps and a comparison with previous microsatellite maps. File S5 contains supplementary figures and tables being: Figure S1, linear model that fits the relation between male and female number of markers; Figure S2, linkage group specific male vs. female recombination lengths; Figure S3, map length and number of markers compared to Swiss map; Figure S4, kernel smoothing results; Table S1, summary of distorted maps, linkage group lengths, number of markers, and sex-specific recombination rates. File S6 contains a figure of the distorted sex-specific map. File S7 contains sex-specific distorted maps and the results of the kernel analysis. File S8 contains the genotypic information of the individuals. The draft genome is available at https://figshare.com/s/c0ff6bacbfc4572e1ff1 and marker sequences are available at https://figshare.com/s/24fa6c7cd2f133467207.p-value < 0.0001), resulting in a final set of 61 informative microsatellites (File S3). In the whole dataset, 19 offspring exhibited three alleles in at least one microsatellite locus, suggesting potential partial trisomy, and were excluded from subsequent analysis.Out of the 113 amplified microsatellites, 77 were heterozygous in at least one parent. Twelve of them possessed null alleles and four loci showed Mendelian segregation violation . The estimated genotyping error inferred based on independent technical replicates was \u223c5% (5.5% sire and 5.3% dam).A total of 162 F1 offspring were used to construct the linkage map, after discarding three individuals with < 5000 reads. Approximately 634 million reads were retained after the quality filtering. Over 10 million reads were obtained from each parent, while on average 3.8 million of reads were sequenced per offspring (ranging from 2 to 6.8 million). The assembly from the parental read alignment resulted in 88,497 contigs. From those, 13,203 contigs were polymorphic and were used for mapping the reads from the progeny and SNP calling. Over 13,600 SNPs were initially identified, from which 7217 markers passed a File S4). Our map presented an observed genome coverage of 99% for both sexes. The average recombination rate was 1.35 times higher and the total length was roughly twofold (1.87 times) larger in the female map than in the male map. Interestingly, all male linkage groups contained a cluster of markers with zero recombination, henceforth referred to as recombination cold spot  ranging from 86 to 159 markers, while 9\u201362 nonrecombining markers were detected in smaller linkage groups in the male map. The nonrecombining regions in the female map were generally smaller (<23 markers). However, the linkage groups Rt4B, Rt7B, and Rt10 showed nonrecombining regions of similar length in both sexes (Sex-specific linkage maps were constructed using 3644 markers (51 microsatellites and 3593 SNPs) and 3180 markers (51 microsatellites and 3129 SNPs), for female and male respectively. After discarding linkage groups formed by <4 markers, 13 linkage groups were recovered in both maps . A totalold spot . The numth sexes .Figure S1). Furthermore, female map showed higher recombination length than the male map but some linkage groups deviated above or below from the general trend . For instance, Rt6 was longer in the male map and Rt10 was shorter in the female map compared to other linkage groups. Rt3 exhibited the lowest recombination rate in the male map. Finally, Rt7B was the shortest linkage group in the male map with the smallest number of markers  except for the length of the male linkage groups  . The lack of correlation between length of male linkage groups and the number of markers supports the independence between number of crossovers and chromosome size in males.Compared with the recent RAD-based linkage map in the common frog (File S7). The resulting map was 663 cM longer in the female and 398 cM longer in the male compared to the maps without including distorted markers. Average recombination rate was similar in distorted and nondistorted maps (Table S1). The distribution of the distorted markers exhibited a nonuniform pattern along and among linkage groups (File S6). For example, a large number of distorted markers was observed on Rt1 in contrast with the low number on Rt4B and Rt7A. Furthermore, there were differences between sexes. Kernel smoothing results showed a higher distortion in the female than in the male map . Only Rt2, Rt5, Rt7B, Rt8B, and Rt9 from the male map exhibited regions with signals of high distortion based on the kernel analysis, while all female linkage groups except Rt9 contained at least one area with an excess of distorted markers. Interestingly, Rt6 and Rt9 from the male map and Rt7B from the female map showed clusters of distorted markers within the recombination cold spots  and 375 SNPs segregating in the sire  were successfully assigned within the 13 linkage groups (ld spots .We used 7278 markers (61 microsatellites and 7217 SNPs) to construct the average linkage map and 7138 markers were successfully assigned to 13 linkage groups. All the linkage groups reached stabilization after five Monte Carlo Expectation Maximization cycles. All markers were located within the same linkage groups as in sex-separated maps. However, as expected, the average map showed less accurate marker order than the female map.R. temporaria draft genome. In 2359 cases (56.7%), the pair of forward and reverse reference sequences aligned onto the same scaffold within 600 bp (range from 190 to 473 bp). From a total of 2331 scaffolds selected, 1184 aligned to the N. parkeri genome (50.8%) and 305 aligned to the X. tropicalis genome (13%). In addition, 81% of homologous sequences between R. temporaria and X. tropicalis were located in the same chromosome and the synteny was also strong, as is shown in the Circos plot . On the other hand, linkage groups 15 and 2 from File S4). Separation of these two linkage groups in File S4), hence, further research is needed to determine if some of these changes can reflect the occurrence of independent chromosomal rearrangements, such as inversions, among the different lineages.The obtained consensus linkage map was consistent with previous microsatellite maps based on the Western and Eastern lineages . HoweverSimilar to earlier studies, we detected large differences in recombination rate between sexes , being isolated in different refugia during the last glaciation. Centromere repositioning events have been observed at similar evolutionary time scales but only at the interspecific level. For example, five centromere repositioning events have occurred between donkeys and zebras, which diverged from each other 1\u20132.78 MYA . If sex determination is strictly genetic and there is no recombination between sex-chromosomes, X and Y are expected to accumulate differences as a result of large rearrangements and/or degeneration of the Y chromosome  and harbored two sex-linked markers in the Finnish population of Kilpisj\u00e4rvi . Therefore, this linkage group warrants further research to determine its potential involvement in sex determination or whether another source of differentiation among the lineages crossed is causing this large male heterozygosity. The possibility of multiple chromosomes participating in the sex determination of the common frog has also been discussed in previous work . The estimated homology between R. temporaria and X. tropicalis in this study (12.8%) is slightly higher than that observed (10%) for the Western lineage of this species (X. tropicalis chromosomes 4, 7, and 8 were split into two pairs in R. temporaria.The comparative genomic analysis between  genomes . As expe genomes , N. parkR. temporaria, facilitating the search for genes of adaptive relevance. In addition, due to the conserved synteny among amphibians, this linkage map represents a valuable tool for further comparative genomic studies. Our work indicates that the genome structure is generally conserved between common frog lineages while the position of the recombination cold spots and marker order can vary. Finally, genomic regions showing strong transmission distortion found here are promising candidates for studying incipient speciation processes.The constructed high-density consensus linkage map provides an important resource for further research in the evolutionary biology of www.g3journal.org/lookup/suppl/doi:10.1534/g3.116.036459/-/DC1.Supplemental material is available online at Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file."}
+{"text": "Due to the increasing availability of individual-level information across different electronic datasets, record linkage has become an efficient and important research tool. High quality linkage is essential for producing robust results. The objective of this study was to describe the process of preparing and linking national Brazilian datasets, and to compare the accuracy of different linkage methods for assessing the risk of stillbirth due to dengue in pregnancy.ReclinkIII), and used manual review to identify further links. Sensitivity and positive predictive value (PPV) were estimated using a subset of gold-standard data created through manual review. We examined the characteristics of false-matches and missed-matches to identify any sources of bias.We linked mothers and stillbirths in two routinely collected datasets from Brazil for 2009\u20132010: for dengue in pregnancy, notifications of infectious diseases (SINAN); for stillbirths, mortality (SIM). Since there was no unique identifier, we used probabilistic linkage based on maternal name, age and municipality. We compared two probabilistic approaches, each with two thresholds: 1) a bespoke linkage algorithm; 2) a standard linkage software widely used in Brazil  and PPV\u00a0=\u00a092.5% . Manual review of uncertain links identified an additional 37 links, increasing sensitivity to 83.7%. The bespoke algorithm with a relaxed threshold identified 132 true matches (sensitivity\u00a0=\u00a069.1%), but introduced 61 false-matches (PPV\u00a0=\u00a068.4%). Despite a lack of unique identifiers for linking mothers and stillbirths, we demonstrate a high standard of linkage of large routine databases from a middle income country. Probabilistic linkage and manual review were essential for accurately identifying cases for a case-control study, but this approach may not be feasible for larger databases or for linkage of more common outcomes. Record linkage is the process used to bring together information recorded in different sources about the same individual or group of individuals . Due to High quality linkage is required so that robust results can be obtained from linked data. However, there are specific challenges in mother-baby linkage, particularly concerning data from low-middle income countries. Firstly, quality of linkage is limited by the availability of unique or well-completed identifiers, and by data quality  prioritize true matches (i.e. high specificity of the exposure), whilst high sensitivity was less important; 2) retain sufficient cases for a reasonable sample size; and 3) verify absence of bias (i.e. understand whether any groups were more or less likely to be linked). This study presents an approach to preparing and linking mortality and morbidity data from routine data sources in Brazil, comparing the performance of different linkage methods for identifying exposure of pregnant women to dengue.SINAN: Notifiable Diseases Information System (Sistema de Informa\u00e7\u00e3o de Agravos de Notifica\u00e7\u00e3o/SINAN), containing individual-level data on all notified diseases.We linked two routinely collected data sets: for dengue, notifications of infectious diseases (SINAN); for stillbirths, mortality (SIM), for 2009 and 2010.The Brazilian Ministry of Health has required notification of all cases of dengue seen in health facilities in Brazil. SINAN captures clinical cases of disease, through forms completed by any health professional who suspects dengue; this notification is compulsory in the country. After a dengue suspected case is identified, the Epidemiological Surveillance Service investigates cases in order to confirm or discard the suspicion based on laboratory results and the Brazilian definition of clinical epidemiological criteria of dengue: presence of fever and two or more of the following symptoms  . Forms iMaternal name was 99.5% complete; municipality was 100% complete. Where age in years was missing (1.3% of records), we derived age from date of birth.n\u00a0=\u00a01,981,912 individuals). We excluded records for 900,054 men, and 398,710 cases discarded by the Epidemiological Surveillance services , containing individual-level data on all deaths including stillbirths.Data were extracted from SINAN for all suspected dengue cases . We kept only the stillbirths .Data on all deaths were extracted from SIM , and those name recorded as numbers: our final study population comprised 678,999 dengue notifications from SINAN and 62,373 stillbirths from SIM Fig. . We searIn Brazil, full names usually have several components, and we aimed to retain the discriminatory power of this variable. In our data, the mean number of names per mother was three (maximum eight). We derived distinct variables for first name, second name, and last name. Similarity between names recorded in SIM and SINAN was compared using the Jaro-Winkler string comparator . The Jarn\u00a0=\u00a05565 municipalities in Brazil in 2010). This process is called blocking; blocking by municipality assumes that records from different municipalities do not belong to the same women. We used this blocking scheme because we considered municipality to be very reliable, and unlikely to have changed between dengue in pregnancy and end of pregnancy.The total number of pairwise comparisons between SINAN and SIM would be prohibitively high: 184,922\u00a0\u00d7\u00a031,867\u00a0=\u00a05,892,909,374 in 2009 and 494,077\u00a0\u00d7\u00a030,506\u00a0=\u00a015,072,312,962 in 2010 (a dengue epidemic year). To decrease the number of pairwise comparisons, we only attempted to link women that resided in the same municipality ) and the probability of agreement given records belong to different mother-baby pairs ).M-probabilities for each identifier were estimated from the true matches in the gold-standard dataset. U-probabilities were calculated based on a list of non-matches, created from all pairwise comparisons of records within SINAN, excluding those belonging to the same individual. We used the SIINAN database to calculate the u-probabilities, as it was the larger of the two databases.Frequency-based weights were calculated for each category of Jaro-Winkler score comparator  (for namSince we were linking different subjects (the mother and her stillbirth), and because the exposure (dengue during pregnancy) could have happened up to 9 months before the outcome (stillbirth) there were timing issues to consider. Two records could differ in time by 9 months and could bridge over the calendar year; some mothers would have the birthday between the data of dengue and the date of the stillbirth. To allow for this, we estimated different weights according to the similarity of age across datasets: equal ages, age differing by 1 year, age differing by 2 years, and ages differing by more than 2 years  Manipulation of names; 2) Blocking ; 3) Match weight calculation .ReclinkIII applies the Levenshtein string comparator to compare names [re names . The Levre names .Records pairs were ordered by match weight and manually inspected to identify threshold values to classify comparison pairs as non-links, links and uncertain links. The number of expected matches was unknown, because we did not know a priori how many of the mothers in SIM should link to a stillbirth in SINAN. Therefore, we explored two different threshold choices for each algorithm. We first chose a conservative threshold, aiming to exclude as many as false-matches as possible . We then chose a relaxed threshold, aiming to capture as many of the true matches as possible (high sensitivity). Any records above the cut-off threshold were classified as links. For the best performing approach, we manually inspected uncertain links to determine whether or not they belonged to the same mother-baby pair.ReclinkIII algorithm, and for each threshold (conservative and relaxed), we estimated the sensitivity and positive predictive value (PPV) by comparing linkage results with the gold-standard dataset. Since we expected the number of links to be very small in comparison to the size of the datasets, we did not calculate specificity or negative predictive value (as these measures would be consistently high). To account for in-sample optimism, we also present average estimates based on \u2018leave one out\u2019 cross classification.For both the bespoke and the 2 test or Fisher\u2019s exact test. A two-sided P value of less than 0.05 was considered to indicate statistical significance. We examined maternal age , maternal education , previous stillbirths or abortions (yes/no), gestational age  and weight when the stillbirth occurred . Stata version 14.1 was used for the statistical analyses.For the best performing algorithm, we examined which characteristics were associated with false-matches and missed matches. Categorical variables were compared between groups with ChiOf the 678,999 eligible dengue cases in SINAN for 2009\u20132010, 191 were linked to a stillbirth record using the nine-step gold-standard algorithm   are available as guidance to others aiming to link similar data sources. Although we did not have a readily available training dataset (where the true match status of each record pair was known), we were able to create a gold-standard dataset from which to derive an appropriate match weight algorithm, and to evaluate the accuracy of both linkage methodologies. This was possible in our study because we had access to identifiable data, and could examine records manually, but is not always the case, when data from clinical and identifiers information are separated to protect the patient privacy .RecLinkIII, which has been widely used to perform linkage in Brazil and which has previously been shown to have high sensitivity and specificity [RecLinkIII appears to work less well when there are a limited number of variables available to perform the linkage, as demonstrated in our study and a similar study by Coutinho et al. [RecLinkIII program uses the Levenshtein string comparator, which according with Freire [The bespoke algorithm created specifically to link this dataset achieved higher linkage quality that the off-the-shelf program cificity , 20\u201322. o et al. , which oh Freire  is not th Freire . Other sh Freire . The impAn important aspect of linkage quality is the choice of threshold. The investigator, keeping in mind that different thresholds are more appropriate for different study questions, must make this choice. In our study, where linkage aimed to provide information on exposure (dengue during pregnancy)  we choseThis study has a number of limitations. Although a gold-standard dataset was used to measure the linkage accuracy, there remains scope for linkage errors to occur: we may not have identified all missed-matches due to missing data on some records. Our estimates of sensitivity should therefore be interpreted with caution, and should not be assumed to apply to other datasets. Due to the low sensitivity of our linkage approach, our study should not be used to estimate stillbirth rates for women exposed to dengue during pregnancy. Our analysis of the association between study characteristics and linkage error may have been limited due to low power. A further consideration for case-control studies is whether linkage of cases and controls (to the exposure) has similar accuracy. We did not address the accuracy of linkage to live births in this study, but will evaluate this in future research.To make best use of linked data, it is important to evaluate the quality of linkage processes and to understand the limitations and bias that errors in linkage could introduce in the research results , 28. Val"}
+{"text": "Gene conversions resulting from meiotic recombination are critical in shaping genome diversification and evolution. How the extent of gene conversions is regulated is unknown. Here we show that the budding yeast mismatch repair related MutL\u03b2 complex, Mlh1-Mlh2, specifically interacts with the conserved meiotic Mer3 helicase, which recruits it to recombination hotspots, independently of mismatch recognition. This recruitment is essential to limit gene conversion tract lengths genome-wide, without affecting crossover formation. Contrary to expectations, Mer3 helicase activity, proposed to extend the displacement loop (D-loop) recombination intermediate, does not influence the length of gene conversion events, revealing non-catalytical roles of Mer3. In addition, both purified Mer3 and MutL\u03b2 preferentially recognize D-loops, providing a mechanism for limiting gene conversion in vivo. These findings show that MutL\u03b2 is an integral part of a new regulatory step of meiotic recombination, which has implications to prevent rapid allele fixation and hotspot erosion in populations.DOI:http://dx.doi.org/10.7554/eLife.21900.001 Meiotic recombination is a key evolutionary process that promotes genome diversification in sexually reproducing organisms. Recombination shuffles parental genomes through genetic exchanges leading to crossovers (COs) or noncrossovers (NCOs). During meiotic prophase, repair of Spo11-induced DNA double-strand breaks (DSBs) by recombination into COs is crucial for the formation of gametes with normal chromosome content. Indeed, COs ensure a physical link between homologs that allows them to properly segregate to opposite poles during meiosis I division . DSBs ocBoth COs and NCOs involve the formation of heteroduplex DNA, which can yield gene conversions after mismatch repair . Gene coTo repair a subset of DSBs into COs, cells mainly employ a meiosis-specific pathway that ensures an even distribution of COs across the genome . For thiMutL\u03b3 (Mlh1-Mlh3), a heterodimer related to the bacterial mismatch repair MutL complex, forms foci on pachytene mouse, human or plant chromosomes in numbers that correspond to CO numbers. In the absence of MutL\u03b3, CO frequency is reduced in mouse meiosis  as well In mismatch repair (MMR), the MutS homologs (Msh2-Msh3 and Msh2-Msh6) are involved in mismatch recognition and recruit either of two MutL heterodimers, MutL\u03b1  and to a lesser degree MutL\u03b3 (Mlh1-Mlh3) . Both Mumlh2\u2206 cells show no increase in mutation rates, except a slight defect in the repair of a subset of frameshift mutations  or MutL\u03b2, has an elusive function. No biochemical activity has been described, and the human MutL\u03b2 has no MMR activity in in vitro complementation assays . Consistutations . In addiutations . PMS1-/-utations . In yeas2\u2206 cells .Here, we used a proteomic approach to identify meiotic partners of Mlh1 in budding yeast. We discovered that MutL\u03b2 controls a new regulatory step of meiotic recombination that limits the length of meiotic gene conversion tracts associated with COs and NCOs. This regulation requires a physical interaction between the MutL\u03b2 complex and the meiosis-specific helicase, Mer3. This work opens new perspectives to investigate mechanisms that control the genetic diversity created by meiotic recombination.MLH1 tagged alleles were fully functional in mismatch repair  Mer3 protein lacking IG-like domain no longer interacted with Mlh1 in vivo , (2) theFollowing principles derived from a statistical analysis of protein complex interfaces , a searcWe next tested the effect of the Mer3R893E mutation on the Mer3 interaction with MutL\u03b2 in meiotic cells. Consistent with the direct interaction between recombinant proteins, Mer3 and Mlh2 were able to reciprocally co-immunoprecipitate each other and Mer3 co-immunoprecipitated both Mlh1 and Mlh2 . The MerOverall, our data demonstrate that in yeast the interaction between Mer3 and Mlh1 is weak, whereas the interaction between Mer3 and Mlh2 is stronger, and required for the complex formation between Mlh1 and Mer3 in vivo. In contrast, for mouse proteins, the interaction between HFM1 and MLH1 is very strong, compared to that between HFM1 and PMS1. In both systems however, Mer3/HFM1 forms a complex with the MutL\u03b2 heterodimer.BUD23 and GAT1 hotspots no longer occurred in the mer3R893E mutant, implying that Mer3 recruits Mlh2 to these sites  formed during recombination between polymorphic parental sequences are maintained due to MMR deficiency, whereas they would lead to gene conversions or restorations of the parental alleles in the MSH2 background  or 3 (mer3R893E) meioses of each genotype  and 2.3 kb (mer3R893E) and tracts associated with NCO went from 0.8 kb to 2.3 kb (mlh2\u2206) and 1.7 kb (mer3R893E)  . In partr3R893E) .mlh2\u2206 and mer3R893E mutants. The two major categories of NCO events, defined depending on the associated strand transfer patterns, occurred with the same frequency as in wild-type and showed a similarly increased length in both mutants , mer3R893E: 434 bp (8 events in 2 meioses)], arguing against Mer3 extending D-loops in this direction in the absence of interaction with Mlh2.Genetic and biochemical evidence suggests that Mer3 extends early recombination intermediates to stabilize them to promote CO formation . We wondmer3K167A (mer3-hd) mutation, which abolishes Mer3 helicase activity  the length of conversion tracts was still strongly increased when deleting MLH2 in the mer3-hd mutant, showing that the helicase of Mer3 is not involved in the increased length of events seen in the absence of Mer3-Mlh2 interaction  are not due to Mer3 helicase activity. Surprisingly, the mer3-hd mutant also still displayed an almost intact \u2018ZMM\u2019 function, with high spore viability and only a short delay in meiotic progression, as opposed to the mer3\u2206 mutant  MerMER3 show meiotic defects typical for the ZMM proteins. Specifically COs, but not NCOs, are reduced, likely as a result of less stable early recombination intermediates .For Mlh1 internal tagging, the indicated HA6 or His6-Flag3 tag sequence, flanked on each side by the GGGGSGGGGS linker sequence, was inserted between aminoacids 710 and 713 of Mlh1. Mlh2 was C-terminally tagged with 13 copies of Myc . The tag\u2206 mutant . The Hismer3R893E mutation in the S288C*SK1 hybrid was obtained by CRISPR-Cas9 mediated cleavage, using a plasmid encoding Cas9 and expressing a guide RNA targeted to the MER3 gene (plasmid generously provided by G. Zhao and B. Fetcher) transformed together with a healing mer3 fragment containing the R893E mutation.The MLH1 alleles were estimated by measuring the spontaneous reversion rate at the lys2::InsE-A14 locus in strains derived from E134  and PMS1 was PCR-amplified from mouse testis cDNA, a gift from J. Barau and D. Bourc\u2019his. PCR products were cloned in pDNOR Gateway plasmids and subcloned in Gateway plasmids derived from the two hybrid vectors pGADT7 (GAL4-activating domain) and pGBKT7 (GAL4-binding domain) creating N terminal fusions (gift from M. Grelon). Point mutations, truncations or internal deletions were introduced by PCR. All plasmid inserts were sequenced.Yeast strains and 2 hybrid experiments were performed as in . Interac10 cells were harvested, washed two times with ice-cold TNG buffer ) and flash-frozen in liquid nitrogen. Frozen cells were mechanically ground in liquid nitrogen. The resulting powder was resuspended in 50 mL of lysis buffer . The lysate was cleared by centrifugation at 8000 g for 10 min and then incubated with 200 \u03bcl of washed and buffer equilibrated anti-Flag magnetic beads  for 2 hr at 4\u00b0C. The beads were washed once with lysis buffer and three times with washing buffer ; 1X PhosSTOP (Roche)). Proteins were eluted with 5 bed volume of elution buffer ; 1X PhosSTOP (Roche); 100 \u03bcg/mL Flag peptide) for 1 hr at 4\u00b0C. Proteins were separated by SDS-PAGE, stained with colloidal blue, and 7 bands covering the entire lane were excised for each sample. In-gel digestion was performed overnight by using trypsin . Peptides extracted from each band were analyzed by nanoLC-MS/MS using an Ultimate 3000 system  coupled to a LTQ-Orbitrap XL mass spectrometer (Thermo Scientific). Raw spectra were processed using Mascot through Proteome Discoverer  and further analyzed in myProMS ; 1X PhosSTOP (Roche); 125 U/mL benzonase (Sigma)) and glass beads three times for 30 s in a Fastprep instrument . The lysate was cleared by centrifugation at 13,000 g for 5 min. 25 \u03bcl of Protein G magnetic beads  (equilibrated 1:1 with lysis buffer) and primary antibodies , 3 \u03bcg of c\u2010Myc monoclonal antibody 9E10  or 5 \u03bcg of HA monoclonal antibody 16B12 ) were added. The tubes were incubated overnight at 4\u00b0C. The magnetic beads were washed four times with 1 mL of wash buffer ; 1X PhosSTOP (Roche)) and resuspended in 30 \u03bcl of 2xSDS protein sample buffer. The beads were heated at 95\u00b0C for 10 min and loaded in duplicate or triplicate onto a 4\u201312% SDS-polyacrylamide gel. The proteins were then blotted to PVDF and probed for Flag, HA or Myc-tagged protein with corresponding antibodies. Signal was detected using the SuperSignal West Pico Chemiluminescent Substrate (ThermoFisher).6.108 cells were processed as described  or 3 mM EDTA (\u2013Mg2+). The reactions were assembled on ice and incubated at 30\u00b0C for 30 min. At the end of incubation, 5 \u03bcl of binding dye (50% glycerol and 0.25% bromophenol blue) was added to each sample. The products were separated on native 4% polyacrylamide gels (acrylamide: bisacrylamide 19:1) at 4\u00b0C. For Mlh1-Mlh2 binding, the reactions were done in the same manner as Mer3 but without competitor DNA and in the presence of 2 mM magnesium (+Mg2+). After separation, the gels were dried and exposed to storage phosphor screens . The screens were scanned using Typhoon FLA 9500  and quantified by Image Quant software.The binding assays for Mer3 was carried out in 15 \u03bcl volume in Tris acetate, pH 7.5, 1 mM DTT, 100 \u03bcg/ml BSA, 1.3 ng/\u03bcl poly(dI-dC)  and DNA substrate . The reactions also contained either 2 mM magnesium  Sigma protease inhibitory mix [P8340]) and MBP-Mlh2-Mlh1 or MBP-Mlh2 proteins were immobilized on amylose resin (New England Biolabs), or GST-Mlh1 was immobilized on gluthatione resin . The resin was washed with wash buffer , 0.2% NP40 detergent, 2 mM EDTA and 1:1000 (v/v) Sigma protease inhibitor mix [P8340]) and incubated for 60 min at 4\u00b0C with 2.5 \u03bcg of recombinant Mer3 protein. The resin was washed five times batchwise with wash buffer and Mer3 was eluted with 100 \u03bcl wash buffer containing 20 mM maltose (Sigma-Aldrich) in amylose pulldown, or 20 mM gluthatione  in GST pulldown. The MBP or GST tag was then cleaved by addition of prescission protease. The samples were separated by 10% SDS-PAGE and visualized by silver staining or Western blot .For the protein interaction assays, MBP-Mlh2-Mlh1, MBP-Mlh2 or GST-Mlh1 were expressed in Smlh2\u2206 meioses, two complete mer3R893E octads, one mer3R893E octad where the sequence of only seven cells was retrieved. We used the two complete octads for tracts lengths and interference, and the three octads for mean CO numbers. For mer3K167A and mer3167A mlh2\u2206, we studied two octads of each genotype. To infer recombination events from octad sequences, we first established a list of 74,911 SNPs by aligning the S288C chromosomes to the SK1 chromosomes using LAGAN (http://cbio.mskcc.org/public/SK1_MvO/) for SK1. Next, sequencing reads from octad were aligned on the S288C and SK1 reference sequences using the BWA software . The shape parameter \u03b3 of the best fitted gamma distribution is indicative of the strength of CO interference  .http://www.ncbi.nlm.nih.gov/Traces/sra/) under the accession number: SRP075437.Raw sequence data have been deposited in the NCBI Sequence Read Archive , and the evaluation has been overseen by a Reviewing Editor and Kevin Struhl as the Senior Editor.Thank you for submitting your article \"Concerted action of the MutL\u03b2 heterodimer and Mer3 helicase regulates the global extent of meiotic gene conversion\" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:The manuscript provides a novel function for the Mlh1-Mlh2 (MutL\u03b2) heterodimer, a conserved protein complex whose primary function has, to date, remained elusive. It shows that Mlh1-Mlh2 interacts with Mer3, a meiosis-specific helicase previously implicated as having crossover-promoting function, and that the two work together to limit the length of heteroduplex tracts formed during meiotic recombination in a manner that appears to be largely independent of Mer3 helicase activity. Because all proteins in this study are conserved in many eukaryotes, including mammals, it is highly likely that these findings will be of general applicability.Essential revisions:A reorganization and better introduction of the key facts are needed, the paper would benefit from having all in vitro interaction data presented in one place so that direct comparisons can be made or model recombination substrate interaction data should be presented independent of meiotic data.In addition, you should consider having someone not immediately in the field read the manuscript to make sure that the big picture and the flow of the data are easy to understand. The following suggestions should also be attended in a revised manuscript.Understanding the larger aspects of the paper are difficult for the non-specialist. A cartoon figure of meiotic recombination and segregation at the beginning would help. For example, in the first two paragraphs of the Introduction, it is not easy to follow how recombination shuffles parental genomes through COs and NCOs or how these concepts extend to allelic shuffling, transmission distortion or gene conversion tract length, few lines later.Interaction between Mer3 and Mlh1 or Mlh2 is well-documented, but not with the intact Mlh1-Mlh2 complex, even though purification of this complex is documented. Demonstrating this interaction should be the primary in vitro interaction data presented. While the paper presents the Mer3-MutL\u03b2 interaction as being mediated through Mlh2, it is clear from the in vitro data  that MlhYeast two-hybrid data will be better interpreted if they are not scattered throughout different supplementary figures and the standards to define an interaction are the same throughout the manuscript. For example, in Figure 2\u2014figure supplement 1A, limited growth on -His plates and none on -His -Ade is taken as a signal of a significant interaction, while later in  the releThere's much more focus on 2-hybrid assays using Mlh1 and Mer3 than on Mlh2 and Mer3, even though authors argue that Mlh2 is responsible for bridging between Mlh1 and Mer3.All two-hybrid data should be presented in a single place and two-hybrid experiments should be presented in parallel with internal standards .Analysis of Mlh1-Mer3 and Mlh2-Mer3 interactions would be greatly strengthened if experiments were done in mlh2- and mlh1- mutants, to directly test bridging interactions.The model intermediate-binding activity of MutL\u03b2 is quite weak, as compared to Mer3, and only poorly discriminates model recombination intermediates from dsDNA. It's hard to directly assess this, since the critical data for MutL\u03b2 (at 10nM) is obscured by the plot in mer3R893E with a mismatch at HIS4-LEU2 should be included in panel D. One possible explanation for this discordance is the use of the NFT1 locus, which is not expected to bind Mer3 or MutL\u03b2, as \"normalization\" standard, which means that all values are \"normalized\" to a background value that might be vulnerable to experimental variation. What happens if un-normalized (i.e. just Mlh2-ChIP/input) is used?Data values in Data presentation for meiotic phenotypes are somewhat disorganized and confusing; given that mer3-hd is analyzed in zmm mutants, while interesting, is of limited effect and possible mechanisms are not clear; moreover, a compelling case for the phenotypic equivalence of mlh2\u0394 and mer3R893E is made on the basis of tetrad data alone, and the msh4 suppression data could easily be omitted without compromising the conclusions.Data regarding partial suppression of the spore viability defect of zmm mutants from other studies.Analysis of gene conversion and crossing over in SK1-S288c hybrids is quite compelling, but the presentation needs to include data on the mean number of noncrossovers per meiosis, and also comparison with \u03b3 values for true  Essential revisions:A reorganization and better introduction of the key facts are needed, the paper would benefit from having all in vitro interaction data presented in one place so that direct comparisons can be made or model recombination substrate interaction data should be presented independent of meiotic data.Following this suggestion, we have reorganized the Results section, so that now the in vitro interaction data are presented in a separate figure, In addition, DNA binding assays of recombinant Mer3 and MutLB are now grouped, together with the MutL\u03b2 endonuclease assay in In addition, you should consider having someone not immediately in the field read the manuscript to make sure that the big picture and the flow of the data are easy to understand.We had the manuscript read by a cell biologist colleague and added explanatory sequences at several places in the manuscript, especially in the Introduction.The following suggestions should also be attended in a revised manuscript.Understanding the larger aspects of the paper are difficult for the non-specialist. A cartoon figure of meiotic recombination and segregation at the beginning would help. For example, in the first two paragraphs of the Introduction, it is not easy to follow how recombination shuffles parental genomes through COs and NCOs or how these concepts extend to allelic shuffling, transmission distortion or gene conversion tract length, few lines later.As suggested, we have added an introductory Interaction between Mer3 and Mlh1 or Mlh2 is well-documented, but not with the intact Mlh1-Mlh2 complex, even though purification of this complex is documented. Demonstrating this interaction should be the primary in vitro interaction data presented.The reviewer may have missed it, but the interaction between purified Mer3 and Mlh1-Mlh2 complex was in While the paper presents the Mer3-MutL\u03b2 interaction as being mediated through Mlh2, it is clear from the in vitro data  that MlhWe have added additional evidence that Mer3 interacts directly with Mlh1 by showing that the 2 hybrid interaction between Mer3 and Mlh1 still occurs in the absence of Mlh2. This and the evolutionary conservation of this interaction is now more extensively discussed (see response to reviewer comment below).Yeast two-hybrid data will be better interpreted if they are not scattered throughout different supplementary figures and the standards to define an interaction are the same throughout the manuscript. For example, in Figure 2\u2014figure supplement 1A, limited growth on -His plates and none on -His -Ade is taken as a signal of a significant interaction, while later in  the releThere's much more focus on 2-hybrid assays using Mlh1 and Mer3 than on Mlh2 and Mer3, even though authors argue that Mlh2 is responsible for bridging between Mlh1 and Mer3.All two-hybrid data should be presented in a single place and two-hybrid experiments should be presented in parallel with internal standards .We group our answer to the last three comments. We have added in Materials and methods a sentence defining \u201cinteraction\u201d: \"interaction is defined compared to the growth seen in the negative control consisting of the combination between the GAL4BD-bait protein in the presence of GAL4AD-only (\u201cempty\u201d on the figures). Any combination that grows better than this control on the selective media is considered as an interaction\".In addition, to compare individual experiments, we have added the interaction between Mlh1 and Mlh3 as an internal standard in each experiment. This is seen in Analysis of Mlh1-Mer3 and Mlh2-Mer3 interactions would be greatly strengthened if experiments were done in mlh2- and mlh1- mutants, to directly test bridging interactions.mlh2\u2206 mutant, and two hybrid interaction between Mlh1 and Mer3R893, defective in the interaction with Mlh2. We still see an interaction between Mlh1 and Mer3, meaning that the two hybrid interaction is not mediated by Mlh2, in agreement with the direct interaction between recombinant proteins.This is a great suggestion, and indeed we have tested two hybrid interaction between Mlh1 and Mer3 in a The model intermediate-binding activity of MutL\u03b2 is quite weak, as compared to Mer3, and only poorly discriminates model recombination intermediates from dsDNA. It's hard to directly assess this, since the critical data for MutL\u03b2 (at 10nM) is obscured by the plot in As suggested, we have moved the Mer3 binding data to the main manuscript, and this data is presented next to MutL\u03b2 DNA binding for direct comparison .2) Use either a log or split-X axis, so that data for 10nM can be clearly discerned.We have changed the scale of the X axis to a log scale, which makes all concentrations easy to distinguish .3) Don't assign Kd values to the interaction between MutL\u03b2 and the various substrates-the current data are just too sparse-instead describe binding in relative terms.We no longer mention Kd values, but just binding in relative terms in the text as suggested Data values in mer3R893E strain that has a mismatch at HIS4LEU2 in panel 5F. This allows reproducing a similar decrease in Mlh2 binding compared to MER3, as the one seen in panels 5C and 5E.The experiments done in Data presentation for meiotic phenotypes are somewhat disorganized and confusing; given that mer3-hd is analyzed in We thank the reviewer for this suggestion. We have moved all the meiotic progression and spore viability data of the mer3-hd mutant to the current Data regarding partial suppression of the spore viability defect of zmm mutants, while interesting, is of limited effect and possible mechanisms are not clear; moreover, a compelling case for the phenotypic equivalence of mlh2\u0394 and mer3-R893E is made on the basis of tetrad data alone, and the msh4 suppression data could easily be omitted without compromising the conclusions.mlh2\u0394 and mer3R893E is also based on the similar delay in meiotic divisions, as well as the increase of spore viability of zmm mutants. We felt that the msh4 data reinforce the findings seen for zip4, another zmm mutant, and we decided to keep both sets of data, which also reinforce the observation that mlh2\u2206 and mer3R893E phenotypes are identical.The phenotypic equivalence between Analysis of gene conversion and crossing over in SK1-S288c hybrids is quite compelling, but the presentation needs to include data on the mean number of noncrossovers per meiosis, and also comparison with \u03b3 values for true zmm mutants from other studies.zmm mutants in a previous study that used the same approach .We have added, in"}
+{"text": "FOXI2, FANK1 and GLRX3 remain candidates for further investigation. This, the largest genetic study of VUR to date, highlights the 10q26 region as a major genetic contributor to VUR in European populations.Vesicoureteric reflux (VUR) is the commonest urological anomaly in children. Despite treatment improvements, associated renal lesions \u2013 congenital dysplasia, acquired scarring or both \u2013 are a common cause of childhood hypertension and renal failure. Primary VUR is familial, with transmission rate and sibling risk both approaching 50%, and appears highly genetically heterogeneous. It is often associated with other developmental anomalies of the urinary tract, emphasising its etiology as a disorder of urogenital tract development. We conducted a genome-wide linkage and association study in three European populations to search for loci predisposing to VUR. Family-based association analysis of 1098 parent-affected-child trios and case/control association analysis of 1147 cases and 3789 controls did not reveal any compelling associations, but parametric linkage analysis of 460 families  under a dominant model identified a single region, on 10q26, that showed strong linkage  to VUR. The ~9Mb region contains 69 genes, including some good biological candidates. Resequencing this region in selected individuals did not clearly implicate any gene but  Other congenital anomalies of the kidney and urinary tract (CAKUT) commonly occur along with VUR. The oft-quoted prevalence of VUR is 1\u20132% but the true prevalence may well be higher4. The disease has been suggested to be twice as common in females as males5 but this most likely reflects an ascertainment bias, and other studies have detected only a slight excess of incidence in females compared to males7. The prevalence of VUR tends to decrease with age5, and serial studies of individual patients show VUR can spontaneously regress during childhood in a subset of initially affected individuals8. Screening studies of first-degree relatives of individuals with VUR identifies VUR in one third to one half of siblings10 and 65% of offspring11. This observation, coupled with the high concordance of primary VUR in identical twins12 and the identification of families with multiple generations affected by primary VUR and RN14, suggests that there may be a substantial genetic component to VUR. However, large-scale genetic studies of VUR carried out to date have been somewhat disappointing and generally rather inconclusive. Although there have been some compelling findings in individual large families, overall, little concordance is seen between the results from different studies23, supporting the notion that the condition is genetically heterogeneous. Here we combined data from the two largest genetic studies of VUR conducted to date22, comprising three separate cohorts , to investigate whether the increased power obtained from use of a larger sample size could help identify genetic contributors operating across multiple affected families/individuals from these three European populations.Primary vesicoureteric reflux (VUR), the retrograde flow of urine from the bladder through the vesicoureteric junction into the upper urinary tract, is the most common renal tract malformation. VUR is usually a benign condition but chronic kidney damage triggered by ascending pyelonephritis and also congenital kidney hypo/dysplasia  can occur and lead to end stage renal disease24 produced no compelling association signals 22 and the UK (2938 Wellcome Trust Case Control Consortium controls)25 similarly produced no compelling association signals. We note that the relatively sparse SNP set available for association analysis (see Methods) provides incomplete genome coverage with levels that are probably, at best, close to the 31% coverage provided by the Affymetrix 111\u2009K array26. Therefore, our results do not preclude the possibility that common variants associated with VUR exist, but we would need to genotype our UK/Slovenian samples  with a much denser genotyping array in order to answer this question definitively.Family-based association analysis carried out using the transmission/disequilibrium test (TDT)27 with the Haplotype Reference Consortium (HRC) reference panel28, treating the Irish and UK/Slovenian cohorts separately (since they had very different numbers of QCed genotyped SNPs available to inform imputation). We then repeated the TDT association analysis at all SNPs passing post-imputation QC  when analysis was restricted to the UK/Slovenian cohort, however this association was not replicated in the Irish or combined cohorts ) than is seen at the lead SNP, suggesting an inconsistency between results that should in reality be highly concordant. A single SNP on chromosome 6 showed genome-wide levels of significance  in analysis of the combined cohorts  of linked families of \u03b1 \u2248 30%. This region had shown some evidence of linkage in the previous separate analyses of these cohorts22 and, indeed, had been highlighted as one of the top findings in the Irish cohort by Darlow et al.22, although the significance achieved in that previous analysis  was not sufficient to definitively conclude the presence of a disease locus in the region. In previous analysis of the UK/Slovenian cohorts19 this region had achieved an HLOD of 2.44 and a ZLRLOD of 1.55 in an expanded set of families where affection status was coded as positive for either VUR or RN. Cordell et al.19 had further noted that the signal of linkage increased to ZLRLOD\u2009=\u20092.32 when analysis was restricted to families where affection status was coded as positive for VUR (regardless of RN status), as in the current analysis.Parametric linkage analysis under a recessive model gave no strong evidence for linkage at any genomic location (data not shown). However parametric linkage analysis under a dominant model, as well as non-parametric linkage analysis, identified a single genomic location (focused around rs7907300 on chromosome 10q26) showing strong evidence  for linkage to an underlying disease locus , resulted in a slightly increased maximum HLOD of 5.07 at the same genomic location (rs7907300).We investigated the sensitivity of our parametric linkage analysis results to slight changes in the assumed parameters of the underlying dominant model, but found this did not have any substantial effect. In particular, changing the assumed disease allele frequency from 0.001 to 0.01 19 produces a reasonably compelling linkage signal. Neither the individual cohorts nor the UK/Slovenian combined cohort achieve the HLOD of 3.3 that would most probably be required to conclude \u2018significant\u2019 linkage31  but the results are certainly consistent with the much stronger (and highly significant) effect seen here when all three cohorts are combined.To clarify the relative contributions of the individual cohorts to the current results, we repeated the parametric (dominant HLOD) analysis within each cohort separately and within the UK/Slovenian cohorts combined Fig.\u00a0. The str29 plot of the equivalent p-values 32 for those SNPs lying within the main peak of significance bounded by red dashed lines in Fig.\u00a033, with genome-wide significance in affected-sib-pair linkage studies corresponding to a p-value of around 2.2\u2009\u00d7\u200910\u22125). The 4 genes omitted from the LocusZoom plot in Fig.\u00a0C10orf88, ACADSB, HMX2 and BUB3) all lie to the left of  GPR26 and thus would probably not be considered good candidates for harboring causal mutations, given the decrease in linkage signal observed within this section of the plot, but any of the genes to the right of GPR26 might be considered good positional candidates.As is often the case in linkage studies, the plausible region within which the underlying disease lies is very large, containing 44 genes in the central ~7\u2009Mb region or 69 genes in the wider ~9\u2009Mb region (Supplementary Table\u00a0\u22126) is seen at around 129.5\u2009Mb (Build 37). This is not replicated in the UK/Slovenian cohorts  within the 12\u2009Mb region spanning the linkage signal , 127.6\u2009Mb , 128.4\u2009Mb  and 131.5\u2009Mb . However these signals are not replicated in the Irish cohort .In the UK/Slovenian cohorts  that was used to generate the linkage signal in the first place. A far more likely scenario is that the linkage signal is caused by heterogeneous mutations operating within different families in a dominant or dominant-like fashion. Given that the TDT can be considered as a test of linkage in the presence of association  we conclude that the apparent TDT association signals seen most likely reflect linkage between alleles at the relevant SNPs and mutations at the underlying disease locus, coupled with perhaps low levels of allelic association (linkage disequilibrium) between alleles at these loci in the founders.40, especially when applied to multiple affected individuals originating from the same family. A caveat in relation to our own study, however, is that we expect that only around 30% of our families will actually harbor mutations at the disease-causing genetic element in the 10q26 region . Prioritizing affected individuals from families where affected sibs shared alleles IBD, we whole-exome sequenced 29 individuals from the 8 families  showing the strongest evidence of linkage in the 10q26 region. There were 37,930 non-synonymous variants called across the whole exome in the 29 individuals. Of these, 139 occurred within the 10q26 region of interest and 37 of these were relatively rare  in the 1000 Genomes and Exome Sequencing Project data sets. Of these relatively rare variants, 23 were found within the same gene in two or more individuals (for 7 different genes) and 6 were predicted to be deleterious by at least one predictor out of SIFT/PolyPhen/MutationTaster43 . Only 7 of the 189 1000 Genomes Project sequences had no rare variants at all, suggesting that the detection of rare variants in FANK1 in our own VUR cases is not, in fact, a rare event, and so may not necessarily be related to their phenotype. Moreover, given a VUR population prevalence of up to 3%, we note that the \u2018rare\u2019 FANK1 variants listed in Supplementary Table\u00a0To interrogate in more detail the genes in the implicated 10q26 region, we performed whole-exome sequencing in a subset of our families. Next-generation sequencing has been extremely successful for the detection of disease-causing mutations in Mendelian, monogenic disordersFANK1, while 11 occurred within the promoter of genes other than FANK1 , a plausible explanation is that such variants may be found within non-coding, regulatory regions. To investigate this possibility, we performed targeted genomic resequencing of the implicated 9\u2009Mb region of 10q26 (from 123\u2009Mb\u2013132\u2009Mb) in 32 unrelated affected individuals from those families in our data set that contributed the most to the linkage signal (the 8 families that had been whole-exome sequenced and 24 others). Of the 34,412 single nucleotide variants (SNVs) passing standard quality control steps, we identified 9170 that were either absent or present at less than 1% in the 1000 Genomes European data, 3625 of which were also shared between affected individuals . We consider this large number of SNVs identified as showing frequency differences between our samples and the 1000 Genomes European samples as most likely to be artefacts arising from the systemic differences in read depth, alignment and variant-calling pipelines between cases and controls. Concentrating on rare SNVs found within promoters in our 9\u2009Mb target region, we found that 69 of 80 promoter-region SNVs occurred within the promoter of FANK1, further interrogation of the\u00a0.bam files showed that the FANK1 region appears to be hypervariable and many of the called SNVs were clearly artefacts  mapping to chromosome 10. This suggests that a number of the reads identified by our pipeline as mapping to 10q26 may in fact really come from chromosome 22, offering a possible explanation for the unusually high level of variability observed , as well as to the desired 10q26 region. This observation led us to re-align our targeted sequences to the GRCh38 assembly. 9 of the 11 previously identified SNVs in the FANK1 and its promoter, which therefore represent artefactual findings unrelated to the VUR phenotype. More sophisticated experimental methods  will therefore be required in order to robustly interrogate this 42.4\u2009kb region, without danger of contamination from other genomic regions.Further investigation revealed that the 42.4\u2009kb region of chromosome 10 from 125,885,884 to 125,928,375 contains segmental duplications from at least 4 other genomic locations  whereas our own sequences have up to 1000 fold coverage in the target region. Analysis of sequence data where there are systemic differences in coverage between cases and controls typically leads to inflated type I errors46, but discarding those samples with insufficient read depth can result in a loss of power . We therefore used the software package TASER47, which effectively re-calls variants in both cases and controls, allowing for systematic differences in read depth (and other factors), at the same time as constructing a test statistic.The analyses described above compared observed allele frequencies in our VUR cases to those in publicly available summary data provided by the 1000 Genomes Project (and similar resources), in order to identify putative causal variants. To achieve a better comparison between our sequenced VUR cases and phenotypically normal controls, we obtained the .bam files from 1106 ALSPAC controls48\u03bb\u2009=\u20090.76). With the MAF filtering threshold set to 0.05 in the base population, we obtained a few signals that were mildly suggestive of association but nothing that reached experiment-wide significance. The output of the current implementation of TASER does not indicate the position of the variants contributing to the test statistic within each window, but by inspecting the input and .bam files we were able to identify the probable variant driving the signal and discount any signals caused by sequencing error . The top findings , a SNP approximately 3\u2009kb upstream of FAM175B (rs533072490) and a further three SNPs in the intergenic region between LINC01163 and MGMT: rs146303503, rs182100352 and an unknown G/A substitution at position 128770839. Given the relatively low frequencies of all these variants seen within the VUR cases, and the relative lack of statistical significance obtained , we do not consider any of these results particularly compelling.In an adaptation of the original implementation of TASER, which splits the entire genome into small windows of interest , we considered our entire target region to be \u2018of interest\u2019. For each of the 1106 ALSPAC control sequences and 32 VUR case sequences, we split the target DNA sequence into consecutive 120\u2009bp windows with 30 windows included within a processing \u2018block\u2019. This resulted in 2490 blocks covering the entire sequence from 121,240,479\u2009bp to 130,201,779\u2009bp (GRCh38) on chromosome 10. Only bases called with a quality score >30 were added to the read count at each position. We found that only counting alleles that are seen at least twice in the low depth control sequences and at least 5 times in the high depth VUR sequences (in order to reduce the expected number of sequencing artefacts) improved the overall distribution of test statistics . Thus this region, containing the key candidate gene FANK1, remains effectively untargeted by our current sequencing endeavours, due to our inability to reliably map the sequence reads in this region.In addition to the findings listed in Supplementary Table\u00a0in vitro-propagated cultures of Normal Human Urothelial (NHU) cells in different differentiation states were obtained from Fishwick et al.49. Results  argue against these being strong positional candidates for explaining the linkage signal. OAT, the only one of the three highest expressed genes in normal urothelium to be moderately near to the centre of the linkage region, encodes the mitochondrial enzyme ornithine aminotransferase, so is an unlikely candidate on grounds of function.To help further prioritize genes in the implicated chromosome 10q26 region, data on gene expression by GLRX3 remains a key region for further investigation. Moreover, given our discovery that the 42.4\u2009kb region of chromosome 10 from 125,885,884 to 125,928,375 contains segmental duplications from other genomic locations, this region (which contains the key candidate gene FANK1) remains effectively untargeted by our current sequencing endeavours. More sophisticated methods  will be required in order to robustly interrogate this region, without danger of contamination with sequence from other genomic regions.Here we have identified, through genome-wide parametric and non-parametric linkage analysis, a region on 10q26 that shows strong evidence of harboring genetic variants contributing to primary nonsyndromic VUR. As is often the case in linkage studies, the plausible region in which the underlying disease locus must lie is very large, and, despite our attempts via association analysis and targeted sequencing (in a subset of samples), we have, as yet, been unable to find variants within the region that plausibly account for the linkage signal observed, although the promotor of 22, four of which lie close to the 10q26 region implicated here, in addition to another, as yet unidentified, gene near the 10q telomere50. However, these previously-implicated genes lie mostly outside our main peak of linkage. According to the gene expression atlas GUDMAP (GenitoUrinary Development Molecular Anatomy Project)52, mouse homologues of most of the human genes in the critical Chr10 locus are normally expressed in at least one site in the developing murine kidney and urinary tract. It is, however, of particular note that: Cpxm2 is expressed in the mature ureter and bladder; Fam53 is expressed in the developing and mature ureter; Dhx32 is expressed in the developing and adult ureter and bladder; Adam12 is expressed in the developing ureter and bladder; and Ptpre is expressed in the developing and adult ureter. Expression during in vitro differentiation of normal human urothelial cells might be considered a more relevant factor than mouse gene expression data obtained from GUDMAP, and indeed there are proven human VUR genes that encode tenascin XB and uroplakins which are expressed in the urothelium54. Cross-referencing the genes in our implicated 10q26 region with expression data from normal human urothelial cells49 highlighted several genes of interest including BUB3, OAT and GLRX3. Of these, only OAT would seem a strong positional candidate, but is a highly unlikely candidate on grounds of function. Our other key candidate, FANK1, was also expressed, albeit at a low level. However, this analysis of human urothelial cells comes with the caveat that epithelium is just one type of tissue found in the urinary tract, and we do not currently have similar data for other components such as nerve and muscle/mesenchyme.There are five genes on chromosome 10 that are already known to be involved in urinary tract developmentFGFR2, which has been implicated in induction abnormalities of the ureteric bud and development of VUR in mice58. However, the localisation of FGFR2 outside our main peak of linkage argues against it being a strong candidate for harboring the mutations that contribute to the linkage signal detected in our families. It is worth mentioning that FGFR2 is involved not only in the embryonic development of the metanephric urinary tract but also in that of the skeleton and skin, and mutations in the gene in humans can cause Crouzon syndrome, Pfeiffer syndrome, Apert syndrome, Jackson-Weiss syndrome, Beare-Stevenson cutis gyrata syndrome, or Saethre-Chotzen syndrome. Demonstration of VUR in mice58 was achieved by conditional knock-out of Fgfr2 very specifically in the peri-Wolffian duct stromal cells only, and therefore any mutation causing non-syndromic VUR or other CAKUT in humans that affects FGFR2 is likely to be in a non-coding regulatory element that affects expression of the gene in the developing urinary tract but has no effect on its expression in other tissues. Since most, if not all, genes already known to be involved in urinary tract development are also known to be involved in the development of other structures, including the eye, the ear, the branchial clefts  the skeleton and the gut, it is quite possible that most mutations that cause VUR will be regulatory mutations and in non-coding DNA, whatever gene they affect.Arguably the best candidate gene in the region is FOXI2. GUDMAP shows that Foxi2 is very highly expressed in the mouse metanephric mesenchyme at the appropriate time to stimulate the growth of the ureteric bud. However, our investigation of expression in human urothelial cells failed to detect FOXI2 expression. In 2009 the Irish group used Sanger sequencing to explore this gene, including all the untranslated sequence, 1\u2009kb of promoter sequence upstream, and several highly conserved non-coding regions in the gene desert at distances up to 1\u2009Mb away, in 232 VUR index cases. Eleven of the variants found in the conserved intergenic elements had \u2018Restricted Substitution\u2019 (GERP) scores >4, of which four have still not appeared in dbSNP, and, of these, one is immediately adjacent to a predicted POU3F1 binding-site and another is within a predicted PRRX2 binding-site. Pou3f1 and Prrx2 are both expressed in mouse metanephric mesenchyme, the latter very highly. However, functional experiments would need to be carried out to determine whether any of the variants actually affect expression and, if so, which gene is affected. FOXI2 itself was sequenced at that time in case there might be a mutation in the gene which would help to confirm its candidacy. Only two novel variants with GERP scores >2 were found in the gene, neither of which were likely pathogenic, and there was not an opportunity to pursue investigation of the intergenic variants at that time. Sequencing right across the whole linkage region with a larger number of patients (and controls) should hopefully enable identification of the correct regulatory element(s), but it is still very likely that functional testing will be required to determine which gene is affected.A stronger positional candidate, given the linkage evidence, is In conclusion, this, the largest genetic study of VUR to date, provides strong evidence for the 10q26 region as the major genetic contributor to primary nonsyndromic vesicoureteric reflux in European populations. Definitive identification of the causal variants contributing to this observation will be required in order to identify the mechanism involved. Removal of families with pathogenic mutations in this region (once identified) from the data set should make it easier to identify, by reanalysis, the next most important of the many loci expected to be responsible for VUR in the 70% or more of families that are not linked to 10q.22. Full details of sample collection, phenotype definition and genotyping can be found in the primary publications from these studies22. In brief, one study22 consisted of 235 Caucasian families  collected from two hospitals in Dublin, Ireland. Families with two or more affected members with primary VUR of any grade were collected; the majority of the families were nuclear (affected-sib-pair) families containing two siblings with VUR, with genotype data available for siblings and both parents. Six family-members (three parents and three siblings) of VUR patients who had CAKUT (without detected VUR) were also counted as affected. The other study19 included two separate cohorts, one from the UK and one from Slovenia. The UK cohort comprised 165 Caucasian families (303 affected offspring) collected from multiple centres across the UK; similar to the Irish cohort the majority of the families were nuclear (affected-sib-pair) families containing two siblings with VUR and with genotype data available for siblings and both parents. The Slovenian cohort comprised 148 Caucasian families  collected from the University Medical Centre, Ljubljana; again the majority of the families were nuclear (affected-sib-pair) families containing two siblings with VUR, with genotype data available for siblings and both parents.We combined data from the two largest genetic studies of VUR conducted to date59 involves classifying the disease into (increasingly severe) grades from I-V according to the site and severity of reflux based on the degree of filling and dilation of the ureter and upper urinary tract, as assessed by cystography. In the Irish cohort, VUR was diagnosed via micturating cystourethrograms allowing VUR grade to be determined in 96% of patients. In the UK/Slovenian cohorts, however, we elected at the start not to include this concept in the analyses. This was because a. the classic grading system was not in fact always used in the micturating cystogram reports and b. the same grading system is not generally used for radioisotope cystograms, a techique increasingly used to diagnose VUR. Thus, for the analyses described here, we considered VUR to be radiologically proven, but did not consider its grade.The classical grading system for VUR60 and 2.04 in the Slovanian cohort. We chose not to reduce our sample size by stratifying the analysis by gender, which for genetic studies would in any case only be likely to have any substantial effect with regards to variants on the X chromosome. We also chose not to formally account for age of diagnosis in the analysis, taking the view that for genetic studies the most relevant measure is simply the fact that an individual is affected, and whether this can be related to their genotype or shared genotype with other affected relatives. However, given that the prevalence of VUR decreases with age (on account of spontaneous resolution), we counted siblings of affected children who were not investigated until later ages as unknown for VUR affection status, if VUR was not found.Because of sample drop-out, affection status and family structure, not all samples/families constributed to all analyses. We estimated that 199 Irish families , 121 UK families  and 140 Slovenian families  contributed to the linkage analyses presented here. Slightly larger numbers of families/individuals contributed to association analysis (family-based and case/control), as association analysis could potentially make use of family structures that were uninformative for linkage  procedures). The female:male gender ratio for patients used in the genetic analyses was 1.49 in the Irish cohort, 1.29 in the UK cohort (slightly higher than the 1.21 ratio seen in the full UK cohort)19, 134,350 SNPs in the UK and Slovenian cohorts remained available for analysis. The Irish cohort was genotyped on the on the Affymetrix Genome-Wide Human SNP Array 6.0, containing 834,482 genome-wide SNPs. Following QC checks22, a final set of 643,691 SNPs in the Irish cohort remained available for analysis. The density of the final set of SNPs available is more than sufficient for genomewide linkage analysis  in all three cohorts; however, for genomewide association analysis, the UK and Slovenian cohorts are most likely underpowered with respect to providing full genome coverage, as illustrated by the fact that in imputation analysis (see below) only 3,600,157 SNPs were successfully imputed for the UK/Slovenian cohorts, in comparison to 6,277,126 SNPs for the Irish cohort.The UK/Slovenian study, intitially conceived as a linkage study, made use of a relatively sparse genotyping array, the Affymetrix NspI array, containing 262,264 genome-wide SNPs. Following \u2018medium-level\u2019 QC checks24, implemented in the program PLINK61 and applied to all affected sibs , was carried out at the subset of 119,548 genotyped autosomal and X-chromosomal SNPs passing quality control checks in all three cohorts.  associations or any significant asociations that replicated across different cohorts).Family-based association analysis using the transmission/disequilibrium test (TDT)22 and the UK (2938 Wellcome Trust Case Control Consortium controls)25 at 108,134 autosomal SNPs passing QC in all case and control cohorts. This analysis was carried out using a linear mixed modelling approach implemented in the software FaST-LMM62 in order to adjust for both relatedness between cases and population differences between cases and controls66.We also performed case/control analysis of our VUR cases together with population-based controls from Ireland (851 Trinity College Dublin/Irish Blood Transfusion Service BioBank controls)67. To construct a suitable SNP set for multipoint linkage analysis, we first pruned our data set to include only those SNPs with minor allele frequencies greater than 0.3 and showing low levels of inter-marker linkage disequilibrium (using the PLINK command \u201c\u2013indep 50 5\u201d), and then thinned the data to use only the two SNPs with the highest heterozygosity in each 1\u2009cM window, using the program MapThin (http://www.staff.ncl.ac.uk/richard.howey/mapthin/). Information content plots from MERLIN and MINX indicated that the resulting SNP set  achieved high levels of information content (\u224895%) across the entire genome (data not shown).We carried out multipoint parametric and non-parametric linkage analysis on the combined Irish/UK/Slovenian cohorts using the software packages MERLIN and MINX68, which we denote here as \u201cZLRLOD\u201d.Parametric linkage analysis allowing for heterogeneity  was carried out assuming a disease allele frequency of 0.001 and either a recessive  or dominant  model. . We chose to assume a slightly rarer disease allele frequency (0.001) than the 0.01 that had been used in previous (separate) analyses of these data sets, in order to better match our expectation that the disease is highly genetically heterogeneous and so the population frequency of any particular causal genetic variant is likely to be small. Non-parametric linkage analysis was carried out using the equivalent LOD score to the Kong and Cox exponential model likelihood-ratio allele-sharing test27 with the Haplotype Reference Consortium (HRC) reference panel28 and pre-phasing performed via Eagle269. \u2018Best-guess\u2019 imputed genotypes were used, subject to the posterior probability of the genotype call exceeding 0.9. To ensure that only high-quality imputed SNPs were included in the final set, imputed SNPs were filtered to include only those with minor allele frequency >0.02 and imputation Rsq >0.8.To improve genome coverage, allowing interrogation of a denser set of SNPs, we carried out SNP imputation followed by TDT association, treating the Irish and UK/Slovenian cohorts separately (since they had very different numbers of QCed genotyped SNPs available to inform imputation). We used the Michigan imputation server70 and variant calling was performed with the GATK HaplotypeCaller algorithm following best practice recommendations73. Sequencing reads were examined via visualisation of the\u00a0.bam files in IGV74.We whole-exome sequenced 29 individuals from the 8 families  showing the strongest evidence of linkage in the 10q26 region. Sequencing was carried out by AROS using Illumina\u2019s Nextera Rapid Capture Exome Kit followed by 100\u2009bp paired end sequencing (6 samples per flow cell lane) on the Illumina HiSeq 2000/2500 platform. Sequences were aligned to the Human GRCh37 assembly using BWA74.We performed targeted genomic resequencing of the implicated 9\u2009Mb region of 10q26 (from 123 Mb-132 Mb) in 32 unrelated affected individuals from the families in our data set that contributed most to the linkage signal. Sequencing was carried out by Oxford Gene Technology (OGT) (whose next generation sequencing services have now been transferred to Source BioScience) using SureSelectXT Custom 9\u2009Mb design and capture followed by 100\u2009bp paired end sequencing (11 samples per flow cell lane) on the Illumina HiSeq 2000 platform. Sequences were again aligned to GRCh37 with BWA, variants were called with GATK HaplotypeCaller following best practice recommendations, and reads were visualised via IGV44 sequenced as part of the UK10K study45. We used the software package TASER47, which effectively re-calls variants in both cases and controls, allowing for systematic differences in read depth (and other factors), at the same time as constructing a test statistic. TASER uses the total number of reads mapped to a variant, and the number carrying the minor allele, to calculate a score statistic at each position in a gene (or window) of interest, thus providing an assessment of the effect of each individual variant on the disease phenotype. A burden statistic is then calculated for each window as the sum of the score statistics for each of the variants within that window, allowing identification of windows that have a higher or lower accumulation of rare variants in the cases than might be expected, compared to controls. This procedure therefore effectively looks for windows containing clusters of rare variants seen in different affected individuals (and not seen - or at least not at comparable frequencies - in controls).We analysed the targeted resequencing data from the 32 unrelated affected individuals together with whole-genome sequence data from 1106 ALSPAC controlsin vitro-propagated cultures of Normal Human Urothelial (NHU) cells were obtained from Fishwick et al.49 for the genes in the implicated chromosome 10q26 region (Supplementary Table\u00a0\u03bcM troglitazone (TZ) as PPAR \u03b3 ligand with concurrent 1 \u03bcM PD153035 to block epidermal growth factor receptor activation. Change in expression of MK167 was used as marker of cell cycle activity.Data on gene expression by The data sets analysed and full sets of results obtained during the current study are available from the corresponding author on reasonable request.Supplementary Information"}
+{"text": "Salvelinus genera , and then identify corresponding chromosome arms among the other available salmonid high-density linkage maps, including six species of Oncorhynchus, and one species for each of Salmo, Coregonus, and the nonduplicated sister group for the salmonids, Northern Pike Esox lucius for identifying post-duplicated homeologs. To facilitate this process, we developed MapComp to identify identical and proximate (i.e. nearby) markers between linkage maps using a reference genome of a related species as an intermediate, increasing the number of comparable markers between linkage maps by 5-fold. This enabled a characterization of the most likely history of retained chromosomal rearrangements post-WGD, and several conserved chromosomal inversions. Analyses of RADseq-based linkage maps from other taxa will also benefit from MapComp, available at: https://github.com/enormandeau/mapcomp/Whole genome duplication (WGD) can provide material for evolutionary innovation. Family Salmonidae is ideal for studying the effects of WGD as the ancestral salmonid underwent WGD relatively recently, \u223c65 Ma, then rediploidized and diversified. Extensive synteny between homologous chromosome arms occurs in extant salmonids, but each species has both conserved and unique chromosome arm fusions and fissions. Assembly of large, outbred eukaryotic genomes can be difficult, but structural rearrangements within such taxa can be investigated using linkage maps. RAD sequencing provides unprecedented ability to generate high-density linkage maps for nonmodel species, but can result in low numbers of homologous markers between species due to phylogenetic distance or differences in library preparation. Here, we generate a high-density linkage map  for the  Whole genome duplication (WGD) can provide the raw material for evolutionary innovation by generating copies of all chromosomes (i.e. producing homeologous chromosome pairs). After WGD, the genome can then undergo rediploidization while retaining all duplicated chromosome arms (homeologs), thereby doubling the pre-duplication chromosome arm number. Rediploidization remains a highly studied topic and probably involves large-scale structural changes between the homeologous chromosomes such as massive repeat element expansion . FurtherEukaryotic genomes with ancestral WGD and residual tetraploidy are challenging to assemble . LinkageEsox lucius  with 25 acrocentric chromosomes  = 100) but differing in total chromosome number (n = \u223c80 chromosomes) have more acrocentric than metacentric chromosomes, whereas Type B species (2n = \u223c60 chromosomes) have more metacentric than acrocentric chromosomes  is typified by centric Robertsonian fusions (hereafter metacentric fusions), whereby two acrocentric chromosomes fuse into one larger metacentric chromosome, retaining the total number of chromosome arms , and genera without high-density maps available , and this increased taxonomic sampling would provide new insights on the timing and process of chromosome arm fusion and fission post-WGD. Considering the important role of metacentric fusions in the rediploidization process, probably due to a higher frequency of tetravalents occuring at meiosis  that have been kept in captivity for three generations at the Station aquicole de l\u2019ISMER , and the F0 male was from a domestic population used in Qu\u00e9bec aquaculture for 100 years, supplied here from the Pisciculture de la Jacques\u2013Cartier . Three biparental crosses of F1 individuals produced three F2 families, and the family with the largest number of surviving offspring was chosen to be the mapping family (n = 192 full-sib F2 offspring).Full details regarding the experimental mapping family were reported previously , 2012b. 2 offspring and F1 parents by high salt extraction  to digest genomic DNA. Digested DNA was then ligated with adapters and barcodes for individual identification then amplified by PCR. For the offspring, uniquely barcoded individuals were then combined in equimolar proportions into eight pools, each pool containing 25 individuals. Pools were each sequenced on a single lane on a HiSeq2000 at G\u00e9nome Qu\u00e9bec Innovation Centre . In order to obtain deeper sequencing of the parents, each parent individual was sequenced using an Ion Torrent at the sequencing platform at IBIS . This platform change between F1 and F2 individuals occurred due to equipment availability, but extra precaution was taken to ensure proper correspondence of loci (see below).Double-digest RADseq  was perfhttp://www.bioinformatics.babraham.ac.uk/projects/fastqc/; last accessed November 8, 2016). Adapters were removed and raw reads were truncated to 80 bp using cutadapt v.1.9.dev.0  were allowed to merge as a single locus when no mismatches were found (cstacks). Loci from parents and offspring were matched against the parental catalog to determine the allelic state at each locus in each individual in sstacks. To improve the quality of the de novo assemblies produced in stacks and to reduce the risk of generating problematic loci with repetitive sequences and paralogs, we used the correction module rxstacks. A haploid cross is required to fully investigate paralogous loci  into fully informative (i.e. informative for both parents) or semi-informative  marker types, specifically the four types of markers that permitted in the outbreeding design: ab\u00d7ac, ab\u00d7ab, ab\u00d7aa and aa\u00d7ab . With heterozygous parents, not all of the markers contribute equally to the construction of the map, because linkage phases change across loci . Subsequently, additional maps were produced  using all markers in an analyses using a CP population type  with the multipoint maximum likelihood mapping algorithm for marker order  were defined by evaluating stability of marker numbers over increasing consecutive LOD values. This number of LGs corresponds to the expected chromosome number of Brook Charr (2n = 84). During mapping, the stabilization criterion was monitored in the session log with the sum of recombination frequencies of adjacent segments and the mean number of recombination events. Default mapping parameters usually performed well with the smaller LG, but for larger LG the stabilization was not always reached, so more EM cycles and longer chains per cycle were used. For full details of parameters used in JoinMap, see The linkage map was first built in JMap v4.1  using thMap v4.1  that onler order . The inier order  due to ler order . MarkersoinMap features  as recommended by Problematic or unlinked markers and small linkage groups were tested using several JIn order to compare the Brook Charr map to other salmonid maps, published linkage map datasets were collected (see the \u201cCode and Pipeline Availability\u201d section), including information on marker name, sequence, linkage group and cM position. Comparisons of linkage group correspondence and synteny between species were investigated using available high-density linkage maps. This included maps generated with haploid crosses for mapping regions exhibiting residual tetraploidy , althougapComp is shown in http://www.genoscope.cns.fr/trout/data/; last accessed November 8, 2016)  that are closest to each other in position on the same contig or scaffold are paired. Pairing occurs without replacement . Each marker pair is then added to an Oxford grid. The pipeline developed for MapComp is available at https://github.com/enormandeau/mapcomp/.The basic workflow of M8, 2016)  or the A8, 2016)  using BW and mapping against more than one locus in the Rainbow Trout or Atlantic Salmon reference genome was permitted.To identify homeologous relationships using MapComp. Chinook Salmon and Coho Salmon were then individually compared with Brook Charr to identify corresponding chromosome arms in Brook Charr. Once these homologous relationships were obtained, the Brook Charr map was compared with Pink Salmon, Sockeye Salmon, Chum Salmon, Rainbow Trout and Atlantic Salmon genetic maps. Chromosome correspondence was identified in Lake Whitefish using a consensus approach, where results from comparisons of Lake Whitefish with multiple different species were considered for unambiguous determination of homology. Homeologs were also identified using a consensus approach, and the original Northern Pike linkage groups were given .1 or .2 designations to represent the duplicated chromosomes. These results were compared with the results of blast was used with Atlantic Salmon linkage groups against the Northern Pike genome to identify salmonid WGD homeologs.Homology of chromosome arms between Chinook Salmon and Coho Salmon maps identified previously using homologous markers  was confapComp were visually inspected for inversions. During linkage mapping, when markers do not fit in the linkage group, they might be placed at the distal ends of the linkage group . To confirm that a metacentric chromosome was completely present, for conserved metacentric identification, we required evidence from both sides of the centromere.The conservation of chromosomal rearrangements among the salmonids was analyzed by using the most taxonomically complete phylogeny of the salmonids  but witheviously , and witeviously . The anaeviously , but re-stacks v1.32  was retained as the final map, as is typical for salmonids due to low recombination rate in males , 954 semi-informative (ab\u00d7ab) and 2,618 fully informative in female parent (ab\u00d7aa). The female map is in aa\u00d7ab) but although these markers were in the correct linkage groups, they did not position well within the linkage group. This is most likely due to the low recombination rate known to occur in male salmonids, as almost complete crossover interference can occur within male salmonids during meiosis  between Coho Salmon and Chinook Salmon with results that used only homologous markers , respectively. In some cases, there are discordances with the p and q designation with the first and second arm present in the linkage map (a and b designation). The reasons for these discrepancies are not clear, but it is worth noting that the two studies used different Atlantic Salmon genetic maps for comparison. This highlights the importance of meta-analyses to collect and analyze this data; here we refer to most arms as the Northern Pike homologs and include the Oxford grids to view the correspondence of all species against Brook Charr in In Brook Charr, a total of 8 metacentric and 34 acrocentric chromosomes were expected from salmonid cytogenetics  and all  markers  and founapComp as described in the Methods. All homeologous pairs in Atlantic Salmon identified by MapComp using the Rainbow Trout intermediate reference genome were concordant with those originally identified (To identify homeologous chromosome arms (i.e. chromosome arms originating from the same pre-duplicated chromosome), the genetic map of Northern Pike was compared with the maps of all species using Mentified , but herentified .Oncorhynchus species is not in fact residually tetraploid in Atlantic Salmon (i.e. 18qa-1qa or Ssa18a-Ssa01b).Homeologous chromosome arms exhibiting residual tetraploidy can be identified by mapping duplicate markers in haploid crosses  in all evaluated species were also identified for each species against Northern Pike using MapComp, with the exception of the aforementioned unidentifiable chromosome arms of Lake Whitefish  are likely to have occurred prior to the diversification of the clade, as demonstrated for nine metacentric fusions in Coho Salmon and Chinook Salmon . Here, cn events . For clan events . For then events , we presOncorhynchus , another is present in all Oncorhynchus species except Pink Salmon  and one underwent fission in Pink and Chum Salmon . Conserved fusions were found within the Oncorhynchus lineage as well, including one fusion in the Chinook/Coho lineage,  followed by an Atlantic Salmon-specific fusion of this metacentric chromosome [4.1\u201315.1] with an additional acrocentric chromosome, 1.2. The initial 4.1\u201315.1 fusion either fused once prior to the divergence of Atlantic Salmon and underwent three different fission events Fi1 in , or fuseSeveral inversions flanked by noninverted regions were revealed between linkage maps, suggesting the presence of chromosomal segment inversions . These pcytokine-like protein 1, solute carrier family 2, facilitated glucose transporter member 9 and cd8 beta, among others  markers, leads to a far greater number of retained marker pairs .Linkage group homology between species are typically identified by finding homologous markers using reciprocal best-hit blast . The metapComp was tested on both the Rainbow Trout and the Atlantic Salmon genome as intermediate references for pairing markers between maps .At least one homeologous chromosome arm fused into a metacentric chromosome is thought to be required for recombination between homeologs . This isAs metacentric formation is thought to be important for ongoing recombination between homeologs, the timing of fusion events may provide additional insight into the rediploidization process in salmonids. From the present study, it is interesting to note that many of the residually tetraploid pairs have at least one homeolog involved in an ancient conserved fusion . The secOncorhynchus spp., 2.1 is the conserved metacentric  or none .The fusion history of the other residually tetraploid pairs are not as simple as the above two examples. Within a residually tetraploid homeolog pair, it is not always the same homeolog in a metacentric fusion across species. This agrees with previous indications that only one of the homeologs must be bound in a metacentric to prevent rediploidization. For example, in Atlantic Salmon, 2.2 is metacentric and 2.1 is acrocentric, whereas in Brook Charr and all Salmo salar as demonstrated by sequence similarity between homeologous chromosomes , preserving the co-occurrence of adaptive alleles within the migratory form (O. clarkii) and Rainbow Trout hybrids compared with collinear regions (Inversions can occur when a segment of a chromosome is cut out by two breakpoints and then reinserted in the opposite orientation . Effectsory form  Addition regions . Recombi regions . Roberts regions . It is papComp helps to solve the issue of low marker homology between reduced representation sequencing (e.g. RADseq) based linkage maps generated with different protocols or restriction enzymes, or from relatively more distantly related species. Synteny is still required in order to pair proximate markers through the intermediate reference genome. Previously, polymorphic microsatellite markers highly conserved among salmonids have enabled exploration of salmonid chromosomal evolution by integrating across species and genera .Supplementary DataClick here for additional data file."}
+{"text": "Proprioception deficits are common post-stroke and predict poor functional outcome. It is unknown if the presence of proprioception deficits is negatively associated with the motor and functional ability of the affected upper extremity and daily living at the chronic stage post-stroke.1) To describe proprioception deficits of individuals with chronic stroke, 2) to correlate the severity of proprioception deficits with the motor and functional ability of the upper extremity, and 3) to compare independence in basic and instrumental activities in daily living , upper extremity motor and functional abilities between individuals with and without proprioception deficits.102 adults aged 29\u201385 years with chronic stroke participated in this cross sectional study. The upper extremity was assessed for proprioception , motor [Fugl-Meyer Motor Assessment (FMA)] and functional ability , grip strength and daily use [Motor Activity Log (MAL)]. Independence in BADL and IADL was also assessed.71 participants had intact proprioception, 31 participants had mild-moderate proprioception deficits. Negative significant (p<.001) correlations were found between the severity of proprioception deficits to the motor ability (FMA) (r = -.41), functional ability (ARAT) (r = -.48), dexterity (BBT) (r = -.43), grip strength (r = -.41) and daily-use   of the affected upper extremity. Significant between-group differences were found for BADL, IADL and upper extremity measures.Proprioception deficits of individuals with chronic stroke are negatively associated with upper extremity motor and functional abilities and independence in daily living. Therefore, proprioception should be assessed at the chronic stage post-stroke. Proprioception is impaired in a large percentage of individuals following stroke \u20134. The pThe presence of proprioception deficits has been found to be an important predictor of poor functional outcome post-stroke \u201316, specA recent review  summarizAdults with chronic stroke who lived in the community were invited to participate in the study. Inclusion criteria were 1. sustained a stroke at least 6 months prior to the study, 2. without a significant cognitive deficit  upper extremity were assessed as well.Thumb Localization Test (TLT) . However, a significant difference (t(100) = 2.6, p<.01) with a medium effect size was found for the dexterity of the less-affected (stronger) hand between groups; participants with intact proprioception transferred more blocks with their less-affected (stronger) hand [52.9 (11.2) blocks] compared to the participants with proprioception deficits [46.6 (10.1) blocks]. This difference did not remain significant after the Bonferroni correction (p = .009).The presence and impact of proprioception deficits at the chronic stage has not been the focus of many studies (e.g. Findlater ). ThirtyThe negative significant correlations found between the severity of proprioception deficits to the motor and functional ability, daily use, grip strength and dexterity of the affected upper extremity, demonstrate the associations (and not cause and effect) between the motor-functional ability and proprioception deficits. These associations of related constructs  together with no association of unrelated constructs, such as age and cognition  both support the construct validity of the TLT to assess proprioception . Since sDemographic characteristics, time since stroke, side of stroke or cognitive status were not different between groups. However, significant differences were found for the affected upper extremity in terms of motor, functional and grip strength between participants with and without proprioception deficits. These differences in motor and proprioception deficits are possibly due to the extended brain damage , 49, 50 These differences are perhaps not only from the initial brain damage caused by the stroke but also from the limited use of the upper extremity during the months since their stroke (as assessed by the MAL). Limited functional use of the upper extremity may have resulted in further restricted motor and functional ability. The effect size for differences between groups for the MAL  were the largest.Interestingly, the differences between groups were not limited to the affected upper extremity. A medium effect size with a trend towards statistical significance regarding the difference in manual dexterity (but not grip strength) of the less-affected (stronger) upper extremity was found. It has been well established that the stroke affects the ipsilateral, as well as the contralateral hand, in terms of motor ability, reaction time, precision , 52 and The participants recruited for this study were overall independent in BADL since intact basic cognition and the ability to walk, were inclusion criteria. Nevertheless, the level of independence in BADL of the participants with intact proprioception was significantly higher than the participants with proprioception deficits (and a large effect size). A vast range of scores on the IADL questionnaire was found for both groups; 3\u201323 and 2\u201321 points/23 points for the participants without and with proprioception deficits. Yet, participants with intact proprioception were significantly more independent in IADL including shopping, use of transportation and cooking, compared to the participants with proprioception deficits (a large effect size). This difference in independence in IADL might be explained by the differences found between groups in mobility  and function of both hands, which was higher for the participants with intact proprioception. In other words, better mobility and function of both hands of the participants with intact proprioception might have translated to more independence in IADL compared to the participants with proprioception deficits.The main limitation of this is study is the fact that proprioception deficits were assessed solely on the Thumb Localization Test , 15. SinTo conclude, the presence of mild-moderate proprioception deficits of the affected upper extremity (as assessed by the TLT) is negatively associated with the motor, functional and daily-use of the affected upper extremity and dexterity of the less-affected upper extremity of individuals with chronic stroke. These participants are also less independent in BADL and IADL. Therefore, at the chronic stage, it is as important to assess proprioception deficits of individuals with stroke. Further studies should assess the effectiveness of treatments that focus on improving proprioception deficits as well as motor deficits to improve hand function and independence.S1 File(SAV)Click here for additional data file."}
+{"text": "Background: The particular function of the left anterior human insula on emotional arousal has been illustrated with several case studies. Only after left hemispheric insula lesions, patients lose their pleasure in habits such as listening to joyful music. In functional magnetic resonance imaging studies (fMRI) activation in the left anterior insula has been associated with both processing of emotional valence and arousal. Tight interactions with different areas of the prefrontal cortex are involved in bodily response monitoring and cognitive appraisal of a given stimulus. Therefore, a large left hemispheric lesion including the left insula should impair the bodily response of chill experience (objective chill response) but leave the cognitive aspects of chill processing (subjective chill response) unaffected.Methods: We investigated a patient (MC) with a complete left hemispheric media cerebral artery stroke, testing fMRI representation of pleasant (music) and unpleasant (harsh sounds) chill response.Results: Although chill response to both pleasant and unpleasant rated sounds was confirmed verbally at passages also rated as chilling by healthy participants, skin conductance response was almost absent in MC. For a healthy control (HC) objective and subjective chill response was positively associated. Bilateral prefrontal fMRI-response to chill stimuli was sustained in MC whereas insula activation restricted to the right hemisphere. Diffusion imaging together with lesion maps revealed that left lateral tracts were completely damaged but medial prefrontal structures were intact.Conclusion: With this case study we demonstrate how bodily response and cognitive appraisal are differentially participating in the internal monitor of chill response. Chill can be experienced in various ways  and is often accompanied by measurable changes in various physiological response systems . IncreasChill experiences often emerge in response to pleasant music e.g.,  but can The PFC has been reported to be associated with emotional control , executiBoth areas, the PFC and the AIC, are anatomically intensely interconnected . The verWe here investigated the response to auditory chill inducing stimuli in a patient with left insula lesion who verbally communicated an animating effect of musical chill. The patient experienced a complete medial cerebral artery infarct 7 years before investigation (chronic stable state) damaging left sided insula together with the ventrolateral PFC, the anterior temporal lobe and the left thalamus. We investigated both the SCR and the subjective chill experience during listening to high chilling auditory stimuli while performing fMRI and compared her data with data of an age, gender and intellectual abilities precisely matched HC. We here used pleasant and unpleasant chill stimuli in order to enable a differentiation of behavior, psychophysiology or functional representation. fMRI was used to investigate possible chill associated functional representation. High resolution structural T1, T2-weighted and diffusion weighted imaging served as the demonstration of the lesion of gray and white matter structure. According to previous reports on AIC and PFC function we expected a mismatch between cognitive evaluation of emotional chill and bodily response, which should be referred to by structural lesion and functional mapping of emotional response representation.3. White matter tracts such as the pyramidal tract and the uncinate fasciculus were no more verifiable using diffusion weighted tractography . She suffered from Broca aphasia, neglect on the right hemifield and a hemiplegia including an upper limp plegia and lower limb paresis.We investigated a 47-year-old, left-handed participant (MC) with an extended stroke of the left middle cerebral artery. The stroke occurred 7 years before fMRI-investigation and the lesion has a volume of 260 cmWe had the opportunity to compare MC with a healthy woman (HC), who had a very similar demographic, social, and intellectual background. Being born in the same year and working in the same profession with a similar level of career as MC, when she experienced the stroke, it makes her the perfect comparator.To evoke pleasant and unpleasant chill reactions we presented musical stimuli, which showed a high ability to induce chills  and recoMC and HC were instructed to listen carefully to the music and sounds and to report intensity, onset, and duration of each chill experience by pressing a foam covered handle device with their right hand. Then they were placed in the scanner and equipped with headphones. The whole experiment lasted about 30 min. Whereas SCR is the optimal psychophysiological parameter for measuring bodily response to chills, heart rate does not reliably change during chill response . TherefoImages were collected with a 3 T Magnetom Verio  using a 12-channel head coil. Echo-planar imaging , phase and magnitude images to calculate a fieldmap aiming at correcting geometric distortions, T1-weighted high resolution anatomical imaging and diffusion weighted imaging were performed over the whole head volume.Data of the handle device and skin conductance were processed with Brain Vision Analyzer 2.0 . Skin conductance data were down-sampled to 10 Hz and differentiated into tonic and phasic activity using Ledalab toolkit . AnalysiLesion volumes were calculated on the basis of the T1-weighted image. Diffusion weighted data were processed utilizing DTIFIT, BEDPOSTX and PROBTRACKX of the FSL software package. Seed masks for tractography were created from the activation maxima of the fMRI statistical maps within the PFC using a spherical kernel with a diameter of 15 mm to ensure the inclusion of white matter. We also applied additional seed masks for the evaluation of the arcuate fasciculus, cingulate gyrus, the posterior limb of the internal capsule, the genu of the Corpus Callosum and the Corona radiata .p < 0.05 corrected for multiple comparisons in the whole brain volume (FWE).FMRI data were analyzed using the SPM8 software . Spatial pre-processing included realignment to the first scan, unwarping, coregistration to the T1 anatomical volume images. T1 images were spatially normalized using the New Segment function of SPM 8 and DARTEL  and funcFor HC we further conducted two psychophysiological interaction (PPI) analyses with SPM8 to investigate, which voxels in the brain increase their interaction with the left and the right AIC during a chill event. Therefore the convoluted time courses (task \u00d7 seed) were analyzed in a GLM while the task time course and the seed time course itself were included as regressors.Most of the applied harsh sounds resulted in chill reports from both participants. Whereas HC reported chills in response to music (chill intensity: 0.12 \u00b1 0.06) and harsh sounds (0.11 \u00b1 0.09) with similar intensities, MC reported higher intensities for chills in response to harsh sounds (0.16 \u00b1 0.08) than for chills in response to music (0.09 \u00b1 0.07).Figure 2A shows the correlation between SCR and the reported chill intensities for the 10 chill passages for MC and HC. For HC the reported chill intensity was positively correlated with SCR , whereas for MC no relevant association was observed . Figures 2B,C show skin conductance level (SCL) and chill reports over the course of the experiment for HC and MC. Though both participants reported chills at a similar range of intensity throughout the entire experiment only HC showed associated autonomic responses regularly. MC showed an attenuated activation of the sympathetic nervous system as expressed in SCR compared to HC.Figure 3). Remarkably, not only the pleasant chills were associated with bilateral ventral striate activation in HC  but also the unpleasant chills showed a right hemispheric ventral striate fMRI-activation . For MC the pleasant chills were not associated with relevant ventral striate activation, but unpleasant chills showed bilateral ventral striate activation .Unpleasant and pleasant chill stimuli evoked fMRI-activations against baseline in different areas of the PFC and temporal cortices, as well as the S2, the AIC, the A1, the thalamus, and bilateral amygdala in both participants. More specifically for the PFC and the AIC we found quite balanced activation for both hemispheres for HC but for MC lateral PFC areas such as AIC and BA 44/45 showed only relevant activation in the right intact hemisphere  of the fractional anisotropy values (FA) of the dlPFC seed was on average  on -0.07 and for the vlPFC -0.03. In contrast, LIFA of the patient was strongly lateralized to the right hemisphere .Table 1) performed for the HC for both pleasant and unpleasant chills demonstrated that the right AIC was interconnected with the bilateral Heschl gyrus but not to prefrontal ROIs . In contrast, the left AIC was not only interacting with the bilateral Heschl gyrus but also with the bilateral prefrontal ROIs .A PPI  we found a comparable subjective chill appraisement but a selective decrease in objective chill parameters (SCR) for both unpleasant and pleasant chill stimuli for the patient. Correspondingly, SCR and assessment scores were positively associated in HC but not in MC. Diffusion imaging discovered selective destruction of more lateral structures such as the uncinate fasciculus whereas medial structure such as the radiate corona and the cingulate were undestroyed. Psychophysiological differences between participants went along with changes in functional representation during listening to stimuli. In MC AIC and BA 44/45 were only relevantly activated in the right hemisphere, but more anterior representation in BA 8/9 and 46/47 was balanced between hemispheres. We interpreted the findings that intact right hemispheric areas for auditory recognition and intact working memory allowed for an adequate evaluation of the stimuli presented but damaged left hemispheric AIC diminished bodily response.We used musical stimuli, which fulfill chill criteria in most healthy participants . We furtWhereas the onsets and duration of aversive chills were constant between participants, those of pleasant chills differed between individuals, which makes an individualized fMRI-design necessary. In HC objective and subjective chill parameters were highly positively associated but dissociated in MCA.Our stimuli not only reliably evoked SCR-changes in HC, they also activated brain areas that have reported in previous functional imaging studies on chill response. Interestingly, in HC not only pleasant chills were associated with bilateral ventral striate response as reported previously  but alsoThe case study at hand highlights the important role of the left AIC for chill experience. With respect to musical stimuli the anterior insula has been especially active during the processing of unexpected chords . OverallA loss of feeling for music after left AIC lesion has already been described  as well We would predict that a loss of bodily response to chilling stimuli is restricted to lesions of the left AIC and will not be present for patients with right AIC lesions, which has to be proven in a study testing groups of right and left anterior insula damaged patients with our paradigm.We showed that MC activated the right AIC and the right BA 44/45, whereas BA 46/47 and BA 8/9 were bilaterally involved. The loss of autonomic changes during perceived chill experiences in this patient suggests that embodiment of this emotional experiences to chills is processed in a network including left AIC and BA 44/45. Lesions within this network seem to reduce the craving response in anticipation of a drug  or the eNevertheless, the appropriate verbal report of a chill experience in MC is noteworthy. The intact regions are therefore critical for an appraisal of the stimulus regardless of the physiological sensation. It might be that intact prefrontal areas allowed for an evaluation of the music but diminished left hemispheric insula function and destroyed connections with lateral prefrontal areas abolished the association between bodily response and chill experience. Especially in BA 46/47, evaluation of emotional valence has been repetitively reported both for visual and acoustic stimuli e.g., . FurtherIt has to be mentioned that the AIC is only one major part of a network monitoring bodily responses and interoception e.g., . ConnectTherefore, lateralized chill response might well be processed in interactions between the AIC and the PFC, but not in single areas. This is underlined by severe structural damage present in our patient for lateral fiber tracts connecting emotional processing and prefrontal cognitive appraisal of stimuli. Our functional interaction analysis (PPI) underlined the network difference between left and right hemispheric interactions of the AIC: in HC only left AIC interacts with the PFC whereas the right insula was not interacting with PFC in our paradigm.This case-control study is only a first step for investigating chill processing for positive and negative emotional assessed auditory material. The patient investigated here showed a complete left side MCA damage including various other areas besides the left AIC. Lesion mapping in a number of patients is necessary in order to specify the role of the left AIC using the same chill parameters and investigating fMRI-response in other areas of the prefrontal lobe. This investigation should again integrate diffusion weighted imaging to define structural intactness of interaction between areas. Up to now the conclusions of this study are very limited and do not allow for specifically pointing to the role of the left AIC for the processing of chill.Whereas left hemispheric lesion after MCA stroke abolished physiological expression (indexed by SCR and SCL-changes) or embodiment of the chill responses, the evaluation of the chill experience was left unchanged. We hereby highlighted the role of left hemispheric limbic areas for emotional response expression and monitoring. Nonetheless, prefrontal regions probably generated a chill experience that could be reported using an analog rating-device in the described patient. Further lesion studies will help to investigate the specific role of each of these areas.This study was carried out in accordance with the recommendations of the Ethics Committee of the German Society of Psychology. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Ethics Committee of the German Society of Psychology .VG helped with measurement, data evaluation and writing of the manuscript. KH worked on study design and writing of the manuscript. KK helped with measurement, data evaluation and writing of the manuscript. JN helped with measurement, data evaluation and writing of the manuscript. UH helped with data evaluation and writing of the manuscript. MD helped with study design, data evaluation and writing of the manuscript. AH worked on the study design and writing of the manuscript. ML worked on the study design and writing of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "Scientific Reports 10.1038/s41598-018-20602-y, published online 01 February 2018Correction to: This Article contains an error in the order of the Figures. Figures 1, 2, 3, 4 and 5 were published as Figures 2, 3, 4, 5 and 1 respectively. The correct Figures"}
+{"text": "Synechocystis sp. PCC 6803 that the Psb29 subunit, originally identified as a minor component of His-tagged PSII preparations, physically interacts with FtsH complexes in vivo and is required for normal accumulation of the FtsH2/FtsH3 hetero-oligomeric complex involved in PSII repair. We show using X-ray crystallography that Psb29 from Thermosynechococcus elongatus has a unique fold consisting of a helical bundle and an extended C-terminal helix and contains a highly conserved region that might be involved in binding to FtsH. A similar interaction is likely to occur in Arabidopsis chloroplasts between the Psb29 homologue, termed THF1, and the FTSH2/FTSH5 complex. The direct involvement of Psb29/THF1 in FtsH accumulation helps explain why THF1 is a target during the hypersensitive response in plants induced by pathogen infection. Downregulating FtsH function and the PSII repair cycle via THF1 would contribute to the production of reactive oxygen species, the loss of chloroplast function and cell death.One strategy for enhancing photosynthesis in crop plants is to improve their ability to repair photosystem II (PSII) in response to irreversible damage by light. Despite the pivotal role of thylakoid-embedded FtsH protease complexes in the selective degradation of PSII subunits during repair, little is known about the factors involved in regulating FtsH expression. Here we show using the cyanobacterium This article is part of the themed issue \u2018Enhancing photosynthesis in crop plants: targets for improvement\u2019. One ofixation . Irreverfixation ,5, but afixation ,6. Only fixation , is a poRepair of PSII occurs in all organisms that carry out oxygenic photosynthesis ,9. AlthoSynechocystis sp. PCC 6803 (hereafter Synechocystis 6803), electron microscopy has revealed the isolated FtsH complex to be hexameric and composed of alternating FtsH2 and FtsH3 subunits [var2 and var1 null mutants [The main pathway for degrading damaged D1 during repair involves proteolysis by specific members of the FtsH family of ATP-dependent metalloproteases in both cyanobacteria ,15 and csubunits , which, subunits ,21. Althsubunits ,21, withthf1 (thylakoid formation 1) mutant of Arabidopsis [Synechocystis 6803 [Synechocystis 6803 psb29 null mutant, but specific effects on FtsH were not examined [psb29 null mutant of the cyanobacterium Synechococcus sp. PCC 7942, but changes to the expression of individual FtsH subunits were not investigated [How expression of FtsH complexes is regulated in response to light stress is unclear. Recent studies of the variegated bidopsis  have indbidopsis ,24. The tis 6803  and a roexamined . A reducstigated . In addistigated .Synechocystis 6803, like THF1 in Arabidopsis, is important for normal accumulation of the FtsH heterocomplex involved in PSII repair. Furthermore, affinity purification data suggest that Psb29 physically interacts with FtsH complexes in vivo. To gain further insights into Psb29, we have determined the crystal structure of Psb29 encoded by Thermosynechococcus elongatus, a thermophilic cyanobacterium widely used to study structural aspects of PSII assembly and repair [Here we show that Psb29 in d repair ,29. Psb22.(a)Synechocystis sp. PCC 6803 [Synechocystis 6803 were grown on an orbital shaker in BG11 medium in 250 ml conical flasks at 29\u00b0C under moderate light conditions (40 \u00b5mol photons m\u22122 s\u22121). For purification of protein complexes, the FtsH2-FLAG strain was grown as described above in 500 ml of medium using 2 l conical flasks. For purification of Psb29-FLAG protein complexes, 4 l of Psb29-FLAG strain were grown in a 10 l flask in BG11 medium supplemented with 1 mM glucose, agitated with magnetic stirrer and bubbled with air. In both cases, surface irradiance was increased to 100 \u00b5mol photons m\u22122 s\u22121 of light to compensate for the longer path length of the flasks. For spot growth tests, 2.5 \u00b5l of mixotrophic culture and 102, 103 and 104 serial dilutions were spotted onto BG11 agar plates and grown for 7 days.All mutants were constructed in the glucose-tolerant WT-P strain of PCC 6803  and growPCC 6803 . For mix(b)psb29 gene in Synechocystis 6803 (Cyanobase designation sll1414) was constructed in two steps. First, the flanking sequencing of sll1414, 445 bp upstream and 555 bp downstream, was PCR amplified with primer set sll1414-1F (AGTTTCTCGTTCTGCCGCCTCAGCTCTT) and sll1414-2R (AATGGGGCCTCATAGTGGGGCATGGATTGAAGATATCAGGGCCGATTACAAAGGGGGGGATAGT), and sll1414-3F (ACTATCCCCCCCTTTGTAATCGGCCCTGATATCTTCAATCCATGCCCCACTATGAGGCCCCATT) and sll1414-4R (ATTAACTCCCCATCCACTTCCACTTCGATGAT). The resulting PCR products were then mixed as DNA template for overlap extension PCR with primer set sll1414-1F and sll1414-4R. The fused PCR fragment containing an EcoRV restriction site instead of sll1414 ORF was then cloned into pGEM-T Easy vector. In the second step, a DNA cassette that confers chloramphenicol resistance was inserted into the EcoRV site. Two transformation vectors were selected due to the nature of blunt-end ligation: pSll1414camA has the chloramphenicol marker integrated in the same direction as sll1414, whereas, pSll1414camB has the marker in the opposite direction. Both plasmids were used to transform the glucose-tolerant WT-P strain of Synechocystis 6803, yielding strains \u0394Psb29camA and \u0394Psb29camB.The transformation vector for disruption of psbA2 locus were generated by cloning PCR fragments into the NdeI and NheI sites of pPD-CFLAG [psb29 (sll1414) was amplified with primer pair CF-Psb29-F (TTTTTTCATATGACTAAAATTCGCACTGTTTCTGACGCCAA) and CF-Psb29-R (TTTTTTGCTAGCGCTTTCGGAACTCTCCGCTGTGGTT) and the coding sequence of ftsH2 (slr0228) was amplified with primer set CF-FtsH2-F (TTTTTTCATATGAAATTTTCCTGG.AGAACTGCCCTACTT) and CF-FtsH2-R (TTTTTTGCTAGCTAGTTGGGGAATTAACTGTTCCTTGACGGGA). The Synechocystis 6803 mutant \u0394Psb29camA was used as background strain to generate Psb29-FLAG/\u0394Psb29 and insertion mutant Rslr0228::cm [The transformation vectors for expressing C-terminal 3xFLAG-tagged derivatives of Psb29 and FtsH2 at the PD-CFLAG . The cod228::cmR  was tran(c)Synechocystis Psb29 conjugated to keyhole limpet haemocyanin .Preparation of membranes by breaking cells using a Mini-Beadbeater-16 (BioSpec) and anti-FLAG pull downs were performed as described in . The chl(d)The MS analyses of protein bands excised from gels were done on a NanoAcquity UPLC (Waters) on-line coupled to an ESI Q-ToF Premier mass spectrometer (Waters), as described in .(e)ftsH2 and ftsH3 transcript levels by quantitative PCR was performed as described in [ftsH2 and ftsH3 and Transcriptor Reverse Transcriptase (Roche). The rnpB gene encoding the B subunit of ribonuclease P was used as a reference and the analysis was performed in triplicate using three independent cultures.Determination of the ribed in  using sp(f)T. elongatus (Cyanobase designation: Tlr1134) was cloned into the BamHI and XhoI sites of the modified pRSETA expression vector [psb29 using primer set Tlr1134-F (GGATCCGTGCAAAATCCTCGAACTGTCTCTGATACCAAACG) and Tlr1134-R (CTCGAGTCAAGCGGGTGCATCGGAGCTGGCAT). The resulting vector pRSETAPsb29 encodes a recombinant protein consisting of a 6xHis tag at the N-terminus followed by a thrombin cleavage site then Psb29. The E. coli strain KRX was used for recombinant Psb29 expression. Psb29 expression in transformed cells was induced at an OD730 of 0.8 with 1 g l\u22121 rhamnose and cells were then grown at 18\u00b0C overnight. Cells were lysed by sonication in lysis buffer . In some preparations, the lysis buffer was supplemented with a Complete Protease Inhibitor Cocktail Tablet \u2013 EDTA . The supernatant was mixed with a Ni-IDA resin . Non-specifically bound proteins were removed by washing three times with wash buffer  and Psb29 was eluted with elution buffer . The protein was concentrated to around 10 mg ml\u22121 in 20 mM Tris-HCl pH 7.9, 500 mM NaCl and used for crystallization trials. Concentrated samples were placed in sitting drop vapour diffusion crystallization screens using a Mosquito\u00ae robot .The coding sequence of Psb29 from n vector  followin322, which diffracted very weakly. If protease inhibitor was omitted, crystals were readily obtained in three crystal forms. Two of these were in P21 (designated A-P21 and B-P21) and the third in I222. The structure was solved by single-wavelength anomalous dispersion (SAD) with the A-P21 crystal form, soaked overnight with 1 mM dipotassium tetraiodomercurate (Jena Bioscience). The P6322 form was soaked overnight in 1 mM 4-(Chloromercuri)benzensulfonic acid sodium salt (Jena Bioscience), but this was not used for phase determination. Crystals were cryoprotected in the mother liquor with 30% glycerol added, and flash-cooled in a loop into liquid nitrogen. Diffraction data were collected at Diamond Light Source and processed using xia2 [1 were found and the structure phased using the autoSHARP [1,I222 and P6322 crystal forms were solved by molecular replacement with Phaser [1 structure as a model. These structures were refined with REFMAC or phenix.refine [For preparations in the presence of protease inhibitor, the only crystals obtained were of needle morphology in P6ing xia2  with XDSing xia2 . See tabutoSHARP  pipelineutoSHARP  and refiutoSHARP . The B-Ph Phaser  using thx.refine . Structux.refine .Table\u00a01(g)Synechocystis 6803 (sll1414 gene product) against UniProt KnowledgeBase Reference proteomes (http://www.uniprot.org). The cut-off threshold was empirically set to 1\u00d710\u22124 after manually examining the resulting hits. 103 records were from cyanobacteria, 84 from plant, 11 from green algae, 12 from red algae and one from a virus that infects the green alga Chlorella sp. strain NC64A. 211 sequences were then aligned using MAFFT version 7 programme with the \u2018G-INS-I\u2019 setting applied [Arabidopsis thaliana THF1, predicted by ChloroP 1.1 Server [211 Psb29 sequences were retrieved by blasting Psb29 from  applied . Gaps wi applied  and then applied  was used applied . The fin applied . Subsets applied . The evo applied . The abo1 Server , were al3.(a)Synechocystis 6803, we performed an immunoblotting analysis of membranes isolated from a psb29 null mutant, \u0394Psb29camA, in which the psb29 gene was replaced by a chloramphenicol-resistance cassette . Cultures grown to late-exponential phase under either photoautotrophic or mixotrophic conditions were analysed. Antibodies specific for each of the four FtsH proteins encoded by Synechocystis 6803 revealed that levels of FtsH2 and FtsH3 were decreased substantially in the mutant compared to the WT control, consistent with a specific effect on the accumulation of the FtsH2/FtsH3 hetero-complex, whereas there was less of an impact on FtsH1 and FtsH4 (a). Similar results were also obtained with a psb29 null mutant, \u0394Psb29camB, containing the chloramphenicol-resistance cassette inserted in the opposite orientation . Reverse-transcription PCR confirmed that ftsh2 and ftsH3 were still transcribed in \u0394Psb29camA so the effect of Psb29 on the expression of FtsH2 and FtsH3 occurred after transcription (b). The 2\u20135-fold increase in ftsH2 and ftsH3 transcripts in \u0394Psb29camA might reflect a compensatory mechanism to increase expression. Importantly, immunoblotting experiments showed that FtsH2 and FtsH3 expression was reduced but not blocked totally in the absence of Psb29 .To test whether Psb29 plays a role in the expression of FtsH in nd FtsH4 a. Similacription b. The 2\u2013(b)Synechocystis 6803 expressing either Psb29 or FtsH2 tagged at the C-terminus by addition of a 3XFLAG tag. Expression of the tagged proteins under the control of the psbA2 promoter in the relevant ftsH2 or psb29 null mutant restored photoautotrophic growth at high irradiances, indicating that the tagged proteins were still functional . Immunoaffinity purification of Psb29-FLAG from detergent-solubilised membranes using anti-FLAG antibodies, followed by 2D gel electrophoresis (clear-native in the first dimension and denaturing in the second) and detection of proteins by protein staining, immunoblotting and mass spectrometry revealed the presence of large complexes containing FtsH2, FtsH3 and FtsH1 (a), which we assign to FtsH2/FtsH3 and FtsH1/FtsH3 heterocomplexes based on previous studies [b). Overall these data support the direct interaction of Psb29 with FtsH2/FtsH3 complexes.To test whether Psb29 interacts with FtsH we generated two strains of nd FtsH1 a, which  studies . Also de studies  and Phb3 studies . Psb29-F(c)T. elongatus as an N-terminal His-tagged protein in E. coli and isolated the protein by Ni-affinity chromatography. Four crystal forms were obtained by hanging drop vapour diffusion; X-ray diffraction data were collected at resolutions from 3.6 \u00c5 to 1.4 \u00c5 and the structure of Psb29 determined by heavy atom SAD . Each Psb29 subunit consists of 9 alpha helices . It is likely that proteolytic cleavage of the C-terminal helix allows more compact higher resolution crystal lattices to form, as there is insufficient space in these lattices to accommodate the C-terminal helix observed in the P6322 crystal form. The I222 crystal form shows a domain-swapping of the N-terminal helix from the N-terminus to residue Ile22, creating a domain-swapped dimer . Given that the domain-swap is not observed in the other crystal forms, this is probably a crystallization artefact.Psb29 in the other crystal forms was proteolytically cleaved at the C-terminus. In the B-P2(d)Chlorella sp. strain NC64A that possesses a Psb29-encoding gene closely related to green algal Psb29 sequences . In the proteome database interrogated on 11th November 2016, 103 out of 106 cyanobacteria were found to encode Psb29 homologues. The genome sequences of the three remaining cyanobacteria, Limnoraphis robusta CS-951, Leptolyngbya valderiana BDU 20041, and Cyanobium sp. PCC 7001 (Synechococcus sp. PCC 7001) are still incomplete and so still yet might encode Psb29.Bioinformatic analyses revealed that Psb29 and its eukaryotic homologue THF1 are found solely in oxygenic photosynthetic organisms . One exception is a virus infecting the green alga T. elongatus shows a mean sequence similarity of 59.2% with the 102 cyanobacterial Psb29 sequences examined and 53.7% with the 84 plant THF1 sequences. Six residues are totally conserved in cyanobacterial and plant Psb29/THF1 sequences : based on the structure described here, F14, V35, L39, G55 and G138 (T. elongatus numbering) appear important for the packing of alpha helices and R133 at the beginning of helix 7 is within H-bonding distance of E36 in the middle of helix 2 . These sequence identities would suggest a high degree of conservation of tertiary structure between Psb29 and THF1 in this region of the molecule. A ConSurf analysis in which all Psb29/THF1 sequences were fitted into the T. elongatus structure revealed high sequence conservation on one face of the molecule, which would indicate an important role for this region in protein function , in the more divergent region of Psb29. The C-terminal end of the protein is also poorly conserved .The alignment of Psb29/THF1 sequences revealed a variety of small insertions and deletions. In the case of plant THF1, these insertion/deletion events correspond to 4.Synechocystis 6803 are likewise reduced in psb29 null mutants (c). These data suggest a conserved role for Psb29/THF1 in fine-tuning the expression of thylakoid FtsH heterocomplexes.Previous work in Arabidopsis has shown that the absence of THF1 leads to a 40\u201380% decrease in the amount of the type A and type B FTSH subunits involved in PSII repair as judged by immunoblotting . We showa), it is possible that Psb29 is also involved in the accumulation of FtsH1/FtsH3 heterocomplexes [psb29 null mutant under the conditions examined  proteins involved in plant immunity [We have also presented the first structural information on Psb29. The first 3 and last 16 residues could not be identified in the most complete crystal structure, possibly because of structural flexibility or because of some proteolytic degradation. The fitting of cyanobacterial and plant Psb29/THF1 proteins into the  figures\u00a0 and 5. Rimmunity . Thus soPSII repair is one of several photoprotective mechanisms used by plants . DespiteWork in cyanobacteria has highlighted D1 synthesis as a weak link in PSII repair due to reactive oxygen species (ROS)-mediated oxidation of elongation factor EF-G required for protein translation . Attemptthf1 null mutant [Chlamydomonas reinhardtii [Although upregulating FtsH activity and the PSII repair cycle would seem beneficial for plant growth, there appear to be situations where plants deliberately downregulate chloroplast FtsH activity, which is known to lead to the enhanced production of ROS even under non-photoinhibitory conditions . The soul mutant  and/or bl mutant  and Chlanhardtii  suggests"}
+{"text": "In an era where the volume of structured and unstructured digital data has exploded, there has been an enormous growth in the creation of data about individuals that can be used for understanding and treating disease. Joining these records together at an individual level provides a complete picture of a patient\u2019s interaction with health services and allows better assessment of patient outcomes and effectiveness of treatment and services. Record linkage techniques provide an efficient and cost-effective method to bring individual records together as patient profiles. These linkage procedures bring their own challenges, especially relating to the protection of privacy. The development and implementation of record linkage systems that do not require the release of personal information can reduce the risks associated with record linkage and overcome legal barriers to data sharing. Current conceptual and experimental privacy-preserving record linkage (PPRL) models show promise in addressing data integration challenges. Enhancing and operationalizing PPRL protocols can help address the dilemma faced by some custodians between using data to improve quality of life and dealing with the ethical, legal, and administrative issues associated with protecting an individual\u2019s privacy. These methods can reduce the risk to privacy, as they do not require personally identifying information to be shared. PPRL methods can improve the delivery of record linkage services to the health and broader research community. Unabating growth in the creation of data, coupled with advances in information technology and Internet connectivity, provides tremendous potential for data-driven breakthroughs in the understanding, treatment, and prevention of disease. These health research innovations are being complemented by data from non-traditional sources . Opportunities include the use of mobile phone records  and GoogA key methodology that has supported health research is record linkage, a process of accurately bringing together records from multiple datasets that belong to the same person. Through record linkage, it has been possible to construct and analyze population-wide datasets comprising \u201clinked\u201d administrative records pertaining to each individual. Health-based record linkage frameworks have been established, which routinely integrate data from hospital admissions, emergency departments, primary care facilities, birth, death, and disease registries , 2, creaPresent models of record linkage use trusted third parties (TTPs) or data linkage units (DLUs) to accurately match records using personal identifiers . IncorpoSharing of public and private datasets also presents privacy and confidentiality challenges. Protecting the privacy of individuals is paramount in the record linkage process and essential to maintain community support and trust. There are serious ethical implications in combining information on individuals  from government and other sources; essentially a form of surveillance of an entire population. For some privacy advocates, this is a bridge too far, conjuring up images of an Orwellian dystopia or the excesses of totalitarian regimes , 14. HeaWhile a number of existing processes and techniques are used to maintain patient privacy during record linkage , the devnot require the release of personally identifying information by data custodians. PPRL methods work on information that has been permanently encoded, encrypted, or transformed before releasing the data for linkage. Through PPRL methods, the benefits of linkage can be realized without the risks associated with disclosure of personal information.This article discusses the emergence and potential benefit of record linkage techniques that limit the release of personal identifiers for linkage. These methods, collectively referred to as privacy-preserving record linkage (PPRL), operate in such a way that they do There is a long history in Australia of record linkage supporting both jurisdictional level and national research and health decision making , 12, 18.The record linkage framework adopted by most of these jurisdictions is a TTP model, whereby dedicated linkage units undertake record linkage to service and support research. Administrative data collections  have typically formed the backbone of enduring record linkage systems , 23. SucLinkage of person-level records through the use of personally identifying information, and generally without consent, has significant ethical and legal implications that have been at the forefront of issues confronted and addressed by DLUs , 24.Commonwealth Privacy Act 1988 s 95). The release of personal data for linkage can be authorized if public benefit outweighs the privacy of individuals . German laws in relation to the disclosure of personal information are particularly restrictive  and, in some cases, only a single data item can be used for anonymous linkage . Protocols may perform matching on a particular set of identifiers, using either exact or similarity comparisons. Similarity matching enables records with slight differences to come together, which is vital for obtaining high-quality linkage results (accuracy). For this reason, PPRL protocols that utilize approximate matching are favored.Efficiency can be often a concern for record linkage and will continue to present challenges to DLUs as the volume of data continues to grow. Although there are no established performance standards, record linkage is computationally slow, and for any PPRL protocol to be practical, it must complete within a reasonable time frame.The extent to which these protocols are used in practice varies. To date, most PPRL implementations use exact matching on particular attributes of a dataset , which aOf all PPRL methods, the Bloom filter method appears to be the most promising for operational use . An advaThe introduction of the Bloom filter method brings new challenges . As wellConsider the  scenario: to attempt to reduce the rate of youth suicide, the government of the day has invested in a comprehensive mental health care package for those who have attempted suicide. The government wishes to see whether their program has worked in reducing the rate of suicide and attempted suicide.To answer this question, two datasets will be required: a hospital admissions dataset and a mortality register. From the hospital admissions dataset, records will be required to be sent to the linkage unit for all those persons who have attempted suicide before and after the start of the health intervention; all records from the mortality register will be required by the linkage unit. The linkage unit will receive only the personal identifying information required for linkage . The linkage unit identifies which records from the supplied hospital dataset have associated mortality records. The linkage unit passes this information back to the data custodians, who then provide the content data  to the researcher for the hospital records, and any linked mortality records, along with a key that identifies which records belong to which individual. The researcher can then use this information to determine whether the intervention reduced suicide and attempted suicide rates.The privacy risk in the aforementioned scenario is the delivery to the linkage unit of personal identifying information from hospital records of those who have attempted suicide. This extremely sensitive information has been made available to a third party. The use of privacy-preserving linkage methods would remove this risk; instead, the linkage unit would receive encrypted personal identifiers; they would have no means of identifying any of these individuals, but would still have the ability to determine which records belong to the same individual between datasets.With a growing demand for linked data from government and the university sector, interest in PPRL, particularly the Bloom filter method, is flourishing. Interest stems from two principal sources: at a technical level, by computer scientists and cryptographers with interests in information and data security, and at an operational level, by groups with interest in and responsibility for delivering record linkage services.Several groups are actively developing and refining PPRL methods at the scientific level including the German Record Linkage Center (University of Duisburg-Essen) , 45, theAt an operational level, PPRL featured prominently in the 2016 International Population Data Linkage Network Conference (Swansea University), with several presentations on the topic including a keynote session that described a collaboration between international research institutions in Canada, Australia, and Wales , 50\u201353.In addition to reducing the privacy risks associated with record linkage, the advent of PPRL protocols potentially heralds a new era of population-focused research using linked data, bridging gaps, and opening up opportunities for new and different forms of linkage-based research. PPRL methods may provide an avenue to access previously \u201chard to get\u201d datasets . PPRL methods may also provide a mechanism for accessing and integrating data from new and emerging sources. As well as data from new technologies , these new sources may include the private health sector that has, to date, had limited exposure to, and engagement with, data linkage frameworks , 55.New methods may require new or adjusted models of operation. Some custodians have expressed a desire to have flexibility in record linkage models to accommodate the features of different data collections . HoweverThe implementation of PPRL methods that do not require the release of personal information but protect privacy through other mechanisms  represents a breakthrough in record linkage, substantially reducing privacy risks without negatively impacting on linkage quality. By utilizing methods that do not require the release of personally identifying information, concerns regarding personal surveillance and government overreach can be allayed. Supplementing traditional linkage methods with PPRL methods will increase the number and type of datasets that can be included in record linkage studies.The advent of PPRL methods to protect patient privacy expands the toolkit of techniques that are available to DLUs. Used in conjunction with traditional linkage methods, PPRL widens the net of record linkage without compromising privacy or linkage quality. These methods will hopefully allow more diverse, patient-centered data sources to be utilized for health research, bringing enormous opportunities to increase our understanding of disease and to tailor interventions and treatment to each individual.AB and AF accept immediate responsibility for the manuscript. AF, AB, SR, JB, and JS each contributed to the conception and design of the paper. AF and AB drafted the first version of the article, with SR, JB, and JS providing important additional input and intellectual content. All authors were involved in revising the manuscript and approving its final form.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "Paralichthys lethostigma, historically support a substantial fishery along the Atlantic and Gulf coasts of the southern United States. Low year-class strengths over the past few years in the western Gulf of Mexico have raised concern that spawning stocks may be overfished. Current management of the resource includes releasing hatchery-raised juveniles to restock bays and estuaries; additionally, there is a growing interest in the potential for commercial aquaculture of the species. Currently, genomic resources for southern flounder do not exist. Here, we used two hatchery-reared families and double-digest, restriction-site-associated DNA (ddRAD) sequencing to create a reduced-representation genomic library consisting of several thousand single nucleotide polymorphisms (SNPs) located throughout the genome.Southern flounder, The relative position of each SNP-containing locus was determined to create a high-density genetic map spanning the 24 linkage groups of the southern flounder genome. The consensus map was used to identify regions of shared synteny between southern flounder and seven other fish species for which genome assemblies are available. Finally, syntenic blocks were used to localize genes identified from transcripts in European flounder as potentially being involved in ecotoxicological and osmoregulatory responses, as well as QTLs associated with growth and disease resistance in Japanese flounder, on the southern flounder linkage map.The information provided by the linkage map will enrich restoration efforts by providing a foundation for interpreting spatial genetic variation within the species, ultimately furthering an understanding of the adaptive potential and resilience of southern flounder to future changes in local environmental conditions. Further,\u00a0the map will facilitate the\u00a0use of genetic markers to enhance restoration and commercial aquaculture.The online version of this article (10.1186/s12864-018-4541-0) contains supplementary material, which is available to authorized users. Paralichthys lethostigma, is a left-eyed, large-tooth\u00a0flounder inhabiting bays and estuaries along the Atlantic coast of the United States from the Carolinas south through Florida and across the northern Gulf of Mexico (Gulf) to Tuxpan, Mexico; the species is notably absent along the southern Florida peninsula [Southern flounder, eninsula , 2. The eninsula , 4, thoueninsula , 5. Curreninsula , 7. It ieninsula , 9 may heninsula . In addieninsula .Genomic resources can be useful in ensuring success of stock enhancement and commercial aquaculture . Developseq), is an ideal approach for creating linkage maps because it allows for simultaneous discovery and genotyping of several thousand, single nucleotide polymorphisms (SNPs) across multiple individuals [Sequencing of reduced representation libraries, using restriction-site associated DNA sequencing  of shared synteny between southern flounder and seven fish species for which chromosome-level genome assemblies are available. The identified syntenic blocks were then used to map microsatellites used to identify QTLs for growth and disease/parasite resistance in Japanese flounder  and tranprocess_radtags [dDocent pipeline [1\u2009=\u20092, and K2\u2009=\u20091.A reduced-representation reference genome comprised of DNA extracted from 24 wild-caught southern flounder was assembled using sequence data from a ddRAD (double digest\u00a0RAD) library generated following . The lib_radtags , and refpipeline  for c\u2009=\u2009DNA was extracted from parents and offspring of two outbred crosses  reared at the CCA Marine Development Center (Texas Parks and Wildlife Department) in Corpus Christi, TX, using Mag-Bind Blood and Tissue DNA kits (Omega Bio-Tek). For each mapping cross, a male and female were strip-spawned. Fertilized eggs/larvae were reared in two separate tanks with standard light/dark cycle, temperature and access to food until they reached approximately 5\u201310\u00a0mm in length at which point they were removed from the tanks and placed in individual tubes with DMSO. CCA Marine Development Center routinely rears southern flounder for augmentation purposes and details of husbandry, spawning and collection procedures adhere to standard operating procedures for Texas Parks and Wildlife Department hatcheries , 38.process_radtags and quality trimmed. Read mapping and SNP calling were performed for each mapping cross, using the dDocent pipeline and the constructed reduced-representation reference genome. Raw SNPs were rigorously filtered using VCFtools [vcfallelicprimitives [rad_haplotyper, [One ddRAD library was constructed per mapping family, following , and seqVCFtools ; contigsimitives  and indelotyper,  was usedr/qtl [Onemap [LPmerge [r/qtl; loci with significantly distorted segregation patterns at the 5% level,\u00a0following Bonferroni correction for multiple comparisons, were removed from the data set. For sets of loci that had identical segregation patterns , only one locus was retained for mapping. Recombination fractions (rf) and log-odds (LOD) scores were calculated for each pairwise combination of the remaining loci; loci were then grouped into linkage groups based on a minimum LOD\u2009=\u20096 and a maximum rf\u2009=\u20090.35. After initial ordering, the quality of the loci, individuals, and locus order was assessed based on the presence of large gaps, excess numbers of crossovers per individual, and genotyping errors indicated by tight double-crossovers. After removing problematic loci and individuals, loci were re-ordered, and a sliding window used to compare alternate orders. The order with the lowest number of crossovers, highest likelihood, and resulting in the shortest chromosome was retained. The final order was reassessed based on the above parameters and sex-specific maps finalized by assigning all loci from co-segregating groups to the same location as the appropriate mapped locus.For each mapping family r/qtl  was used [Onemap  was used[LPmerge  to creatonemap which implements algorithms for simultaneous maximum-likelihood estimation of linkage and linkage phase in full-cross data sets [Family maps were created for each mapping cross, using loci mapped in male- and/or female-specific maps. Again, for sets of loci that had identical segregation patterns, only one locus was retained for mapping. Two-point recombination fractions were calculated between all pairs of loci, using ata sets . Loci weLPmerge was used to create a consensus linkage map by merging corresponding linkage groups from mapping crosses A and B, using linear programming to minimize the mean absolute error between the resulting consensus linkage map and the individual family maps.Before creating a consensus map, family maps were compared to identify incongruent ordering. Problematic loci always fell into a cluster of loci with zero observed recombination in at least one of the family maps. To resolve conflicts, these loci were removed from that family map. If a locus was in a cluster of loci with zero observed recombination in both maps, it was removed from the map with the larger cluster, and the remaining markers re-ordered and mapped. This process was repeated three times to ensure the best possible merging and ordering of loci, given the constraints of sample size and possible genotyping error. A total of 125 and 160 loci were removed from Family A and B maps, respectively, during this process to eliminate conflicts; they were not removed from the final consensus map. Finally, synteny_mapper pipeline [Paralichthys olivaceus, European seabass, Dicentrarchus labrax (assembly accession no. GCA_000689215.1), barramundi, Lates calcarifer (assembly accession no. GCA_001640805.1), three-spined stickleback, Gasterosteus aculatus (assembly accession no. GCA_000180675.1), Nile tilapia, (GCA_001858045.2) Oreochromis niloticus (assembly accession no. GCA_001858045.2), fugu, Takifugu rubripes (assembly accession no. GCA_000180615.2), and green spotted puffer, Tetraodon nigroviridis (assembly accession no. GCA_000180735.1), following [Paralichthys to more distantly related puffers), most of which have been successfully used in the past as comparison genomes for the synteny_mapper.pl pipeline [The pipeline  was usedollowing . This apollowing . Order mpipeline .Edwardsiella tarda [Two data sets containing sequences from expressed transcript studies of European flounder and three data sets from QTL studies of Japanese flounder were downloaded and synteny mapped as described in . In briela tarda .ggplot2 [circos [https://github.com/sjoleary/SFL_LinkageMap.Linkage maps and other figures were generated using ggplot2  and circ [circos . An exteParalichthys [The final reduced-representation reference genome consisted of 52,831 RAD fragments  totaling 14\u00a0Mb. Based on the range (500\u2013800\u00a0Mb) of genome sizes in three other species in the genus lichthys , 14\u00a0Mb rThe total number of SNP-containing loci mapped, total map length, mean number of loci, mean length per linkage group (LG), and mean distance between loci for linkage maps by family and by sex are summarized in Table\u00a0Syntenic blocks identified between the southern flounder consensus map and each of the sequenced fish genomes are shown in Fig.\u00a0https://github.com/sjoleary/SFL_LinkageMap/tree/master/results.Overall, 73% of the loci in the downloaded data set consisting of microsatellites and differentially expressed genes blasted against the seven genomes fell into synteny blocks and were then successfully mapped onto the southern flounder linkage map. The efficiency\u00a0of the synteny mapping approached varied by genome from approximately 12%\u00a0(green spotted puffer) to 67%\u00a0(Japanese flounder) of loci being successfully located. Over 68% of loci fell into homologous synteny blocks on more than one genome. Of the loci mapped by only\u00a0one genome, 48% were mapped using the Japanese flounder genome. A total of 1780 microsatellites from mapping studies of Japanese flounder , 29 wereComparative genomics is a powerful tool for transferring, integrating, and linking genomic information gathered from functional genomics, proteomics, and metabolomics between model and non-model species. For exploited and cultured non-model species, these approaches can be used to augment understanding of reproduction, growth, development, and disease resistance , 13, 50.seq-derived, SNP-containing loci allows generating high-density linkage maps without requiring a separate marker development step, making the procedure more time- and cost-efficient than previous approaches, e.g. microsatellites or RFLPs. By using paired-end sequencing, sequences of approximately 300\u00a0bp in length were incorporated into the map; thus\u00a0optimizing downstream comparative genomics [Creating a linkage map consisting of RADgenomics . To accogenomics , 41.In general, creating a consensus map from multiple mapping families increases the number of informative loci that can be successfully mapped , but comThe effectiveness of the comparative bioinformatic framework applied here varied depending on the comparison genome. The highest coverage of synteny blocks (and the largest degree of conservation of the location of the synteny blocks) was found between southern flounder and Japanese Flounder (46.7%), European seabass (43.6%), and barramundi (44.9%). However, coverage of syntenic blocks across all comparison species was 83%, emphasizing a benefit of using multiple, assembled genomes for synteny mapping. The success of identifying blocks of synteny between genomes and a linkage map is likely a function of multiple factors: quality of the sequenced genome, density of the linkage map, size of genomes available for comparison, number of chromosomes in comparison species relative to the target species, and phylogenetic relationships of comparison species relative to one another. Aside\u00a0from Japanese flounder (congeneric with southern flounder) European seabass and barramundi are related\u00a0comparatively to flounders , 60,\u00a0havEdwardseilla tarda, an enteric bacteria, has been implicated in mortality of several fishes undergoing culture, including flatfishes [in silico to guide experimental study design for characterizing genes that control traits of interest. Further, comparative approaches can be used to integrate and compare results of similar experiments performed in multiple species to understand shared genes/pathways and differences across taxa. Around 30% of all mapped transcripts from the studies of response to anthropogenic pollution and salinity changes in European flounder [Using synteny mapping, we localized five QTLs from Japanese flounder that were associated with resistance to viral/bacterial infection and to growth characters, as well as, transcripts from European flounder that were up and down regulated in response to a variety of anthropogenic pollutants found in the environment and to differences in salinity. Lymphocystis, a viral disease, has been found in wide range of cultured marine and freshwater fishes, including flounders , 62. Ideatfishes . The QTLatfishes .\u00a0All thrflounder , 33 wereflounder  and obseflounder , 67 thatOur study demonstrates that genetic information useful to culture and management of fish species can be compared and integrated among species by using linkage maps and synteny mapping in combination with high quality chromosome-level genome assemblies. A substantial amount of genomic resources are now available for cultured flatfishes, including chromosome-level\u00a0genome\u00a0assemblies , 69, traAdditional file 1:Detailed Methods & Materials. (DOCX 40\u00a0kb)Additional file 2:Filtering parameters for each filtering step, and number of SNPs and contigs remaining for Families A and B. (DOCX 15\u00a0kb)Additional file 3:Excel file containing a spreadsheet with locus, linkage group, and position for the consensus map, family map A and B, and sex-specific maps for male and females for each mapping family. (XLSX 251\u00a0kb)Additional file 4:Comparison of number of loci and linkage group length (in parentheses) for corresponding linkage groups for consensus map (LG), family maps , and sex-specific maps . (DOCX 16\u00a0kb)Additional file 5:Comparative view of location of syntenic blocks on consensus linkage map of southern flounder and European seabass. Solid black rectangles represent chromosomes of European seabass. Black ticks indicate the positions of loci mapped on southern flounder linkage groups (colored rectangles); loci mapped to the same location are stacked. Syntenic blocks are connected by ribbons; the color corresponds to the color of each linkage group. Width of the ribbon represents size of the syntenic block on a linkage group and its corresponding location on the chromosome of each comparison species. (PNG 3116\u00a0kb)Additional file 6:Comparative view of location of syntenic blocks on consensus linkage map of southern flounder and fugu. Solid black rectangles represent chromosomes of fugu. Black ticks indicate the positions of loci mapped on southern flounder linkage groups (colored rectangles); loci mapped to the same location are stacked. Syntenic blocks are connected by ribbons; the color corresponds to the color of each linkage group. Width of the ribbon represents size of the syntenic block on a linkage group and its corresponding location on the chromosome of each comparison species. (PNG 2296\u00a0kb)Additional file 7:Comparative view of location of syntenic blocks on consensus linkage map of southern flounder and barramundi. Solid black rectangles represent chromosomes of barramundi. Black ticks indicate the positions of loci mapped on southern flounder linkage groups (colored rectangles); loci mapped to the same location are stacked. Syntenic blocks are connected by ribbons; the color corresponds to the color of each linkage group. Width of the ribbon represents size of the syntenic block on a linkage group and its corresponding location on the chromosome of each comparison species. (PNG 584\u00a0kb)Additional file 8:Comparative view of location of syntenic blocks on consensus linkage map of southern flounder and nile tilapia. Solid black rectangles represent chromosomes of nile tilapia. Black ticks indicate the positions of loci mapped on southern flounder linkage groups (colored rectangles); loci mapped to the same location are stacked. Syntenic blocks are connected by ribbons; the color corresponds to the color of each linkage group. Width of the ribbon represents size of the syntenic block on a linkage group and its corresponding location on the chromosome of each comparison species. (PNG 2546\u00a0kb)Additional file 9:Comparative view of location of syntenic blocks on consensus linkage map of southern flounder and green spotted puffer. Solid black rectangles represent chromosomes of green spotted puffer. Black ticks indicate the positions of loci mapped on southern flounder linkage groups (colored rectangles); loci mapped to the same location are stacked. Syntenic blocks are connected by ribbons; the color corresponds to the color of each linkage group. Width of the ribbon represents size of the syntenic block on a linkage group and its corresponding location on the chromosome of each comparison species. (PNG 1925 kb)"}
+{"text": "Integrating medical data using databases from different sources by record linkage is a powerful technique increasingly used in medical research. Under many jurisdictions, unique personal identifiers needed for linking the records are unavailable. Since sensitive attributes, such as names, have to be used instead, privacy regulations usually demand encrypting these identifiers. The corresponding set of techniques for privacy-preserving record linkage (PPRL) has received widespread attention. One recent method is based on Bloom filters. Due to superior resilience against cryptographic attacks, composite Bloom filters  are considered best practice for privacy in PPRL. Real-world performance of these techniques using large-scale data is unknown up to now.Using a large subset of Australian hospital admission data, we tested the performance of an innovative PPRL technique (CLKs using multibit trees) against a gold-standard derived from clear-text probabilistic record linkage. Linkage time and linkage quality  were evaluated.Clear text probabilistic linkage resulted in marginally higher precision and recall than CLKs. PPRL required more computing time but 5 million records could still be de-duplicated within one day. However, the PPRL approach required fine tuning of parameters.We argue that increased privacy of PPRL comes with the price of small losses in precision and recall and a large increase in computational burden and setup time. These costs seem to be acceptable in most applied settings, but they have to be considered in the decision to apply PPRL. Further research on the optimal automatic choice of parameters is needed. Linking databases is a valuable and cost-effective technique, increasingly used in public health [In medical research, information on patients is often scattered across different databases of several data holders. The task of finding records referring to the same person across one or more datasets is, in medical contexts, denoted as c health , 2, offic health , 4, medic health , 5, pharc health  and democ health . Applicac health , increasc health  or mortac health .For many research endeavors, linking the information needed would be trivial if a unique personal identifier (PID) is available. However, in many settings, legal and administrative issues prevent the use of PIDs, restricting data linkage to personal identifiers such as names. Since this requires the release of personally identifying information to trusted third parties , privacyprivacy-preserving record linkage, allow linkages using encrypted identifiers. Although no personal identifying information is released by data custodians, record linkage is still possible.A number of new record linkage methods have been developed to overcome this problem at a technical level. These methods, known collectively as A summary of privacy-preserving record linkage techniques notes that each method differs in its accuracy, maturity, practicality and suitability for large-scale linkages . Few of Bloom filters to enable linkage [One notable method for privacy-preserving record linkage utilises  linkage . The Blo linkage . As encr linkage .Alternate methods of privacy-preserving record linkage using Bloom filters have been developed, with a single Bloom filter composed from many identifiers. Reasons for using only a single Bloom filter for linkage include legal constraints in some jurisdictions  and atteBoth of these composite Bloom filter methods have been shown to increase privacy by reducing the chance of a successful, malicious attack , 22. HowIn this paper, we test the accuracy and efficiency of the multibit tree technique on CLKs generated from large real-world medical data, for which the true links (which records belong to the same person) are already known. Testing multibit trees on real-world data is an important step in verifying its viability for linking record-level Bloom filters in public health settings.Ten years of Western Australian (WA) Hospital Admissions data, along with ten years of New South Wales (NSW) Admitted Patient Data were used in this evaluation. For each of these datasets, we had pre-existing and accurate information about which records belonged to which person.The datasets had been de-duplicated previously (by the WA Data Linkage Branch (WADLB)  and the A summary of these datasets can be found in Table Linkage quality was evaluated using pairwise precision, recall, and F-measure. Precision refers to the proportion of incorrect links found from all the found links and thus provides a measure of false positives. Recall is the proportion of all correct links found, and thus measures false negatives. The F-measure is the harmonic mean between precision and recall, giving a single figure from which we can compare results. These measures are widely used in the record linkage literature , 30.The CLK encryption method is based on the idea of hashing all available personal identifiers into a single structure called a Bloom filter (a binary vector). Each Bloom filters is used as an encrypted linkage key and can then be compared with other keys, resulting in a score which describes how similar the Bloom filters  are.Four different parameter sets were tested, which corresponded to different choices of personal identifiers to combine into each CLK, and are outlined in Table Consistent with the CLK construction method suggested by Schnell et al. , each daPairs of Bloom filters are compared using the Jaccard, or Tanimoto, similarity. The intersection of the bit positions set to one in both Bloom filters is divided by the union of the bit positions set to one in the two Bloom filters. This results in a similarity score between 0 and 1, where a higher score reflects a greater similarity measure: The desirable property of all Bloom filter-based encryptions is that they are similarity-preserving. This presents security considerations, as this property can be exploited to attack the encryption and potentially reveal personal identifiers. In recent years, several attacks have been published. The first attack, proposed by Kuzu et al. , revealeA second attack was devised by Niedermeyer et al.  and extesplit-half technique is repeated until a user-defined minimum number of records in each leaf is reached . For our experiments, a leaf limit of one was used.Searching for similar pairs is computationally expensive. To reduce the search space and thus improve linkage speeds, tree-based structures can be used for blocking. One prominent method is the use of multibit trees, as suggested by Kristensen et al.  and suggTo find similar pairs in terms of Tanimoto-similarity, every record in the second dataset is queried sequentially. For each record, an upper bound of the Tanimoto-similarity can be estimated before the actual similarity calculation, by comparing the values at the bit positions of each leaf in the tree. Leaves with a similarity under a user-defined Tanimoto threshold are disregarded in the calculation of the similarities. This way, the search space can be reduced drastically.For our de-duplication linkages, the same dataset was used for the multibit tree and for the sequential queries. We applied a construction method for multibit trees similar to Bachteler et al. , testingAll NSW and WA datasets were encrypted into CLKs for each parameter set as described above. For testing of linkage quality and blocking ability on data with few missing values, the WA CLK dataset was then de-duplicated, using multibit trees as the blocking method, at a range of Tanimoto thresholds. For testing of linkage quality on data with many missing values, a random sample of 5 million records was taken from the combined NSW CLK datasets using parameter set 1 . This represents a reasonable sample size for a real-world operation, the name identifiers resulting in approximately 30% missing values. The pair-wise precision, recall and F-measure scores were calculated by comparing results to the \u2018truth set.\u2019For testing of performance, the NSW  and WA CLK datasets were combined for a dataset with a total of approximately 20 million records. From this combined dataset, random samples were taken to create datasets of 5, 10 and 15 million records. All of these datasets were then de-duplicated, using multibit trees with a single Tanimoto threshold of 0.85, as this has previously been shown to be a reasonable value for most applications . The exeAll de-duplication linkages used multibit trees with a leaf limit value of one. The multibit tree outputs all candidate pairs, where the criterion for a pair is that it exceeds the given Tanimoto threshold value.The evaluation was run on a Windows Server 2012 R2 Virtual Machine, running under ESXi on a Cisco UCSC-C240-M3S Server with Intel Xeon CPU E5-2609@2.40GHz. The VM was assigned 48GB RAM and 6 vCPUs. The evaluation code was assigned 4 vCPUs.Results for the de-duplication of the WA CLK dataset can be found in Fig. Maximum F-measure varied considerably across the different parameter sets. Highest F-measure was 0.978 from parameter set 1 while lowest F-measure was 0.781 for parameter set 3. The inclusion of address information (parameter sets 2 and 3) tended to reduce overall scores. This can be explained by the varying recall: including addresses introduces unstable identifiers, which either differ in the datasets  or are missing. This will lead to a reduction in the amount of true pairs found, which is why sets 1 and 4 show superior linkage quality with respect to recall.All parameter sets but set 1 show high precision scores. Since adding middle names allows for better discrimination between records that would otherwise exhibit the same values across all identifiers, the amount of false positive classifications will decrease, leading to increased precision values for these parameter sets.The de-duplication linkage of the 5 million sample CLK dataset of the combined NSW Public and Private Hospital datasets  was abandoned after 2 weeks of elapsed execution time. Analysis of the pairs created to that point showed that the number of missing identifiers in the CLKs was leading to the creation of an inordinately large number of false positives; a large portion of rows with only values for date of birth and sex appeared to be linking to each other. The anticipated poor linkage results and excessive processing time led to the decision to abandon all linkage quality tests with this particular dataset.The time taken to complete the de-duplication of the samples of our combined dataset was a monotone function of the sample size see Fig. . The smaThe results in Fig. Overall, the use of CLK with multibit trees for a full linkage was not as high quality as could be achieved using either unencrypted linkage or with field level Bloom filters . Using tOur results show that the use of multibit trees for indexing/blocking of CLK data has great potential. The best recall was achieved using parameter set 1, with a value of 0.9858 at a threshold of 0.8. The unencrypted linkage on the same dataset, mentioned previously, had a recall of just 0.981, using standard blocking. The worst results for recall were for parameter sets 2 and 3, with values at all thresholds below 0.75. This is unacceptably low for any linkage, but the inclusion of all identifiers, especially with volatile address information, precludes the ability to match individuals that have changed their address. This shows, that while including more identifiers in the CLKs will usually increase the discriminative power, leading to higher precision, stable identifiers without missing data fields are needed in order to avoid sacrificing recall. While using multibit trees for indexing of CLK data has the ability for a very high coverage of possible links, its quality is ultimately determined by the identifiers used to create the CLK and the quality of the data.In terms of performance, the linkages were reasonably slow. While operational linkages are commonly performed on an ad-hoc basis, and there are tight processing deadlines to meet, linkages which take more than a few days processing time are probably not feasible. As such, the multibit tree method, as it is currently implemented, could not be recommended for large-scale linkages. As a comparison, an unencrypted linkage of the same 20 million records can be completed within a day.An alternate approach to using the multibit tree method may be to create a set of hashed blocking variables alongside the CLK, referred to as external blocking . Our simFurther testing is required to improve the CLK linkage results. One factor which is likely to improve results is the use of methods of weighting different personal identifiers based on how likely they are to identify an individual. The impact that a field has within a Bloom filter is directly proportional to how many bits that field encodes. However, in this paper, we used the baseline approach, where the number of bits was solely based on the number of bigrams in the identifier. For example, addresses usually contain many bigrams but are far less useful in identifying an individual over time when compared to date of birth or name. Testing Bloom filters which weight individual fields (by hashing bigrams more or less often) according to their usefulness in identifying individuals (discriminating power) may be an important avenue of further research.The results reported here are heavily dependent on parameter settings. For these methods to be useful in practice, where \u2018truth sets\u2019 are usually not available, tried and tested parameter settings that are robust across different kinds of datasets are required. Missing values were also shown to be a major factor affecting the quality of the indexing and linkage. Since CLKs do not account for the number of identifiers for which valid information is present, calculation of similarities based on CLKs will be attenuated by asymetrically missing identifiers. However, handling missing identifiers in PPRL is a largely unexplored field of research.Demand for privacy-preserving record linkage is increasing . SecuritHowever, very few techniques for PPRL suitable for large data sets are available. One of these few techniques are Bloom filter-based methods for PPRL. These methods are increasingly used for a wide variety of medical research projects, such as linking mammography data  or build"}
+{"text": "Bombus terrestris), we demonstrate that both diffraction grating and multilayer iridescence impair shape recognition , supporting the idea that both strategies can be effective means of camouflage. We conclude that iridescence produces visual signals that can confuse potential predators, and this might explain the high frequency of iridescence in many animal taxa.Iridescence is a taxonomically widespread and striking form of animal coloration, yet despite advances in understanding its mechanism, its function and adaptive value are poorly understood. We test a counterintuitive hypothesis about the function of iridescence: that it can act as camouflage through interference with object recognition. Using an established insect visual model ( Iridescence can be generated though a wide range of mechanisms, including thin-film interference, diffraction gratings, multilayers and photonic crystals6. However, the two most common mechanisms underlying iridescence in both animals and plants are multilayers and diffraction gratings7 and in all cases, the result of these optical phenomena is that the hue and intensity of the reflected light will vary depending on the angle of view or angle of illumination, as illustrated by the shimmering fruits of Pollia condensata, vivid green and orange breast feathers of the male Lawes\u2019s parotia (Parotia lawesii), a bird of paradise, the green and purple stripes of the Japanese jewel beetle (Chrysochroa fulgidissima), and the brilliant wings of the blue morpho butterflies (Morpho sp.)9.Iridescence is a form of structural coloration caused by interference of light reflected from nanostructures within a surface2. In species with strong sexual dimorphism, such as the Indian peafowl (Pavo cristatus), iridescence may serve an important function in mate choice12. However, it is also common in monomorphic animals, such as beetles, and in non-reproductive life stages such as butterfly chrysalises3. Here, the evolution of iridescence by sexual selection is a priori unlikely. Instead, the American artist and father of modern camouflage theory, Abbot Thayer, proposed that iridescence was an anti-predator adaptation. Thayer described his observations of iridescent insects: \u201cBrilliantly changeable or metallic colors are among the strongest factors in animals\u2019 concealment\u201d13 (p.87). He continued: \u201cEven without motion, the animal\u2019s surface, which would show all in its true place and plane if it were plainly colored, is by its iridescence made to appear \u2018dissolved\u2019 into many depths and distances\u201d.Due to its unique optical properties and striking appearance, iridescence in animals is, understandably, usually assumed to be a signal15. The latter may impede identification by predators searching for a typical shape: even if individual colour patches are detected, they are not recognised as belonging to a single object and so the prey remains unrecognised16. In perceptual terms, disruptive coloration interferes with feature binding17. In a similar way, iridescence creates changing colour and intensity boundaries, thereby disrupting the stable edge features normally used in object recognition: the brightness of iridescence may make (varying) parts of objects more conspicuous, but the changing colour patterns and boundaries could also deceive and confuse potential predators. This effect might be particularly acute in animals that lack the extensive upstream processing characteristic of the primate visual cortex.Thayer\u2019s statement is counter-intuitive: how can something that is both brilliant and changeable contribute to concealment? Camouflage generally works through matching the background, mimicking inedible objects (masquerade), or by utilizing colour patterns that break up the otherwise recognisable shape of a prey (disruptive coloration)Bombus terrestris as models for insect perception, we tested whether three forms of iridescence  impair shape recognition. Bees are clearly not predators, but other hymenopterans are predators and parasitoids of many insects; furthermore, much is known about the bee visual system20, and they can easily be trained to perform discrimination tasks21.Here, using artificial discs as prey and na\u00efve 21, we compared the recognition of oval and circular discs that displayed matte, floral diffraction grating iridescence, synthetic diffraction grating iridescence or multilayer iridescence on their surface  visits before undergoing the experiment. After conditioning, the bees returned to the hive while the arena and discs were cleaned with ethanol to remove scent marks. For the experiment, which took place within minutes after the conditioning phase, a conditioned bee was released back into the arena, where eight unrewarded discs  had been randomly positioned. A total of 20 visits to the discs was observed for each bee. At this stage a visit was defined as either landing with all feet on the discs or drinking. The shape of the disc was recorded for each visit .Using absolute conditioning\u03c72\u2009=\u200911.32, d.f.\u2009=\u20093, P\u2009=\u20090.0101). Tukey-type pair-wise comparisons showed that the bees had a stronger preference for this stimulus when targets were matte, compared to both the synthetic diffraction grating  and multilayer iridescent targets . However, there was no difference in discrimination when the matte and floral iridescent conditions were compared , nor synthetic diffraction grating vs multilayer , synthetic diffraction grating vs floral  or multi-layer vs floral . For matte , and floral iridescent targets  shape recognition was significantly higher than would be expected for random foraging, but this was not the case for targets with iridescence produced by synthetic diffraction gratings , or multilayer .There was a significant effect of treatment on the bees\u2019 ability to identify the previously rewarded stimulus  a colored disc made out of \u20182-Ton\u2019 epoxy  and mixed with \u201cultramarine blue\u201d color pigment , and (2) a sucrose well made of an upturned lid of 1.5\u2009ml Eppendorf containers and glued onto the disc.Following an established protocolWe used Elite HD\u2009+\u2009Light Body silicone dental mould  to make the negative copies of the target discs, with equal amounts of base and catalyst. The matte copy was made by gluing a paper disc  on top of a glass disc, and then gently push it down the dental mould with the paper-covered side of the glass disc faced downwards. The subtle \u201cfloral\u201d diffraction-grating iridescent copy was made by gently pushing down a tulip (\u201cQueen of the Night\u201d) tepal in the dental mould. To make perfectly regular iridescence diffraction grating discs, the dental mould was instead cast from 1000 lines per mm unmounted holographic diffraction grating film .When the silicone dental mould mixture had hardened, the specimen was removed, leaving an inverted mould. The positive replicas of the floral iridescent, diffraction grating iridescent and matte discs were then made of the specimen by using the \u20182-Ton\u2019 epoxy. Equal amounts of the resin and catalyst were combine with pigment  and mixed. The inverted silicone mould was then filled with the pigmented epoxy. Once the cast was fully hardened (overnight at room temperature) it was removed from the mould. The multilayer iridescent targets were made by adding a sheet of multilayer thin film  on top of the epoxy resin immediately after pouring it into a dental mould cast by a glass disc .\u03b8\u2009=\u20090\u00b0, and we also measured the targets, but not the background at \u03b8\u2009=\u200915\u00b0, 30\u00b0 and 45\u00b0 . A deuterium - halogen tungsten lamp  was used as a standardized light source, and measurements were taken using a premium-grade reflection probe . We used a Stanley 1-77-153 FatMax CL2 Cross Line Laser to set the axis of the illuminating and reflection probe so that it was perpendicular (i.e. 90\u00b0) to the plane of the targets and background (BG) for targets at et al.24, each colony of bees were housed in plastic nesting boxes connected to a flight arena via a transparent plastic tube. The tube was regulated by a series of gates such that we could control the bees\u2019 access to the flight arena. We used seven identical (l\u2009\u00d7\u2009w\u2009\u00d7\u2009h: 112\u2009\u00d7\u200975\u2009\u00d7\u200930\u2009cm) wooden framed flight arenas topped with UV-transparent acrylic (PerspexTM) sheet for these experiments. The floor of each flight arena was covered in green AT 202 Advance Gaffa tape . Illumination in the laboratory was provided by 46 Sylvania Activa 172 Professional 36\u2009W fluorescent tubes  powered by Philips high frequency-ballasts  with a flicker frequency higher than 20 KHz, on a 12:12\u2009hour light/dark schedule. Temperature was automatically regulated and held between 21\u201323\u2009\u00b0C. The bees were fed daily ad libitum with 30% sucrose solution and 3 times weekly with 15\u2009g of pollen/colony. In order to motivate the bees to forage from the targets during the behavioural experiments, the sucrose solution was provided using wells (1.5\u2009ml Eppendorf containers) fitted to epoxy targets of varying colours . The flowers were randomly distributed inside the flight arena and redistributed each day so that the bees wouldn\u2019t learn any specific positions of the flowers within the arena. For each behavioural experiment, a \u201crewarded\u201d target means that it contained a 30% sucrose solution, whereas an unrewarded target means that it contained only water. Targets that were re-used in experiments were always cleaned with pure ethanol , Sigma-Aldrich Ltd., St. Louis, Missouri, USA) to remove any possible scent marks from the bees25.Prior to the experiments, each bee  was individually marked using coloured Queen Marking Paints  for identification. Following the protocol of Whitney 26. To test for the effect of target type  on the response variable \u201clands on conditioned and unconditioned stimuli\u201d over the consecutive visits , we used Generalized Linear Mixed Models (function glmer in the package lme4)27 to model the binomial error distribution. Individual bumblebee (N\u2009=\u200960) was treated as a random effect. We then carried out pair-wise comparisons between treatment groups with Tukey-type adjustment for multiple testing (function glht in the multcomp package)28. Finally, we tested whether the probability of landing on the pre-conditioned stimuli deviated from that expected by random choice  for each treatment separately using Binomial GLMM\u2019s.All data were analyzed using R v. 2.13The dataset supporting this article will be made available from the Dryad Digital Repository upon acceptance."}
+{"text": "Linked datasets are an important resource for epidemiological and clinical studies, but linkage error can lead to biased results. For data security reasons, linkage of personal identifiers is often performed by a third party, making it difficult for researchers to assess the quality of the linked dataset in the context of specific research questions. This is compounded by a lack of guidance on how to determine the potential impact of linkage error. We describe how linkage quality can be evaluated and provide widely applicable guidance for both data providers and researchers. Using an illustrative example of a linked dataset of maternal and baby hospital records, we demonstrate three approaches for evaluating linkage quality: applying the linkage algorithm to a subset of gold standard data to quantify linkage error; comparing characteristics of linked and unlinked data to identify potential sources of bias; and evaluating the sensitivity of results to changes in the linkage procedure. These approaches can inform our understanding of the potential impact of linkage error and provide an opportunity to select the most appropriate linkage procedure for a specific analysis. Evaluating linkage quality in this way will improve the quality and transparency of epidemiological and clinical research using linked data. Errors in data linkage are a potential source of bias in results of studies using linked data, yet researchers using linked data often find it difficult to assess the extent of such bias, due to the separation of linkage and analysis processes.We describe three methods for evaluating data linkage quality and identifying potential sources of bias: applying the linkage algorithm to a subset of gold standard data to quantify linkage error; comparing characteristics of linked and unlinked data to identify potential sources of bias; and evaluating the sensitivity of results to changes in the linkage procedure.These methods are relevant, however and by whoever the linkage is conducted, and can provide a better understanding of the pattern of any bias, the extent to which linkage error may affect our results, and determinants of the amount of bias that is likely to be introduced.When linkage error is identified as a possible source of bias, methods to adjust for these biases should be used, which can help provide more robust results.,,Epidemiological and clinical research is increasingly based on datasets created by linking data from different sources such as administrative hospital datasets, clinical databases and national death registers.Studies that evaluate linkage quality are therefore often restricted to estimates of the match rate (the proportion of records that were linked), sensitivity (the proportion of true links that were detected), or positive predictive value (the proportion of detected links that were true), which can be obtained, for example, by comparing a linked dataset with a \u2018gold standard\u2019 or reference dataset where true match status is known.,There is a lack of guidance on how to explore the extent to which error impacts upon analysis, and this area has been identified as a priority for research.,Linkage error can occur in two ways: false matches and missed matches. False matches occur when records belonging to different individuals are erroneously linked together. False matches typically  add noise to estimates, diluting the association between variables captured in different datasets and biasing effect estimates towards zero.,An important further issue is that linkage errors do not always occur randomly, meaning that particular subgroups of individuals are often over- or under-represented amongst records affected by linkage error. Systematic reviews of studies comparing the characteristics of linked and unlinked records have identified that more vulnerable or hard to reach populations are often missed, with the probability of a missed match being associated with a range of characteristics including gender, age, ethnicity, deprivation and health status.,If unlinked records are to be excluded from analysis, selection bias (or collider bias) can occur if selection into the linked dataset is related to both an exposure and an outcome of interest.using a gold standard dataset to quantify false matches and missed matches;comparing characteristics of linked and unlinked data to identify potential sources of bias;using sensitivity analyses to evaluate how sensitive results are to changes in linkage procedure.The following sections describe three approaches to evaluating linkage quality  methods and probabilistic (or score-based) methods.,,If data are available where the true match status of each pair of records is known, these \u2018gold standard\u2019 data can be used to test linkage algorithms and estimate rates of linkage error. There are various ways in which gold standard datasets can be derived, for example from an additional data source with complete identifiers, from a subsample of records that have been manually reviewed or otherwise determined to be matches (or non-matches), or from a representative synthetic dataset (e.g. generated through simulating data).IJE online), the MIS-HES links provided a gold standard dataset that could be used to validate the same subset of births in the linked HES mother-baby cohort  within 15 English obstetric units for births between April 2012 and March 2013.y cohort .We compared linked records in the gold standard dataset (linked using direct patient identifiers including NHS number) with those in our HES mother-baby cohort (linked using indirect identifiers captured for birth and delivery records). To enable this comparison, we applied our original linkage algorithm to the same set of HES records captured in the MIS data. This allowed us to quantify the number of false matches or missed matches that our linkage algorithm produced, and to derive standard measures of linkage quality .The gold standard dataset comprised records for 72\u2009817 babies . Of thesThe low error rates observed in this evaluation demonstrate that the original linkage algorithm was highly accurate. We might therefore assume that the impact of linkage error will be negligible, since, for example, excluding such a small proportion of the target population is unlikely to affect the generalizability of results or to dramatically reduce precision. However, further evaluation is needed to assess whether selection bias could be present, e.g. if records from a particular subgroup were more likely to be missed, or whether the 0.9% of false matches could have introduced enough noise to bias results.In order to identify particular subgroups of records that are differentially missed during linkage, we can compare the characteristics of linked and unlinked records. This method of quality appraisal can only be implemented when all of the records in at least one of the files (or within the target sample within one of the files) are expected to link; it would not be useful, for example, when linking to a register of deaths to determine mortality, as there would be expected systematic differences between those who link and those who do not. This approach can be implemented if researchers have access to record-level or aggregate information on the characteristics of unlinked records.P-values for comparing unlinked and linked records. Standardized differences are calculated as the mean difference divided by the standard deviation, and can be easily calculated in statistical software packages (e.g. using the \u2018stddiff\u2019 command in Stata).,,Since data linkage studies are often characterized by large sample sizes, standardized differences can be more informative than Compared with true matches in the gold standard, the 297 records that failed to link (missed matches) and 636 baby records that linked to the wrong mother  were more likely to be: multiple births; or babies with lower gestational age, lower birthweight or more neonatal medical conditions; or babies born by caesarean section; or those of non-White ethnic background . RecordsThe results in this example indicate that although linkage error rates were low, there was still some potential for bias, as particular subgroups of records were more often affected than others. Whether these differences were large enough to introduce bias into results depends on the relationship between these variables and the parameters of interest. It is therefore helpful to explore how results of interest might change according to different levels of error.,In order to assess the sensitivity of results to different linkage procedures, we can perform sensitivity analyses, aiming to assess the extent to which results vary and the direction of likely bias. This can involve changing the linkage algorithm or varying the match weight threshold for probabilistic linkage, and re-running analyses to evaluate any impact on results.We conducted a sensitivity analysis to evaluate the mother-baby linkage by changing the threshold used in our linkage algorithm, and comparing results across different sets of linked records. We compared linkage results from our original probabilistic algorithm using a threshold weight of 20 for classifying records as links, with results from an algorithm that minimized false links by using a considerably higher threshold of 45. These thresholds were selected based on examination of the observed distribution of weights in our analysis; this distribution can differ substantially depending on the number and quality of matching variables, so thresholds are generally selected in the context of a specific linkage or analysis. The aim of this type of sensitivity analysis is to select thresholds that are likely to reflect plausible limits for the trade-off between false matches and missed matches. We also compared results with those from the initial deterministic linkage only, i.e. where records agreed exactly on hospital, maternal age, gestational age, baby\u2019s sex, birth order and GP practice code.As expected, increasing the match weight threshold in probabilistic linkage, or using deterministic linkage only, produced linkages that introduced fewer false matches but more missed matches . This isIJE online). Analysis was performed in Stata 14.We expected that impact of linkage errors in each of these linkage scenarios would depend on the research question, and therefore assessed four different outcomes: proportion of stillbirths; the proportion of preterm births (< 37 weeks of gestation); the association between neonatal survival to discharge and delivery risk factors; and the association between delivery risk factors and ethnic group. Odds ratios were estimated from logistic regression models, adjusting for a number of maternal and neonatal risk factors  of the data would be affected by missed matches. In particular, we expected that records of preterm births or stillbirths would be less likely to link than those of later gestations or live births, and that the ascertainment of these outcomes would therefore be lower in datasets more affected by missed matches.By comparing results across different linkage algorithms , we obseWe expected that statistical power would be affected either through missed matches (due to a reduction in the size of the study population) or a lack of precision introduced by false matches (leading to increased noise in the association between variables). Given the large sample size, we assumed that power implications would be most important for identifying associations with rare outcomes .As expected, we found that as the number of linked records decreased due to more missed matches at higher thresholds , there wWe expected that selection bias could be introduced if selection into the linked dataset were associated with both the outcome and exposure. For example, earlier comparisons between linked and unlinked data had indicated that that records for mothers with delivery risk factors were less likely to link, and also that mothers in the Black ethnic group were less likely to link. If this was the case, mothers with delivery risk factors who were successfully linked would be more likely to be from other ethnic groups . Conditioning on linked records could therefore induce a spurious protective relationship between Black ethnicity and delivery risk factors.In the gold standard data, we observed that 6.5% of mothers with delivery risk factors were from the Black ethnic group, whereas in the deterministic linkage only 4.7% of mothers with delivery risk factors were from the Black ethnic group. There was no true association between ethnicity and delivery risk factors. However, within the deterministically linked data there appeared to be a protective effect  are often only available to the data linkers and not to researchers.However, when gold standard datasets are not available, researchers can consider alternative approaches: comparisons of characteristics of linked and unlinked data, and sensitivity analyses. These methods can be easily implemented but require data linkers to provide information on the characteristics of unlinked records and/or on the quality of each potential link.Sensitivity analyses can be performed if measures of linkage certainty  are provided by data linkers alongside a linked dataset.In our example, we expected all babies to link with a mother, which made comparisons between linked and unlinked records easily interpretable and allowed us to directly estimate the proportion of missed matches. However, careful consideration needs to be given to appropriate reference populations when all records are not expected to link. For example, we would not expect all hospital records to link with a mortality record and vice versa; rather, a successful link indicates that an individual has died (\u2018informative linkage\u2019). If that is the case, comparing the characteristics of the individuals whose records were and were not linked would also be affected by differences in the groups for whom no linked records were available (i.e. the difference between those who died and those who survived). In such situations, external reference data  can allow us to assess how linkage rates might differ for different subgroups.,Further methods not covered in this article can also be used to evaluate linkage quality in the absence of a gold standard. For example, estimates of false match rates can be derived by applying linkage algorithms to records known to have no match .Evaluation of linkage quality can guide decisions about appropriate study design. For example, if linkage is used to identify individuals with a particular condition or disease (informative linkage), high levels of missed matches will lead to under-ascertainment, meaning that cohort study designs may be unsuitable . Where linkage rates are too low, researchers may conclude that linked data are not fit for these purposes. On the other hand, a case-control study may still be valid, whereby a high threshold is used to identify cases and a low threshold is used to identify controls (assuming no other biases are present).,An alternative, which still makes use of all available records, is to use multiple imputation to handle missing values due to unlinked or equivocal records.,In situations in which we have information about how linkage error affects the distribution of outcomes and exposures in our data, it may be possible to use well-established techniques for quantitative bias analysis, to adjust for these errors.,Studies of linked data are often based on administrative data that have not been collected primarily for research. In addition to linkage error, researchers should also consider other issues specifically relevant to these types of data , and explore methods to handle any potential bias that is identified.We describe three methods for evaluating linkage quality: applying the linkage algorithm to a subset of gold standard data to quantify linkage error; comparing characteristics of linked and unlinked data to identify potential sources of bias; and evaluating the sensitivity of results to changes in the linkage procedure. These methods are generalizable to many other linkage situations and can be used as a guide for evaluating the quality of linkage for population-based analyses of linked data. Researchers using linked data should collaborate with data providers to understand the data linkage process, including data extraction and cleaning, linkage methods and resulting data quality.IJE online.This work was supported by the Wellcome Trust [grant number 103975/Z/14/Z]. JvM is supported by the NIHR CLAHRC North Thames. The work was also supported by the Economic and Social Research Council through the Administrative Data Research Centre for England (ES/L007617/1).Supplementary DataClick here for additional data file."}
+{"text": "Populus species. However, there is also the need for high-density genetic linkage maps for the European aspen (P. tremula) as a tool for further mapping of quantitative trait loci (QTLs) and marker-assisted selection of the Populus species native to Europe.Restriction-site associated DNA sequencing (RADseq) technology was recently employed to identify a large number of single nucleotide polymorphisms (SNP) for linkage mapping of a North American and Eastern Asian P. trichocarpa. 2055 SNPs were employed for the construction of maternal and paternal linkage maps. The maternal linkage map was assembled with 1000 SNPs, containing 19 linkage groups and spanning 3054.9\u00a0cM of the genome, with an average distance of 3.05\u00a0cM between adjacent markers. The paternal map consisted of 1055 SNPs and the same number of linkage groups with a total length of 3090.56\u00a0cM and average interval distance of 2.93\u00a0cM. The linkage maps were employed for QTL mapping of one-year-old seedlings height variation. The most significant QTL (LOD\u00a0=\u00a05.73) was localized to LG5 (96.94\u00a0cM) of the male linkage map, explaining 18% of the phenotypic variation.We established a hybrid F1 population from the cross of two aspen parental genotypes diverged in their phenological and morphological traits. We performed RADseq of 122\u00a0F1 progenies and two parents yielding 15,732 high-quality SNPs that were successfully identified using the reference genome of P. tremula intra-specific cross will provide a valuable source for QTL mapping and identification of candidate genes facilitating marker-assisted selection in European aspen.The set of 15,732 SNPs polymorphic in aspen and high-density genetic linkage maps constructed for the The online version of this article (10.1186/s12870-017-1127-y) contains supplementary material, which is available to authorized users. Populus species are the fast-growing trees appreciated worldwide due to their rapid growth and quality of wood, which is amenable to a variety of mechanical and chemical processing. They are of particular importance for the pulp and paper industries and the fuel and energy sectors, providing valuable raw materials in a short time. In Europe, the most frost-resistant and productive Populus species that has adapted to unfertile and acidic soils is aspen (Populus tremula L.) 7P. tremuP. tremula and P. trichocarpa, our results suggest the presence of several chromosome inversions between the two taxonomically remoted Populus species. P. tremula belongs to the section Populus (syn. Leuce Duby), whereas balsam poplars P. trichocarpa and P. simonii are from the same section Tacamahaca Spach. Even for P. simonii genetic maps, several linkage groups exhibited inverse orders of some SNPs relative to the P. trichocarpa genome [P. deltoides and P. simonii one or more local regions have been reported where SNP order was inconsistent with reference genome positions [P. tremula  chromosome inversions could be exposed relatively to the SNP marker order on P. trichocarpa chromosomes. An inversion occurs when a chromosome breaks at two points and the segment bounded by the breakpoints is reinserted in the reversed orientation [P. tremula with the reference genome of P. trichocarpa would make it possible to determine whether the observed inconsistency in the order of SNP markers on two genomes is due to the inversion of chromosomes, rather than the distortion of the genetic map.Despite the highly conserved synteny and collinearity observed between SNP order on the chromosomes of a genome . Moreoveositions . We hypoentation . A key eentation . The comP. tremula, a plant that has the widest-known geographic distributions of any tree species. The SNPs were obtained by RADseq genotyping of two parental aspen trees and their 122\u00a0F1 progenies followed by mapping of 20.17 million Illumina reads to the P. trichocarpa reference genome. 2055 of the SNP markers were successfully employed for the construction of high-resolution maternal and paternal linkage maps of the intraspecific aspen cross. The constructed linkage maps were successfully employed for mapping of QTLs related to plant height variation in the full sibling family. The high resolution linkage map and the set of SNP markers will provide a valuable source for QTL mapping and association studies in P. tremula facilitating marker-assisted selection in European aspen.In the present study we describe a set of 15,732 SNPs polymorphic within Additional file 1: Figure S1.Figure S2. Two-weeks-old F1 hybrid aspen seedlings placed in lining-out nursery. (PPTX 1375\u00a0kb)Germination of hybrid seeds (F1) obtained via artificial crossing of two parental aspen genotypes. Additional file 2: Figure S3.P. trichocarpa reference genome. (PDF 391\u00a0kb)Information about 138 sequenced ddRAD libraries and reads successfully mapped to the Additional file 3: Figure S4.Individual ancestry of 122\u00a0F1 aspen offspring estimated by maximum likelihood method based on 16,234 SNPs with ADMIXTURE software. (JPEG 1846\u00a0kb)Additional file 4:List of 15,732 robust SNPs available for aspen genotyping. (TXT 8857\u00a0kb)Additional file 5:P. tremula intra-specific cross. (XLSX 427\u00a0kb)Detailed information on genetic distances and linkage phase between adjacent SNP markers in the maternal linkage map constructed for Additional file 6:P. tremula intra-specific cross.  (XLSX 463\u00a0kb)Detailed information on genetic distances and linkage phase between adjacent SNP markers in the paternal linkage map constructed for Additional file 7: Figure S5.Figure S6. Chi-square goodness of it test for Normality of seedling height values among the 122\u00a0F1 progenies derived from P. tremula intra-specific cross. (PPTX 939\u00a0kb)Evaluation of one-year-old seedling height variation before the plants were planted in the field."}
+{"text": "Electrocardiographic measures of left ventricular hypertrophy (LVH) are used as predictors of cardiovascular risk. We combined linkage and association analyses to discover novel rare genetic variants involved in three such measures and two principal components derived from them.The study was conducted among participants from the Erasmus Rucphen Family Study (ERF), a Dutch family-based sample from the southwestern Netherlands. Variance components linkage analyses were performed using Merlin. Regions of interest (LOD\u2009>\u20091.9) were fine-mapped using microarray and exome sequence data.We observed one significant LOD score for the second principal component on chromosome 15 (LOD score\u2009=\u20093.01) and 12 suggestive LOD scores. Several loci contained variants identified in GWAS for these traits; however, these did not explain the linkage peaks, nor did other common variants. Exome sequence data identified two associated variants after multiple testing corrections were applied.MAPK3K11 on chromosome 11 (MAF\u2009=\u20090.01) that helped account for the suggestive linkage peak observed for the first principal component. Conditional analysis revealed a drop in LOD from 2.01 to 0.88 for MAP3K11, suggesting that this variant may partially explain the linkage signal at this chromosomal location. MAP3K11 is related to the JNK pathway and is a pro-apoptotic kinase that plays an important role in the induction of cardiomyocyte apoptosis in various pathologies, including LVH.We did not find common SNPs explaining these linkage signals. Exome sequencing uncovered a relatively rare variant in The online version of this article (10.1186/s12920-018-0339-9) contains supplementary material, which is available to authorized users. Left ventricular hypertrophy (LVH) is a predictor of increased cardiovascular morbidity and mortality . Those wPTGES3 and NMB, reached genome-wide significance. IGF1R and SCN5A were identified and replicated without reaching genome-wide significance [Genome-wide linkage analyses, candidate gene association studies, genome-wide association studies (GWAS) and gene mapping have been conducted to identify genes influencing LVH. In the first GWAS of these traits, two loci, ificance . Among tificance  and chroificance , 12 wereExome sequencing has been successfully used for Mendelian disorders . More re2) was computed. Blood pressure was measured twice on the right arm in a sitting position after at least 5\u00a0min rest, using an automated device . The average of the two measures was used for analysis. Hypertension status was identified through the use of antihypertensive medication and/or through the assessment of blood pressure measurements according to the guidelines of the World Health Organization [The ERF study is a family-based study including over 3000 participants descendant from 22 couples that lived in the Rucphen region in the southwest Netherlands in the nineteenth century . All desnization . The MedExaminations included 12-lead ECG measurements. A 10\u00a0s 12-lead ECG  was recorded with an ACTA-ECG electrocardiograph  with a sampling frequency of 500\u00a0Hz. Digital measurements of the ECG parameters were made using the Modular ECG Analysis System (MEANS) , 19. BriMEANS was used to measure QRS complex duration and the three LVH proxies. Sokolow-Lyon was defined as the sum of the S wave in V1 plus the R wave in V5 or V6, Cornell as the sum of R in aVL and S in V3, and 12-lead as the sum of R to S in all 12 leads; these three voltages were then multiplied by QRS duration to obtain voltage-duration products as an approximation of the area under the QRS complex \u201323. Prin6\u00a0K Illumina Linkage IV Panels\u00ae) was used for genotyping for the linkage analyses. All genotyping procedures were performed according to the manufacturer\u2019s protocols. Only markers with a minor allele frequency (MAF)\u2009>\u20090.05 were selected for further analysis. Genotyping errors leading to Mendelian inconsistencies were detected using PedCheck [Illumina\u2019s HumanHap6k Genotyping BeadChip  around the linkage peaks. Genes within the LOD-2 SI were annotated using SCAN (SNP and CNV Annotation Database).P-value >\u200910\u2212\u20096 were used for imputations. To account for relatedness, a genomic kinship matrix was computed in GenABEL [P-values were adjusted with the FDR-based q-value technique [Of 2385 phenotyped people, dense genotypes were available for 2128 subjects, typed on 3 different genotyping platforms , which were merged first  and then ~\u20092.54 million SNPs were imputed using MACH (v1.0.16) , with th GenABEL . This mahttp://broadinstitute.github.io/picard/) to remove systematic biases and to recalibrate the PHRED quality scores in the alignments. Genetic variants were called using the Unified Genotyper tool of the GATK. About 1.4 million Single Nucleotide Variants (SNVs) were called and, after removing the low quality variants (QUAL <\u2009150), we retrieved 577,703 SNVs in 1309 individuals. ECG and covariate data were available for 1072 of these samples. Further, for comparison and to predict the functionality of the variants, annotations were also performed using the dbNSFP  and Seattle (http://snp.gs.washington.edu/SeattleSeqAnnotation138/) databases. These databases gave functional prediction results from four different programs, PolyPhen-2, SIFT, MutationTaster and LRT, apart from gene and variant annotations.The exomes of 1336 individual from the ERF population were sequenced \u201cin-house\u201d at the Center for Biomics of the Department of Cell Biology of the Erasmus MC, the Netherlands, using the Agilent version V4 capture kit on an Illumina HiSeq 2000 sequencer using the TruSeq Version 3 protocol. Mean depth base was 74.23\u00d7 (median\u2009=\u200957\u00d7) and mean depth region was 65.26\u00d7 (median\u2009=\u200952.87\u00d7). The sequence reads were aligned to the human genome build 19 (hg19) using BWA and the NARWHAL pipeline . The aliP-value\u2009=\u20094.9\u2009\u00d7\u200910\u2212\u20094), 98 for CV  and 60 for 12 LS . For the PCs, the numbers were 141 for PC1  and 71 for PC2 .We employed a Bonferroni correction for the number of deleterious mutations selected for each trait to correct for multiple comparisons in the exome data: 101 for SL  were selected for replication in the Rotterdam Study (RS). The Rotterdam Study is a prospective cohort study ongoing since 1990 in the city of Rotterdam in the Netherlands [P-value <\u200910\u2212\u20096) were also removed from the data. The final dataset consisted of 635,814 SNVs in 1450 individuals with complete phenotype and covariate data.Exomes from 1764 individuals from the RS population were sequenced at an average depth of 20\u00d7 using the Nimblegen SeqCap EZ V2 capture kit on an Illumina HiSeq 2000 sequencer and the TrueSeq Version 3 protocol. The sequence reads were aligned to hg19 using BWA. Subsequently, the aligned reads were processed further using Picard, SAMtools and GATK. Genetic variants were called using the Unified Genotyper Tool from GATK. Samples with low concordance to genotyping array (<\u200995%), low transition/transversion ratio (<\u20092.3), high heterozygote to homozygote ratio (>\u20092.0) and low call rate (<\u200980%) were removed from the data. SNVs with a low call rate (<\u200990%) and out of HWE (P\u2009<\u20091\u2009\u00d7\u200910\u2212\u20096), call rate (<\u200998%), MAF (<\u20090.01), and Mendelian errors (>\u2009100), MACH was used to perform the imputations.One SNP, rs139580877, was not available in the Rotterdam Study exome data. This variant was imputed using the GIANT 1000 Genomes Phase I Version 3 All reference panel, as previously described . In brieP-values and MAF for each SNP. None achieved statistical significance after correction for multiple comparisons.Table\u00a0P-value \u22640.05 after regressing out the effects of age, BMI, height and sex. This effort uncovered an A\u2009>\u2009G variation (rs139580877) in the SPEF2 gene on 5p13.2, which was significantly associated with 12LS when adjusted for multiple testing . This variant, with 108 carriers in ERF, is predicted to be probably damaging by PolyPhen-2 with a score of 0.972 and as deleterious by SIFT with a score ranging between 0.02 and 0.03. It is a missense variant, among more than 2000 described for this gene. In the principal components analysis, rs138968470, on 11q13.1 in the MAP3K11 gene, was associated with PC1 adjusted for multiple testing . SKAT-O and burden tests provided some supporting evidence for the association of this gene with LVH proxy measures . A second, more common intragenic variant inside PRSS12 was nominally associated . We re-ran the linkage analyses conditioning on these variants to see if they explained the observed linkage signals. For PC1, the LOD score in the 11q13.4 linkage region dropped in the conditional analysis (from 2.01 to 0.88), suggesting that the associated variant (rs138968470), or neighbouring variants in linkage disequilibrium (LD), explained the linkage signal. This variant also showed evidence of association with the two traits (12LS and SL) underlying PC1 . Using Gene Network (http://genenetwork.nl/gene/ENSG00000173327), to perform in-depth analyses of the expression of MAP3K11, demonstrated that its expression is strongly linked to rho signalling  . There was no evidence of association for any of these variants in the Rotterdam Study.Summary statistics for the Rotterdam Study sample are provided in Additional\u00a0file\u00a0MAP3K11 on 11q134 for PC1; the MAP3K11 variant substantially decreased the LOD score for this peak. The 24 carriers of this missense mutation clustered into five pedigrees in the ERF population  and 10 suggestive regions . Exome variant analysis in these regions uncovered a missense coding variation in 5q11.2 anMAP3K11 gene, associated with PC1. Conditional linkage analysis, including the MAP3K11 variant, reduced the LOD score (from 2.01 to 0.88), suggesting that this variant largely explained the linkage signal at this chromosomal location. The SNP is located in the first exon of a gene encoding a protein that belongs to the serine/threonine kinase family of mitogen-activated protein kinases. MAP3K11 ) [MAP3K11 may be related to regulation of JNK and the subsequent JNK controlled pathway.Genetic variants discovered by GWAS, based on individual single-nucleotide polymorphisms (SNPs), explain only a small proportion of the heritability of complex traits , 39, 40; (MLK3)) , works a (MLK3)) . MAP3K11 (MLK3)) . JNK, an (MLK3)) . Apoptos (MLK3)) . In thisPEF2), which has been postulated to play an important role in spermatogenesis and flagellar assembly [PRSS12 gene, underlying the SL locus on chromosome 4, which approached significance , but did not replicate in the Rotterdam Study. Absence of replication could be related to imputation quality for rs139580877 and the low number of carriers for the other SNPs  linked to LVH, for which there are a number of potential explanations. Linkage peaks are not precise in highlighting the location of the causal variant; even the region of interest cannot be easily pinpointed. Additionally, we did not take into account alternative mechanisms, such as structural and copy number variations (CNVs) or repeats in the linkage regions. Lastly, causal rare variants may be located outside the coding sequence, which we did not include in our sequencing analyses.MAP3K11 (11q13) in LVH through the regulation of JNK. However, we cannot exclude the presence of other variants that are in linkage disequilibrium with the MAP3K11 variant (rs138968470) that might explain the observed association.In conclusion, 13 loci were identified for ECG LVH proxy measures and PCs using linkage analysis in a large pedigree; these were subsequently fine-mapped with microarray and exome sequence data. Common variation from the microarrays did not explain these peaks. The exome data, though, suggested the involvement of Further analysis will need to be performed to demonstrate the involvement of this protein in LVH. A number of other suggestively linked peaks were determined. We could not explain these with microarray or exonic sequence variants at present, asking for more extensive follow-up outside the coding regions.Additional file 1:Table S1. Coding variants under the linkage peaks for LVH proxy measurements. Table S2. Selected damaging variants in the coding regions contained in the linkage regions. Table S3. SKAT and burden tests for genes of interest. Table S4. Results of linkage analyses before (LOD1) and after (LOD2) regression on GWAS SNPs under the linkage peaks. Table S5. Descriptive statistics of the Rotterdam study population. Table S6. Replications results in the Rotterdam Study. Figure S1. Venn diagram showing the overlap between the different ERF genotyping experiments. Figure S2. Pedigrees segregating rs138968470. (DOCX 119\u00a0kb)"}
+{"text": "Takifugu rubripes) as a test model, we propose a new strategy for ultrahigh-density genetic linkage map construction using low-coverage whole-genome sequencing of a haploid/doubled haploid (H/DH) population without above requirements. Low-coverage (\u22481\u00d7) whole-genome sequencing data of 165 DH individuals were used for de novo assembly and further performed single nucleotide polymorphisms (SNPs) calling, resulting in the identification of 1,070,601 SNPs. Based on SNP genotypes and de novo assembly, genotypes were associated with short DNA segments and an ultrahigh-density linkage map was constructed containing information of 802,277 SNPs in 3090 unique positions. Comparative analyses showed near-perfect concordance between the present linkage map and the latest published torafugu genome (FUGU5). This strategy would facilitate ultrahigh-density linkage map construction in various sexually reproducing organisms for which H/DH populations can be generated.Next-generation sequencing enables genome-wide genotyping of a large population and further facilitates the construction of a genetic linkage map. Low-coverage whole-genome sequencing has been employed for genetic linkage map construction in several species. However, this strategy generally requires available high-quality reference genomes and/or designed inbred pedigree lines, which restrict the scope of application for non-model and unsequenced species. Here, using torafugu ( A genetic linkage map is a powerful tool in genetic and genomic research. It lays a strong foundation for comparative genomics and provides vital clues toward understanding genome evolution and divergence ,2,3,4. MOwing to the rapid development of next-generation sequencing (NGS) in the last decade, the ability to simultaneously sequence a large number of individuals in a multiplex manner has now become possible, so that an entire population can be rapidly genotyped for linkage mapping . Since 2Takifugu rubripes) through gynogenesis.To resolve this issue, we hypothesized that an ultrahigh-density genetic linkage map could be constructed using low-coverage whole-genome sequencing of haploid/doubled-haploid (H/DH) population without requiring a high-quality reference genome and the laborious establishment of inbred lines. H/DH individuals can be generated via natural or artificial uniparental reproduction, which is found in a wide range of species in several kingdoms. Indeed, in recent years, the H/DH population has been exploited as an ideal population type for genetic linkage map construction, particularly in plants ,18,19,20Torafugu is a popular species with economic importance in the waters of East Asia, and has emerged as an ideal model in genomic studies owing to its compact genome . In factBased on the proposed strategy, an ultrahigh-density linkage map of torafugu was constructed. The accuracy of the obtained linkage map was validated with comparison to the published genome FUGU5. The proposed strategy represents a cost-effective and less complex tool for genetic linkage map construction and can be widely applied in a wide diversity of sexual organisms, especially non-model and unsequenced species, for which H/DH populations can be generated.2. After fertilization for 3 h, the eggs were subjected to 45 min of cold-shock treatment at 0.6 \u00b0C, followed by incubation in aerated tanks with fresh seawater at 18.0 \u00b0C. Several days after artificial insemination, hundreds of eggs were observed to contain embryonic bodies, which were selected for further analysis.A wild female torafugu was purchased from a market in Akita Prefecture and was subjected to mito-gynogenesis for generating a DH population according to the process described in detail in our previous paper . In brieGenomic DNA was extracted from each of the selected 192 eggs using Agencourt DNAdvance Kit  after homogenization. An average of 125 ng DNA was obtained from each sample. DNA libraries of these individuals were prepared and barcoded according to the Nextera DNA Library Prep Reference Guide  and were then subjected to sequencing in two lanes of Illumina HiSeq 2000 system. A total of 74.43 Gb of sequencing data, consisting of 2 \u00d7 100-bp paired-end reads with an average insert size of 230 bp, were obtained from 192 samples of the generated DH torafugu population. Potential remnants of adapter sequences were removed, low-quality bases with a Phred quality score below 20 were trimmed, and the 23 samples with very low sequencing coverage were removed. After these processes, a total of 71.32 Gb of sequencing data from 169 samples were ultimately reserved and applied to further analysis.The obtained sequencing data were utilized to perform de novo assembly on the SOAPdenovo2  assembleOwing to the low-coverage (\u22481\u00d7) sequencing, most of the SNPs in each sample were detected once or less, which led to a large quantity of missed genotypes and insufficient data for genotyping calibration. As shown in We used a modified version of an approach designed for phase-unknown genetic linkage mapping in ants to construct a genetic linkage map using the phase-unknown SSGs, described in brief as follows . In the \u2212100 and identity of at least 95%. Genomic synteny was visualized using CIRCOS 0.69 software [\u221226.The sequences of the SSGs located on the genetic linkage map obtained with the strategy outlined above were aligned to the published torafugu genome (FUGU5) using BLAST (version 2.2.29) with an e-value cut-off of 1 \u00d7 10software . The flaIn total, 69.58 Gb of sequencing data from 165 samples of the generated DH torafugu population were used to perform de novo assembly. The total sequencing data coverage was 174, whereas the average coverage for each sample was approximately 1. After performing de novo assembly using the sequencing data, a relative low-quality assembly of a total size of 356.59 Mb and N50 size of 22,235 bp was generated, which was composed of 54,127 scaffolds with the length ranging from 200 to 264,568 bp. After SNP calling from the sequencing data of each DH individual, a total of 1,070,601 SNPs were discovered in the population using the above de novo assembly as reference. Despite the existence of a large quantity of missed genotypes and insufficient data for genotyping calibration due to the low-coverage (\u22481\u00d7) sequencing of each sample, the genotypes of adjacent SNPs could be testified/compensated by each other based on the above de novo assembly. Therefore, as shown in An ultrahigh-density genetic linkage map  was consThe sequence information of the 37,343 SSGs of the linkage map obtained with the proposed strategy was subjected to BLASTN analyses against the latest published genome FUGU5. Overall, 31,822 SSGs could be mapped to the 22 chromosomes of FUGU5. As shown in We successfully developed an effective strategy for the construction of an ultrahigh-density genetic linkage map of torafugu based on low-coverage (\u22481\u00d7) whole-genome sequencing of each individual of a DH population generated through mito-gynogenesis. The sequencing data were used for de novo assembly and further SNP calling to generate a low-call-rate SNP dataset with unknown phase. Based on the relatively low-quality de novo assembly, an SSG was designed as a high-call-rate genetic marker to assign a genotype to a short DNA segment after combing the information of its constituent low-call-rate SNPs. The high-call-rate SSG dataset enabled the construction of an ultrahigh-density genetic linkage map containing most of the information of SNPs (sub-million in this case) of the mapping population. The accuracy of the present linkage map was verified by subsequent analyses of recombination fractions and assessment of LOD scores for all marker pairs, along with comparative analyses between the linkage map and FUGU5. In addition, integration with the present linkage map allowed for validation and further refinement of FUGU5. Based on these indicators, an improved genome assembly of torafugu will be achieved in our future work.In the whole-genome assembly project, de novo assembly and linkage map construction are independent works contributing to chromosome-scale assembly. However, in our case, both of them can be achieved from the low-coverage whole-genome sequencing of the mapping population. Thus, for a non-sequenced species, de novo assembly, linkage map construction, and further chromosome-scale assembly can be efficiently completed by adopting our strategy.The strategy of the present study was developed for genetic linkage map construction based on an H/DH dataset with phase-unknown format. This strategy would be ideally implemented for various types of sexually reproducing organisms that could be used to generate large numbers of H/DH individuals, especially plants, teleosts, and fungi, without requiring other complex crossing schemes and designed inbred pedigree lines to identify the linkage phase. Notably, our strategy based on SSG could be applied to the construction of an ultrahigh-density genetic linkage map using only single gamete cells, the ubiquitously existing haploids, combined with single-cell sequencing technology, which further extends the application range. One of the most important advantages of our strategy is that it does not require the parental genetic phases and/or a high-quality reference genome, which are necessary for existing single-gamete sequencing strategies ,40,41,42The lack of a requirement of a high-quality reference genome for low-coverage whole-genome sequencing expands the application of our proposed strategy to a wide range of non-model and non-sequenced species. Moreover, our approach has an advantage of simplicity, in that whole-genome sequencing was applied to each individual sample, whereas existing techniques such as specific-locus amplified fragment sequencing (SLAF-seq) , reduced"}
+{"text": "SH3GL2 (Src homology 3 (SH3) domain GRB2\u2010like 2) is mainly expressed in the central nervous system and regarded as a tumour suppressor in human glioma. However, the molecular mechanism of the SH3GL2 protein involved in malignant behaviours of human glioma has not been elucidated. In this study, we tried to investigate the role of SH3GL2 in glioma cell migration and invasion and explore its underlined molecular mechanism. Firstly, we discovered that the protein level of SH3GL2 was widely decreased in the human glioma patients, especially in high\u2010grade glioma tissues. Then, we determined the role of SH3GL2 in migration and invasion of glioma cells upon SH3GL2 knocking down and overexpressing. It was showed that knocking down of SH3GL2 promoted the migration and invasion of glioma cells, whereas overexpression of SH3GL2 inhibited them. Further study on molecular mechanism disclosed that silencing of SH3GL2 obviously activated the STAT3  signalling thereby promoting the expression and secretion of MMP2. On the contrary, overexpression of SH3GL2 had opposite effect. Taken together, the above results suggest that SH3GL2 suppresses migration and invasion behaviours of glioma cells through negatively regulating STAT3/MMP2 signalling and that loss of SH3GL2 may intensify the STAT3/MMP2 signalling thereby contributing to the migration and invasion of glioma cells. Glioma is the most universal type of primary intracranial tumours et al. in 1996 and mapped to chromosome 9p22 SH3GL2 gene has nine exons and encodes a 352 amino acid protein also named Endophilin\u20101 The SH3GL2 gene was cloned by Sparks STAT3 is abnormally activated in glioblastoma and has been considered as a valuable therapeutic target in this disease and numerous other human cancers via a STAT3\u2010binding element Recent studies have suggested that STAT3 enhances MMP2 expression by directly interacting with the promoter of MMP2 In this study, we firstly examined the protein expression of SH3GL2 in glioma patients and glioma cell lines by Western blotting and immunohistochemistry. Then, the role of SH3GL2 in the migration and invasion glioma cells was investigated through silencing or overexpressing approaches. Finally, we studied the effect of SH3GL2 on STAT3/MMP2 signalling.SH3GL2 antibody was purchased from Abcam . Antibodies specific for MMP2, STAT3, p\u2010STAT3, FLAG and \u03b2\u2010actin were purchased from Cell Signaling Technology ; SU9516 , a potent and selective CDK2 inhibitor, was purchased from Tocris Bioscience .Thirty\u2010three specimens of human glioma tissues  and nine specimens of non\u2010tumorous brain tissues  were collected at the Affiliated Hospital of Xuzhou Medical University . All glioma specimens had confirmed pathological diagnosis and were classified according to the World Health Organization (WHO) criteria. Written informed consent was obtained from each patient, and the study was approved by the Research Ethics Committee of Xuzhou Medical University.2 at 37\u2103.Glioma cell lines U251, U87, A172, U118, C6 and human embryonic kidney cell line HEK293T were bought from Shanghai Cell bank, Type Culture Collection Committee, Chinese Academy of Sciences. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM)  supplemented with 10% foetal bovine serum  and grown in a humidified incubator with 5% COFor silencing of SH3GL2, three short hairpin RNA (shRNA) sequences were designed as follows:SH3GL2 #1\u2010F: 5\u2032\u2010GATCGGATGAAGAGCTTCGTCAATTCAAGAGATTGACGAAGCTCTTCATCCTTTTTTG\u20103\u2032,SH3GL2 #1\u2010R: 5\u2032\u2010AATTCAAAAAAGGATGAAGAGCTTCGTCAATCTCTTGAATTGACGAAGCTCTTCATCC\u20103\u2032;SH3GL2 #2\u2010F: 5\u2032\u2010GATCGGAGATGGATATTGAACAATTCAAGAGATTGTTCAATATCCATCTCCTTTTTTG\u20103\u2032,SH3GL2 #2\u2010R: 5\u2032\u2010AATTCAAAAAAGGAGATGGATATTGAACAATCTCTTGAATTGTTCAATATCCATCTCC\u20103\u2032;SH3GL2 #3\u2010F: 5\u2032\u2010GATCGCCTAGAAGGGAATATCAATTCAAGAGATTGATATTCCCTTCTAGGCTTTTTTG\u20103\u2032,H3GL2 #3\u2010R:5\u2032\u2010AATTCAAAAAAGCCTAGAAGGGAATATCAATCTCTTGAATTGATATTCCCTTCTAGGC\u20103\u2032;Control\u2010F,5\u2032\u2010GATCTTCTCCGAACGTGTCACGTTTCAAGAGAACGTGACACGTTCGGAGAATTTTTTG\u20103\u2032,Control\u2010R, 5\u2032\u2010AATTCAAAAAATTCTCCGAACGTGTCACGTTCTCTTGAAACGTGACACGTTCGGAGAA\u20103\u2032.BamH I and EcoR I cloning sites. Cell transfection was carried out with PolyJet  as described in the manufacturer's protocol. The lentiviruses were produced in HEK293T cells by cotransfecting the corresponding plasmids with the helper plasmids. For overexpression of SH3GL2, the SH3GL2 construct was generated by the human SH3GL2 cDNA was cloned into the vector p3 \u00d7 FLAG\u2010CMV\u201014 (3*FLAG) at the Hind III and BamH I restriction sites.The shRNA and control oligomers were annealed and then subcloned into the pLV\u2010shRNA plasmid by the The construction of stable cell lines was carried out as we previously described The cells were seeded in six\u2010well plates under normal conditions for 24 hrs. Then, the scratches were developed in the middle of the wells with a pipette tip. Thereafter, the cells were cultured for an additional 48 hrs in the presence of SU9516. Photographs were harvested with an inverted microscope  at the designated time\u2010points. The number of cells crossing the wound was normalized to the control.5) in serum\u2010free culture media was added into the inserts, and each insert was placed in the lower chamber filled with culture media containing 10% foetal bovine serum as a chemoattractant. After 24 hrs of incubation at 37\u00b0C, the non\u2010invasive cells were removed from the upper chamber by wiping with cotton\u2010tipped swabs. Then, the filters were fixed with methanol for 15 min. and stained with a 0.1% crystal violet solution for 10 min. Five fields of adherent cells in each well were randomly photographed with an inverted microscope and counted. The same experimental design was used for migration experiments except that filters were not pre\u2010coated with Matrigel.Transwell assays were performed with a polycarbonate filter membrane with a diameter of 6.5 mm and pore size of 8 \u03bcm (Invitrogen) according to the manufacturer's protocol. To assess invasion ability, the filters were pre\u2010coated with 10 \u03bcg of Matrigel (BD). The cell suspension  according to the manufacturer's instruction. Quantitative RT\u2010PCR was carried out by an ABI7300 real\u2010time PCR instrument  using SYBR Green. Primers for the amplification of SH3GL2, MMP2 and GAPDH were as follows:SH3GL2\u2010F: 5\u2010AAGCAGTTCCATAAAGCCACT\u20103,SH3GL2\u2010R: 5\u2010TCCTGGCCACGGATTTTTGA\u20103;MMP2\u2010F: 5\u2010CAGGATCATTGGCTACACACC\u20103,MMP2\u2010R: 5\u2010CCATACTTCACACGGACCACT\u20103;GAPDH\u2010F: TGGAGTCCACTGGCGTCTTC,GAPDH\u2010R: CATTGCTGATGATCTTGAGGCT.MMP2 secretion into the conditioned media was determined by gelatin zymography. The cells were cultured in serum\u2010free media for 48 hrs, and the conditioned media was harvested, centrifuged and resuspended in SDS loading buffer without \u03b2\u2010mercaptoethanol. In each condition, equal amounts of protein were loaded on a 10% SDS\u2010PAGE, supplementing 1 mg/ml gelatin as substrate. Zymograms were isolated in a Tris/glycine SDS running buffer under non\u2010denaturing conditions. After electrophoresis, SDS remnants were removed by washing gels with 2.5% Triton X\u2010100. Zymograms were subsequently reacted with a MMP substrate buffer  for 16 hrs at 37\u00b0C. After incubation, the gels were stained with 0.25% Coomassie brilliant blue R250 for 3 hrs at room temperature and then destained by 30% methanol and 10% acetic acid until the bands of lysis become clear. The proteolytic activity was visualized as the development of clear bands under a blue background.Immunohistochemical (IHC) staining was carried out according to the protocol supplied by the S\u2010P immunohistochemistry kit . The sections were fixed with 4% paraformaldehyde and blocked with 10% goat serum. Then, the sections were incubated with SH3GL2 antibody , followed by Biotin\u2010conjugated anti\u2010rabbit IgG and HRP\u2010conjugated streptavidin. Subsequently, the reactions were developed by 3\u2032\u2010diaminobenzidine (DAB) chromogenic reagent (Zhongshan Goldenbridge Biotech Co.). Then, the sections were counterstained with haematoxylin and dehydrated by incubation in gradient concentrations of alcohol, followed by 100% xylene. Finally, the coverslips were mounted onto the slides with neutral gum. The photographs were collected under an Olympus IX\u201071 microscope (Olympus).At the designated time, the cells were lysed and equal amounts of protein were isolated on a 10% SDS\u2010PAGE and then transferred to 0.45\u2010\u03bcm pore size PVDF membrane . After blocking with 5% non\u2010fat milk, the membrane was probed with primary antibodies  at 4\u00b0C overnight. On the following day, membranes were incubated in a horseradish peroxidase\u2010labelled goat anti\u2010rabbit/mouse IgG and detected by enhanced chemiluminescence detection system . Band densities were quantified by ImageJ Software . The relative amount of each protein was determined by normalizing the densitometry value of interest to that of the loading control.t\u2010test. P values <0.05 were considered statistically significant (*P < 0.05).The results were representative of experiments repeated at least three times, and quantitative data were expressed as means \u00b1 S.E.M. Statistical analyses were carried out using the SPSS Version 13.0 . Differences in multiple groups were determined by a one\u2010way analysis of variance (ANOVA) followed by post hoc test. Comparison between two groups was performed by Student's To investigate the possible role of SH3GL2 in the development of human glioma, total lysates were extracted from 33 specimens of human glioma tissues  and nine specimens of human non\u2010tumorous brain tissues, and the SH3GL2 protein level was evaluated by Western blotting. As shown in Figure As high\u2010grade glioma usually has a strong ability of invasion, we focused on exploring the possible roles of SH3GL2 in the migration and invasion of glioma cells. For this purpose, we employed knocking down approach in U251 cell line that has a high level of SH3GL2 and used overexpressing strategy in U87 cell line that has a low level of SH3GL2. Firstly, we down\u2010regulated SH3GL2 expression using its specific shRNA and observed the effects on cell migration and invasion. For silencing of SH3GL2, three shRNA targets  were screened for their efficacy in suppressing SH3GL2 expression, and a negative non\u2010targeting shRNA was used as a control. We found that using three shRNA alone did not have an ideal silencing efficiency; however, three shRNA mixtures could successfully knock down the expression of SH3GL2 (Data not shown). The off\u2010target effect of the shRNAs was excluded by testing their effect on human and mouse SH3GL2 Fig. . ThereafTo further determine the role of SH3GL2 in glioma cell migration and invasion, we then transiently transfected 3*FLAG\u2010tagged SH3GL2 cDNA into U87 cells to achieve the gain\u2010of function. The expression efficiency of SH3GL2 was confirmed by Western blotting and quantitative RT\u2010PCR experiments Fig. A and B. Recent studies have indicated that silencing of SH3GL2 activated STAT3 signalling, while overexpressing of SH3GL2 inhibited it SH3GL2 is mainly distributed in central nervous system, particularly enriched in the presynaptic ganglion i) multiple signalling pathways changed in glioma converge to STAT3 and (ii) it involves in multiple characteristics of glioma aggressiveness, via regulation of genes especially implicated in cell proliferation, growth, apoptosis, migration, invasion and neoangiogenesis Inhibition of specific tumour suppressor genes and activation of specific oncogenes have been demonstrated to be important for the occurrence and malignant progression of glioblastoma In conclusion, we have presented the first evidence linking SH3GL2 to malignant behaviours of human glioblastoma through STAT3/MMP2 pathway. These findings will provide some basis for further investigation of SH3GL2\u2010mediated signalling pathway and for evaluation of the prognostic utility of SH3GL2 status in patients with malignant glioma. However, more deeply studies are needed to clarify the precise mechanisms of SH3GL2\u2010mediated migration and SH3GL2\u2010mediated invasion in human malignant glioma.The authors declare that they have no conflict of interest.Fig. S1 The off\u2010target effect of the shRNAs was excluded by testing their effect on human and mouse SH3GL2.Click here for additional data file."}
+{"text": "FCRL2 gene associated with QT . Heritability analysis demonstrated that the SNP explained 2.42% of the trait\u2019s genetic variability in ERF (P = 0.02). Pathway analysis suggested that the gene is involved in cytosolic Ca2+ levels (P = 3.3 \u00d7 10-3) and AMPK stimulated fatty acid oxidation in muscle (P = 4.1 \u00d7 10-3). Look-ups in bioinformatics resources showed that expression of FCRL2 is associated with ARHGAP24 and SETBP1 expression. This finding was not replicated in the Rotterdam study. Combining the bioinformatics information with the association and linkage analyses, FCRL2 emerges as a strong candidate gene for QT interval.Electrocardiogram (ECG) measurements play a key role in the diagnosis and prediction of cardiac arrhythmias and sudden cardiac death. ECG parameters, such as the PR, QRS, and QT intervals, are known to be heritable and genome-wide association studies of these phenotypes have been successful in identifying common variants; however, a large proportion of the genetic variability of these traits remains to be elucidated. The aim of this study was to discover loci potentially harboring rare variants utilizing variance component linkage analysis in 1547 individuals from a large family-based study, the Erasmus Rucphen Family Study (ERF). Linked regions were further explored using exome sequencing. Five suggestive linkage peaks were identified: two for QT interval , one for QRS interval  and two for PR interval . Fine-mapping using exome sequence data identified a C > G missense variant  in the  The electrocardiogram (ECG) is an important tool for diagnosing, monitoring and evaluating risk in patients with cardiovascular disease CVD; . ECG meaARHGAP24, SETBP1, LRIG1, CREBBP, MEIS1. TBX20, and TBX5. Some ion channel encoding genes, such as SCN5A, HERG, KCNE1, and KCNE2, have been associated with long QT syndrome  identified at least 71 common variants associated with their variability . A numbeme LQTS; , atrial me LQTS; . Collectme LQTS; .Genome-wide association studies generally interrogate only common variants, typically of small effect. Families, in addition to being robust against population stratification, may be enriched for less frequent variants, which can potentially be identified by linkage and fine mapping. The aim of this study, therefore, was to discover less frequent variants using linkage analysis in a large family-based study, the Erasmus Rucphen Family Study (ERF).2) was computed. Blood pressure (BP) was measured twice on the right arm in a sitting position after at least five minutes rest, using an automated device . The average of the two measures was used for analysis. Hypertension was defined through the use of antihypertensive medication and/or through the assessment of BP measurements according to the The ERF study, which is a part of the Genetic Research in Isolated Populations (GRIP) Program, is a family-based study including over 3000 participants descendant from 22 couples that lived in the Rucphen region in the southwest Netherlands in the 19th century . All desExaminations included 10 s 12-lead ECG measurements, recorded with an ACTA-ECG  with a sampling frequency of 500 Hz. Digital measurements of the ECG parameters were made using the Modular ECG Analysis System MEANS; . Briefly6K Illumina Linkage IV Panels\u00ae) was used for genotyping for the linkage analyses. All genotyping procedures were performed according to the manufacturer\u2019s protocols. Only markers with minor allele frequency (MAF) > 0.05 were selected for further analysis. Genotyping errors leading to Mendelian inconsistencies were detected using PedCheck (FCRL2 variant (rs74608430) was performed using Merlin in one large, single pedigree.Illumina\u2019s HumanHap6k Genotyping BeadChip .Regions of interest with LOD > 1.9 were selected for further study . Borders2 to remove systematic biases and to recalibrate the PHRED quality scores in the alignments. Genetic variants were called using the Unified Genotyper tool of the GATK. About 1.4 million Single Nucleotide Variants (SNVs) were called and, after removing the low quality variants (QUAL < 150), we retrieved 577,703 SNVs in 1,309 individuals. Linear regression analyses, with SNVs in an additive model, were conducted on ECG measures, adjusted for age, sex, BMI, and height. To reduce the burden of multiple testing, we assessed only damaging variants in the LOD-2 SI; we found 324 such variants for QT, 52 for QRS and 61 for PR. We employed a Bonferroni correction for the number of deleterious mutations selected for each trait . The proportion of trait variance explained by the SNP was calculated using the Merlin software  and mean depth region was 65.26x (median = 52.87x). The sequence reads were aligned to the human genome build 19 (hg19) using BWA and the NARWHAL pipeline . The alisoftware .We sought to replicate our findings in the Rotterdam Study (RS) cohort. The RS is an ongoing prospective cohort study conducted since 1990 in the city of Rotterdam in The Netherlands . The Ill3 and Seattle4 databases. These databases gave functional prediction results from four different programs  (Table 2) were analyzed using Ingenuity Pathway Analysis . Several IPA modules were implemented: the \u201ccore analysis\u201d was used to assess pathways, relationships, and mechanisms relevant to the dataset; the \u201cupstream regulator analysis\u201d was implemented to identify molecules (including microRNA and transcription factors) that may affect expression levels; and the \u201cdownstream effects analysis\u201d was utilized to predict downstream biological processes that are increased or decreased.5 The GEO2R6 tool was used to analyse microarray-based expression data in the GEO database (GEO Accession numbers: GSE2240 and GSE41177). The Gene Network tool7 was used to describe co-expression networks and to assess potential functional effects of identified genes.To predict the functionality of genetic variants, and for comparison to BWA and NARWHAL, annotations were also performed using the dbNSFP  , in addiTable 1 shows the characteristics of the participants included in the discovery linkage analyses and exome sequencing, as well as the exome chip replication sample. There were no significant differences between the largely overlapping linkage and exome sequence groups. The replication sample was considerably older, and was characterized by increased frequency of hypertension (and BP differences), increased PR interval and decreased QT interval compared to the discovery samples. The three ECG traits studied  demonstrated only modest pair-wise correlations in the discovery dataset (Supplementary Table 1).Supplementary Table 2 shows the linkage results for the ECG traits, which yielded a total of five regions with suggestive LOD scores (LOD > 1.9). QT was suggestively linked to two regions, on chromosome 1 (LOD = 2.63) and on chromosome 2 (LOD = 2.05). A suggestive LOD score for QRS was observed on chromosome 1 (LOD = 2.52) and, for PR, two suggestive regions were located on chromosomes 9 and 14 with LOD scores of 2.20 and 2.29, respectively (Supplementary Table 2). Plots of the linked regions are shown in Figure 1.Table 2. Of these mutations, 1334 had a frequency less than or equal to 5%, 437 were predicted to be damaging by at least two of the prediction software packages used, and six were nonsense variants. By linkage peak, there were 207 missense damaging mutations and two nonsense mutations on 14 and 113 missense damaging mutations and two nonsense mutations on 2q32 for QT; 51 missense mutations and one nonsense mutation on 1p36 for QRS; and 29 missense mutations on 9q21 and 31 missense mutations and one nonsense mutation on 14q12 for PR. In total, 21 variants had nominal regression P-values less than 0.05  without reaching the significance levels needed to account for multiple comparisons (Supplementary Table 3). Looking for known genes under the linkage peaks (Supplementary Table 4), we found two variants previously related to heart failure, TTN  and HSD3B1 (P = 3.9 \u00d7 10-2 MAF = 1.1 \u00d7 10-2). Neither achieved statistical significance after Bonferroni correction, although both genes were marginally associated with QT. Only a single variant, a C > G (Ser > Cys) variant in FCRL2 , approached the Bonferroni threshold for multiple-testing (P = 1.5 \u00d7 10-4). This variant, under the linkage peak on chromosome 1q23.1 for QT, is highly conserved (scorePhastCons = 0.998) and also predicted by PolyPhen-2 to be damaging (0.999). In the whole ERF population, rs74608430 explained 2.42% of the heritability of QT . This finding was not replicated in the RS . A sequence kernel association test analysis of the gene also failed to achieve significance in the replication sample (P = 0.44).Our analysis of coding variants in these linked regions revealed 55,050 variants in coding regions of genes under the peaks, as described in FCRL2. Among the functions predicted by Gene Network are the regulation of cytosolic Ca2+ levels (P = 3.3 \u00d7 10-3) and AMPK stimulated fatty acid oxidation in muscle (P = 4.1 \u00d7 10-3). In the GEO database, FCRL2 expression was higher in AF ; MPL is in a module with MEIS1, associated with PR ; and CEP350 interacts with CREBBP, associated with QT . These three genes are not in linkage disequilibrium with each other. At the chromosome 2q34 locus linked with QT, a heart failure gene, TTN, was under the linkage peak. According to Gene Network analysis, expression of TTN is related to expression of three previously known QT genes  and two QRS and PR associated genes (TBX20 and TBX5)  .Not much is known about the function of er in AF . Supplemwith QRS . In the gure 1E) . AdditioTPM1 mutations with sudden death is a clear example of a locus discovered by linkage analysis (TTN (P = 5.5 \u00d7 10-2) and HSD3B1 (P = 3.9 \u00d7 10-2) and one gene with unknown cardiac function FCRL2 (P = 2.8 \u00d7 10-4). None of them reaches statistical significance level after correction for multiple comparisons.Linkage analysis is an important tool for the identification of genomic regions influencing trait variability. The role of analysis . The advanalysis . We perfThis study was conducted in a large, well-characterized family-based cohort, ascertained on the basis of genealogy and not phenotype. Multiple levels of genetic data, including a linkage panel and exome sequence data, provided a powerful dataset for identifying variants that may not be easily discovered with GWAS. Unfortunately, exome data was not available in the whole cohort, which could limit our ability to identify causal variants. Additionally, the sequence data did not include extra-genic or intronic variants that may be responsible for the observed linkage peaks.TTN and HSD3B1, which have been previously related to CVDs. HSD3B, a gene on chromosome 1 (1p13.1), has two isoforms (HSD3B1 and HSD3B2) that were found to be associated with an increase in plasma aldosterone .Another interesting gene covered by these variants was  failure  and hear failure . AdditioFCRL2 gene under the linkage peak on chromosome 1p23.1. This variant explains 2.42%  of the total genetic variance of QT (h2 = 36%) in the ERF population. FCRL2 has not been previously described with respect to cardiac function. Bioinformatics resources, however, showed that FCRL2 expression is associated with ARHGAP24 and SETBP1 expression, two genes implicated in ECG variability by GWAS. This suggests that FCRL2 may be relevant for heart function. FCRL2 is expressed mostly in liver, heart, testis and kidney8. Gene Network predicts that it may be relevant for cytosolic Ca2+ levels and AMPK stimulated fatty acid oxidation in muscle. These are plausible pathways for QT function. This finding for rs74608430, however, was not replicated in the RS, in which the MAF was 2.9 \u00d7 10-2. The absence of replication could be related to environmental differences influencing complex gene-environment interactions between these two study groups  in the y groups . AnotherFCRL2 is correlated with some microRNAs . Among these, miR-337-5p is known to be differentially expressed in patients with valvular heart disease and patients with chronic AF  or repeats in the linkage regions. Lastly, causal rare variants may be located outside the coding sequence, which we did not include in our sequencing analyses.FCRL2. TTN, MYH6, MYH7, TNNT2, and HSD321. Further analysis will need to be performed to demonstrate the involvement of these proteins in ECG measurements. We could not explain these with exonic sequence variants, so they will require more extensive follow-up, but provide potentially important indicators of the location of variation influencing ECG.Although the combination of linkage and exome sequencing did not lead to the identification of a causal variant, suggestive linkage regions contain a number of plausible candidate genes, including CS: Formal analysis, writing \u2013 original draft preparation; IZ: Formal analysis; NA: Formal analysis; AD: Formal analysis; EvL: Formal analysis; JK: Formal analysis, investigation, software; MvB: Formal analysis; BS: Investigation, resources; AU: Investigation, resources; AK: Formal analysis, software; JW: Investigation, resources; RW: Writing \u2013 original draft preparation, supervision; BO: Investigation, resources; TA: Formal analysis, supervision; CvD: Conceptualization, formal analysis, investigation, resources, writing \u2013 original draft preparation, supervision; AI: Conceptualization, formal analysis, writing \u2013 original draft preparation, supervision.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "Figures"}
+{"text": "To compare the antibacterial activity of alexidine (ALX) alone or as a final irrigant in combination with sodium hypochlorite (NaOCl), with the most common canal irrigants, NaOCl and chlorhexidine (CHX).Enterococcus faecalis for 24\u2009h and then distributed into 4 groups of 20 fragments each. The NaOCl, CHX and ALX groups were immersed in 1\u2009ml of 2.5% NaOCl, 2% CHX, and 1% ALX for 10\u2009min, respectively. The samples of the NaOCl+ALX group were immersed in 1\u2009ml of 2.5% NaOCl for 10\u2009min followed by 1% ALX for 10\u2009min. Bacteriological samples were taken, cultured, and the colony-forming units were counted.Ninety-four root fragments from extracted human teeth were infected with P>0.05) except for the comparisons CHX versus ALX and NaOCl+ALX versus ALX (P=0.004). ALX alone was the worst irrigant. CHX and NaOCl+ALX eradicated all bacteria. All experimental groups were significantly more effective than the control group immersed in saline (P<0.05).There was no significant differences among the experimental groups (The antibacterial effect of ALX alone was inferior to 2% CHX and 2.5% NaOCl. However, the combination of NaOCl with ALX as a final irrigant eradicated the biofilms. Bacteria are the main microorganism implicated in the apical periodontitis.5 NaOCl is the most common root canal irrigant due to its tissue-dissolving capability, its broad antimicrobial action, as well as its ability to neutralize toxic products.7 However, NaOCl has many disadvantages, including cytotoxicity, reduced efficacy in the presence of organic matter, and interference with pulp regeneration procedures.8\u201310 These limitations stimulate the search for safer and more effective irrigants. An alternative to NaOCl is chlorhexidine digluconate (CHX). This irrigant is a bisbiguanide disinfectant that has high antimicrobial activity, substantivity, and biocompatibility. However, CHX has been shown to have no tissue-dissolving activity and, when combined with NaOCl, produces para-chloroaniline, a toxic precipitate.11\u201313During the root canal treatment, mechanical debridement is of utmost importance to remove microorganisms and organic content that might serve as nutrients for residual bacteria. Nonetheless, studies have demonstrated that although instrumentation and irrigation are effective in substantially reducing the number of bacteria in infected canals, in many cases bacteria remain in the main root canal even when sodium hypochlorite (NaOCl) is used as the irrigant.15 Alexidine is used as a disinfectant in contact lens solutions17 and as an antiseptic in mouthwashes.18\u201320 A recent study showed that the antibacterial activity of alexidine against E. faecalis infecting dentin blocks was superior to CHX.21 Also, while there are many reports of allergic reactions, including anaphylaxis, following exposure to chlorhexidine, there is a lack of reports for ALX.22\u201324 Another important advantage of alexidine is that its combination with NaOCl does not produce any precipitate or para-chloroaniline.25 Therefore, the combination of NaOCl as the main irrigant with ALX as the final irrigant may be of great utility for the treatment of endodontic infections.The search for the ideal root canal irrigant revealed another candidate - alexidine (ALX). This substance is a bisbiguanide disinfectant similar to CHX, it contains two hydrophobic ethylhexyl groups in its structure and it has a higher affinity for major bacterial virulence factors such as bacterial lipopolysaccharide and lipoteichoic acid than CHX.The purpose of this study was to compare the efficacy of ALX alone or as a final irrigant in combination with NaOCl with the most common canal irrigants, NaOCl, and chlorhexidine2 fragments. The 94 specimens generated were immersed in 2.5% NaOCl solution for 5\u2009min and then in 17% EDTA  for 5\u2009min, followed by washing with 2.5% NaOCl for 5\u2009min to remove the smear layer formed by the cutting action of the disks and any pulp tissue remaining. During these procedures, all solutions were agitated in an ultrasonic bath at a frequency of 50\u2009Hz . Finally, the root fragments were washed with distilled water and sterilized by autoclaving.Forty-seven upper canines were obtained from the Tooth Bank of the Est\u00e1cio de S\u00e1 University, Rio de Janeiro, RJ, Brazil. The teeth were extracted for orthodontic or prosthetic reasons. The study was approved by the Ethical Committee at Est\u00e1cio de S\u00e1 University . The coronals and the apical thirds of the teeth were removed using diamond disks . Thereafter, the middle thirds of the roots were split along the long axis and cut into 25\u2009mmE. faecalis (ATCC 29212) using an apparatus described by Luppens26 and specially adapted by the authors for the present study  of a 24-well plate . The manipulation of root fragments during the experiment was performed aseptically in a laminar flow hood . The quality control of the materials sterilization process was attested by the Institutional Sterilization Center.Before inoculation, the cementum surfaces of the 94 root fragments were bonded onto the internal acrylic base of the apparatus. Afterwards, the medium Tryptic Soy Broth  supplemented with 10% glucose . was pumped through the system for 30\u2009min after which it was removed. Then a 24\u2009h culture (20\u2009ml) of 2 and sputter-coated with gold under vacuum and analyzed in a scanning electronic microscope at 10.00\u2009Kv and at 5000 magnification .Two samples were used to confirm the biofilm formation. On removal from the device, they were immediately fixed in freshly prepared 2% glutaraldehyde  and then dried in ascending ethanol concentrations. They were then dehydrated to their critical point in COThe root fragments were divided randomly into 4 groups  of 20 blocks each and 12 samples were separated for the control group. The root fragments of the NaOCl, CHX, and ALX groups were immersed in 1\u2009ml of 2.5% NaOCl, 2% CHX and 1% ALX for 10\u2009min, respectively. The 1% solution of ALX was prepared by dissolving ALX dihydrochloride powder  in sterile distilled water (1\u2009g/100\u2009ml). The samples of the NaOCl+ALX group were immersed in 1\u2009ml of 2.5% NaOCl for 10\u2009min followed by 1% ALX for 10\u2009min. In all groups, except the control group, a neutralizer solution was used for 5\u2009min after the action of the irrigants. This solution was composed of 3% Tween 80, 0.3% lecithin, 0.1% histidine and 0.5% sodium thiosulfate. In the control group, the root fragments were immersed in 1\u2009ml of sterile saline for 10\u2009min.Microbial samples were obtained from root fragments by agitation in ultrasound for 3\u2009min. Tenfold serial dilutions were carried out in saline. Then, aliquots of 20\u2009\u03bcl of each dilution were plated onto Mitis-Salivarius agar  plates, and incubated at 37\u00b0\u2009C for 24\u2009h. The colony-forming units (CFU) that grew were counted and then transformed into actual counts based on the known dilution factors.P<0.05. The statistical analysis was performed using SPSS 17.0 computer software .Bacterial counts were analyzed via Kruskal\u2013Wallis and Mann\u2013Whitney tests. The significance level was established at E. faecalis biofilm was observed by electron microscopy on both fragments analyzed (P>0.05) except for the comparisons CHX versus ALX, and NaOCl+ALX versus ALX (P=0.004). ALX alone was the less effective irrigant. CHX and NaOCl+ALX eradicated all bacterial cells in all samples. The NaOCl group showed bacterial growth only in one of the 20 samples while ALX showed bacterial growth in seven of the 20 samples (P<0.05).An analyzed . Intergr samples . All exp27 There are reports showing that microorganisms grown in biofilms could be 1000\u20131500 times more resistant to antimicrobials than planktonically grown bacteria.28 This in vitro study compared the antibacterial effect of ALX, a promising root canal irrigant, alone or as a final irrigant in combination with NaOCl, with the most common root canal irrigants: NaOCl and CHX.Biomechanical cleaning with files and antibacterial irrigants reduces the bacteria load in infected root canals; however, microbial communities grown in biofilms are remarkably difficult to eradicate with antimicrobial agents.E. faecalis was chosen as a bacterial marker since its resistance to many intracanal disinfectants is well documented30 Gram-positive facultative anaerobe bacterium is commonly found in endodontically treated root canals that failed2. The persistence of E. faecalis may stem, in part, from its ability to form biofilms in root canals and its capability to invade dentinal tubules.32 Additionally, this bacterium possesses a plethora of virulence factors, highlighting: aggregation substances, surface adhesins, sex pheromones, lipoteichoic acid, extracellular superoxide, gelatinase, hyaluronidase, and cytolysin (hemolysin).4in vivo has not been determined. This fluid exchange provides proteins, glycoproteins and other nutrients to the bacteria growing as a biofilm. This not only provides a sustainable nutrient source but also exerts a shear force on the bacterial biofilm.33In the present study, the inoculation apparatus allowed the formation of the biofilm under a slow turbulent flow to facilitate the adhesion of cells. When a tooth undergoes pulpal necrosis and subsequently develops periradicular periodontitis, exudates may cycle in and out of the canal. However, the exact flow rate that occurs E. faecalis infected bovine dentin34 and the second compared these irrigants against Streptococcus mutans biofilm cultivated on human dentin blocks.35 Methodological differences such as the substrate and the bacterium tested could have influenced these results. Contrary to these results, another study21 found a better antibacterial substantivity against E. faecalis using 1% ALX in comparison to 2% CHX. However, it is important to emphasize that in this substantivity assay, the antimicrobial action was evaluated over a period of 80 days. Also, the dentin fragments were immersed in the antimicrobial solution first and after transferred to the bacterial suspension, which is the opposite sequence from the other studies. In the present and previous studies, the antibacterial action was analyzed only once, immediately after the irrigant contact time.Contrary to expectations ALX alone was the less effective irrigant, but its combination with NaOCl was similar to CHX. Two previous studies compared the antibacterial activity of ALX and CHX, in the same concentration, and neither study found any significant difference. The first tested the canal irrigants against 36, which found that 5.25% NaOCl was highly effective against E. faecalis compared with CHX and ALX. There was no significant difference between 1% ALX and 2% CHX. Despite both studies used different concentrations of NaOCl, it is not expected significant differences in the antimicrobial activity of NaOCl varying its concentration.37\u201339The results from the present study are in accordance with a recent studyThe best results were obtained with 2% CHX and with the combination of 2.5% NaOCl+1% ALX as a final irrigant. In fact, both substances completely destroyed the bacterial biofilms. However, CHX in not able to dissolve organic tissues. Thus, the combination of NaOCl+ALX has a good potential for endodontic treatment to eliminate biofilms: the solvent capability of NaOCl, the high biocompatibility of ALX, the advantage that it does not form any precipitate when in combination with NaOCl and now, the confirmed antibacterial efficacy of the tested protocol, compatible with CHX and NaOCl alone, justify this potential. However, it is important to highlight that the group NaOCl+ALX was privileged by a higher contact time between the root fragments and irrigant solutions (20\u2009min) in comparison with the other groups (10\u2009min). This difference was necessary since ALX was used in this group as a final irrigant. Certainly, further studies are required to compare this final irrigation protocol with others.E. faecalis was inferior to 2% CHX and 2.5% NaOCl. However, the combination of NaOCl with ALX as a final irrigant has potential to be used in endodontic treatment to eliminate biofilms.Under the conditions of the present study, it was concluded that 1% ALX alone should not be indicated as an intracanal irrigant since its antibacterial effect against Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations."}
+{"text": "Older adults are disproportionately targeted by fraud schemes that advertise unlikely but large returns (positively skewed risks). We examined adult age differences in choice and neural activity as individuals considered risky gambles. Gambles were symmetric (50% chance of modest win or loss), positively skewed (25% chance of large gain), or negatively skewed (25% chance of large loss). The willingness to accept positively skewed relative to symmetric gambles increased with age, and this effect replicated in an independent behavioral study. Whole-brain functional magnetic resonance imaging analyses comparing positively (vs. negatively) skewed trials revealed that relative to younger adults, older adults showed increased anticipatory activity for negatively skewed gambles but reduced activity for positively skewed gambles in the anterior cingulate and lateral prefrontal regions. Individuals who were more biased toward positively skewed gambles showed increased activity in a network of regions including the nucleus accumbens. These results reveal age biases toward positively skewed gambles and age differences in corticostriatal regions during skewed risk-taking, and have implications for identifying financial decision biases across adulthood.The online version of this article (10.3758/s13415-017-0545-5) contains supplementary material, which is available to authorized users. Given an uncertain future, nearly all long-term financial choices involve some degree of risk. Although research on financial risk-taking has explored the effects of expected value (mean) and risk (variance) on financial choice , researchers have begun to investigate how skewed gambles influence affective responses and brain activity, which modulate choice , it has produced mixed results  and neural activity (Study 1) before individuals chose to accept or reject symmetric, positively skewed, or negatively skewed gambles as they underwent fMRI scanning. Our first aim was to determine whether age influenced the choice of risky skewed gambles. Based on an anticipatory affect account of financial risk-taking  were recruited from the San Francisco Bay Area community to complete a study at the Stanford Center for Cognitive and Neurobiological Imaging. Five were excluded from analyses because of head motion >2 mm during the fMRI scan, leaving a total sample of 32. Our a priori sample size calculation suggested that we needed to collect data from at least 27 subjects in order to have a power level of at least .8, to observe an effect at least as large as the linear effect of age on brain activity  reported in the most similar published study of financial risk-taking across adulthood . Participants completed 24 trials of each gamble type, presented in pseudorandom order. In other words, the three gambles described above were repeated 24 times in a mixed order, for a total of 72 trials.Three types of gambles were included: symmetric, positively skewed, and negatively skewed. Symmetric gambles featured an equal probability (50%) of winning or losing a moderate amount of money ($3.05). Positively skewed gambles featured a low probability (25%) of winning a large amount ($5.25) paired with a high probability (75%) of losing a small amount ($1.75). Negatively skewed gambles were the opposite, and featured a low probability (25%) of losing a large amount ($5.25) paired with a high probability of gaining a small amount ($1.75). Critically, the expected value of each gamble was set to $0, making it equivalent to both the alternative conditions and to a \u201cno gamble\u201d option, which was to accept $0 for certain, and variance was also equated across all gamble types  extending from the mid-pons to the top of the skull were acquired with an axial interleaved scheme. Functional scans were acquired using a T2*-weighted gradient pulse sequence . Anatomical scans, which were used for localization and coregistration of functional data, were acquired using T1-weighted spoiled grass sequence .Analysis of functional neuroimaging data was conducted using Analysis of Functional Neuroimages (AFNI) software Cox, . PreprocMultilevel binary logistic regressions were carried out using the lme4 package (Version 1.11.1)  and its interactions, were included in the model  skewed versus symmetric , and (2) positive versus negative skew . Before inclusion in the regression models, these contrasts were convolved with a single gamma function to model the hemodynamic response. Subject-level regression models also included nine covariates of noninterest: residual motion (in six dimensions), white matter and cerebral spinal fluid time series, and polynomial trends across the experiment. For each contrast of interest, T-statistic maps were transformed to Z scores and spatially normalized by warping into Talairach space. The residual error time series from these subject-level models were used to estimate the noise smoothness values for cluster estimation. Statistical maps were then generated to examine three covariates of interest across the entire sample: (1) age, (2) positive-skew bias, and (3) the interaction between these two covariates. For follow-up verification of these analyses, regions of interest were specified around the significant clusters of activation that emerged in group analyses. Follow-up activity time-course analyses examined whether activation prior to choice, or anticipatory activation,  differed by gamble condition. Bootstrapped statistics with 2,000 replications were conducted to verify each significant effect to confirm that outliers were not overly influencing the analysis.Analyses focused on changes in brain activation during anticipation . Based on extensive prior research that suggests that there are no adult age differences in neural activation following the receipt of gains versus losses  to evaluate the reliability of the age effects on choice. Detailed methods and results are presented in the A separate behavioral study was conducted with a much larger sample , but did not confirm that older adults were more likely to reject negatively skewed than symmetric gambles . To ensure that these age effects were not driven by outliers, we compared the difference between the proportion of positively skewed and negatively skewed gambles chosen by each participant. This individual difference measure\u2014skew bias\u2014varied from 1 to \u22121, with those who chose a higher proportion of positively skewed gambles (positive-skew bias) having positive scores and those who chose a higher proportion of negatively skewed gambles (negative-skew bias) having negative scores. While young participants varied widely on this measure, with similar numbers of young individuals showing negative and positive skew biases, almost all of the older participants displayed a positive skew bias .First-level general linear models quantified differences between skewed versus symmetric trials  and positively skewed versus negatively skewed trials  across the brain. Second-level regression analyses examined individual differences by age and behavioral bias scores on the effects of general skew and valenced skew. Both main and interaction effects of age and behavioral bias were included together in second-level models because of the behavioral interaction between age and skew bias. Whole-brain analyses revealed significant age differences in anticipatory neural activity related to both skew and valence  and a bias-related decrease in anticipatory activation for negatively skewed gambles .Individual differences in the behavioral measure of positive skew bias from the task were also related to individual differences in anticipatory brain activity related to skew valence. In clusters along the cingulate gyrus, as well as bilateral regions of the caudate and posterior insula, a behavioral positive-skew bias was associated with see Fig. . The revb = 0.01, 95% CI ). Within older adults, individuals showed increased activity in the right inferior frontal gyrus on trials where they were more likely to accept the gamble: those with a positive-skew bias showed greater activity on positively skewed trials, while those with a negative-skew bias showed greater activity on negatively skewed trials. Follow-up bootstrapped correlations of bias on anticipatory activity in right inferior frontal gyrus VOI in older adults confirmed that this interaction was driven by a bias-related increase in activation in anticipation of negatively skewed gambles . These associations were not present in younger adults  gambles and more likely to reject negatively skewed (compared to symmetric) gambles , where again we found an age-related increase in acceptance of positively skewed gambles relative to symmetric gambles , AIns, and anterior cingulate cortex (ACC), regions commonly associated with executive function and cognitive control  is a significant and growing problem for people of all ages. While some evidence suggests that older adults might not necessarily be more susceptible to fraud"}
+{"text": "Recent advances in nucleic acid sequencing technologies have led to a dramatic increase in the number of markers available to generate genetic linkage maps. This increased marker density can be used to improve genome assemblies as well as add much needed resolution for loci controlling variation in ecologically and agriculturally important traits. However, traditional genetic map construction methods from these large marker datasets can be computationally prohibitive and highly error prone.TSPmap, a method which implements both approximate and exact Traveling Salesperson Problem solvers to generate linkage maps. We demonstrate that for datasets with large numbers of genomic markers  and in multiple population types generated from inbred parents, TSPmap can rapidly produce high quality linkage maps with low sensitivity to missing and erroneous genotyping data compared to two other benchmark methods, JoinMap and MSTmap. TSPmap is open source and freely available as an R package.We present TSPmap will be a useful tool to handle such large datasets into the future, quickly producing high quality maps using a large number of genomic markers.With the advancement of low cost sequencing technologies, the number of markers used in the generation of genetic maps is expected to continue to rise. The online version of this article (10.1186/s13040-017-0158-0) contains supplementary material, which is available to authorized users. Genetic maps are the foundation of genotype to phenotype mapping and a critical component in the discovery of the molecular basis of both simple and complex traits. Increased sample size and number of markers in a map improves the resolution of chromosomal regions underlying quantitative trait loci (QTL) and reduces the number of possibly causal variants for further investigation. Additionally, genetic maps are a valuable tool for constraining and validating the assembly of eukaryotic genomes because genetic linkage map construction is robust to repetitive regions and paralogs, which can confound assembly algorithms based purely on sequence data. These motivations, and recent advances in genotyping by sequencing (GBS) technologies, have led to dramatic increases in the number of markers used to generate genetic maps. Indeed, recent reports demonstrate the use of maps composed of more than 10,000 markers .While next generation sequencing technologies have allowed for the identification of increasing number of genetic markers, the rate of erroneous calls, on the order of 1\u20132% \u20138, affecTo date, software methods for generating genetic maps have used strategies such as simulated annealing to maximize a likelihood function \u201312, grapn edges, and that also results in the lowest cost, meaning that the sum of the weighted edges on the Hamiltonian circuit is minimized. Each genetic marker corresponds to a vertex in the graph G, and the recombination frequency (rf) values between markers represent the weights between the vertices. This allows the recombination frequency matrix to serve as the weight matrix for the TSP instance, and the solution will give us the lowest-cost path through the markers. However, no genetic mapping tool using an exact TSP solver has been developed to date. This is likely because only recently has the computational power required to implement exact TSP solvers for large datasets been achieved for personal computers, finally unlocking the potential to apply this approach toward generating genetic maps [The TSP is formulated as a problem in which an agent wishes to visit all of the vertices of a graph, G. The edges of the graph are weighted. The goal is to find a Hamiltonian circuit (a \u201ctour\u201d) that visits all of the vertices using tic maps .minimal spanning tree (MST). Finding the minimal spanning tree of a graph, G, has a polynomial time complexity of O(V\u2009+\u2009E). By contrast, the TSP is an NP-Hard problem, meaning there is no known deterministic algorithm for solving all TSP instances in polynomial time. Thus it would seem better to use a minimal spanning tree if possible to generate genetic linkage maps. Under perfect conditions, one can indeed use a minimal spanning tree to generate accurate genetic linkage maps. By perfect conditions, we mean that the recombination frequencies exactly and precisely capture the distance between genetic markers. This means, for example, if m1, m2, m3, m4, m5 are genetic markers that are already in the correct order, then the distance between m3 and m4 must be less than the distance between m3 and m5, and the distance between m2 and m3 must be less than the distance between m3 and m1. We can think of these markers as being points on a straight line. In this case, a minimal spanning tree will link the markers in the proper sequence. However, we can\u2019t precisely know the true recombination frequencies of a set of genetic markers. If we think of the recombination frequencies as being two numbers, the true recombination frequencies plus a noise term, then as the level of \u201cnoise\u201d increases the minimal spanning tree is increasingly corrupted by this noise. At a certain low level of noise it is no longer possible to properly order the genetic markers, but it may still be possible to separate the genetic markers that are on different linkage groups. At higher levels of noise, it may not even be possible to correctly separate the genetic markers that are on different linkage groups. There is a commonly used tool, MSTmap, which attempts to use information from the minimal spanning tree to construct genetic linkage maps [Another computational framework that can be applied to the problem of finding genetic linkage maps is the age maps . HoweverBy posing genetic linkage map construction as a Traveling Salesperson Problem, we add the additional constraint that we are selecting small weights and imposing an ordering on all of the genetic markers. Methods that only look at the minimal spanning tree do not impose an ordering on all of the genetic markers. By imposing an ordering on all of the genetic markers, we also obtain a solution that is more robust to noise, errors and missing data in the recombination frequencies.TSPmap: an R package that applies TSP algorithms to the generation of genetic linkage maps from genetic marker data. We compared this method with commonly used tools, JoinMap and MSTmap, with simulated datasets of varying marker number and missing/erroneous markers, and found that this new tool generates maps in less time and with equal or higher quality.Here we present An important element in our strategy for constructing genetic linkage maps is to exploit problem decomposition. A fundamental motivation for exploiting problem decomposition is to break large problems into smaller problems. This is particularly important if we want to use an exact solver for the TSP. An exact solver might perform reasonably for 2000 markers, but then require an unreasonable amount of time to solve a TSP with 10,000 markers. A natural form of problem decomposition is to separate different groups of markers that are on different chromosomes. For example, if we construct the minimal spanning tree, we may not be able to determine the ordering of all of the genetic markers. However, we may be able to determine that certain groups of genetic markers are on different linkage groups with high probability by examining the minimal spanning tree. In other cases, we may run a heuristic TSP solver, which is less sensitive to problem size, to generate an initial high quality solution. From this initial solution, we may determine that different groups of markers either are on different linkage groups or might be on different linkage groups. By separating the markers into different groups, we can solve each group as a separate instance of a TSP, and then reassemble the solutions for each group into one overall solution. Thus, the proposed solver uses a mix of heuristic methods to generate initial solutions to the TSP instances, and then uses an exact solver on groups of markers that are clearly on the same linkage group.TSPmap uses two TSP solvers. Lin-Kernighan-Helsgaun (LKH) is an heuristic local search algorithm based on the Lin-Kernighan algorithm [TSPmap, LKH is used in the early stages of the mapping process, namely to identify and separate the linkage groups. Once these groups are identified, the final order of markers on each linkage group is determined using the exact solution implemented by Concorde  between markers to identify linkage groups and order markers. In mapping populations where genotypes are almost entirely homozygous, such as those comprised of recombinant inbred lines (RILS) or generated through double haploidization, TSPmap includes a pipeline for quickly generating rf matrices. First, genotype data in a matrix format are filtered by removing duplicate and heterozygous markers, as well as markers above a user-defined threshold of similarity across all individuals. Since smaller rf values indicate higher correlation between markers, the smaller recombination frequency values are most relevant to the construction of the linkage map. The larger values represent unlinked markers and thus are of limited utility. Because the performance of the TSP solver is slowed by the inclusion of these values, the user may input a cutoff threshold (default set to 0.4) above which all rf values are inflated to 0.5, preventing the solver from spending computation time optimizing these non-informative values. However, setting the threshold too low could begin to affect the clustering and the final ordering of markers within the linkage group. This is especially true for noisy datasets where relatively high rf values may still be informative.rf value range. This prevents the TSP solver from erroneously linking pairs of markers because of missing calls in the data set.Missing data can cause recombination frequencies to be underestimated or overestimated, depending on whether the missing call represents a difference or similarity between the two markers. To account for this uncertainty, recombination frequency values for markers with missing data are adjusted to lie at the midpoint of the possible TSPmap can also take as input rf matrices generated by R/QTL [2 intercrosses and backcrosses. These R/QTL formatted rf matrices can then be used for downstream steps in the TSPmap algorithm. However, it should be noted that creating rf matrices in R/QTL can be time intensive with large numbers of markers, and may slow the overall workflow of generating linkage maps with TSPmap.by R/QTL  for diffrf value between the beginning node and ending node are expected to be large since they are on opposite ends on the linkage group. To eliminate this effect we convert TSP problem into a Hamiltonian path problem (HPP). To accomplish this, we introduce into the TSP weight matrix a dummy vertex that has a zero-weight connection to all other vertices. The inclusion of this vertex allows the TSP solver to connect the last vertex in the tour with the first without incurring the large-weight penalty associated with the rf value between two distant vertices, in essence allowing the solver to construct a non-cyclic path [In the typical TSP formulation, the solution is a Hamiltonian cycle. That is, the solution is a tour in which each vertex is visited exactly once and ends at the beginning vertex, thus forming a complete cycle. In the case of a genetic linkage map, we have no need to consider the linkage between the last marker in the linkage group and the first. In fact, allowing the TSP to run in this standard configuration will negatively impact the result, since the algorithm will include the value of this last edge when attempting to minimize the total tour cost, and the lic path .k, into the algorithm as a parameter. Along with the number of markers, m, this is used to define the minimum size of a cluster (s) according to the formulaUsing the recombination frequency matrix, the first step is to connect the unordered dataset into a minimum spanning tree and then break the spanning tree into large clusters that may contain markers from more than one linkage group. This is because we only want to break the spanning tree at a location that has a very high probability of being a transition between chromosomes. To further aid decomposition, the user inputs an initial estimate of the number of linkage groups (chromosomes), s in this way the algorithm is meant to penalize small clusters, but allow for the possibility of large variation in physical size and marker density among chromosomes. However, we acknowledge that the assigned minimum chromosome size may not hold true in species with extreme variation in chromosome size, e.g. birds and some other vertebrates.By calculating .5*k recombination frequency values. If this does not produce k significant clusters, the next-largest rf value is added to the list of cut points between clusters. This is repeated until at least k significant clusters have been produced. In this way, more cuts are made than by simply breaking into the number of known chromosomes, because some chromosomes might not neatly form a single linkage group while other pairs might not have a nice break between linkage groups. Therefore, the algorithm is designed make more cuts, and later test which mergers are best.The algorithm begins by constructing the minimum spanning tree and breaking it apart at the largest 1rf values that exceed a user-specified threshold. When such a value is detected, the cluster is broken apart to ensure that the final clusters do not contain markers from more than one linkage group  are processed using Concorde to produce the optimal linkage map for the given data. Because Concorde is an exact solver it is able to find the exact TSP solution to the order of markers in within each linkage group. Finding an exact solution does not guarantee that the order of the markers is absolutely correct; instead, the exact solution is the best solution possible given the available recombination frequencies. Experimental datasets can contain a few markers that are highly erroneous across the entire population. Once TSPmap has determined the marker order, these highly erroneous markers can be identified and dropped using tools available in R/QTL (i.e. droponemarker function)  prior toTSPmap R package is freely available and includes a user tutorial vignette and example datasets.This procedure was implemented in R, version 3.2.5 . The algm) of 1000 or 4000, evenly distributed across five linkage groups or 10,000 distributed across 10 linkage groups, a genotype error rate (\u03b7) of 0.0, 0.01, or 0.05, and a missing genotype rate (\u03b3) of 0.0, 0.05, or 0.10. Five replicates of each configuration were created.Datasets representing a mapping population of 300 recombinant inbred lines were simulated using the R/QTL package . DatasetTSPmap. An example script for implementation of TSPmap with these data can be found in Additional\u00a0file\u00a0JoinMap\u00a04.0 [MSTmap [JoinMap, the maximum likelihood algorithm was used, as the regression method gave unreasonably long runtimes for the 4000-marker datasets, and the results for 1000-marker datasets were determined to be higher quality using the maximum likelihood algorithm. Default parameters were used, except values were increased to 5000 for chain length, 10,000 for number of chains without improvement before stopping, and 1000 for chain length per Monte Carlo EM cycle. Additionally, we created linkage maps with these simulated data using MSTmap, which uses a minimum spanning tree algorithm [MSTmap can be found in Additional\u00a0file\u00a0MSTmap.For each simulated marker dataset we generated linkage maps with nMap\u00a04.0  and MSTm [MSTmap . For Joilgorithm . The exac, to the true number of chromosomes in the simulated data. Additionally, we calculated the number of erroneous pairs, E, in each solution. This number is the number of pairs of markers which appear in reversed order in their estimated positions as compared to their true positions in the simulated data [To quantitatively measure the quality of a solution, we compared the final number of linkage groups (chromosomes) ted data .TSPmap correctly identified the linkage groups for all simulated data sets  for MSTmap in Table\u00a0c values are not reported where MSTmap was unable to separate the majority of the linkage groups, as the value is not meaningful. The c values in Table\u00a0MSTmap produced 4 or 5 linkage groups . JoinMap failed to correctly identify the linkage groups for only one data set and generally performed similarly to TSPmap, albeit with much longer run times.ts Table\u00a0; Fig.\u00a04.TSPmap with MSTmap and JoinMap, we tested the number of miss-ordered markers relative to simulated positions across a number of marker dataset sizes, error rates and missing data contents  and missing genotype rate (\u03b3) increased, TSPmap and JoinMap dramatically outperformed MSTmap, which exhibited very high E values in the datasets with the most missing and erroneous data. It appeared that linkage group assembly represented the error-prone step in the MSTmap protocol, where an inability to fully separate the linkage groups led to substantial marker mis-ordering  x (C x D)), F2, and backcross. We simulated five sets of 27 genotype matrices for each cross type, representing three error probabilities , three missing data probabilities  and three marker densities . Partially informative (e.g. dominant) markers were not simulated, because recombination fractions cannot be calculated among pairs of markers in 4-way mapping populations that are only informative in alternative crosses. Marker positions of each genotype matrix were randomized, recombination fractions were calculated in R/qtl, and the resultant matrix was fed into TSPmap. For each dataset, the correlation coefficient between the true marker order and the simulated marker order generated by TSPmap was >0.999 Example script for generating linkage maps with Additional file 2:MSTmap. (TXT 593\u00a0kb)Example parameters for generating linkage maps with Additional file 3: Figure S1.TSPmap with simulated datasets of different types of mapping populations , marker number , proportions of missing data  and genotyping error rates . The accuracy of the TSPmap solution was measured by the correlation coefficient between the true marker order and marker order generated by TSPmap for each simulated dataset. Note the scale of the y-axis is 0.99935\u20131.000. (DOCX 40\u00a0kb)Performance of Additional file 4: Figure S2.TSPmap using marker datasets from A. Arabidopsis thaliana [JoinMap. (DOCX 213\u00a0kb)Linkage maps generated by thaliana  and B. &thaliana  compared"}
+{"text": "Lithium-sulfur (Li-S) batteries have become promising candidates for electrical energy storage systems due to their high theoretical specific energy density, low cost and environmental friendliness. However, there are some technical obstacles of lithium-sulfur batteries to be addressed, such as the shuttle effect of polysulfides. Here, we introduced organically modified carbon nanotubes (CNTs) as a coating layer for the separator to optimize structure and enhance the performance of the Li-S battery. The results showed that the cell with a CNTs-coated separator exhibited an excellent cycling performance. Compared to the blank separator, the initial discharge capacity and the capacity after 100 cycles for the CNTs-coated separator was increased by 115% and 161%, respectively. Besides, according to the rate capability test cycling from 0.1C to 2C, the battery with a CNTs-coated separator still released a capacity amounting to 90.2% of the initial capacity, when the current density returned back to 0.1C. It is believed that the organically modified CNTs coating effectively suppresses the shuttle effect during the cycling. The employment of a CNTs-coated separator provides a promising approach for high-performance lithium-sulfur batteries. Nowadays, rechargeable lithium batteries have been widely used in portable electronics, electric vehicles and various energy storage devices. Lithium-sulfur (Li-S) batteries have been considered to be one of the most promising choices for next generation, high-energy rechargeable batteries due to their high theoretical energy density. For lithium-ion batteries with graphite as negative electrodes, the theoretical energy densities are typically limited to around 400 Wh/kg or 1400 Wh/L , while t2S are electronically and ionically insulating. On the other hand, due to the soluble characteristics and thus the shuttle behavior of the polysulfide intermediates , which are generated during the charge/discharge process, irreversible capacity loss is commonly unavoidable. This consequently leads to low coulombic efficiency and poor cycle life for Li-S batteries  in methanol (40%) was provided by Gelest . Sulfonate salt NPES [C9H19C6H4O(CH2CH2O)10SO3\u2212K+] was obtained from Aldrich . Sublimed sulfur was purchased from Sinopharm Chemical Reagent Co., Ltd. . Celgard 2325 from Celgard, LLC  was adopted as separator. Bis(trifluoromethane) sulfonamide lithium(LiTFSI), lithium nitrate , n-butyl alcohol (99%), N-methyl-2-pyrrolidone , 1,3-dioxolane (DOL) and 1,2-dimethoxyethane (DME) were obtained from Aladdin .Multi-walled carbon nanotubes (CNTs) were provided by Chengdu Organic Chemicals Co., Ltd.  Ethanol, chloroform, concentrated sulfuric acid . FTIR analysis was carried out using a Nicolet 6700 Fourier transform infrared spectrometer . The surface morphologies of the virgin and coated separators were observed using a field emission scanning electron microscope . The water contact angles of the separators were measured by a JC 2000C contact angle measuring instrument . The thermal shrinkage ratio V/V = 1:1) with 1 wt % LiNO3 additive. The cathode was prepared by mixing elemental sulfur, acetylene black and poly(vinylidene fluoride) with a weight ratio of 3:2:1 in N-methyl-2-pyrrolidinone. The slurry was coated on the aluminum foil and dried under vacuum to form the working cathode. Lithium metal foil was adopted as the anode. Cycling performance of the batteries was analyzed using a cell testing system . Both the cyclic voltammetry curves (CV) and AC impedance of the battery were measured using a CHI 660E electrochemical workstation . The voltage range was controlled in 1.5~3 V and the scanning rate was 0.1 mV/s. For the AC impedance measurement, the scanning frequency range was controlled from 0.01 to 100 kHz, with an AC potential amplitude of 5 mV at the open-circuit potential.The CR2032 coin cells were assembled in an argon-filled glove box to evaluate the electrochemical performance of the virgin and CNTs-coated separator. The electrolyte was made up of 1 M bis(triuoromethanesulfonyl)imide (LiTFSI) in dioxolane/dimethoxyethane solution (DOL/DME, In summary, we proposed a novel and effective method for the modification of separators in lithium-sulfur batteries. Grafted long-chain molecules and tube-like CNTs enable the formation of micropores in CNTs, which could accommodate polysulfides. The hydrophilic groups help trapping polysulfides to suppress shuttle effect. The organically modified CNTs coating also facilitate the battery with a high temperature stability. In addition, the conductive nature of CNTs also contributes to a decrease in internal resistance. The performance of the battery is improved significantly after adopting our method, indicating new avenues for practical lithium-sulfur batteries."}
+{"text": "A mathematical model explains saturating axon guidance responses to molecular gradients. Published 2, February 2016eLife paper cited above. In attempting to reproduce the quantitative analysis of the experimental axon trajectory data we encountered a number of errors and inconsistences. The first author (HN) could not supply the source data for the control experiments contributing to Figures 4, 5, 6, 8, and 11, or the final version of the code that generated these figures. We found that the Excel spreadsheets supplied with the figures were incorrect, as were the boxes drawn in Figures 9 and 10. Although we found no problem with the mathematical model or theoretical analysis, Figures 12 and 13 rely on parameters obtained from fitting data that is no longer available for verification.We are retracting the We hope to be able to address these issues with new experimental data in the future. However, the changes that would be required to correct the paper are sufficiently extensive that its overall conclusions must currently be considered to be in doubt. We are therefore in agreement that retraction is the appropriate course of action at this time. We sincerely apologise."}
+{"text": "Ovis ammon polii), a subspecies of argali (Ovis ammon) that is distributed mainly in the Pamir Mountains, provides a mammalian model to study high altitude adaptation mechanisms. Due to over-hunting and subsistence poaching, as well as competition with livestock and habitat loss, O. ammon has been categorized as an endangered species on several lists. It can have fertile offspring with sheep. Hence, a high-quality reference genome of the Marco Polo Sheep will be very helpful in conservation genetics and even in exploiting useful genes in sheep breeding.The Marco Polo Sheep  has an N50 contig size of 30.7 Kb and a scaffold N50 of 5.49 Mb. The repeat sequences identified account for 46.72% of the genome, and 20 336 protein-coding genes were predicted from the masked genome. Phylogenetic analysis indicated a close relationship between Marco Polo Sheep and the domesticated sheep, and the time of their divergence was approximately 2.36 million years ago. We identified 271 expanded gene families and 168 putative positively selected genes in the Marco Polo Sheep lineage.Caprinae, and for the future conservation of the Marco Polo Sheep.We provide the first genome sequence and gene annotation for the Marco Polo Sheep. The availability of these resources will be of value in the future conservation of this endangered large mammal, for research into high altitude adaptation mechanisms, for reconstructing the evolutionary history of the  Ovis ammon polii) is a subspecies of argali (Ovis ammon), named after the explorer Marco Polo. It was first described scientifically in 1841 by Edward Blyth [O. ammon has been categorized in several protection lists, such as Appendix II of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) and the International Union for Conservation of Nature and Natural Resources (IUCN) Red List, as a vulnerable or nearly threatened species. Conservation and restoration measures are therefore needed in order to safeguard the species, and information about its genome will be a key element in formulating an appropriate conservation strategy.The Marco Polo Sheep  to 15 kilobase pairs (kb) were constructed using the standard protocol provided by Illumina . To construct small-insert libraries , DNA was sheared to the target size range using a Covaris S2 sonicator  and ligated to adaptors. For long-insert libraries , DNA was fragmented using a Hydroshear system . Sheared fragments were end-labeled with biotin, and fragments of the desired size were gel purified. A second round of fragmentation was then conducted before adapter ligation. All libraries were sequenced on an Illumina HiSeq 2000 platform (Table S1). A total of 1022.43 Gb of raw data was generated, and 624.74 Gb of clean data was retrieved after removal of duplicates, contaminated reads (reads with adaptor sequence), and low-quality reads using the sickle software tool [High\u2013molecular weight genomic DNA was extracted from fibroblast cells cultured from the ear skin biopsy sample of a male are tool , a k-merApproximately 65 Gb of clean reads were randomly selected from all short libraries to estimate the genome size using the k-mer-based method and the formula G = k-mer_number/k-mer_depth. In this study, a total of 52 413 427 492 k-mers were generated and the peak k-mer depth was 17. The genome size was estimated to be approximately 3 Gb , and all the clean data correspond to a coverage of \u223c208-fold.RRID:SCR_015531) [RRID:SCR_015026) [de novo assembly for the Marco Polo Sheep has a total length of 2.71 Gb, including 116.91 Mb (4.3%) of unknown bases. The assembly is slightly larger than that of the domestic sheep  [Capra hircus, ARS1, 2.92 Gb) [The assembly was performed using Platanus v1.2.4  , which i_015026) . The fin2.61 Gb)  and smal2.92 Gb) . The N50RRID:SCR_015055, v2.5) [RRID:SGR_015008, v2.0.1 [de novo assembled scaffold during the assembly process. The FRC curve was calculated for the Marco Polo Sheep, sheep, taurine cattle, and 2 versions of goat assemblies . We found that the curve for our assembly was similar to that for the sheep and the 2 goat assemblies, with taurine cattle slightly different from the others, indicating that the level of contiguity and correctness of the Marco Polo Sheep genome assembly is comparable to those of sheep and goat.We assessed the quality of the genome assembly with respect to base-level accuracy, integrity, and continuity. More than 99.65% of the short-insert paired-end reads could be mapped to the assembly, and more than 98.35% of the sequence has a coverage depth greater than 20-fold (Table S4); thus the assembly is of high-level single-base accuracy. A core eukaryotic genes (CEG) mapping approach  [RRID:SCR_002105) [We mapped the reads from short-insert length libraries to the Marco Polo Sheep reference genome with BWA   and perf_002105) . Applyin_002105) . We furt_002105)  in R to _002105)  and wise_002105) , the InDde novo\u2013 and homology-based approaches. Transposable elements were identified at both the DNA and the protein levels, based on known sequences contained within the DNA repeat database (RepBase v21.01) [RRID:SCR_012954) [de novo prediction, first RepeatModeler V1.0.8  was employed to construct a de novo repeat library, and then RepeatMasker was used to identify repeats using both the de novo repeat database and RepBase. We then combined the de novo prediction and the homolog prediction of transposable elements according to the coordination in the genome. Tandem repeats were annotated with RepeatMasker and Tandem Repeats Finder  [The transposable elements present in Marco Polo sheep sequences were identified using a combination of  v21.01) , using R_012954)  and Repe; v4.07) . In summde novo prediction to annotate protein coding genes. For homology-based prediction, protein sequences from 5 different species  (Table S12) were mapped onto the repeat-masked Marco Polo sheep genome using TblastN with an E-value cutoff of 1e-5; the aligned sequences as well as the corresponding query proteins were then filtered and passed to GeneWise  [de novo prediction, we first randomly selected 1500 full-length genes from the results of homology-based prediction to train the model parameters for Augustus v3.2.1  [de novo approaches and generated a consensus gene set (Table S13). The final gene set was produced by removing low-quality genes of short length (proteins with fewer than 50 amino acids) and/or exhibiting premature termination. The final total gene set consisted of 20 336 genes, and the number of genes, gene length distribution, and exon number per gene were similar to those of other mammals, while the intron length was slightly larger than goat (CHIR_1.0), sheep (Oar_v3.1), and taurine cattle (UMD3.1) . The repeat content was annotated by RepeatMasker v4.0.5 [RRID:SCR_006695) (87.17%), gene ontology , Swiss-Prot (91.67%), TrEMBL (92.33%), and Kyoto Encyclopedia of Genes and Genomes  (Table S15). In addition, we identified 2978 non-coding RNAs in the Marco Polo Sheep genome (Table S16).We used homology-based and _015054)  to searc_008417)  and gene_008417) . GenScan_008417) , Augustu_008417) , and gen_008417)  were ther v4.0.5  with uniRRID:SCR_006119) [First, large-scale variations among Marco Polo Sheep, sheep, and goat were identified by synteny analysis using the program LAST  . A totalRRID:SCR_002344) [GigaDB  (Table S12) [RRID:SCR_007839) [To analyze gene families, we downloaded the protein sequences of 8 additional species  from Ensembl   and Gigable S12) . The con_007839) . The expRRID:SCR_006086)  [RRID:SCR_014932) MCMCtree program v4.5 [Next, we selected 5788 single-copy gene families from the above-mentioned 9 mammalian species and used PRANK v3.8.31  with theig. S14) . The divram v4.5  and caliram v4.5 . The conram v4.5  and confRYR1) was located in the pulmonary artery smooth muscle cells, which could subserve coupled O2 sensor and NO regulatory functions to respond to the tissue hypoxic decrease [P2RX3) is reported as a potential new target for the control of human hypertension, which could reduce the arterial pressure and basal sympathetic activity and normalize carotid body hyperreflexia in conscious rats with hypertension during P2RX3 antagonism [IGF1) encodes the growth-promoting polypeptide mainly involved in body growth and differentiation as well as the glucose, lipid, and protein metabolism [IDE) encodes a zinc metallopeptidase that degrades intracellular insulin, which could accelerate glycolysis, pentose phosphate cycle, and glycogen synthesis in the liver [PHF6) encodes a protein with 2 PHD-type zinc finger domains, and its function was associated with B\u00f6rjeson-Forssman-Lehmann syndrome, which is 1 of the syndromic obesities in humans [PROX1) could occupy promoters of metabolic genes on a genome-wide scale to control energy homeostasis [We further used the free ratio model to calculate the average Ka/Ks values and the branch-site likelihood ratio test to identify positively selected genes in the Marco Polo Sheep lineage. A total of 10 353 high-confidence single-copy genes were identified by InParanoid and MultiParanoid within the human, dog, taurine cattle, goat, sheep, and Marco Polo Sheep. We found that the Marco Polo Sheep has a regular level of the average Ka/Ks values, but containing more outliers Fig. . A totaldecrease ; purinertagonism . Four getabolism ; insulinhe liver ; PHD finn humans ; the proeostasis . In addieN) of Marco Polo Sheep shows a peak at \u223c1 Mya, followed by 2 distinct declines. The most recent decline involved at least a 7-fold decrease in eN, and it occurred \u223c60 000 years ago .Finally, we inferred the demographic history of the Marco Polo Sheep using the Pairwise Sequentially Markovian Coalescent (PSMC) model . ConsensCaprinae, and they will have relevance for the future conservation of the Marco Polo Sheep.In summary, the novel genome data generated in this work will provide a valuable resource for studying high altitude adaptation mechanisms within mammals and for investigating the evolutionary histories of the http://bigd.big.ac.cn/gsa). The assembly and annotation of the Marco Polo Sheep genome are available in the GigaScience database, GigaDB  [The sequencing reads of each sequencing library have been deposited at NCBI with the Project ID PRJNA391748, Sample ID SAMN07274464, and the Genome Sequence Archive  in BIG D_004002) . SupplemFigure S1. 21-mer-based analysis carried out to estimate the size of the Marco Polo Sheep genome.Figure S2. GC content distribution for the genomes of Marco Polo Sheep, goat, and sheep.Figure S3. FRCurve of 5 genome assemblies.Figure S4. The distribution of observed heterozgosity stats within Marco Polo Sheep genome.Figure S5. Counts of InDels in coding regions, showing an enrichment of multiples of 3 bases.Figure S6. Comparison of gene structure characteristics with those of other mammals.Figure S7. Comparison of gene structure characteristics of the 1:1 orthologs in the 5 mammals.Figure S8. Comparison of the repeat content in the intron regions among Marco Polo Sheep, sheep (Oar_v3.1), and goat (CHIR_1.0).Figure S9. Summary of the number of chromosomes to which a given scaffold of the Marco Polo Sheep genome could be aligned.Figure S10. Divergence between Marco Polo Sheep, sheep, and goat.Figure S11. Synteny relationship between Marco Polo Sheep and sheep.Figure S12. Synteny relationship between Marco Polo Sheep and goat.Figure S13. Density of breakpoints (number per million bases) in different regions of the genome.Figure S14. Phylogeny relationships between Marco Polo Sheep and other mammals.Figure S15. Demographic history of Marco Polo Sheep.Table S1. Summary of sequenced reads.Table S2. Estimation of genome size based on 21-mer statistics.Table S3. Statistics for the final assemblies of the Marco Polo Sheep genome.Table S4. Numbers of reads mapped to the assembled Marco Polo Sheep genome.Table S5. Summary of CEGMA analysis results.Table S6. Summary of BUSCO analysis results obtained by counting matches to 4104 single-copy orthologs .Table S7. The distribution of SNVs in the Marco Polo Sheep genome.Table S8. Genes located in the low-heterozygosity regions.Table S9. The distribution of InDels in the Marco Polo Sheep genome.Table S10. Prediction of repetitive elements in the assembled Marco Polo Sheep genome.Table S11. Classification of interspersed repeats in the assembled Marco Polo Sheep genome.Table S12. Data on all species used during the genome analysis.Table S13. Prediction of protein-coding genes in the Marco Polo Sheep.Table S14. Comparative gene statistics.Table S15. Functional annotation of predicted genes in the Marco Polo Sheep.Table S16. Summary statistics of non-coding RNAs in the Marco Polo Sheep.Table S17. Summary of synteny alignments.Table S18. Summary of breakpoints between Marco Polo Sheep, sheep, and goat.Table S19. Summary statistics of gene families in 9 species.Table S20. Candidate PSGs in the Marco Polo Sheep lineage.BLAST: basic local alignment search tools; bp: base pair; BUSCO: Benchmarking Universal Single-Copy Orthologs; CDS: coding sequence; CEGMA: a core eukaryotic genes mapping approach; CITES: the Convention on International Trade in Endangered Species of Wild Fauna and Flora; FRC: feature-response curve; InDels: insertion and deletions; Gb: giga bases; kb: kilo bases; GO: gene ontology; IUCN: the International Union for Conservation of Nature and Natural Resources; KEGG: Kyoto Encyclopedia of Genes and Genomes; LINE: long interspersed nuclear elements; Mb: mega bases; Mya: million years ago; Ne: effective population size; PSMC: pairwise sequentially Markovian coalescent; SINE: short interspersed nuclear element; SNV: single-nucleotide variants; SRA: Sequence Read Archive.The authors declare that they have no competing interests.K.W. and W.W. conceptualized the research project. K.W., W.W., and H.W. designed analytic strategy and coordinated the project. Y.W., H.W., and W.W. collected the samples and led the genome sequencing. Y.Y. and K.W. led the bioinformatics analysis. Y.Y., Y.W., and Y.Z. generated the genome assembly and the genome annotation. Y.Y., R.L., and L.C. finished the synteny analysis. Y.W., Y.Z., and G.Z. performed the gene family construction and the phylogeny analysis. Y.Y. and Q.Q. detected the PSGs and carried out data submission. Y.Y., W.W., and K.W. wrote the paper. All authors read and approved the final manuscript.GIGA-D-17-00160_Original-Submission.pdfClick here for additional data file.GIGA-D-17-00160_Revision-1.pdfClick here for additional data file.GIGA-D-17-00160_Revision-2.pdfClick here for additional data file.Response-to-Reviewer-Comments_Original-Submission.pdfClick here for additional data file.Response-to-Reviewer-Comments_Revision-1.pdfClick here for additional data file.Reviewer-1-Report-.pdfClick here for additional data file.Reviewer-2-Report-.pdfClick here for additional data file.Reviewer-2-Report-(Revision-1).pdfClick here for additional data file.Supplementary figures and tablesClick here for additional data file."}
+{"text": "Genomic prediction accuracy within a large panel was found to be substantially higher than that previously observed in smaller populations, and also higher than QTL-based prediction.TM Affymetrix SNP array. A high-quality consensus map was also constructed, allowing the linkage disequilibrium present in the germplasm to be investigated. Using the complete SNP array, genomic prediction accuracies were found to be substantially higher than those previously observed in smaller populations and also more accurate compared to prediction approaches using a finite number of selected quantitative trait loci. Multi-trait genetic correlations were also assessed at an additive and residual genetic level, identifying a negative genetic correlation between grain yield and protein as well as a positive genetic correlation between grain size and test weight.In recent years, genomic selection for wheat breeding has been widely studied, but this has typically been restricted to population sizes under 1000 individuals. To assess its efficacy in germplasm representative of commercial breeding programmes, we used a panel of 10,375 Australian wheat breeding lines to investigate the accuracy of genomic prediction for grain yield, physical grain quality and other physiological traits. To achieve this, the complete panel was phenotyped in a dedicated field trial and genotyped using a custom AxiomThe online version of this article (doi:10.1007/s00122-017-2975-4) contains supplementary material, which is available to authorized users. Plant breeding has been successful in producing significant yield gains in wheat since the beginning of the twentieth century  mapping studies, and applied in breeding programmes through marker-assisted selection (MAS)  assess the level of LD using a constructed high-quality genetic consensus map; (2) investigate genetic correlations between traits at an additive and residual genetic level; (3) investigate the improvement in selection accuracy that is achieved by incorporating a genomic relationship matrix into the analysis model; (4) investigate the improvement in genomic prediction accuracy that is achievable with a germplasm of this size and compare it to a simplified prediction approach based on selection of finite QTL.A panel of diverse bread wheat lines was provided by Australian Grain Technologies Pty Ltd (AGT). The panel consists of lines from preliminary yield testing (PYT) and advanced yield testing (AYT) stages of the AGT breeding programmes. Online Resource 1 summarises the panel and its subsets. The PYT-South and AYT-South sets are comprised of lines bred for southern Australia, and the AYT-Other set represents lines from the north eastern, eastern, and western growing regions. PYT material is a combination of FTM Affymetrix array containing 18,101 SNP markers. To build the customised array, SNPs were selected from previous variant identifications and SNP screenings in a range of genotyping platforms. The most prominent platform was a high-density AxiomTM array developed in the collaborative French BreedWheat project  consisting of 420,000 diverse SNPs. This was used to genotype a panel of approximately 200 wheat accessions from a range of geographic regions  for use in SNP selection. To achieve adequate and even coverage of the genome, SNPs were clustered into 20,000 groups based on a linkage disequilibrium threshold of TM 384 layout array from Affymetrix. Arrays were read using the GeneTitan Multi-Channel Instrument, and allele calls were made using AxiomTM Analysis Suite software by Affymetrix.Marker genotyping was performed using a custom AxiomTM Affymetrix array. The DH populations represent key families of Australian wheat germplasm and were chosen to maximise polymorphic markers across the genome. The individual SNP DH linkage maps were constructed using a synergistic combination of the R/qtl (Broman and Sen To provide an accurate assessment of LD between SNP markers in the AWP a consensus map was constructed using nine doubled haploid (DH) populations (Online Resource 1) genotyped on the custom Axiomkth marker in the jth linkage group of the ith bi-parental linkage map and jth linkage group of the consensus map. The optimal scaling factor jth consensus linkage group was then derived usingjth observed consensus linkage group and profiling jth linkage groups from the bi-parental linkage maps. This procedure was repeated for all 21 chromosomes and the consensus map was scaled accordingly. Assessment of LD was then based on these scaled positions within each of the chromosomes. Table The complete set of nine DH linkage maps (marker names and positions) were then used in MergeMap   3) occurUsing mixed model results, genomic best linear unbiased predictions of the additive genetic effects nt}gp in  were immTM Math Kernel Libraries. Given a new set of lines with marker data From the marker linear mixed model , predictgiven by . Inversikth marker outlier statistic iskth marker effect obtained directly from  and ith validation set were then predicted using To provide an informative comparison with genomic prediction results discussed in the plant research literature, the predictive ability of the fitted additive genotype model , as welled using  and markTM i7-6700K (4.00Ghz) with 64Gb RAM.All linear mixed modelling was conducted using the ASReml-R package . A negative relationship between grain protein and grain yield has frequently been identified at a phenotypic level  and residual error sources. The strong positive correlation between improvement in model fit and narrow sense heritability demonstrates that the additive relationship matrix improves the model by more accurately capturing additive genetic variance. Traits with a high proportion of additive genetic variance will, therefore, benefit most from the inclusion of the marker relationship matrix in the model.The efficacy of genomic prediction is typically assessed by means of cross-validation, where predictions of the validation set are correlated to the corresponding phenotypic estimated breeding values Supplementary material 2 (PDF 396\u00a0kb)Supplementary material 3 (XLS 1146\u00a0kb)Supplementary material 4 (CSV 425000\u00a0kb)Supplementary material 5 (XLS 26\u00a0kb)Below is the link to the electronic supplementary material."}
+{"text": "Gadus morhua), is known to vary genetically across the North Atlantic, Greenland, and Newfoundland. This genetic variation occurs both spatially and temporally through decades of heavy fishing, and is concentrated in three linkage disequilibrium blocks, previously defined by pedigreed linkage mapping analysis. Variation within these genomic regions is correlated with both seawater temperature and behavioral ecotype. The full extent and nature of these linkage groups is important information for interpreting cod genetic structure as a tool for future fisheries management.Atlantic cod . We show that pairwise linkage analysis among these SNPs is a powerful tool to detect linkage disequilibrium clusters by recovering the three previously detected linkage groups and identifying the 1031 genes contained therein. Across these genes, we found significant population differentiation among spawning groups in the Gulf of Maine and between Georges Bank and Gulf of Maine. Coordinated divergence among these genes and their differentiation at both short and long spatial scales suggests that they are acting as linked supergenes in local adaptation of cod populations.Differentiation between SNPs in linkage disequilibrium blocks is the major signal of genetic differentiation between all groups tested within the Gulf of Maine. Our data provide a map of genes contained in these blocks, allowing an enhanced search for neutral genetic structure for demographic inference and fisheries modeling. Patterns of selection and the history of populations may be possible to identify in cod using this description of linkage disequilibrium blocks and future data sets to robustly separate neutral and selected genetic markers.The online version of this article (doi:10.1186/s12864-017-3660-3) contains supplementary material, which is available to authorized users. Genomic islands of divergence are the result of selection on regions within the genome undergoing adaptive divergence between subgroups within a species , 2. ThesDrosophila species have shown that haplotype frequencies of these inversions change along environmental clines. For example, the In(3R)Payne inversion in D. melanogaster quickly evolved different frequencies across latitudes in Australia. Moreover, allele frequencies of the more equatorial haplotype of the inversion polymorphism have increased across the entire cline, suggesting a population adaptive response to increased temperature over 30\u00a0years [subobscura, where the more southerly haplotype of the O inversion has increased in frequency over the last 15 to 30\u00a0years in all European populations tested [Mimulus guttatus), populations show differential fitness across coastal and inland habitats based on a set of genes linked within an inversion [Within species, islands of divergence are associated with differential selection across variable environments. In the threespine stickleback within Lake Constance in Germany, for example, strong differentiation between migratory lake and resident stream ecotypes is associated with differentiation across a set of strongly linked genes . Chromos30\u00a0years \u201317. A sts tested . In the nversion . All in nversion \u201322.Gadus morhua. Borza et al. (2010) and Hubert et al. (2010) developed a panel of ~1500 SNPs derived from expressed sequence tags (EST) and used them to create linkage maps from pedigreed cod populations [The relationship between environmental conditions and genomic islands has also been studied in the Atlantic cod, ulations , 24. Theulations , 26. Theulations , as wellulations , 29. Usiulations , genomiculations  and the ulations . In all ulations . Most ofulations , 35.To date, the loci within the cod linkage groups have been identified through breeding studies and classic quantitative trait loci (QTL) approaches. Moreover, the search for neutral genetic differentiation, outside the known linkage groups, has been based on relatively few loci compared to genome-level data sets. Both the searches for selected genes and those that might reflect neutral genetic differentiation have not yet taken advantage of full-genome approaches . Here, wUsing complete genome sequencing, we describe a panel of 3,390,654 single nucleotide polymorphisms (SNPs) throughout the cod genome. Pairwise linkage analysis shows SNPs within 1031 genes falling into three cod \u2018supergenes\u2019, two of which show population divergence within the Gulf of Maine. Genes within these linkage blocks include DNA structural proteins and chromatin assembly genes, metabolic and catabolic genes, meiosis regulation and oocyte maturation genes, odorant receptors, egg coat structural proteins, heat shock proteins and many cell signaling genes that might be involved in environmental adaptation, habitat choice or mating. Once linkage disequilibrium blocks were identified and excluded from the analysis, our limited population data show no signs of neutral genetic differentiation in the three populations we sampled. Though our power to detect neutral structure is low with such few samples, comparison of many SNPs suggests that the signature of population differentiation is largely driven by shifts in the supergenes, possibly driven by present or past patterns of natural selection.Fin-clip samples from adult Atlantic cod in spawning condition were collected and stored frozen prior to preparation of DNA libraries. A total of 31 individuals were sampled: 10 from Georges Bank, and 21 from the Gulf of Maine. Within the Gulf of Maine, the sample was subdivided into two groups, based on spawning season: 10 cod were sampled from a winter spawning group, and 11 cod from a spring spawning group  in 500\u00a0\u03bcL of Digestion Mixture  at 50\u00a0\u00b0C for 1\u00a0h. After digestion, the samples were centrifuged at 13,000\u00a0rpm for 10\u00a0min; the supernatant was recovered, and then mixed with 50\u00a0\u03bcL of 3\u00a0M potassium chloride to precipitate the SDS. An equal volume of isopropyl alcohol was added to precipitate nucleic acids, and the pellet was washed with 70% ethanol. DNA/RNA was resuspended in 100\u00a0\u03bcL of 10\u00a0mM Tris-HCl pH\u00a08.0 and treated with 5\u00a0\u03bcL of 10\u00a0mg/mL RNase A. Another isopropyl alcohol precipitation was performed, then genomic DNA was isolated using AMPure XP beads. Total gDNA was resuspended and stored in TE buffer .Genomic DNA (gDNA) from each fin clip was prepared by digesting entire fin clips  following their standard protocol. Samples were individually barcoded to allow for multiplexing during sequencing. Each library was independently size selected using Ampure XP beads (0.6 volume beads to 1.0 volume DNA) and checked for adequate sizing via gel electrophoresis. Libraries were quantified by qPCR (KAPA Illumina Quantification Kit), then normalized and mixed in equimolar fashion.One set of samples, consisting of five individuals from each sampled location, was sequenced on an Illumina HiSeq 2000 using 100 base paired end reads. Each batch of five samples from the original sample groupings were mixed and multiplexed across a distinct eight-lane HiSeq flowcell, resulting in approximately 80x coverage across the genome (predicted size ~800\u00a0Mb). The remaining samples  were pooled and sequenced to less depth of coverage (~25x) on three runs of an Illumina NextSeq 500 using 150 base paired end reads.http://broadinstitute.github.io/picard) for downstream SNP discovery and analysis.Raw reads were trimmed of any residual adapter sequences, and low-quality base calls were removed using a baseline Phred score of 20 or less. Individual reads were retained if the length was\u2009>\u200970 bases, and reads were re-paired using Trimmomatic . Read paSNP discovery was performed on individual samples using FreeBayes version 0.9.10-3-g47a713e, using the default settings . Variantr2 value to remove any arbitrary sign introduced by intermediate calculations. To examine the genomic extent of linkage disequilibrium effects within each LG, mean r2 was calculated for every 250th SNP within each LG, as was pairwise FST. The edges of each linkage disequilibrium block (hereafter LD block) were located by finding the regions with high mean LD (mean r2\u2009>\u20090.1) and determining the first and last SNP with similar mean r2 values to the block. As there are 250 SNPs between the first high LD SNP and its subset neighbor and untested linked loci are located within this space, we placed the LD block edges on the SNPs adjacent to the ones with the first and last high LD value.We limited our analysis of linkage disequilibrium to linkage groups (hereafter LG) 2, 7, and 12, as these regions contain the three \u201cislands of divergence\u201d studied in this species. Within each LG, we extracted every 250th SNP, and performed pairwise linkage disequilibrium analysis using the LD function in the \u2018genetics\u2019 package (version 1.3.8.1) in R version 3.03 (R development core team 2015). We determined the correlation coefficient (as r) between the paired genotypes of all 31 individuals, and reported the Using the gene model information from the genome, we determined which genes were in each LD block, and performed enrichment tests of Gene Ontology (GO) categories of the genes within the resultant LD block using the weight01 algorithm within the \u2018topGO\u2019 package version 2.24.0  in R. AdST was calculated among the three populations using the wc function from the \u2018hierfstat\u2019 package [To test for population differentiation, we filtered all SNPs for those found within exons and created subsets for analyses. For exonic SNPs on linkage groups 2, 7, and 12 we used all available exonic SNPs, each partitioned into two groups: those within the LD block and those outside of the block. For the remainder of the genome, we created a subset of every 10th exonic SNP. We focused solely on SNPs within exons for this analysis, as we are interested in both neutral population structure and adaptive divergence between spawning sites/times, and SNPs within the coding regions of genes can be used to examine both processes. From this subset of SNPs, Weir and Cockerham\u2019s pairwise F package , 43 withST and PCA analysis above. To avoid potential confounding selection with population structure, SNPs within the linkage blocks on LG02, LG07, and LG12 were removed from this dataset and tested separately. With these sets of SNPs, we tested for population structure using the admixture algorithm NGSadmix [To test for neutral population structure, we utilized the same subsets of exonic SNPs used for FNGSadmix . For eacNGSadmix .2) and FST was calculated for every 250th SNP in LG02 (589 SNPs), LG07 (816 SNPs), and LG12 (559 SNPs). From these data, mean pairwise r2 values were determined for each SNP  in comparison with the surrounding SNPs (mean r2\u2009~\u20090.03 to 0.04). FST was less useful in determining the edges of LD blocks, as this varied over wide ranges on SNPs within linkage groups 2 and 7. Interestingly, FST and LD appear correlated in linkage group 12 , 9.3\u00a0Mb (LG07) and 11.6\u00a0Mb(LG12) in length (Table\u00a0r2\u2009>\u20090.1), while 170 of the 312 SNPs (~54%) in LG07 and 42 of the 246 SNPs (~17%) in LG12 were strongly linked. Our findings agree with regions of linkage disequilibrium identified previously by EST-derived SNPs [To capture potentially linked loci between the subset SNP loci, the starting base for each LD block was identified as the SNP immediately before the first SNP with a mean ved SNPs , 25\u201327 aved SNPs . Howeverp-adj\u2009<\u20090.01 after false-discovery rate correction) centered on genes involved with DNA/chromatin structuring . This number is slightly smaller than the total number of SNPs passing our filters  due to unused SNPs located on the few remaining unscaffolded contigs as well as mitochondrial DNA, yet still represents 97% of all identified SNPs. Proportions of each SNP type were calculated within each LD block, as well as the remaining SNPs not on linkage groups 2, 7, or 12  of all SNPs located on linkage groups 1 through 23 , linkage group 7 used 13,839 SNPs (4920/8919) and linkage group 12 used 8835 SNPs (4343/4492). The remainder of the genome used a subset of exonic SNPs . Mean pairwise FST for all loci tested  ranged between 0.0062 (spring spawners vs. Georges Bank) and 0.0297 (winter spawners vs. Georges Bank). Mean pairwise FST analysis of each SNP subset tested shows a trend of increased FST values within some LD blocks as compared to the exonic SNPs on the same linkage group. Mean pairwise FST for each comparison is shown in Table\u00a0The subsets of exonic SNPs used for population structure analyses contained a total of 54,030 SNPs. FST and LD along each of the chromosomes with linkage blocks. FST among all three populations was highly correlated with linkage disequilibrium in LG02 and LG12  as well as between winter spawners and Georges Bank cod  using a Kolmogorov-Smirnov two-sided test on the FST distributions . The LD block on linkage group 7 was also genetically divergent between winter spawners and Georges Bank cod . However, the highest differentiation was found between winter spawners and Georges Bank cod within the LD block on LG12 , with a broader, longer-tailed distribution of FST values. Mean pairwise FST values for exonic SNPs on the same linkage groups but not within LD blocks ranged between 0.0001 and 0.0036, and mean FST values for the remainder of the genome ranged between 0 and 0.0003, which is consistent with one large, outbreeding population.The LD block on linkage group 2 showed a significant increase in higher FST values when comparing spring spawning cod to the winter spawning cod  at small spatio-temporal scales within the Gulf of Maine and Georges Bank. Through sequencing and linkage disequilibrium analysis, we found three large clusters of linkage disequilibrium spanning multiple genes, in agreement with the known relative positions of these LD clusters from previously published studies of genomic \u201cislands of divergence\u201d in cod [de novo by deep sequencing of wild-caught individuals.In this study, we used whole genome resequencing and single nucleotide polymorphism discovery to describe patterns of genome-wide linkage disequilibrium in the Atlantic cod  categories, suggesting that the LD blocks on linkage groups 7 and 12 may not have functionally linked genes within them.PanI locus). When Kovach et al. [ST analyses show that divergence between the sampled populations is limited solely to the linkage blocks within LG 2, 7, and 12, and not throughout the genome. This suggests that some level of adaptive divergence may be responsible for the amount of differentiation historically seen in these genomic regions, though caution must be employed in interpretations of selection in this study due to a low per-population sampling size.Previous work has shown that genetic differentiation is present within the Gulf of Maine and Georges Bank populations of cod , 35. MosMytilus edulis in southern Long Island Sound is generated by natural selection each summer [Strong neutral genetic differentiation between populations implies very low genetic or demographic exchange between them, and as a result, neutral genetic boundaries in fisheries species have long been used to suggest the existence of different stocks , 61. Theh summer . Howeverh summer , 29, impPrevious studies of cod within this system  have detOur data set was designed to provide high coverage of whole genome sequences for relatively small numbers of individuals. Our analysis shows that large regions of select linkage groups appear to be under adaptive divergence between spawning groups. Furthermore, this genetic divergence does not extend to loci on the same linkage map groups within the Gulf of Maine. Together, these results suggest that extensive study of neutral gene divergence within the Gulf of Maine could be accomplished by choosing markers strictly outside known regions of linked polymorphisms. Furthermore, patterns of nucleotide differentiation among linked gene regions might suggest the way natural selection has acted on these regions, and if it continues to shape population genetics of modern cod.Additional file 1:List of genes found within LD block of LG02. GFF file containing the gene ID, positions, and gene names of the 294 genes found within this linkage disequilibrium block. (TXT 108 kb)Additional file 2:List of genes found within LD block of LG07. GFF file containing the gene ID, positions, and gene names of the 306 genes found within this linkage disequilibrium block. (TXT 116 kb)Additional file 3:List of genes found within LD block of LG12. GFF file containing the gene ID, positions, and gene names of the 437 genes found within this linkage disequilibrium block. (TXT 166 kb)"}
+{"text": "OE (overexpression), GTPase mutants RabF1Q93L (constitutively active) and RabF1S47N (dominant negative) lines show longer root growth than wild-type, rabF1 knockout and N-myristoylation deletion  complementary overexpression mutant plants under salt induced stress, which indicates that N-myristoylation of RabF1 is indispensable for salt tolerance. Moreover, RabF1 is highly expressed during senescence and RabF1OE lines were more tolerant of dark-induced senescence (DIS) than wild-type and rabF1.Arabidopsis small GTPase RabF1 (ARA6) functions in endosomal vesicle transport and may play a crucial role in recycling and degradation of molecules, thus involved in stress responses. Here we have reported that complementary overexpression lines RabF1 Small GTPase Rab proteins are mainly involved in membrane transport between organelles, through vesicle transport of cargo proteins to their destinations. They also play a role in activating effector proteins to regulate different stages of membrane transport within the organelles and promote the downstream application of other proteins for a variety of regulatory roles ,2. DurinArabidopsis thaliana RabF1 (ARA6) has been extensively studied for its regulatory parts in endosomal transport via the assembly of a distinct SNARE  complex in [SOS3  which contains a consensus N-terminal myristoylation sequence showed functional role in salt tolerance to salt [N-myristoylated protein in vivo and in vitro [Chara australis\u2014has been found to be localized at the plasma membrane, the TGN, and multivesicular endosomes (MVEs), suggesting involvement in endosomal transport [AtRabF2b suppresses abnormal phenotypes of atvps9a-2. In contrast, the overexpression of AtRabF1 with an equivalent mutation does not suppress abnormal phenotypes of atvps9a-2. AtRabF1 plays a specific role in transport from endosomes to the plasma membrane, while conventional Rab5 proteins, AtRabF2a and AtRabF2b mainly work in transport to vacuoles through endosomes [AtRabF1 is responsible for starch and sugar homeostasis through the function of Qua-Quine Stach gene (QQS), and the proliferation of Pseudomonas syringae pv. tomato DC3000 was repressed in AtRabF1 knockout mutant [In mplex in ,11,12. Implex in . An Arab to salt . RabF1 hin vitro ,15. RabFin vitro ,11. RabFransport . Moreoveransport . Overexpndosomes ,11,12,18t mutant .N-myristoylation site. Moreover, RabF1 is highly expressed during senescence, thus showed its protective effect in dark-induced senescence (DIS) leaves and photosynthetic parameter such as chlorophyll content.However, the complete picture of the function of RabF1 is not yet fully understood, although several recent works on RabF1 have highlighted the unique properties of the protein ,11,19. WRabF1, rabF1-1 and rabF1-2, were collected from NASC for genotypic and phenotypic analysis (rabF1-1 and rabF1-2 had T-DNA insertions disrupting the RabF1 gene and were homozygous for this mutation (RabF1 in both mutant lines (rabF1-1 mutant background with overexpressing RabF1-EYFP (RabF1OE), constitutively active 35S-RabF1Q93L-EYFP (RabF1Q93L), dominant negative 35S-RabF1S47N-EYFP (RabF1S47N) and 35S-RabF1\u03941\u221229-EYFP (RabF1\u03941\u221229) plants were used for salt stress. Expression level was checked by RT-PCR for two lines of each construct (http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi), RabF1 responds to osmotic stress, and recently, it was reported that RabF1Q93L lines were more tolerant to salt stress [OE, RabF1Q93L, RabF1S47N and RabF1\u03941\u221229 overexpression lines under conditions of 100 mM NaCl salt stress. Without added salt all lines exhibited similar root lengths i.e., no differences were observed  showed significantly longer root lengths  of RabF1 that generated the root phenotypic effect on salt stress condition. Therefore, it is more likely that the N-myristoylation ability of RabF1 is responsible for its salt stress related phenotype, as it was observed in the salt tolerance gene SOS3  where salt tolerance is dependent on N-myristoylation [In this study, both the RabF1er roots b. The rooylation .OsRab7B3 overexpression line. During the normal senescence process, over-expressing OsRab7B3 transgenic plants had yellowing leaves earlier than wild-type plants. Thus, it was suggested that OsRab7B3 is a positive factor, which promotes senescence in rice [RabF1 gene using a publicly available microarray database and Genevestigator v3 [RabF1 has a high expression level throughout all tissues, with a slightly higher level in senescing tissues. In senescing tissue, the expression value is ca. 14 Units, whereas in other tissues it is ca. 13 Units  exhibits increased expression throughout the progression of senescence and is highest at the final stage of senescence; SEN1 (senescence-associated protein1) expression has been found to be highest at the beginning of DIS and to be responsive to plant defence signals in Arabidopsis; RBCS1A  is associated with accumulation of Rubisco in Arabidopsis leaves and works additively with other Rubisco small subunit genes to yield sufficient Rubisco for photosynthesis and is expressed in leaf tissues throughout the developmental period, but is generally down-regulated in old plants; and LHCB1.3 (CAB1) a subunit of the light-harvesting complex II (LHCII) of Arabidopsis is generally expressed constitutively [Over recent decades several genes have been characterized with respect to their roles in natural and induced senescence studies. Some of them are used as good marker genes for senescence, for example; tutively ,30,31,32SAG12 and SEN1, were highly expressed in leaves from rabF1 mutants compared to the wild-type plants , the RabF1 GTPase function can be linked to the senescence phenotype  for ARA6/RabF1 (At3g54840), SAIL_98_E08 (rabF1-1) and WiscDsLox481-484C9 (rabF1-2) were obtained from NASC (http://arabidopsis.info/). The background line for all of these mutant lines, ecotype Col-0 , was used as the control for all experiments. The rabF1-1 T-DNA band was detected by LB1 (5\u2032-GCCTTTTCAGAAATGGATAAATAGCCTTGCTTCC-3\u2032) and atrabF1-1RP (5\u2032-AACGAGGCTCCAACAGTTACC-3\u2032), while the RabF1 gene was detected by atrabF1-1LP (5\u2032-TTGGAGAAACCGAATTGATTG-3\u2032) and atrabF1-1RP  and atrabF1-2RP (5\u2032-TTCACTCACATCAGAGCATGG-3\u2032), while the RabF1 gene was detected by atrabF1-2LP (5\u2032-TTTCCGAAGGTGTAATCATCG-3\u2032) and atrabF1-2RP , 26 cycles for SEN1 (senescence-associated protein 1), and 27 cycles for SAG12 (senescence-associated gene 12) genes. Specific primers to respective genes were as follows: Actin ; RBCS1A ; LHCB1.3 ; SEN1 ; SAG12 .An RNeasy plant mini-kit (Qiagen AB Sweden) was used to purify total RNA with an in-column DNase treatment. One microgram of total RNA/sample was used as a template while performing RT-PCR using an illustra Ready-To-Go RT-PCR Beads (0.5 mL tubes) kit ; the RT-PCR procedure was performed according to the manufacturer\u2019s instructions. For semi-quantitative reverse transcription-PCR, two mg of total RNA was used for the synthesis of the first-strand of cDNA with a RevertAid\u2122 H Minus First Strand cDNA Synthesis Kit  with oligo(dT)18. PCR cycle was terminated after 25 cycles for RabF1 coding sequence, corresponding to the Arabidopsis Genome Initiative (AGI) accession number At3g54840, was cloned with GatewayTM technology . The sequence was amplified by PCR from a complete cDNA clone (Gene bank accession no. BT002860). The PCR fragment was inserted into the pDONRTM vector and transferred into destination vectors . Two lines of each construct were used ; and for RabF1\u03941\u221229 .Plants were grown on MS plates (0.5% sucrose) for four days. Four-day-old seedlings were moved to vertical plates containing 100 mM NaCl, and the plates were transferred into long day conditions for another six days. Ten-day-old seedlings were used to measure root length. The root lengths of seedlings were measured and analysed using Image J 1.46r . The leaf was placed with its adaxial side facing up and incubated in a dark chamber at 22 \u00b0C for the indicated time. The chlorophyll was determined photometrically as previously described after DIS treatment ,41. The"}
+{"text": "This study investigated the characteristics of arm elevation via principal component analysis in symptomatic overhead athletes with scapular dyskinesis. One hundred-thirty-four overhead athletes with scapular dyskinesis  were evaluated by three-dimensional electromagnetic motion and electromyography to record the scapular kinematics (upward rotation/posterior tipping/exterior rotation) and muscle activation  during lowering phase of arm elevation. The results showed: (1) for pattern I and II, the first 3 principal component (PCs) explained 41.4% and 42.6% of total variance of movement; (2) the first PCs were correlated with MT, LT activity (r\u2009=\u20090.41~0.61) and upward rotation, posterior tipping (r\u2009=\u2009\u22120.59~\u22120.33) in pattern I, and UT, MT, SA (r\u2009=\u20090.30~0.70) activity in pattern II; (3) contour plots of muscle activity demonstrated that muscle activities varied with dyskinesis patterns. In summary, for the pattern I, the major characteristics are coactivation of MT and LT and corresponding scapular posterior tipping and upward rotation. For the pattern II, the major characteristics are coactivation of UT, MT and SA without corresponding scapular external rotation. Previous research has found that the prevalence of scapular asymmetry in scapular plane elevation and flexion did not differ in asymptomatic and symptomatic participants 3. Other studies have demonstrated that various shoulder disorders, including rotator cuff injuries, glenohumeral instability, and labral tears, are associated with scapular dyskinesis, with prevalence rates of 33 to 100%7. One of the reasons for these non-specific results may be the methods used to assess scapular dyskinesis/asymmetry. With only video/visual observation of dyskinesis, lack of adequate reliability and validity has been reported10. Recently, the classification of patterns of dyskinesis based on visual observation and palpation has been shown to have sufficient reliability11. It is likely that patterns of scapular kinematics are specific to shoulder disorder mechanisms.Scapular dyskinesis has been recognized as a non-specific response to a painful condition in the shoulder12. Excessive protraction during arm elevation may result in decreased subacromial space and impingement13. Investigation of scapular kinematics in subjects with and without shoulder injuries identified decreased scapular upward rotation, decreased scapular posterior tilt, and increased scapular elevation in the subjects with shoulder injuries16. It was proposed that sufficient scapular movements may prevent the greater tuberosity of the humeral head from passing smoothly under the acromion during humeral elevations and lead to impingement syndrome19. Additionally, excessive activity of the upper trapezius (UT) muscle combined with reduced activity of the lower trapezius (LT) muscle and serratus anterior (SA) muscle have been observed in patients with shoulder impingement21. Moreover, lack of control of the functional kinetic chain of the scapula can result in an imbalance of force transmission from the lower extremities and trunk to the upper extremities. This imbalance can also influence skilled shoulder function, as occurs in athletic performance24. Therefore, scapular movement strategies specific to patterns of dyskinesis should be identified to facilitate the assessment of clinical pathologies and physiological adaptions, as well as that of treatment or rehabilitation effects.Several mechanisms have been proposed to explain shoulder injuries in terms of scapular dyskinesis. Unlike a retracted position of the scapula , a protracted position of the scapula  decreases the subacromial space19. Understanding the movement characteristics in patients performing overhead sports may help the treatment and prevention of shoulder disorders in overhead athletes with scapular dyskinesis. The aim of this study was to assess the scapular kinematics and associated muscle activities in overhead athletes with different patterns of scapular dyskinesis to determine whether scapular characteristics were specific to patterns of dyskinesis.Lack of appropriate scapular kinematics and muscle imbalance may significantly influence movement in overhead athletes such as throwing in baseball or spiking in volleyball because of the cumulative effect of repetitive overhead motions and forces on shoulder complexThe scapular dyskinesis pattern distribution in raising and lowering phases were demonstrated as follow: 1) raising phase ; (2) lowering phase . Because most of participants did not show scapular dyskinesis in raising phase, we would only focus on lowering phase in further analyses. As mentioned above, data with pattern I\u2009+\u2009II dyskinesis were included and analyzed in both the pattern I and the pattern II groups. As a result, the classification of dyskinesis patterns identified 88 participants with inferior angle prominence of the scapula in pattern I and mixed pattern dyskinesis  and 110 participants with medial border of the scapula prominence in pattern II and mixed pattern dyskinesis  in lowering phase. There were no differences in the demographic data among the 2 patterns. Kinematics and EMG data are presented in Table\u00a0 raising For pattern I dyskinesis, 3 PCs explained a total of 41.4% of the variance of movement (KMO\u2009=\u20090.579). The first PC (17.5%) was correlated with MT, LT activity (r\u2009=\u20090.41~0.61) and upward rotation, posterior tipping (r\u2009=\u2009\u22120.59~\u22120.33); the second PC (13.4%), with MT activity and scapular external rotation (r\u2009=\u20090.40~0.67); and the third PC (10.5%), with LT activity (r\u2009=\u20090.44~0.57). For pattern II dyskinesis, 3 PCs explained a total of 42.6% of the variance of movement (KMO\u2009=\u20090.632). The first PC (17.0%) was correlated with UT, MT, SA activity (r\u2009=\u20090.30~0.70); the second PC (14.3%), with posterior tipping and LT (r\u2009=\u20090.55~0.72); and the third PC (11.3%), with MT activity and scapular upward rotation and external rotation (r\u2009=\u2009\u22120.6~0.47)  of the UT, MT, LT, SA shared the major components. This finding implies that these muscles are activated together as a force couple during arm movement.Principle component analysis of the scapular kinematics and muscle activities in different patterns of dyskinesis has not been reported previously. Theoretically, scapular kinematics and associated muscle activities during arm movements should share similar variance. However, our findings indicated that the first PC was described as primary mover/movement characteristic  in pattern I versus stability characteristic  for pattern II. External rotation was described as accessory movement characteristics in third PC in pattern II dyskinesis. Thus, change of muscle activation may not be obviously corresponding to scapular movement during arm elevation in pattern II dyskinesis. The results indicated that activation of the scapular muscles plays primary roles as stabilizers and secondary roles as movers of the scapula in patients with pattern II dyskinesis. This may explain why specific muscle activities and theoretically associated scapular kinematics were not found to be highly related in past research30. For medial border of scapula prominence (pattern II), UT, MT and SA were demonstrated as principal component without scapular kinematics included in the first PC, which indicated the role of stabilizers were essential in the UT, MT and SA in pattern II dyskinesis. For inferior angle prominence of scapula (pattern I), muscle function of MT, LT and shared variance in the first PC with upward rotation and posterior tipping can be explained to stabilize the axis for scapular upward rotation by MT and to function posterior tipping of the scapula by LT during arm elevation. However, the activation of LT cannot generate adequate scapular posterior tipping against the scapular inferior angle in pattern I dyskinesis. On the other hand, activated SA was not associated with generating scapular external rotation against the scapula medial border in pattern II dyskinesis.Scapular muscle activations are unique to each pattern of dyskinesis. MT and LT activities should be considered as characteristics of pattern I dyskinesis while UT, MT, SA activities can be considered as characteristics of pattern II dyskinesis. Additionally, the contour lines and plots expressed the functions of the observed or hypothetical muscle activations on two kinematic variables (upward rotation and posterior tipping). The UT has previously been shown to elevate the scapula and extend the neck, while the MT acts to retract the scapula. Additionally, the SA functions as scapular upward rotator and external rotatorIn clinical implication, evaluation of MT and LT muscle activations, as shared the same major variance with scapula kinematics, should be considered to correct inferior angle prominence of pattern I scapula dyskinesis subjects. On the other hand, without shared the variance with scapular kinematics in medial border prominence pattern II dyskinesis subjects, UT, MT and SA activations are likely to play major roles in stabilization of scapula instead of movement of the scapula. Instead of scapular muscle training, correction of medial border prominence dyskinesis may consider other factors, like soft tissue tightness or posture. Validation of this assumption should be further investigated.32. Third, movement artifacts and crosstalk cannot be excluded with the use of surface electrodes during dynamic movements. However, the 6-Hz filter and less than 10 k\u03a9 of skin resistance of the EMG data reduced the possible impact33. Placing electrode pairs 2\u2009cm apart can minimize the effect of crosstalk from other muscles34. Third, results from testing arm elevation in scapular plane may not represent results during functional movement or overhead sport activity. Additionally, participants were generally young and participated in overhead sports in this study. Sedentary or elderly people may have different scapular movement and muscle activation.The limitations of this study should be noted. First, the KMO values in both dyskinesis patterns are at the borderline of acceptance for PCA analysis. Further studies need to confirm our findings of movement characteristics in different scapular dyskinesis patterns. Second, the kinematics data were measured with humeral elevation of less than 120 degrees to reduce the error of the skin-based method. Validation studies have demonstrated that scapular motion can be accurately estimated for humeral elevation inferior to 120\u00b0 for elevation in the sagittal planePCA demonstrated that the three PCs accounted for 41% and 43% of variance for pattern I and II dyskinesis, respectively. For the inferior angle prominence dyskinesis, the major characteristics are coactivation of middle and lower trapezius and corresponding scapular posterior tipping and upward rotation. For the medial border prominence dyskinesis, the major characteristics are coactivation of upper/middle trapezius and serratus anterior without corresponding scapular external rotation.This study was a cross-sectional study. All participants performed arm elevation in the scapular plane. The characteristics of the scapular kinematics and associated muscle activities specific to patterns of dyskinesis were identified.One hundred thirty-four subjects with unilateral shoulder pain and scapular dyskinesis were recruited for this study. The inclusion criteria were (1) age of 20 to 40 years old, (2) with unilateral shoulder pain of less than 5 on a 10-point visual analog scale during arm elevation and (3) demonstration of inferior angle or medial border of scapula prominence during arm elevation. All of the participants performed recreational overhead sports . Exclusion criteria were a history of stroke, diabetes mellitus, rheumatoid arthritis, rotator cuff tear, surgical stabilization of the shoulder, osteoporosis, or malignancies in the shoulder region. Participants who had pain or disorders of the cervical spine, elbow, wrist, or hand, who had pain radiating from the shoulder to the arm, or who could not elevate their arms to 150 degrees were also excluded. They received written and verbal explanations of the purposes and procedures of the study. All subjects gave written informed consent to the Research Ethics Committee of the National Taiwan University Hospital  following a complete explanation of this study, and in accordance with the Declaration of Helsinki.et al. validated scapular kinematics between skin-based sensor and bone-pinned methods and confirmed that the skin-based method is valid when arm elevation is below 120 degrees31. The details of the methodology can be found in a previous paper26. Three sensors were placed in locations where the skin motion artifact was minimized . Anatomic landmarks  were palpated and used for subsequent receiver mounting and landmark digitization. The investigator with 7 years\u2019 clinical experience in musculoskeletal palpation did the anatomic landmarks palpation.The Polhemus 3Space FASTRAK system , an electromagnetic-based motion analysis system, was used for collecting 3-dimensional kinematic data of the scapula. Karduna The sEMG assembly comprised pairs of silver chloride circular (recording diameter of 10 mm) surface electrodes  with an interelectrode (center- to center) distance of 20 mm, and a Grass AC/DC amplifier  with a gain of 1000, a common mode rejection ratio of 86\u2009dB at 60\u2009Hz, and a bandwidth (\u22123 dB) of 10 to 500\u2009Hz. The sEMG data were collected at 1,000\u2009Hz/channel using a 16-bit analog to digital converter . Surface EMG electrodes were placed on the upper trapezius , middle trapezius , lower trapezius  and serratus anterior  of the involved shoulder. The referenced electrode was placed on the ipsilateral clavicle. Maximal voluntary isometric contraction (MVIC) was tested and used to normalize the sEMG data during the task . The MVICs were collected for 5\u2009seconds in each of 3 trials, with 1\u2009minute of rest separating trials.11. The inter-rater reliability of the classification test was moderate to substantial 11. Two single patterns with inferior angle of the scapula prominence (pattern I) and medial border of the scapula prominence (pattern II) and the mixed pattern with combination of the two single patterns were selected in this study.Visual combined palpation was used to classify scapular position and movement patterns (single patterns or mixed patterns) in both the raising and the lowering phases, modified by Kibler\u2019s method35. Scapular orientation relative to the thorax was described using a Euler angle sequence of rotation about Zs (protraction/retraction), rotation about Y\u2019s (downward/upward rotation), and rotation about X\u2019s (posterior/anterior tipping). Full bandwidth sEMG data captured by the data acquisition software  were reduced using a root mean square (RMS) algorithm to produce sEMG envelopes with an effective sampling rate of 50 samples. Then the data were normalized to the MVIC trials. The EMG data of each muscle were the average of the middle 3 trials. The mean sEMG amplitude of each muscle, reported as a percentage of MVIC, was used to assess the activity of the muscle. For the analysis, 4 levels at 30\u00b0, 60\u00b0, 90\u00b0, and 120\u00b0 of arm elevation for kinematics and 5 levels during 0\u201330\u00b0, 30\u201360\u00b0, 60\u201390\u00b0, 90\u2013120\u00b0 and above 120\u00b0 of arm elevation for EMG data were used.Kinematics and sEMG data were collected during arm elevation in the scapular plane. Participants were asked to elevate the arms that the dumbbells in each hand weighed 2.3\u2009kg (5\u2009lb) or 1.4\u2009kg (3\u2009lb), depending on each participant\u2019s ability to elevate the arm. In general, male participants lifted 5\u2009lb and female participants used 3\u2009lb of resistance. Raw kinematic data were low-pass filtered at a 6-Hz cutoff frequency and converted into anatomically defined rotations. In general, we followed the ISB guidelines for constructing a shoulder joint coordinate system36. Additionally, the characteristics of movements of the dyskinesis patterns were demonstrated by contour plots of 4-muscle EMG with scapular upward rotation and posterior tipping in the X and Y axes, respectively.The Statistical Package for the Social Sciences (SPSS) 17.0 was used for data analysis. Descriptive statistics of kinematics and EMG data were calculated. Data of participants with pattern I\u2009+\u2009II dyskinesis were included and analyzed in both the pattern I and the pattern II groups. To identify the characteristics of each dyskinesis pattern, principal component analysis (PCA) was performed on all data  for each dyskinesis pattern with varimax transformation. This analysis yielded the amount of variance explained by each principal component, and also the correlations between principal components (PCs) and the 3-dimensional scapular kinematics and 4 muscles EMG. The Kaiser-Meyer-Olkin (KMO) Measure of sampling adequacy was used to test the appropriateness of using PCA"}
+{"text": "A large share of agriculturally and horticulturally important plant species are polyploid. Linkage maps are used to locate associations between genes and traits by breeders and geneticists. Linkage map creation for polyploid species is not supported by standard tools. We want to overcome this limitation and validate our results with simulation studies.We developed PERGOLA, a deterministic and heuristic method that addresses this problem. We show that it creates correct linkage groups, marker orders and distances for simulated and real datasets. We compare it to existing tools and demonstrate that it overcomes limitations in ploidy and outperforms them in computational time and mapping accuracy. We represent linkage maps as dendrograms and show that this has advantages in the comparison of different maps.PERGOLA can be used successfully to calculate linkage maps for diploid and polyploid species and outperforms existing tools.The online version of this article (doi:10.1186/s12859-016-1416-8) contains supplementary material, which is available to authorized users. Polyploidy describes the condition of having more than two chromosome sets and is common in flowering plants. A large share of agriculturally and horticulturally important plant species are polyploid. Among them are wheat and sugar cane, which are the most planted  and most fecund  crops, respectively [Linkage mapping describes the process of calculating the genetic relation between markers. The general concept is used for decades and established in the fields of plant and animal breeding. During meiosis recombinations occur along the chromosomes. Investigating these provides information about the genetic distances of markers (e.g. SNPs). Comparing recombinations between multiple offspring in a mapping population (e.g. F2 or backcross) allows to calculate similarities between markers. The more similarly two markers co-segregate the higher the linkage is between them and the more likely it is that they are located closely together \u20135. Consehttps://cran.r-project.org/package=pergola) [We developed PERGOLA, a linkage mapping tool for polyploids implemented as R package (pergola) . We demoPERGOLA is much faster than existing mapping tools and therefore also provides an alternative for linkage mapping in diploids.We applied PERGOLA to simulated and real datasets of varying ploidy levels. We simulated a hexaploid F2 population with 100 offspring with PedigreeSim . The inpThe allotetraploid data set was obtained from a peanut experiment with 89 offspring . For ourSimilarity of genotypes was used to predict recombination frequencies and linkage between markers. This information was then used to estimate linkage maps. Linkage mapping was divided into the steps grouping, ordering, and spacing. The former two are visualized in Fig. \u03b8 of two markers (e.g. SNPs) is the frequency of one crossover between them. The concept of linkage mapping differs for polyploids because the calculation of recombination frequencies is more complex than for diploids [\u03b8 between two markers m and n of ploidy p with Genetic recombination describes the exchange of DNA between two chromosomes during meiosis. The recombination frequency diploids . In the diploids \u201314. All A and B are sums of recombination events for the two possible allelic configurations defined as mi and ni are the allele counts for individual i for each pair of markers m and n. For instance the allele counts at tetraploid loci AAAA, AABB and ABBB would be 4,2 and 1, respectively. The two different allelic configurations account for the unknown parental origin of the alleles. Two markers m=AAAA (4) and n=AAAT (3) indicate Am,n=1 and Bm,n=3 recombination events and where  mapping .Our heuristic approach is fast and ignores some biological details (e.g. double reduction) . PERGOLAThe heuristic calculation of recombination frequencies overestimates linkage because it always assumes the lowest possible number of recombinations. This is not necessarily the actual number of recombination events. For instance AABB/AABB can have 0, 2, 4 or even more recombinations due to double crossovers, but we always approximate 0 in that case. If two markers are closely linked we assume no recombination and the approximation is correct. For distant markers the genotypes are different for a large proportion of the population by chance and their A linkage group is a subset of co-segregating markers. Ideally each linkage group represents one chromosome, but that cannot always be achieved . Markersdistance . Single mentclass2pt{minimn\u22121 internal nodes. The decision whether a node is flipped or not is based on the best global ordering. Each tree has 2n\u22121 possible orderings where n is the number of leafs. OLO finds an order that minimizes the sum of adjacent recombination frequencies (SARF) with a worst-case complexity of O(n4) [s=, the SARF criterion is defined as PERGOLA applies the optimal leaf ordering (OLO) algorithm to determine the marker order within each linkage group . First, f O(n4) . Assuminai and its adjacent SNP ai+1 [i and i+1 identify the SNPs in order s. OLO includes an early termination step, which avoids unnecessary calculations, if the result cannot be improved. That usually reduces the runtime, but the worst case remains unchanged.where NP ai+1 . The subl is indicated as subscripted number: Given high marker density datasets the marker order according to the SARF criterion is not always unique. Multiple close markers or single distant markers can result in varying linkage maps with the same SARF value. Subsequently the same input leads to different results, which is the definition of a non-deterministic algorithm. In these cases we stepwise extend the SARF criterion to neighboring markers until the ordering is resolved unambiguously. For real-world datasets our extension leads to unique results, but theoretically it is possible to construct worse-case scenarios, where only ambiguous orders can be found. This size of the neighborhood C and D in Table Two markers have equal distances to their neighbors, but a smaller distance between each other. These can be swapped without changing the SARF value , s2= and s3= have the same SARF1 value of 15. Extending the neighborhood n to 2 leads to SARF2(s1)=32, SARF2(s2)=36 and SARF2(s3)=34. Thus, s1 is the best order and s2 and s3 can be discarded.An example is provided in Table The determinism nature of our method refers to the outcome of the marker ordering step. The general process of linkage mapping is still a stochastic approximation of the real linkage based on an SNP markers.For spacing we applied the Haldane mapping function  to the rJoinMap\u00ae; 4.1 by Kyazma\u00ae; B.V., Wageningen, Netherlands  is one oMapMaker Macintosh version 2.0  was usedR/qtl version 1.33-7 is an R package that supports linkage mapping .We compare PERGOLA with three other linkage mapping tools: n2 pairs of markers. The second method is cophenetic correlation [Further we recalculate the maps of hexaploid simulated data and autotetraploid potato data. In addition to visual comparison we applied two computational correlation measurements. Our first method to compare two dendrograms is the Goodman-Kruskal-gamma index . It calcrelation . It measWe applied PERGOLA to simulated hexaploid datasets. First, the datasets were randomized to remove any prior information (e.g. haplotypes) that is not available for a real dataset. Second, we calculated the pairwise recombination frequencies for all markers. An example is visualized in Fig. The results should be interpreted with caution because the data is simulated. PedigreeSim simulates the genotypes based on one model, which has been intensively discussed in the community , 27, 28.We applied PERGOLA to allotetraploid genotypes of a peanut crossing population . The datHowever, the diploid nature of the peanut dataset allowed us to compare the results and performance of PERGOLA to linkage mapping tools, which do not support polyploids. We selected MapMaker, JoinMap\u00ae; and R/qtl. MapMaker was used by the authors of the peanut dataset  and the Comparing linkage mapping tools is difficult because depending on the parameter settings each tool can output different maps. We used the default parameters of each tool and the Haldane function to calculate the spacing between the markers. The results gave a general impression of the performance and should not be overinterpreted. All tools could be applied in multiple ways and lead to different maps. The motivation of the comparison was to find out if PERGOLA performs worse than the other tools for a diploid-like data set. For polyploid data sets the other tools can not be applied and PERGOLA is the method of choice.orderMarkers, Calculate map and sortLeafs, respectively. R/qtl is the slowest one and took 16 min and 47 s. JoinMap\u00ae; had a similar runtime of 14 min and 47 s. PERGOLA was the fastest method and took 0.011 s. The better performance results from the use of the OLO algorithm compared to the sliding window approach in R/qtl and the large overhead in JoinMap\u00ae;. Runtime performance is important because linkage maps have many parameters (e.g. filter criteria) that influence the result. Faster methods allow for systematic optimization of linkage maps. For instance, usually the number of chromosomes is known. If a parameter setting results in a number of linkage maps that differs from the expected chromosome number, the setting should be changed. The runtime of PERGOLA allows for computationally expensive resampling methods (e.g. jackknifing or bootstrapping) to be used. That can improve the interpretability of linkage maps and related QTL detections.The runtime of MapMaker is unknown because the authors of the peanut dataset did not provide computation times. Data preparation is unique for every tool and depends on the format of the given data. Thus, we excluded that step from the time measurement. Linkage grouping was at most a matter of seconds for all methods and has been ignored. The computational time comparison focuses on marker ordering because it is the most expensive and distinctive step. In R/qtl, JoinMap\u00ae; and PERGOLA these are the commands In PERGOLA and JoinMap\u00ae; we manually selected ten linkage groups because they were suggested in the grouping step. R/qtl created these linkage groups automatically. We used the Haldane mapping function in all tools. R/qtl applies a sliding window approach where all possible permutations of markers are calculated and compared. That approach leads to locally optimized solutions, but can fail to find the best marker order within the linkage group. The default window size is seven, but performs better if the window size is increased. However, this would lead to even slower computation times and was not tested. JoinMap\u00ae; performs similar to R/qtl, but uses a more sophisticated approach. It calculates and compares different solutions internally and outputs the best solution to the user.To compare the general linkage maps we transformed all maps into dendrograms. We aligned the chromosome orders and orientation between the maps. Dendrograms maintain the grouping, ordering and spacing of the maps and allow manual  and computational comparisons. The root line connects the multiple linkage groups at the same height. In our implementation of PERGOLA its height is 0.2 times higher than the highest connection within the linkage groups. It does not reflect their similarity, but supports the readability of the map. The marker order and spacing in the map equal the leaf order and branch height in the dendrogram. We created tanglegrams from the dendrograms for a pairwise comparison of all maps . They alThe pairwise tanglegrams show that the maps are generally similar. All maps consist of ten linkage groups, mainly containing the same markers. The maps by R/qtl and MapMaker contain five and six markers more than PERGOLA and JoinMap\u00ae;, respectively. This information is not illustrated in the tanglegrams. The markers have been filtered out and could not be integrated into the ten linkage groups. The total number of markers in the dataset is 459. It is unknown how many have been filtered out for the MapMaker map because they have not be provided together with the map. However, the marker density is not significantly reduced by the filtering. The quality of the map is more important, than a small number of additional markers. Thus, noisy markers should be filtered out rather than creating large gaps in a linkage group.In our experiment, the Goodman-Kruskal-gamma index for all pairs of maps is almost 1, indicating perfect correlation. This contradicts the observations we made in the tanglegrams where we observe differences between the linkage maps. Marker grouping has a much larger effect on the Goodman-Kruskal-gamma index than ordering or spacing and if many markers are grouped similarly, differences in the latter steps are not represented by it. We conclude that the Goodman-Kruskal-gamma index is too insensitive for the allotetraploid data set. This is also supported by our simulation study. In contrast the cophenetic correlation coefficient provides reasonable measurements between the maps, as shown in Table The results show that PERGOLA calculates linkage maps in a fraction of the time of the other methods. That makes it not just a useful method for polyploid crops, but also as an alternative for diploid datasets. The heuristic approach of the recombination calculation leads to minor rearrangements in the grouping. They can be neglected given the overall map similarity and performance advantages of PERGOLA. The tanglegrams suggest a higher similarity between R/qtl, JoinMap\u00ae; and MapMaker because the grouping is identical. On the contrary, the cophenetic correlation indicates that the map by JoinMap\u00ae; is more similar to the PERGOLA map. That supports our aforementioned hypothesis, that there is not one correct linkage map and we can only estimate the biological situation from different directions. Depending on the input data, filtering parameters, linkage mapping tools and validation criteria, multiple maps are valid. Currently, it is impossible to discard one map or choose one over the other.We conducted a simulation study to validate the results of PERGOLA and R/qtl for diploids where the real map is known. JoinMap\u00ae; was excluded because it is limited to a graphical interface and could not be automatically applied to the hundreds of simulated datasets. We used two different numbers of markers (10 and 20 per chromosome) and three population sizes , which resulted in 6 different combinations per tool. Each combination was repeated 100 times. The input linkage maps consisted of two chromosomes and randomly spaced markers. We compared the reference maps with the calculated ones using cophenetic and Goodman-Kruskal correlation. The mean values and standard errors of the cophenetic correlation are shown in Fig. p-value of 0.01 and similar results as the previous permutation test (Additional file We did another map comparison with the second real data set, a population of 190 offspring from an autotetraploid potato cross . The autPERGOLA allows the creation of linkage maps for polyploid crops. The application to simulated data showed that it leads to reasonable linkage maps. Further, we demonstrated that it can be successfully applied to real datasets with different polyploidy types. PERGOLA outperformed existing programs for diploids in terms of computation time and mapping accuracy. The transformation of linkage groups into a two dimensional dendrogram has been shown to be a valuable alternative to the currently dominating bar scheme. It is more structured and allows to evaluate the three steps of grouping, ordering and spacing separately. The Goodman-Kruskal index is too insensitive to compare linkage maps and the cophenetic correlation index should be used instead. Taken together, PERGOLA is a valuable extension not only to the polyploid genetic toolbox, but for geneticists in general."}
+{"text": "Clostridium pasteurianum is emerging as a prospective host for the production of biofuels and chemicals, and has recently been shown to directly consume electric current. Despite this growing biotechnological appeal, the organism\u2019s genetics and central metabolism remain poorly understood. Here we present a concurrent genome sequence for the C. pasteurianum type strain and provide extensive genomic analysis of the organism\u2019s defence mechanisms and central fermentative metabolism. Next generation genome sequencing produced reads corresponding to spontaneous excision of a novel phage, designated \u03c66013, which could be induced using mitomycin C and detected using PCR and transmission electron microscopy. Methylome analysis of sequencing reads provided a near-complete glimpse into the organism\u2019s restriction-modification systems. We also unveiled the chief C. pasteurianum Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) locus, which was found to exemplify a Type I-B system. Finally, we show that C. pasteurianum possesses a highly complex fermentative metabolism whereby the metabolic pathways enlisted by the cell is governed by the degree of reductance of the substrate. Four distinct fermentation profiles, ranging from exclusively acidogenic to predominantly alcohologenic, were observed through redox consideration of the substrate. A detailed discussion of the organism\u2019s central metabolism within the context of metabolic engineering is provided. Clostridium pasteurianum is an obligately anaerobic, endospore-forming soil bacterium that is emerging as an attractive industrial host owing to its unique fermentative metabolism12C. pasteurianum on conventional sugars, such as glucose or sucrose, yields a butyric acid fermentation characteristic of the clostridia557C. pasteurianum has drawn significant attention to the organism in light of the recent growth in global biodiesel production, which has generated an abundance of crude glycerol, now considered a waste-stream, rather than a valued co-product9101112Klebsiella, Citrobacter, and ClostridiumE. coliC. pasteurianum is the only organism known to convert glycerol as a sole carbon and energy source into butanolC. pasteurianum glycerol fermentation is currently one of the most poorly understood glycerol-to-biofuel processes.Bacteria defend against bacteriophages (phages), plasmids, and other invading nucleic acids through the use of primitive cellular immune systems. The chief prokaryotic defence mechanisms are restriction-methylation (RM) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems, both of which utilize endonuclease-mediated attack and afford host cells with self versus non-self discrimination17C. pasteurianum to produce butanol from crude glycerol232829C. pasteurianum3132C. pasteurianum (strain BC1) (unpublished data) and two completed genome sequences have recently been announced for the type strain [ATCC 6013 (DSM 525)]35C. pasteurianum is paramount to the advancement of this organism for biotechnological valorization of crude glycerolC. pasteurianum type strain ATCC 6013 (DSM 525) and provide detailed analysis of the organism\u2019s unique fermentative metabolism. We show that the organism exhibits a highly flexible, branched central metabolism, where product distribution varies considerably between carbon sources and is dictated chiefly by redox characteristics of the fermentation substrate. To stimulate a more thorough understanding of C. pasteurianum genetics and general biology, we also provide insight into the organism\u2019s defence mechanisms through analysis of the restriction-modification methylome and identification of a chief Type I-B CRISPR system. Finally, we provide evidence that the genome of C. pasteurianum encodes an intact prophage that is spontaneously excised under standard growth conditions and induced using mitomycin C. The detailed genomic analysis of C. pasteurianum presented herein will contribute to our understanding of substrate utilization and biofuel production by this promising organism, as well as provide a genetic and metabolic framework for rational strain engineering.A small number of studies have investigated the potential of C. pasteurianum comprised of 12 contigsC. pasteurianum genome and a concurrent genome sequencing effortC. pasteurianum. It is possible that contig 2 (13.2\u2009kb) corresponds to sequence generated directly from the excised phage (i.e. the phage genome) or phage-like product. Further, the 28\u2009bp overlap sequence between the 6.2\u2009kb and 7.0\u2009kb regions in contig 2 was found to be preserved within the 5\u2032 terminus of the 6.2\u2009kb region and the 3\u2032 terminus of the 7.0\u2009kb region of contig 1 (see attP) and the corresponding 28\u2009bp sites within the 6.2\u2009kb and 7.0\u2009kb regions of contig 1 are the respective left (attL) and right (attR) phage attachment sites within the chromosome of C. pasteurianum (i.e. prior to phage excision) , whereas subsequent excision of the prophage proceeded via recombination between attL and attR. Based on this hypothesis, phage excision should result in a single chromosomal \u201cscar\u201d site (attB) within the phage-less C. pasteurianum genome. We confirmed this hypothesis by successfully amplifying the phage-less bacterial chromosome region (attB), as well as products corresponding to the unexcised prophage (attL and attR) and the excised circular phage product (attP)  . Chromost (attP) . All foue genome . FurtherC. pasteurianum prophage occurred spontaneously in the aforementioned analysis, prophage excision can be artificially induced using ultraviolet irradiation or mitomycin C, a potent antibiotic38C. pasteurianum to varying concentrations of mitomycin C  and monitored culture turbidity for signs of phage release and cell lysis, characterized by a dramatic decline in OD600\u22121 mitomycin C exhibited a dramatic decrease in OD600 approximately four hours following induction. Imaging via TEM of the 5.0\u2009\u03bcg\u2009ml\u22121 mitomycin C phage lysates revealed an abundance of phages with long tails possessing short terminal fibers (Siphoviridae family of bacteriophagesC. pasteurianum chromosome (29.9%). A total of 52 protein-coding genes, possessing homology to both phage and bacterial genes, are annotated in the phage genome, 48 of which are transcribed in the same direction  and RNA polymerase sigma factor (sigE), indicating a potential relationship between \u03c66013 and sporulation of C. pasteurianum. Spore titers have been reported to differ between seemingly identical strains of C. pasteurianum obtained from different culture collections (ATCC and DSM)Although activation of the l fibers . We measirection . Phage gC. pasteurianum genome sequences35C. pasteurianum contig gap is part of a large 93.5\u2009kb chromosomal duplication , which was found to be approximately 22\u2009kb larger than previously reported tion see . This duC. pasteurianum genome is 4,444,510\u2009bp. Our current draft genome is comprised of a single 4,373,654\u2009bp contig possessing a GC content of 29.9% (C. pasteurianum is most closely related to (from most related to least related) C. acidisoli, C. akagii, C. arbusti, and C. carboxidivoransC. acetobutylicum, C. botulinum, C. autoethanogenum, and C. ljundahlii are the closest relatives with fully-sequenced genomes 35C. pasteurianum sequencing projects is provided in C. pasteurianum genome is predicted to possess 3,803 protein coding genes, 81 tRNA genes, and 30 rRNA genes  flagella loci were identified in the genome and together encode all core flagellar structural genes, including genes involved in filament [fliC ], hook [flgE (CP6013_1358)], and rod  formation. A collection of chemotaxis genes are located immediately downstream of the flagellar loci, while motA (CP6013_3023) and motB (CP6013_3022) chemotaxis genes are encoded at a distant location in the genome. C. pasteurianum is believed to be the first isolated nitrogen-fixing organism and nitrogen fixation from cell-free lysates was first observed using C. pasteurianum2 into ammonia using ferredoxin as electron donor and ATP to drive the reactionnif), including genes encoding the MoFe dinitrogenase  and Fe dinitrogenase reductase  protein componentsC. pasteurianumnifE (CP6013_1736), nifN-B (CP6013_1735), nifC (CP6013_1733), nifV1 (CP6013_1731), and nifV2 (CP6013_1732) genes involved in nitrogenase assembly and Mo/Fe insertion. It has been reported that C. pasteurianum possesses a total of six nifH and nifH-like genes48nifK (CP6013_3827) and nifE (CP6013_3826 and CP6013_3832) genes, as well as two nifB or nifB-like genes (CP6013_3829 and CP6013_3830). Interestingly, three nifH-like genes, nifH5 (CP6013_2037), nifH3 (CP6013_3385), and nifH4 (CP6013_3825), are found adjacent to genes encoding putative transposases . NifH3 (CP6013_3385) is the only NifH-like protein that is not transcribed under nitrogen fixation conditions, as it exemplifies a Mo-independent Fe-nitrogenaseanfDGK; CP6013_3387\u20133389) are close to the Fe dinitrogenase reductase gene . In addition to Mo-dependent (Nif) and Mo-independent (Anf) nitrogenases, it has been reported that C. pasteurianum harbors a vanadium-dependent nitrogen-fixing (Vnf) systemvnfD (CP6013_1748), vnfG (CP6013_1747), and vnfK (CP6013_1746), which are positioned adjacent to nifH6 (CP6013_1749).C. pasteurianum and guide future genetic work with this organism, we analyzed the organism\u2019s methylome using SMRT sequencing dataClostridium genusC. pasteurianum. This system, designated CpaAI (5\u2032-CGCG-3\u2032)To gain further insight into the cellular defence mechanisms of C. pasteurianum is predicted to encode a total of eight methyltransferase genes, which ostensibly includes the single Type I RMS system, two Type II RM systems, a single Type II protein with dual R\u2009+\u2009M activities, and two lone Type II M proteins lacking associated R activity. The CpaAII Type I RMS system (5\u2032-AAGNNNNNCTCC-3\u2032) is encoded by genes CP6013_0336, CP6013_0337, and CP6013_0338, respectively, while genes CP6013_2557 and CP6013_2558 represent the CpaAI (5\u2032-CGCG-3\u2032) Type II RM proteins, respectively. Whereas gene CP6013_0098 corresponds to the R protein of the Type II CpaI system (5\u2032-GATC-3\u2032), three consecutive adjacent genes (CP6013_0095\u20130097) could putatively encode the associated M activity. Based on REBASEC. pasteurianum appears to restrict DNA substrates possessing CpG (5\u2032-CG-3\u2032) or GpC (5\u2032-GC-3\u2032) methylation based on plasmid transformation assaysC. pasteurianum. While wild-type E. coli cleaves DNA substrates containing m5CC. pasteurianum could not be deciphered based on the genomic analysis performed in this study.Based on analysis by REBASEC. pasteurianum for putative CRISPR arrays and Cas-encoding genes using CRISPRfinderC. pasteurianum genome, the 30\u2009bp direct repeat sequences between the two CRISPR loci are identical, suggesting that the same set of Cas proteins are employed for spacer acquisition and interference. The 37-spacer locus was found to be associated with several cas genes (CP6013_0534\u20130541), while no such genes could be identified in proximity to the 8-spacer array. Based on the proposed classification of CRISPR-Cas systemsC. pasteurianum CRISPR system belongs to the Type I-B subtype owing to the presence of the signature Type I cas3 gene (CP6013_0538) and Type I-B cas8b (csh1) gene (CP6013_0535). Furthermore, the C. pasteurianum CRISPR-Cas system possesses the same cas gene arrangement (cas6-cas8b-cas7-cas5-cas3-cas4-cas1-cas2) found in other prokaryotic Type I-B systemscas genes are transcribed in the same direction and are located downstream of the 37-spacer CRISPR array. Analysis of similar Type I-B systems suggests that transcription occurs in the same direction as the cas genes, indicating existence of a CRISPR leader sequence possessing an active transcriptional promoter immediately upstream of the 37-spacer CRISPR array. We were unable to identify sequences at the 5\u2032 or 3\u2032 ends of the 8-spacer CRISPR array possessing homology to the presumed 37-spacer CRISPR leader. Accordingly, it is unclear if the 8-spacer CRISPR locus possesses a leader sequence, and, therefore, functionality of this CRISPR array is uncertain.Approximately 45% of bacterial genomes encode CRISPR-associated (Cas) proteins and putative CRISPR arrays comprised of repetitive repeat-spacer units22C. thermocellum and Methanococcus maripaludis have been recently elucidatedC. pasteurianum and, as expected, identified a putative hairpin secondary structure , as well as Bacillus coagulans and Eubacterium limosum. Most CRISPR arrays harbored by these organisms specify only a small number of spacers (<7), compared to 37 in C. pasteurianum. It is likely that C. pasteurianum has been subjected to a greater degree of phage predation compared to other organisms employing similar CRISPR systems, leading to extensive acquisition of novel spacer sequences by C. pasteurianum. Although BLAST analysis of the 45\u2009C. pasteurianum CRISPR spacers provided no perfect matches to potential protospacer sequences, a number of spacers returned protospacer hits with five or fewer mismatches, which could be sufficient to confer immunity, since spacer-protospacer sequences are often imperfectC. pasteurianum Type I-B CRISPR-Cas machinery against plasmid-borne protospacer sequences.Processing of CRISPR RNAs (crRNAs) and subsequent binding to specific Cas proteins differs significantly between Type I and Type II CRISPR systems5754tructure . MoreoveC. pasteurianum is unprecedented in nature, as the organism combines metabolic pathways found independently in other clostridia  pathway . A complete complement of genes corresponding to the non-oxidative phase of the PPP, including multiple copies of ribulose-5-phosphate isomerase , transketolase , and transaldolase , were identified in the genome. Like most Gram-positive bacteria, C. pasteurianum also lacks a full Entner-Doudoroff (ED) pathway. Instead, gluconate is catabolized via a modified ED pathway through conversion into 2-keto-3-deoxy-6-phosphogluconate, which is subsequently siphoned into glycolysis via glyceraldehyde-3-phosphate and pyruvate (see section below on gluconate fermentation)C. acetobutylicum, C. ljungdahlii, C. autoethanogenum)2567C. pasteurianum possesses a non-cyclic, or branched, citrate \u201ccycle\u201d. Cell-free extracts have been shown to generate glutamate from oxaloacetateC. pasteurianum, it is assumed that glutamate formation from oxaloacetate proceeds via the aforementioned pathway. The second branch of the citrate cycle is exemplified by malate dehydrogenase (CP6013_0066 and CP6013_0670) and fumarate hydratase (CP6013_3554 and CP6013_3555). However, genes corresponding to \u03b1-ketoglutarate dehydrogenase, succinate dehydrogenase, and succinyl-CoA synthetase could not be identified in the genome of C. pasteurianum. pathway . The orgC. pasteurianum, the fermentation of glycerol is unique owing to production of 1,3-propanediol, a signature product of glycerol metabolismdhaBCE; CP6013_0898\u20130900) converts glycerol into the toxic intermediate, 3-hydroxypropionaldehyde, which is then reduced to 1,3-propanediol by 1,3-propanediol dehydrogenase C. pasteurianum genome, both enzymes are encoded within a 7\u2009kb reductive glycerol regulon, the structure and organization of which is distinct from that of other organisms capable of growth on glycerol, such as Citrobacter freundiidhaBCE and dhaT genes from C. pasteurianum have been cloned and characterized72C. pasteurianum and various species of Klebsiella and Citrobacter (65\u201381% identity). Further, DhaT from C. pasteurianum was found to share 86% of amino acid identities with the same enzyme from C. butyricum. The reductive 1,3-propanediol pathway requires NADH, in addition to vitamin B12C. pasteurianum involves conversion of glycerol into the glycolytic intermediate dihydroxyacetone phosphate by the concerted action of glycerol dehydrogenase (DhaD) and dihydroxyacetone kinase (DhaK). Dihydroxyacetone phosphate is then further oxidized to pyruvate via the standard glycolytic pathway. The genome of C. pasteurianum encodes at least five putative dhaD genes  and one potential dhaK gene (CP6013_1936), whereby the chief dhaDK regulon (CP6013_1936 and CP6013_1937) precedes a glpF gene (CP6013_1935) encoding the glycerol uptake facilitator protein  without detectable growth inhibitionCompared to other fermentations carried out by  protein . An abunC. pasteurianum involves oxidation to acetyl-CoA, certain culture conditions can result in significant accumulation of lactate via direct reduction of pyruvate by NADH, catalyzed by lactate dehydrogenase12C. cellulolyticumC. pasteurianum harbors two l-lactate dehydrogenase genes (CP6013_1427 and CP6013_0421), which share 43% of amino acid identities. Under standard growth conditions where iron is not limiting, pyruvate is oxidized to acetyl-CoA through pyruvate:ferredoxin oxidoreductase, referred to as the phosphoroclastic reaction owing to the formation of ATP and acetate from acetyl-CoA during growth on glucoseC. pasteurianum contains three putative pyruvate:ferredoxin oxidoreductase genes . The organism also harbors a potential alternative route of pyruvate oxidation via pyruvate formate lyaseClostridiumC. pasteurianumC. pasteurianum, including the bidirectional hydrogenase I (CP6013_3094)2-oxidizing, uptake hydrogenase (CP6013_3784 or CP6013_3422)Although the preferred outcome of pyruvate catabolism in C. pasteurianum is largely substrate-dependent and dictated by redox. The cell relies on the analogous acetate- and butyrate-formation pathways as the major source of ATP synthesis, since production of either metabolite results in substrate-level phosphorylation (C. pasteurianum harbors a single acetate-formation operon comprised of pta (CP6013_1096) and ackA (CP6013_1097) encoding phosphoacetyltransferase and acetate kinase, respectively. Interestingly, the coding sequences corresponding to pta and ackA in C. pasteurianum are separated by 134\u2009bp, whereas the analogous genes in C. acetobutylicum are separated by only 11\u2009bp  and alcohol dehydrogenases  for reduction of acetyl-CoA to ethanol via acetaldehyde. C. acetobutylicum harbors two bifunctional aldehyde-alcohol dehydrogenases, encoded by adhE (aad) and adhE2, that play major roles in the production of butanol and, to a lesser extent, ethanol8687C. pasteurianum  were found to possess substantial similarity (62\u201381%) to both AdhE and AdhE2 from C. acetobutylicum. It is probable that genes encoding these enzymes are involved in the production of ethanol and butanol in C. pasteurianum.When 4 metabolites through the condensation of two molecules of acetyl-CoAthl), 3-hydroxybutyryl-CoA dehydrogenase (hbd), crotonase , and butyryl-CoA dehydrogenase (bcd), and results in the generation of butyryl-CoA and oxidation of two moles of NADH per mole of butyryl-CoA formed. Thiolase, the first enzyme of this trunk pathway, condenses two molecules of acetyl-CoA, yielding acetoacetyl-CoA. C. pasteurianum harbors two putative thiolase genes (CP6013_2289 and CP6013_3617), one of which (CP6013_3617) has been cloned and characterizedC. acetobutylicum, referred to as the butyryl-CoA synthesis (bcs) operon  subunits, etfA and etfB, required for reduction of crotonyl-CoA by NADH in the enzymatic step catalyzed by Bcdcrt-bcd-etfB-etfA-hbd; CP6013_0322\u20130326) was found to be highly conserved between C. pasteurianum and C. acetobutylicum (80% nucleotide identity across the entire 4.8\u2009kb operon), as well as most other clostridia. Based on amino acid identities, the proteins of the bcs operon are most similar to those from C. arbusti, C. acetobutylicum, C. tetani, and C. botulinum (69\u201394% amino acid identity). In particular, 92\u201394% of amino acids of the bcs enzymes are common between C. pasteurianum and C. arbustii. In addition to the bcs operon, additional copies of crt (CP6013_2054), bcd (CP6013_2052 and CP6013_2324), etfB (CP6013_1682), etfA , and hbd (CP6013_1378 and CP6013_1968), could be identified in the genome of C. pasteurianum (C. carboxidivorans and C. beijerinckiirex gene (CP6013_0321) encoding a putative redox-sensing transcriptional regulator upstream of the bcs operon in C. pasteurianum. The C. pasteurianum Rex protein was found to possess 76% identity to the corresponding protein from C. acetobutylicum+ ratio.Butyrate- and butanol-forming clostridia produce C) operon . In addieurianum . MultiplC. pasteurianum generate reductant in the form of NADH and reduced ferredoxin+ oxidoreductase/NADH:ferredoxin oxidoreductase, which catalyzes the reversible reduction of NAD+ by reduced ferredoxin9394+ is evident under certain non-standard culture conditions, such as inhibition of hydrogenase by carbon monoxide+ oxidoreductase reaction is inhibited by low levels of NADH930\u2032\u2009=\u2009\u2212320\u2009mV) to ferredoxin (E0\u2032\u2009=\u2009\u2212400\u2009mV), is highly unfavorable, spawning considerable skepticism surrounding the thermodynamic feasibility of this pathway in vivoC. pasteurianum evolve more molecular hydrogen than can be accounted for by the phosphoroclastic reaction (determined by the combined amount of acetate and butyrate formed), indicating that under certain conditions NADH serves as reductant through operation of the unfavorable NADH:ferredoxin oxidoreductase reactionC. pasteurianum produce hydrogen gas from acetyl-CoA and NADH, again implying electron transfer from NADH to ferredoxinet al.C. pasteurianum, C. kluyveri, and possibly other solventogenic clostridiaC. pasteurianumC. pasteurianum was found to possess similarity to both bcd (CP6013_0323) and etfA (CP6013_0325), suggesting presence of a Bcd-EtfA fusion protein. It has been suggested that redox partners evolve into a single fusion protein to promote more efficient conversion of unstable intermediates and rapid transfer of electronsbcd-etfA fusion ortholog could only be identified in C. kluyveri (76% nucleotide identity), which could provide insight into the recently-proposed electron bifurcation mechanism of the Bcd-EtfAB enzyme complex in C. pasteurianum and C. kluyveri97Growing cultures of C. pasteurianum harbors a full acetone-formation pathway consisting of CoA transferase subunits A and B (encoded by ctfAB) and an acetoacetate decarboxylase (encoded by adc) for conversion of acetoacetyl-CoA to acetone via acetoacetate37C. acetobutylicumctfAB genes  are preceded by a putative adhE (aad) gene (CP6013_1661). Additional copies of the ctfAB genes  and two genes encoding putative CtfAB fusion proteins (CP6013_2053 and CP6013_3216) were also identified in the genome , as found in C. acetobutylicumC. pasteurianum CtfAB and Adc enzymes possess a high degree of similarity (71\u201384%) to the corresponding proteins of C. acetobutylicum, a significant acetone-producer. Despite these similarities, acetone is not a common metabolite of C. pasteurianumC. acetobutylicum as a means of preventing acid crash under low pH conditionsC. pasteurianum to uptake and reassimilate acids, as acid levels generally increase throughout the course of fermentation1C. acetobutylicum. It is also possible that the acetone pathway remains inactive in C. pasteurianum due to a lack of pathway induction under standard growth conditions, poor enzymatic activities, or lack of a functional transcriptional promoter to drive expression of the sol operon or adc gene. Induction of acetone production in C. acetobutylicum has been studied extensively and inducers include low pH and elevated concentrations of acetate and butyratee genome . AnalysiC. pasteurianum. Butyryl-CoA can be converted into butyrate or further reduced to butanol in pathways that mimic the C2 fermentative pathways leading to production of acetate and ethanol. C. pasteurianum harbors a single butyrate-formation operon, consisting of phosphotransbutyrylase  upstream of butyrate kinase   to the corresponding enzymes from C. acetobutylicum. Unlike C. acetobutylicum, however, we were unable to identify a second copy of buk [i.e. buk2C. pasteurianum. The other pathway from butyryl-CoA is the reductive butanol formation route, where consecutive dehydrogenation steps convert butyryl-CoA first to butyraldehyde, then butanol. In addition to the aforementioned adhE (aad) and adhE2 genes, two butanol dehydrogenases, encoded by bdhA and bdhB, have been implicated in butanol formation in C. acetobutylicumC. pasteurianum genome using BdhA and BdhB protein queries returned a large array of alcohol dehydrogenases  possessing similarity to the C. acetobutylicum isozymes. Notably, protein products corresponding to genes CP6013_2711 and CP6013_1579 produced the highest degree of identity to both BdhA  and BdhB  from C. acetobutylicum. Although bdhA and bdhB occur in tandem within the chromosome of C. acetobutylicumC. pasteurianum. Surprisingly, disruption of bdhA or bdhB in C. acetobutylicum had no effect on solvent formation, while disruption of adhE nearly abolished production of solventsadhE from C. acetobutylicum, specifically CP6013_1661, CP6013_2575, CP6013_0292, and CP6013_1611, are likely to be the greatest contributors to butanol, as well as ethanol, formation in C. pasteurianum.Similar to acetyl-CoA, butyryl-CoA serves as a major branch point in the central metabolism of 13_3581) . Ptb andi.e. buk2 within tC. pasteurianum readily utilizes glucose, fructose, mannitol, sorbitol, and sucrose, among other substratesC. pasteurianum genes encoding putative phosphotransferase system (PTS) components could be identified for most of these fermentable substrates109C. pasteurianum, product distribution varies dramatically and is dictated foremost by the degree of reductance of the substrateC. pasteurianum, where fermentation of glucose generates a predominantly acidogenic metabolism, while fermentation of mannitol or glycerol yields almost exclusively alcohols5C. pasteurianum grown on substrates of varying degrees of reductance, thereby allowing manipulation of the intracellular NADH/NAD+ ratio\u22121 of substrate .C. pasteurianum. The substrate is first dehydrated to 2-keto-3-deoxy-gluconate (KDG) by gluconate dehydratase (CP6013_2550)C. pasteurianum static flask cultures grown on sodium gluconate yielded an entirely acidogenic fermentative metabolism yielding 8.5\u2009\u00b1\u20090.6\u2009g\u2009L\u22121 acetate and 5.9\u2009\u00b1\u20090.3\u2009g\u2009L\u22121 butyrate, as the butyrate pathway alone was sufficient to oxidize NADH. The NADH-consuming ethanol and butanol pathways were not induced during gluconate catabolism, presumably due to a low intracellular NADH/NAD+ ratio. With a degree of reductance of 4\u22121) as the predominant fermentation product, with equal quantities of acetate (2.4\u2009\u00b1\u20090.4\u2009g\u2009L\u22121) and butyrate (2.4\u2009\u00b1\u20090.8\u2009g\u2009L\u22121). The relatively high levels of butanol detected in this study may be explained by growth medium formulation, as we utilized a medium optimized for production of butanol from glycerolC. pasteurianum109110C. pasteurianum leads to a product profile characterized by high butanol selectivity, as cultures produced 6.0\u2009\u00b1\u20091.7\u2009g\u2009L\u22121 butanol and only trace amounts of acetate (0.4\u2009\u00b1\u20090.1\u2009g\u2009L\u22121), butyrate (0.5\u2009\u00b1\u20090.3\u2009g\u2009L\u22121), and ethanol (0.7\u2009\u00b1\u20090.4\u2009g\u2009L\u22121), indicating that the cell relies almost exclusively on the butanol pathway for oxidation of NADH. Similar products have been detected from the fermentation of mannitol by C. pasteurianum in continuous cultureC. pasteurianum using a unique glycerol facilitator protein channel (CP6013_1935), where it is then converted into 1,3-propanediol or siphoned into glycolysis via dihydroxyacetone phosphate. Owing to its high degree of reduction, glycerol catabolism by C. pasteurianum leads to substantial quantities of reduced end products, specifically butanol and 1,3-propanediol103\u22121) surpassed that of 1,3-propanediol (5.2\u2009\u00b1\u20091.8\u2009g\u2009L\u22121), while ethanol (1.3\u2009\u00b1\u20090.4\u2009g\u2009L\u22121), acetate (0.7\u2009\u00b1\u20090.2\u2009g\u2009L\u22121), and butyrate (0.2\u2009\u00b1\u20090.1\u2009g\u2009L\u22121) represented minor co-products. The highly reduced product profile of C. pasteurianum during growth on glycerol underscores the immense industrial potential of this organism in producing butanol from crude glycerol. Since substantial quantities of 1,3-propanediol are produced along with butanol, the 1,3-propanediol pathway represents a key target of rational metabolic engineering. Note that fundamental genetic engineering technologies have only recently been developed for this organism313228in situ removal of butanol.With a degree of reductance of 3.67, gluconate is the most oxidized substrate fermented by tase CP603_2550C. C. pasteurianum, carbon and electron flow can be manipulated using a number of strategies. The effect of carbon monoxide on anaerobic fermentations has been widely documented in Clostridium+ oxidoreductase reaction to oxidize ferredoxin, resulting in NADH formation and subsequent production of butanol and ethanolC. pasteurianum is able to utilize electrons derived directly from a supplied electric currentC. pasteurianum is a rare exception capable of uptaking electrons directly from a cathode. Moreover, cells were found to utilize substrate and exogenous electrons concomitantly, thus building on the biotechnological potential harnessed by C. pasteurianum. On the other hand, utilization of externally-supplied electrons manifested in increased titers of 1,3-propanediol, rather than butanol, which is in line with the role of the 1,3-propanediol pathway in maintaining redox poiseC. pasteurianum metabolism, electrosynthesis of butanol represents an important and challenging target of future strain engineering efforts. In this context, it is anticipated that the genomic analysis presented herein, as well as previous studies of C. pasteurianum metabolism1C. pasteurianum.While the redox state of the fermentation substrate represents the chief factor governing product distribution in C. pasteurianum type strain ATCC 6013 (DSM 525) was obtained from the American Type Culture Collection (ATCC). Oligonucleotides 2SO4, 5.08\u2009g Bacto yeast extract, 0.2\u2009g MgSO4\u2009\u00b7\u20097H2O, 0.02\u2009g CaCl2\u2009\u00b7\u20092H2O, 0.06\u2009g FeSO4\u2009\u00b7\u20097H2O, 1\u2009mg resazurin, and 2\u2009ml trace element solution SL 7. The initial pH of the medium was 6.0\u20136.1 prior to sterilization. Carbon sources  were sterilized separately as 100\u2009g\u2009L\u22121 stock solutions and added to culture flasks to achieve a final concentration of 40\u2009g\u2009L\u22121. Cysteine-HCl (0.5\u2009g\u2009L\u22121) was used to reduce growth medium prior to inoculation. Static cultures were grown in 125\u2009ml Erlenmeyer flasks containing 50\u2009ml medium within an anaerobic containment chamber  consisting of an environment of 85% N2, 10% H2, and 5% CO2. Seed cultures were prepared by heat-shocking single sporulated agar plate colonies at 80\u2009\u00b0C for 10\u2009minutes in 10\u2009ml 2\u00d7YTG medium, pH 6.4  as described previously31ides see  were pur600). Culture supernatants were analyzed for metabolite production 60\u201390\u2009h after inoculation. Product concentrations were determined by LC-10AT HPLC analysis  equipped with a RID-10A refractive index detector  and Aminex HPX-87H column . Column temperature was maintained at 65\u2009\u00b0C. The mobile phase consisted of 5\u2009mM H2SO4 (pH 2.0) at a flow rate of 0.6\u2009mL\u2009min\u22121. RID signal data processing was performed using Clarity Lite . End product titers reported represent the average of two or three biological replicates.Cell growth was monitored by measuring optical density at 600\u2009nm  and divided into six 25\u2009ml cultures. Mitomycin C was added to a final concentration of 0, 0.5, 1, 2.5, 5, or 10\u2009\u03bcg ml\u22121 and OD600 was monitored until a sharp decline in turbidity was observed approximately 4\u2009h post-induction. One ml of the resulting phage lysates was centrifuged at 10,000\u00d7\u2009g for 10\u2009minutes and the supernatants were filtered through a 0.45\u2009\u03bcm filter. Following washing of phage particles twice with 0.1\u2009M ammonium acetate, pH 7.5, five \u03bcl of lysate was pipetted onto 200-mesh Formvar/carbon-coated copper grids and incubated for approximately five minutes. Excess lysate was blotted with Whatman filter paper and grids were allowed to dry overnight. Grids were then stained for 10\u2009minutes using a saturated uranyl acetate solution, followed by washing with 50% ethanol and drying in air for approximately three hours. Imaging was performed at 60\u2009kV using a Philips CM10 transmission electron microscope equipped with a digital camera. Phage images were captured using 245,000\u00d7 magnification.Phage excision and transmission electron microscopy (TEM) were performed in a manner similar to previous methodsC. pasteurianum ATCC 6013 according to a previous methodC. pasteurianum ATCC 6013 was sequenced, assembled, and annotated as describedC. pasteurianum draft genomeTotal DNA was isolated from Taq DNA Polymerase . Large PCR products and intact genomic DNA were separated using 0.3\u20130.5% agarose gels and low voltage (12\u201315\u2009V) electrophoresis for 12\u201318\u2009h. Restriction endonucleases were obtained from New England Biolabs  and utilized according to the manufacturer\u2019s guidelines.Long range PCR (15\u201335\u2009kb) was performed using LongAmp Accession numbers: This Whole Genome Shotgun project has been deposited at GenBank under the accession JPGY00000000. The version described in this paper is version JPGY02000000.How to cite this article: Pyne, M. E. et al. Genome-directed analysis of prophage excision, host defence systems, and central fermentative metabolism in Clostridium pasteurianum. Sci. Rep.6, 26228; doi: 10.1038/srep26228 (2016)."}
+{"text": "Acrophobia, an abnormal fear of heights, is a specific phobia characterized as apprehension cued by the occurrence or anticipation of elevated spaces. It is considered a complex trait with onset influenced by both genetic and environmental factors. Identification of genetic risk variants would provide novel insight into the genetic basis of the fear of heights phenotype and contribute to the molecular-level understanding of its aetiology. Genetic isolates may facilitate identification of susceptibility alleles due to reduced genetic heterogeneity. We took advantage of an internal genetic isolate in Finland in which a distinct acrophobia phenotype appears to be segregating in pedigrees originally ascertained for schizophrenia. We conducted parametric, nonparametric, joint linkage and linkage disequilibrium analyses using a microsatellite marker panel, genotyped in families to search for chromosomal regions correlated with acrophobia. Our results implicated a few regions with suggestive evidence for linkage on chromosomes 4q28 (LOD\u2009=\u20092.17), 8q24 (LOD\u2009=\u20092.09) and 13q21-q22 (LOD\u2009=\u20092.22). We observed no risk haplotypes shared between different families. These results suggest that genetic predisposition to acrophobia in this genetic isolate is unlikely to be mediated by a small number of shared high-risk alleles, but rather has a complex genetic architecture. Acrophobia is a pervasive mental disorder, also known as an irrational fear of heights, affecting approximately five percent of the world\u2019s population24The symptoms of individuals suffering from acrophobia involve changes in behavioural, cognitive and physiological functioning, such as confusion and dizziness, when exposed to heightsAlthough the biological mechanisms of acrophobia have been thoroughly investigated12To reduce genetic heterogeneity we have used an isolated homogenous population from north-eastern Finland with small number of founders. This population, described elsewhere1517Our sample was composed mostly of large multigenerational pedigrees with multiple individuals affected with acrophobia and at least one parent born in the isolate . It incl17We first carried out parametric two-point linkage analysis to identify genetic regions linked with acrophobia. The strongest evidence for linkage in the acrophobia sample (including cases with comorbid schizophrenia) was obtained with marker D5S2115 (LOD\u2009=\u20092.16) using a dominant model. In the pure acrophobia sample, the same marker yielded a LOD score of 0.58 (dominant model), suggesting that this finding may be mainly driven by the schizophrenia phenotype. This was further confirmed by analysing all individuals with schizophrenia without comorbid acrophobia . For the pure acrophobia sample, we obtained the highest LOD score with marker D8S373 (LOD\u2009=\u20092.09) adopting a recessive inheritance model. Since in the acrophobia with comorbid schizophrenia sample the same marker yielded a LOD score of 0.51 (recessive model) and in the pure schizophrenia sample a LOD score of 0.00 (recessive model), this signal may be produced mainly by the acrophobia phenotype. We carried out parametric multipoint analysis for chromosomes with markers and models which yielded LOD score of >2.0 in at least one parametric two-point analysis . In the acrophobia sample (including cases with comorbid schizophrenia), marker D5S2115 yielded a maximum LOD score of 0.054 . In the pure acrophobia sample the highest LOD score on chromosome 8 was with marker D8S373 .We next carried out multipoint nonparametric genome-wide linkage analysis with empirical NPL_ALL model , measuriWe hypothesized that reduced genetic heterogeneity in the genetic isolate would lead to the majority of the cases carrying the same predisposing variant, detectable as linkage disequilibrium (LD). Therefore, we performed PSEUDOMARKER analysis of LD conditional on linkage . The strTo test the statistical power of the analysed sample, we performed simulation with SLINKWe aimed to find genetic loci predisposing to acrophobia in a genetically isolated population with the hypothesis that due to reduced genetic heterogeneity, a few risk alleles predisposing to the phenotype might be identified. While several loci attained LOD score of >2.0, we observed no genome-wide significant evidence for linkage at any of the studied markers.PCDH20) gene in this region, has been previously connected to positive symptom dimension in a genome-wide association study (GWAS) of schizophrenia (P\u2009=\u20093\u2009\u00d7\u200910\u22126)We detected the strongest evidence of linkage to acrophobia on chromosomal region 13q21-q22 with a peak on marker D13S162 . To our knowledge, this region has not been previously associated with phobias or other anxiety disorders. SNP rs2323266, located 14.01\u2009Mb from marker D13S162 and close to the protocadherin 20 . Again, in our study, this signal appeared to be mainly coming from acrophobia and not schizophrenia phenotype . This region has not been previously associated with anxiety disorders or schizophrenia.KCNQ3)24ADCY8)LYNX1)313233ARC)Chromosomal region 8q24.2-q24.3 with marker D8S373 (LOD\u2009=\u20092.09) was the third region most strongly linked to acrophobia. It has previously been associated with bipolar disorder2425Several of the chromosomal regions we identified seemed mainly specific for schizophrenia. The most significant of those regions were 5q31 and 1q32, discussed above for markers D5S2115 and D1S2817. Both of them have been previously associated with schizophrenia in numerous studies353637Furthermore, several markers on the long arm of chromosome 13, localized between 13q31 and 13q33, showed evidence of linkage (LOD\u2009>\u20092.0) to both pure acrophobia and acrophobia with comorbid schizophrenia, and also weak linkage to pure schizophrenia (maximum LOD\u2009=\u20091.26). Therefore, this region may harbour variants influencing susceptibility to both phenotypes. As specific phobia subtypes have high comorbidity with other anxiety disordersWe recognise the hypothesis-generating character of our study. It included a large number of genotyped markers (570) and analysed models (6). Although this serves to strengthen the study, it inevitably lead to multiple statistical testing. However, as the tests carried out are not completely independent, the adjustment for multiple testing is not straightforwardIn recent years, single nucleotide polymorphisms (SNPs) have replaced microsatellite markers due to their lower genotyping costs. This technical advance has enabled genome-wide association studies (GWASs) in which thousands of individuals are genotyped. Consequently, the usage of microsatellite markers, a class of short tandem repeats (STRs), has severely decreased. However, due to their highly polymorphic nature microsatellites are still considered more informative than the diallelic SNPs1117In conclusion, our findings suggest that the genetic basis of acrophobia is highly complex, even in this genetic isolate, as we were not able to identify high-risk variants shared by several families. However, we identified several chromosomal regions with suggestive evidence for linkage which could be investigated further in other acrophobia samples and meta-analyses of such datasets having an increased statistical power.The sample was composed mostly of large multigenerational pedigrees with multiple affected individuals and at least one parent born in the isolate . It comp171846th edition (DSM-IV) criteriaData from psychiatric case notes and treatment facilities concerning affected individuals and blood samples were collected between 1991 and 2002We analysed 575 autosomal microsatellite markers across the genome that had been genotyped as a part of earlier linkage-based gene mapping studies17unknown, due to the fact that unaffected family members were not systematically assessedWe performed statistical analyses separately for acrophobia with comorbid schizophrenia and pure acrophobia sample to discriminate between the possible shared and separate genetic signals associated with the phenotypes. We further followed with statistical analysis of the pure schizophrenia sample for the most interesting results. We first checked all genotypes for Mendelian inconsistencies with PedCheck softwareWe performed two-point parametric linkage analysis with statistical software package FASTLINK 4.1\u2009P under a recessive and dominant mode of inheritance with, respectively, penetrance of 0.001% and 90%, disease allele frequency of 0.00001 and 0.01, and phenocopy rates of 0 and 0.01. FASTLINK 4.1\u2009P program was implemented in a helper program AUTOGSCANWe carried out multipoint parametric and nonparametric linkage analysis with SimWalk2 version 2.965455We performed the linkage disequilibrium conditional on linkage analysis with PSEUDOMARKER software package under default dominant and recessive models58The power of the analysed pedigrees to detect linkage was estimated with SLINK simulation program and the replicates were analysed with ISIM analysis program implemented in SLINK packageHow to cite this article: Misiewicz, Z. et al. A genome-wide screen for acrophobia susceptibility loci in a Finnish isolate. Sci. Rep.6, 39345; doi: 10.1038/srep39345 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations."}
+{"text": "Porous Si/eumelanin hybrids are a novel class of organic\u2013inorganic hybrid materials that hold considerable promise for photovoltaic applications. Current progress toward device setup is, however, hindered by photocurrent stability issues, which require a detailed understanding of the mechanisms underlying the buildup and consolidation of the eumelanin\u2013silicon interface. Herein we report an integrated experimental and computational study aimed at probing interface stability via surface modification and eumelanin manipulation, and at modeling the organic\u2013inorganic interface via formation of a 5,6-dihydroxyindole (DHI) tetramer and its adhesion to silicon. The results indicated that mild silicon oxidation increases photocurrent stability via enhancement of the DHI\u2013surface interaction, and that higher oxidation states in DHI oligomers create more favorable conditions for the efficient adhesion of growing eumelanin. Organic\u2013inorganic hybrid devices attract growing research and industrial interest for a broad range of applications since they combine the low fabrication costs and versatility of the organics with the usually higher efficiency of the inorganics ,2,3. A sRecently, we reported a promising organic\u2013inorganic hybrid material for photovoltaic applications consisting of a bulk heterojunction of porous silicon (PSi) and eumelanin obtained by polymerization of the key precursor 5,6-dihydroxyindole (DHI) . DHI wasn+-doped porous silicon (n-PSi) was produced in situ by ammonia-induced solid state polymerization (AISSP) [\u22122 was measured upon irradiation with visible light. The photocurrent was not affected by acetic acid vapors but was irreversibly abated by gaseous ammonia, supporting the potential of eumelanin as a powerful enhancer of PSi photoresponse to visible light via hole-type electrical conduction. However, despite the promising features of the PSi\u2013eumelanin interface produced by the AISSP protocol, several issues remained to be settled including the low efficiency, a marked instability, and a limited reproducibility.In a subsequent variant of that methodology, the polymerization of DHI in  (AISSP)  after th (AISSP) . With thTo unravel the complexity of the hybrid eumelanin/Si interface to design efficient and stable PSi\u2013eumelanin-based devices, it is therefore essential to elucidate the molecular mechanisms of eumelanin buildup and adhesion on the inorganic substrate. One main goal is to improve the adhesion, interaction and electron-ion transfer at the organic/inorganic interface in order to optimize the electronic performance for photovoltaic applications.The aim of this paper is to address the factors responsible for the main interface issues, including temporal instability ,12 of thOne of the main issues with eumelanin\u2013PSi hybrids is their short lifetime (a few days) was photocurrent generators and a significant fluctuation of the photocurrent values for different samples (about +/\u2212 a factor of three). Although the high reactivity of the developed surface of pristine PSi samples is a well-known characteristic of this material ,24, thisSeveral factors can contribute to this behavior, including the formation of the organic/inorganic interface in a porous structure, the polymerization process of eumelanin from DHI, and the loss of hydration water. To address the complexity of hybrid formation in a porous structure, this issue was addressed by investigating the PSi/eumelanin interface and analyzing the polymerization process from DHI to eumelanin.Interface instability can be related to the lack of formation of stable bonds between Si and eumelanin. Based on this assumption, one possible explanation is that the volume reduction accompanying polymerization to eumelanin would weaken the initial electrical contact between PSi and the organic eumelanin component, lowering photocurrent generation.To test this hypothesis, Electrochemical Impedance Spectroscopy (EIS) was used to gain information on the interface stability. EIS is very sensitive to surface and interface processes, and this makes it very useful for porous materials because of their high developed inner surface .In the hypothesis of a significant role played by the PSi/melanin interface, a different behavior should be obtained when modifying the interface formation process. As a reference measurement before any surface modification, we studied the behavior of standard PSi/melanin samples. In The first information that can be obtained from In the day 1 curves, in black in the figure, we can see a large semicircle on the left, corresponding to higher frequencies, a small part of a middle semicircle  and, on the right, another semicircle, smaller than the first one, for lower frequencies. In the measurements made on days 2, 4, and 7, we can see the disappearance of the middle circle (at about 4300 Ohm) and the rapid growth of the last one. We can interpret this rapid evolution of the circles as a sign of the instability of the porous silicon/DHI interface, which significantly changes in the first 4\u20137 days from preparation and confirms the instability in the photocurrent measurements.To probe the interface formation process, we used two approaches. The first was based on a slight electrochemical oxidation of the silicon surface, the second used a 50/50 mixture of DHI and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) instead of pure DHI to impregnate the sample.2 and DHI. The potential advantages of a thin oxide layer in hybrid and Si/organic photovoltaic heterojunction have already been described in several publications [Partial oxidation of the surface was expected both to exert a stabilizing effect and to facilitate a stronger bonding of DHI to the surface, thanks to better affinity between SiOications ,33,34,35ications , the maxA few more comments are needed for the comparison of the results in The second strategy tested as a possible means of stabilizing the junction was based on the use of mixed solutions of DHI and DHICA to impregnate the PSi matrices. The rationale was that DHICA combines a DHI skeleton with a carboxyl function that could improve the anchoring on the PSi surface via oxygen bridges akin to those observed for Ti oxide surfaces . MoreoveOn the basis of these results it was concluded that a modest stabilization of the PSi/melanin interface can be achieved by mild oxidation of the PSi layer before impregnation with the monomer, and that the addition of DHICA slightly modifies the GEIS semicircle profile but does not increase the stability.To gain an insight into the molecular mechanisms leading to interface formation between growing eumelanin on the PSi surface, in model experiments the evolution of the absorbance spectra in air-equilibrated solutions of DHI in ethanol at various concentrations was examined. In From the data in In From the experiments reported herein, it can be concluded that DHI also polymerizes in an air-equilibrated organic medium, and that the process leads to the formation and evolution of more defined chromophoric species compared to the same reaction in aqueous neutral buffer, with less pronounced scattering at longer wavelengths. Such differences can be explained by considering that in ethanol oligomer aggregation and precipitation are not so pronounced as in an aqueous buffer, where significant scattering is observed. Moreover, the lower dielectric constant in an organic solvent would not favor oligomer ionization by deprotonation as in an aqueous buffer. On this basis, the higher absorption wavelength and flatter appearance of the main visible band in the aqueous buffer could be ascribed to the deprotonated forms of the oxidized oligomers. This conclusion was supported in a separate experiment in which the oxidation mixture of DHI in aqueous buffer pH 7.0 was acidified to pH 2, causing a marked blue shift of the broad absorption band at 560 nm to about 500 nm. It is possible that the transient species absorbing at 780 nm is an unstable precursor of the main band at 460 nm, but no evidence supports this conclusion. The longer persistence of the main chromophores in ethanol with respect to the aqueous buffer can be explained by the stronger solvation effects in the organic solvent, limiting aggregation and precipitation of dark material.Based on this background of experimental and model studies, further insights into the process of interface buildup were gained by computational modeling of the formation and adhesion of single and doubly stacked molecules reported in this work, allowing for a direct comparison of the stacking and covering processes of eumelanin on Si. To this aim, the chromophoric changes observed in ethanol were used as a basis to validate the model structure selected to probe eumelanin adhesion on Si by computational methods.The structural variety of the isomers that populate each oligomer level contributes to the molecular disorder of eumelanin type materials ,9,19,39.For the purposes of this study, 2,4\u2032:2\u2032,4\u2032\u2032:2\u2032\u2032,4\u2032\u2032\u2032-tetraindolyl (Model 1) was considered as a model representative component of eumelanin for computational analysis . SelectiA more complete analysis of disorder in eumelanins would encompass the enormous number of possible molecular models and alternative redox states, and was outside the scope of the present investigation because of the huge computational effort required. All the results were obtained from Density\u2013Functional Theory, employing the plane\u2013wave Quantum Espresso package with the PBE  exchThe formation energies of these molecules are given in Formation energies are calculated as The results show a clear dependence of the formation energies on the solvent used in the synthetic process. The presence of solvent, here taken into account via the dielectric permittivity of the medium, increases the formation energy, causing a decrease in the molecular stability. Some of the structures considered are not formed in methanol  groups yield improved stability, as opposed to \u2013OH groups, which determine a higher formation energy. However, in that case the result can be at least partially due to the fact that for steric reasons two nitrogen atoms at most can be present as N\u2013H groups. Looking more carefully at the results of For the molecular structures outlined above, stacking energies have also been computed. Values are given in To assess whether structures related to the model tetramer may be responsible for the spectrophotometric course of DHI oxidation in ethanol and hence for the buildup of the eumelanin\u2013silicon interface, the absorption spectrum of the model tetramer in In the study by Meng and Kaxiras in , a calcuUnder the specific experimental conditions described above, DHI monomers undergo an aggregation process that causes changes in the absorption spectrum of the solution as a function of time.Within the gross limitations of the present approach, due to the marked complexity of DHI polymerization and the notorious difficulty of modeling the myriad structures and redox states available to each aggregate of DHI oligomers, it appears that calculated model structures show absorption bands spanning the entire visible range, in line with what is observed in a DHI polymer in ethanol. A superposition of the computed spectra is shown in the As is clear from In The PT tetramer shows a strong peak A  number of possible correspondences with the experiments.This point is of particular importance because it can support the hypothesis that the Model 1 tetramer of ,48 couldOf course, given the high degree of structural heterogeneity in eumelanin-type polymer and the lack of experimental evidence on the structure and redox state of main building blocks in DHI melanin, we must consider these data as being largely speculative. Further work is necessary to assess the effect of the solvent on the structural and optical properties of the model tetramers under investigation.Studies of the adhesion properties of eumelanin building blocks on the silicon surface were then carried out using Model 1-d. Stronger adhesion properties are determined as the number of C=O groups in the molecules increases. Carbonyl (C=O) groups tend to bind to the surface, probably because of their expectedly greater polarization by resonance effects (the oxygen atom should be more negatively charged) and more efficient \u03c0\u2013electron overlap . If verified in future experiments, the computational finding that more oxidized forms of the oligomers exhibit greater adhesion properties can be of potential interest for improving the current coating/impregnation protocols for porous silicon. For example, the addition of oxygen gas or an enhancement of the oxidizing power inducing DHI polymerization over pSi may promote a faster and more efficient adhesion. This may be a valuable strategy to address current interface issues. It will also be of interest to assess whether interaction between oxidized DHI oligomers and the PSi surface can promote tautomerism between QM and IQ with an increase in the proportion of C=O groups at each degree of oxidation and further stabilization. These and other issues are the focus of ongoing work.Moreover, from the comparison with the adhesion energies of stacked molecules , it can Future theoretical and experimental work aimed at completing these preliminary model experiments will hopefully improve our understanding of the eumelanin\u2013silicon interface for possible applications in photovoltaics.n+ doped bulk Si wafers  according to the procedure indicated in [PSi structures were fabricated by electrochemical etching in the dark of cated in . The porcated in .\u00ae and a PerkinElmer Lambda950 UV-Vis-NIR spectrophotometer .The electrochemical Impedance Spectroscopy measurements were performed using a PARSTAT 2273 potentiostat by Princeton Applied Research . Each measurement was made using a constant current of 0.1 mA and a superimposed oscillating signal, whose amplitude was 10 \u00b5A, in the frequency range 100 kHz\u2013100 mHz. The electrochemical solution was a 0.1 M KCl in EtOH. For the absorbance measurements, we prepared several DHI/EtOH solutions by adding DHI powder to EtOH in several concentrations: 0.5, 0.25, and 0.12 mg/mL. The measurements were performed at room temperature, using a quartz cell sealed with Parafilm3 in EtOH solution according to the procedure described in [The electrochemical oxidation was performed using a 0.1 M KNOribed in . The maxr = 2.5 mm. More details about the procedure can be found in [The photocurrent measurements were performed using a halogen lamp as a white source and a Keithley  6487 multimeter to measure the current intensity. The illuminated spot radius on the sample was found in .Density\u2013functional theory (DFT) calculations are performed using the plane\u2013wave Quantum Espresso package .The PerdIn order to describe the effect of the solvent on the formation energy, a continuum solvation model was used: the self-consistent continuum solvation model (SCCS) proposed by Andreussi, Dabo, and Marzari  and implDFT ,56 and TThe B3LYP exchange-correlation (XC) functional has stable behavior, with some well-documented limitations . The strTo obtain the singlet\u2013singlet excitation energies and the electronic absorption spectrum in the visible/UV region for each tetramer, TD-DFT calculations were performed at the same level B3LYP/6-31G* employed for its electronic ground-state. The frequency\u2013space implementation of the TD-DFT scheme based on the linear response of the density matrix was used. In this computational approach, the poles of the linear response function are associated with vertical excitation energies and the pole strengths with the corresponding oscillator strengths .For each molecule only the first 120 singlet\u2013singlet roots, which in all cases were sufficient to cover the energy window considered here, were taken into account.A similar computational scheme has been successfully applied on several other families of organic molecules and to another class of molecules that are the basic constituents of eumelanin ,65,66,67By combining model experiments with different theoretical approaches, it has been possible to demonstrate that the eumelanin/PSi interface is partially stabilized by mild oxidation of the inorganic surface, probably generating an expanded range of functional groups amenable to efficient interaction with growing eumelanin building blocks. Model computational studies indicated, moreover, that the highest redox states of oligomer components in eumelanins are more susceptible to adhesion than the reduced forms. These results provide an improved background for the development of advanced eumelanin\u2013PSi interfaces with better photocurrent stability properties for photovoltaic applications."}
+{"text": "UBE2Z and SNP rs6725887 in WDR12 by GWAS, were found within the 17q21.2 and 2q33.3 loci. These studies lay a foundation for future identification of causative variants and genes for CAD.Coronary artery disease (CAD) is the leading cause of death, and genetic factors contribute significantly to risk of CAD. This study aims to identify new CAD genetic loci through a large-scale linkage analysis of 24 large and multigenerational families with 433 family members (GeneQuest II). All family members were genotyped with markers spaced by every 10\u2009cM and a model-free nonparametric linkage  analysis was carried out. Two highly significant CAD loci were identified on chromosome 17q21.2 (NPL score of 6.20) and 7p22.2 (NPL score of 5.19). We also identified four loci with significant NPL scores between 4.09 and 4.99 on 2q33.3, 3q29, 5q13.2 and 9q22.33. Similar analyses in individual families confirmed the six significant CAD loci and identified seven new highly significant linkages on 9p24.2, 9q34.2, 12q13.13, 15q26.1, 17q22, 20p12.3, and 22q12.1, and two significant loci on 2q11.2 and 11q14.1. Two loci on 3q29 and 9q22.33 were also successfully replicated in our previous linkage analysis of 428 nuclear families. Moreover, two published risk variants, SNP rs46522 in  Existing knowledge of genetic components affecting the risk of CAD is largely based on results from genome-wide association studies (GWAS), a systematic, unbiased and powerful approach to identify disease-associated variants using population samples. Although the majority of GWAS have focused on European ancestry populations14, several GWAS were also reported in African Americans15, East Asians21 and South Asians22. Due to newly developed SNP imputation methods25 based on the HapMap project (https://www.ncbi.nlm.nih.gov/probe/docs/projhapmap/) and the 1000 Genome project , meta-GWAS is becoming a more popular strategy for CAD and other complex diseases. The largest meta-GWAS recently analyzed 9.4 million imputed SNPs among >185,000 samples and identified 10 novel CAD loci14. To date, there have been 65 independent CAD susceptibility loci reported at a genome-wide significance level . The heritability of CAD has been estimated from 40% to 60% by genetic-epidemiologic studies26. However, recent studies strongly indicate that GWAS variants cannot fully explain the heritability of CAD, and all published risk variants explained only 10\u201320% of heritability27.Genetic factors contribute to the risk of developing coronary artery disease (CAD) and its major complication, myocardial infarction (MI), which is the result of the accumulation of atherosclerotic plaques in the walls of the coronary arteries28. Since then, over ten GWLAs, including our own studies, have identified additional genetic loci for CAD or MI, including 1p34\u201336,1q25, 2q14.3, 2q36\u201337.3, 2q13, 3q13, 5q31, 7p14, 8p22, 13q12\u201313,14q32.3, 15q26.3, 16p13, and 17p11.2\u201317q2139. Recently, we have completed a genome-wide linkage scan in a well characterized U.S GeneQuest cohort with 428 nuclear families and identified six novel CAD loci on chromosomes 3p25.1, 3p29, 9q22.3, 9p34.11, 17p12, and 21q22.340. In contrast to aforementioned GWASs, the number of genetic loci identified by GWLA was much smaller and independent, suggesting that many linkage loci remain to be identified in new CAD or MI families40.Genome-wide linkage analysis (GWLA) is another systematic and unbiased approach to identify genetic loci for human complex diseases and to search for evidence of major genetic effects. The first GWLA for CAD, conducted in 2000, involved an analysis of 156 affected sibling pairs and revealed two genetic loci on chromosomes 2q21.1\u201322 and Xq23\u201326 that were linked to premature CAD41. In this study, we performed a large scale GWLA in a well-characterized U.S. cohort of 24 large, multigenerational CAD families (mean pedigree size\u2009=\u200918). This cohort, referred to as GeneQuest II, was independent from our previously reported GeneQuest cohort with 428 nuclear families40. The most attractive feature of the GeneQuest II cohort is the inclusion of extended family members of affected siblings or trios. To our knowledge, this is the largest linkage analysis of multiple large pedigrees to identify genetic loci for CAD, and significant susceptibility loci were identified.Most GWLAs for CAD have been conducted in either single large pedigrees or a large number of nuclear families. Increasing the number of family members within families can improve the power of linkage analysisThe 24 large and multigenerational families with CAD and MI were genetically characterized Table\u00a0. The pedA full set of 410 microsatellite markers spanning the entire human genome by every 10\u2009cM were initially genotyped for all 433 family members in the 24 CAD families. 36 markers were excluded for further analysis, including 9 autosomal markers with genotype and pedigree errors and 27 makers on X and Y chromosomes. Therefore, after quality control, 374 microsatellite markers on autosomes 1\u201322 from 433 family members were subjected to subsequent statistical analysis.As shown in Table\u00a0Multipoint NPL analysis was further performed. Multipoint NPL scores were plotted along the genetic map for each of 22 chromosomes Figs\u00a0. MultipoMoreover, both two-point and multipoint NPL analyses were carried out in individual families. Each of the 6 significant CAD loci was found to occur in at least one individual family Table\u00a0. NPL sco43. Counts of RefSeq genes and gene-disease pairs with score of >0.001 are summarized in Table\u00a0To explore candidate genes for CAD under the six significant genetic loci identified for CAD in the combined GeneQuest II families, we annotated all genes underlying each linkage. Genetic intervals of the six linkages were converted to physical locations according to the genetic maps generated by the HapMap 2 project (lifted over to hg19). RefSeq genes located under the six linkages were retrieved from the UCSC database , and then evaluated for potential relationship with cardiovascular diseases using the online program DisGeNET40. Suggestive evidence of linkage to the 3q29 CAD locus (P\u2009=\u20092.0\u2009\u00d7\u200910\u22124) was also found in a meta-analysis of four GWLS in Finnish, Mauritan, Germany, and Australian cohorts44. Therefore, the present study provides strong validation of the 3q29 and 9q22.33 linkages for CAD using an independent, large family-based linkage scan, suggesting that these two loci can be prioritized for identifying the underlying causative genes for CAD. Candidate genes for CAD at the 3q29 and 9q22.33 loci are listed in Table\u00a0UTS2B gene encodes Urotensin IIB and was shown to play a role in the acceleration of atherosclerosis development. Increased human Urotensin II levels were observed in hypertension, diabetes, atherosclerosis and CAD45. There are 20 unique genes within the 9q22.33 locus and three genes  were linked to cardiovascular diseases (Table\u00a0TGFBR1 encodes transforming growth factor beta receptor 1 (TGF\u03b21) and an increase in active TGF\u03b21 levels were correlated with both the occurrence and severity of CAD46.Identification of new genetic loci for CAD is critical for addressing the important issue of \u201cmissing heritability\u201d in the field of genetics, and in fully elucidating the genetic basis of CAD. In this study, we report a unique genome-wide linkage scan for CAD in 24 large, multigenerational families from a well-characterized U.S cohort (GeneQuest II). We carried out a model-free NPL-all scan and identified six susceptibility loci for CAD on chromosomes 2q33.3, 3q29, 5q13.2, 7p22.2, 9q22.33 and 17q21.2. It is interesting to note that the 3q29 and 9q22.33 loci were previously identified by us in a genome-wide linkage scan for CAD in 428 nuclear families in the GeneQuest population12 and located about 8\u2009Mb away from D17S1299. SNP rs46522, located in the UBE2Z-GIP-ADTP5G gene cluster, exhibited a strong cis-eQTL (expression quantitative trait locus) to UBE2Z in whole blood samples and to ATP5G1 in left ventricle samples according to the GTEx database v647. On the other hand, we identified a set of 514 unique RefSeq genes within the 17q21.2 CAD locus; 77 of them were linked to cardiovascular diseases based on data from DisGenNET . DisGenNET analysis identified 9 genes related to cardiovascular diseases  with two-point NPL score of 5.19 and a multipoint NPL score of 4.74 -dependent pathway of blood coagulation59. An elevated plasma TFPI level was significantly associated with the presence and severity of CAD61. TFPI expression can be regulated by ADTRP, a CAD susceptibility gene identified by our group17.The 2q33.3 locus, represented by marker D2S1384 at 200.43\u2009cM , covers a genomic region of 15.14\u2009Mb  of CAD patients63. CCNB1 encodes a regulatory protein involved in mitosis and a recent study showed that genetic variants in CCNB1 contributed to risk of the restenosis of intracoronary stents64.The 5q13.2 locus was mapped at marker GATA138B05 at 78.80\u2009cM (or 71.40\u2009Mb) and spanned an interval of 5.1\u2009cM (4.95\u2009Mb)  and represented by D2S1384 and D17S1299, respectively, contain CAD-risk SNPs identified by GWAS (rs6725887 at 2q33.3 and rs46522 at 17q21.2)  and eventually reduce the number of candidate genes for some loci. Moreover, fine mapping with SNP arrays may allow us to compare the SNP linkage data with the top hits from previous GWAS and identify new SNPs associated with CAD. Similarly, ongoing whole genome sequencing may be another powerful approach to capture SNPs or causal variants associated with CAD in the 24 GeneQuest II families. Second, we highlighted 3\u201377 genes at each CAD locus based on the evidence from existing literature with a purpose to illustrate the relevance of each CAD locus to etiological process of CAD. However, the CAD causal genes being responsible for each linkage were possibly overlooked in this study . Future fine mapping studies may be carried out with additional markers surrounding the microsatellite polymorphisms used for linkage analysis or SNP microarrays with a much increased marker density. Single SNPs may not as informative as microsatellite markers for linkage analysis due to their bi-allelic status, but haplotypes constructed using multiple SNPs may be considered as multi-allelic markersdy Table\u00a0. Third, In summary, we report the results of a genome-wide linkage scan of 24 large GeneQuest II families and uncover six genetic loci for CAD on chromosomes 2q33.3, 3q29, 5q13.2, 7p22.2, 9q22.33 and 17q21.2. Our study identifies four novel CAD loci . Similar analysis in individual families confirmed the six significant CAD loci and also identified nine new significant linkages on 2q11.2, 9p24.2, 9q34.2, 11q14.1, 12q13.13, 15q26.1, 17q22, 20p12.3, and 22q12.1. Our study also independently confirms the 3q29 and 9q22.33 CAD loci identified by our earlier genome-wide linkage scan for CAD in 428 nuclear families. Two loci on 2q33.3 and 17q21.2 contain GWAS risk variants identified from population samples. These studies may provide a new framework for uncovering causative variants, genes and biological pathways involved in the pathogenesis of CAD.Twenty-four large, extended, and multigenerational CAD families were recruited at the Center for Cardiovascular Genetics of the Cleveland Clinic. The study was referred to as GeneQuset II to distinguish it from the original GeneQuest study which recruited more than 428 nuclear families, mostly for sib-pair analysis. The GeneQuest II study started in the year of 2001 and is completely independent from the earlier GeneQuest study carried out between 1995 and 2000. This study was reviewed and approved by the Cleveland Clinic Institutional Review Board (IRB) on Human Subject Research, and conformed to the guidelines set forth by the Declaration of Helsinki. Written informed consent was obtained from all participants.69. Families or patients with hypercholesterolemia, insulin-dependent diabetes, childhood hypertension, and congenital heart disease were excluded from this study. Each family has at least four definitely diagnosed CAD patients; and the average pedigree size was 18. Clinical and demographic features of the 24 GeneQuest II CAD families with 433 family members are summarized in Table\u00a040, genome-wide linkage analysis was carried out using all family members instead of sibling pairs only, given the large pedigrees collected in GeneQuest II  or coronary artery bypass (CABG), and a previous diagnosis of myocardial infarction (MI) as describedWhole blood samples were drawn from each study participant. Genomic DNA was isolated using the Gentra Puregene blood . All DNA samples were quantified using NanoDrop 2000  and inspected for quality by agarose gel electrophoresis.Genome-wide genotyping was performed by Mammalian Genotyping Service of the National Heart, Lung, and Blood Institute directed by Dr. James L. Weber at Center for Medical Genetics at Marshfield Clinic (http://research.marshfieldclinic.org/genetics/GeneticResearch/screeningsets.asp) using Screening Set 11. The screening set consists of 410 microsatellite markers spanning the whole human genome by every 10\u2009cM on average.40. In brief, genotypes with non-consensus calls were re-genotyped or deleted. Microsatellite markers on sex chromosomes were excluded. Missing parental genotypes were added and treated as missing values to complete family pedigrees  within each family was verified by the RELTEST program included in the S.A.G.E software page70. The RELTEST program did not detect any inconsistent family relationship. Allele frequencies for all microsatellite markers were estimated by module FREQ in S.A.G.E in the pooled samples containing all of our existing family studies. Program Mega272 was used to generate the input format required for Genehunter version 2.1_r2 beta71. Affected and unaffected individuals were coded as \u201c2\u201d and \u201c1\u201d, respectively, whereas individuals with uncertain phenotype were coded as \u201c0\u201d.Prior to linkage analysis, raw genotyping data were cleaned as described in our previous studiesees Fig.\u00a0 for link73 allele-sharing between all affected subjects within a family. We used the NPL-all statistic within Genehunter version 2.1_r2 beta for linkage analysis, which examines all individuals in the 24 GeneQuest II families simultaneously and provides a more powerful test (www.broad.mit.edu/ftp/distribution/software/genehunter/). Without specifying the disease transmission model for all markers, non-parametric linkage (NPL) analysis was carried out to jointly analyze genotype data of all 24 GeneQuest II families. The linkage between CAD and a genetic marker was evaluated by calculating NPL score Z, which is the summation of standardized identity-by-descent allele-sharing scores across multiple families. Under a null hypothesis of no linkage, Z has mean 0 and variance 1 by choosing appropriate weighting factors. Statistical significance of Z can be inferred by comparing the observed Z against to its null distribution. Two types of NPL scores were calculated for each marker: 1) A two-point NPL score examined whether a single marker was linked to CAD; 2) A multipoint NPL score investigated whether a group of markers were linked to CAD. The advantage of the multipoint approach is its capability of incorporating the information of adjacent markers into linkage analysis (making markers more informative). The NPL-all linkage analysis was also carried out individually in each of the 24 GeneQuest II families. The larger a NPL score is, the stronger the linkage it indicates. As suggested by Lander and Kruglyak74, linkage peaks were defined in three categories: (1) Highly significant linkage: NPL of 4.99 ; (2) Significant linkage: NPL of 4.08 ; (3) Suggestive Linkage: NPL of 3.18 .The principle of the Genehunter linkage analysis is to examine any excess of identity-by-decenthttp://genome.ucsc.edu/.UCSC database: 42: http://www.disgenet.org/web/DisGeNET/menu/home.DisGenNET47 portal: http://gtexportal.org/home/.GTExGenetic map of microsatellite markers: http://research.marshfieldclinic.org/genetics/GeneticResearch/screeningsets.asp.https://github.com/joepickrell/1000-genomes-genetic-maps.Physical map based on hg19: www.broad.mit.edu/ftp/distribution/software/genehunter/.Genehunter version 2.1_r2 beta:"}
+{"text": "AHNAK), previously associated with blood biomarkers in COPD, phospholipase C Beta 3 (PLCB3), shown to increase airway hyper-responsiveness, solute carrier family 22-A11 (SLC22A11), involved in amino acid metabolism and ion transport, and metallothionein-like protein 5 (MTL5), involved in nicotinate and nicotinamide metabolism. Association of SLC22A11 and MTL5 variants were confirmed in the meta-analysis of 9,888 cases and 27,060 controls. In conclusion, we have identified novel rare variants in plausible genes related to COPD. Further studies utilizing large sample whole-genome sequencing should further confirm the associations at chromosome 11 and investigate the chromosome 15 and 5 linked regions.Chronic obstructive pulmonary disease (COPD) is a complex and heritable disease, associated with multiple genetic variants. Specific familial types of COPD may be explained by rare variants, which have not been widely studied. We aimed to discover rare genetic variants underlying COPD through a genome-wide linkage scan. Affected-only analysis was performed using the 6K Illumina Linkage IV Panel in 142 cases clustered in 27 families from a genetic isolate, the Erasmus Rucphen Family (ERF) study. Potential causal variants were identified by searching for shared rare variants in the exome-sequence data of the affected members of the families contributing most to the linkage peak. The identified rare variants were then tested for association with COPD in a large meta-analysis of several cohorts. Significant evidence for linkage was observed on chromosomes 15q14\u201315q25 [logarithm of the odds (LOD) score = 5.52], 11p15.4\u201311q14.1 (LOD = 3.71) and 5q14.3\u20135q33.2 (LOD = 3.49). In the chromosome 15 peak, that harbors the known COPD locus for nicotinic receptors, and in the chromosome 5 peak we could not identify shared variants. In the chromosome 11 locus, we identified four rare  <0.02), predicted pathogenic, missense variants. These were shared among the affected family members. The identified variants localize to genes including neuroblast differentiation-associated protein ( SERPINA1 gene at chromosome 14q32.13, encoding AAT, was in fact the first gene identified to be associated with COPD  and Family with sequence similarity 13 member A (FAM13A), chromosome 5 \u2013 5-hydroxytryptamine receptor 4 (HTR4), chromosome 15 \u2013 Nicotinic cholinergic receptors (CHRNA3/5) and Ion-responsive element binding protein 2 (IREB2) and chromosome 19 \u2013 Cytochrome P450 family gene (CYP2A6), member RAS oncogene family gene (RAB4B) and Egl-9 family hypoxic-inducible factor 2 (EGLN2)  . HoweverDespite the undeniable progress in understanding the genetic origins of COPD, a major part of its heritability remains unexplained. A complicating factor in studies on the genetics of COPD is that COPD is considered a complex genetic trait, i.e., multiple, possibly interacting, genetic and environmental factors are involved. Therefore, there is a need for fine mapping techniques that can identify functional, rare variants with large effects explaining specific types of COPD. Rare variant association studies can be carried out in relatively small sample sizes when using family-based settings . In a geThis study uses the ERF study, a Dutch genetically isolated population, to localize and identify rare genetic variants and subsequently shows the relevance of these variants in the general population by performing an association analysis in a large sample.1/FVC) < 0.7, with or without medication use (n = 116). If the information on FVC was missing (n = 14), the following criteria for COPD were used: FEV1 < 80%, use of respiratory medication and a COPD diagnosis in the report of the respiratory specialist to the general practitioner. If no lung function measurement was available (n = 15), COPD diagnosis was based on: medication use with CT-scan of the lungs indicating COPD and/or a history of frequent COPD exacerbations mentioned in the medical documents. Thus, the COPD diagnosis could be confirmed for 145 participants, of which 3 did not have genotyping data, resulting in the final sample size for the linkage study of 142 COPD cases.The linkage study was performed in 142 related participants from the ERF study. ERF is a family-based cohort study, studied as part of the Genetic Research in Isolated Population (GRIP) program. It is based in a genetically isolated community from the south-west area of the Netherlands, set up to investigate genes underlying different quantitative traits and common diseases . The parThe association analysis was performed using data from the RS , the LLS , the VlaVla study  and the data from the study of 1/FVC < 0.7), assessed either by spirometry in the research center or by reviewing medical histories of the participants. Spirometry was performed by trained paramedical personnel, according to the guidelines of the American Thoracic Society/European Respiratory Society (ATS/ERS). In absence of interpretable spirometry measures, all medical information of subjects regularly using respiratory medication was reviewed, including files from specialists and general practitioners, to confirm a diagnosis of COPD. Both ERF and RS have been approved by the Medical Ethics Committee of the Erasmus Medical Center. All participants provided written informed consent to participate in the study and to obtain information from their treating physicians.Rotterdam Study is a prospective, population-based study , focusinLifelines study is a multi-disciplinary prospective population-based cohort of the Northern provinces of the Netherlands with a three generation design, focusing on the onset of common complex diseases . COPD wa1/FVC < 0.7. Data of the last survey in 1989/1990 were used and spirometry data were collected by performing a slow inspiratory maneuver, using a water-sealed spirometer . The Committee on Human Subjects in Research of the University of Groningen reviewed the study and affirmed the safety of the protocol and study design and all participants gave their written informed consent.The VlaVla is a prospective, Dutch population-based cohort including individuals from Vlagtwedde  and Vlaardingen (an urban area), aimed to gain insight into the risk factors for chronic airway diseases and lung function . COPD waIn the study by For all participants, DNA was extracted from venous blood using the salting out method .P < 10-8) and X-chromosome variants and participants with an overall call rate <96%. Mendelian inconsistencies were designated as missing genotypes. The final dataset comprised 5,250 autosomal SNVs in 3,018 participants.For the linkage analysis genotyping was performed using the 6K Illumina Linkage IV panel . Further, QC was performed involving exclusion of the variants with call rate <98%, those diverging from Hardy\u2013Weinberg equilibrium  families using PEDCUT software . We used2 from the National Heart, Lung and Blood Institute (NHLBI) and with MAF < 0.05 in the general population (1000 Genomes). As frequencies in a genetically isolated population may be inflated or deflated due to genetic drift  based on the FunctionGVS column of the SeattleSeq Annotation databaseic drift , we usedN = 636) and in exome-chip (N = 572) data, in three RS cohorts , using the HRC imputed data , the LLS , the VlaVla cohort  and the N = 11,797). For this analysis, in ERF we used \u201cseqMeta\u201d package in R  . FurtherTable 1. All 27 families included in the linkage analyses in ERF are depicted in Supplementary Figure Table 2 and Figure 1, we identified significant evidence for linkage of COPD to chromosomes 15q14\u201315q25 (HLOD = 5.52), 11p15.4\u201311q14.1 (HLOD = 3.71), and 5q14.3\u20135q33.2 (HLOD = 3.49).The general characteristics of the study samples are presented in Figure 2). Exome-sequence data were available for 8 of 17 COPD cases from these two families. We identified four missense variants including rs116243978 (AHNAK), rs35169799 (PLCB3), rs141159367 (SLC22A11), and rs146043252 (MTL5), shared among five of the eight affected family members (Table 3). Each of these variants was predicted to be highly pathogenic  which suggests their relevance for the disease development. Of these four variants, one (rs141159367 in SLC22A11) showed a significant association  with COPD in the meta-analysis (Table 4). The variant rs146043252 in MTL5 showed a nominal association signal .We next searched for rare, deleterious and shared variants by most (>50%) of the affected family members in the three identified regions mentioned above. In the linked regions of chromosomes 5 and 15 we could not identify any variants that passed mentioned filtering criteria. For the linked region on chromosome 11, we identified two families that were contributing most (LOD > 1) to the linkage score , shared by at least five family members. One of these four variants, i.e., rs141159367 in SLC22A11, was significantly associated with COPD in 9,888 cases and 27,428 controls (P = 0.002) while another variant (rs146043252 in MTL5) showed nominal association with COPD (P = 0.04).In this study, we found significant evidence for extensive linkage of COPD to the chromosomes 15q14\u201315q25 (40.1 Mb), 11p15.4\u201311q14.1 (73.9 Mb), and 5q14.3\u20135q33.2 (64.1 Mb). We were able to identify four rare and predicted pathogenic variants under the chromosome 11 peak, in plausible genes , which encodes an integral membrane protein and part of the family of OATs, known to mediate the absorption and elimination of endogenous and exogenous organic anions and as such, are involved in the pharmacokinetic, pharmacodynamic and safety profiles in a wide range of drugs (SLC22A11 (OAT4) is mainly expressed in kidney and placenta. However, it is also shown to be expressed in lung tissue, fibroblasts and T-lymphocytes (P < 5 \u00d7 10-7), among other tissues/cells reported in the Gene network  encodes testis expressed metallothionein like proteins (TESMIN). They are highly conserved, low-molecular-weight cysteine-rich proteins induced by and binding to heavy metal ions, and they do not have enzymatic activity. They play a central role in the regulation of cell growth and differentiation, and are involved in spermatogenesis, differentially regulating meiosis in male and female cells , based on the Gene network (Our linkage analysis yielded different regions compared with those identified earlier. However, the fact that both le cells . MTL5 wa network . Metallo network .The main strength of our study is the genetically isolated family-based population, which can display increased frequencies of some variants found at very low proportions in panmictic populations. This allowed us to perform a genome-wide linkage scan and identify rare coding variants. However, even though we identified linkage of three regions to COPD, a limitation of our study is the low power to explain the peaks at chromosomes 5 and 15, possibly due to the use of exome data. As intronic regulatory variants may play a significant role, in the future, faster and cheaper whole-genome sequencing will allow us to improve identification of rare variants and our understanding of their involvement in COPD. As our sample consists of high percentage of current or ex-smokers, it is possible that we are demonstrating genetic effects on smoking which further affects the development of COPD. Nevertheless, we were able to demonstrate a positive association, independent of smoking, of two variants in the association meta-analysis comprising 9,888 cases and 27,060 controls. Yet, studies with very large sample sizes utilizing mediation or mendelian randomization techniques are needed to disentangle these relationships and confirm our results in the general population.AHNAK, PLCB3, SLC22A11 and MTL5. The variants in SLC22A11 and MTL5 were significantly associated with COPD in our meta-analysis. Further studies pooling large sample sizes could confirm the role of the identified rare variants at chromosome 11 in the general population. Similarly, large studies utilizing whole-genome sequencing should further investigate the role of linked regions in chromosomes 5 and 15 in COPD.Using the powerful genome-wide linkage scan in a Dutch genetic isolate, we have confirmed the implication of the 15q25 region in COPD and identified regions at chromosomes 5 and 11. Within the region on chromosome 11 we identified four deleterious rare variants shared between most of the affected family members in IN, NA, NT, LL, JV, DvdP, BH, DQ, and MC were involved in the analysis of the data. IN, JV, DvdP, CCvD, DvdP, HB, CMvD, and NA contributed to the conception and design of this work and were involved in the interpretation of the results. IN, JV, DvdP, LL, GB, and DvdP were involved in data collection/preparation. All authors were involved in writing and critically revising the manuscript, approved the final manuscript, and agreed to be accountable for it.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "They are assembled from iron and cysteine sulfur on protein scaffolds. Iron is typically stored as iron oxyhydroxide, ferrihydrite, encapsulated in 12\u2009nm shells of ferritin, which buffers cellular iron availability. Here we have characterized IssA, a protein that stores iron and sulfur as thioferrate, an inorganic anionic polymer previously unknown in biology. IssA forms nanoparticles reaching 300\u2009nm in diameter and is the largest natural metalloprotein complex known. It is a member of a widely distributed protein family that includes nitrogenase maturation factors, NifB and NifX. IssA nanoparticles are visible by electron microscopy as electron-dense bodies in the cytoplasm. Purified nanoparticles appear to be generated from 20\u2009nm units containing \u223c6,400 Fe atoms and \u223c170 IssA monomers. In support of roles in both iron\u2013sulfur storage and cluster biosynthesis, IssA reconstitutes the [4Fe-4S] cluster in ferredoxin  Pyrococcus furiosus.The biosynthesis of iron-sulfur clusters in anaerobic organisms has not been extensively investigated. Here, the authors identify and characterize a multi-subunit protein that stores iron and sulfur in thioferrate for the assembly of the clusters in  Iron is an essential nutrient for almost all known organisms. It functions as a protein cofactor in fundamental pathways including respiration, photosynthesis and the biogeochemical cycling of sulfur and nitrogen. There are two major types of iron-containing protein cofactors, haemes and iron\u2013sulfur clusters. The most common iron\u2013sulfur cluster is the cubane-type [4Fe-4S] cluster, which is involved in electron transfer, catalysis, DNA repair and small molecule sensing2+ clusters will lose Fe reversibly upon oxidation, to form cubane-type [3Fe-4S]+ clusters or cysteine persulfide-ligated [2Fe-2S]2+ clusters. Repair of such degraded forms is efficient because they can be restored simply by reduction and the addition of ferrous iron without the need for the full biosynthetic machinery.Iron\u2013sulfur cluster biosynthesis is carried out by two main systems in microorganisms. Bacteria (and mitochondria) typically use the ISC system while archaea and some bacteria (and some plastids) use the SUF system42 while maintaining sufficient cellular iron in spite of the insolubility of free ferric iron at neutral pH72+ and O2 in the interior of the sphere using catalytic iron sites, but the mechanism of Fe release from the mineral core is still largely unresolved. Although ferritin homologues exist throughout the three domains of life, including anaerobes, an anaerobic oxidant that facilitates oxidation of Fe2+ has yet to be identified, and the physiological role of ferritin in many organisms is not clear2 g\u22121) and metastability While the identity or need for a specific Fe donor for iron-sulfur cluster biosynthesis is still under debatePyrococcus furiosus, which grows optimally near 100\u2009\u00b0C in hydrothermal marine vents0) and uses it as an (insoluble) electron acceptor to generate (soluble) hydrogen sulfideP. furiosus does contain a homologue of the SufS cysteine desulfurase1P. furiosus grown with and without S0 revealed upregulation in the expression of numerous genes during hydrogen sulfide production, including those involved in iron and iron-sulfur cluster metabolismsipA is only upregulated by sulfide in the presence of sufficient ironTo investigate the process of iron and sulfur storage and their incorporation into iron-sulfur clusters in an anaerobic micro-organism that cannot use oxygen to oxidize ferrous iron, we examined the archaeon P. furiosus cells expressing IssA shows naturally electron-dense particles that co-locate with IssA immunolabelling. TEM of purified IssA reveals particles up to 300\u2009nm, which appear to be comprised of \u223c20\u2009nm spheres. X-ray absorption spectroscopy (XAS) strongly supports a thioferrate-type linear (FeS2\u2212)n structure of iron and sulfur, and the EPR of IssA is in accord with this assignment. Finally, we show that IssA is capable of assembling a [4Fe-4S] cluster on P. furiosus ferredoxin (Fd), an abundant electron carrier in this organism, in the presence of the small molecule thiol, dithiothreitol (DTT). These properties of IssA together with the conditions under which it is expressed13P. furiosus stores excess Fe and S, when they are both highly abundant, in IssA-bound thioferrate, an iron\u2013sulfur structure not previously known in biology. The stored thioferrate can subsequently be mobilized for assembly of [4Fe-4S] clusters, which are widely used in P. furiosus. Phylogenetic analyses suggest that homologues of IssA in many archaea, and possibly bacteria as well, may also store iron as thioferrate.In this study, we present characterization of IssA. Transmission electron microscopy (TEM) of 0 was added to a growing P. furiosus culture, IssA could be detected in cells by TEM and immuno-gold labelling after 20\u2009min  of the protein was too large to enter the chromatography column and was retained on the pre-column filter (diameter 1\u2009\u03bcm). IssA that entered and eluted from the chromatography column did so in a peak at the exclusion limit (40\u2009MDa) or just after the exclusion limit (100\u2009MDa dextran or 400\u2009nm spheres) from Superose 6 and Sephacryl S-1000 SF columns, respectively . DynamicNegative stain TEM of the purified protein showed IssA assemblages with dimensions ranging from 20 to 300\u2009nm . Incubat+ cluster in the high pH form of the enzyme aconitases electron to unfilled molecular orbitals involving both sulfur 3p and metal 3d orbitals, with vacancies due to covalency of the Fe\u2013S bond 1616XAS was conducted at both Fe K-edge and S K-edge absorption energies to characterize the iron and sulfur bound by IssA. R values, using data to 18\u2009\u00c5\u22121. However, it shows behaviour characteristic of real EXAFS, rather than a noise peak in the Fourier transform, so that the feature persists irrespective of the k-ranges, and moreover fits to a very similar Fe\u00b7\u00b7\u00b7\u00b7Fe distance with different k-ranges. The use of multiple scattering EXAFS reproduces many weaker features in the EXAFS n polymer with two sulfur atoms bridging each pair of Fe(III) ions. Compounds with such a structure are known as thioferrates193+ and S2\u2212 in anionic chains of edge-sharing FeS4 tetrahedra separated by charge-balancing cations n structure. This is also supported by the measured acid-labile iron, sulfide and sulfane sulfur content of IssA . Degradation of synthetic thioferrates in acid (the conditions used for the assays) has been shown to produce Fe2+, S2\u2212 and S0 in a 2:3:1 ratio0\u2019 in a 1:2 ratio with iron. Since S0 will also be produced under the acidic conditions of the S2\u2212 assay, formation of polysulfides further reduces the amount of sulfur available for detection as H2S  Fe3+ or magnetically isolated linear [3Fe-4S]+ clusters (S=5/2)g=2 region (<0.01 spin/IssA monomer) is unknown. The anomalous temperature dependence of the very broad isotropic signal is unique among protein-derived EPR signals, but is in agreement with the EPR signal observed for synthetic thioferrates203+ ions in thioferrate polymers results in decreased intensity at temperatures below the N\u00e9el temperatureg=2  was investigated. P. furiosus almost exclusively contains [4Fe-4S] cluster-containing Fe\u2013S proteins, and P. furiosus Fd is an abundant protein that is used as electron donor for numerous enzymes. Reconstitution experiments were carried out anaerobically under a variety of conditions. IssA with stoichiometric or a twofold excess of bound Fe and S (as thioferrate) was mixed with apo-Fd at pH 6.8  and incubated at room temperature for 24\u2009h or at 80\u2009\u00b0C for 1\u2009h. Since a [4Fe-4S]2+ cluster is more reduced than the all-Fe3+ thioferrate iron and sulfide donor, sodium dithionite (Em\u223c\u2212420\u2009mV versus NHE), DTT (Em\u223c\u2212330\u2009mV versus NHE), or tris(2-carboxyethyl) phosphine  were included in the reaction mixture as a reductant, a disulfide-cleaving reagent (DTT and TCEP) and a dithiol-metal chelating agent (DTT). After centrifugation to remove unreacted IssA, the Fd was purified and the cluster content and integrity were assessed compared with native holo-Fd based on ultraviolet\u2013visible absorption and CD spectra quantified based on protein determinations. No reconstitution occurred in the presence of dithionite or TCEP. However, reconstitution of the [4Fe-4S]2+ cluster was observed in the presence of DTT , that is replaced in vitro by DTT, plays a role in disassembling the thioferrate polymer into transferrable pieces by chelating Fe, [2Fe-2S]2+, or linear [3Fe-4S]1+ fragments under reducing conditions that are then assembled into [4Fe-4S]2+ clusters in acceptor proteins such as Fd.To test the hypothesis that IssA functions as a storage protein for Fe and S that can be used for the assembly of iron-sulfur clusters, the ability of IssA to reconstitute the [4Fe-4S] cluster in the apo-form of e of DTT . ReconstP. furiosus is composed of 179 amino acids and has a predicted molecular weight of 19\u2009kDa from its gene sequence (PF2025). The N-terminal 109 residues comprise an IPR003731 InterPro globular domainP. furiosus protein.IssA from 7S9C)8S9C S9C ref. . NifB prS9C ref. . Due to E. coli, and purified by affinity chromatography (apo-IssA) to investigate whether the domain binds iron and sulfide and how this affects oligomerization. When colourless monomeric apo-IssA was incubated with a 40-fold excess of iron (ferrous ammonium sulfate) and sulfur (sodium sulfide), the black product eluted from a Superose 6 SEC column with molecular weights ranging from \u223c200\u2009kDa to \u223c900\u2009kDa with up to 25 Fe per IssA monomer. Hence, the IPR003731 globular domain of IssA clearly binds Fe and S, stimulating oligomerization in vitro, although the product is very heterogeneous and it was not characterized further.A gene encoding the polyhistidine-tagged globular IPR003731 domain of IssA (residues 1\u2013109) was constructed, expressed in P. furiosus IssA contains a proline-rich region  followed by a flexible region comprised predominantly of cationic (7 of 70), aromatic (14 of 70) and glycine (15 of 70) residues. Secondary structure predictionP. furiosus protein is highly conserved only in IssA proteins in species within the Thermococcales. Indeed, some members of the IssA clade have a shortened tail (<70 residues) and some have little or no tail region, which includes the IssA-type protein from Methanothermobacter thermautotrophicus that was used to model the structure of the IPR003731 domain of P. furiosus IssA  of sus IssA . In spitsus IssA  that shain vivo  or a cellular thiol (in vivo) to bind Fe3+ and/or [2Fe-2S]2+/[3Fe-4S]1+ thioferrate fragments , and both clusters can be readily converted to [4Fe-4S]2+ clusters in biological and in synthetic chemistry372+ clusters to generate a [4Fe-4S]2+ cluster occurs via two-electron reductive coupling37de novo cluster assembly in the ISC system381+ clusters can be converted to a [4Fe-4S]2+ cluster by the addition of Fe2+ and one electronP. furiosus, the in vitro cluster assembly results presented here, coupled with the high Fe and S content of IssA and its iron- and sulfide-dependent expression, strongly support a role for IssA in storing Fe and S that can be used for the biosynthesis of Fe\u2013S clusters. Moreover, the in vitro results raise the possibility of spontaneous [4Fe-4S] cluster assembly from Fe3+,2+ and S2\u2212 on acceptor proteins under Fe and sulfide replete conditions in some strictly anaerobic hyperthermophilic archaea such as P. furiosus. Except for SufCBD and two putative SufS cysteine desulfurases, P. furiosus does not encode any other known Fe\u2013S cluster assembly protein. Moreover, SufC and SufD contain no cysteine residues and the putative SufB scaffold protein has only two cysteines (compared to 13 in E. coli SufB), which are both rigorously conserved in other SufB proteins. Hence, the scaffolding hypothesis that constitutes the current paradigm for Fe\u2013S cluster assemblyP. furiosus and related organisms.We have also demonstrated that IssA can provide Fe and S for assembly of [4Fe-4S]e of DTT . The mecragments . For exae2+ ref.  and the P. furiosus when the organism is grown in the presence of abundant iron and sulfide, we propose that the thioferrate structure is synthesized directly from these inorganic precursors. The fact that apo-IssA binds Fe and S from inorganic salts also supports this idea. Interestingly, expression of the genes encoding the two cysteine desulfurase homologues in P. furiosus (PF0164 and PF1066) are strongly down-regulated (5.1- and 3.2-fold) in response to S0, while the sufBD homologues, which are likely to be involved with some aspect of Fe\u2013S cluster trafficking, are strongly upregulated along with issA when S0 is presentP. furiosus. Consequently, Fe\u2013S clusters synthesized from thioferrate may require less energy than canonical ATP-driven scaffold-assembled Fe\u2013S clusters.Because IssA is only produced in 2\u2212)n at the IPR003731 domain and that the tail region stabilizes the structure through electrostatic interactions. The 70 amino-acid C-terminal region of IssA contains 7 cationic residues (mostly arginine) that are sufficiently close to each other to preclude a folded structure without a negatively charged counterpart such as thioferrate. In addition, the abundance of glycine residues further indicates a lack of secondary structure in the absence of thioferrate. Hence we propose a model in which the cationic tail may bind the anionic thioferrate chain by wrapping around thioferrate in perhaps a helical arrangement, which is further stabilized by interactions between the tail\u2019s aromatic residues. This interaction would confer a defined structure on the otherwise disordered IssA tail. Since apo-IssA is purified as a monomer, but oligomerizes in the Fe\u2013S bound state, we suggest that formation of the observed \u223c20\u2009nm spherical IssA particles is also dependent on association with thioferrate. According to the estimated Fe-protein ratio, additional cations are needed to completely balance the negative charge on thioferrate, and we expect these are provided by loosely bound cations as well as the single zinc ion per protein , 2\u2009mM dithionite and 2\u2009mM DTT (buffer A) containing 1% w/v sodium dodecyl sulfate, then twice in buffer A and applied to a caesium chloride gradient  to remove precipitated material from the media. Fractions containing IssA were pooled and dialyzed against 4\u2009l buffer A and concentrated using a Centricon centrifugal concentrator with a 10\u2009kDa cut-off (Millipore). Protein concentration was estimated using the bicinchoninic acid method at 60\u2009\u00b0C  and applied 3\u2009\u03bcl to a glow discharged carbon-coated TEM grid. The grid was washed with 3\u2009\u03bcl dH2O and stained with 3\u2009\u03bcl of 2% uranyl acetate. Microscopy was conducted on a JEOL 2010\u2009F TEM operated at 200\u2009kV high tension and 50\u2009kX magnification. 100 electron micrographs were recorded in a Gatan Ultrascan 4\u2009K by 4\u2009K CCD camera. We automatically selected 30,000 raw particles and performed 2D image classification in EMAN 2  to 20\u2009\u03bcl IssA sample. This mixture was shaken at room temperature for 4\u2009h after which the sample was fully dissolved. We diluted the solution six fold with deionized water (dHN 2 ref. . Composi\u03c7(k) were quantitatively analysed as previously describedab initio theoretical phase and amplitude functions calculated using the programme FEFF version 8.25 (ref. Details of iron and sulfur K-edge data collection are described in .25 ref. . No smoog. The supernatant was removed and the X-band (\u223c9.6\u2009GHz) EPR spectrum was obtained using a Bruker ESP-300E EPR spectrometer equipped with an ER-4116 dual-mode cavity and an Oxford Instruments ESR-9 flow cryostat.100\u2009mg purified IssA was loaded into a quartz EPR tube and centrifuged for 30\u2009min. at 1,000P. furiosus was prepared using the method of Moulis and MeyerApo-Fd from Details of design, expression and purification of the apo-IssA construct are described in IssA reconstitution was carried out by adding ferrous ammonium sulfate (10\u2009mM) and sodium sulfide (10\u2009mM) in buffer A to apo-IssA (0.25\u2009mM) and incubating with shaking for 1\u2009h at 80\u2009\u00b0C. Excess iron and sulfide were removed by buffer exchange using a Centricon concentrator (Millipore) with a 10\u2009kDa cut-off.Details of IPR003731 sequence selection and alignment, phylogenetic tree generation and refinement, construction of the structural model of IssA, and related analyses are described in Additional data that support the findings of this study are available from the corresponding author upon reasonable request.How to cite this article: Vaccaro, B. J. et al. Biological iron-sulfur storage in a thioferrate-protein nanoparticle. Nat. Commun.8, 16110 doi: 10.1038/ncomms16110 (2017).Publisher\u2019s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations."}
+{"text": "Linkage analysis produces two separate complementary marker linkage maps that have little use in disease association analysis and breeding. There is a need to develop efficient statistical methods and computational algorithms to construct or merge a complete linkage dominant marker maps. The key for doing so is to efficiently estimate recombination fractions between dominant markers in repulsion phases.Dominant markers in an F2 dominant and codominant marker data, respectively. The results obtained from simulated and real genotype datasets showed that the ELS algorithm was able to accurately estimate frequencies of gametes and outperformed the EM algorithm in estimating recombination fractions between dominant loci and recovering true linkage maps of 6 dominant loci in coupling and unknown linkage phases. Our BAT method also had smaller variances in estimation of two-point recombination fractions than the EM algorithm.We proposed an expectation least square (ELS) algorithm and binomial analysis of three-point gametes (BAT) for estimating gamete frequencies from F2 population and BAT is a computationally efficient and fast method for estimating frequencies of three-point codominant gametes.ELS is a powerful method for accurate estimation of gamete frequencies in dominant three-locus system in an\u00a0FThe online version of this article (10.1186/s12859-017-1804-8) contains supplementary material, which is available to authorized users. In a natural population, for example, human population, the frequencies of these gametes are not purely derived from recombination events but may be due to selection, genetic drift, migration and mutation. If, however, sister gametes are found to be equal in statistics, then these frequencies can still be used to inference recombination fractions between loci and recombination inference.BATII given in Additional file 2 population and BAT is a computationally efficient and powerful method for estimating frequencies of non-sister three-point codominant gametes.Accurate estimation of recombination fractions between loci is given by methodologies developed for accurate estimation of gamete frequencies in a population. Analyses of simulated and real dominant and codominant data show that the ELS method proposed here is a powerful algorithm for accurate estimation of frequencies of gametes with unknown phase in dominant three-locus system in FAdditional file 1:Source code: three R functions: BAT.R, ELS.R, simulatF2.R. (ZIP 4\u00a0kb)Additional file 2:Appendix A. Binomial analysis of three-point method (BATII) is described in detail. BATII is used to estimate frequencies of sister gametes at codominant loci in natural populations. (DOCX 184\u00a0kb)Additional file 3:Appendix B. A proof of a proposition that equal weights of two datasets combined into a dataset have maximum linkage information and minimum error for linkage analysis is given. (DOCX 41\u00a0kb)"}
+{"text": "Endodontic irrigating solutions may interact chemically with one another. This is important, because even when solutions are not admixed, they will come into contact with one another during an alternating irrigation technique, forming unwanted by-products, which may be toxic or irritant. Mixing or alternating irrigants can also reduce their ability to clean and disinfect the root canal system of teeth by changing their chemical structure with subsequent loss of the active agent, or by inducing precipitate formation in the root canal system. Precipitates occlude dental tubules, resulting in less penetration of antimicrobials and a loss of disinfection efficacy. Sodium hypochlorite is not only a very reactive oxidizing agent, but is also the most commonly used endodontic irrigant. As such, many interactions occurring between it and other irrigants, chelators and other antimicrobials, may occur. Of particular interest is the interaction between sodium hypochlorite and the chelators EDTA, citric acid and etidronate and between sodium hypochlorite and the antimicrobials chlorhexidine, alexidine, MTAD and octenisept. The use of sodium hypochlorite (NaOCl) dates back over 100 years, when Dakin, recognizing its great reactivity and its ability to chemically digest organic matter, reported the use of an 0.5% solution for wound disinfection . The uniWhen tested on mature biofilms of clinical isolates of endodontic infection, NaOCl has superior antimicrobial activity compared to several other irrigants; however, it neither totally eradicates biofilm volume, nor achieves complete killing of bacteria . Strateg\u2212) concentration vary significantly according to brand, and can range between 10.9 to 12.0 for pH, and from 0.03\u20131.1% for OH\u2212 concentration [\u2212 that have a higher pH. It is also worth noting that endodontics is often performed with commercial bleaches used for general cleaning, such as Chlorox, which may result in the use of inappropriately high levels of NaOCl [\u2212) predominates. At neutral pH, hypochlorous acid (HOCl) is mostly present, while below pH 4, chlorine gas starts to form [\u2212 is present, tissue dissolution is maximized [NaOCl solutions used in endodontic irrigation range in concentration from 0.5\u20136% [of NaOCl . The rea to form . The antaximized . The pH aximized  or when aximized , and thuE\u00b0/V [\u2212 is a nucleophile, and can drive chemical reactions [NaOCl is a strong oxidizing agent with a reduction potential of 1.6 E\u00b0/V . The oxiE\u00b0/V . The reaE\u00b0/V . Additioeactions . EDTA is used commonly at concentrations of 15% or 17%. Aqueous solutions are prepared by dissolving the di- or trisodium salts, resulting in a neutral to slightly alkaline pH ,20. At tThe remaining free available chlorine in a 1:1 mixture of 1% NaOCl and 17% EDTA at 1 min is 10%, but this declines to 0% at one hour after mixing . However1H spectroscopy (1H NMR) studies have identified unspecified and slowly formed EDTA breakdown products [In the above reactions, the EDTA molecule is left intact . Howeverproducts , these aproducts , which w4EDTA) with NaOCl. This combination results in the longer maintenance of both free available chlorine and desirable pH conditions [4EDTA falls from 11.9 at 10 min after mixing to 9.0 at one hour after mixing, during which period the remaining free available chlorine declines from 90% to 62% [Recently, more alkaline solutions have been achieved by mixing the tetrasodium salt of EDTA  is a cationic bisbiguanide with broad spectrum antimicrobial actions, particularly against Gram-positive bacteria . The twoanalysis , whilst  [1H NMR ,38, no Pcipitate . Chloropcipitate .Enterococcus faecalis may be present [CHX has been suggested for use in endodontic irrigation as a final rinse following NaOCl irrigation to achieve more bacterial killing than NaOCl alone . This is present . The pre present . Additio present  and in a present ,42, redu present . Lastly,Alexidine (ALX) is a bisbiguanide that is similar in structure to CHX, except that the side chains consist of 2-ethylhexyl groups instead of the chlorophenyl groups of CHX . In its The yellow reaction by-product has not been identified, and thus its toxicity is yet to be determined. Compared to the blocked dentinal tubules observed from the CHX/NaOCl combination, scanning electron microscopy (SEM) shows root canal irrigation with an ALX/NaOCl mixture results in largely patent dentinal tubules, although it is unclear how this situation compares to alternating NaOCl and EDTA solutions .MTAD is an endodontic irrigant containing 3% doxycycline, 4.25% citric acid and 0.5% polysorbate 80 ,45. The Because of their calcium chelation ability, tetracyclines have a high affinity for tooth structure , and darTM is an antimicrobial containing 0.1% octenidine and 2% phenoxyethanol [Octeniseptyethanol . Octenidyethanol ,49 with yethanol . The cloyethanol . MixtureRecent research on the NaOCl/octenisept combination has highlighted that there are few clinical implications, because of the lack of interactions between these. Moreover, phenoxyethanol precipitates only minimally occlude dentinal tubules ,48. FurtThe mixing of sodium hypochlorite with other endodontic irrigants can result in not only a lowering of the pH of the hypochlorite component and its decomposition to chlorine gas, but also in the formation of unwanted by-products originating from the added irrigant. These two processes have implications in terms of clinical performance and toxicity. Rapid loss of free available chlorine inactivates the tissue dissolution capacity of sodium hypochlorite and precipitate formation can occlude dentinal tubules. Complete chemical analysis of breakdown products remains an important objective whenever considering combining agents with sodium hypochlorite."}
+{"text": "Sodium hypochlorite (NaOCl) remains the most used irrigation solution during root canal preparation because of characteristics such as wide-spectrum antimicrobial activity and organic tissue dissolution capacity. However, these solutions can alter dentin composition and there is no consensus on the optimal concentration of NaOCl to be used.  To determine the organic matter dissolution and changes in dentin chemical composition promoted by different concentrations of NaOCl over time. Material and Methods: Fragments of bovine muscle tissue were weighed before and after 5, 10, and 15 min of immersion in the groups (n=10): G1- 0.9% saline solution; G2- 1% NaOCl; G3- 2.5% NaOCl; and G4- 5% NaOCl. Bovine dentin fragments were subjected to the same irrigants and absorption spectra were collected by Attenuated Total Reflectance of Fourier Transform Infrared Spectroscopy (ATR-FTIR) before and after 0,5, 1, 2, 3, 5, 8, and 10 min of immersion in the solutions. The ratios of the amide III/phosphate and carbonate/phosphate absorption bands were determined. The tissue dissolution and carbonate/phosphate ratios were submitted to the two-way analysis of variance (ANOVA) with Tukey\u2019s multiple-comparison test (\u03b1<0.05) and to the one-way analysis of variance with Tukey\u2019s (\u03b1<0.05). The amide III/phosphate ratio was analyzed by Friedman test (\u03b1<0.05) and the Kruskal-Wallis test with Dunn\u2019s post-hoc (\u03b1<0.05). The increase in NaOCl concentration and contact time intensified the dissolution of organic matter and dentin collagen with reduction in the amide III/phosphate ratio. Significant differences between all groups (p<0.05) were observed in the dissolution of organic matter at 10 min and in the amide III/phosphate ratio between the saline solution and 5% NaOCl at 5 min. The carbonate/phosphate ratio decreased significantly in G2, G3, and G4 after 0,5 min of immersion (p<0.05), but more alterations did not occur in the subsequent periods (p>0.05). Intergroup differences were not observed in this ratio (p>0.05). The increase in the exposure time and in the concentration of NaOCl solution lead to an increase in the tissue dissolution and dentin collagen deproteination. Furthermore, some carbonate ions are removed from the dentin inorganic phase by the NaOCl. Therefore, it is important to know how much the increase in NaOCl concentration, with the objective to enhance sanitization, improves the organic matter dissolution without causing much undesirable alterations of the chemical composition of the dentin. The aim of the present study was to determine the dissolution capacity of organic matter and the chemical alterations on the composition of the dentin surface produced by different concentrations of NaOCl at different exposure times. The null hypothesis tested was that the different concentrations of NaOCl solutions have similar capacity of tissue dissolution and effects on dentin composition and act similarly over time.Concentrated (10-15%) NaOCl solution  was diluted in distilled water to produce solutions with 1%, 2.5%, and 5% concentrations that were confirmed by iodometric titration. The solutions obtained were stored, protected from the light in airtight plastic bottles in a refrigerator at 4\u00b0C, and removed one hour before the experiments to reach room temperature. A 0.9% physiological saline solution was used as a control. The pHs of the solutions were determined before the experiments using a calibrated pH meter.Bovine muscle tissue was acquired on the day of the experiment and kept refrigerated in 100% humidity. The muscle was cut with scalpel blades in pieces with 2x2x6 mm (width x thickness x length) and the specimens obtained were weighed on the FX-300 electronic balance . To do the sample calibration, the data obtained were submitted to statistical analysis to verify and ensure that all groups were statistically similar before the beginning of the experiment. Next, the samples were submitted to one of the following solutions (n=10): G1\u2013 0.9% physiological saline solution (control); G2\u2013 1% NaOCl; G3\u2013 2.5% NaOCl; and G4\u2013 5% NaOCl.Specimens from each group were immersed for 5 min in individual containers filled with 10 mL of the test solution. All the containers were placed in an ultrasonic tub to agitate the irrigants for 15 s per each minute. Next, the specimens were submerged in distilled water for 0,5 min to remove the irrigation solutions. They were then blotted with filter paper and re-weighed. This procedure was repeated 3 times to obtain data of 5, 10, and 15 min of immersion. The solutions were renewed before each immersion period to simulate clinical conditions and to prevent saturation. All test procedures were done at room temperature (25\u00b0C).Crowns of bovine teeth were removed at the cementoenamel junction using a diamond disc at low-speed under water cooling. Then, the incisal of the crowns were removed in the same way. Each crown was then longitudinally sectioned in the mesiodistal direction in the Isomet 1000 cutting machine  to obtain the buccal and lingual portions. Slices with approximately 0.8 mm thicknesses were obtained from these crown halves. The slices were then cut again with a diamond disc at low-speed to remove the surrounding enamel and to obtain twenty specimens with approximately 4 mm x 4 mm x 0.8 mm (length x width x thickness) .-1 at resolution of 1 cm-1 using 32 scans.One surface of the dentin specimens was wet polished with 4000 grain silicon carbide abrasive papers  and alpha alumina suspensions with 1 and 0.3 microns  until a flat and smooth surface was obtained. Finally, the specimens were immersed in distilled water and ultrasonicated for 1 min to remove any residue from the polishing. They were then dried with absorbent paper and the polished surface positioned on the diamond crystal that was the internal reflection element from the Fourier Transform Infrared (FTIR) Spectrometer Nicolet 380  and the absorbance spectra were collected by the technique of Attenuated Total Reflection (ATR), between wavenumbers of 4000 and 400 cmThe specimens were randomly assigned to the previously described four groups (n=5). Each specimen was placed inside a microtube containing 1.5 mL of the solutions for 0,5 min and ultrasonicated for 15 s. Next, they were transferred to a microtube containing 1.5 mL of distilled water and rinsed for 1 min with 15 s of ultrasonic agitation. They were then dried with absorbent paper and the ATR-FTIR spectra recorded again. Specimens were replaced in the solutions for additional 0,5 min following the same protocol described to collect the new spectra. This process was sequentially repeated to obtain the spectra at time intervals of 0, 0.5, 1, 2, 3, 5, 8 and 10 min. However, after obtaining the 1 min spectrum, the ultrasonic agitation was performed for 15 s per each minute of immersion in the irrigants. To ensure the effectiveness of the solutions, they were renewed after the time intervals of 2, 5, and 8 min.-1 were identified. The areas of the absorption bands of phosphate (PO3-), carbonate (CO3-) , and amide III of each spectrum were determined. The wavenumber values employed for the area integrations for the amide III were between 1298\u20131216 cm-1 spectral range, between 888\u2013816 cm-1 for the carbonate, and between 1170\u2013780 cm-1 for the phosphate. Inside the phosphate spectral range there is the carbonate band at 888\u2013816 cm-1, whose value was subtracted to obtain the real value of the area of phosphate band.A typical absorbance spectrum obtained from a disc of untreated dentin is shown in -1 there is no overlapping with bands of other dentin components. Instead, in bands of amides A and B at 3115 and 2860 cm-1 and amide I at 1645 cm-1, overlapping occurs with water bands, and in bands assigned to amide II, present at around 1550 cm-1, the overlapping occurs with carbonate bands3-)/(CO3-)ratio, which was the second parameter. The ratios were obtained by taking the quotient between the areas of the bands.To evaluate the effects of NaOCl solutions on the chemical composition of dentin, two parameters were calculated. The first was the amide III/phosphate ratio that was used to determine the collagen deproteination by NaOCl. The amide III band was chosen, for in this region of 1298\u20131216 cmThe amide III/phosphate ratio was measured to evaluate how the amide III or the phosphate changed when immersed in NaOCl solution. For example, when this ratio decreases, it means that the amount of amide III (organic matter) decreased compared with the phosphate (inorganic matrix). However, when employing a chemical agent that removes organic matter and inorganic matter simultaneously, this ratio could stay unaltered. The carbonate/phosphate ratio is employed to evaluate the dissolution of the inorganic matrix; this ratio measures the carbonate dissolution in relation to the phosphate radical.The collected data of tissue dissolution and carbonate/phosphate ratios showed normal distribution, and were submitted to the two-way analysis of variance (ANOVA) with Tukey\u2019s multiple-comparison test (\u03b1<0.05) to detect intragroup differences over time and the one-way analysis of variance with Tukey\u2019s (\u03b1<0.05) to detect any differences between the groups at the same time period.post-hoc (\u03b1<0.05) test was used to detect intergroup differences in the same period.The amide III/phosphate ratio exhibited abnormal distribution. The nonparametric Friedman test (\u03b1<0.05) was used to detect intragroup differences among different periods of immersion and the Kruskal-Wallis test with Dunn\u2019s Regarding the carbonate/phosphate ratio, all irrigants caused a decrease in its initial proportion . HoweverIn the present study, the tissue dissolution capability and the changes in the dentin chemical composition by different concentrations of NaOCl solutions were assessed. The results demonstrated that NaOCl can dissolve the organic matter and deproteinate the collagen of dentin in high quantities; and otherwise, it can cause a small reduction in the carbonate component of the inorganic phase of the dentin.The null hypothesis tested has to be rejected, since there were differences between the concentrations of NaOCl solutions in the ability of tissue dissolution and in the effects on dentin composition over time.,,,,,-),- has a powerful oxidative effect that promote higher tissue dissolution and is more abundant in alkaline solutions,A concentration and time-dependent organic tissue dissolution capacity was observed for the NaOCl solutions , as prev,,,,,,Tissues from different sources were used in studies about the tissue dissolving ability of irrigation solutions,The bovine incisor dentin has a similar structure and number of tubuli of human molar dentin,,,,,In the dentin vibrational spectrum, it is possible to observe bands related to water, to hydroxyapatite, that originate from the carbonate and phosphate groups and to the organic matrix from the groups present in the collagen such as amides I, II, and III,,,,,In the present study, a time-dependent effect in the reduction of the amide III/phosphate ratio was identified in all NaOCl concentrations . The res,,Carbonate groups may occupy phosphate and hydroxyl ions sites in bone and teeth apatite. These substitutions affect the crystallinity of the apatites and can accelerate the dissolution process of the tooth structureThe design of the present study does not directly reflect the clinical conditions, but allows quantitative evaluations regarding the different concentrations and time exposure of NaOCl solutions. This study confirmed the advantage of using a longer contact time and higher concentrations of NaOCl to promote tissue dissolution. However, it increases the alterations in dentin composition and the risks of periapical tissue damage from inadvertent extrusion. Based on these results, the use of NaOCl at lower concentrations, such as 1 and 2.5%, demonstrates to be effective in promoting a suitable dissolution of organic tissue present in the root canal system and preventing a pronounced damage to the dentin structure.The findings of this study indicated that the increase in the exposure time and in the concentration of NaOCl solution lead to an increase in the tissue dissolution and dentin collagen deproteination. Moreover, some carbonate ions are removed from the dentin inorganic phase by the NaOCl."}
+{"text": "N-methyltransferase (HMT) has so far prevented the direct analysis of HMT proteins in man and other mammals.The lack of suitable antibodies for the histamine inactivating enzyme histamine A series of monoclonal antibodies was produced by immunizing mice with human and porcine HMT expressed in vitro. Antibodies were characterized by immunoblotting and immunohistochemical staining.Six different monoclonal antibodies specific for human HMT and four different monoclonal antibodies specific for porcine HMT were obtained that can detect HMT with up to tenfold greater sensitivity than the most sensitive enzymatic assays currently available. Using these antibodies allowed us to confirm the expression and cellular localization of HMT in various human and porcine tissues, where the presence of the enzyme had previously been deduced from activity measurement and HMT mRNA analysis. Immunohistochemical staining of human and porcine tissue sections clearly showed that HMT is a cytosolic protein, which is localized in specific cells of most mammalian tissues.The new monoclonal antibodies not only allow a comprehensive quantitative evaluation of the expression of HMT at the cellular level in man and other mammals but will also facilitate sensitive analyses of disease-associated alterations of this protein. Slides were incubated for 16\u00a0h at 4\u00a0\u00b0C with the mouse monoclonal antibodies for human or porcine HMT diluted 1:100\u20131:1500 in TNB and then for 2\u00a0h a 25\u00a0\u00b0C with horseradish peroxidase-conjugated anti-mouse immunoglobulins  diluted 1:100 in TNB. The Tyramide Signal Amplification System  was used according to manufacturer\u2019s instructions for signal amplification. For staining of immunocomplexes, slides were incubated for 5\u00a0min with DAB substrate  and counterstained with Mayer\u2019s hemalum . Slides were dehydrated by incubation for 3\u00a0min each in 70\u00a0% ethanol, 80\u00a0% ethanol, 96\u00a0% ethanol, 2\u00d7 100\u00a0% ethanol, and 2\u00d7 xylol and coverslips were mounted with Entellan .For immunohistochemical staining, tissues were fixed for 16\u201324\u00a0h in 4\u00a0% paraformaldehyde and embedded in paraffin wax. Sections of 5\u00a0\u00b5m were cut, mounted on silanized glass slides , and dried 16\u00a0h at 50\u00a0\u00b0C. Slides were dewaxed 4\u00a0\u00d7\u00a03\u00a0min in xylol, 1\u00a0\u00d7\u00a03\u00a0min each in 100\u00a0% ethanol, 96\u00a0% ethanol, and 80\u00a0% ethanol, rinsed for 5\u00a0min with water, and autoclaved for 10\u00a0min at 121\u00a0\u00b0C in 10\u00a0mM sodium citrate pH 6.0 for antigen retrieval. Slides were washed 2\u00d7 with TBS  and mounted on Coverplates in Coverplate Racks  for subsequent incubations. Each incubation step was followed by three washes with TNT (TBS containing 0.05\u00a0% Tween 20). Endogenous peroxidase activity was blocked by incubation in 1\u00a0% HUsing GST fusion proteins of recombinant human and porcine HMT expressed in bacteria as antigens for immunizations of mice Fig.\u00a0, six clopI values calculated for the human and porcine HMT polypeptide sequences, respectively. However, no spots were visible at the respective positions of parallel Silver-stained gels , which is in accordance with previous studies analyzing HMT enzymatic activity and HMT mRNA in various porcine tissues [Histamine  tissues . In agreWe next tested whether the monoclonal antibodies could detect HMT in tissue sections. When performing immunohistochemical staining on human and porcine kidney sections, identical staining patterns were obtained with all antibodies at dilutions of 1:100\u20131:1500 as shown exemplary for HYB372-04, HYB372-05, and HYB372-07 in Fig.\u00a0A comprehensive study investigating the expression and cellular distribution of HMT in various human and porcine tissues is under way and will be presented in a separate communication. The results available so far confirm earlier analyses of HMT activity measurement and HMT mRNA expression in porcine tissues  showing To test the utility of the monoclonal antibodies for investigations of HMT in human samples, we analyzed the presence of HMT in a series of consecutive control biopsies of kidney and liver transplant tissue obtained for routine diagnostic procedures Fig.\u00a0. Using bA series of monoclonal antibodies exhibiting a high specificity and sensitivity for human and porcine HMT, respectively, was obtained by immunizing mice with HMT proteins expressed in vitro. All antibodies recognized a single protein band of 33\u00a0kDa corresponding to monomeric HMT on immunoblots of tissue homogenates and except for HYB373-03 did not bind to any other human or porcine protein in the samples analyzed. With these antibodies it was possible to detect HMT with tenfold higher sensitivity than with the most sensitive radioenzymatic assay in use . FurtherThe specificity of the antibodies was demonstrated by detection of a single band of the expected size of 33\u00a0kDa on immunoblots of different human and porcine samples Fig.\u00a0a, b, corAll antibodies detected HMT by immunohistochemical staining of tissue sections and produced identical staining patterns when used at optimum concentration Fig.\u00a0. In all We also included a small exploratory study analyzing HMT in minute amounts of samples from control biopsies of kidney and liver grafts Fig.\u00a0 to demonIn conclusion, the ten monoclonal HMT antibodies described here represent excellent tools for the sensitive and specific detection of the human and porcine HMT proteins allowing a comprehensive evaluation of the expression and cellular localization of the enzymes in man and pig. Furthermore, these antibodies will facilitate a better understanding of the role of HMT in histamine inactivation in different cell types as well as sensitive analyses of disease-associated alterations of the enzyme."}
+{"text": "AbstractCurculionoidea are newly recorded from the Canadian province of Quebec: Coelocephalapionemaciipes ; Ischnopterapionvirens ; Omphalapionhookerorum ; Perapionpunctinasum  ; Anthonomusrobustulus LeConte, 1876; Pseudanthonomushelvolus ; Bagousmagister LeConte, 1876; Bagoustanneri O\u2019Brien, 1979; Buchananiusstriatus ; Ceutorhynchusbolteri Dietz, 1896; Ceutorhynchuspallidactylus ; Ceutorhynchuspauxillus Dietz, 1896; Conotrachelusbuchanani Schoof, 1942; Conotracheluspusillus LeConte, 1878; Conotrachelusrecessus ; Curculiorubidus ; Cylindrocopturuslongulus ; Hadroplontuslitura ; Hyperarumicis ; Lixusterminalis LeConte, 1876; Myosidesseriehispidus Roelofs, 1873; Phloeotribusdentifrons ; Plocamusechidna ; Scolytusmuticus Say, 1824; Sirocalodessericans ; Smicronyxsculpticollis Casey, 1892 . Among these, Buchananiusstriatus, Conotrachelusbuchanani, Conotracheluspusillus, and Curculiorubidus  are also recorded from Canada for the first time. The latter is also newly reported from Ontario. Collecting data are provided for Lixuspunctinasus LeConte, 1876, previously reported to occur in Canada without any further information, and for Choragussayi LeConte, 1876 (Anthribidae) and Rhyssomatusaequalis Horn, 1873 (Curculionidae), both previously recorded from Quebec, also without further details.The following species of  Brentidae and 29 Curculionidae species new to Quebec , increasing the total number of species of each family known to occur in the province to 22 and 386, respectively  reported herein (4 Brentidae and 23 Curculionidae), listed according to the classification of Ischnopterapionvirens , Omphalapionhookerorum , Ceutorhynchuspallidactylus , PageBreakCurculiorubidus , Hadroplontuslitura , Myosidesseriehispidus Roelofs, 1873, and Hyperarumicis  are all adventive species (sensuectively . Recent es sensu that werSpecimens belonging to species recorded or referred to in the present article were identified (or their identity was confirmed) by recognized specialists listed henceforth under each species name by their name, or if an author of this paper, by their initials.Label data are provided in chronological order for every species. These data were translated from French to English, and various details , when known, have been added between brackets.Specimens were either swept or beaten from various plant species, attracted to mercury vapour, ultraviolet or porch lights or handpicked from various substrates or from a flight interception trap made of tulle fabric  held between two wood piles or set up in a suburban backyard.VASCAN) (http://data.canadensys.net/vascan/search).Plant family, generic and specific names follow the classification used in Database of Vascular Plants of Canada , Varennes, Quebec, CanadaCCOB Charles W. O\u2019Brien Insect Collection (private collection), Green Valley, Arizona, United StatesCCTE Claude Tessier Insect Collection (private collection), Quebec, Quebec, CanadaCHMS Henri Miquet-Sage Insect Collection (private collection), Mont-Saint-Hilaire, Quebec, CanadaCMNCCanadian Museum of Nature, Ottawa, Ontario, CanadaCNCICanadian National Collection of Insects, Arachnids, and Nematodes, Agriculture and Agri-Food Canada Research Centre, Ottawa, Ontario, CanadaCPTO Pierre de Tonnancour Insect Collection (private collection), Terrasse-Vaudreuil, Quebec, CanadaCRVI Robert Vigneault Insect Collection (private collection), Oka, Quebec, CanadaCSDU St\u00e9phane Dumont Insect Collection (private collection), Montreal, Quebec, CanadaCSLA Serge Laplante Insect Collection (private collection), Gatineau, Quebec, CanadaTaxon classificationAnimaliaColeopteraAnthribidaeLeConte, 1876, new data supporting first record for Quebec Species identification confirmed by RSA, 2015 and 2016 CCCH); [MRC Marguerite-d\u2019Youville] Varennes, 16VII1999, attracted to UV light, C. Chantal ; same except: 29VI2006 ; [MRC Brome-Missisquoi] Saint-Armand, 2VIII2007, understory, on foliage, C. Chantal ; [MRC Marguerite-D\u2019Youville] \u00cele Sainte-Th\u00e9r\u00e8se, 1IX2009, C. Chantal ; [MRC Deux-Montagnes] Parc national d\u2019Oka, La Grande Baie, 19VII2014, beaten from dead branches over forest litter, R. Vigneault ; [MRC Coaticook] Compton, 25VIII2014, C. Levesque ; [MRC Deux-Montagnes] Parc national d\u2019Oka, La Grande Baie, 27VI2015, beaten from dead branches over forest litter, R. Vigneault ; same except: 30VI2015 (16:00), P. de Tonnancour ; same except: 2VII2015, R. Vigneault ; same except: 5VII2015 (16:00), beaten from dead branches of Acersaccharum, P. de Tonnancour & R. Vigneault ; same except: 9VII2015, R. Vigneault ; [MRC Deux-Montagnes] Parc national d\u2019Oka, Calvaire, 25VI2016, beaten from dead branches over forest litter, R. Vigneault ; same except: 1VII2016 ; same except: La Grande Baie, 6VII2016, beaten from dead branches of Acersaccharum, R. Vigneault ; same except: La Grande Baie, 6VII2016, beaten from dead branches over forest litter, R. Vigneault , 12VII2016 , and 1VIII2016 .[Agglom\u00e9ration de Longueuil] Longueuil, 18VII1992, C. Chantal , new to Quebec Species identification confirmed by RSA, 2016 Apioninae known to occur in Quebec by the conspicuous elongate postscutellar spot of white vestiture and spot of dense white scales at the base of elytral interstriae 2 and 3. Nothing is known of its habits or life history, except that adults were collected in August on dock, Rumex L. spp., including golden dock, Rumexpersicarioides L. (Polygonaceae) (bugguide.net (http://bugguide.net/node/view/1077586/bgpage).This native species is easily separated from all other onaceae) . Ontarioonaceae) . A photoCRVI).[MRC Deux-Montagnes] Parc national d\u2019Oka, composting site, 29V2015, white tulle fabric flight interception trap, R. Vigneault , new to Quebec Species identification confirmed by RSA, 2015 inTripleurospermuminodorum (L.) Sch.Bip. (= Matricariaperforata M\u00e9rat) (Asteraceae) in British Columbia, Alberta, Saskatchewan, and Manitoba (Anthemiscotula L. (Asteraceae), in Nova Scotia ; [MRC Marguerite-D\u2019Youville] Varennes, 30VI2008, C. Chantal ; [MRC La Vall\u00e9eduRichelieu] Mont-Saint-Hilaire, 2VII2008, H. Miquet-Sage ; [MRC Marguerite-D\u2019Youville] Varennes, 2V2010, C. Chantal ; same except: 20V2010 , and 9VI2010 ; [MRC La Vall\u00e9eduRichelieu] MontSaintHilaire, 13VI2010, H. Miquet-Sage ; [MRC Marguerite-D\u2019Youville] Varennes, 30V2011, C. Chantal ; same except 9VI2012 , and 21V2014 ; [MRC La Vall\u00e9eduRichelieu] MontSaintHilaire, 12V2014, H. MiquetSage ; same except: 20VI2014 , and 25VI2014 ; [MRC Marguerite-D\u2019Youville] Varennes, 7VI2015, C. Chantal .[MRC La Vall\u00e9eduRichelieu] Saint-Charles-sur-Richelieu, 29VI2003, H. Miquet-Sage , new to Quebec Species identification confirmed by RSA, 2016 PageBreak1994, in Pennsylvania (Trifolium L. spp. (Fabaceae). It can be distinguished from the superficially similar Stenopterapionmeliloti Kirby, 1808, by its smaller size and the bluish colour of its pronotum and venter (black in S.meliloti). As indicated by Widely distributed through most of the Palaearctic region , this adsylvania . Until nsylvania . It is cCCCH); same except: 3X2013 ; MRC Vaudreuil-Soulanges, Terrasse-Vaudreuil, 15IX2014 (15:00), white tulle fabric flight interception trap, P. de Tonnancour ; same except: 7X2014 (15:00)  and 12X2014 (17:00) ; [MRC Coaticook] Waterville, 11VII2015, H. Miquet-Sage ; MRC Vaudreuil-Soulanges, Terrasse-Vaudreuil, 21IX2015 (12:30), white tulle fabric flight interception trap, P. de Tonnancour ; [MRC Marguerite-D\u2019Youville] Varennes, 21IX2015, C. Chantal ; MRC Vaudreuil-Soulanges, Terrasse-Vaudreuil, 22IX2015 (15:00), white tulle fabric flight interception trap, P. de Tonnancour ; same except, 22IX2015 (15:00), beaten from Oidium infected foliage of Helianthusstrumosus, ; same except: 27IX2015 , white tulle fabric flight interception trap ; same except: 6X2015 (16:15) , and 7X2015 (14:30\u201315:30) ; same except: 11X2015 (15:00), beaten from Oidium infested foliage of Helianthusstrumosus ; same except: 12X2015 (11:00\u201315:00), white tulle fabric flight interception trap ; same except: 5XI2015 (14:00\u201315:00), climbing on pale house exterior wall ; same except: 6XI2015 (15:00) , 9XI2015 (15:00) , 19XI2015 (12:00) , 26XI2015 (13:00) , 27XI2015 (12:30) , 11XII2015 (13:00\u201315:00) , and 12XII2015 (12:00) ; Montreal, Parc Zotique-Racicot , 11V2106, swept from Trifolium sp., S. Dumont ; same except: 12-V-2016 ; MRC Haut-Richelieu, Henryville, dike adjacent to R\u00e9serve \u00e9cologique Marcel-Raymond], 12V2016 (13:00\u201316:00), swept from grasses, Equisetum and Solidago, P. de Tonnancour ; MRC Vaudreuil-Soulanges, Notre-Dame-de-l\u2019\u00cele-Perrot, 20V2016 (17:00), swept from Trifoliumpratense, P. de Tonnancour ; Montreal, Parc Zotique-Racicot , 23V2106, swept from Trifolium sp., S. Dumont ; same except: 24V2016 ; MRC Brome-Missisquoi, SaintArmand, 25V2016 (16:00), swept from Trifoliumpratense, P. de Tonnancour ; MRC Vaudreuil-Soulanges, Saint-Lazare, 29VI2013 (16:00\u201317:00), swept from Trifoliumpratense, P. de Tonnancour ; MRC Laval, Laval, rue des Charmes , 20VII2016 (15:00), swept from Trifoliumpratense, P. de Tonnancour ; MRC Vaudreuil-Soulanges, Terrasse-Vaudreuil, 10XI2016 (15:00), climbing on pale house exterior wall .[MRC Haut-Richelieu] Henryville [dike adjacent to R\u00e9serve \u00e9cologique Marcel-Raymond], 29IX2012, C. Chantal , new to Quebec Species identification confirmed by RSA, 2016 Desmodium Desv. sp. (Fabaceae), based on the very few available data at the time (Scirpus L. spp. (Cyperaceae) in wet habitats.The occurrence of this small native pale-legged species in the province was expected as it was previously known in Canada from Ontario and the Maritime Provinces. Although this species has been tentatively associated with tick-trefoil, the time , it is wCCCH); same except: 15V2015 ; MRC Vaudreuil-Soulanges, Notre-Dame-de-l\u2019\u00cele-Perrot, 11V2016 (13:00), swept from Scirpusatrovirens ; same except: 14V2016 (15:00) ; [MRC Deux-Montagnes] Parc national d\u2019Oka, 19V2016, swept from herbs in field ; MRC Brome-Missisquoi, SaintArmand, 25V2016 (15:00), swept from Scirpus sp. .[MRC Marguerite-D\u2019Youville] Varennes, 30VI2014, C. Chantal   . It was eraceae) .CCCH); same except: 3VII2006 , and 2VII2008 ; MRC Vaudreuil-Soulanges, Notre-Dame-de-l\u2019\u00cele-Perrot, 31V2011 (13:00), meadow, swept from Solidago/Aster, P. de Tonnancour ; same except: 1VI2011 (14:00) ; [MRC Brome-Missisquoi] Saint-Armand, 3VIII2011, C. Chantal ; [MRC Haut-Richelieu] Henryville [dike adjacent to R\u00e9serve \u00e9cologique Marcel-Raymond], 28V2013, (14:00\u201317:00), swept from grasses, Equisetum and Solidago, C. Chantal and P. de Tonnancour ; MRC HautSaintLaurent, Saint-Anicet , 14VI2013 (18:00), beaten from Cornusstolonifera, P. de Tonnancour ; same except: 15VI2013 (13:00), wet meadow, swept from various herbaceous plants, P. de Tonnancour ; [MRC La Vall\u00e9eduRichelieu] Mont-Saint-Hilaire, 24VI2013, H. Miquet-Sage ; MRC Haut-Saint-Laurent, Franklin, roadside opposite to R\u00e9PageBreakserve \u00e9cologique du Pin-Rigide, 17VII2013 (14:00), beaten from Lythrumsalicaria, P. de Tonnancour ; MRC Haut-Richelieu, Henryville, dike adjacent to R\u00e9serve \u00e9cologique Marcel-Raymond, 8VI2014 (14:00\u201316:00), swept from grasses, Equisetum and Solidago, P. de Tonnancour ; [MRC Brome-Missisquoi] Saint-Armand, 16VI2014, C. Chantal ; MRC Haut-Richelieu, Henryville, dike adjacent to R\u00e9serve \u00e9cologique Marcel-Raymond, 4VI2015, P. de Tonnancour (16:00\u201318:00) ; [MRC Brome-Missisquoi] Saint-Armand , 25V2016, S. Dumont .[MRC Brome-Missisquoi] Saint-Armand, 7VI2004, C. Chantal , new to Quebec Species identification confirmed by RSA, 2016 Hamamelisvirginiana L. (Hamamelidaceae)  . Adults idaceae) .Hamamelisvirginiana, P. de Tonnancour & R. Vigneault ; same except: 2VII2015 (18:00)  and 5VII2015 (18:00) ; same except: 5IX2015 (17:00), R. Vigneault  and 20VIII2016 ; same except: 27-VIII-2016 (14:00), P. de Tonnancour ; same except: , 27VIII2016 (16:00) .MRC Deux-Montagnes, Parc national d\u2019Oka , 30VI2015 (17:00), beaten from Taxon classificationAnimaliaColeopteraCurculionidae, new to Canada Species identification confirmed by RSA, 2015 Curculio species by its very small size (<3.5mm), lack of femoral teeth and association with birch, Betula L. spp. All specimens recorded in Quebec were collected in a stand of gray birch, Betulapopulifolia Marshall (Betulaceae), and most were directly beaten from gray birch. Adults are said to be active from May to October in Europe , but all specimens reported herein were captured in August . This species is also newly recorded from Ontario, based on a specimen photographed by Burke Korol in Barrie, Simcoe County, on August 21, 2015 and posted on bugguide.net (http://bugguide.net/node/view/1127147).This record comes three years after the first North American detection of the species in Michigan  and is bQuercusrubra, P. de Tonnancour ; same except: 8VIII2015 (15:00), beaten from Alnusrugosa , beaten from Betulapopulifolia  or swept from various herbaceous plants in gray birch stand ; same except: 10VIII2015 (13:00), swept from various herbaceous plants in gray birch stand ; same except: 16VIII2015 (15:00), beaten from Betulapopulifolia ; 17VIII2015 (14:00) ; 17VIII2015 (14:00 and 18:00) ; 18VIII2015 (19:00) ; 22VIII2015 (14:00), C. Chantal ; 20VIII2016 (16:00), P. de Tonnancour ; 23VIII2016 (16:00) ; 28VIII2016 (16:00) ; 29VIII2016 (18:00) ; 1IX2016 (13:00) .MRC Vaudreuil-Soulanges, Notre-Dame-de-l\u2019\u00cele-Perrot, 10VIII2013 (17:00), beaten from Taxon classificationAnimaliaColeopteraCurculionidaeCasey, 1892, new to Quebec Species identification confirmed by RSA, 2015 Cuscuta L. spp. (Convolvulaceae)  , obligatCCCH, 1); [MRC Deux-Montagnes] Parc national d\u2019Oka, La Grande Baie, 30VI2015 [swept from low vegetation in swampy area], R. Vigneault .[MRC Pierre-De Saurel] Saint-Roch-de-Richelieu, 20VI2005, C. Chantal  . It was previously known in Canada only from Ontario ; same except 30V2013 ; same except 23VI2013 ; MRC Deux-Montagnes, Parc national d\u2019Oka, 30VII2012 (18:00), swept from Cyperaceae, Polygonum sp., Pontederiacordata, and Sagittaria sp., P. de Tonnancour .MRC Vaudreuil-Soulanges, Terrasse-Vaudreuil, 31V2013 (21:30), UV + porch light, P. de Tonnancour ; [MRC Haut-Richelieu] Henryville, 24VI2003, attracted to UV lamp, C. Chantal ; [MRC Deux-Montagnes] Parc national d\u2019Oka, La Grande Baie, 30VI2015 , R. Vigneault ; same except: 2VII2015 (16:00), beaten from dead branches, edge of swampy bay, P. de Tonnancour .[MRC Brome-Missisquoi] Saint-Armand, 2VII2001, attracted to UV lamp, C. Chantal , new to Canada Species identification confirmed by RSA, 2015 Baridinae occurring in Quebec by its extremely wide and convex shape, its tiny size  and its vestiture of sparse but long erect scales. The only other North American congener, Buchananiussulcatus , has been recorded as developing in the fruiting bodies of the fungus Trichodermapeltatum (Berk.) Samuels, Jaklitsch & Voglmayr (Hypocreaceae) growing on American Beech, Fagusgrandifolia Ehrh. (Fagaceae), in Maryland (This minute native species (1.4\u20131.6mm) is easily distinguished from all other Maryland . This spCCCH); [MRC Deux-Montagnes] Parc national d\u2019Oka, La Grande PageBreakBaie, 28VI2014 [beaten/swept from undergrowth/fallen branches in deciduous stand], R. Vigneault ; same except: composting site, 28-V-2016 (19:00), white tulle fabric flight interception trap, R. Vigneault ; same except: Calvaire d\u2019Oka, 1VII2016, beaten from fallen dead branches of deciduous tree, R. Vigneault .[MRC Joliette] Joliette, 7IX2013 [swept from forest understory], J.-F. Roch , new to Quebec Species identification confirmed by RSA, 2016 bugguide.net (http://bugguide.net/node/view/1078735/bgimage).This remarkable native species was previously known in Canada only from Ontario . The cirCRVI); MRC Deux-Montagnes, Parc national d\u2019Oka, La Grande Baie, 3VII2016, brushed from trunk of a recently dead Fagusgrandifolia, R. Vigneault ; same except: 6VII2016 (12:00), P. de Tonnancour ; same except: 13VII2016 (17:00), P. de Tonnancour & R. Vigneault ; same except: 24VII2016 (17:00), P. de Tonnancour, R. Vigneault & S. Laplante ; same except: 1VIII2016, R. Vigneault ; same except: 20VIII2016 .[MRC Deux-Montagnes] Parc national d\u2019Oka, composting site, 04VI2015 (18:00), white tulle fabric flight interception trap, R. Vigneault  was collected on spotted water-hemlock, Cicutaoccidentalis Greene (now Cicutamaculata L.) (Apiaceae).This native species was previously recorded in Canada only from British Columbia , but it CCCH); MRC Deux-Montagnes, Parc national d\u2019Oka, 29V2015 (18:00\u201320:00), white tulle fabric flight interception trap, P. de Tonnancour ; MRC PageBreakHaut-Richelieu, Henryville, dike adjacent to R\u00e9serve \u00e9cologique Marcel-Raymond, 4VI2015 (16:00\u201318:00), swept from grasses, Equisetum and Solidago, P. de Tonnancour ; same except: 12V2016 (13:00\u201316:00) .[MRC Haut-Richelieu], Henryville, 28V2013, sweeping, C. Chantal , new to Quebec Species identification confirmed by PB, 2014 Brassicaceae and Resedaceae and is occasionally associated with Cannabissativa L. (Cannabaceae)  . It was abaceae) , where iabaceae) .CNCI); [MRC Nouvelle-Beauce] East of St-Lambert-de-Lauzon, Rd. 218, 18VII2001, 46\u00b036,133'N; 71\u00b011.412'W, corn field with radish, Mason, Sarazin & Boudreault, QC 2001\u2013110 ; same except: QC 2001\u2013100 ; [MRC de l\u2019\u00c9rable] NE of Plessisville, Road 116, 18VII2001, 46\u00b018.796'N; 71\u00b040.129'W, small canola field, Mason, Sarazin & Boudreault, QC 2001-330 ; [MRC Arthabaska] Saint-Albert, Hwy 122, 12VII2002, 46\u00b000.455'N; 72\u00b006.016'W, wild radish along edge of corn field, Mason, Boudreault & Farmakis, QC 2002-213 ; same except: QC 2002-214 ; [MRC Drummond] Domaine-Descoteaux, 22VII2003, 45\u00b049.142'N; 72\u00b013.983, J. Miall & P. Mason, wild mustard, QC03-121 ; [MRC Drummond] StGuillaume, 22VII2003, 45\u00b054.909'N; 72\u00b044.660'W, J. Miall & P. Mason, wild mustard, QC03-116 ; [MRC Drummond] S[ain]t-Cyrille-de-Wendover, north-east, 45\u00b057.049'N; 72\u00b023.877'W, 22VII2003, J. Miall & P. Mason, wild radish, QC03-119 ; [MRC Pierre-De Saurel] S[ain]teVictoire, Hwy 239, 2km east, 45\u00b056.580'N; 73\u00b004.189'W, 22VII2008, ex. stem of Raphanusraphanistrum, em[ergence] 26VIII2008, Mason, Miall & Brauner, Sitre QC 08-710 ; same except: 22VII2003, 45\u00b057.744'N; 73\u00b006.760'W, J. Miall & P. Mason, wild radish, QC 03-114 (1 CNCI); Centre-du-Qu\u00e9bec, [MRC Arthabaska] Saint-Rosaire, 19VII2012, swept from canola ; [MRC Coaticook] Compton, 27VI2014, C. Levesque ; same except: 24VII2014 ; same except: 1VIII2014 .[MRC Coaticook] Missisquoi Co., Mont le Pinacle, 10VI1984, Larochelle, Larivi\u00e8re ; MRC Marguerite-D\u2019Youville, Verch\u00e8res, 4VI2010, C. Chantal ; MRC Vaudreuil-Soulanges, Saint-Lazare, 9VI2013 (15:00), sandpit, beaten from Erysymum sp., P. de Tonnancour ; same except: 12VI2013 (14:00), beaten from Brassica sp. ; same except: 14VI2013 (13:00) , 19VI2013 (14:00) ; same except: 6VI2014 (13:00), swept from Equisetum and grasses , 10VI2014 (17:00) ; same except: 23VI2014 (17:00), swept from Equisetum .[MRC de D\u2019Autray] Lanoraie, 26VIII1986, sweeping Sphagnum bog, L. LeSage, on Taxon classificationAnimaliaColeopteraCurculionidae, new to Quebec Species identification confirmed by RSA, 2016. Cirsiumarvense (L.) Scop. (Asteraceae)  , an invaeraceae) . All speCirsiumarvense, P. de Tonnancour and/or S. Dumont, 8VII2015 (13:00) ; 9VII2015 ; 10VII2015 ; 12VII2015 (16:00) ; 14VII2015 (15:00) ; 26VII2015 ; 25VIII2015 ; 01IX2015 (13:00) ; same except: , 28VI2016 (13:00) ; 30VI2016 ; same except: 4VII2016 ; MRC Vaudreuil-Soulanges, Ville de l\u2019\u00cele-Perrot, 11VII2016 (15:00), beaten from Cirsiumarvense, P. de Tonnancour ; MRC Laval, Laval , 20VII2016 (14:00), beaten from flowering Cirsiumarvense, P. de Tonnancour ; Montreal, Parc Zotique-Racicot , 26VII2016, beaten from Cirsiumarvense, S. Dumont ; same except: 28VII2016 ; MRC Laval, Laval , 17IX2016, beaten from flowering Cirsiumarvense, P. de Tonnancour .Montreal, Parc Zotique-Racicot , beaten from Taxon classificationAnimaliaColeopteraCurculionidae, new to Quebec Species identification confirmed by Hiraku Yoshitake, 2014 This native species was previously known in Canada from Manitoba and Ontario , but itsBrassica sp., P. de Tonnancour .MRC Vaudreuil-Soulanges, Saint-Lazare, 12VI2013 (14:00), sandpit, beaten from Taxon classificationAnimaliaColeopteraCurculionidae, new to Quebec Species identification confirmed by Hiraku Yoshitake, 2014, and RSA, 2016. Podapiongallicola Riley, 1883, on pine , which has been reared from various pines and several other conifers ; MRC Vaudreuil-Soulanges, Mont Rigaud, 31V2013 (13:00), beaten from Asclepiassyriaca, P. de Tonnancour ; same except: 5VI2013 (13:00), rocky outcrop, swept from Rumexacetosella ; same except: 2V2015 (15:00), rocky outcrop, beaten from Pinusstrobus, P. de Tonnancour ; MRC Collines-de-l\u2019Outaouais, Luskville (Sentier des chutes), 26V2015 (13:00), beaten from small Amelanchier sp., P. de Tonnancour .MRC Vaudreuil-Soulanges, Notre-Dame-de-l\u2019\u00cele-Perrot, 30IV2013 (16:00), beaten from flowering shoots of Taxon classificationAnimaliaColeopteraCurculionidaeRoelofs, 1873, new to Quebec Species identification confirmed by RSA, 2016 Trachyphloeus Germar, 1817, and was reported by This adventive species, originally from Asia, has gone undetected for many years in collections under the genus CCCH).[MRC Brome-Missisquoi], Saint-Armand, 6VII2015 (afternoon), C. Chantal , new to Quebec Species identification confirmed by Hiraku Yoshitake, 2014 Hyperarumicis is associated with various Polygonum L. spp. and Rumex spp. (Polygonaceae), especially the invasive curled dock, Rumexcrispus L., also introduced from Europe. Its potential as a biological control agent against this weed was recently assessed , this Palaearctic species has expanded its range considerably in North America. Surveys conducted from 1997 to 1999 in two Quebec vineyards failed to detect its presence , but itsassessed . This spassessed .CCTE); MRC Vaudreuil-Soulanges, Notre-Dame-de-l\u2019\u00cele-Perrot, 3VII2011 (17:00), beaten from Rumexcrispus, P. de Tonnancour ; MRC Vaudreuil-Soulanges, Terrasse-Vaudreuil, 6VII2011 (2:00), UV + porch light, P. de Tonnancour ; MRC Haut-Saint-Laurent, Saint-Anicet , 15VI2013 (13:00), wet meadow, swept from various herbaceous plants, P. de Tonnancour ; Montreal, \u00cele-Bizard (Parc-nature du Bois-de-l\u2019\u00cele-Bizard), 17VI2013, \u2265 5 cocoons on Rumex sp. (one emergence on 22VI2013), C. Pilon (observation documented by photos); MRC Haut-Saint-Laurent, Saint-Anicet , 26VI2015 (15:00), beaten from Rumexcrispus, P. de Tonnancour ; MRC Coaticook, Waterville (45.27993 N 71.89987 O), 10VII2015 (20:00), beaten from Rumexcrispus, P. de Tonnancour ; same except 11VII2015 (10:00), H. Miquet-Sage, P. de Tonnancour, S. Dumont ; MRC Haut-Richelieu, Henryville, dike adjacent to R\u00e9serve \u00e9cologique Marcel-Raymond, 12V2016 (13:00\u201316:00), swept from grasses, Equisetum and Solidago, P. de Tonnancour 12V2016 .[MRC Brome-Missisquoi], Saint-Armand, 15VI2003, C. Tessier . Numerous CMNC specimens from Texas were collected on Polygonum.CPTO); same except: 23VIII2014 (15:00), small pond margin, beaten from Bidenscernua, P. de Tonnancour .MRC Vaudreuil-Soulanges, Notre-Dame-de-l\u2019\u00cele-Perrot, 3VII2008 (17:00), handpicked from building wall, P. de Tonnancour  Delarbre) (Polygonaceae) more than a century ago ; same except: 9V1993 , 30V1995 ; [MRC Deux-Montagnes] Parc national d\u2019Oka, 26V2002, flowers of Prunusvirginiana, C. Chantal ; [MRC Deux-Montagnes] Parc national d\u2019Oka, La Grande Baie, 28V2002, R. Vigneault ; same except: 4V2003 ; [MRC Deux-Montagnes] Parc national d\u2019Oka, Calvaire d\u2019Oka, 15VII2007, R. Vigneault ; [MRC Deux-Montagnes] Parc national d\u2019Oka, 16VI2011, R. Vigneault ; [MRC Deux-Montagnes] Parc national d\u2019Oka, 1VIII2012 (16:00\u201317:00), swept from Polygonum sp., P. de Tonnancour & R. Vigneault ; same except: 19VIII2012 (17:00), swept from Polygonumamphibium, P. de Tonnancour ; same except: 26VIII2012 (17:00) ; same except: 18V2013 (15:00), beaten from Crataegus sp., P. de Tonnancour ; same except: 25V2014, composting site, white tulle fabric flight interception trap, R. Vigneault .[MRC Deux-Montagnes] Parc national d\u2019Oka, 4V1993, R. Vigneault  R. Br. (Convolvulaceae)). Based on these label data and on those of most specimens reported henceforth, R.aequalis appears to be associated with C.sepium.This native species was known in Canada only from Ontario  until BoCPTO; 2, CSLA); Montreal, Parc Zotique-Racicot , 8VII2015 (13:00), beaten from Castylegiasepium + Cirsiumarvense, P. de Tonnancour ; same except: 9VII2015, S. Dumont , 24VII2015 ; same except: 26VII2105, beaten from Castylegiasepium + Cirsiumarvense, S. Dumont ; same except: 25VIII2015, beaten from Castylegiasepium + Cirsiumarvense, S. Dumont ; same except: 1IX2015 (13:00), beaten from Castylegiasepium, P. de Tonnancour & S. Dumont ; same except: 7VI2016 S. Dumont ; same except: 10VI2106 ; 14VI21016 ; MRC Vaudreuil-Soulanges, Ville-de-l\u2019\u00cele-Perrot, 15VI2016 (12:00), beaten from Castylegiasepium, P. de Tonnancour ; Montreal, Parc Zotique-Racicot , 16VI2016, beaten from Castylegiasepium, S. Dumont ; MRC Vaudreuil-Soulanges, Notre-Dame-de-l\u2019\u00cele-Perrot, 17VI2016 (12:30), beaten from Castylegiasepium, P. de Tonnancour ; MRC Vaudreuil-Soulanges, Ville-de-l\u2019\u00cele-Perrot , 18VI2016 (12:00), beaten from Castylegiasepium, P. de Tonnancour ; MRC Vaudreuil-Soulanges, Notre-Dame-de-l\u2019\u00cele-Perrot, 21VI2016 (17:00), beaten from Castylegiasepium, P. de Tonnancour ; Montreal, Parc Zotique-Racicot , 23VI2016, beaten from Castylegiasepium, S. Dumont ; same except: 28VI2015 (13:00), P. de Tonnancour ; same except: 30VI2016, S. Dumont ; same except: 4VII2016 ; same except: 26VII2016 ; same except: 28VII2016 ; same except: 18VIII2016 .MRC Haut-Saint-Laurent, Saint-Anicet , 15VI2013, wet meadow, swept from various herbaceous plants, P. de Tonnancour & S. Laplante   , specifiand 1984 , obvioussylvania .C.buchanani.Specimens from southern USA were examined and found to be consistently larger than the northern forms from Quebec and northern USA, but dissections failed to reveal any further significant differences between the two groups. The status of the Canadian and northern USA forms needs further study. For the time being specimens reported herein will be considered as CCCH); MRC Vaudreuil-Soulanges, Terrasse-Vaudreuil, 21V2009 (21:00\u201322:00), mercury vapour light, P. de Tonnancour ; same except: 18VI2010 (23:00), mercury vapour + UV + porch light, P. de Tonnancour ; same except: 6VII2011 (23:00), UV + porch light, P. de Tonnancour ; MRC Marguerite-D\u2019Youville, Contrec\u0153ur, 8VII2012 (0:30), mercury vapour + UV light, P. de Tonnancour ; [MRC La Vall\u00e9eduRichelieu] MontSaintHilaire, 2-VI-2013, H. Miquet-Sage ; MRC Vaudreuil-Soulanges, Terrasse-Vaudreuil, 21IX2014 (21:00), UV + porch light, P. de Tonnancour ; Montreal, Parc Zotique-Racicot , 19VI2015, beaten from Celtisoccidentalis, S. Dumont ; same except: 2VII2015 ; Montreal, 11875, rue Zotique-Racicot , beaten from Celtisoccidentalis, 8VII2015, P. de Tonnancour & S. Dumont ; same except: 12VII2015 (16:00), P. de Tonnancour ; same except: 9VII2015, S. Dumont ; same except: 16VIII2015 ; 21VIII2015 ; 25-VIII-2015 ; same except: 1IX2015, P. de Tonnancour & S. Dumont ; same except: 12X2015, S. Dumont ; same except: 22V2016 ; same except: 24V2016 ; MRC Vaudreuil-Soulanges, Terrasse-Vaudreuil, 30V2016 (01:00), UV + porch light, P. de Tonnancour ; Montreal, Parc Zotique-Racicot , 7VI2016, beaten from Celtisoccidentalis, S. Dumont ; same except: 14VI2016 ; same except: 27VI2016, UV light ; MRC Vaudreuil-Soulanges, Terrasse-Vaudreuil, 27VI2016 (22:45), UV + porch light, P. de Tonnancour ; Montreal, Parc Zotique-Racicot , 30VI2016, beaten from Celtisoccidentalis, S. Dumont ; same except: 8VIII2016 ; same except: 18VIII2016 .[MRC Brome-Missisquoi], Saint-Armand, 5VI2007, C. Chantal ; same except: plage d\u2019Oka, 2VIII2011 ; [MRC Marguerite-D\u2019Youville] Varennes, 8IX2015, attracted to UV lamp, C. Chantal ; MRC Deux-Montagnes, Parc national d\u2019Oka, 21VII2015 (1:00), beaten from foliage of Caryaovata, P. de Tonnancour .[MRC Deux-Montagnes] Parc national d\u2019Oka, composting site, 23VII2011, R. Vigneault , new to Quebec Species identification confirmed by RSA, 2015 Conotrachelus Dejean, 1835 was previously known in Canada only from Ontario  as the type of the monobasic genus Loceptes Casey, 1910 .MRC Vaudreuil-Soulanges, Terrasse-Vaudreuil, 19VI2014 (0:00), attracted to UV + porch light, P. de Tonnancour , new to Quebec Species identification confirmed by Hume Douglas, 2016 CNCI Canadian specimens are from Point Pelee National Park). As for the above-mentioned Conotrachelusbuchanani, this native species is associated with Celtis spp.  from dead branches of Celtisoccidentalis in Almonte, Ontario, ca. 30 km from the Quebec border .This minute native species (1.2\u20131.6mm) was previously known to occur in Canada only in the southernmost part of Ontario ; same except: 20V2016 (16:30) ; Montreal, rue Zotique-Racicot , 21VIII2015, beaten from Celtisoccidentalis, S. Dumont ; same except: 21V2016 ; same except: 22V2016 ; same except: 23V2016 ; same except: 24V2016 ; same except: 7VI2016 ; MRC Laval, Laval, rue des Charmes , 20VII2016 (15:00), beaten from Celtisoccidentalis, P. de Tonnancour ; MRC Vaudreuil-Soulanges, Terrasse-Vaudreuil , 20VII2016, ex-larva from dead branch of Celtisoccidentalis, P. de Tonnancour ; same except: 28-VII-2016 ; same except: 14-VIII-2016 ; same except: 15-VIII-2016 ; same except : 18-III-2017 ; MRC Laval, Laval, rue des Charmes , 2IV2017, ex-larva from dead branch of Celtisoccidentalis, P. de Tonnancour .MRC Vaudreuil-Soulanges, Terrasse-Vaudreuil , 20IX2013 (18:00), white tulle fabric flight interception trap, P. de Tonnancour . It occurs in association with common hackberry, Celtisoccidentalis, in Quebec, but also with dwarf hackberry, Celtistenuifolia Nutt. ; MRC Laval, Laval, rue des Charmes , 6VII2016 (15:00), beaten from Celtisoccidentalis, P. de Tonnancour .MRC Vaudreuil-Soulanges, Terrasse-Vaudreuil, 14VI2016 (14:00), white tulle fabric flight interception trap, P. de Tonnancour (1,"}
+{"text": "Pay What You Want (PWYW) and Name Your Own Price (NYOP) are customer-driven pricing mechanisms that give customers (some) pricing power and that have been used in service industries with high fixed costs to price discriminate without setting a reference price. This paper describes buyer and seller data in a series of induced-value laboratory experiments that compare PWYW and NYOP in monopoly and competitive situations. Sellers are in a one-shot interaction with buyers. Sellers using customer-driven pricing mechanisms may exogenously or endogenously receive additional promotional benefits, for instance through word-of-mouth effects. The major findings based on the data presented here are reported in the paper \"Delegating Pricing Power to Customers: Pay What You Want or Name Your Own Price?\"   Specifications TableValue of the data\u2022This dataset compares buyer and seller behavior in two customer-driven pricing mechanisms: Name Your Own Price and Pay What You Want.\u2022Researchers can combine the analysis of buyer and seller behavior in one-shot interactions of PWYW and NYOP (this dataset) in comparison to the data related to the paper by Schmidt et al. \u2022This dataset provides a baseline on buyer and seller behavior in controlled interactions to compare with in case of related field experiments.1The data comprise buyer and seller behavior of a total of 384+144 subjects who participated in 8 different treatments of an experiment reported in Kr\u00e4mer et al., 2017: 6 treatments with exogenous benefits  and 2 treatments with endogenous benefits . The data captures for each treatment, round and subject the experimental conditions, subsequent decisions and outcome.In the first set of experiments, sessions lasted about two hours and subjects earned on average 18 Euros (about 24 US Dollars at the time of the experiment), including a show-up fee of 4 Euros. In the endogenous benefit treatments average earnings amounted to 25 Euros (about 27 US Dollars at the time of the experiment), including a show-up fee of 4 Euros. All sessions were conducted at the experimental laboratory of the University of Munich (MELESSA). The subject pool consisted mainly of students from a wide range of majors. Treatments were implemented using zTree variable_description_pwyw_nyop.xlsx.22.1In each treatment, subjects are randomly assigned to a role  that remains fixed throughout the experiment. Each session consists of 24 participants resulting in three markets in the competition treatments (two sellers facing six buyers in each market) and six markets in the monopoly treatments (one seller facing three buyers). All treatments are repeated for 20 periods, and subjects are randomly re-matched every period. The data comprise eight sessions of the competition treatments and another eight sessions of the monopoly treatments. In order to perfectly control the valuations of the buyers and the costs of the sellers we use an induced-value design Conditional on using a customer-driven pricing mechanism, sellers may receive a benefit (b) per unit sold. In the first set of experiments the benefit is exogenously given. In the second set of experiments, benefits are created endogenously in the lab (see below).The exogenous benefit is proportional to the number of units sold. In order to identify the effect of b, we assigned a strictly positive benefit b randomly to 50% of all markets and the remaining 50% of markets have a benefit of zero. Sellers know whether they enjoy a positive benefit from using a customer-driven pricing mechanism while customers know only that these benefits exist in half of all cases. At the end of each session we elicit information about risk preferences and social preferences of the participants and their demographic characteristics.2.22.2.1In treatments PCFlex  and NCFlex  one of the two sellers can choose whether to use posted prices or to use PWYW  while the other  seller has to use a posted price. At the beginning of each period all subjects observe the per-unit cost of the good which is the same for both sellers and drawn from c\u2208{10, 30, 50}. The flexible seller privately learns the per-unit benefit b\u2208{0, 40} from using a customer-driven pricing mechanism and buyers privately learn their valuations of the good drawn independently from v\u2208{10, 25, 40, 60, 120, 200}. Then each seller decides whether to enter the market. Additionally, the flexible seller decides which pricing mechanism to use. All buyers and sellers subsequently learn about the market structure. Now the posted-price sellers set their prices, and a NYOP seller sets the (secret) threshold price above which s/he is willing to accept all bids. Finally buyers decide whether and if so from which seller to buy. If they opt for a posted-price seller they have to pay the posted price. If they decide to shop with a PWYW seller they get the good with probability 1 and are free to decide how much to pay for it voluntarily (including a price of zero). If they go for a NYOP seller they are asked to submit a bid. If that bid exceeds the secret threshold price set by the seller in advance, they pay their bid and a transaction occurs. In case the bid is below the seller\u05f3s threshold price, they do not receive the good and do not have to pay either. Finally, payoffs are realized.2.2.2The competition treatments with fixed roles (treatments PCFix and NCFix) are set up identically to the treatments with flexible roles except for the fact that one of the sellers is constrained to use either PWYW (in treatment PCFix) or NYOP (in treatment NCFix) if s/he decides to enter the market.2.3In two monopoly treatments  and  there is only one seller who is forced to use PWYW  if s/he enters the market. In these treatments, a market consists of only three buyers. Costs and benefits are parameterized as in the competition treatments, while buyers\u2019 valuations are drawn from a restricted set, v\u2208{40, 60, 120, 200}. All sellers have the same cost-benefit combination in a given period as in the competition treatments and learn that combination before market entry.2.4The exogenous benefit treatments varied our previous designs as follows: Each market consists of two sellers and six buyers. The demand is now split into two groups: There are two well-informed buyers, who are fully aware of the market structure, and four follow-up buyers, who have to rely on word of mouth to learn which seller(s) entered the market. The purchasing decisions of the well-informed buyers directly affect the market structure for the follow-up buyers, modeling word-of-mouth advertising in a reduced fashion. For example, if both well-informed buyers purchase at the PWYW seller, only the PWYW seller is visible to the follow-up buyers. However, follow-up buyers may become fully informed about which sellers are in the market if they pay an additional search cost. Furthermore, in the new NYOP treatment buyers can still buy from the posted-price seller if their submitted bid was not successful.With the beginning of a new period, buyers and sellers are informed about costs . In addition, buyers learn their valuations . Then, sellers decide on entry. Having observed the sellers\u2019 entry decisions, the two well-informed buyers decide whether and if so with which seller to transact. If at least one of the well-informed buyers has opted for the PWYW/NYOP seller, this seller is available for follow-up buyers at no additional cost. The same applies for the posted-price sellers. If only one seller is available to follow-up buyers, they can invest search costs (csinvest=10) to find out whether the other seller has entered the market. If this is the case, they can also purchase from the discovered seller. If the market for well-informed buyers is split equally, both sellers are available at no additional search cost. After learning about the market structure follow-up buyers make their purchasing decisions. In case of submitting an unsuccessful bid to a NYOP seller, the buyer can turn to the posted-price seller if this seller is available. Thus, the availability of sellers for follow-up buyers in the NYOP treatment depends on the interaction between the well-informed buyers and the sellers.To illustrate this consider the following situation: A well-informed buyer submits a bid to the NYOP seller which is rejected. The buyer subsequently purchases the good from the posted-price seller, making both sellers available to the follow-up buyers. However, in all cases where both well-informed buyers interact with the same seller only this seller is available to the follow-up buyers."}
+{"text": "Salvelinus fontinalis). The consensus map was constructed from three full-sib families totaling 176 F1 individuals. The map consisted of 42 linkage groups with a total female map size of 2502.5 cM, and a total male map size of 1863.8 cM. Synteny was confirmed with Atlantic Salmon for 38 linkage groups, with Rainbow Trout for 37 linkage groups, Arctic Char for 36 linkage groups, and with a previously published Brook Trout linkage map for 39 linkage groups. Comparative mapping confirmed the presence of 8 metacentric and 34 acrocentric chromosomes in Brook Trout. Six metacentric chromosomes seem to be conserved with Arctic Char suggesting there have been at least two species-specific fusion and fission events within the genus Salvelinus. In addition, the sex marker  was mapped to Brook Trout BC35, which is homologous with Atlantic Salmon Ssa09qa, Rainbow Trout Omy25, and Arctic Char AC04q. Ultimately, this linkage map will be a useful resource for studies on the genome organization of Salvelinus, and facilitates comparisons of the Salvelinus genome with Salmo and Oncorhynchus.Next generation sequencing techniques have revolutionized the collection of genome and transcriptome data from nonmodel organisms. This manuscript details the application of restriction site-associated DNA sequencing (RADseq) to generate a marker-dense genetic map for Brook Trout ( Their use in comparative genomics between nonmodel and model organisms is important as linkage maps can facilitate the identification of candidate genes for traits of interest . MoreoveSalmo salar; Oncorhynchus mykiss; O. nerka; O. kisutch; O. keta; O. tshawytscha; O. gorbuscha; Salvelinus alpinus; S. fontinalis; O. mykiss  genome duplication that occurred early in salmonid evolution . AlthougS. fontinalis) is a species of salmonid native to the northern United States and Canada. Although research on Brook Trout has been less intensive than on other salmonids, recent studies have described variation in several evolutionary traits of interest such as morphology, size, age of sexual maturation, and water temperature tolerance  feeding in Lake Superior before returning to natal spawning grounds. Both populations have been used in restocking efforts in Lake Superior since the 1990s , and the Siskiwit River population was founded from adfluvial fish . The two strains are genetically distinct from each other  and placed in 100% ethanol. DNA was extracted from tail tissue via a modified Phenol\u2013Chloroform extraction protocol described in 1) samples and RAD libraries were prepared following SbfI-linked Illumina sequencing has been used for SNP discovery in multiple salmonid linkage maps , and SNPs scored in <80% of F1 samples removed. These filtering criteria produced a total of 12,961 candidate SNPs.Raw sequences were quality filtered (minimum Q score of 20) and trimmed (3\u2032 end) to 76 bp using the program using the process RADtags script in STACKS , allowinSeperateChromosome command was run with a minimum LOD score of 12, a maximum recombination fraction of 0.4, and a minimum number of markers per linkage group of 10. Any marker that showed evidence of segregation distortion (\u03c72 test P < 0.001) was removed. Unmapped markers were then rerun against the threshold map using the command JoinSingles, with a minimum LOD score of 5, a minimum LOD difference of 3, a maximum recombination fraction of 0.4, and Mendelian inheritance (\u03c72 test of segregation distortion P > 0.001). Markers were ordered using the OrderMarkers command using default parameters. This command rearranges the order of the markers on a linkage group and reports the \u201cbest\u201d order (lowest LOD likelihood). Linkage groups were drawn using the program MAPCHARTv2.1  than the Rainbow Trout genome. Synteny between linkage groups was only inferred if >5 RADseq loci matched a specific linkage group.To determine chromosomal organization with Brook Trout and other salmonid linkage maps we used a comparative approach using the program MapComp. This program compared the markers placed on the linkage map reported herein to the Brook Trout linkage map reported in sdY could be used to accurately determine sex in the F1 samples. PCR conditions followed those reported in sdY and biological sex for any of the parents, therefore all F1 individuals were genotyped using the methods described above. PCR amplification for 10 candidate males and 10 candidate females was repeated twice to determine accurate assignment of sex. In no incidence was there a mismatch. sdY was added to the mapping dataset by scoring females as homozygotes and males as heterozygotes.The parents of all three mapping crosses were used as positive controls to validate whether File S1 contains the input file for Lep-MAP showing genotypes for all three families for the 1990 mapped markers. File S2 contains the consensus sequence, marker ID, female map position, male map position, and linkage group for all mapped markers. File S3 shows the position of mapped markers in the Brook Trout linkage map. File S4 shows Oxford plots comparing the Brook Trout linkage map to (A) the Rainbow Trout linkage map, (B) The Atlantic Salmon linkage map, (C) The Brook Trout linkage map published in Supplemental Material, Illumina RADseq produced a total of 427,823,712 quality-filtered sequences for the F1 samples and 39,938,749 quality filtered reads for the parents. The number of quality filtered reads varied from 780,935 to 12,936,817 for the F1 individuals  and from 1,755,890 to 16,643,600 for the parents .File S1, sequence and position of RAD loci provided in File S2). The number of mapped markers varied per family , of which 701 loci were shared between all three families, 728 were shared between two of the three families, and 561 loci were specific to a family. Linkage groups built from female informative meioses ranged from 0 to 185.1 cM with a total map size of 2502.5 cM. The male map totaled 1863.8 cM with individual linkage groups ranging in size from 0 to 112.9 cM .A total of 12,961 unique RAD loci were discovered in three F1 families of Brook Trout. The final linkage map consisted of 1990 markers located on 42 linkage groups  matched three Brook Trout linkage groups , of which five  are homologous with metacentric chromosomes in Brook Trout , suggesting conservation of these metacentric chromosomes among Salvelinus  or one metacentric and one acrocentric chromosome (type 2). The population used by Materials and Methods). These homologies were used to identify potential tetrasomic inheritance patterns in Brook Trout , Ssa10 (fused acrocentric), Ssa13 (fused acrocentric), Ssa14 (fused acrocentric), and Ssa20 (fused acrocentric)] each matched two Brook Trout linkage groups in the study herein. The same chromosomes also matched two linkage groups in Salmo split from the ancestor of Oncorhynchus and Salvelinus. Two metacentric Brook Trout linkage groups (BC01 and BC08) each matched two Atlantic Salmon chromosome arms . The alignment of linkage group BC01 to multiple Atlantic Salmon chromosomes appears to represent true karyotype differences between Salvelinus and Atlantic Salmon, as the same result was seen with AC18 in Arctic Char  and BC05 (AC27), were not seen in other salmonids. Interestingly, both of these chromosomes are metacentric, suggesting that the fusion of these chromosomes happened after Salvelinus diverged from the other salmonids.Comparison with Atlantic Salmon supported previous observations of genome evolution in salmonids. Chromosomes Ssa01 and Ssa09 in Atlantic Salmon were each formed through a fusion of three ancestral chromosome arms (sdY) mapped to BC35, in an area of reduced recombination in both males and females. This linkage group matched Ssa09qa in the Atlantic Salmon genome and AC04q in Arctic Char (sdY in salmonids has received a lot of interest, as sdY is part of a cassette that has moved to different chromosomes between species (Salvelinus, as chromosome painting has determined that the sex marker has moved to different linkage groups in different populations of Arctic Char (AC04 in North American and AC01/21 in European Arctic Char; sdY in Salvelinus has not been determined. Follow-up studies that document patterns of gene expression in Brook Trout are necessary to determine if sdY is the master sex-determining gene in this species.Our mapping results determined that the sex marker ( species  and betwSalvelinus and Salmo split from a common ancestor, and between Arctic Char and Brook Trout. Using comparative genomic approaches with software such as MapComp increased our understanding of salmonid genome evolution, particularly in chromosome arms that are undifferentiated and can exhibit tetrasomic inheritance.Here, we present a linkage map for Brook Trout comprised of 42 linkage groups. Comparisons with other salmonid linkage maps confirmed some of the many fusion and fission events that have occurred both after www.g3journal.org/lookup/suppl/doi:10.1534/g3.117.300317/-/DC1.Supplemental material is available online at Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file."}
+{"text": "H3F3B and colocalizes with potential gene target miR-616. A custom miRNA target prediction program predicted that the binding of miR-616 to H3F3B transcripts would be altered by the allelic variants of rs1060120. We used dual luciferase assays to experimentally validate this interaction. The rs1060120 A allele significantly reduced luciferase expression, indicating a stronger interaction with miR-616 than the G allele (p = 0.000412). These results provide functional validation that this SNP could alter schizophrenia epigenetic mechanisms thereby contributing to schizophrenia-related disease risk.Despite much progress, few genetic findings for schizophrenia have been assessed by functional validation experiments at the molecular level. We previously reported evidence for genetic linkage of broadly defined schizophrenia to chromosome 17q25 in a sample of 24 multiplex families. 2,002 SNPs under this linkage peak were analyzed for evidence of linkage disequilibrium using the posterior probability of linkage (PPL) framework. SNP rs1060120 produced the strongest evidence for association, with a PPLD|L score of 0.21. This SNP is located within the 3'UTR of the histone gene  Schizophrenia is a complex disorder that is believed to be caused by the interaction of multiple genetic risk factors, within and between individuals. Environmental factors may also play a role. Considerable advances have been made in schizophrenia genetics. This includes recent work by the Psychiatric Genomics Consortium (PGC) that has reported 108 independent associations with common genetic variants (SNPs), 83 of which were newly implicated in schizophrenia, from studies involving over 35,000 cases . In otheH3F3B.miRNAs are small endogenous RNA molecules that are able to bind to specific regions of the 3\u2019 UTR of coding mRNAs thereby decreasing expression of their protein product . This isIn 2000 we published a traditional parametric linkage analysis using genome-wide microsatellite markers to search for linkage to schizophrenia and related phenotypes within a sample of 22 Canadian extended families recruited for study because multiple relatives were clinically diagnosed with schizophrenia or schizoaffective disorder . Both anA meta-analysis of schizophrenia linkage studies identified a suggestive level peak on 17q, within 4 Mb of the linkage peak in our sample . While tH3F3B is associated with a broad schizophrenia spectrum phenotype in our family sample and also alters the function of a miRNA target site within the 3\u2019 UTR of the gene.Given the evidence in our family sample for a locus related to a broad schizophrenia phenotype on chromosome 17, we conducted additional studies to identify and validate a functional variant under the linkage peak that exhibited strong linkage disequilibrium with our broad phenotype. Because of the importance of miRNAs in schizophrenia, we elected to interrogate the region under our linkage peak with SNPs predicted to alter miRNA targets in addition to the stock SNP content pulled from a dense GWAS mapping array. Here we present evidence that a SNP in the gene NOS1AP [The subjects analyzed for this study are the same that were previously used in our association studies of NOS1AP . The samNOS1AP ,25,31,32The linkage peak previously identified on chromosome 17q25  was bounTM 100 flow cytometry [DNA from subjects was extracted from blood samples or lymphoblastoid cell lines using the GenePure system (Gentra Systems). DNA samples were genotyped using Affymetrix 6.0 arrays at The Centre for Applied Genomics at the University of Toronto using standard procedures. The MirSNPs were genotyped by ligase detection reaction (LDR) and Luminexytometry ,38. Reacytometry . A list Before data cleaning, the Affymetrix 6.0 array data had an average completion rate of 99.0% across all SNPs, genome-wide. Genotype cleaning of the 2,002 SNPs of interest was first conducted with PLINK v1.07 and inclSNPs were analyzed for evidence of linkage disequilibrium using the posterior probability of linkage (PPL) framework as previously described  and as iHT-1080 fibrosarcoma cells (ATCC) were cultured in Eagle's Minimum Essential Medium Alpha, supplemented with fetal bovine serum to a final concentration of 10% and 50 to 100 I.U./mL penicillin and 50 to 100 (\u03bcg/mL) streptomycin (Thermofisher). 96-well plates were seeded with HT-1080 fibrosarcoma cells (ATCC) 24 hours prior to transfection to achieve 80% confluence at the time of transfection.H3F3B containing either the G (reference) or A  allele of the SNP rs1060120, were transfected using the manufacturer\u2019s protocol in conjunction with the Cypridina TK control construct driven by an HSV-TK constitutive promoter (SwitchGear Genomics). The transfection was performed using FuGENE HD* Transfection Reagent (Promega). The LightSwitch Dual Assay System (Switchgear Genomics) was used to normalize for variation between experimental replicates due to transfection variability by utilizing a co-transfection control.Luciferase reporter constructs containing RenSP and the human 3\u2032-untranslated region (3\u2032UTRs) for Wells were co-transfected with either the miR-616-3p mimic  or the non-targeting scramble miRNA  to yield a final concentration of 20 nm. Triplicate independent transfections were performed using a minimum of three replicate wells for each reaction experimental condition. The three independent transfections were performed consecutively over the course of several weeks.Luciferase expression levels were measured using a Veritas Microplate luminometer (Turner Biosystems) following the protocol from the BioLux Cypridina Luciferase Assay (New England Biolabs).The luciferase expression levels were normalized following manufacturer\u2019s instructions for the Dual Luciferase Assay by dividing the LightSwitch Reagent values for each condition by the Cypridina co-transfection control values (Active Motif). To normalize the luciferase datacall values were divided by the luciferase measurement for the G UTR.The data was analyzed in accordance with the randomized complete block design by using an additive term for trial in the ANOVA. Doing so accounts for trial-to-trial variability. Tukey's HSD (honestly significant difference) test was used to compare treatment differences; \u201cadjusted p-values\u201d in the text refer to the Tukey HSD adjustment. The R statistical environment was used for analysis.H3F3B, rs4969391 in gene BAIAP2, rs7211218 in predicted gene LOC283999, rs1663196 in gene TBC1D16, and rs1128687 in gene CHMP6 . A total of five MirSNPs were predicted from the 17q25 region of this linkage peak, chr17: 74,684,647 to 83,257,441 (GRCh38): rs1060120 in gene ne CHMP6 .H3F3B, produced a score of 0.21. For rs1060120, the A allele was found to be associated with illness. Notably, rs1060120 was predicted to colocalize with potential miRNA target miR-616  was on the array and was a predicted MirSNP, thus, in total 1,544 SNPs were tested. Overall, 1,338 SNPs (87%) of the 1,544 tested from the 17q25 region produced PPLD|L scores < 0.02, indicating evidence against LD. Only three SNPs produced PPLD|L scores > 0.10. SNP rs894941 and rs7220244 from the Affymetrix 6.0 Array each produced scores of 0.12; SNP rs1060120, the MirSNP in the 3\u2019UTR of gene  miR-616 . All bro miR-616  and listH3F3B with either the A allele or the G allele. In the absence of added miRNA, expression of the two UTR variants was observed to be equivalent. Co-transfection of a scramble miRNA resulted in a slight decrease in expression, likely due to non-specific minor toxic effects of the construct. Co-transfection with the miR-616 mimic produced significantly decreased expression for both 3\u2019 UTR variants . The rs1060120 A-allele containing variant however exhibited a significantly greater decrease in luciferase expression (p adjusted = 0.000412) than the 3\u2019UTR G-allele variant   . These dophrenia \u201351. Suchophrenia \u201354. was confirmed, using a dual luciferase reporter assay in a mammalian cell culture system, to functionally alter the targeting of miR-616. It is therefore possible this common variant could alter the function of miR-616 and thereby contribute to risk of schizophrenia spectrum diagnoses through downstream effects.Searching under a previously reported linkage peak on 17q25 , we haveS1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file."}
+{"text": "Studies involving the use of probabilistic record linkage are becoming increasingly common. However, the methods underpinning probabilistic record linkage are not widely taught or understood, and therefore these studies can appear to be a \u2018black box\u2019 research tool. In this article, we aim to describe the process of probabilistic record linkage through a simple exemplar. We first introduce the concept of deterministic linkage and contrast this with probabilistic linkage. We illustrate each step of the process using a simple exemplar and describe the data structure required to perform a probabilistic linkage. We describe the process of calculating and interpreting matched weights and how to convert matched weights into posterior probabilities of a match using Bayes theorem. We conclude this article with a brief discussion of some of the computational demands of record linkage, how you might assess the quality of your linkage algorithm, and how epidemiologists can maximize the value of their record-linked research using robust record linkage methods. Key MessagesUnderstanding probabilistic record linkage is essential for conducting robust record linkage studies in routinely collected data and assessing any potential biases.Match weights are based on likelihood ratios and are derived from concepts familiar to epidemiologists, such as sensitivity and specificity, and match weights can be converted into probabilities using Bayes theorem.Only a basic understanding of conditional probability is required to understand the fundamentals of probabilistic record linkage.,With the increasing use and availability of routinely collected \u2018big\u2019 data, it is becoming more useful to undertake research that involves linking data from multiple sources. Therefore, the importance of fully understanding and developing robust record linkage procedures is becoming increasingly necessary, as is fully recognizing and reporting the limitations and biases of the methods used, emphasized by the imminent publication of the record linkage study extension  deterministic and (ii) probabilistic. Deterministic record linkage is the process of linking information by a uniquely shared key(s). Records are matched if linkage fields agree or unmatched if they disagree. For example, in a longitudinal cohort study, deterministic linkage is often used to link multiple waves of data collection together. Probabilistic record linkage attempts to link two pieces of information together using multiple, possibly non-unique, keys. For example, in a registry-based study, disease events may be linked to mortality data using non-unique first and last name combinations. Despite the apparent simplicity of the task, the process is always complicated by errors in the linkage key(s) or lack of unique key(s) linking both pieces of information together.In this article we describe: (i) the process of performing record linkage; (ii) pre-merge data cleaning; (iii) the Fellegi-SunterIJE online), annotated Stata code which recreates all the analyses described.For the sake of clarity, we assume a simple scenario where a researcher is attempting to link data from two files. The first file is known as the \u2018master file\u2019 (MF) and the second file contains information with which the researcher would like to supplement the master file. This file is known as the \u2018file of interest\u2019 (FOI). The information which is used to link the two files together is contained within fields or variables and known as the \u2018key\u2019. For pedagogic reasons, we include, as Deterministic record linkage is commonly performed in many research studies and assumes there is a known key which links two files together\u2014the MF and FOI, as defined above. The results from a deterministic record linkage procedure will result in two mutually exclusive categories of \u2018matched\u2019 and \u2018unmatched\u2019 records. Unmatched records can then be further defined as \u2018in the master file\u2019 or \u2018in file of interest\u2019.Suppose, for example, that we are interested in investigating the association between an individual\u2019s gender and highest educational qualification, which requires a single data set containing both pieces of information. If we assume gender and education are stored in two distinct files, i.e. \u2018the master file\u2019 and the \u2018file of interest\u2019, and the linking key is composed of the individual\u2019s first and last name, we can attempt to deterministically link the files. Despite the relatively trivial task of linking two data sets by first and last name, the results are somewhat disappointing and there is only one matched record, see However, it is also clear that there is partial agreement between the linking keys of first name and last name. For example, Fiona Steele and Fiona Steel only disagree on a single character in the field last name. Whether the linking fields partially agree or completely disagree is not reflected when conducting a deterministic record linkage. The researcher is then left with the following choices: (i) accept that only a single record is matched between the master file and file of interest; (ii) conduct data cleaning to reduce the heterogeneity in the linkage and reattempt the linkage; or (iii) adopt some form of probabilistic matching.Despite the name, the first stage of probabilistic record linkage is not a statistical issue. If you are attempting to link the two files illustrated in In order to do this, you must first ensure all matching fields are uniquely identifiable across both files. Following the merge  the agreement pattern between the two sets of keys is determined see . The firThe results of joining the two files and calculating agreement patterns between the linking keys indicates that there maybe more commonality between the two files than previously indicated by deterministic linkage. Whereas only one record is indicated as matching on both the first and the last name fields, there is partial agreement between the first and last name on other records in the data set.In comparison with deterministic record linkage, the researcher is now in a comparatively informed position. If the choice is to accept only records with identical fields on both the first name and the last name, this will result in a matched data set equivalent to that identified using deterministic linkage. However, the researcher now has the choice of accepting a lower threshold for determining the linkage status of any two records, such as allowing a link to be established on either the first or the last name field.The simple dichotomy presented by the agreement pattern presented in ,Assuming that 0 indicates complete disagreement, 1 indicates complete agreement and a value between 0 and 1 indicates partial agreement, a more complex agreement pattern can be constructed. For example, the edit distance,,There are many other methods of comparing the dis-similarity between strings. Common methods include name phonetic algorithms such as SOUNDEXDespite the wide variety of methods of comparing strings, any heterogeneity introduced by punctuation, capitalization, abbreviations and alternative spellings emphasizes the need for data cleaning.st January 1960, 31/1/1960, 31\u20101\u201060); (x) checking for transposition in dates ; (xi) finding automatically filled dates or dates too far in the future or in the past ; and (xii) using checksums to find invalid unique identifiers such as those embedded in NHS numbers. Depending on the topic of interest, there may be many other data cleaning procedures which are applicable.As emphasized previously, matching on names can be problematic due to typographical differences. Fortunately this problem can be mitigated by data cleaning routines. Examples of common data cleaning procedures can include: (i) changing the case on all the strings; (ii) removing punctuation; (iii) deleting consecutive spaces; (iv) trimming trailing or leading spaces; v5) removing prefixes (Mr/Mrs/Dr/Prof.) and suffixes ; (vi) ignoring middle initials; (vii) looking for transpositions in words ; (viii) identifying nicknames  (ix) unifying date formats \u00a0 see . In pracIt is then necessary to assign a numerical value which reflects the (dis-)similarity of the two records. The (dis-)similarity of two records is expressed as the ratio of two conditional probabilities that the two records have the same agreement pattern across the variable of interest.thj comparison by thi linkage field of the thj comparison is denoted by Suppose we attempt to match two files on first and last name, and we denote the agreement pattern for the m- and u-probabilities,The probabilities m- and u-probabilities can be difficult. The m-probability can be conceptualized as an indicator of data quality. Suppose, for example, that the data error rate  in records which were truly matches was known, the linkage field was binary (e.g. sex) and this data error rate was approximately 20% in the master file and file of interest. In that case, you would expect 64% (0.8 \u00d7 0.8\u2009=\u20090.64) of matching fields to correctly agree and 4% of matching fields to incorrectly agree (0.2 \u00d7 0.2\u2009=\u20090.04), leading to an m-probability of 68% for the sex field. If matching fields are not binary, then the probability of two matching fields incorrectly agreeing is probably closer to zero than 4%. Disagreement in the remaining 32% of pairs of records, i.e. the false negatives, may be due to typographical/data entry errors, missing data and or changes in sex.Interpreting u-probability is defined as chance agreement between two records which are truly unmatched. This can be conceptualized and simplified as chance agreement using the following logic. Assume two files  contain 1000 records each. Then a full comparison between FileMaster and FileFOI will result in 1\u2009000\u2009000 potential comparisons, of which 1000 comparisons can be true matches. Therefore, the 999\u2009000 comparisons are non-matches. As unmatched pairs make up the majority of comparisons, it is often assumed that all comparisons form part of the unmatched set. Assuming that the linkage keys are not unique identifying numbers and have some repetition, it then becomes quite natural to investigate the frequencies within each matching key of FileMaster and FileFOI and how likely it is that a pair of records will match by chance alone.The m- and u-probabilities can be adjusted depending on the uniqueness (frequencies) of the linking fields. Consider a simple scenario of linkage between two files  of equal size  without duplicates. Of the 100 comparisons created by joining FileMaster and FileFOI, there will be at most 10 true matches. If the linkage key of interest is surname and there are 7 Smith and 3 Sayers, the m-probability of Smith and Sayers is 7/10\u2009=\u20090.7 and 3/10\u2009=\u20090.3, respectively. The remaining 90 comparisons are therefore non-matches. We know that there will be 49 comparisons where Smith agrees between the two files of interest; 7 of those comparisons are true links, whereas the remaining 42 are incorrect links. Similarly, there are 9 matches for Sayers of which 3 are correct. The u-probability of Smith and Sayers is 42/100\u2009=\u20090.42 and 6/100\u2009=\u20090.06, respectively. SeeBoth the Correspondingly, the likelihood ratios for agreement on \u2018Smith\u2019 and \u2018Sayers\u2019 are 1.6 (0.7/0.42) and 5 (0.3/0.06), respectively, which indicates that a match on \u2018Smith\u2019 is less discriminating than a match on \u2018Sayers\u2019.Master is defined as FOI as Master is FOI is m- and u-probabilities are equal to Formally, if the frequency of names in Filem- and u-probabilities for errors and missingness in the linkage fields, and assume u-probabilities are an unconditional probability of chance agreement such that m- and u-probabilities are either estimated using previous experience, an assumed \u2018gold standard\u2019 data set, or by more complex computerized methods.,et\u00a0al. calculated m-and u-probabilities by deterministically linking a subset of individuals that were matched on either hospital number or NHS number, i.e. they assumed that if a pair of records linked on either field this is a gold standard or at least a reasonable starting point before further refinement.m-probability for the year of birth would be 0.95.In Fellegi and Sunters\u2019 original paper they illustrate how to adjust the m- and u-probabilities are analogous to the results from a diagnostic testing scenario,m-probability is equivalent to sensitivity, and the u-probability is equivalent to 1 minus the specificity. Furthermore, it is easy to see how the positive predictive value and negative predictive value can be also calculated,From an epidemiologist\u2019s perspective, the m- and u-probabilities of the agreement indicator for the thi field for the thj comparison, it is then possible to construct an overall match weight for the thj comparison, denoted m- and u-probabilities, where After estimating the m- and u-probabilities across the agreement indicators for the thj comparison.m- and u-probabilities of 0.95 and 0.25 for first and last name fields, respectively, yields the weights shown in Assuming the linkage fields are conditionally independent, the matching weight can be expressed as the ratio of the product of the Furthermore, the match weights can be adjusted for complex agreement patterns, Despite the somewhat difficult interpretation of the linkage weights, it is very clear which records are likely to be a match. For example, we can see that records 3, 7, 11 and 15 are 4, 5.83, 5.05 and 3.74 times more likely to match than the next nearest matching record, respectively.The final operation is to define two thresholds which classify the potential links into three categories: links, non-links and potential links. It is possible to generate two different thresholds using the distribution of linkage weights, The prior probability of a match is defined as:Applying these results to the complex agreement weights presented in Despite exact agreement of linkage fields, the probability of linking records between the master file and the file of interest is less than 1,reflecting the possibility of inconsistencies in the data quality and chance agreement. However, by eyeballing the data, it is clear that the majority of correct links are identified with probability greater than 0.8.The exact placements of thresholds used to define link status can be a matter of trial and error.6 or more. Therefore, the use of blocking or stratification is employed. This process involves splitting the database into smaller blocks or strata, which was originally described as the \u2018restriction of explicit comparisons to a subspace\u2019.6 potential links, in contrast to the unblocked comparison which would result in 10 \u00d7 107 potential links. Nevertheless, when blocking there is a clear trade-off between the size of the blocks and the ability to fully explore the data set looking for potential matches, with the explicit assumption that individuals not in the block will not be a match.Despite the trivial example presented previously, it is very easy to see how the size of linked datasets can quickly expand. Even with a modestly large master file and file of interest of 10\u2009000 individuals, the resulting linked file would result in 100\u2009000\u2009000 potential links. In projects using routinely collected data, the number of individuals of interest can be 1 \u00d7 10,Following the creation of a linked data set, it is important to consider the quality of linkages and how this might influence your resultsThere are a number of different approaches which can be used to quantify the rate of linkage errors including: (i) comparison with a gold-standard sub-sample; (ii) sensitivity analysis; (iii) comparison of linked and unlinked data; and (iv) identification of implausible matches.Using a gold-standard sub-sample is probably the most intuitive method of establishing linkage errors. Comparing the probabilistically linked data set to the gold-standard sub-sample will give rise to a simple 2 \u00d7 2 table of linkage errors. Following creation of the 2 \u00d7 2 table, simple statistics such as sensitivity, specificity and positive/negative predictive values can be calculated and reported.m- and u-probabilities influence the number of potential links. Comparing linked and unlinked data can also be useful in establishing if some groups of records are easier to link than others. For example, assuming there are some common fields not used as linkage keys, such as socioeconomic status (SES), it is possible to compare the linkage rates within the SES group of the master file. Similarly, investigating how linkage rates vary across time might be a useful indicator of time-dependent biases.Structured sensitivity analyses can also be used to see how the changes to the Identifying implausible matches may only be possible in specific scenarios. Suppose, for example, that probabilistic linkage was being used to ascertain patient mortality within routinely collected medical records, and a trajectory indicated the following mortality pattern: alive, dead, alive. There may be some question about the quality of the linkages or the veracity of the source data.We have described the process underlying deterministic and probabilistic record linkage using a simple exemplar. Despite the apparent complexity of probabilistic linkage, it can be broken down into a relatively small number of simple data manipulation operations with relatively little statistical knowledge. Furthermore, the statistical principles underpinning the weight calculations used to define links in probabilistic linkage can be derived from Bayes\u2019 theorem, which may be covered on epidemiology courses and only requires a rudimentary understanding of conditional probability.Using a simple exemplar we have illustrated the critical steps and assumptions that underpin probabilistic record linkage. These include: (i) the inequity of the edit distance when comparing long and short strings; (ii) the assumption of conditional independence when calculating the match weights; (iii) the choice of block size which influences the computational burden of the linkage exercise; (iv) the choice of selection thresholds before accepting or rejecting pairs of records as links or non-links, and those requiring clerical review; and (v) the somewhat arbitrary pre-merge data cleaning processes that occur in the hope of finding more matching records.The benefits of probabilistic record linkage are simple . However, despite the simplicity of the exemplar, there are many complex issues of current research in the record linkage field including privacy preserving record linkage,Record linkage, whether probabilistic or deterministic, will become increasingly important as the breadth and scope of routinely collected data rapidly expand. We have illustrated that it is simple to conduct robust probabilistic record linkage using standard statistical software, and that sensitivity of results can be easily explored using different matching assumptions. Furthermore, probabilistic record linkage has the potential to maximize the value of routinely collected data by improving the linkage between the linked files of interest, which in turn will reduce the volume of missing data and improve the classification within the linked variables of interest, thereby strengthening the inferences from linkage studies.IJE online.A.S. is funded by an MRC Fellowship (MR/L01226X/1).Supplementary DataClick here for additional data file."}
+{"text": "Drosophila (During postembryonic development, the nervous system must adapt to a growing body. How changes in neuronal structure and connectivity contribute to the maintenance of appropriate circuit function remains unclear. Previously , we measured the cellular neuroanatomy underlying synaptic connectivity in osophila . Here, w Drosophila larva grows from a first instar just out of the egg to a third instar ready to pupariate, its body wall surface area grows by two orders of magnitude  reconstructions from l system  or bilatl system , visual l system , and olfl system  systems.l system  or high l system .Drosophila larvae. Despite considerable growth in body size between hatching and pupariation, almost no new functional neurons are added to the larval nervous system  . Both th31\u00b10.20)  and the 31\u00b10.20)  increaseMeasuring twigs requires painstaking visual inspection of EM imagery, so we also looked at a purely topological measure of neuronal arbor structure, Strahler order , that maTo get better insight into how twigs changed between the first and third instar, we measured the properties of individual twigs on LNs. Typical dendritic twigs in both the L1v and L3v are small. They were short in both total length and maximum depth from twig root, had few branch points, and few post-synaptic sites . HoweverWe next asked how the input from a pre-synaptic sensory neuron is distributed across the twigs on an LN\u2019s dendrite for each mdIVr system , mdIV\u2192LN the L3v . Within contacts .A practical consequence of numerically strong but anatomically distributed synaptic connectivity is that EM reconstruction becomes robust to random errors. The vast majority of manual errors in previous larval reconstructions was the omission of single dendritic twigs . To measng arbor . For conbor  was used on five adjacent sections to find optimal parameters for the elastic registration pass. Elastic alignment was applied with local smoothness filter approximating an affine local transformation. The resulting aligned image stack was exported to an image tile pyramid with six scale levels for browsing and circuit reconstruction in CATMAID , SG , CMSM , AC , and Waleed Osman . Comprehensive reviews of arbors and synapses in the L3v were performed by SG, AC and CMSM.https://github.com/ceesem/Larva_development_structure_2017  from the LN dendrites and measuring the number of pre-synaptic sites that were within a distance  and three reviewers, one of whom, Ronald L Calabrese (Reviewer #2), is a member of our Board of Reviewing Editors. The following individual involved in review of your submission has agreed to reveal their identity: Hermann Cuntz (Reviewer #1).Thank you for submitting your article \"Conserved neural circuit structure across The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:eLife in 2016, which reported new methods to apply quantitative arbor and network context to iteratively proofread and reconstruct circuits and create anatomically enriched wiring diagrams in Drosophila. They used these methods to measure the morphological underpinnings of connectivity in new and existing reconstructions of Drosophila sensorimotor (larva 1st instar L1v) circuits in the brain and VNC. They extend this work here by similarly reconstructing nociceptive networks in segment A3 of the VNC in new [encompassing segment A3 of the third instar larva (L3v)] and existing serial section electron microscopy volumes. They report a remarkable conservation of connectivity  across instars despite considerable neuronal growth and increases in total number of synapses. Five-fold increases in size of interneurons were associated with compensatory structural changes that maintained cell-type-specific synaptic input as a fraction of total inputs. They argue that their new data on A3 VNC in L3V validates the use of the L1V total nervous system volume to determine connectivity across larval stages, and that patterns of structural growth in larval development act to conserve the computational function of a circuit, for example determining the location of a nociceptive stimulus. The work is carefully done with appropriate quantification and statistics. The connectome established and the atlas of neurons will be very useful for fly workers who wish to pursue behavioral or developmental studies.This is a strong advance on an important paper in Essential revisions:1) The study is written in a very compact manner and would gain from substantial rearranging of the story and adding descriptive text. The story constantly switches between mdIVs and LNs, and it is very difficult to understand why. All sections of the text are too short.2) The Materials and methods section on the data analysis is completely missing.3) The figures need some rethinking and corrections. The terms/labels in the figures are often not described (not even used) in the main text, which makes them difficult to understand. Some of the supplemental figures could also be incorporated into the main article . Many figure panels mentioned in the text are entirely missing in the figures. Essential revisions:1) The study is written in a very compact manner and would gain from substantial rearranging of the story and adding descriptive text. The story constantly switches between mdIVs and LNs, and it is very difficult to understand why. All sections of the text are too short.We were pleased to have the room to expand the manuscript. More descriptive text has been added to nearly every paragraph discussing results. We believe the revised manuscript motivates its structure considerably more and will be clearer to the reader.2) The Materials and methods section on the data analysis is completely missing.We have greatly expanded the data analysis section of Materials and methods to detail our representation of neuronal reconstructions, filling fraction analysis, receptive field analysis, and error simulation. We have also posted analysis scripts on Github.3) The figures need some rethinking and corrections. The terms/labels in the figures are often not described (not even used) in the main text, which makes them difficult to understand. Some of the supplemental figures could also be incorporated into the main article . Many figure panels mentioned in the text are entirely missing in the figures.We have addressed these concerns by expanding the number of figures, making terminology more consistent and clear, and fixing typos in the text figure references. Throughout all figures, we increased text size and spacing of panels and labels. Major changes included:Previous New Previous Figure 2\u2014figure supplement 4 is now Previous All figure panels referenced in the text that were previously missing in figures were reference errors and intended to point to panels in the original set of figures. We have carefully checked figure references in the revised manuscript.As an additional point, on re-evaluation of the data in response to reviewer comments, we also noticed that two cell types (A09a and A09c) in the L3v had axons that were truncated by the volume boundary. As a result, we removed them from analysis that required a complete count of the cable length or synapse count . While s"}
+{"text": "To screen susceptibility loci for ankylosing spondylitis (AS) using an affected-only linkage analysis based on high-density single nucleotide polymorphisms (SNPs) in a genome-wide manner.AS patients from ten families with Cantonese origin of China were enrolled in the study. Blood samples were genotyped using genomic DNA derived from peripheral blood leukocytes by Illumina HumanHap 610-Quad SNP Chip. Genotype data were generated using the Illumina BeadStudio 3.2 software. PLINK package was used to remove non-autosomal SNPs and to further eliminate markers of typing errors. An affected-only linkage analysis was carried out using both non-parametric and parametric linkage analyses, as implemented in MERLIN.Seventy-eight AS patients  were enrolled in the study. The mean age of onset was 23\u00b110 years and mean duration of disease was 16.7\u00b112.2 years. Iritis , dactylitis , hip joint involvement , peripheral arthritis , inflammatory back pain (IBP)  and HLA-B27 positivity  were observed in these patients. Using non-parameter linkage analysis, we found one susceptibility locus for AS, IBP and HLA-B27 in 6p21 respectively, spanning about 13.5Mb, 20.9Mb and 21.2Mb, respectively No significant results were found in the other clinical trait groups including dactylitis, hip involved and arthritis. The identical susceptibility locus region spanning above 9.44Mb was detected in AS IBP and HLA-B27 by the parametric linkage analysis.Our genome-wide SNP linkage analysis in ten families with ankylosing spondylitis suggests a susceptibility locus on 6p21 in AS, which is a risk locus for IBP in AS patients. Ankylosing spondylitis (AS) is a subtype of spondyloarthritis (SpA), a group of inflammatory axial and peripheral joint diseases. Although AS is a hereditary disease, some environmental factors have also been shown to be involved in the pathogenesis of the disease ; 2. In aIn recent years, genome-wide single nucleotide polymorphism (SNP) scan has become a popular approach for detecting new genetic variants in diseases including AS \u20139. For eA recent linkage analysis by microsatellites suggested that AS is an autosomal dominant disease in some large pedigrees with a penetrance of 0.56 in risk allele carriers. Accordingly, a susceptibility locus on chromosome 2q36.1-2q36.3 was identified later on . AlthougFour hundred and one subjects from ten Chinese Han ethnic families were surveyed by experts in AS face to face. AS was diagnosed according to the modified New York criteria for AS . PatientGenomic DNAs were isolated from whole blood using a commercial DNA extraction kit (Qiagen). DNA was diluted to a concentration of 50 ng/ml (in 10mM Tris/1mM EDTA). Each sample was genotyped using approximately 200 ng of genomic DNA and Illumina HumanHap 610-Quad SNP Chip according to the recommended manufacture\u2019s procedures . The Illumina HumanHap 610-Quad beadchip consists of a panel of 620, 901 SNPs, among which 576, 112 are autosomal SNPs.2 is greater than 0.3. A total of 117 158 SNPs were included in linkage analysis ultimately.Genotype data were generated using Illumina BeadStudio 3.2 software. PLINK package was used Firstly, for disease (AS) trait, we conducted an affected-only non-parametric and parametric linkage analysis in all 10 families. For parametric linkage analysis, MERLIN software was implemented ; 19. An PLINK package was used for general statistical analysis. P value < 0.05 was considered to be statistically significant.Among the 78 patients, the mean age of onset was 23\u00b110 years and the mean duration of disease was 16.7\u00b112.2 years. Iritis , dactylitis , hip joint involvement , peripheral arthritis , inflammatory back pain  and HLA-B27 positivity  were observed in these patients.  Data wasUsing non-parameter affected-only linkage analysis for AS disease, a susceptibility locus was found on 6p21, spanning a 13.5 Mb region (from 22296903 to 35831395) where the LOD scores were above 3 with the highest one reaching 5.164. No other locus was detected with LOD > 3 in other chromosomes Figs , Table 2A similar result was obtained in HLA-B27 trait by non-parameter linkage analysis. A susceptibility locus on 6p21 was identified, spanning a 21.2 Mb region (from 20334049 to 41550354) where the LOD scores were above 3 with the highest one reaching6.141. No other locus was detected with LOD score > 3 in other chromosomes , Table 2No locus with LOD score > 3 was detected in any chromosome in the non-parameter linkage analysis for other clinical trait groups including dactylitis, hip involved and arthritis.The above identified susceptibility loci on 6p21 were verified by a parametric linkage analysis using an autosomal dominant model, with a LOD score above 3 . SimilarTo the best of our knowledge, this study is the first one that used high-density single nucleotide polymorphisms (SNPs) to screen susceptibility loci for AS with an affected-only linkage analysis in ten AS families and validated the susceptibility locus with an association analysis between sporadic AS patients and healthy controls in the same array. In previous studies, microsatellite marker was the most common method for linkage analysis in the study of genetic diseases, especially in AS families ; 13; 15.Secondly, the significant heritability of IBP and HLA-B27 was identified in the current study. For the first time, we confirmed the risk locus for IBP in AS patients by linkage analysis, demonstrating that the susceptibility locus on 6p21 was linked to IBP trait in AS. However, the possible susceptibility gene(s) for IBP on 6p21 remains unknown and are needed to be further explored. Previous studies pointed out that IBP was a frequent clinical trait of AS and manifFurthermore, only a small proportion of B27-positive Caucasians develop AS , indicatIn conclusion, our genome-wide SNP linkage analysis in ten families with ankylosing spondylitis suggests a susceptibility locus on 6p21 for AS, which is a risk locus for IBP in AS patients.S1 File(DOCX)Click here for additional data file.S2 File(DOCX)Click here for additional data file."}
+{"text": "It has long been recognized that there is an association between enlarged head circumference (HC) and autism spectrum disorder (ASD), but the genetics of HC in ASD is not well understood. In order to investigate the genetic underpinning of HC in ASD, we undertook a genome-wide linkage study of HC followed by linkage signal targeted association among a sample of 67 extended pedigrees with ASD.HC measurements on members of 67 multiplex ASD extended pedigrees were used as a quantitative trait in a genome-wide linkage analysis. The Illumina 6K SNP linkage panel was used, and analyses were carried out using the SOLAR implemented variance components model. Loci identified in this way formed the target for subsequent association analysis using the Illumina OmniExpress chip and imputed genotypes. A modification of the qTDT was used as implemented in SOLAR.p\u2009=\u20091.72E\u221207).We identified a linkage signal spanning 6p21.31 to 6p22.2 (maximum LOD\u2009=\u20093.4). Although targeted association did not find evidence of association with any SNP overall, in one family with the strongest evidence of linkage, there was evidence for association  contains supplementary material, which is available to authorized users. PTEN  [Autism spectrum disorder (ASD) is characterized by phenotypic and genetic heterogeneity, and an association with abnormal brain growth has long been recognized. For example, there is an association between ASD and specific Mendelian disorders, such as Rett\u2019s syndrome , a microdeletion syndrome at 16p11.2  [cephaly) \u20135. Crosscephaly) , 5. In acephaly)  and therHMGA2 (12q15) and CRHR1 (17q21). None of these loci, however, overlap previously identified ASD genes.Beyond the known rare single gene associations for ASD, little is known about the genetic architecture of HC variance in ASD. For the population more generally, evidence from genome-wide association (GWA) studies indicates loci at 12q15 and 12q24 are associated with infant 6\u201318\u00a0months) HC  while va\u00a0months HUnderstanding the genetic architecture of abnormal brain growth in ASD may shed light on the pathogenesis of ASD, as well as identifying new ASD genes and those involved in brain development more generally. With this in mind, we examined the genetic underpinning of HC using QTL-based genome-wide linkage combined with targeted association analysis. HC has already been identified as a highly heritable trait , and so N\u2009=\u2009198) or without (N\u2009=\u2009469) ASD, had data on HC from at least one time point, as well as height and genotypic data. Diagnoses of ASD were made according to a combination of ADI-R and ADOS assessments [Families were part of the Utah collection of multiplex ASD families (the \u2018Utah sample\u2019 ). SubjecOne thousand three hundred ninety-seven multiplex ASD families that were part of the Autism Genome Project (AGP) provided an opportunity for replication. This consortium of international researchers comprised scientists from ~50 centers in North America and Europe (the \u2018AGP sample\u2019). From the complete sample, subjects from the Utah discovery sample were excluded, and of the remaining families, only those with available HC data were included. This final sample comprised 973 families among which 1041 individuals  had both HC and genotype data. For each site, diagnosis was based on the ADI-R or ADOS or best clinical estimate as described previously [In both samples, HC was measured from the occipital protuberance to the forehead using standardized procedure. If multiple measurements were available on any one individual, one was selected randomly. Reliability of HC measurements was established across raters at the Utah site; intraclass correlations for reliability measurements were >0.95. Additionally, reliability was established across sites participating in AGP (within- and across-site intraclass correlations were >0.90). Data were visualized graphically to identify the distributional characteristics and presence of potential outliers. Height was measured on all participants using a stadiometer.r2 threshold of 0.5  which removed 1207 SNPs. As part of the validation procedure, 115 SNPs with a minor allele frequency (MAF) of <0.1 were removed and 4 SNPs that were not in Hardy-Weinberg Equilibrium  using genotype data from founders. The total number of SNPs left at this stage was 4718. AGP sample: Genotypes were obtained using the Affymetrix  10K SNP arrays at the Translational Genomics Research Institute. A total of 5371 tagSNPs were selected having removed those in strong linkage equilibrium with each other (maximum D\u2019\u2009=\u20090.6), those not in HWE, and those with MAF\u2009<\u20090.1 were also removed [Utah sample: Genotyping was provided by the Center for Inherited Disease Research (CIDR) using the Illumina 6K SNP linkage panel. Methods and quality control procedures have been described in detail previously . After qp\u2009<\u20090.05, samples with <95% call rate, SNPs with <97% call rate), of which 2647 were shared with the 6K chip. As not all members of families had OE genotypes, but most did have the sparser set of 6K SNPs, we used a family imputation approach as implemented in GIGI (v1.06.1) [We obtained additional Illumina OmniExpress (OE) data on 335 members from 14 of the 67 Utah families. These 14 families comprise most of the extended families having three or more generations. In total, 716,503 SNPs passed QC (removed: SNPs with HWE v1.06.1)  to imputN\u2009=\u2009505) were also genotyped using the Illumina HumanCoreExome chip, providing dense SNP genotypes across the genome.Many individuals from the Utah sample  in conjunction with genome build hg38. QTL analysis on LD pruned SNP data was then performed separately for each dataset, and then using the datasets combined, using SOLAR v7.6.4 [2 and height as covariates. Multipoint estimates of IBD sharing using MCMC methods were computed using LOKI v2.3 [Targeted association: The qTLD test [R v3.2.2 . Linkageetic map . Base paR v7.6.4 , with daR v7.6.4 . SOLAR iOKI v2.3  and thenTLD test , as implTLD test , is a moTLD test . In thisp\u2009=\u20090.05/number of SNPs). Investigation of pattern of family-specific variant segregation: Illumina HumanCoreExome data were formatted as a vcf file and imported into GEMINI (v0.18) [In our analysis, HC was specified as the trait of interest. Targeted association was performed for a region defined as -1-LOD around signals that were significantly indicative of linkage as indicated by a genome-wide LOD score of >3.3. We first ran the analysis for the complete sample and then again using only the family contributing most strongly to the linkage signal. As our association analyses were not genome-wide, we calculated level of significance using the conservative Bonferroni adjustment ( (v0.18)  for analThe 67 Utah pedigrees comprised 667 individuals who had both genotype and phenotype information . This region comprised 10,817 OE SNPs, and we therefore set our Bonferroni corrected p values of 4.3E\u221205 (rs9295654) and 6.9E\u221205 (rs2690129). Other SNPs with marginal evidence of association are summarized in Table\u00a0p\u2009=\u20091.72E\u221207, Additional file No evidence of association was observed for HC using the complete sample  and rs1367731 .Three variants were shared among the five members of Family 1 with HC >1.88 SD from the mean. None were exonic however. These included rs1076829  a 2 and height. One family accounted for much of the linkage evidence. These signals were neither identified in our non-overlapping replication sample, nor in the combined discovery and replication sample, although nearby \u2018suggestive\u2019 loci were identified. Additionally, in our replication sample, several other loci were identified with \u2018suggestive\u2019 evidence of linkage.The aim of this study was to identify genetic loci for HC in families segregating ASD. In the Utah families, one locus with two signals was identified with significant evidence of linkage to HC residualized for the effects of sex, age, ageBy also carrying out linkage-signal targeted association, we were also able to identify an allele of one SNP associated with HC in the one family driving the linkage signal. This combination of linkage and targeted association is an attractive strategy for the identification of familial segregating genetic risk for complex disorders. Although we also had the opportunity to examine allele sharing in this family by way of available Illumina HumanCoreExome data, only three SNPs demonstrated minor alleles segregating among individuals with HC\u2009>\u20091.88 SD from the mean, and none were in coding regions.None of the linkage signals in our study overlapped the population-level GWAS association results for HC at 18\u00a0months of age  or for iSimilarly, none of these linkage peaks overlapped those demonstrated in previous studies of these same samples using autism and related phenotypes , 21, 22.The fact that our linkage signals did not overlap those for ASD in the same samples needs some explanation, as this does not support an etiological relationship between HC and ASD in these families. On the one hand, much variation in HC was seen from family to family, with some families segregating larger heads. Among such families, therefore, there may be a more intimate relationship between the aetiological factors for ASD and head size. However, even for the most significantly linked family, no overlap was seen for ASD and HC linkage signals. This does not, of course, rule out the possibility that more than one genetic mechanism, acting in tandem, is involved in the expression of the ASD phenotype. For example, a combination of one locus, influencing brain size, and another, influencing some other brain mechanism, could raise vulnerability to ASD. Additionally, power is low in both analysis, and so false negatives are highly likely.NRSN1 and DCDC2, and both are of potential interest. NRSN1 codes for a protein involved in nerve growth and has a possible role in neurite extension [DCDC2 is a highly brain expressed gene with a role in neuronal migration [Although 6p21.31 is a gene-rich region, our associated SNP does not overlap any expressed or regulatory elements. The most proximal genes are xtension . Associaxtension . Similarigration  and withigration . The widigration , schizopigration  and specigration . There iigration .While confounding any simple explanation for the possibility of shared genes underlying HC and ASD in these families, the study does illustrate the potential utility of the family design in targeting genetics of complex phenotypes, as well as the importance of considering a family-by-family as well as pooled approach. The identification of linkage signals for HC also raises the ongoing need to consider HC as a biomarker for brain growth that may inform the search for genes and regulatory elements that harbor susceptibility to ASD and other developmental disorders of brain growth.NRSN1 and DCDC2; both genes are potential candidates for brain growth. Head circumference is a marker of brain growth and represents an important, easily measurable trait for future studies of the genetics of neurodevelopmental disorders.Head circumference, as an index of brain growth, was found to be linked to a chromosomal region at 6p21.31 that neither overlaps with any previously identified ASD loci, nor linkage signals for ASD and ASD-related phenotypes in these same families. This signal was found to be principally driven by one family, and by further investigation of this family by way of linkage signal-targeted association, a significant association signal was identified near to"}
+{"text": "Fragaria \u00d7\u00a0ananassa) populations with the goal of evaluating this technique in a species with a complex octoploid genome. GBS sequence data were aligned to the F. vesca \u2018Fvb\u2019 reference genome in order to call SNPs. Numbers of polymorphic SNPs per population ranged from 1,163 to 3,190. Linkage maps consisting of 30\u201365 linkage groups were produced from the SNP sets derived from each parent. The linkage groups covered 99% of the Fvb reference genome, with three to seven linkage groups from a given parent aligned to any particular chromosome. A phylogenetic analysis performed using the POLiMAPS pipeline revealed linkage groups that were most similar to ancestral species F. vesca for each chromosome. Linkage groups that were most similar to a second ancestral species, F. iinumae, were only resolved for Fvb 4. The quantity of missing data and heterogeneity in genome coverage inherent in GBS complicated the analysis, but POLiMAPS resolved F. \u00d7\u00a0ananassa chromosomal regions derived from diploid ancestor F. vesca.Genotyping-by-sequencing (GBS) was used to survey genome-wide single-nucleotide polymorphisms (SNPs) in three biparental strawberry ( Genotyping-by-sequencing (GBS) is a powerful, cost-effective method for identifying single-nucleotide polymorphisms (SNPs) on a whole-genome scale. The GBS technique commonly used involves a form of reduced representation genome sequencing based on partial restriction enzyme digestion, usually with a methylation-sensitive restriction enzyme, followed by barcoded adaptor ligation and next-generation sequencing of highly multiplexed samples, typically 48, 96, or 384 samples per lane . ApplicaFragaria consists of 20 species that range in ploidy from diploid to decaploid , having arisen in Europe in the 18th century from hybridization between two octoploids: North American F.\u00a0virginiana Mill. and South American F. chiloensis (L.) Duchesne ex Weston  test the efficacy of the POLiMAPS pipeline in resolving sub-genome contributions from the ancestral diploid Fragaria species.The objectives of this study were to (1) evaluate the utility of GBS by developing linkage maps for three bi-parental 3V 1420 Multilabel Counter . The DNA concentration was adjusted to 100 ng/\u03bcL per sample for subsequent genotyping by sequencing (GBS) library preparation.The strawberry samples analyzed in this study consisted of: parents and 24 offspring from the \u2018Holiday\u2019 \u00d7 \u2018Korona\u2019 population from the Netherlands ; parents\u00ae fluorometer , checked for adequate size distribution (150\u2013350 bp) with the Bioanalyzer 2100 HS-DNA chip , and sequenced with the Illumina HiSeq2000 .Preliminary testing with three restriction enzymes  for fragment size range led to selection of ApeKI for GBS library construction. Three GBS libraries were constructed at the USDA-ARS National Clonal Germplasm Repository (NCGR) and one was constructed at Clemson University according to the procedure previously described  for 96 sFvb genome assembly (https://github.com/listonlab/POLiMAPS). POLiMAPS identifies markers with approximately Mendelian segregation by requiring a minimum number of offspring displaying each of the two possible genotypes . It also sets a maximum value for number of offspring with missing genotypes . Default parameters were used with the following exceptions. Because there were relatively few offspring in the \u2018Holiday\u2019 \u00d7 \u2018Korona\u2019 cross (24), we decreased \u2212o to 6. Conversely, because there were more offspring in \u2018Redgauntlet\u2019 \u00d7 \u2018Hapil\u2019 (51) and \u2018Tribute\u2019 \u00d7 \u2018Honeoye\u2019 (63), we increased \u2212m to 4 for \u2018Redgauntlet\u2019 \u00d7 \u2018Hapil\u2019 and to 5 for \u2018Tribute\u2019 \u00d7 \u2018Honeoye\u2019.Adapter sequences were removed at the sequencing facilities prior to our receiving the data. Reads were quality-filtered by converting to \u2018N\u2019 sites with Phred-scaled quality scores lower than 20, and by excluding reads with fewer than 50 retained  sites. SNPs were called using the POLiMAPS pipeline . In brieassembly  using BWassembly . SAMtoolassembly  to generSNPs that were segregating in both parents were excluded from linkage mapping, as tri-or quad-allelic markers were expected to be rare, and difficult to distinguish from sequencing errors. Segregating loci were organized into parental sets, which were subjected to linear regression mapping using JoinMap\u00ae v. 4.1 . A minimFragaria  and the F. \u00d7 ananassa linkage groups which aligned to the homologous chromosome and had at least 300 phylogenetic sites were included. The objective of this analysis was to identify the LG from each population that belonged to each of the sub-genomes. When more than four linkage groups per parental map were found, an attempt was made to merge some of the linkage groups in each parental map using the following criteria: (1) continuous marker position on the Fvb reference; (2) a chi-squared (\u03c72) test supporting linkage between the last SNP at the end of one linkage group and the first SNP at the beginning of another linkage group (P\u00a0<\u00a00.05); (3) similar phylogenetic position of the two linkage groups; and (4) strongest cross-link (SCL) metric between JoinMap groups.Dendrograms were constructed for each linkage group using the genetic information for each cultivar and the diploid congeners following the previously described POLiMAPS approach for octoploid Fragaria . This meomosomes . Two rouFragaria comparators F.\u00a0nipponica and F.\u00a0bucharica, and with Rubus coreanus to serve as an outgroup. The minimum number of phylogenetic sites per JoinMap linkage group was lowered to 150 for all parents and sites were allowed to be missing in any of the diploids.Once some of the LGs were merged, the phylogenetic analysis was repeated, with the additional Fvb assembly to visualize genetic rearrangements between F.\u00a0vesca ssp. bracteata and F. \u00d7 ananassa.An integrated linkage map for \u2018Holiday\u2019 chromosome 6D was created utilizing the 90 K Axiom data  and the Fvb reference genome ranged from 992,053 (\u2018Hapil\u2019) to 1,754,838  to 65 (\u2018Holiday\u2019). Over all three populations, there were 178 JoinMap groups, with 46 (26%) consisting of fewer than ten SNPs, and 25 with gaps of 15 cM or larger. For a given chromosome, the number of aligned linkage map groups ranged from three to seven , Table 2Fragaria species, there was a median of 8% missing data in the phylogenetic character matrix , and for linkage groups there was a median of 95% missing data in the phylogenetic character matrix . The resulting trees\u2013one for each chromosome\u2013were examined to identify JoinMap groups that could potentially be merged according to proximity on a tree. Merging reduced the overall number of chromosome-aligned groups by six or fewer for \u2018Tribute\u2019 (from 29 to 27), \u2018Honeoye\u2019 (from 33 to 28), \u2018Redgauntlet\u2019 (from 39 to 34), and \u2018Hapil\u2019 , and F. iinumae (Bi). The remaining two sub-genomes, B1 and B2, could not be distinguished and were un-assigned and referred to as B12. The Av sub-genome corresponds to sub-genome A of F. vesca as A, B, C and D, in order of most to least divergence.GBS and related reduced-representation sequencing methods have recently been used to study  iinumae , and F. ananassa . Both oflueberry  and red lueberry . The POLlueberry  was usedF. iinumae and F. \u00d7 ananassa in the aforementioned experiments. However, the numbers of polymorphic SNPs used for mapping varied widely among the three analyzed populations. The \u2018Redgauntlet\u2019 \u00d7 \u2018Hapil\u2019 population had by far the greatest number of polymorphic SNPs, with 1,253 and 1,096 derived from each parental genotype, while the \u2018Holiday\u2019 \u00d7 \u2018Korona\u2019 population had 735 and 1,096, and the \u2018Tribute\u2019 \u00d7 \u2018Honeoye\u2019 population had 358 and 468 SNPs. Relative levels of homozygosity may account for this variability. The number of restriction enzymes employed, choice of restriction enzymes, and sequence coverage depth per individual over the sampled portion of the genome can also affect the number of polymorphic SNPs detected . Homozygous genome regions can account for large gaps in linkage maps, and the fragmented linkage groups can be attributed to heterogeneity in genome coverage of GBS data, which results in an overall high quantity of missing data. Low marker density and large gaps can also result from conserved chromosomal regions with high homozygosity. \u2018Holiday\u2019 and \u2018Korona\u2019 have common ancestors in their pedigrees, making regions of shared homozygosity more likely in that population (Many of the JoinMap groups that aligned to the pulation . A studypulation , however\u00ae IStraw90 Axiom Array, the number of SNPs mapped were >6,500 in \u2018Holiday\u2019 \u00d7 \u2018Korona\u2019 (F. iinumae (F. vesca (Fragaria species. Cost per sample for GBS is considered an advantage over array-based genotyping. However, the availability of the Axiom\u00ae IStraw35 384HT array at a cost of $50 per sample has decreased the price of high throughput genotyping which now is closer to that of GBS. The IStraw35 array is expected to be just as useful as the IStraw90 for linkage mapping in F. \u00d7 ananassa but its usefulness in other species awaits further evaluation. However, since SNPs in both arrays targeted SNPs in the octoploid domestic strawberry and were enriched for the F. vesca sub-genome (The numbers of SNPs mapped by GBS in this study ranged from 2,663 in the \u2018Tribute\u2019 \u00d7 \u2018Honeoye\u2019 population to 3,912 in \u2018Redgauntlet\u2019 \u00d7 \u2018Hapil\u2019. When using the Axiom\u2018Korona\u2019 , >8,400 \u2018Korona\u2019 , and 11,\u2018Korona\u2019 . At this iinumae , and in F. vesca , but hasb-genome  the arraFragaria species has reported synteny and high colinearity along chromosomes (Fragaria species in the phylogenetic analysis increased the robustness of the F. vesca clade. We expected all sub-genomes to have numerous regions sufficiently similar to the reference genome to produce correctly-aligning reads, given the previous success of this alignment pipeline (F. vesca sub-genome aligned given its higher similarity to the reference genome, leading to an enrichment for F. vesca sub-genome markers in our analysis. Because one of our goals was to connect linkage groups with Fvb chromosomes, we prioritized aligning reads with high confidence to the reference genome, and discarded reads that could not align given the BWA \u2212n 0.001 parameter. Other analyses of GBS data in Fragaria may seek to maximize the number of markers even if their reference genome position is less certain. When high-quality assemblies of the F. \u00d7 ananassa sub-genomes, and of other Fragaria diploids, become available, the issue of aligning divergent reads will be less of a concern. Overall, the greatest number of SNPs were assigned to the F. vesca sub-genome. Each parental genotype had linkage groups that could be assigned to the F. vesca sub-genome on chromosome Fvb 1, and all parental genotypes except for \u2018Holiday\u2019 had F. vesca-like groups on chromosomes Fvb 2 and Fvb 7. Fvb 4 was the only chromosome for which all parental genotypes had linkage groups that could be assigned to the F. iinumae sub-genome, however, most of the linkage groups from parental genotypes could not be assigned to any sub-genome. These results are consistent with the findings of F. \u00d7ananassa assembly to F. vesca pseudomolecules, and concluded that approximately 20% of the scaffolds were F. \u00d7 ananassa-specific.The goal of the POLiMAPS phylogenetic analysis was two-fold: First, to resolve homoeologs in cases where more than four JoinMap linkage groups from any crossing parent aligned to the same haploid reference chromosome; and second, to identify ancestral diploid sub-genome contributions. Comparative genomic mapping between octoploid and diploid omosomes . The incpipeline . HoweverFvb 6, comprising over 3Mb and over 700 genes, are included in a linkage group containing markers from across Fvb 3. This gene-dense region of Fvb 6 contains genes and QTLs linked to important traits, such as sex phenotype (One linkage group derived from \u2018Holiday\u2019 aligned to both chromosome 3 and chromosome 6. This region may represent a translocation event. Markers from the distal end of henotype . HoweverF. ananassa chromosomal regions derived from the diploid F. vesca. However, the large number of missing data in GBS experiments, combined with the complex relationships among homoeologs in polyploid plants, complicate such analyses and may limit the usefulness of GBS in these plants. Furthermore, the availability of array-based high throughput genotyping at a reduced cost in the form of the IStraw35 array provides another useful and easy tool for linkage mapping in F. \u00d7 ananassa. Use of an F. \u00d7 ananassa reference sequence for SNP detection and higher coverage of GBS libraries developed after cutting with two restriction endonucleases may address the challenges observed in this study and require further evaluation.In summary, POLiMAPS was employed with genotyping-by-sequencing data in three small families to resolve 10.7717/peerj.3731/supp-1Supplemental Information 1Mapping data for Axiom markers was obtained from Click here for additional data file.10.7717/peerj.3731/supp-2File S1Click here for additional data file.10.7717/peerj.3731/supp-3File S2Click here for additional data file.10.7717/peerj.3731/supp-4File S3Click here for additional data file."}
+{"text": "Dental caries is a common chronic disease among children and adults alike, posing a substantial health burden. Caries is affected by multiple genetic and environmental factors, and prior studies have found that a substantial proportion of caries susceptibility is genetically inherited.PRIM: dichotomized as zero versus one or more affected primary teeth, (2) QTOT1: age-adjusted quantitative caries measure for both primary and permanent dentitions including pre-cavitated lesions, and (3) QTOT2: age-adjusted quantitative caries excluding pre-cavitated lesions. Genotyping was conducted for approximately 600,000 SNPs on an Illumina platform, pruned to 127,511 uncorrelated SNPs for the analyses reported here.To identify such genetic factors, we conducted a genome-wide linkage scan in 464 extended families with 2616 individuals from Iowa, Pennsylvania and West Virginia for three dental caries phenotypes: (1) PRIM, 2.38 at 6q25.3 for QTOT1, and 2.76 at 5q23.3 for QTOT2. Some overlap in linkage regions was observed among the phenotypes. Genes with a potential role in dental caries in the eight chromosomal regions include CACNA1E, LAMC2, ALMS1, STAMBP, GXYLT2, SLC12A2, MEGF10, TMEM181, ARID1B, and, as well as genes in several immune gene families. Our results are also concordant with previous findings from association analyses on chromosomes 11 and 19.Multipoint non-parametric linkage analyses generated peak LOD scores exceeding 2.0 for eight genomic regions, but no LOD scores above 3.0 were observed. The maximum LOD score for each of the three traits was 2.90 at 1q25.3 for These multipoint linkage results provide evidence in favor of novel chromosomal regions, while also supporting earlier association findings for these data. Understanding the genetic etiology of dental caries will allow designing personalized treatment plans based on an individual\u2019s genetic risk of disease.The online version of this article (10.1186/s12903-018-0559-6) contains supplementary material, which is available to authorized users. Dental caries is one of the most common chronic diseases among children and adults alike. Childhood caries is associated with failure to thrive, and it can affect self-esteem and school performance . For botCaries is known to have a genetic component. Detection of genetic factors is complicated by the fact that numerous diverse environmental factors influence the incidence and severity of this disease, including the microbiome, dietary habits, fluoride exposure, salivary factors and tooth structure.Prior studies have shown that caries experience in humans is determined by genetic causes with heritability values between 20 and 60% . To dateOur present study is the first to apply genome-wide multipoint linkage analysis to explore the genetic etiology of caries (whether in childhood or adulthood) using densely spaced SNPs on a population previously analyzed by genome-wide association. Genome-wide linkage analysis is a complementary strategy to genome-wide association analysis for gene-discovery. Whereas association identifies specific marker alleles correlated with the caries phenotype, linkage analysis strategies identify genomic regions shared between related individuals who show similar disease characteristics. The advantage of linkage analysis is that it makes full use of familial inheritance, is less sensitive to allelic heterogeneity, and, unlike association, can be used to detect rare disease-causing mutations. Furthermore, multipoint linkage utilizes genotypes from SNPs neighboring the test location, while association conducts tests at each location independently.In this study, multipoint non-parametric linkage analysis was conducted, i.e., no assumptions were made with respect to the mode of inheritance of dental caries , and thehttps://www.ncbi.nlm.nih.gov/gap; accession number phs000095.v3.p1). Details on genotyping and quality control protocols are also presented on dbGaP, or can be found in earlier studies [The families and individuals included in this study are from western Pennsylvania, West Virginia, and Iowa. Subjects from Pennsylvania and West Virginia were ascertained through the Center for Oral Health Research in Appalachia . Additi studies , 20. TabCaries scores were assessed on the COHRA, IFC, and IHS subjects in accordance with the COHRA study protocol . For subPRIM), and two that combine primary and permanent dentitions . PRIM was coded as a binary primary dentition caries phenotype based on the count of decayed and/or filled primary teeth (dft) score. An individual with a dft score of 1 or more was designated as being affected. The primary teeth from all subjects with primary or mixed dentition were assessed for PRIM. These individuals included adults with over retained primary teeth. QTOT1, an age-adjusted quantitative caries phenotype, is based on the sum of the dft score (primary teeth) and D1MFT score . QTOT1 scores were generated by adjusting this raw sum for age and age-squared effects using locally fitted splines. Scores for 113 individuals below 2\u00a0years of age and 5 individuals above 60\u00a0years, were excluded from linkage analysis due to a very low caries experience in the 0\u20132\u00a0years age group, and the presence of very few subjects above 60\u00a0years of age. Age-adjusted QTOT2, the second quantitative caries phenotype, is based on the sum of the dft score and the D2MFT score . Age-adjustment was performed as for the QTOT1 phenotype; and QTOT2 scores for 115 individuals between 0 and 2\u00a0years of age and 44 individuals above 60\u00a0years were set to missing.We defined three dental caries phenotypes, one based on primary dentition . SNPs with residual LD were clustered into super-markers. The procedures used for filtering SNPs, map creation, and LD-based SNP pruning and clustering are described below.The Genetic Map Interpolator (GMI) program,  was used\u2212\u20095 for rejecting the null hypothesis of no deviation from HWE proportions.In addition to the quality control and cleaning steps detailed on dbGap, we filtered SNPs on the basis of low genotyping success rate and deviation from Hardy-Weinberg equilibrium (HWE) proportions using the software PLINK . SNPs wi2 (a measure of LD based on the square of the correlation coefficient between loci) value among remaining SNPs fell below 20%. In PLINK, LD pruning consists of creating blocks of 50 consecutive SNPs followed by recursive removal of SNPs within blocks, until the LD r2 value among the remaining SNPs is below the desired threshold (20% in our case). Only the unrelated genotyped individuals in our data \u2013 pedigree founders and unrelated cases/controls -- were used to calculate LD in this step. Any remaining LD was then accounted for using LD-based clustering in Merlin [2 value greater than a specified threshold (in our case 10%), is analyzed collectively as a super-marker.The genotyping panel available to this study was designed for genome-wide association analysis. When conducting linkage analysis on densely spaced SNP marker loci, the presence of substantial marker-to-marker LD is known to inflate linkage signals, especially if parental genotypes are missing . In thisn Merlin . In clus2\u2009\u2264\u200920%) were retained. LD-based SNP clustering combined 92,495 SNPs into 20,634 super-markers, leaving 35,016 SNPs to be analyzed individually. The average genetic map distance between the final set of markers (super-marker index and singleton SNPs) is approximately 0.07\u00a0cM on the autosomes and 0.13\u00a0cM on the X-chromosome. Super-marker and singleton SNP allele frequencies were generated as maximum likelihood estimates using Merlin. The SNP clustering and allele frequency estimation steps utilized 1022 informative families.Table\u00a0Table PRIM phenotype using the SAll statistic [In non-parametric linkage (NPL) analysis, affected individuals within each pedigree are examined to detect whether affected relatives share genomic regions identical-by-descent (IBD) more often than expected due to their relatedness alone. This IBD sharing is tested at locations along each chromosome. Genome-wide NPL analysis was carried out for the tatistic  as impletatistic .DMFT) and sample-based means and variances for QTOT1 and QTOT2. All results, NPL and QT linkage, are reported as LOD (logarithm of the odds of linkage) scores.The quantitative trait (QT) regression-based linkage method, Merlin-regress,  was utilThe most commonly used LOD score threshold of 3.0 used to test for significant linkage (Morton) was derived for parametric linkage analysis of a single locus on a binary trait phenotype. Subsequent research (e.g. those reviewed in ) that adFor super-markers, the NPL and QT LOD scores correspond to the index (first) SNP of each cluster. In the linkage scan for each phenotype, maximizing markers in regions with LOD score\u2009\u2265\u20092.0 were identified as linkage peaks. A support interval of one LOD drop was used for exploring genes under selected linkage peaks. The one LOD drop support interval is the interval where the LOD score is within one unit of its maximum.Regions with LOD scores \u22651.0 were identified for trait. Overlap of linkage signal among the three traits was determined based on overlap of peak support intervals or secondary peaks(s) of at least 1.0 LODs, lying within the primary peak support interval. In the event peaks for multiple phenotypes overlapped, the resulting support intervals were reduced to the region of overlap.Genes within these support intervals were examined for a potential etiologic role in dental caries incidence. Genes identified as causal would include, for example, genes related to blood glucose levels, secretory function of the salivary glands, and host immune response. Proximity of genes to SNPs corresponding to linkage peaks was determined by physical map positions obtained from UCSC Genome Browser corresponding to the March 2006 (NCBI36/hg18) Assembly . When noA systematic search of literature was conducted to compile caries risk-conferring genes and genomic regions from previous studies utilizing some portion of our data, as well as from studies of other populations. Physical positions for these genes and genomic regions were then mapped to our linkage scans. Linkage regions with a LOD score of 1.0 or greater have been reported as indicative of concordance or replication, as appropriate.QTOT1 and QTOT2.For multifactorial diseases such as caries, the true underlying genetic model for disease is difficult to ascertain. In this study, model-free linkage methods were used to detect linkage. The QT methods are sensitive to the misspecification of the required programmatic input values. We conducted a sensitivity analysis for the heritability parameter (HP), since published literature provides a wide range of heritability values (40\u201360%), and our work utilized HP\u2009=\u200950%. In the sensitivity analysis, HP values were set at 40, and 60% for Mega2  was usedPRIM (panels A and B), QTOT1 , and QTOT2 . There were 287 individuals with known PRIM phenotypes (panel A), of which 243 individuals were affected for PRIM. Of these 243 subjects, 242 were 18\u00a0years or younger in age (panel B). Subjects with primary or mixed dentition included in the PRIM NPL analysis ranged from 15\u00a0months to 22.5\u00a0years of age, with a mean of 7.4\u00a0years. These subjects with primary dentition caries constitute mainly the youngest generation. The distribution of the raw caries index by decade, age-adjusted index by decade, and age-adjusted caries index within all phenotyped individuals compared to those between 2 and 60\u00a0years of age are shown for QTOT1  and QTOT2 . The number of phenotyped individuals, range, mean and standard deviation are presented in panel I for both quantitative traits. A larger number of individuals were phenotyped for D2MFT as compared to D1MFT in this study. For QTOT1 and QTOT2, there were 2484, and 2868 phenotyped individuals in the 2\u201360 age range. Both of the age-adjusted phenotypes follow an approximately normal distribution, with a mean of zero. The QTOT1 and QTOT2 mean and standard deviations for the 2\u201360 age group were included as distribution parameters within quantitative trait linkage.Figure\u00a0PRIM, QTOT1, and QTOT2. The empirical 5% genome-wide significance level, indicated as a solid horizontal line, was 3.48, 3.61, and 3.76 for PRIM, QTOT1, and QTOT2, respectively. Overlapping LOD score peaks for multiple phenotypes were observed in a few regions.Figure\u00a0PRIM, 2.38 for QTOT1, and 2.76 for QTOT2. Detailed results for all SNPs that lie within the support region for peaks reported in Table Table\u00a0QTOT2; LOD 2.30) and the other on chromosome 3 , no such etiologic genes were identified within the support intervals. In these intervals, genes BCL11A (60.538\u201360.634\u00a0Mb) and KAT2B (20.056\u201320.171\u00a0Mb) were found to be closest to the respective LOD score peak SNPs. The genes within linkage peak regions that may play an etiologic role in dental caries are described in the sections below.For each linkage peak, the table also reports the closest gene, if found, with a potential role in caries incidence. For two of these peaks, one on chromosome 2 (CACNA1E (179.719\u2013180.037\u00a0Mb) gene has been shown to be involved in glucose-evoked insulin secretion in mice [LAMC2 (181.422\u2013181.481\u00a0Mb) laminin gene are known to cause non-Herlitz form of junctional epidermolysis bullosa, which includes hypodontia and dental caries among its phenotypes [The highest LOD 2.90 across all three traits was observed on chromosome 1 for PRIM Table . Under t in mice . Poor glenotypes .QTOT2 peak on chromosome 2 includes the ALMS1 (73.466\u201373.691\u00a0Mb) gene. Mutations in this gene causes Alstr\u00f6m syndrome, where gingivitis and discolored enamel are two clinical phenotypes [STAMBP (73.910\u201373.944\u00a0Mb) gene have been reported to have cleft palate and facial dysmorphology [The second enotypes . Individrphology .GXYLT2 (73.020\u201373.107\u00a0Mb) gene is located within the chromosome 3 QTOT1 peak. GXYLT2 acts on epidermal growth factor, which is expressed in human submandibular and parotid glands, and important for the maintenance of oroesophageal and gastric tissue.The QTOT2. This QTOT2 peak contains the SLC12A2 (127.447\u2013127.553\u00a0Mb) gene, whose protein product helps the movement of chloride ions in saliva, thereby assisting in salivary function. Also within the support interval are genes from the IL family, which code for cytokines involved in blood production and immune system function. Defects in these genes result in autoimmune diseases and immune deficiency. A third gene, MEGF10 (126.654\u2013126.825\u00a0Mb) has been implicated in MARDD , with cleft palate as an associated phenotype [The highest genome-wide quantitative trait LOD was observed for henotype .TMEM181 (158.877\u2013158.976\u00a0Mb) gene under the QTOT1 linkage peak codes for a putative G-coupled protein receptor which mediates reaction to cytolethal distending toxins secreted by many pathogenic bacteria. ARID1B (157.141\u2013157.572\u00a0Mb) mutations result in mental retardation along with minor teeth anomalies [The nomalies .NLRP, KIR, and LILR immune gene families that code for various receptors within immune cells. NLRP2 (60.170\u201360.204\u00a0Mb), and NLRP7 (60.127\u201360.151\u00a0Mb) were closest to the peak.This region harbors several genes from the Table\u00a0PRIM LOD of 1.23 was observed 8500\u00a0bp from the MPPED2 (30.338\u201330.558\u00a0Mb) gene. A suggestive association of primary teeth caries was reported by a previous study on 1305 children aged 3\u201312, some of whom are also part of this analysis . The phenotype was defined similarly to our PRIM phenotype.A NLRP12 (58.989\u201359.019\u00a0Mb) in 1142 children aged 3\u201313, a subset of whom are also included in our study. QTOT1 and QTOT2 LOD scores \u22651.0 were observed 0.4\u20131.2\u00a0Mb from this gene.A gene-set enrichment analysis study  reportedQTOT1 and QTOT2, Fig.\u00a0QTOT1. For QTOT2, the corresponding range is \u2212\u20090.05 to 0.1 LOD. Although in percentage terms they represent exponential changes as compared to the baseline, none of the deviations in the 0 to 0.5 baseline LOD score range result in the LOD score approaching significance. Panels E and F break down for each trait, the percentage of all SNPs that drop below\u2014or exceed\u2014the 2.0 LOD score threshold with a change in HP. For both traits, a decrease in HP to 40% results in a minimal percentage of SNPs changing status (be it an increase or decrease in LOD score). In contrast, SNPs with LOD scores of 2.0 or greater at HP 50% are more likely to drop below the 2.0 LOD threshold when the HP is increased to 60%.For each of the two quantitative traits, QTOT1 and QTOT2 LOD scores due to a change in HP for only the linkage peaks reported in Table Table\u00a0To our knowledge, this study was the first to apply genome-wide multipoint linkage analysis to explore the genetic etiology of caries using densely spaced SNPs.We defined two new quantitative phenotypes which combine childhood and adulthood caries indices while also accounting for variability by age. The linkage findings in this study nominated genes on six chromosomes  with potential involvement in caries etiology. Some of the genes are known to cause syndromes with a dental or oral phenotype, while others have a role to play in human immune and host defense response, blood glucose levels, and secretory function of the salivary glands all of which may have a potential impact on incidence of dental caries  for the relationship between diabetes and dental caries). After a comprehensive review of the literature, we also detected linkage to regions on chromosomes 11 and 19 previously reported as associated to caries.As expected, we do not recapitulate all findings from all prior association studies published by our group although this linkage study and the previous association studies utilized data from the same sources . As mentioned previously, linkage and association are complementary strategies for gene-discovery. In linkage, similarities and differences in pairs of phenotypes are modeled in terms of genetic similarity over related pairs from families. In association, this modeling is performed at the level of individuals. Our linkage uses multi-point analysis, i.e., the LOD score at any specific location is influenced by linkage at neighboring loci. Association generally uses a set of independent one-locus tests. Finally, as described in methods, this study differs from prior published work, both in the number, and the type (in terms of family composition) of individuals included in the analysis. Linkage utilizes family data and all related pairs (affected or phenotyped) within a pedigree whereas association generally is conducted on unrelated cases and controls, or at most parent-offspring trios.The genotyping panel was designed for association analyses, and therefore, is far denser than a linkage SNP panel. Although dense bi-allelic SNP panels may allow extraction of more information, a concern for this study was existing linkage disequilibrium between SNPs. We pruned SNPs based on marker-to-marker LD, and then exploited any remaining LD among the pruned set to create clusters which served as polymorphic markers. Despite the pruning and clustering, our analysis was conducted on a much denser set of markers  compared to a typical linkage panel with 6000 SNPs. The use of multi-allelic super-markers also had the potential of increasing power of linkage studies in such a setting.Genome-wide significance for each phenotype was empirically assessed through a series of simulations, which provides an approximation of the true underlying distribution of a statistic since not all features of the data can be completely replicated. In an exploratory study, adhering strictly to genome-wide significance thresholds may be overly conservative. Furthermore, of the 4727 subjects, only 2616 contributed to the linkage analysis, providing a comparatively small number of relative pairs given the large sample size.The sensitivity analysis conducted for the parameter HP explores the impact of parameter value selection on a model-free QT method. The results from this analysis indicated that the non-parametric quantitative trait linkage method, as implemented in Merlin, was robust to variation in HP, and that changing the HP parameter had a minimal impact on LOD scores. Even more importantly, the linkage peaks were insensitive to parameter misspecification.Environmental factors are not accounted for in this study due to unavailability of such data on many of our subjects, which would have drastically reduced the cohort size. We also did not attempt to analyze gene-by-gene interaction. The available methods for detection of gene-gene interaction that are applicable to our study design are computationally complex, thus making whole-genome interaction analysis beyond the scope of the current work (e.g. see the review of the various classes of interaction detection methods by Li ).This study presents two new quantitative measures for dental caries which combine both the primary and permanent dentition, while adjusting for age effects. Genes identified in peak linkage regions underline the importance of exploring potential relationships between caries and other traits. We did not include environmental factors in this study. The interaction between putative caries risk conferring genes and factors including fluoride exposure, dietary habits, and the microbiome need to be investigated, as do interactions between the genes themselves. From a clinical perspective, individuals would be at an elevated lifetime risk of developing caries in both primary and permanent dentition, given increased genetic susceptibility. Understanding the genetic etiology of dental caries will allow health providers to design personalized treatment plans based on an individual\u2019s genetic risk of disease.Additional file 1:. Detailed results for all SNPs that lie within the support region for peaks with a LOD score of 2.0 or more, as summarized in Table SNPs within support regions of reported peaks"}
+{"text": "Linkage and quantitative trait loci (QTL) maps are critical tools for the study of the genetic basis of complex traits. With the advances in sequencing technology over the past decade, linkage map densities have been increasing dramatically, while the visualization tools have not kept pace. LinkageMapView is a free add-on package written in R that produces high resolution, publication-ready visualizations of linkage and QTL maps. While there is software available to generate linkage map graphics, none are freely available, produce publication quality figures, are open source and can run on all platforms. LinkageMapView can be integrated into map building pipelines as it seamlessly incorporates output from R/qtl and also accepts simple text or comma delimited files. There are numerous options within the package to build highly customizable maps, allow for linkage group comparisons, and annotate QTL regions.https://cran.r-project.org/web/packages/LinkageMapView/ Linkage studies over the years have been producing genetic maps (linkage maps) that act as invaluable tools in areas of genetic disease detection, anchoring genome sequence assemblies and elucidating genetic mechanisms of agronomical important traits in plants. The advent of high-throughput sequencing has allowed for better marker identification and greater map densities. This is a desirable outcome for solving many genetics based problems but visualizing these maps creates a new challenge. As the marker density increases, it becomes difficult to add labels, QTL regions and centimorgan values and still maintain readability.There is existing software that generates linkage and QTL maps and these fall into three categories. Rudimentary maps are available in software where the primary objective is QTL analysis. For example, R/qtl . MapCharLinkageMapView uses R base graphics for plotting, labels and optional colored segments. The main function, lmv.linkage.plot, is the only function a user needs to directly invoke. The generated maps are output in Adobe Portable Document Format (PDF). Because linkage maps can be dense with loci, LinkageMapView has many options that allow the user to reduce extraneous detail, minimize map dimensions, and highlight areas of importance.Input to LinkageMapView is a delimited text file with the first three columns containing linkage group name, cM position, and locus. Alternatively, input can be an R/qtl , loci with the same name and in adjacent linkage groups will be connected with a line.conndf: An optional data frame containing the homologs to connect with a line. If autoconnadj is TRUE, this list will be merged with the automatic list.revthese: An optional vector of linkage group names to reverse. The end position becomes position 0 and position 0 becomes the largest position.posonleft: An optional boolean vector the length of the number linkage groups to map, indicating if the positions should be plotted on the left (TRUE) or on the right (FALSE).The maps from two use cases are shown in The first case  demonstrListing 1. Sample commands to generate linkage group map from R/qtl cross object.library(qtl)data(hyper)lmv.linkage.plot)The second case  demonstr-density linkage maps. These characteristics make LinkageMapView more useful than currently available linkage map plotting tools.LinkageMapView is a freely available and open source tool to produce publication quality linkage and QTL maps. It is implemented in R to provide easy integration with R QTL analysis programs and so it can be run on any platform. The plethora of options provides the user with flexibility in dealing with high"}
+{"text": "Verbal trait disorders encompass a wide range of conditions and are marked by deficits in five domains that impair a person\u2019s ability to communicate: speech, language, reading, spelling, and writing. Nonword repetition is a robust endophenotype for verbal trait disorders that is sensitive to cognitive processes critical to verbal development, including auditory processing, phonological working memory, and motor planning and programming. In the present study, we present a six-generation extended pedigree with a history of verbal trait disorders. Using genome-wide multipoint variance component linkage analysis of nonword repetition, we identified a region spanning chromosome 13q14\u2013q21 with LOD\u00a0=\u00a04.45 between 52 and 55\u00a0cM, spanning approximately 5.5\u00a0Mb on chromosome 13. This region overlaps with SLI3, a locus implicated in reading disability in families with a history of specific language impairment. Our study of a large multigenerational family with verbal trait disorders further implicates the SLI3 region in verbal trait disorders. Future studies will further refine the specific causal genetic factors in this locus on chromosome 13q that contribute to language traits.The online version of this article (doi:10.1007/s00439-016-1717-z) contains supplementary material, which is available to authorized users. Verbal trait disorders are comorbid, developmentally associated disorders and deficits in communication. These include clinical and subclinical disorders of speech, language, reading, spelling, and writing , which loads onto several cognitive processes critical for language-related ability including auditory processing, receptive language ability, and motor planning and programming  it is a robust endophenotype for verbal trait disorders ; (2) is highly heritable; (3) has a Mendelian model of inheritance . All subjects were assessed by one of two experienced examiners in the participants\u2019 homes or hotel sites in five states within the continental US. All oral instructions and audio-recorded stimuli were presented at comfortable listening levels based on findings from a conventional hearing screening. The assessment protocol included the following measures and instruments: Kaufman Brief Intelligence Test-2  and the vowel/\u0251/Shriberg . This NWStudies of speech-language disorders using the NRT have supported its validity and reliability  was defined as performing greater than one standard deviation below the mean on either the NRT or SRT. Preliminary studies indicated that a cutoff below one standard deviation on either the NRT or the SRT was maximally sensitive and specific to subjects with only mild, subclinical difficulty in one or more of the five verbal traits based on parent- and self-reported histories of children and adults. Of the 41.9\u00a0% of participants in the present study who met the nonword criteria for a verbal trait disorder (see Table\u00a0Z\u00a0=\u00a0\u22121.82). Significantly fewer females met criteria for VT+ (31.4\u00a0%) than VT\u2212 , but the proportion of males who met criteria for VT+ (55.6\u00a0%) compared to the proportion who met criteria for VT\u2212 (44.4\u00a0%) was non-significant (Z\u00a0=\u00a00.82). Among the four age groups, the only age group within which affection status differed significantly was the school-aged participants, who had a significantly lower percentage of participants who met criteria for VT+ (33.3\u00a0%) than VT\u2212 .Tables\u00a0Z\u00a0=\u00a02.89). Using conventional criteria for statistical significance, the percentage of VT+ participants who had at least one test score or questionnaire entry indicating a concern with any one of the five verbal traits (73.1\u00a0%) was not significantly larger than the percentage of VT\u2212 participants with such histories .Last, Verbal Trait History for problems in verbal trait domains of speech, language, reading, spelling, and/or writing were determined by test scores in any of the relevant domains lower than one standard deviation below standardized means, or any self- or parent-reported difficulty in any of the five domains . Of the Verbal Trait History variables in Table\u00a0DNA was extracted from whole blood using the Gentra Puregene Blood Kit (Qiagen) at the University of Nebraska Medical Center. Genotyping across 551,839 single nucleotide polymorphism (SNP) markers was performed using the Illumina Infinium HumanCoreExome-24-v.1 at the Yale Center for Genome Analysis . Genotypes were called using Illumina GenomeStudio with a total of 547,644 (99.24\u00a0%) passing quality control (QC). One individual failed QC due to low genotyping call rate and was excluded from the analysis.Reference map files for the HumanCoreExome dense marker panel were obtained from the Rutgers maps  threshold equal to 0.04 in the EUR reference population; (2) MAF 0.2\u20130.5 in the EUR reference population; (3) non-monomorphic marker within the pedigree;( 4) minimum intermarker distance of 0.5\u00a0cM; and (5) restricted to the 22 autosomes. A separate marker sub-panel was generated for pedigree structure validation using similar criteria as above except maximum LD threshold was equal to 0.25 and MAF from 0.3 to 0.5 in the EUR reference population. For genome-wide linkage analysis (excluding sex chromosomes), a total of 5448, 5493, and 5498 markers for sub-panels 1, 2, and 3, respectively, were created. A sub-panel of 5454 genome-wide markers was created for pedigree structure validation.We used the pedigree based analysis pipeline (PBAP) to sub-select genetic markers for pedigree quality control (QC) and for interfacing with MORGAN Thompson  to calcuQC for appropriate parent\u2013offspring relationships within the larger pedigree was assessed by comparing expected kinship coefficients (based on pedigree structure) and estimated coefficients computed by maximizing the likelihood from available genotype data across the 5454 marker sub-panel  were removed from the final model. In addition, a variance component for household random effects that further controlled for shared environment among nuclear families within the larger pedigree was included. The final model representing the log likelihood when the additive genetic variance was equal to 0 (no linkage elements) and covarying for household and age*sex effects, was used as the null model for hypothesis testing during linkage analysis.All statistical analyses were conducted using the SOLAR software package .Genome-wide multipoint variance component linkage analyses were conducted to examine linkage between VT+ and MIBDs. Multipoint linkage analysis considers recombination along a chromosome to determine the probability that a trait locus is located within a genomic region. Maximum likelihood estimates for linkage were calculated at approximately 0.5\u00a0cM intervals across the 22 autosomes and compared against the null model (no linkage) using a likelihood ratio test  between 52 and 55\u00a0cM on chromosome 13q14.2\u2013q14.3 , the linkage signal expands to 46\u201361\u00a0cM across 22 linkage markers spanning a 23.2\u00a0Mb region on chromosome 13q14.11-21.32, encompassing approximately 77 genes.Genome-wide multipoint linkage analyses for VT+ revealed a peak LOD score of 4.35  were observed on chromosome 2q37.1, 4q12\u201313.2, 4q25, 7q22.3\u201331.2, 8q24.3, and 12p13.33  derived from performance on NWR in an extended pedigree with a history of verbal trait disorders. This region encompasses SLI3 on chromosome 13q21, a SLI locus previously identified by Bartlett et al. , using aWithin this pedigree, there are at least three distinct haplotypes segregating with VT+, of which, only Haplotype 1 originated with a founder in the oldest generation\u2014the other two are more recently married into, consistent with assortative mating. Within the EUR reference population of 1000 genomes project, Haplotype 1-Recombined Fig.\u00a0 is commoITM2B encodes a transmembrane protein that helps to inhibit the accumulation of beta-amyloid, but mutations have been implicated in Familial British Dementia and Familial Danish Dementia with similar pathology to Alzheimer disease  with specific variants linked to reduction in laterality  identify the gene(s) in this region contributing to the linkage signals observed in the present study and others that have been identified this same region, and (2) elucidate the complex genetic and environmental interactions that may increase susceptibility.Supplementary material 1 (DOCX 13 kb)Supplementary material 2 (DOCX 20 kb)Supplementary material 3 (DOCX 18 kb)Supplementary material 4 (DOCX 30 kb)Below is the link to the electronic supplementary material."}
+{"text": "Linkage of medical databases, including insurer claims and electronic health records (EHRs), is increasingly common. However, few studies have investigated the behavior and output of linkage software. To determine how linkage quality is affected by different algorithms, blocking variables, methods for string matching and weight determination, and decision rules, we compared the performance of 4 nonproprietary linkage software packages linking patient identifiers from noninteroperable inpatient and outpatient EHRs. We linked datasets using first and last name, gender, and date of birth (DOB). We evaluated DOB and year of birth (YOB) as blocking variables and used exact and inexact matching methods. We compared the weights assigned to record pairs and evaluated how matching weights corresponded to a gold standard, medical record number. Deduplicated datasets contained 69,523 inpatient and 176,154 outpatient records, respectively. Linkage runs blocking on DOB produced weights ranging in number from 8 for exact matching to 64,273 for inexact matching. Linkage runs blocking on YOB produced 8 to 916,806 weights. Exact matching matched record pairs with identical test characteristics  for the highest ranked group, but algorithms differentially prioritized certain variables. Inexact matching behaved more variably, leading to dramatic differences in sensitivity (range 0.04\u201393.36%) and positive predictive value (PPV) (range 86.67\u201397.35%), even for the most highly ranked record pairs. Blocking on DOB led to higher PPV of highly ranked record pairs. An ensemble approach based on averaging scaled matching weights led to modestly improved accuracy. In summary, we found few differences in the rankings of record pairs with the highest matching weights across 4 linkage packages. Performance was more consistent for exact string matching than for inexact string matching. Most methods and software packages performed similarly when comparing matching accuracy with the gold standard. In some settings, an ensemble matching approach may outperform individual linkage algorithms. Linkage among medical databases such as electronic health records (EHRs), health insurer claims, and patient-generated data is becoming increasingly important for delivering high-quality, high-value healthcare; conducting valid and generalizable research; and evaluating healthcare policy. In countries with fragmented healthcare systems, such as the United States, linkage of EHRs across multiple healthcare settings and institutions enables clinicians to access information arising from care provided in other systems, which can improve continuity and efficiency of care and reduce redundancy.Additionally, linkage among different kinds of data\u2014such as EHR, registries, and claims\u2014can provide clinicians and researchers with access to complementary sources of information. For example, EHR-derived data on prescribed drugs, vital signs, laboratory results, and smoking and alcohol histories combined with claims-based data on dispensed drugs and out-of-system diagnoses and encounters, can provide a more comprehensive picture of a patient\u2019s care than information from either dataset alone.Consequently, database linkage can help create comprehensive, longitudinal datasets with information on patients\u2019 conditions and treatments over time. In addition to their utility in clinical care, such approaches can be applied to research, allowing investigators to access richer datasets and in the process overcome selection, information, and confounding biases \u20134. Also,linking variables is designated that is common to both datasets, which provides a basis for comparing individual records from each dataset. Second, a numerical weight is calculated for each compared pair (one record from each database), which is interpreted as the degree of confidence that paired records represent the same person or entity. Finally, a matching threshold is calculated or specified, and pairs whose weights exceed the designated threshold are declared to be matches.Conceptually, all record linkage algorithms operate similarly. First, a set of Typically, linkage variables are either numeric or text, such as string variables, where matching of these variables is done using exact or inexact methods. Exact string matching requires that two strings match exactly, character-by-character, including capitalization and any other characters such as hyphens, accents, or spaces. By contrast, inexact string matching, which has multiple versions, assigns a numerical similarity based on criteria such as the number of insertions, deletions, and replacements needed to convert one string to the other.deterministic or probabilistic [Methods for converting string comparisons to weights may be classified as either bilistic . In the bilistic . The M-pbilistic ) or calcbilistic ).blocking variables are compared. If the blocking variable has n values, both time and memory requirements are reduced by a factor of n.Numerous record linkage programs exist, which differ with respect to cost and methodologic transparency (open-source as compared with proprietary), operating system/hardware requirements, and scalability. Conceptually, all linkage programs perform string comparison, weight determination, and match determination. Data preprocessing is a key step in record linkage, including purging of duplicate records, harmonization of linkage variables , and common representation of missing values. Blocking is a common strategy to reduce computational burden, where only pairs of records that agree on one or more Most studies on linkage performance use only one software package to link synthetic or real-world databases \u201315. AlthWe compared the performance of 4 linkage software packages applied to real patient data from university-affiliated institutions. We focused on variables typically available in real healthcare data ) that contain actual errors but with very low levels of missingness see also . The RutWe used data for the three years 2013\u20132015 contained in noninteroperable EHRs from two neighboring, clinically affiliated but administratively separate institutions. The inpatient dataset (IPD) came from the Robert Wood Johnson University Hospital, a 965-bed urban teaching hospital with approximately 30,000 admissions per year. As received, the IPD included demographic data on all patients admitted overnight to the hospital during the study period. Each hospital admission resulted in a distinct entry; consequently, individuals with repeated hospitalizations had multiple entries. The outpatient dataset (OPD) came from the Rutgers Robert Wood Johnson Medical School, which has a multispecialty outpatient medical practice of over 500 affiliated physicians. The OPD contained information about all patients seen at least once during the study period, with only one record per person, based on a unique outpatient medical record number (MRN), representing the most recent set of demographic data. Both the IPD and OPD included first name, last name, DOB, gender, race, street address, city, state, and ZIP Code. Because only the OPD contained information on ethnicity, we excluded this variable from linkage experiments. Because the datasets were from clinically affiliated institutions, administrators used a proprietary linkage package to assign common MRNs. An inpatient MRN accessible within the OPD was used as a gold standard to evaluate linkage accuracy.We preprocessed both datasets to harmonize the variable names and values. Preprocessing entailed dropping variables not used in the linkage runs or other analyses , reclassifying race , and extracting year of birth (YOB) from DOB. We converted implausible values\u2014such as ZIP Codes containing letters\u2014to missing values, but we did not standardize names.After data preprocessing, we proceeded with deduplication. The OPD contained 176,154 records of purportedly unique individuals, making deduplication unnecessary. We deduplicated the original 104,289 IPD records by removing entries that matched exactly on 6 variables: MRN, last name, first name, gender, YOB, race, and ZIP Code. Records containing a missing ZIP Code were retained only if no other record matching on all other identifiers had a valid ZIP Code. The final IPD and OPD datasets contained the following variables: last name, first name, gender, DOB, YOB, age, race, ZIP Code, and MRN. We also assigned a unique study identifier to each record.RecordLinkage package), Merge ToolBox , Curtin University Probabilistic Linkage Engine , and Link Plus  (Table A in We selected software packages based on multiple criteria: (1) available for a Windows-based computer, (2) nonproprietary, (3) described in prior publications, (4) containing reasonable documentation with some transparency regarding default settings, (5) capable of operating in scripting/batch mode, and (6) capable of saving weights for compared pairs. Based on these criteria, we chose 4 software packages: R  and the other using YOB as the blocking variable . First name, last name, and gender comprised matching variables for all linkage runs. Aside from the software package, we varied linkage runs by the string matching method (exact or inexact); for inexact string matching, we applied the most common method, Jaro-Winkler. We also varied the weight determination method, using 3 probabilistic approaches , as welWe prepared 2 de-identified analysis files, one for each linkage experiment, with each file containing one row of information for each compared record pair. Analysis files included columns for IPD and OPD record identifiers, gender, age (upper limit 90), and race; a variable indicating whether the record pair matched on inpatient MRN; and 17 sets of weights corresponding to each linkage run. We assembled the analysis files using R (Version 3.4.0) and SAS (Version 9.4).To compare results across runs, we scaled the 17 sets of weights to range from 0 to 1, corresponding to the lowest and highest weights respectively. The scaling was linear and was done using the following equation:Additionally, we ranked the weights within runs from highest (ranked as 1) to lowest. Declaring matches based on weight rank, such as rank 1 or rank 2, also allowed for comparability across algorithms.Using this analysis file, we investigated the 17 sets of weights and scaled weights from multiple perspectives. We conducted descriptive analyses of the weights, including display of their empirical cumulative distribution functions. We also evaluated relationships among the weights, including their correlation, principal components analysis, and accuracy with respect to the gold standard, inpatient MRN. We also compared the performance of using matching weights as decision rules, including the area under the receiver operating characteristic (ROC) curve (AUC).The deduplicated datasets contained 69,523 inpatient records and 176,154 outpatient records, respectively. The total number of possible record pair comparisons, without blocking, was 12,199,192,962 pairs. Blocking on DOB reduced the number of record pair comparisons to 400,490. Datasets were similar based on gender distribution but distinctly different based on age and race .The empirical cumulative distribution functions of scaled weights varied considerably across the 17 linkage runs, confirming that these methods behaved differently Fig A in .Although there was substantial agreement among the 9 algorithms that use exact string matching, they did not produce identical rankings . All runAll runs using exact methods were highly correlated . Among rAmong 30,536 record pairs matching on DOB, first name, last name, and gender across most runs, 809 pairs  did not match on MRN . Manual We also noted a small number of record pairs with very low weights despite matching MRNs, representing either different people with the same MRN or errors in the matching variables . Record We compared the performance of the packages and algorithms using scaled weights and the gold standard, inpatient MRN, to identify declared matches, false positive matches, and false negative matches as the weight threshold varied. Across of range of scaled weights, the number of declared matches of record pairs varied among different packages and algorithms  Fig E in .An alternative ensemble method was based on the number of linkage runs that assigned the highest or second highest weight to a record pair see .In recognition that DOB is not available in all databases or accurate in all circumstances, we performed additional experiments blocking on YOB . These aUsing real data from noninteroperable EHRs, we performed a comprehensive assessment of the behavior and usability of nonproprietary available linkage software, evaluating the decision-making capability of specific linkage methods such as type of string comparison and weight determination and output from the linkage runs. Across multiple runs, we found relatively few perceptible differences in matching results, specifically with respect to ranks of the highest weights. Performance among software packages using exact string matching varied much less than that for methods using inexact string matching. From other perspectives, such as declaring matches to be pairs assigned the highest weight, linkage runs with inexact string matching were notably less efficient. As seen in The performance of most software packages and algorithms was similar, although not identical, with respect to matching accuracy as compared with our gold standard. In our linkage runs, exact matching using EM algorithms for weight determination appeared to be slightly less reliable than other exact matching algorithms: EM algorithms prioritized matching on first name over matching on last name, a more diverse and specific matching variable. Why some linkage runs prioritized first name over last name or vice versa is unclear. Compared with exact matching, linkage runs of inexact matching algorithms led to more variability in both the number of discrete weights assigned  and the number of record pairs receiving the highest weight . Our linkage runs also revealed more variability in assigning low weights than high weights when using both exact string matching and inexact string matching. This diversity among low-weight record pairs is unlikely to affect declared matches at common matching thresholds; however, it does underscore the differences in weight determination among approaches.We focused on the weights associated with the record pairs evaluated for each linkage program, evaluating linkage programs and algorithms as decision tools rather than the actual matching decisions. The findings suggest that the selection of weight thresholds for declaring matches can have a substantial impact on both operational and inferential uses of the linked data , 21. ChoA prior study similarly compared the performance and accuracy of different linkage approaches at different matching thresholds . HoweverEven small numeric differences in weighting could be more important in some settings, such as work with low-quality, incorrect data, or high levels of missingness. Prior work has shown that probabilistic approaches, which are often more time consuming, generally perform better in settings with low-quality data or high levels of missingness . AlthougUnlike prior work , we did The extent to which our findings can be generalized to other datasets is uncertain. Like all real datasets, the two datasets we used had some data quality problems. As one such indicator, nearly 3%  of the hAnother factor that may limit the generalizability of the findings is that we chose not to do extensive data cleaning beyond deduplication , 25, 26.We also did not explore the effect of using additional linking variables such as address because of the challenges in standardizing address text and concerns about the reliability of address variables, which can and do change over time. Identifying and accounting for identifier errors when linking data, especially on ZIP Code, helps to reduce bias caused by linkage errors . FurtherWe assessed the behavior and performance of various linkage algorithms using nonproprietary available linkage software and real data from two EHR systems. In settings in which levels of missing data are low and data quality is high, exact string matching approaches vary little across software packages, although approaches using exogenous weight determination, such as Fellegi-Sunter, may outperform those with endogenous methods, such as EM algorithms. With few exceptions, most linkage runs with either exact string matching or inexact string matching yielded similar groups of higher-weighted record pairs with high accuracy. Where possible, blocking on DOB seems preferable to blocking on YOB, given its greater computational efficiency and greater accuracy. Certain ensemble methods appear to improve overall performance of the algorithms.S1 File(PDF)Click here for additional data file."}
+{"text": "We compared the sample volume of endoscopic ultrasound-guided fine-needle aspiration and biopsy (EUS-FNAB) specimens obtained by 22-gauge (22G) and 25-gauge (25G) needles, and the accuracy rate.This was a retrospective study in a single tertiary referral center. We investigated 153 patients with pancreatic ductal adenocarcinoma (PDAC) who underwent diagnostic EUS-FNAB before neoadjuvant gemcitabine-based chemoradiotherapy between October 2006 and November 2015. We performed immunohistochemical (IHC) analysis of human equilibrative nucleoside transporter 1 using the remnant cell blocks following pathological PDAC diagnosis. We compared the sampling rate, accuracy rate, and success rate of IHC analysis between 22G and 25G.P\u200a=\u200a.60).There were 70 patients in the 22G group and 83 patients in the 25G group. The overall sampling rates on cytology and histology were 100% and 98.0%, respectively. The sampling rate did not differ between the 22G and 25G groups. The overall diagnostic accuracy rates on cytology and histology were 94.8% and 79.7%, respectively. The accuracy rates of 22G and 25G groups on cytology were 94.3% and 95.2%, respectively, whereas those on histology were 80.0% and 79.5%, respectively. The diagnostic accuracy on cytology and histology did not differ significantly between the 22G and 25G groups. Of 153 histology specimens, 69.3% of those with PDAC provided sufficient samples for IHC analysis. The success rate of IHC analysis did not differ significantly between the 22G (67.1%) and 25G (71.1%) groups (Both 22G and 25G provided a high diagnostic yield with equivalent accuracy rates on histology. EUS-FNAB specimens obtained using 22G or 25G can be equally adequate for IHC analysis and may be suitable for diagnostic examination. Further investigations such as EUS-FNAB needle design and novel cell block preparation are needed to obtain adequate samples for use in \u201cprecision medicine.\u201d Median overall survival in patients with advanced PDAC who receive gemcitabine is <7 months. Even with the recent new combination chemotherapy regimens , the overall survival is <1 year. and allowed the identification of patients who may respond to targeted therapies.,6 There are several hurdles to this approach, such as the acquisition of adequate amount of tumor sample for molecular profiling in the clinical setting.,8 For this novel therapy to be successful, the initially obtained specimen should contain viable tumor cells, not only for diagnosis but also for any potential therapeutic examinations.The recent development of next-generation sequencing technologies is expected to make therapy by this novel approach feasible. Whole genome association studies have clarified the mutations associated with prognostic valueEndoscopic ultrasound-guided fine-needle aspiration and biopsy (EUS-FNAB) can be a suitable method for obtaining such specimens. EUS-FNAB is considered as a safe and useful technique for the diagnosis of solid pancreatic tumors based on many reports evaluating the diagnostic yield. However, only a few reports have evaluated its ability to obtain a sufficient sample. In this study, we used remnant EUS-FNAB materials following cytological and/or histological diagnosis. Some of these samples might have been adequate as they were used before IHC analysis. In this situation, hENT1 IHC analysis was successfully performed in 68.4% cases of PDAC.We reported that immunohistochemical (IHC) expression of human equilibrative nucleoside transporter 1 (hENT1) in pretreatment PDAC specimens obtained with EUS-FNAB was associated with the prognosis of patients with PDAC receiving gemcitabine-based chemoradiotherapy.,11 Generally, 22G may be suitable for pathological diagnosis because they collect larger sample volumes than 25G. Nevertheless, a recent report suggested that 25G are more sensitive than 22G for diagnosing pancreatic malignancy. It is suggested that 25G is easier for puncture, and result in fewer bloody and contaminated specimens than 22G.,14As for needle size, several reports have compared the diagnostic yield of 22-gauge needles (22G) and 25-gauge needles (25G) in facilitating the cytological diagnosis of PDAC.Hence, we aimed to evaluate the adequacy of sample quantity for \u201cprecision medicine\u201d by comparing the success rates of IHC analysis in samples obtained by 22G and 25G.22.1This was a retrospective study in a single tertiary referral center.2.2We investigated 153 patients with PDAC who underwent diagnostic EUS-FNAB before neoadjuvant gemcitabine-based chemoradiotherapy between October 2006 and November 2015. We compared the sampling, accuracy, and success rates of IHC analysis in samples obtained by 22G and 25G.We obtained written informed consent for EUS-FNAB from all patients. The study protocol was approved by the Institutional Review Board for Human Research of Mie University Hospital.2.3A convex-array echo-endoscope  was used for EUS-FNAB. We punctured the pancreatic mass under endoscopic ultrasonographic guidance, after identifying the tumor using B-mode imaging, and confirming the absence of vessels in the target area. We used 2 types of needle: 22G  and 25G . The different needles were used according to their availability. We defined the success group of IHC analysis using remnant cell blocks as the available group. In terms of cellularity of the cell block, remnant specimens included more than 50 lesional cells. IHC staining was performed using the labeled streptavidin\u2013biotin peroxidase complex method with the Benchmark XT auto-immunostaining system . Rabbit polyclonal anti-SLC29A1 (ENT1) antibody  was used as the primary antibody.Cytologists immediately examined the specimen with rapid on-site evaluation using rapid staining  to verify that the obtained sample was sufficient. If the sample was insufficient, further punctures were performed. We confirmed the diagnosis of PDAC by cytological and/or histological analyses with EUS-FNAB specimen. The both cytological and pathological diagnoses were based on the review of all these materials by the cyto-pathologists. Thereafter, we retrospectively evaluated 153 stored cell blocks using IHC analysis for hENT1 expression as shown in our previous study.Final diagnoses were made according to surgical histology and clinical follow-up for a minimum of 6 months.2.4The primary outcome was the adequacy of the obtained sample volume using 22G and 25G determined according to the success rate of IHC analysis. The secondary outcome was the sampling and accuracy rates of procedures performed using 22G and 25G. The sampling rate of cytology was defined as the rate of obtaining pancreatic cells regardless of whether malignant cells could be confirmed. We defined the sampling rate of histology as the rate of obtainability of gray-whitish, worm-like tissue samples that were visible macroscopically. Diagnostic accuracy was defined as the rate of diagnosis as adenocarcinoma. Atypical and benign were defined as negative.P value < .05 was considered to represent a statistically significant difference.Statistical tests were performed using SAS University Edition . We performed the Fisher exact test to compare categorical variables and the Wilcoxon rank-sum test to compare continuous variables. A 3P\u200a=\u200a.003, Table In total, 153 patients underwent EUS-FNAB: 70 patients in the 22G group and 83 patients in the 25G group. No statistically significant differences in the characteristics of the patients (age and sex), tumor location, tumor size, or resectability were found between both groups (Table P\u200a=\u200a.003). The sampling rates of the 22G and 25G on cytology were 100% (70/70) and 100% (83/83), and those on histology were 98.6% (68/70) and 97.6% (82/83).The 22G and 25G groups had high sampling rates on cytology and histology with no difference in the sampling rate , surgical pathology (n\u200a=\u200a1), and follow-up examination of tumor metastasis (n\u200a=\u200a3).P\u200a=\u200a.60) . The success rate of IHC analysis did not differ significantly between the 22G 67.1%, 47/70) and 25G  groups (7/70 and P\u200a=\u200a.012).No significant difference was observed in factors other than age between the available and unavailable groups Table . The mea4We demonstrated that EUS-FNAB using both 22G and 25G have a high diagnostic yield. Furthermore, we evaluated the feasibility of IHC analysis for hENT1, and showed that both 22G and 25G performed well for sample acquisition. In our study, EUS-FNAB had a higher diagnostic yield than that reported in previous studies; the accuracy rates on cytology and histology were 94.8% and 79.7%, respectively. The accuracy rates on histology did not differ between 22G and 25G. In addition, the remnant specimens of 69.3% patients were available for IHC analyses, the rate of suitability for IHC analysis did not differ significantly between 22G and 25G.\u201312 The ease of puncture due to the flexibility of the thinner 25G might influence the accuracy of these needles. However, the accuracy rate of these different needle gauges on histology is controversial. Sakamoto et al reported that the accuracy rate on histology was significantly different between 22G (62.5%) and 25G (45.8%). By contrast, Kida et al reported that there were no differences between 22G (68.0%) and 25G (69.6%). Park et al also showed similar evidence: 22G (68.2%) and 25G (68.3%). In agreement with these 2 later reports, our results showed no difference between 22G and 25G (80.0% and 79.5%). However, our study showed higher diagnostic accuracy than the previous studies. We suggest that rapid on-site evaluation by pathologists increased the accuracy rate.Several studies showed the diagnostic accuracy of EUS-FNAB and compared the accuracy rates of 22G and 25G. In these studies, 25G showed a higher accuracy rate than 22G on cytology. Navina et al evaluated the cellularity of EUS-FNAB material by scoring the number of lesional cells; only 12.4% of specimens had more than 100 cells, which was deemed to represent sufficient cellularity for \u201cprecision medicine.\u201d From these results, it can be inferred that the success rate of \u201cprecision medicine\u201d using EUS-FNAB samples obtained from PDAC specimens remained lower than that desired. In other reports, most samples used for molecular analysis, including next-generation sequencing, were obtained via surgery rather than EUS-FNAB. Specimens obtained via EUS-FNAB were deemed to contain poor-quality DNA due to the presence of intratumor heterogeneity and desmoplastic changes in PDAC.,19In addition to diagnosis, the remnant specimens of 69.3% patients were available for IHC analyses. Furthermore, the rate of suitability for IHC analysis did not differ significantly between 22G and 25G. We demonstrated that both 22G and 25G performed well for sample acquisition. Other than the present study, only a few reports have evaluated whether EUS-FNAB can be a reliable source of sufficient material for \u201cprecision medicine,\u201d with special emphasis on needle size. Boone et al reported the correlation between SMAD4 expression in pretreatment PDAC tissues and the prognosis of PDAC patients. Their study design was similar to that of the present study, which used the remnant sample after diagnosis to evaluate SMAD4 protein: only 44.4% of samples could be analyzed.\u201322 Young et al reported that next-generation sequencing on EUS-FNAB cell blocks yielded genomic profiles in all cases (17/17). Furthermore, new core biopsy needles have been introduced in clinical practice, which are expected to acquire more quantity of cells and to be more useful for \u201cprecision medicine.\u201d Therefore, future studies should evaluate new EUS-FNAB needle designs. In addition, novel technologies with the potential to yield useful material from small sample volumes should be investigated to facilitate the development of personalized treatment approaches for patients with PDAC.Recently, there are a few challenges to using next-generation sequencing on samples obtained via EUS-FNAB, including molecular aberrations.There are a few key limitations to the present study that should be discussed. First, this was a retrospective and single-center study. Therefore, the possibility of selection bias could not be excluded. Second, the adequacy rate of IHC was not high enough for clinical use in \u201cprecision medicine\u201d applications. Therefore, the quality and quantity of samples obtained via EUS-FNAB should be assessed and further improved.In conclusion, the results of this study showed that both 22G and 25G needles provided a high diagnostic yield and that EUS-FNAB specimen obtained using either 22G or 25G needles can be equally adequate for IHC analysis. It is envisaged that EUS-FNAB will be a reliable source of \u201cprecision medicine.\u201dConceptualization: Reiko Yamada.Data curation: Naohiko Yoshizawa, Reiko Yamada, Takashi Sakuno, Hiroyuki Inoue, Hiroshi Miura, Toshifumi Takeuchi.Formal analysis: Naohiko Yoshizawa.Funding acquisition: Reiko Yamada.Investigation: Naohiko Yoshizawa, Reiko Yamada, Takashi Sakuno, Hiroyuki Inoue.Methodology: Reiko Yamada.Project administration: Reiko Yamada.Resources: Reiko Yamada, Takashi Sakuno, Hiroyuki Inoue, Hiroshi Miura, Toshifumi Takeuchi.Supervision: Kyosuke Tanaka, Noriyuki Horiki, Yoshiyuki Takei.Validation: Misaki Nakamura, Yasuhiko Hamada, Masaki Katsurahara.Writing \u2013 original draft: Naohiko Yoshizawa, Reiko Yamada.Writing \u2013 review and editing: Reiko Yamada."}
+{"text": "Dione juno juno  and described at ultrastructural and pathological levels. In this study, the complete genome sequence of DijuNPV was determined and analyzed. The circular genome presents 122,075 bp with a G + C content of 50.9%. DijuNPV is the first alphabaculovirus completely sequenced that was isolated from a nymphalid host and may represent a divergent species. It appeared closely related to Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV) and other Choristoneura-isolated group I alphabaculoviruses. We annotated 153 open reading frames (ORFs), including a set of 38 core genes, 26 ORFs identified as present in lepidopteran baculoviruses, 17 ORFs unique in baculovirus, and several auxiliary genes . The thymidylate kinase (tmk) gene was present fused to a dUTPase (dut) gene in other baculovirus genomes. DijuNPV likely lost the dut portion together with the iap-3 homolog. Overall, the genome sequencing of novel alphabaculoviruses enables a wide understanding of baculovirus evolution.Baculoviruses are capable of infecting a wide diversity of insect pests. In the 1990s, the Dione juno nucleopolyhedrovirus (DijuNPV) was isolated from larvae of the major passionfruit defoliator pest  Baculoviruses are capable of infecting a wide diversity of insect hosts, including larvae of lepidopterans, hymenopterans, and dipterans . The virBaculoviridae is classified into four genera: Alphabaculovirus (which contains lepidopteran-specific nucleopolyhedroviruses [NPVs]), Betabaculovirus (which contains lepidopteran-specific granuloviruses [GVs]), Gammabaculovirus (which contains hymenopteran-specific NPVs), and Deltabaculovirus (which contains dipteran-specific NPVs) Alphabaculication  for thisiap-3 ortholog. When the CfMNPV genome was not included, the DijuNPV genome presented strict collinearity with the OpMNPV and HycuNPV genomes (data not shown).We performed a genomic comparison among some of the DijuNPV-related virus genomes using a progressive MAUVE algorithm, including Hyphantria cunea nucleopolyhedrovirus (HycuNPV), OpMNPV, and CfMNPV. Seven Locally Collinear Blocks (LCB) were found, and most were shown to be strictly conserved among these genomes . LCB1 anbaculovirus repeated ORF (bro) multigene family called by bro-a (DijuNPV-ORF-95), bro-b (DijuNPV-ORF-96), and bro-c (DijuNPV-ORF-148), cathepsin (DijuNPV-ORF-37), and chitinase (DijuNPV-ORF-38) that are related to virus horizontal transmission; genes associated with apoptosis control such as iap-1 (DijuNPV-ORF-120) and iap-2 (DijuNPV-ORF-88); and genes related to nucleotide metabolism like a thymidylate kinase (DijuNPV-ORF-126). Importantly, as characteristic of all group I alphabaculoviruses, the DijuNPV genome presents a gp64 ortholog (DijuNPV-ORF-36).The DijuNPV genome contains the 38 currently defined core genes ,31 and tbro-a), an ac45-like homolog, and other six hypothetical genes found in other baculovirus genomes, including DijuNPV-ORF-4, ORF-9, ORF-10, ORF-25, ORF-75, and ORF-124. The best hits found for each gene is shown in bro-c (DijuNPV-148). When compared by pairwise alignment, this gene presents a deletion at the amino-terminal portion in the DijuNPV homolog (data not shown). A tmk homolog (DijuNPV-ORF-126) is shared only by DijuNPV and OpMNPV, which will be discussed later on. Interestingly, a conotoxin-like 2 (clt-2) homolog was found in the DijuNPV, OpMNPV, HycuNPV, and AnpeNPV-L2 genes and lacked by CfMNPV, whereas a ctl-1 homolog was found in all related viruses but not in the DijuNPV.We also performed an ORF content comparison among DijuNPV and its closest relatives , and we plotted the result in a Venn diagram . A totalac30-like and fgf gene orthologs carry the iap-3 homolog gene in other DijuNPV relatives . The OpMNPV and DapuNPV genomes contain four putative nucleotide metabolism-related genes, including ribonucleotide reductase 1 (rr1), ribonucleotide reductase 2 (rr2), and a fusion of tmk and dut. By alignment and phylogenetic analysis, we found that the DijuNPV tmk (ORF-126) was related directly to the tmk portion of the fused genes found in both OpMNPV and DapuNPV genomes , OpMNPV, DapuNPV, and Perigonia lusca single nucleopolyhedrovirus (PeluSNPV), suggesting a common ancestry (tmk genes (except ErelGV) clustered together, and the same occurred with alphabaculoviruses except for Malacosoma neustria nucleopolyhedrovirus (ManeNPV) and Operophtera brumata nucleopolyhedrovirus (OpbuNPV), which clustered together with betabaculoviruses. Therefore, the DijuNPV genome seemed to have lost a region that would harbor homologs of iap-3, rr1, rr2, and dut, retaining only the tmk homolog.The loci flanked by elatives . Howevervolution  in a sim genomes . In the ancestry . The cloD. juno and A. vanilla and could be potentially used for passionfruit crop protection [DijuNPV is highly pathogenic to nymphalid caterpillars of both species otection ,14. The otection , and molotection . Other botection , Catopsiotection , three iotection , and NeoAlphabaculovirus [gp64 homolog as the major BV envelope fusion protein that replaced the ancient f protein during clade divergence [We found that DijuNPV is an alphabaculovirus specifically related to the still monophyletic group I inside genus ulovirus ,36. Grouvergence . The stavergence . The bravergence .Autographa californica nucleopolyhedrovirus. Gene functions tend to be extended to homologous proteins in less studied viruses, such as DijuNPV. Despite the diversity in gene content and organization of baculovirus genomes, a set of 38 core genes are conserved across their genomes and play important roles in the viral cycle [iap-3 homolog that is present in other closely related viruses. Although other iap genes is found in the alphabaculovirus genomes such as iap-1 and iap-2, usually the product of iap-3 is the functional IAP that plays role in blocking apoptosis during baculovirus infection [p35-deficient AcMNPV to replicate in Sf9 cells. Based on that observation, it would be valuable to characterize the ability of iap-1 and 2 products to block apoptosis. Importantly, in our work, we used a more liberal ORF annotation criterion  that allowed finding 17 unique genes not reported before in baculovirus. This is criterion is justified based on the empirical data found in Ref. [We annotated 153 genes in the DijuNPV genome. Baculovirus genes are divided into regulatory, structural, and accessory proteins according to its functional characterization in members of model types, for instance Autographa californica multiple nucleopolyhedrovirus (AcMNPV) [al cycle ,31. Surpnfection . Only An in Ref. . Many puInterestingly, the OBs from DijuNPV were isolated in-field and represent a population of viruses. Therefore, the sequencing data generated by the Illumina HiSeq were used to search for intrapopulation virus diversity. We found several synonymous and non-synonymous SNVs in the DijuNPV virus population at an average frequency of 38 \u00b1 4.6 %. In a similar way, the sequencing of an AcMNPV isolate generated identified 118 SNVs with average frequencies of 33\u201336% . In contiap-3 homolog should be found in the DijuNPV, there is a homolog of tmk. In a previous work, we described the evolutionary history of this tmk homolog (cp016 homologs). The gene might be found in three different manners in baculovirus genomes: fused to either a polynucleotide kinase 3\u2032-phosphatase , a dut homolog, or alone. In alphabaculoviruses, the gene is usually fused to the N-terminal portion of pnk, and the unique exception takes place in the Clanis bilineata nucleopolyhedrovirus (ClbiNPV) genome where no pnk is found. Nucleotide metabolism-related genes are not essential for baculovirus infection given that several species lack them [tmk-dut from PeluSNPV and ErelGV were shown to increase viral DNA replication, virus progeny production, and occlusion body formation during in vitro infection when compared to the parental AcMNPV virus that lacks dut and tmk genes [At the same locus in which the ack them . Howeverack them . For insmk genes .D. juno. The virus may represent a novel species into genus Alphabaculovirus, which is closely related to other group I members. The genome was shown to have five hrs and 153 ORFs with 17 as unique. Furthermore, several auxiliary genes were encountered, such as homologs of iap-1 and 2, chitinase, cathepsin, gp37, and tmk. The later was present fused to a dut gene in other baculovirus genomes. DijuNPV lost the dut portion together with the usually functional iap-3 homolog. Overall, the genome sequencing of novel alphabaculoviruses enables a wide understanding of baculovirus evolution.In this work, we have described the genome of the first baculovirus isolated from a nymphalid host, the passionfruit pest"}
+{"text": "The VetStat analyzer yielded results that were in agreement with the cobas b 123 analyzer for determination of pH, pCO2, bicarbonate ([HCO3-]) and potassium concentration [K+], while the epoc analyzer achieved acceptable agreement for [HCO3-] and [K+]. The VetStat analyzer may be useful in performing blood gas analysis in equine samples but analysis of [Na+], [Cl-] and pO2 should be interpreted with caution. The epoc delivered reliable results for [HCO3-] and [K+], while results for pH, pCO2, pO2 and [Na+] should be interpreted with caution.Portable blood gas analyzers are used to facilitate diagnosis and treatment of disorders related to disturbances of acid-base and electrolyte balance in the ambulatory care of equine patients. The aim of this study was to determine whether 2 portable analyzers produce results in agreement with a stationary analyzer. Blood samples from 23 horses hospitalized for various medical reasons were included in this prospective study. Blood gas analysis and electrolyte concentrations measured by the portable analyzers VetStat and epoc were compared to those produced by the cobas b 123 analyzer via concordance analysis, Passing-Bablok regression and Bland-Altman analysis. Limits of agreement indicated relevant bias between the VetStat and cobas b 123 for partial pressure of oxygen (pO Owners of the horses gave informed consent for data collected from their horses to be used for scientific purposes. Blood samples from 23 horses admitted to the equine clinic of the University of Li\u00e8ge between May and June 2015 were included in this study. Patients with diverse medical conditions were included to represent a wide range of measured variables. Horses were sampled for diagnostic purposes only as determined to be necessary by a veterinary clinician unrelated to the present study.Both the VetStat and epoc analyzers utilize disposable, single-use cartridges covering a range of analytical capabilities. The epoc analyzer requires a sample volume of 95 \u03bcL and takes less than 1 minute, while the VetStat analyzer requires a sample volume of 125 \u03bcl and takes less than 2 minutes for sample analysis.2 and electrolyte concentrations via a potentiometric method using ion selective electrodes and the Nernst-equation. The pO2 is measured via an amperometric method with ion-selective electrodes of the Clarke type. The POC blood gas analyzer VetStat determines the blood gas partial pressures and electrolyte concentrations by measuring their optical fluorescence using optodes. The [HCO3-] is calculated by means of the Henderson-Hasselbalch equation in all 3 instruments.The stationary POC system cobas b 123 and the POC analyzer epoc determine pH, pCOAll analyzers were calibrated, maintained and operated according to their manufacturer\u2019s instructions. All machines were kept at constant room temperature for at least 24 hours before and during the experiments. The epoc reader runs a calibration cycle for each cassette, containing an integrated quality control system with specific reference liquids for this machine. Each batch of VetStat sample cassettes is calibrated during the manufacturing process with the calibration information contained in the bar code that is scanned before each analysis. Internal calibration and quality control processes are performed by the reader with the VetStat reader calibrated with standard reference cassettes, standard hemoglobin cassettes and standard gas cartridges at prescribed intervals. The cobas b 123 system runs automated calibration cycles at defined intervals. The system calibration runs once a day in the morning with a 1 point-calibration running every 60 minutes, and a 2-point-calibration, running every 8 hours; these are postponed if the system starts a cycle during analyses.Arterial or venous blood samples were taken with pre-filled heparinized single-use disposable syringes  during standard examination of the horses or as part of the anesthetic monitoring. Venous blood samples were collected anaerobically by venipuncture of the jugular vein. Arterial blood samples were collected from the carotid or transverse facial artery. The patient\u2019s rectal temperature was noted to allow for temperature correction.www.random.org). All measurements were undertaken by 1 of 2 people trained to use the equipment. The devices were situated adjacent to each other in order to minimize the time interval between measurements. A maximum of 5 minutes delay for introducing samples to all 3 analyzers was considered acceptable. In order to determine repeatability of the measurements with the three compared instruments, seven serial measurements of the same blood sample were performed on each analyzer. The procedure of these serial measurements was conducted as fast as possible within a time range less than 25 minutes in order to minimize the changes in sample composition over time.Analysis was immediately performed after blood sample collection. Each blood sample was analyzed once by the stationary analyzer and the 2 POC analyzers, with the order of the analyzers pre-determined by an online randomization program (ccc) contains a measure of precision (Pearson\u2019s \u03c1) and accuracy  and was used as an indicator for the strength of concordance between measurements  were only compared between VetStat and cobas b 123 since the epoc cartridges do not provide [Cl-] values. The differences between compared results were normally distributed.A total of 41 blood samples (1 to 4 samples per horse) including 32 arterial and 9 venous samples were collected and analyzed. Due to cartridge calibration failures, 8 epoc results and 2 VetStat results were incomplete. Furthermore, 3 results for , [Na+], [K+] and [Cl-], respectively.. The cusum test indicated no significant deviation from linear relationship between compared results for all determined parameters (P > 0.05).In 2 (B), and pO2 (C) and for comparison of epoc versus cobas b 123 analyzer for blood pH (G), pCO2 (H), and pO2 (I). The dashed line is the line of identity, the solid line is the best fit.Passing-Bablok regression analysis for comparison of VetStat versus cobas b 123 analyzer for blood pH (A), pCO2 (E), and pO2 (F) and for comparison of epoc versus cobas b 123 analyzer for blood pH (J), pCO2 (K), and pO2 (L). The dashed lines are upper and lower limit of agreement, the solid line is the mean difference (Bias).Bland-Altman plot analysis for comparison of VetStat versus cobas b 123 analyzer for blood pH (D), pCOFor the determination of pH, Bland-Altman analysis indicated a significant positive systematic bias between the VetStat and cobas b 123 analyzers (P = 0.0009). However, the estimated 95% limits of agreement indicated that the amount of systematic and random bias remained within the pre-set limits of acceptance. For pH determined by the epoc and the cobas b 123 analyzer, Bland-Altman analysis detected a significant negative systematic bias (P < 0.0001) while the 95% limits of agreement indicated that the underestimation of pH by the epoc analyzer ranged outside the limits of acceptance. The Passing-Bablok regression indicated a significant constant as well as proportional bias.2, no significant systematic bias between the VetStat and cobas b 123 analyzers was detected with the 95% limits of agreement within the limits of acceptance. However, a significant positive systematic bias between the epoc and the cobas b 123 analyzer for the determination of pCO2 was detected (P = 0.0005). The 95% limits of agreement indicated that the epoc analyzer overestimated the pCO2 beyond the pre-set limits of acceptance.For the determination of pCO2, no significant systematic bias between the VetStat and cobas b 123 analyzer as well as between the epoc and the cobas b 123 analyzer was identified. However, the 95% limits of agreement for both instruments indicated random deviations between the analyzers, which were higher than the pre-set limits of acceptance, especially for very high pO2 values. Due to the subjection of the magnitude of random error on the level of the pO2, the 95% limits of agreement were additionally calculated for pO2 values up to 100 mmHg for the comparison between the VetStat and the cobas b 123 analyzers  and between the epoc and the cobas b 123 analyzers . For pO2 values up to 100 mmHg, the 95% limits of agreement were considerably closer, although the random error for the determination of pO2, including exclusively lower values, still ranged outside the pre-set limits of acceptance.For the determination of pO3-], a significant negative systematic bias between the VetStat and cobas b 123 analyzers (P = 0.0001) was found. However, the 95% limits of agreement ranged within the limits of acceptance. For [HCO3-] calculated by the epoc and cobas b 123 analyzers, no significant systematic bias was found.For the determination of [HCO+], significant positive systematic bias between the VetStat and cobas b 123 analyzers as well as between the epoc and cobas b 123 analyzers (P < 0.0001) was identified. The 95% limits of agreement ranged outside the pre-set limits of acceptance. Between the epoc and the cobas b 123 analyzers, significant constant as well as proportional bias was detected.For the determination of [Na+], significant positive systematic bias between the VetStat and the cobas analyzers (P = 0.0009) as well as between the epoc and the cobas b 123 analyzers (P = 0.0004) was found. The 95% limits of agreement remained within the pre-set limits of acceptance. Between the VetStat and the cobas b 123 analyzer, significant proportional bias was detected.For the determination of [K-], significant positive systematic bias between the VetStat and the cobas b 123 analyzer (P < 0.0001) was found. The 95% limits of agreement indicated an overestimation of [Cl-] by the VetStat analyzer which ranged outside the pre-set limits of acceptance. Besides the constant bias, significant proportional bias was detected.For the determination of [Cl2 and [K+], the VetStat failed to meet precision targets for [Cl-], while the epoc failed to meet the precision targets for pCO2, pO2, and [K+].Repeatability results for each analyzer are summarized in Determination of blood gas partial pressures and electrolyte concentrations at stall-side and under field conditions requires that portable analyzers yield results in agreement to analyzers used in veterinary clinics or similar institutions. To verify whether this condition is met for the POC analyzers VetStat and epoc, their performances were compared to the stationary cobas b 123 analyzer. Even though the cobas b 123 analyzer does not have to be permanently installed and therefore may be considered a mobile unit, it is not specifically intended for field use.By evaluating the types and magnitudes of the differences as compared with pre-set clinical limits of acceptance, decisions can be made about the acceptability of methods . The decUse of Pearson\u2019s correlation coefficient as a sole measure of agreement between methods is not appropriate. Furthermore, high correlation does not necessarily mean agreement since the correlation coefficient cannot detect systematic bias ,24,25. TSince measurement errors had to be assumed in both comparison and test methods, the Passing-Bablok regression was preferred over ordinary linear regression . In cont2, a substantial agreement for pH, pCO2 and [K+] and a poor agreement for [HCO3-], [Na+] and [Cl-]. The agreement between the epoc and the cobas b 123 analyzer was substantial for pO2 and [K+], moderate for pCO2 and poor for pH, [HCO3-] and [Na+]. Based on the reported 95% limits of agreement and their comparison to pre-set limits of clinical acceptance, the systematic bias between the VetStat and the cobas b 123 analyzer for the determination of [Na+] and [Cl-] was clinically relevant, with the [Na+] considerably overestimated by the VetStat analyzer. The identified proportional bias meant that the slight overestimation of [K+] by the VetStat analyzer would increase with increasing [K+], while the overestimation of [Cl-] would increase with decreasing [Cl-]. Systematic bias between the epoc and the cobas b 123 analyzers was found to be clinically relevant for pH, pCO2 and [Na+] and [K+], with the epoc analyzer underestimating the pH and overestimating pCO2 and [Na+]. The identified proportional bias means that the underestimation of the pH by the epoc analyzer would increase with decreasing pH values, while the overestimation of [Na+] would increase with decreasing [Na+]. Since the determination of [Cl-] is not provided by the epoc analyzer, this parameter could not be included in the comparison.Concordance correlation indicated an almost perfect agreement between the VetStat and the cobas b 123 analyzers for pO2, the 95% limits of agreement for both analyzers indicated a high random error ranging outside clinically acceptable limits, especially for high pO2 values.Although no significant systematic bias was detected for the determination of pO+], pH, pO2, hematocrit, hemoglobin and base excess (BE)  and [K+] but less agreement for paCO2 and [Cl-]  . In dogsrelevant . The varn = 39), with each sample analyzed only once on each analyzer instead of in duplicate as recommended by CLSI guidelines. However, when adopting the null hypothesis that there is no difference between the analyzers, setting the alpha error at 0.05 and the (1-\u03b2) error at 0.8, the minimal sample size for a Cohen d effect size of 0.8  was determined to be n = 21. A small sample size was also a limitation in the repeatability analysis. Repeatability or within-run precision was measured by analysis of a single sample 7 consecutive times for each machine. Although the method chosen for the present study was similar to what is suggest by the ICSH , [K+], glucose, lactate, [HCO3-], BE, and saturation of oxygen (SO2) while it failed to meet precision targets for pCO2, [Ca2+], hematocrit and hemoglobin. However, if minimal acceptable values were used instead of optimal values, all measures values bar hematocrit and hemoglobin met the precision targets. We therefore consider the epoc analyzer to be a reasonably precise machine. Unfortunately, no such data is available for the VetStat or cobas b 123 analyzer.There are numerous limitations in this study. Due to limited time and resources, the sample size for the present study was relatively small (the ICSH , only 7 the ICSH . As compthe ICSH , the epo2, pO2, and [K+] was higher than the pre-set limits of acceptance. The epoc analyzer therefore failed to achieve the required precision for these parameters, while the precision for the remaining parameters was acceptable. This is contradictory to the results obtained in a previous study, where the epoc analyzer met all precision targets except for pCO2 [+] may arise during sample handling, and although no gross hemolysis was recognized it can not be excluded to have occurred during the consecutive measurements leading to greater variation in the results. The same observation was made for the the cobas b 123 analyzer. Due to technical failure, serial measurements of pO2 were not performed with the VetStat analyzer precluding the determination of the precision of this parameter. For the remaining parameters the precision of the VetStat analyzer was considered to be acceptable.The imprecision of the epoc analyzer for the determination of pCOfor pCO2 . FalselyThe assessment of precision in this study is limited in part because only 1 blood sample was repeatedly measured, limiting evaluation of the effect of different ranges of values of precision. Furthermore, only the short-time imprecision was assessed. To achieve a more comprehensive assessment of precision, serial measurements of samples with different partial pressures/concentration ranges and over an extended period would be necessary.2 values measured in this study were partly higher than would be expected under usual circumstances due to the partial collection of blood samples under general anesthesia. Therefore, the measured range of pO2 values may not represent the expected range for healthy standing unsedated horses. The choice of inclusion of samples under general anesthesia was made in compliance with CLIA guidelines, which suggest inclusion of a wide range of samples , [Na+] and [Ca2+] [+] measurements in samples analyzed for the within-run precision data of the cobas b 123 and the epoc. Hemolysis is known to increase blood [K+] measurements  ,33. We eurements  with higurements . Althoug2, [HCO3-] and [K+] and may therefore be a useful POC analyzer for equine blood gas analysis. However, measurement of [Na+], [Cl-] and pO2 should be interpreted with caution. The epoc analyzer achieved acceptable results for the determination of [HCO3-] and [K+], while results for pH, pCO2, pO2 and [Na+] should be interpreted with caution.In conclusion, this study investigated agreement of 2 POC blood gas analyzers against a stationary analyzer which, although widely used in practice, cannot be considered as a gold standard reference. The VetStat analyzer delivered reliable results for the determination of pH, pCO"}
+{"text": "Modern SNP genotyping technologies allow measurement of the relative abundance of different alleles for a given locus and consequently estimation of their allele dosage, opening a new road for genetic studies in autopolyploids. Despite advances in genetic linkage analysis in autotetraploids, there is a lack of statistical models to perform linkage analysis in organisms with higher ploidy levels. In this paper, we present a statistical method to estimate recombination fractions and infer linkage phases in full-sib populations of autopolyploid species with even ploidy levels for a set of SNP markers using hidden Markov models. Our method uses efficient two-point procedures to reduce the search space for the best linkage phase configuration and reestimate the final parameters by maximizing the likelihood of the Markov chain. To evaluate the method, and demonstrate its properties, we rely on simulations of autotetraploid, autohexaploid and autooctaploid populations and on a real tetraploid potato data set. The results show the reliability of our approach, including situations with complex linkage phase scenarios in hexaploid and octaploid populations. These multiple chromosome sets can originate from the combination of genomes from different, but related species, or from duplicated genomes from the same species  segregation, since homologous chromosomes, or homologs, tend to form bivalents within each sub-genome. Autopolyploids, however, have more than two homologs per homology group, forming either random bivalents or multivalents during meiosis, resulting in polysomic segregation  analysis, as well as the assembly of reference genomes and the study of evolutionary processes  estimation of pairwise recombination fractions and associated statistical tests; v) if the order is optimal, the map is complete, otherwise, return to step single-dose or simplex markers (double simplex). Given this level of simplification, it is possible to use the five-step procedure coupled with a standard software suitable for diploid populations. Nevertheless, it is well accepted that the use of single-dose markers imposes limitations on the construction of adequate genetic maps. These approaches sub-sample the genome  evaluation have opened the door for further genetic mapping studies in high-level autopolyploids. It is now possible to measure the abundance of specific alleles within a locus in a polyploid genome . This teGenetic linkage maps can be constructed based on two-point or multipoint estimates of the recombination fraction. Two-point methods use information on pairs of markers, and even though they are less computationally demanding than multipoint methods, they require a higher amount of information in the markers to provide reliable results. Only recently, using a two-point-based method, i and i using clustering algorithms, as proposed by , the initial state function (Equation 7), the emission probability function (Equations 8 and 9). In the genetic mapping context, the first connects adjacent marker loci in function of their recombination fraction, the second is the prior probability of the genotypes in the mapping population, and the third connects the observed marker dosage to the complete multi-allelic hidden genotypic states. While these ideas are widespread in the genetic mapping literature, for instance in In this section, we define the notation used throughout this article and present the probabilistic model for the gamete formation in autopolyploids. The mathematical derivation of the HMM, including the estimation of the model parameters, is based on the work of P and Q with the same ploidy level (full-sib family). The ploidy level is denoted by m, and can be any even number greater than zero. Let the vectors k and P and Q, respectively. The superscript i indicates one of the possible alleles for the loci, and each locus has m different alleles in each parent. For example, for a cross between two autohexaploid individuals, e.g., i) there is only formation of bivalents during the meiosis; k and v) there is separation of sister chromatids during meiosis II and Consider a mapping population derived from a cross between two autopolyploid individuals regation  and no dregation : i) theri.e., the number of possible bivalent chromosomal pairings for a given homology group during meiosis can be obtained by sequentially choosing pairs out of m homologs without replacement, divided by all possible permutations of the chosen pairsBivalent formation occurs during meiosis I . In diploid cells, there is only one possible pairing configuration: two duplicated homologs from a homology group pair to form one bivalent. However, in autopolyploid cells, given the previous assumptions, the expected number of possible pairing configurations, P. Since parent Q undergoes a similar process, it is possible to combine the expected gametic frequencies to obtain the expected genotypic frequency in the full-sib population. Each of the bivalents obtained for a given configuration k and parental, which results from bivalents with zero or any other even number of recombinations between k and recombinants, which results from bivalents with any odd number of recombinations. As presented by k and l denotes the number of total recombinant bivalents between loci k and k and P. Consistent means that the gamete can be produced from bivalent configuration We will present the expected gametic frequencies considering parent l can be obtained by a simple examination of superscripts of elements contained in i.e., it is not consistent with Since we assume that alleles with the same superscript are in the same homolog, 6)} m=6, . If one consistent with the observed gamete, and consequently In reality The probability of observing a specific gamete is always the same for each l can change from zero to l recombinant chromosomes, the number of possible pairing configurations is For every gamete, i.e., linkage phase) for the parents of the mapping population is unknown, it also needs to be estimated. For several years, hidden Markov models have been proven to be an excellent avenue for obtaining these estimates  the gametic transition probabilities k, is simplyk. Also, assume that genotypes in k. After some simplifications  the transition probability, i.e., the conditional probability of a gametic genotype k, isThe construction of a genetic map involves the estimation of the genetic distance and order between markers within linkage groups. If the origin of the haplotypes , which are essentially biallelic. Due to this lack of identity between the observed data and the full transition space, we make use of the emission function, which is defined as the probability of observing a molecular phenotype given a genotype The initial state distribution is the probability of observing a specific genotype. Given the assumption that there is no preferential pairing during the formation of bivalents, a uniform probability density function can be employed as the initial state probability functiondosage of a SNP at a specific locus. The dosage of a SNP can be estimated using the ratio between the abundance of its two allelic forms. Several methods were proposed to perform this task including , the information contained in these markers does not propagate into the rest of the chain. Thus, based on the dosage and linkage phase configuration of the markers involved in the analysis, the m. The number of possible genotypic states in the progeny for a given locus at position k is k in parent P is two , In a two-point context, the likelihood function derived from any of the configurations belonging to the same partition . To be in accordance with molecular data that have been made available through sequence technologies, we simulated bi-allelic markers that can be observed in terms of dosage in parents and progeny. Three different linkage phase scenarios were simulated. In scenario A, for each marker, one of the allelic variants was assigned to the first homolog in the homology group and the remaining variants of the same type were assigned to the subsequent homologs. In scenario B, the allelic variant was randomly assigned to one of the first m homologs. Thus, it is expected an increasing difficulty to detect recombination events from scenario A, where the allelic variants were concentrated in the same homologs, to scenario C, where they were randomly distributed.the aim of this simulation study was to evaluate the local performance of the algorithm considering three ploidy levels . For each parental configuration, we simulated 200 full-sib populations of 200 offspring considering a combination of three levels of preferential pairing  and three levels of cross-like quadrivalent formation proportion  with the position of the pairing partner switch varying across simulations. No hexavalents were simulated in this study. For autohexaploids, the multivalent configurations were always composed by a cross-like quadrivalent plus a bivalent. The centromere was positioned at 20.0 cM from the beginning of the chromosome (subtelocentric centromere with arms ratio 1:4) to study the effect of the double reduction which is more pronounced at the end of both chromosome arms. All simulations were conducted using the software PedigreeSim . In addiP and Q. In scenario (A) the method was capable of recovering the correct linkage phase configuration in all situations for all ploidy levels. In scenarios (B) and (C) there was a slight decrease in the ability to correctly estimate the linkage phase configuration, especially for The proportion of correctly estimated linkage phase configurations for the dense chromosome-wise map is shown in Q in configuration 1. Again, the use of a higher two-point threshold level, Within the preferential pairing level 0.25, results showed decay of correctly estimated linkage phases, which was more pronounced for hexaploid cases with threshold level Given a correctly estimated linkage phase, the recombination fractions were consistently estimated for all levels of preferential pairing with no quadrivalent formation . HoweverAtlantic and B1829-5. The population comprises 160 offsprings genotyped with the SolCAP Infinium 8303 potato array. The genotype calling was performed using fitTetra R package . Furthermore, our algorithm estimated the same linkage phase in both cases, indicating the robustness of the phasing method to local marker misplacement. To address the genotyping errors, we used the approaches presented in Equations 8 and 9, i.e., ad hoc error rate of 5%. We applied this prior information in both de novo MDS-based and the genome-based orders. The result also can be observed in Table S8.1 and Figure S8.1. Both approaches produced smaller maps when compared to their relative original maps. However, since de novo MDS-based and the genome-based order when modeling a global error was less than 10 cM.While obtaining a Among the available methods to construct maps in high-dose autopolyploids, namely, pergola , and poligreeSim  consider\u03b7 were used: 3 and 5; the same levels were used for the multilocus LOD threshold. The phase search was limited to the last 50 markers inserted at the end of the map. To construct maps using polymapR, we first applied the function cluster_SN_markers to perform a grid search from LOD Scores 1 to 20 and chose the lowest one that yields six homologs based on simplex markers in coupling linkage phase. In the next step, we assigned double simplex and duplex markers to the linkage group using the function assign_linkage_group, and the remaining marker types were assigned using the function homolog_lg_assignment. This procedure was performed for both parents assuming LOD Score thresholds of 3 and 5. All remaining pairwise recombination fractions were computed, and the MDS algorithm . Although in general, our method yielded denser maps, it is worthwhile to mention that, polymapR\u2019s method is substantially faster than ours, notably when our method uses high values for \u03b7, in which case the HMM computations play a significant role in the phasing procedures . Nonetheless, it was precisely the multipoint procedure that allowed our method to position more markers when compared to polymapR.Table S10.2, Supplementary Information, shows the average map length and the associated standard deviation obtained in all simulations. In cases with a high number of single-dose markers, the average map length produced by polymapR ranged from 87.0 to 89.9 cM, while in the case where the dosages where uniformly assigned, map lengths ranged from 76.7 and 80.1 cM. These results confirm the underestimation tendency in the MDS algorithm when using when using Although the concept of linkage mapping is relatively simple, the combinatorial properties and increasingly missing information that arise from the multiple sets of chromosomes make the construction of genetic maps in high-level autopolyploids challenging. In this work, we frame and solve two fundamental steps toward the construction of such maps, namely multipoint recombination fraction estimation and linkage phase estimation. We showed that, combined with standard grouping and ordering procedures , these mlgorithm ; conside\u03b7) improved the linkage phase estimation in all cases. This fact indicates that the haplotype phasing is more accurate when HMM-based likelihood is used as objective function to evaluate linkage phases. We also observed that quadrivalent formation yield overestimated recombination fractions between adjacent markers located further away from the centromere. Interestingly, To assess the statistical power of our method, we conducted two simulation studies. In simulation 1, we demonstrated that our model was capable of correctly estimating the majority of parental linkage phase configurations and recombination fractions in a limited number of markers, even for complex linkage phase configurations and high ploidy levels. Since other methods are based on single-dose markers to assemble homology groups, to the best of our knowledge, this is the only method capable of phasing markers in high-dose autopolyploid genomes in small regions. These well-assembled regions could function as multiallelic codominant markers which propagate their information through the HMM to the rest of the chain, improving the quality of the final map. In simulation 2, we analyzed a sequence of 200 markers in combinations of different levels of preferential pairing and rates of quadrivalent formation. In this situation, quadrivalent formation rate had a marginal effect on the phasing procedure, whereas preferential pairing reduced its performance, especially for autohexaploids. The usage of a higher two-point threshold  the outbred nature of the experimental crosses and (ii) the incomplete information of the markers based on dosage . In experimental population derived from inbred lines, the origin of the haplotypes can be easily inferred from the genetic design. However, obtaining pure inbred lines in high-level autopolyploids has been proven to be impractical due to the high number of crosses and generations necessary to achieve homozygous genotypes and to the inbreeding depression which some species undergo  and between loci (epistatic effects). Therefore, the present study provides a sound basis for unveiling the complex structure of autopolyploid genomes through genetic mapping."}
+{"text": "Previous studies on linkage disequilibrium have investigated second order linkage disequilibrium in animal and plant populations. The objective of this paper was to investigate the genome-wide levels of third order linkage disequilibrium in a composite line founded by admixture of four Iberian pig strains. A model for the generation of third order linkage disequilibrium by population admixture is proposed. A computer Expectation-Maximization algorithm is developed and applied to the estimation of third order linkage disequilibrium at inter- and intra-chromosomal level using 26,347 SNPs typed in 306 sows. The relationship of third order linkage disequilibrium with physical distance was investigated over 35 million triplets in SSC12. Basic and normalized estimates of inter and intra-chromosomal third order linkage disequilibrium are reported.Genome-wide analyses revealed that third order linkage disequilibrium is rather common among linked loci in this Iberian pig line. It is shown that population admixture of multiple populations may explain the observed levels of third order linkage disequilibrium although it could be generated by genetic drift. Third order linkage disequilibrium decreases rapidly up to 4\u00a0Mb and then declines slowly. The short distances between consecutive markers explain the maintenance of the observed third order linkage disequilibria levels when using a model incorporating the break-up of disequilibrium by recombination. Genome-wide testing also revealed that only 3.6% of the normalized estimates were different from 1, \u2212\u20091, 0, or from a not well-defined situation in which there is only one possible value for the third order linkage disequilibrium parameter, given allele frequencies and pairwise linkage disequilibria parameters.Third order linkage disequilibrium is common among linked markers in the analyzed pig line and may have been generated by population admixture of multiple populations or by genetic drift. As with second order linkage disequilibrium, the absolute value of the third order linkage disequilibrium decreases with physical distance. Normalization of third order linkage disequilibrium should be avoided for closely linked bi-allelic loci.The online version of this article (10.1186/s12863-018-0661-4) contains supplementary material, which is available to authorized users. Linkage disequilibrium is defined as the non-random association of alleles at two or more loci. In many instances it is due to the physical organization of DNA sequences in which each nucleotide follows another in one single chain. It can also be due to genetic drift or selection. Linkage disequilibrium is a key parameter to understand evolution and as stated by Slatkin, it is an indicator of the population genetic forces that structure a genome , 2. In tThe classification and number of parameters characterizing linkage disequilibrium for any number of loci is given in Table\u00a0All this research was carried out in the eighties and nineties when the development of genetic markers was still in its infancy (with microsatellites at their peak use) but with little coverage of animal genomes for today\u2019s standards. The development of array technologies incorporating from thousands to hundreds of thousands of Single Nucleotide Polymorphisms (SNPs) has provided new tools to uncover the associations between alleles at different loci located elsewhere in the genome. Kim et al.  proposedThe objective of this paper is to investigate third order linkage disequilibria using a 60\u00a0K SNP array of Illumina in a closed population of Iberian pigs. This is the first report on the extent of third order linkage disequilibria in animal genomes. In order to carry out extensive third order linkage disequilibrium estimation, a simple and efficient EM algorithm for the estimation of third order linkage disequilibrium of biallecic markers was also developed. In addition, the way that third order linkage disequilibrium is generated by population admixture was also investigated.K with haplotypes TM and tm, and allele k with haplotypes Tm and tM. For example,\u00a0if the three loci are very closely linked (assuming no recombination) and each arrow between haplotypes represents a mutation then TMK, tmK, tMk, and Tmk\u00a0must exist for full third order linkage disequilibrium. This requires two mutations (arrows) for\u00a0creation of each haplotype\u00a0and also the\u00a0loss of intermediate haplotypes; for unlinked loci, a combination of haplotypes could also\u00a0exist by the loss of the other haplotypes by either genetic drift or selection, which is accelerated by free recombination. Population admixture could facilitate the creation of third order linkage disequilibrium by disconnecting the processes of genetic drift and selection in the two populations. A population admixture model to generate third order linkage disequilibrium is introduced in the next section.Second order linkage disequilibrium in closely linked loci exists because one mutated allele is associated to a short stretch of an ancestral haplotype. Breaking down of associations by recombination erodes second order linkage disequilibrium but genetic drift can generate linkage disequilibrium again if populations are small. In addition, second order linkage disequilibrium exists because of co-selection of close or distant loci affecting quantitative traits. On the contrary, third order linkage disequilibrium cannot be generated itself by mutation between closely linked loci unless several haplotypes are lost by genetic drift. Figure\u00a0T/t, M/m, and K/k be located in that order on a chromosome. We will assume that these loci are not affected by selection.It is well established that the crossing between two populations differing in allele frequencies at two loci may generate second order linkage disequilibrium . In this\u03c4 is the mixing proportion of the two populations at crossing. The haplotype frequencies at the two populations at crossing can be put in terms of allele frequencies and second and third order linkage disequilibrium parameters:The frequency of haplotype TMK at the cross Z isX; . The same coefficients but with superscript Y are for population Y.where T/t, M/m and K/k in the crossed populations are given by:The allele frequencies at Then, the third order linkage disequilibrium in the crossed population, Z, is:After substituting eqs. , 2), an, an2), a\u03b3T, \u03b3M, and \u03b3K represent the difference in allele frequency in the two populations at crossing for loci T/t, M/m, and K/k, respectively. A full derivation of eq.  or two-locus haplotype (one dot) frequencies\u00a0corresponding to\u00a0haplotype ijk. For exmple\u00a0f.jk is the frequency of haplotype with alleles jk \u00a0at\u00a0the last two loci, M/m and K/k.\u00a0This model assumes no interference.where t, by making use of the dynamics of haplotype and allele frequencies. For example, for ijk\u2009=\u2009TMK the disequilibrium isThird order linkage disequilibrium can be obtained in each generation, \u03b4TM\u2009=\u2009\u03b4TK\u2009=\u2009\u03b4MK\u2009=\u20090). In this situation, \u03b4TMK must range between TMK, tmK, Tmk, tMk), none of them with alleles complementary to each other. We investigated the break-up of linkage disequilibrium in this situation, in which third order linkage disequilibrium is the highest possible. The break-up of third order linkage disequilibrium was computed with the equation of third order linkage disequilibrium , second order , and third order linkage disequilibrium parameters (\u03b4TMK) are:Consider again three SNPs, eiringer , Bennet eiringer  and Thomeiringer , the hapfT\u00a0=\u2009fM\u00a0=\u2009fK\u2009=\u20090.5) and zero second order disequilibria between all pairs (\u03b4TM\u2009=\u2009\u03b4TK\u2009=\u2009\u03b4MK\u2009=\u20090). After using equation (6), the haplotype frequencies are: \u03b4TMK must range between fTmk\u2009=\u2009ftMK\u2009=\u2009fTMk\u2009=\u2009ftmk\u2009=\u20090. Then, only four haplotypes are segregating , none of them with complementary alleles to each other.Intermediate allele frequencies at the three loci (fT\u00a0=\u2009fM\u00a0=\u2009fK\u2009=\u20090.5), and maximum second order LD (\u03b4TM\u2009=\u2009\u03b4TK\u2009=\u2009\u03b4MK\u2009=\u20090.25). Then, the haplotype frequencies are fTmK\u2009=\u2009\u2009\u2212\u2009\u03b4TMK, ftMK\u2009=\u2009\u2009\u2212\u2009\u03b4TMK, ftmK\u2009=\u2009\u03b4TMK, fTMk\u2009=\u2009\u2009\u2212\u2009\u03b4TMK, fTmk\u2009=\u2009\u03b4TMK, ftMk\u2009=\u2009\u03b4TMK, and \u03b4TMK\u2009=\u20090 and ,Intermediate allele frequencies for the three loci  at intermediate allele frequencies and in absence of any second order linkage disequilibrium. That is, allele K will be associated to haplotypes TM and tm, and allele k to haplotypes Tm and tM. A similar argument can be done for \u03b4TMK\u2009=\u2009\u2212\u20091/8 with allele k is associated to haplotypes TM and tm, and allele K to haplotypes Tm and tM.Summarizing, the range of possible values of the third order linkage disequilibrium are between \u2212\u20090.125 and\u2009+\u20090.125 and equal to zero when second order linkage disequilibria are at their maximum values. Only haplotypes The likelihood to estimate linkage disequilibrium parameters and allele frequencies for three loci when using genotypic information data from diploid individuals is:K is a constant, NG is the triplet genotypic information on diploid individuals, i-th triplet genotype, and ni is the number of individuals (counts) having the i-th triple genotype in the population. By triple genotype, we mean the joint genotype at the three loci T/t, M/m, and K/k. The 27 triplet genotype probabilities, \u03c6i can be obtained from Table\u00a0TtMmKk, is \u03c6TtMmKk\u00a0=\u20092(fTMKftmk\u00a0+\u2009ftMkfTmK+ fTmkftMK+ fTMkftmK) and the corresponding number of observed triple heterozygotes is nTtMmKk.where entclass1pt{minima.. [I)Set initial haplotype frequencies (arbitrarily),II)Expectation step in which genotype frequencies are estimated based on haplotype frequencies from step I. In order to resolve to which haplotypes may correspond observed genotype counts the proportion of double or triple heterozygotes in coupling or repulsion for each haplotype needs to be computed. In programing in Fortran we used code 3 for individuals with the heterozygote genotype. For example, for haplotype \u201c111\u201d, the proportion of individuals with genotype heterozygote at the two first loci and homozygote with allele 1 at the third loci is:The likelihood equation is maximized for a third order linkage disequilibrium parameter, three second order linkage disequilibrium parameters, and three allele frequencies corresponding to each of the three SNPs. Therefore, the model has seven degrees of freedom corresponding to the eight haplotypes minus one. Solving equation  is not t..  pointed .. , require..  and justSimilarly, the proportion for homozygote  individuals at the first locus and heterozygote at the last two loci is:In the same way, the proportion of individuals with other genotypes are:W. This process is done for the eight haplotype frequencies.III)The maximization step consists in estimating haplotype frequencies using genotype counts observed or as estimated in step II. For example for the haplotype \u201c111\u201d, the frequency to be estimated is:The products between frequencies of haplotypes in the denominator correspond to all possible combinations of complementary haplotypes that could result in genotype as the subscript of wijk represents the counts for genotype ijk with values 1 or 2 for alleles 1 or 2, and 3 for the heterozygote. All other seven haplotypes are constructed in the same way.IV)Go to step II until convergence of haplotype frequencies is reached.where This algorithm is simple and suitable for fast computing when implemented in a computer language such as Fortran90. In this implementation, the number of individuals for each triple genotype is stored in an array with three dimensions. Each one corresponds to one locus and there are three alternatives: \u201c1\u201d and \u201c2\u201d are used for homozygotes, and \u201c3\u201d for heterozygotes. Source code for estimating haplotype frequencies in Fortran90 is provided in the Additional\u00a0file\u00a0Genotypes from 306 sows belonging to a composite line  genetically isolated between 1963 and 2013 and resulting from the blending of four ancient Spanish and Portuguese Iberian breed strains , were usDNA samples were isolated from blood using a standard phenol/chloroform protocol and genotyped with the Illumina Porcine SNP60 BeadChip  and the Estimation of the third order linkage disequilibrium parameter was carried out for all triplets of three consecutive SNPs for each of the autosomal chromosomes. This will be referred to as short range intra-chromosomal third order linkage disequilibrium. The total number of triplets was 26,311. In addition, inter-chromosomal third order linkage disequilibrium was estimated by randomly drawing three out of the 18 autosomal chromosomes and by selecting randomly one SNP within each chromosome. In order to make an easier comparison between inter and intra-chromosomal third order linkage disequilibrium, the process was repeated 26,311 times, the same number as for the intra-chromosomal third order linkage disequilibrium. Similar to the second order linkage disequilibrium, the third order linkage disequilibrium is expected to be negligible in most inter-chromosomal situations.\u03b2TMK, with the number of haplotypes and a likelihood ratio test was investigated. The likelihood ratio test was computed using the full model, i.e., estimating all parameters versus a model with only allele frequencies  was carried out in order to investigate the relationship between third order linkage disequilibrium and physical distance. Third order linkage disequilibria were estimated among all possible triplets in a rolling chromosomal fragment of 400 SNPs. The total number of analyses was 35,784,200. In addition, the relationship of the proposed normalized third order linkage disequilibrium g et al. ).The range of possible values of the third order linkage disequilibrium is between \u2212\u20090.125 and\u2009+\u20090.125 and is equal to zero when second order linkage disequilibria are at their maximum values. Table\u00a0c) is assumed between every two consecutive markers. The break-up of third order linkage disequilibrium is quick at a high recombination fraction but much lower for markers with low recombination fraction. It may take many generations for the LD to be fully eroded. For example, for c\u2009=\u20090.001 (~100kb), third order linkage disequilibrium reduces only marginally in 20 generations.Once third order linkage disequilibrium is generated by population admixture, selection or genetic drift, its value is eroded by recombination. Figure\u00a0Figure Plots of both inter- and intra-chromosomal third order linkage disequilibria versus two of the second order linkage disequilibria parameters illustrate the relationship of second and third order disequilibria Fig.\u00a0. There iFollowing Robinson et al. (1991), the results of the estimates of normalized third order linkage disequilibrium were 0 (14.9%), \u00b1 1 (20%), or an undefined situation in which the third order disequilibrium parameter is restricted to one single value given allele frequencies and second order disequilibrium parameters (61.5%). Only 3.6% of all the analyses resulted in a third order parameter different from 0, 1, \u2212\u20091 or the undefined situation. This may be related to the number of haplotypes  because there was not a single estimate of undefined situation, 1, \u2212\u20091 or 0 among the 309 triplets with eight haplotypes. There were only seven out of 1648 triplets for which estimates of normalized third order linkage disequilibrium were different from undefined situation, 1, \u2212\u20091 or 0 among triplets with just two haplotypes. Therefore, normalizing third order linkage disequilibrium as proposed by Robinson et al., (1991) is not useful for deciphering situations involving closely linked markers.Figure In addition, the use of the newly proposed measure to estimate the proportion of third order linkage disequilibrium revealed that a high proportion of third order linkage disequilibrium versus all disequilibria seems to be associated to a larger number of haplotypes Fig.\u00a0.Fig. 8HeK must be associated to haplotypes TM and tm, and allele k to haplotypes Tm and tM. A fictive metabolic pathway that could represent this situation is represented in Fig.\u00a0M increase products A and B, respectively. Alleles t and m reduce products A and B. Allele K at the third loci would only function for intermediate levels of product B to react to product C. Individuals with haplotypes TM would generate large amounts of product B. Individuals with haplotypes tm would generate small amounts of product B. Individuals with haplotypes Tm or tM would generate intermediate amounts of product B. Therefore, genetic systems with an excess of haplotypes TmK and/or tMK with respect to equilibrium would be selected when product C provides increased adaptation in a given environment. Thus, some metabolic pathways may trigger reactions for intermediate values of a product since merely second order linkage disequilibrium may not be enough to unravel the complexity of living organisms.The importance of gene interactions in the expression of phenotypic traits is gaining interest in the scientific community . The bioThe results obtained in this study are based on the Expectation Maximization algorithm , 21, whiNormalization of third order linkage disequilibrium  for closIt has been proposed that the difference between normalized pairwise disequilibria estimated from analyses of third and second order disequilibria should shed light on hitchhiking selection in a method called \u201cconstrained disequilibrium values\u201d , 23. RobIf interested in testing the third order linkage disequilibrium at specific locations, one would carry out hypothesis testing by means of a likelihood ratio test within the maximum likelihood framework. This is not straightforward because the expected haplotype frequencies in the reduced model  are notThird order linkage disequilibrium can be generated by population admixture. It follows a similar pattern as second order linkage disequilibrium. Needing, in addition to a difference in allele frequencies of the two populations at crossing, differences in their pairwise linkage disequilibrium parameters. The observed levels of third order linkage disequilibrium in the analyzed Torbiscal line led us to conclude that this disequilibrium might have been generated after the crossing of multiple strains of Iberian pigs some 50\u00a0years ago. Alternatively, genetic drift may have also had a role given the small population size of this strain. More research using other animal, plant or human populations with a different population history may help to understand if the levels of third order linkage disequilibrium are high, when relating to the crossing history of the populations in question.Once population admixture has generated third order linkage disequilibrium, the three-locus disequilibrium declines exponentially over time by recombination . HoweverThe use of composite lines in pig breeding schemes is becoming quite popular for both sire and dam lines . The levThe main conclusions of this paper are: a) the existence of third order linkage disequilibrium is substantial in a composite Iberian pig line, b) third order linkage disequilibrium in this strain might have been generated by admixture of four strains of Iberian pigs, c) the absolute value of the third order linkage disequilibrium decreases rapidly with a physical distance above 4\u00a0Mb, d) the number of haplotypes is much reduced for linked loci due to the mutual constraints of pairwise and third order linkage disequilibrium parameters, and e) normalization of third order disequilibria is not advised for closely linked biallelic loci. High order linkage disequilibrium might shed light on our understanding of the complex metabolic pathways in which multiple loci are involved. Much of the actual variation in quantitative traits might go unnoticed when analyzing just one or two loci at a time.Additional file 1:Appendix 1. Derivation of third order disequilibrium in population admixture. (DOCX 19 kb)Additional file 2:Appendix 2. Subroutine to estimate third order linkage disequilibrium. (DOCX 16 kb)"}
+{"text": "The figures and captions are in the incorrect order for Figs"}
+{"text": "Gait speed is an indicator of health and function with aging. The potential genetic contributions to gait speed and its decline with aging are not well characterized. We sought to better quantify the genetic contributions to and identify potential genes and genetic variants underlying change in gait speed among older adults. To accomplish these aims, we used data from 2379 individuals belonging to 509 families in the Long Life Family Study . Gait-speed was measured over 4 meters at baseline and after an average of 7\u00b11.1 years. Quantitative trait linkage analyses were completed using pedigree-based maximum-likelihood methods with logarithm of the odds (LOD) scores > 3.3 indicating genome-wide significance. We also performed linkage analysis in the top 10% of families contributing to LOD scores to allow for heterogeneity among families (HLOD). Data were adjusted for age, sex, height and field center. At baseline, 26.9% of individuals had \u201clow\u201d gait-speed <1.0 m/s (mean: 1.1\u00b10.2 m/s) and gait speed declined at a rate of -0.02\u00b10.03 m/s per year (p<0.0001). Baseline and change in gait-speed were significantly heritable . We did not find significant evidence for linkage for baseline gait speed; however, we identified a potentially novel locus for change in gait speed on chromosome 16p (LOD 4.2). A subset of 21 families contributed to this linkage peak (HLOD = 6.83). Sequence analysis of the chromosome 16 region may yield new insight on the biology of age-related mobility decline."}
+{"text": "Moreover, the expression of rNod2 was significantly upregulated in the heart, liver, and spleen induced by enterohemorrhagic Escherichia coli (EHEC). Overexpression of rNOD2 induced the expression of pro-inflammatory cytokine, including Il1\u03b2, Il6, Ifn-\u03b3, and Tnf, as well as defensins, including Defb124, Defb125, and Defb128 through the nuclear factor (NF)-\u03baB signaling pathway. Furthermore, overexpression of rNOD2 inhibited the growth of EHEC, and knockdown of rNOD2 or inhibition of the NF-\u03baB pathway promoted its replication. In addition, our results suggest that rNOD2 can significantly activate NF-\u03baB signaling and trigger antibacterial defenses to increase the expression of pro-inflammatory cytokine and defensins after stimulation by EHEC. These findings are useful to further understanding the innate immune system of rabbits and providing a new perspective for the prevention of bacterial diseases in rabbits.Nucleotide-binding oligomerization domain 2 (NOD2), a member of the NOD-like receptors (NLRs) family that is well-known to play a key role in innate immune responses and is involved in innate antibacterial responses. In this study, rabbit NOD2 (rNOD2) was cloned from rabbit kidney (RK) cells. It was distributed in various tissues, and the highest level of  The innate immune system plays a crucial role in the non-specific immune response, and is essential for triggering the acquired immune response against microbial pathogens. The typically conserved exogenous microbial components termed pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns are recognized by host germline-encoded pathogen recognition receptors, which can be grouped into four families: nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), toll-like receptors, retinoid acid-inducible gene-1-like receptors and C-type lectin receptors (CLRs) . Moreover, an increase in bacterial colonization and a reduction in neutrophil, cytokine, and chemokine were also detected , which is produced by all bacteria  in weaned rabbits results in large losses in rabbit breeding. To date, the rabbit immune system and the relationship between it and pathogen recognition receptors are rarely studied. Although the crystal structure of rabbit NOD2 (rNOD2) has been studied  was obtained from MedChem Express . MDP was obtained from InvivoGen .The EHEC used in this study was isolated from clinically acute diarrhea rabbits and grown in a nutrient broth medium at 37\u00b0C for 12 h.2.RK-13 cells were cultured in Dulbecco's modified Eagle medium  supplemented with 10% fetal bovine serum  at 37\u00b0C and 5% (v/v) COThirty-five-day-old healthy New Zealand White rabbits were purchased from a farm, and given sufficient water and feed. All animal experiments were handled in accordance with the appropriate biosecurity guidelines. This study was carried out in accordance with the recommendations of the Shandong Agricultural University Animal Care and Use Committee (no. SDAUA-2015-005).TransZol up (Transgen) and cDNA synthesis was carried out with TransScriptR One-step gDNA Removal and cDNA Synthesis SuperMix (Transgen). To obtain the sequence of rNOD2, degenerate primers were designed based on the predicated gene (NCBI XM_008261590.2) .8 CFU per rabbit). Five rabbits from each group were randomly selected and killed, and the kidney, liver, and spleen were collected at 1, 2, and 3 days post infection (dpi). Primers of qdrNOD2 F and qdrNOD2 R were selected for analysis expression of rNOD2  for 24 h. pCDNA3.1 (+) vector was used as a control.The rNOD2 was amplified with the primers in Table Cells were fixed with 4% paraformaldehyde and then permeabilized with 0.1% Triton X-100 for 10 min. After blockade of nonspecific binding with 5% bovine serum albumin in PBS for 30 min, cells were incubated with HA Tag Monoclonal antibody , and then incubated with fluorescein isothiocyanate (FITC)-goat anti-mouse IgG. Images were visualized with a Leica fluorescence microscope.Cells were lysed with ice-cold RIPA buffer containing a protease inhibitor (Beyotime). Protein samples were boiled in SDS sample buffer, and then run on SDS-PAGE. The separated proteins were transferred to a PVDF membrane. Membrane was blocked with 5% skim milk for 1 h. HA tag monoclonal antibody was used to detected rNOD2. Then Membrane was incubated with secondary antibodies. \u03b2-actin was used as control. Protein bands were visualized with a ChemiDoc XRS  by a Western ECL Substrate kit.TransIL-LT1 Transfection Reagent. NC siRNA was used as a control. The si-RNA sequences that we used were as follows: si-rNOD2-1, sense 5\u2032-CCUGGACACUGUCUGGAAUTT-3\u2032, antisense 5\u2032-AUUCCAGACAGUGUCCAGGTT-3\u2032; si-rNOD2-2, sense 5\u2032-GCAGGACUUUCAGGAAUUUTT-3\u2032, antisense 5\u2032-AAAUUCCUGAAAGUCCUGCTT-3\u2032; si-rNOD2-3, sense 5\u2032-GCACUGAGUUCAACCUCAATT\u22123\u2032, antisense 5\u2032-UUGAGGUUGAACUCAGUGCTT\u22123\u2032;NC, sense 5\u2032-UUCUCCGAACGUGUCACGUTT-3\u2032, antisense 5\u2032-ACGUGACACGUUCGGAGAATT-3\u2032.The siRNA sequences  targeting the rNOD2 and negative control (NC) siRNA were synthesized by company . One microgram of siRNA and NC siRNA were transfected with TransIL-LT1 Transfection Reagent. After 24 h, the cells were lysed and harvested, and luciferase activities were examined by a dual-luciferase reporter assay system .The RK-13 cells were cultured in 24-well plates and grown in standard conditions for 12 h prior to transfection. The luciferase reporter plasmids (pGL3-NF-\u03baB and pGL3-IFN-\u03b2) were purchased from Agilent . The pRL-TK plasmid  acted as an internal control to normalize transfection efficiency. 1 \u03bcg of pC-rNOD2 or pCDNA3.1 (+) vector and 500 ng of si-rNOD2 or NC siRNA with 100 ng of reporter plasmid and 50 ng of pRL-TK plasmid were transfected into cells using TransIL-LT1 Transfection Reagent. pCDNA3.1 (+) vector and NC siRNA were used as control, respectively. After 24 h, EHEC (1 \u00d7 107 CFU) were incubated with RK-13 cells for 2 h. Then, the bacteria were removed and washed 3 times with PBS containing 100 \u03bcg/ml gentamicin. The RK-13 cells were cultured in DMEM containing 100 \u03bcg/ml gentamicin for 3 h. Then RK-13 cells were lysed in 1% Triton X-100. After 20 min, cell lysates were plated onto nutrient agar for intracellular CFU determination. Other RK-13 cells were harvested for RNA extraction.The RK-13 cells were cultured in 24-well plates and grown in standard conditions for 12 h prior to transfection. One microgram of pC-rNOD2 or 500 ng of si-rNOD2 was transfected into cells using Tnf and Il6 were as previously reported  based on published target sequences  as an endogenous reference gene.Total RNA was extracted from the tissues of rabbits and RK-13 cells and reversed as described above. qRT-PCR was carried out using the 7500 Fast Real-Time PCR System  with TransStartR Tip Green qPCR SuperMix (Transgen Biotech). The primers of P < 0.05.Data represent the means \u00b1 standard deviations. Statistical analyses were performed by SPSS 19.0 software , the significant difference between control and the treated group were evaluated by non-parametric Mann-Whitney U test. We adopted a level of statistical significance of MF125932). As observed in Table Ochotona princeps (91.7%) and Macaca nemestrina (86.5%). Larimichthys crocea and Takifugu rubripes were at the bottom of the table.The full-length sequence of rNOD2 was obtained from RK-13 cells. The open reading frame contains 3,042 bp and encodes a protein of 1,013 amino acids. The nucleotide sequence of rNOD2 has been deposited to the GenBank  of expression 1 dpi in the liver, and peaked at 2 dpi   at 3 dpi  at 3 dpi in the kidney  after 24 h transfection. In addition, our results also showed that rNOD2 could drive IFN-\u03b2 expression  by rNOD2 overexpression. When the expression of \u03b2-defensin Defb124, Defb125, Defb128, Defb135, and \u03b1-defensin Np5 was investigated, all of these genes exhibited significant upregulation with the overexpression of rNOD2 except for Defb124.To study the gene expression level of cytokines and defensins by rNOD2, RK-13 cells were transfected with pC-rNOD2 or pCDNA3.1 (+) vector. As shown in Figure P < 0.05, Figure P < 0.05, Figure P > 0.05, Figures si-rNOD2-1, si-rNOD2-2, and si-rNOD2-3 were designed to target the different positions of rNOD2. The knockdown efficiency of Si-rNOD2-3 was 37.25% Figure . rNOD2 iIl1\u03b2  by rNOD2 overexpression . Similarly, the expression of defensins Defb125 (2.18-fold) and Defb128 (2.66-fold) was significantly increased.As shown in Figure Defb125 and Defb128 was significantly downregulated by 0.18-fold and 0.12-fold (P < 0.05). Although the expression of all pro-inflammatory cytokines was reduced, only the expression of Ifn-\u03b3 was significantly downregulated by 0.32-fold (P < 0.05; Figure To further investigate whether NF-\u03baB pathway was activated by rNOD2, cells were pretreated with inhibitors of NF-\u03baB. After inhibition of the NF-\u03baB pathway, overexpression of rNOD2 unable increase the expression of pro-inflammatory cytokines and defensins induced by EHEC. The expression of all defensins was significantly decreased. Especially, the expression of To explore the ability of rNOD2 to inhibit EHEC, RK-13 cells were infected with EHEC after transfection with rNOD2 and si-rNOD2, and the EHEC counts were calculated. As shown in Figure O. princeps, M. nemestrina, Pan troglodytes, and Homo sapiens NOD2 indicated 91.7, 86.5, 86.4, and 86.4% identity, respectively. To understand the evolutionary relationship of rNOD2, phylogenetic tree was generated based on NOD2 protein of various species, and rNOD2 belonged to the branch of mammals. Similar results indicate that rNOD2 was closer related to O. princeps NOD2 than to M. nemestrina, P. troglodytes, and H. sapiens NOD2.In this report, we cloned rNOD2 in RK-13 cells, and its open reading frame encodes a protein of 1,013 amino acids. Similar to human and mouse NOD2, rNOD2 does not contain signal peptide so that in can release the mature proteins into intracellular space, indicated that rNOD2 is intracellular cytosolic sensors and expressed mainly in the cytosol (Inohara and Nu\u00f1ez, rNod2 in order to replicate in the spleen. The upregulation of the rNod2 at 3 dpi indicates the rNod2 likely participates in the innate immune defense against EHEC infection.NOD2 is an essential protein for innate immune responses, results of tissue distribution showed a broad expression spectrum. The highest level of rNOD2 was detected in the spleen, duodenum, and jejunum. Similar results have been observed in newborn swine, the NOD2 expressed in varies tissues with different level, and the highest expression in the mesenteric lymph nodes and spleen (Tohno et al., Il6, Il8, Tnf, and Defb135 significantly induced by overexpression of rNOD2. The defensins contribute to innate immunity response and are crucial part of the first line of defense for various pathogenic microbes. They display a broad range of antimicrobial activities and are effective against viruses, gram-negative bacteria, gram-positive bacteria, and fungi (Tavares et al., Il1\u03b2, TNF-a, Il6, and Il8, I- and II-IFN, as well as the antibacterial peptide cathelicidin-2 (Chang et al., In humans, NOD2 induces the activation of NF-\u03baB, which is mediated through homophilic CARD-CARD interactions with receptor-interacting protein 2 (Ogura et al., Il1\u03b2, Il6, Ifn-\u03b3, and Tnf, as well as \u03b2-defensins, including Defb124, Defb125, and Defb128. In contrast, knockdown of rNOD2 inhibited the expression of pro-inflammatory cytokines and \u03b2-defensins. Our results are consistent with a previous research that macrophages from NOD2\u2212/\u2212 mice could not produce pro-inflammatory cytokines or undergo NF-\u03baB activation after MDP challenge (Kobayashi et al., It has been demonstrated that NOD2 is mainly associated with antibacterial immune responses in mammals (Meylan et al., in vivo. In addition, rNOD2 might activate NF-\u03baB signaling and promote the expression of pro-inflammatory cytokine and defensins in RK-13 cells infected with EHEC. Most important is that rNOD2 can inhibit the growth of EHEC by activating NF-\u03baB in vitro. In the future, it is believed that rNOD2 will be provided novel therapeutic perspective against bacterial infections based on its antibacterial ability.In conclusion, we cloned the rNOD2 gene from RK-13 cells and demonstrated that it was widely distributed in the tissues of rabbits. rNOD2 was involved in immune response to the EHEC infection MG and RL conducted the study and wrote the manuscript. QX and XF collected samples. NL and YS analyzed data. LW and TC designed the study and revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer SM and handling Editor declared their shared affiliation."}
+{"text": "Record linkage is an important tool for epidemiologists and health planners. Record linkage studies will generally contain some level of residual record linkage error, where individual records are either incorrectly marked as belonging to the same individual, or incorrectly marked as belonging to separate individuals. A key question is whether errors in linkage quality are distributed evenly throughout the population, or whether certain subgroups will exhibit higher rates of error. Previous investigations of this issue have typically compared linked and un-linked records, which can conflate bias caused by record linkage error, with bias caused by missing records (data capture errors).Four large administrative datasets were individually de-duplicated, with results compared to an available \u2018gold-standard\u2019 benchmark, allowing us to avoid methodological issues with comparing linked and un-linked records. Results were compared by gender, age, geographic remoteness  and socioeconomic status.Results varied between datasets, and by sociodemographic characteristic. The most consistent findings were worse linkage quality for younger individuals  and worse linkage quality for those living in remote areas (seen in three of four datasets). The linkage quality within sociodemographic categories varied between datasets, with the associations with linkage error reversed across different datasets due to quirks of the specific data collection mechanisms and data sharing practices.These results suggest caution should be taken both when linking younger individuals and those in remote areas, and when analysing linked data from these subgroups. Further research is required to determine the ramifications of worse linkage quality in these subpopulations on research outcomes.The online version of this article (10.1186/s12913-018-3495-x) contains supplementary material, which is available to authorized users. Record linkage is a set of methodologies designed to bring together information relating to the same person from within or across datasets . This teStudies using linked data will generally contain linkage error. There are two types of record linkage errors; false positives, where two records are designated as belonging to the same individual when in truth they do not; and false negatives, where two records are designated as belonging to different individuals when in truth they belong to the same individual. Linkage error can occur due to both legitimate changes in a person\u2019s particulars (i.e. change of address) or due to data fields being missing or in error (i.e. poor recording practices in the data collection in question). Researchers using linked data will generally not have the personally identifying information made available to them for privacy reasons . This meA number of studies have illustrated how poor linkage quality can cause bias and distort results. A study of the effect of linkage methods (two deterministic strategies and one probabilistic strategy) on mortality rate estimates showed relative differences of up to 25% between the true estimate and that found through record linkage . In a stA key question is whether errors in linkage quality are distributed evenly across a study population. In other words, do the linked records of people from certain subgroups  contain a greater proportion of errors? If a certain subgroup was found to have lower linkage quality, this would suggest research results might be systematically biased against this group.Thi), which will make their data harder to link. Different ethnic groups may also display different rates of identifier reporting; for instance, in the United States, African American adults are less likely to report social security number, a highly identifying attribute, making their data harder to link [There are a number of plausible reasons why linkage quality may vary between subgroups. Key causes of linkage error are changing or incorrect identifiers . Given t to link . Recordi to link . Differe to link . A recen to link , indicatA number of studies have explored the relationship between linkage quality and sociodemographic factors, although typically as part of a wider study. A review by Bohensky et al.  found diTwo key issues arise when using this research design to explore bias caused by poor linkage quality. Firstly, the approach focusses only on false negative errors (records which incorrectly did not find a match) and does not consider the issue of false positive errors, an equally important type of linkage error. By only reporting on one of the two error types, we cannot gain an accurate understanding of the relationship between linkage quality and sociodemographic factors.A second and more fundamental problem in comparing sociodemographic subgroups using this methodology is that we often cannot distinguish whether an individual is unmatched due to linkage quality error, or due to the fact that these matched records do not exist. In many of these cases, differences in linkage bias arise from differences in subgroup data capture. Such differences can be expected to be highly dataset dependent. For example, in a recent US study linking a clinical surgical registry to US Medicare inpatient claims, the unmatched individuals in the clinical surgical registry were more likely to be those who did not have coverage under Medicare, rather than individual whose correct match could not be found . ConsequIn this paper, we attempt to address these methodological shortcomings through the use of an alternate research design. Four large, real-world Australian administrative datasets were de-duplicated, with results and compared to an available \u2018truth-set\u2019, allowing comparison of both false positive and false negative errors.n\u2009=\u20096,772,949), ten years of New South Wales (NSW) hospital admissions data , three years of NSW public emergency department (ED) presentation data  and three years South Australian (SA) ED presentation data . Each dataset contained errors typical of administrative data, including missing and incorrect identifiers, and identifiers that change over time. Each dataset had previously been de-duplicated  to a high quality by jurisdictional linkage units . These linkage units utilised a variety of deduplication methods including probabilistic record linkage, intensive manual review of created links and quality assurance procedures to analyse and review potential errors [Each of the four separate administrative health datasets used in the evaluation contained multiple event-based records per person. The datasets comprised: ten years of Western Australian (WA) hospital admission data , geographical region and socioeconomic status. Year of birth was split into three categories; those born prior to 1950, those born from 1950 to 1979, and those born from 1980 onwards . Indices of geographic remoteness  were usA de-duplication linkage was conducted on each of the four datasets, using the record linkage method described above, resulting in a series of matched record-pairs. Linkage quality was calculated for each of the sociodemographic characteristics by ignoring those record-pairs that did not include at least one record with that sociodemographic category. For example, linkage quality for individuals for remote areas was calculated by evaluating the linkage quality on all pairs of records of which at least one record was from a remote area. Pair-based linkage quality was calculated directly on these record-pairs. Additionally, the metrics were calculated for a range of threshold scores to investigate how linkage bias may change as the threshold is either increased or decreased.As clinical content data for each dataset was not available, statistics on the incidence of hospital encounters  were calculated for each socio-demographic- category, with results compared against results from the gold-standard benchmark.The number of records within each sociodemographic category for each dataset is shown in Table\u00a0Overall, the linkage quality achieved in the deduplication of each dataset was very high. Linkage results at the optimal threshold  are shown in Table\u00a0male\u2009=\u20090.981, recall female\u2009=\u20090.962). There was an identifiable effect of age on linkage quality, with persons born after 1980 having a lower linkage quality  for all four datasets, as compared to those born before 1950 . The effect of geographic remoteness on linkage quality was not as clear. In the NSW emergency, SA emergency and WA hospital datasets, records of persons in remote areas had lower recall compared with those in major cities  was only noticeably greater for those in remote areas in the WA hospital dataset . For socioeconomic status, the effect on linkage quality, in terms of precision and recall, was even less clear. In the WA hospital dataset, precision and recall both generally decreased as socioeconomic status decreased, with those in the upper quintiles having the highest linkage quality. For the NSW hospital dataset, however, the effect was the opposite, with precision and recall generally decreasing as socioeconomic status increased. Results for the NSW emergency dataset showed little difference in precision and recall between socioeconomic quintiles, while the results for SA emergency data did not show any clear trend.Variations in linkage quality by sociodemographic category for each of the four datasets, calculated at the optimal threshold score, are shown in Figs.\u00a0We further determined the optimal threshold score for each particular sociodemographic factor . However, this resulted in no substantial change in linkage quality .Comparisons of incidence rates for each dataset and sociodemographic category are shown in Table\u00a0There was some evidence that linkage quality varied across and within different sociodemographic categories. While some differences were found for gender and socioeconomic status, these results occurred only in particular datasets. The finding of poorer linkage quality for those in remote areas was more consistent, appearing in three of the four datasets. The most reliable finding was decreased linkage quality for younger individuals as compared to older individuals, found in all four datasets.There are a number of reasons why poorer linkage quality may be expected in younger individuals and individuals who live in more remote areas. Younger individuals are more mobile and so change address more often . Birth rThe results found in this study suggests differences in linkage error across sociodemographic categories are highly dataset dependent. Specific quirks in recording standards in some data collections account for some of these findings. For instance, the NSW hospital admissions dataset did not contain name information for individuals attending private hospitals (containing only date of birth and address information). This is likely the reason for the finding in this dataset that linkage quality decreases as individuals have higher socioeconomic status, since those with higher socioeconomic status are more likely to attend private hospitals, and so have missing name data. It is quite possible that other findings in our dataset also reflect specific recording anomalies that we are not aware of.The differences found in linkage quality were generally small, with a few larger differences  found in remote areas. These differences in linkage quality had little notable effect on incidence rates. The effect of these errors on other derived clinical indicators is an important area of further study.The use of subgroup-specific threshold settings made little difference to overall linkage quality, and based on these results cannot be recommended. Other published techniques such as the use of graph theory methods  or alterA key strength of our study was the use of validated, real world datasets where the \u2018answer\u2019 in relation to linkage results is known. This allowed us to assess both false positive and false negative errors in record linkage. The use of a gold standard benchmark avoids the difficulty associated with determining whether non-matched records are the result of linkage error or some other reason (i.e. data capture error), a key weakness in many previously published studies. It should be noted that while the gold-standard results used in this study were of very high quality due to extensive clerical review and quality assurance, errors may still exist within the gold-standard results.This study utilised a \u2018default\u2019 probabilistic linkage strategy, which we consider a relatively standard approach, and which achieves high quality results. As such, the level of error found in this study we consider to be small. While we could deliberately degrade the linkage strategy to reduce linkage quality and therefore amplify the level of bias in the results, we would risk introducing bias that would not typically be found in practice.Information on additional subpopulations that may have higher risk of lower linkage quality, such as Indigenous Australians, immigrant groups, those with poorer health status, and those with mental health conditions, was not available but further research in these areas is warranted. Our study used structured and defined administrative datasets, with high quality data collection standards; as a result, overall linkage quality was very high. Data collections with overall poorer quality may produce magnified or alternate results to those found in this study. Interactions are likely to exist between several variables that influence linkage quality. For instance, Indigenous Australians make up a greater proportion of the population in remote areas as compared to major cities and are more likely to be from lower socioeconomic status areas. In addition, regional and especially remote areas generally have lower socioeconomic status as compared to major cities. Interaction effects such as these were not explored in this study. It is also not clear whether the number of existing records per person influences the difficulty of linking data related to that individual; this may interact with the tested sociodemographic categories, particularly those related to age.Our results suggest linkage quality in those younger and those living in remote areas may be lower, as a result, caution should be taken when analysing differences in linked Australian hospital data by age or geographic remoteness. Further research is required to determine the ramifications of these results for researchers utilising linked data.Additional file 1:Table S1. Proportion of missing values in each dataset, stratified by sociodemographic variable. (DOCX 21 kb)"}
+{"text": "Escherichia-coli-based reconstituted translation system, we have studied how the antibiotic viomycin affects the accuracy of genetic code reading. We find that viomycin binds to translating ribosomes associated with a ternary complex (TC) consisting of elongation factor Tu (EF-Tu), aminoacyl tRNA and GTP, and locks the otherwise dynamically flipping monitoring bases A1492 and A1493 into their active conformation. This effectively prevents dissociation of near- and non-cognate TCs from the ribosome, thereby enhancing errors in initial selection. Moreover, viomycin shuts down proofreading-based error correction. Our results imply a mechanism in which the accuracy of initial selection is achieved by larger backward rate constants toward TC dissociation rather than by a smaller rate constant for GTP hydrolysis for near- and non-cognate TCs. Additionally, our results demonstrate that translocation inhibition, rather than error induction, is the major cause of cell growth inhibition by viomycin.Applying pre-steady state kinetics to an  Mycobacterium tuberculosis strains resistant to first-line drugs  are delivered to the A site of the ribosome in ternary complex (TC) with elongation factor Tu (EF-Tu) and GTP. For fast and accurate protein synthesis, the ribosome must select for GTP hydrolysis those TCs which contain an AA-tRNA with a base triplet (anticodon) cognate to the base triplet on the mRNA (codon) displayed in the ribosomal A site. Those cognate AA-tRNAs will then be selected for A-site accommodation and peptidyl transfer. In quantitative terms, this means that cognate codon selection of AA-tRNAs for GTP hydrolysis and peptidyl transfer must be characterized by much higher catalytic efficiency (reaction .E. coli numbering) of the decoding center  or RP-HPLC (f[3H]Met-Phe) with on-line radiation detection.To study the impact of viomycin on translational accuracy, we designed experiments to measure its effect on the kinetic efficiency  and near-cognate GTP hydrolysis (0.053\u00a0\u00b1\u00a00.005 \u00b5M\u22121 s\u22121) in the absence of viomycin is due to proofreading selection. The ratio of these two The large difference between the 115\u00a0\u00b1\u00a015 . In contdentical . This meH84A) with a GTPase-deficient mutant of EF-Tu (EF-TuH84A). In this EF-Tu mutant, an essential histidine in the G-domain has been replaced by alanine  or near-cognate (CUC) codon in the A site were equilibrated with TCH84A containing\u00a0H84As were chased from the A site by addition of GTPase proficient TC, containing WT EF-Tu (EF-TuWT) and either H84A s from the A site, defined as the inverse of the average dissociation time, was then estimated from the rate of f[3H]Met-Phe or f[3H]Met-Leu formation (supplementary methods).Viomycin is known to strongly stabilize peptidyl-tRNA in the ribosomal A site . To addr alanine , but fordrolysis . InitiatH84A dissociation from ribosomes displaying the cognate UUC codon (\u22121 in the absence of viomycin and decreased from 0.616\u00a0\u00b1\u00a00.044 s\u22121 in the presence of 1 \u00b5M viomycin to 0.198\u00a0\u00b1\u00a00.0275 s\u22121 at 10 \u00b5M viomycin. The corresponding viomycin-induced increase in dissociation mean time (H84A from ribosomes displaying the near-cognate CUC codon (\u22121 and decreased modestly to 0.209\u00a0\u00b1\u00a00.066 s\u22121 at 200 \u00b5M viomycin.The rate of TCUC codon  was 1.21UC codon  was too UC codon . In the catk/mK) observed above . We quantified the viomycin sensitivity of each codon by estimating its AUC, 29.0 \u00b1 2.7 mM for UAC and 3.25 \u00b1 0.20 mM for UUA and accuracies of initial selection of 2400 \u00b1 120 for AUC, 6400 \u00b1 670 for UAC and 580 \u00b1 47 for UUA.The model presented above predicts that viomycin sensitivity :Phe reads the codon CUC; this would roughly double the error rate assuming that there were equal concentrations of UUC and CUC displaying ribosomes in the reaction mixture.Here, Based on the results presented above, we have constructed a kinetic model for how viomycin reduces the fidelity of mRNA decoding . We alsosociates . Viomyci4k is fully compatible with earlier kinetics results showing that the maximal rate of GTP hydrolysis (catk) is lower in near-cognate than cognate cases and becomes equal upon addition of aminoglycosides   is fullyInc) 2, 1 mM dithioerythritol and 5 mM HEPES pH 7.5). All reaction mixes contained 10 mM phosphoenolpyruvate (PEP), 1 \u00b5g/ml pyruvate kinase (PK) and 0.1 \u00b5g/ml myokinase (MK). His-tagged initiation factors IF1, IF2 and IF3, elongation factor Ts, and phenylalanine and leucine aminoacyl tRNA-synthetases were purified using nickel-affinity chromatography . Wild-type elongation factor Tu was prepared as in E. coli MRE600) and f[3H]Met-tRNAfMet were prepared according to Phe was prepared as in 3H]Met and [3H]GTP were from Perkin-Elmer, viomycin was from USP, all other chemicals were from either Merck or Sigma-Aldrich.All experiments were performed at 37\u00b0C in HEPES-polymix buffer  and purified using nickel-affinity chromatography . The identity and purity of the H84A mutant protein was confirmed by mass spectrometry.The wild type fMet(1.5\u20133.0 \u00b5M), mRNA (3 \u00b5M), GTP (1 mM) and ATP (1 mM). The TC mixture contained EF-Tu (0.3\u20130.6 \u00b5M), phenylalanine (200 \u00b5M), PheRS (0.5 \u00b5M), tRNAPhe (2 \u00b5M), viomycin (0\u20132000 \u00b5M), [3H]GTP (0.3\u20130.6 \u00b5M) and ATP (2 mM). After 15 min incubation at 37\u00b0C, equal volumes of the two mixes were rapidly mixed and the reaction quenched at different time points with formic acid  using a quench-flow instrument (RQF-3 KinTek corp.). After quenching, the samples were centrifuged at 20,800 g. The supernatant, containing the [3H]GTP and [3H]GDP was analyzed by anion exchange chromatography with on-line scintillation counting (\u03b2-RAM model 4 IN/US systems). A Mono-Q GL column  was used and the mobile phase was a multistep gradient of 0\u20132 M NaCl in 20 mM Tris (pH 7.5).Two mixtures were prepared. The ribosome mixture contained 70S ribosomes (1.0\u20132.0 \u00b5M), IF1, IF2 and IF3 (2 \u00b5M each), fMet-tRNA3H]Met-tRNAfMet (1 \u00b5M), mRNA (2 \u00b5M), GTP (1 mM) and ATP (1 mM). The TC mixture contained EF-Tu (1\u201310 \u00b5M), EF-Ts (1 \u00b5M), phenylalanine (200 \u00b5M), PheRS (0.5 \u00b5M), tRNAPhe (12 \u00b5M), viomycin (0\u20132000 \u00b5M), GTP (1 mM) and ATP (1 mM). After 15 min incubation at 37\u00b0C, equal volumes of the two mixes were rapidly mixed and the reaction quenched at different time points with formic acid  using a quench-flow instrument (RQF-3 KinTek corp.). After quenching, the samples were centrifuged at 20,800 g and the supernatant discarded. The pellet was dissolved in 165 \u00b5l 0.5 M KOH to cleave the peptides from the tRNA. After 10 min 13 \u00b5l of 100% formic acid was added, the samples were centrifuged at 20,800 g and the radioactive peptides in the supernatant were analyzed by RP-HPLC using a H2O/MeOH/trifluoroacetic acid (58/42/0.1 by volume) mobile phase and a C-18 column (Merck) with on-line scintillation counting (\u03b2-RAM model 4 IN/US systems) to quantify the relative amounts of f[3H]Met and f[3H]Met-Phe.Two mixtures were prepared. The ribosome mixture contained 70S ribosomes (0.5 \u00b5M), IF1, IF2 and IF3 (1 \u00b5M each), f[3H]Met-tRNAfMet (1 \u00b5M), mRNA (2 \u00b5M), GTP (1 mM) and ATP (1 mM). The first TC mixture contained EF-TuH84A (15 \u00b5M), phenylalanine (200 \u00b5M), PheRS (0.5 \u00b5M), tRNAPhe (15 \u00b5M), viomycin (0\u2013400 \u00b5M), GTP (1 mM) and ATP (1 mM). The second TC mixture contained EF-Tu (1.5 or 12 \u00b5M), EF-Ts (1 \u00b5M), phenylalanine (200 \u00b5M) or leucine (200 \u00b5M), PheRS (0.5 \u00b5M) or LeuRS (0.5 \u00b5M), tRNAPhe (2 \u00b5M) or bulk tRNA of which 2 \u00b5M was tRNALeu2 and an additional 10 \u00b5M were other leucine tRNA isoacceptors, viomycin (0\u2013600 \u00b5M), GTP (1 mM) and ATP (1 mM). All three mixes were incubated at 37\u00b0C for 15 min. During the experiment, one volume of the ribosome mixture was mixed with one volume of the first TC mixture, the resulting mixture was incubated for 5\u201310 s and then one volume of the second TC mixture was added. The reaction was quenched at different time points after the addition of the second TC mixture using formic acid . The samples were treated as the quench flow peptide samples above.Three mixtures were prepared. The ribosome mixture contained 70S ribosomes (0.75 \u00b5M), IF1, IF2 and IF3 (1 \u00b5M each), feLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor, Alan Hinnebusch, and a Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your work entitled \"Insights into the fidelity mechanism of mRNA decoding from characterization of viomycin induced miscoding in translation\" for consideration by eLife.Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in Summary:cat/Kmk) for cognate versus near-cognate codons in the A-site in the presence of increasing concentrations of viomycin, for both GTP hydrolysis by the EF-Tu-GTP-aatRNAPhe ternary complex in the step of initial selection prior to proof-reading and for peptide-bond formation following proof-reading. The results show no effect of viomycin on cat / Kmk for cognate UUC, but a marked increase in cat / Kmk for near-cognate CUC; which leads to a large reduction in accuracy, defined as the ratio of (cat / Kmk)-cognate to (cat / Kmk)-near-cognate. Rather than finding that the near-cognate codon exhibits a much smaller cat / Kmkfor peptide bond formation versus GTP hydrolysis owing to proof-reading, they are identical in the presence of drug, which means that proof-reading is disabled by the drug. They conclude, reasonably, that drug binding increases initial selection of the near-cognate complexes and that all near-cognates that pass initial selection go on to form peptide bonds.This paper examines the mechanism of miscoding in translation by the drug viomycin, concluding that drug binding increases initial selection of the near-cognate tRNA complexes rather GTP hydrolysis by the near-cognate ternary complex. Based on the results obtained and various theoretical considerations, the paper also challenges two aspects of the current induced-fit model for tRNA selection accuracy, concluding that (i) accuracy in the tRNA selection phase of decoding is enforced primarily by increased rates of near-cognate tRNA dissociation resulting from a higher rate of the back reaction from the codon recognition state to the initial binding state, rather than from a reduced rate of GTP hydrolysis for non-cognate versus cognate tRNAs; and (ii) interactions of bases A1492/A1493 with the tRNA anticodon stem loop is not restricted to cognate tRNA, with the unstated implication that these interactions play no important role in monitoring codon:anticodon helices in the A-site. Using pre-steady-state kinetic analysis with a quench-flow apparatus, they measure the catalytic efficiency (cat / Kmk)-cognate/(cat / Kmk)-near-cognate and IK for the four different near-cognates. They claim that this relationship indicates that the accuracy of initial selection for different near-cognates is determined primarily by different rates of tRNA rejection  rather than different rates of GTP hydrolysis in the manner proposed by the current model.The authors construct a kinetic model for drug action based on a current model in which the initial binding of cognate and near-cognate occurs at the same rate and only after transition to a codon-recognition state is the near-cognate tRNA rejected, returning to the initial binding state if GTP hydrolysis does not occur first. They propose that viomycin binds to the codon-recognition state and traps the tRNA in the A-site, preventing dissociation to the initial binding state and allowing GTP hydrolysis to occur. This has little effect on cognate tRNA because the rate of conversion to the initial binding state is slow and/or GTP hydrolysis is fast; but it dramatically increases the efficiency for near-cognate by preventing conversion to the initial binding state. In principle the drug could also increase the rate of GTP hydrolysis by near-cognate, but they approximate the equations that give a complete theoretical description of the kinetics with an expression in which the drug concentration required to increase the efficiency of each near-cognate reaction to one-half that of the cognate reaction, Ki, is governed by only the forward and back rates of the conversion from initial binding to codon-recognition states, and is independent of the rate of GTP hydrolysis. They go on to repeat the previous experiment for three different near-cognate codons and find a linear relationship between accuracy,  or that viomycin accelerates GTP hydrolysis for near-cognates. It is difficult to follow the logic of your arguments, and it is questionable whether the approximation of the full set of equations required for a complete theoretical description of the kinetics you adopted in order to analyze the relationship between accuracy and There was also objections to your second argument, that because viomycin binding requires A1492/A1493 to be in the flipped-out conformation, its binding to near-cognate/ribosome complexes necessitates that A1492/A1493 be flipped out for near-cognate as well as cognate tRNA-at odds with proposals that flipping out of A1492/A1493 is restricted to cognate tRNAs as a key step of the induced fit mechanism that accelerates GTP hydrolysis for cognate tRNAs. Your conclusion has been criticized because there is no crystal structure of ribosome/drug/near-cognate tRNA ternary complexes, and so the mode of viomycin binding in the presence of near-cognate tRNA is unknown; and also because there is no way to exclude that viomycin simply binds first and flips out the bases prior to tRNA binding. It was also noted in the discussion session that the available structural data do not assess the codon-recognition state, which is necessarily transient and distinct from the state probed by Ramakrishan and colleagues by stalling the ribosome with non-hydrolyzable GTP analogues or GTPase deficient mutants. Thus, we do not yet know the nature of the codon-anticodon interaction in the codon recognition state and viomycin's interactions with the ribosome and/or the codon-anticodon pair. Again, it was stated in the reviews that you should experimentally measure the conformational dynamics of A1492/A1493 during accommodation and viomycin binding to make mechanistic claims on this topic.Finally, there was the criticism that extended into the discussion session among the reviewers, that you have failed to cite or discuss the previous study of Pape et al. (Rodnina) on the mechanistically similar antibiotic paromomycin, which involved separate measurements of the effect of paromomycin on GTP hydrolysis and aa-tRNA dissociation (measured using non-hydrolyzable GTP), leading to the conclusion that both steps are affected in the case of near-cognate aa-tRNA. It was objected that your study argues with that work without citing it, but with less experimental data  to make the argument and using complex mathematical analyses based on questionable assumptions. While it was noted that there are reasons to question the conclusions of Pape et al., challenging them should be justified by the appropriate experimental data, such as measuring tRNA disassociation rates using a GTPase-deficient mutant of EF-Tu.eLife.The reviewing editor originally recommended that the paper be sent out for review primarily because if your speculative arguments were judged to be compelling by expert reviewers, then they would have the impact of swaying the field towards accepting a revision of the tRNA selection mechanisms proposed by others in the field. However, with these speculations being strongly challenged by two of the three reviewers, it is now judged that the solid aspects of the paper concerning the mechanism of viomycin action and implications for its biological impact are not of sufficient general interest on their own for Reviewer #2:The manuscript by Holm et al. presents a clear kinetic description of the established miscoding impact of viomycin on tRNA selection (REF 5 and Wurmbach and Nierhaus Eur. J. Biochem 1983) using modern pre-steady state methods. A big picture message of the manuscript is that viomycin operates by preventing the ribosome from efficiently rejecting near-cognate tRNA at early stages of the selection process, which has larger implications regarding the fidelity mechanism. The authors' key conclusion from their finding is that viomycin impacts tRNA selection at a step prior to GTP hydrolysis and after conserved decoding site residues have contacted the mRNA-tRNA pair . This conclusion seems to be well supported by the data presented and fits with some, but not other, group's interpretations of the selection mechanism. Thus, the presented findings may have the broader impact of swaying the field towards accepting revision of the tRNA selection mechanisms proposed by Rodnina and colleagues that currently serves as the most widely accepted mechanistic framework. The experiments appear to have been rigorously performed, properly analyzed and kinetically modeled. The manuscript is well written and the findings appear to be appropriately interpreted in the context of existing literature. For these reasons, publication is recommended.Reviewer #3:Holm and Sanyal performed kinetic analysis of ribosome miscoding induced by the antibiotic viomycin. The authors measured the rate of GTP hydrolysis by EF-Tu and the rate of dipeptide formation to examine the effect of viomycin on initial tRNA selection and complete accommodation of tRNA to the A site, respectively. The authors found that accommodation of cognate tRNA is not affected by viomycin. By contrast, viomycin dramatically enhances rates of GTP hydrolysis and dipetide formation in the case of near-cognate tRNA. Kinetic data also suggest that viomycin completely abrogates proofreading step of tRNA selection. The experiments are well-executed. Observations made by the authors are of substantial interest to the protein synthesis community.eLife.However, the authors went further and made a number of additional conclusions that are not related to experimental results in an obvious way. . These conclusions are inferred from kinetic analysis of the authors' data. The authors acknowledge that they did not directly measure most of the rate constants required to obtain the complete kinetic description of tRNA accommodation. In particular, the authors did not directly measure the rate of tRNA disassociation of the codon recognition complex . Hence, the authors' experiments cannot directly delineate whether viomycin slows down the dissociation of the EF-Tu ternary complex from the ribosome (as the authors conclude) or viomycin accelerates GTP hydrolysis. Furthermore, the authors did not directly examine the conformational dynamics of bases A1492 and A1493 of 16S rRNA during accommodation and viomycin binding. I could not completely follow the line of theoretical arguments that the authors used in support of their conclusions. I also could not fully understand the authors' justification for the \"approximation\" of complete kinetic description of the tRNA accommodation process to a simpler kinetic equation (equation 3) that the authors used. Hence, I might not be able to adequately judge the validity of main conclusions of the manuscript. Nevertheless, it seems to me that the authors' conclusions are made based on a number of assumptions that are not directly drawn from experimental results. Besides, the manuscript does not seem very accessible to a wide audience of readers and is rather addressed to a narrow group of scientists specialized in the kinetics of the tRNA accommodation process. I therefore doubt that this manuscript is suitable for publication in Reviewer #4:Holm and Sanyal employ pre-steady-state kinetics to study how the translation inhibitor viomycin affects the accuracy of aminoacyl-tRNA selection. The miscoding effect of viomycin has been reported before, e.g. by Marrero et al., 1980, also by Wurmbach and Nierhaus, 1982 (the authors do not cite the latter paper). The current study provides a more detailed assessment of miscoding of near-UUC codons by Phe-tRNAPhe. The authors find that the rates of GTP hydrolysis and dipeptide formation on near-cognate tRNA increase with the increasing concentrations of viomycin. The authors interpret that the concentration dependence reflects an equilibrium shift in the binding of near-cognate aminoacyl-tRNA to the A site. The authors find that the inhibition of selection accuracy (Ki) is in the millimolar range. This contrasts the effect of viomycin on tRNA translocation, which viomycin inhibits at low \u03bcM concentrations . As such, the study suggests that the main effect of viomycin on cellular translation is via the translocation step and not via decoding errors.In addition to the conclusion about the mechanism of action of viomycin, the authors attempt to extend their findings to understand the mechanism of aa-tRNA decoding. Specifically, they ask whether the accuracy of selection is achieved via (1) \"induced fit\" of the ternary complex resulting in slow GTP hydrolysis on near-cognate aa-tRNA or (2) dissociation of near-cognate aa-tRNA. The authors also discuss the involvement of the decoding-center nucleotides A1492 and A1493 in tRNA selection. This part is speculative because the authors' findings do not directly report on tRNA association/dissociation or dynamics of the decoding center or even the binding mode (site and conformation) of viomycin in the presence of near-cognate tRNA. The rationalization of decoding accuracy in this work is not robust because it relies heavily on previous work, including crystal structures of near-cognate tRNAs or structures of viomycin-bound ribosomes. A mechanistic scheme is presented, in which viomycin binds following the ternary complex \u2013 but how was the order established, in which viomycin and the ternary complex bind? Surprisingly, the authors do not discuss a kinetic study of aa-tRNA miscoding by paromomycin , which addresses individual steps of ternary complex acceptance/rejection. That earlier work is particularly relevant because paromomycin' and viomycin's binding sites within h44 overlap and these antibiotics induce nearly identical rearrangements of A1492-A1493, so their mechanisms of tRNA miscoding are likely similar.In summary, the detailed biochemical dissection of viomycin action on decoding in this work is interesting as it suggests that miscoding is not the primary mode of viomycin's antimicrobial action. However the implications for a general mechanism of translation accuracy are indirect and would require substantial additional work (e.g. direct measurements of ternary complex and/or aa-tRNA association/dissociation rates).eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your work entitled \"The mechanism of error induction by the antibiotic viomycin provides insight into the fidelity mechanism of translation\" for consideration by eLife.Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in Although both reviewers felt that the work was well done, noting improvements over the original version of the paper, they also agreed that the findings add only incrementally to knowledge regarding the mechanisms of viomycin action and decoding in relation to previously published biochemical and structural studies on these topics. As such, neither felt that the paper satisfies the journal's standards for publishing work of the greatest importance to the field, and indicated that it would be of interest to only a very small group of specialists.Reviewer #1:Holm et al. performed kinetic analysis of ribosome miscoding induced by antibiotic viomycin. This manuscript is much improved in comparison to the earlier version of the paper. The manuscript is well written. Experiments and data analysis were meticulously done. The authors' conclusions are compelling. Important experiments with GTPase deficient mutant of EF-Tu that were missing from the previous version of the paper are now included. However, my enthusiasm is dampened because the manuscript does not offer many new insights regarding the mechanism of viomycin action or the mechanism of decoding. Most of authors' observations are consistent with previously published biochemical and structural studies of decoding/viomycin including authors' own works . Furthermore, the authors conclude (and state this in the Abstract of manuscript) that viomycin-induced miscoding is not the cause of cell growth inhibition by viomycin. This conclusion further diminishes biological and medicinal significance of the study.Reviewer #2:The authors biochemically dissect how viomycin induces miscoding. They demonstrate that viomycin increases the rates of (1) EF-Tu-catalyzed GTP hydrolysis and (2) peptide bond formation for the near-cognate ternary complexes. Viomycin sensitivity (Ki) of different near-sense codons correlates with the accuracy for cognate tRNA over these codons , consistent with interference of viomycin with the decoding step(s). A competition assay is used to demonstrate that viomycin stabilizes near-cognate tRNA on the ribosome with catalytically-inactive EF-Tu, suggesting that the mechanism for GTPase activation on near-cognate tRNAs is due to stabilization of the ASL in the decoding center. These findings are consistent with previous biochemical and structural work showing that viomycin potently stabilizes tRNA in the A site, thus inducing miscoding and inhibiting translocation.Although this work describes a well-designed and detailed biochemical study of viomycin's effect on near-cognate tRNA, these findings only incrementally add to what's been known. Increased binding of near-cognate tRNA to the A site in the presence of viomycin was demonstrated earlier . Next, the effect of viomycin on A1492 and A1493 constitutes bulk of the mechanistic insights discussed in the paper. But these insights are based almost entirely on previous structural studies because present biochemical assays do not directly report on the conformational changes in the decoding center. This discussion could as well be part of a review article, in the absence of the presented data. Furthermore, in vivo relevance of increased miscoding is not shown in this work. It is possible that these in vitro findings are not relevant because translocation inhibition is the prevalent mode of action of viomycin, so that mistranslated proteins do not contribute to cellular toxicity.A minor note: The authors somewhat unexpectedly (for a broader readership) bring up aminoglycosides in the Introduction of the decoding mechanism. How relevant is this given that viomycin is not an aminoglycoside? If the modes of aminoglycosides and viomycin are deemed similar, what is the rationale for this study in the light of the known mechanism of miscoding by aminoglycosides? [Editors\u2019 note: the author responses to the first round of peer review follow.]Summary of the review: Our manuscript concerns the mechanism of action of a ribosome targeting antibiotic drug, viomycin. In this work we show specifically how viomycin acts on selection of tRNA by the ribosome to induce missense errors in reading of the genetic code. We propose a complete mechanistic description of this mode of action of the drug and additionally, through comparison with our previous work on inhibition of translocation by viomycin, suggest that this is not the primary mode of action of the drug. This part of our manuscript was well received by the reviewers.We then go further and show that the effects of viomycin on tRNA selection constrain the possible biochemical mechanisms through which the high accuracy of translation is achieved. We argue that our results contradict the prevailing \u2018induced fit\u2019 model. We also argue based on our results and on the published structures of the viomycin-bound ribosome that the so-called \u2018monitoring bases\u2019 A1492 and A1493 of the 16S rRNA are much more dynamic than has been proposed previously based on structural studies. These points raised in our manuscript were strongly criticized in the review.What has been done: We have carried out a new set of experiments using a GTPase deficient mutant of EF-Tu to directly measure stabilization of ternary complexes on the ribosome by viomycin. These experiments address several concerns brought up in the review. We now show directly that viomycin strongly stabilizes both cognate and near/non cognate ternary complexes on the ribosome before GTP hydrolysis. We show that this stabilization is enough to completely explain the accuracy reducing effects of the drug. This provides direct experimental proof for the approximations made in our model.IK value.A key argument in our manuscript concerns a correlation between the accuracy of a particular mismatched codon\u00b7anticodon pair during initial selection and the sensitivity of that pair to viomycin. We find that the more accurate a given mismatch is the more viomycin is required to yield a given misreading frequency for that mismatch. That is, the easier it is for the ribosome to reject a tRNA from the A site the harder it is for viomycin to bind to the ribosome while that tRNA is present. This propensity of viomycin to bind, while a tRNA is present in the ribosomal A site but before GTP hydrolysis by EF-Tu has occurred, is quantified in our manuscript by a single kinetic parameter, a IK values from our experimental data we rely on an approximation that was criticized in the review. We have updated the main text and that of the supplementary material to further clarify the nature of this approximation. To state it shortly we ignore the effect of the forward rate of GTP hydrolysis for near-cognate tRNA on the size of the IK value because it is so much smaller than the effective backwards rate of tRNA rejection. This assumption is true by definition for near/non cognate tRNA, it is also independent of the exact structure of the kinetic model used, if it were not true then the tRNA in question would be more likely to be accepted by the ribosome than to be rejected, and would by definition be a cognate tRNA.When estimating these IK value, and the accuracy of initial selection are strongly correlated. This is the evidence we rely on to strengthen our claim that the accuracy must be determined by tRNA rejection rates, rather than tRNA acceptance rates.Therefore, what we find is that the effective backwards rate of tRNA rejection, as measured by the In our manuscript we also propose a hypothesis for the role of the monitoring bases A1492 and A1493 during tRNA selection. Since directly measuring the dynamics of these bases during tRNA selection on the ribosome is currently impossible by any experimental technique that we are aware of, we base our suggestion on our biochemical results and preexisting structures of viomycin-bound ribosomes.We suggest that the conformation of the decoding center with A1492, A1493 and G530 interacting with the codon anticodon helix, is a necessary prerequisite for GTPase activation. To proceed to GTP hydrolysis any tRNA, be it cognate or non-cognate has to pass through this state. The propensity for a given tRNA to trigger GTP hydrolysis is then directly proportional to the lifetime of this state. The monitoring bases enhance the accuracy of tRNA selection by greatly decreasing the energy level of this state for cognate but not near/non-cognate tRNA. This same hypothesis has now been suggested by the Ramakrishnan lab  and demonstrated by Korestelev lab . Our lab together with Joachim Frank\u2019s lab also reached the same structural conclusion . We do not claim to have directly observed the proposed behavior by the monitoring bases, but we do suggest that such a mechanism is compatible with our experimental data as well as all existing structural data on both viomycin-bound ribosomes and ribosomes with cognate or near-cognate tRNAs in the A site.References:Shao, S., Murray, J., Brown, A., Taunton, J., Ramakrishnan, V., and Hegde, R.S. (2016). Decoding Mammalian Ribosome-mRNA States by Translational GTPase Complexes. Cell 167, 1229-1240 e1215. 10.1016/j.cell.2016.10.046Loveland, A.B., Demo, G., Grigorieff, N., and Korostelev, A.A. (2017). Ensemble cryo-EM elucidates the mechanism of translation fidelity. Nature 546, 113-117.Fislage, M., Zhang, J., Brown, Z.P., Mandava, C.S., Sanyal, S., Ehrenberg, M., and Frank, J. (2018). Cryo-EM shows stages of initial codon selection on the ribosome by aa-tRNA in ternary complex with GTP and the GTPase-deficient EF-TuH84A. Nucleic Acids Res 46, 5861-5874.[Editors' note: the author responses to the re-review follow.]Although both reviewers felt that the work was well done, noting improvements over the original version of the paper, they also agreed that the findings add only incrementally to knowledge regarding the mechanisms of viomycin action and decoding in relation to previously published biochemical and structural studies on these topics. As such, neither felt that the paper satisfies the journal's standards for publishing work of the greatest importance to the field, and indicated that it would be of interest to only a very small group of specialists.Reviewer #1:Holm et al. performed kinetic analysis of ribosome miscoding induced by antibiotic viomycin. [\u2026]. This conclusion further diminishes biological and medicinal significance of the study.Reviewer #2:The authors biochemically dissect how viomycin induces miscoding. They demonstrate that viomycin increases the rates of (1) EF-Tu-catalyzed GTP hydrolysis and (2) peptide bond formation for the near-cognate ternary complexes. [\u2026] If the modes of aminoglycosides and viomycin are deemed similar, what is the rationale for this study in the light of the known mechanism of miscoding by aminoglycosides?eLife in 2016, with title \u201cInsights into the fidelity mechanism of mRNA decoding from characterization of viomycin induced miscoding in translation\u201d. In that manuscript, we presented with careful and precise biochemical work, how the antibiotic viomycin affects the accuracy of decoding. More importantly, we presented two vital insights about the fidelity mechanism of the decoding process on the ribosome, which were new to the field and not in line with views presented in the literature. These were, in brief:As you know, the current manuscript roots from an earlier manuscript submitted to i) The widely accepted \u2018induced fit\u2019 model for initial selection is incorrect; instead the accuracy in initial codon selection by ternary complex is decided by differential rejection rates of the cognate and near-cognate tRNAs before GTP hydrolysis by EF-Tu rather than by varied GTP hydrolysis rates for cognate and near-cognate tRNAs as proposed earlier.ii) The monitoring bases in the decoding center, A1492 and A1493 flip out dynamically and interact with the codon anticodon helix irrespective of whether the tRNA is cognate or non/near cognate. We hypothesized that the propensity for a given tRNA to trigger GTP hydrolysis by EF-Tu and being accepted is directly proportional to the lifetime of this flipped-out state.Two of the three referees and the reviewing editor reacted strongly against both of these findings (in 2016). The reviewing editor writes \u2013 \u2018you find\u2026.that flipping out of the bases is not restricted to cognate codons, at odds with the recent model that these bases mediate an induced fit in the A site that triggers GTP hydrolysis\u2019, and also, \u2018its (viomycin\u2019s) binding to near-cognate/ribosome complexes necessitates that A1492/A1493 be flipped out for near-cognate as well as cognate tRNA-at odds with proposals that flipping out of A1492/A1493 is restricted to cognate tRNAs as a key step of the induced fit mechanism that accelerates GTP hydrolysis for cognate tRNAs.\u2019 The main complaint from one reviewer was that without a proper structure our hypothesis about the decoding mechanism could not stand. The reviewing editor criticized our conclusion because there was no crystal structure of ribosome/drug/near-cognate tRNA ternary complexes. We feel that two of the reviewers and the reviewing editor may have been preoccupied with the idea of the \u2018induced fit\u2019 model and were not open to results opposing it in the absence of high-resolution structures. There was however, no criticism concerning viomycin\u2019s mode of action.While we worked to gather more biochemical results to support our model of decoding, a paper presenting high-resolution structures of these very decoding states was published by the lab of Andrei Korostolev . The results in this paper are fully in line with our proposed mechanism  that irrespective of the cognate or near/non cognate nature of the codon the monitoring bases are dynamically flipping in and out of h69. Now in 2019, when we submitted a more complete work presenting this mechanistic model for the fidelity of initial selection, supported by this recent structural data, our manuscript was rejected on the grounds that \u2018the findings add only incrementally to knowledge regarding the mechanisms of viomycin action and decoding in relation to previously published biochemical and structural studies on these topics\u2019.We strongly object to the statement quoted in the paragraph above. We would like to point out that our state-of-the-art quantitative biochemical data are complementary to the recent structural data, not mutually exclusive. While the proposed mechanism of decoding can be speculated qualitatively from the now published structures, the quantitative aspects of our work go far beyond what can be deduced from just these structures. We were surprised that the reviewers and the reviewing editor minimized the importance of biochemical validation and extensions of the hypotheses proposed from structure, and imply that such biochemical experiments are useless.We also question the referees\u2019 reasoning based on comparison with our previous study on translocation inhibition by viomycin , that our manuscript is unimportant due to the demonstration that viomycin induced decoding error is not the main cause of the drug\u2019s antimicrobial effect. We disagree with this argument. First, we feel both pathways need to be studied mechanistically to determine which of the two inhibition mechanisms is the strongest. Second, it has been demonstrated for other drugs , which cause both misreading and inhibition of translocation that the induction of decoding errors is correlated with the strength of the side effects observed in patients. Hence the demonstration of the fact that error-induction is not relevant to the antimicrobial activity of viomycin, a drug with severe side effects, is in our mind highly important for drug-development purposes.Reviewer #2 is surprised that we mention aminoglycosides in the paper since the paper is about viomycin (which is not an aminoglycoside). What we have discussed is that in spite of the fact that these two classes of drugs share overlapping binding sites on the ribosome their modes of action are strikingly different, something that was not suggested from previous studies. In fact, the current opinion in the field is misleading. It is believed that as the drugs share a common binding site they must operate in the same way. Hence, we explicitly compare viomycin with aminoglycosides in our manuscript. Here the referee\u2019s reasoning itself demonstrates why biochemical studies such as ours are an essential complement to structural studies as none of these questions could be addressed by the previous structural studies mentioned by both referees.Reviewer #2 also suggests that our finding was already known from an article by Wurmbach and Nierhaus, Eur J. Biochem, 1983. We are aware of this paper, which presented a semi quantitative study on an incomplete translation system. We therefore feel it provides little mechanistic insight about viomycin\u2019s actions on bacterial protein synthesis and chose not to discuss it.This extended review and the editorial process has led us losing our priority in solving pertinent issues regarding fidelity mechanism of decoding on the ribosome. As both the past and the present rejections appear to be based on bias from the reviewers, we feel obliged to appeal the editorial decision and ask for a new review of the current manuscript."}
+{"text": "Caenorhabditis elegans and humans. We showed previously that C. elegans ABCB6/HMT-1 detoxifies cadmium, copper, and arsenic, and is expressed in liver-like cells, the coelomocytes, head neurons and intestinal cells, which are the cell types that are affected by heavy metal poisoning in humans. The subcellular localization of ABCB6/HMT-1 proteins is unclear. ABCB6/HMT-1 proteins have a distinguishing topology: in addition to one transmembrane domain and one nucleotide-binding domain, they possess a hydrophobic N-terminal extension (NTE) domain encompassing five to six transmembrane spans. The role of the NTE domain in the function of ABCB6/HMT-1 in the native organism remains to be investigated. We used a versatile, multicellular model system, C. elegans, to establish the subcellular localization of ABCB6/HMT-1 and refine its structure-function studies in the native organism. We show that ABCB6/HMT-1 localizes mainly to the apical recycling endosomes and, in part, to early and late endosomes of intestinal cells. We also show that ABCB6/HMT-1 lacking the NTE domain is mistargeted to the plasma membrane and is unable to confer cadmium resistance. Although the NTE domain is essential for ABCB6/HMT-1 interaction with itself, the absence of NTE does not fully prevent this interaction. As a result, ABCB6/HMT-1 lacking the NTE domain, and expressed in wild-type worms or co-expressed with the full-length polypeptide, inactivates and mistargets the full-length ABCB6/HMT-1. We also show that the 43 amino acid residue stretch at the COOH-terminus is required for the ABCB6/HMT-1 interaction with itself and cadmium detoxification function. These results suggest that both NTE and COOH-terminus must be present to allow the protein to interact with itself and confer cadmium resistance. Considering that ABCB6/HMT-1 proteins are highly conserved, this study advances our understanding of how these proteins function in cadmium resistance in different species. Furthermore, these studies uncover the role of the endosomal-recycling system in cadmium detoxification.The chronic exposure of humans to toxic metals such as cadmium from food and air causes dysfunction of vital organs, neurodegenerative conditions, and cancer. In this regard, members of the ABCB sub-family of the ATP-binding cassette (ABC) transporter superfamily, ABCB6/HMT-1, are acutely required for the detoxification of heavy metals and are present in genomes of many organisms including the nematode worm,  Cadmium (Cd) is a highly toxic transition metal that poses a threat to human health and environment . It is nSaccharomyces cerevisiae, Ycf1p (Yeast Cadmium factor 1), detoxifies heavy metals including Cd and As by sequestering metal-GS complexes into a lysosomal-like compartment, the vacuole, and confers heavy metal tolerance in heterologous systems ], are acutely required for the detoxification of heavy metals in different species including Schizosaccharomyces pombe, Chlamydomonas reinhardtii, Caenorhabditis elegans, Drosophila melanogaster, Rattus norvegicus, and Homo sapiens , and As and is expressed in liver-like cells, the coelomocytes, as well as head neurons and intestinal cells, which are the cell types that are affected by heavy metal poisoning in humans (Members of the highly conserved family of ATP-binding cassette (ABC) transporters are recognized for their contribution to heavy metal detoxification in different species . For exa systems . ABCB6-l sapiens . For exan humans . It has n humans . HoweverC. elegans at a minimum homodimerizes (The eukaryotic ABC transporters are organized either as \u201cfull-molecule\u201d transporters consisting of two polytopic transmembrane domains (TMD1 and TMD2) and two ATP-binding domains [nucleotide-binding domain (NBD)1 and NBD2], or as half-transporters, containing one TMD and one NBD . In ABCBimerizes .Figure 1A). The NTE domain consists of five to six transmembrane spans (TMD0) and a cytosolic linker (L0), which connects the TMD0 with core domains of ABC transporters (TMDs or NBDs).In addition to the four-domain structure in full-transporters or two-domain structure in half-transporters, some ABC transporters possess a hydrophobic N-terminal extension domain termed NTE . Structure-function analyses established that the NTE domains of YCF1p and ABCC2/MRP2 play an important role in membrane trafficking, whereas the NTE domain and the COOH-terminal region of ABCC1/MRP1 contain redundant trafficking signals, which only become essential when one or the other region is missing or is mutated  was initially identified in the mitochondria . SubsequC. elegans is a non-mammalian model system that provides the advantage of tinkering these questions in the intact multicellular organism. Thus, here, we used C. elegans to determine the subcellular localization of ABCB6/HMT-1. We also continued the structure-function studies of ABCB6/HMT-1 in C. elegans with the goal of identifying other functional regions in the polypeptide that play a role in localization, interactions, and ability to confer Cd resistance. We show that C. elegans ABCB6/HMT-1 localizes to the endosomal recycling system in intestinal cells, while the ABCB6/HMT-1 lacking the NTE domain tends to associate with the plasma membrane. We also show that ABCB6/HMT-1 lacking the NTE domain exerts a dominant negative effect on the localization and function of the full-length ABCB6/HMT-1 polypeptide suggesting that the C-terminal part of the protein is involved in protein\u2013protein interactions as well. Thus, here we also identified the C-terminus region of HMT-1 that is necessary for ABCB6/HMT-1 dimerization and function in Cd resistance.In this regard, Caenorhabditis elegans strains used in this study are listed in Table 1. Worms were maintained at 20\u00b0C on Solid Nematode Growth medium (NGM) using the Escherichia coli OP50 strain as a food source . Briefly, the RfA (reading frame cassette A) was inserted into the AgeI restriction site localized after the phmt-1 and prior to 5\u2032 of the GFP in the pPD117.01-phmt-1::GFP. The newly generated vector was designated pPD117.01-phmt-1::GFP-Gate. To enable co-localization studies, an open reading frame of RFP was PCR amplified using the primer pairs with engineered AgeI and NheI restriction enzymes recognition sites at the 5\u2032 and 3\u2032, respectively (Table 2). The resulting PCR fragment was cloned into AgeI and NheI sites of the pPD117.01-phmt-1::GFP-Gate to replace GFP with RFP and generate pPD117.01-phmt-1::RFP-Gate. Truncated hmt-1 fragments  were generated by PCR using primer pairs listed in Table 2 and introduced into the pDONR222 entry vector before recombination with pPD117.01-phmt-1::GFP-Gate or pPD117.01-phmt-1::RFP-Gate.A 10 kb genomic fragment, consisting of the promoter and the genomic sequence of 1 vector  to genert-1::GFP  was modipPD117.01-Gate carrying \u0394NTE-HMT-1::GFP or NTE::GFP, or HMT-1\u039443::GFP into the gonadal syncytium of adult hermaphrodites of hmt-1(gk161) mutant worms to generate VF23, VF24, and VF46 strains, respectively (Table 1). We also injected pPD117.01-phmt-1; hmt-1::rfp into the hmt-1(gk161) mutant to create VF37 worm strain expressing the full-length HMT-1::RFP fusion under the control of hmt-1 promoter. N2 (wild-type) worms expressing the full-length HMT-1::GFP or \u0394NTE-HMT-1::GFP, NTE::GFP, or HMT-1\u039443::GFP were generated by crossing with males of VF12, VF23, VF24, VF46 the newly generated strains were designated VF31, VF32 VF33, and VF50 (Table 1). Using genetic crosses, we also generated hmt-1(gk161) strains co-expressing ABCB6/HMT-1\u039443::GFP with the full-length ABCB6/HMT-1::RFP, both controlled by hmt-1 promoter region . VF57 was generated by crossing wild-type worms expressing LMN-1::GFP with VF3 (hmt-1(gk161)). We used Cd sensitivity assays to select for homozygous hmt-1(gk161) expressing different truncated or full-length GFP- or RFP-fusions. The genotype was confirmed by worm PCR. Briefly, 20 worms from each strain were collected into PCR lysis buffer containing 60 \u03bcg/ml proteinase K, 10 mM Tris-Cl, pH 8.2, 50 mM KCl, 2.5 mM MgCl2, 0.45% Tween-20, 0.05% gelatin and frozen at -80\u00b0C for 10 min to facilitate the release of genomic DNA (gDNA). A drop of mineral oil was placed on the top of the buffer prior to lysing worms for 1 h at 65\u00b0C followed by enzyme inactivation at 95\u00b0C for 15 min. The subsequent mixture containing gDNA served as a temple for PCR using primer pairs listed in Table 2. The expression of GFP or RFP was verified using a Leica MZ16FA automated fluorescence stereozoom microscope with the Leica EL6000 metal halide illuminator. To maintain stable inheritable expression of transgenes, the injected plasmids were integrated into the genome by \u03b3-irradiation as described . Cadmium sensitivity was assessed by comparing the percentage of progeny that have reached the adult stage under cadmium vs. control conditions. Morphological changes in intestinal cells were observed under the Zeiss Axio Imager M2 microscope. Results are presented as mean values from three independent experiments each of which had three replicates. The total number of worms tested (n) is presented above each graph bar.Cadmium sensitivity assays were performed as described . Briefly-mediated fluorescence does not co-localize with autofluorescent gut granules , we established the emission peaks of GFP and gut granules autofluorescence. We determined that when excited with the 488 nm laser line, GFP emission peaked at 517 nm, whereas autofluorescence emission peaked at 530 nm. Therefore, to eliminate contributions from autofluorescent lysosome-related gut granules, in subsequent studies we collected GFP signal at 517 nm. In addition, to eliminate crosstalks between the emission from GFP and autofluorescence, or GFP and lysosomal dye, Lysotracker Red or GFP and RFP signals, we used 2-channel fast line-by-line multitracking technique.The initial characterization of the full-length ABCB6/HMT-1::GFP localization was done using the Zeiss LSM 710 confocal microscope system and ZEN 2009 software . Live worms (young adults) were mounted on 2% agarose pads and immobilized with 20 mM sodium azide as described previously . To examrab-5::RFP  or rab-7::RFP , or rme-1::RFP , or rab-11:mCherry  from the intestine-specific vha-6 promoter , L4 stage HMT-1::GFP expressing worms were placed on OP50 seeded NGM plates containing 4 \u03bcM LysoTracker Red. Animals were grown for 20 h without exposure to light before young adults were removed and examined by confocal microscopy. To determine whether HMT-1-GFP co-localizes with markers for different endocytic compartments, males of VF12 strain were crossed into worms expressing promoter . These wpromoter . The locAfter establishing the localization of the full-length ABCB6/HMT-1, the subsequent studies were done using Zeiss Axio Imager M2 microscope equipped with the motorized Z-drive as well as FITC and Texas Red filter sets for capturing GFP, RFP, and mCherry-mediated fluorescence. Images were collected with AxioCam MR Camera and the Zeiss AxioVision 4.8 software.E. coli seeded NGM plates  under standard condition for 3.5 days (until the progeny of inoculated worms have reached the adult stage and sufficient amount of embryos were visible on plates). The adult worms and eggs were washed with M9 buffer and collected by centrifugation at 3,500 \u00d7 g. Worm and embryo pellets were resuspended in 5 ml of alkaline hypochlorite solution  and incubated with frequent agitation for 3\u201310 min at room temperature. This procedure dissolved gravid adults but remained embryos intact. The release of embryos was observed under a dissecting microscope. When \u223c90% of worms were dissolved, unaffected embryos were collected by centrifugation at 3,500 \u00d7 g, the supernatant was aspirated and embryos were washed free from alkaline bleach by five rounds of centrifugation /resuspension in M9 buffer. The final embryo pellet was resuspended in 2\u20133 ml of sterile M9 buffer, embryos were allowed to hatch, and were synchronized at the L1 stage in M9 during incubation at 20\u00b0C for 18 h with shaking. Approximately 10,000 L1s were then inoculated onto 150 mm \u00d7 15 mm NGM plates  seeded with OP50 E. coli. Worms were grown for 2.5 days at 20\u00b0C until L1 became young adults.Age-synchronized young adult worms were used for the protein isolation and fractionation. To obtain the sufficient amount of synchronized worms, 100 adult worms were grown on 10 OP50 E. coli by two rounds of centrifugation  and resuspension in M9 medium. To replace M9 medium with lysis buffer, worm pellet from the second centrifugation was resuspended in a lysis buffer containing 50 mM TRIS-HCl, pH 7.6, 2 mM 2-mercaptoethanol 1 mM phenylmethylsulfonyl fluoride, and 1 \u03bcg/ml each of leupeptin, aprotinin, and pepstatin. After centrifugation at 3,500 \u00d7 g for 2 min, the final worm pellet was resuspended in the same lysis buffer (1/1.5 of V worms/V buffer ratio) and transferred into eppendorf tubes. Worms were then broken by sonication at 4\u00b0C in the lysis buffer and worm debris was cleared by low-speed centrifugation at 3,500 \u00d7 g for 10 min. The supernatant, containing microsomal and soluble proteins was collected and subjected to ultracentrifugation at 100,000 \u00d7 g for 1 h using Beckman bench-top ultracentrifuge. The supernatant, containing soluble proteins was collected, frozen in liquid N2 and kept at -80\u00b0C for subsequent manipulations. The microsomal pellet , was washed, re-pelleted at 100,000 \u00d7 g and reconstituted in the same lysis buffer. Membranes were fractionated by sucrose step density gradient centrifugation using 16, 22, 28, 34, 40% sucrose solutions prepared in the same lysis buffer. About 500 \u03bcg of total microsomal membrane protein were subjected to ultracentrifugation for 2.5 h at 100,000 \u00d7 g using Beckman SW41Ti rotor. Protein fractions were collected from each sucrose interface and washed free of sucrose by three rounds of centrifugation /resuspension in the lysis buffer. The resulting membrane fractions were frozen in liquid N2 and stored at -80\u00b0C.Young adult hermaphrodites were collected from plates with M9 medium and washed free from SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analyses were done as described . BrieflyThe suspension of membrane proteins in the IP buffer  was pre-cleared with Protein G beads  for 1.5 h at 4\u00b0C, Protein G beads were spun down and the supernatant was incubated for 4 h at 4\u00b0C with anti-GFP conjugated beads  in the IP buffer. Supernatant was removed by low-speed centrifugation, beads were washed with IP buffer by three rounds of centrifugation/resuspension, and proteins were eluted in Laemmli buffer  and used for the immunoblot analysis.Arabidopsis Biological Resource Center http://www.arabidopsis.org/abrc/index.jsp. Interaction between bait and prey constructs were detected by growing yeast cells on SC medium lacking adenine, histidine, leucine, tryptophan. \u03b2-galactosidase assay was conducted to confirm interactions.Yeast-two-hybrid assays were performed using the mating-based split-ubiquitin system (mbSUS) as described . The Nubp-values of the data. Specifically, \u2217 indicates p < 0.05, \u2217\u2217 indicates p < 0.01, and \u2217\u2217\u2217 indicates p < 0.001.At least three independent experiments each of which had three replicates were performed in all cadmium sensitivity assays. The number of animals tested is indicated above each graph bar in figures. We utilized two-way ANOVA with Bonferroni post-test to evaluate the statistical significance for our data. The asterisk above the graph bar indicates phmt-1::gfp reporter construct have shown that ABCB6/HMT-1 is expressed in multiple tissues of C. elegans including head neurons, intestinal cells, and coelomocytes (phmt-1::hmt-1::gfp). To test whether the GFP-tagged ABCB6/HMT-1 is functional, we used the hmt-1 (gk161) mutant allele (from here on hmt-1) and integrated the translation ABCB6/HMT-1::GFP construct into the hmt-1 mutant genome. The hmt-1::gfp construct fully rescued the sensitivity of hmt-1 mutants to low concentration of Cd (5 \u03bcM CdCl2) . Nearly 80% of hmt-1 worms were rescued by hmt-1::gfp when worms were grown at higher Cd concentration (25 \u03bcM CdCl2), while only 20% were rescued at 50 \u03bcM CdCl2 . We concluded that phmt-1::hmt-1::gfp is partially functional and proceeded with the analysis of its subcellular localization.Our past studies using the transcriptional omocytes . To estaFigures 1C,D) and was located at the periphery of the internal vesicular structures . These vesicles were reminiscent of lysosomes and of the lysosome-related fat-storing gut granules that exhibit autofluorescence in GFP and rhodamine filter-sets . Therefore, to eliminate the contribution of autofluorescence, from then on we collected hmt-1::GFP signals at 517 nm as detailed in Section Materials and Methods. In addition, instead of using the classical way of acquiring the fluorescent signal for both, GFP and rhodamine channels simultaneously, we used 2-channel fast line-by-line multitracking technique. We then tested if ABCB6/HMT-1::GFP would localize to the lysosomes as does its homologs HsABCB6 and rAbcb6 from human and rats, respectively .Confocal microscopy revealed that the bulk of ABCB6/HMT-1::GFP-mediated fluorescence was present in intestinal cells  and only a minor fraction of ABCB6/HMT-1::GFP co-localized with markers for early and late endosomes, RAB-7 and RAB-5 . SDS-PAGE and immunoblot analysis further confirmed that ABCB6/HMT-1 did not co-fractionate with lysosomal, basolateral recycling endosome or Golgi membrane markers . Therefore, we concluded that ABCB6/HMT-1::GFP is expressed mainly in the apical recycling endosomes in C. elegans intestinal cells and, in part in early and late endosomes.To establish the identity of ABCB6/HMT-1::GFP-localized subcellular organelles, we co-expressed this construct with marker proteins for different endocytic compartments . To do sC. elegans. To test that we used co-immunoprecipitation and co-localization assays in C. elegans. We generated hmt-1 mutants expressing ABCB6/HMT-1::RFP, and hmt-1 mutants co-expressing ABCB6/HMT-1::GFP and ABCB6/HMT-1::RFP. We then evaluated whether ABCB6/HMT-1::RFP construct was functional by testing its ability to rescue the Cd sensitivity of the hmt-1 mutant. We found that similar to ABCB6/HMT-1::GFP, the expression of ABCB6/HMT-1::RFP rescued the sensitivity of hmt-1 mutant worms to low (5 \u03bcM) Cd and was also effective in rescuing the sensitivity to higher concentrations of Cd in the medium . Co-expression of both ABCB6/HMT-1::GFP and ABCB6/HMT-1::RFP in the hmt-1 mutant increased Cd tolerance of hmt-1 worms even more , and ABCB6/ HMT-1::RFP localized to the same endomembrane as ABCB6/HMT-1::GFP . Therefore, animals expressing ABCB6/HMT-1::RFP or ABCB6/HMT-1::GFP or co-expressing both constructs were used to test whether ABCB6/HMT-1 interacts with itself in its native organisms. Based on the ability of the anti-GFP antibody to co-immunoprecipitate both ABCB6/HMT-1::GFP and ABCB6/HMT-1::RFP , we concluded that ABCB6/HMT-1 interacts with itself in C. elegans. GFP has a tendency to dimerize  and a construct containing only the NTE domain (NTE::GFP) . In all cases ABCB6/HMT-1::GFP variants were expressed under the control of the hmt-1 promoter.The NTE domain was previously shown to play a critical role in Cd resistance of r, Ycf1p . Also, or, Ycf1p . Here, whmt-1 mutants. We found that while wild-type and hmt-1 mutant worms expressing the full-length ABCB6/HMT-1::GFP were not sensitive to 5 \u03bcM CdCl2 , the hmt-1 mutant expressingABCB6/HMT-1::GFP without the NTE domain, or only the NTE domain were as sensitive to 5 \u03bcM CdCl2 as hmt-1 mutant worms . The truncated proteins were expressed and were associated with the membrane fraction of proteins in C. elegans as evident by the immunoblot analysis . These results are consistent with our past findings in S. cerevisiae . To ensure that the diffuse distribution of fluorescence was not due to the degradation of \u0394NTE-HMT-1::GFP that would lead to cytosolic localization of GFP, total cellular lysates from the hmt-1 mutants expressing ABCB6/HMT-1::GFP, or \u0394NTE-HMT-1::GFP, or NTE::GFP were fractioned into soluble and membrane protein fractions and subjected to SDS-PAGE and immunoblot analysis. These studies showed that all fusion protein variants were associated with the membrane fraction of proteins and thus, the \u0394NTE-HMT-1::GFP or NTE::GFP constructs did not degrade .The NTE domain of ABCB6 in humans plays a role in targeting the protein to the lysosomal membrane . SimilarFigures 5A,C). To test whether HMT-1 variants are associated with the apical recycling endosome, we co-expressed truncated ABCB6/HMT-1::GFP variants with a marker for apical recycling endosomes, RAB-11::mCherry. We found that \u0394NTE-HMT-1::GFP and NTE::GFP did not co-localized with RAB-11::mCherry . Unexpectedly, the co-expression of the \u0394NTE-HMT-1::GFP with the full-length HMT-1::RFP altered the pattern of the expression of the full-length polypeptide . Specifically, while the full-length ABCB6/HMT-1 localized to the internal vesicular structures that corresponded to apical recycling endosomes, regardless whether it was tagged with the RFP or GFP , the full-length ABCB6/HMT-1::RFP was predominantly localized to the plasma membrane when it was co-expressed with the \u0394NTE-HMT-1::GFP . This result suggested that two constructs may have interacted in vivo and that this interaction altered their localization pattern compared to the localization pattern of individually expressed constructs . This suggestion is consistent with our finding that \u0394NTE-HMT-1 weakly interacts with the full-length ABCB6/HMT-1 in yeast-two-hybrid assays   .hmt-1 mutant worms co-expressing the full-length ABCB6/HMT-1::RFP with ABCB6/HMT-1::GFP variants. We found that the co-expression of the full-length ABCB6/HMT1::RFP with the NTE::GFP rescued the Cd-sensitivity of the hmt-1 mutant . This finding is consistent with the minor effect of the NTE::GFP on the expression pattern of ABCB6/HMT-1::RFP . By contrast, the co-expression of ABCB6/HMT-1::RFP with \u0394NTE-HMT-1::GFP significantly altered the ability of the full-length polypeptide to rescue the Cd sensitivity of hmt-1 mutant worms . Specifically, nearly all of hmt-1 mutant worms co-expressing full-length ABCB6/HMT-1::RFP and ABCB6/HMT-1::GFP or hmt-1 mutant worms co-expressing full-length ABCB6/HMT-1::RFP with NTE::GFP reached the adult stage in the presence of 25 \u03bcM of Cd. By contrast, only 38 \u00b1 3.5%, of the hmt-1 mutants co-expressing the \u0394NTE-HMT-1::GFP with the full-length ABCB6/HMT-1::RFP reached the adult stage under this condition and only 8.3 \u00b1 1.5% of worms have reached the adult stage under 50 \u03bcM of Cd . These findings suggested that \u0394NTE-HMT-1 exerts a dominant negative effect on the full-length polypeptide. Consistent with this suggestion, the expression of \u0394NTE-HMT-1::GFP in wild-type N2 worms significantly increased their Cd sensitivity  . Refractile inclusions also appeared in wild-type worms expressing \u0394NTE-HMT-1::GFP but not in worms expressing the full-length ABCB6/HMT-1::GFP or NTE::GFP only . These results are consistent with the suggestion that the function of endogenous ABCB6/HMT-1, but not other ABC-transporter pathways or other metal-detoxification pathways, was disabled in wild-type worms co-expressing \u0394NTE-HMT-1.As we showed previously, refractile inclusions appeared in intestinal cells of Cd-grown es 7A,B) . We alsohmt-1 mutant worms and exposed the transgenic worms to Cd. These analyses revealed that refractile inclusion indeed might be associated with the nucleus since they resided within the LMN-1::GFP marker . Thus, the C-terminal part of ABCB6/HMT-1 is also involved in ABCB6/HMT-1 protein interactions. Finally, our findings also show that in addition to lysosomes, the recycling endosomes are essential for Cd detoxification processes.The nature of these retractile inclusions is unknown. We noticed, however, that these inclusions usually reside near the nucleus. To test whether indeed these inclusions are associated with the nucleus, we expressed the nuclear envelop marker, LMN-1::GFP marker that labels the nuclear envelope, in hmt-1 mutant worms expressing ABCB6/HMT-1\u039443::GFP or hmt-1 worms co-expressing ABCB6/HMT-1\u039443::GFP and full-length ABCB6/HMT-1::RFP in Figure 8A. Western blot analysis confirmed that ABCB6/HMT-1\u039443::GFP was expressed in worms . We then tested whether ABCB6/HMT-1\u039443::GFP would rescue the Cd sensitivity of hmt-1 mutant worms and found that hmt-1 mutants expressing HMT-1\u039443::GFP were acutely sensitive to Cd . These findings suggested that the 43 amino-acid region in the C-terminus of ABCB6/HMT-1 is required for its function in Cd resistance. However, unlike \u0394NTE-HMT-1::GFP, ABCB6/HMT-1\u039443::GFP did not increase Cd sensitivity of wild-type or hmt-1 mutant worms expressing full-length ABCB6/HMT-1::RFP . This finding suggested that the modified polypeptide did not exert a dominant negative effect on the function of the full-length ABCB6/HMT-1 and that the deleted 43 amino acid residues might be needed for the protein\u2013protein interactions. To test whether the inability of ABCB6/HMT-1\u039443::GFP to confer Cd resistance of the hmt-1 mutants was due to a loss of ABCB6/HMT-1 homo-oligomerization, we conducted the yeast two-hybrid assay. The results revealed that ABCB6/HMT-1\u039443 does not interact with the full-length ABCB6/HMT-1 . Together, our results indicate that the 43 amino-acid region in the C-terminus of ABCB6/HMT-1 is important for the function of ABCB6/HMT-1 in Cd resistance and the ability to interact with itself.To identify additional protein motif(s) in the ABC core region that might be involved in protein\u2013protein interaction, we tested the function of the C-terminal end, in particular, 43 amino acid residues beyond the Walker A ATPase motif (759-KGIILERGNHKELLAQQGTYASMWEAQIAEQRAKSIELGEELP-801), because this part was shown to be essential for localization of ABCC1/MRP1 from humans . We deleThe ABC transporters are a conserved group of integral membrane proteins that mediate the Mg\u22c5ATP-energized transport of a wide range of substrates including anticancer drugs, cellular toxins, and heavy metals . The funFigure 1A). Studies of the functional significance of NTE showed that in some topologically similar full-molecule ABC transporters, NTE is required for proper membrane trafficking and oligomerization, whereas in others, acts redundantly with the COOH-terminal region and thus, is dispensable for membrane trafficking  suggesting that translational ABCB6/HMT-1::GFP or RFP fused proteins reside at the correct endomembrane. We then found that the bulk of ABCB6/HMT-1::GFP or ABCB6/HMT-1::RFP fluorescence is associated with intestinal cells  and is localized to the periphery of intracellular vesicles that resembled lysosomes . To our surprise, ABCB6/HMT-1::GFP did not associate with the lysosomes as evident by a distinct fluorescent pattern from ABCB6/HMT-1::GFP and a lysosomal dye, lysotracker . The majority of ABCB6/HMT-1::GFP co-localized with the marker for apical recycling endosomes, RAB-11 . We also found that a minor fraction of ABCB6/HMT-1::GFP was co-localized with markers for early and late endosomes, RAB7 and RAB5 . Based on these results, we concluded that ABCB6/HMT-1 localizes to the apical recycling endosomes and, in part, to early and late endosomes in C. elegans. It is noteworthy that early, late, and recycling endosomes comprise the endosomal-recycling system that is involved in sorting, re-exporting, and degradation of membrane constituents and extracellular solutes including minerals and associated ligands  that are internalized via the endocytic uptake at the plasma membrane , 6, and discussed below]. The fact that the functional ABCB6/HMT-1 localizes to recycling endosomes and, in part, to early and late endosomes suggests that the endosomal-recycling system in C. elegans is involved in Cd detoxification processes.Here, we used a versatile multicellular organism, the nematode worm, membrane . Early rmembrane . It is uC. elegans is a heme auxotroph and relies on the dietary heme uptake and import into intestinal cells but not on the de novo synthesis in mitochondria . Furthermore, we found that the truncated ABCB6/HMT-1 polypeptide lacking the NTE domain exerted a dominant negative effect on the full-length ABCB6/HMT-1. This conclusion was based on finding that the full-length ABCB6/HMT-1 failed to rescue the Cd sensitivity of hmt-1 mutant worms and mislocalized to the plasma membrane when it was co-expressed with ABCB6/HMT-1 lacking the NTE . The dominant negative effect of ABCB6/HMT-1 without the NTE domain occurred via the protein interaction with the full-length ABCB6/HMT-1 and not with other ABC transporter-mediated cadmium detoxification pathways. This conclusion was based on the appearance of hmt-1-specific refractive inclusions in intestinal cells of Cd-grown wild-type worms expressing ABCB6/HMT-1 without the NTE domain . The latter phenotype is specific to Cd-grown hmt-1 mutant worms and is not manifested by other Cd-sensitive C. elegans mutants . The latter finding is consistent with our previous observations that the NTE domain is not sufficient for the protein\u2013protein interactions of ABCB6/HMT-1 . Taken together, the results of structure-function studies suggested that ABCB6/HMT-1/HMT-1 possess interaction motifs within the NTE and the C-terminal 43 amino acids and that both NTE and C-terminal must be present to allow the protein to interact with itself and confer Cd resistance.We have also refined our past structure-function studies of ABCB6/HMT-1 in its native organism. Specifically, we showed that the NTE domain is essential but not sufficient for the localization and function of ABCB6/HMT-1. This conclusion was based on findings that the NTE domain alone or truncated ABCB6/HMT-1 lacking the NTE were not able to rescue the Cd sensitivity of the  mutants , 2005. IB6/HMT-1 . To idenB6/HMT-1 . We founIn conclusion, we substantiated our past findings that ABCB6/HMT-1interacts with itself in its native organisms and, at a minimum, homo-dimerized. Further, we showed that ABCB6/HMT-1 resides at the endosomal recycling system and that the NTE domain is essential for routing the protein to the correct endomembrane. We also found that C-terminus is required for ABCB6/HMT-1 interaction with itself. Lastly, both homo-dimerization and correct localization of HMT-1 is required for Cd-detoxification function.SK, AS, and OV designed and carried out the experiments, analyzed the data, and wrote the manuscript. All authors gave final approval for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "Propionibacterium acnes, Staphylococcus epidermidis, Actinomyces radicidentis, Streptococcus mitis and Enterococcus faecalis strain OMGS3202), bovine serum albumin 4%w/v and their combination. NaOCl was obtained from a stock solution with iodometric titration. Ultrapure water served as negative control. Samples were stirred at 37\u00b0C in aerobic and anaerobic conditions for 30min to approximate a clinically realistic time. Centrifugation was performed and the supernatants were collected and stored at -800 C until analysis. The reaction products were analysed in real time by selected ion flow tube mass spectrometry (SIFT-MS) in triplicates. SIFT-MS analysis showed that the released VOCs included chlorinated hydrocarbons, particularly chloroform, together with unexpected higher levels of some nitrogenous compounds, especially acetonitrile. No difference was observed between aerobic and anaerobic conditions. The chemical interaction of NaOCl with NOM resulted in the formation of toxic chlorinated VOCs and DBPs. SIFT-MS analysis proved to be an effective analytical method. The risks from the rise of toxic compounds require further consideration in dentistry.Root canal irrigation with sodium hypochlorite (NaOCl) is an indispensable part of the chemomechanical preparation of infected root canals in Endodontology. However, there is limited information on the emergence of toxic or hazardous volatile compounds (VOCs) from the interaction of NaOCl with the infected content of tooth biomaterials. The aim of this study was to assess the formation of VOCs and disinfection by-products (DBPs) following the interaction of NaOCl 2.5% v/v with a model system of different sources of natural organic matter (NOM) present in infected root canals, including dentine powder, planktonic multi-microbial suspensions ( Sodium hypochlorite (NaOCl) is one of the of the most widely practised public health components of disinfection of drinking water, sewage-water plants, water supply and distribution systems, swimming pools and industrial applications with the aim of preventing the spread of infection and contamination , 2. Chloet al.  and distribution width (relative span = d90-d10 / d50) . D50 valThe available chlorine content of a 10\u201315% v/v stock solution of NaOCl  was verified with a standard iodine/thiosulfate method . The cheo C until further use.Titrations (n = 3) of sodium thiosulphate consumption were carried out after the standardisation of the titrating agent with potassium iodate (0.05M) as a primary standard at 0.22M. The concentration of NaOCl stock solution was 1.474M. Stock NaOCl solution was diluted with HPLC water to obtain 2.5% v/v NaOCl (0.34M). The NaOCl solutions were stored in air-tight dark containers at 5Propionibacterium acnes, Staphylococcus epidermidis, Actinomyces radicidentis and Streptococcus mitis, recovered in previous study as predominant taxa from the root canals of teeth with refractory endodontic infections, were selected [Enterococcus faecalis strain OMGS 3202 was also included [7 cells mL-1 .selected . Enterocincluded . To esta0C under both aerobic and anaerobic conditions for 30 min to approximate a clinically realistic time of root canal irrigation procedures and ensure homogeneous contact between the reactants [0C until analysis. The preparation of the aliquots and their associated samples as well as the experimental procedures were conducted in triplicates.To study the interaction of NaOCl with combined sources of NOM and their effect on the formation of any chlorinated DBPs and other VOCs, four experimental groups containing aqueous suspensions of NaOCl 2.5% v/v and one control group containing ultrapure water and not NaOCl were developed . The toteactants . After t3O+, NO+ and O2+) are generated in a microwave discharge. Each of these reagent ions can be selected by a quadrupole mass filter and separately injected into a fast-flowing helium carrier gas in a flow tube. The sample gas to be analysed naturally flows into the helium at a controlled rate via a calibrated capillary by virtue of the atmospheric pressure of the sample gas and the much lower pressure of the helium . The chosen reagent ion then reacts with the trace components in the sample (to the exclusion of the major air components) to generate product  ions. The reagent ions and analyte ions are mass analysed by a quadrupole mass spectrometer and counted by a detector. Thus, the characteristic analyte ions identify the neutral trace components present in the sample and their count rates provide their concentrations in real time.The SIFT-MS technique has been extensively described elsewhere , 32; how0C. After equilibrium between the liquid and headspace above it (30 min), the headspace was sampled directly into the SIFT-MS via a heated, calibrated capillary that defines the headspace sample flow rate, as is necessary for absolute quantification of VOCs. The analytical downstream quadrupole mass spectrometer was scanned over the range of mass-to-charge ratio, m/z, using the three reagent ions H3O+, NO+ and O2+ independently. From the m/z values of the analyte ions and their count rates, and using the kinetics database stored in the instrument library, the concentrations of the identified VOCs were immediately obtained [For analysis, the samples were defrosted in air. Analysis of the headspace volatile compounds was carried out in real-time by SIFT-MS. Prior to analysis, three replicate 2.5 mL aliquots of each sample were placed into a sample bag constructed from 50 cm length, 65 mm diameter Nalophan NA , which was then filled with purified air and sealed prior to incubation at 37 obtained , 32.3O+ reagent ion for the control group and experimental group 4 are presented in 3), acetaldehyde (CH3CHO), acetone (CH3COCH3), methanol (CH3OH), ethanol (C2H5OH) can be seen. In the NO+ reagent ion spectra (not reproduced), the characteristic ions for acetic acid (CH3COOH) were present. The characteristic ions of NH3 were also detected in the O2+ reagent ion spectra . It is well known that serum albumin and hypochlorite interaction lead to protein injury and degradation [3 release [2Cl) formation is prominent due to interaction of NaOCl with higher levels of organic matter in group 4, we can suggest that NH3 formation may also originate from the decomposition of chloramine in alkaline aqueous conditions according to the reaction: 3NH2Cl + 3OH\u2212 \u2192 NH3 + N2 + 3Cl\u2212 + 3 H2O [3, a higher level of CH3CHO release in group 4 than the control or groups 1\u20133 was observed, under anaerobic conditions. This implies that a specific reaction of NaOCl with BSA may occur.The presence of ammonia in the control group is as a result of the emission from the bacterial population, which is produced from the metabolism of peptides and amino acids, especially via L-aspartate catabolism . Group 4radation . One pos release . Assumin + 3 H2O . Apart f3O+ spectrum  is most probably either C5H13N (pentylamine) or C4H9NO (butyramide). The minor peak at m/z 102 (plus its monohydrate m/z 120) is most probably either C6H15N (dipropylamine) or C5H11NO (pentanamide). However, there is no doubting the identity of the much more pronounced characteristic analyte ions of acetonitrile (CH3CN) in the headspace of all groups containing NaOCl (not present in the to the control group headspace). The reaction of CH3CN with H3O+ reagent ions in humid media almost uniquely produces four analyte ions CH3CNH+(H2O)0,1,2,3 with m/z values of 42, 60, 78, 96, which are clearly seen in Evidence for the presence of amines or amides in the Hspectrum  and othe3CN is by the reaction of NaOCl with aldehydes and monochloramines, which has previously been investigated using solid phase microextraction (SPME) and GC-MS [A major question that needs further investigation is how these volatile nitrogenous compounds are formed and whether their direct analysis by SIFT-MS in the headspace may offer insight into the reactions of NaOCl with proteins and amino acids. One possible explanation for the formation of CHnd GC-MS . Note thnd GC-MS .3CN can be absorbed into the body by inhalation of its vapour and by dermal absorption through the skin and by ingestion. No data are available on its carcinogenic effects in humans; EPA has classified it as non-carcinogenic to humans [CHo humans . Its rel3O+ and NO+ and so these reagent ions are not very useful for the analysis of these compounds. However, they usually react rapidly with O2+ ions [35Cl to 37Cl isotopes which is statistically 3:1 (75%:25% peak intensities). Hence, by recognising analyte ions that differ by 2 m/z units that have peak intensity ratios of 3:1, it is possible to identify monochlorinated ions and suggest molecular formulae. For ions containing two chlorine atoms, three isotopologue ions will be formed separated by 2 m/z units with the peak height intensities in the ratio 9:6:1 (56%:38%:6%). Even though the spectra obtained using O2+ reagent ions are very \u201cbusy\u201d, because these energetic reagent ions can fragment analyte neutral molecules and produce multiple mass spectral peaks, as can be seen in 2+. This closed shell ion is known to be the product of the O2+ reaction with chloroform (CHCl3) [3 is present in the headspace of all the samples containing NaOCl at easily measured concentrations that exceed that of NH3 in groups 1\u20134  . Thus, Coups 1\u20134 . To deteet al. [3, following the 15-min interaction of NaOCl with bovine dentine powder and pulp tissue fragments. In another study, the formation of trihalomethanes and other DBPs was documented using gas chromatography with electron capture detector (GC-ECD), following the 1-h interaction of chlorine (1\u20133 mgL-1) with E. coli and Pseudomonas aeruginosa bacterial cells [3 may derive from the interaction of NaOCl with acetone according to the historically known haloform reaction: 3NaOCl + C3H6O \u2192 CHCl3 + 2NaOH + NaOCOCH3 [As mentioned in the Introduction, Varise et al.  reportedal cells . The comal cells . This eval cells . An alteNaOCOCH3 .3, a trihalomethane, may have implications in dentistry [3 at 10 mg (m3)-1 or 2 mg L-1 per 8 working hours [3 is still used in endodontics as a solvent of root canal sealers and gutta-percha in cases of root canal retreatment [3 has no negative health effects under careful and controlled use [3 formation is evident with all combined components in our experimental group samples and further risk assessments are essential in dentistry.The production of CHClentistry . It has entistry . The Unientistry . The Worentistry , but theng hours . CHCl3 ireatment  as well reatment . Despitereatment , risk aslled use , 59. TheThe results of this study offer an overview of the chemical interactions within chlorinated aliquots of endodontic origin. The detection of VOCs and DBPs and their relevant concentrations should be interpreted with caution as they do not entirely represent conditions of endodontic practice. However, they do indicate that additional chemical compounds that are encountered when NaOCl is used as main root canal irrigant. So, the outcomes of this study cannot be directly extrapolated in clinical conditions, but the formation of DBPs and VOCs implies that the risks and drawbacks from the use of NaOCl in dental clinical procedures require critical review and further appraisal with respect to human and environmental exposure.Patients and dental staff may accidentally inhale the volatile phase of DBPs formed during the contact of NaOCl with organic substrates in chemo-mechanical and irrigation procedures, representing a potential threat to human health . There iFurther studies are required for the examination of the cumulative effects on dental staff and the degree of patient exposure through accidental inhalation of the volatile phase of DBPs during endodontic treatment procedures, under conditions of good practice.ex vivo study and in the rejection of the null hypothesis, the chemical interaction of NaOCl 2.5% with dentine powder, bacteria, bovine serum albumin and their combination resulted in the formation of toxic DBPs and VOCs under both aerobic and anaerobic conditions.SIFT-MS is shown to be an effective technique for the real-time analysis of volatile compounds released by the reaction of NaOCl and representative components of an infected root canal system. Within the limitations of this"}
+{"text": "Studies based on high-quality linked data in developed countries show that even minor linkage errors, which occur when records of two different individuals are erroneously linked or when records belonging to the same individual are not linked, can impact bias and precision of subsequent analyses. We evaluated the impact of linkage quality on inferences drawn from analyses using data with substantial linkage errors in rural Tanzania.Semi-automatic point-of-contact interactive record linkage was used to establish gold standard links between community-based HIV surveillance data and medical records at clinics serving the surveillance population. Automated probabilistic record linkage was used to create analytic datasets at minimum, low, medium, and high match score thresholds. Cox proportional hazards regression models were used to compare HIV care registration rates by testing modality (sero-survey vs. clinic) in each analytic dataset. We assessed linkage quality using three approaches: quantifying linkage errors, comparing characteristics between linked and unlinked data, and evaluating bias and precision of regression estimates.n\u00a0=\u2009263) and clinics (n\u00a0=\u2009142). Automated probabilistic linkage correctly identified 233 individuals  at the low threshold and 95 individuals (PPV\u2009=\u200990%) at the high threshold. Significant differences were found between linked and unlinked records in primary exposure and outcome variables and for adjusting covariates at every threshold. As expected, differences attenuated with increasing threshold. Testing modality was significantly associated with time to registration in the gold standard data . Increasing false matches weakened the association . Increasing missed matches  was strongly correlated with a reduction in the precision of coefficient estimate .Between 2014 and 2017, 405 individuals with gold standard links were newly diagnosed with HIV in sero-surveys (Similar to studies with more negligible levels of linkage errors, false matches in this setting reduced the magnitude of the association; missed matches reduced precision. Adjusting for these biases could provide more robust results using data with considerable linkage errors. A growing number of demographic and epidemiological research studies are conducted using linked datasets from multiple sources . In the A recent Wellcome Trust report detailed how record linkage adds to the value of medical research in low- and middle-income countries . A uniquThe linked data infrastructure created by PIRL includes gold standard links between HIV serological survey data and manually digitised medical records from three clinics serving the surveillance population, two of which offer HIV testing services while the third enrols HIV-positive individuals into care. As an illustrative example to evaluate linkage errors, we tested whether individuals who receive their first HIV diagnosis during a village-based HIV serological survey enrol for HIV care services quicker than those who receive their first HIV diagnosis in a clinic that also offers HIV testing. For this analysis, we first assessed the relationship between diagnosis location and time to enrolment into HIV care in the gold standard linked data. We then conducted automated record linkage, a process that included no human interaction or involvement like PIRL, to create four test datasets based on varying levels of match score threshold. Linkage errors, including false and missed matches, were quantified in each test dataset, overall and by individual characteristics. Finally, we determined whether and how linkage errors impacted the analysis of the primary research question by comparing the characteristics of linked and unlinked records and the bias and precision of regression coefficients.The Kisesa observational HIV cohort study was established in 1994 and is located in a rural ward in the Magu district of Mwanza region in northwest Tanzania ) of individuals diagnosed in a clinic subsequently registered for HIV care by the study cut-off date, compared to 42 (16%) of those diagnosed in the sero-survey (p\u00a0<\u20090.0001).During the study period, 263 and 142 individuals with gold standard links received their first positive HIV diagnosis in the sero-survey and clinics, respectively  Table\u00a0. A majorOf the 405 gold standard links, automated linkage correctly identified 248 records, falsely matched 157 records, and missed 157 records at the minimum match score threshold. This resulted in a sensitivity and positive predictive value (PPV) of 61% and a false match rate of 39% .There was a significant association between testing modality and time to registration at the CTC in the linked gold standard data in favour of those receiving their diagnosis at a walk-in clinic   introduced bias at all match score thresholds. False matches reduced the magnitude of the association between the tested exposure and outcome while increasing numbers of missed matches reduced the precision of these estimates, which is comparable to analyses in settings with higher quality data , 11, 29.We used standardised differences to identify variables that were more affected by linkage error and potential sources of bias as was done in previous studies . We founWe found measurable bias in the regression coefficient of the primary exposure at every match score threshold. Selection bias is likely to have impacted the analyses given that selection into the linked datasets was related to both the exposure and outcome , 33. TheOur findings suggested the optimal point that balanced trade-offs between false and missed matches  was at the minimum match score threshold. However, optimisation in this context is inappropriate because there were large biases in the primary regression coefficient at this and all other match score thresholds, which would have led to misleading interpretations of the results. While the consequence of optimisation in our primary analysis would have resulted in a biased measure of the association between variables found in different data sources, a potentially more appropriate use of optimisation may have been to obtain a count outcome from a single data source. The proportion of individuals who registered at the CTC (outcome) was 29% in the gold standard data and ranged between 17 and 19% in the automated linked datasets. Therefore, if our research question was to obtain the proportion or rate of individuals who registered at the CTC our conclusions would also have been meaningfully different at every match score threshold. While optimisation was possible with our data, we conclude there was no optimal threshold that balanced trade-offs between PPV and sensitivity as well as resulted in unbiased associations between two variables or count outcomes of a single variable.A strength of this analysis was the access to individual-level data collected in the PIRL software, clinics, and sero-surveys. This information is often only available to individuals performing the linkage and not to researchers conducting analyses \u201339 and aThere were some limitations. First, the magnitude of the tested association between the selected exposure and outcome was large in the gold standard data, which was probably why the conclusions of the primary regression analysis were similar in the automated linked datasets at the lower match score thresholds even after measurable attenuation in the estimate. It is likely that a more modest association found in the gold standard data would have resulted in a null association and therefore different conclusions as has been found in other studies . Second,Recently, there has been increased attention on how errors arising during the linkage process impacts inferences drawn from analyses using imperfectly matched data, but predominately in high-income countries with negligible linkage errors. We provided original evidence that the impact of linkage quality is similar in a low-income country setting with substantial linkage errors. We plan to investigate methods that minimise or correct for these biases and provide more robust results using data with considerable linkage errors. Until these analyses are complete, our results suggest that researchers in similar settings desiring to perform probabilistic record linkage should allocate resources toward PIRL or similar system[s]."}
+{"text": "Vibrio mimicus is a foodborne pathogen, which is widely distributed in the aquatic environment. Moreover, it is often involved in aquatic animal diseases. In recent years, V. mimicus is an emerging pathogen in some species of Siluriformes. The strain SCCF01 was isolated from yellow catfish (Pelteobagrus fulvidraco). In this study, we aimed to perform genomic analysis of V. mimicus strain SCCF01 to identify genetic features and evolutionary relationships. Information on gene function and classification was obtained by functional annotation, and circular graph of strain SCCF01 genome, which was created by Circos v0.64. Information on virulence genes  was obtained by virulence genes annotation. Genome element prediction showed that most of the mobile elements were distributed in chromosome I. Therefore, chromosome I of SCCF01 genome has more plasticity than chromosome II and might be larger in size. Genomic linear relationship between the strain of V. mimicus and strain SCCF01 was analyzed by linear pairwise comparison but was unable to determine the relationship. Gene family analysis predicted that the evolutionary direction of strain SCCF01 was: clinical strain \u2192 environmental strain \u2192 SCCF01 strain. Phylogenetic analysis showed that the strain SCCF01 was more closely related to environmental strains. According to gene family analysis and phylogenetic analysis, we speculated that strain SCCF01 has probably diverged from environmental strains. Vibrio mimicus (V. mimicus) was initially considered as an atypical V. cholerae, which is closely related to Vibrio cholerae. V. mimicus is a widely distributed aquatic bacterium that can cause disease in humans and massive death of aquatic animals. It is a foodborne pathogen   were detected in chromosome I. These mobile elements are connected closely to horizontal gene transfer contribution to acquire genes ,35\u201337. TV. mimicus SCCF01 genome, suggested that the evolution of the strain SCCF01 genome structure is marked by interchromosomal rearrangements . Our analysis of the ngements . StructuVibrio, and the gene gain events in the evolutionary process of V. mimicus and V. cholerae were more frequent than the gene loss events [A gene family is a set of several similar genes, formed by duplication of a single original gene. The statistics of gene family numbers were obtained according to the cluster of orthologous group based on protein sequences of SCCF01, ATCC33654, ATCC33655, VM223, and MB-451. Gene family analysis showed that the number  of strain SCCF01 were calculated more than other strains . In addis events . TherefoV. mimicus species  based on locally collinear block searching. The phylogenetic tree from V. mimicus species  and environmental V. mimicus . The evolutionary relationships inferred by this tree suggest that SCCF01 is more closely related to the environmental isolate.In order to determine the phylogenetic relationship of the SCCF01, genome tree analysis was performed from  species  and Vibr species  showed tVibrio genus  showed that the strain SCCF01 was classified into the V. mimicus cluster and further proved that the strain SCCF01 was determined to be V. mimicus on the genome level.The phylogenetic tree from V. mimicus strain SCCF01 revealed common basic features. The information of virulence  was obtained by virulence genes annotation and will be useful for the development of gene attenuated vaccine and pathogenesis study for this pathogen. Secondly, chromosome I of the SCCF01 genome has more plasticity than chromosome II and might be larger in size. Finally, we speculate that the strain SCCF01 has probably diverged from environmental strains based on gene family analysis and phylogenetic analysis.First, Genome analysis of"}
+{"text": "Pyrophosphate modulates plant stress responses via SUMOylation. Published 20, February 2019eLife paper cited above based on irregularities associated with the western blots shown in Figures 3D and E as well as Figures 4C and D performed by Gorkem Patir-Nebioglu (GPN). Initiated by reports on PubPeer, the authors have investigated the matter and have found that in both Figures 3 and 4 the blots were spliced together from different original images without indication and a single lane was copied twice in Figure 3E. In addition, GPN has admitted to having intentionally loaded less protein for fugu5 at 4\u00b0C  and 40\u00b0C . The authors are currently running independent repetitions of the experiments in question and hope to be able to provide appropriate data in the near future. However, with the currently available data, the conclusion that SUMOylation is reduced in fugu5 mutants is not justified and all authors have agreed to retract the paper.The authors are retracting the"}
+{"text": "Past experimental research has shown that when rating systems are available, buyers are more generous in accepting unfair offers in ultimatum bargaining. However, it also suggests that, under these conditions, sellers behave more fairly to avoid receiving negative feedback. This paper experimentally investigates which effect is stronger with the use of a rating system: buyers\u2019 inflated inequity acceptance or sellers\u2019 disapproval aversion. We explore this question by varying the information condition on the buyers\u2019 side. Our experiment shows that in a setup where the size of the pie is common knowledge for both buyers and sellers, when a rating system is present, the sellers exhibit disapproval aversion but the buyers do not display greater acceptance of inequity. By contrast, when only sellers are aware of the size of the pie, sellers behave aggressively to exploit buyers and their behavior does not change in the presence of a rating system; however, buyers display greater acceptance of inequity when a rating system is present. We discuss how these results can be explained by a theoretical model that includes sellers\u2019 social disapproval aversion and buyers\u2019 disappointment aversion in addition to the players\u2019 inequality aversion.The online version of this article (10.1007/s10683-017-9554-z) contains supplementary material, which is available to authorized users. However, which effect is more dominant when there is a rating system present in ultimatum bargaining: buyers\u2019 inflated inequity acceptance or sellers\u2019 disapproval aversion? Does the relative strength of these two effects differ by information condition? This paper is the first to study how the impact of expressing emotions differs according to whether the size of the pie is common knowledge to all players (complete information) or is only known to sellers (incomplete information).In recent decades, economists have devoted considerable efforts to studying the impact of expressing emotions on people\u2019s behavior when there is complete information and have found that emotional expression may affect both the senders and the recipients of the expression. On the one hand, it has been documented that people have preferences against receiving disapproval from others. Consequently, they behave pro-socially to ensure that they do not receive negative feedback.Although past studies have used complete information setups to study the effects of expressing emotions, understanding such effects under the incomplete information setting is equally important for two particular reasons. First, the incomplete information setup is more realistic under some circumstances, in which sellers are better informed than buyers about the products they sell. On the one hand, such price settings as foods in grocery stores and standard items such as pens, university textbooks and music CDs in physical stores or on the online marketplace  can be described as buyers having complete information. On the other hand, some transactions can be best described by containing incomplete information on the buyers\u2019 side. Examples include used products, medical services, and education services such as higher education. Users usually become aware of the quality of the goods and services only after they have purchased or consumed them. A rating system is available in some cases , but not for other cases .Second, asymmetry of information between sellers and buyers is known to change the picture of the bargaining between them. Many experiments with complete information have demonstrated that people prefer fair outcomes in ultimatum games  and buyer i submits a purchase threshold (pbi). If psj\u00a0\u2264\u00a0pbi, then the transaction between i and j is closed.Our experiment is based on a finitely repeated ultimatum game. We design four treatments by varying two dimensions. The first dimension is constituted by whether buyers are given the opportunity to rate sellers or not. Sellers are informed of their own ratings after the transactions have been completed. The ratings are not disclosed to other group members and are not carried over from period to period.j and buyer i could result in more unequal divisions under incomplete information than under complete information conditions. We then describe how a fairer or a less fair situation could hold as an equilibrium outcome with a rating system if players are inequality averse and sellers exhibit disapproval aversion. We then show that with a rating system present, buyers\u2019 inflated inequity acceptance could dominate sellers\u2019 disapproval aversion when the buyers are not aware of the size of the pie (incomplete information), because buyers dislike disappointment resulting from possibly a lower-than-expected size of the pie.We first theoretically describe how bargaining between seller Our experiment results largely confirm the theoretical analyses regarding social disapproval aversion and disappointment aversion. First, the divisions of the pies were much more unequal with incomplete than with complete information. Second, sellers exhibited disapproval aversion with complete information, which is consistent with past research. Specifically, the sellers attempted to keep smaller shares of the pies when the rating system was available, compared with when it was not available. Third, and in sharp contrast, with incomplete information, sellers\u2019 disapproval aversion did not affect their bargaining behavior. Whether or not the rating system was present, sellers aggressively attempted to take more from their buyers. Instead, buyers displayed greater acceptance of inequity when the rating system was present than otherwise. The enhanced buyers\u2019 acceptance of unfair offers increased the inequality in the divisions of the pies. In short, our paper suggests that a rating system may have opposite effects depending on the information conditions.The rest of the paper proceeds as follows: Sect.\u00a0Our experiment is based on a finitely repeated ultimatum game. At the onset of the experiment, subjects are randomly assigned to an interaction unit (group) with another nine subjects. A group of ten subjects is then randomly divided into two subgroups of five subjects. Five subjects in one subgroup are assigned the role of seller (proposer) and the five in the other subgroup are assigned the role of buyer (responder).t, each seller is randomly matched with a buyer in his group. Since there are five buyers and five sellers in a group, the probability that a seller is matched with the same buyer both in period t and period t\u00a0\u2212\u00a01 is 20%. In each period, every seller has one commodity whose quality is the same across all five sellers in the group. The quality  of the commodity, qt, is randomly  drawn from the set of integers ranging between 0 and 40 in each period. The random drawing process is independent across periods. The experimental design follows the standard ultimatum games with a strategy method. Each seller proposes a price, psj, to sell a commodity to his matched buyer. They can sell at most one commodity. psj must be an integer ranging from 0 to 40. Each buyer simultaneously submits a purchase threshold, pbi, to her matched seller.psj\u00a0\u2264\u00a0pbi, the deal between buyer i and seller j is closed; seller j obtains a payoff of psj\u00a0\u2212\u00a0qt/2, and buyer i obtains a payoff of qt\u00a0\u2212\u00a0psj. Here, we can interpret qt/2 as the production cost of a commodity or the value of it for the seller. If psj\u00a0>\u00a0pbi, the deal is not closed, and the payoffs for both players are zero in that period. Note that when a deal is closed but qt\u00a0\u2212\u00a0psj\u00a0<\u00a00, the buyer incurs a loss. Each player is informed of their own interaction outcome at the end of each period. Buyer i is then made aware of the seller\u2019s offering price. However, seller j is not informed of the matched buyer\u2019s purchase threshold; the seller is only informed of whether the offer was accepted. Subjects are paid based on the sum of their payoffs earned during all 50 periods.qt, and the interaction rules, such as the formula for the payoffs, are common knowledge to the subjects.The structure of each period is identical. At the onset of a period qt) is known to both sellers and buyers, or is only known to sellers, before the transactions in each period. In the incomplete information condition, the buyers learn the realized value of qt at the end of each period.No Rating, Complete Information\u201d (N\u2013C) treatment, the \u201cRating, Complete Information\u201d (R\u2013C) treatment, the \u201cNo Rating, Incomplete Information\u201d (N\u2013IC) treatment, and the \u201cRating, Incomplete Information\u201d (R\u2013IC) treatment.We design four treatments by varying two dimensions in the experiment. The first dimension is whether the value of the commodity . Buyers are instructed that the lowest number (0) means \u201cvery unfair,\u201d 3 means \u201cunfair,\u201d 7 means \u201cfair,\u201d and the highest number (10) means \u201cvery fair.\u201dAt the end of each treatment, demographic information, such as gender, is collected. These responses are used as control variables in the data analysis.Each seller is randomly assigned an identification number, is randomly paired with a buyer, and then these interact with each other in each period. As discussed in Sect.\u00a0qt, any division of the pie (p\u00a0\u2212\u00a0qt/2)/(qt/2)\u00a0\u00d7\u00a0100[%] for the seller and (qt\u00a0\u2212\u00a0p)/(q/2)\u00a0\u00d7\u00a0100[%] for the buyer, where p\u00a0\u2208\u00a0, can be realized as an equilibrium outcome. Note that the size of the pie in this experiment is qt/2 (=\u00a0qt\u00a0\u2212\u00a0qt/2). Under a Nash equilibrium, the same p is offered by a seller and is also set as a purchase threshold by the buyer, and their transaction is closed. In addition, there are many equilibrium outcomes where deals are not closed, and both sellers and buyers receive nothing (see Appendix A.1).Our experiment uses the standard ultimatum game with a strategy method. Thus, there are multiple equilibrium outcomes in each treatment. First, in the N\u2013C and R\u2013C treatments, for a given qt, the matched buyer selects a purchase threshold pb irrespective of qt, as the buyer is not informed of the value of qt. We write psj: \u00a0\u2192\u00a0 as the strategy of seller j, and pbi \u2208  as the strategy of buyer i.ps\u00a0>\u00a020, and the buyer sets her purchase threshold at 0.Second, in the N\u2013IC and R\u2013IC treatments, while a seller can condition his strategy on Equilibrium Analysis Based on the Standard Theory.There are multiple equilibria in all of the four treatments. In an equilibrium where a deal is closed, both the buyer and seller can obtain positive shares of the pie in the N\u2013C and R\u2013C treatments. However, in the N\u2013IC and R\u2013IC treatments, only a very unequal division of the pie is observed in a Bayesian Nash equilibrium where a deal is closed.Based on Summary 1, we now have the following testable hypothesis for the impact of the information condition on subjects\u2019 divisions of the pies.A\u00a0more unequal division of the pie is realized in the N\u2013IC and R\u2013IC treatments than in the N\u2013C and R\u2013C treatments.Players\u2019 Inequality Aversion and Sellers\u2019 Disapproval Aversion\u00b5i indicates the utility weight of the inequality of buyer i and it differs by buyer. In our theoretical analysis, we use the following quadratic function as f(.)Unlike as given in Summary 1, buyers obtain some positive payoffs and thus the divisions of the pies can be less unequal in equilibrium even in the incomplete information treatments, if we assume that people have other-regarding preferences. As an illustration, assume that all subjects have inequality-averse preferences  payoffs in all the treatments.qt, the degree of inequality with regards to the division of the pies is mitigated to some degree.As shown in Appendix A.2, regardless of the information condition, the seller\u2019s best response strategy (price) would depend on c\u00b7(r\u00a0\u2212\u00a05), where c is a positive constant and r is the rating . With this setup, the seller\u2019s payoff is re-written by Next, we consider how the presence of a rating system may affect the players\u2019 behaviors using the model with inequality aversion [Eqs.\u00a0\u20133)]. As]. As3)].j selects psj to maximize his utility i selects pbi and then r in the later rating stage to maximize her utility pbi\u00a0\u2265\u00a0psj), the buyer\u2019s rating scores are negatively correlated with the seller\u2019s offering prices .We also assume that there are no costs on the buyer\u2019s side because giving ratings is mandatory.Buyers give positive (negative) ratings to sellers who take less (more) from the pies when their transactions are closed.pbi with the seller\u2019s offering price whenever As the disutility the buyer incurs from material inequality is diminished by acts of expressing emotions, the buyer shows more willingness to accept a higher price , compared with when the rating system is not available. Note that the buyer would accept an offer by matching Equilibrium Analysis Based on Inequality Aversion and Disapproval Aversion.(a) Given q, buyers exhibit more willingness to accept unfair offers in the R\u2013C (R\u2013IC) treatment than in the N\u2013C (N\u2013IC) treatment. However, (b) there exists not only a fairer equilibrium but also a less fair equilibrium in the R\u2013C (R\u2013IC) treatment than in the N\u2013C (N\u2013IC) treatment.Buyers\u2019 Disappointment Aversionq; but (b) disappointment aversion would not affect buyers\u2019 behaviors in the R\u2013IC treatment because of the rating opportunity.qt when making decisions. The likely impact of buyers\u2019 disappointment aversion with incomplete information suggests that the difference between buyers\u2019 inequity acceptance with and without rating could be greater in the incomplete information setup than in the complete information setup.Under which information condition could buyers exhibit stronger inequity acceptance: complete or incomplete information? We now explain that stronger inequity acceptance may be observed in the incomplete information setting due to buyers\u2019 disappointment aversion. A large body of literature suggests that a subject could incur a disutility from disappointment if realized outcomes are lower than his/her certainty equivalent in risky decisions ). One may wonder to which equilibrium subjects\u2019 interactions could converge through adjustments to their strategy over the course of repetition.(a)\u00a0Sellers exhibit strong disapproval aversion in the R\u2013C treatment. (b) By contrast, in the R\u2013IC treatment, buyers exhibit strong inequity acceptance.qt) was randomly selected in each period, and the same value was used for qt for both groups.Four sessions per treatment\u2014two at Brown University and two at the National Taiwan University\u2014were conducted. A total of 320 students participated in the experiment. All instructions were neutrally framed (see the Appendix for the instructions).Section\u00a0qt/2 (=\u00a0qt\u00a0\u2212\u00a0qt/2), the seller j\u2019s keep value and keep share are each calculated as (qt\u00a0\u2212\u00a0pbit); which we call the \u201ckeep\u201d of the buyer. We also define buyer i\u2019s \u201ckeep share\u201d as (qt\u00a0\u2212\u00a0pbit)/(qt/2).Table\u00a0,,Among others, four clear findings, each of which holds both for the USA and Taiwan sessions, were obtained. First, consistent with Hypothesis 1, the divisions of the pies drastically differed according to the information condition (see columns (7) and (15)). The average realized shares of sellers for closed deals were around 45% to 52% in the N\u2013C and R\u2013C treatments. However, sellers became more selfish with incomplete information. Unlike the N\u2013C and R\u2013C treatments, the average keep shares of sellers were significantly higher than 1 in the N\u2013IC and R\u2013IC treatments (columns (4) and (12)).Hypothesis 1 holds. This can be explained by sellers\u2019 attempts to take more from buyers with incomplete information. Due to the sellers\u2019 aggressive behavior, buyers\u2019 acceptance rates were lower with incomplete information than with complete information.larger with rating than without rating in the incomplete information setting. This suggests that sellers did not exhibit disapproval aversion in the R\u2013IC treatment. However, consistent with Hypothesis 3(b), buyers\u2019 behaviors were significantly affected by the presence of the rating system. Both buyers\u2019 keep and keep shares were far lower in the R\u2013IC than in the N\u2013IC treatment (see again columns (5), (6), (13), and (14)).Second, consistent with Hypothesis 3(a), the average sellers\u2019 keep and keep share were both lower in the R\u2013C than in the N\u2013C treatment (columns (3), (4), (11), and (12)).(i) With complete information, consistent with Hypothesis 3(a), sellers\u2019 keep values and keep shares were both lower in the R\u2013C than in the N\u2013C treatment. (ii) With incomplete information, consistent with Hypothesis 3(b), buyers\u2019 keep values and keep shares were both lower in the R\u2013IC than in the N\u2013IC treatment.increased realized the shares of sellers and, accordingly, the division of the pies became more unequal between sellers and buyers.Fourth, the impact of the rating system on subjects\u2019 bargaining outcomes differs according to the information condition (columns (7) and (15)), driven by Result 2. With complete information, the presence of the rating system reduced the realized shares of sellers from the closed trades. With incomplete information, by contrast, it instead q (see Sect.\u00a0As explained in Sect.\u00a0i to seller j (Table\u00a0j\u2019s keep (columns (1) and (2)) or keep share (columns (3) and (4)) is included as an independent variable.We take a regression approach in which the dependent variable is a rating score given by buyer  j Table\u00a0. In thisSecond, Table\u00a0Whether transactions were closed or not, buyers were more likely to give negative ratings to sellers who attempted to take more from the pies.We saw that the information condition has a clear impact on sellers\u2019 bargaining behaviors in Sect.\u00a0pbi, but not buyers\u2019 keep shares, as buyers were not aware of q when submitting pbi. First, buyers\u2019 purchase thresholds were consistently higher with rating (the R\u2013IC treatment) than without rating (the N\u2013IC treatment). By contrast, the trends of sellers\u2019 keep shares were on average at very high levels and were similar between the N\u2013IC and R\u2013IC treatments. This resonates with the idea that buyers become more inequity-acceptable when they have an ex-post opportunity to rate as the buyers do not need to care about disappointment due to a possibly lower q in the incomplete information setting.We first give an overview of the trends of subjects\u2019 bargaining behaviors. Figure\u00a0The results we obtained in Sect.\u00a0Results 1 and 2 hold also when we test the impact of each treatment factor while controlling for the panel data structure.This paper investigated the effects of expressing emotions in a finitely repeated ultimatum game. In the treatments where both sellers and buyers were aware of the value of the commodity, sellers exhibited disapproval aversion with a rating system present. In contrast, buyers did not display greater acceptance of inequity in that condition. The picture changed drastically once buyers were uninformed of the value of the commodity. With the incomplete information setup, sellers no longer exhibited disapproval aversion, and they attempted to take much larger shares from buyers regardless of the presence of a rating system. Buyers, who were put in weaker positions, became more open to accepting unfair offers if a rating system was available.ps\u00a0>\u00a0q in our design. Our setup is reasonable for a wide variety of circumstances, but it would also be a useful follow-up study to examine the same question in a setup where sellers are required to split the pie so that both sellers and buyers obtain non-negative payoffs. Second, it would also be useful to perform a robustness check using different games, such as prisoner\u2019s dilemma games, to establish the behavioral regularity of our findings. It is possible that the relative strength of disapproval aversion may differ in other games. Finally, needless to say, more replication studies are essential as results may depend on various factors such as culture and populations, although we found similar patterns between the USA and Taiwan.As a final remark, we note that although our results are clear, there are many avenues for future research. For example, details of the experimental setups may affect the direction or degree of the effects of expressing emotions. For instance, the payoffs of buyers were negative if Supplementary material 1 (PDF 544\u00a0kb)Supplementary material 2 (PDF 641\u00a0kb)Below is the link to the electronic supplementary material."}
+{"text": "All supported samples showed a very good dispersion of the carbide or nitride phases.In this work, we studied the effect of molybdenum precursors and the synthesis conditions on the final phase composition of bulk and supported molybdenum carbides and nitrides. Ammonium heptamolybdate, its mixture with hexamethylenetetramine, and their complex were used as the precursors at different temperatures. It was investigated that the synthesis of the target molybdenum nitrides strongly depended on the structure of the precursor and temperature conditions, while the synthesis of carbide samples always led to the target phase composition. Unlike the carbide samples, where the \u03b1-Mo Using this method, crystalline Mo2N was synthesized at the temperature higher than 650 \u00b0C, while Wang et al. [2C by mixing HMT with HMT-AHM at the 7:1 molar ratio and at 700 \u00b0C in an argon atmosphere.In a number of studies, molybdenum nitride and carbide have been prepared by a thermal treatment of single-source precursors. It is a simple and effective method to obtain Mon source ,44,45,46n source  studied 2C, while in the second, they used mechanically mixed AHM and HMT with a molar ratio of 1:4 with obtaining metallic Mo and MoO2. The researchers also described an impact of different parameters in hydrogen thermal treatment preparation of HMT and AHM, namely, AHM:HMT molar ratios, temperature, or heating rates [2C was obtained when the molar ratio of precursors AHM and HMT reached 1:4, and nitride, carbide, and carbonitride composite materials were obtained when the molar ratio was 1:2. They showed that Mo2N became a major product phase at a lower temperature (500 \u00b0C), and for Mo2C a higher temperature (650 \u00b0C) was needed.The main purpose of this study is to gain an understanding of the influence of various preparation methods of a precursor on its final physical, chemical, and textural characteristics. Wang et al.  have alrng rates . They re2O3 spheres (diameter 2.5 mm) were purchased from Sasol , and extrudates of zeolite Beta, TiO2, and ZrO2 were purchased from Euro Support Manufacturing Czechia, s.r.o. . Mesoporous silica SBA-15 (powder) and pellets of alkali-activated zeolite foam (AZF) with diameter 5 mm were synthesized in the laboratory.Analytical grade ammonium heptamolybdate (AHM), hexamethylenetetramine (HMT), and ammonia solution (25 wt%) were supplied by Lach-Ner s.r.o. . The \u03b3-AlBased on the method reported by Afanasiev , the ini3 was prepared by calcination of AHM at 450 \u00b0C for 6 h.Mechanically mixed samples of the HMT+AHM (2:1) and HMT+AHM (8:1) precursors were prepared by grinding in a mortar and pestle for 10 min of HMT with AHM in the corresponding molar ratios 2:1 and 8:1. HMT+AHM-S (1:1) and HMT+AHM-S (2:1) precursors with molar ratios 1:1 and 2:1 were synthesized by evaporation of the ammonia solution of HMT with AHM. MoO2O3, TiO2, ZrO2, SBA, BEA, and AZF. Al2O3, impregnated with HMT+AHM-S (2:1), was signed as Al2O3#. To obtain a high content of molybdenum nitride or carbide phase, the impregnation was performed with a saturated solution. The impregnated support from the same batch was used for the carbide and nitride synthesis. In the case of the AZF support after drying at 120 \u00b0C for 6 h, the impregnation was repeated once more.Precursors for the supported samples were prepared by incipient wetness impregnation of the HMT-AHM 7.5\u00d7 ammonia solution on the supports Al2 flow (75 cm3/min) at 200 \u00b0C for 12 h;Heating (10 \u00b0C/min) of the precursor in the N3/min). A mixture of 20 vol% H2 in N2 was used to prepare nitride samples and a mixture of 20 vol% CH4 in H2 for the carbide preparation. After reaching the desired temperature, the reaction was run for 3 h;Heating to the desired temperature (of 10 \u00b0C/min) in a working gas flow (75 cm3/min);Cooling to the room temperature in the working gas flow (75 cm3/min) for 30 min;Flushing the reactor with nitrogen (400 cm2 in Ar (75 cm3/min) for 2 h.Passivation in 1 vol% OThe synthesis of the final nitrides and carbides was carried out in a vertical quartz tubular reactor  with an internal diameter of 27 mm and length of 1 m , heated The chemical composition of the supported samples was determined by X-ray fluorescence analysis (XRF) of powder materials using S8 Tiger  with the Rh cathode. The results were interpreted using the Spectra plus software. The non-supported samples were analyzed by the ICP method using ICP-EOS Agilent 725 . The carbon and nitrogen content was determined by the elemental analysis of the catalyst powder using Flash2000 Elemental Analyzer .The crystallography of all synthesized material catalysts in powder form was analyzed by X-ray diffraction (XRD) analysis using D8 Advance ECO , applying CuK\u03b1 radiation (\u03bb = 1.5406 \u00c5) with a resolution of 0.02\u00b0 and a period of 0.5 s. The patterns were collected in the 2 theta range of 5\u201370\u00b0 and evaluated by using the DIFFRAC.EVA software  with the Powder Diffraction File database .2 physisorption and mercury porosimetry. The specific surface area (BET) was measured by N2 adsorption/desorption at 196 \u00b0C by using Autosorb iQ . All the samples were dried under vacuum before the analysis in a glass-cell at 200 \u00b0C for 16 h.The textural properties of the samples were determined by NThe visual appearance was studied by scanning electron microscope (SEM) JSM-7500F  with a cold cathode-field emission SEM . Additional images were obtained by using an optical microscope Jenavert  equipped with a Canon EOS 1200D camera . Images with different focusing were folded by the QuickPHOTO CAMERA software .Physical properties and stability of the precursors and samples were studied by thermogravimetric analysis (TGA) using TGA Discovery series  operating in the temperature range of 40\u2013900 \u00b0C (heating 10 \u00b0C/min) in the nitrogen flow . A Quadrupole mass detector OmniStar GSD320  was used for detection of fragments in SCAN mode with 1450 V voltage of the electron multiplier. Thermal behavior of the samples was analyzed by differential scanning calorimetry (DSC) using Q2000 . Approximately 5\u201310 mg of a sample was placed into Tzero pierced aluminum pans. An initial temperature was equilibrated at 0 \u00b0C, then the samples were cooled down to \u221250 \u00b0C at a rate of 10 \u00b0C/min and held for 1 min. After this, the samples were heated up to 450 \u00b0C (10 \u00b0C/min).Initially, before the synthesis, all of the precursors were characterized by several analytical methods. As seen from 3 sample had typical diffraction patterns corresponding to molybdenum trioxide (molybdite).The precursor complexes, synthesized using HMT+AHM-S, were similar, but not completely the same to those reported by Afanasiev. The MoOThe influence of the HMT excess on the HMT-AHM complex prepared by the Afanasiev method was investigated by varying the molar ratio of HMT:AHM in the sequence of 15:1 (sample HMT-AHM 7.5\u00d7), 10:1, and 20:1 (samples HMT-AHM 5\u00d7 and 10\u00d7). The TGA results  show thaDecomposition of other precursors differed from the HMT-AHM 7.5\u00d7 sample. Thus, in the case of HMT+AHM-M (2:1) and HMT+AHM-M (8:1), a conspicuous signal at 200 \u00b0C inherent to HMT decomposition was observed. The crystallization of the HMT and AHM ammonia solution with a molar ratio of 1:1 in the sample HMT+AHM-S (1:1) led to the formation of large transparent crystals that decomposed differently than when only tiny crystals were formed in the sample HMT+AHM-S (2:1). In spite of the fact that the TGA curves of the latter and HMT-AHM 7.5\u00d7 went through the same trajectory and were very similar, a slight difference in their structures was noticeable.2O and NH3, giving transition polymolybdate phases. At the same time, fragments corresponding to NO, N2O, O2, and N2 were also observed, which may have been caused by the presence of air in the thermogravimetric furnace, which was not hermetically sealed. The HMT-AHM complex differed by the signal of CO2 detected in the range of 190\u2013275 \u00b0C, which further gradually released up to 700 \u00b0C.TGA decompositions of AHM and HMT-AHM were determined using a mass spectrometer. Using the literature data ,48, it wDSC results displayed in HMT+AHM-M (2:1) had a sharp endothermic peak, which occurred in the temperature range of 110\u2013130 \u00b0C, and the subsequent endothermic peaks at 250\u2013350 \u00b0C correspond to AHM. The change in the molar ratio of HMT:AHM in HMT+AHM-M (8:1) sample also changed the DSC curve. Only two distinct peaks were observable on the record: the first endothermic peak at 170.51 \u00b0C corresponded to AHM decomposition and the second one at 248.73 \u00b0C to HMT sublimation.According to the DSC records, the complexes obtained by crystallization of the HMT and AHM ammonia solutions differed among themselves and also in comparison with the HMT-AHM complex prepared by the Afanasiev method. Both HMT+AHM-S (1:1) and HMT+AHM-S (2:1), as well as HMT+AHM-M (2:1) complexes, were characterized by the endothermic peak around 120 \u00b0C, which was probably related to the sudden formation of another type of a complex. Despite the fact that the HMT-AHM complex had the same peak, but not quite at intensely, we can conclude that the complex is very similar, but not exactly identical to HMT+AHM-S (2:1). This difference also affected the final structure of molybdenum nitrides and carbides.3 sample has small molybdite crystals produced by thermal decomposition of AHM.SEM results  show tha4 in H2 with a flowrate of 75 cm3/min. All the prepared samples showed a high carbon content  molybdenum carbides were synthesised using 20 vol% CH content , indicat for AHM a, while  for AHM b gave het 700 \u00b0C .The prepared carbides showed a relatively wide range of specific surface area values . The sam2C phase. The exception was AHM-800, the crystallite size of wich was 40.2 nm. The size of the \u03b2-Mo2C crystalline phase was possible to determine only for the sample HMT-AHM-600, where this phase appears separately. The size of \u03b2-Mo2C crystallites in this sample was 4.9 nm. In the case of AHM-700 and HMT-AHM-700, crystallite sizes could not be measured due to the overlap of corresponding diffraction lines. The crystallites sizes were calculated from the reflection of 39.5\u00b0 2 theta for \u03b1-Mo2C and 37.1\u00b0 2 theta for \u03b2-Mo2C.The crystallites sizes  were relThe structure of the prepared non-supported molybdenum carbides was analysed using scanning electron and optical microscopes. The samples synthesised from the HMT-AHM and HMT+AHM-S (2:1) complexes showed a well-crystallized structure, while the microcrystalline structure was peculiar to the samples obtained from AHM . Evaluat2 in N2 was used as a working gas. The specific surface area of the prepared materials and the nitrogen content were very dependent on the content and type of the nitride phase (2/g for the sample prepared from HMT+AHM-M (8:1), followed by HMT-AHM-700. Nitrogen content  molybdenum nitrides were synthesised using the same conditions as for carbides. A mixture of 20 vol% Hde phase . The max content  clearly 2N modification, the cubic phase of metallic molybdenum, and monoclinic MoO2 started to occur at 700 \u00b0C. Higher temperatures resulted in metal molybdenum and MoO2. Nitridation of HMT-AHM  precursor at 700 \u00b0C was cubic Mo3N2 with a small amount of \u03b1-Mo2C. The other precursors examined by XRD  . An exception was for the previously mentioned HMT+AHM-S (2:1), where cubic Mo3N2 was formed with a small proportion of \u03b1-Mo2C. The MoO3 precursor was only partially reduced to MoO2 and produced a very small proportion of metallic molybdenum.The use of the AHM precursor a did not HMT-AHM b gave a d by XRD  produced2N phase and determined at 37.4\u00b0 2 theta. The value was in two ranges of about 18 and 27 nm. Crystallite sizes of other presented phases \u03b3-Mo2N (13.8 nm) and \u03b2-Mo2N (11.7 nm) were calculated at 37.5\u00b0 and 37.7\u00b0 2 theta, respectively.The crystallite sizes of the nitride phases are shown in 2) and sponge phases consisted of \u03b2-Mo2N phases. The same crystalline phase was observed when using the MoO3 precursor  at 700 \u00b0C as a concomitant phase. Basing on these results, we can conclude that HMT-AHM and HMT+AHM-S (2:1) were the most effective precursors of molybdenum carbides and nitrides synthesis. Even though they were very similar, they were not identical materials.Another fact, confirming that the carbides were produced more easily (compare to nitrides), was evidenced by the presence of \u03b1-Mo2O3# was used for the comparative study. The preparation temperature was 700 \u00b0C as a compromise between the formation of the desired phase and the avoidance of structural changes in the supports. The samples exhibited high molybdenum contents ranging from 22.1 to 38.4 wt% (x) or nitride (MoNx) phases decreased the initial specific surface area of the SBA-15 and BEA supports.Basing on the synthesis of the non-supported molybdenum carbides and nitrides, the HMT-AHM precursor was chosen to prepare supported samples. HMT+AHM-S (2:1) on Al38.4 wt% . As a co2/g), determined mainly by the surface of the bulk nitride or carbide phases located in macroporous cavities of the AZF support. The significant decrease of the area from the original was due to clogging micropores, as is the case of the BEA samples. However, there was no noticeable difference in comparing nitride and carbide samples in SBET reduction. Both groups showed very similar surface area values without any prevalence in blocking pores  filled with molybdenum carbide or nitride crystal particles. This was reasoned by the fact that the larger particles of carbide and nitride phases have better stability during the reactions and increased resistance to complete oxidation to crystalline metal oxides ,50. Thesng pores .2 (0.16% of N) contained no nitrogen, or below the detection limit of the device. The carbon content varied between 0.49 and 2.57% for all samples. A small amount of carbon (0.08\u20130.45%) was inherent for each nitride sample and nitrogen was in similar values (0.21\u20132.38%) as carbon in carbides  and crystalline \u03b1-Mo2C and \u03b2-Mo2C (approximately 60:40) and \u03b2-Mo2N, respectively. The influence of the support acidity on the formation of carbide or nitride phases was not clear. On the more acidic supports, in the case of carbides, a mixture of \u03b1- and \u03b2- phases was produced, while in the mixed nitride phase, the crystalline cubic phase of Mo3N2 was observed.XRD results  showed tmplicity ,52,53. Hreserved . The obtx nanocrystals inside the cylindrical pores and outside on the catalyst surface. Moreover, the dramatic decrease in the surface area of SBA-15 was confirmed by TEM, as the blockage caused by the formation of crystals inside the long cylindrical mesoporous structure of the support , and less often the mixture of HMT with AHM, while the preparation of supported catalysts by the impregnation of the HMT-AHM complex is not practically used. AHM impregnated supports are initially calcined in the air to form MoO3 on the surface of the support. The most commonly used one is Al2O3 followed by SiO2/SBA-15. In the case of Mo2C containing catalysts, carbon  is preferably used, which contributes to the formation of the carbide phase on the support surface.According to the literature ,67,68,692O3, TiO2, ZrO2, SBA-15, and zeolite Beta, and also on the less common AZF support. It was investigated that the synthesis of target molybdenum nitrides strongly depends on the structure of the precursor and temperature conditions, while the synthesis of carbide samples always led to the target phase composition. Unlike the carbide samples, where the \u03b1-Mo2C phase was predominant during nitridation, the mixture of \u03b2-Mo2N and MoO2 with a small amount of metal molybdenum was generally formed. However, using the precursor complex obtained from the mixture of hexamethylenetetramine with ammonium heptamolybdate (HMT-AHM), the pure phase of molybdenum nitride was achieved at 800 \u00b0C. At 700 \u00b0C, \u03b3-Mo2N with a small amount of \u03b1-Mo2C was formed from the same precursor. A similar situation occurred when using the precursor synthesized by evaporation of the ammonia solution of HMT with AHM at a molar ratio of 2:1, where the resulting cubic Mo3N2 phase was also accompanied by a small amount of \u03b1-Mo2C at 700 \u00b0C. Supported samples, even at the high molybdenum content, had the high dispersion of the phases. All carbide samples were composed of \u03b1- and \u03b2-Mo2C mixtures. Nitrides supported on Al2O3 consisted of \u03b2-Mo2N, and on TiO2 and ZrO2 consisted of \u03b2- and \u03b3- Mo2N mixtures, SBA-15, and BEA, despite the \u03b2- and \u03b3-phases also containing Mo3N2 and Mo3N4. The composite samples prepared on the foamed AZF support included cavities filled with crystalline \u03b1- and \u03b2-Mo2C in the case of carbides and the \u03b2-Mo2N crystalline phase for nitrides.This study set out the possibility of molybdenum carbide and nitride synthesis using various precursors and reaction conditions. Based on the analysis of the studied supported and bulk materials, the most suitable preparation conditions to obtain the desired phases were considered. The ability to prepare carbide and nitride phases was demonstrated on the commonly used supports Al"}
+{"text": "Modern studies have shown that adaptogens can non-specifically enhance the resistance of human body under a wide range of external stress conditions with a multi-targeted and multi-channel network-like manner, especially by affect the immune-neuro-endocrine system and the hypothalamic\u2013pituitary\u2013adrenal axis. This review article draws the attention to the relationships of adaptogens, tonics from traditional Chinese medicine (TCM) and ginseng-like herbs worldwide, which all have similar plant sources and clinical applications. To clarify the sources and pharmacological mechanisms of these plant-originated adaptogens, which will provide useful information for the utilization of adaptogens to improve the human health. Meanwhile, the TCMs and the world-wide ginseng-like herbs from each region\u2019s ethnopharmacology will be beneficial modernization and globalization. Schisandra chinensis (Turcz.) Bail. and other herbs with the following definition: plant-originated adaptogens that can non-specifically the enhance human body. According to the primary definition of adaptogens, these substances must meet three criteria: first, adaptogens must to be non-specific and must assist the human body in resisting a wide range of adverse conditions, such as physical, chemical or biological stress. These may include environmental pollution, climate change, radiation, infectious diseases, and interpersonal disharmony. Second, adaptogens must maintain homeostasis in humans, that is, these substances can offset or resist physical disorders caused by external stress. Third, adaptogens must not harm the normal functions of the human body.The term of adaptogen was first proposed in 1940 by a scientist from the USSR, namely, N. Lazarev, when he described Then I.Brekhman, a Soviet scientist, studied ginseng in approximately 1950, he extended the concept of adaptogens as follows: medicines that have similar functions as adaptogens can help the body maintain ideal homeostasis under adverse or stressful conditions.Again, Brekhman and Dardymov further defined plant-originated adaptogens in 1969 [In the 1990s, a group of scientists, comprised of Hildebert Wagner, George Wikman and Alexander Panossian, performed many studies on adaptogens and proposed the following definition: adaptogens are natural bioregulators that increase the ability to adapt environmental factors and avoid the damage caused by those factors. In fact, the advantage of adaptogens are they minimized the bodily response to stress, reducing the negative reactions during the alarm phase and eliminating, or at least decreasing, the onset of the exhaustion phase that is part of the so-called general adaptation syndrome .With continuous research for more than half a century, the concept of adaptogens has been continuously modified and perfected. In 1998, the American Food and Drug Administration (FDA)  defined Yance, an American herbal doctor, held a view that adaptogens can improve our ability to recognize, respond, recover, and restore or regenerate. He divided adaptogens into three categories, including primary adaptogens, secondary adaptogens, and adaptogen companions, based on his clinical experience . PrimaryFurthermore, some precise scientific experiments demonstrated that adaptogens can enhance the resistance of human body against various external stimuli as non-specific regulators. Adaptogen function mainly by affecting the hypothalamic\u2013pituitary\u2013adrenal (HPA axis) in response to stimulation by external stress. Primary adaptogens can not only maintain or recover homeostasis and allostasis but can also promote anabolic recovery. Primary adaptogens can produce positive stress response and the associated hormone expression. Primary adaptogens strengthen the functioning of each systems, promote optimal response, promote recovery of function, and help regulate energy use by improving the function of neuroendocrine system and enhancing cellular energy transfer, which can make body utilize oxygen, glucose, lipids and proteins very effectively, thus providing us with a steady supply of energy .Another category of adaptogens is secondary adaptogens, which are consistent with most traditional definitions of adaptogens but not all of the criteria of primary adaptogens. Secondary adaptogens cannot influence the HPA axis directly; however, these adaptogens can affect the immune, nervous and endocrine systems. Secondary adaptogens share several common features: first, these adaptogens typically exert influence on the immune, nervous and endocrine systems; second, these adaptogens do not influence the HPA axis, directly; third, these plant-originated adaptogens include fatty acids, sterols and phenols; fourth, these substances can enhance anabolism. While the secondary adaptogens may meet most of the qualifications of primary adaptogens, but they have yet to be studied extensively .Another category is adaptogen companions, which may not satisfy all the traditional standards but can have beneficial effects on the HPA axis and on anabolism to support the functions of adaptogens. Although these kinds of medicinal plants have similar functions as the other two kinds of adaptogens mentioned above, these plants can not formally be called adaptogen. Thus, these plants are classified as adaptogens companions because they can act synergistically with the other two kinds of adaptogens mentioned above, thereby improving the effects of the adaptogens .Panax ginseng C.A.Mey, Schisandra chinensis (Turcz.) Baill., Acanthopanax senticosus (Rupr. et Maxim.) Harms, Rhodiola crenulata (Hook. f. et Thoms) S.H.Fu, and Lepidium meyenii Walp.Currently, studies have confirmed that the following plants are true adaptogens: According to web of science database, we searched the key word, adaptogen. Based on the analysis of results, we got a conclusion that more and more researches focus on this field over past 10\u00a0years and it showed an increasing tendency from 1999 to 2018, which profiles that it is still a worthwhile direction to explore. Furthermore, we also found out that the many published articles concentrated on the direction of pharmacology pharmacy, biochemistry molecular biology, plant sciences and agriculture. It is still worthy to show its historical development and find out the relationships among adaptogen and tonics and ginseng-like herbs worldwide, which can give a hint to further research in plant-originated adaptogen. Summarized the results of previous studies and our own researches focused on tonics from TCM, we launched out our understanding of adaptogen: Herbs which can non-specific and non-toxicity help human body resist the environmental stress to maintain a homeostasis. The mechanism may multi-targets and multi-channel network to the neuroendocrine system. The adaptogens with tonics from TCM and the world-wide ginseng-like herbs have similar phytochemical and pharmacological properties.Adaptogens can affect different tissues and organs, and adjust each of these parts to attain homeostasis.Adrenals, the glands of stress, mobilize various stress responses to each stress, including physical, biochemical, hormonal, thermal, internal, external, emotional and mental. Stress rather than pathological damage is the primary cause of adrenal fatigue. Excessive stress may be caused by a single strong stimulatory event or by the accumulation of chronic or repeated stress. When the capacity of the adrenals to secrete enough hormones that can make the necessary physiological, and biochemical compensations for that level of stress cannot meet the requirements of continually excessive pressure, adrenal fatigue occurs. The adrenal gland continues to work under adrenal fatigue but cannot maintain normal homeostasis. On the one hand, if the adrenals can deal well with this circumstance and cortisol levels remain adequately elevated to handle the various stresses, over time, the signs and symptoms of metabolic syndrome, such as muscle wastage, hyperglycemia, and suppresses immune or inflammatory responses , begin tEleutherococcus senticosus inhibits catechol-O-methyl transferase, both of which reside in close proximity to stress hormone receptors and catalyze the degradation of stress hormones into inactive compounds [Panax quinquefolius L. [Withania somnifera [Panax ginseng C.A.Mey. [Codonopsis pilosula (Franch.)Nannf. [Eleutherococcus senticosus (Rupr. & Maxim.) Maxim. [Gynostemma pentaphyllum (Thunb.) Makino, Glycyrrhiza uralensis Fisch.ex DC. [Ganoderma Lucidum Karst [Sedum rosea (L.) Scop [The amount of stress hormone produced by the human body increases under external pressure. Adaptogens can increase the effectiveness of adrenal gland secretion, thereby abolishing excess hormone production . Other sompounds . In the olius L. , Withaniomnifera , Panax gC.A.Mey. , Codonop.)Nannf. , Eleuthe) Maxim. , Gynosteh.ex DC. , Ganoderum Karst , and SedL.) Scop .Arthritis is caused by tissue damages and joint diseases, which is typically accompanied by pain and swelling. The most common types of arthritis are osteoarthritis and rheumatoid arthritis. Fibromyalgia maybe an accompanying condition of arthritis; however, it is not considered a form of arthritis because it does not cause inflammation or joint damage.Withania somnifera [Panax ginseng C.A.Mey [Gynostemma pentaphyllum (Thunb.) Makino [Ganoderma Lucidum Karst [Sedum rosea (L.) Scop. [Glycyrhiza uralensis Fisch. ex DC. [Adaptogens can reduce arthritis-associated inflammation and pain effectively , 18. Theomnifera , Panax g C.A.Mey , Gynoste) Makino , Ganoderum Karst , Sedum r.) Scop. , and Gly. ex DC. .Many people suffer from insomnia and other sleep-related problems. External stress perturbs the normal secretion of circadian cortisol, which is the main cause of sleep-related problems. The secretion of cortisol follows the biological clock and external circadian rhythms. The secretion of cortisol peaks in the morning and then decreases, reaching a minimum value at night. Proper exercise, diet and sleep can help maintain stable cortisol levels in the human body.Panax quniquefolius L. [Withania somnifera (L.) Dunal. [Schisandra chinensis (Turcz.) Baill. [Gynostemma pentaphyllum (Thunb.) Makino [Sedum rosea (L.) Scop [Adaptogens help produce cortisol and relieve stress , 25. Stuolius L. , Withani) Dunal. , Schisan) Baill. , Gynoste) Makino , and SedL.) Scop .Panax quniquefolius L., Panax ginseng C.A.Mey, Gynostemma pentaphyllum (Thunb.) Makino, Schisandra chinensis (Turcz.) Baill., and Sedum rosea (L.) [The following plant-originated adaptogens can alleviate the effects of the time difference syndrome, caused by disruption of physiological rhythm of the human body: sea (L.) , 32.One of the most important functions of adaptogens is their ability to help stabilize the internal environment of the human body by affecting the neuroendocrine system. The chemicals in plant-originated adaptogens enhance the ability to adapt to external environments and avoid damage \u201335. A unResearchers have found that plant-originated adaptogens have a positive influence on all aspects of the health of animals and humans . CancersPlant-originated adaptogens play key roles in anti-tumor and multifaceted anticancer mechanisms, such as inhibition of cancer cells production, stabilization of the functions of human body, and promotion of cell repair , 40. TheThe most common chemical anti-tumor medicines currently on the market have side effects such as cytotoxicity and immune suppression. Plant-based immune regulators, for example, plant-originated adaptogens, are generally used as auxiliary treatments to reduce the side effects of these chemical medicines and regain health , 45. NotPanax ginseng C.A.Mey can substantially reduce multidrug resistance by inhibiting Pgp, and it has been shown that Panax ginseng C.A.Mey can prolong the life of the cancer-bearing mice in animal experiments [Eurycoma longifolia can inhibit the expression of Bcl-2 to prevent the development multidrug resistance [When cancer cells adapt to chemotherapy, the main consequence is the development of multidrug resistance. The most simple mechanism of the development of multidrug resistance is as follows: anti-tumor drug molecules flow out from cancer cells through membrane channel proteins driven by ATP, especially by adjustment of P-glycoprotein pump (Pgp-pump) and due to the effects of breast cancer resistance protein-1 (BCRP/ABCG-2) and multidrug-resistance-associated protein-1 (MRP-1) from thymic cancer cells . Among periments , 53. In sistance .In the case of various stress modes, adaptogens can activate the adjustment of different responses to cope with different forms of stress. Adaptogens are the material basis of the bodily response to the external environment and can act on the immune system and the stress response system, showing in Fig.\u00a0Recent studies have shown that the inhibitory effects and long-term overexpression of endogenous glucocorticoids cause stimulatory effects that are adjusted by SNS under stress. The secretion of CRH and AVP increases, which is stimulated by external pressure, thereby promoting the secretion of cortisol and adrenocorticotropic hormone. Furthermore, angiotensin, cytokines and arachidonic acid metabolites participate in the stress response. SNS provides the human body with a fast response mechanism to external stress. In addition to catecholamine, the sympathetic and parasympathetic nervous systems can also secrete a variety of neuropeptides, ATP and nitric oxide (NO) . The folSchisandra chinensis (Turcz.) Baill, can prevent and resist stress because these substances can activate the secretion of cortisol and NO in the plasms and saliva, allowing the body to adapt to heavier loads. After the consumption of plant-originated adaptogens, physical exercises do not increase the level of cortisol and NO in the human body; in fact, the levels decrease, comparing to those present prior to physical exercise. Thus, adaptogens can increase the level of messenger substances that activate stress (NO) and suppress stress (cortisol).Adaptogens do not increase the levels of cortisol and NO in the human body under acute physiological loads , 31, 61.Adaptogens can improve the stress response system to respond to high levels of external signals in the normal or abnormal states. We should determine the similarities or differences between adaptogens and known classical metabolic regulators. According to the above description, the main difference may be that adaptogen can stimulate the CNS. It is now possible to obtain additional information at the biochemical level and identify analogues adrenal cortex hormone analogue, catecholamine analogue and so indicating that the active ingredients have similar structures.There is much overlap between these medicines and many tonics are internationally recognized as plant-originated adaptogens .In traditional Chinese medicine, it is believed that harmony and balance are indispensable for health and the concepts of yin and yang are used to diagnose and cure disease . MedicinPanax ginseng C.A.Mey, Panax quniquefolius L., Panax notoginseng (Burkill) F.H.Chen, Eleutherococcus senticosus (Rupr. & Maxim.) Maxim, Sedum rosea (L.) Scop., and Schisandra chinensis (Turcz.) Baill. According to the terminology used in traditional Chinese medicine, the mechanism of action of plant-originated adaptogens is to achieve equilibrium in both yin and yang, showing great vitality [Tonics have wide range of applications in traditional Chinese medicine and can be used under the conditions of low body resistance and weak constitution or when the human body is finding it difficult to fight severe diseases, this function of tonics is similar to that of plant-originated adaptogens . Tonics vitality .Based on the chemical composition, common tonics can be divided into following several categories in Table\u00a0Withania somnifera), cucurbitacin-R-saponin (Bryonia dioica). The second category includes aromatic compounds with structures similar to that of catecholamine: (a) lignans: eleuteroside E (Acanthopanax senticosus (Rupr. et Maxim) Harms), schisandrin b (Schisandra chinensis (Turcz.)); (b) phenylpropane derivatives: syringin (Acanthopanax senticosus (Rupr. et Maxim) Harms), cinnamyl glycoside (Rhodiola crenulata (Hook.f. et Thoms) S.H.Fu); (c) phenylethane derivatives: salidroside.According to Panossian\u2019s conclusion, the main active chemical components can be divided into the following two categories. The first category includes terpenoids with four-ring skeletons that are similar to cortisol: sitoindosides , regulating cellular and humoral immunity, and affecting cytokine activity. These functions are similar with those of the plant-originated adaptogens mentioned above. Plant-originated adaptogens are generally considered to be the \u201celite of herbs\u201d , and in Many regions, ethnic groups and countries, they have their own medical histories and habits. Continuous development and transfer of knowledge through generations leads to the formation of unique medicinal systems such as traditional Chinese medicine and Indian ayurvedic medicine. Coincidentally, in different regions and medical systems, there are several medicinal plants that are considered to be national treasures or that are called ginseng locally. There is much overlap between plant-originated adaptogens and ginseng species worldwide. Furthermore, most ginseng species worldwide are internationally recognized as plant-originated adaptogens. Both ginseng-like herbs worldwide and plant-originated adaptogens have very similar clinical applications. Ginseng species are widely used by local communities world-wide because these plants can enhance the resistance of the human body and can have various beneficial effects, such as anti-fatigue, anti-ageing, anti-stress, anti-anxiety, anti-inflammatory, and anti-depression. Furthermore, ginseng species may improve the circulatory system and immune system , which cWithania somnifera, which belongs to Slanaceae, is called Indian ginseng; this plant has nourishing and strengthening effects and can delay ageing [Panax japonicus, which belongs to Solanaceae is called Japanese ginseng; this plants have tonifying, strengthening and anti-fatigue effects [Eurycoma longifolia, which belongs to Simaroubaceae is called Malaysian ginseng; this plant can be used as a postpartum tonic or aphrodisiac [Lepidium meyerii, which belongs to Brassicaceae, is called Peruvian ginseng; this plant can be used for natural nutrient, can enhance fertility effectively, and has anti-fatigue effects. The common ginsengs species from different countries and their functions are listed in The results of modern pharmacological studies show that these medicinal plants called ginseng have effects on the neuroendocrine and immune systems, which is similar to the mechanism of action of plant-originated adaptogens, the elite of herbs. However, there have been few studies on the chemical compositions, mechanisms of action, traditional curative effects and similarities of these types of medicinal plants. Comparisons with ginseng species from around the world are helpful for widening the spectrum of plant-originated adaptogen species and for in-depth analysis of the mechanism of action of ginseng species worldwide. The functions and the main functional ingredients of common ginseng-like herbs worldwide were showed in\u00a0Table\u00a0Most medicinal plants called ginseng belong to Araliaceous, but there are also other medicinal plants that belong to other families. For example, y ageing . Panax j effects . Eurycomrodisiac . LepidiuPlant-originated adaptogens have been demonstrated to regulate stress-related changes, at least in animal experiments. However, even after more than 40-years of herbal research, there are very few drugs that have been successfully introduced as adaptogens in modern medicine. Most of these kinds of plant-based medicines are considered to be plant-originated adaptogens and the remaining few are immune enhancers, anabolic agents and antioxidants, which are the same as plant-originated adaptogens. Therefore, there are many difficulties associated with judging whether a plant is a plant-originated adaptogen or not .Adaptogens are stress response modifiers that non-specifically increase resistance to various stressors, thereby promoting adaptation and survival. Adapting to environmental challenges are multistep processes that involve diverse mechanisms and interactions. Multiple molecular networks are involved that coordinate both intracellular and extracellular stress signaling. The metabolic regulation of homeostasis by adaptogens at the cellular and systems levels is associated with multiple targets . To dateTherefore, stress is known to lead to high blood pressure, myocardial ischemia, depression and even cancer. Briefly, stress performance is the manifestation of the final effect of the stress response on the target organ. However, it remains challenging to study various steps of this stress response . First, To date, various studies and practical applications have shown that plant-originated adaptogens are a kind of elite herbal medicine, playing an important role in human health and helping the human body resist various stress factors. However, the clinical application of plant-originated adaptogens and their use in health care products remains in the preliminary stage. Categorization of plant-originated adaptogens, clarification of their pharmacological functions, and the determination of the similarities and differences between adaptogens, tonics and ginseng species worldwide, will help in effective utilization of plant-originated adaptogens, and provide a new way to guarantee human health."}
+{"text": "The Collaborative Study on the Genetics of Alcoholism (COGA) is a large-scale family study designed to identify genes that affect the risk for alcoholism  and alcohol-related characteristics and behaviors  were analyzed independently; this approach allows investigators to examine the reproducibility of the initial study findings.Diagnostic and Statistical Manual of Mental Disorders, Third Edition, Revised [DSM\u2013III\u2013R] of the International Classification of Diseases and Related Problems (ICD\u201310) of the Because of the complexity of the risk factors for alcoholism and of the disorder itself, the COGA project was designed to gather extensive data from the participants. Although standard diagnostic systems for alcoholism can reliably determine who needs treatment, the diagnostic criteria used in these systems comprise problems in many domains of functioning. This means that two people with the same diagnosis . This process is called genotyping. More than 1.2 million genotypes have been generated on 2,310 people from families of alcoholics and 1,238 people from control families. By monitoring the inheritance patterns of such marker alleles within families with alcoholic members, the investigators could identify chromosomal regions that influence  certain alcohol-related traits.The methods used in these genetic analyses and other aspects of the COGA study are described in more detail in the article by Bierut and colleagues, pp. 208\u2013213, in this issue.Genetic analyses using the diagnostic criteria for alcohol dependence as the phenotype have revealed regions on several chromosomes that appear to contain genes affecting the risk for alcoholism. The primary analyses were based upon determining the extent of allele sharing among siblings who meet diagnostic criteria for alcoholism. The primary COGA definition of being affected with alcoholism requires a person to meet both DSM\u2013III\u2013R criteria for alcohol dependence and the Feighner criteria  for defiAnalyses of 987 people from 105 families in the initial sample provided evidence that regions on 3 chromosomes contained genes that increase the risk for alcoholism . The strThe data from the second part of the split sample\u2014the replication sample, which comprised 1,295 people from 157 families\u2014generally supported the initial findings . Thus, tADH) genes.ADH alleles are known to affect the risk for alcoholism; however, the known protective alleles occur at high frequency in Asian populations but are rare in the Caucasian population that makes up most of the COGA sample . This phenotype is quantitative and heritable, and a low number of drinks consumed in a 24-hour period may reflect a reduced tolerance for high levels of alcohol. An advantage of a quantitative phenotype is that everyone in a study can contribute to the genetic analysis, not just people who meet diagnostic criteria. Analysis of the MAXDRINK phenotype in both the initial and replication data sets (and in the combined sample) showed the strongest evidence for linkage in the same region of chromosome 4 where the ADH genes reside  to more social symptoms . Each person diagnosed with alcoholism exhibits a unique mix of those symptoms; therefore, a diagnosis of alcoholism does not reflect a uniform phenotype. This lack of uniformity complicates genetic analyses. Consequently, researchers have constructed other, more defined phenotypes from the data obtained in the COGA interviews. These include an analysis of symptoms related to alcoholism that produced phenotypes which appeared to reflect the severity of alcohol problems. Analysis of these phenotypes provided evidence for a DNA region on chromosome 16 that was associated with an increased risk for more severe alcohol problems .or depression\u201d phenotype, with very strong evidence for linkage to the same region of chromosome 1 that was linked to alcoholism alone . Analysis of such electrophysiological data may reveal a subset of genes that affect these quantitative, biological phenotypes related to alcoholism , 2002. OIn addition to these findings, recent analyses demonstrate strong evidence for a locus that affects brain wave oscillations as measured by electroencephalography . Thus, a2 receptor gene (DRD2) and a serotonin transporter gene (HTT). However, the analyses found no evidence that DRD2 affected the risk for alcoholism  within those genes. Where the available data are incomplete or insufficient, COGA researchers are seeking these polymorphisms themselves. Of particular value are single-nucleotide polymorphisms (SNPs)\u2014sites at which people differ in a single base pair\u2014in or near genes within the regions of interest. COGA investigators are doing additional genotyping of SNPs in and near candidate genes in the regions of linkage for further analysis of linkage and linkage disequilibrium . This should allow the investigators to greatly narrow the regions and to identify individual genes in which variations affect the risk for alcoholism and the other phenotypes they are studying.The COGA data set is a rich resource for further research. For example, it has already provided a test of new methods for genetic analysis, as presented at the Genetic Analysis Workshop 11 . In addiFinally, the large number of children and adolescents in the original sample will prove invaluable as these young people pass through the age of greatest risk for developing alcoholism. The value of the COGA data as a national resource for studies of alcoholism should increase with the re-interviews and with the development of new methods for both the determination and analysis of various genotypes. These efforts ultimately are expected to lead to the identification of genes that affect the risk for alcoholism and related phenotypes."}
+{"text": "Community Paramedics (CPs) require access to timely blood analysis in the field to guide treatment and transport decisions. Point of care testing (POCT), as opposed to laboratory analysis, may offer a solution, but limited research exists on CP POCT. The purpose of this study was to compare the validity of two devices (Abbott i-STAT\u00ae and Alere epoc\u00ae) by CPs in the community.In a CP programme responding to 6000 annual patient care events, a split sample validation of POCT against traditional laboratory analysis for seven analytes  was conducted on a consecutive sample of patients. The difference of proportion of discrepant results between POCT and laboratory was compared using a two sample proportion test. Usability was analysed by survey of CP experience, a linear mixed effects model of Systems Usability Scale (SUS) adjusted for CP clinical and POCT experience, an expert heuristic evaluation of devices, a review of device-logged errors, and coded observations of POCT use during quality control testing.p\u2009=\u20090.323) difference between i-STAT\u00ae  compared with epoc\u00ae . There were no instances of the laboratory reporting a critical value when POCT did not. In 88 of 1046 (8.4%) comparisons the a priori defined acceptable difference between POCT and the laboratory was exceeded; occurring more often in epoc\u00ae  compared with i-STAT\u00ae (p\u2009=\u20090.007). Eighteen of 19 CP surveys were returned, with 11/18 (61.1%) preferring i-STAT\u00ae over epoc\u00ae. The i-STAT\u00ae had a higher mean SUS score (higher usability) compared with epoc\u00ae . There were no statistically significant differences in device logged errors between i-STAT\u00ae and epoc\u00ae (p\u2009=\u20090.063).Of 1649 episodes of care screened for enrollment, 174 required a blood draw, with 108 episodes (62.1%) enrolled from 73 participants. Participants had a mean age of 58.7\u2009years (SD16.3); 49% were female. In 4 of 646 (0.6%) comparisons, POCT reported a critical value but the laboratory did not; with no statistically significant  contains supplementary material, which is available to authorized users. The traditional role of Emergency Medical Services (EMS) systems is to respond to emergency calls. Paramedics\u2019 traditional role in EMS is changing, including where Community Paramedics (CPs) provide a bridge between the hospital and the community by offering specialized primary care services for individuals with chronic diseases or difficulty accessing traditional healthcare services. While there is heterogeneity in the structure and process of CP programmes, in general these programmes focus on high needs patients such as the frail elderly.  CPs receOne of the challenges of providing care to these patients in the community is timely access to diagnostic tests such as blood analyses. Presently the primary option for many CP programmes is to collect blood specimens and transport them to a laboratory service for analysis. The process involves the CP collecting a blood sample in the community, transporting the sample to a blood testing laboratory, and following-up on results, often hours later. This process is resource intensive, presents multiple opportunities for misidentification of patients or results, and may delay timely treatment. An alternative process for CP programmes may be point of care testing (POCT).POCT technology has advanced considerably in the last decade, resulting in the commercial availability (at the time of this study design) of two portable devices that can provide a variety of blood tests quickly at the patient\u2019s bed side from a venous blood sample (Abbott i-STAT\u00ae and Alere epoc\u00ae).A systematic review completed in 2013 on CP care did not identify any peer reviewed studies that assessed the use of POCT technology in this setting, although technology assessment was not the explicit purpose of the review. \u20136 One ofThe purpose of this study was to assess the validity of two commercially available devices (Abbott i-STAT\u00ae and Alere epoc\u00ae) in the CP setting against the reference standard of laboratory analysis and compare the usability of these devices by CPs.This study was conducted in a mature CP programme that responds to approximately 6000 patient care events per year. Patients can be generally described as medically fragile and seen in a home setting . At the time of this study there were 19 active CPs in the programme using five vehicles that cannot convey patients and one that can. CPs must be registered as an Advanced Care Paramedic with the Alberta College of Paramedics, and have at least five years of clinical experience.  In additIn routine practice CPs draw blood specimens and transport the sample to twelve different laboratory service locations for analysis. The CP will then follow-up on results several hours later, discuss results with a physician, and if required re-visit the patient to implement or modify a treatment plan.Consecutive patients meeting inclusion criteria were enrolled by CPs into a modified single subject (split-sample) study between September 1 and November 30, 2016. Inclusion criteria consisted of patients who had sufficient capacity to be their own decision maker, age was greater than or equal to 18\u2009years, at least one study analyte was ordered for testing, and the patient was able to provide informed consent. Patients were not excluded if they already had been consented into the study. In other words, one patient may have been enrolled multiple times in the study if they had multiple episodes of care that required a blood draw.After informed consent, a blood draw was carried out and the specimen transported for laboratory blood testing in a \u201cBD vacutainer PST tube\u201d with 56\u2009units of lithium heparin as per routine practice. On scene a portion of the drawn blood was also used for POCT testing (split-sample).POCT testing involved the use of both i-STAT\u00ae and epoc\u00ae devices. The analytes sodium (Na), potassium (K), chloride (Cl), creatinine (Crea), hemoglobin (Hgb), hematocrit (Hct), and glucose (Glu) were included in the study. The rationale for choosing these particular analytes was the high frequency of occurrence in the CP programme and availability on each of the test cartridges or cards for the two POCT devices.For comparing device usability, four assessment methods were used to increase the validity of the collected data. They were an online preference and feedback survey for CPs (with a standardized device usability survey embedded), usability testing, device-logged error analysis and heuristic evaluation of the two devices. The latter three methods elucidate upon and can objectively validate the online survey responses.The online survey was developed to gather CP experiences, preference, and feedback regarding both POCT devices. The survey was pilot tested on a CP team lead and one of the investigators and refined accordingly prior to sending to all CPs involved in the study .To reduce the effect of device order influencing survey responses, participants were randomly assigned the survey order for each device (either i-STAT\u00ae or epoc\u00ae first) using R sample command. , 10 AnswA portion of the survey required participants to complete the Systems Usability Scale (SUS) for each device. The SUS is a validated reliable measuring scale of technology learnability and usability. The scores are normalized and can be compared to a benchmark of quartile ranges, acceptability ranges and adjective ratings. In addition, two Human Factors consultants reviewed device usability with both heuristic evaluation and usability testing methodologies. Heuristic evaluation is a method of device interface evaluation that uses broad categories of design principles to systematically evaluate usability problems.  The consUsability testing was completed by analysing video from the CP\u2019s performing quality control (QC) procedures outlined below. Three observation sessions were used to video record six CPs using the devices. The observations occurred at weeks nine and 10 of exposure to the devices. Participants were video recorded by researchers standing in the room where QC testing normally occurred. Any device errors, including test card or cartridge errors that were encountered, issues running the tests, steps missed and feedback from the staff were incorporated into the human factors review.The study was approved by the University of Calgary, Conjoint Health Research Ethics Board (REB16\u20131000). Two populations were identified as participants in this study, the patient and the CP. Each population provided written informed consent as a condition of enrollment into the study.CPs received one day (eight hours) of training in the week prior to the start of the study. The curriculum included training on the operation of i-STAT\u00ae and epoc\u00ae devices and troubleshooting strategies. CPs also received an overview of the research study, ethics, consent procedures, additional equipment, documentation and data collection. Since drawing blood was already in the CP scope of practice and routinely being performed, no additional training was necessary. Each CP received an additional two-hour, device quality control testing training session. While an optimal process for using two POCT devices during a patient event was suggested to CP participants, it was left to each individual CP on how they managed both devices as long as both devices were used as closely as possible to each other.Six i-STAT\u00ae and six epoc\u00ae devices were purchased and systematically tested prior to use in the study. The devices, associated test cards or cartridges, and analytes underwent initial laboratory validation using split sample testing of patient blood comparatives to the laboratory reference instruments, with in run and day to day precision testing using liquid QC solutions and calculation verification tests using liquid calculation verification solutions as per standards set by the laboratory service that works with the CP programme. All devices passed the validation, quality control, and calculation verification testing.While in service, all devices were housed in a temperature controlled and shock resistant environment. Test cartridges for i-STAT\u00ae and test cards for epoc\u00ae were also stored in the temperature controlled containers. Temperature monitors were placed on the inside and outside of the device containers to ensure an operating temperature of between 18\u2009\u00b0C and 30\u2009\u00b0C. All QC, calculation verification solutions and additional i-STAT\u00ae test cartridges were stored in two fridges that were both temperature monitored throughout the study period. Additional epoc\u00ae test cards were stored at room temperature. Devices underwent weekly QC testing and if applicable daily electronic simulation testing as per the manufacturers\u2019 and local laboratory recommendations.For the device validation objective, a sample of at least 100 patients was the target to provide a margin of error for point estimates of 6.2% on a 95% confidence interval assuming at least a 10% prevalence for out-of-range blood results and a targeted sensitivity of 99% for the device.Data were downloaded from the two POCT devices and linked to the appropriate electronic patient care records (ePCR) and laboratory values. Data in the ePCR were verified for missing data shortly after the patient contact, and if applicable sent to the author of the ePCR for final completion. All data were manually entered into a Microsoft Excel spreadsheet by one investigator and independently verified by a research associate. Each patient and CP was given a unique study identifier as was each event. All identifying patient data were then removed and the data analyzed using Stata version 11 . Descriptive data are reported as means and standard deviations for normally distributed data, or medians and inter-quartile ranges for data that clearly diverge from normality.POCT results were compared with the reference standard laboratory values using the methods described by Bland and Altman (2009).  CriticalThe proportion of out-of-range results between i-STAT\u00ae and laboratory and epoc\u00ae and laboratory were compared by a two sample test of proportion. A Chi-squared test and logistic regression with a Wald test were used to explore if one device contributed more out-of-range results compared with others.For the contrasting device usability objective, the SUS analysis consisted of a linear regression mixed effects model. The participants were considered as a random intercept effect taking into account their paramedic experience, experience in this specific CP programme and previous exposure to the devices in a work environment.All statistical tests were considered significant at the 0.05 level.Of 1649 episodes of care screened for enrollment, 174 episodes of care had a blood draw, with 108 episodes of care enrolled in the study, from 73 participants  (Table\u00a0In 4 of 646 (0.6%) individual comparisons between i-STAT\u00ae and laboratory and epoc\u00ae and laboratory, POCT reported a critical value but the laboratory did not; occurring more often in i-STAT\u00ae  compared with epoc\u00ae , although these results were not statistically significant p\u2009=\u20090.33 (Table\u00a0p\u2009=\u20090.007)(Table In 88 of 1046 (8.4%) individual comparisons between i-STAT\u00ae and laboratory and epoc\u00ae and laboratory, the a priori defined acceptable difference between POCT and the laboratory was exceeded; occurring more often in epoc\u00ae  compared with i-STAT\u00ae  appeared to give more results outside of acceptable comparative ranges than others Table\u00a0. When i-n\u2009=\u200911.4\u2009years, SD\u2009=\u20096.4) and a range of CP programme experience of 0.2 to 4.1\u2009years .All 19 CPs were sent the survey, 17 complete surveys and one partially complete survey were received (94.7% response rate). The respondents had a range of EMS experience from 5 to 32\u2009years  than the epoc\u00ae device. Using the means from the linear mixed effects model , the i-STAT\u00ae mean score was 84.0 and the epoc\u00ae 59.6. Figure\u00a0p\u2009=\u20090.122). The i-STAT\u00ae logged a statistically significant larger proportion of errors during the quality check procedures  compared with the epoc\u00ae (p\u2009=\u20090.001). However, the i-STAT\u00ae experienced fewer errors during the blood testing in the field  compared with the epoc\u00ae , although these results were not statistically significant (p\u2009=\u20090.063)  compared with the epoc\u00ae device, which logged 53 errors out of 469 tests  although these results were not statistically significant  there were discrepant critical results in three out of 401 individual comparisons (0.7%), and 38 out of 599 (6.3%) individual comparisons outside of comparative standards. If agencies within the same system use devices from different manufacturers, discrepant results should be anticipated.This study quantified that CPs will get their results considerably quicker using POCT compared with transportation to laboratory , however as POCT may not be capable of running all ordered tests, it should be assumed that POCT will not replace all laboratory testing. For example, in the sample of 108 episodes of care there were 88 episodes (82%) where a white blood cell (WBC) test was also ordered meaning that these episodes would still require transport of blood to the laboratory. Based on these results, it may be reasonable to assume that implementing a POCT programme will not replace transporting blood for laboratory analysis, but rather be an \u2018add-on\u2019 process. Other tests were found to have been ordered in the sample of 108 episodes of care that POCT devices are currently unable to test, however the scope of this study did not allow for further analysis of these data. It is not known whether the implementation of a POCT programme may change the ordering habits of physicians. For example, in this sample, physicians were accustomed to ordering through the laboratory analytes they knew were available, and may have ordered WBC because it was convenient or part of a \u2018panel\u2019 not because it was required. It could be that with a more limited menu of test options for POCT, physicians modify their ordering practices.For the device usability, i-STAT\u00ae was the preferred device of CPs in this study. The i-STAT\u00ae had a lower error rate (than epoc\u00ae) during actual patient use but a higher error rate (than epoc\u00ae) in QC testing. An issue not automatically logged in the devices\u2019 error logs, but observed in the QC testing, was that the epoc\u00ae cartridges needed to be removed and retried 11 out of 27 times (41%) before they would work. Ongoing frustrations with these non-logged issues may be the reason why the users preferred the i-STAT\u00ae over the epoc\u00ae during the trial. Field observations of POCT use were unable to be conducted during this study, but would provide important information on why the device error rates changed between QC and patient testing and some of the outliers found in the comparison of the POCT with laboratory tests.The reasons given by people who preferred i-STAT\u00ae in general were related to the function of the device. For example, the device was simple, it was easy to clean and use with fewer errors. But in general, the reasons for preferring epoc\u00ae were related to the logistics of using the device. For example, the test cards do not need to be refrigerated, there was no daily electronic simulation test and one card performed all the blood tests. There are important differences between these two systems, which should be reviewed prior to selection , and between i-STAT and epoc. All results reported in mmol/L. Figure S2. Results for potassium from i-STAT and epoc compared to gold standard , and between i-STAT and epoc. All results reported in mmol/L. Figure S3. Results for chloride from i-STAT and epoc compared to gold standard , and between i-STAT and epoc. All results reported in mmol/L. Figure S4. Results for creatinine from i-STAT and epoc compared to gold standard , and between i-STAT and epoc. All results reported in umol/L. Figure S5. Results for hematocrit from i-STAT and epoc compared to gold standard , and between i-STAT and epoc. All results reported in %. Figure S6. Results for hemoglobin from i-STAT and epoc compared to gold standard , and between i-STAT and epoc. All results reported in g/L. Figure S7. Results for glucose from i-STAT and epoc compared to gold standard , and between i-STAT and epoc. All results reported in mmol/L. Figure S7. Results for glucose from i-STAT and epoc compared to gold standard , and between i-STAT and epoc. All results reported in mmol/L. (DOCX 773 kb)"}
+{"text": "The epithelial-mesenchymal transition (EMT) shifts epithelial cells towards a malignant mesenchymal phenotype, enhancing their invasiveness and chemoresistance. Cancer cells initiate an \u201cEMT program\u201d for their metastatic dissemination and this in turn is governed by several transcription factors including Snail1, Slug Snail2), Twist and Zeb that repress epithelial and activate mesenchymal transcriptional signatures , Twist a and in tAn extensive work has been done during the last years describing Snail1 lability in epithelial cells pushed by the action of several E3-ubiquitin ligases   cells showed impaired cell invasion and metastasis formation, being both effects partially rescued by Snail1 . HoweverRecently, Dub3 was also described as a deubiquitinase of Snail1 . WhereasIn summary, DUBs targeting Snail1 are an attractive target for chemotherapy. The description of USP27X as a TGF\u03b2-activated DUB and its role on EMT provides new clues to specifically counteract the pathological stabilization of Snail1. The development of selective USP27X inhibitors will offer new possibilities to fight against metastasis formation."}
+{"text": "PNPT1 gene under the linkage peak at chromosome 2 that is likely to have a regulatory function. The variant was associated with quantitative cIMT in the family-based study population . Furthermore, we identified several genes under the linkage peak at chromosome 21 highly expressed in tissues relevant for atherosclerosis. To conclude, our linkage analysis identified four genomic regions significantly linked to cIMT. Further analyses are needed to demonstrate involvement of identified candidate genes in development of atherosclerosis.Carotid intima-media thickness (cIMT) is an established heritable marker for subclinical atherosclerosis. In this study, we aim to identify rare variants with large effects driving differences in cIMT by performing genome-wide linkage analysis of individuals in the extremes of cIMT trait distribution (>90th percentile) in a large family-based study from a genetically isolated population in the Netherlands. Linked regions were subsequently explored by fine-mapping using exome sequencing. We observed significant evidence of linkage on chromosomes 2p16.3 , 19q13.43 , 20p13 , and 21q22.12 . Fine-mapping using exome sequencing data identified a non-coding variant (rs62165235) in  ZHX2), 19q13 (near APOC1), and 8q23.1 (PINX1) and an additional suggestive region on 6p22 . Maximum cIMT was measured on the three still, longitudinal, two-dimensional ultrasound images of the near and far wall from both left and right arteries, as described previously . The mea2) and WHR was computed by dividing the waist and hip circumferences with each other. Hypertension was defined as systolic blood pressure above 140 mmHg, diastolic blood pressure above 90 mmHg, or use of medication for treatment of hypertension. Dyslipidemia was defined as total cholesterol above 6.2 mmol/L or use of lipid-lowering medication, whereas diabetes was defined as fasting plasma glucose levels above 7 mmol/L, random plasma glucose above 11.1 mmol/L, or use of medication indicated for treatment of diabetes.Information on covariates included age, sex, and smoking status. BMI was defined as weight divided by the square of height  were removed during the quality control process. In total, 5,250 autosomal variants were available for analysis.Genomic DNA was extracted from peripheral venous blood of all study participants using the salting out procedure . Genotyp1. To further assess the functionality of the variants, we used RegulomeDB database that annotates SNVs with known and predicted regulatory elements and CADD tool for scoring the deleteriousness of variants . Descriptive characteristics of the selected individuals are presented in Table 1. The selected individuals were older and higher cIMT measurements compared to all ERF study participants (Table 1). They also had a higher prevalence of hypertension, dyslipidemia, and diabetes than all ERF study participants, whereas the BMI and WHR were comparable (Table 1). These 103 affected individuals were connected to each other in a large pedigree consisting of 5,083 individuals. To facilitate linkage analysis, the 103 affected individuals were clustered into 21 smaller non-overlapping sub-pedigrees with a maximum bit size of 24 using the PEDCUT software version 1.19  using the RVtests software  evidence of linkage in either the non-parametric or the parametric analyses are shown in Table 2. Significant evidence of linkage for cIMT was observed to chromosomes 2p16.3, 19q13.43, 20p13, and 21q22.12 in the parametric linkage analysis under the dominant model, and to chromosome 19q13.43 and 20p13 in the parametric linkage analysis under the recessive model. The families contributing predominantly to these linkage peaks and the distribution of their per-family HLOD scores are shown in Supplementary Figures S4.The results of affected only genome-wide NPL and parametric linkage scans are illustrated in Table 3). The most interesting finding is a variant (rs62165235) with MAF 0.038 in 1kG mapping to PNPT1. The variant, shared by six out of eight affected relatives, is likely having a regulatory function and affecting transcription factor binding and matched DNase Footprinting and DNase sensitivity . The variant was sequenced at a read depth of 37x and it showed significant association with quantitative cIMT in the ERF  after applying Bonferroni correction . The effect estimate of the minor allele C on untransformed cIMT suggested a mean increase of 0.04 mm for each minor allele . This variant explained 0.3% of variation in the ERF.We next determined to what extent the affected members in these families shared rare variants under the linkage peaks. Sharing analyses under the base to base linkage peak at chromosome 2 (family specific HLOD = 3.63) identified intronic and coding-synonymous variants (Supplementary Tables S3). There are, however, several potentially interesting candidate genes for atherosclerosis in each of these linked regions, for instance, among the genes under the base to base linkage peaks at chromosome 19 and 20, several genes have been implicated in the pathogenesis of cardiovascular disease, including FCAR, TNNT1, OSCAR, FPR2 under the peak at chromosome 19 and ADAM33, TRIB3, HSPA12B under the peak at chromosome 20. The linkage peak at chromosome 21 harbors several genes that are highly expressed in tissues relevant for atherosclerosis . According to the Ingenuity Pathway Analysis (IPA) tool (QIAGEN Inc.4), which exposes possible functional relationship between the genes by expanding upstream analysis to include regulators that are not directly connected to targets in the dataset, these genes connected to a network illustrated in Supplementary Figure S5.The search for shared variants within the linkage regions at chromosome 19, 20, and 21 identified several variants to be shared among the affected family members; however, none of the variants showed significant association with quantitative cIMT . Search for shared variants within this region identified no variants that can explain linkage signal.There were several regions that showed suggestive evidence of linkage, including 1q31.1, 5q35.3, 7p21.3, 8p22, 12q24.33, 14q22.2, 15q21.3, and 17q25.3. As the region at chromosome 12 has previously been linked to cIMT, we have explored it further  gene which encodes a protein predominantly localized in the mitochondrial intermembrane space and is involved in import of RNA to mitochondria5. PNPT1 has been characterized as a type I interferon-inducible early response gene . However, the variant was not available in the exome sequencing data of the Rotterdam Study.Identification of variants under the base to base peak at chromosome 2 using exome sequence data revealed a variant that lies in DNase sites, promotes histone marks and protein binding regions, and changes regulatory motifs based on the variant allele change. The variant is mapped to intron 1 of polyribonucleotide nucleotidyltransferase 1 (nse gene , 2003. Tnse gene . Even thIFNAR1, DYRK1A, SON, IFNGR2, MORC3, MRPS6, IL10RB, TMEM50B, CBR1, RCAN1, and TTC3. DYRK1A signaling pathway is linked to homocysteine cycle which is associated with an increased risk of atherosclerosis (IFNGR2 and RCAN1 also play a role in atherosclerosis (TP53. TP53 encodes a tumor suppressor gene p53 involved in regulation of cell proliferation and apoptosis. Numerous studies implicated p53 in development of atherosclerosis and vascular smooth muscle cell apoptosis (The other interesting region includes the linkage peak at chromosome 21 which is also known as a Down critical region. Interestingly, persons with Down syndrome are protected against atherosclerosis, in spite of increases in metabolic disturbances and obesity in Down syndrome . Even thclerosis . IFNGR2 clerosis . Notablypoptosis . Higher poptosis . HoweverFCAR and TNNT1 genes associated with coronary heart disease (OSCAR and FPR2 genes associated with atherosclerosis plaque phenotype (ADAM33 and TRIB3 associated with extent and promotion of atherosclerosis (HSPA12B which is found to be enriched in atherosclerotic lesions (Furthermore, we identified 19q13.43 and 20p13 regions with significant evidence of linkage to cIMT. Several genes under the linkage peak have previously been implicated in the pathogenesis of cardiovascular disease. The base to base peak at chromosome 19 encompassed  disease  and OSCAhenotype , whereasclerosis  and HSPA lesions .Our study presents the linkage analysis using extreme phenotype approach that was designed to capture region with genetic variants that have large effects on cIMT. Combination of linkage analysis in a large family-based study and exome sequence data provide a unique opportunity to explore the variants in the linkage regions. However, despite these distinct advantages, we were able to identify a genetic variant for only one of the several linked genomic regions, for which, there may be several reasons including structural variants, and intronic or intergenic single-nucleotide variants that were not evaluated in the current study. Interestingly, the 19q13.43, 20p13, and 21q22.12 linkage peaks were previously associated with various phenotypes in our study population including personality traits and depressive symptoms , 2017b.Our linkage analysis identified four genomic regions at 2p16.3, 19q13.43, 20p13, and 21q22.12 for cIMT. The significant linkage regions contain several plausible candidate genes. Further analyses are needed to demonstrate their involvement in atherosclerosis.The raw datasets are available on request.DV, CvD, and NA contributed to the conceptualization and design of this work and were involved in interpretation of the results. DV and MK were involved in the analysis of the data. DV and NA were involved in writing and revising the manuscript. MG, RB, JvR, MvdH, RK, WvI, AU, and CvD were involved in data collection/preparation. MK, MG, RB, JvR, MvdH, RK, AU, WvI, and CvD contributed to the interpretation of the data and read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "Background: Subgenotype C4 of enterovirus 71 (EV71) is the predominant agent of Hand Foot and Mouth disease (HFMD) circulating in the mainland of China. For the first time, a subgenotype C2 of EV71 named SY30-2 was isolated from a HFMD case in Beijing, China. Since it is uncertain whether antibodies raised against subgenotype C4 of EV71 can protect C2 EV71, it is important to monitor and check the presence of cross-reactive antibodies against new EV71 subgenotypes. To find out the causes for the different NtAb, this study is to investigate the relationships between amino acid residue variations and cross-reactive antibodies against EV71 subgenotypes C2 and C4.Methods: Nucleotide and amino acid sequences from full-length genome sequence of SY30-2 were compared to EV71 reference strains. A microneutralization test was used to detect neutralizing antibody (NTAb) in the sera of subgenotype C4 of EV71 infected cases against SY30-2 and FY17 (a C4 isolate). The 3D structure of the viral capsid protein of SY30-2 was constructed.Results: Genome sequence and similarity plot analyses showed that SY30-2 shared the highest identity with subgenotype C2 of EV71 strains in every fragment of the genome. While the microneutralization test result showed that children infected with subgenotype C4 of EV71 had higher NTAb titers against FY17 than SY30-2 (p < 0.001). The amino acid sequence comparison revealed that four amino acid residues VP1-22, VP1-31, VP1-249 and VP3-93 were highly conserved in subgenotype C4 of EV71 compared with the corresponding amino acid residues on subgenotype C2 of EV71 (p < 0.05). Furthermore, the 3D-structure of viral capsid protein showed that VP1-22, VP1-31 and VP3-93 were located on the surface of virion.Conclusion: This is the first report of an EV71 subgenotype C2 isolated from HFMD in Beijing, China. Only a few antigenic variations on subgenotype C2 of EV71 could have led to a great decrease in NTAb titer. Thus, imported new genotypes and subgenotypes of EV71 should be closely monitored. The efficacy of available vaccines against new viruses should be evaluated as well. Picornaviridae family (Hand Foot and Mouth Disease (HFMD) is a very common infectious disease usually associated with children younger than 5 years old. EV71 is a member of the Enterovirus genus of the e family . It is oThe EV71 particle has a non-enveloped, icosahedral capsid comprising 60 protomers. Each protomer consists of four viral proteins VP1, VP2, VP3 and VP4 . VP1, VPIn this current study, based on the identification of a EV71 subgenotype C2, we attempted to combine the result of NTAb titer against subgenotypes C4 and C2 of EV71 with corresponding viral genetic sequence data to identify relationships between cross-reactive antibodies and amino acid residue variations. Understanding of the antigenicity of diverse EV71 isolates is crucial for the surveillance strategy formulation, especially after EV71 vaccine is widely used in China.In this study, the SY30-2 strain was isolated from a boy of 3 years of age. He traveled to Thailand with his parents from April 5 to 11 in 2015. On April 11, he got the symptom of fever and rash on hands and feet, and had no neurological complications. On April 13, his parents took him to see a doctor. His throat swab and information were collected by pediatrician after obtaining the informed consent of the parents. The throat samples were kept in minimum essential media (MEM) and stored at 4\u00b0C until transfer to Beijing CDC for laboratory test. FY17 is a subgenotype C4 of EV71 strain isolated in FuYang, China in 2008. It was obtained from China CDC.Non-anticoagulated whole blood and information were collected from the index case of each HFMD outbreak in Beijing after obtaining the informed consent of the guardians. The blood samples were first stored in a 4\u00b0C refrigerator overnight. The next morning, the serum of each sample was dispensed into serum preservation tubes and stored at -20\u00b0C.Total nucleotide extraction was carried out with a Roche MagNA Pure LC 2.0 nucleic extraction system  using MagNA Pure LC Total Nucleic Acid Isolation Kit\u2013Large Volume , according to the manufacturer\u2019s instructions.Complete nucleotide sequences of the VP1 gene were amplified using specific primers as previously described . PCR proHuman rhabdomyosarcoma (RD) cells were used to isolate enterovirus from the supernatant of the throat swab specimen. The RD cells were cultured using MEM with 10% fetal bovine serum till 75% of flask bottom was covered by monolayer RD cell. Then the cell culture medium was removed and the supernatant of the specimen was inoculated on the RD cells. After 1 h of incubation at 36\u00b0C, the specimen supernatant was replaced by MEM with 2% fetal bovine serum. Cytopathic effect of the enterovirus infected RD cells was observed every day. Cells were collected when 75\u201390% covered cells showed cytopathic effect.TM 1st strand cDNA Synthesis Kit, Takara) using the RNA as the template and the primer SY30-2R-3\u2032 as the specific reverse transcription primer. A DNA fragment containing approximately 3 kb of the 5\u2032region of the viral genome was amplified with primers SY30-2F-5\u2032 and SY30-2-R3296-3\u2032. The DNA fragment containing approximately 4 kb of 3\u2032 region of SY30-2 was amplified by PCR with primers SY30-2-F3036-5\u2032 and SY30-2R-3\u2032. Both PCRs were performed under the following conditions: initial denaturation for 1min at 94\u00b0C; 33 cycles of 30s at 94\u00b0C, 30s at 60\u00b0C and 8 min at 72\u00b0C; with a final extension of 72\u00b0C for 10 min. Obtained PCR products were sequenced with SY30-2F-5\u2032 and SY30-2R-3\u2032, respectively. Based on the obtained sequence, new primers were designed and PCR products were sequenced until the end of the product.The full-length genome of EV71 was amplified from the throat sample with the primers of SY30-2-F (5\u2032-TTA AAA CAG CCT GTG GGT TGC-3\u2032), SY30-2-R3296 (5\u2032-TGG ATT GGC TTT GAA TAG ATA-3\u2032), SY30-2-F3036 (5\u2032-CCC ACA TTC GGT GAA CAC AAG C-3\u2032) and SY30-2R-3\u2032 (TTT TTT TTT TTT TTT TTT TTG CTA TTC TGG). The reverse transcription reaction was performed at 42\u00b0C for 1h with reverse transcriptase . Bayesian Markov chain Monte Carlo (BMCMC) methods were used to analyze the phylogenesis of the VP1 genes of EV71 using BEAST v1.8. Bayesian analyses were performed using a relaxed molecular clock model. Phylogenetic trees were displayed and annotated by using FigTree. Pairwise distances of nucleotide and amino acid sequences between different gene fragments were calculated with MEGA software (version6). Similarity plot analysis was performed using the Simplot program (version 3.5.1). A sliding window of 200 nucleotides was used, moving in 20 nucleotide steps.Virus titer was determined as the median end-point of the tissue culture infectious dose (TCID50) by a microtitration assay using a standard protocol . NTAb agThe amino acid property classes were defined for the hydropathy, volume and chemical characteristics. According to the Kyte and Doolittle amino acid hydropathy index , three \u2018The 3D structure of the EV71 capsid protein was constructed using SWISS-MODEL by homology modeling. The crystal structure of EV71 (PDB: 4CEW) was used as template for the model. Specific amino acid residues were located using the Pymol software.t-test or Wilcoxon signed rank test were used to compare the difference in the NTAb between FY17 and SY30-2. Pearson\u2019s \u03c72 test was used to compare the difference of constituent ratio of amino acid residue between subgenotypes C2 and C4. Odd Ratio (OR) was calculated to determine the association between the subgenotype and amino acid residue. A p-value of < 0.05 (2-sided significance testing) was considered statistically significant in above analyses.Statistical analysis was conducted by SPSS19 software . NTAb titers were log transformed to calculate the geometric mean titers (GMTs). Undetectable titer was assigned a level of 0 for the calculation of GMT. Paired This study was carried out in accordance with the recommendations of institutional review board and human research ethics committee of the Beijing CDC. The approval number of the local ethics committee was 201702. Samples (throat swabs and serum specimens) collection in this study was agreed by the patient\u2019s guardian with prior written informed consent which was in accordance with the Declaration of Helsinki. The protocols were approved by the institutional review board and human research ethics committee of the Beijing CDC.Figure 1). The nucleotide and amino acid identity of the VP1 sequence between SY30-2 and a former isolated EV71subgenotype C2 (JQ326306) in China were 92.0 and 99.0%, respectively. The nucleotide and amino acid identities of the VP1 sequences between SY30-2 (MG214681) and C4 reference strains  were 85.8\u201387.6% and 98.6\u201399.0%, respectively. The phylogenetic placement of the SY30-2 strain was further confirmed by sequencing the whole genome and calculating the evolutionary distance with other subgenotypes of EV71. Results showed that SY30-2 genome sequence (GenBank accession number: MG214681) shared 91.6\u201392.9% nucleotide identity with subgenotype C2 reference strains . The highest identity in each gene fragment was observed between SY30-2 and C2 reference strains compared with other subgenotypes of EV71 (Table 1). Similarity plot analyses result showed no recombination for SY30-2 isolate when compared with other subgenotype C2 of EV71 .The whole VP1 sequence (891bp) of SY30-2 was obtained and aligned with EV71 reference strains including genotype A, B0-B7, C1-C5, D, E, F strains (GenBank accession numbers were listed in the phylogenetic tree). The isolates of each genotype and subgenotype clustered together on the phylogenetic tree based on the VP1 whole sequence. According to the phylogenetic tree and the nucleotide identity between SY30-2 and C2 reference strains , SY30-2 belonged to subgenotype C2 (P < 0.001) . The NTAb titers of all serum samples against FY17 were significantly higher than those against SY30-2. NTAb titers of serum samples against FY17 and SY30-2 were substantially correlated . Five serum samples from children without HFMD symptoms were collected as controls. Four children had no detectable NTAb titers against both FY17 and SY30-2. One serum sample had the NTAb titer of 2048 against FY17 and 32 against SY30-2, which we speculated having a latent infection.A total of 52 serum samples within 5 days of HFMD onset were collected from subgenotype C4 of EV71 infected children. All EV71 infected cases had detectable NTAb titers against FY17 and ranged from 16 to 32768 with the median NTAb titer of 512 . Cross-reactive NTAb titers against SY30-2 ranged from 0 to 256 with the median NTAb titer of 8 . There were no NTAbs against SY30-2 in 11 (20.4%) serum samples. Significant difference in the NTAb titers was found between FY17 and SY30-2 (P = 0.049), in this group the median NTAb titer against FY17 was 192 .Serum samples were divided into two groups based on whether there were NTAbs against SY30-2. In the group with positive NTAbs against SY30-2, the median NTAb titer against FY17 was 512 , and it was significantly higher than that in the group with negative NTAbs against SY30-2 and one residue in VP3 (S93N).p < 0.05) (Table 2).In order to find out if these five amino acid residues were highly conserved on different subgenotypes, 28 VP1 sequences and VP3 sequences from EV71 subgenotype C4 (including three strains of EV71 vaccines available in China) and 21 VP1 sequences and VP3 sequences from subgenotype C2 of EV71 were collected .The 3D structure of EV71 capsid (PDB: 4CEW) was obtained to locate the amino acid residues VP1-22, VP1-31, VP1-98, VP1-249, and VP3-93. As Subgenotype C2 of EV71 has been widely detected in Australia, the Netherlands, Japan, Singapore and Thailand for several years . HoweverTo our knowledge, this was the first time a subgenotype C2 of EV71 from HFMD was isolated in Beijing, and it was the second time a subgenotype C2 of EV71 was isolated in China. The first isolate in China was reported in 2012. It was obtained from an AFP case in 1996 . These tIn this study, we found serum from local people infected with C4 EV71 didn\u2019t show high NTAb titer against SY30-2. This was inconsistent with a previous study, in which serum from C4 EV71-induced HFMD and subclinical patients all contained NTAbs against subgenotype C2 of EV71 strains . We specIn this study, we found that serum from subgenotype C4 of EV71 infected people could provide NTAbs against subgenotype C2 of EV71 infection. This result was in line with other studies . HoweverIn this study, significant difference was found in the NTAb titers against FY17 and SY30-2 which were likely due to the structure difference caused by four amino acid residues on VP1 and VP3. According to a previous study , potent In conclusion, this is the first report of an EV71 subgenotype C2 isolated from HFMD in Beijing, China. Only a few antigenic variations on EV71 could lead to a great change in NTAb titer. We suggest that the epidemiology of new genotype and subgenotype of EV71 should be closely monitored and the efficacy of available vaccines against newly introduced viruses should be evaluated from time to time.JL and YL carried out the experimental design, participated in the experiment, and drafted the manuscript. SZ, HM, and XL participated in the experimental design, performed the data analysis, and reviewed the manuscript. ZL, WZ, HJ, and DH participated in the data collection and analyzed and reviewed the manuscript. YD, YY, and LC participated in the experiment and reviewed the manuscript. QW contributed to the experimental design and provided a final review of this manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "Stenotaphrum secundatum  Kuntze] is a warm-season, perennial turfgrass species well adapted for home lawns and commercial landscapes with economic and ecological value. However, a lack of genomic resources in St. Augustinegrass has hindered the full utilization of genetic variance for maximizing genetic gain and limited our understanding of the species\u2019 evolution.St. Augustinegrass [In this study, we constructed the first high-density linkage map for St. Augustinegrass using a genotyping by sequencing (GBS) approach. The integrated linkage map consists of 2871 single nucleotide polymorphism (SNP) and 81 simple sequence repeat (SSR) markers, spanning 1241.7\u2009cM, with an average distance of 0.4\u2009cM between markers, and thus represents the densest genetic map for St. Augustinegrass to date. Comparative genomic analysis revealed inter-chromosome arrangements and independent nested chromosome fusion events that occurred after St. Augustinegrass, foxtail millet, sorghum, and rice diverged from a common ancestor. Forty-eight candidate quantitative trait loci (QTL) were detected for turf quality-related traits, including overall turf quality, leaf texture, genetic color, and turf density. Three hot spot regions were identified on linkage groups LG3 and LG8, where multi-QTL for different traits overlapped. Several leaf development related genes were contained within these identified QTL regions.This study developed the first high-density genetic map and identified putative QTL related to turf quality, which provide valuable genetic resources for marker-assisted selection (MAS) in St. Augustinegrass.The online version of this article (10.1186/s12870-018-1554-4) contains supplementary material, which is available to authorized users. Stenotaphrum secundatum [Walt.] Kuntze) is a warm-season turfgrass that is well adapted to tropical and subtropical regions of the world /4 for female  additivity; Am\u2009=\u2009[(\u03bcac\u2009+\u2009\u03bcbc) - (\u03bcad\u2009+\u2009\u03bcbd)]/4 for male (Seville) additivity and D\u2009=\u2009[(\u03bcac\u2009+\u2009\u03bcbd) - (\u03bcad\u2009+\u2009\u03bcbc)]/4 for dominance where \u03bcac, \u03bcad, \u03bcbc and \u03bcbd are estimated phenotypic means associated to each of the 4 possible genotypic classes ac, bc, ad and bd, deriving for an ab \u00d7 cd cross [Turf quality-related traits evaluated by Kimball et al. were used for QTL mapping . All hybcd cross . FurtherAdditional file 1:Table S1 to S12. Table S1. Sequence of barcodes and adapters in St. Augustinegrass GBS library. Table S2. Number of sequencing reads for St. Augustinegrass parent lines and hybrids. Table S3. Detail of linkage group and marker sequences of \u2018Raleigh\u2019 St. Augustinegrass genetic map. Table S4. Detail of linkage group and marker sequences of \u2018Seville\u2019 St. Augustinegrass genetic map. Table S5. Detail of linkage group and marker segregation in integrated St. Augustinegrass genetic map. Table S6. Genomics comparison between \u2018Raleigh\u2019 St. Augustinegrass linkage groups and foxtail millet genome. Table S7. Genomics comparison between \u2018Seville\u2019 St. Augustinegrass linkage groups and foxtail millet genome. Table S8. Genomics comparison between \u2018Raleigh\u2019 St. Augustinegrass linkage groups and sorghum genome. Table S9. Genomics comparison between \u2018Seville\u2019 St. Augustinegrass linkage groups and sorghum genome. Table S10. Genomics comparison between \u2018Raleigh\u2019 St. Augustinegrass linkage groups and rice genome. Table S11. Genomics comparison between \u2018Seville\u2019 St. Augustinegrass linkage groups and rice genome. Table S12. Gene ontology analysis of sequence within QTL regions related to leaf development. (XLSX 346 kb)"}
+{"text": "Aspergillus fumigatus is the most prevalent airborne fungal pathogen that causes invasive fungal infections in immunosuppressed individuals. Adaptation to iron limited conditions is crucial for A. fumigatus virulence. To identify novel genes that play roles in adaptation to low iron conditions we performed an insertional mutagenesis screen in A. fumigatus. Using this approach, we identified the tptA gene in A. fumigatus, which shares homology with the Saccharomyces cerevisiae thiamine pyrophosphate (ThPP) transporter encoding gene tpc1. Heterologous expression of tpc1 in the tptA deletion mutant completely restored the ThPP auxotrophy phenotype, suggesting that Tpc1 and TptA are functional orthologues. Importantly, TptA was required for adaptation to low iron conditions in A. fumigatus. The \u0394tptA mutant had decreased resistance to the iron chelator bathophenanthroline disulfonate (BPS) with severe growth defects. Moreover, loss of tptA decreased the expression of hapX, which is a major transcription factor indispensable for adaptation to iron starvation in A. fumigatus. Overexpression of hapX in the \u0394tptA strain greatly rescued the growth defect and siderophore production by A. fumigatus in iron-depleted conditions. Mutagenesis experiments demonstrated that the conserved residues related to ThPP uptake in TptA were also required for low iron adaptation. Furthermore, TptA-mediated adaptation to low iron conditions was found to be dependent on carbon sources. Finally, loss of tptA resulted in the attenuation of virulence in a murine model of aspergillosis. Taken together, this study demonstrated that the mitochondrial ThPP transporter TptA promotes low iron adaptation and virulence in A. fumigatus. Although iron is highly abundant in the earth\u2019s crust, the bioavailability of iron is very low due to its oxidation into sparingly soluble ferric  . Controldstream) . Howeverdstream) . Thus, vAspergillus fumigatus is an ubiquitous airborne fungal pathogen of humans [A. fumigatus infection, invasive aspergillosis (IA), occurs when inhaled A. fumigatus spores germinate into hyphae and invade the lung tissue of immuno-compromised patients [A. fumigatus primarily employs two high affinity iron uptake systems, reductive iron assimilation (RIA) and siderophores  to survive in low iron environments [ftrA gene, which encodes an iron permease, does not affect A. fumigatus virulence [A. fumigatus SidA, which catalyzes the first committed step of hydroxamate siderophore biosynthesis, is essential for virulence, suggesting that siderophore-mediated iron uptake plays a major functional role during fungal infection [f humans . The mospatients . Despitepatients \u201312. A. firulence . In contnfection .Aspergillus is regulated by two central transcription factors HapX and SreA. Under low iron conditions, HapX activates siderophore-mediated iron acquisition and represses iron-consuming pathways to conserve iron [A. fumigatus virulence in murine models of aspergillosis [Iron homeostasis in rve iron . During rve iron . These tgillosis ,15, emphAgrobacterium-mediated transformation study in A. fumigatus. Transformants exhibiting specific defects under iron-limited conditions were selected for further study. Here, we present the identification and functional characterization of a novel low iron adaptation related gene, tptA, a homolog of the Saccharomyces cerevisiae thiamine pyrophosphate (ThPP) transporter encoding gene tpc1. In S. cerevisiae, Tpc1 mediates mitochondrial uptake of the essential cofactor ThPP, which is required for the activity of acetolactate synthase (ALS), pyruvate dehydrogenase (PDH) and oxoglutarate dehydrogenase (OGDH) [A. fumigatus.To identify novel genes crucial for growth under low iron conditions, we carried out a large-scale e (OGDH) . The stuA. fumigatus transformants was constructed. Transformants were screened for their ability to grow and/or conidiate on minimal media (MM) in the presence of the iron chelator bathophenanthroline disulfonate (BPS). Seven mutants impaired by low iron conditions were identified. The T-DNA flanking sequences in four of the mutants were cloned successfully using Thermal-asymmetric interlaced (TAIL)-PCR in the mutant genomes [sidC (AFUA_1G17200), sidF (AFUA_3G03400) and sidI (AFUA_1G17190) were previously reported to be involved in low iron adaptation through mediating siderophore biosynthesis [To identify novel genes involved in adaptation to low iron conditions, a T-DNA insertional mutagenesis library containing 5,000  genomes . Among tynthesis ,19.tptA (thiamine pyrophosphate transporter), as it encodes a predicted protein that showed 28.61% amino acid identity with S. cerevisiae Tpc1, a mitochondrial transporter of ThPP [A. fumigatus tptA allele restored the conidiation defect in the presence of BPS suggesting that dysfunction of tptA is responsible for the growth defect in this strain under low iron conditions . The resulting \u0394tptA mutant was confirmed by PCR and Southern blotting ). In contrast to the T421 mutant, complete deletion of tptA resulted in a mutant that did not grow on MM, even in the presence of 1 or 5 mM FeCl3 . Since TptA has been reported to play a role in ThPP transport in S. cerevisiae [tptA resulted in ThPP auxotrophy. As expected, the addition of ThPP (>10\u00a0\u03bcM) or thiamine (> 1\u00a0\u03bcM) restored growth of the tptA deletion mutant on MM ). The addition of other cofactors such as pyridoxine, riboflavin or inositol did not improve growth of the \u0394tptA mutant on MM ). In addition, it has been reported that the simultaneous addition of valine and isoleucine fully restored the growth of a \u0394tpc1 mutant when grow with glucose as a carbon source in S. cerevisiae [\u0394tptA mutant ).To explore the function of mM FeCl3 . The difS. cerevisiae, contain three tandem repeat homologous domains of approximately 100 amino acids, and are predicted to have six transmembrane helices (TM1-TM6) with N- and C-termini located in the intermembrane space [A. fumigatus under all tested conditions, indicating that TptA::GFP is fully functional (data not shown). As expected, the TptA::GFP fusion protein co-localized with the mitochondrial marker MitoTracker Red, suggesting that TptA is located within mitochondria  in the presence of ThPP. The tptA complemented strain tptAc had an identical phenotype to the wild-type A. fumigatus strain under all tested conditions, suggesting that the phenotypes exhibited by the \u0394tptA mutant were caused only by the loss of tptA  iron siderophore triacetylfusarinine C (TAFC), production of TAFC by \u0394tptA mutant was only 70% of the WT  completely restored the TAFC production defect in the \u0394tptA mutant ). Collectively, the above results indicate that mitochondrial ThPP level affects the expression of hapX and the subsequent TAFC production under low iron conditions.In agreement with the important role of HapX in mediating the production of the secreted low molecular weight ferric  recognition/interaction. The positively-charged residues His82, His137, Lys231, and Lys291 also play important roles in transport [53, Asp60 , Lys255, Lys315  Gly153 and Gly205 , were found to be conserved between SLC25A19 and TptA . A schematic diagram of TptA showing the location of the above-mentioned residues is shown in A link between mitochondrial ThPP transport and the regulation of iron adaptation has not been previously reported. To confirm that these two biological processes were directly linked, we sought to construct TptA proteins with mutations in key residues required for ThPP transport and evaluated the effects of these mutations on adaptation to low iron conditions. Structural characterization of hMTPPT (SLC25A19), a human mitochondrial ThPP transporter, has shown that the conserved residues Thrransport . The hMTransport . Among ttptAD60A and tptAG153S exhibited severe growth and conidiation defects on MM without ThPP, suggesting that residues Asp60 and Lys153 are essential for ThPP transport. In contrast, the tptAR53A, tptAK255A and tptAK315A mutants exhibited only mild growth or conidiation defects on MM without ThPP. The tptAG205A mutant was indistinguishable from wild-type A. fumigatus on MM without ThPP. The addition of 30\u00a0\u03bcM ThPP completely restored the growth of all five site directed mutants that showed growth defects on MM  , \u0394tptA exhibited strict auxotrophy for ThPP. In comparison, slight growth was observed when grown on GMM (1% glycerol) in the absence of ThPP. Moreover, slight growth of the \u0394tptA mutant was also seen with 2 % potassium acetate, 0.4% acetic acid or 0.4% ethanol as sole carbon sources in the absence of ThPP .In  acetate . In cont\u0394tptA mutant on different carbon sources. Consistent with the above observations, the \u0394tptA mutant showed a dramatically decrease in conidiation, biomass and TAFC production when grown on MM in the presence of 30\u00a0\u03bcM ThPP  a leukopenic mouse model with induced immunosuppression from both cortisone acetate and cyclophosphamide, and (ii) a non-leukopenic model with immunosuppression from cortisone acetate [A. fumigatus, \u0394tptA and ctptA strains was observed (\u0394tptA mutant survived significantly longer than mice infected with either wild-type A. fumigatus or the tptA-complemented strain ctptA (P\u00a0<\u00a00.001), although more than 90% mortality of \u0394tptA infected was observed in this model. No statistically significant differences in survival were observed between mice infected with wild-type A. fumigatus and the ctptA mutant  led to thiamine auxotrophy and attenuated virulence in both pulmonary models (neutropenic and non-neutropenic) of infection and in disseminated infection [Adaptation to iron starvation is crucial for the virulence of pathogenic fungi . Much ofire iron . T-DNA iumigatus . Here, wrevisiae . Thiaminrevisiae . Most banfection .via acetyl-CoA, ODGH catalyzes a rate-limiting step of the citric acid cycle, and ALS catalyzes the first step in the synthesis of branched-chain amino acids . Since ThPP is produced in the cytosol by thiamine pyrophosphokines, the primary function of Tpc1 is most likely to catalyze the uptake of ThPP into mitochondria, where it is required for the activity of ALS, PDH and OGDH [Drosophila melanogaster ThPP carrier have been identified as being responsible for mitochondrial transport of ThPP and ThMP [Aspergillus have not yet been functionally characterized. Our results indicated that TptA in A. fumigatus fulfills similar cellular roles to Tpc1. Firstly, TptA contains conserved mitochondrial carrier motifs and is located in the mitochondria. Secondly, the addition of ThPP, but not other cofactors, partly restored growth defects in the \u0394tptA mutant. Moreover, Tpc1 fully restored ThPP auxotrophy in the \u0394tptA mutant. However, some functional differences between yeast and A. fumigatus ThPP carrier proteins were also found. Firstly, in S. cerevisiae, \u0394tpc1 cells exhibited thiamine auxotrophy on synthetic MM supplemented with glucose or galactose, but not with the non-fermentative carbon source glycerol, suggesting that ThPP is imported into the mitochondria by a different transporter under these conditions [tptA is required for the utilization of various carbon sources (including glucose and glycerol). However, it seems that tptA plays a more important role in glucose utilization and low iron adaptation than it does in glycerol utilization. Secondly, growth defects in the \u0394tpc1 mutant were restored by the simultaneous addition of valine and isoleucine along with the fermentative carbon source glucose, suggesting that ThPP is mainly required in the mitochondria for synthesis of branched chain amino acids [\u0394tptA mutant on glucose, suggesting that tptA may play additional essential roles, rather than simply branched chain amino acid synthesis in A. fumigatus.ThPP is an essential coenzyme for acetolactate synthase (ALS), pyruvate dehydrogenase (PDH) and oxoglutarate dehydrogenase (OGDH), found in mitochondria . PDH briand OGDH . To dateand ThMP ,30,31. Hnditions . In contno acids . HoweverhapX expression and subsequent TAFC production. HapX is a bZIP transcription factor that has been shown to mediate adaptation to both iron starvation and iron excess via interaction with the CCAAT binding complex (CBC) [tptA indirectly regulates hapX expression. Considering that TptA is involved in energy metabolism in the cell, its absence may cause an effect on the expression of genes involved in different metabolic pathways, which may include hapX mediated iron adaptation. It has been reported that increased heme synthesis can activate CCAAT-binding to the Hap complex and to genes required for the tricarboxylic acid (TCA) cycle, electron transport chain and oxidative phosphorylation in yeast [A. fumigatus, it has been reported that hapX negatively regulates TCA expression and heme synthesis under iron limited conditions [hapX expression requires further exploration. On the other hand, overexpression of hapX did not completely restore the BPS-sensitive phenotype in the \u0394tptA mutant, suggesting an additional function of tptA apart from hapX regulation in low iron adaptation.The function of TptA in low iron adaptation is partly due to the regulation of ex (CBC) . As TptAin yeast . Howevernditions . WhetherA. fumigatus caused by the loss of tptA may reflect the role of ThPP in mitochondria, such as in the TCA cycle and branched chain amino acid biosynthetic pathway, which have been reported to be crucial for both pulmonary and systemic aspergillosis [\u0394tptA mutant had attenuated virulence only in the cortisone-acetate model but not in the leukopenic mouse model. In the cortisone-acetate model, the presence of neutrophils and monocytes may increase extracellular iron starvation or impose iron starvation by internalization. Interestingly, the \u0394hapX mutant also appeared to be slightly more virulent in the leukopenic mouse model compared to the cortisone-acetate model [hapX and tptA, play a more important role in fungal virulence. Taken together, this study established a link between the mitochondrial ThPP transporter TptA and fungal low iron adaptation using a forward genetics method. TptA was involved in not only ThPP transport into the mitochondria, but also the regulation of hapX expression, TAFC production and fungal virulence.Virulence attenuation in gillosis . Additiote model . The aboA. fumigatus strains used in this study is provided in Table S1. A. fumigatus strains were grown on minimal medium (MM) containing 1% (w/v) glucose, 70 mM NaNO3 and 18\u00a0\u03bcM FeSO4. Alternatively, YAG medium  was used as a nutrient source. For iron starvation, iron in MM was omitted. To increase iron starvation, the iron-specific chelator bathophenanthroline disulfonate (BPS) was added to MM. Supplementation with iron (FeCl3) and/or ThPP was carried out as described in the figure legends. To perform the carbon sources assay, 1% glucose in the MM media was replaced with 2% potassium acetate, 1% glycerol (GMM), 0.4% acetic acid or 0.4% ethanol.A list of 7 conidia/ml) of the indicated strains was spotted onto the relevant media. All plates were incubated at 37\u00b0C for 48\u00a0h except where indicated. The colonies were observed and imaged and the total conidial production of each colony was harvested in 1 ml 0.02% Tween 80 and counted using a hemocytometer.To analyze the mutant phenotypes, two microliters of conidia from a stock suspension  on induction medium supplemented with 200\u00a0\u03bcM acetosyringone, a phenolic compound that induces transfer-DNA (T-DNA) to enter the recipient strain. After co-cultivation for 48\u00a0h at 24\u00b0C, YAG medium supplemented with hygromycin (300 \u03bcg/ml) and cefotaxime (200 \u03bcg/ml) was used to select transformants. Transformants possessing hygromycin resistance were inoculated onto MM containing 200\u00a0\u03bcM BPS at 37\u00b0C for 48\u00a0h. The transformants shown to be more sensitive to iron starvation conditions compared to A. fumigatus A293 were isolated for further analysis.escribed . In brieA. fumigatus database. Primers used in this study are shown in Table S 2.To determine the T-DNA insertion sites, thermal asymmetric interlaced (TAIL)-PCR was performed as previously described . TAIL-PCtptA knockout strain, fusion PCR was used as previously described [tptA gene were amplified using primers tptA P1/P3 and tptA P4/P6. The selective marker pyr4 (approximately 2 kb in length) from the plasmid pAL5 was amplified using primers Pyr4 F/R. Next, the three previously mentioned PCR products were used as a template to generate the tptA deletion cassette using primers tptA P2/P5 and then transformed into the A. fumigatus strain A1160 [To construct a escribed . In briein A1160 . Transfo\u0394tptA complemented strain CtptA, a PCR-generated DNA fragment including the tptA open reading frame (ORF) plus approximately 1 kb upstream of the ATG start codon and 1 kb downstream of the stop codon, was obtained using primers tptA-up-XbaI and tptA-down-HindIII. Subsequently, this fragment was cloned into XbaI and HindIII digested pAN7-1, which contains the hygromycin B resistance gene hph, to generate the tptA complementation plasmid, pTptA-com-hph. The plasmid pTptA-com-hph was then transformed into the tptA deletion strain and transformants were selected on YAG media supplemented with 200 \u03bcg/ml hygromycin.For the construction of the A. fumigatus tptA, fusion PCR was used as previously described. In brief, an approximately 3 kb fragment, including the native promoter, the 5\u02b9 UTR, tptA ORF and the 3\u02b9 UTR, was amplified using primers tptA P1/tptA-com P6. The selective marker ptrA from the plasmid pCH008 was amplified with primers PtrA F/R. The two fragments were then fused and subcloned into pEASY-Blunt zero (TransGen Biotech) to obtain the plasmid pTptA-com-ptrA. The plasmid pTptA-com-ptrA was then transformed into the T421 mutant and transformants were selected on MM supplemented with 0.1 \u03bcg/ml pyrithiamine to obtain the tptA::T421 strain.To complement the T421 mutant with the wild-type S. cerevisiae tpc1 (Sctpc1) in the tptA mutant background, the hygromycin B resistance gene hph was amplified using primers hph-up-SpeI and hph-down-SpeI and then cloned into the SpeI site of pBARGPE to generate pBARGPE-hph. The Sctpc1 ORF was amplified from S. cerevisiae S288c genomic DNA with the primers OE::Sctpc1-up-EcoRI and OE::Sctpc1-down-EcoRI and then subcloned into the EcoRI site of pBARGPE-hph to generate a tpc1 overexpression plasmid, pOEtpc1-hph. The plasmid pOEtpc1-hph was transformed into the tptA deficient strain to obtain the OESctpc1::tptA strain. To overexpress Sctpc1 in the T421 mutant background, an approximately 1.8 kb fragment including the tpc1 ORF and constitutive promoter gpdA from the plasmid pOEtpc1-hph was amplified with primers gpd F and Sctpc1\u00a0R. The aforementioned selective marker ptrA was amplified with Sctpc1-PtrA F/PtrA R. The two fragments were then fused using primers gpd F/PtrA R and the resultant PCR product was transformed into the T421 mutant to obtain the OESctpc1::T421 strain.To overexpress hapX gene were amplified with the primer pairs hapX P1/P3 and hapX P4/P6. The selective marker hph from pAN7-1 was amplified with primers Hph F/R. The same approach to that described previously was taken to create a hapX deletion cassette with primers hapX P2/P5 and then the resulting PCR product was transformed into the \u0394tptA mutant background and parental wild-type A. fumigatus to generate the \u0394hapX\u0394tptA strain and \u0394hapX strain, respectively.1 kb sections of the flanking regions of the hapX ORF from A. fumigatus A1161 genomic DNA was amplified with the primers OEhapX-up-ClaI and OEhapX-down-ClaI. The generated fragment was then subcloned into the ClaI site of pBARGPE-hph to obtain pOEhapX-hph. The plasmid pOEhapX-hph was transformed into the tptA deletion strain and parental wild-type to generate the strains OEhapX::\u0394tptA and OEhapX, respectively.The tptA stop codon and a 1 kb fragment immediately downstream of the tptA stop codon were amplified using primer pairs TptA-GFP P1/P3 and TptA-GFP P4/P6, respectively. The TptA::GFP fusion PCR cassette (amplified using primer pairs TptA-GFP P2/P5) was transformed into strain A1160 and homologous recombination was verified by PCR using primers TptA-GFP P1/GFP + Pyr4\u00a0R and GFP + Pyr4\u00a0F/TptA-GFP P6.To create a TptA::GFP cassette, a GFP + pyrG fragment was amplified from the plasmid pFNO3 using primer pairs GFP + PyrG F/R. The same approach to that described previously  was usedA. fumigatus tptA gene, was used as a template. Two fragments containing the desired mutation were amplified using tptA-up-XbaI or tptA-down-HindIII with their respective primer pairs and the resulting PCR products were treated with DpnI to remove the template. The two fragments were then cloned into XbaI and HindIII digested pAN7-1 using the ClonExpressII One Step Cloning Kit (Vazyme). The related plasmids were transformed into the \u0394tptA mutant to obtain the strains referred to as R53A, tptAD60A, tptAG153S, tptAG205A, tptAK255A and tptAK315AtptA.For site-directed mutagenesis, complementary primers, approximately 20 bp in length that included the desired mutation in the center position, were designed and synthesized. The plasmid pTptA-com-hph, harboring the wild-type 3 was added  to convert desferri-siderophores to the ferri-forms. Ferric-TAFC was absorbed with Amberlite XAD-16 resin (CWG) equilibrated with 50 mM potassium phosphate buffer (pH 7.5). The resin was subsequently washed with 10 ml of phosphate buffer and TAFC was eluted with 5 ml of methanol [To identify and quantify siderophore (TAFC) production, strains were cultured under iron depleted conditions (MM-Fe) for 24\u00a0h. The supernatants were then filtered and excess FeClmethanol . Methanomethanol .XhoI, separated by electrophoresis at 80\u00a0V for 1.5\u00a0h and transferred to a nylon membrane. A 0.7 kb fragment amplified with the primers tptA probeF and tptA probeR was used as a probe. Labeling and visualization were performed using a DIG DNA labeling and detection kit (Roche Applied Science), according to the manufacturer\u2019s instructions.Genomic DNA from the appropriate strains was digested with 7 conidia of the parental wild-type were inoculated into 100 ml MM or MM-Fe liquid media and incubated at 37\u00b0C for 24\u00a0h. To compensate for the reduced growth rate and to yield the same biomass, 1\u00a0\u00d7\u00a0108 conidia of \u0394tptA and tptAK255A mutants were cultured under the same conditions. Total RNA was isolated from mycelia with TRIzol (Roche) as described in the manufacturer\u2019s instructions. Genomic DNA digestion and cDNA synthesis were performed using HiScript II Q RT SuperMix for qPCR kit (Vazyme), according to the manufacturer\u2019s instructions. The qRT-PCR was performed using an ABI One-step fast thermocycler (Applied Biosystems) with AceQ qPCR SYBR Green Master Mix (Vazyme). Tubulin was used as an internal control for qRT-PCR. All qRT-PCR primers are shown in Table S2 in the supplementary data.3\u00a0\u00d7\u00a010To localize TptA::GFP, fresh conidia were inoculated onto sterile glass coverslips overlaid with 1 ml of liquid MM for 14\u00a0h before observation. The coverslips with hyphae were gently washed three times with phosphate buffered saline (PBS). The mitochondrial marker MitoTracker Red (Invitrogen), dissolved in PBS, was added at a final concentration of 50\u00a0nM and incubated for 5\u00a0min at room temperature . Images A. fumigatus strains was tested in two different murine models of invasive pulmonary aspergillosis [3A. fumigatus conidia resuspended in PBS. Mice were monitored daily and moribund animals were euthanized. For determination of pulmonary fungal burden, the lungs were harvested at day 3 post-infection and were either homogenized to determine the fungal burden or fixed in formalin for histopathology. The fungal burden in lung homogenates was quantified by determining relative galactomannan content as previously described [Virulence of the gillosis . In the escribed . For pul"}
+{"text": "Practice-based research (PBR) is of pivotal importance for hospital pharmacists which not only up-grades the profession but also improves the patient care. This study aimed to evaluate the attitude, perception, willingness, motivation and barriers to PBR among hospital pharmacists in Pakistan.st December, 2017 and 1st March, 2018 from 130 hospital pharmacists employed in 41 hospitals of Lahore, Pakistan. A survey instrument comprising of six sections was designed to determine the attitude, perception, willingness, motivation and barriers to PBR. Data were analyzed by using Statistical Package for Social Sciences . The normality of the data was determined through Shapiro-Wilks and Kolmogorov-Smirnov tests. Independent Samples Mann-Whitney U Test and Independent Samples Kruskal-Wallis Test were carried out to test if there were differences among the characteristics of the hospital pharmacists. Logistic regression analysis was used to figure out the factors associated with attitude, perceptions, willingness and motivation towards PBR. A p-value <0.05 was used for statistical significance of differences.A descriptive, cross sectional study design was employed. Data were collected between 1p-value = 0.040) as compared to those practicing in the outpatient settings. The male hospital pharmacists , those practicing in the outpatient  and inpatient settings had increased motivation towards PBR .A total of 141 pharmacists were approached. Among them, 130 responded to the survey (response rate 92%). Out of a maximum score i.e., 5 (100%) the respondents obtained a median score of 4 (IQR = 0) for attitude, perception and motivation towards PBR; whereas, a median score of 4 (IQR = 1) was obtained for willingness thus demonstrating fair positive attitude, good perceptions, increased motivation and willingness towards PBR. The most common barrier limiting the pharmacists\u2019 participation in PBR was lack of time (23.8%) followed by lack of incentives (16.2%) and lack of support (14.6%). Results of the logistic regression analysis revealed that hospital pharmacists practicing in the inpatient settings had 4.56 times more positive attitude towards PBR (OR = 4.56, 95%CI = 1.07\u250019.42, Despite the presence of several barriers, the respondents had fair positive attitude, good perceptions, increased motivation and willingness towards PBR which is a promising finding. Medical research demands continuous up-gradation of professional practice and knowledge . Thus, iThe social cognitive theory states that researchers are greatly influenced by the environmental and personal factors . Environnd most populated city in the world and second most populated city in Pakistan, with the population of 15,245,000 [A descriptive, cross sectional study design was employed. Out of the total 66 hospitals present in Lahore, hospital pharmacists are employed in 41 hospitals . Lahore is the 32,245,000 . This ciStudy population consisted of hospital pharmacists employed in private and government hospitals of Lahore. The total sampling strategy was used for the current study. All the hospital pharmacists (n = 141) working in Lahore were approached out of which 130 hospital pharmacists consented to participate in this study.st December, 2017 and 1st March, 2018. An additional note was attached with each questionnaire which defined the aim of the research. An extensive literature review of previously published findings was executed [Data were collected between 1executed , 16, 17 executed . Three eThe survey instrument had six distinct sections. The first two sections sought the attitude, perception and willingness of hospital pharmacists towards PBR and were measured on a five point Likert scale . The third section included the factors that motivated the pharmacist to be a part of research and was also measured by the Likert scale. The fourth section examined the barriers in executing research such as lack of time, support and knowledge etc. The fifth section explored the main areas of interest for research and the final section comprised of the participant\u2019s demographics.Data were analyzed by using Statistical Package for Social Sciences . Internal consistency of the questionnaire was measured by Cronbach\u2019s alpha, while reproducibility was evaluated using intra-class correlation for each item in the attitude, perceptions, willingness, and motivation scales, with acceptable values \u22650.6. Calculation for Cronbach\u2019s alpha was made as 0.77 for attitude, 0.73 for perceptions, 0.75 for willingness, and 0.73 for motivation section. Descriptive statistics such as frequencies, percentages, median and interquartile ranges were used to analyze the data. Moreover, the normality of the data was determined through Shapiro-Wilks and Kolmogorov-Smirnov tests. Outcomes regarding attitude, perceptions, willingness and motivation towards PBR were dichotomized as \u201cPositive\u201d versus \u201cNegative\u201d, \u201cGood\u201d versus \u201cPoor\u201d, \u201cMore\u201d versus \u201cLess\u201d, and \u201cHigh\u201d versus \u201cLow\u201d, respectively. Median scores of \u22653 were considered as \u201cPositive\u201d, \u201cGood\u201d, \u201cMore\u201d, and \u201cHigh\u201d; whereas, scores <3 were considered as \u201cNegative\u201d, \u201cPoor\u201d, \u201cLess\u201d, and \u201cLow\u201d, respectively. Independent Samples Mann-Whitney U Test and Independent Samples Kruskal-Wallis Test were carried out to test if there were differences among characteristics of the hospital pharmacists with regard to their attitudes, perceptions, willingness and motivation towards PBR. Logistic regression analysis was performed to figure out the factors associated with attitude, perceptions, willingness and motivation towards PBR. Results were expressed as Odds Ratio (OR) accompanied by 95% Confidence Intervals (95%CI) and a p-value <0.05 was used for statistical significance of differences.The ethical approval was obtained from the Pharmacy Research Ethics Committee (PREC) at the Akhtar Saeed College of Pharmaceutical Sciences . Before initiating the study, the purpose and protocols were thoroughly explained to participants and written consents were also obtained.A total of 141 hospital pharmacists were approached. Out of them, 130 participants agreed to participate in the survey (response rate = 92%). Most of the respondents were male  and had a professional experience of less than 2 years . Most of the respondents had neither any clinical training  nor other board certifications . Majority of them were interested in surgical unit , employed in inpatient departments , and had major research interest in the field of therapeutics  .Out of a maximum score i.e., 5 (100%) for the attitude towards PBR, the respondents obtained a median score of 4 (IQR = 0), demonstrating a fair positive attitude towards PBR.\u201cPharmacy practice research is significant in recognizing and examining complications in pharmacy\u201d . Similarly, most of the respondents  agreed (Strongly agreed (SA) + Agreed (A)) with Item no. 2 . For details please refer to Majority of the respondents  agreed (Strongly agreed (SA) + Agreed (A)) with the statement Out of a maximum score i.e., 5 (100%) for the perceptions towards PBR, the respondents obtained a median score of 4 (IQR = 0), demonstrating good perceptions towards PBR.\u201cIt is crucial to be well-informed of the research fitting to the practice of pharmacy\u201d . Similarly, most of the respondents  agreed (Strongly agreed (SA) + Agreed (A)) with Item no. 5 . For details please refer to Majority of the respondents  agreed (Strongly agreed (SA) + Agreed (A)) with the statement Out of a maximum score i.e., 5 (100%) for the willingness towards PBR, the respondents obtained a median score of 4 (IQR = 1), demonstrating high willingness towards PBR.\u201cI have the required abilities to participate in research\u201d . Similarly, most of the respondents  agreed (Strongly agreed (SA) + Agreed (A)) with Item no. 4 . For details please refer to Majority of the respondents  agreed (Strongly agreed (SA) + Agreed (A)) with the statement Out of a maximum score i.e., 5 (100%) the respondents obtained a median score of 4 (IQR = 0) for the motivation towards PBR thus demonstrating increased motivation towards PBR.\u201cProvide better services and increased patient care\u201d . Similarly, most of the respondents  agreed (Strongly agreed (SA) + Agreed (A)) with Item no. 1 . For details please refer to Majority of the respondents  agreed (Strongly agreed (SA) + Agreed (A)) with the statement A number of barriers limiting pharmacists\u2019 participation in PBR were identified. The most common barrier reported by most of the pharmacists was lack of time  followed by lack of incentives , lack of support  and lack of knowledge . Please refer to p-value <0.05) between gender and attitude, perceptions, and willingness of hospital pharmacists towards PBR. Independent samples Kruskal-Wallis Test showed that area of practice was found to be statistically associated  with the motivation of hospital pharmacists  as compared to those practicing in the outpatient settings. Furthermore, male hospital pharmacists had 8.86 times increased motivation towards PBR  as compared to female hospital pharmacists. Similarly, hospital pharmacists practicing in the outpatient settings had 23.51 times increased motivation towards PBR  and hospital pharmacists practicing in the inpatient settings had 12.24 times increased motivation towards PBR  as compared to those practicing in other settings  where only 32 to 50% of the pharmacists were interested in conducting research [Respondents in the current study demonstrated good perceptions towards PBR. Many participants disagreed with the statement an study . The finresearch . A possiresearch , 17, 21.\u201cProvide better services and increased patient care\u201d and \u201cThe pharmacy profession would be uplifted\u201d. The factors that motivated the pharmacists to take part in PBR included the desire of uplifting the pharmacy profession, provision of better services and increased role in patient care, continuous medical education and for sustaining research activities. Similar motivational factors have been reported in other studies [Study participants also showed an increased motivation towards PBR. Participants strongly agreed/agreed with the statement  studies , 23. FurOur study revealed that the major barriers that hindered the participation of pharmacists in research were the lack of time, lack of incentives, lack of support, and lack of knowledge to conduct PBR. Lack of time has been reported to be a major barrier in various previously published studies , 21, 24.This study has few limitations as well. First, the study was conducted in a single city of Pakistan, so results could not be generalizable to the entire country. However, the healthcare system and curriculum of healthcare professionals is similar across the country and the findings are likely to be similar for entire country. Second, some estimations of CI in The respondents had fair positive attitude, good perceptions, increased motivation and willingness towards PBR which is a promising finding. The most common barrier limiting pharmacists\u2019 participation in PBR was lack of time followed by lack of incentives and lack of support. Male gender was found to be statistically associated with positive attitude, perceptions, and willingness towards PBR. Similarly, area of practice was found to be statistically associated with the motivation of hospital pharmacists.S1 Appendix(DOCX)Click here for additional data file.S1 File(SAV)Click here for additional data file."}
+{"text": "WRKY transcription factors (TFs) participate in various physiological processes of plants. Although WRKY genes have been well studied in model plants, knowledge of the functional roles of these genes is still extremely limited in cotton.GhWRKY42, was isolated and characterized. Our data showed that GhWRKY42 localized to the nucleus. A transactivation assay in yeast demonstrated that GhWRKY42 was not a transcriptional activator. A \u03b2-glucuronidase (GUS) activity assay revealed that the promoter of GhWRKY42 showed fragment deletion activity in Nicotiana tabacum and was mainly expressed in the roots, stems and leaves of ProGhWRKY42::GUS transgenic Arabidopsis plants. Quantitative real-time PCR (qRT-PCR) analysis indicated that GhWRKY42 was up-regulated during leaf senescence and was induced after exposure to abiotic stresses. Constitutive expression of GhWRKY42 in Arabidopsis led to a premature aging phenotype, which was correlated with an increased number of senescent leaves, reduced chlorophyll content and elevated expression of senescence-associated genes (SAGs). In addition, virus-induced gene silencing (VIGS) was used to silence the endogenous GhWRKY42 gene in cotton, and this silencing reduced plant height.In this study, a group IId WRKY gene from cotton, GhWRKY42 is involved in abiotic stress responses, premature leaf senescence and stem development. This work establishes a solid foundation for further functional analysis of the GhWRKY42 gene in cotton.Our findings indicate that The online version of this article (10.1186/s12863-018-0653-4) contains supplementary material, which is available to authorized users. Plants are constantly challenged by various factors that affect plant growth and development throughout their life cycle. To combat these challenges, some responsive genes, including WRKY transcription factors (TFs), are induced to help plants adapt through physiological and morphological changes . WRKY TFArabidopsis  in the promoter of target genes to modulate stress responses, plant development and leaf senescence , 41. LiuGhWRKY42 was found to be up-regulated during natural senescence and exhibited significantly higher expression in the early-aging cotton variety CCRI10 than in the non-early-aging variety Liao4086. It has been reported that the GhNAC12 gene, which is more highly expressed in CCRI10 than in Liao4086 during leaf senescence, causes an early-aging phenotype in Arabidopsis [GhWRKY42 may be involved in the aging process and may play a positive regulatory role during leaf senescence. Consistent with our prediction, overexpression of GhWRKY42 did lead to an advance of leaf senescence in transgenic Arabidopsis. In a previous study, overexpression of AtWRKY45 in Arabidopsis was observed to up-regulate expression of representative SAGs during age-triggered leaf senescence\u00a0[Arabidopsis lines and RNAi cotton lines show that GhNAP positively regulates leaf senescence through ABA-mediated pathways [Arabidopsis [GhWRKY42 transgenic lines, suggesting that GhWRKY42 may be associated with leaf senescence via ABA-mediated pathways.Senescence is a natural phenomenon and prevails among all living organisms, including plants. During leaf senescence, genetic and environmental factors affect mature leaves, leading to the initiation of leaf senescence; this senescence is accompanied by chlorophyll, membrane, protein and nucleic acid degradation as well as nutrient relocation from senescing leaves to growing organs or storage tissues \u201345. Cropbidopsis . Therefonescence\u00a0. Phenotypathways . Phytohopathways . ABA-resbidopsis . In our LP1 in foxtail millet and OsWRKY78 in rice have all been shown to play an important role in stem elongation and plant height [GhWRKY42 in the stem and reduced height in VIGS plants. Therefore, we hypothesized that GhWRKY42 might be related to stem development. Plant height is an important plant architecture trait, and decreased height is beneficial for mechanical harvesting and lodging resistance [GhWRKY42-mediated mechanism.Previous studies have shown that some genes are closely associated with plant height. Wei et al. identified the QTL DTH8 in rice, which includes the HAP3 gene and regulates yield, plant height and flowering time . WRKY TFt height , 52. In sistance . Our finGhWRKY42, a group IId WRKY member, is closely associated with leaf senescence and plant development. GhWRKY42 is located in the nucleus and exhibits no transcriptional activity. GhWRKY42 is induced by leaf senescence and various stresses. Ectopic expression of GhWRKY42 in Arabidopsis promotes leaf senescence, and VIGS cotton plants exhibit a decreased plant height phenotype. Our work could lead to a better understanding of the functional roles of WRKY genes in cotton. However, how the GhWRKY42 gene regulates leaf senescence and plant height development requires further study and clarification.Two early-aging cotton varieties, CCRI10 and CCRI74, and a non-early-aging variety, Liao4086, were used in our experiments. The cotton varieties were cultivated in the field of the Cotton Research Institute of the Chinese Academy of Agricultural Sciences . Different tissues were collected from CCRI10 plants. Roots and stems were collected from two-week-old seedlings. Leaves were collected from newly flattened leaves. Petals, pistils and stamens were sampled at anthesis, and fiber and ovules were harvested at 10\u00a0days post anthesis.GhWRKY42 during leaf senescence, cotyledons were collected from two cotton varieties, CCRI10 and Liao4086, which exhibit different aging characteristics. We collected cotyledon samples weekly at eight different developmental stages, ranging from the flattened cotyledon stage to the completely aged stage. The expression patterns of GhWRKY42 were further evaluated in true leaves of\u00a0the early-aging cotton variety CCRI74\u00a0at five aging stages, as described previously [To evaluate the expression pattern of eviously . Each saGhWRKY42 in cotton, 10-day-old CCRI10 cotton seedlings were planted in pots for subsequent stress treatments. The CCRI10 cotton seedlings were planted in a growth chamber at 25\u00a0\u00b0C, with a 16\u00a0h light/8\u00a0h dark cycle. For the abiotic stress treatment, the seedlings were irrigated with 15% polyethylene glycol 6000 (PEG6000) and 200\u00a0mM sodium chloride (NaCl); for the signaling molecule treatment, the seedlings were sprayed with 100\u00a0\u03bcM methyl jasmonate (MeJA) and 200\u00a0\u03bcM abscisic acid (ABA). Each cotyledon sample included material collected from eight uniform plants, and each treatment was repeated three times. The samples were harvested at 0\u00a0h, 2\u00a0h, 4\u00a0h, 6\u00a0h, 8\u00a0h and 12\u00a0h. All samples were quickly frozen in liquid nitrogen for subsequent RNA extraction.To evaluate the stress response of GhWRKY42, primers were designed based\u00a0on the coding sequence of GhWRKY42 (accession KF669797) submitted to NCBI by Dou et al. [GhWRKY42 were amplified from cDNA and DNA, respectively, obtained from CCRI10 leaves at the five-leaf stage. The fragments were subsequently inserted into the pMD18-T vector  and transformed into Escherichia coli competent cells (E. coli DH5a) for sequencing. The genomic and coding sequences of GhWRKY42 were submitted to Gene Structure Display Server online software (GSDS2.0) (http://gsds.cbi.pku.edu.cn/) to predict gene structures. Multiple sequence alignment was conducted using DNAMAN software, and a phylogenetic tree was built by using MEGA 7 software. The GhWRKY42 promoter fragment was amplified from DNA, and the online software PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) was employed to predict cis-acting elements.To amplify the full-length cDNA and genomic sequences of u et al. . The pri2O. The optimal PCR amplification procedure used was as follows: a pre-denaturation step at 95\u00a0\u00b0C for 30\u00a0s; 40\u00a0cycles of 95\u00a0\u00b0C for 5\u00a0s and 60\u00a0\u00b0C for 34\u00a0s; and a melting curve step at 95\u00a0\u00b0C for 15\u00a0s, 60\u00a0\u00b0C for 1\u00a0min and 95\u00a0\u00b0C for 15\u00a0s. GhActin and AtActin2 were used as reference genes. The 2\u2212\u0394\u0394CT method was applied to calculate relative expression levels [Total RNA was isolated using RNAprep PurePlant Kit (Polysaccharides & Polyphenolics-rich) . One microgram of total RNA was prepared for cDNA synthesis in a 20\u00a0\u03bcl reaction system using a PrimeScript\u2122 RT reagent kit with gDNA Eraser. The cDNA was diluted 5 times for qRT-PCR. Transcript levels were detected using a 7500 Real-Time PCR system (Applied Biosystems) and SYBR\u00ae Premix Ex Taq\u2122 II (Tli RNaseH Plus) (TaKaRa). The 20\u00a0\u03bcl reaction volume contained the following components: 10\u00a0\u03bcl of SYBR Premix Ex Taq II (Tli RNaseH Plus) (2\u00d7), 0.8\u00a0\u03bcl of the PCR forward primer (10\u00a0\u03bcM), 0.8\u00a0\u03bcl of the PCR reverse primer (10\u00a0\u03bcM), 0.4\u00a0\u03bcl of ROX Reference Dye II (50\u00d7), 2\u00a0\u03bcl of cDNA and 6\u00a0\u03bcl of ddHn levels . Three iGhWRKY42 without the termination codon was cloned into the pBI121-GFP vector to generate the 35S-GhWRKY42::GFP construct, driven by the cauliflower mosaic virus 35S promoter. The 35S-GhWRKY42::GFP plasmid was extracted to obtain a plasmid concentration of at least 1\u00a0\u03bcg/\u03bcl. The inner epidermis of a fresh onion was cut into approximately 1.5\u2009\u00d7\u20091.5\u00a0cm pieces with a scalpel on a clean bench. The epidermal pieces were then transferred to solid Murashige and Skoog (MS) medium and cultivated at 28\u00a0\u00b0C for 3\u20136\u00a0h in darkness. The gene gun device was sterilized and was placed on a clean bench, and the bombarding chamber and some accessories were cleaned with 75% alcohol. After the particulate carrier membrane was washed with 70 and 100% alcohol, plasmids encased in gold powder were added to the middle of the particulate carrier membrane. After the membrane dried slightly, the onion epidermis was bombarded using the gene gun with the following parameters: particle bombardment running distance, 9\u00a0cm; rupture disk pressure, 1300\u00a0psi; and vacuum degree, 28\u00a0mmHg. The epidermis after bombardment was transferred to fresh MS agar medium at 25\u00a0\u00b0C for 12\u00a0h in darkness. The resulting green fluorescence was detected using a confocal laser scanning microscope (Zeiss LSM 700) at a wavelength of 488\u00a0nm.The open reading frame (ORF) of GhWRKY42 was cloned into the pGBKT7 vector to construct pGBKT7-GhWRKY42. The pGADT7-largeT+pGBKT7-GhWRKY42 , pGADT7-largeT+pGBKT7-p53 (positive control) and pGADT7-largeT+pGBKT7-laminC (negative control) plasmids were transformed into Y2HGold yeast competent yeast cells. The transformed yeast products were spread on corresponding dropout selective medium plates that did not contain tryptophan or leucine (SD/\u2212Trp/\u2212Leu) and incubated for 3\u20135\u00a0days at 30\u00a0\u00b0C. Positive clones were identified and streaked on SD/\u2212Trp/\u2212Leu medium plates and plates containing medium without tryptophan, leucine, histidine or adenine (SD/\u2212Trp/\u2212Leu/-His/\u2212Ade). The plates were inverted and incubated at 30\u00a0\u00b0C for 3\u20135\u00a0days to identify transcriptional activity.The ORF of GhWRKY42 was inserted into the binary expression vector pBI121 driven by the 35S promoter to generate the 35S::GhWRKY42 construct. The GhWRKY42 promoter fragment was also inserted into the pBI121 vector by replacing the 35S promoter to generate the ProGhWRKY42::GUS construct. The 35S::GhWRKY42 and ProGhWRKY42::GUS constructs were individually introduced into Agrobacterium tumefaciens strain LBA4404 and transformed into Arabidopsis ecotype Columbia using the floral-dip method [\u2212\u20092\u00a0s\u2212\u20091. Two weeks later, the green seedlings on the plates were selected and transplanted into the nutrient soil in a growth chamber. The positive plants were further verified using PCR, and selfed seeds harvested from the positive plants were employed as the T1 generation. Using the same method, the seeds were screened until the T3 homozygous generation. The phenotypic characteristics of the transgenic and WT plants were observed at different developmental stages.The ORF of p method . For thecis-elements in the GhWRKY42 promoter, four promoter deletion fragments were delimited. The four fragments were amplified from the pMD18-T vector containing the GhWRKY42 promoter and inserted into the pBI121 vector by replacing the 35S promoter. As a result, four promoter deletion plasmids, ProGhWRKY42::GUS (\u2212\u20091943\u00a0bp to \u2212\u20091\u00a0bp), ProGhWRKY42\u20131::GUS (\u2212\u20091407\u00a0bp to \u2212\u20091\u00a0bp), ProGhWRKY42\u20132::GUS (\u2212\u2009778\u00a0bp to \u2212\u20091\u00a0bp) and ProGhWRKY42\u20133::GUS (\u2212\u2009391\u00a0bp to \u2212\u20091\u00a0bp), were constructed and transformed into LBA4404. Transient expression in N. tabacum was performed in accordance with previously described methods [Arabidopsis plants harboring the ProGhWRKY42::GUS construct were used to analyze organizational expression characteristics. GUS staining was performed as follows: the prepared materials were soaked in the GUS dye solution, after which the materials were placed in darkness at 25\u201337\u00a0\u00b0C overnight; the materials were then decolorized approximately 2\u20133 times using 70% alcohol until the negative control materials turned white, and the blue dots in the white background observed under microscopy were identified as GUS expression sites.Based on the position of stress response  methods . TransgeGhWRKY42 gene were integrated into the pCLCrVA vector to construct pCLCrVA-GhWRKY42, which was then transformed into LBA4404. The LBA4404 strains carrying pCLCrVA-GhWRKY42, pCLCrVA (negative control) or pCLCrVA-PDS (positive control) were mixed with the strain harboring pCLCrVB (helper vector)  and co-injected into two fully expanded cotyledons of CCRI10 plants. In the VIGS assay, at least 20 seedlings were used per group. For qRT-PCR detection, samples from at least 6 uniform injected plants were used. The cotton plants were then cultivated at 22\u00a0\u00b0C with a 16\u00a0h light/8\u00a0h dark cycle in a greenhouse. The experiment was repeated three times. The detailed VIGS procedure was performed as previously described [For the VIGS assay, approximately 300-bp fragments amplified from the pMD18-T vector containing the escribed , 58.Determination of the\u00a0chlorophyll content was performed as described by Shah et al. .Additional file 1:Table S1. Predicted cis-acting elements in the promoter region of GhWRKY42. (DOCX 36\u00a0kb)Additional file 2:Table S2. Primers used in this study. (DOCX 32\u00a0kb)"}
+{"text": "Fasting triglyceride levels were available at 4 time points (visits), 2 pre- and 2 post-fenofibrate intervention. Multipoint identity-by-descent (MIBD) matrices were derived from genotypes using IBDLD. Variance-component linkage analyses were then conducted using SOLAR . We found evidence of linkage (logarithm of odds [LOD] \u22653) at 5 chromosomal regions with triglyceride levels in plasma. The highest LOD scores were observed for linkage to the estimated genetic value (additive genetic component) of the log-normalized triglyceride levels in plasma. Our results suggest that a chromosome 10 locus at 37\u00a0cM (LOD Triglyceride levels in plasma are highly heritable traits that have been consistently associated with obesity, cardiovascular disease, coronary heart disease, Type 2 diabetes, and metabolic disease. The genetics of triglycerides has been the subject of extensive research, including both genome-wide association and genome-wide linkage studies in a variety of cohorts.As a result, several loci have been either associated or linked with changes in triglyceride levels. Li et al.  found liBesides design and methodological differences, those linkage studies share the characteristic of being observational studies. The Genetics of Lipid Lowering Drugs and Diet Network (GOLDN) study  conducteIn this study, we conduct genome-wide linkage scans to map loci that influence fasting triglyceride levels in plasma before and after fenofibrate was administered to GOLDN participants. To do so, we follow a different approach than the one used by Hidalgo et al. . We usedWe used real data from the GOLDN study that was made available to participants of the GAW20. Specifically, SNP dosages from 822 individuals genotyped at 718,407 loci, phenotypes from 1106 individuals, and genealogies for 4151 individuals in 188 families. Quality control of the genotype and genealogical information was conducted using PREST-plus , to asseFasting triglyceride (TG) levels in plasma (mg/dL) from peripheral blood, drawn at visits 1 and 2 (pre-) and at visits 3 and 4 (post-fenofibrate intervention), were used to derive phenotypes consisting of the averaged and log-normalized pre (log_tg_pre) and post (log_tg_post) fenofibrate TG levels. In addition, we used the additive genetic component of the phenotypic variance  of log-normalized pre (egv_log_tg_pre) and post (EGV_log_tg_post) TG levels, as described by Porto et al. . Age, seOne thousand heritable traits (SIMQTs) were simulated using SOLAR version 8.1.1) [.1.1 [8] Physical coordinates and annotations for genes and marker loci were set to be relative to release 19 of the human genome (hg19) from UCSC. Genetic coordinates were interpolated, accounting for the local base pair/cM rate, from a sex-average combined physical and genetic map .2\u2009\u2265\u20090.9 followed by a filter of minor allele count (MAC) >\u20095 using PLINK (version 1.90p) [Multipoint estimates of identity-by-descent (MIBD) for each SNP locus and chromosome-wide empirical kinship estimates were obtained using IBDLD (version 3.33)  using thn 1.90p) . A genomn 1.90p)  estimateThe pedigree-based multipoint variance component approach built into SOLAR  was usedTwo individuals were excluded from downstream analyses based on genealogy mismatches (see ). One inHeritability estimates for the TG level in plasma before and after the fenofibrate intervention, and across SIMQTs ranged between 33 and 48% Table\u00a0. BlackbuLinkage was detected for log_tg_post in chromosome 10 at 30\u00a0cM, with a peak LOD score of 3.35, within a 12-cM region of LOD \u22652 support  instead of the expected long-range MIBD estimates, we shifted the point of MIBD estimation by half a centimorgan in the genetic map. Then we estimated new MIBD matrices and repeated the linkage analyses of the log_tg_pre and log_tg_post traits. The resulting linkage scans contained the same features as those appearing in Fig.\u00a0All evidence of linkage disappeared with the removal of the genetic signal from log_tg_post, accomplished by the introduction of egv_log_tg_post as covariate into its linkage model see Fig.\u00a0.Across a thousand SIMQT linkage scans we observed 1 scan with LOD scores \u22653.35, 3 scans with LOD scores \u22653, and 33 scans with LOD scores \u22652. The maximum LOD score observed in the averaged linkage scan from all SIMQTs was 0.119 .Hidalgo et al.  reportedWe found linkage between the fasting TG levels in plasma after the intervention with fenofibrate (log_tg_post) and a locus that maps to chromosome 10 at 30\u00a0cM (LOD\u2009=\u20093.35; see Table\u00a0There are precedents in the literature for some of our findings. The analysis from Liu et al.  found suWe demonstrated that conducting linkage scans with MIBD matrices estimated from dense SNP loci is feasible. Focusing our analyses on the additive genetic component of the trait allowed us to improve our power to detect linkage. Our results identified loci that appear to influence TG levels in plasma and seem consistent with a differential response to the presence of fenofibrate."}
+{"text": "Prochloraz is a fungicide that is widely used on vegetables to maintain freshness during storage. To ensure that prochloraz is used in a safe way that reduces the levels of residue on the product, we evaluated two treatment methods (soaking and spraying) that are commonly used for garlic sprouts. An ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for prochloraz residue on garlic sprouts. The linear range of the method was 5\u2013500 \u03bcg/kg and the correlation coefficient was 0.9983. The average recovery range was 88\u201394%, and the relative standard deviation range was 2.6\u20139.7%. Garlic sprout samples that had been soaked in or sprayed with prochloraz were collected from cold storage facilities in Laixi and Pingdu, China. For the soaked samples, the ranges for the levels of prochloraz residue on the whole garlic sprouts and stems (edible portion) were 15.76\u201325.14 mg/kg and 0.58\u20131.62 mg/kg, respectively. For the sprayed samples, the ranges for the levels of prochloraz residue on the whole garlic sprouts and stems were 1.85\u20137.89 mg/kg and 0.01\u20131.29 mg/kg, respectively. The results of this study provide a scientific basis for rationalizing the use of prochloraz and improving the safety of edible garlic sprouts.  They arutrients  and garlutrients .http://tianqi.2345.com/wea_history/54823.htm). In these temperatures, garlic sprouts can lose water, leading to aging and decay [Of the types of vegetables kept in cold storage facilities in China, garlic sprouts make up the largest proportion and are stored for longer periods than other vegetables . Garlic nd decay ,8,9,10. nd decay ,12,13,14nd decay ,16. Prochloraz is a broad-spectrum imidazole fungicide with high efficiency and low toxicity. However, at the same time, prochloraz has been suspected of acting as an endocrine disrupter ,18. It iTo study the scientific use of prochloraz and undertake accurate assessments of prochloraz residue on garlic sprouts, in this study, we developed an ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for detecting prochloraz residue on garlic sprouts. We compared the levels of prochloraz residue on garlic sprouts after spraying and soaking treatments, and evaluated the health risk of prochloraz residue on garlic sprouts. Stock solutions (1000 mg/L) of prochloraz, for the preparation of prochloraz solutions of different concentration gradients, were stored in capped glass vials in the dark at 2 \u00b0C until required for use. A working solution (50 \u03bcg/kg) for calibration was prepared by diluting the stock solutions. Petroleum ether and acetone were obtained from Pharmco Products Inc.  and acetonitrile was obtained from Sigma-Aldrich . Sodium chloride was purchased from J. T. Mallinckrodt Baker Inc. .Garlic sprout samples that had been treated with prochloraz were collected from cold storage facilities in Shandong Province . The garlic sprouts were treated by either soaking or spraying, and we collected two sprayed samples and two soaked samples from each of four cold storage facilities. A prochloraz aqueous suspension was prepared by diluting a commercial formulation of 25% prochloraz emulsifiable concentrate  with tap water. All garlic samples were treated with a prochloraz solution containing 500 mg of active ingredient per liter. Soaking treatments were carried out by dipping the flower buds of the garlic sprouts in the prochloraz aqueous suspension for 2 min. Spraying treatments were carried out by spraying the flower buds of the garlic sprouts with the prochloraz aqueous suspension. All treated samples were dried in a ventilated area and then stored at 2 \u00b0C in a cold storage facility. All samples were treated with prochloraz on 22 May 2016. Samples (50 g each) for residue analysis were collected on 1 June, 29 June, and 30 July 2016. Because the fungicide was mainly applied to the flower buds of the garlic sprouts, the samples used for residue analysis were divided into whole plant samples and stem (edible portion) samples without the flower buds.g for 5 min. An aliquot (10 mL) of the supernatant was removed from each centrifuge tube and placed in a 150 mL flask. An additional 20 mL of acetonitrile was added to each 100 mL centrifuge tube, and the above extraction steps with NaCl were repeated. A 10 mL aliquot of the supernatant from the second extraction for each sample was transferred to the corresponding 150 mL flask. The combined extracts for each sample were concentrated close to dryness at 40 \u00b0C.The garlic sprouts samples were finely chopped and homogenized. The homogenized garlic sprout samples were weighed into 100 mL centrifuge tubes (5.0 g per tube), then 20 mL of acetonitrile was added to each tube and the tubes were thoroughly shaken for 1 min. Next, 5.0 g of NaCl was added to each tube, and the tubes were ultrasonicated for 15 min and then centrifuged at 4000\u00d7 2 solid-phase extraction (SPE) column was pre-leached with 5 mL of petroleum ether:acetone . Next, two 3 mL portions of petroleum ether:acetone  were added to each sample in a flask. The upper liquid was removed and added to the SPE column, the SPE column was rinsed with 5 mL of petroleum ether:acetone , and the eluate was discarded. Then, the SPE column was rinsed with acetonitrile (3 \u00d7 5 mL), and the eluate was collected into a flask and concentrated close to dryness at 40 \u00b0C. The volume was set to 5 mL and placed in a volumetric flask with acetonitrile:water  to await measurement.A NHCalibration curves and garlic sprout samples were prepared using working standard solutions. Samples that were verified by UPLC-MS/MS to not contain prochloraz were used as blank samples. The blank samples were spiked with 5, 50, and 500 \u03bcg/kg of prochloraz, and subjected to the UPLC-MS/MS analysis to evaluate the accuracy and precision of the method. The slopes of the calibration curves for the samples prepared in solvent and in the matrix were compared. To minimize matrix effects, the linearity was studied using calibration curves prepared with matrix-matched standards. The limit of quantification  was determined as the lowest concentration meeting the method performance criteria for trueness and precision for a given compound. Recovery tests were repeated five times for each spiking level.v/v) + 0.05% formic acid) and B (methanol + 0.05% formic acid) at 40 \u00b0C. The mobile phase flow rate was 0.45 mL/min. A gradient elution was performed as follows: 0\u20130.25 min, 5% B; 0.25\u20138.50 min, 100% B and 8.50\u201310 min, 5% B. The total run time was 10 min. The sample injection volume was 3 \u00b5L.The MS/MS transition was undertaken according to an established method , and theThe mass spectrometry source temperature was 150 \u00b0C, and the nitrogen gas flow rates for the cone and desolvation gases were 50 and 800 L/h, respectively. The desolvation temperature was 500 \u00b0C. Argon was used as the collision gas with a flow rate of 0.15 mL/min. The mass spectrometer was operated in the multiple reactions monitoring mode, and two precursor/product ion transitions were monitored for each analyte. The target ion transition with the highest intensity (primary ion transition) was used for quantitation, and the second target ion transition was used for confirmation. Further confirmation was obtained through a product ion scan for each peak, which was matched to a reference spectrum for each analyte. The quantification and confirmation calculations were performed using the software Target Lynx 4.1 . Ion transitions, cone voltages, collision energies, and dwell times for the analytes are shown in The limit of detection (LOD) was calculated as three times the value of the instrument background signal generated by the blank matrix, and the LOQ was calculated as 10 times the background signal generated by the blank matrix . The ionLinearity was evaluated using blank extracts at seven concentration levels  . For quaIn the chemical analysis, the matrix refers to components other than the analyte in the sample. The matrix can greatly interfere with analysis of the analyte, and the accuracy of the analytical results. There are many ways to eliminate matrix effects, such as the removal of matrix components by purification, special injection methods, standard addition, or the use of a stable-isotope labeled internal standard or analyte protectant ,25,26. OProchloraz is widely used in agriculture to maintain vegetable and fruit freshness. To rationalize the use of prochloraz in cold storage and evaluate the safety of edible garlic sprouts, we determined the levels of residue of prochloraz on garlic sprouts . A graduThese results show that the levels of prochloraz residue present on the whole plant after spraying (1.85\u20137.89 mg/kg) are lower than those after soaking (15.76\u201325.14 mg/kg). Therefore, the use of spraying over soaking for treatment during storage could reduce the levels of prochloraz residue on garlic sprouts. Furthermore, lower prochloraz residue levels were found for the stem samples than the whole plant samples for both soaking  and spraying . To date, a maximum residue limit has not been established for prochloraz on garlic sprouts. However, according to the European Union Pesticides database, the maximum residue limit for prochloraz on garlic is 0.5 mg/kg, and that for chives or shallots is 5.0 mg/kg. In addition, the maximum residue limit for prochloraz on flowering Chinese cabbage is 2.0 mg/kg, as established by Chinese regulation. Therefore, we suggest that the buds of garlic sprouts are removed before consumption to reduce the intake of prochloraz residue.In this study, we developed a rapid and efficient UPLC-MS/MS method to determine the levels of prochloraz residue on garlic sprouts. A comparison of garlic sprouts treated by spraying and soaking showed that the levels of prochloraz residue were lower after spraying than after soaking. Prochloraz residue levels for the stems were lower than those for the whole plant. Therefore, to reduce the intake of prochloraz by humans and for environmental safety, we recommend the spraying method is used for prochloraz application during storage, and that the flower buds of the garlic sprouts are not eaten."}
+{"text": "After publication of this article , concernFigures 3A and 3B appear highly similar in quadrants P1, P2, P3, and select areas of P4.Within Figures 3C and 3F, there are areas that appear to be duplicated within the plots in R2 and R4 quadrants.Similarities were noted between Figures 3D and 3E , and between Figures 3D, 3E, and 3G .Similarities between Figures 5C and 5D suggest they may be derived from the same image, although they reportedly show kidney capsule samples isolated at different timepoints .In response to queries about these concerns, the corresponding author requested retraction, apologized for the figure issues, and indicated that the original data underlying these results are no longer available.PLOS ONE Editors retract the article.In light of concerns about the integrity of these data, the SYZ, FRL agreed with the retraction. YW, HJY, HQ, CYD did not respond."}
+{"text": "Enterococcusfaecalis biofilm. Biofilms were formed inside the root canals and divided into 7 groups (n13): 0.5% NaOCl + Er,Cr:YSGG; Saline + Er,Cr:YSGG; 0.5% NaOCl + syringe irrigation(SI); 2.5% NaOCl + SI; 5% NaOCl + SI; positive and negative controls. Bacterial survivors were counted and specimens visualized under scanning electron and confocal laser scanning microscopy. Treatments with 0.5% NaOCl + Er,Cr:YSGG and 2.5% NaOCl + SI gave a significant reduction in the number of CFU/mm2. Moreover, scanning electron microscopy and confocal laser scanning microscopy imaging confirmed and reinforced bacteriological data. Thus, Er,Cr:YSGG LAI proved to be able to improve the intracanal distribution of 0.5% NaOCl after 60 s of activation, reaching the same level of effectiveness than 2.5% NaOCl. This is regarded as of clinical interest, since working with lower concentrations may contribute to reduce undesired effects.The onset and persistence of endodontic infections due to residual biofilm after chemical disinfection promotes secondary bacterial infection. Alternative methods to disinfect operated root canals are a matter of great interest. The aim was to evaluate the antibacterial effectiveness of sodium hypochlorite (NaOCl) at low concentrations activated by the Er,Cr:YSGG laser-activated irrigation (LAI) against 10-day-old intracanal  Enterococcus faecalis is a frequently recovered bacterium from persistent infections [E. faecalis is an aerotolerant anaerobic Gram-positive coccus, expressing several virulence factors, such as aggregation substances, enterococcal surface protein (Esp), pili ) and cytolysin [The onset and persistence of endodontic infections due to residual biofilm after chemical disinfection promotes secondary bacterial infection . Environfections . E. faecytolysin . Furtherytolysin . A biofiytolysin . Theoretytolysin . The sucConventional syringe irrigation (SI) is widely accepted. Yet it has been argued that in SI the irrigant may not reach the apical region of the canal  nor the Recently, laser-activated irrigation (LAI) has been proposed as an alternative method to achieve cleaning and disinfection of the root canal system being much more efficient than SI or passive ultrasonic irrigation (PUI) . The LAIE. faecalis biofilm ex vivo, in extracted teeth. Effectiveness was estimated by both bacteriological and microscopy approachesSodium hypochlorite (NaOCl) is the most widely used endodontic irrigant; it has a broad antibacterial spectrum and dissolves dental pulp tissue . It is up < 0.05) and the non-parametric Kruskal\u2013Wallis test confirmed significant differences between different groups (p < 0.05). The bactericidal index is shown in 2 (p < 0.001). Moreover, reduction of CFU was significantly greater for 5% NaOCl + SI group (p < 0.001). Lower efficiencies were achieved by saline solution +\u2018LAI and 0.5% NaOCl delivered by SI. Values were compared with the ones obtained in inoculated untreated teeth.Bacterial counts and interquartile range (IQR) values are shown in E. faecalis formed on the dentin surface, occluding the dentin tubules . The images revealed the presence of both alive and dead bacteria in the passive irrigation group with 0.5% NaOCl . Longer bacterial incubations afford more relevant characteristics thanks to the formation of mature biofilms. The time needed for colonization by cubation , others cubation , or evencubation ,23 mimicE. faecalis biofilms. Radcliffe et al. [E. faecalis planktonic cells, while 5.25% NaOCl required only 2 min to achieve complete disinfection. In our case, 5% NaOCl released with the SI protocol was significantly more effective at removing E. faecalis biofilm than the other treatments with or without activation (p < 0.001). The main negative fact is that NaOCl at such high concentrations is extremely irritating to the periapical tissue [There is no consensus regarding the actual time needed to completely eradicate e et al.  demonstrl tissue . Thus, tl tissue .2 count. The SEM revealed a large part of the canal wall and tubules as being free of microorganisms. This finding is encouraging because its effect equaled that of 2.5% NaOCl administered by conventional irrigation. This is relevant as it demonstrates the existence of a synergic effect between the laser and low concentrations of NaOCl. Similar results were obtained by Jaramillo et al. [It has been reported that laser-activated irrigation significantly enhances the effectiveness of root canal disinfection ,27. The o et al. , who cono et al.  observedE. faecalis biofilm since most of dentin tubules exhibited a high number of bacteria. The inability to achieve good results by NaOCl at low concentration without activation stands out the relevant interest of laser energy in the disinfection of the root canal at these low concentrations.Teeth treated with conventional irrigation and 0.5% NaOCl showed a minimum alteration of the E. faecalis remained within the dentin tubules and on the dentin surface. It should be noted that the combination of the laser and saline improved the elimination of the smear layer, supporting the observations made by Di Vito et al. [Despite obviously saline is not bactericidal, some bactericidal effects were observed when used as an irrigant with LAI. The related bacterial death may be due to the intense flow action created within the irrigant . Althougo et al. .It has been reported that NaOCl extrusion increases during activation by the laser. Peeters & De Moore  demonstrE. faecalis biofilm in extracted teeth, the Er,Cr:YSGG LAI proved to be able to improve the antibacterial properties of 0.5% NaOCl after 60 s of activation, reaching the same level of effectiveness as 2.5% NaOCl. This is of great clinical interest, because it demonstrates that a lower concentration of NaOCl may be useful diminishing undesired secondary effects. It should be taken into account that laser has some disadvantages as is the price of equipment and may have some risks when non-expert dentists use inappropriate parameters. It has been seen that the increase in temperature caused by laser energy can produce undesirable effects in the dentin, such as cracks, small fissures or carbonization . In our The study protocol was approved by the Clinical Research and Ethics Committee of the University of Barcelona (#2016-23). A total ninety-one human single-rooted teeth extracted for therapeutics purposes were collected. To eliminate periodontal ligament remnants and calculus from the root surface, the specimens were subjected to cleaning using endodontic tips  and a Gracey 7/8 curette . The specimens were stored in formalin solution 10% at 4 \u00b0C until use.\u00ae, Ballaigues, Switzerland). After the use of each instrument, the root canals were irrigated with 1 mL of 2.5% NaOCl using a syringe and a 30-gauge side-vented needle  to the WL. The canals were irrigated with 1 mL ethylenediaminetetraacetic acid (EDTA)  for 1 min, followed by 1 mL of 2.5% NaOCl and 1 mL of saline. The apical foramen and the root surface were sealed with a double layer of nail polish  to prevent the extrusion of the irrigant through the apex and to provide a closed system [All teeth were decoronated under the cemento-enamel junction to a standardized length of 14 mm as described by Christo et al. . A corond system . The denE. faecalis ATCC 29212 (American Type Culture Collection) was maintained by weekly subculturing on trypticase soy agar (TSA) plates . A single colony was inoculated in 40 mL of tryptic soy broth (TSB) medium and incubated at 37 \u00b0C. After 24 h of incubation, the culture was diluted 100 times in fresh TSB, and adjusted spectrophotometrically (Unicam UV-2 at 600 nm) at OD600 = 1.3 . Root surfaces were coated with 0.01% (w/v) poly-L-lysine hydrobromide  to enhance bacterial adhesion and inoculated with 10 \u00b5l of bacterial culture using a 30-gauge syringe and needle . The dental roots were placed in Eppendorf tubes and incubated at 37 \u00b0C for 10 days. Re-inoculation at days 1, 4 and 7 were performed to ensure the presence of live bacteria during the incubation period [n period . Finallyn = 13). Each group was submitted to a different treatment: (I) 0.5% NaOCl + Er,Cr:YSGG LAI (II) Saline + Er,Cr:YSGG LAI (III) 0.5% NaOCl + SI (IV) 2.5% NaOCl + SI (V) 5% NaOCl + SI (VI) Positive control (no treatment) (VII) Negative control (no bacteria). Eighteen teeth were then randomly divided in to two subgroups for investigation with CLSM (n= 8) and SEM microscopy (n = 10) techniques.The teeth were randomly distributed into seven groups  at a wavelength of 2780 nm. The laser operating parameters were 1 W of power, 10 Hz of repetition frequency, 100 mJ energy per pulse and 140-\u03bcs of pulse duration. The coaxial water spray from the Gold Handpiece  was switched off throughout the treatment. An RFT 2 tip  was used. It is a conical tip with a 50\u00b0 angle, designed for endodontic treatment. The real power was 0.55 W at 10 Hz, 55 mJ per pulse. Autoclaved tips were positioned only in the coronal reservoir during activation. During the laser irradiation cycles, irrigant was added as the coronal reservoir was empty; thus, LAI was permanently carried out in the presence of irrigant. The Er,Cr:YSGG laser was activated for 30 s, followed by a rest phase of 30 s and ending with 30 s of activation . Finally, sodium thiosulfate and saline were used as before. 2.Bacteria were suspended in Ringer \u00bc by using an ultrasonic cleaner  at maximum power followed by vortex agitation for 3 min. Colony-forming units (CFU) per ml were enumerated by plating tenfold serial dilutions on TSA plates incubated for 24 h at 37 \u00b0C. Values were transformed to CFU/mmA water-cooled diamond cutting blade mounted on a precision cutting machine  was used to cut the specimens longitudinally. The two parts were mounted on the microscope supports by means of a conductive double-sided adhesive disc. Next, they were covered with a fine graphite layer to improve their electrical conductivity (Emitech K950X high vacuum evaporator) and examined in a Jeol J-7100F scanning electron microscope  at 15.0 kV. Visualizations were done at 1000\u00d7 and 10,000\u00d7 to assess the bacterial biofilm and the smear layer in the coronal (10\u201312 mm from the apex), middle (6\u20137 mm from the apex) and apical (1\u20132 mm from the apex) parts. To stain the biofilms, a mixture of SYTO 9 and propidium iodide prepared at a dilution ratio of 1:2 (1.5 \u00b5L of SYTO 9 and 3 \u00b5L of propidium iodide (PI) in 1 mL of Ringer \u00bc) was applied to the whole biofilm. After 30 min of incubation in the dark at 37 \u00b0C, the stained biofilms were washed once with Ringer \u00bc to remove nonspecific staining. Fluorescence was observed using a Zeiss LSM 880 spectral confocal laser scanning microscope  equipped with a 488-nm argon laser and 561-nm diode lasers. The reconstruction of whole teeth was performed with stitched images of different focal planes obtained with 10x magnification objective  using the Zen black software . The zoom images were obtained with 40\u00d7 immersion oil objective . The image resolution was 1024 \u00d7 1024 pixels with both magnifications. ImageJ software  and IMARIS software  were used to obtain LSM images. \u00ae, College Station, TX, USA). Data were transformed logarithmically. The bactericidal effects were expressed as a bactericidal index (BI); i.e., the difference between the logarithm of the bacterial counts of the control and the treatment groups. The normality of the scale variables was explored using the Shapiro\u2013Wilk test and the visual analysis of the P-P graph and the box plot. When normality was rejected, both the interquartile range (IQR) and the median were calculated. A statistical analysis was performed to compare the UFC/mm2 values using the Kruskal\u2013Wallis nonparametric test and Bonferroni\u2019s post hoc test for multiple comparisons. The level of significance was set at p < 0.05.Statistical analysis was performed with Stata14 (StataCorp"}
+{"text": "Accurate determination of working length (WL) is crucial for the success of endodontic therapy. The aim of this study was to determine the influence of 2% chlorhexidine digluconate solution, 2% chlorhexidine digluconate gel and 2% hypochlorite solution on the accuracy of two devices: the Raypex 5 and the ApexDal.Twenty-nine single-rooted human teeth were used in this study. The crowns were cut horizontally and embedded in an alginate mass. In each tooth, six endodontic measurements were made using two electronic apex locators (EALs): a Raypex 5 and an ApexDal. For each EAL, measurements were taken with the following products: 2% chlorhexidine solution (CHX-S group), 2% chlorhexidine gel (CHX-G group) and 2% NaOCl (NaOCl group). After performing an endodontic measurement, the endodontic instruments were stabilized with flow resin composite. Afterwards, the roots were removed from the alginate mass, and the apical one-third of each root was cut lengthways to recover the canal system. Last, the distance between the file tip and the apical foramen was measured under a microscope at 60 x magnification.Statistically significant differences were found between CHX-S and NaOCl and CHX-G and NaOCl, but no significant differences were detected between CHX-S and CHX-G during the testing of both devices. No statistically significant differences were observed between the Raypex 5 and ApexDal for all intracanal media tested.The EALs Raypex 5 and ApexDal had higher accuracy in the anatomical foramen of the root containing chlorhexidine in the gel or in the solution form than in the canal containing sodium hypochlorite. Accurate working length (WL) determination is a challenge for dentists . The proIrrigation is presently the best method for the removal of tissue remnants and dentine debris during instrumentation . All oveEnterococcus faecalis and Candida albicans [Sodium hypochlorite (NaOCl) and chlorhexidine are the most popular irrigation solutions used. Chlorhexidine gluconate (CHX) can replace NaOCl during endodontic treatment . CHX hasalbicans \u201329, subsalbicans , tolerabalbicans . The conalbicans , 32. Thealbicans \u201337. Howealbicans \u201340. Amonalbicans , 39. To The aim of this study was to determine the influence of 2% chlorhexidine digluconate solution (CHX-S), 2% chlorhexidine digluconate gel (CHX-G) and 2% NaOCl solution on the accuracy of two devices: the Raypex 5 and the ApexDal. The null hypotheses tested were as follows: (i) the accuracy of the contemporary EAL measurements would not depend on the kind of solution used, and (ii) the accuracy of the contemporary EAL measurements would not depend on the kind of equipment used.Twenty-nine single-rooted vital human teeth (incisors and upper second premolars) that had been scheduled for extraction from patients for periodontal or prosthetic reasons were selected. The patients were aged 35\u201355\u2009years. The selected teeth were tested with an Analytic Technology pulp tester  to confirm that they contained vital pulp tissue. Immediately after extraction, the teeth were placed in a 10% formalin solution for 48\u2009h. After fixation, the teeth were stored in 2.5% NaOCl solution for 48\u2009h, and the root surfaces were cleaned to remove all organic debris and deposits. The mesiodistal and buccolingual radiographs were taken to determine that the selected teeth had noncomplicated root canal anatomy, single straight root canals and mature root formation. All roots were inspected under an operating microscope at 12.5x magnification  to determine any sign of external resorption, cracks or fractures along the roots. The research was carried out with the consent of the Ethics Committee of Pomeranian University of Medicine  and was conducted in accordance with the Declaration of Helsinki ethical principles. To take part in this research, all 29 study participants signed a voluntary written consent form (KB- 0012/10/19).First, the crowns were cut horizontally with a high-speed diamond flame bur REF F 0250 343, no 16  at 2-mm coronal to the CEJ. Then, on the flat occlusal surfaces, edges were made as reproducible reference points. Special edges resembling a cube were made with a round diamond bur REF F 0001 343, no 18  placed in a turbine. Afterwards, the orifice and 1/3 of the coronal part of each canal were flared with Gates-Glidden drills (sizes 2 to 4) . The patency of the canals was checked by inserting of a size 10\u2009K-file  until the tip was visible at the apical foramen.The teeth were embedded up to the CEJ in an alginate mass , which was prepared according to the manufacturer\u2019s instructions and poured into a plastic container. A metal lip clip was also placed into the alginate mass to close the current circulation. The measurements were always performed in the moist alginate mass, according to the model developed by Kaufman and Katz, i.e., within 15\u201320\u2009min for one tooth. When not in use, the container with the alginate mass was wrapped with wet paper and refrigerated . Measure2 test. A probability of less than 0.05 was considered significant.Statistical analysis was performed using STATISTICA for Windows 9.0 . To evaluate differences between values, the following nonparametric tests were used: Friedman\u2019s ANOVA, Wilcoxon\u2019s matched-pair test and chip\u2009<\u20090.05) were found between CHX-S and NaOCl and CHX-G and NaOCl, but no significant difference was detected between CHX-S and CHX-G  was located 79.3% of the time for CHX-S, 86.2% of the time for CHX-G and 53.2% of the time for NaOCl. There was no significant difference between CHX-S and CHX-G, but there were differences when CHX-S or CHX-G were compared with NaOCl (Table\u00a0p\u2009<\u20090.05) between CHX-S and NaOCl as well as CHX-G and NaOCl, but no significant difference was found between CHX-S and CHX-G  EALs measure the resistance and capacitance separately, and there can be different combinations of values of capacitance and resistance that provide the same impedance and thus the same foraminal reading. Therefore, this generation of EAL solved the problem of fourth generation EALs, which must be operated in relatively dry or partially dried canals. The effects of various irrigants, such as saline, hydrogen peroxide, sodium hypochlorite solution, and ethylenediaminetetraacetic acid (EDTA) solution, on fifth generation EAL performance have been investigated. Numerous studies indicate that endodontic measurement can be performed in the presence of any conductive fluid, but the type of irrigant solution may affect the accuracy of the EAL. The most tested irrigants are sodium hypochlorite and chlorhexidine solutions. Erdemir et al. showed no significant difference in file tip position between 2.5% NaOCl and 0.2% chlorhexidine gluconate using a Tri Auto ZX . There wE. faecalis. In Dammaschke\u2019s research, 2% CHX-G was as effective as camphorated-and-mentholated chlorophenol (ChKM) against E. faecalis [E. faecalis, and the long-lasting bactericidal effect observed in this study, a 2% concentration of chlorhexidine in solution and gel form was used.The literature review revealed that in accurate EAL measurements, usually 0.1, 0.2, 0.8% , 45 CHX faecalis . Thus, wfaecalis . CHX-G, faecalis . It was faecalis . Due to 2O2. The researchers did not find an influence of the irrigant solution on the performance of the device, but Root ZX worked least precisely in the presence of 5.25% NaOCl. In this study, in addition to 2% NaOCl, different solutions were tested than those in the Jenkis study, but both EALs achieved the poorest results in the presence of NaOCl. Kaufman et al. [Many studies assessed the accuracy of endometric measurements performed with different equipment. In Jung et al. , an in vn et al.  tested aThe apex locators Raypex 5 and ApexDal locate with the highest accuracy the anatomical foramen of the root containing chlorhexidine in the gel or in the solution form than in canal containing the sodium hypochlorite."}
+{"text": "Lysine crotonylation is a newly discovered post\u2010translational modification, which is structurally and functionally different from the widely studied lysine acetylation. Recent advances in the identification and quantification of lysine crotonylation by mass spectrometry have revealed that non\u2010histone proteins are frequently crotonylated, implicating it in many biological processes through the regulation of chromatin remodelling, metabolism, cell cycle and cellular organization. In this review, we summarize the writers, erasers and readers of lysine crotonylation, and their physiological functions, including gene transcription, acute kidney injury, spermatogenesis, depression, telomere maintenance, HIV latency and cancer process. These findings not only point to the new functions for lysine crotonylation, but also highlight the mechanisms by which crotonylation regulates various cellular processes. Acylation of lysine neutralizes the positive charge of the amino group and may change the conformation of proteins.Lysine crotonylation is a newly discovered histone PTM, which is specifically enriched at active gene promoters and potential enhancers in mammalian cell genomes.2Tan et al3Although initially identified on histones, lysine crotonylation has expanded to large number of non\u2010histone proteins. To characterize the global crotonylation proteome, the proteomic method based on sensitive immune\u2010affinity purification and high\u2010resolution liquid chromatography\u2010tandem (LC\u2010MS/MS) was applied to identify new crotonylated proteins and modification sites. The utilization of antibodies with high specificity to the crotonylated peptides involved in immunoprecipitation significantly improved the ability to enrich and identify crotonylated lysine residues. In recent years, several landmark studies have revealed dramatically improved number of crotonylated lysine residues and crotonylated proteins Table , and the4Lysine crotonylation is enzymatically regulated by the dynamic balance between crotonyltransferases and decrotonylases.Sabari et al5Histone deacetylases (HDACs) were also reported to have histone decrotonylase (HDCR) activity Figure ; Table 2HDAC3 was firstly reported to exhibit HDCR activity in vitro. By utilizing a collection of fluorogenic substrates, HDAC3\u2010NCoR1 was exhibited decrotonylase activity with a catalytic efficiency that is comparable to the deacetylase activity of other KDAC isoforms.6Double PHD finger (DPF), bromodomain and YEATS are three major classes of acetylation and non\u2010acetyl acylation readers.77.1Tan et al7.2Acute kidney injury (AKI) is a potentially lethal condition with no available therapy beyond replacement of renal function.7.3Genome\u2010wide removal of histones from chromatin and their replacement is a unique epigenetic event during spermatogenesis.7.4Histone crotonylation was found decreased in the medial prefrontal cortex of susceptible rodents exposed to chronic social defeat stress.7.5Telomere elongation with increasing passage depends on the mechanisms of both telomerase and recombination\u2010based alternative lengthening of telomeres.7.6Latent HIV reservoirs in the host are established early before viral infection.7.7Cancer is a life\u2010threatening malignancy that has become a global healthcare problem.8Lysine crotonylation is recently identified as a novel evolutionarily conserved histone PTM.The developments in high\u2010resolution LC\u2010MS/MS approaches have enabled the crotonylome\u2010wide mapping of lysine crotonylation.In general, one protein may occur simultaneously multiple PTMs.Over the past years, we have witnessed the tremendous advances in understanding of the mechanisms and cellular functions on protein crotonylation. However, it still exists many limitations in the published studies. Despite a growing understanding of crotonylation function in regulating diverse physiological function, it remains unclear how such biochemical changes occur and whether they play crucial roles in more disease progression. Besides, current crotonylation studies have mainly focused on a small number of histones, limiting the ability to clarify the relationship between crotonylation and human physiological processes. Moreover, the number of writers, erasers and readers of lysine crotonylation found so far is still very limited, more studies on the discovery and identification of writers, erasers and readers will be helpful for understanding the function of crotonylation.In the further research, we should focus on how human physiological processes and related diseases mechanistically regulated by crotonylation. Beside, more studies should search for understanding the role of crotonylation in non\u2010histones. We can also discovery more sites about lysine crotonylation, including the mechanisms by which they occur. Therefore, future studies are needed to uncover the effects of crotonylation on regulating protein functions and to interpret the underlying mechanisms behind protein crotonylation's ability to modulate diverse physiological and pathological processes.The authors declared no conflict of interest.JHW and HQZ provided direction and guidance throughout the preparation of this manuscript. JHW draw the graph. HYL and JC collected and prepared the related literature. All authors have read and approved the final manuscript."}
+{"text": "Acute appendicitis is the most common indication for pediatric abdominal emergency surgery. Determination of the severity of appendicitis on clinical grounds is challenging. Complicated appendicitis presenting with perforation, abscess or diffuse peritonitis is not uncommon. The question remains why and when acute appendicitis progresses to perforation. The aim of this study was to assess the impact of water permeability on the severity of appendicitis. We show that AQP1 expression and water permeability in appendicitis correlate with the stage of inflammation and systemic infection parameters, leading eventually to perforation of the appendix. AQP1 is also expressed within the ganglia of the enteric nervous system and ganglia count increases with inflammation. Severity of appendicitis can be correlated with water permeability measured by AQP1 protein expression and increase of ganglia count in a progressive manner. This introduces the question if regulation of water permeability can present novel curative or ameliorating therapeutic options. Written consent of the patient\u2019s legal guardian was obtained prior to the operation. The use of human tissue was in accordance with the ethics approval by the Ethics Commission North-West-Central Switzerland in Basel (EKNZ 2015-263). The stage of inflammation was assessed by routine scoring at the time of surgery by the surgeon as well as by routine H.E. staining-based pathological evaluation and categorized as acute, phlegmonous, or perforated.The appendix specimens for this study were prepared according to a modification of the \u201cSwiss role\u201d as described by Meier-Ruge and Bruder for histomorphological examination of the intestineThe samples were cut to a thickness of 7\u2009\u03bcm were cut on a cryotome and mounted onto microscope slides displaying the entire appendix role from base to tip . For the\u00ae Gold Antifade Mountant with DAPI . Slides were analyzed with an Olympus BX63 microscope using CellSens software. For the analysis, three consecutive pictures of each base and tip of the resected specimens were recorded at a magnification of 10\u00d7, and the ganglia of the enteric nervous system were counted by two independent examiners.For AQP1 and MAP2 immunofluorescence double staining, the polyclonal rabbit anti-AQP1 antibody  and the monoclonal mouse MAP2 antibody  both at a dilution of 1:200 were used following a standardized protocol. As secondary antibodies goat anti-mouse IgG1 AlexaFluor 488 and goat anti-rabbit AlexaFluor 647 were used. Negative controls missing the primary antibody were included in every staining cycle. Slides were mounted using ProLongp values of less than 0.05 was defined as significant. For analyzing the correlation of the diagnosis with AQP1 expression and infection parameters, the Spearman\u2019s-rank-correlation-coefficient was used.Data were analyzed using the SPSS version 23.0 , and a We examined samples of 40 patients, including 21 males and 19 females, with a mean age of 10\u2009years old ranging from 2.9 to 17.7\u2009years . Neithern\u2009=\u200914 (35%), as phlegmonous n\u2009=\u200919 (47.5%), and as perforated n\u2009=\u20095 (12.5%). A significantly positive correlation between the AQP1 expression and the stage of the disease was found  (p\u2009\u2264\u20090.001) (p\u2009=\u20090.028)  . Further\u2264\u20090.001) . Moreove=\u20090.028) . Taking Furthermore, prominent changes in the enteric nervous system were observed in the inflamed specimen regarding number and localization of ganglia. 23 inflamed appendices were further examined. Ganglia count showed that 65.2% of the specimens had a higher ganglia count in the inflamed region, namely the tip of the appendix, compared to the not inflamed region , Table 2In this study, we found a positive correlation of AQP1 expression in progressive appendicitis, and with this water permeability of the entire enteric wall, with the stage of inflammation. The increase of water permeability appears to eventually be associated with perforation of the appendix wall. Furthermore, AQP1 is expressed within the ganglia of the enteric nervous system of the appendix and inflammation appears to be associated with an increase of ganglia within the inflamed region. Moreover, a significant positive and continuous correlation between CRP and leucocyte count and AQP1 expression was found.,The expression of the water channel AQP1 has been shown to be essential in many different physiological processes throughout the whole body. The major function of APQ1 is maintaining and regulating water homeostasis. There is previous evidence that APQ1 plays a role in some aspects of the peripheral nervous systemIn tumor disease, for example, there is evidence that up-regulation of AQP1 expression is linked to hypoxiaWhile the function of several AQP isotypes in the central nervous system is being actively researched, their expression and physiological function in the digestive tract and especially in the enteric nervous system is still largely unknown and results are controversial. Gao et\u00a0al. first reported a strong AQP1 protein expression in the submucosal and myenteric nerve plexus in the human esophagus. AQP1 is co-localized with S-100, indicating glial cell-specific AQP1 expression, while neurons in the ganglia were not positive for AQP1The distribution of the AQP1 water channels in the enteric nervous system could suggest a functional involvement of AQP1 in regulating water homeostasis, intestinal motility, mucosal transport and enteric pain perceptionIn our pediatric patient appendices we found AQP1 positive ganglia most prominently but not exclusively in the myenteric plexus. Within the ganglia, AQP1-positive cells did not co-localize with MAP2-positive neurons, which is in agreement with earlier findings by Gao et\u00a0al.A further functional aspect of nerve function evolves around pain perception. AQP1 has been found to be an important factor in axonal growth and the regeneration of dorsal root ganglion neurons which transduce peripheral pain signals through small-diameter, non-myelinated C-fibers,There are several inhibitors of AQP1 that are currently being evaluated. Amongst the quaternary ammonium based compounds TEA is the lead component in blocking AQP1. It is more selective for AQP1 than for potassium channels and is highly effective regarding water permeationSeverity of appendicitis and of systemic inflammation parameters can be positively correlated with water permeability, as measured by AQP1 protein expression in a progressive manner. Inflammation correlates with an increased ganglia count in the enteric nervous system, in which AQP1 is present in MAP2-negative cells. Both, increase in AQP1 expression and in ganglia count, strengthen the theory of a progressive course of the disease. This might in the future lead to novel curative or ameliorating treatment options."}
+{"text": "Corrigendum:After publication, the authors noted that Figures 1 and 2 are not the final versions of the figures. Both figures have now been amended to include: correct representations of the upper and lower quartiles, outliers, minimum and maximum values. In particular, in the new boxplots, the whiskers extend up from the top of the box to the largest data element that is less than or equal to 1.5 times the interquartile range (IQR) and down from the bottom of the box to the smallest data element that is larger than 1.5 times the IQR. Values outside this range are considered to be outliers and are drawn as points. Additionally, each measure has a different pattern and each interval in the Y axis is labelled with extended lines. In Figure 2, the Y axis has been renamed Absolute Laterality Index."}
+{"text": "During alcoholic hepatitis (AH) monocytes traverse the vascular boundaries and massively invade the liver. In principle, tissue extravasation can be limited through shedding of CD18 integrins from leukocytes, including monocytes. The soluble (s) product sCD18 conceals adhesion receptors on the endothelium, which reduces monocyte extravasation. In AH, monocytes are dysfunctional, but whether this involves their self-generated anti-migration is unknown. Our aim was, therefore, to investigate monocyte CD18 dynamics in AH.We studied 50 AH patients and 20 healthy controls. We measured monocyte expression and conformational activation of CD18, plasma (P)-sCD18, stimulated in vitro CD18 shedding and P-sCD18 in a short-term chronic-binge mouse model.p\u2009<\u20090.01), but the sCD18 concentration per monocyte was reduced in vivo by 30% and in vitro by 120% (p\u2009<\u20090.01). Ethanol reduced the in vitro shedding of CD18 in the patients only. TNF\u03b1 increased sCD18 concentration per monocyte, but less so in the patients (p\u2009<\u20090.04). P-sCD18 per monocyte was inversely related to disease severity. In early alcoholic liver disease, P-sCD18 was decreased in the mouse model.AH-derived monocytes had a 30\u201360% higher expression of active CD18 receptors (The monocyte CD18 integrins are highly activated in AH and the single monocyte shedding of CD18 was decreased favoring tissue extravasation. Alcohol in itself and altered monocyte responsiveness to TNF\u03b1 may explain this lowered shedding.The contribution of this mechanism to the excessive monocyte liver infiltration in AH should be further explored as it may serve as a potential therapeutic target to limit liver inflammation. Alcoholic hepatitis is the most severe type of alcoholic liver disease associated with high morbidity and mortality. The lack of effective treatment options in these patients underlines the need for a better pathophysiological understanding of the disease.2. Circulating monocytes may be divided into subsets based on their expression pattern of CD14 and CD16, with the CD14+CD16+, intermediate monocytes, having an inflammatory phenotype and being the subset most predominantly recruited during inflammation5. The CD18 (\u03b22) family of heterodimeric integrins, comprising CD11a/CD18, CD11b/CD18, CD11c/CD18 and CD11d/CD18, are central for leukocyte extravasation from blood into tissues. CD18 integrins require conformational changes to bring the receptors to an active state with high affinity for ligands6. This can be detected on the cell surface by conformation-sensitive antibodies7.In alcoholic hepatitis, neutrophil liver infiltration is a hallmark, but also monocyte and macrophage numbers in the liver increase dramatically and contribute towards the florid inflammatory state8. In ICAM-1 knock-out mice, leukocyte recruitment to the liver is reduced in models of alcoholic liver injury and also antibodies targeting CD18 reduce liver injury in these models12. This supports a dominant role of this system in alcohol induced liver inflammation.CD11a/CD18 and CD11b/CD18 are expressed on monocytes and mediate adhesion to the endothelium via intercellular adhesion molecule (ICAM)-1 as part of the extravasation process. In alcoholic hepatitis, ICAM-1 is up-regulated in the liver along with the presence of a CD18 positive inflammatory infiltrate13. TNF\u03b1 can induce shedding of CD18 in vitro leading to the production of sCD18 complexes16. In the circulation of alcoholic hepatitis patients, levels of pro-inflammatory cytokines such as TNF\u03b1 are elevated17. The sCD18 complexes antagonize monocyte adhesion to ICAM-1 in a concentration-dependent manner and also promote macrophage efflux from sites of inflammation18. Shedding of the soluble complex may thus be a tissue inflammation-limiting mechanism. However, monocytes from patients with alcoholic hepatitis are dysfunctional in their production of reactive oxygen species and their killing of bacteria19. We hypothesized an up-regulation of active CD18 receptors on monocytes in patients with alcoholic hepatitis and that their shedding of CD18 is defective, which together could potentially promote and aggravate hepatic inflammation in AH.Recently, soluble (s) complexes of the CD18 integrin receptor have been discovered and their plasma concentration shown to change as part of inflammatory diseasesFollowing this hypothesis, we investigated the expression of activation-dependent epitopes in monocyte CD18 in patients with alcoholic hepatitis and measured the levels of sCD18. The findings were related to clinical characteristics. To support a mechanistic understanding, we compared the influence of ethanol, TNF\u03b1 and lipopolysaccaride. Our findings now highlight previously unanticipated aspects of CD18 integrin activation and shedding in inflammatory disease, namely the dysregulation of these molecular mechanisms in alcoholic hepatitis.In this prospective cohort study, we consecutively recruited 50 patients with alcoholic hepatitis from four hospitals in central Denmark. We included 20 age-matched healthy persons with no previous history of liver disease as controls. The patients were followed for 30 days with samples obtained at the day of diagnosis and day 14 and day 30 after diagnosis. Between diagnosis and day 14 four patients died and five were lost to follow-up or denied ongoing participation. Between days 14 and 30, the numbers were two and eight, respectively. Patients were stratified using the Glasgow Alcoholic Hepatitis Score (GAHS). Patients with GAHS\u2009\u2265\u20099 on admission were treated with 400\u2009mg pentoxifyllin t.i.d. in accordance with Danish national guidelines at time of recruitment in addition to nutritional therapy. Patients with GAHS\u2009<\u20099 were treated solely with nutritional support and standard medical care. The pentoxifyllin-treated patients do not differ significantly from the un-treated patients with regards to clinical and biochemical characteristics. No patient received prednisolone. Patient characteristics are presented in Table\u00a020. The diagnosis was based on a set of clinical and biochemical variables; a history of excessive alcohol consumption with less than 3 weeks of abstinence leading up to admission; acute jaundice (developed within the previous 2 weeks with serum bilirubin >80\u2009\u03bcmol). Patients were excluded if they had biliary stones, other liver diseases, gastrointestinal bleeding within the past 3 months, signs of an infectious focus , hepatocellular carcinoma or other malignancy or had received immune-modulating therapy within the 8 weeks leading up to admission. When in diagnostical doubt biopsy was performed (n\u2009=\u200910) and none of these refuted the diagnosis.Patients between the age of 18 and 75 years were included when diagnosed with alcoholic hepatitis in similar manner to the criteria later formulated in the established guidelinesThe study was conducted in accordance with the Helsinki Declaration and approved by the Central Denmark Region ethical committee (j.no. 20100281). Informed, written consent was obtained prior to study inclusion. The study was registered at ClinicalTrials.gov (NCT00992888). Clinical and biochemical data were collected on all study days. Disease severity was scored by GAHS, Modified Maddrey Discriminant function, Model of Endstage Liver disease (MELD) and Child Pugh Score.Venous sampling was performed in EDTA vacuum tubes. Peripheral blood mononuclear cells (PBMCs) were isolated within 2\u2009h of sampling using Ficoll\u2013Hyperpaque centrifugation  and stored at \u2212140\u2009\u00b0C. Plasma was obtained from centrifugation of whole blood 3000\u2009g, 10\u2009min at 4\u2009\u00b0C and stored at \u201380\u2009\u00b0C for later side-by-side analyses.6 cells/setup) were blocked for unspecific binding in phosphate-buffered saline with 1 % (w/v) bovine serum albumin  together with 10\u2009\u03bcg/ml murine IgG . PBMCs were stained with either 10\u2009\u00b5g/ml biotinylated IgG1 antibody to CD18 (KIM127) or 10\u2009\u00b5g/ml biotinylated IgG1 isotype control in combination with secondary antibody streptavidin-FITC (Dako). The KIM-127 antibody to CD18 is an established marker for the ligand-binding active conformation of CD18 integrins7. The following antibodies were used for leukocyte subset stratification of the PBMCs: anti-CD18 BV421 (BD Horizon), anti-CD14 V500 (BD Horizon), anti-CD16 PE/Cy7 (Biolegend). Anti-CD56 APC-eFlour780 (eBioscience) and LiveDead staining  were used for CD56\u2212dump  and viability estimation. All samples were analyzed immediately after staining using BD LSRFortessa Cell Analyzer. Data analyses were performed in FlowJo . Monocytes were divided into three subsets based on their expression of CD14 and CD16; classical (CD14+CD16\u2212), intermediate (CD14+CD16+) and non-classical (CD14lo CD16+) (supplementary figure\u00a0PBMCs (1\u2009\u00d7\u20091021. In- house ELISA assays were utilized to measure sCD163 in human plasma as previously described22. Plasma lipopolysaccharide (LPS) levels were measured using the Limulus amebocyte lysate assay . When correcting for the number of monocytes, total leukocytes and neutrophils, we appointed the healthy controls a value of 0.45\u2009\u00d7\u2009109, 6.75\u2009\u00d7\u2009109 and 4.5\u2009\u00d7\u2009109/l, respectively, i.e. the mean of the reference intervals. In patients with alcoholic hepatitis we used leukocyte differential counts collected on all study days.In house TRIFMA assays where utilized to measure sCD18 in human plasma and mouse plasma as previously publishedin vitro culture experiments with PBMCs, cells (4\u2009\u00d7\u2009105 cells/setup) were cultured in RPMI medium supplemented with 10% (v/v) heat-inactivated FCS , 1 % (v/v) penicillin\u2013streptomycin\u2013glutamine . PBMCs were stimulated with either 10\u2009ng/ml TNF\u03b1 , 10\u2009ng/ml LPS , 25\u2009mmol/l ethanol (Sigma-Aldrich) or left untreated for 48\u2009h at 37\u2009\u00b0C in a humidified incubator with 5% CO2. After incubation, supernatants were stored at \u221280\u2009\u00b0C.For 23. Histological haematoxylin-eosin and masson trichrome stains were performed on paraffin embedded tissue sections and markers of liver injury  were\u00a0measured in plasma to verify the development of liver injury. Plasma was stored for later sCD18 analyses. The study was approved by the national animal ethics committee (protocol no. 2013-15-2934-00872/BES) and conducted in accordance with local and international animal welfare guidelines.We employed the National Institute on Alcohol Abuse and Alcoholism (NIAAA)\u00a0chronic-binge model of ethanol mediated injuryp-value\u2009<\u20090.05 was considered statistically significant.We utilized non-parametric testing of our experimental data. Analyses of differences between groups, alcoholic hepatitis and healthy controls, were carried out by Wilcoxon rank-sum test and Wilcoxon signed-rank test was used for paired data. Patient characteristics, which were normally distributed, were compared using parametric tests. We used Spearmans rho for correlation analyses. Results are presented as median (interquartile range) unless specified otherwise, and a two-tailed In all three monocyte subsets examined, we observed an increased expression of the ligand-binding active conformation of CD18 in alcoholic hepatitis in comparison with the expression in healthy controls  (Table\u00a0p\u2009<\u20090.01). The level stayed low throughout the 30 days\u2019 follow-up. Other leukocyte subsets especially neutrophils may also contribute to the plasma pool of sCD18. As both the neutrophil count and the total leukocyte count was elevated in the\u00a0alcoholic hepatitis\u00a0patients, we assessed whether taking this into account changed the pattern. Indeed, both P-sCD18 per neutrophil and P-sCD18 per leukocyte were reduced in alcoholic hepatitis compared with healthy controls (p\u2009<\u20090.05) (supplementary figure\u00a0p\u2009<\u20090.05). The corrected P-sCD18 per monocyte values did not correlate with monocyte CD18 expression.We assessed whether the elevated surface expression of CD18 was accompanied by a higher plasma (P)-sCD18 in alcoholic hepatitis. Because the patients with alcoholic hepatitis had elevated numbers of monocytes in their peripheral blood . The concentration of sCD18 did not change significantly during the 30 days follow-up period and continued to be higher than levels in healthy controls (p\u2009<\u20090.05). PBMCs from patients with alcoholic hepatitis likewise spontaneously produced more sCD18 than cultures of healthy PBMCs . There were no correlations between the monocyte surface expression of CD18 and P-sCD18 or CD18 shedding in vitro. However, the frequency of circulating intermediate monocytes tended to correlate with P-sCD18  and with spontaneous in vitro sCD18 concentration .The total concentration of P-sCD18 was 30% higher in patients with alcoholic hepatitis than in healthy controls  (data not shown). The correlation was present also at day 14 and day 30 after diagnosis.We observed a positive correlation between the plasma marker of macrophage activation; sCD163, and sCD18 in patients with alcoholic hepatitis (p\u2009<\u20090.05). TNF\u03b1 and LPS was able to increase the shedding of CD18 per monocyte and the total sCD18 level both in alcoholic hepatitis and in healthy controls . However, the TNF\u03b1-mediated enhancement of CD18 shedding per monocytes was lower in alcoholic hepatitis patients than in healthy controls (p\u2009<\u20090.05). LPS increased CD18 shedding per monocyte equally in alcoholic hepatitis patients and in healthy controls. P-LPS levels correlated with P-sCD18 levels in patients with alcoholic hepatitis .We examined which mediators could be responsible for moderating the shedding of CD18 in alcoholic hepatitis. Interestingly, ethanol only had an impact on CD18 shedding in alcoholic hepatitis patients. In these patients, it reduced CD18 shedding per single monocyte as well as the total sCD18 level . P-sCD18 correlated with Child Pugh Score  and tended to correlate with GAHS  and MELD . P-sCD18 also correlated with ascites  and pp . The spontaneous shedding of CD18 correlated positively with GAHS  and tended to also correlate with Child Pugh Score . Monocyte expression of CD18 was not related to disease scores at diagnosis. When comparing the change in P-sCD18 per monocyte with the change in disease scores through the 30 days follow-up, we observed an increase in P-sCD18 per monocyte with decreasing GAHS  and a similar tendency for Child Pugh Score  and MELD . P-sCD18 did not correlate with changes in disease scores.We looked at correlations between sCD18 and clinical measures at diagnosis. The correlations between P-sCD18 per monocyte and disease scores were inverse (GAHS p\u2009=\u20090.65). We also verified that performing our analyses on the pentoxifyllin treated and the un-treated patients separately did not change our results on P-sCD18, monocyte expression of CD18 or our in vitro findings.As TNF\u03b1 induce the shedding of CD18, pentoxifyllin treatment may theoretically lower P-sCD18 by reducing shedding, and this could affect our results. We therefore tested whether P-sCD18 at diagnosis was different comparing pentoxifyllin treated to un-treated patients and found no difference (1930\u2009\u00b1\u20091460 vs. 1907\u2009\u00b1\u2009652, To study the dynamics of sCD18 in the early phase of ethanol-induced liver injury, we employed the NIAAA chronic-binge mouse model. We verified that the mice with this model developed ethanol-induced liver injury in our hands by measuring AST and ALT levels both of which were elevated in ethanol fed mice Fig.\u00a0. Liver iMonocytes and macrophages have long been granted a central role in the pathogenesis of alcoholic hepatitis. Yet, our understanding of their involvement is still inadequate and especially human data is scarce. In this study, we report activation of the integrin CD18 on monocytes from patients with alcoholic hepatitis, but diminished shedding per monocyte of the integrins despite elevated P-sCD18. This may be related to direct effects of alcohol or an attenuated anti-inflammatory response to pro-inflammatory mediators.25. To strengthen the functional understanding of CD18 expression, we now selectively measured ligand-binding active and total CD18\u00a0separately7. To our knowledge, the strong up-regulation of CD18 activation epitopes, which we here demonstrate, has not previously been reported in human inflammatory disease. In this way, our study adds to a currently limited number of studies relating CD18 integrin conformational change directly to inflammatory disease. This important finding reflects that monocytes in alcoholic hepatitis are primed for binding to adhesion molecules expressed by the endothelium, i.e., ICAM-1.Previous studies have measured the cellular expression of total CD18, i.e., CD18 integrins in the active and inactive conformations. In patients with Child Pugh C liver cirrhosis and acute liver failure, monocyte expression of CD11b is elevated14. In very early rheumatoid arthritis and animal models of arthritis induction, the systemic sCD18 levels shows a biphasic temporal pattern with an initial increase in serum sCD18 levels at arthritis induction followed by a secondary decrease. Further, disease remission in early rheumatoid arthritis is associated with increased sCD18 levels26. Finally, very high and very low levels of sCD18 levels were recently associated with fatal outcomes in critically ill sepsis patients27. Therefore, changes in sCD18 levels seems to be part of the inflammatory response in general. However, the changes seen in AH indicate that sCD18 levels are primarily altered in diseases with involvement of monocytes.Soluble CD18 decreases leukocyte adhesion to the endothelium and thereby limits tissue inflammation. The total P-sCD18 pool seems to be a result of shedding from leukocytes and depletion by ligand binding. In chronic rheumatoid arthritis and spondyloarthritis, the plasma levels of sCD18 are decreased and inversely associated with disease activity15. In patients with alcoholic hepatitis the shedding of CD18 per leukocyte is decreased despite elevated total P-sCD18. This suggests that the increased monocyte and neutrophil count in alcoholic hepatitis is a likely explanation for the high P-sCD18. Especially, there was an association between high numbers of intermediate monocytes and P-sCD18 levels and in vitro CD18 shedding suggesting that this cell subset is also essential for CD18 shedding in alcoholic hepatitis. The lowered shedding per monocyte, in combination with monocyte CD18 activation, tilts the balance towards adhesion to the endothelium as the first step in extravasation. Consequently, we hypothesize that this may contribute to increased monocyte migration and partly explain the increased number of monocytes in the liver during alcoholic hepatitis (Fig.\u00a028. Unfortunately, we do not have liver specimens from these patients to verify that this indeed translates into a large, CD18-positive liver infiltrate. However, the population of intermediate monocytes, which is the predominant sCD18 shedder amongst PBMCs, is also the monocyte population, which increases the most in the liver during inflammation29. This further supports a dominant role of the CD18 system in recruitment of monocytes to the liver. The decreased total plasma sCD18 level in our mouse model may reflect that shedding of CD18 indeed is low in the early stages of alcoholic liver disease contributing to the initiation of liver inflammation or simply be explained by the fact that it is a model without monocytosis or marked monocyte infiltration in the liver12.Previous studies have shown shedding of CD18 from especially intermediate monocytes and neutrophils31. The lowered P-sCD18 observed in our mouse model, where the animals are gavaged with ethanol only 9\u2009h prior to being sacrificed lends support to the ethanol depressing mechanism also being present in vivo. Although most alcoholic hepatitis patients were abstinent from alcohol in the weeks leading up to disease presentation in hospital, monocyte function is only partially restored in subjects with alcohol use disorders after 2 weeks of abstinence. Effects of alcohol consumption are therefore likely still impacting immune functions in our study patients30. In response to the pro-inflammatory mediator TNF\u03b1, alcoholic hepatitis patients fail to raise sCD18 equivalent to that of healthy subjects. The lack of such anti-inflammatory response is again in accordance\u00a0 with several studies showing a pro-inflammatory skewing of monocytes in alcoholic liver disease, which aggravates the effects of alcohol on this system34. This tolerance to TNF\u03b1 may also explain why we do not observe any differences between the pentoxifyllin-treated and the un-treated patients in the CD18 system, and why excluding either group from our analyses does not change our results. However, we cannot in this design exclude an impact of pentoxifyllin on CD18 shedding as the two groups differ also in disease severities.The decreased single monocyte shedding of CD18 may arise from direct effects of alcohol, as we observe in vitro the ability of ethanol to lower CD18 shedding. Such dampening effects of ethanol on immune functions have been demonstrated in several studies of other monocyte functions26. The fact that the uncorrected P-sCD18 levels increase with increasing disease severity most likely reflect that leukocytosis increase with increasing disease severity.The inverse relation between the P-sCD18 and in vitro CD18 shedding per monocyte and disease severity scores are in line with findings from arthritis patients and further supports decreased CD18 shedding being detrimental in alcoholic hepatitis35. Today, antibodies that blocks T cell integrin receptor binding to mucosal adhesion molecules are used as therapy in inflammatory bowel disease37. Thus, this study brings forward data on a mechanism that could be utilized as a therapeutic target in alcoholic hepatitis and therefore deserves further investigations into its mechanistic.Our study is obviously limited by its sample size. To further elucidate whether our findings reflects alcoholic liver disease per se or is predominantly to be attributed to the inflammation of alcoholic hepatitis, additional control groups are required. Nevertheless, this study provides novel insights into the role of monocyte CD18 integrin expression in alcoholic hepatitis. Twenty years ago, antibodies antagonizing the CD11b/CD18 complex were demonstrated to strongly reduce neutrophil adhesion to inflamed endothelium in vitroIn conclusion, we provide data to suggest that the CD18 receptor is involved in recruiting monocytes to the liver in alcoholic hepatitis and that regulation of monocyte adhesiveness such as production of the anti-migration complex sCD18, is dysfunctional. As this relates to disease severity, strategies to influence this system may be an option to reduce morbidity and mortality in alcoholic hepatitis and should be explored.Alcoholic hepatitis holds high mortality and no targeted treatments existMonocyte liver infiltration is dominant but mechanistically poorly elucidatedMonocytes are primed for extravasation with marked CD18 integrin activation and decreased sheddingAlcohol and altered responsiveness to TNF\u03b1 may be responsibleThese changes in CD18 may contribute to monocyte liver recruitment in AHConsequently the CD18 system may be a therapeutic target to limit liver inflammationSupplementary Figures"}
+{"text": "Myopia is one of most common eye diseases in the world and affects 1 in 4 Americans. It is a complex disease caused by both environmental and genetics effects; the genetics effects are still not well understood. In this study, we performed genetic linkage analyses on Ashkenazi Jewish families with a strong familial history of myopia to elucidate any potential causal genes.Sixty-four extended Ashkenazi Jewish families were previously collected from New Jersey. Genotypes from the Illumina ExomePlus array were merged with prior microsatellite linkage data from these families. Additional custom markers were added for candidate regions reported in literature for myopia or refractive error. Myopia was defined as mean spherical equivalent (MSE) of -1D or worse and parametric two-point linkage analyses (using TwoPointLods) and multi-point linkage analyses  were performed as well as collapsed haplotype pattern (CHP) analysis in SEQLinkage and association analyses performed with FBAT and rv-TDT.MYP14) but not myopia. Another genome-wide significant locus was found on 8q24.22 with a maximum two-point LOD score of 3.75. CHP analysis also detected the signal on 1p36, localized to the LINC00339 gene with a maximum HLOD of 3.47, as well as genome-wide significant signals on 7q36.1 and 11p15, which overlaps with the MYP7 locus.Strongest evidence of linkage was on 1p36(two-point LOD\u2009=\u20094.47) a region previously linked to refractive error  contains supplementary material, which is available to authorized users. Myopia is a common, complex trait with both genetic and environmental factors influencing risk , 2. As rGenotype data were available for 527 Ashkenazi Jewish individuals (64 extended families) selected due to their strong information content for linkage studies of myopia from among the 105 Ashkenazi Jewish families included in the Penn Family Study. Details of the recruitment of these families has been previously described . This stCIDR standard quality control procedures were applied to the entire dataset. Blind duplicates and HapMap controls were distributed across plates for concordance checking. Cases and controls were evenly distributed across plates, but family members were kept on the same plate. Samples with suspected mixtures or unusual X and Y patterns or gender mismatch identified and dropped before release. SNP clustering was performed on all SNPs in project and SNP genotypes with genotype quality (GC) score less than 0.15 recoded as missing genotypes. Autosomal SNPs with less than 85% call rate, cluster separation of less than 0.3 and heterozygote rate greater than 80% were dropped. Subsets of SNPs manually reviewed are detailed in Supplementary Methods and details of SNPs not released due to technical failure can be found in Additional\u00a0file\u00a0After receiving data from CIDR, additional quality control measures were applied. Genotype and phenotype data were combined and an additional 85 ungenotyped individuals were added to the pedigrees to complete family relationships. Detailed Mendelian error checking was performed in Sib-pair , sex disWe did not filter SNPs based on Hardy-Weinberg equilibrium (HWE), instead SNPs that were not in HWE were flagged. All significant and suggestive SNPs reported here were in HWE. We did initially find a single SNP that was out of HWE at 16q22.1 that had a highly significant for two-point logarithm of the odds (LOD) score of 7.76. This SNP had an excess of heterozygotes (approximately 70%) and a decrease in both homozygotes. We later found that this SNP was within a known copy number variant (CNV), which is responsible for the heterozygote inflation. This SNP was removed from all analyses and is not reported as significant.After cleaning, we merged the exome variant data with older microsatellite (sequence tagged site (STS)) data from previous linkage studies in the same population , 14, 28.A full description of the phenotyping has been previously described  but brieTwo-point linkage analyses were performed using the program TwoPointLods . This is2 value greater than 0.2, one of the SNPs in the pair was removed. Because of their high information level, no STS markers were removed in pruning analyses. Thus, after cleaning we were left with 3764 markers.It is well-known that including markers that are in strong linkage disequilibrium (LD) in multi-point linkage analyses that assume linkage equilibrium can cause inflation of false positive rates. Previous analyses have allowed us to determine that even multi-point linkage analyses that attempt to adjust for intermarker LD are often inaccurate for very dense marker maps, so the data were pruned. All SNPs were condensed into 1\u2009cM bins. The SNP with the highest minor allele frequency (MAF) in the bin was chosen to then represent the bin in the multi-point analyses. We performed further LD analysis on the binned SNPs in Haploview . For anyMulti-point linkage analyses were performed using SimWalk2 \u201334, withA new approach to deal with intermarker LD without pruning is the collapsed haplotype pattern (CHP) method by Wang et al.  and implWe also performed two types of association analyses. The family-based association test FBAT , 38 was Variants were annotated using Annovar to get the most up to date predictions of function. Predicted microRNA targets were identified using miRanda  and scorFour samples were not released due to poor performance on the array. After quality control, there were 67,451 polymorphic variants and the mean call rate was 99%. Additional family members without DNA for genotyping were included to define family relationships. Demographic and clinical characteristics can be found in Table\u00a0A summary of the four genome-wide significant chromosomal regions identified by either the two-point or CHP linkage analyses can be found in Table\u00a0Two-point parametric linkage analysis was performed genome-wide Fig.\u00a0 and compLINC00339 , which does not overlap with the published chromosome 7p15 myopia locus MYP17, which is on the opposite chromosomal arm. The third significant linkage was to the NCR3LG1 gene on chromosome 11p15.1 . This latter linkage is within the same region as the suggestive 11p linkage observed in the two-point linkage analysis above. NCR3LG1 is about 14\u2009kb from PAX6. PAX6 itself did not have any HLOD scores above 0.6 in the CHP analysis . Suggestive CHP linkage was found at 11p14.1  and 11p15.2 .  linkage analysis of these data using SEQLinkage and Merlin identified three genome-wide significant genes. The first significantly linked gene was 339 Fig.\u00a0, in the The association analyses using FBAT and rv-TDT found no genome-wide significant signals.Here we report significant linkage with myopia at 1p36.12, 8q24, 7q36.1, and 11p15.1. The loci on chromosomes 1 and 11 are replications, while the loci on chromosomes 8 and 7 are novel. All of these linkage signals are cumulative effects across families. However, the families do not share identical linked haplotypes; if they did we should have seen significant association within these regions as well. This suggests that several different causal variants may exist across the linked families, with these causal variants possibly all being in the same gene .LINC00339, a long non-coding RNA gene known to be associated with endometriosis [CDC42, a GTPase directly downstream of LINC00339, contained three significant variants in the two-point analysis, but has not previously been implicated in myopia either. However, one of its activation targets, LAMA1, has been found to cause myopia in the presence of other phenotypes [FRAP1 and PDGFRA (both located on 1p36.2) have both been found to be associated with corneal curvature and eye size in Asian and European populations [Our strongest signals occurred in the 1p36.12 region, identified as significant in both the two-point and CHP analyses. Linkage of refractive error (but not myopia) to markers on 1q36  has beenetriosis \u201348 but wenotypes . Slightlulations , 51. NeiMYP17 [SSPO). The subcommissural organ (SCO) is one of the circumventricular organs, a set of brain structures that form the linkage between the central nervous system and the peripheral blood stream. It is one of the first differentiated brain structures to form and its function is largely unknown. SCO-spondin is a large glycoprotein from the thrombospondin. This protein is highly expressed during CNS development and is believed to be important in cellular adhesion, axonal pathfinding and homeostasis. The Pax6 mutation which causes a small eye and is known as Sey also causes abnormalities in the SCO [Sey/Sey mice die at birth with numerous defects including an inability to properly form the SCO and Sey/+ mice demonstrate a mosaic of SCO cells, some of which are not expressing the Reissner\u2019s fiber, a fibrous aggregation of the secreted molecules of the SCO and is formed by secretion of SCO-spondin, and other abnormalities related to normal development of this important brain region. This admittedly tenuous link to PAX6 is an intriguing addition to the complex story of myopia and its relationship to early brain development.We report the discovery of a novel locus linked to myopia on 7q36.1, distinct from another known chromosome 7 locus, MYP17 \u201354, loca the SCO . HomozygWNT1-inducible pathway protein 1 (WISP1). This gene is a member of the WNT1-inducible signaling pathway family of genes, all of which belong to the connective tissue growth factor family. It is a downstream regulator in the Wnt/Frizzled signaling pathway, is associated with cell survival by attenuating p53-mediated apoptosis in response to DNA damage through activation of AKT kinase and is widely expressed in many tissues. No prior eye disease associations currently exist for this gene, but the Wnt pathway is important in development of the eye. Significant linkage was only reported on a single variant in the two-point analysis, and this region was not significantly linked to myopia in either the multipoint or the CHP analyses. Thus it is possible that this is a false-positive two-point signal. However, linkage for this variant is driven by three families with LOD scores of 1.5, 1.0, and 0.76 and in each of those families the variant is part of a small linked haplotype across the 8q24 region, making it less likely that the signal is a false positive  cell cytotoxicity receptor ligand and when it interacts with NKp30 results in NK activation and cell death. It interacts exclusively with NCR3 but not with other NK cell activating receptors. It has only been reported as expressed in tumor tissues. None of these facts make NCR3LG1 a particularly attractive candidate for myopia development however, and there are many other candidate genes in the region that, based on biological function, may be more likely to be causal genes  or identical by descent (IBD). Linkage by contrast tracks the co-segregation of haplotypes and the trait within a pedigree, but is not concerned with whether those segregating haplotypes contain alleles IBS across different families. Using a founder population such as the Orthodox Ashkenazi Jewish families in this analysis increases the likelihood that there may be shared risk alleles across linked families, but this is not guaranteed. Therefore, this result, combined with the annotation of the significantly linked variants/genes discussed above, suggests that even using the exome-targeted array, we have likely not genotyped the actual causal allele(s) and instead are only able to detect its presence via linkage to specific haplotypes in each linked family.WISP1 on 8q and SSPO on 7q, though with our limited exome-based array we were unable to resolve the signal further than the chromosomal regions. We plan to perform either targeted sequencing on the regions of interest or whole genome sequencing (WGS) on the most highly linked families to unequivocally identify the causal variants that account for the linkages to myopia detected here.This study found significant linkage to myopia in Ashkenazi Jewish families at four chromosomal loci - 1p36.12, 8q24, 7q36.1, and 11p15.1. The signals at 7q and 8q were novel, while the signals at 1p and 11p are replications of previously identified signals, albeit ones where the causal genes have yet to be identified. We were able to identify several potential causal genes, including Additional file 1:Table S1. SNPs not released due to technical failure. A detailed list of the number of SNPs that failed at different points of the technical quality control. (XLSX 10 kb)Additional file 2:Table S2. Two-point, Multipoint and CHP LODs and HLODs for Genome-Wide Suggestively Linked Regions Additional file 4:Table S4. Annotation of Suggestive and Significantly Linked Variants Sorted by Chromosome and then by HLOD - Two Point Linkage Results. A list of all variants that were either significantly or suggestively linked, along with annotations provided by annovar. (XLSX 43 kb)Additional file 5:Table S5. Annotation of Suggestive and Significantly Linked Variants in the 1p36 region sorted by base pair start location - Two Point Linkage Results. A list of all variants on 1p36 that were either significantly or suggestively linked, along with annotations provided by annovar. (XLSX 15 kb)Additional file 6:Table S6. Annotation of Suggestive and Significantly Linked Variants on chromosome 8 sorted by base pair start location - Two Point Linkage Results. A list of all variants on chromosome 8 that were either significantly or suggestively linked, along with annotations provided by annovar. (XLSX 11 kb)Additional file 7:Table S7. Annotation of Suggestively Linked Variants on chromosome 11 sorted by base pair start location - Two Point Linkage Results. A list of all variants on chromosome 11 that were either significantly or suggestively linked, along with annotations provided by annovar. (XLSX 11 kb)"}
+{"text": "The Polo kinase is an essential regulator of cell division. Its ability to regulate multiple events at distinct subcellular locations and times during mitosis is remarkable. In the last few years, a much clearer mechanistic understanding of the functions and regulation of Polo in cell division has emerged. In this regard, the importance of coupling changes in activity with changes in localization is striking, both for Polo itself and for its upstream regulators. This review brings together several new pieces of the puzzle that are gradually revealing how Polo is regulated, in space and time, to enable its functions in the early stages of mitosis in animal cells. As a result, a unified view of how mitotic entry is spatio-temporally regulated is emerging. These Ser/Thr kinases are defined by the presence of an N-terminal kinase domain (KD) and an additional C-terminal domain, termed the Polo-box domain (PBD), which engages in protein interactions [Discovered in nactive) ,2. Drosoractions ,4. In alractions . During ractions \u20139.To regulate these different functions, Polo must be activated and dynamically recruited to distinct subcellular structures, in space and time . The loc2.Xenopus extracts, Plx1 (Polo) was isolated as a kinase required to phosphorylate and to activate Cdc25C, the phosphatase required for activation of Cyclin B\u2013Cdk1 [polo mutants isolated in Drosophila suggested that Polo was needed for cells to assemble functional mitotic centrosomes and a bipolar spindle, but mutant neuroblasts did enter mitosis [The Polo kinase has long been known to promote mitotic entry in various systems. In n B\u2013Cdk1 ,16. As Cn B\u2013Cdk1 . However mitosis . However mitosis \u201322. In a mitosis . Thus, tRecently, two studies revisited the question and demonstrated that complete inhibition of Plk1 can prevent mitotic entry for hours in a vast majority of hTERT-RPE1, HeLa or RKO cells, even without prior activation of the DNA damage checkpoint ,25. Inte3.C. elegans [Drosophila [C. elegans embryos and Drosophila cells in culture [et al. [The importance of Polo activity for mitotic entry is reflected by its activation and relocalization during this transition. During mitotic entry, Polo kinase is activated by phosphorylation of an evolutionarily conserved residue located in the activation loop (Thr210 in human Plk1) ,30. Phos elegans ,34, and osophila . Aurora  culture \u201338. More [et al.  showed tet al. [Drosophila, Kachaner et al. showed that phosphorylation of Polo is required for the exposure of the NLS and nuclear import of Polo  and trigger its relocalization from the nucleus to the cytoplasm, where Cdc25 activates Cdk1 [Drosophila somatic cells. In vertebrates, three genes encode Cdc25 paralogues , and they have all been shown to respond to Plk1 activity [Xenopus extracts established that Plx1 phosphorylates and activates Cdc25C, and that this event is required for activation of Cyclin B\u2013Cdk1 [Drosophila Cdc25 which is excluded from nucleus following its activation by Polo, human Cdc25C and Cdc25B have been reported to be imported into the nucleus upon phosphorylation by Plk1 [C. elegans, CDC-25.1, which is essential for embryogenesis, is enriched in the nucleus in early embryos, where it is activated by PLK-1 during the early embryonic divisions [Once in the nucleus, what does Polo do? In tes Cdk1 . String activity . Pioneern B\u2013Cdk1 ,16. Recen B\u2013Cdk1 . It is n by Plk1 ,48. That by Plk1 \u201353. In Civisions . Thus, tIn the nucleus, Polo could also contribute to mitotic entry by additional mechanisms. Wee1 is a Cdk1-inhibitory kinase that resides predominantly in the nucleus . In humaDrosophila and in human cells, Gwl is nuclear in interphase, but in prophase it re-localizes to the cytoplasm where PP2A-B55 is enriched. This localization dynamics is important for Gwl function [Drosophila, phosphorylation of Gwl by both Polo and CDK activities promotes Gwl relocalization to the cytoplasm just before NEB [Xenopus [Drosophila suggest that Polo can function against Gwl and with PP2A-B55 in regulating mitosis [Polo has been linked to Greatwall (Gwl), a kinase that promotes mitotic entry by antagonizing protein phosphatase 2A in complex with its B55 regulatory subunit (PP2A-B55). As the cell enters mitosis, Gwl becomes active and phosphorylates endosulfine proteins (ENSA and Arpp19 in humans). Once phosphorylated, endosulfines selectively inhibit PP2A-B55, a phosphatase that efficiently targets several mitotic phosphosites . In Drosfunction ,62. In Dfore NEB . However[Xenopus . While t mitosis ,65. The C. elegans revealed that PLK-1 is required for NEB in both meiosis and mitosis [C. elegans and in human cells . In worms and flies, this localization of Polo is striking ,66. Work mitosis ,68. Recean cells , point 8an cells , point 9ng sites . PLK-1 rng sites ,70. Plk1In summary, it is now clear that Polo relies on built-in mechanisms by which its activation triggers changes in subcellular localization that are required for Polo to coordinate multiple events in space and time as the cell enters mitosis.5.Drosophila in a genetic screen for regulators of asymmetric cell division [C. elegans [C. elegans embryos and in human cells [Now that we understand that Polo kinase activation by phosphorylation also induces its localization to the nucleus, where Polo promotes mitotic entry, it becomes even more crucial to understand the mechanism leading to Polo activation. In human cells, Plk1 phosphorylation on its activation loop requires Bora, which was originally discovered as a cofactor of Aurora A in division . Bora acdivision ,32. Impo elegans ,34,72,73an cells ,72,73. Tan cells . Thus, Pin vitro [Which is the physiological Cyclin\u2013CDK complex phosphorylating Bora? Bora contains a conserved cyclin-binding site (Cy) and both Cyclin B\u2013Cdk1 and Cyclin A2\u2013Cdk2 can phosphorylate Bora in vitro ,75. In hin vitro , it is uin vitro ,78. Cyclin vitro ,80. By ain vitro ,75. ThesXenopus extracts confirmed this hypothesis and revealed that Cyclin A\u2013Cdk1 phosphorylates Bora to trigger Plk1 activation and mitotic entry [Xenopus Bora produced in Escherichia coli but not mutated versions of Bora defective in Cyclin binding (mutated on the Cy motif) or lacking the three evolutionarily conserved CDK-dependent SP phosphorylation sites. However, exogenous Bora failed to rescue mitotic entry in extracts also depleted of Cyclin A. By contrast, Bora thiophosphorylated in vitro, and thus resistant to endogenous phosphatases, readily supported mitotic entry in extracts depleted of Bora and Cyclin A. These observations unequivocally demonstrate that Bora is the necessary and sufficient Cyclin A target required for Plx1 activation and mitotic entry. Once Cyclin B\u2013Cdk1 is activated in mitosis, it likely sustains Polo activation by phosphorylating Bora as Cyclin B co-immunoprecipitates with Bora in mitotic cells [Recent work in ic entry . In thisic entry ,82. Striic cells . KnowledIn addition to Bora, the multi-functional adaptor protein WAC contributes to Plk1 activation by Aurora A and to timely mitotic entry in human cells . In cont6.Xenopus extracts [\u03b1 is mainly in the cytoplasm [During mitosis in human cells, protein phosphatase 1 (PP1) dephosphorylates Thr210 of Plk1 and thereby antagonizes its activity . This evextracts . As PP2Aytoplasm , it may 7.Although still incomplete, the new knowledge of the complex mechanisms enforcing spatio-temporal control of Polo kinase during mitotic entry constitutes a major leap forward in our understanding of cell division at the molecular level. Much work remains to be done to comprehend the regulation and precise functions of Polo kinase in late phases of cell division, including cytokinesis. Understanding the mechanisms regulating Polo opens possibilities to selectively disrupt specific regulatory modes or downstream functions of Polo in order to dissect their importance experimentally. This can be achieved by mutations at specific sites in Polo, inactivation of upstream regulatory enzymes or even the development of small molecule modulators as tool compounds. Several efforts have been deployed to develop PBD inhibitors that could be used to assess the importance of PBD interactions with targets for specific functions of Polo . The dev"}
+{"text": "Fine-needle aspiration biopsy (FNAB) cytology is a simple, inexpensive, andaccurate diagnostic test for benign, infectious, and malignant lesions ofthe breast, thyroid, lymph nodes, and other organs. Similarly, bone marrowaspiration and trephine (BMAT) biopsy procedures are relatively simple andinexpensive techniques that are important for diagnosing and monitoring manyhematologic diseases including leukemias and lymphomas. However, thescarcity of pathologists in Kenya limits patient access to these simplediagnostic tests. We describe a task sharing and shifting program thatsought to improve the provision of FNABs and BMAT biopsies in tertiarypublic hospitals in Kenya.Between January 2016 and February 2017, we trained pathologists, pathologyresidents, and technologists from the University of Nairobi and Aga KhanUniversity Hospital, Nairobi, in FNAB and BMAT biopsies, who in turn trainedpathologists, medical officers (MO), clinical officers (CO), andtechnologists at five tertiary public hospitals. The program involvedcurriculum development, training workshops, the establishment of new andstrengthening existing FNAB and BMAT biopsy clinics, interim site visits,audits, and stakeholder workshops.Fifty-one medical personnel at the tertiary hospitals were trained. The FNABnumbers increased by 41% to 1,681, with 139 malignant diagnoses (7.1%). BMATbiopsy numbers increased by 268% to 140, with 34 malignant cases. Between60% and 100% of the FNAB and BMAT biopsy procedures were performed by MO andCO over the project period. One new FNAB and two new BMAT biopsy clinicswere established.This project demonstrates a successful model of task sharing and shiftingfrom specialist pathologists to MO and CO that improved access to importantFNAB and BMAT biopsy services in a low-resource setting. Broader access to FNABs and BMAT biopsies isthus limited, largely because there are few well-trained personnel to perform thesediagnostic techniques.8 Task sharingand shifting has been demonstrated previously to be useful for training laboratorytechnologists to process tumor specimens9 and in the delivery of HIV care.10Task sharing and shifting through training nonpathologist medical and paramedicalstaff to perform procedures such as FNAB and BMAT biopsy may overcome some of thechallenges associated with the scarcity of pathologists in low- and middle-incomecountries (LMICs).11 We evaluated the effects of thisprogram by the change in the number of skilled personnel performing FNAB and BMATbiopsy procedures, the number of FNAB and BMAT biopsy procedures performed, the rateof unsatisfactory FNAB and BMAT biopsy samples, the diagnosis turnaround time (TAT),and the effect on the capacity for quality cytology processing at thesefacilities.We describe a task sharing and shifting program for FNAB and BMAT biopsy proceduresthat was developed by the pathology subtrack members at a National CancerStakeholders\u2019 meeting held in 2014 in Naivasha, Kenya, jointly supported byKenya\u2019s Ministry of Health and the US National Cancer Institute.This was a partnership between Aga Khan University Hospital, Nairobi (AKUHN) andthe University of Nairobi (UoN), supported by collaborators from the Center forGlobal Health at the US National Cancer Institute and St. Vincent\u2019sHospital and Notre Dame University Medical School, Sydney, Australia. Theprogram was implemented over a 14-month period from January 2016 throughFebruary 2017 and initially included four participating county healthfacilities: Coast Provincial General Hospital (CPGH) in Mombasa, NyeriProvincial General Hospital (NPGH) in Nyeri, Jaramogi Oginga Odinga Teaching andReferral Hospital in Kisumu, and Embu Provincial General Hospital (EPGH) inEmbu. A fifth site, the Kisii Teaching and Referral Hospital (KTRH) in Kisii,was added in July 2016 .The overall aim of the program was to provide diagnostic support for cancer care,using a two-step approach: first, a training-of-trainers workshop trainedpathology residents from the UoN and AKUHN and practicing pathologists from thefive participating hospitals to become trainers in FNAB and BMAT biopsytechniques; and second, these new trainers trained additional pathologists,medical officers (MO), and clinical officers (CO) in the five county hospitalsto perform quality FNAB and BMAT biopsy procedures. MO are first- andsecond-year postinternship doctors who work in county hospitals for a mandatory3 years after their medical graduation. CO are career paramedical officers whowork in county hospitals after completing a 3-year diploma or degree.In parallel, laboratory technologists were trained in the handling and processingof FNAB and BMAT biopsy samples by experienced laboratory technologists from UoNand AKUHN. The program overview is outlined in The project team developed pre- and post\u2013FNAB and BMAT biopsy trainingsurvey tools that assessed the status of FNAB and BMAT biopsy services inparticipating facilities between September and November 2015.Training curricula on FNAB and BMAT biopsy procedures were developed throughonline consultations by the project team over 3 months beginning inSeptember 2015. The curriculum included the theory and technical aspects ofFNAB and BMAT biopsy procedures, indications for the procedures, and qualityassurance requirements.A 3-day training workshop was conducted in January 2016 at UoN to train thepathology residents and county pathologist trainers. The training startedwith a precourse multiple-choice test and a practical evaluation of thetrainees\u2019 ability to perform FNAB and BMAT biopsy procedures,followed by hands-on workbench and clinical training in FNAB and BMAT biopsytechniques, with trainees performing one to two FNABs in an organized FNABclinic under the direct supervision of an international instructor (AF).Each participant also performed one to two BMAT biopsy procedures onprebooked patients under the supervision of faculty. A parallel course inthe preanalytic and analytic techniques needed for quality FNAB and BMATbiopsy specimen processing was conducted for senior technologists from boththe UoN and AKUHN. Then there was a teach-back session to assess theteaching ability of the workshop participants, which was followed byadministration of a confidence rating tool that was based on the Likertscale (0 is not confident and 5 is very confident) to evaluate theconfidence levels of the newly trained trainers. Postcourse evaluation ofthe participants (practicing and resident pathologists) was conducted at theend of the 3-day training through practical assessment of competency in theFNAB technique. In addition, the trained residents each completed at least10 BMAT biopsy and 20 FNAB procedures between January and March 2016 atKenyatta National Hospital under the supervision of faculty, which wasfollowed by a final work-based competency assessment.Pathology resident trainers and on-site county pathologists used the samecurriculum and 3-day training program to train interested MO and some COattached to the participating health facilities. The trainers also undertooka follow-up visit 6 weeks later to provide supervision and, where necessary,revision of parts of the training.The program was evaluated through pre- and post-training surveys and mid- andend-of-project audits and quality-assurance challenges . QualityThe pathology residents administered the previously developed baseline andpostproject surveys to MO, CO, pathologists, technologists, and hospitalleadership at all four original participating sites. The surveys examinedthe impact of the program on the provision of FNAB and BMAT biopsy serviceat these facilities.Two audits were conducted, a midterm audit at all sites and an end-of-projectaudit at CPGH and EPGH. Fewer facilities were included in the end-of-projectaudit because of time constraints. The auditors  used FNAB and BMATbiopsy audit checklists developed by the audit teams and the project leads.Each audit visit to a county hospital incorporated the following: a preauditmorning meeting with the trainees and medical administrator, theauditors\u2019 practical structured assessment of each MO, remedialinstruction as required, work-based assessment by the auditors of the MO asthey performed FNAB and BMAT biopsy procedures in clinics, and a feedbackand discussion meeting at the end of each audit dayTwo technical challenges, consisting of unstained direct FNAB smears andformalin-fixed paraffin-embedded trephine biopsies, were sent toparticipating sites to assess the technical staff on proficiency in samplehandling and processing.A stakeholders\u2019 meeting of the county health directors, medicalsuperintendents, and pathologists from all four original participating siteswas held on March 1, 2016, at AKUHN to obtain a commitment for the project.The agenda included introducing the project to the stakeholders, presentingthe expected benefits and the roles and responsibilities of theparticipating teams, and proposing an income-generation model that thecounty hospitals could adopt.A stakeholder and participant workshop was held in January 2017 to review theperformance of participating sites, the achievements of the project, theshared challenges, the lessons learned, and the proposed changes for animproved model for the project going forward.A total of 23 participants, including nine pathologists, six pathology residents,and eight technologists, were trained as trainers of trainers in the FNAB andBMAT biopsy techniques. All 23 trainers showed improvement between their initialand post-training assessments of FNAB and BMAT biopsy technique . The traThe number of FNABs performed between March/April and November 2016 showed anoverall increase of 41% across all facilities compared with the same period in2015 . In addiThere was an even more marked increase in the number of BMAT biopsies at thetraining sites, from a total of 38 procedures between March/April and November2015 to 140 procedures over the same period in 2016, an increase of 268% . FurtherOverall, the rate of unsatisfactory FNABs decreased from an average of 14% beforeon-site training to 8% soon after the training and to 4% several months laterafter the auditing process and retraining . These uThe TAT for FNAB in the project period was compared with a similar period theprevious year . The TATAt baseline, all the FNABs were performed by a pathologist, and by the end of theproject, the 33 MO and two CO were performing 60% to 100% of the FNAB and BMATbiopsy procedures. The FNAB smear-making technique was described before trainingas a squash and smear technique, and this was replaced by the far superior splitsample specimen procedure taught by the international instructor (AF).12 and a study from Rwanda demonstrated how task sharing andshifting of technical skills in anatomic pathology laboratories can influence TATthrough the efficient use of available staff.13Our program provides an example of how task sharing and shifting can address some ofthe challenges associated with the shortage of pathologists in LMIC. Task sharingand shifting is a well-established strategy in other areas of health care,Involving pathology residents as trainers was a strength of this project and hadseveral benefits. The residents reinforced and improved their procedural skills andbecame empowered as trainers, while also learning project implementation and reportwriting. For county hospital pathologists, the project reinforced their proceduralskills and potentially improved their reporting ability in FNAB and BMAT biopsy.Importantly, one site (NPGH), where the pathologist left, was able to continueproviding FNAB and BMAT biopsy services using a project-trained MO and two CO whomhe had trained to procure samples, which were then evaluated at a referrallaboratory.Where there were deficiencies noted during the interval audits, remedial training wasprovided. For BMAT biopsies, the lack of adequate numbers of patients throughout thetraining period meant that experiential improvement was still a challenge.Furthermore, to have an impact on TAT, local pathologists will require additionaltraining in reporting BMAT biopsy specimens.Quality assurance was a major component of this project. The audits conducted by theexternal team, the two external quality-assurance challenges for technical staff,and the feedback sessions were integral to ensuring that the quality chain from thetime of sample requisition to the time of result availability was maintained forappropriate and timely patient management.In all the county hospitals, the training of MO and CO in these procedures added totheir workload and challenged their routine work rosters. Initially, no allowanceswere made by the medical administration for MO to perform these procedures. Coverhad to be provided by their fellow MO, and this situation continued throughout theprogram, but the motivated MO found time to service the FNAB clinics effectively.During audit meetings, these matters were discussed to a degree, and at the finalstakeholders\u2019 meeting there was apparent recognition by the medicaladministrators of the increased workload.Some pathologists also viewed the enhanced FNAB and BMAT biopsy service as anincrease in their workload and, particularly with BMAT biopsies, a challenge toreport. In these referral hospitals, the pathologists provide forensic autopsyservices that require frequent court attendances, which poses a challenge forincreasing any of their other duties. Similarly, some medical administratorsperceived increasing diagnostic tests as an increase in costs, although adultpatients were charged an equivalent of 5 to 7 USD for FNAB and BMAT biopsy services,which should have covered the costs. Hospital administrators should be encouraged toreinvest income from billing for FNAB and BMAT biopsy procedures into theirpathology laboratories for purchase of consumables that are needed to sustain theservice. The study demonstrated that there is a need during program initiation foradministrators to fully understand the benefits and local requirements of theprogram to encourage buy in.In the county hospitals, the CO are the most stable cadre of staff, and training onlya few from the outset limited the impact of the project, especially when MOtransferred out or left for postgraduate training or were on annual leave. Thesituation was compounded in late 2016 by a nationwide doctors\u2019 strike thatlead to massive cancellations of routine diagnostic procedures.There were no data collected on the impact of improved and timely diagnosis onpatient management in terms of how soon patients could schedule surgicalappointments, what procedures were performed, and identification of the short andmidterm clinical outcomes. This was a significant limitation in this study, becauseadoption and funding of such a program more widely will require the demonstration ofa favorable benefit-to-cost ratio.Additional monitoring, training, and mentorship of MO, CO, interns, and techniciansare needed on a regular basis to maintain the skills and interest in such a program.Establishing a cohort of MO well trained in FNAB and BMAT biopsy procedures, who canthen train other MO and CO, would lead to increased numbers of those able to performthese procedures. Training in FNAB and BMAT biopsy procedures should be regarded asa basic requirement for all interns and MO, and these procedures should be recordedin logbooks as a requirement for continuing medical education. Training in FNAB andBMAT biopsy techniques should also be extended to residents in surgery, medicine,and radiology, so that as future consultants they will understand the roles andadvantages of these procedures and be able to perform them. Furthermore, training inthe use of increasingly inexpensive ultrasound imaging to guide FNABs is alsoneeded, to increase the range of lesions that can be accessed by FNAB.14 shouldbe a long-term goal for the FNAB clinics, to enhance patient care through immediateprovisional reporting and triage of specimens and to reduce patient call-backs forinsufficient material. This evaluation requires pathologists, or at least traineepathologists, to be available to perform rapid on-site evaluation. To further reducecosts, FNAB should be available in all outpatient clinics, ideally on thepatient\u2019s first visit, to enhance patient care and avoid multiple returnvisits. In time, enhanced molecular and other ancillary testing of FNAB and BMATbiopsy materials can be incorporated to maximize the diagnostic usefulness of thesesimple tests.Rapid on-site evaluationWe strongly recommend that a monitoring evaluation on framework be implemented at thesites of this program to encourage sustainability and to assess the impact of theprogram over a 3- to 5-year period. This would provide the data needed forappropriate recommendations for a wider adoption of this model.15 With additionalgovernment support and the collaboration of Kenyan and international pathologists,this project can potentially be rolled out across Kenya and similar LMICsettings.This project has demonstrated that a model of task-shifting diagnostic procedures andskills from pathologists to MO and other medical personnel can be implementedsuccessfully in a low-resource setting, and this can address some of the challengesassociated with the shortage of pathologists. Furthermore, the project has shownthat this can be accomplished by training pathology resident trainers centrally,using an experienced faculty, and then supporting these resident trainers to teachregional county hospital MO and CO. The project has decentralized and improved theFNAB and BMAT biopsy services in the county hospitals in line with government healthpolicy."}
+{"text": "Aquaporin-1 (AQP1) gene and the AQP1-Aquaporin channel. Regulation of water flow across cell membranes is essential for supporting inter- and intracellular fluid balance, which is critical for health and exercise performance. The transmembrane water channel AQP1 is important for cardiorespiratory endurance (CE) because it influences fluid transfers in erythrocytes, endothelial, and pulmonary cells and is vital for transport of ammonium, bicarbonate, carbon dioxide, glycerol, nitric oxide, potassium ion, water, and trans-epithelial and renal water. Very recent publications suggest the association between a DNA sequence variant, rs1049305 (C > G), in the 3\u2032-untranslated region of the AQP1 gene and CE performance. Other reports indicate further significant associations between AQP1 channel and CE phenotypes. The purposes of this systematic review were to examine the extent of the associations between the AQP1 rs1049305 genotype and CE exercise performance and body fluid loss in long-distance runners and AQP1 channel associations with other CE phenotypes.There is abundant and mounting information related to the molecular and biological structure and function of the AQP1 gene and AQP1 channel structure and function, associations between the AQP1 gene sequence variant rs1049305 (C > G)\u00a0 and CE performance, body fluid loss in long-distance runners, and other studies reporting on the AQP1 gene and channel CE phenotype associations. Synthesis methods: For each selected study, the following data were extracted: authors, year of publication, sample size and number of cases and controls, CE definition, exclusion criteria, inclusion criteria for cases and controls, methods used for genotyping, genotype, allele frequencies and HWE for genotype frequencies in cases and control groups, and method of AQP1 gene and AQP1 channel analysis.Data sources: A comprehensive review was conducted using PubMed, EMBASE, CINAHL, and Cochrane electronic databases. The search ranged from January 1, 1988, to December 31, 2018. Studies reported in English, French, and Spanish were considered. Eligibility criteria: The criteria for inclusion in the review were (a) case-control study; (b) unequivocal definition of cases and controls; (c) CE was defined as performance in endurance events, laboratory tests, and/or maximal oxygen consumption; (d) exclusion criteria of known causes; (e) genotyping performed by PCR or sequencing; (f) genotype frequencies reported; and (g) no deviation of genotype frequencies from Hardy-Weinberg equilibrium in the control group. Study appraisal: The systematic review included studies examining the AQP1 gene sequence variant rs1049305 (C > G) and CE performance; (b) the association of the rs1049305 C-allele with faster CE running performance; (c) in knockout model, using a linear regression analysis of distance run as a function of Aqp1 status (Aqp1-null vs. wild-type mice) and conditions of hypoxia (ambient [O2] = 16%), normoxia (21%), and hyperoxia (40%) indicated that the Aqp1 knockout ran less distance than the wild-type mice (p < 0.001); (d) in vitro, a reduced AQP1 expression was associated with the presence of the rs1049305 G-allele; (e) AQP1 null humans led normal lives and were entirely unaware of any physical limitations. However, they could not support fluid homeostasis when exposed to chronic fluid overload. The limited number of studies with \u201cadequate sample sizes\u201d in various racial and ethnic groups precluding to perform proper in-depth statistical analysis.The initial databases search found 172 pertinent studies. Of those, 46 studies were utilized in the final synthesis of the systematic review. The most relevant findings were (a) the identification of an independent replication of the association between AQP1 gene and AQP1\u00a0channel seems to support homeostatic mechanisms, yet to be totally understood, that are auxiliary in achieving an advantage during endurance exercise. AQP1 functions are vital during exercise and have a profound influence on endurance running performance. AQP1s are underappreciated structures that play vital roles in cellular homeostasis at rest and during CE endurance running exercise. The outcome of the present systematic review provide support to the statement of hypotheses and further research endeavors on the likely influence of AQP1 gene and AQP1 channel on CE performance. Registration: The protocol is not registered.The  AQP1 gene and AQP1 channel functions are vital during exercise and have profound influence on cardiorespiratory endurance performance.The This is significant because genetic (molecular) mechanisms and their effects on cardiorespiratory endurance performance phenotypes are major areas of inquiry in the science and medicine of sport and exercise.Regulation of water flow across cell membranes is essential for supporting a proper fluid balance within the cells, which is a critical factor in health status and endurance performance.2max). The latter is the highest rate at which the body takes up, transports, and uses oxygen at sea level. CE and the VO2max measures are high, significant, and positively correlated, and both are highly influenced by genetic mechanisms  treating [O2] categorically referring to 21% O2 indicated that the Aqp1 knockout reduced the distance run by 4.7 \u00b1 0.5 km (p < 0.001), adjusting for [O2]. Compared to 21% O2, reducing O2 to 16% reduced the distance run by 1.6 \u00b1 0.6 km (p = 0.01), while increasing O2 to 40% increased the distance run by 1.2 \u00b1 0.6 km (p = 0.04), adjusting for Aqp1 status. These findings led to the conclusion that the Aqp1-null mice have a major effect in voluntary exercise tolerance (CE performance), consistent with the hypothesis that Aqp1 plays an important physiological role in O2 transport across plasma membranes. It is well accepted that in humans the execution of prolonged exercise (like distance running) depends highly on molecular mechanisms mostly related to the management of O2.Xu et al. , using m2 management. In humans, prolonged exercise capacity as that required by long-distance running is highly influenced by VO2max, metabolic economy, lactate threshold, temperature regulation, and fatigue resistance. Abundant information indicates the genetics mediate the magnitude of these mechanisms [2max [2max are strongly and positively associated. New findings [2max absolute values and those adjusted for body weight and for fat-free mass were 0.68 (95% CI 0.59\u20130.77), 0.56 (95% CI 0.47\u20130.65), and 0.44 (95% CI 0.13\u20130.75), respectively. The meta-regression analysis revealed that sex could partially explain the heterogeneity in the VO2max heritability estimates adjusted by body weight. The heritability estimates reported among the studies were statistically significant. Last, for submaximal endurance, phenotypes and endurance performance heritabilities were 0.49 (95% CI 0.33\u20130.65) and 0.53 (95% CI 0.27\u20130.78), respectively.The present observational study of the AQP1 channel shed further light on the possible role of a molecular mechanism, like that related to the AQP1 channel presence or absence and the acute response to exercise and Oms [2max . One of findings  arising AQP1 gene and CE performance was shown by Rivera et al. [AQP1 gene and the CE performance-related phenotype. In this occasion, elapsed running time in a 10-km event was compared by AQP1 C-allele carrier status, e.g., carriers  and heterozygous for C-allele (CG); n = 50) and non-carriers ; n = 41). The main findings indicated that AQP1 C-allele carries ran an average of 13.4% faster (p < 0.05) than non-carriers during the 10-km race, which is approximately 16.12 km/h for carriers and 13.9 km/h for non-carriers. There was no difference in training status between the two groups . These findings provide further support to the notion that inter-individual variability in CE performance could be partly explained by molecular mechanisms, such as DNA sequence variations. The findings of Rivera et al. [AQP1 rs1049305 CC and CG genotype in promoting endurance running performance level.In humans, the second line of support for the hypothesis of an association between the a et al.   variant within the 3\u2032 UTR region of the AQP1 gene was evaluated in South African Caucasian male (n = 504) finishers in either the 2000 (n = 112), 2001 (n = 222),\u00a0and 2006 (n = 170) South African Ironman Triathlons [AQP1 rs1049305 C-variant was associated with the duration of marathon running segment in three Ironman events. Triathletes who carried the C-allele completed the 42.2-km run stage faster  than triathletes with the GG genotype . That study also contended that their findings and those of Martinez et al. [AQP1 rs1049305 C-variant contributes to a physiological state receptive to training and beneficial to endurance (long distance) running performance. Some further argue that the weakness of observing a similar genotype effect on performance in the swim and bike stages likely reflects the differing physiological requirements of these activities [In humans , Asians (0.38 % ), and Caucasians (0.42 %), but a striking prevalence of the AQP1 C-allele in African Americans (0.86 %) and Sub-Saharans (0.98 %). Others [Kenyan and Ethiopian runners have dominated Olympic middle- and long-distance running events since the 1968 games in Mexico City . The popormation  found sm. Others  reportedAQP1 gene and endurance exercise. They examined the effects of aerobic interval training (AIT) and moderate continuous training (MCT) on osmotic stress-mediated rheological function and AQP1 channel activity of human erythrocytes under hypoxic exercise (HE) stress in humans. Thirty healthy sedentary males were randomly assigned to either the AIT group which performed 3-min intervals at 40% and 80% VO2max, n = 15, or the MCT group required to perform sustained exercise at 60% VO2max, n = 15, for 30 min/day, 5 days/week for 6 weeks. Erythrocyte rheological responses to HE (100 W under 12% O2 for 30 min) were determined before and after various regimens. The findings revealed that acute HE increased osmotic fragility and decreased deformability of erythrocytes, and depressed erythrocyte AQP1 activity as indicated by increased magnesium chloride (HgCl2-) induced instability of erythrocyte membrane under hypotonic conditions. After the 6 weeks of exercise intervention, the AIT group exhibited higher maximal power output and VO2max than the MCT group. Both AIT and MCT diminished the extents of enhanced osmotic fragility, reduced deformability, and AQP1 activity of erythrocytes caused by HE. They concluded that AIT was superior to MCT for enhancing aerobic capacity. Either AIT or MCT effectively alleviated the impairments of erythrocyte rheological characteristics and AQP1 function evoked by HE.Huang and Wang  used a dAQP1 rs1049305 (C > G) variant during endurance runs. Rivera et al. [AQP1 rs1049305 C-allele had a greater adjusted body fluid loss (3.7 \u00b1 0.9 kg) than non-carriers (1.5 \u00b1 1.1 kg) (P < 0.05). Saunders et al. [Along these lines, Rivera et al.  and Sauna et al.  reporteds et al.  determinn = 50; r = \u2212\u20090.31; p = 0.023). Faster runners lost more body mass compared with slower runners while also drinking more [The exercise-induced body fluid loss differences, by AQP1 genotype, observed by Rivera et al.  may alsoing more .2 transport [A relevant finding of this systematic review is that during osmotic stress, such as intense exercise , 42, AQPransport , two facransport , 29. Wakransport  hypothesransport , 45, 46.In humans, AQP1 null individuals led normal lives and were entirely unaware of any physical limitations . HoweverAQP1 gene sequence variant rs1049305 (C > G) in the 3\u2019 UTR and CE performance [AQP1 gene and channel are related to CE performance was substantiated by peer-reviewed publications [AQP1 gene rs1049305 (C > G) have three forms: CC, CG, and GG. The present systematic review findings favor the hypothesis that CC and CG forms are apparently positive contributors to the complex physiological state receptive to training and beneficial to CE (long distance) running performance level, while other form of the gene (GG) was seemingly less functional or poorly associated [The central finding of the present systematic review was the independent replication of the association between formance \u201330. Furtications , 42\u201344. sociated \u201330.The main limitation of the present review was the limited number of studies with \u201cadequate sample sizes\u201d in various racial and ethnic groups precluding to perform proper in-depth statistical analysis.AQP1\u00a0gene rs1049305 C-allele appears to provide for the enhancement of homeostatic mechanisms, yet to be understood, that are auxiliary in achieving an advantage during CE running exercise. The C-allele seems to allow for a greater fluid loss and fluid intake in fast versus slow runners. Perhaps, the AQP1 gene rs1049305 C-allele and the AQP1 channel might facilitate more intense training, faster recovery, and enhanced temperature regulation. Greater AQP1 [2 transport. AQP1s are underappreciated structures that play vital roles in cellular homeostasis at rest and during CE running exercise.The ter AQP1  activityAQP1 gene and channel seems to support homeostatic mechanisms, yet to be totally understood, that are auxiliary in achieving an advantage during endurance running\u00a0exercise. AQP1 functions are vital during exercise and have a profound influence on endurance running performance.The"}
+{"text": "Biofilm heterogeneity has been characterized on various scales for both natural and engineered ecosystems. This heterogeneity has been attributed to spatial differences in environmental factors. Understanding their impact on localized biofilm heterogeneity in building plumbing systems is important for both management and representative sampling strategies. We assessed heterogeneity within the confined engineered ecosystem of a shower hose by high-resolution sampling  on varying scales (\u03bcm to m). We postulated that a biofilm grown on a single material under uniform conditions should be homogeneous in its structure, bacterial numbers, and community composition. A biofilm grown for 12 months under controlled laboratory conditions, showed homogeneity on large-scale. However, some small-scale heterogeneity was clearly observed. For example, biofilm thickness of cm-sections varied up to 4-fold, total cell concentrations (TCC) 3-fold, and relative abundance of dominant taxa up to 5-fold. A biofilm grown under real  use conditions developed considerably more heterogeneity in all variables which was attributed to more discontinuity in environmental conditions. Interestingly, biofilm communities from both hoses showed comparably low diversity, with <400 taxa each, and only three taxa accounting for 57%, respectively, 73% of the community. This low diversity was attributed to a strong selective pressure, originating in migrating carbon from the flexible hoses as major carbon source. High-resolution sampling strategy enabled detailed analysis of spatial heterogeneity within an individual drinking water biofilm. This study gives insight into biofilm structure and community composition on cm-to m-scale and is useful for decision-making on sampling strategies in biofilm research and monitoring. Small-scale heterogeneity was assessed by comparing (1) biofilm structure and thickness, (2) total cell concentrations, and (3) bacterial community composition of a total of 200 sections of 1.2 cm. Additionally, a biofilm grown in an identical hose under real-use conditions was analyzed in the same way to assess the impact of more variable environmental conditions on biofilm spatial heterogeneity. Our sampling design enabled a high-resolution assessment of drinking water biofilms on small-scale, and the combination of quantitative and qualitative tools for biofilm characterization. This study provides a deeper insight into biofilm formation on building plumbing materials and consequently informs on biofilm sampling strategies.Biofilms were grown inside commercially available flexible shower hoses, purchased from the same batch of production. The hoses were made from plasticized polyvinyl chloride (PVC-P), with an inner diameter of 0.8 cm, a total length of 1.80 m, and originally with a metal cover outer sheath.In the laboratory setup, the metal sheath was removed and the hose was horizontally aligned in a dark container, preventing any motion or physical disruption (further referred to as \u201ccontrol hose\u201d). The installation was connected to a warm water tap with automated flushing events realized by a time-controlled magnetic valve. Over the course of one year, the hose was automatically flushed for 15 min with warm water (35\u201342\u00b0C) twice per day with consistent stagnation times of 8 and 16 h, respectively . A flow Complementary to the control hose, an identical PVC-P hose was installed in a real shower , with the aim to assess the impact of more variable environmental conditions on biofilm heterogeneity. Usage habits  varied over the course of one year, with three residents sharing the shower. For showering, mixtures of warm and cold water lines were used with varying and higher flow velocities compared to the control hose (average use: 8\u201312 L/min), and random stagnation times that went up to 14 days. The water was also non-chlorinated, but originating mostly from untreated groundwater (95%) with a minor addition of pre-treated spring water  .\u00ae, Advanced Power) into a total volume of 10 mL of 0.2 \u03bcm filtered bottled water . For this, each section was covered with 5 mL of filtered water in a petri dish and brushed for approximately 45 s, depending on the stickiness of the biofilm matrix. The remaining 5 mL were used to remove residuals of biofilm from the toothbrush head and from the surface of the petri dish (approximately 20 s brushing) and transferred to the sample tube. The 10 mL biofilm suspension was then needle sonicated to disrupt cell clusters . The needle was submerged to the upper third of the sample volume and sonication occurred for 30 s, with 5 \u00d7 10% pulses, and 40% power. The biofilm suspensions were measured with flow cytometry (FCM) to quantify total cell concentrations . Finally, biofilm suspensions were filtered for DNA analysis . For all sampling steps, pieces were randomized to minimize the impact of processing errors.Both hoses were processed, sampled, and analyzed in the same way . For the2 to make results more comparable within this study and to other studies.For data analysis, the terminology \u201ccm-sections\u201d refers to the 1.2 cm sections, representing a total of 200 cm-sections per experimental hose. Furthermore, for bacterial cell concentrations and the analysis of sequencing data, units were converted to values per cmTM) Software was used to move the pieces in distinct steps of 2 mm without disrupting the alignment. Biofilm thickness was then determined using an analysis software in MATLAB\u00ae (Version R2016b) which has previously been reported by Derlon and colleagues (1.50i). Finally, these interfaces were used to create binary images, which were used for further image analysis. Potential problems that arose during image processing where solved as follows: (1) Detached biofilm structures floating around were creating artificially high values for biofilm thickness. For correction, these parts were masqued manually with black boxes (ImageJ). (2) If no clear detectable line was indicating the biofilm-water interface, it could result in wrong values for minimal biofilm thickness. For this, white lines were drawn manually indicating the biofilms surface (ImageJ). For better comparison between the different quantitative measurements, average thickness values were used for combined 1.2 cm sections .For characterizing the structure, biofilms were imaged using a Spectral Domain OCT Imaging System , with an axial detection limit of 4.4 \u03bcm. The 6 cm pieces were horizontally aligned and covered with a thin layer of 0.2 \u03bcm filtered water for optimal imaging. Along the length of each piece, images of 2 mm (length) \u00d7 1 mm (height) were captured, which equals 30 images per piece or 1\u2019200 per hose, respectively. The Advanced Positioning Technology  to detect TCC. Finally, samples were incubated at 37\u00b0C for 10 min and then measured using a BD Accuri C6\u00ae flow cytometer , with an instrumental threshold set at 800 (FL1-H) and a volume of 50 \u03bcL measured at a high flow velocity of 66 \u03bcL/min. For analysis, one gate was applied for all samples.Total bacterial cell concentrations (TCC) were quantified for each 1.2 cm section by FCM. Sample preparation, measurements, as well as data analysis were performed as described elsewhere . First, \u00ae membrane filters , using sterile bottletop filter units attached to a vacuum pump . DNA filters were immediately frozen in liquid nitrogen and stored at \u221220\u00b0C until DNA extraction.Prior to DNA extraction, biofilm suspensions were concentrated on 0.22 \u03bcm polycarbonate Nucleopore\u00ae Kit . Extracts were stored at \u221220\u00b0C until 16S rRNA gene amplification for sequencing.DNA extraction was performed according to the protocol of the DNeasy PowerWaterTM DNA Broad Range Assay in duplicates, using the Spark\u00ae 10M Multimode Microplate Reader . The amount of DNA was normalized between all samples (1 ng) and primers were added in a final concentration of 0.3 \u03bcM  were incorporated. In the course of sample processing, some biofilm sections needed to be excluded due to low quantities of extracted DNA, poor amplification, or poor number of reads after sequencing. Data on community composition was generated in collaboration with the Genetic Diversity Centre (GDC), ETH Zurich.For 16S rRNA gene sequencing, the V3-V5 region of the gene was amplified by polymerase chain reaction (PCR) using the primers Bakt_341F and Bakt_805R . First, f 0.3 \u03bcM . After af 0.3 \u03bcM . Purifiein silico PCR was performed and primer sites trimmed  of the National Center for Biotechnology Information (NCBI): Accession number Ten centimeters from the beginning and end of each hose were immediately prepared for scanning electron microscopy. For this, biofilms were fixed with 2.5% Glutaraldehyde in Cacodylate buffer  at room temperature for 60 min and stored in Cacodylate buffer at 4\u00b0C afterwards. Final preparation and imaging was done by the Center for Microscopy and Image Analysis (University of Zurich).top and bottom, reflecting the actual spatial orientation of the control hose in the laboratory setup. The real hose was used vertically in a shower, hence the longitudinal top and bottom do not represent any specific orientation. Data from 200 biofilm sections was analyzed on various scales (from \u03bcm\u2013m) for each individual hose. Here, large-scale refers to the complete hose . Small-scale refers to the differences between adjacent 1.2 cm-sections.We analyzed in detail biofilms that formed inside two identical shower hoses under controlled use and real use conditions, with both exposed to non-chlorinated warm water during approximately 12 months. The purpose of this study was to assess the degree of spatial heterogeneity within each individual biofilm by high-resolution sampling, with the communities developing under supposedly uniform (control hose) or more variable  environmental conditions. In 9 bacteria, at an average distribution of 2.4 \u00b1 0.5 \u00d7 107 cells/cm2 (n = 200)  , with thn = 200) .n = 200) (n = 100) compared to the top  . Moreover, biofilm thickness increased notably over the length of the 120 cm piece following the flow direction; approximately 100% in the top (linear regression with R2 = 0.43) and 255% in the bottom (linear regression with R2 = 0.40). This amounts to an average increase of 0.83 \u03bcm/cm (top) and 2.13 \u03bcm/cm (bottom). In addition to the spatial trend, variability in biofilm thickness was already evident on small-scale. Adjacent cm-sections of the top varied 11.7 \u00b1 8.9% (n = 99), ones in the bottom varied even more with 23.9 \u00b1 28.5% (n = 99), with the standard deviations suggesting higher variation/heterogeneity throughout the bottom part of the hose. On an even smaller scale , variations of up to 50% could be observed (10 \u03bcm3/cm2 (n = 100) in the top and 3.9 \u00b1 1.2 \u00d7 1010 \u03bcm3/cm2 (n = 100) in the bottom part of the hose.The biofilm topography was sinuous, with uneven protrusions and depressions resembling hills/dunes . The aven = 200) . On largobserved . In addi7 cells/cm2. Interestingly, average TCC values were the same at the top  and at the bottom , in stark contrast to the thickness data presented above. Correlations between TCC and biofilm thickness were weak, but higher for the top  compared to the bottom biofilm . On large-scale, linear regressions suggest an increasing trend in TCC along the length of the hose for both top (R2 = 0.36) and bottom (R2 = 0.17). However, this trend is mainly driven by lower concentrations in the first 30 cm-sections of the control hose, with on average 34% lower concentrations in the top and 17% in the bottom part compared to the rest of the hose (n = 99) and bottom . The combination of the TCC data and an estimated average cell volume of 0.3 \u03bcm3 [calculation based on average cell size from SEM imaging, 6 \u03bcm3/cm2 (n = 100) in the top and 7.0 \u00b1 1.1 \u00d7 106 \u03bcm3/cm2 (n = 100) in the bottom. This, in turn, allows the calculation of the relative contribution of bacterial cell volume to the overall biofilm volume (Vcells:Vbiofilm), which was notably small with approximately 0.03 \u00b1 0.01% (n = 100) for the top and 0.02 \u00b1 0.01% (n = 100) for the bottom biofilm.Total cell concentrations (TCC) of 1.2 cm-sections ranged between 1.1\u20133.4 \u00d7 10the hose . Fluctuap < 0.001). Following this, taxa richness (S) was higher in the top (S = 335) compared to the bottom (S = 288), both with an Evenness index (J\u2032) of 0.4. On small-scale, richness ranged from 55 to 92 taxa/cm-section (J\u2032 = 0.5\u20130.6), with on average 72 \u00b1 6 taxa/cm-section (n = 95) in the top and 67 \u00b1 6 taxa/cm-section (n = 100) in the bottom. In addition, richness showed variations between adjacent cm-sections of 9 \u00b1 7% (n = 92) in the top and 7 \u00b1 6% (n = 99) in the bottom. Regarding beta-diversity, Bray-Curtis revealed compositional dissimilarities in the communities of adjacent cm-sections between 0.05\u20130.38 , arguing in favor of a rather similar community composition along the length of the biofilm on small-scale. Interestingly, only few dominant taxa  made up the majority of the community composition. In fact, the 10 most dominant taxa accounted for 89.3% of the total biofilm community  and bottom (R2 = 0.45) (n = 11) to 25.8 \u00b1 3.1% (n = 11) following sections 63\u201384 in the bottom (n = 11) to 10.6 \u00b1 2.2% (n = 11) (2 < 0.14), (2) TCC (R2 < 0.1), or (3) richness (R2 < 0.2) were weak.The overall biofilm community comprised 384 ZOTUs (henceforth referred to as taxa). On large-scale, ordination by non-metric multidimensional scaling, based on the Bray-Curtis dissimilarity, showed a clear trend in sample clustering in the control hose . Here, oommunity , coverinommunity , green, . 23.4%, , red, an. 23.4%, , blue. T further . Due to  = 0.45) . Also, re bottom , green. (n = 11) , blue. O9 bacteria at an average distribution of 3.8 \u00b1 1.4 \u00d7 107 cells/cm2 (n = 200) (top vs. bottom) were not analyzed separately.A comparatively thin biofilm established on the inner surface of the real hose during 12 months of random usage and handling , 5A. Then = 200) . The bacn = 200) , comparan = 200)  this doen = 200) (n = 27)  compared to the rest of the hose . On small-scale, we observed considerable heterogeneity between adjacent cm-sections .The real hose biofilm was considerably thinner than the control hose biofilm, often below the OCT detection limit (\u223c4 \u03bcm), but also showing uneven protrusions and depressions throughout . The aven = 200) . On larg7 cells/cm2  compared to the control hose biofilm (above). On large-scale, linear regression showed an ongoing decreasing trend over the length of the entire hose (R2 = 0.73). Small-scale heterogeneity between adjacent cm-sections was on average 17.2 \u00b1 15.2% (n = 198), thus comparable to results from the control hose biofilm. The combination of TCC and an average cell volume (0.3 \u03bcm3) accounted in this hose biofilm for an average bacterial cell volume of 1.1 \u00b1 0.4 \u00d7 107 \u03bcm3/cm2 (n = 200). This, in turn, allows the calculation of the relative contribution of bacterial cell volume to the overall biofilm volume (Vcells:Vbiofilm) which was about 1.2 \u00b1 0.5% (n = 200) and therefore considerably higher than in the control hose.Total cell concentrations of 1.2 cm-sections ranged between 1.5\u20138.1 \u00d7 10ells/cm2 , thus bep < 0.001). It should be noted that input water varied between these two locations, in addition to the differences in operation (n = 183). Also, random fluctuations between adjacent cm-sections showed variations in richness, with 15 \u00b1 14% (n = 165). These were less pronounced compared to ones in the control hose. Bray-Curtis dissimilarity showed variations in beta-diversity of adjacent sections, ranging from 0.04 to 0.55 , and again highlighting a similar pattern in community composition heterogeneity as the control hose biofilm. Moreover, only few taxa dominated the community composition , which was consistent with the control hose biofilm. Here, the 10 most dominant taxa accounted for 90.4% of the entire community composition  to the following sections 89\u201399 , corresponding to an increase of 27%  decreased to an average of 8.3 \u00b1 4.2% (n = 11) within the range of sections 79\u2013100 (2 < 0.2), with an exception in the relative abundance of Bradyrhizobium spp. which positively correlated with TCC (R2 = 0.37).On large-scale, no significant heterogeneity in the community composition was caused by the orientation of the hose , as was peration . Regardi. 34.7%, , purple,. 24.2%, , red, an. 14.2%, , yellow.bacteria . Consiste of 27% , purple.s 79\u2013100 , yellow.Microbial heterogeneity within large, but connected, ecosystems was previously characterized in both natural  and engiMicrobial heterogeneity within drinking water pipes was previously ascribed to variations in material properties, e.g., surface structure and adhesion characteristics , as welldispersal of cells from the source water and selection based on growth . As Swiss tap water is usually carbon limited . The inclusion of a second hose biofilm from an environment with arguably more variability in environmental conditions expanded the broader applicability of the findings to other systems. Both setups comprised identical material but showed differences in usage and incoming water compositions. As a result, the extent of the individual small-scale heterogeneity was different between the two biofilms, but also considerable differences between the two similar but disconnected  ecosystems were detected. Firstly, the biofilm of the real hose was ten-fold thinner than the one of the control hose , 4. It wBradyrhizobium spp.) and only two were similar on family level   , S5. Itagaceae) , based oagaceae) . The comagaceae) , 5C. Whiagaceae) .The differences between the control and the real hose were interesting. However, these hoses represent single examples from each environment  and thus provide insufficient replication for (1) representing biofilms of these environments in general and (2) for drawing definitive conclusions on the role of the environment on biofilm formation. Rather, the focus of this study was on the small-scale variations within the biofilms of each hose.Heterogeneity in microbial assemblages of connected ecosystems has been widely attributed to localized variations in environmental conditions . PatchinIn the real hose, which was installed vertically, gravity obviously impacted biofilm thickness differently, with particles likely accumulating in the lower bend. Here, we observed clear heterogeneity with thicker patches of biofilm in the lower section and a continuously decreasing gradient in TCC along the length of the hose . In addiIn both the control and the real hose biofilm, community composition showed heterogeneity on both large- and small-scale. For example, the relative abundance of some of the most dominant taxa changed on large-scale along the length of the hose, gradually as well as fluctuating. On small-scale, localized heterogeneity was observed for dominant taxa of both control and real hose biofilms , 5C, bei2; The assessment/characterization of small-scale heterogeneity within individual biofilms allows us to draw several conclusions regarding sampling and analysis strategies on a broader scale. Sample size and the required number and spatial distribution of sampling points within a given system are some of the most critical issues when considering biofilm sampling strategies. Across disciplines, biofilm characterization is often limited by the accessibility of the relevant surface which necessarily results in diverse sampling approaches. Consequently, sample sizes in biofilm studies range from microscopic analysis on \u03bcm-scale  to microIn the present study, the combination of individual results  allowed us to simulate larger sample sizes and to compare these results. For example, the average of ten adjacent samples provides the  outcome of sampling the length of 12 cm as one single sample. It is obvious that a sampled biofilm area should be as large as possible to obtain a characterization as close as possible to the average of an entire system . HoweverWhile smaller sampling areas result in large deviations from the overall average  and redu\u2022High-resolution sampling of shower hose biofilms (200 samples/120 cm) in addition to detailed analysis on various scales (\u03bcm\u2013m), enabled the assessment of small-scale spatial heterogeneity in biofilm structure, bacterial numbers, and community composition.\u2022A biofilm grown inside a flexible hose under controlled laboratory conditions, was likely uniformly exposed to processes such as dispersal, carbon migration, growth, and selection along its length. Accordingly, the respective biofilm was homogenous on large-scale, but showed notable localized heterogeneity on small-scale.\u2022A biofilm grown under real  use conditions showed considerably more variations in all variables on both large- and small-scale, with particularly clear spatial fluctuations in the relative abundance of dominant taxa.\u2022The control hose biofilm was different to the real hose biofilm with respect to thickness and community composition, which was most probably influenced by different operational conditions and water sources. However, both hoses showed impressively low biofilm community diversity, which was attributed to the selective force of the migrating carbon from the flexible PVC-P hoses.\u2022In addition, our results show that the adequate biofilm sample size strongly depends on the research question: whether the small-scale uniqueness of an ecosystem is explored , or whether an average overview of an entire system is required .The datasets generated for this study can be found in the NCBI Sequence Read Archive (SRA) number PRJNA554997.LN: experimental design, experimental work, data analysis, and manuscript writing. CP: experimental design, experimental work, and data analysis. J-CW: sequencing data processing and manuscript writing. FH: experimental design, experimental work, and manuscript writing.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "The data presented in this article are related to research article \u201cInvestigation on thermal and fire propagation behaviors of multiple lithium-ion batteries within the package\u201d . This data article provides the data information including the experiment pictures, flame temperatures, pressure and heat flux sensors temperatures, and gas concentrations of 6\u00a0\u00d7\u00a06 batteries and 10\u00a0\u00d7\u00a010 batteries. The video of the whole thermal and fire propagation behaviors of 6\u00a0\u00d7\u00a06 batteries is also provided. A plurality of thermocouples are placed above the battery pack to measure the flame temperatures of multiple lithium-ion batteries fire. The thermocouples are arranged as shown in The ambient temperature at which the pressure and heat flux sensors probes are located is an important parameter for examining their operating conditions. The positions of the pressure and heat flux sensors probes are specifically described in Ref Three simultaneous gas stream measurements of oxygen, carbon dioxide, and carbon monoxide are analyzed in the Servomex 4100 gas analyzer."}
+{"text": "Hirundo rustica, a migratory species, we found that plasma testosterone levels increased with increasing the proportion of eumelanin pigments compared with pheomelanin pigments, but not with the amount of pheomelanin pigments, during the mating period. In the Pacific swallow Hirundo tahitica, a nonmigratory congener, we found that, during severe winter weathers, survivors had a proportionally smaller amount of eumelanin pigments compared with pheomelanin pigments than that in nonsurvivors, but no detectable difference was found in the pheomelanin pigmentation itself. These results indicated that a minor pigment, eumelanin, matters at least in some physiological measures and viability. Because the major pigment, pheomelanin, has its own physiological properties, a combination of major and minor pigments provides multiple information to the signal receivers, potentially enhancing the signaling function of pheomelanic coloration and its diversification across habitats.Pigment\u2010based plumage coloration and its physiological properties have attracted many researchers to explain the evolution of such ornamental traits. These studies, however, assume the functional importance of the predominant pigment while ignoring that of other minor pigments, and few studies have focused on the composition of these pigments. Using the pheomelanin\u2010based plumage in two swallow species, we studied the allocation of two pigments  in relation to physiological properties and viability in populations under a natural and sexual selection. This is indispensable for studying the evolution of pheomelanin\u2010based plumage coloration. Pheomelanin and eumelanin share the same pathway only during their initial stages of development, which can be a key to unravel the functional importance of pigment allocation and thus of plumage coloration. Using the barn swallow,  Becauseh Figure , making h Figure . In facth Figure , and theh Figure . Then, wh Figure , is link22.1SE\u00a0=\u00a00.73\u00a0\u00b1\u00a00.33, F1,23\u00a0=\u00a04.81, p\u00a0=\u00a00.039). We also collected approximately 10\u201320 throat feathers to quantify the pheomelanin and eumelanin contents. Detailed information on the measurements can be found in previous publications , from March to May 2015 and 2016. In this area, barn swallows nest under the eaves of houses along streets  using the Scion Image software . The throat patch of each bird was traced twice, and the mean of the two measurements was used for further analyses , from 28 January to 6 February 2016. Pacific swallows inhabit this island throughout the year, and no barn swallows are wintering or breeding there. As in barn swallows, Pacific swallows . Sensitivity (80% maximum binding) and midpoint (50%) were 14.01 and 67.93\u00a0pg/ml, respectively. As recommended by the manufacturer's protocol, any sample outside the assay range, 3.9\u2013500\u00a0pg/ml, was discarded . The remaining samples were run in a single assay (using split with two mounts), and intra\u2010assay variation, calculated as the mean coefficient of variance of the duplicates, was 13% in the data set of 2015 . Because duplicates showed highly repeatable values, and because we lost some samples due to small volume per tube, we did a single analysis  for the samples collected in 2016 to prevent further sample loss. In 2016, mean blood plasma volume per tube was 20\u00a0\u00b5l (range: 7\u201333\u00a0\u00b5l) and standard curve was well fitted to the data (r\u00a0=\u00a00.99). Sensitivity (80% maximum binding) and midpoint (50%) were 7.97 and 56.22\u00a0pg/ml, respectively, in 2016. No samples fell outside the assay range of 3.9\u2013500\u00a0pg/ml in 2016. Cross\u2010reactivity of the assay is generally low, except for 5a\u2010dihydrotestosterone (5a\u2010DHT: 27.4%) as shown in the manufacturer's protocol, although the 5a\u2010dihydrotestosterone concentration is very low in barn swallows . For samples of very small volume per tube , we could not adequately separate sediments from the plasma sample, and thus, we discarded these samples  were added. The mixture was mixed vigorously at 25\u00b0C  for 20\u00a0hr. The residual H2O2 was decomposed by adding 20\u00a0\u03bcl 10% Na2SO3, and the mixture was then acidified with 56\u00a0\u03bcl 6\u00a0M HCl. After vortexing, the reaction mixture was transferred to a 1.5\u2010ml Eppendorf tube and centrifuged at 10,000\u00a0g for 1\u00a0min, and an aliquot (80\u00a0\u03bcl) of the supernatant was directly injected into the HPLC system. Given the amount of dilution used for the current study, amounts of degradation product from pheomelanin, thiazole\u20102,4,5\u2010tricarboxylic acid (TTCA), and that of eumelanin, pyrrole\u20102,3,5\u2010tricarboxylic acid (PTCA), were measured  according to the method described previously , which is direct selection on each trait , while other variables remained nonsignificant . When we added body condition as another explanatory variable, plasma testosterone level marginally increased with decreasing body condition , F\u00a0=\u00a07.88, p\u00a0=\u00a00.01, and nonsignificant remaining variables; F\u00a0<\u00a02.83, p\u00a0>\u00a00.11). Body condition was not significantly related to study year, pigmentation, and other male ornaments in the current data set, both in univariable and in multivariable linear models .The measure of eumelanin pigmentation, log(PTCA/TTCA), was significantly positively related to plasma testosterone levels in male barn swallows during the mating period Table . Pheomel3.2After statistically controlling for flight apparatus (wing and tail length), which were the main predictors of survival ), measure of eumelanin pigmentation levels (log(PTCA)) was significantly explained by sex and species (Table H.\u00a0tahitica had higher eumelanin levels than females and H.\u00a0rustica, respectively (Figure In 2016, we captured both es Table : After cy Figure .4The main finding of the current study is that the eumelanin, not pheomelanin pigmentation levels, consistently explained a physiological property, plasma testosterone level, during the mating period in barn swallows and survival of wintering Pacific swallows. Thus, some properties of pheomelanic plumage coloration, that is, its link with testosterone and survivorship, are at least in part due to higher amount of eumelanin in relation to pheomelanin pigmentation. Because some previous studies have demonstrated that pheomelanin pigmentation is linked to other physiological properties (e.g., resistance to oxidative stress: Roulin et al., l\u2010tyrosine or a higher level of tyrosinase activity than those of birds with less eumelanin. Because testosterone influences tyrosinase stimulation (e.g., male willow ptarmigan Lagopus lagopus lagopus; Stokkan, Theoretically, the positive link between eumelanin deposition and plasma testosterone level is predictable, because the eumelanin production pathway is linked to testosterones via melanocortin systems (Ducrest et al., The winter survival in relation to eumelanin pigments in the Pacific swallow is also consistent with the theoretical expectation of the effects of testosterone levels on survival (Ketterson & Nolan, In conclusion, we found that a minor pigment, that is, eumelanin pigment level in pheomelanic plumage, could indicate some physiological properties together with the major pigment, pheomelanin, rather than mere by\u2010product of major pigments. Because pheomelanin pigmentation has its own properties (e.g., glutathione\u2010mediated antioxidant capacity: Arai et al., None declared.MA and EA conceived the ideas, designed methodology, collected field data, and wrote the drafts. SI and KW helped EA to do pigment analysis. MS and HS helped MA and EA to do ELISA. All authors contributed to the drafts and gave final approval for publication.\u00a0Click here for additional data file."}
+{"text": "Here we present both sex-averaged and sex-specific linkage maps for Coregonus sp. \u201cAlbock\u201d, a member of the European whitefish lineage (C. lavaretus spp. complex), containing 5395 single nucleotide polymorphism (SNP) loci across 40 linkage groups to facilitate future investigation into the genomic basis of whitefish adaptation and speciation. The map was produced using restriction-site associated digestion (RAD) sequencing data from two wild-caught parents and 156 F1 offspring. We discuss the differences between our sex-averaged and sex-specific maps and identify genome-wide synteny between C. sp. \u201cAlbock\u201d and Atlantic Salmon , which have diverged following the salmonid-specific whole genome duplication. Our analysis confirms that many patterns of synteny observed between Atlantic Salmon and Oncorhynchus and Salvelinus species are also shared by members of the Coregoninae subfamily. We also show that regions known for their species-specific rediploidization history can pose challenges for synteny identification since these regions have diverged independently in each salmonid species following the salmonid-specific whole genome duplication. The European whitefish map provided here will enable future studies to understand the distribution of loci of interest, e.g., FST outliers, along the whitefish genome as well as assisting with the de novo assembly of a whitefish reference genome.Genomic datasets continue to increase in number due to the ease of production for a wider selection of species including non-model organisms. For many of these species, especially those with large or polyploid genomes, highly contiguous and well-annotated genomes are still rare due to the complexity and cost involved in their assembly. As a result, a common starting point for genomic work in non-model species is the production of a linkage map. Dense linkage maps facilitate the analysis of genomic data in a variety of ways, from broad scale observations regarding genome structure  However, due to the \u223c50 million-year divergence time between Salmoninae and Coregoninae, and the limited number and density of whitefish linkage maps, the analysis of genomic whitefish datasets to answer questions about the physical distribution of loci and their function is limited  . We identify rearrangements present between the two species which reflect the occurrence of fission and fusion events following the Ss4R whole genome duplication, some of which were confidently identified to be shared only between members of the Salmoninae subfamily in past studies. We also discuss the results of our synteny mapping in the context of the rediploidization history of salmonids. This Coregonus linkage map will facilitate future research regarding the genomic basis of adaptation in the adaptive radiation of Swiss whitefish and assist with the ongoing de novo assembly of the whitefish genome.In this study we produce a detailed linkage map for Alpine whitefish using a RAD sequencing approach. We produced both sex-specific and sex-averaged linkage maps for Coregonus sp. \u201cAlbock\u201d, a formally undescribed species which is one member of the European whitefish lineage (C. lavaretus spp. complex). Coregonus sp. \u201cAlbock\u201d likely originates from an introduction of whitefish from Lake Constance into Lake Thun and taxonomic description of the species is in progress. The parental whitefish collected from Lake Thun in December 2016 were crossed in vitro by mixing sperm and eggs  together before adding cold water to harden successfully fertilized eggs. Fertilized eggs were then placed in a flow-through system which ran 5\u00b0 lake water over the eggs for 11 weeks until they began to hatch. Before larvae had fully utilized their yolk sac they were sedated and killed with MS222  and preserved in 100% ethanol .One F1 family consisting of two parents and 156 offspring was used for linkage map construction. Both parent whitefish were sexually ripe, adult, Sbf-1 enzyme, which has been shown to digest salmonid DNA effectively . The DNA concentration of each extract was measured using the Qubit 1.0 Fluorometer (Thermo Fisher). In total five RAD libraries were made, with 44 F1 samples pooled into each of the four offspring RAD libraries and the two parental samples pooled into a fifth library. Each library was produced following the protocol of process_radtags module in Stacks version 1.40  resulting in individual alignment files. The GATK Haplotype Caller . Offspring can therefore be heterozygous or homozygous  and the phasing/origin of each allele is known. In addition to these highly informative loci, loci for which both parents are heterozygous can also provide information in the offspring in certain linkage mapping programs . In these cases, three offspring genotypes may be observed e.g., AA, Aa, aa in an expected ratio of 1:2:1 with only homozygous offspring being informative since we know that one copy of each allele is from each parent . Heterozygous offspring genotypes are uninformative since the origin of each allele is unknown . Loci were then filtered in R . Linkage groups were then identified using SeparateChromosomes2 with a logarithm of odds (LOD) score of 16 (lodLimit = 16) and the minimum number of markers per linkage group set to 25, resolving 40 linkage groups , three times on each linkage group to produce a male and a female linkage map. This procedure was then repeated specifying a sex-averaged map (sexAveraged = 1). The marker orders with the highest likelihoods for each linkage group for each type of map were combined to produce the final most likely male and female sex-specific maps and one final sex-averaged map, each positioning the same 5395 SNP markers. A custom R script was used to calculate differences in the marker densities and lengths between maps and the sex-averaged map was plotted using MapChart , the Lep-MAP inpute file and all three linkage maps are available at Figshare: https://doi.org/10.25387/g3.7093799.Fastq files for all 156 offspring and both parents are deposited in the NCBI short read archive (SRA accession PRJNA478121). All R, Python and bash scripts used can be accessed at C. sp. \u201cAlbock\u201d adults. Both parents and 156 F1 offspring were successfully genotyped using a RAD-seq approach. In total 9757 SNPs were retained following stringent quality control and loci filtering steps, with 7800 identified as informative in Lep-MAP3  from 0.31 cM in Calb04 to 0.99 cM in Calb35. The average inter-marker distance in the male map was 0.46 cM with the smallest and largest ratios found in Calb12 and Calb39 respectively with 0.18 cM and 1.05 cM.Our sex-averaged map has high resolution, with a low average distance between adjacent markers of 0.46 cM, varying from 0.27 cM in Calb04 to 0.77 cM in Calb34. The linkage map of the close relative C. sp. \u201cAlbock\u201d. Comparing total map lengths for the female and male maps gives a female:male recombination ratio of 1.09, however, this does not account for the two whitefish linkage groups which have length 0 cM in our female map . Calculating this female:male recombination ratio for each linkage group separately, including only those > 0 cM in both maps, results in a ratio of 1.25. Salmonid species have been shown to have sexual dimorphisms in recombination rate with published female:male recombination ratios varying from 1.38 in Atlantic Salmon (Sex differences can be observed by comparing our sex-specific linkage maps for P = 5.468x10\u221211) and conclude that this is the result of the mapping parameters we used , meaning mappings are evenly distributed between AORe chromosome arms. Mappings to chromosome arms which make up collinear LORe blocks are not expected to be unique, lowering the mapping confidence  of loci there, which resulted in the filtering out of these mappings. Confident mappings within LORe regions are therefore scarce because these regions do not follow the 1:1 ohnologue orthology that we required through our mapping parameters to keep markers.Synteny analysis was carried out to investigate broad scale genome structural variation, such as fission and fusions of chromosomes or chromosome arms, within the Salmonidae family. Stringent filtering of mapped RAD loci to the salmon genome was applied to identify synteny while excluding uncertain mappings. From 5395 loci included in our linkage map we retained 839 mappings of high quality, which were spread across all 40 whitefish linkage groups . Synteny we used . These pC. clupeaformis linkage map and these regions, carried out by The prevalence of delayed rediploidization is likely the reason that three salmon chromosomes, Ssa02, Ssa08 and Ssa26 were not identified as homologs to any of our whitefish linkage groups, with Ssa08 having no significant mappings at all. All three of these chromosomes, specifically the Ssa08q, Ssa02p, Ssa02q and Ssa26 arms, are LORe regions and the lack of markers mapped to these regions in our analysis is likely caused by an abundance of 2:2 orthology between salmon and whitefish. Markers which might have mapped to these salmon chromosomes have likely been filtered out due to their poor mapping scores. This may also underpin the similarly uncertain assignment of synteny between the Only a small number of markers on each whitefish linkage group mapped to a different salmon chromosome than the identified homologous chromosome  the patterns of synteny we observe as well as those identified by While multiple salmonid linkage maps, including those of C. clupeaformis. It is therefore possible that a fission event has occurred in the European whitefish lineage, however, due to relatively low number and density of markers on W38 and W39 future investigation should aim to clarify this pattern.We also identify one possible European whitefish-specific fission event with markers from both W38 and W39 mapping to Ssa28, an AORe dominated chromosome which is homologous to only one linkage group in each salmonid species compared by Oncorhynchus species, however, synteny with C. clupeaformis, the only member of Coregoninae included in these comparisons, was less clear , Ssa10 (to W14 and W15), Ssa13 (to W19 and W20), Ssa15 (to W22 and W23), Ssa16 (to W24 and W25), Ssa18 (to W27 and W28) and Ssa20 (to W30 and W31)  . In addiand W31) .C. clupeaformis but only one linkage group in all other salmonids. Our C. sp. \u201cAlbock\u201d map identifies synteny from only one linkage group, W10, to Ssa07 and similarly from W32 to Ssa21 suggesting the pattern of synteny may not be conserved between Coregonus species. Since Ssa07q is a LORe dominated chromosome arm the lack of synteny identified to a second whitefish linkage group may be the result of the lack of 1:1 ohnolog orthology and therefore a lack of confident mappings. The pattern of Ssa21 on the other hand most likely represents a difference between C. cluepeaformis and C. sp. \u201cAlbock\u201d since Ssa21 has an expected/observed mappings ratio of 0.94 (close to 1) and a high density of markers. Further work must therefore be carried out to better identify potential genome structural variation between C. sp. \u201cAlbock\u201d and C. clupeaformis.Two salmon chromosomes, Ssa07 and Ssa21 were shown by e.g., with regards to Ssa14, Ssa19, Ssa21 and Ssa28) represents true variation between these species or variation in linkage mapping resolution and accuracy. A comparison of synteny between our C. sp. \u201cAlbock\u201d map and the Atlantic Salmon (using our synteny mapping approach) and the C. clupeaformis map to the Atlantic Salmon , Arctic Charr (Salvelinus alpinus; NCBI BioProject: PRJNA348349; Thymallus thymallus; A wealth of genomic resources used to study adaptation and speciation are now available for a variety of systems. Multiple species from popular model radiations including Galapagos finches (Coregonus genus. Our linkage map can also be paired with future resources to investigate the outcome of whole genome duplication including estimations of the rediploidized proportion of the genome, already calculated in Atlantic Salmon. Future work should further aim to identify regions of the genome which may underpin reproductive isolation in whitefish to better understand the speciation mechanism in this adaptive radiation.Our linkage map fills a gap in the resources available to analyze European whitefish genetic data allowing investigation into this species rich, ecologically diverse, lineage. The patterns of synteny between European whitefish and Atlantic Salmon reported here should be further investigated once whitefish genomes become available to identify synteny at a finer scale, identifying chromosome fission and fusion events and possible inversions also within the Coregonus linkage map to date, with a total sex-averaged map length of 2293.86 cM containing 5395 SNP loci. We have found evidence of sex-specific recombination rate variation within C. sp. \u201cAlbock\u201d by calculating the female:male recombination ratio i.e., a ratio of female and male linkage map lengths. The level of heterochiasmy inferred by this ratio is reflected in other species with known sex-specific recombination variation, including other salmonids (C. sp. \u201cAlbock\u201d linkage groups exhibit synteny with Atlantic Salmon chromosomes, in some cases following a pattern of synteny shared with other salmonid species. This linkage map will facilitate a host of future studies into the genomic basis of adaptation in Alpine whitefish including those on the identification of QTL for traits of interest, the interpretation of genome-wide divergence data and the colocalization of regions under selection e.g., FST outliers identified from genome scans. It also has the potential to assist in the ongoing assembly of Alpine whitefish reference genomes.In conclusion, we have produced the densest"}
+{"text": "Melanin denotes a variety of mammalian pigments, including the dark electrically conductive eumelanin and the reddish, sulfur-containing, pheomelanin. Organic (bio)electronics is showing increasing interests in eumelanin exploitation, e.g., for bio-interfaces, but the low conductivity of the material is limiting the development of eumelanin-based devices. Here, for the first time, we report an abrupt increase of the eumelanin electrical conductivity, revealing the highest value presented to date of 318 S/cm. This result, obtained via simple thermal annealing in vacuum of the material, designed on the base of the knowledge of the eumelanin chemical properties, also discloses the actual electronic nature of this material's conduction. In the 1974, McGinness et al. reported the first experimental evidence of the semiconducting behavior of the eumelanin . We name the obtained material as High Vacuum Annealed Eumelanin, HVAE.Conductive eumelanin films were prepared via the preliminary oxidative polymerization of the solid state form of DHI , MALDI-MS analysis was done using a positive reflectron MALDI and LDI spectra were recorded on a Sciex 4800 MALDI ToF/ToF instrument. Grazing Incidence Wide Angle X-ray Scattering (GIWAXS) was run with a Fr-E+ SuperBright rotating anode microsource  equipped to a three-pinhole camera (Rigaku SMAX-3000) through a multilayer focusing optics . Elemental composition  was estimated using a Perkin\u2013Elmer 2400 CHNSO elemental analyzer. Measurements of electrical resistance vs. temperature were performed measuring the two-terminals devices of one type of the HVAE  in a probe station CASCADE Summit 11000B-M, featuring a closed chamber with thermal chuck, keeping the samples in constant flow (10 L/min) of pure dry Nitrogen, allowing the temperature to stabilize within \u00b11\u00b0C before each measurements run, and using a Keithley 4200 SCS Semiconductor Characterization System to acquire the electrical data.All the commercially available reagents and materials were used as received. All the solvents were analytical grade quality. The DHI was prepared according to a reported procedure  in a sealed chamber at 1 bar pressure. The material so obtained is here named DHI-eumelanin, to distinguish it from the starting DHI, and from the final HVAE. The thicknesses of the DHI-eumelanin films were 260 \u00b1 6 nm. Films showed the typical dark brown color of the eumelanin (The eumelanin formation was obtained by the oxidation of the DHI films thanks to the Ammonia-Induced Solid State Polymerization (AISSP) method, a recently developed solid state protocol ; some samples were also annealed at various time lengths (from 30 min up to 6 h). The processes were performed in a dedicated high vacuum chamber using a turbomolecular pump to obtain the vacuum level, and doing preliminary leak detection and samples temperature verifications. The mean thickness of the HVAE films was dependent on the annealing conditions, with the smallest values down to 110 \u00b1 2 nm for the processes at 600\u00b0C longer than 1 h  in high vacuum conditions   inspection confirmed the retaining of the high quality morphology of the HVAE films , showingUV-Vis spectra, observed at the different process steps , show anStrong support to the picture of a structural reorganization and an enhanced packing order  following the reduction of O and N contribution. Consistent information is provided by the FTIR spectra of the DHI-eumelanin and the HVAE films (\u22121) and to the water  , cleaning the storage chamber each time it was opened using pure dry nitrogen (oxygen and water vapor content below 5 ppm).The electrical properties of the materials were measured using two set-ups, because of the different conductivity values that samples presented. Between the various measurements runs, the samples were stored in mild vacuum . The observed values of R and the trend of R vs. T reveal that not simple mechanisms are operating for the conductivity of the material: the small values of R indicate that it is a good electronic conductor (Le et al., Measurements of electrical resistance vs. temperature were also performed , measuriad-hoc eumelanin-based active layers for a wide range of applications in organic electronics and bioelectronics, deserving further extensive investigations to get a conclusive picture about the conductor vs. semiconductor behavior of the eumelanin and insights about the mobility of the charge carriers.Results here reported indicate a radical modification of the actual picture of the eumelanin charge transport properties, reversing the paradigm according to which eumelanin conductivity increases with the water content of the material. Indeed, if the eumelanin films are rearranged into conductive layers, thanks to a simple thermal annealing in vacuum which succeeds in inducing a structural reorganization of their molecular constituents, the contribution of the electronic current is here demonstrated to be largely preeminent with respect to the reported ionic one (Mostert et al., All datasets generated for this study are included in the manuscript and/or the All authors conceived the experiments. LM and PM with contributions by AP, PT, and DA carried out the measurements. LM, AP, and PT processed and analyzed experimental data. LM fabricated all the samples. All the authors discussed the results and wrote the main manuscript. AP, PT, CG, MGM and CM contributed to refine the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "Salmonella biofilms act as a continuous source for cross-contamination in the food processing environments. In this study, a stable rugose morphotype of Salmonella was first induced by sequential exposure to subinhibitory concentrations (SICs) of sodium hypochlorite (NaOCl) (ranging from 50 to 300 ppm over 18-day period) in tryptic soy broth. Then, rugose and smooth morphotypes of Salmonella Typhimurium ATCC 14028 and Salmonella Heidelberg ATCC 8326 were characterized for biofilm forming abilities on polystyrene and stainless steel surfaces. Rugose morphotype of both ATCC 14028 and ATCC 8326 exhibited higher Exopolysaccharide (EPS) formation than smooth morphotype (p \u2264 0.05). Also, the SICs of NaOCl (200 or 300 ppm in broth model) increased the biofilm formation ability of rugose morphotype of ATCC 8326 (p \u2264 0.05) but decreased that of ATCC 14028. The 2-day-old Salmonella biofilms were treated with biocidal concentrations of 50, 100, or 200 ppm NaOCl (pH 6.15) in water for 5, 10, or 20 min at room temperature. The biofilm reduction in CFU/cm2 for the rugose was lower than the smooth morphotype on both surfaces (p \u2264 0.05) by lethal NaOCl in water. Scanning electron micrographs on both polystyrene and stainless steel surfaces demonstrated that the rugose morphotype produced a denser biofilm than the smooth morphotype. Transmission electron micrographs revealed the cell wall roughness in rugose morphotype, which may help in tolerance to NaOCl. The gene expression data indicate that the expression of biofilm regulator (csgD), curli , and cellulose (bcsE) was significantly increased in rugose morphotype when induced by sequential exposure of NaOCl SICs. These findings reveal that the rugose morphotype of S. Typhimurium and S. Heidelberg produced significantly denser biofilm on food contact surfaces, which also increased with sequential exposure to SICs of NaOCl in the case of S. Heidelberg, and these biofilms were more tolerant to biocidal NaOCl concentrations commonly used in the food processing plants. Salmonella is responsible for 380 deaths and 19,000 hospitalizations in the USA , quaternary ammonium compounds (QAC), and organic acids such as peroxyacetic acid and acetic acid are frequently used . PreviouEscherichia coli are in the shorter rod shaped form. Previous study shows the ability of Salmonella to switch to rugose morphotype in tryptic soy agar within 3 days at 25\u00b0C , which are associated with morphological adaptation . Liu et  at 25\u00b0C . Rugositcolonies . This mocolonies .Salmonella (csgBAC operon) and cellulose  are the two major components of Salmonella biofilm. CsgD regulates the biofilm synthesis by activating the transcription of curli and cellulose synthesis genes , and histone-like nucleoid structuring (H-NS) proteins in response to environmental cues and metabolic stress. Other genes such as spiA    also plaSalmonella and Vibrio cholerae confers tolerance to their planktonic cells against NaOCl  and abiotic factor  . These fSalmonella to sublethal NaOCl induced rugose morphotype  to determine the efficacy of lethal NaOCl in water for inactivating such rugose biofilms on two different food contact surfaces, polystyrene and stainless steel; and (3) to determine the biofilm- and rugose-related gene expression in S. Typhimurium ATCC 14028 induced by sublethal NaOCl.In our previous study, we reported that the adaptation of rphotype . HoweverSalmonella serotypes, S. Typhimurium ATCC 14028 and S. Heidelberg ATCC 8326, were used in the present study. These strains were stored as a stock culture in tryptic soy broth  supplemented with 25% glycerol at \u221280\u00b0C. Before experiments, cells from frozen stock were streaked on tryptic soy agar  plates and incubated for 24 h at 37\u00b0C. Later, a subculture was obtained after inoculating a single colony into 10 ml of TSB and incubation at 37\u00b0C for 24 h.Two NaOCl solution  was used as a stock solution for available chlorine. This stock was stored in dark and at 4\u00b0C. Fresh solution of NaOCl concentrations of 200, 300, 400, 500, and 600 ppm in TSB for MIC assay and the 50, 100, and 200 ppm of NaOCl concentrations in sterile water for biofilm inactivation assay were prepared using the 5% stock NaOCl solution before each experiment.Salmonella cell concentration of 6 log CFU/ml was obtained from serial 1/10 dilution of smooth morphotype grown at 37\u00b0C for ~22 h, followed by addition of different NaOCl concentrations  in 15 ml polypropylene tubes. Thereafter, the tubes were incubated for 24 h at 37\u00b0C. The lowest concentration of NaOCl in TSB, which inhibited the visible turbidity after 24 h as per CLSI 2016, was determined as MIC, while below the MIC concentrations, which did not inhibit bacterial growth, were selected as SIC.The determination of minimum inhibitory concentration (MIC) and subinhibitory concentration (SIC) of NaOCl were determined using the macrodilution method in TSB as per CLSI, 2016. To determine MIC, Salmonella serotypes were exposed to increasing NaOCl concentration to induce rugosity development. The inoculum of 106 cells/ml was sub-cultured with 50 ppm of NaOCl in TSB for 3 consecutive days at 37\u00b0C for 24 h. Thereafter, a similar subculture pattern was followed to induce stress in 50 ppm NaOCl increments every 3 days to a maximum concentration of 300 ppm (18 days of stress). Thereafter, the rugose morphotype  was exhibited on TSA at 37\u00b0C after 24 h. For control cells, Salmonella smooth cells were subcultured in a similar manner but without NaOCl exposure in TSB. The stability of rugose morphotype was determined after subculturing cells in TSB and TSA without NaOCl. Rugose cells developed characteristic corrugated, dry and rough morphology on TSA after 24 h at 37\u00b0C. Later, rugose cells were grown overnight in TSB at 37\u00b0C and harvested by centrifugation. A log of 109 cells/ml were stored as frozen in 25% glycerol stock at \u221280\u00b0C for further experiments. The MIC for both morphotypes (smooth and rugose) of both Salmonella serotypes for NaOCl was determined using the macrodilution method in TSB as previously described. Further, to observe difference in rugose and smooth colony formation, an aliquot of bacterial load (103 CFU/ml) of both the morphotypes was plated on LB agar containing Congo red (40 mg/L) and coomassie brilliant blue (20 mg/L). The plate was incubated at 37\u00b0C for up to 48 h. Thereafter, images of Salmonella smooth and rugose morphotype colony edge pictures were acquired and analyzed by EVOS XL Core inverted digital microscope with 4X objective.Smooth morphotype of both 6 CFU/ml. Biofilm was developed after inoculation of 200 \u03bcl TSB culture containing 6 log CFU/ml of bacteria in individual wells of a 96 well polystyrene microtiter plate  and incubation at 25\u00b0C for 48 h. After 48 h of incubation, bacterial inoculum was aspirated out from each well of a plate. Thereafter, the wells were washed five times with sterile distilled water to remove all loosely attached cells. The plate was air dried for 45 min and biofilm was stained with 250 \u03bcl of 0.41% w/v crystal violet dye  for 45 min. The dye was pipetted out from individual wells and to remove excess of dye, wells were washed five times with sterile distilled water. Plates were air dried for 45 min and thereafter, stained wells were destained with 95% ethyl alcohol. The OD600 was measured using a micro-quant microplate spectrophotometer . The experiment was replicated four times with eight replicate wells used for each treatment.Biofilm formation of rugose and smooth morphotypes was measured by crystal violet assay . An over6 CFU/ml was added into individual well of a 96-well polystyrene microtiter plate  in duplicate. Plate was incubated for 48 h at 25\u00b0C. Each well was aspirated, and loose or unbound cells were removed by washing wells three times with sterile PBS. An aliquot 200 \u03bcl of 0.1% buffered peptone water was added into individual wells and strongly attached biofilm cells were removed with sterile cotton swab method  and 5 ml of 0.1% buffered peptone water (Stainless steel (SS) coupons  were used for biofilm formation and enumeration. SS coupons were autoclaved at 121\u00b0C for 15 min prior to use. The SS coupons were immersed completely in individual wells of 24 well plates with 2 ml of ne water . Tubes wg for 10 min at 4\u00b0C. The obtained pellet was resuspended in fresh TSB and serially diluted to obtain a cell concentration of 106 CFU/ml. Thereafter, cells (200 \u03bcl) were transferred to each one of the eight wells of a 96-well microtiter plate. The plate was incubated for 48 h at 25\u00b0C and loose or unattached cells were removed by pipetting out the bacterial inoculum. Each well of the plate was washed three times with sterile PBS to remove loosely or unbound cells. Plate was air dried at room temperature for 45 min and wells were added with 250 \u03bcl of ruthenium red (0.1%) stain solution. The biofilm was stained for 45 min at room temperature. Wells without Salmonella biofilms were also added with 250 \u03bcl of ruthenium red (0.1%) stain solution to act as blanks. After staining, the residual ruthenium red stain solution from each well was carefully transferred to individual wells in a new polystyrene 96-well microtiter plate. The EPS production was measured by subtracting the OD600 of a blank well (ODB) value to the OD600 value of the residual stain (ODS) collected from sample wells respectively. The OD600 was measured using a micro-quant microplate spectrophotometer . The experiment was replicated four times with eight replicate wells used for each treatment.Exopolysaccharide (EPS) formation by rugose and smooth morphotypes of each strain was measured by ruthenium red assay . OvernigSalmonella biofilm formation of each strain was obtained in a 96-well microtiter plate in the presence of subinhibitory NaOCl of 200 or 300 ppm in TSB (treatment) or in the absence of NaOCl exposure (control) at 25\u00b0C for 48 h. The organic content of TSB interacts with NaOCl and reduces its bioavailability. Thus, the NaOCl concentrations of 200 or 300 ppm were used in TSB. Biofilm formation after subinhibitory NaOCl exposure was measured by crystal violet assay  in sterile water for 5, 10, or 20 min at room temperature. The pH 6.15 was adjusted with 1 N HCl in these aqueous NaOCl solutions. While NaOCl of 200\u2013300 ppm was considered sublethal concentration in broth, but even 50 ppm was found to be biocidal in water since there is no organic content to neutralize NaOCl. After chlorine exposure, NaOCl solution was pipetted out from individual wells and they were washed with 250 \u03bcl of sterile PBS three times to remove any remnant of NaOCl. An aliquot of 200 \u03bcl of 0.1% buffered peptone water was added in each well and cells were quantified following removal from the well surface with a sterile cotton swab  at 25\u00b0C for 48 h. The SS coupons were immersed completely in individual wells of 24-well plates with 2 ml of Salmonella 6 log CFU/ml culture/well. After biofilm formation, planktonic cells were removed by pipetting out and coupons were washed three times with sterile PBS to remove loosely attached cells. Each biofilm on coupons was exposed to 0, 50, 100 and 200 ppm NaOCl (pH 6.15) concentration for 5, 10, or 20 min. Biofilm cells on SS surfaces, which survived after inactivation assay were quantified as discussed before. The minimum detection limit by this method was 50 CFU/SS coupon. The experiment was replicated four times.The biofilm of rugose and smooth morphotype of each strain of S. Typhimurium ATCC 14028 was grown to early stationary phase in TSB at 37\u00b0C for 15 h. The total RNA was extracted using TRIzol as described before . Biofilm was fixed using 1/2 strength Karnowsky\u2019s fixative (pH 7.2). Fixed biofilms were stained with 250 \u03bcg/ml alexa flour 647 conjugate, 10 \u03bcM syto 9 and 10x sypro red  stains successively for 30 min each to identify formation of different components of biofilm such as EPS, nucleic acid and proteins, respectively. Fluorochromes were excited using a kryptoneargon mixed-gas laser with a PMT2 filter. Randomly selected four different fields were analyzed to observe the biofilm formation of rugose and smooth morphotype.To observe the three-dimensional distributions of cellular and extracellular components of both morphotypes of both Salmonella biofilm was developed on Nunc Thermanox polystyrene cover slips  and the SS coupons at 25\u00b0C for 48 h. Coverslips and coupons were washed three times with sterile saline to remove loosely attached cells and fixed in 1/2 strength Karnowsky\u2019s fixative (pH 7.2) overnight at 4\u00b0C. Both coverslip and coupons were washed three times with sterile distilled water and post fixed in 2% buffered (0.1 M sodium cacodylate) osmium tetroxide, followed by dehydration through a graded ethanol series . The coverslips and coupons were later dried using a critical point dryer  and sputter-coated with platinum (20 nm). Thereafter, coverslips were analyzed on a scanning electron microscope  to obtain micrographs. Four randomly selected areas were analyzed to study rugose and smooth morphotype biofilm formation.Rugose and smooth morphotype biofilm morphology was also studied by using scanning electron microscopy. S. Typhimurium (ATCC 14028) was prepared for transmission electron microscopy (TEM) by previously described methods (g for 10 min at 4\u00b0C and the resulting concentrated cell pellets were prepared for TEM. Pellets were fixed using 1/2 strength Karnovsky fixative in 0.1 M sodium cacodylate buffer (pH 7.2) overnight at 4\u00b0C. Fixed cells were washed in buffer, post fixed in 2% buffered osmium tetroxide, dehydrated through a graded ethanol series, and embedded in Spurr\u2019s resin. Ultra-thin sections were cut using a Riecher Jung Ultra cut microtome, collected on copper grids and stained with uranyl acetate and lead citrate and viewed on a JEOL JSM-1230  at 80 kv. Four randomly selected areas were analyzed to study the ultrastructure changes in rugose and smooth morphotype.Ultrastructural morphology of rugose and smooth morphotype of  methods . Overnig600 values were analyzed in a 2 (strain) \u00d7 2 (morphotype) factorial arrangement of treatments in a randomized complete block design. Biofilm measurement by CFU enumeration by plate counting was replicated three times for each strain independently and CFU/cm2 values were analyzed in 2 (surface) \u00d7 2 (morphotype) factorial arrangement of treatments in a randomized complete block design. The biofilm formation in the presence of subinhibitory NaOCl experiment was replicated independently for each strain four times and OD600 values were analyzed in a 2 (morphotype) \u00d7 4 (NaOCl concentrations) factorial arrangement of treatments in a randomized complete block design. The biofilm inactivation experiment was replicated independently for each strain (three times on polystyrene and four times on SS) and log-transformed counts were analyzed in a 2 (morphotypes) \u00d7 4 (NaOCl concentrations) \u00d7 3 (incubation times) factorial arrangement of treatments in a randomized complete block design. The means were separated using Fisher\u2019s protected LSD when p \u2264 0.05. The gene expression data was analyzed by using Student\u2019s t-test for the comparisons between smooth and rugose morphotypes. The data was analyzed using Statistical Analysis Software (SAS V 9.4) .The crystal violet biofilm measurement assay and ruthenium red EPS measurement assay were replicated four times and ODS. Typhimurium ATCC 14028 and S. Heidelberg ATCC 8326 smooth morphotype was 400 ppm, while it was 500 ppm for rugose morphotype in TSB. Results showed that higher concentration of NaOCl was required to inhibit planktonic rugose morphotype growth in TSB. Regardless of these differences between morphotypes, the SICs used were 200 and 300 ppm of NaOCl in TSB for both smooth and rugose of ATCC 14028 and ATCC 8326.The MICs of NaOCl for Salmonella cell aggregates and pellicle formation were observed after daily passages for 18 days in the presence of subinhibitory NaOCl in TSB at 37\u00b0C. An aliquot of overnight incubated rugose culture in TSB was plated on TSA and characteristic corrugated and dry morphology was formed after 24 h at 37\u00b0C. Rugose morphotype was stable at 37 and 25\u00b0C and did not revert to smooth morphotype after several subculturing on TSA without NaOCl. Smooth morphotype formed homogenous suspension in TSB, however, rugose colonies were aggregated and elastic even after suspending in TSB. On Congo red agar, smooth morphotype formed smooth edge colonies with no corrugations while rugose morphotype formed characteristics red, dry and rough (rdar) colonies and developed corrugate edged patterns at 37\u00b0C after 48 h. Both S. Typhimurium ATCC 14028 and S. Heidelberg ATCC 8326 formed rdar morphology on Congo red agar (red agar .S. Typhimurium ATCC 14028 and S. Heidelberg ATCC 8326 produced significantly higher biofilm biomass than the smooth morphotype on polystyrene surface at 25\u00b0C (p \u2264 0.05)  where the rugose morphotype formed biofilm with cell density of 7.2 log CFU/cm2 and smooth morphotype formed biofilm with cell density of 6.2 log CFU/cm2 (S. Typhimurium ATCC 14028 biofilm of rugose morphotype showed more exopolysaccharides (violet color), protein compounds (red color) and nucleic acid (green color) than smooth morphotype  . The OD6photypes . The CFU CFU/cm2 . Scanninrphotype .S. Typhimurium ATCC 14028 and S. Heidelberg ATCC 8326 produced significantly higher EPS compounds after 48 h on the polystyrene surfaces than smooth morphotype (p \u2264 0.05)  . EPS prorphotype . EPS staS. Typhimurium ATCC 14028 reduced significantly in the presence of SICs (200 and 300 ppm) of NaOCl (p \u2264 0.05), while the smooth morphotype formed significantly lower biofilm at 300 ppm but not at 200 ppm (p \u2264 0.05)  of NaOCl (p \u2264 0.05), while these SICs did not affect biofilm formation ability of smooth morphotype (The SICs of NaOCl can significantly affect biofilm formation ability of both rugose and smooth morphotypes depending on the tested strain. Biofilm formation of rugose morphotype of  \u2264 0.05) . For S. rphotype .S. Typhimurium ATCC 14028 and S. Heidelberg ATCC 8326 48 h old biofilms on 96-well polystyrene microtiter plates at 25\u00b0C is presented in Salmonella morphotypes, (2) NaOCl concentrations, and (3) exposure time was investigated. A significant three-factor interaction (NaOCl concentrations \u00d7 exposure time \u00d7 morphotypes) was found for S. Typhimurium ATCC 14028 . By contrast, 3\u20134 logs of rugose biofilm cells survived even after 200 ppm NaOCl treatment for 20 min on polystyrene surface  was found for TCC 8326 . Biofilmreatment .S. Typhimurium ATCC 14028 and S. Heidelberg ATCC 8326 biofilms on SS coupons at 25\u00b0C is presented in S. Typhimurium ATCC 14028, 2 logs of smooth morphotype biofilm cells survived after 50 ppm NaOCl for 5 min, and were then non-detectable after 100 or 200 ppm NaOCl regardless of exposure times. By contrast, the rugose morphotype biofilm cells of S. Typhimurium ATCC 14028 survived by 2\u20133 logs after 50 ppm NaOCl treatment at all exposure times, and by 1\u20132 logs survival after 5 or 10 min exposure to 100 ppm NaOCl, and were then non-detectable at 200 ppm of NaOCl  was found for biofilm inactivation of pm NaOCl ,B.envZ, csgD, csgA, csgB, csgC, bcsE, hns, rpos, and csrA by \u223c 139.5, 100.3, 2.84, 11.03, 11.3, 2.35, 3.06, 6.36, and 5.67 fold, respectively  temperature stress  formed rugose after NaOCl adaptation at 25 and 37\u00b0C in nutrient rich medium. In another study 4% ethanol in growth medium induced higher transcription of csgD resulted in formation of rugose during logarithmic growth phase. Similarly, microaerophilic conditions in nutrient rich medium  induced higher tendency to form rugose compared to aerobic and anerobic conditions  pH e stress , and (3)e stress . Anrianynditions .Salmonella rugose strains were also isolated from meat sources. Previous finding shows that the Salmonella rugose morphotype is commonly prevalent (56.4%) in strains isolated from turkey meat  produced by Salmonella rugose morphotype. In the present study, the curli formation by Salmonella rugose was determined by culturing cells on Congo red agar after 48 h at 37\u00b0C. The \u03b2 strands of curli subunits of rugose morphotype binds with hydrophobic Congo red dye to form red dry and rough (rdar) colonies, however, in the absence of curli formation smooth morphotype produced smooth edge colonies on Congo red agar  assay and CFU enumeration method. The crystal violet assay is the standard, simple and high throughput method to assess biofilm formation to an abiotic surface such as polystyrene or polypropylene and measures all components of biofilm such as EPC(s) and cellular content. The CFU of biofilm on TSA was used to enumerate viable, strongly attached biofilm cells on polystyrene and SS surface. In poultry abattoir, the temperature in reception and evisceration area is around 25\u00b0C and also there was higher biofilm formation by Salmonella at ambient temperature  of perature . Therefo surface . Rugose  surface . These rSubsequently, the actual EPS production in biofilm was estimated by ruthenium red assay which measures carbohydrate dye binding ability for two morphotypes. The ruthenium red assay showed rugose morphotype biofilm produced significant higher amount of carbohydrate than smooth morphotype. Confocal scanning micrographs validated the findings from CV and ruthenium red assay that rugose cells are in a dense aggregate form as compared to the monolayer of smooth morphotype . The resSalmonella to survive in the food processing environmental conditions  did not influence the biofilm formation ability of the smooth morphotype of S. Heidelberg ATCC 8326 but at 300 ppm exposure, biofilm formation was decreased for S. Typhimurium ATCC 14028. The rugose morphotype biofilm formation ability was influenced by the presence of subinhibitory concentration of NaOCl (200 or 300 ppm in TSB) in an opposite manner between the two strains. Thus, S. Heidelberg ATCC 8326 biofilm formation ability significantly increased and S. Typhimurium ATCC 14028 biofilm formation ability was significantly decreased (S. Typhimurium strain (S175), there was enhancement of biofilm formation after contact with subinhibitory concentrations of NaOCl  and stronger biofilm which may help nditions . In the ecreased ,B. In a of NaOCl . HoweverSalmonella biofilm grown at 25\u00b0C was exposed to three different NaOCl concentrations  in water for three different time intervals  at pH 6.15. In a previous study, the exposure time of 10\u201315 min at 100\u2013200 ppm NaOCl was used for Salmonella biofilm inactivation (The ability of the rugose morphotype to produce more EPC(s) such as curli and cellulose may provide resistance of its biofilm form against antimicrobials such as NaOCl . To invetivation . Our restivation , 5. The tivation .Listeria monocytogenes . The gene expression data in our study indicate that the expression of csgD reguhlator (hns), biofilm regulator (csgD), curli synthesis  and cellulose formation (bcsE) was significantly increased in rugose morphotype when induced by sequential exposure of sodium hypochlorite SICs. Sodium hypochlorite induced oxidative stress in S. Typhimurium may have resulted in higher expression of environment stress related genes such as envZ and csrA  may aid in its survival and persistence in the food processing environments, thereby increase its transmission between surfaces and hosts. Exposure to subinhibitory NaOCl may enhance Salmonella rugose morphotype biofilm formation. Also, the higher curli and cellulose formation in rugose morphotype protects its biofilm which provides an increased tolerance and survival against disinfectants such as NaOCl.These findings show that The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.The research was designed and led by RN from FSNHP and by CS, CM, and AK from POSC and was completed by MB and DK. SC contributed to reagents and materials for the gene expression study.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "Datasets concerning some user-scale Smart Grids, named Nano-grids, are reported in this paper. First several Solar Home Systems composed of a photovoltaic plant, a backup generator and different types of lithium-ion batteries are provided. Then, the inventory analysis of hybrid Nano-grids integrating batteries and hydrogen storage is outlined according to different scenarios. These data inventory could be useful for any academic or stakeholder interested in reproducing this analysis and/or developing environmental sustainability assessment in the field of Smart Grids. For more insight, please see \u201cEnvironmental analysis of a Nano-Grid: a Life Cycle Assessment\u201d by Rossi F, Parisi M.L., Maranghi S., Basosi R., Sinicropi A. [1]. Several Solar Home Systems composed of a photovoltaic plant, a backup generator and different types of lithium-ion batteries are described. Then, the inventory analysis of hybrid Nano-grids integrating batteries and hydrogen storage is outlined according to different scenarios . The inv2\u2022The first column collects the Ecoinvent 3.2 reference flows;\u2022The second column contains the amount of energy or material whose evaluation is based on the Nano-grid design and modelling as described in Ref.\u00a0. A negat\u2022The third column contains the unit of measurement of inputs and outputs;\u2022The fourth column contains the provider process for the flows;\u2022The fifth column contains sources and comments. The whole inventory is based on Ecoinvent 3.2 but when a component is not available in the database, information has been gathered from scientific papers in the literature. Based on literature data, the inventory of the missing components has been built using Ecoinvent 3.2 . Other cData are represented in Tables divided in two sections: Inputs and Outputs.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors."}
+{"text": "Mycobacterium kansasii subtypes (I\u2013VI) should be elevated, each, to a species rank. Consequently, the former M. kansasii subtypes have been denominated as Mycobacterium kansasii (former type I), Mycobacterium persicum (II), Mycobacterium pseudokansasii (III), Mycobacterium innocens (V), and Mycobacterium attenuatum (VI). The present work extends the recently published findings by using a three-pronged computational strategy, based on the alignment fraction-average nucleotide identity, genome-to-genome distance, and core-genome phylogeny, yet essentially independent and much larger sample, and thus delivers a more refined and complete picture of the M. kansasii complex. Furthermore, five canonical taxonomic markers were used, i.e., 16S rRNA, hsp65, rpoB, and tuf genes, as well as the 16S-23S rRNA intergenic spacer region (ITS). The three major methods produced highly concordant results, corroborating the view that each M. kansasii subtype does represent a distinct species. This work not only consolidates the position of five of the currently erected species, but also provides a description of the sixth one, i.e., Mycobacterium ostraviense sp. nov. to replace the former subtype IV. By showing a close genetic relatedness, a monophyletic origin, and overlapping phenotypes, our findings support the recognition of the M. kansasii complex (MKC), accommodating all M. kansasii-derived species and Mycobacterium gastri. None of the most commonly used taxonomic markers was shown to accurately distinguish all the MKC species. Likewise, no species-specific phenotypic characteristics were found allowing for species differentiation within the complex, except the non-photochromogenicity of M. gastri. To distinguish, most reliably, between the MKC species, and between M. kansasii and M. persicum in particular, whole-genome-based approaches should be applied. In the absence of clear differences in the distribution of the virulence-associated region of difference 1 genes among the M. kansasii-derived species, the pathogenic potential of each of these species can only be speculatively assessed based on their prevalence among the clinically relevant population. Large-scale molecular epidemiological studies are needed to provide a better understanding of the clinical significance and pathobiology of the MKC species. The results of the in vitro drug susceptibility profiling emphasize the priority of rifampicin administration in the treatment of MKC-induced infections, while undermining the use of ethambutol, due to a high resistance to this drug.Only very recently, has it been proposed that the hitherto existing  Mycobacterium genus, except those aetiologically implicated in tuberculosis (TB) and leprosy, that is members of the M. tuberculosis complex and M. leprae or M. lepromatosis, respectively. More than 180 NTM species have been recognized to date  comprise all species of the Mycobacterium kansasii is one of the most virulent and prevalent NTM pathogen in human medicine. It was first described by Buhler and Pollak in 1953 from a series of respiratory samples of patients with a TB-like pulmonary disease  was proposed by Hauduroy in 1955 and refers to where its first isolations were performed   and infection may be diagnostically challenging. Moreover, the incidence rates are influenced by a combination of demographic and clinical factors, including patient geographical origin and HIV status. Pulmonary M. kansasii infections tend to cluster in specific geographical areas, such as central Europe or metropolitan centers of London, Brasilia, and Johannesburg types, based on the analysis of restriction fragment length polymorphisms (RFLPs) using the MPTR probe, pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphism (AFLP) analysis, and PRA of the hsp65 gene  region  and atypical type II (IIb) have been reported. Whereas, the former had type I-specific sequence of the hsp65 gene and type II-specific sequence of the spacer region, the latter displayed type II-specific spacer sequence and a unique hsp65 gene sequence  and the translational elongation factor Tu (tuf), successfully applied for the differentiation between the subspecies I-VI  mass spectral profiles have recently been established PCR profiling , Poland (n = 5), the Czech Republic (n = 4), Spain (n = 2), Belgium (n = 1), Germany (n = 3), South Korea (n = 3), and Italy (n = 1). Patients from whom the strains had been collected were classified as having, or not, a pulmonary NTM disease according to the criteria of the American Thoracic Society (ATS) , M. gastri (DSM 43505T), M. marinum (DSM 44344T), M. szulgai (DSM 44166T), M. conspicuum (DSM 44136T), M. riyadhense (DSM 45176T). and M. tuberculosis H37Rv were also included in the study.A total of 27 he study . IncludeAll strains were maintained as frozen stocks and cultured on L\u00f6wenstein-Jensen or Middlebrook 7H10 agar  medium, supplemented with oleic acid, albumin, dextrose, and catalase, and incubated at either 30 or 37\u00b0C.M. kansasii by using high pressure liquid chromatography (HPLC) of cell wall mycolic acids, in accordance with the Centers for Disease Control and Prevention (CDC) guidelines  for 13 drugs, including isoniazid (INH), rifampicin (RMP), streptomycin (STR), ethambutol (EMB), clarithromycin (CLR), amikacin (AMK), rifabutin (RFB), moxifloxacin (MFX), ciprofloxacin (CFX), co-trimoxazole (SXT), linezolid (LZD), doxycycline (DOX), and ethionamide (ETO), were determined by the microdilution method, using the Sensititre\u00ae SLOMYCO plates , following the Clinical and Laboratory Standards Institute (CLSI) recommendations  disruption by using zirconia ceramic beads in a FastPrep-24 instrument  and further extracted chemically followed with a DNAzol\u00ae reagent .For the whole-genome sequencing, chromosomal DNA from all 27 DNA concentration, its purity and integrity was determined using a Qubit high-sensitivity (HS) assay kit .http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/), and de novo assembled with the SPAdes Genome Assembler ver. 3.10.0 . The libraries were sequenced on a HiSeq 2500 or a NextSeq 500 instrument  at a read length of 2 \u00d7 150 bp. The quality of reads before and after pre-processing was assessed using FastQC (v0.11.5) Andrews, . The rawM. kansasii strains, sequenced in this study, genomes of other 53 Mycobacterium sp. strains were analyzed. The genomic sequences of these strains were retrieved from the GenBank database (http://www.ncbi.nlm.nih.gov/genbank/) and their appropriate accession numbers were provided in In addition to genomes of 27 The assembled genomes were deposited under NCBI Bio-Project accession numbers: PRJNA374853 and PRJNA317047. The genomes were deposited in the GenBank under accession numbers provided in Gene identification and annotation were achieved using the DFAST pipeline v1.0.5 with default settings  method, based on a combination of genome-wide average nucleotide identity (gANI) and alignment fraction (AF) of orthologous genes  was used. This algorithm was designed to replace standard DNA\u2013DNA hybridization (DDH) by calculating DNA-DNA relatedness  , rpoB , tuf  genes, and the 16S-23S rRNA intergenic spacer region (ITS)  were extracted from the whole-genome sequence of the M. tuberculosis reference strain H37Rv, using CLC Genomic Workbench 10 , and were used, each, as a reference for alignments with the respective sequences of other mycobacterial species. Sequences from the same loci of the draft genomes analyzed were identified using the blastn algorithm. Due to a significant variation of the sought sequences, whenever necessary, they were extracted manually or downloaded from the NCBI database (https://www.ncbi.nlm.nih.gov/).Full-length sequences of the 16S rRNA , M. tuberculosis reference strain H37Rv (Accession no.: NC_000962.3) and listed by Brosch et al.  and -s2 0.5 (length difference cut-off) parameters was used .Core-genome single-ortholog tree was built as described previously   corresponding to an average N50 score of 4933 kb .Sequencing of 27 M. kansasii genomes (The genome sizes and GC contents ranged from 5.6 to 6.6 Mbp (avg. 6.2 Mbp \u00b1 0.3 Mbp) and from 65.86 to 66.38 (avg. 66.14 \u00b1 0.10), respectively. These values were consistent with the data reported for previously assembled  genomes .M. kansasii strains under the study was assessed with three species identification-relevant parameters, namely the alignment fraction (AF) of orthologous genes, the average nucleotide identity (ANI), and the genome-to-genome distance (GGD)  and M. tuberculosis complex species (The whole-genome-level relatedness among the 27 ce (GGD)  and 4. Tatabases . Whereas species .M. kansasii strains ranged between 0.6 and 1.0. The lowest AF value recorded for two strains, members of the same M. kansasii genotype was 0.69 . Within all other types, the AF values were 0.85 or higher.The AF values for all M. kansasii genotypes showed AF values equal to or below 0.75 with strains of other Mycobacterium species, except for M. gastri and M. persicum, which yielded AF values as high as 0.86 with M. kansasii type IV and V  or 0.99 with M. kansasii type II , respectively , commonly used as a boundary of species delineation  except between M. kansasii genotype II and M. persicum  and between M. kansasii genotype IV and M. gastri (95.2%)  .M. kansasii strains were clearly divided into six cliques, each containing strains of a distinct M. kansasii subtype (I\u2013VI) only. All the remaining NTM species had their genomes clustered within separate cliques, except that four genomes of strains classified as M. persicum were allocated in the M. kansasii genotype II clique. The genomes of 19 strains, representing five M. tuberculosis complex species were accommodated in a single cluster (clique).The AF and ANI metrics were also analyzed combinatorially, using the Microbial Species Identifier (MiSI) algorithm. The MiSI method sorts the analyzed genomes into species-like taxa or cliques, based on the AF and ANI species-level cut-off values set at 0.6 and 96.5%, respectively   or between any M. kansasii and any other Mycobacterium species . The only exception was when comparing genomes of M. kansasii type II to any of M. persicum, for which the GGD values were between 0.0003 and 0.0044 , both within and between the subtypes  were observed between three M. kansasii type I strains  and strains of M. kansasii type II. The type II-specific ITS sequences of those three type I strains accounted for the high intra-type heterogeneity (92.1\u2013100% sequence similarity).Comparisons of the 277-bp ITS sequences from milarity . The ITShsp65 gene fragments showed similarities of 90.3\u201392.3% between M. kansasii and M. tuberculosis, and 90.9\u201397.9% between M. kansasii and other NTM species, except M. persicum which shared 99.5\u2013100% similarity with M. kansasii type II  of M. kansasii type I sharing up to 99.8% similarity with M. kansasii type II strains.Sequence analysis of the 644-bp  type II . Alignmetuf  gene analysis were similar to those obtained with the hsp65 gene  had the same tuf sequences as M. kansasii type II strains.The results of the partial p65 gene . The tufrpoB  gene sequences found all M. kansasii strains to share <89% sequence similarity with M. tuberculosis and <96% similarity with NTM species, but again not M. persicum, whose sequences were identical or nearly identical with those of M. kansasii type II , which displayed high similarity or identity with type II strains.Finally, alignments of the partial  type II . The levM. kansasii subtypes, phylogenetic trees inferred from five individual loci were constructed  clusters, according to their subtype affiliation  were always present in the M. kansasii type II-M. persicum cluster, whereas another two belonged to that cluster only in the trees based on the ITS region (strain no. NLA00100521), 16S rRNA gene (NLA00100521), and hsp65 gene (strain no. K4). Having the type I-specific hsp65 gene sequence (sequevar I) and type II-specific ITS sequence (sequevar II), strain no. NLA00100521 represents the so-called intermediate type I (I/II), considered a transitional form between environmental type II and human-adapted type I .To better illustrate the phylogenetic relatedness of structed \u20137. A sepoB genes , since soB genes . In all iliation \u20138. The 1 strains . NoteworM. kansasii subtypes to represent distinct species. This was also the conclusion of the recent study by Tagini et al., who based their results upon ANI and GGD analysis of the genomes of 21 M. kansasii strains comprising all six subtypes , if the authors had included M. kansasii type II in the genome-based comparative analysis  M. kansasii type IV strains, analyzed in our study, fully satisfied the genomic criteria for a separate species. This was also implied by our predecessors, but in the absence of any type strain, they could not formally establish the species. Here, we propose a new species name, Mycobacterium ostraviense sp. nov., to accommodate M. kansasii type IV strains, with a strain no. 241/15, as a type. The description of this new species is given at the end of the article.The two M. kansasii type VII remains an enigma. Neither the strain nor its genomic sequence is available, precluding any relevant phylogenetic analyses. This type was reported only once  has been widely used for the identification of a plethora of NTM species, including M. kansasii and its subtypes , if they had not been inspected at the whole genome-level. Thus, neither PCR-RFLP profiling nor single or multilocus sequencing allows unequivocal identification of M. persicum. A definite diagnosis should be supported by the genome-wide analysis. Still, the two type IIb strains, under this study, displayed the type I-specific hsp65 RFLP patterns, upon digestion with HaeIII . This feature can be exploited for differentiation between types I and II, if whole-genome sequencing is not affordable. Nevertheless, a new, robust genetic marker allowing for fast and accurate identification of all M. kansasii-derived species (former types I-VI), bypassing the need for whole-genome analysis, would be of great benefit. For this, a more in-depth, comparative analysis of the genomes of more strains representing the six M. kansasii-derived species, and other NTM species, will have to be undertaken.Since the mid-1990s, the PCR-RFLP analysis, based on single-copy, orthologous genes . In this context, the newly proposed species names for types V (M. innocens) and VI (M. attenuatum) may not reflect the true phenotype of those bacteria.There has been a continuing debate on how the differences between M. tuberculosis virulence, known as \u201cregion of difference 1\u201d (RD1) was interrogated across the genomes of M. kansasii subtypes for its functional integrity . The RD1 encodes a secretory apparatus (ESX-1 type VII secretion system) responsible for exporting two highly potent antigens and virulence factors\u2014the 6-kDa early secreted antigenic target (ESAT-6) and the 10-kDa culture filtrate protein (CFP-10)  and cfp-10 (esxB) genes have also been demonstrated in some NTM species, including M. kansasii (types I-V), M. szulgai, M. marinum, and M. riyadhense , including the ESAT-6 and CFP-10-coding genes and two other genes (rv3871 and rv3877) coding for the essential components of the ESX-1 secretion system, in all types of M. kansasii and other NTM species  and VI (M. attenuatum). Neither it was present in M. szulgai and M. riyadhense. The protein encoded by this gene is an ESX-1 secretion-associated protein EspI. It was shown that inactivation of the rv3876 gene did not impair secretion of ESAT-6 , required for virulence and growth in macrophages  did not show any consistent  species-specific pattern of RD genes . It was It has been canonically accepted, upon description of new species, to provide a detailed phenotypic characterization. However, in the era of genomic-based bacterial taxonomy, the significance of the phenotype has much eroded and the use of conventional biochemical testing has been increasingly abandoned. The algorithm for routine differential diagnostics of NTM species should obligatorily include only growth rate and pigment production  were, unlike all other species, unable to reduce nitrate and that their catalase was heat-liable. Whether these features are stable within the species need to be verified on a larger set of strains. Interestingly, both these features are typical for M. gastri  (M. kansasii). Of these drugs, only RIF and CLR showed interspecies differences in their activity, with the MICs for M. kansasii and M. persicum slightly higher than for other M. kansasii-derived species. More than 80% of strains were resistant to EMB. Among these, were all strains of M. kansasii (former type I), M. persicum (II), and M. ostraviense (IV). Single strains of M. kansasii, M. pseudokansasii, and M. attenuatum were resistant to CIP. The MICs of STR and DOX varied widely (<0.5\u201316 mg/L vs. 1\u2013>16 mg/L), yet the highest values (16 vs. >16 mg/L) were observed only for strains of M. kansasii, M. persicum, and M. attenuatum. The INH and ETO MICs were low, and within relatively narrow ranges (<0.25\u20132 mg/L vs. <0.3\u20130.6 mg/L). These findings confirm the key observations from previous studies, on the susceptibility of M. kansasii strains to RIF and their high resistance to EMB  . ShortlyM. kansasii. Not only does it further substantiate the delineation of new species from the M. kansasii group to replace the former subtypes I\u2013VI, but consolidates the position of five of the so erected species, and provides a description of the sixth one, M. ostraviense, a successor of the subtype IV. By showing a close genetic relatedness, a monophyletic origin, and overlapping phenotypes, our findings support the recognition of the M. kansasii complex (MKC), accommodating all M. kansasii-derived species and M. gastri. Neither of the most commonly used taxonomic markers can accurately distinguish all the MKC species. Likewise, no species-specific phenotypic characteristics exist that would allow identification of the species, except the non-photochromogenicity of M. gastri. In the context of the previously proposed polyphasic strategy in resolving species boundaries and their interrelatedness, FAME (fatty acid methyl ester) analysis, as an adjunct typing method, might be useful  catalase. Strongly resistant to ethambutol (> 16 mg/L) but susceptible to amikacin, clarithromycin, co-trimoxazole, linezolid, fluoroquinolones, and rifamycins.The species name refers to the former  at 37\u00b0C . No growM. kansasii (former subtype I) and M. gastri, yet it displays unique sequences at the hsp65, tuf, and rpoB genes, and the ITS locus. At the genomic level, it is most closely related to M. gastri, with pairwise ANI and GGD values of 95.2 and 0.056, respectively.The species has the same 16S rRNA sequences as T was isolated from a sputum of a patient with no NTM disease, based in Karvin\u00e1, near Ostrava, in the Moravian-Silesian Region of the Czech Republic. The type strain has been deposited in the Leibniz Institute German Collection of Microorganisms and Cell Cultures  under the accession number DSM 110538.The type strain, 241/15All datasets generated for this study are included in the article/Analyses were based on data that did not contain any sensitive personal information. Therefore, informed consent and ethical approval were not required in like with local legislation.M. kansasii strains. BM carried out analysis on regions of difference 1-14 (RD1-14) with a homemade script Diffind. AB carried out DNA isolations for whole-genome sequencing. JD analyzed the results on the distribution of the RD1-14 genes in M. kansasii genomes. MD constructed the core-genome phylogenies. LP performed drug susceptibility testing. JI provided 13 M. kansasii strains of subtypes I-VI and critically reviewed the manuscript. MZ-D co-performed phenotypic assays.TJ conceptualized and supervised the study, provided the funding, organized and integrated the data, and wrote the manuscript. PB, JL, and DS performed bioinformatic analyses including AF-ANI, GGD, and phylogenetic tree analysis. ZB performed culturing, subtyping, and phenotypic profiling of The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "Band 3 is the most abundant membrane protein in human red blood cells (RBCs). Our understanding of its physiological functions mainly came from clinical cases associated with band 3 mutations. Severe reduction in band 3 expression affects blood HCO3\u2014/CO2 metabolism. What could happen physiologically if band 3 expression is elevated instead? In some areas of Southeast Asia, about 1\u201310% of the populations express GP.Mur, a glycophorin B-A-B hybrid membrane protein important in the field of transfusion medicine. GP.Mur functions to promote band 3 expression, and GP.Mur red cells can be deemed as a naturally occurred model for higher band 3 expression. This review first compares the functional consequences of band 3 at different levels, and suggests a critical role of band 3 in postnatal CO2 respiration. The second part of the review explores the transport of water, which is the other substrate for intra-erythrocytic CO2/HCO3\u2014 conversion . Despite that water is considered unlimited physiologically, it is unclear whether water channel aquaporin-1 (AQP1) abundantly expressed in RBCs is functionally involved in CO2 transport. Research in this area is complicated by the fact that the H2O/CO2-transporting function of AQP1 is replaceable by other erythrocyte channels/transporters . Recently, using carbonic anhydrase II (CAII)-filled erythrocyte vesicles, AQP1 has been demonstrated to transport water for the CAII-mediated reaction, CO2(g) + H2O \u21cc HCO3\u2014(aq) + H+(aq). AQP1 is structurally associated with some population of band 3 complexes on the erythrocyte membrane in an osmotically responsive fashion. The current findings reveal transient interaction among components within the band 3-central, CO2-transport metabolon . Their dynamic interaction is envisioned to facilitate blood CO2 respiration, in the presence of constantly changing osmotic and hemodynamic stresses during circulation.The Cl Band 3 e kidney . Band 3 e kidney ; (2) blohrocytes , 2011.Figure 1). GP.Mur evolved from homologous gene recombination of glycophorin B and glycophorin A, and is essentially glycophorin B with a piece of glycophorin A inserted in the middle  (a) are naturally occurred in \u223c0.5% local Taiwanese. So if a person bearing such an alloantibody is accidentally transfused with GP.Mur RBCs, an acute intravascular hemolytic reaction might occur (6 glycophorin A (GPA) protein molecules and 1.7\u20132.5 \u00d7 105 glycophorin B (GPB) molecules in an average human RBC (GYP.Mur (hetGYP.Mur), GP.Mur replaces half of GPB protein expression; in people with homozygous GYP.Mur (hoGYP.Mur), GP.Mur substitutes all the expression of GPB.GP.Mur, commonly known as Miltenberger subtype III (Mi.III) in Southeast Asia, is an erythrocyte antigen of the MNS blood group system . The preigure 1) . Alloantht occur . There auman RBC . In peopThe early proteomic work from my group identifies unique structural features of GP.Mur-associated protein complexes on the RBC membrane (\u201cghosts\u201d). Importantly, GP.Mur red cells express significantly more band 3. The protein-protein interaction between band 3 and AQP1 on the GP.Mur RBC membrane is substantial, compared to that on the GP.Mur-negative cell membrane . Band 3 glycophorin A in GYP.Mur (GYP.B-A-B), we hypothesized that the glycophorin A-derived peptide in GP.Mur promoted the protein expression of band 3. We took the experimental approach similar to an early study done in Xenopus oocytes, which demonstrates increase of band 3 levels upon GPA co-expression   .kMk, and Mi.V  (The interaction between GPA and band 3 begins in the endoplasmic reticulum (ER) where GPA facilitates protein synthesis of band 3 . GPA-banand Mi.V . On the n band 3 . Therefoulfate\u2014) . By fluoulfate\u2014) , though ulfate\u2014) . The disulfate\u2014) .2 metabolite from tissues are carried in the form of soluble bicarbonate until exhalation in the lungs. By utilizing CO2(g)/HCO3\u2014(aq) exchange, the capacity of one\u2019s CO2 tolerance can be expanded substantially. This chemical conversion, CO2(g) + H2O \u21cc HCO3\u2014(aq) + H+(aq), is primarily facilitated by intraerythrocytic carbonic anhydrase II (CAII), as the rate of its spontaneous conversion outside RBCs (t1/2 = 14 s) is too slow to meet physiological demands  (6 molecules/RBC) facilitates hydration of CO2(g) to HCO3\u2014(aq). Bicarbonate permeates through band 3 dimers or dimerized dimers (tetramers), following its concentration gradient across the RBC membrane . Extracellular bicarbonate needs to enter red cells via band 3 to be converted into CO2(g) by intraerythrocytic CAII .When blood circulates to the capillary bed, COnd RhAG) . Abundanmembrane . Because2 transport and respiration (catK or its turnover number (\u223c6 \u00d7 105/sec) at least 10 times faster than the rate of Cl\u2014/HCO3\u2014 exchange of an erythroid AE1 molecule (\u223c5 \u00d7 104 ions/sec) (6 molecules/RBC), the efficiency of CAII-catalyzed CO2/HCO3\u2014 conversion is about an order higher than the rate of AE1-conducted Cl\u2014/HCO3\u2014 flux through the cell membrane. For comparison, the rate of water permeation through an AQP1 is \u223c3 \u00d7 109 H2O/sec. Intraerythrocytic water is used in CO2/HCO3\u2014 exchange. With 160,000\u2013200,000 AQP1 on the erythrocyte membrane, the rate of water transport via AQP1 is estimated 480\u2013600 times faster than the enzymatic activity of intraerythrocytic CAII. Therefore, the anion exchange activity of erythroid AE1 is the rate-limiting step for blood CO2 transport  . Since eransport , which idividual .in utero transfusion at 22 weeks gestation, and then delivered at 29 weeks gestation -associated mutations in band 3, their band 3-mediated HCO3\u2014 transport is further reduced to less than 5% of the transport efficiencies   . We did 2 and bicarbonate due to this mild exercise challenge were smaller in GP.Mur-positive subjects   . Intraerhrocytes . Does thWe later employed a sensitive, biophysical approach\u2014fluorescence resonance energy transfer by fluorescence lifetime imaging microscopy (FLIM-FRET), to verify AQP1-band 3 interaction that was initially identified by proteomics . FLIM me2/HCO3\u2014 conversion. After the discovery of the protein-protein interaction between CAII and band 3  heterologous expression system, or by the direct binding assay with individual purified proteins  and tetrameric forms (ankyrin complexes); the other half of band 3 molecules  are mobile dimers located within the corrals set by the submembranous spectrin mesh . AQP1 is2-transport metabolon, first proposed by Reithmeier, is based on the experimental observation that the major proteins supporting this intraerythrocytic reaction (CO2 + H2O \u21cc HCO3\u2014 + H+) are spatially adjacent to one another to maximize the efficiency of blood CO2 transport  (2 fluxes out of RBCs and CO2 fluxes in. Deoxygenated hemoglobin (deoxy Hb) preferentially binds to the N-terminal, cytoplasmic domain of band 3  absorption of proton by negatively charged deoxy Hb which is transiently bound to band 3.The idea of a COigure 3) . This fof band 3 . The for2 flux in or out of RBCs is via AQP1 gas channel. The rest of CO2 flux is likely through another gas channel, RhAG, and/or direct diffusion across the lipid bilayer  the entry of the substrates CO2 and H2O into RBCs through AQP1, (2) intraerythrocytic hydration of CO2 by CAII, (3) the exit of one of the two reaction products, bicarbonate, from erythrocytes via band 3, and (4) the absorption of the other reaction product, proton, by nearby deoxy Hb\u2014 . This transient arrangement of having H2O/CO2 channel AQP1, adjacent to band 3, CAII and proton-absorbing deoxy Hb\u2014, conceivably allows an almost uninterrupted \u201cchanneling\u201d of reactant influx and product outflow , the network of ordered water allows CO2 loosely bound to CAII (2) binding allows a nearly complete occupancy of CO2 at the active site, contributing to the ultra-high efficiency of CAII. Indeed, the concentration of CO2 at the active site of CAII is very high (0.45 M)  . This im2O/CO2 channel is functionally replaceable by other channels/transporters (2O/CO2-transporting function might not be exclusively carried out physiologically by AQP1 alone. The only known symptom in AQP1 null people shows upon water deprivation; AQP1 deficiency reduces their capability to concentrate urine or reabsorb free water at the medullary collecting duct of the kidney (AQP1 as a Hsporters . The firsporters . This ste kidney . Similare kidney .2O permeability. The RBCs from UT-B null mice are similarly water-permeable as the RBCs from wild-type mice. But water permeability in the RBCs of AQP1 and UT-B double-knockout mice is further reduced. Double knockout of AQP1 and UT-B show reduced survival and retarded growth, in addition to reduced urinary concentrating ability (The second clue came from comparing the single knockouts of AQP1 or urea transporter UT-B to the double knockout of AQP1 and UT-B. The RBCs from AQP1 null mice exhibit significantly reduced osmotic H ability . This su ability . Therefo ability .2-transporting function of APQ1 is replaceable by RhAG gas channel (2 transport is not observed in AQP1 knockout mice (2 respiration and physical tolerance. Unlike the clear-cut monogenic effect of kidney AQP1 in urine concentration, the role of AQP1 in red cell functions remains to be explored.The CO channel . The funout mice . Interesout mice ,b. TheirThe author confirms being the sole contributor of this work and approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "Inonotus obliquus using DEAE-52 cellulose and Sephadex G-100 column chromatography. The structural characterization and in vitro and in vivo hypoglycaemic activities of these molecules were investigated. HPLC analysis HIOP1-S was a heterpolysaccharide with glucose and galactose as the main compontent monosaccharides . However, HIOP2-S was a heterpolysaccharide with glucose as the main monosaccharide . The average molecular weights of HIOP1-S and HIOP2-S were 13.6 KDa and 15.2 KDa, respectively. The \u03b2-type glycosidic bond in HIOP1-S and HIOP2-S was determined using infrared analysis. 1H-NMR spectra indicated that HIOP2-S contains the \u03b2-configuration glycosidic bond, and the glycoside bonds of HIOP1-S are both \u03b1-type and \u03b2-type. The ultraviolet scanning showed that both HIOP1-S and HIOP2-S contained a certain amount of binding protein. Congo red test showed that HIOP1-S and HIOP2-S could form a regular ordered triple helix structure in the neutral and weakly alkaline range. HIOP1-S and HIOP2-S showed strong \u03b1-glucosidase inhibitory activities and increased the glucose consumption of HepG2 cells. In addition, Streptozotocin (STZ)-induced hyperglycaemic mice were used to evaluate the antihyperglycaemic effects of HIOP1-S and HIOP2-S in vivo. The results showed that HIOP2-S had antihyperglycaemic effects. Taken together, these results suggest that HIOP1-S and HIOP2-S have potential anti-diabetic effects.In the present study, two polysaccharides (HIOP1-S and HIOP2-S) were isolated and purified from  Diabetes mellitus (DM) is a chronic metabolic disorder caused by insulin deficiency or functional disturbance of the receptors. Diabetes mellitus has become a worldwide epidemic, with the number of diabetes mellitus patients increasing annually. In 2013, 382 million individuals worldwide suffered from diabetes, and the total number of individuals with diabetes is expected to reach 592 million by 2035 ,5. Thus,Inonotus obliquus (Chaga) is a rare edible fungus that is parasitic on birch trees, belonging to the family Hymenochaetaceae of Basidiomycetes. Chaga is a typical tree fungus mainly distributed in cold regions of 45\u00b0 N to 50\u00b0 N latitude, such as in Northern Russia, Europe, North America and Hokkaido, Japan. I. obliquus has been documented to contain poly-saccharides, polyphenols, triterpenes, melanin and steroid, showing various biological activities . After one week of adaptive feeding, fasting but only water for 12 h, a small dose of pre-cooled STZ solution was injected intraperitoneally with a single injection dose of 35 mg/kg, every 4 times a day for 3 days, during the intragastric administration of the mice, fresh water was replaced every day to ensure the fresh water supply; the litter was changed every day to ensure the clean environment of the mice; the temperature of the breeding room was kept at 22 \u00b1 1 \u00b0C; the humidity was 50\u201365%. 48 h after the last injection of STZ, the mice were fasted for 6 h, the blood was taken from the tail vein and the fasting blood glucose was determined, when the blood glucose level was \u226512 mmol/L, we considered the model construction was successful. All animal handling procedures were carried out in strict accordance with the Chinese People\u2019s Republic of China Laboratory Animal Use and Care Regulations and guidelines developed by the Laboratory Animal Center and approved by the Ethics Committee, and the permit number is KY110023.The successful modeled mice were randomly divided into a model group (STZ induction), a positive control group (125 mg/kg metformin hydrochloride) and a polysaccharide treatment group , 12 mice in each group, and 12 healthy mice were used as the blank control group to ensure that the mice were given the same daily gavage time. The normal control group and the model group were given the same amount of sodium chloride solution. During the gavage, the mice were weighed weekly and the blood glucose level was measured.p values of less than 0.05 or 0.01 were considered statistically significant.SPSS 17.0 software  was used for the statistical analysis. The data are expressed as the means \u00b1 SD. In the present study, the water-extracted crude polysaccharide (HIOP) was fractionated through DEAE-52 cellulose and Sephadex G-100 column chromatography. Two major polysaccharide fractions (HIOP1-S and HIOP2-S) were obtained. The chemical and physical properties of these polysaccharides were determined using chemical methods, including GPC, IR, HPLC, and so on. The in vitro and vivo hypoglycaemic activities of the two major polysaccharides were also evaluated. The results indicated that HIOP1-S and HIOP2-S have high potential to act as a substitute for the standard oral hypoglycaemic agents metformin and acarbose, reflecting the high hypoglycaemic effects and safety of these polysaccharides, and HIOP1-S and HIOP2-S can be developed as potential new anti-diabetic drugs for further treatment. Further chemical and pharmacological investigations are needed to evaluate the mechanism underlying the hypoglycaemic actions of these compounds."}
+{"text": "Polo is an equestrian sport that requires two teams of four players to score goals at opposing ends of a 150 m \u00d7 275 m pitch. Each player is rated on a handicap system (\u22122 to +10) that quantifies their abilities and permits their inclusion in different levels of Polo play; the cumulative handicap of the four players sets the level of play. Using GPS technology, we investigated how levels of Polo differ regarding distance covered, speeds achieved and high-intensity activities performed. As cumulative Polo handicap increased, so too did the distances and average speeds attained, decelerations performed and impacts encountered during each period of play. These findings suggest that as each player improves and increases their handicap, they will need to ensure the ponies they play have sufficient aerobic, anaerobic and speed capacities to perform effectively at that level. This information provides valuable insights to Polo players, grooms and equine vets, as to how they can best prepare their ponies for game-day and how they may be able to maintain pony longevity in the sport.Global positioning systems (GPS) have recently been shown to reliably quantify the spatiotemporal characteristics of Polo, with the physiological demands of Polo play at low- and high-goal levels also investigated. This study aims to describe the spatiotemporal demands of Polo across 0\u201324 goal levels. A player-worn GPS unit was used to quantify distance, speed and high-intensity activities performed. Data were divided into chukkas and five equine-based speed zones, grouped per cumulative player handicap and assessed using standardized mean differences. Average distance and speed per chukka increased in accordance with cumulative player handicap, with the magnitude of differences being trivial\u2013large and trivial\u2013very large, respectively. Differences between time spent in high-intensity speed zones (zones 4 and 5) show a linear increase in magnitude, when comparing 0 goal Polo to all other levels of play . High-intensity activities predominantly shared this trend, displaying trivial\u2013large differences between levels. These findings highlight increased cardiovascular, anaerobic and speed based physiological demands on Polo ponies as playing level increases. Strategies such as high-intensity interval training, maximal speed work and aerobic conditioning may be warranted to facilitate this development and improve pony welfare and performance. The use of global positioning systems (GPS) in sport and animal research is increasingly prevalent and can provide valuable data pertaining to activity type, distance covered, speeds attained and location ,2,3,4. DIn order to advance the application of GPS data in equestrian sports, consistent GPS use in training and competitive scenarios is to be encouraged ,12. A grPolo presents an ideal model to apply GPS, as Polo ponies are required to perform high-intensity movements and tolerate impacts in a manner that is unique to Polo, and players are required by Polo regulations to interact with a relatively large number of ponies per game in comparison to other equestrian pursuits . Polo isThis study aims to assess the spatiotemporal demands of Polo, across a range of handicap levels, to accurately describe the performance requirements placed upon Polo ponies, with a view to informing training practices and identifying points of distinction between levels of play. It is hypothesized that as cumulative player handicap  increases, average speed and distance covered per chukka (period of play) will also increase.All data were gathered during the 2018\u20132019 New Zealand Polo Season, on the north island of New Zealand. Data were obtained from a total of 338 chukkas of outdoor field Polo. All players had a current New Zealand Polo Association handicap . The cumulative handicap for each team (four players) was used to define the level of play  for the tournament . All games were contested under Hurlingham Polo Association rules  and wereThe present investigation utilized VX Sport 350 GPS units , sampling at 10 Hz, with a speed range of 0\u201360 km/h, in equestrian mode. The speed range permits for derivation of speed zones  but does not set an absolute upper limit upon data captured. These devices have previously been reported as reliable independent of unit position (CV < 10% and ICC > 0.70 ), for usGPS units were turned on 30 minutes prior to the start of each game to allow sufficient time for satellites to be located and a secure connection to multiple satellites established. As players use multiple ponies per game, possibly per chukka, with limited time between chukkas, it is neither feasible nor representative of typical Polo play to mount a GPS unit per horse or record data per horse; hence the use of a player-worn unit. Each player was fitted with one GPS unit in a pouch on the player\u2019s belt; this position has previously been shown to produce reliable results of speed and distance in Polo , with thData were extracted using specialist software  and were trimmed to remove the initial satellite lock period. The game period was divided into chukkas as per notational analyses that accompanied each game. Speed zones were assigned a priori based upon an estimated maximum speed of 60 km/h, which is within the tolerable limits of the manufacturer\u2019s equestrian mode. Using in-built software thresholds, the following speed zones were constructed: Zone 1: 0\u201319.2 km/h; Zone 2: 19.2\u201323.4 km/h; Zone 3: 23.4\u201328.2 km/h; Zone 4: 28.2\u201347.4 km/h; and Zone 5: 47.4\u201360 km/h.Distance covered (m) and time (min:sec) in each speed zone per chukka were selected as primary dependent variables, with the number of sprints (a positive or negative acceleration > 3m/s/s), impacts, and acceleration and deceleration counts, collectively termed high-intensity activities, provided as secondary dependent variables that further describe the load placed upon Polo ponies. Data were presented per chukka to allow comparison between levels of play.g) \u00b1 90% confidence intervals (C.I.), between handicap levels . Standardized mean differences were described using the following magnitudes: trivial 0\u20130.2, small 0.2\u20130.6, moderate 0.6\u20131.2, large 1.2\u20132.0, and very large >2.0 [Data were exported to Microsoft Excel and variables were analyzed using a customized spreadsheet to calculate standardized mean differences (Hedge\u2019s rge >2.0 .The median chukka duration from the sample (n = 338) was 11:09 \u00b1 0:10 (min:sec), with absolute minimum and maximum values of 6:33 and 19:27, respectively.Distance characteristics for each level of play are shown in Distance covered in each speed zone per chukka at each level of play is shown in Average speed per chukka increases in accordance with increasing cumulative player handicap , with thTime spent in each speed zone per chukka at each level of play is shown in Differences between time spent in speed zones 4 and 5 show a linear increase in magnitude, when comparing 0 goal Polo to all other levels of play , with a similar trend seen when 6 and 10 goal play were compared to 16 and 24 goal levels ; confidence intervals for 6 and 10 goal play overlap zero in speed zone 4, and they differ trivially to one another in time spent in speed zone 5. Confidence intervals also overlap zero when time in speed zone 4 is compared between 16 and 24 goal play, yet moderate differences are also seen when comparing time spent in speed zone 5 between these levels. Collectively, these findings emphasize the findings outlined in All effect sizes, confidence intervals and descriptors for high-intensity activities can be found in Differences in accelerations only occur in 50% of comparisons; 16 goal play requires moderately more accelerations than 0, 6, 10 and 24 goal Polo, with 24 goal Polo only demonstrating a small increase in acceleration count compared to 10 goal play, whereas small to moderate differences in decelerations are seen between all level comparisons, except for 0 and 6 goal levels , when 16 and 24 goals are compared . Moderately fewer impacts were sustained in 0 and 10 goal play compared to the 24 goal level (1.2 \u00b1 0.3); this difference decreased in accordance with handicap, and when 0 and 10 are compared to 16 goal (1.2 \u00b1 0.2) play the difference is small. Confidence limits overlap zero between all levels of play and 6 goals, likewise for 0 and 10 goal impact counts.The aim of this research was to assess the spatiotemporal demands of Polo and to accurately describe and compare the performance requirements placed upon Polo ponies across varying levels of Polo play. It was hypothesized that as cumulative player handicap increased, average speed attained and distance covered per chukka would also increase. The findings of this investigation support this initial hypothesis, with overall trends displaying a rise in distance and speed metrics as level of play increased. Further to this, speed zones 4 and 5 showed a linear increase in magnitude when compared across level of play; a trend also shared by decelerations and impacts. These findings provide valuable insight into the horse management and tactical demands of Polo, as they afford a greater understanding of potential horse welfare considerations and may also mitigate potential injuries to ponies or Polo players.The use of the cumulative team handicap to categorize Polo encourages creativity and variety in approaches to best satisfy this constraint, whilst maximizing a team\u2019s effectiveness. For example, a 0-goal team may be made up of three players with a -2 handicap, and one 6 goal player; or, equally, it may comprise two 1 goal players, a 0-goal player and one -2 goal player. As cumulative player handicap increases to \u226510 goals, it prompts the inclusion of higher handicapped individuals in order to be competitive. Based on the HPA handicap guidelines , a higheGondin et al.,  concludeBy understanding the requirements of the level of Polo being played and the physical capabilities of a player\u2019s string, pony management strategies can be further individualized to maximize the effectiveness of each pony and ultimately improve their contribution to the team\u2019s performance whilst ensuring pony and player safety ,26. PracSpeed zone  and highA possible limitation of this study is the use of a player worn GPS unit, which indirectly but reliably measures the characteristics outlined in this paper . HoweverThe aim of this research was to assess the spatiotemporal demands of Polo and to accurately describe and compare the performance requirements placed upon Polo ponies across varying levels of Polo play. Key findings of this investigation were that as cumulative player handicap increased, so too did distance covered per chukka, with a greater proportion of time spent at higher velocities and a greater number of high-intensity activities also performed. As the level of play increases, the increased average speed and distance covered require ponies to possess the cardiovascular and anaerobic performance/fitness to match the physiological demand of the level of Polo they are playing. Strategies to facilitate this development may include the incorporation of high-intensity interval training, maximal speed work, and aerobic conditioning. GPS presents a tool that can effectively quantify the spatiotemporal demands of Polo, and is capable of detecting changes in activities that are indicative of the level of Polo played."}
+{"text": "OBJECTIVES/SPECIFIC AIMS: Develop a plain language informed consent template that met IRB and regulatory requirements. Evaluate the effectiveness of the template at improving the readability of informed consents. Field test the informed consent with low health literacy. METHODS/STUDY POPULATION: We conducted a retrospective analysis of over 200 UAMS IRB approved, investigator initiated informed consents from 2013 to 2015 to determine the readability before intervention. The mean grade level readabilities were derived from the results of 3 readability formulas  using open-source readability tools. A plain language informed consent template that meets IRB and regulatory requirements was developed, adhering to health literacy best practices for written communication. The template was made available to investigators as an optional resource, and IRB committees were trained on use of the template. In addition, a focus group will be conducted to qualitatively assess understandability of the template with study participants identified as having inadequate health literacy. Data analysis will include readability assessment of IRB approved informed consents post intervention with and without use of the plain language template, as well as qualitative feedback from focus group participants. RESULTS/ANTICIPATED RESULTS: The retrospective analysis revealed a mean readability of 10th grade for IRB approved informed consents from 2013 to 2015 (n=217). The readability of the developed plain language template was 5th grade. Preliminary post-intervention results show adoption of the template by investigators (n=16) resulted in informed consents with a mean readability of 7th grade (range 6\u20139th grade), compared to a mean of 10th grade (range 7\u201311th grade) for the comparator . Data collection will continue through May 2017. The focus group is forthcoming and results will be included in the poster. DISCUSSION/SIGNIFICANCE OF IMPACT: Low health literacy is common in individuals with healthcare disparities and can limit their participation in clinical research. Few studies have examined interventions to address this barrier to research. Preliminary results of this study support the utilization of a plain language informed consent template in investigator-initiated research. Moreover, this study demonstrates the importance of stakeholder engagement among CTSA leadership, health literacy experts, the institutional review board, investigators, and research subjects in the development and testing of this intervention to make informed consents \u201cunderstandable to the subject\u201d while containing all required elements."}
+{"text": "Chrysophrys auratus (snapper) to 1) construct the first linkage map for this species, 2) scan for growth QTL, and 3) search for putative candidate genes in the surrounding QTL regions. The newly constructed linkage map contained \u223c11K SNP markers and is one of the densest maps to date in the fish family Sparidae. Comparisons with genome scaffolds of the recently assembled snapper genome indicated that marker placement was mostly consistent between the scaffolds and linkage map (R = 0.7), but that at fine scales (< 5 cM) some precision limitations occurred. Of the 24 linkage groups, which likely reflect the 24 chromosomes of this species, three were found to contain QTL with genome-wide significance for growth-related traits. A scan of 13 candidate growth genes located the growth hormone, myogenin, and parvalbumin genes within 5.3, 9.6, and 25.0 cM of these QTL, respectively. The linkage map and QTL found in this study will advance the investigation of genome structure and aquaculture breeding efforts in this and related species.Characterizing the genetic variation underlying phenotypic traits is a central objective in biological research. This research has been hampered in the past by the limited genomic resources available for most non-model species. However, recent advances in sequencing technologies and related genotyping methods are rapidly changing this. Here we report the use of genome-wide SNP data from the ecologically and commercially important marine fish species  New QTLe.g., a linkage map or high quality genome assembly) is an important prerequisite for QTL mapping, as it allows the relative positioning of different marker loci. High-quality genome assemblies are most effective because they allow genetic markers to be positioned at a base-pair level, while also providing sequence information for the surrounding area. However, most non-model species do not currently have chromosome-level genome assemblies and instead rely on linkage maps to ascertain the relative position of markers in the genome  in a wide range of taxa , commonly referred to as the Australasian snapper (henceforth referred to as \u201csnapper\u201d). Snapper supports a valuable recreational and commercial inshore fishery around the northern parts of New Zealand, southern Australia, and some of the Pacific Islands  construct a high density linkage map, 2) conduct QTL mapping for three measures of growth , and 3) investigate the position of 13 candidate growth genes and their relative position to growth QTL.Here we focus on the marine teleost 1 = 70 individuals, F2 = 577 individuals) were investigated in this study. Uncontrolled mass spawning of the F1 generation in a single tank was used to produce the offspring F2 generation. This resulted in a complex pedigree, meaning that we obtained a combination of full-siblings, half-siblings, and unrelated individuals in the F2 generation (Supplementary Table 1). The F2 offspring were held in a single tank until they were approximately one year old and then split evenly among four tanks with comparable feeding, light, water flow, aeration, tank design. All research carried out in this study was reviewed and approved by the animal ethics committee of Victoria University of Wellington in New Zealand (Application number 2014R19).A snapper breeding program was started at The New Zealand Institute for Plant & Food Research Limited in 2016 and includes a population with three generations held at the Nelson Research Centre in New Zealand. Data from the two most recent generations  and again when they were approximately three years old (1045-1131 days). Length measurements were made by collecting images of each individual and then making measurements from those images. A ruler was included in each image to provide a scale. The number of individuals measured differed between year one and year three as a result of natural mortality during the study.Samples of fin tissue were collected for all fish and DNA was extracted from these samples using a modified salt extraction protocol . QuantifPstI and MspI. The adaptor ligation step was done after digestion, without allowing the DNA/adaptor mixture to dry out. The barcoded adaptors were designed by Deena Bioinformatics and bound to the PstI cut sites. Adaptors were subsequently annealed according to the method of Ko et al. (2003). The high fidelity enzyme AccuPrime Taq DNA polymerase High Fidelity (Life Technologies) was used for amplifications. Each library was amplified separately and its quality assessed by capillary electrophoresis prior to sequencing . All GBS libraries were prepared in parallel in plates. Duplicate or triplicate samples were prepared for each of the parent and grandparents and single samples for each of the offspring . Each plate  was pooled, then cleaned up, quantified and sent to the Australian Genome Research Facility (AGRF) in Melbourne, Australia, for sequencing. Each pool was sequenced on a single lane with the Illumina HiSeq 2500 platform in single end (SE) mode, with a read length of 100 bases. In total, eight pools of libraries were sequenced in eight lanes for this project.Only high quality genomic DNA was used for the preparation of Genotyping By Sequencing (GBS) libraries based on the protocol described by FastQC was used to conduct an initial check of the sequencing data quality. Sequences were then de-multiplexed and cleaned. Adapters and primers were removed and the sequencing data were cleaned using Fastq-mcf in the ea-utils package . Genotyp2 individual in the dataset were identified using CERVUS v3.0.7  were used, and included a total of 269 offspring and 14 parents, and reduced the total number of available SNPs for this analyses to 20,311 SNPs. Markers were separated into chromosomes with the SeparateChromosomes module (logarithm of odds (LOD) limit = 14, minimum markers per linkage group = 50). The marker order was then generated with the OrderMarkers module. Markers near the start and end of each linkage group (start and end 10% based on centimorgan (cM) distance) were removed if they were more than 3 cM from the next closest marker. The accuracy of the final linkage map was investigated by comparing the linkage map position (cM) with the position of markers on available genome scaffolds (base-pairs) from the genome assembly (number of scaffolds 5998). The scaffold and base-pair position for each marker in the linkage map was retrieved from the STACKS v1.40 output files. Using this information the correlation between linkage map (cM) and scaffold (base-pair) position was tested for all scaffolds that contained >50 SNPs. The mean and 95% confidence interval of the correlation residuals was then calculated. Whether scaffolds were placed uniquely on one of the 24 expected linkage groups was also investigated as well as the number and total base-pairs of scaffolds able to be positioned on the linkage group. The extent of linkage disequilibrium across the linkage groups was reviewed by calculating the pairwise linkage disequilibrium results for each set of markers using PLINK v1.9 . The sex-specific recombination rate was calculated by comparing the linkage map distance (cM) and genome scaffold distance (bp) between individual marker pairs for males and females.The parents for each FINK v1.9 . This wa1 and F2 generations and phenotyping data from the F2 generation were used. QTDT used 10,716 markers which had been placed on the linkage map. GRIDQTL used a subset of markers (n = 1007), which were filtered randomly to a minimum spacing of 1 cM. Before running the analysis, the genotype data were filtered for Mendelian errors by dropping loci for any individual that contained alleles not observed in either of the two parents. The phenotype measurements used for the analysis were standardized by tank and date collected to correct for temporal and tank effects. The QTL scan results from QTDT were visualized using the ggplot2 library v3.1.0 in the R statistical environment v3.2.3  and using the built in permutation procedure in GRIDQTL with 1000 permutations.Quantitative trait loci identification was carried out using the general model implemented in QTDT v2.6.1 . Strong positive correlation was also observed between year one and year three for each measure .Peduncle length, fork length, and weight were recorded when individuals where 436-487 days old (year one) and 1045-1131 days old (year three). The distribution and relative sizes of fish in year one and year three are illustrated in 1) and 23.9x in the parents (F2).A total of 1.6 billion reads were produced for all eight pooled GBS libraries with approximately 2, 4 or 6 million reads for each single, duplicate, or triplicate individual library respectively. Using the STACKs pipeline a total of 20,311 SNPs were found after filtering for >7x coverage, present in 75% of the individuals in the population, and a minor allele frequency (MAF) of 0.05. The average coverage per SNP was 15.6x in the offspring  and 30.2 cM from the nearest QTL peak (GRIDQTL). Myogenin on linkage group 3 was 9.6 cM from the nearest QTL marker (QTDT) and 18.4 cM from the nearest QTL peak (GRIDQTL). Paravalbumin on linkage group 16 was 25 cM from the nearest QTL marker (QTDT) and 7.8 cM from the nearest QTL peak (GRIDQTL).The base-pair position on the genome scaffolds were found for all 13 candidate genes including valbumin . Based oChrysophrys auratus. Proof checking the marker order against the snapper de novo genome assembly indicated that the linkage groups were of high quality. QTL mapping revealed eight markers on three linkage groups that were significantly associated with growth. Three candidate genes for growth were located on the same linkage groups as these QTL. These genomic resources will be used to inform the selective breeding program in New Zealand and will form the basis of further genomic investigation in snapper.We assembled the first chromosome level linkage map for the Australian snapper Salmo salar) includes \u223c96K markers, although it should be noted that the genome size in this salmonid species is also significantly larger (2.97 Gb genome size)  . ComparaGasterosteus aculeatus, 3.11 cM/Mb, . It is not known how sex is being determined in snapper, but if the heterochiasmatic sex commonly has a lower recombination rate, then this may indicate that males are heterogametic in this species. Further work is needed to explore this in more detail.Using the newly constructed linkage map and available genome scaffolds, we were able to calculate the sex-specific recombination rates for snapper, which showed that females have a higher recombination rate compared to males . This reflects observations in other fish species, with females often . Other factors that can affect growth include feed amounts, fish density in tanks, and tank design . We attempted to control for these factors in the current study by standardizing the conditions between tanks and by standardizing measures from each tank.The target trait for this study was growth , which wSalmo salar: 0.06 to 0.08, , brill to 0.23, .e.g., candidate genes or causative alleles) are likely to be located. If high rates of linkage are present between markers, a confidence interval for the QTL region can be estimated - as seen in R/QTL , growth hormone inhibiting hormone (GHIH), growth hormone (GH), and insulin-like growth factors (IGF-1 and \u2013II) (growth hormone and insulin-like growth factor I and II were able to be mapped to the linkage map in the current study. Growth hormone was located near (within 5.3 cM) a QTL of genome-wide significance. In Sparus aurata, a close relative of C. auratus, a microsatellite repeat in the promoter region has previously been implicated for differences in growth  are another set of potential candidate genes  was located on the same linkage group as a genome wide significant QTL, but was much further away from a putative growth QTL (25 cM) than the previous two genes. A mutation in the promoter region of this gene was previously found to be involved in weight differences in the finfish species Lates calcarifer.Previous studies have outlined a range of genes and molecular networks that are thought to be candidates for further investigation in teleost species . We locaand \u2013II) . Of theste genes . These rWhile the linkage map constructed in this study can confidently place SNPs in \u223c5 cM regions, further work is needed to improve the accuracy of marker placement. More accurate placement of SNPS would help with future work to fine-map and further characterize the QTL and candidate gene locations described in this study. Improved precision should be possible in the near future using the genome assembly that is being further improved by our group. Future work should also aim to detect possible sex-linked markers, to identify regions associated with sex determination, and to investigate sex-specific recombination patterns across the genome. While sex-specific information was not investigated in the current study, this is an area of particular interest in snapper and the data from this study could be used to further investigate it. In conclusion, this study provides valuable genetic and genomic resources for future evolutionary studies and aquaculture breeding programs in this and related species."}
+{"text": "Pseudostellaria heterophylla is one of the well-known traditional Chinese medicines and has been used in clinics for 100\u00a0years in China. The chemistry and pharmacology of P. heterophylla were reviewed to understand its active compounds. Then analysis of these compounds related to quality control of this herb was discussed. For the analysis of chemicals, three aspects have been discussed in this review. The first two aspects focused on the methodologies for analysis of cyclic peptides and carbohydrates in P. heterophylla, respectively. The last one dealt with the other methods used for identification of P. heterophylla. Some rich chemicals such as oligosaccharides in this plant were rarely evaluated. Many analyses were performed on this plant, however, few of them were accepted as quality control method. Pseudostellaria heterophylla, tai-zi-shen (\u592a\u5b50\u53c2) or hai-er-shen (\u5b69\u513f\u53c2) in Chinese, is a well-known traditional Chinese medicines (TCMs) first officially recorded in Ben Cao Cong Xin, which contains 721 kinds of herbs, by Wu Yiluo in 1757 [P. heterophylla was considered as one of the precious medical material from ancient China and now is one of the most commonly used TCMs in clinic, which invigorating spleen, replenishing qi, moistening lung and benefiting blood. It has been used for treatment of fatigue, spleen asthenia, anorexia, asthenia after severe illness and cough due to lung dryness [ dryness .Pseudostellaria heterophylla mainly distributed in Liaoning, Hebei, Shandong, Anhui and Sichuan provinces. Ningde (Fujian Province) and Shibing (Guizhou Province) in China offer the most suitable envionment for P. heterophylla cultivation [P. heterophylla can only be replanted once every 4\u00a0years [P. heterophylla with high quality in geo-authentic production zone are limited and the demand for this medicinal material is rising annually, the government has established a large-scale cultivation areas for it in Jurong (Jiangsu Province), Zherong (Fujian Province), Shibing (Guizhou Province) and Xuancheng (Anhui Province) of China [P. heterophylla and their quality have shown significant differences [P. heterophylla before establishing an effective quality control method to ensure its safety and efficacy [tivation . However 4\u00a0years , 9. As tof China . Howeverferences , 12. Theefficacy .P. heterophylla, including cyclic peptides (pseudostellarin), polysaccharides, amino acids, saponins, and sapogenins based on chemical studies [P. heterophylla have attracted many researchers\u2019 interest. And high-speed counter-current chromatography (HSCCC) was demonstrated to be an efficient separation method for cyclic peptides [P. heterophylla [P. heterophylla, have been reported to exhibit multiple pharmacological activities [P. heterophylla [Various components were found in  studies . In recepeptides \u201317. Up trophylla \u201321, whictivities . Lectinsrophylla , 24. Therophylla .Fig.\u00a01StP. heterophylla has multiple pharmaceutical activities including immunomodulatory [Based on the abundant chemical constituents, dulatory , 25, antdulatory \u201329, antidulatory  activitidulatory \u201333.P. heterophylla, effectively suppressed the adhesion and invasion of human esophageal carcinoma cells by mediating PI3\u00a0K/AKT/\u03b2-catenin pathways and regulated the expression levels of adhesion- and invasion- associated genes [Plant cyclopeptides comprise a large group of small molecules from natural medicines, which exhibit various pharmacological activities, such as immunomodulatory, anti-inflammatory, antioxidant, anti-aging and antitumor effects , 35. Preed genes . Furthered genes .P. heterophylla. The fraction riched with polysaccharides of P. heterophylla has protective effects against cobalt chloride-induced hypoxic injury in H9c2 cell [P. heterophylla also can improve exercise endurance and have protective effects against oxidative stress [P. heterophylla have been proved their benefits to chronic fatigue syndrome. That may be why P. heterophylla is usually used as a tonic herb [P. heterophylla are commonly used. A water-soluble, pectic polysaccharide with molecular weight of 48\u00a0kDa, composed of rhamnose, galactose, arabinose and galacturonic acid and 1,4-linked galacturonic acid as main chain with small amount of 1,2-linked rhamnose, could obviously stimulated insulin secretion [P. heterophylla polysaccharide. The mean molecular weight of H-1-2 was 14\u00a0kDa and it was only composed of d-glucose monosaccharide. In vitro, HepG2, 3T3-L1, and L6 cells were used to assess cellular glucose consumption and cellular glucose uptake. The results showed that H-1-2 could clearly increase glucose uptake and utilization in muscle and adipose cells, which is beneficial for screening leading compounds of anti-diabetes [P. heterophylla has also been demonstrated to have protective effects on retinal laser injuries [P. heterophylla exhibited a dose-dependent antitussive effect [In recent years, increasing studies have been focused on the bioactivities of polysaccharides from 9c2 cell . Crude pe stress \u201333. Polye stress . Polysacnic herb . Howeverecretion . A noveldiabetes . The sape effect .P. heterophylla. High performance liquid chromatography (HPLC), thin layer chromatography (TLC), gas chromatography (GC), matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), near infrared (NIR) spectroscopy and nuclear magnetic resonance (NMR) etc. have been applied for characterization of components in P. heterophylla, which were summarized in Table\u00a0P. heterophylla, and their bioconcentration factors (BFs) of investigated heavy metals are not higher than 0.5 except for Cd, where Pb and As were especially low. Only Cd could be enriched slightly in P. heterophylla while others could not [Various methods have been developed to analyze the components in ould not .Table\u00a02CP. heterophylla, and heterophyllin B , 12 nucleosides, and 16 amino acids were simultaneously quantified by ultra-performance liquid chromatography tandem triple quadrupole mass spectrometry (UPLC-QQQ-MS/MS) . The othrophylla \u201346. 1H-Nrophylla , which hP. heterophylla. Their pharmaceutical activities have been discussed in \u201cP. heterophylla were not well investigated to date, even if some of their beneficial effects such as antioxidant, immunostimulant and antitumor activities have been demonstrated. In fact, few types of polysaccharides have been identified in structure. However, the biological activities of polysaccharides are closely correlated to their molecular size, types and ratios of constituent monosaccharides, and features of glycosidic linkages  [P. heterophylla [Polysaccharides are the main bioactive macromolecule components in harides) , 49. Recrophylla , 27, 50.P. heterophylla [P. heterophylla [P. heterophylla from different cultivated fields [P. heterophylla, and significantly differentially expressed genes in P. heterophylla from different fields were found [The isobaric tags for relative and absolute quantification (iTRAQ) MS/MS have been applied for discrimination of different habitats of rophylla , 52. Furrophylla . Near inrophylla . NMR hasd fields , 53. In re found .P. heterophylla were quantified by QTRAP LC\u2013MS/MS for evaluating the processing methods and discriminating different idioplasm resources of P. heterophylla [P. heterophylla [Besides, nucleosides and nucleobases in rophylla \u201357. HPLCrophylla \u201360.Pseudostellaria heterophylla is one of the well-known TCMs with multiple pharmacological activities in last decades. Even researchers evaluated the chemicals especially cyclic peptides in this plant, the methods for quality control of P. heterophylla are still not reasonable. Some chemicals such as oligosaccharides, which are rich in this plant based on our research (data will be published in others), were rarely evaluated. The investigation of oligosaccharides, with high amount in aqueous extract of P. heterophylla, may lead to develop a rational and scientific quality control methods for this herb."}
+{"text": "Journal of Experimental Botany, Vol. 68, No. 13 pp. 3529\u20133539, 2017; doi:10.1093/jxb/erx200The original manuscript contained errors in some of the figures used.In These figures have now been updated with corrected versions."}
+{"text": "Choline kinase \u03b11 (ChoK\u03b11) has become an excellent antitumor target. Among all the inhibitors synthetized, the new compound Ff35 shows an excellent capacity to inhibit ChoK\u03b11 activity. However, soluble Ff35 is also capable of inhibiting choline uptake, making the inhibitor not selective for ChoK\u03b11. In this study, we designed a new protocol with the aim of disentangling whether the Ff35 biological action is due to the inhibition of the enzyme and/or to the choline uptake. Moreover, we offer an alternative to avoid the inhibition of choline uptake caused by Ff35, since the coupling of Ff35 to novel biomimetic magnetic nanoparticles (BMNPs) allows it to enter the cell through endocytosis without interacting with the choline transporter. This opens the possibility of a clinical use of Ff35. Our results indicate that Ff35-BMNPs nanoassemblies increase the selectivity of Ff35 and have an antiproliferative effect. Also, we demonstrate the effectiveness of the tandem Ff35-BMNPs and hyperthermia. Chemoresistance to cancer is a major concern ,2 that hHowever, it has been described that certain inhibitors of ChoK\u03b11, besides hemicholinium-3, are also capable of inhibiting choline uptake, thus making it more difficult, if not impossible, for their use in systemic clinical treatments . For thiMagnetococcus marinus MC-1 magnetosome membrane protein MamC, have demonstrated themselves to be promising nanocarriers, able to couple with drugs forming stable nanoassemblies at physiological pH, while efficiently releasing the drug in acidic (tumor) environments in response to pH changes. In fact, these BMNPs present novel features compared to other nanoparticles produced inorganically and/or other biomimetic nanoparticles ).The kinetics of Ff35 adsorption on the magnetic nanoparticles was studied to determine the time required for this adsorption to reach equilibrium. In these experiments, an aliquot of 5 mg of BMNPs was mixed with 1 mL of Ff35 100 \u00b5M in HEPES buffer  for several time intervals up to 48 h. After the incubation time, the Ff35-BMNPs nanoassemblies (here referred as Ff35-BMNPs) were collected with a magnet and washed twice with HEPES buffer. Then, the supernatants were measured by UV-Vis spectroscopy at a wavelength of 304 nm and these measurements were used to calculate the percentage of the absorbed compound. The molar absorptivity of Ff35 in HEPES buffer at 304 nm was determined as 2677.5 L molQ) is the amount of adsorbed drug per mass unit of adsorbent, (eC) is the amount of nonadsorbed compound, (LFK) is the LF affinity constant, and (r) is the cooperativity coefficient. A value of r < 1 means a negative cooperativity whereas a value of r > 1 means a positive cooperativity choline into phosphocholine, both in the absence (control) or presence of different inhibitor concentrations. Briefly, the final reaction mixture contained 100 mM Tris-HCl (pH 8.5), 10 mM MgCl2, 10 mM ATP, and 20 ng of purified ChoK\u03b11. The reaction was initiated with 1 mM [methyl-14C]choline (4500 dpm/nmol) and incubated at 37 \u00b0C for 10 min. The assay was stopped by immersing the reaction tubes in boiling water for 3 min. Aliquots of the reaction were applied to the origin of silica gel plates in the presence of phosphocholine (0.1 mg) and choline (0.1 mg) as carriers. The chromatography was developed in methanol/0.6% NaCl/28% NH4OH in water  as solvent, and phosphocholine was visualized under exposure to iodine vapour. The corresponding spot was scraped and transferred to scintillation vials for measurement of radioactivity by a Beckman 6000-TA  liquid-scintillation counter. The 50% inhibitory concentrations  were determined from the % activity of the enzyme at different concentrations of synthetic inhibitor by using a sigmoidal dose-response curve (the ED50plus v1.0 software).The effect of Ff35 on ChoK was assayed in purified ChoK\u03b11 as previously described by Schiaffino et al. , by dete\u22121), or Ff35-BMNPs (Ff35 1 \u03bcM and BMNPs 300 \u00b5g mL\u22121) were added for different times. Cell viability was assayed by the MTT -2,5-diphenyltetrazolium bromide). Formazan crystals were dissolved in 100 \u03bcL of DMSO, and the absorbance was read at a wavelength of 570 nm using a microplate reader . The GI50  value was determined from the % cell viability at different concentrations of synthetic inhibitor by using a sigmoidal dose-response curve (the ED50plus v1.0 software), referencing this value to an untreated cells control taken as a 100% of the viability.HepG2 cells were seeded onto 96-well plates (10000 cells/well) and grown in MEM/10% FBS for 24 h. After 24 h, the medium was removed and 100 \u03bcL of fresh medium containing Ff35 , BMNPs (300 \u00b5g mL\u22121), or Ff35-BMNPs (concentration of Ff35 was 0.5 \u03bcM and BMNPs was 150 \u00b5g mL\u22121 or that of Ff35 was 1 \u03bcM and BMNPs was 300 \u00b5g mL\u22121). After 10 min, 24 h, and 48 h of treatment, the cells were immediately exposed to a pulse of [methyl-14C]choline  for 5 min at 37 \u00b0C. The incorporation of choline was stopped by medium aspiration followed by two washes in ice-cold PBS containing 580 \u00b5M choline. Then, the cells were solubilized in NaOH 0.1 N and an aliquot was used to determine the total amount of radiolabeled choline taken up by the cells, measured by liquid scintillation using a Beckman 6000-TA counter .Choline uptake was assayed as previously reported . HepG2 c\u22121) or Ff35-BMNPs (concentration of Ff35 was 1 \u03bcM and BMNPs was 300 \u00b5g mL\u22121) or only MEM/10% FBS medium, as a control, were added for 24 h. Cells were collected using trypsin and centrifuged at 1500 rpm for 5 min in MEM/10% FBS. Cell pellets were fixed in 2.5% glutaraldehyde and 2% paraformaldehyde in 0.05 M cacodylate buffer for 4 h at 4 \u00b0C. The samples were washed three times with cacodylate buffer and postfixed in an aqueous solution of 1% OsO4 containing 1% potassium ferrocyanide for 1 h at 4 \u00b0C in darkness. The following washes were done (25 \u00b0C): 0.15% tannic acid in cacodylate buffer, cacodylate buffer, and H2O. The samples were left in 2% uranyl acetate for 2 h and washed several times with H2O. Then, dehydration in ethanol solutions rising from 50% to 100% was done at 4 \u00b0C. The samples were embedded in resin (EMbed 812/100% ethanol (1/1)) for 60 min at room temperature, the same resin at a 2/1 ratio for 60 min, and then resin without ethanol overnight. For polymerization, the samples were incubated in pure resin for 48 h at 60 \u00b0C. Ultrafine sections (50\u201370 nm) were cut using a Leica Ultramicrotome R and contrasted using 1% aqueous uranyl acetate for 5 min and lead citrate in a CO2-depleted atmosphere for 4 min [HepG2 cells were grown in six-well dishes for 24 h. Then, BMNPs (300 \u00b5g mLor 4 min . A ZeissH = 21 kA/m (B = 26.4 mT in air) at the center of the coil, where the samples were placed, measured with a NanoScience Laboratories Ltd., Probe , with 10 \u00b5T resolution. All samples were previously prethermostated at 37 \u00b0C. Prior to any determination, the adiabatic condition of the system was verified by subjecting a sample of Milli-Q water as control, in order to ensure that any temperature changes in the samples under study were due to the action of the magnetic field, and not a consequence of environmental temperature gradient. A preliminary experiment was performed with a suspension of bare BMNPs (300 \u00b5g mL\u22121) to set the conditions (frequency and strength of the field and time of application) needed to guarantee that a temperature of 43 \u00b0C was reached. The sample temperature was determined with an optical fiber thermometer . For actual hyperthermia experiments, HepG2 cells were incubated for 24 h at 37 \u00b0C with either 300 \u00b5g mL\u22121 BMNPs or Ff35-functionalized BMNPs and exposed to the AC magnetic field for time lapses ranging between 1 and 3 h. Immediately after the hyperthermia treatment, the cells were processed for the MTT test.Magnetic hyperthermia experiments were carried out using a laboratory-built AC current generator, based on a Royer-type oscillator. The AC source was connected to a double five-turn coil built with a copper tube 4 mm in diameter. This allowed control of the temperature of the coil by flowing thermostated water. The coil was 20 mm in diameter and 45 mm long. The magnetic field frequency was 197 \u00b1 3 kHz, and its strength was p < 0.05 is considered statistically significant.The results are shown as averages \u00b1 SEM. A one-way ANOVA was done with post hoc comparisons by Scheff\u00e9\u2019s test (SPSS 13.0). B was 150 K for BMNPs. According to Prozorov et al. [B and slow magnetization increase is related to particles that expose a large magnetic moment per particle and high crystallinity.BMNPs exhibited well-developed faces and a size ranging from 20 to 50 nm, with an average crystal size of 35 \u00b1 8 nm, according to TEM analyses A,B. The v et al. , this hiQ) increased with the equilibrium concentration of Ff35 in the supernatant (Ce) at a higher rate at the lowest Ce values. Such a rate decreased as Ce increased (R2 = 0.93402), showing a drug loading capacity (maxQ) of 0.0026 \u00b1 0.0003 mg Ff35 mg magnetite\u22121. This model introduces the effects of energetic heterogeneity of the surface and the cooperativity between Ff35 molecules, meaning that once a Ff35 molecule is coupled to BMNPs, it lowers the energy required for the coupling of the next ones. The values of the LF affinity constant (LFK) and cooperativity coefficient (r) parameters, calculated by means of this model were of 40 \u00b1 10 mg of Ff35 per mg of magnetite and 1.5 \u00b1 0.6, respectively.The kinetics of Ff35 adsorption on BMNPs over time shows that the system reached equilibrium at ~6 h A. The amncreased B. The admaxQ). Due to the fact that this is the first study on Ff35 adsorption on magnetite nanoparticles (or on any other nanoparticles), comparisons of the maxQ of the present study are done in reference to other studies involving either the BMNPs used here or other nanocarriers. The maxQ value obtained in the present manuscript was lower than those values obtained for the coupling of other drugs like doxorubicin (DOXO) to BMNPs. For example, the adsorption of DOXO on the same BMNPs was 0.69 \u00b1 0.03 mg DOXO/mg BMNPs [maxQ for Ff35 is about two orders of magnitude lower than that for other compounds, this amount that is coupled to the BMNPs is enough to have a cytotoxic effect as it will be shown below.Our results show that BMNPs are able to carry 0.0026 mg of Ff35 per mg of magnetite (mg BMNPs  or 0.41 mg BMNPs . Even thr > 1) demonstrates a strong positive cooperativity between the molecules of Ff35 during the adsorption process. This type of interaction has been previously described in DOXO adsorption on citrate-coated apatite nanocrystals [The BMNPs\u2019 loading capacity could be explained, on the one hand, by the electrostatic interaction occurring at pH 7.2  between Ff35, which exposes two basic groups, positively charged and the negatively charged magnetite surface (isoelectric point (iep) = 4.4) , which acrystals  and on Bcrystals .50 values derived from the growth inhibition curves were of 6.23 \u00b1 0.39, 1.37 \u00b1 0.004, and 0.45 \u00b1 0.10 \u03bcM for 24, 48, and 72 h, respectively. Lactate dehydrogenase (LDH) activity in the culture medium was not detected after any of the treatments up to 10 \u03bcM of Ff35 (data not shown), so the decrease in cell proliferation observed after the treatment with soluble Ff35 could not be attributed to any acute cytotoxicity produced by plasma-membrane leakage. Ff35-BMNPs also significantly decreased cell growth after 24 or 48 h of treatment. HepG2 cell proliferation was not affected by the presence of BMNPs, which demonstrates the cytocompatibility of the BMNPs.Soluble Ff35 show a negative effect on HepG2 cell growth in a time and concentration-dependent manner A. The IC50 value of 0.46 \u00b1 0.079 \u03bcM. In addition, choline uptake was also inhibited in the presence of soluble Ff35 in 55% and 75% at 0.5 \u03bcM and 1 \u03bcM, respectively, after only 10 min of exposure. The effect was even more noticeable after 24 or 48 h of treatment, reaching inhibition levels of up to 68% and 88% at Ff35 0.5 \u03bcM and 1 \u03bcM, respectively, after 48 h of treatment . However, the amount of compound released from Ff35-BMNPs (Ff35-BMNPs 0.5 \u03bcM and BMNPs 150 \u00b5g mL\u22121) was not sufficient to inhibit choline uptake.Interestingly, the treatment with Ff35-BMNPs for 10 min did not produce significant changes in the incorporation of choline into the cells compared with cells treated with BMNPs B or in cHowever, despite the fact that Ff35-BMNPs had little effect on the uptake of choline, it produced a marked decrease in cell proliferation, as mentioned above, similar to that observed in cells treated with soluble Ff35 at the same concentration B. This i\u22121), and to determine the possible morphological changes caused by Ff35-BMNPs exposure. Ultrastructural analysis by TEM showed that BMNPs were internalized in the cells by endocytosis (\u22121. Both the BMNPs (via a method that does not depend on choline transporters. Again, these results open the possibility for a clinical use of ChoK\u03b11 inhibitors by making their uptake by the cells independent of choline transporters. Moreover, the experiments of the present study provide a protocol that allows us to disentangle whether the cytotoxic activity of a ChoK\u03b11 inhibitor such a Ff35 is due to the selective inhibition of the enzyme, or on the contrary, due to the nonselective inhibition of the choline uptake by the cell as an undesirable secondary effect. This protocol could be standardized and extended to other molecules.TEM was used to visualize the internalization of BMNPs or Ff35-BMNPs (concentration of Ff35 was 1 \u03bcM and BMNPs was 300 \u00b5g mLocytosis . Controlocytosis A,B showeocytosis C,D, confhe BMNPs C,D and Fhe BMNPs E,F were We consider now the effect of hyperthermia and the combination hyperthermia-Ff35 on cell proliferation. In all cases, the HepG2 cells were subjected to the magnetic field for periods of 1, 2, and 3 h. We assume that, according to generally accepted results, tumor cells are more sensitive than healthy ones to a temperature increase , and, alTherefore, these results open the door, not only to the potential use of ChoK\u03b11 inhibitors without the secondary effect linked to the inhibition of choline uptake, but also, to the application of a dual therapy based on targeted drug release and hyperthermia to potentiate the cytotoxic activity of these compounds on tumor cells.In summary, this study shows that the coupling of the ChoK\u03b11 inhibitor Ff35 to BMNPs offers great advantages. On one hand, since the nanocarrier is a magnetic nanoparticle, it opens the possibility of a magnetic guidance to the target site through the application of a continuous magnetic field. Also, it offers the possibility of also using the nanocarrier as a hyperthermia agent, thus combining the cytotoxic effect of the molecule with the cytotoxic effect induced by hyperthermia and the triggering of drug release from the BMNPs that hyperthermia induces. Finally, and no less important, it offers the potential of Ff35 entering the cell independently of the choline transporters. This is crucial, as the coupling of Ff35-BMNPs allows the compound to exert a cytotoxic activity comparable to that of the soluble compound while avoiding the secondary effect linked to the inhibition of choline uptake. Also, it provides a protocol that could be extensible to other molecules to disentangle the cytotoxic effect of the drug in terms on enzyme inhibition and/or inhibition of choline uptake. Therefore, the novel design of the nanoassemblies of Ff35-BMNPs showed in this study and the demonstration of the activity of such a compound without interfering in the choline uptake are crucial results, as they would allow the potential use of these ChoK\u03b11 inhibitors as antitumoral drugs that would otherwise be compromised."}
+{"text": "Panicum virgatum L.) is a perennial warm-season C4 grass identified as a model species for bioenergy feedstock production. Lanthanum (La) as a rare earth element can stimulate the physiological processes of plant growth. The purpose of this study was to investigate the effect of lanthanum on seed germination of switchgrass. However, no significant differences in seed germination were found. The energy dispersive X-ray analysis showed that abundant lanthanum deposits resided on the pericarp and testa of the seed while few lanthanum deposits were present on the aleurone and endosperm. This phenomenon demonstrates that a semi-permeable layer, which could restrict or impede solute exchange, while allowing the permeability of internal and external water and gas, may be located between the testa and aleurone. Light microscopy and histochemical analysis revealed that the main chemical composition of the semi-permeable layer would be expected to be suberin because the layer was stained yellow with aniline blue. The quantum chemical calculations predict that the intervals between adjacent carbon chains in suberin molecule are so small that lanthanum ([La(H2O)8]3+) cannot pass through the suberin molecule. In conclusion, the seed germination of switchgrass is not affected by lanthanum because the semi-permeable layer restricts the penetration of lanthanum into the embryo.Switchgrass ( Panicum virgatum L.) is a perennial warm-season C4 grass identified as a model species for bioenergy feedstock production 3+ [2O)8]3+ and suberin-[La(H2O)8]3+ complexes are optimized at the density functional theory with Becke-3-Lee-Yang-Parr functional (DFT-B3LYP) level 3+  22] in  in 3+. IH2O)8]3+ . The strP) level  with a mExperimental data were analyzed with SPSS 19.0 software. Means were compared by one-way analysis of variance(ANOVA), and Duncan\u2019s test was used for multiple comparisons of the different treatments.3)3 reduced the seed germination of switchgrass at day 2 compared to the control (P>0.05) at and after day 7.Soaking treatments with different concentrations of La , and itsThe main structure of the seed includes the pericarp (a), testa (b), aleurone (c) and endosperm (d) . Based o2O)8]3+ cannot penetrate suberin molecule. Moreover, if suberin molecules are stacked one by one in the suberized semi-permeable layer, [La(H2O)8]3+ would not pass through it, which is consistent with our experimental observation. There are many binding sites in the suberin molecule for La(H2O)83+, but these binding sites can be divided into three kinds. The first kind of binding site (I) is composed of one phenolic-OH and one phenolic-OCH3 group. The second one (II) consists of one C-OH and one C-O-C group. The third one is made up of two C-O-C groups. The calculated results show that La(H2O)83+ is anchored to these binding sites by H-bonds. The hydrogen atoms in La(H2O)83+ could form H-bonds with the O atoms at the binding sites, and the lengths of the H-bonds range between 1.44 and 1.72 \u00c5. Our calculations also predict that the binding energies (E(suberin-[La(H2O)8]3+ complex)\u2014E(suberin)\u2014E(La(H2O)83+)) are -268.85, -297.47 and -375.71 kJ mol-1 for the first, second and third kind of binding sites, respectively, which demonstrates that the suberin-[La(H2O)8]3+ complexes are very stable. From 2O)8]3+, and these phenomena explain why the suberin-[La(H2O)8]3+ complexes are stable although the polarities of the binding sites are weak.The optimized geometry of suberin molecule has a layered structure , and theTriticum durum, seed pre-soaking for 8 h with lanthanum inhibited seed germination at low concentrations (0.01 mM and 0.1 mM), while pre-soaking for only 2 and 4 h inhibited seed germination when higher concentrations (1 mM and 10 mM) of lanthanum were used 3+ could not penetrate it because the intervals between adjacent carbon chains were very small. Our finding further explained and confirmed the conclusion that the semi-permeable layer is an important structure for restricting the penetration of toxic solutes into embryos during imbibition from the soil  [38 [3837]  further the soil .3+ (in the form of La(H2O)83+) into embryo, which explains the lack of effect on seed germination of switchgrass by lanthanum.In this study, the seed of switchgrass is determined to have a semi-permeable layer, which is located between testa and aleurone layer, and the main chemical composition of the semi-permeable layer is suberin. The suberin molecule restricts the penetration of LaS1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file."}
+{"text": "Linking routinely-collected data provides an opportunity to measure the effects of exposures that occur before birth on maternal, fetal and infant outcomes. High quality linkage is a prerequisite for producing reliable results, and there are specific challenges in mother-baby linkage. Using population-based administrative databases from Brazil, this study aimed to estimate the accuracy of linkage between maternal deaths and birth outcomes and dengue notifications, and to identify potential sources of bias when assessing the risk of maternal death due to dengue in pregnancy.We identified women with dengue during pregnancy in a previously linked dataset of dengue notifications in women who had experienced a live birth or stillbirth during 2007\u20132012. We then linked this dataset with maternal death records probabilistically using maternal name, age and municipality. We estimated the accuracy of the linkage, and examined the characteristics of false-matches and missed-matches to identify any sources of bias.Of the 10,259 maternal deaths recorded in 2007\u20132012, 6717 were linked: 5444 to a live birth record, 1306 to a stillbirth record, and 33 to both a live and stillbirth record. After identifying 2620 missed-matches and 124 false-matches, our estimated sensitivity was 72%, specificity was 88%, and positive predictive value was 98%. Linkage errors were associated with maternal education and self-identified race; women with more than 7 years of education or who self-declared as Caucasian were more likely to link. Dengue status was not associated with linkage error.Despite not having unique identifiers to link mothers and birth outcomes, we demonstrated a high standard of linkage, with sensitivity and specificity values comparable to previous literature. Although there were no differences in the characteristics of dengue cases missed or included in our linked dataset, linkage error occurred disproportionally by some social-demographic characteristics, which should be taken into account in future analyses. Research involving record linkage has increased in recent years with the growing availability of administrative population-based databases and the relatively low cost of joining information from different sources. Linkage has been particularly important for research on maternal, fetal and infant health. Maternal mortality in Brazil is still relatively high, at 62 maternal deaths per 100,000 live births in 2015. Therefore, studying potential risk factors associated with this health indicator is a priority in the public health agenda[Linking information on women\u2019s social contexts and health status prior to and during pregnancy to fetal and childhood outcomes can yield knowledge beyond the limits of traditional cohort studies. Due to the large scale and availability of population-based datasets, and the routine collection of such data , rare or long-term outcomes in particular can be investigated. In the past decade, many high-income countries have used linkage to identify maternal deaths and to investigate the health of mothers and their babies\u20135. HowevThe quality of the linkage is important for producing reliable results, and there are specific challenges in mother-baby linkage, mainly arising from to the limited availability of common identifiers for linkage . The impThis linkage study is part of a series of studies measuring the association between dengue and pregnancy outcomes in Brazil,9. BraziIn this article, we aimed to validate the linkage strategy we used in Paixao et al 2017 to creata priori information on the expected number of matches gives us information about the likely quality of the same linkage strategy when applied to datasets where the expected number of matches is not known . The purpose of our current analysis  was to investigate whether any linkage errors were differently distributed amongst the group with the outcome  and the comparison group (women who survived). This would establish any potential bias in the linked dataset, which might influence subsequent analyses. We aimed to estimate the accuracy of the linkage and identify potential sources of bias, where any subgroups of records were more or less likely to link.Evaluating a linkage strategy with a dataset  for which we have We linked three routinely collected Brazilian datasets: a) Notifiable Diseases Information  for dengue; b) Live Births Information System  for live births; and c) Mortality Information System  for stillbirths and maternal deaths. We linked the datasets in two stages . First, In both linkage processes, we used variables common in all three datasets: maternal name, maternal age, and place of residence. To link with death records, we also used information on the date of the maternal death and the birth outcome, because maternal deaths are likely to occur on the same day as the birth outcome or soon after (and by definition must occur within 42 days after the end of pregnancy). Therefore, the difference in dates contributed information on the likelihood of two records being a match.The Brazilian Notifiable Diseases Information System (SINAN) contains records on notifiable diseases, characteristics of the individual with the disease , symptoms of the disease, laboratory tests, and disease severity.After excluding men, and non-dengue records, we retained 1,725,943 cases of dengue from 2007\u20132012. This is likely to underestimate the burden of disease, as less than 10% of dengue cases are reported to SINAN in Brazil. For every notified case, there are an estimated 12 dengue cases in the community[The Ministry of Health of Brazil uses the WHO definition of a live birth: the complete expulsion or extraction from the body of the pregnant woman of a product of conception, independent of the duration of pregnancy, who, after the separation, breathes or shows any other signs of life, such as heartbeat, umbilical cord pulsation, or definite movement of voluntary muscles, whether or not the cord is cut and whether or not the placenta is attached. The Brazilian Live Birth Information System (SINASC) contains records of all live births in Brazil; these data come from birth registration, a legal document completed by the health worker who attends the birth. It includes information on the mother ; the pregnancy ; and the neonate . An evaluation of the birth registration system in Brazil found 97% of Brazilian live births are registered.The Ministry of Health of Brazil uses the WHO definition of maternal death: the death of a woman while pregnant or within 42 days of termination of pregnancy, irrespective of the duration and site of the pregnancy, from any cause related to or aggravated by the pregnancy or its management but not from accidental or incidental causes. Maternal mortality is widely underreported worldwide. Since 2008, the health surveillance system in Brazil has investigated deaths in women of 15\u201349 to enhance detection of maternal deaths. In 2010, 76% of deaths occurred in this group were investigated[The Brazilian Mortality Information System (SIM) contains records of all deaths in Brazil, including stillbirths. Stillbirth in Brazil is defined as the death of a product of conception before the expulsion or complete extraction from the body of the pregnant woman, occurring from 22 weeks or weighing more than 500g). These data come from the Death Certificate, a required legal document. We retained all maternal deaths, defined as those coded under obstetric causes of death by ICD-10, the \"O\" group), and all stillbirths.It is possible that stillbirths are under-reported in the national system, howeverWe obtained ethical approval from the Research Ethics Committee, Public Health Institute, Federal University of Bahia, Brazil (CAAE: 26797814.7.0000.5030 CEP-ISC) and the London School of Hygiene and Tropical Medicine, UK (Ethics Ref: 10269).We derived our pre-processing and blocking schemes, gold-standard data and match weights based on previous linkage of Brazilian birth data, as described by Paixao et al (2017). First wTo estimate parameters for linkage weights and to validate the quality of the linkage, we created a gold-standard dataset. Firstly, we linked using deterministic linkage with exact agreement on full name and age. In subsequent steps we relaxed the rules by allowing matches with differences in age or in the ways names were recorded. Each step was followed by manual review to exclude false-matches.Match weight calculations were based on the Fellegi-Sunter method. For eacFrequency-based weights were calculated for each category of Jaro-Winkler score comparator (for name of the woman) and year of age, so that rarer values were given higher weights. Separate weights were also calculated for the five most frequently occurring names in the data .Since we were linking different subjects (the woman and her live or stillbirth), and because the exposure (dengue during pregnancy) could have happened up to nine months before the outcome , there were timing issues to consider. Two records could differ in time by nine months and could bridge calendar years; some women would have a birthday between the date of dengue notification and the date of the live birth/death. To allow for this, we estimated different weights according to the similarity of age across datasets: equal ages, age differing by one year, age differing by two years, and ages differing by more than two years. In the maternal mortality linkage, we included the time between the birth outcome and death of the mother as a linkage variable (occurred on the same day as the birth outcome (day 0 or 1); between 2\u20137 days; between 8\u201315 days; between 16\u201330 days; after 30 days).Match weights were calculated by summing the log of the ratio of m-probabilities and u-probabilities across different identifiers. The algorithm was implemented in Stata 14.1 and R 3.4.1.Records pairs were ordered by match weight and manually inspected to identify a conservative threshold values aiming to exclude as many as false-matches as possible . We classified any records above the cut-off threshold as links.a priori, as we did not know how many pregnant women were expected to link with a dengue notification registered in SINAN. These findings are described by Paixao 2017[For the first linkage stage (of women with dengue notification to women with live births/stillbirths), we did not know the expected number of matches ixao 2017.For the second linkage stage , we expected all maternal deaths would link with a live birth or a stillbirth after excluding women with maternal deaths coded under pregnancy with abortive outcome . We therefore expected around 90% of maternal morbidity records to have a pregnancy outcome (stillbirth or live birth) and therefore to link with one of our datasets. We used this value to identify the number of missed matches (records from the same mother-baby pair that failed to link) and to estimate the sensitivity (true links among the matches) of the linkage.To estimate the proportion of false matches (records that were erroneously linked from different mother-baby pairs), we looked at women who were coded as having died in a pregnancy with an abortive outcome (i.e. those who should not have linked with a live birth/stillbirth record). In Brazil, if women had an abortive outcome, the product of conception would not be recorded in either SINASC or SIM, therefore we did not expect a link from these women. Those who linked were assumed to be false-matches, as were those who linked to a live birth and a stillbirth simultaneously, unless they were multiple births.2 test or Fisher\u2019s exact test, and means were compared with a t-test. A two-sided P value of less than 0.05 was considered to indicate statistical significance. Stata version 14.1 was used for the statistical analyses.We then examined which characteristics were associated with missed matches in the maternal mortality dataset. We compared the characteristics of maternal deaths classified as having dengue through linkage, with the maternal deaths where dengue was coded (using ICD-10 codes) as an underlying cause of death but that were missed by our linkage. We examined maternal age in years (continuous variable), maternal education , maternal marital status , self-identified race  and classification of deaths occurring during pregnancy or puerperium. Categorical variables were compared between groups with Chi6717 of the 10,259 maternal deaths in SIM from 2007\u20132012 were linked: 5,444 (53%) linked to a live birth record, and 1,306 (13%) to a stillbirth record. Of these linked records, 33 linked to both a live and stillbirth simultaneously: 20 were multiple pregnancies (with a live birth and stillbirth outcome); 3 were duplicate records (stillbirths misreported as live births and notified in both datasets); and 10 were false matches. After excluding these, 3542 maternal deaths remained unlinked .Of the 10,259 maternal deaths in SIM, 1,046 (10%) were coded as having an abortive outcome, which we did not expect to link to a live or stillbirth. Of these pregnancies with an abortive outcome, 124 (12%) linked ; 55 linked with a live birth and 71 with a stillbirth (two linked with both). This gave an estimated specificity of 922/1046 = 88% . HoweverMissed matches were not associated with age or marital status . RecordsWe identified 23 maternal deaths that were coded in SIM with ICD-10 codes for dengue as the underlying cause of death, but that had not linked to the dengue notification cases in our linkage. The reasons were:the maternal death was in a pregnancy with an abortive outcome ;the maternal death record did not link to a live birth or stillbirth ;the maternal death linked with a live birth or stillbirth, but not to the dengue notifications .We investigated these latter 12 cases further, finding that in 5/12 cases, the symptoms of dengue began in the puerperium, more than 42 days after the fetus was born, and therefore were classified as no dengue during pregnancy in the previous linkage; 1/12 cases were classified as an uncertain link; the remaining 6/12 were false-matches in the original linkage.Excluding the three abortive outcomes, there were no differences in socio-demographic characteristics between the 20 records that our linkage failed to identify, and the 14 cases of dengue identified in our linkage of maternal mortality and dengue datasets.We implemented the linkage methods described by Paixao et al (2017) in a datLinkage to bring together information from two different sources about the same individual can achieve high rates of sensitivity, even when using records containing truncated or ambiguous matching variables\u201316. HoweA very important aspect of linkage is how linkage error might impact on inferences from the linked dataset. The analyst should know and report any evaluation of linkage accuracy and be aware of groups disproportionately affected by linkage error ,20. In tBeyond socioeconomic status, there are other possible explanations for the distinctive characteristics observed in the non-linked group. First, completion of the forms by health care professionals and the process of digitalization in private and public health facilities may result in differential quality of records. Second, in Brazil, unsafe induced abortion has been associated with low education and income,22, and To further evaluate the linkage accuracy on the dengue status of the pregnant women (exposure variable), we compared the dengue cases that the algorithm captured, with those that it could not . We found no difference in socio-demographic characteristics between the two groups. It is therefore unlikely that the linkage process introduced bias in our previous linkage of live births/stillbirths and dengue, since missed matches occurred randomly in relation to the exposure variable. However, this analysis had limited power because the sample size was small (34 cases of dengue). Therefore, we interpret our results showing no difference between the two groups cautiously.This study has a number of limitations. First, maternal deaths are under-reported. However, as this error probably affected both those exposed and not exposed to dengue, it is unlikely to bias the study results. Second, we did not know the gestational age at which each maternal death occurred, and so we cannot conclude whether the missed-matches occurred due to linkage errors, or if they should not have been linked . It is possible that we underestimated the number of false-matches, but we expected all the maternal deaths to link, it is unlikely that records have liked to the wrong record, because that record would also have been linked. Due to the restricted number of variables, we could not improve the sensitivity of the algorithm, and so our linked dataset should not be used to estimate rates of live births or maternal mortality rates for women exposed to dengue during pregnancy. Our analysis of the association between study characteristics and linkage error may have been limited due to low power. Finally, we assumed that linkage quality metrics in our maternal mortality linkage would be representative of those in our initial linkage between birth outcomes and dengue notifications, since the data are derived from similar sources and contain the same matching variables.It is important to understand the quality of the linkage and the potential bias that can be introduced in results of analyses of linked data. In this study, sensitivity and specificity of our linkage strategy were comparable with previous literature. Although there were no differences in the characteristics of dengue cases missed or included in our linked dataset, linkage error occurred disproportionately according to some social-demographic characteristics, which introduces bias and should be taken into account in future analyses. These results reinforce the need to evaluate linkage quality and to take linkage error into account within analyses of the following studies when necessary."}
+{"text": "Deep Eutectic Solvents/Complex Salts-Based Electrolyte for Next Generation Rechargeable Batteries focuses on the effects of electrolytes on the electrochemistry/chemistry of rechargeable batteries and cells.Recent years have seen an expansion of renewable energy technologies driven by global demands for energy alongside social and environmental concerns. One of the most significant solutions, rechargeable batteries have promising features which include high capacity, energy density, rate capability, long lifetime, and cost-effectiveness. As the key component in energy storage devices, the electrolyte has had a major impact on the chemistry/electrochemistry of rechargeable batteries/cells for a number of reasons. These include its potential window, which limits the redox potential of an electrochemical reaction. Its electrochemical activity and conductivity also influence the electrochemical reaction and consequently the battery performance. The composition, as well as the stability, of rechargeable batteries, shapes the electrolyte-electrode interface. Furthermore, its corrosivity cannot be neglected. For these reasons, researchers are highly motivated toward breakthroughs in battery performance, exploring the fundamental properties of electrolytes based on novel formulation/synthesis. Hence, this special issue of Pan et al.). Other submissions discuss the compositing strategy for a polyethylene oxide-based solid state electrolyte for flexible device and lithium metal batteries . Another study considers the emerging ionic conductor for an Mg ion solid state electrolyte , where another describes how electrolyte modification can be used to depress zinc dendrite in zinc-based flow batteries . The electrolyte concentration, as well as cation/proton types of diffusion kinetics in a vanadium flow battery  are also interest. Another contribution examines the molecular design of organic electroactive molecules via functional group modification for redox flow batteries . Furthermore, the electrolyte-electrode interface is also emphasized in this topic, where its material characteristics and stability strongly influence the kinetics and cycling stability of batteries/cells for example in an in situ Scanning Electrochemical Microscopy study, which revealed that the SEI formation on carbon depends on voltage range , or, as described elsewhere, regulating the nature of ionic liquid and the interface compatibility . Another study designed a novel 3D MoSe2 template for constructing stable SEI for reversible stripping/plating of metallic Mg , where others discuss regulating the interface between electrolyte and Zn for preventing zinc dendrites , or investigating the interfacial charge transfer in aqueous zinc electrolytes for zinc ion batteries .In this Research Topic, representative types of electrolytes are discussed in relation to next-generation rechargeable batteries, including ionic liquid, solid state electrolyte (polymeric and inorganic), multivalent electrolyte (aqueous and non-aqueous) for multivalent ion batteries, aqueous, and a novel organic electroactive molecule-based electrolyte for flow batteries. Recent developments build upon the correlation between design and synthesis of the electrolyte, its properties, and the related electrochemical properties and performance of batteries and cells. From the perspective of electrolyte design, contributions include exploration of formulating ionic liquid toward designed ion separation for tuning the performance of supercapacitors (We would like to thank all the authors for their valuable contributions and the reviewers for their thoughtful insights and suggestions. It is essential to further examine and share the fundamentals of the chemistry and electrochemistry of electrolytes, which serves as the basis for the successful creation of next-generation rechargeable energy storage devices.Although the papers included here provide significant insights, there are still important developments and points for the future development of this area of research. Safety is a long-term pursuit in rechargeable batteries, and further insights into safe electrolyte design with promising electrochemical properties are essential. The pursuit of high performance and long lifetime in rechargeable batteries will also need to go further than constructing a database of electrolytes, and future explorations on the relation between electrolytes and battery chemistry are essential. It would also be promising to uncover the correlation between electrolyte and charge storage mechanism for any novel electrodes designs, which will synergistically guide the development of new generation battery systems. Moreover, the complexity of the electrolyte-electrode interface creates room for exploring battery chemistry, which will drive cutting-edge characterization and analysis tools and in parallel, boost the development of new battery systems, especially those for utilizing metal anode based or multivalent ion batteries.We hope that this special issue will inspire future research in the development of novel electrolytes and drive the development of new generation rechargeable energy storage devices. These endeavors will pave the way for realizing a green and sustainable society.DY, GC, CJ, and HZ co-edited this special issue. All authors contributed to the article and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "Suberin is a hydrophobic biopolymer of significance in the production of biomass-derived materials and in biogeochemical cycling in terrestrial ecosystems. Here, we describe suberin structure and biosynthesis, and its importance in biological , ecological (soil organic carbon) and economic (biomass conversion to bioproducts) contexts. Furthermore, we highlight the genomics and analytical approaches currently available and explore opportunities for future technologies to study suberin in quantitative and/or high-throughput platforms in bioenergy crops. A greater understanding of suberin structure and production in lignocellulosic biomass can be leveraged to improve representation in life cycle analysis and techno-economic analysis models and enable performance improvements in plant biosystems as well as informed crop system management to achieve economic and environmental co-benefits. Quercus suber (cork) bark is about 30\u201350% of the dry weight mass [The survival of terrestrial plants depends on their ability to control water loss and solute transport, insulate from climatic extremes and variations, protect against pathogenic attacks and to recover from mechanical damage. Suberin is a lipophilic bio-macromolecule that is integral for the ability of plants to withstand and recover from such stresses and challenges \u20137. Suberght mass , 9 whereght mass . Hollowaght mass . Given tght mass \u201314. Subeght mass \u201318.Phytophthora sojae [2) and temperature, have been reported to result in altered suberin chemistry of roots [The production of suberin can change in response to drought and other abiotic stresses to prevent water loss and enhance water retention capacity within root systems . Suberinra sojae . Suberinra sojae , 25 and ra sojae . Changesof roots . A recenof roots . SuberinSuberized cells can be found in the stem periderm and the root periderm, exodermis and endodermis, and other specialized tissues such as seed coat, fruit and vegetable skin, and abscission zone , 28\u201330. Arabidopsis mutant characterization studies [Genetic understanding of suberin biosynthesis in stem and root and in other specialized tissues such as seed coats, and their functional roles in water movement regulation, defense and mineral accumulation properties has been greatly aided by  studies , 28, 29. studies , 29, 31.Arabidopsis enhanced suberin1(esb1) mutant provided clear evidence not only for root suberin in water and solute transport, but also that higher root suberin content had ramifications on whole plant function including a reduction in water loss and wilting under drought-like conditions, and differential shoot ionome composition [Altered levels of suberin content can result in significant impacts on plant health and productivity . A detaiposition . Given tLolium plants\u00a0 [Considering drought, a die-back of cortical and epidermal tissues and increased suberization of\u00a0endodermis protecting the stele from desiccation has been reported from  plants\u00a0 . In resp plants\u00a0 , 35. Wat plants\u00a0 , 36, 37. plants\u00a0 , 38, 39.2 levels in the environment can influence tissue development as well as cell wall chemistry, including suberization [Toxins, nutrient status and COrization , 31. Forrization . Suberinrization . The ext\u03c9-hydroxyacids and \u03b1,\u03c9-diacids are typically the most abundant long chain lipids found in suberin [Suberin is a nonlinear, irregular, poly(acylglycerol) macromolecule built from poly-functional long chain fatty acids, fatty alcohols and glycerol which are covalently linked to phenolic moieties. A general structure of suberin was proposed by Kolattukudy in which a cross-linked aromatic subdomain is covalently linked to long-chain diacids and hydroxyacids through ester bonds , 42. \u03c9-h suberin , 8. It w suberin \u201345. In s suberin .Suberin (primary association with cork) and cutin (primary association with cuticle) are both complex macromolecules that serve as protective barriers in plants. While suberin is a biopolymer consisting of both aromatic and aliphatic domains, cutin is a polyester consisting primarily of omega hydroxy acids (C16 and C18 families) and has lower abundance of longer chain fatty acids (C20\u2013C30) than suberin . In contDue in part to its heterogeneous, irregular and diverse nature, unaltered suberin isolation from plant tissues and characterization remains an analytical challenge in research applications. Additionally, the nature and properties of suberin and derived moieties make detailed characterization tedious and laborious, often requiring many steps, chromatographic separation and sophisticated detection technology, rendering high-throughput and accurate analyses difficult to achieve. Typically, acid or base-catalyzed transesterification or methanolysis are used to remove or isolate and determine suberin content and its lipid, phenolic and glycerol components in biomass but many of these methods may induce changes on the structure and may not accurately reflect relative composition of specific constituents and moieties \u201310. The Arabidopsis [Arabidopsis roots using confocal microscopy [Arabidopsis roots [Arabidopsis seed coats, but not root suberin biosynthesis or cutin biosynthesis, based on supporting data from histochemical staining  in conjunction with confocal microscopy, SEM and TEM [A working hypothesis of suberin superstructure and high-level domain architecture as it exists in plant cells arises from a number of dominant spectroscopic and microscopic observations. First, transmission electron microscopy (TEM) images of suberized plant cell walls reveal a poly-lamellar structure with repeating dark and light bands, with 30\u201360 repeating layers found in cork . Isolatebidopsis . The autbidopsis . Classiccroscopy . Cohen eis roots . AtMYB10 and TEM .\u03c9-hydroxyacids, \u03b1,\u03c9-diacids, epoxy-substituted lipids, phenolics and glycerol have been characterized by various mass spectrometry techniques, particularly on depolymerized or otherwise isolated or extracted suberin material  acids, fatty  alcohols, mono- and di-l Tables  and 2. AGC/MS analyses require that the suberin be depolymerized and monomeric constituents are derivatized prior to analysis. Additionally, liquid chromatography with mass spectrometry (LC/MS) may be used to identify and quantify suberin-derived products. Thiombiano et al. developed a workflow to characterize flax seed coat hydrolysates, including suberin and cutin-derived species as well as lignans, using LC/MS and GC/MS . Their mOther techniques such as matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) are important for analyzing biopolymers such as suberin given their ability to probe structural information. For example, MALDI-MS was used to analyze fruit cuticles to image the surface heterogeneity and to understand the structural features associated with cutin and suberin in tissues , 56. TheQuercus suber cork and its isolated suberin as well as lignin fractions in conjunction with NMR experiments [While GC/MS, LC/MS and MALDI-MS are powerful and important techniques used to analyze biopolymers such as suberin, they often require laborious sample preparation and chemical depolymerization steps that are not easily adaptable to high-throughput analyses that may be needed for population-scale and multi-omic studies. Mass spectrometry techniques such as pyrolysis-mass spectrometry (py-MS) offer the advantage of potential minimal sample preparation, although these techniques often require time intensive chromatographic separation for speciation and quantitative, unbiased characterization. Additionally, py-MS techniques may not necessarily provide structural insights but could potentially be implemented in a high-throughput platform. Py-MS techniques use a pyrolysis step  to produce vapors from materials prior to chromatography and/or MS analysis. Marques et al. demonstrated how py-GC/MS can be used to analyze suberin in biomass and the complications that can arise related to the presence of lignin and the analytical conditions and parameters used . Py-GC/Meriments . The autPy-MS techniques such as pyrolysis field ionization mass spectrometry (Py-FIMS) are particularly useful for analyzing soils and have been used to characterize soils based on the suberin species detected in the analyses , 60\u201363. Pyrolysis metastable atom bombardment time-of-flight mass spectrometry (Py-MAB-TOF-MS) is a fingerprinting method that has been used to analyze lipids in soils that originate from a variety of sources in an effort to expand the profile range of species and hence variability detected amongst different soils . Pyrolys2 environments within the aliphatic moieties at different chemical shifts with different dynamics properties [2\u2013O\u2013 groups and might be physically closer to ester linkages [2 domains exist, Yan and Stark used two-dimensional 1H\u201313C Wide-Line SEparation (WISE) NMR to study dynamics and domain architecture within wounded potato tissue [13C chemical shift in the direct dimension with the 1H profile in the indirect dimension, thus providing insight into the rigidity as measured by the shape of the 1H profile. The majority of water-hydrated suberin remains rigid, but about 20% of its CH2 groups are quite dynamic with molecular motion in the 50\u00a0kHz frequency range [Liquid and solid-state NMR techniques have also been used to probe suberin architecture as well as compositional and structural information related to the specific constituents that comprise suberin biopolymers. Early solid-state NMR measurements first from potato skins, and later on cork suberin, revealed two distinct methylene CHoperties \u201371. It wo tissue . The WIScy range .1H solution-state and 13C solid-state NMR techniques [\u03b1,\u03c9-alkanedioic acids, whereas aliphatic components that require harsher methanolysis conditions are richer in mid-chain modified \u03c9-hydroxyalkanoic acids. The findings also help explain the observation of two distinct methylene CH2 domains; it was proposed that a more rigid, partially ordered and repeating aliphatic acylglycerol region with various mid-chain modifications comprises a dominant central aliphatic structure, while hydrocarbon chains protrude into less ordered regions including the ferulate-rich polyaromatic region. Mild methanolysis conditions show that mostly \u03c9-hydroxyacids and ferulates are released, but when harsher conditions are applied, mid-chain modified fatty acids are identified. Later, the stereochemistry of mid-chain modified hydroxyacids  were identified using solution-state NMR [Lopes et al. characterized extracts from cork suberin obtained from sequentially harsher alkaline methanolysis using GC/MS, chniques . Togethetate NMR .1H and 13C HR-MAS data provide evidence of covalent linkages between polymers.Key solid-state NMR measurements have helped elucidate the spatial distributions of suberin subdomains. When suberized potato tissues are extensively solvent-extracted and enzymatically digested to remove unbound sugars and waxes, solid-state NMR data shows clear evidence of recalcitrant structural sugars, which are suggested to be bound to suberin , 75\u201377. Like other biopolymers, improvements in analytical technologies for suberin are important for biomass optimization efforts both for its impacts on plant and ecosystem health but also in regard to its impacts on biomass designed for applications in renewable energy and chemicals.The presence and structure of suberin in biomass clearly impacts plant growth, composition and survival and potentially has ecological ramifications on soil composition and health, including soil microbial composition. Additionally, suberin has implications on biomass conversion platforms related to its role in impacting the yield of desired products either due to the direct role of suberin abundance and structure on lignocellulosic feedstock conversion efficiency and/or in its indirect role impacting the productivity and composition of biomass used for biochemical or bioenergy production. As lignin composition in belowground biomass may have a relationship with total biomass yield of aboveground tissue that is used in conversion processes, and may also impact the ability of a feedstock to sequester C in soil ; suberinDue to its important role in the health and sustainability of plants, crop systems, and ecosystems as well as its own valorization potential and impact on conversion processes, it is imperative that suberin production in plants is considered in designing and cultivating crops intended for biochemical, bioproduct and bioenergy production Fig.\u00a0. Here weStudies employing plant genetic variants or mutants and characterization of associated\u00a0suberin pathway or phenotype modification(s) have been foundational for understanding and validating the physiological importance of suberin in plants . Another\u03c9-hydroxylase CYP86 and CYP94 subfamily were proposed to be involved in catalyzing fatty acid \u03c9-hydroxylation. Experimental validation studies showed that CYP86A1/HORST is expressed particularly within suberized tissues of roots and further alterations in suberin observed in compositional analysis of the mutant demonstrated the gene\u2019s involvement in suberin biosynthesis [CYP86A1/(horst) gene mutant, providing evidence of genetic control on root suberin levels [CYP86B1 gene is characterized as having a similar expression pattern in the root endodermis, and the corresponding ralph mutant shows a monomer specific alteration of very long chain \u03c9-hydroxy acids, diacids, although total suberin content was not significantly affected [CYP86B1/RALPH encodes a very long chain fatty acid (VLCFA) \u03c9-hydroxylase in plants [Solanum tuberosum down-regulated in CYP86A33 gene expression was found to be more fragile compared to control plant, and the RNAi-downregulated lines were also reported to have a reduction in weight, by 50% [CYP86A33 gene\u00a0had alterations in suberin ultrastructure showing a significant reduction in the thickness of suberin, the secondary wall of the periderm, and a significant decrease in \u03c9-functionalized monomers in aliphatic suberin which correlated with disappearance of the characteristic alternating dark and light lamellae [Using a transcriptomics analysis approach, root-expressing genes belonging to the cytochrome P450 fatty acid ynthesis . An obsen levels , 89. CYPaffected . Based on plants . In a se, by 50% . Plants lamellae .Arabidopsis suberin monomers. Seven KCS genes have been described as having a prolific and specific expression in various tissues/organs including roots [Arabidopsis, at least five KCS family genes have been associated with elongation of very long chain monomers in root suberin (C22) [KCS2/DAISY and KCS20 genes allude to potential redundancy within the 21-membered KCS family of Arabidopsis, a likely involvement with other bioprocesses requiring very-long-chain fatty acids and in turn, an\u00a0impact on the fatty acid pool for biosynthesis of the suberin monomer [ Fatty acid elongation involves \u03b2-ketoacyl-CoA synthases (KCSs) \u201396. Therng roots . In Arabin (C22) . The mil monomer , 98.\u03c9-hydroxyacid:hydroxycinnamoyltransferase) belonging to the BAHD family of acyltransferases) [Arabidopsis knockout lines of FATTY ALCOHOL:CAFFEOYL-CoA CAFFEOYL TRANSFERASE (FACT), an acyltransferase closely related to ASFT, was reported to be dramatically reduced in alkyl caffeate content while alkyl coumarate content was unaffected in suberized tissues [Knowledge of these empirically validated suberin biosynthesis pathway genes has allowed for new validated connections/candidate genes identified via network analysis, such as the fatty acyl reductases (FAR); FAR1, FAR4, and FAR5, and feruloyl transferases (ALIPHATIC SUBERIN FERULOYL TRANSFERASE (ASFT) and RWP1/HHT (ferases) , 99\u2013101. tissues , 99, 100FHT) gene via RNAi in potato periderm caused a reduction in ferulate esters, impacted developmental and functional water permeability properties, however, lamellar structure was apparently unaffected [Populus PtFHT1 gene in Arabidopsis resulted in higher root ferulate levels but not p-coumarate [Capsicum annuum (heterologous expression in rice) [Arabidopsis have led to identification of an enhanced suberin mutant, esb1, opening up the potential and promise of the approach in discovering additional suberin pathway genes from among the endodermis specific genes [ESB1 gene locus, At2g28670, is expressed within the endodermis [esb1 mutant roots had a two-fold increase in suberin aliphatic content and a disordered Casparian strip relative to control roots [esb1 has led to improvements in understanding the relationship between suberin content and root permeability, providing the first genetic evidence of suberin\u2019s role in both ion translocation to shoots and water balance [ESB1 gene, driven by tissue specific or designer promoters and use of advanced analytical technologies can deepen the understanding of the role of suberization and formation of Casparian strip barriers in plant nutrient acquisition, growth and biomass quality and productivity, as well as advance knowledge on the genetic underpinnings.Presence of a high degree of hydroxycinnamic acids and their derivatives such as feruloyltyramine distinguishes polyaromatic components of suberin from lignin. A complex network of feruloyl transferase and conjugation enzymes catalyze phenylpropanoid pathway-derived ferulate (p-hydroxycinnamic acid)  and tyroaffected . Ectopicoumarate . Distincoumarate , potato oumarate , 107 andin rice)  to be imin rice) . Mutant\u00a0ic genes , 110. Thdodermis . The\u00a0esbol roots , 22. TheArabidopsis atabcg1 mutants  with a particular reduction in fatty alcohols and acids and long chain dicarboxylic acids [Arabidopsis ANAC058, has been shown to impact suberin deposition, though the target/direct downstream genes in this process have yet to be uncovered [Membrane proteins of the ABC family  are shown or suggested to aid in the transport of suberin monomers through the plasma membrane , 111. Suic acids . A similic acids . Abscisiic acids . Potato ncovered .MYB family genes have been shown to act on or regulate suberin. Roles of AtMYB107 and AtMYB9 in suberin biosynthesis pathway are well documented [MYB93 genes from rice, tomato, apple, potato and grape suggests potential cross-species conservation of functional roles in suberin synthesis [A number of cumented , as wellcumented . MYB41 hcumented . Conservynthesis . A\u00a0MYB fynthesis . TobaccoArabidopsis, SUB has a regulatory role by transactivating promoters of suberin biosynthesis genes [SUBERMAN (SUB) transcription factor has been shown to increase the root suberin lamellae formation. In is genes . Beyond is genes .Populus stem periderm led to the discovery of several candidate suberin pathway genes [viz., suberin, lignin, and other surface waxes were characterized at four developmental stages [CYP86A7, were exclusively expressed in developed stage 3 of cork. Transcriptome analyses showed that this stage corresponds to highest number of genes responding to FAE, wax biosynthesis and lipid polyester biosynthesis [GPAT6, 7, and 8\u00a0genes, which encode cutin-specific acyltranferases in Arabidopsis were also upregulated in Populus bark transcriptomes, possibly\u00a0playing functional roles within the Populus suberin biosynthesis pathways [Populus homologs of Arabidopsis SHINE1 (SHN1)/WAX INDUCER1 (WIN1) [Populus periderm suberization [Studying suberin and wax composition through four developmental stages of hybrid ay genes . Chemical stages . Microscl stages . Some gepathways . Express1 (WIN1) , regulatGenomics and genetic studies have shown that modification of genes involved in suberin biosynthetic pathways can significantly alter suberin composition and structure and have implications on plant growth and stress adaptability. Further expansion of the fundamental suberin biosynthesis knowledge base and integration with validations in bioenergy crop species under applied economic and environmental contexts will be useful towards\u00a0sustainable bioenergy crop improvements efforts.Various studies support suberin\u2019s potential implications on carbon (C) input/storage/persistence belowground and on soil organic matter and soil properties such as aggregation which may in turn relate to biomass decomposition and microbial interactions below ground , 60, 64.2 [2 levels and temperature\u00a0can result in increases in suberin content by 28%, and by 36%, respectively [2 studies [2 coupled with environmental warming can result in greater specific root length and specific root area, and\u00a0potentially increase the content of suberin per unit of mass [Fine root turnover as well as root exudates contribute to large inputs of organic C into soil, which supports soil health and microbial diversity, and in turn plant growth and biomass productivity, in a feedback loop , 124. Ro2 \u2013127. A sectively . These r studies , 126. El14C) suberin molecular markers were used, which correlated with root biomass [14C content with fine root necromass, which suggested their greater contribution to SOC, in part due to suberin, and also that root necromass acts as a major source of SOC at soil depths greater than 60\u00a0cm. The weaker correlation between suberin and root necromass in surface soil profiles (between 10 and 35\u00a0cm deep) may be attributed to a higher level of degradation of root biomass, and a lower suberin content [While it is known that soil organic C has horizontal  and vertical (rooting and soil depth paths)\u00a0gradients, an important aspect in understanding the decomposition of organic C in soil is to distinguish between above- and below-ground contributing sources. C source can be distinguished, relatively, in studies tracing cutin and suberin as suberin is primarily root-derived, while cutin is primarily leaf-derived. Based on a study that used field and lab incubation experiments to track contributions of plant biopolymers to SOM, it was reported that in the\u00a0deciduous forest type studied, relative to leaf, root-contributed aliphatic compounds are a source of SOM with greater stability . As summ biomass . A posit content . In anot content  focusing content . The stu content . In agre content , 129. To content . Even wi content , 129 andA study by Sumiyoshi et al.  aimed toIn a study of rice and the rape crops rotation, bulk and rhizosphere soil samples were analyzed for suberin diacids using GC\u2013MS and compared to infer differences in C inputs across growth stages and cultivar types . The stuWhile the fundamental genetics and genomics studies have yet to foray into implications in ecosystem settings and interconnections among the relevance of suberin in C contributions to soil, exciting recent discoveries in suberin biology such as discovery of a key suberization regulator  and evidBiological and thermochemical conversion of lignocellulosic biomass focuses primarily on methods used to convert and valorize the biopolymer cell wall components lignin, cellulose and hemicellulose. Suberin occurs in specialized tissues and certain types and physiological fractions of biomass including roots and bark that may occur in abundance in forestry and agricultural waste streams. Relevancy of bark and the significant suberin component in biomass harvested from woody bioenergy feedstock crops have received limited attention relative to lignin and cellulose. The indirect impacts of suberin abundance and composition on stem biomass conversion are related to the effects of root and/or bark suberin variation on plant growth, physiology and chemistry including composition of lignin, cellulose and hemicellulose or on overall agronomic performance  of the feedstocks . The dirmHn conversion than their respective individual fractions [Thermochemical conversion methods such as pyrolysis are used to convert biomass to solid, liquid and gaseous products that could be used for chemical and energy production. Various types of catalysts and process conditions can be used to tune the distribution and properties of products derived from biomass where the biomass composition and pretreatment considerations are key factors in the conversion methodology. Thermochemical conversion of biomass high in suberin content relative to low-suberin biomass may result in various property and compositional differences related to water content, high-heating value and specific lipid-derived species present in bio-oils. For example, silver birch bark pyrolyzed in a series of thermal stages and subsequent fractionation generated a variety of suberin-derived products in the organic fractions but overall lower liquid yield (37.1 wt% vs. 60\u201365 wt%) and higher yields of certain oxygenated compounds such as fatty acids and aqueous fractions; which are substantially less favorable qualities in comparison to products generated from pyrolysis of lower suberin birch woody xylem . Studiesractions . Also, bractions .Quercus bark (cork) has undergone reductive catalytic fractionation (RCF) for production of bio-oil and specific chemicals including 4-ethylguaiacol derived from lignin and suberin [Catalysts have been incorporated in other thermochemical routes such as hydrogenolysis and depolymerization processes that have been implemented on barks and suberin-rich materials. Garrett et al. performed catalytic hydrogenolysis on various types of biomass barks using two different catalysts to understand the chemistry associated with the production of lipid and aromatic species derived from the suberin and lignin in the barks . Their s suberin . RCF has suberin . Bark oi suberin .Other non-biological conversion strategies such as acid hydrolysis and liquefaction have been used to convert barks to chemical intermediates and products. Two stage acid hydrolysis of birch wood and bark was investigated by Kim et al.  to find Direct thermochemical depolymerization of suberin has been used to produce biofuels particularly in order to take advantage of its high energy content. Kumaniaev et al. isolated and depolymerized suberin from birch bark in an optimized system and subsequently upgraded the oligomeric products by hydrotreatment to produce diesel and aviation fuel ranges . Oil yie4 yield, higher than that produced from wood, in part due to the high lipid contents of the potato peel and corresponding fermentation residues (being 2\u20138 wt%). Utilization of Pinus patula bark in enzymatic saccharification and fermentation processes with implications on a biorefinery concept have been demonstrated, however, suberin was not specifically considered in the study [Biological conversion methods used to convert biomass include enzymatic saccharification and hydrolysis, fermentation and anaerobic digestion (AD). Like thermochemical processes, biomass composition and pretreatment are important variables that can impact the yield and type of products generated. Suberin and/or bark presence in biological conversion methods targeted for sugar-derived chemicals by enzymatic hydrolysis has generally been shown to negatively impact the yield of desired products. For example, black locust bark suberin with known biocidal activity  must be he study .Combinations of thermochemical and biological conversion platforms can improve economics and utilization of waste materials in biorefinery concepts. Like the individual approaches, combined conversion platforms may still be impacted and be necessarily adaptable to differences and changes in feedstock properties and composition. However, most studies have focused on feedstock quality attributes such as lignin content, ash content, cellulose crystallinity, surface area, etc. without considering suberin which would otherwise be related to bark content, energy content and other attributes known to impact pretreatment and conversion economics . Rasi etThe significance of suberin in plant performance is unequivocal, and there are several lines of evidence supporting significance of suberin in potential economic  and ecological  contexts. Substantial progress has been made in gaining genomics insights and developing analytical methods to understand suberin biosynthesis in plants and deposition in plants and soil. However, there is a need to increase the pace and expand the breadth and depth of these studies, particularly for bioenergy-relevant crops.Arabidopsis and food crops (Table First, our current understanding of suberin genomics is derived primarily from plant growth and adaptation studies using ps Table  and is cCo-considerations of above- and below-ground plant chemistry and productivity will be necessary, especially in the context of suberin. Studies show that there is a strong genetic component and cultivar specificity to suberin quality and quantity, while also showing that as part of plant\u2019s adaptive mechanisms, suberin biosynthesis can be influenced by external abiotic and biotic factors. Applications of genomics/genetics and analytical approaches to characterizing stems and roots of large replicated populations under field conditions are needed to understand and quantify the interactive effects of the genetic and environmental components and improve crop\u00a0performance for future climate scenarios. Advancements in systems and synthetic biology approaches can be leveraged to design plants with precise and differential gene expression in above and belowground tissues to generate plants that are co-optimized for enabling a carbon\u2013neutral bioeconomy . ExtendiCorrelation of suberin chemistry to soil health, microbial activity,\u00a0and persistence and sequestration of soil C needs to be evaluated using improved analytical technologies in order to quantify the content, structure, composition and significance of suberin , 129 and13C-enriched plant and fungal cell walls, gaining key information on their detailed molecular structure and high-level architecture [13C isotopic enrichment, have not been applied to characterize suberized tissues. 13C enrichment of suberin should be possible, albeit expensive, by growing select plants in a 13CO2 atmosphere using a controlled growth chamber. Solution-state NMR methods should also be established for spectroscopic phenotyping. A few examples of HSQC NMR fingerprinting applied to suberin have been used by various groups, but established analytical protocols, comparable to those developed by the Ralph lab for lignin analysis are lacking [While many wet chemistry, microscopy and spectroscopic techniques are used to isolate and/or analyze suberin in biomass successfully at various scales and with varying degrees of changes induced on the native structure and composition, there is not consistent or standardized and validated methodology that is universally used to characterize and define suberin content, structure and composition in biomass. Suberin architecture, spatiotemporal dynamics and macromolecular structure in cell walls are particularly primed for new advancements in knowledge. For example, in recent years multiple groundbreaking studies have applied multi-dimensional and other advanced solid-state NMR methodologies to itecture , 158. Wh lacking , 160.The majority of suberin analysis methods require a number of steps to prepare samples and while analytical techniques can provide detailed speciation of suberin moieties, high-throughput suberin analyses for large sample populations are lacking. One possible solution would be to adapt high-throughput pipelines used to analyze sugars and lignin in biomass to analyze suberin content and/or composition . It may Additionally, there are not specific, validated methodologies used for the characterization of suberin in soils, particularly to analyze isolated species that can differentiate biomass origins and are separate from microbial contributions, particularly lipid moieties , 13. SubOptimizing suberin in biomass for conversion platforms can be approached by designing biomass with suberin that improves biomass yields, conversion potential and/or consists of suberin in plant tissues with favorable characteristics for direct valorization. Additionally, it will be important to establish relationships between biomass conversion and suberin abundance in bark, roots and other bioenergy feedstock tissues, with or without the presence of suberin in biomass being converted, thereby measuring the impacts of suberin on lignocellulosic conversion methodology. Further, conversion methods themselves can be optimized for biomass to include or account for suberin conversion.Direct conversion of high-suberin biomass has been demonstrated using a number of different approaches as outlined here and in other reviews that have provided a brief history and review of some suberin utilization and conversion strategies for production of a variety of materials with various applications , 84. HigTaken together, suberin exists at a high impact vantage point, and deeper and broader studies tracking suberin chemistry, underlying genes and associated economic and environmental impacts are urgently needed to undertake informed\u00a0co-optimization of both above- and below-ground plant tissues and to enable the vision of a circular, carbon-neutral\u00a0and sustainable\u00a0bioeconomy. Harnessing plants and their\u00a0chemistry for environmental and economic co-benefits will require us to address key gaps in our fundamental knowledge base, integrate above- and below-ground aspects and better\u00a0model impacts across scales. Cross-disciplinary perspectives and expertise will be needed to cover plant biology, systems and synthetic biology, analytical chemistry, processing, agronomy, forestry, ecology, data analytics and modeling aspects for assessing and optimizing plant performance and productivity, and evaluating impacts on\u00a0ecosystem and biorefinery performance. For population-scale studies and higher resolution characterization, there is a need for consistent, standardized and high-throughput analytical characterization with links to genome science and technology to enable predictive systems biology models. Last, but not the least, integration of suberin chemistry with multiple lines of evidence from genomics, phenotyping, biogeochemistry and conversion assessments into TEA and LCA models will be needed in holistically considered biorefinery operations and management of dedicated bioenergy crop plantations in order to enable a sustainable bioeconomy."}
+{"text": "MARF1) gene is essential for meiotic progression in animals; however, its detailed function remains unclear. In this study, we examined the molecular mechanism of dMarf1, a Drosophila homolog of MARF1 encoding an OST and RNA Recognition Motif (RRM) -containing protein for meiotic progression and oocyte maturation. Although oogenesis progressed in females carrying a dMarf1 loss-of-function allele, the dMarf1 mutant oocytes were found to contain arrested meiotic spindles or disrupted microtubule structures, indicating that the transition from meiosis I to II was compromised in these oocytes. The expression of the full-length dMarf1 transgene, but none of the variants lacking the OST and RRM motifs or the 47 conserved C-terminal residues among insect groups, rescued the meiotic defect in dMarf1 mutant oocytes. Our results indicate that these conserved residues are important for dMarf1 function. Immunoprecipitation of Myc-dMarf1 revealed that several mRNAs are bound to dMarf1. Of those, the protein expression of nanos (nos), but not its mRNA, was affected in the absence of dMarf1. In the control, the expression of Nos protein became downregulated during the late stages of oogenesis, while it remained high in dMarf1 mutant oocytes. We propose that dMarf1 translationally represses nos by binding to its mRNA. Furthermore, the downregulation of Nos induces cycB expression, which in turn activates the CycB/Cdk1 complex at the onset of oocyte maturation.Meiosis and oocyte maturation are tightly regulated processes. The meiosis arrest female 1 ( Drosophila melanogaster is an ideal model organism for the detailed study of oogenesis. Drosophila females have one pair of ovaries that consist of 18\u201320 ovarioles . In gerDrosophila melanogaster Polo kinase plays an important role in inducing oocyte maturation. Its activity is tightly regulated by several proteins, including Endos, Matrimony, and Greatwall (reviewed in [iewed in ). The aciewed in . The Cyciewed in . The reliewed in . AlthougMARF1  gene was first reported to be essential for meiotic progression in mouse [MARF1 mutant female mouse is sterile due to its disability to release the first meiotic arrest following gonadotropin-induced oocyte maturation. The catalytic subunit of protein phosphatase 2A (PP2A) is upregulated in the absence of MARF1 activity, and results in the reduction of MPF activity and failure of oocyte maturation. Although the molecular mechanism associating ectopic activity of PP2A and defective meiotic progression remains elusive, mouse MARF1 functions to maintain the protein phosphorylation cascade for meiotic maturation. In addition, Yao et al. have recently reported that the ribonuclease activity of the NYN nuclease domain of mouse MARF1 regulates meiotic progression and genome integrity in oocytes [Drosophila homolog of MARF1. In order to understand the molecular mechanism of meiotic progression and oocyte maturation, we investigated the activities of dMarf1, a Drosophila homolog of MARF1, in this study. Although the mutant females carrying a dMarf1 loss-of-function allele laid eggs, they never hatched due to incomplete meiosis.The in mouse . The MAR oocytes . HoweverdMarf1 [cyclin A was one of the targets of the dMarf1/CCR4-NOT function and proposed the mechanism of the temporal regulation of cyclin A reduction during late oogenesis. In this paper, we propose an additional layer of dMarf1 function. We identified a few mRNAs including nanos (nos) bound by dMarf1. Nos protein, which functions as a translational repressor of CycB, is downregulated during late oogenesis in the control. By contrast, high expression of Nos was found even in the mature oocytes of the dMarf1 mutant females. Our results suggest that dMarf1 can repress nos to induce cycB expression, and activate CycB-Cdk1 complex (MPF) at the onset of oocyte maturation.During our study, Fukunaga\u2019s group at Johns Hopkins reported a characterization of dMarf1 ; dMarf1 yw strain or the respective heterozygote was used as a control. A TRiP line, HMS04468 , was used to knockdown dMarf1 expression. The following drivers were used for transgene or RNAi expression: NGT40 and nosGal4-VP16, Mat\u03b1 Gal4-VP16 (BL#7063) (for germline cells) and traffic jam-Gal4 (for somatic follicle cells). A loss-of-function allele of dMarf1, dMarf1KO321, was generated using the CRISPR-Cas9 system  were PCR amplified using the cDNA clone RE61489 as a template. Triple repeats of the Myc-epitope tag were added to the N-terminal region. Purified PCR products were assembled using the In-Fusion system  with an XbaI-digested pUASp-attB vector (Drosophila Genomics Resource Center). The resultant plasmids were injected into embryos for insertion at the phiC31 integration site\u2014attP-3B\u2014at 65B2 (BL#32543) using the standard protocol [All fly stocks were maintained at 25\u00b0C or 18\u00b0C. Either the protocol . The pri2, 1 mM CaCl2) to prevent oocyte activation [Female flies were fed with wet-yeast paste for 2 to 3 days, and their ovaries were dissected in oocyte isolation buffer  were immunostained as described previously . For stant males , which cMat\u03b1 driver. Approximately 300 ovaries were collected and homogenized using a hand pestle in the oocyte isolation buffer containing proteinase inhibitor cocktail  and 0.1% Triton X-100 (Wako). The lysate was cleared by two rounds of centrifugation at 20,000 Relative Centrifugal Force (RCF). Myc-dMarf1-FL protein was immunoprecipitated from the cleared ovary lysate using anti-c-Myc antibody-conjugated magnetic beads (Thermo Fisher Scientific). Anti-HA antibody magnetic beads (Thermo Fisher Scientific) were used as a negative control. After washing the beads with phosphate-buffered saline (PBS) containing 0.1% Triton X-100, TRIzolTM  was added to extract the bound RNA.Myc-tagged full-length dMarf1 (Myc-dMarf1-FL) was expressed in germline cells by crossing with the TM/D-TOPO (Invitrogen), and recombined into pDEST17 to generate a His-tag fusion protein, His-dMarf1-C, using the Gateway system (Invitrogen). The His-dMarf1-C protein expressed in E. coli was purified using Ni SepharoseTM 6 beads  according to the manufacturer\u2019s protocol. The purified protein combined with complete adjuvant was injected into guinea pigs. Serum was obtained by centrifugation and heat inactivated at 56\u00b0C for 30 min.The C-terminal nucleotide sequence of dMarf1 (901\u20131305 residues) was amplified and ligated into pENTRTM reagent (Invitrogen), according to the manufacturer's protocol. The purified RNA (~1 \u03bcg) was treated with DNase I  to remove any contaminating genomic DNA. DNase I was inactivated at 70\u00b0C for 10 min in the presence of EDTA, and the complementary DNA was synthesized using superscript III (Invitrogen). Real-time PCR was performed using Fast SYBR Green system (Thermo Fisher Scientific) with StepOnePlus . Act5C was used for normalization.RNA was extracted from the ovaries or immunoprecipitates using TRIzoldMarf1KO321/CyO heterozygous and dMarf1KO321 homozygous females using TRizol (ThermoFisher) in accordance with the manufacturer\u2019s instruction. Quality of the purified RNA was examined by Agilent 2100 Bioanalyzer (Agilent) prior to the library construction. RNAs with poly(A) tails were enriched by using oligo-dT beads. RNAs containing poly(A) tails were purified with oligo-dT beads, fragmented and reverse transcribed. Adapters were ligated and the libraries were amplified by PCR. DNA fragments with small size (less than 200 bp) were removed by AMPure XP beads (Beckman Coulter). Libraries were sequenced with Illumina HiSeq sequencer (Illumina) on a 100 cycle Single End Read sequencing run. The cDNA library preparation and next-generation sequencing were performed at Hokkaido System Science . Total reads obtained by the Illumina HiSeq sequencing were 23,867,733 and 23,608,608 reads for dMarf1KO321 heterozygous control and homozygous mutant, respectively. Reads were mapped to the Drosophila genome (Release 6) using the TopHat2 program [dMarf1KO321 heterozygous control and homozygous mutant, respectively. The differentially-expressed genes were detected by Cuffdiff program from the Cufflinks package [Total RNAs were extracted from hand-dissected ovaries from  program . The map package .TACACTCTTTCCCTACACGACGCTCTTCCGATCT, PE1_rc; AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTA, PE2; GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT, PE2_rc; AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC) were trimmed by Trimmomatic software [Drosophila genome (Release 6) using the TopHat2 program [Myc-dMarf1 was immunoprecipitated from the ovarian lysate using anti-c-Myc antibody-conjugated magnetic beads as described in Materials and Methods. Immunoprecipitate with anti-HA antibody-conjugated magnetic beads was used as a negative control. RNAs bound to the magnetic beads were isolated by TRizol (ThermoFisher) in accordance with the manufacturer\u2019s instruction. Quality of the purified RNA was checked by Agilent 2100 Bioanalyzer prior to the library construction. The following RNA purification and the library construction were conducted as described above. Libraries were sequenced with Illumina HiSeq sequencer on a 150 cycle Pair-End Read sequencing. The cDNA library preparation and next-generation sequencing were performed at Gene-Nex (Japan). Total reads obtained by the Illumina HiSeq sequencing were 14,273,308 pairs and 10,686,509 pairs for Myc-dMarf1 bound and control fraction, respectively. Among reads, low-quality reads and adaptor sequences  buffer containing 2% deoxycholate and proteinase inhibitor cocktail. The egg chambers were homogenized by the plastic pestle, and the lysates were cleared by centrifugation at 20,000 RCF for 1 min at 4\u00b0C. Homogenization and centrifugation were repeated twice more. After third homogenization, lysates were further cleared by centrifugation at 20,000 RCF for 15 min at 4\u00b0C. The supernatants (~30 \u03bcl) were collected in low-protein binding tubes . The protein concentrations of the lysates were between 0.95 to 1.37 \u03bcg/ml. Proteins extracted from egg chambers were reduced, alkylated, and digested with trypsin. The peptides were labeled using the Tandem Mass TagTM system (Thermo Fisher Scientific). The pooled peptides were separated using the UltiMate 3000 RSLCnano system and analyzed by Q-Exactive mass spectrometry (Thermo Fisher Scientific). The differential protein expression levels between control and mutant were determined based on the signal strengths of the corresponding peptides detected for each protein. We had two biological replicates for each control and mutant samples. P-value was calculated for proteins with more than two peptides detected by using a standard software, Scaffold  and shown in The heterozygous and homozygous ovaries of 2, pH 6.4) [Mature but inactive egg chambers were isolated from dissected ovaries in the oocyte isolation buffer at 25\u00b0C. Oocyte activation was initiated by exchanging the isolation buffer with a hypotonic buffer  ,18. AfteProtein lysates from ovaries  were electrophoresed on a 7.5% or 10% sodium dodecyl sulfate-polyacrylamide gel. The following primary antibodies were used: guinea pig anti-dMarf1 , mouse anti-Myc 9E10 , mouse anti-CycA , mouse anti-CycB , rabbit anti-Nanos , rabbit anti-Dhd , mouse anti-Polo , mouse anti-glorund , and mouse anti-\u03b1Tubulin DM1A . Immunoreactive bands were visualized using HRP-conjugated goat anti-guinea pig , anti-rabbit, or anti-mouse secondary antibodies , and immunoblots were detected using LAS-1000  with an ECL chemiluminescent substrate (Thermo Fisher Scientific).The next generation sequencing data shown in this study are deposited to the DDBJ sequence Read Archive (DRA). The submission IDs for RNA-seq and RIP-seq data are SSUB012974 and SSUB012977, respectively.Drosophila melanogaster was screened for the mouse homolog of MARF1 using the method of polypeptide similarity search. The protein encoded by the CG17018 gene showed the highest score. The full-length CG17018 polypeptide consists of 1,305 amino acid residues and shares 31% sequence identity with mouse MARF1 in their middle regions that contain the RNA Recognition Motif (RRM) and OST/Lotus domains  (Drosophila homolog of MARF1.The genome of  domains . The OSTin (NYN) . HereaftXenopus, and Zebrafish , as a control. The mutant females in which dMarf1 expression was downregulated using either germline driver line laid a similar number of eggs compared to the control females , an M-phase marker. The fertilized eggs of the control females exhibited the characteristic rosette structure containing three pronuclei, indicating the completion of meiosis [Next, we examined late-stage oogenesis defects in criteria  and coun ovaries . In addi control . These rGV stage).dMarf1 plays a role in the maturation process after the release of the first arrest point, we performed the egg activation assay in vitro [dMarf1KO321 mutant eggs were resistant to bleach, indicating the failure of vitelline membrane maturation in the absence of dMarf1. This result prompted us to examine meiotic spindles of the metaphase I-arrested oocytes. Symmetric spindles  were detected in 97% of the control oocytes at stage 14, and only 3% showed abnormally shaped spindles. However, 22% of the stage 14 dMarf1KO321 oocytes exhibited broken spindles or misaligned chromosomes ; however, none of the dMarf1KO321 mutant eggs hatched  whose expression was downregulated , transformer 2 (tra2), and abnormal oocyte (abo) mRNAs in the Myc-dMarf1 bound fraction was further confirmed by qRT-PCR (tra2 encodes an RNA-binding protein that functions together with transformer (tra) to promote sex-specific splicing of doublesex (dsx) mRNA [abo is a negative regulator of histone gene transcription [tra2 and abo are not directly involved in meiosis or oocyte maturation, dMarf1 is suggested to function by binding with tra2 and abo mRNAs, which will be discussed later.It is known that dMarf1 harbors a conserved RRM domain; hence, we screened for RNA molecules bound by dMarf1 to identify its direct effectors. Myc-dMarf1-FL protein expressed in the germline cells was immunoprecipitated. To obtain the potential genes whose mRNAs bound by dMarf1 protein, the co-precipitated RNAs were analyzed by next-generation sequencing. Six different RNAs were enriched in the immunoprecipitated fraction ( qRT-PCR . tra2 ensx) mRNA . abo is cription . Althougnos, encodes a translational repressor of cycB, the protein of which complexes with Cdk1 to form maturation promoting factor (MPF) [nos expression by binding to its RNA, we analyzed Nos protein expression in the late stages of oogenesis. Consistent with the results of the previous study, Nos was highly expressed in stage 10 egg chambers and its expression rapidly decreased in the later stages [dMarf1KO321 egg chambers were comparable to that in stage 10 control egg chambers  . To examr stages   ,34,35. Hosophila ; moreoveMARF1 gene is evolutionarily conserved in animals; most proteins of the MARF1 family contain three major domains: NYN, OST , and RRM , Tejas (Tej), and Tapas (Tap) [nos mRNA. nos may not be the only target of dMarf1; other mRNAs such as tra2 and abo can also be targeted by dMarf1, although the biological significance of these interactions remains unclear. Zhu et al. have recently reported that dMarf1 can bind to cyclin A mRNA via its RRM domain [cyclin A in the dMarf1-bound mRNA fraction  ,36,37. Aas (Tap) . Interesas (Tap) . In addiM domain . Howeverfraction . FurtherDrosophila [dMarf1KO321 mutant phenotype , NP_001119538 for Xenopus tropicalis (western clawed frog), XP_020900751 for Exaiptasia pallida (sea anemone), and XP_011405091 for Amphimedon queenslandica (sponge).(A) Domain structures and multiple sequence alignments of the MARF1 family members. The polypeptide length is shown on the right. The domain structure is accessed from the NCBI. The NYN, RRM, and OST domains are denoted by blue, orange, and green boxes, respectively. (B) The phylogenic tree of MARF1 family members was generated using neighbor-joining method in CLC Genomics Workbench (Qiagen). The distance scale is also indicated. The NCBI reference sequences of the proteins are as follows: NP_724394 for (PDF)Click here for additional data file.S2 FigSecondary structure of dMarf1 polypeptide was predicted using the PSIPRED method. The vertical blue bar represents prediction confidence for each residue, and alpha helices and beta sheets are shown in pink barrels and yellow arrows, respectively.(PDF)Click here for additional data file.S3 FigThe C-terminal regions of MARF1 in various animal species are aligned. The bar under the alignment indicates sequence conservation . The alignment and schematic representation were designed in CLC Genomics Workbench (Qiagen).(PDF)Click here for additional data file.S4 FigDrosophila melanogaster (DROME), A0A0J9R6W8_Drosophila simulans (DROSI), B3NKL7_Drosophila erecta (DROER), B4IT21_Drosophila yakuba (DROYA), B4LQH5_Drosophila virilism (DROVI), B3MX08_Drosophila ananassae (DROAN), A0A1A9UH46_Glossina austeni (GLOAU), A0A1A9XT70_Glossina fuscipes fuscipes (GLOFF), A0A1A9W5J8_Glossina brevipalipis (9MUSC), and A0A0L0C5F0_Lucilia cuprina (LUCCU).The multiple sequence alignment for the C-terminal region of the insect MARF1 family members is shown. The bar under the alignment indicates sequence conservation at each residue . The alignment and schematic representation were designed in CLC Genomics Workbench (Qiagen). The NCBI reference sequences of the proteins are as follows: Q7KWG9_(PDF)Click here for additional data file.S5 FigdMarf1KO321 homozygous mutant males expressing a loss-of-function allele did not show significant defects in fertility. (B) dMarf1KO321 homozygous females laid eggs, but they did not hatch. (C-D) The expression of dMarf1 was knocked down using an shRNA under three different drivers: germline drivers, NGT40; nosGal4 and Mat\u03b1, and a somatic driver, tj-Gal4. The number of eggs laid by dMarf1-KD females within 24 h is plotted in (C). The hatching rate of eggs laid by dMarf1-KD females within 24 h is plotted in (D). The p-value of the student\u2019s t-test is shown in each graph.(A) (PDF)Click here for additional data file.S6 FigdMarf1 mutant females and rescue females expressing full-length Myc-dMarf1 in germline cells within 24 h. The p-value of the student\u2019s t-test is shown in graph. (B) Embryos from the control, mutant, and rescue females are stained with DAPI (blue), anti-\u03b1-tubulin (green), and anti-phospho-histone H3 (red). Scale bar, 5 \u03bcm (C) The progression of embryo development 0\u20136 h after laying eggs for control, mutant, and rescue females. The developmental stage (before or after blastoderm) was analyzed by the assessing the distribution of chromosomes in the DAPI-stained embryos.(A) The hatching rates of eggs laid by (PDF)Click here for additional data file.S7 FigThe dotted parts represent the truncated regions. RRM and OST denote RNA recognition motif and OST/Lotus domain, respectively.(PDF)Click here for additional data file.S8 FigdMarf1KO321 compared to the control is plotted. The p-values of student\u2019s t-test are indicated by ns  or * . (B) mRNA bound to Myc-dMarf1 was quantified using qRT-PCR. The fold enrichment compared to the control is plotted. The p-values of student\u2019s t-test are indicated by ns  or * .(A) mRNA expression in stage 14 egg chambers was quantified using qRT-PCR. The fold enrichment of (PDF)Click here for additional data file.S9 FigdMarf1KO321 females. (B) Quantitative analysis of CycB3 and Gnu protein expression in stage 14 egg chambers. The mean and standard deviation values are shown in terms of arbitrary unit. The p-value of the student\u2019s t-test is also shown in the graph.(A) Western blot showing protein expression at each stage of egg chambers in the heterozygous control and (PDF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S1 Protocol(DOCX)Click here for additional data file.S1 File(PDF)Click here for additional data file."}
+{"text": "Enterococcus faecalis and Streptococcus mutans and in vivo toxicity using embryonic zebrafish assays of sodium hypochlorite (NaOCl) and electrolyzed oxidizing (EO) water (containing hypochlorous acid (HOCl))-based root canal irrigating solutions. Methodology: Using 100 \u03bcL microbial count of 1 \u00d7 108 cfu/mL Enterococcus faecalis to mix with each 10 mL specimen of NaOCl or HOCl for designed time periods. The above protocol was also repeated for Streptococcus mutans. The concentration of viable microorganisms was estimated based on each standardized inoculum using a plate-count method. Zebrafish embryo assays were used to evaluate acute toxicity. Results: All the HOCl or NaOCl treatment groups showed > 99.9% antibacterial efficacy against Enterococcus faecalis and Streptococcus mutans. Zebrafish embryos showed almost complete dissolution in 1.5% NaOCl within 5 min. Both survival rates after being treated with 0.0125% and 0.0250% HOCl for 0.5 min or 1.0 min were similar to that of E3 medium. Conclusions: Both NaOCl and HOCl revealed similar antibacterial efficacy (> 99.9%) against Enterococcus faecalis and Streptococcus mutans. While 1.5% NaOCl fully dissolved the Zebrafish embryos, both 0.0125% and 0.0250% HOCl showed little in vivo toxicity, affirming its potential as an alternative irrigation solution for vital pulp therapy.The objective of this study was to evaluate the antibacterial efficacy against  The removal of the pathologic pulp, cleaning, and shaping of the root canal system are essential for successful endodontic treatment to disinfect pulpal space or prevent its reinfection of reference . The comAmong the current available endodontic irrigating solutions, sodium hypochlorite (NaOCl) is known for its ability to digest organic tissues during chemomechanical debridement of the root canals . HoweverThe electrolyzed oxidizing (EO) water can be produced by the electrolysis of a dilute NaCl aqueous solution through an electrolysis chamber. Similar to NaOCl as a chlorine-based sanitizer, EO water comprised of hypochlorous acid (HOCl) usually possesses an extraordinary bactericidal effect due to its high reduction/oxidation potential (ORP) over 1100 mV . WhereasStreptococcus mutans and Enterococcus faecalis were the bacteria of choice in this study due to their prevalence in caries (S. mutans) and in teeth needing endodontic retreatment  [Besides its usage as a standard irrigant in endodontic procedures, NaOCl is commonly used in procedures trying to preserve pulp\u2019s vitality, like in the case of deep carious lesions where the pulp could be inflammed but is not infected. aecalis) .Among piscine models, the zebrafish is a popular vertebrate model to study the toxicity of various xenobiotic agents . No liceVital pulp therapy is a well-defined treatment initiated to preserve and maintain pulp tissue in a healthy state . Vital pUnless otherwise specified, all chemicals were purchased from Sigma-Aldrich  and used without further purification. The ANK-Neutral Anolyte GH-40  was used to produce 50 L/h EO water by mixing DD water with an over-saturated solution of sodium chloride under 110 V. The output concentration of (HOCl) was set at the range of 330\u2013350 ppm. The residual chlorine concentration of the EO water was measured using a HI96771 chlorine photometer  first, then diluted to 0.0125% and 0.0250%, respectively.Enterococcus faecalis  cultures were inoculated on the surface of tryptic soya agar with polysorbate 80, lecithin , and 5% defibrinated sheep blood at 37 \u00b0C for 48\u201372 h, then adjusted microbial count to about 108 colony-forming units (cfu)/mL. Same method applied to obtaining Streptococcus mutans  cultures. One hundred microliters microbial count of 1 \u00d7 108 cfu/mL E. faecalis and S. mutans, respectively, was used to mix with each 10 mL specimen of NaOCl (1.5% and 5.25%) and HOCl (0.0125% and 0.0250%) to give an inoculum of 105 to 106 cfu/mL for designed contact time periods. A suitable sample was removed immediately from each suspension and the number of cfu/mL in each suspension was determined by plate-count method according to United States Pharmacopeia (USP) Chapter < 51 > antimicrobial effectiveness testing.Prior to the test, 2, and 0.33 mM MgSO4) at a temperature of 28 \u00b0C. To evaluate acute toxicity and developmental defects caused by NaOCl and EO water, zebrafish embryos were exposed to sodium hypochlorite (NaOCl) (1.5%) for 5 min as well as EO waters (0.0125% and 0.0250%) for 0.5 and 1.0 min using a yellow 100 \u00b5m cell strainer , then transferred to Petri dishes (n = 10) and three replications and recorded data at representative stages . The survivability and conditions of the embryos were captured under a light microscope  and a digital camera  under 40\u00d7 and 100\u00d7 magnifications. Percentage survival of the embryos was evaluated and scored for lethal or teratogenic effects.The animal ethic approval (LAC-2019-0243) from Taipei Medical University ethics committee was obtained. One hundred fifty fertilized wild-type zebrafish (Danio rerio) eggs 1-h post fertilization (1 hpf) were moved to Petri dishes and incubated within the zebrafish embryo E3 medium  in the measured data of each group.One-way analysis of variance (ANOVA) was used to evaluate the statistical significance of the measured data. Duncan\u2019s test comparison was used to determine the significance of deviations .The survival rates of Zebrafish embryos exposed to various concentrations of HOCl or NaOCl at different soaking times were investigated. S. mutans and E. faecalis. Results from S. mutans and E. faecalis in all the NaOCl and HOCl treatment groups, suggesting low concentration such as 0.0125% and 0.0250% HOCl is equally effective as 1.5% and 5.25% NaOCl. S. mutans is one of the prevalent species found in complex microflora of dental caries [S. mutans in oral infection greatly reduces dental caries in vivo [S. mutans. While S. mutans plays a major role in dental decay that may require vital pulp therapy depending on the amount of tooth structure damage, E. faecalis on the other hand is a common persistent pathogen in endodontic infections and the dominant species recovered in failed endodontic cases [E. faecalis phenotype and duration of contact with the agent [E. faecalis cells in the treatment of 5.25% NaOCl group for 10 min was 94.14% for 3-week-old biofilm model [E. faecalis in low concentration, thus making it a potential replacement for NaOCl as irrigating solution in failed endodontic treatment.Vital pulp therapy is aimed to remove source of infection from dental caries during early stages of the inflammation, thus healthy pulp tissue can be preserved to prevent complete necrosis. Various degrees of vital pulp therapy are available, such as indirect pulp capping, direct pulp capping, or pulpotomy, which all depend on stringent aseptic techniques to be successful . NaOCl il caries , and stu in vivo . Due to ic cases . Variouslm model  but 96.3lm model . From th2O2-Cl system of phagocytes [E. faecalis and S. mutans, when compared with 5.25% NaOCl. Similar outcomes for using HOCl as an endodontic irrigating solution for root canal cleanliness and smear layer removal in the root canals of ex vivo human teeth were verified [Several studies have depicted antimicrobial mechanism of HOCl as an innate substance produced to fight infection. Similar to intrinsic HOCl produced from the human body\u2019s immune cells by the myeloperoxidase-Hagocytes , EO wateagocytes . Generalagocytes . The 0.0verified . In addiverified , and thiverified  is elimiCurrent research in regenerative endodontics uses mainly improved materials, instruments, and medications . The conGrossman and Meiman  reportedOne could argue that when cytotoxicity of pulpal tissue is not of concern in cases of pulpectomy of necrotic pulp or when cleaning endodontic space from previous endodontic-treated teeth with persistent infection, NaOCl can continue to serve as the standard irrigant. However, severe complications can occur when NaOCl comes into contact with soft tissue if it is extruded out of apical foramen. These complications may include but are not limited to chemical burns leading to tissue necrosis, tissue swelling that may be oedematous or hemorrhagic, or both due to severe acute inflammatory reaction . ParaestThe concept of \u201cminimally invasive endodontics (MIE)\u201d has been practically adapted by passive ultrasonic irrigation , contracE. faecalis and S. mutans. Unlike the Zebrafish embryo fully dissolved in 1.5% NaOCl, both 0.0125% and 0.0250% HOCl showed similar survival rate to E3 medium with little or mild in vivo toxicity, revealing the potential as an alternative irrigation solution for vital pulp therapy.Within the limitations of this study, it can be concluded that both EO waters containing 0.0125% and 0.0250% HOCl revealed a remarkable but similar bactericidal effect (> 99.9%) to that of conventional NaOCl against"}
+{"text": "Background: Sodium hypochlorite (NaOCl) is the most commonly used irrigant in endodontics. The purpose of this study was to evaluate the effect of NaOCl solution (2.5%) and gel (3%) with/without passive ultrasonic irrigation (PUI) onEnterococcus faecalis, Escherichia coli, and their endotoxins, lipopolysaccharide (LPS) and lipoteichoic acid (LTA).Methods: 40 human lower premolars were contaminated withE. coli (ATCC 25922) for 28 days andE. faecalis (ATCC 29212) for 21 days. Specimens were randomly divided into four groups: (1) 2.5% NaOCl irrigating the canals without PUI activation; (2) 2.5% NaOCl with PUI; (3) 3% NaOCl gel irrigating the canals without PUI; and (4) 3% NaOCl gel with PUI. 40 mL of irrigant was used for each group. PUI activation was carried out using E1-Irrisonic stainless-steel tip at 10% frequency. After treatment, all specimens were filled with 3mL of 17% ethylenediaminetetraacetic acid (EDTA) for 3min and then washed with nonpyrogenic saline solution. Three samples were collected from the canals: S1, at baseline to confirm biofilm formation; S2 after treatment; and S3 after EDTA. Samples were assessed forE. coli andE. faecalis colony forming units, and LPS and LTA were assessed using chromogenic kinetic LAL assay and ELISA, respectively. Data were analyzed by Kruskal-Wallis, Friedmann and Dunn tests with \u03b1\u22640.05.Results: All groups were effective in reducing the microbial load ofE. coli andE. faecalis after treatment without a significant difference among the groups. NaOCl and NaOCl gel groups had no significant difference in reducing LPS and LTA. Statistically increased reduction was seen for NaOCL + PUI and NaOCl gel + PUI compared for groups without PUI.Conclusions: NaOCl gel has the same antimicrobial action of NaOCl solution and can partially detoxify endotoxins. PUI improves NaOCl (gel or solution) action overE. faecalis and E. coli and their endotoxins. It has century  because century  and disscentury .Enterococcus faecalis is a Gram-positive bacterium found in the root canal system (RCS) and can be disinfected by NaOCl  in Gram-positive bacteria  and lipoNaOCl is a cytotoxic substance . PassiveE. faecalis, E. coli, and their endotoxins, LTA and LPS, respectively.The purpose of this study was to evaluate the effect of NaOCl solution (2.5%) and gel (3%) with/without PUI ono1.504.995). The teeth used in this study were obtained from clinics where teeth are donated during routine procedures and following authorization of the patients. The research team presented the terms of donation by the clinics from which the teeth where obtained to the research ethics committee when submitting the study methodology. A total of 40 human lower premolars were collected .This study was approved by the research ethics committee of S\u00e3o Paulo State University, Institute of Science and Technology  and irrigated with 5 mL of NaOCl 1% for each file used. The canals were dried with sterile paper points  and the apical region was sealed with light-cured resin composites . The outer surfaces of the specimens were covered with two layers of epoxy adhesive , except the cervical opening (E. coli (ATCC 25922) for 28 days andE. faecalis (ATCC 29212) for 21 days and incubated at 37\u00b0\u00b11\u00b0C, following the protocol ofSpecimens were contaminated withSpecimens were divided into four experimental groups (n=10/group) as follows: (1) 2.5% NaOCl  irrigating the canals without PUI activation; (2) 2.5% NaOCl irrigating the canals with PUI; (3) 3% NaOCl gel  irrigating the canals without PUI; and (4) 3% NaOCl gel irrigating the canals with PUI. All specimens were instrumented as a part of the biomechanical preparation by Reciproc R40  following the protocol of each experimental group .PUI activation was performed using an E1-Irrisonic stainless-steel tip  (0.10mm in diameter) at the working length using CVDente 100 ultrasound activator  at 10% frequency.After treatment, all specimens were filled with 17% ethylenediaminetetraacetic acid (EDTA)  for 3min and then washed with nonpyrogenic saline solution.Three samples were collected during the experiment, as in  forE. coli. The plates were kept at 37\u00b0C for 24h and then CFU/mL were counted.Serial dilutions of all samples were performed with sterile saline solution and aliquots of 30\u00b5l of each sample were seeded in two different culture medias: Enterococcosel agar  forLPS levels in each sample was assessed as inLTA was assessed using enzyme-linked immunosorbent assay using ELISA 96-well plates  sensitized with anti-LTA monoclonal antibody (manufacturer) and kept overnight at relative humidity. Next day, the plates were washed with a wash buffer (PBS with 0.05% Tween 20) and incubated with a blocking buffer  for 1 h at room temperature. Then, they were washed with a wash buffer and received 100 \u03bcL of the samples collected and 100 \u03bcL of the LTA standard followed by serial 2-fold dilutions (standard curve) and maintained for 2 hours at room temperature. Afterwards, the plates were washed again and 100 \u00b5L of anti-LTA antibody was added for 1 hour at room temperature. The plates were washed again and 100 \u03bcl of horseradish peroxidase HRP conjugated rabbit IgG antibody was added for 1 hour at room temperature. Lastly, the plates were washed, and the reaction was developed using tetramethylbenzidine (TMB). After 20 min under the light, 50 \u03bcL of stop solution (2 N sulfuric acid) was added to each well of the plate and optical densities were read in the microtiter plate reader  at 450 nm absorbance E. coli  ofE. coli  andE. faecalis  in S2 . In addition,E. faecalis inoculated in the RCS. There have also been more recent studies that have the same results .In conclusion, our study showed that NaOCl gel has the same antimicrobial action of NaOCl solution and can partially detoxify endotoxins. PUI improves NaOCl (gel or solution) action overhttps://doi.org/10.7910/DVN/JNK3TH .Data are available under the terms of the The manuscript evaluates the effect of NaOCl solution (2.5%) and gel (3%) with/without passive ultrasonic irrigation (PUI) on Enterococcus faecalis, Escherichia coli, and their endotoxins, lipopolysaccharide (LPS) and lipoteichoic acid (LTA). The article is very well written, responding adequately to the objectives proposed in this study. The language should be revised. The present study has a great clinical relevance. The title of the work is clear, concise, short and objective. Furthermore, it summarizes the authors conclusions, which is a positive point. The abstract proposed by the authors clearly summarizes the objective, materials and methods, statistical analysis, results and conclusions. The introduction presents a background based on previous studies and at most recent literature. The authors could add a paragraph about the use of PUI in endodontics to address all of the research factors. The authors did not report power calculation of the sample. Was the sample size based on previous studies? They should answer this question in the text. The authors well described the specimens\u2019 preparation, the tests performed and the statistical analyses used in the study. The results section is well described and synthetizes the results obtained in the present study. The tables and figures are descriptive. Table 2 will be better viewed if it is before the discussion and the results present in this table could be better explored in the written presentation of the results. In the discussion, the authors could add one more paragraph about the use of PUI, as suggested in the introduction. The conclusions of this paper drawn adequately supported by the results.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:EndodonticsI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. This is a well designed study that evaluated the effect of sodium hypochlorite as an irrigant solution and gel over one Gram-positive bacterium and another Gram-negative one, as well over their endotoxins with and without passive ultrasonic irrigation (PUI).\u00a0 This study may be the first study that evaluated the effect of sodium hypochlorite gel over the endotoxins of endodontic pathogens and this has a great clinical relevance. The introduction:1 and others in the study of Lukic et al (2020)2. It\u00a0presented briefly the current literature and a historic background about the use of sodium hypochlorite. As a suggestion for the researchers in their future projects, I think that it is more appropriate to test another Gram-negative bacterium than e.coli. Even e.coli was found in the root canal system, however, I think that there are a variety of microorganisms that may be tested and have more relevance in the literature as example: Porphyromonas gingivalis  as in the study of Wang et al (2019) The methods: The study design was carefully planned, I think that the study in its current version is accepted to be indexed, all the cited articles in the methodology section have more details about the execution of this study. I am just wondering why the researchers did not use the sodium hypochlorite in the same concentration in both solution and gel forms? It is not a big deal here as the concentration is almost the same (2.5 and 3 %), however, I think this should be explained in the discussion section, or at least mentioning a previous study that used different concentrations.\u00a0 The discussion: Why did the authors not take advantage of the positive results obtained from using PUI in this study to be discussed furtherly in the discussion section or even in the introduction section? I think another paragraph will improve the discussion section. The conclusions of this paper drawn adequately supported by the results.Is the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Endodontics and operative dentistryI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard."}
+{"text": "Gymnema sylvestre\u201d [ Spanish Journal of Agricultural Research [ AgroFOOD industry hi-tech [ Acta Chromatographica [ KMITL Science and Technology Journal [The Scientific World Journal has retracted the article titled \u201cProduction of Gymnemic Acid Depends on Medium, Explants, PGRs, Color Lights, Temperature, Photoperiod, and Sucrose Sources in Batch Culture oflvestre\u201d . As raisResearch , ProtocoResearch , AgroFOO hi-tech , Acta Chgraphica , 2014 4tgraphica , and KMI Journal . The autDetails of the main concerns, in which images are reused though they do not represent the same experiments, are as follows: Spanish Journal of Agricultural Research 2011 9(4), 1262-1270 [ Gymnema and Phyla.(i) Figures 1(b), 1(g), and 1(k) in this article appear to be the same as Figures 2F, 2H, and 2C, respectively, in A. B. A. Ahmed, R. Pallela, A. S. Rao, M. V. Rao, R. Mat Taha, \u201cOptimized Conditions for Callus Induction, Plant Regeneration and Alkaloids Accumulation in Stem and Shoot Tip Explants of Phyla nodiflora,\u201d(ii) Figure 1(i) in this article appears to be the same as Figure 1(m), though flipped horizontally.(iii) Figure 1(j) in this article appears to be the same as Figure 4(i).Additionally, the following figures and results may represent the same experiments, but the earlier articles were not cited and the reuse was not indicated: Gymnema sylvestre (Retz) R. Br. Ex Roemer and Schultes through Callus Culture Under Abiotic Stress Conditions,\u201d Protocols for In Vitro Cultures and Secondary Metabolite Analysis of Aromatic and Medicinal Plants, 2009, Volume 547 of the series Methods in Molecular Biology pp 93-105 [(i) Figures 5(a)\u20135(d) in this article appear to be the same as Figures 2a\u20132d, respectively, in Abdul Bakrudeen Ali Ahmed, Adhikarla Suryanarayana Rao, Mandali Venkateswara Rao, \u201cIn Vitro Production of Gymnemic Acid from Gymnema sylvestre,\u201d AgroFOOD industry hi-tech - May/June 2012 - vol 23 n 3, pp. 31-34 [(ii) Figures 1(i), 4(a)\u20134(d), 5(a), and 5(d) in this article appear to be the same as Figures 2b, 2a\u20132d, 5, and 6, respectively, in A. Bakrudeen Ali Ahmed, A.S. Rao, M.V. Rao, R.M. Taha, \u201cDifferent Wavelengths Light to Induce Physiological Changes Callus for the Biosynthesis of Gymnemic Acid in Gymnema sylvestre Methanolic Extracts,\u201d Acta Chromatographica 25(2013)2, 339\u2013361, 0231\u20132522 [(iii) Figures 1(i), 1(j), 4(a), 4(h), 4(j), 4(l), 5(a), and 5(b) in this article appear to be the same as Figures 1c, 1d, 1b, 1f, 1e, 1g, 4b, and 4e, respectively, in A.B.A. Ahmed, A.S. Rao, M.V. Rao, R.M. Taha, \u201cHPTLC/HPLC and Gravimetric Methodology for the Identification and Quantification of Gymnemic Acid from Gymnema sylvestre,\u201d 2014 4th International Conference on Biotechnology and Environment Management, IPCBEE vol.75 (2014) [(iv) Figures 4(a)\u20134(d), 5(a), 5(b), and 5(d) in this article appear to be the same as Figures 1a\u20131d, 2, 3, and 4, respectively, in Bakrudeen Ali Ahmed Abdul, Rao, M.V, Rao, A.S, Rosna Mat Taha, \u201cOptimization of Gymnemic Acid Production with Anti-Diabetic Studies and Regeneration of Langerhans Cells from5 (2014) . Gymnema sylvestre (Retz) R. Br. Ex Roemer & Schultes,\u201d KMITL Science and Technology Journal Vol. 9 No. 1 Jan. - Jun. 2009, pp. 18-26 [(v) Figures 1(a) and 1(d) in this article appear to be the same as Figures 1a and 1d (right), respectively, in Abdul Bakrudeen Ali Ahmed, Adhikarla Suryanarayana Rao, Mandali Venkateswara Rao, \u201cSomatic Embryogenesis and Plant Regeneration from Cell Suspension Culture ofp. 18-26 ."}
+{"text": "The goal of this survey by the Medical Library Association (MLA) Diversity and Inclusion Task Force was to have a better understanding of the demographics of the association as well as ascertain how the membership feels about MLA's diversity efforts.A survey was created with the input of both task force members as well as MLA professional staff. It was administered via SurveyMonkey and distributed through email over the course of two weeks in October 2019.The demographics portion of the survey\u2014beyond asking the usual questions about race or ethnicity (72% white), age (65% between 30 and 59), and so on\u2014also asked questions that were more specific to diversity including, but not limited to, gender representation , sexuality , military service (97% have never served), ability (26% have anxiety sometimes or in certain situations), and college financial aid . Diversity-specific questions asked about diversity, equity, and inclusion (DEI) in the association: 59% strongly agreed or agreed that MLA has a strong commitment to DEI; 54% felt that the amount of time that association was spending on DEI issues was just about right; and 56% were very satisfied or satisfied with the DEI environment at MLA. Members also reported feeling like they belonged in MLA (59%), they were treated with respect (77%), and they were valued by MLA (59%)The survey paints a picture of the membership that is much deeper than any previously conducted membership survey. It shows the diversity of membership, especially in terms of ability and religion. Generally, the membership feels that MLA is right on target with the level of focus that MLA is giving issues of diversity. This survey reinforces the diversity work that has been done and supports diversity work in MLA in the future. In fall 2019, the Diversity and Inclusion Task Force administered an online survey to the membership of the Medical Library Association (MLA) to ascertain not only the demographic makeup of the association, but also to have a better understanding of how the membership felt about MLA itself, as well as the diversity, equity, and inclusion (DEI) efforts of the association. Because respondents did not necessarily answer every question, the data in this report are promulgated as percentages rather than as raw numbers.In terms of survey respondents' attitudes about the association's efforts regarding DEI, the results were largely positive:59% strongly agreed or agreed that MLA has a strong commitment to DEI54% felt that the amount of time that the association is spending on DEI issues is just right56% were very satisfied or satisfied regarding the DEI environment in MLARespondents were also asked how they felt about MLA as an association and their places in it.26% have been members of MLA for less than 4 years; 17% have been members of MLA for 25 years or more50% paid their own annual membership fees59% strongly agreed or agreed that they felt valued as members of MLA59% strongly agreed or agreed that they felt a sense of belonging in MLA77% strongly agreed or agreed that they were treated with respect in MLA64% strongly agreed or agreed with the statement, \u201cI feel welcomed and included in MLA Annual Meetings.\u201d76% strongly agreed or agreed that they were able to participate in MLA at the level that they desire69% strongly agreed or agreed that they were able to find at least one community or group in MLA in which to belong76% strongly agreed or agreed that being a member of MLA has had a positive effect on their professional growthAs a representation of MLA membership, survey respondents generally demographically corresponded with the relative norms seen across librarianship as a profession.65% were between the ages of 30 and 5972% were white95% lived and worked or went to school in the United States97% had never served in any branch of the US military79% were female67% were heterosexual26% stated that they had anxiety sometimes or in certain situations42% were Christian77% were caregivers in some capacity92% had master's degrees in library and information science49% used federal student loans to pay for their education21% were categorized as medical librarians or librarians39% earned between $50,000 and $70,999There were some surprises in the data. For instance, Christians represented the largest group in the association at 42%, but as a group they were under 50% of the membership. Likewise, the data for the question that asked about ability was very surprising in that the data reflected far more people with disabilities than had ever been recorded in a library association. Generally speaking, the membership was happy with the efforts of the association to be more diverse, equitable, and inclusive. The membership also generally felt respected and included in the association. Respondents had many suggestions across the questions where commenting was available, which indicated involvement and interest in the goings on of the association by the membership. There is, of course, always room for improvement, and data from this survey will be examined closely by association leadership.In fall 2019, MLA determined that the association's Diversity and Inclusion Task Force would complete an online survey of the membership with the purpose of better understanding not only the demographic makeup of the association, but also how members felt about the association's DEI efforts, as well as how they felt about the association itself and their position in it.A subcommittee of the whole was formed to develop the questions. Working with MLA's resident survey expert, Kate Corcoran, questions were formulated by members of the task force, tested, and then loaded into SurveyMonkey. The survey was active from October 15, 2019\u2013October 31, 2019, and was sent to 2,691 email addresses. Nine hundred eighteen people answered the first question, which equals a response rate of 34%. Several rounds of emails were sent to the membership over that period to encourage participation. Because not all respondents answered all questions, percentages are used throughout this report rather than raw data.One of the primary purposes of the survey was to find out what the membership thought about the association's DEI efforts that have been ongoing for the last several years. To that end, the questions in this section were designed to ascertain how well MLA is perceived to be doing regarding DEI efforts.Respondents were asked to what degree they felt that MLA had a commitment to DEI. Forty-four percent stated that they agreed that MLA has a strong commitment, with 59% agreeing or strongly agreeing with the statement. Only 8% strongly disagreed or disagreed with the statement, and 33% neither agreed nor disagreed.Another question asked respondents to what degree they felt that MLA focused on issues of DEI. Fifty-four percent felt that the amount of time spent on these issues was \u201cabout right\u201d; 37% felt that MLA is spending \u201ctoo little\u201d or \u201cfar too little\u201d time and energy on DEI issues; and 8% felt that MLA is spending \u201ctoo much\u201d or \u201cfar too much\u201d time on these topics.Respondents also had an opportunity to leave comments on this question. There were 197 comments, of which 82 were negative and reflected the mentality that MLA is either doing too much or not enough. For instance, one respondent stated that:\u201cI think MLA has jumped on the diversity and inclusion bandwagon without considering whether it's pertinent to our organization. We can embrace the goals without spending our limited time and resources on something that in my opinion is peripheral to the mission of the Medical Library Association.\u201dIn contrast, there were seventy positive comments, of which the majority reflected that MLA was doing well and that there was more to be done. For example:\u201cAt first I thought the recent emphasis was too much. Then I realized that was probably because I'm part of a privileged group and it's time to get over it.\u201dThe forty-five neutral comments ran the gamut, ranging from \u201cno opinion\u201d to listing suggestions for improvement in the organization, at MLA annual meetings, and so on.Respondents were asked to rate their satisfaction with MLA's environment regarding DEI since becoming a member of MLA. Fifty-six percent were either satisfied or very satisfied; only 10% were very dissatisfied or dissatisfied; and 34% were neither satisfied nor dissatisfied.Several questions pertained specifically to individuals' relationships with the association, such as how long respondents had been members of MLA as well as whether the respondents felt valued in MLA.When asked how long the member had been part of the association, the largest percentage was in the 0\u20134 years range, at 26%. There was also a fairly significant percentage at the completely opposite end of the spectrum, with 17% of respondents having been members for 25 years or more. Survey takers were asked whether or not their employer paid for their annual MLA membership. Fifty percent responded that their employer did not pay for their annual MLA membership; 27% selected \u201cYes, full membership outside of professional development funds\u201d; 13%: \u201cYes, full membership if I use professional development money\u201d; and 6%: \u201cYes, part of my membership.\u201d There were 35 comments with this question, of which the majority elaborated on how their memberships have been paid, or not, in the past.Forty-five percent of respondents agreed that they felt valued as individuals in MLA; 59% strongly agreed or agreed that they felt valued in MLA. Only 7% disagreed or strongly disagreed, and 34% neither agreed nor disagreed.Respondents were asked to what degree they felt that they belonged in MLA. Fifty-nine percent strongly agreed or agreed that they felt they belonged. Only 11% strongly disagreed or disagreed that they belong, and 30% neither agreed nor disagreed.Respondents were asked whether they felt that they were treated with respect in MLA. Fifty-six percent agreed that they were treated with respect; 77% strongly agreed or agreed that they were treated with respect in MLA. Only 4% strongly disagreed or disagreed, and 19% neither agreed nor disagreed.There was a follow up question for those who felt that they were not treated with respect in MLA with a total of nineteen comments. A variety of issues were raised, including but not limited to, cliquishness in the organization, prohibitive costs, lack of diversity, and ageism (both older to younger and younger to older).Respondents were asked to indicate to what degree they agreed with the statement: \u201cI have felt welcomed and included in MLA annual meetings.\u201d Sixty-four percent strongly agreed or agreed; 6% strongly disagreed or disagreed; and 16% neither agreed nor disagreed.Respondents were able to leave a comment if they indicated that they felt unwelcome or excluded when they attended MLA annual meetings. There were thirty-six comments, which included topics ranging from MLA annual meetings being cliquish, to the annual meeting being too expensive to attend, to difficulty connecting to others because of the way the annual meeting was structured, and other factors.Another question inquired whether or not respondents were able to participate in MLA at the level that they desired. Seventy-six percent strongly agreed or agreed that they were able to participate at the level that they wanted to; 8% strongly disagreed or disagreed; and 16% neither agreed nor disagreed.Respondents were able to follow up negative responses with comments about why they felt they had not been able to participate in MLA to the degree to which they wanted to. There were forty-nine comments in total, with similar answers to the previous question. Respondents commented about cliquishness, cost, and communication problems between the association and the membership.Respondents were asked whether they had been able to find 1 or more communities or groups in MLA that they felt that they belonged to. Sixty-nine percent strongly agreed or agreed that they were able to find at least 1 community or group in which to belong; 10% strongly disagreed or disagreed; and 21% neither agreed nor disagreed.There was a question of whether or not respondents agreed with the statement: \u201cMy experience in MLA has had a positive influence on my professional growth.\u201d Seventy-six percent strongly agreed or agreed with the statement; 6% strongly disagreed or disagreed; and 18% neither agreed nor disagreed.There were multiple questions designed to capture demographic information from the membership. Questions varied from \u201cWhere do you work\u201d and \u201cHow long have you been in the profession\u201d to more inclusive questions about gender, sexuality, and religious identities.The respondents had a main age range of 30\u201339 (21%), 40\u201349 (21%), and 50\u201359 (23%). The largest group was \u201cwhite or Caucasian\u201d at 73%. Ninety-five percent of respondents marked that they resided in the United States for work or school. Two percent indicated that they worked or learned in Canada, and 2% marked outside of the United States or Canada. The other 2% of respondents indicated that they were from all over the world, including Africa, Europe, Australia, the Caribbean, South America, and the Middle East.Ninety-seven percent of respondents to the survey indicated that they had never served in the US military; 2% indicated that they were veterans; and 0.3% indicated that they were currently serving.Seventy-nine percent indicated female, and 13% indicated male. One percent marked gender queer, and 0.1% each indicated non-binary, transgender female, and transgender male, respectively. Of the 4 comments received for this question, 2 of them were negative toward the question and 2 further elucidated their gender identity.Sixty-eight percent of people marked heterosexual as their sexual orientation. The rest of respondents fell across the spectrum of responses in the single digits, with 13% preferring not to respond to the question. While the bulk of respondents answered \u201cnot applicable\u201d in terms of ability, the fact that so many people responded to the question at all is groundbreaking and indicates that there are more librarians with disabilities in the profession than believed to be in the past. Twenty-six percent of respondents marked that they had anxiety sometimes or in some situations. While the bulk of respondents identified as Christian at 42%, a large percentage of respondents marked \u201cnone\u201d for this question at 32%. There were 40 comments with this question of which many delineated specific sects of Christianity, such as Catholicism. There was a follow-up qualitative question regarding religion that asked, \u201cHow, if at all, should MLA make allowance for individual religious considerations when planning events, meetings, or other activities?\u201d A total of 437 comments responded to this question. After coding responses, of which there might be more than 1 code per comment, 188 comments indicated that there should be some allowance for religious considerations, and this most often took the suggested form of MLA providing some space for prayer or meditation and/or providing a list of local religious institutions if members wanted to visit them for religious observance. One hundred seventy-three comments focused on not scheduling the meeting during major holy days. Eighty-eight comments mentioned food options in some way, including kosher and halal options. Fifty-five responses indicated that the association should not take religion into consideration at all, and 28 comments were neutral, had no opinion, or were suggestions unrelated to the question. Finally, 19 responses asked that Sunday mornings be kept unscheduled so that members can follow religious observances.Forty-two percent of respondents indicated that they were not caregivers in anyway. This indicated that the bulk of those who answered the question were, in fact, caregivers. The members of the association who took the survey were extremely well educated. However, discrepancies in the data suggested that many respondents did not accurately interpret the meaning of the question, \u201cPlease indicate the degree(s) you have earned.\u201d Ninety-three percent indicated they had a master's degree in library and information science, but only 58% indicated they had a bachelor's degree. Since a bachelor's degree is usually a prerequisite to postgraduate degrees, the data for this question are suspect. There were 39 comments for this question, most of which focused on types of degrees or mentioned different types of certificates achieved.This question, in part, helped answer the question the task force had on the socioeconomic status of the membership. Most respondents (50%) took out federal student loans for their educations. Survey respondents were asked what their current employment status was. Ninety percent indicated that they were employed full time; 4% indicated part time; 4% stated retired; 0.7% stated they were students or not currently employed; and 0.6% marked other. There were 5 comments with this question, of which all of the responses fell under the above-mentioned categories.There were 25 options for respondents to choose from when selecting what their job role was. The largest selection was for medical librarian/librarian at 21%. The results of the pay range question had a cluster between $50,000\u2013$80,999, of which the highest percentage was 21% between $61,000\u2013$70,999. This survey represented the first opportunity for MLA to survey the membership regarding substantial demographics that included ability, gender, sexuality, and ethnicity. The results of the survey represent a snapshot in time of the membership that took the survey. It is the hope of the Diversity and Inclusion Task Force that this membership survey will be repeated every three years in order to build a demographic history of the association and how it changes over time."}
+{"text": "Record linkage is the process of identifying and combining records about the same individual from two or more different datasets. While there are many open source and commercial data linkage tools, the volume and complexity of currently available datasets for linkage pose a huge challenge; hence, designing an efficient linkage tool with reasonable accuracy and scalability is required.We developed CIDACS-RL , a novel iterative deterministic record linkage algorithm based on a combination of indexing search and scoring algorithms (provided by Apache Lucene). We described how the algorithm works and compared its performance with four open source linkage tools  in terms of sensitivity and positive predictive value using gold standard dataset. We also evaluated its accuracy and scalability using a case-study and its scalability and execution time using a simulated cohort in serial (single core) and multi-core (eight core) computation settings.Overall, CIDACS-RL algorithm had a superior performance: positive predictive value  and sensitivity . In the case study, using a ROC curve to choose the most appropriate cut-off value (0.896), the obtained metrics were: sensitivity = 92.5% (95% CI 92.07\u201392.99), specificity = 93.5% (95% CI 93.08\u201393.8) and area under the curve (AUC) = 97% (95% CI 96.97\u201397.35). The multi-core computation was about four times faster (150 seconds) than the serial setting (550 seconds) when using a dataset of 20 million records.CIDACS-RL algorithm is an innovative linkage tool for huge datasets, with higher accuracy, improved scalability, and substantially shorter execution time compared to other existing linkage tools. In addition, CIDACS-RL can be deployed on standard computers without the need for high-speed processors and distributed infrastructures. Linking records from big health and non-health related administrative data sources has been popular in many countries, including Australia, Brazil, Canada, United Kingdom, and the USA. It overcomes the limitations of using an isolated data source and has contributed significantly to the advancement of knowledge \u20134, healtRecord linkage, also called data linkage, is the process of combining records about the same individual or entity from two or more different data sources , 14. It There are two main types of linkage algorithms: deterministic and probabilistic. Deterministic linkage methods vary from a one-step procedure using a single unique identifier or a set of several attributes  to step-wise algorithmic linkages involving a series of progressively less restrictive steps to allow variations between record attributes . A record pair is classified as \u201clinked\u201d if it meets the criteria or parameters at any step; otherwise it is classified as \u201cnon-linked\u201d . ProbabiSeveral variations of record linkage methods and computerized tools in the literature, have emerged to meet different requirements and challenges, such as accuracy, speed, and scalability. Many of these tools have a general purpose, allowing a combination of existing configurations and methodologies \u201327. WhilThe pre-processing step involves data cleaning and standardization whereby incomplete and incorrectly formatted data is converted into a well-defined, consistent form , 25. SpeExecuting a linkage routine between data sets A and B will result in allenges , 29. To allenges , 30, andallenges , 27, 31.The blocking and indexing step generates pairs of candidate records pertaining to the same comparison block . These mThe field comparison and classification step measures the similarity of attributes for each record pair using different functions, hence calculates a matching weight. This step classifies candidate record pairs using weight vectors as \u201cmatches\u201d, \u201cnon-matches\u201d, and \u201cpossible matches\u201d. The \u201cposible matches\u201d group can be manually assessed and further classified into \u201cmatches\u201d or \u201cnon-matches\u201d using a clerical or manual review process , 31. TheThe final step\u2013accuracy assessment\u2013evaluates the linkage algorithm and the quality of the linkage . Linkage accuracy is often assessed using a gold standard dataset where the true match status of each pair of records is known , 25. ComIn this paper, we aim to describe the architecture and design CIDACS-RL, a record linkage tool that utilizes a novel application of combined indexing search and scoring algorithms (provided by Apache Lucene). In addition, we provide evidence on its accuracy and scal-ability compared to existing open source linkage tools. The motivation behind the development of CIDACS-RL arise from the need to link huge datasets in the range of millions in a reasonable time for which the other tools in this comparison are not designed. Both indexing and scoring algorithms have long been used in many different application domains including record linkage. However, to our knowledge, the combined implementation of indexing search and scoring  as a blocking procedure, using Apache Lucene , with hiThis paper is structured as follows: \u201cCIDACS-RL is a tool developed at the Centre for Data and Knowledge Integration for Health (CIDACS) to link administrative electronic health records and socioeconomic datasets hosted at the centre. All datasets are identifiable at individual level and cover a significant period of time (from 2003 to 2018), storing data on demographics, health episodes, and participation in social protection programmes in thousands of records. The main dataset (CadUnico) has more than 100 million records and is the main baseline for linking to other health and socioeconomic datasets to produce bespoke data (\u201cdata marts\u201d) for epidemiological analysis. Detailed description of data sources in Brazil used in linkage for epidemiological studies can be found elsewhere .Linkage imposes a significant challenge in terms of execution time: from one day to one week, depending on the databases. Besides feasible execution times, CIDACS-RL also aims to achieve high accuracy . This is because the linked dataset is mostly used for epidemiological studies to evaluate associations between exposures and health outcomes, and potential sources of bias due to linkage should be avoided . Within A and B were to be linked and A is the largest database), CIDACS-RL indexes the largest database (A) and then each record in B is searched based on this index. Thus, instead of comparing each record of B with all records of A, only a small portion of A is compared.To address the execution requirement, CIDACS-RL makes use of combined indexing search and scoring algorithms provided by Apache Lucene , an openA  and builds an index Ai. CIDACS-RL uses Lucene\u2019s tools during the indexing processes: strings are stored as text fields and written to the index. Through the I/O module, CIDACS-RL reads each line in dataset A, creates one instance of text field class for each attribute, and writes the attribute set corresponding to each line to a Lucene document.The Indexing module take as input the linkage attributes from dataset B with every record from dataset A, we query a small subset of similar records from Ai and apply comparison functions on them. CIDACS-RL uses a mixture of exact, semi-exact, and fuzzy queries provided by Apache Lucene to overcome different errors that may exist in data linkage attributes, such as missing or duplicate characters and abbreviations.A challenging issue in linking huge datasets is to reduce the number of pairwise comparisons. Blocking strategies used for this purpose must be carefully developed, as they have a direct impact on the final result. This is because blocking restricts the comparisons made by the linkage system - true records might not be compared if the blocking process is too restrictive or if there are any errors on the variables used for blocking . TherefoThe Query module performs the query in three different ways: (i) exact, (ii) semi-exact, and (iii) fuzzy. Exact query takes each linkage attribute as a parameter and returns only records in which every attribute is equal to those used for querying. Semi-exact query is a modification of exact query being composed of an arrangement of Apache Lucene has its own query language that takes a search string as input with the following format: \u201c<attribute name>: <text to be searched>\u201d. Class QueryParser is used by CIDACS-RL to build the query and then IndexSearcher class retrieves the records that match the query. For example, an exact query with name and date of birth would have the following format:enshtein  is used ng names . Hence, CIDACS\u2019 datasets also have date and categorical attributes that can be used for linkage. Some attributes may have semantic meaning which the TF-IDF does not account for; therefore, CIDACS-RL relies on a custom scoring function tailored for Brazilian data sources to compare record pairs. This function is based on different metrics and approaches, depending on the type of the attribute. CIDACS-RL supports four kinds of attributes: string, categorical, date, and municipality code/IBGE (\u201cInstituto Brasileiro de Geografia e Estatistica/Brazilian Institute of Geography and Statistics\u201d). IBGE code is a 7-digit numeric code where the first two digits represent one of Brazil\u2019s 27 states, the following four digits represent one of 5570 municipalities, and the last digit is used for verification purposes.In CIDACS-RL, blocking is implemented by the indexing search function during the comparison process. All attributes present in the linkage are used for searching. Thus, records returned from the search function are very similar to records used as parameter to the search function. To compare a pair of records, CIDACS-RL first compares each pair of attributes present in those records. A set of empirically derived weights is passed as parameters to the system based on the discriminatory power of the attributes, which is used to summarize all scores into one value Fig.\u00a0. In ordeJaro-Winkler  was usedB to a record in dataset A. Algorithm 1 shows the cascade approach used to combine the three kinds of search described in \u201cIndexing and query processes\u201d section PairWiseComparison function receives both the Ai index generated by the indexing module and the dataset B. Each query function  takes each record in dataset B to query in Ai and returns a set of similar records based on TF-IDF (similarRecordsArray). Another function (findMostSimilar) uses the score function to compare the record in dataset B (which was used as a source for the query) with all records retrieved from Ai and finds the most similar record based on the custom scoring function. If any record with a score greater than the threshold is found by the exact query, the pair is added to the result and the semi-exact query is not executed. The same occurs for semi-exact and fuzzy. The steps described are performed for each record on dataset B and the function returns all pairs matched along with the score obtained. CIDACS-RL assumes that duplicates are removed earlier during data cleaning and standardization and, if still exist, the frequency is minimal given the nature of the datasets. In addition, when there is more than one record matched in the query, the algorithm selects the one that has the highest similarity score. Inconsistencies might arise if multiple records in B match best with one in A. This issue is addressed in the post-processing step by retaining the matches with the highest score. The code and implementation of CIDACS-RL is available on GithubWe used combined scoring and query modules to link every record in dataset We compared CIDACS-RL with other four well-established open source record linkage tools available in the literature: RecLink (version 3.1) , FRIL . We have derived a gold standard dataset from live births and mortality records to assess the performance of the different linkage tools. We also used a case study dataset, much larger than the gold standard dataset, from socioeconomic and demographics data linked to notifications of tuberculosis cases. This study was used to describe the performance of CIDACS-RL in big, real datasets. Finally, we created a synthetic dataset containing common attributes present in most Brazilian public health databases to assess the scalability of the CIDACS-RL tool. The case study and synthetic datasets were used only for CIDACS-RL. Detailed description of the data sets used to build the gold standard and case study datasets are summarised in the Supplementary Material 2.A gold standard dataset was created using two administrative data sources from the Brazilian Ministry of Health (MoH): the Mortality Information System  and the Live Birth Information System . Both SINASC and SIM contain individual-level data with five attributes in common: name, mother name, date of birth, municipality and sex. These attributes have been of good quality, in terms of completeness of recording, in the last decade. SINASC collect information on live births throughout Brazil using the \u201cStatement of Live Birth\u201d (DN \u2014 Declara\u00e7\u00e3o de Nascido Vivo) form with a unique identifying number, the DN number. SIM collects information on mortality using a death certificate. For children less than one year old, death certificate also includes the DN number.From both datasets, records from one calendar year (2015) with complete and valid information on DN number and attributes were used. Then, the two datasets were linked using exact matching on the unique DN number, which is also used during the experiment to assess the performance of each tool. A number of non-linked records from SIM and SINASC were added to the final linked dataset in order to simulate a number of matches and non-matches usually found in CIDACS datasets Fig.\u00a0. Using tThis case study, used to assess the performance of CIDACS-RL tool in big real data, comprises data from two governmental databases: the Unified Registry for Social Programmes (CadUnico) and the Information System for Notifiable Diseases . This dataset was used only for CIDACS-RL to show its performance using huge dataset much larger than the gold standard dataset. Hence, the other linkage tools used on the gold standard dataset are not included here, in part because the tools do not run on such a size of dataset except AtyImo, at least in our experiment.CadUnico is a database from the Ministry of Social Development built with the aim of identifying low-income families who could be eligible for social protection programmes, including conditional cash transfers (\u201cBolsa Fam\u00edlia\u201d), housing, and water wells (\u201cCisternas\u201d). It has a wide range of demographic and socioeconomic information of more than 100 million records of Brazilian citizens, covering the period 2001\u20132015. The CadUnico dataset used in our experiment consolidated the whole period into a single dataset, which has 114,008,179 records . SINAN contains clinical cases of notifiable diseases collected by health professionals who attend patients with suspected diseases of compulsory notification within the Brazilian Public Health System (SUS). In this case study, we used a tuberculosis (TB) notification dataset from SINAN containing 1,182,777 reported tuberculosis cases from 2001 to 2013 in Brazil. Both datasets, CadUnico and SINAN, lack unique identifiers between them unlike the data sources used for the construction of the Gold Standard dataset for the comparison of the different linkage tools.AFor each SINAN-TB record, there was a record in the CadUnico baseline which corresponds to the candidate pair with the highest score among all possible candidates. In addition, each record pair should contain information on the attributes used for linking the two datasets laid side by side;BManual verification was performed only for a random sample comprising 30,000  hence, 29,816 pairs contained in the resulting dataset, since the total number of record pairs in the dataset was very large . The outcome of manual verification was established as a \u201cgold standard\u201d for further evaluating the performance of CIDACS-RL in linking these two datasets. Note that this \u201cgold standard\u201d is different from the gold standard dataset, described in the previous section, used for comparison of the five linkage tools.CSensitivity, specificity, positive predictive value and receiver operating characteristic (ROC curve) were estimated to assess the accuracy of the linkage.Five attributes  from each dataset were used to link these two datasets (CadUnico baseline with the SINAN-TB dataset). The dataset generated from the linkage procedure  was analyzed to assess its quality, according to the following procedure: Simulated datasets of 1-20 million records were obtained from  and are We assessed the accuracy of our linkage algorithms through standard metrics: sensitivity, specificity, positive predictive value (PPV), and area under receiver operating characteristic curve (AUC-ROC), as described in Supplementary Material 3. The main purpose of accuracy assessment in record linkage is identify how many matches and non-matches were retrieved by the linkage tools. True matches  and true non-matches  are usually unknown prior to linkage. In our gold standard dataset, it was possible to know in advance all true matches and non-matches since we used the unique key identifier, the SINASC registration (DN) number to flag true matches. In the gold standard dataset, we also recorded the time taken (in minutes) to complete the linkage process.We also assessed two types of errors that could appear during record linkage: false negative (FN), which is a missed match that can impact linkage sensitivity , and false positive (FP), which is a false match and will impact PPV .In the case study, we evaluated the performance of CIDACS-RL in huge databases larger than the gold standard dataset. We calculated the sensitivity, specificity, True matches, false matches, and missed matches for different thresholds.Scalability of a record linkage tool is a critical challenge when dealing with huge datasets. The naive approach in record linkage involves a large number of comparisons equivalent to the product of the sizes of the two datasets; however, comparison of all record pairs using expensive functions has proven infeasible in most real-world applications . To reduWe compared two scenarios: (i) the linkage was performed serially, using only one processor (single core), and (ii) we adapted CIDACS-RL to run over Spark  and usedSince each linkage tool produces a dataset containing a similarity score for each pair, ROC curves were plotted to find the best cut-off point that maximizes accuracy for each tool. Figure\u00a0Table\u00a0In the case study dataset. after manual verification of the sample, 17,355 pairs of records were identified as true matches and 12,461 as false matches. Based on this result, we analyzed accuracy of CIDACS-RL in huge dataset through ROC curves to identify an appropriate cut-off point to classify matched pairs, as summarized in Table Applying this cut-off point (0.896), pairs of records in the complete dataset were classified as linked if their scores were greater than or equal to the cut-off point and not linked if their scores were lower.Description of tuberculosis (TB) notifications as true matches, false matches, and missed matches using the different thresholds is presented in Table\u00a0Figure\u00a0CIDACS-RL has proved to be a very powerful and accurate tool both in controlled (using the gold standard dataset) and uncontrolled (using the case study datasets) experiments. The novelty presented by this tool is the combined implementation of indexing search and scoring as a blocking step for record linkage. Such implementation aims to improve speed, scalability, and accuracy over huge datasets where common unique key attributes are not available, such as the 100 Million Brazilian Cohort. Compared to AtyImo, a linkage tool previously developed at CIDACS, CIDACS-RL has shown superior accuracy, measured using positive predictive value and sensitivity, and a shorter execution time.The current implementation of CIDACS-RL is an iterative deterministic linkage based on five attributes and involving different queries  in the pairwise comparison step. The semi-exact query, like the classical deterministic linkage algorithms, was developed to work with a small number of columns  is another critical challenge in record linkage initiatives involving big health datasets which contain massive amounts of personal and sensitive data. In its current version, CIDACS-RL does not implement privacy preservation techniques; however, we aim to use a similar approach implemented in AtyImo to provide such capability in the future. While privacy preservation is required through the entire linkage process, it has proved difficult to find a linkage tool that optimizes the main linkage challenges . For example, high linkage quality and/or privacy could be achieved through computationally complex approaches such as secure multi-party computation techniques, machine learning techniques or graph-based approaches; however, these methods might not be scalable to large databases \u201348. Privn AtyImo . DevelopOur linkage tool has several strengths. It is very fast hence has reasonably short execution time compared to other linkage tools; it can link large databases with tens of millions of records over standard computers without the need for high speed processors; and it is scalable to distributed infrastructures. In addition, it has high accuracy and sensitivity compared to other open source linkage tools. This is mainly due to the implementation of Lucene\u2019s indexing search and scoring instead of classical blocking and filtering methods. The later usually do not use all attributes present in linkage datasets for indexing , which cThe optimal choice between deterministic and probabilistic linkage methods needs consideration of several factors that influence performance, which include database quality, availability of unique identifiers, file size, acceptable trade-offs between positive predictive value and sensitivity for a specific linkage project, and resource availability (software programs and high speed computers). More importantly, data standardization, cleaning, flexibility on approximate matches are important in both approaches. For example, when there is substantial missing values and/or error in the linkage attributes, both methods may not be suited for linkage. A future work is planned to extend the CIDACS-RL with probabilistic implementation and compare it to the current step-wise deterministic version.CIDACS-RL algorithm utilizes combined indexing search and scoring for linking huge datasets that pose tremendous computational challenges to other linkage tools. This innovative application of existing technologies instead of traditional blocking has resulted in higher accuracy, scalability, and substantially shorter execution time when compared to other linkage tools. The tool can also be employed on standard computers without the need for high speed processors and it is also scalable to parallelised or distributed infrastructures. It has made possible linking CIDACS\u2019 huge datasets with tens of millions of records in few days to build large cohorts such as \u201cthe Brazilian 100 Million Cohort\u201d within the CIDACS environment. This enables epidemiological research on associations and impact of social protection policies on a large range of health outcomes, with a degree of detail  never done before. Further developments of the tool are currently in progress.Additional file 1: Description of: (1) the different linkage tools; (2) the datasets and the creation of the gold standard dataset; (3) the comparison metrics; (4) the descriptive analysis of the linkage between CadUnico and SINAN-TB; (5) the determination of Threshold in Weight Vector Classification."}
+{"text": "The Australian Collaboration for Coordinated Enhanced Sentinel Surveillance (ACCESS) was established to monitor national testing and test outcomes for blood-borne viruses (BBVs) and sexually transmissible infections (STIs) in key populations. ACCESS extracts deidentified data from sentinel health services that include general practice, sexual health, and infectious disease clinics, as well as public and private laboratories that conduct a large volume of BBV/STI testing. An important attribute of ACCESS is the ability to accurately link individual-level records within and between the participating sites, as this enables the system to produce reliable epidemiological measures.The aim of this study was to evaluate the use of GRHANITE software in ACCESS to extract and link deidentified data from participating clinics and laboratories. GRHANITE generates irreversible hashed linkage keys based on patient-identifying data captured in the patient electronic medical records (EMRs) at the site. The algorithms to produce the data linkage keys use probabilistic linkage principles to account for variability and completeness of the underlying patient identifiers, producing up to four linkage key types per EMR. Errors in the linkage process can arise from imperfect or missing identifiers, impacting the system\u2019s integrity. Therefore, it is important to evaluate the quality of the linkages created and evaluate the outcome of the linkage for ongoing public health surveillance.Although ACCESS data are deidentified, we created two gold-standard datasets where the true match status could be confirmed in order to compare against record linkage results arising from different approaches of the GRHANITE Linkage Tool. We reported sensitivity, specificity, and positive and negative predictive values where possible and estimated specificity by comparing a history of HIV and hepatitis C antibody results for linked EMRs.Sensitivity ranged from 96% to 100%, and specificity was 100% when applying the GRHANITE Linkage Tool to a small gold-standard dataset of 3700 clinical medical records. Medical records in this dataset contained a very high level of data completeness by having the name, date of birth, post code, and Medicare number available for use in record linkage. In a larger gold-standard dataset containing 86,538 medical records across clinics and pathology services, with a lower level of data completeness, sensitivity ranged from 94% to 95% and estimated specificity ranged from 91% to 99% in 4 of the 6 different record linkage approaches.This study\u2019s findings suggest that the GRHANITE Linkage Tool can be used to link deidentified patient records accurately and can be confidently used for public health surveillance in systems such as ACCESS. The Australian Collaboration for Coordinated Enhanced Sentinel Surveillance (ACCESS) of blood-borne viruses (BBVs) and sexually transmissible infections (STIs) monitors diagnostic testing and other episodes of care for priority BBVs and STIs ,2. ACCESA key challenge for ACCESS (and similar sentinel surveillance systems) is that patient outcomes can be inaccurately measured if individuals attend multiple health services, leading to potential reporting bias. For example, markers of testing frequency, an important indicator for BBV/STI prevention and management -5, may bThe linkage of deidentified ACCESS records across sites relies on specialized health data extraction software GRHANITE, which is installed at participating clinics and laboratories. GRHANITE interfaces with patient databases, securely extracting line-listed consultation, demographic, BBV and STI clinical and pathology data . Before The GRHANITE Linkage Tool has been validated to perform large-scale population-level record linkage  to achieTypically, when a patient first attends a medical facility, an EMR is created in the facility\u2019s patient database, containing the patient\u2019s identifying information, including the name, date of birth, contact details, and Medicare number . Most clinics will also have recorded other demographic information, such as preferred language, country of birth, and indigenous background in the EMR.Every individual\u2019s EMR has a unique medical record number generated by the patient database, linking all of a patient\u2019s consultations, tests, and prescription records. Multiple EMRs may be created for one patient at the same facility if the patient\u2019s details change and are not updated, leading to the creation of a new EMR; if the patient uses an alias; or if the patient attends a clinic that allows anonymous or free testing.Data were extracted from participating ACCESS clinical sites that included an EMR for every patient available in their databases at the time of extraction. GRHANITE generated a new unique record ID and up to four irreversible hash-coded linkage keys for each EMR. Personal identifying information  in the patient\u2019s EMR was passed through advanced encryption to generate both record ID and linkage keys . The recWilliam in one clinic with a full date of birth and Bill in another clinic with only a year of birth recorded. GRHANITE utilizes data preprocessing to remove unwanted characters and words and to resolve nicknames utilizing an Australian national nickname list. Phonetic encoding (double metaphone) is then employed, which permits fuzzy matching based on misspellings of the surname and forename. Transposition of day and month of birth is also supported. After preprocessing, identifiers are combined and then encrypted utilizing secret seeding keys and cryptographic hashing to generate the GRHANITE privacy-preserving cryptographic hashed linkage keys . Fo. Fo11]. Linkage key and components of base identifying information:Type 1: 5 Medicare digits; date of birth; and sexType 2: 5 Medicare digits; postcode; first three characters of first name; and year of birthType 3: Last name and first name (either order permitted) and fuzzy matching used; date of birth with day/month (transpositions permitted)Type 4: Last name and first name (either order permitted) and fuzzy matching used; 5 Medicare digitsThere are three steps in the record linkage process in ACCESS when applying the linkage tool. The first step finds pairs of EMRs based on at least one linkage key matching and records the linkage key type/s used to match each record pair. The second step examines the strength of the link using other available data within the matched pair of records to accept or reject linked records as described in To evaluate the record linkage in ACCESS, we generated two gold-standard datasets, using a deterministic record linkage method, where the true match status could be identified . To assePrEPX is a population-level intervention study in Victoria in which HIV pre-exposure prophylaxis was made available to eligible individuals, and the study used ACCESS data to monitor participants\u2019 BBV and STI testing . Eight cA second and much larger gold-standard dataset was generated from the EMRs extracted from 7 clinics and 4 laboratories participating in ACCESS between January 2009 and April 2018. To be included in this dataset, patients had to have at least one specimen sent from one of the ACCESS clinics to one of the ACCESS laboratories. A unique laboratory specimen ID was assigned to the specimen at the laboratory, and when laboratories returned pathology results to the clinic, this specimen ID was also recorded at the clinic. To create the gold-standard dataset, clinic and laboratory records were matched using the laboratory specimen ID, year of birth, and test date. We allowed for a 7-day difference in test dates, as in medical records, the recorded date can commonly vary for the same specimen. Only matched records were included in the gold-standard dataset and linked using an arbitrarily assigned link identifier .An EMR in the pathology results gold-standard dataset may match to many other EMRs for several reasons, including the following: individuals may have had multiple specimens sent to multiple laboratories for testing, individuals may have attended different clinics and therefore had the same test result sent from the laboratory to more than one clinic, or individuals may have had multiple EMRs at the laboratory or clinic as a result of outdated or incomplete personal identifiers.The sensitivity was calculated as the number of correctly linked EMRs, as identified using the GRHANITE Linkage Tool, as a percentage of the total number of linked EMRs in the gold standard dataset.In the PrEPX gold-standard dataset, the specificity was calculated as the number of single EMRs correctly identified as unlinked using the GRHANITE Linkage Tool as a percentage of the total number of unlinked EMRs. The positive predictive value (PPV) and negative predictive value were also calculated to provide probabilities of true matches and missed matches.Given the deidentified nature of the ACCESS data, it was not possible to include unmatched specimen IDs in the pathology results gold-standard dataset because there was no way to confirm whether they belonged to different individuals (correctly unmatched), making it impossible to calculate specificity. Therefore, to evaluate specificity, we assessed the concordance of chronological HIV and hepatitis C test records to identify EMRs that should not have been linked. By identifying the linked EMRs with discordant results, the PPV (the proportion of linked records with concordant antibody results) could be determined. The specificity was then estimated using the PPV and the sensitivity for each linkage approach as summarized in Following infection, any HIV or hepatitis C antibody test that subsequently occurs should always return a positive result. Using the pathology results gold-standard dataset provided only a small number of HIV and hepatitis C results; therefore, a dataset of linked EMRs was derived using all available EMRs from the same clinic and laboratory sites used to create the gold-standard dataset. Two datasets were created, one that contained any HIV western blot or antibody result and one that contained any hepatitis C antibody result. EMRs containing discordant results before record linkage were excluded from the sample so as not to confuse it with discordance resulting from record linkage. Records within each dataset were then linked using all six approaches  of the GTo calculate the PPV, the linked EMRs were then inspected for negative antibody results occurring at least seven days after a positive test result, which were then classified as incorrectly matched. Where most subsequent antibody tests were negative, the initial and any subsequent positive results were considered incorrectly matched records.The PrEPX gold-standard dataset identified 28 joins among 56 EMRs, indicating 28 study participants had attended two different clinical sites during the PrEPX study period. The remaining 3644 EMRs were from participants who only attended a single clinic during the study and therefore did not have any linked records.Over 99% of EMRs had all four linkage key types present in 8 of the 9 sites, indicating that the patient-identifying information to generate those linkage keys was near fully recorded at the clinics. One site was missing data needed to generate linkage types 1, 2, and 4  in 11% (8/76) of their EMRs .In all linkage approaches, except the approach requiring two or more linkage keys, all pairs of EMRs from the 28 individuals who attended two sites were correctly joined (100% sensitivity). With the approach which required two or more linkage keys for matching, one pair was not identified (96% sensitivity). Specificity was 100% using all linkage approaches, without any of the remaining 3644 EMRs in the dataset being falsely linked .Using the GRHANITE Linkage Tool on the pathology results gold-standard dataset created 50,484 linked records among 86,538 EMRs, with a maximum of six EMRs identified as belonging to the same individual.A total of 99.69%  of EMRs contained at least one linkage key type, and all four linkage key types were present in 73.51%  of records, suggesting that the completion of patient-identifying information in the patient database was very high overall. However, 21.62%  of EMRs had only linkage key type 3 available for matching. One or more of linkage types 1, 2, and 4  was missing in 97.42% (7914/8124) of EMRs from one public laboratory, 53.95%  of EMRs from the sexual health clinic, 48.25% (1403/2908) of EMRs from a private laboratory, and 23.42%  of EMRs from another public laboratory .For the first 4 linkage approaches, the GRHANITE Linkage Tool correctly linked 94% to 95% of EMRs in the pathology results gold-standard dataset, dropping to 66%  where two or more linkage keys are needed to form a match . In the In the derived HIV dataset, the number of linked EMRs containing an initial positive Western blot result ranged from 1090 to 1427 with all linkage approaches except when two or more linkage keys are needed. The linkage approach which requires two or more linkage keys to match resulted in 257 linked EMRs. The PPV was between 87% and 91% for the first 4 linkage approaches and estimated specificity ranged from 90% to 93%. When fewer EMRs were linked because of the different linkage approaches, both PPV and specificity improved .In the derived hepatitis C dataset, with the first 4 linkage approaches, in excess of 3700 linked EMRs contained an initial positive hepatitis C antibody result, with a drop to 2809 records when two or more linkage keys are needed. The PPV was greater than 98.9% and an estimated specificity was over 99% for all six linkage approaches .This paper describes a comprehensive evaluation of a system of probabilistic record linkage using a privacy-preserving software tool within a large-scale health surveillance system. The results showed that this software provides a highly reliable and accurate system for linking routinely collected EMRs through the generation of linkage keys reliant on available identifying information. Optimizing the record linkage involves an appropriate balance between the sensitivity (correctly identifying records belonging to the same person) and specificity (ensuring records that belong to different people are not linked) as well as what will best suit the study design objectives and populations under study without impeding the interpretation of study results.The high performance of the linkage tool when applied to the relatively small PrEPX gold-standard dataset was related to the data completeness for EMRs in the PrEPX trial compared with the completeness of data in the pathology results gold-standard dataset  and 4. PWhen the linkage tool was applied to the larger pathology results gold-standard dataset, sensitivity ranged between 89% and 95% where the linkage approach relied on a single linkage key matching. However, with the approach that requires records to link on two or more linkage key types, sensitivity was reduced to 66%. This is attributable to 22% of EMRs only having a single linkage key type available for linkage, which is mostly because of the Medicare number not being available. The inclusion of laboratory records in the pathology results gold-standard dataset may contribute to a lower sensitivity as a result of patient identifier errors such as mislabeling and recording of laboratory samples , compareThe main challenge in evaluating the GRHANITE Linkage Tool was the development of gold-standard datasets given the deidentified nature of EMRs in ACCESS. Researchers rarely have access to gold-standard datasets on which to perform linkage validation outside large administrative health data sources, and our gold-standard dataset of 86,538 records was comparable with other published studies . The golBeyond the false-positive record linkages identified by examining the concordance of linked test results for HIV and hepatitis C, there is potential for other false-positives to occur in cases where individuals share common patient identifiers, such as twins. Given the deidentified nature of ACCESS data, without the actual identifying demographic values, these niche cases cannot be identified. The small impact of these false-positives is not expected to impact the main purpose of public health surveillance using ACCESS. For other research projects that require a lower level of false-positive record linkage, particularly if it is known to contain a high proportion of individuals sharing common patient identifiers, then using a linkage approach that only accepts linkage based on a match of multiple linkage keys would minimize false-positives. In addition, ensuring concordance of other extracted data, such as sex, year of birth, HIV, and hepatitis C testing history, can reduce the level of false-positive record linkages to acceptable levels.Evaluating record linkage is an important part of assessing the utility of surveillance and research systems for answering key population-level research questions or for accurately describing population-level trends using linked data. Our findings suggest that the GRHANITE Linkage Tool is appropriate for accurately linking individuals\u2019 episodes of care and underpins the ability for ACCESS to perform privacy-preserving linkage of patient medical records."}
+{"text": "The recent delivery of a fluorescent quinolizidine\u2010substituted spiropyran, which is able to switch in vivo and bind to guanine quadruplexes (G4) at physiological pH values, urged us to elucidate its molecular switching and binding mechanism. Combining multiscale dynamical studies and accurate quantum chemical calculations, we show that, both in water and in the G4 environment, the switching of the spiropyran ring is not promoted by an initial protonation event\u2014as expected by the effect of low pH solutions\u2014but that the deprotonated merocyanine form is an intermediate of the reaction leading to the protonated open species. Additionally, we investigate the binding of both deprotonated and protonated open forms of merocyanine to c\u2010MYC G4s. Both species bind to G4s albeit with different hydrogen\u2010bond patterns and provide distinct rotamers around the exocyclic double bond of the merocyanine forms. Altogether, our study sheds light on the pharmacophoric points for the binding of these probes to DNA, and thereby, contributes to future developments of new G4 binders of the remarkable family of quinolizidine\u2010substituted spiropyrans. Protonate me, if you can: Multiscale calculations show that the opening of the fluorescent quinolizidine\u2010substituted spiropyran is not proton\u2010assisted and that the deprotonated merocyanine open form is an intermediate, both in solution and in guanine quadruplexes. Guanine quadruplexes G4s) are non\u2010canonical secondary structures that can be adopted by particular guanine\u2010rich sequences.s are nonKa values than QSP (pKa\u22485.9) making pH\u2010mediated opening under physiological conditions inviable. Thus, quinolizidine\u2010spiropyrans constitute a unique family of probes to be used in vivo.Quinolizidine\u2010substituted spiropyran  has been reported to be the most stable form of both MC and MCH species.The protonation mechanism of SPs has been extensively discussed. Wojtyk and co\u2010workersd Scheme\u2005\u2009a. The aspiro\u2212O and the protonation of this oxygen. Thus, depending on the order of these two processes, there will be at least two possible reaction mechanisms \u2005explicitly include the effect of the water or DNA environment in the reaction, and (iii)\u2005explore two reaction coordinates independently. The systematic exploration of the potential energy surfaces provided stationary points and the minimum free energy pathway. The two reaction coordinates are defined as the bond breaking between the carbon Cspiro and the oxygen O atoms of the spiro\u2010junction (RC1) and the proton transfer to O from a water molecule (Hwater) of the solvent  semiempirical level of theoryThe reaction mechanism was investigated by using steered QM/MM MD simulations together with two\u2010dimensional umbrella sampling (2D\u2010US) calculations, both in explicit water solution and bound to c\u2010MYC G4 . These simulations allowed us to (i)\u2005observe the C1 and QMC2), and the product QMCH  are almost degenerate in energy  in agreement with the fact that the merocyanine species can adopt different isomers with respect to the double bond (\u03b2\u2010bond). The two obtained rotamers correspond to the E\u2010isomer of the double bond and differ in their relative orientation of the phenyloxy group with respect to the nitrogen of the indoline . To obtain an accurate estimation of the energetics of all stable geometries, four structures, corresponding to possible combinations of rotations around the \u03b1/\u03b3\u2010bonds, were optimized at a higher level of theory  and data points .We then investigated what happens in the G4 context: does the polynucleotide change the reaction mechanism? To answer this question, we studied the reaction in the local environment of G4 with the same methodology. As no structure of QSP bound to G4 is available, we superimposed QSP with the ligand position of a quindoline/c\u2010MYC G4 complex (PDB id: 2L7V)QSP\u2010QMC, as well as the relative energies of the QMCH and QMC minima are comparable to those in water. However, in the presence of G4, only the TTC isomer of the QMC form is identified. Further, a new local minimum, Z\u2010QMCH  is found in the 2D free energy surface , the distance between the Cspiro and the O atoms is 2.71\u2005\u00c5. In this geometry, the two rings around the exocyclic double bond (\u03b2\u2010bond) are not co\u2010planar, with a dihedral angle of 14\u00b0. The steric repulsion of the two methyl groups on C3 of the indoline ring with the oxygen atom of Z\u2010QMCH prevents the co\u2010planarity of both \u03c0\u2010systems. This angle is slightly larger in the geometry obtained in our 2D\u2010US exploration  may be responsible for the relative stabilization of this positively charged Z\u2010isomer intermediate.The obtained 2D free energy surface is shown in Figure\u2005E\u2010QMC species, which, in the presence of protons in solution , evolves to the more stable QMCH species. In the DNA context, QSP follows the same reaction mechanism. However, the electrostatics of the environment allows the isomerization between Z\u2010QMCH and E\u2010QMCH, the latter species being the most stable one. In both cases, the pH value of the solution will control the QMC/QMCH ratio. At low pH values or physiological pH, the equilibrium will be shifted to QMCH. At high pH values, it is expected to have a representative population of QMC. The role of the DNA may be the same as the one of an excess of protons: as G4 binds more strongly to QMCH species over QSP or QMC, it will shift the equilibrium towards the QMCH:G4 complex. This chemical equilibrium is different from other SP derivatives. The quinolizidine substitution increases the thermodynamic stability of the QMC with respect to the closed QSP isomer, owing to its structural and chemical nature , which was the result of a structure\u2010based G4 probe design.In summary, our results indicate that the thermal opening of QSP to form QMCH is not a proton\u2010assisted process neither in water nor bound to G4. As soon as QSP opens, it forms the To validate the former hypothesis, we performed all\u2010atom MD simulations, binding the three species QSP, QMC, and QMCH to the parallel\u2010stranded DNA G4 of the promoter c\u2010MYC. To this end, the three species QSP, QMC, and QMCH were positioned on the surface of the external G\u2010tetrads at the 3\u2032\u2010end of the DNA by superimposition of each of the compounds with one of the quindoline molecules present in the solution structure of a 2:1 quindoline\u2010c\u2010MYC G4 (template structure: PDB id: 2L7V).cis conformation whereas for QMCH mostly trans \u03b1\u2010bond conformers are found. In contrast, the dihedral angle controlling the \u03b3\u2010bond is fixed as cis in QMCH but varies to cis and trans in QMC. This means that the major conformer of QMCH is TTC and both CTC and TTC isomers dominate QMC. The hydrogen bond between the quinolizidine hydroxyl group of QMCH (absent in QMC) and G4 . Looking at the energy decomposition, the van der Waals component (vdW) is the largest term in both complexes. Both species present comparable values for this term, but the electrostatics make a difference, with an extra stabilization of approximately 8\u2005kcal\u2009mol\u22121 in QMCH. In contrast to QMC, QMCH is positively charged, and as a general trend in other merocyanine species, this favors its interaction with DNA.In all the simulations, QMCH binds more strongly to G4 than QMC  should be addressed to the authors.SupplementaryClick here for additional data file."}
+{"text": "Right hand grip strength, isometric mid-thigh pull values were found to significantly correlate to and explain variance within Polo player handicap . Whereas left hand grip strength  and reaction time  were non-significant, moderate and trivial correlates and predictors of handicap respectively. Practically, these findings highlight the differing roles between rein and mallet hands of Polo players and emphasise the importance of a strong and stable platform when riding and striking the ball. Lack of association with reaction time may be explained in part by higher handicapped Polo players employing a more proactive approach to the game.Polo is an equestrian team sport consisting of four players per team, with level of play determined by cumulative player handicap , with a higher handicap denoting a better player. There is minimal literature investigating Polo players\u2019 physical attributes, hence the understanding of the physical characteristics that may contribute to an improved handicap are unknown. This study sought to identify the relationship between pertinent strength measures  and reaction time in Polo handicap in 19 New Zealand Polo players, and ascertain whether handicap could be predicted by these measures. Correlation coefficients were expressed using R values, accompanying descriptors and 90% confidence intervals (C.I.). Variance explained was expressed via the R Polo is one of the oldest equestrian sports in the world and requires the synchronisation of both equine and human athletes in a dynamic and high-paced environment . PreviouOne common factor previous studies have acknowledged, is the subjective handicap rating system used to provide Polo players a quantitative measure of their ability (between \u22122 and +10) . This syHorse riding requires physical strength through both the upper and lower limbs, general cardiovascular endurance, balance, reaction time, and flexibility ,9,10,11.Current literature has shown greater biomechanical asymmetries within experienced riders than in lesser trained equestrians , as PoloThe aim of this study is to quantify the strength and reaction time characteristics of Polo players, and to assess the relationship of these characteristics to player handicap. Findings will provide evidence to inform Polo player training programmes and advise how physical attributes may contribute to improving player handicap. For the reasons outlined above, it is hypothesised that left and right grip strength, and lower limb strength, will possess high correlations to player handicap. It is also hypothesised that reaction time will show little correlation to handicap, as a proactive tactical awareness becomes better developed as experience in the sport increases.Testing consisted of an opportunity sample of Polo players at a licensed New Zealand Polo Association tournament in March 2019 in Cambridge, New Zealand. Testing was conducted pitch-side beyond the requisite safety zone, prior to Polo play. Awareness of this study was raised prior to the tournament through social media posts, with recruitment taking place over the two-day tournament in person. Participants self-reported as being recovered from previous day\u2019s play, which consisted of two four-chukka Polo games. Player handicap was selected as the independent variable, as this is a measure of players\u2019 Polo ability that is awarded by the local Polo governing body  and reviewed annually; therefore, it could not be manipulated by the researchers. Strength assessments related to horse riding skill or body position ) and that mimicked the anticipatory requirements of Polo (reaction time) ,11 were Nineteen participants  were recruited for this investigation , all of which had a minimum of two seasons playing experience. Participants\u2019 height was not recorded due to the variability in heel height of players\u2019 Polo boots; performing testing unshod would have breached testing location health and safety regulations due to the close working proximity to horses. Ethical approval for this investigation was awarded by the Waikato Institute of Technology (Wintec) Human Ethics Research Group , on the 25 March 2019. Participants provided written informed consent prior to undertaking the testing battery and retained the right to withdraw themselves and their data from the study at any time.Left and right-hand grip strength was assessed via a hand grip dynamometer , calibrated up to 100 kg. Grip strength procedures need to mimic the specific demands of the sport to improve the validity of the recording . As suchIsometric mid-thigh pull (IMTP) was assessed using a customised testing rig, consisting of two Pasco force plates  and perpendicular vertical poles drilled at 1 cm increments to allow appropriate grip adjustment and positioning of the bar to the participants\u2019 mid-thigh. Intraclass correlation coefficient (ICC) statistics in similar protocols have shown reliable measures both within (ICC = 0.97) and between (ICC = 0.89) sessions . A demonReaction time was assessed via Fitlight reaction lights  set at 30 sec sample duration, with a 0.1 sec delay between lights. Eight lights were mounted on two tables positioned in a right angle and arranged in a fan-like shape around the participant; lights were not placed behind the participants as when mounted on a horse a player cannot leave the confines of the saddle, and to play behind the saddle is considered dangerous. The Fitlight system would randomly activate one of the eight lights that the participant had to wave their hand directly over (approx. 1\u20133 cm distance from light) to record a single point, this in turn randomly activated another of the eight lights. The total number of lights successfully recorded per 30 s trial was recorded.Participants were permitted three attempts for each test, following a demonstration by a researcher; participants\u2019 best efforts were used for analysis.p > 0.05), meaning parametric tests could be employed. Pearson correlation coefficients were used to assess the relationship between Polo handicap and measures of strength and reaction time, with statistical significance set a priori at p \u2264 0.05. Ninety percent confidence intervals (C.I.) were used to describe the uncertainty in the data and magnitudes of relationships were described using the following intervals: Trivial 0\u20130.2, Small 0.1\u20130.3, Moderate 0.3\u20130.5, Large 0.5\u20130.7, Very Large 0.7\u20130.9 and Nearly Perfect >0.9 [2 statistic.Data were assessed for normality via the Shapiro Wilks test and found to be normally distributed ; confidence intervals for correlation coefficients were calculated using a customised spreadsheet [Linear regression was used to determine the predictive ability of Polo handicap upon strength variables and reaction time, with relationships described using the formula eadsheet .p = 0.019). Right hand grip strength was significantly different (p = 0.019) to left hand grip strength .Group means identified handgrip strength was greater in the right hand (50.9 \u00b1 16.6 kg) when compared to the left (46.3 \u00b1 15 kg). As depicted in p = 0.004) and IMTP-R (p = 0.035), which displayed correlations to player handicap of 0.609 and 0.484, respectively. Reaction time was shown to have a non-significant relationship (p = 0.889) to player handicap, with a group mean of 23.3 \u00b1 2.7 light responses per 30 s testing window.Mean values for IMTP and mid-thigh isometric pull relative to player bodyweight (IMTP-R) were 1888.3 \u00b1 597.2 N and 23.9 \u00b1 5.52 N/kg, respectively. Significant relationships to player handicap were also demonstrated by IMTP  also demonstrated significant Rmoderate relationship with player handicap, which was contrary to the initial hypothesis.The purpose of this study was to characterise strength and reaction time attributes of Polo players and assess the relationship between these factors and player handicap. This study shows that right-hand grip strength, IMTP, and IMTP-R have significant relationships to player handicap. However, reaction time neither correlates to nor is predictive of player handicap, therefore supporting the hypothesis of this paper. Left-hand grip strength presented a non-significant p = 0.102) and decreased grip strength were observed in the left hand. This may be explained by the riding style required for Polo [Previous literature has highlighted the range of handgrip strength values that are present across various sporting codes , with drfor Polo , where ffor Polo ,30,31. Wmoderate to large relationships with player handicap and significant R2 values of 0.235 and 0.371, respectively, highlighting the predictive qualities of these measures, with respect to player handicap. Mean values for IMTP and IMTP-R in Polo players are comparable to those of recreationally strength trained males [In jockeys, leg strength and power are more positively associated with falls than in track-work riders , despiteed males , but ~10ed males . To dateThere is a clear need for a strong base of support, and the ability to produce high levels of force on the stirrups through both legs whilst Polo players are riding at speed, playing shots out of their saddle  and absoThere is a paucity of literature surrounding lower limb strength in horse riders, therefore the need to discuss the relationship between lower limb strength and handicap warrants further investigation within the Polo context. The methods utilised in the current study using the IMTP provide a typically static, yet reliable measure of outright lower limb strength, but the oscillatory nature of riding and stochastic nature of Polo presents a unique opportunity for future research to explore various methods of measuring this strength in a Polo specific manner. The use of the legs as opposed to the reins (left hand grip strength) to decelerate and turn the horse will preserve the integrity of horses\u2019 mouths and tongues, which are prone to oral injury through Polo participation . Indeed,trivial non-significant relationship to handicap, which is dissimilar to previous literature pertaining to reaction time in equestrian pursuits, and sports requiring high speed and agility characteristics [Reaction time data showed a eristics ,43. It ieristics . It is iWithout consistent and objective handicap profiling procedures, it is difficult to make conclusive statements about how players may be able to utilise these findings to improve their handicap. However, results of this study suggest practitioners working with Polo players, or other equestrian pursuits, should focus on the development of grip strength, as well as the riders\u2019 ability to transfer force through their lower limb as this provides a stronger platform on the stirrup when playing on-ball. Time spent developing players\u2019 ability to read the game and make proactive moves may be a more effective use of time than training reactive components. Future research should further investigate the bilateral differences between left and right hands of Polo players, and the motor nuance required to perform most effectively. Lower limb strength and endurance capacities should also be investigated within Polo, and could be used in conjunction with player heart rates to clarify central or peripheral limitations . Further"}
+{"text": "A total of 27.1% of the isoflurane vaporizers and 35.9% of the sevoflurane vaporizers were incorrect. Machine learning techniques showed that the most important variables in the classification of the anesthetic machines as conforming or non-conforming were mostly the scavenger system and the canister, followed some way behind by the APL valve, source of oxygen, reservoir bag, vaporizer, and connections.The objective of this retrospective study was to review the results of a 4-year audit performed on anesthetic machines and vaporizers used in veterinary clinics in Spain and Portugal. Data was collected between July 2016 and April 2020. Inspections were carried out by a team of seven veterinarians, using a human-modified system of checks that was adapted to a veterinary practice. The evaluation of each item was noted as \u201ccorrect\u201d or \u201cincorrect\u201d. The vaporizers' performance was evaluated using a self-calibrating gas analyzer. The vaporizer was classified as \u201ccorrect\u201d or \u201cincorrect\u201d when the vaporization error was less than or equal to, or more than 20%, respectively. The anesthetic machine was classified as \u201cconforming\u201d if all its components were noted as \u201ccorrect\u201d and no leaks were detected, or as \u201cnon-conforming\u201d if any of the components was noted as \u201cincorrect\u201d or if a leak was detected. If the inspector was able to repair on-site the item malfunctions detected and the machine was fit for use, they issued a final report as \u201cconforming.\u201d On the contrary, if such malfunctions persisted, the final report was \u201cnon-conforming,\u201d and a recommendation to remove the machine from service until its final repair was provided. To perform statistical analysis, each inspected item was used as predictor, classification and regression trees were built, and a random forest analysis was performed. A total of 2,001 anesthetic machines and 2,309 vaporizers were studied. After inspection, 42.7 and 26.4% of the machines were non-conforming and conforming, respectively, whereas 30.9% could be repaired  Modern anesthesia workstations are an integration of several components required to safely administer anesthesia to a patient. They consist of the anesthetic machine, vaporizers, ventilator, breathing systems, scavenging system, and monitors. Medical gases  are supplied through central units or oxygen concentrators, pipeline systems, terminal units, and hoses that connect these to the anesthesia machine. Breathing systems connect the anesthetic machine to the patient and dispense a controlled composition of medical gas mixture . AlthougIt has been widely demonstrated in human medicine that the malfunctioning of anesthetic equipment can lead to severe complications and fatal outcomes \u20135. Some Unlike in human medicine, veterinary clinics in Spain and Portugal are not obliged to follow safety protocols in regard to the anesthetic equipment, and these remain mere recommendations for a safe practice. To the Authors' knowledge, the only audit of veterinary anesthetic equipment to date was performed in New Zealand, it was published in 1995, and detected that 91% of anesthetic machines were faulty . TherefoAnesthetic machines, vaporizers and other anesthetic equipment from veterinary clinics that had a commercial relationship with two veterinary pharmaceutical companies  and that were inspected between July 2016 and April 2020 were analyzed in this report. Data collection included date of inspection, name and address of the clinic, and the name of the veterinarians who commonly used the anesthetic equipment. However, this data was partially anonymized for privacy, in accordance with the General Data Protection Regulation EU 2016/679 , and onlInspections were carried out by a team of seven veterinarians experienced in clinical anesthesia, employees of the aforementioned companies, who had additionally received a specialized training in anesthetic equipment. The inspection team developed its own checking-up protocol based on the guidelines of the Sociedad Espa\u00f1ola de Anestesiolog\u00eda y Reanimaci\u00f3n  and adapFirstly, the anesthetic machine was inspected, beginning with a visual evaluation that consisted of noting its brand and model, absence or presence of components, state of conservation, location within the clinic and marks of previous revisions. Additionally, the main user of the machine was asked if leaks, component malfunctions, excessive consumption of oxygen or anesthetic agent, or any notable adverse events were detected during use.2O. Then, the flow was interrupted, and the pressure gauge visualized to ensure the pressure was maintained. In the case of a decrease in pressure, the flow required to maintain 30 cm of H2O of pressure was recorded to quantify the leak and this number noted as the total leakage of the system. In accordance to standard recommendation . This analyzer, which has a self-calibration system, has a measurement range of 0\u20138.5% for isoflurane and 0\u201310% for sevoflurane, with a precision of one decimal place and an anesthetic agent accuracy \u00b1(0.2 vol % + 15% relative). In addition, this analyzer was periodically calibrated following manufacturer's recommendations. The inhalant anesthetic agent used in the agent-specific vaporizers, whether isoflurane or sevoflurane, was noted. A system that consisted of an airway adapter, a filter, and a sampling line connected to the gas analyzer was used for sidestream analysis. Depending on the machine analyzed, the inspector selected the location where to take the sample from . The measured vaporization (M) displayed on the analyzer was compared to three selected vaporization percentages  on the vaporizer dial (V) at four different fresh gas flows . The vaporization error (E) was calculated as E = (M \u2013 V)/V. The vaporizer was classified as \u201ccorrect\u201d or \u201cincorrect\u201d when E was less than or equal to, or more than 20%, respectively . The vapThe anesthetic machine was classified as \u201cconforming\u201d if all its components were noted as \u201ccorrect\u201d and no leaks were detected, or as \u201cnon-conforming\u201d if any of the components was noted as \u201cincorrect\u201d or if a leak was detected. If the inspector was able to repair on-site the item malfunctions detected and the machine was fit for use, they issued a final report as \u201cconforming.\u201d On the contrary, if such malfunctions persisted, the final report was \u201cnon-conforming,\u201d and a recommendation to remove the machine from service until its final repair was provided.The statistical language R 4.0.2 was used. Firstly, a descriptive study of data was carried out. Parametric variables are shown as mean \u00b1 standard deviation. Non-parametric numerical variables are shown as median and interquartile range. Qualitative variables are expressed as number of observations and frequency tables. Secondly, machine learning analysis, such as classification and regression trees and random forest analysis were performed to study the outcome of the inspection (conforming/non-conforming), using the result of the inspection of each part of the anesthetic machine (correct/incorrect) as predictors.A total of 573 veterinary clinics from Spain and 119 from Portugal participated in this study, and 2,001 anesthetic machines and 2,309 vaporizers were inspected . The medAt initial evaluation, 528 machines (26.4%) and 1,473 (73.6%) were classified as conforming and non-conforming, respectively. The median (range) of incorrect items detected in non-conforming machines was 2 . One incOut of the 2,001 machines, 1,407 used a concentrator as oxygen source, 485 an oxygen cylinder and 109 lack of an oxygen source, which were mobile secondary equipment. Most of the clinics used activated charcoal canisters as scavenger system and only seven veterinary clinics had an AGSS.A total of 2,309 vaporizers were revised, 1,994 of which used isoflurane and 315 sevoflurane. In total, 1,656 were found to be correct while the remaining 653 were found to be incorrect , 5.The classification tree analysis showed that the most important variables in the classification of the anesthetic machines as either conforming or non-conforming were, in descendent order, the scavenger system, the canister, the APL valve, the reservoir bag, the oxygen source, connections and the vaporizer . In the in-situ in 30.9% of the machines. A total of 27.1% of isoflurane and 35.9% of sevoflurane vaporizers were found to be non-conforming. Machine learning techniques showed that the most important variables in the classification of the anesthetic machines as either conforming or non-conforming were mostly the scavenger system and the canister, followed by the oxygen source, the expiratory and APL valves, the reservoir bags and the vaporizers.This audit detected that 73.6% of the anesthetic machines were non-conforming. During revision, the inspector was able to solve the malfunction In a study of 64 anesthetic machines in veterinary clinics in New Zealand, the percentage of malfunctions was as high as 91% . In thisThe use of anesthetic equipment checklists is recommended by most human and veterinary anesthesia clinical guidelines \u201311, 14. The classification tree and the random forest analysis agreed in that malfunctions were usually found in the scavenger system, the canister, the APL valve, the reservoir bag, the oxygen source, connections, and the vaporizer. The algorithms obtained thereof may help to optimize the inspection of anesthetic machines. The component in which most problems were observed was the scavenger system, 37.9% of which were found to be non-conforming. Most malfunctions were due to the absence of a scavenger system or an exhausted activated charcoal canister. Chronic exposure to an environment contaminated with inhalational anesthetics can cause chronic toxicity in exposed personnel , 21. WheThe second most frequent component to malfunction was the canister 25.1%). Canisters require regular inspections, as the absorbent contained within must be changed when it is exhausted. Improper sitting of the canister, accumulation of dust in the junctions, or malfunctioning of the absorbent may result in adverse events to the patient and personnel %. Canist.The reservoir bag was the third most frequent component to malfunction (11.3%). In veterinary medicine, reusable latex, silicone or rubber bags are normally used. Over time, when exposed to high oxygen concentrations, the material loses its elasticity and becomes damaged, especially in the area where the bag is attached to the anesthesia breathing system . Reservoin situ by the inspector. If the APL valve cannot be fully open, it may cause from a small increase in airway resistance to barotrauma. Conversely, if it cannot be fully closed, it may preclude positive pressure ventilation. This malfunction has been also frequently observed in humans  and the failures detected could never be repaired n humans .2 concentrators. In recent years, the use of oxygen concentrators in veterinary anesthesia has become popular in Spain and Portugal due to their versatility and low cost and as an alternative to medicinal oxygen bottles. These devices concentrate ambient oxygen and provide FiO2 up to 95% (2 concentration and the fresh gas flow they provide. A concentrator malfunction can result in a hypoxic mixture, which can cause adverse effects to the patient, and even death (The oxygen source failed in 10.0% of the machines. Malfunctions were recorded in only 1.9% of the cylinders, compared to 12.9% of the Op to 95% . HoweverThe inspiratory and expiratory valves are two of the most important components of the circle system. Their malfunction can result in adverse effects such as rebreathing of expired air, respiratory collapse and barotrauma . MalfuncIn this audit, 27.1% of the isoflurane vaporizers and 35.9% of the sevoflurane vaporizers had vaporization errors >20% with flows of 2 and 3 L/min and therefore required recalibration by a technical service. An error rate ranging from 12.8\u201314.2% to 16.5\u201319.0% in isoflurane and sevoflurane vaporizers was observed, respectively. The VAMOS anesthetic gas analyzer has an accuracy of one decimal place and overestimates the error at low percentages of vaporization, which could be considered a limitation of the study. For example, if the vaporizer dial is set to 0.5% and the actual vaporization percentage is 0.55%, the analyzer will display 0.6%, which represents an error of 20%. In this study, VAMOS was used because it can detect a 20% error. However, a more accurate analyzer would be required to evaluate the performance of the vaporizer at low percentages. In this audit, the minimal studied flow was 0.5 L/min which is the minimal flow recommended for accurate vaporization . TherefoOur study has several limitations. Firstly, there is a sample bias. This study was not randomized, since only veterinary clients that had a commercial relationship with Ecuphar Veterinaria SLU in Spain or Belphar Lda in Portugal have been included. Based on a total count of 6,228 and between 1,400 and 1,600 veterinary clinics present in Spain and Portugal in 2019 , respectin situ. In conclusion, a regular revision of anesthetic equipment by qualified personnel and the daily implementation of routine checklists are key to ensure proper functioning and to avoid adverse effects on the patient, personnel, and environment.In this study, most audited machines malfunctioned during the inspection and the inspector was able to repair a significant number of them The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.JR and LD contributed to the conception of the study, interpretation, drafted and revised the manuscript, and approved the final version. CM, AB, AM, and DL contributed to the data acquisition, drafted and revised the manuscript, and approved the final version. All authors contributed to the article and approved the submitted version.The authors declare that this study received funding from Ecuphar Veterinaria SLU and Belphar Lda. The funders were not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication. The handling editor declared a past co-authorship with one of the authors JR."}
+{"text": "The Relatives Education And Coping Toolkit (REACT) is an online supported self-management toolkit for relatives of people with psychosis or bipolar designed to improve access to NICE recommended information and emotional support.Our aim was to determine clinical and cost-effectiveness of REACT including a Resource Directory (RD), versus RD-only.ISRCTN72019945). Participants were UK relatives aged >\u2009=\u200916, with high distress (assessed using the GHQ-28), and actively help-seeking, individually randomised, and assessed online. Primary outcome was relatives\u2019 distress (GHQ-28) at 24\u2009weeks. Secondary outcomes were wellbeing, support, costs and user feedback.A primarily online, observer-blind randomised controlled trial comparing REACT (including RD) with RD only (registration n\u2009=\u2009292 (73.2%); RD-only n\u2009=\u2009307 (76.6%)). Distress decreased in both groups by 24\u2009weeks, with no significant difference between the two groups . Estimated cost of delivering REACT was \u00a362.27 per person and users reported finding it safe, acceptable and convenient. There were no adverse events or reported side effects.We recruited 800 relatives  with high distress at baseline . Median time spent online on REACT was 50.8\u2009min (IQR 12.4\u2013172.1) versus 0.5\u2009min (IQR 0\u20131.6) on RD only. Retention to primary follow-up (24\u2009weeks) was 75%  than a comprehensive online resource directory.ISRCTN72019945 prospectively registered 19/11/2015. Relatives and friends of people experiencing psychosis or bipolar provide much unpaid care , 2, but Within this context, our aim was to test the clinical and cost effectiveness of an online self-management intervention, based on the principles of psychoeducation and family intervention . The RelWe conducted an online, two-arm, pragmatic, observer-blind, randomised controlled superiority trial open to relatives of people with psychosis or bipolar across the UK. Inclusion criteria were broad and relatives could self-refer into the trial. A nested qualitative study examined user experiences of REACT. Prior to the end of data collection, a trial protocol  and a coAged 16 or overLiving in the UKRelative or close friend of someone with psychosis or BipolarCurrently experiencing distress . This was included to avoid a distress floor effect at baseline Internet accessSufficient English fluency to comprehend intervention contentInclusion criteria were (according to self-report):Living in any of six geographical areas by postcode taking part in a parallel implementation study of the same intervention (IMPART) .Only one relative per service user was allowed to participate, to avoid a clustering effect.Exclusion criteria were:n\u2009=\u200955) of relatives in the REACT arm who had completed the 24\u2009week follow-up at the time of interviewing were invited to take part in qualitative interviews about their experiences of using REACT, with the aim of recruiting approximately 25 interviewees.Recruitment took place from 22 April 2016 to 30 September 2017. We used a range of online  and offline recruitment strategies , all directing potential participants to the study home page, including information about how to take part. At registration, all participants gave online written informed consent, indicated how they had found out about REACT, and provided postal, email and telephone contact details. A convenience sub-sample (Eligible participants were randomised using a 1:1 ratio to \u201cREACT (including RD) plus Treatment As Usual (TAU)\u201d or \u201cRD only plus TAU\u201d using web-based variable block randomisation, in which the unit of randomisation was the relative. Participants then received an email indicating which arm of the trial they had been allocated to, and a link to the REACT website, with their username and password. Those in the RD-only arm had access only to the directory pages. All participants were aware that the RD was one component of the REACT intervention, and therefore were likely to have perceived REACT as the \u201cintervention of interest\u201d and the RD as the comparator.All data were self-reported and predominantly entered online by participants. Data sets submitted by post at follow-up were inputted by the trial manager, blind to allocation. Data were uploaded directly to the Clinical Trials Research Centre (CTRC) database. To prevent bias, the chief investigator, trial manager (TM) and statisticians were blinded to treatment assignment. REACT supporters, clinical supervisors, qualitative interviewer, one CTRC analyst of web usage data and technical staff were unblinded. To minimise unwanted unblinding, all contact with participants was prefaced by a reminder not to disclose trial arm. If the TM was unblinded regarding a particular participant, another blind team member delivered any non-automated reminders and carried out any data entry for that participant.reacttoolkit.uk  people who had had \u2026 much worse experiences than my experience. So it was that ability to connect with people who kind of have some empathy with what\u2019s going on in your life and how difficult it can be in those moments.Mother, 65: The videos were really helpful because it wasn\u2019t constant reading so I like that. I loved the depth of information that was available. The layout itself was absolutely great as well, it was easy to read, it was eye-catching enough and quite interactive as well \u2026 The opportunity for me to be able to write notes and things like that, I thought that was really, really good.Sister, 26: A consistent message was that REACT would be most useful to relatives early in the recovery journey, when they were likely to be seeking information and strategies.We only had a diagnosis last year, so actually I was really desperate for any resources and any further information that I could find, so I was literally soaking everything up as much as I could, and I found REACT through Bipolar UK and \u2026 it has been really helpful because I think what I really struggled to find was anybody else in a similar situation who had a recent diagnosis, you know early forties and [with] a young familyFemale partner, 43: Some relatives found seeking help for their own needs difficult, and most relatives found prioritising time to use REACT difficult.I was engaging with it [REACT] and then he then went into crisis and then went into hospital and in fact he was in hospital until the following January, and I was then caught up in that. And then you know after that I needed reminders. So I think that\u2019s your difficulty really, is that the very people that you\u2019re trying to help have so much on their plates really.Mother, 57: Twenty four of fifty-five participants who were invited to take part in a qualitative interview did so; 10 declined, 10 did not respond and 11 were unavailable within the narrow time period available for this data collection. Consistent with the whole trial sample, participants were typically middle aged (median age 54\u2009yrs. (range 26\u201369); predominantly female , including 12 psychoeducation modules, a moderated online forum, confidential direct messaging service, and a comprehensive Resource Directory was compared to the Resource Directory only. Relatives reported high levels of distress (GHQ-28 primary outcome) at baseline, which reduced significantly in both groups over the 24\u2009weeks follow-up period, but there was no difference between the groups at follow up. Carer wellbeing and support scores (CWS) were very low at baseline and increased significantly in both groups, with no significant differences between groups. Changes over time may reflect regression to the mean. There were no adverse events: relatives using REACT reported feeling safe and supported, and qualitative experiences of using REACT were positive.REACT offers an inexpensive, safe and acceptable way to deliver NICE recommended information and support to relatives of people with severe mental health problems, but there was no evidence that it reduces distress more effectively than a comprehensive resource directory. These findings are consistent with previous studies showing that in general, interventions designed to improve outcomes for relatives are less effective for those with higher levels of distress , This maThis trial was rigorously conducted, with a large, broadly recruited sample, clearly defined and theoretically based supported intervention, an active control group, good follow-up rate for an online trial, web-based randomisation, robust blinding, and a pre-published analysis plan that appropriately addressed missing data. The key limitations were: failure to recruit more men and people from ethnic monitory groups, which limits the generalisability of the findings; and (with hindsight) the inclusion of GHQ-28 minimal score as an inclusion criterion, which limited the sample to highly distressed relatives, increasing the likelihood of regression to the mean in both arms of the study over the follow-up period. Non-random dropout (greater in participants with higher baseline GHQ-28 scores) further limited the potential to identify group differences, though this was robustly dealt with using a joint modelling approach.The findings highlight two key lessons for research in digital health interventions. The first is that we cannot assume that online interventions adapted from those delivered face-to-face will be equally effective: REACT draws on evidence based cognitive behavioural interventions , and wasThe second is that we need new methodologies appropriate to the rigorous evaluation of digital health interventions. They must be controlled: without an active control group, a pre-post evaluation of REACT would have made it appear very effective. They must account for higher levels of dropout and missing data: without accounting for non-random missing data, REACT would have appeared a more effective intervention than RD-only in improving relatives\u2019 support. However, they also need to allow a more flexible iterative development of the technology in response to feedback, to test the technology as one component of a much broader care package within context, and to establish which part of an intervention has what effect on which people. In this study, REACT remained relatively fixed throughout the trial (excluding updating directory for accuracy), despite ongoing feedback about ways it could have been improved, and general advances in website design. We also do not understand exactly what relatives did in response to using REACT or the RD. In particular, whether or not they sought support from organisations recommended in the RD or how effective this was. Alternative methodologies such as iterative testing and adaptation suggested by Mohr et al.,  or thoseRelatives need access to information and emotional support. REACT offers an inexpensive, safe and acceptable way to deliver this, even if it does not reduce their distress. Therefore, REACT should continue to be developed in light of user feedback, and offered and evaluated as one component of a comprehensive care package, which includes face-to-face support."}
+{"text": "In the article titled \u201cTuina Massage Improves Cognitive Functions of Hypoxic-Ischemic Neonatal Rats by Regulating Genome-Wide DNA Hydroxymethylation Levels\u201d , there wIn Figures p values. These are replaced with a series of bubble diagrams, which can display the degree of functional enrichment from three indicators: the depth of the color corresponds to the p value in the original figure, the size of the point is gene count, and the Rich factor on the x-axis indicates the proportion of genes that falls into this GO term.The bar chart in Figures Moreover, Figures As shown in Figures"}
+{"text": "In this design challenge by the Texas Space Grant Consortium, the researchers design a cooling system for a lithium-ion battery. Lithium-ion batteries are an effective and reliable source of energy for small, portable devices. However, similar to other existing sources of energy, there is always a problem with overheating. The objective is to design a cooling system for lithium-ion batteries that will work in a zero gravity environment for orbital and interplanetary space systems. The system is to serve as a backup battery and a signal booster that can be incorporated into a spacesuit. The design must be able to effectively cool the batteries without the use of an atmosphere to carry away heat but also be a lightweight and reliable design. The design incorporates carbon nanotubes suspended in distilled water creating a nano-fluid environment. This design must include a failsafe in the event of thermal runaway, a problem common to lithium-ion batteries. This failsafe will completely shut off the system if the batteries reach a certain temperature. A cooling system that incorporates nano-fluids will achieve a lightweight and efficient way of cooling batteries. In the early 1970s, the first non-rechargeable/disposable lithium-ion batteries became commercially available. Today, rechargeable lithium-ion batteries are being used as a power source for many portable devices and in consumer electronics. In space, lithium-ion batteries provide power to the astronaut\u2019s spacesuit, portable electronic devices, and satellites. A few benefits of lithium-ion batteries are as follows: it is the lightest of all the elemental metals that have a low maintenance battery, possesses the greatest electrochemical potential, and provides the largest energy density for weight. By ensuring certain precautions are met when charging and discharging, lithium-ion batteries prove to be safe. While there are several benefits of lithium-ion, a disadvantage in the continuous charging and discharging of these batteries is overheating  and 86\u00a0\u00b0F (30\u00a0\u00b0C) to be the optimal range for lithium-ion battery performance. External temperatures may cause short-circuiting of the battery because of Joule heating (I2R), the cell then begins to produce heat by internal chemical reactions. Battery temperatures exceeding 86\u00a0\u00b0F (30\u00a0\u00b0C) begin to accelerate the rate of performance, which may result in thermal runaway. Thermal runaway occurs when an exothermic reaction goes out of control, that is, the reaction rate increases due to increasing temperature resulting both temperature and rate. This phenomenon will cause the lithium-ion battery to possibly combust and catch on fire. In addition, NASA has a set of battery guidelines that exist to prevent the propagation of thermal runaway. Therefore, with NASA\u2019s battery guidelines in mind, the team\u2019s goal is to integrate the use of nanotubes within the cooling system to enhance battery performance.The main purpose is to power a space suit through a backup battery and signal booster with an incorporated cooling system. To increase the surface area for heat extraction, nanotubes are integrated into the cooling system to surround the batteries, while it is suspended in distilled water  and cylindrical (3.7\u00a0V 3000\u00a0mA\u00a0h). Due to its versatility, the design is expected to function with either batteries and is also anticipated to be used for other batteries systems. Sheets of aerogel surrounding the enclosure serve as the perfect insulator that will help negate external variables in a zero gravity environment. The aerogel prevents the external heat transfer to the environment by trapping the heat inside the model  with 0.05 vol\u00a0% of nanotube used, which increases by 9.36\u00a0% compared to distilled water without nanotube. These data indicate higher volume of SWCNT contributes to higher thermal conductivity of the nano-fluid. High concentration of nanotube tends to promote agglomeration; however, agglomeration can be reduced to minimal using the sonication method to improve the stability of the nanoparticles (Sabiha et al. t test, there is a mean difference of about three degrees and the result is 95\u00a0% confident that in the given time interval, the nanotube solution would be between 1.71 and 4.29\u00a0\u00a0F warmer than it would be if the de-ionized water was used. With the p value of 0.0001, our results are statistically significant.The data points from all ten trial tests were averaged out to create a linear relationship between the changes in temperature over time. As shown in the table, the nano-fluid extracts heat at a faster rate than di-ionized water. The nano-fluid reached 80\u00a0\u00a0F in 15\u00a0min, while di-ionized water took ~20\u00a0min to reach the same temperature. According to the statistical The group designs a cooling system for a cylindrical and flat battery and then examines results for best use to avoid overheating the battery and creating malfunctions (Loyselle et al. Future project plans may include, but are not limited to, performing further experiment of multiple nano-fluids with variation in concentration of the nanotubes, creating a system of more efficient batteries to control more than just small components, and reducing the cost of battery cooling system while maintaining or increasing its integrity. With this prototype, the researchers pursue further experimentation with different types of materials to improve the system efficiency."}
+{"text": "Globally, HIV testing services (HTS) have been scaled up resulting in 79% of all people with HIV aware of their status in 2018 . Since 2As with any HTS, HIVST needs to provide a pathway to appropriate HIV treatment, care and prevention services. Because no single test, including HIVST, can provide an HIV\u2010positive diagnosis, all individuals with reactive HIVST results must receive further testing by a trained provider before initiating antiretroviral therapy (ART) . MeasuriEvidence from randomized trials shows that the proportion of people linked to ART following HIVST is comparable to that of standard facility\u2010based HTS . HoweverMonitor ART initiations at treatment centres/facilities before and during HIVST distribution in the relevant catchment area [ent area , 9. For Include questions in clinic registers that can help to ascertain if the present clinic visit/testing was prompted by prior HIVST use. It is important to note that these data are subject to recall bias and some people may not disclose prior HIVST use and/or results for reasons such as stigma or to get a result from the provider without biases. These data also do not provide a denominator to measure linkage following HIVST. Nonetheless, such data can provide useful information on the proportion of ART initiations prompted by HIVST.Population\u2010based surveys such as demographic and health surveys, integrated bio\u2010behavioural surveys and other special surveys provide opportunities to monitor HIVST use and linkage at the population level. These surveys are particularly useful for monitoring trends over time, such as awareness, use, coverage and linkage, provided appropriate questions are included. Because these surveys are usually repeated every three to five years, they may not be useful for ongoing programme monitoring.Digital tools, such as messaging Apps, websites, hotlines and social media platforms, can also be leveraged to collect HIVST usage and linkage information. For example in South Africa, a survey of a random sample of self\u2010testers through mHealth platforms such as interactive voice response and SMS was found feasible for estimating HIVST usage and linkage [ linkage .individual\u2010level follow\u2010up to confirm linkage may be considered in the context of small\u2010scale demonstration projects or within research studies to assess the effectiveness of linkage interventions. One example of such an approach is when women in antenatal care distribute HIVST kits to their partners. Women can be given an invitation letter for their partner along with a self\u2010test kit. The male partners are asked to show the invitation letter when they attend clinics for HTS and women can also be interviewed at their second visit [Lastly, nd visit . The linGiven these challenges, measuring linkage in programmes at the individual level following HIVST may not be feasible in many low\u2010income settings. Such an effort may require repeated follow\u2010up with self\u2010testers which can be resource intensive in the absence of client information and linked electronic records. Attempts by providers to ascertain the client\u2019s HIVST results through repeated follow\u2010up can also be perceived as being against client autonomy and could deter HIVST utilization in the future. Concerns about monitoring linkage have kept several national HIV programmes from making HIVST available to clients. We believe that resource intensive monitoring efforts should not come in the way of making HIVST widely available at the earliest. We give below a few pragmatic approaches to measuring linkage to treatment services that programmes can consider and adapt depending on their local context.No single method would give an accurate measure of linkage following HIVST due to the limitations of each of them. However, using data and information from diverse sources, such as survey and programme data, can increase confidence in linkage estimates and minimize missing information. WHO is developing guidance for countries to monitor and evaluate HIVST, including linkage.HIVST is an important testing approach for meeting the global goals of diagnosing 95% of all people with HIV by 2025. Effective linkage to appropriate services following HIVST is important. Given the privacy of HIVST, which allows autonomy, fosters empowerment and reaches people who may not otherwise test, a resource\u2010intensive approach to monitor linkage is neither feasible nor desirable as programmes scale up HIVST. The need to collect in\u2010depth linkage data should not delay the wider availability of HIVST. Programmes, donors and implementers should consider pragmatic and innovative ways to measure linkage.The authors declare no conflicts of interest.MSJ, CJ, ATC and PM conceived the idea. ATC wrote the first draft of manuscript. MSJ, PM, EC, EC, LC, HI, EBA, MdE, MDC, MM, TS, VW, RB and CJ reviewed, provided input and approved the final draft of manuscript.Bill and Melinda Gates Foundation OPP1177903 and Unitaid (PO# 10140\u20130\u2010600 and PO# 8477\u20130\u2010600). PM is funded by the Wellcome Trust (206575/Z/17/Z). EC is funded under a Wellcome Trust Senior Research Fellowship in Clinical Science (grant number: WT091769) and by Unitaid\u2010STAR Initiative (NCT02718274).The authors alone are responsible for the views expressed in this article and they do not necessarily represent the views, decisions or policies of the institutions with which they are affiliated including the World Health Organization, the U.S. President's Emergency Plan for AIDS Relief, the U.S. Agency for International Development and the U.S. Government. The corresponding author had final responsibility for the decision to submit for publication."}
+{"text": "The X chromosome has historically been one of the most thoroughly investigated chromosomes regarding intellectual disability (ID), whose etiology is attributed to many factors including copy number variations (CNVs). Duplications of the long arm of the X chromosome have been reported in patients with ID, short stature, facial anomalies, and in many cases hypoplastic genitalia and/or behavioral abnormalities.Here, we report on a large Iranian family with X\u2010linked ID caused by a duplication on the X chromosome identified by whole genome sequencing in combination with linkage analysis.SLC16A2, RLIM, and NEXMIF, if impaired, can lead to syndromes presenting with ID. Of note, this duplicated region was located within a linkage interval with a LOD score >3.Seven affected males in different branches of the family presented with ID, short stature, seizures, facial anomalies, behavioral abnormalities , speech impairment, and micropenis. The duplication of the region Xq13.2q13.3, which is ~1.8\u00a0Mb in size, includes seven protein\u2010coding OMIM genes. Three of these genes, namely Our report indicates that CNVs should be considered in multi\u2010affected families where no candidate gene defect has been identified in sequencing data analysis. We report a duplication on the X chromosome encompassing Xq13.2q13.3 in a large Iranian family with X\u2010linked intellectual disability. There are seven affected males in ones and twos within two generations of the pedigree who presented with a similar phenotype, including ID, short stature, seizures, facial anomalies, behavioral abnormalities, speech impairment, and micropenis.  After obtaining written informed consent, approved by the Ethics Committee of USWR, the family was enrolled in an ongoing research project aiming to clarify the genetic basis of hereditary ID and patients underwent detailed clinical evaluation.2.2After chromosome analysis and Fragile X syndrome screening, X\u2010exome sequencing and whole exome sequencing (WES) were performed for individual (V:1) as described before   were genotyped by Affymetrix Axiom Precision Medicine Research Array (PMRA). Multipoint parametric linkage analysis based on an X\u2010linked recessive model was done by using the Merlin program . Data were analyzed using the Agilent Cytogenomic software v4.33.1The family originating from the Northern East part of Iran comprises seven affected males in two generations related through the maternal lineage Figure . The aff3.2FMR1 repeat expansion . X\u2010exome and whole exome sequencing did not provide evidence for a potentially disease\u2010causing deletion or single nucleotide variant (SNV). Subsequent SNP genotyping followed by linkage analysis delineated a linkage interval on chromosome X (~18\u00a0Mb), located between heterozygous SNP markers rs241748 and rs2157410 (GRCh37/hg19), with a significant LOD score of 3.879  had a normal karyotype and a negative test for 9 Figure . WES dat9 Figure  and excl4CHIC1 (OMIM *300922), TSIX (OMIM *300181), XIST (OMIM *314670), JPX (OMIM *300832), FTX (OMIM *300936), SLC16A2 (OMIM *300095), RLIM (OMIM *300379), NEXMIF (KIAA2022) (OMIM *300524), ABCB7 (OMIM *300135), UPRT (OMIM *300656), and ZDHHC15 (OMIM *300576) , Tonne\u2010Kalscheuer syndrome , and Mental retardation, X\u2010linked 98 , respectively. The genes CHIC1 and ZDHHC15 are located at the borders of the duplicated region. Loss of expression of the latter, which is involved in neuronal connectivity  carried a duplication at Xq13.2q13.3. The duplicated region contains approximately 40 genes , comprising 11 OMIM genes, namely  carried ATRX gene (OMIM *300032) . However, a whole NEXMIF duplication, leading to reduced expression, has been reported in a family with XLID and autism  within the duplicated region contributed to the phenotype of the affected individuals and provide a wider perspective on the underlying genetic defect. Our report clearly shows that CNVs should be considered for all families with several affected individuals and no promising single gene variants in WES data re/analysis, even in consanguineous societies. It should also be noted that SNP genotyping followed by linkage analysis is still a powerful tool to narrow down the region of interest in WGS data analysis.Authors declare no conflict of interest.SM: drafting the manuscript, FL: providing linkage analysis, HH: running WES data, ZF: analysis of WES data, MB: interpretation of CNV analysis, SSA: segregation study, SA: providing samples and obtaining the informed consent form, HHR: providing WES data, VMK: providing X\u2010exome sequencing data, DA: running WGS data, KK: clinical examination and genotype\u2010phenotype correlation, YR: analysis of WGS data, HN: study design, providing financial support for the project, supervision.Fig S1Click here for additional data file.Table S1Click here for additional data file."}
+{"text": "Background: Linkage of administrative data sources provides an efficient means of collecting detailed data on how individuals interact with cross-sectoral services, society, and the environment. These data can be used to supplement conventional cohort studies, or to create population-level electronic cohorts generated solely from administrative data. However, errors occurring during linkage  can lead to bias in results from linked data.Aim: This paper provides guidance on evaluating linkage quality in cohort studies.Methods: We provide an overview of methods for linkage, describe mechanisms by which linkage error can introduce bias, and draw on real-world examples to demonstrate methods for evaluating linkage quality.Results: Methods for evaluating linkage quality described in this paper provide guidance on (i) estimating linkage error rates, (ii) understanding the mechanisms by which linkage error might bias results, and (iii) information that should be shared between data providers, linkers and users, so that approaches to handling linkage error in analysis can be implemented.Conclusion: Linked administrative data can enhance conventional cohorts and offers the ability to answer questions that require large sample sizes or hard-to-reach populations. Care needs to be taken to evaluate linkage quality in order to provide robust results. Data linkage is an important tool for generating longitudinal data that can be used to understand the development and causes of variation in outcomes across the life course  or missed matches  . False mDespite advances in linkage methods and improvements in data quality over time, some level of linkage error or uncertainty is almost always inevitable in linkage of administrative datasets that were not collected primarily for research  algorithms and probabilistic linkage techniques involving \u201cmatch weights.\u201d Deterministic methods typically make use of a set of pre-specified rules for classifying pairs of records as belonging to the same individual or not. For example in national hospital data in England , admissions for the same individual are linked over time using a three-step algorithm involving NHS number, date of birth, postcode and sex , can be used to inform linkage  supplementation of primary data collection in conventional cohorts with linked data that has been collected for other purposes (often population-level administrative and registry data), and (ii) construction of electronic cohorts solely from secondary data sources, usually retrospectively and relying on de-identification in place of consent. Data linkage is supporting new models of efficient cohort research such as UK Biobank, in which large-scale collection of biological specimens provides the main source of primary data for a cohort, with most other data provided through routine linkage to population-level datasets . Most administrative data cohorts are created from event-based datasets  and even when only one such dataset is used, the records within it have to be linked internally to create a longitudinal record for each individual  files. This can either be done using one primary \u201cspine\u201d dataset (e.g. a cohort) and linking each new file to the spine, or by sequentially linking pairs of files together. For simplicity, we start by considering how data from one or two files can be combined and analysed, and represent these scenarios using Venn diagrams  to define a study sample; (ii) to define a variable of interest when the value of that variable is inferred through linkage itself (e.g. linking with a disease registry to infer disease status); or (iii) to provide information about additional variables of interest obtained through linkage. In administrative data cohorts, linkage error can also result in double-counting , or undercounting . A detailed discussion of how linkage error can impact on results from linked data is provided elsewhere . Then, once a linkage strategy has been implemented, we can estimate linkage error rates to help us understand whether and how linkage error might impact on analysis results. Available techniques for assessing rates and distributions of linkage error are discussed in Results, using examples from published literature.uncertainty in linkage, as well as bias. These approaches are discussed further in the following sections.Development of methods to handle bias due to linkage error has been identified as a priority for research Jorm . There aA range of methods aiming to assess linkage quality can be found in the literature, and examples of these are provided in could there be, and how strongly might the error be correlated with variables of interest? This type of quantitative bias analysis can be sufficient to demonstrate the sensitivity of results to the range of plausible assumptions that could be made about linkage error. For example, based on a linked electronic cohort of children with Down\u2019s syndrome, we specified plausible ranges for a set of \u201cbias parameters\u201d that were relevant to a given analysis, including the numbers of missed matches and false matches, and the proportion of false matches that occurred between comparable records , and people who were known to have died in prison and therefore should link (\u201cpositive controls\u201d). By examining match rates among the positive controls, the authors were able to estimate the sensitivity of survival classification (the proportion of cohort deaths linked) (Although the expected number of matches was not known  linked) . By examIn complex linkage scenarios, or where there are multiple variables of interest, estimating linkage error rates across subgroups may not be straightforward. In these situations, imputation-based methods can provide a useful approach to handling linkage error and representing uncertainty in linkage. In generating one version of a linked dataset in the presence of imperfect identifiers, errors will be inherent; different versions of a linked dataset could be constructed according to how data are pre-processed, how linkage is conducted, and how decisions about thresholds are made. Imputation based approaches re-frame linkage as a missing data problem, and the aim moves away from identifying definite links between records, to carrying through the correct values into analysis, along with appropriate measures of uncertainty.Consider a \u201cNested\u201d design in which we expect all records in one file to link , but records with missing data are excluded from analysis. In this setting, the problem is analogous to complete case analysis . This should include input from both those who know how the data have been generated, and those who know how the linked data will be analysed. An iterative process, where initial linked datasets are created and evaluated, allows analysts to feedback information about any implausible links, and to understand the balance between false and missed matches.Secondly, it is important to retain as much information about the linkage process as possible. If deterministic linkage has been used, then a match rank, or description of the linkage step achieved (i.e. an agreement pattern for a set of known identifiers), can be provided alongside each record pair. In probabilistic linkage, match weights can be provided for each record pair. It is also helpful to provide information on multiple candidate links, especially where there is a small margin of certainty about which is the most likely match. This allows the researcher both to perform quality assurance  and to incorporate this uncertainty into analysis (e.g. using imputation-based approaches as described above). Methods and software to handle linkage error within analysis are currently being developed under a Wellcome Trust grant (212953/Z/18/Z). Guidelines are available to provide advice on the information that can and should be shared between data providers, data linkers and data analysts, in order to facilitate careful evaluation of linkage quality  can help researchers understand the trade-off between false and missed matches. Regardless of the linkage methods used, some errors are likely to remain, reflecting the dynamic and imperfect nature of administrative data that are generated as individuals interact with different services over time. It is vital that the mechanisms by which these errors might impact on results are considered, so that potential biases can be identified and mitigated in analysis."}
+{"text": "IAA8 loss-of-function mutant iaa8-1 exhibited delayed seed germination. The phenotype of iaa8-1 was restored by ectopic expression of IAA8. Interestingly, IAA8 accumulated to high levels during seed germination, which was achieved not only by increased protein synthesis but also by the stabilization of IAA8 protein. We also showed that IAA8 down-regulates the transcription of ABSCISIC ACID INSENSITIVE3 (ABI3), a negative regulator of seed germination. Our study, thus strongly suggest that the auxin signaling repressor IAA8 acts as a positive regulator of seed germination in Arabidopsis thaliana.Seed germination is a complex biological process controlled by various regulators, including phytohormones. Among these, abscisic acid and gibberellic acid inhibit and promote seed germination, respectively. Many studies have addressed the biological roles of auxin in plant growth and development, but very few have considered its role in seed germination. Here, we identified a novel function of the auxin signaling repressor Aux/IAA8 during seed germination. The  Seed maturation is the final stage of embryogenesis. The embryo becomes protected by a hard outer cover of dead tissue, the testa, underneath which the endosperm is deposited . Upon geReleasing dormancy is a prerequisite to germination that can be induced by various external and internal stimuli . GerminaABSCISIC ACID INSENSITIVE3 (ABI3), ABI4, and ABI5 was identified as negative regulators of seed germination. Among these, ABI3 acts as a major downstream components of ABA signaling  display arrested germination (yuc1/yuc6) showed enhanced seed germination rate  was used in all experiments. T-DNA insertion mutants iaa8-1 (CS25210) and iaa8-2 (SALK_202296) were obtained from SALK. T-DNA insertion was confirmed by genotyping PCR using IAA8 gene-specific and T-DNA border primers  medium supplemented with 2% sucrose and 0.25% Phytagel. Plates were then transferred to a growth chamber at 22 \u00b1 2\u00b0C under long day conditions (16-h-light/8-h-dark photoperiod) with 100 E mCauliflower mosaic virus (CaMV) 35S::3xflag-IAA8 construct in binary vector pCAMBIA 1300 was introduced into Agrobacterium tumefaciens strain GV3101 and used for transformation of iaa8-1 mutant plants by floral dipping. Transformed lines were selected on \u00bd MS medium containing hygromycin (40 g/mL). Three independent homozygous lines overexpressing IAA8 were selected from the T3 generation and used for all experiments.The Seeds were carefully harvested after siliques were fully mature. The germination assay was performed according to the method of 2O2. Tissues were ground in liquid nitrogen to fine powder, and total proteins were extracted using extraction buffer containing 50 mM HEPES, pH 7.5, 5 mM EDTA, 5 mM EGTA, 2 mM DTT, 25 mM NaF, 1 mM Na3VO4, 50 mM \u03b2-glycerophosphate, 20% glycerol (v/v), 2 mM PMSF, 1% Triton X-100 (v/v), and protease inhibitor cocktail . Following two rounds of centrifugation at 12,000 \u00d7 g for 15 min, supernatants were transferred to pre-chilled micro centrifuge tubes. Protein concentration was determined using a protein assay kit  with bovine serum albumin (BSA) as a standard. For immunoblot analysis, 80 \u00b5g of total protein from each sample was separated by 10% Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes . Proteins were probed using mouse anti-flag  as primary antibody and horseradish peroxidase (HRP) conjugated anti-mouse as secondary antibody (1:5000) and visualized using an ECL kit .Immunoblot analysis was performed according to the method of Tubulin2 as an internal control. Semi-quantitative RT-PCR was carried out as described by Total RNA was extracted from seeds by the LiCl/phenol method according to the protocol of iaa8-1 and iaa8-1/IAA8 OX seedlings were treated with cold (4\u00b0C) and H2O2 for 12 h. Chromatin immunoprecipitation (ChIP) was carried out as described (Ten-day-old escribed  using moiaa8-1 (CS25210) and iaa8-2 (SALK_202296) from SALK . Three independent homozygous lines were selected from the T3 generation. To verify ectopic expression, semi-quantitative RT-PCR and immunoblot analyses were performed (iaa8-1/IAA8 OX plants from three independent lines were treated with or without 10 \u00b5M MG132 (a proteasome inhibitor) for 3 h. Proteins were extracted and immunoblot analysis was performed. As expected, IAA8 protein was almost undetectable under mock conditions (control); however, high levels of IAA8 protein accumulated in the presence of MG132  for further experiments, and hereafter referred to as iaa8-1/IAA8 OX.We next tested seed germination phenotypes using Col-0, iaa8-1, and iaa8-1/IAA8 OX seeds in the presence and absence of ABA  and found that the IAA8 transcript is induced by seed imbibition. We therefore proposed that IAA8 transcription might be induced during seed germination. To determine the expression pattern of IAA8 during seed germination, we performed RT-qPCR using freshly harvested Col-0 seeds. The IAA8 transcript level peaked in germinating seeds at day 2  and 5 mM H2O2 alone or together for 12 h. Total proteins were then extracted and immunoblot analysis was performed  with or without 20 \u00b5M 1-naphthaleneacetic acid (NAA) for 1, 3, and 6 h. As expected, IAA8 protein accumulated rapidly in the presence of MG132; however, preventing de novo protein synthesis by treatment with CHX caused significant depletion of IAA8  assay was performed to confirm the binding ability of IAA8 with ABI3 promoter using anti-flag antibody. We treated the iaa8-1 and iaa8-1/IAA8 OX plants with mock, cold and H2O2 for 12 h, and then ChIP-qPCR analysis was performed. The results showed that IAA8 associates to AuxRE motif on ABI3 promoter under mock condition in iaa8-1/IAA8 OX plants , shy2 (iaa3), iaa6 (shy1), axr2-1(iaa7), iaa8, iaa12 (bdl), iaa14 (slr), iaa16, axr3-1(iaa17), iaa18, iaa19 (msg2), and iaa28 reported to display auxin related phenotypes  showed delayed seed germination compared to the wild type (axr2-1 (IAA7) and axr3-1 (IAA17) show enhanced seed germination and exhibit stronger ABA insensitivity than the wild type  funded by RDA and the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIP) (no. 2019R1A2C1009932), partly by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (no. 2018R1A6A3A11042628), and by the Vietnam National Foundation for Science and Technology Development (NAFOSTED) under grant number 106.02-2017.09. SH and SB were supported by the Brain Korea 21 Plus (BK21+) fellowship program.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "Evidence that Hepatitis D virus (HDV) genotype is involved in HDV infection pathogenesis is increasing. Indeed, HDV genotypes have been shown to be linked to different outcomes in terms of liver fibrosis and treatment response. Herein, we show that the promising HDVdb genotyping tool available online can lead to wrong genotyping results. The current HDVdb algorithm should be carefully considered as a \u201cbeta-version\u201d and warrants algorithm core corrections, as soon as possible, for an optimal and beneficial use. HDVdb is based on classic BLAST algorithm [https://talk.ictvonline.org/ictv-reports/ictv_online_report/negative-sense-rna-viruses/w/deltavirus.We read with great interest the manuscript \u201cHDVdb: a comprehensive Hepatitis D Virus Database\u201d by Usman et al. published in Viruses . The autlgorithm  with a dhttp://hdvdb.bio.wzw.tum.de/hdvdb/bio/blast). In the HDVdb detailed output reports, maximum alignment scores were obtained with JA417556 and AX741159, HDV-6 reference sequences according to HDVdb. Other HDV-6 sequences namely JA417546, JA417551, AX741149 and AX741154 also showed high identity scores. JA417556, JA417546, and JA417551 from patent EP2343374 are identical to AX741159, AX741149 and AX741154 from patent WO03027291, and correspond to AJ584848|dFr910 (ICTV HDV-5 reference sequence), dFr47 and dFr73 strains, respectively. These sequences are the three original HDV genotype 5 reference sequences characterized [Two output consensus sequences  that had been clearly affiliated to HDV genotype 5 (HDV-5) using our local bioinformatic pipeline were surprisingly assigned to genotype 6 (HDV-6) using HDVdb genotyping tool (n = 95) are available upon request. Further discordances were noted for HDV genotype 2 (HDV-2). Indeed, one HDV-2 sequence, namely LT604953, corresponding to the first HDV-2 described in Yakutia in 2001 (Strain \u201cYakut-6\u201d) [http://hdvdb.bio.wzw.tum.de/hdvdb/Downloads/Dataset/hdvdb_dataset_page/Genome_6.fasta). Here, again, a mistake concerning LT604953 affiliation was evidenced, which may lead to a wrong genotyping result for the query sequence. Additionally, three other HDV-2 sequences\u2014namely X60193  accurately characterized by the French National Reference Center for HDV  [As a confirmation, a phylogenetic tree, including the 10 potentially mis-genotyped sequences by the HDVdb online tool and 74 other sequences  after a Genotyping of HDV may be crucial for the future management of HDV-infected patients, as it has been shown to be associated with the severity of liver disease as well as the treatment response ,13. Howe"}
+{"text": "Linked data are increasingly being used for epidemiological research, to enhance primary research, and in planning, monitoring and evaluating public policy and services. Linkage error  can manifest in many ways: as missing data, measurement error and misclassification, unrepresentative sampling, or as a special combination of these that is specific to analysis of linked data: the merging and splitting of people that can occur when two hospital admission records are counted as one person admitted twice if linked and two people admitted once if not. Through these mechanisms, linkage error can ultimately lead to information bias and selection bias; so identifying relevant mechanisms is key in quantitative bias analysis. In this article we introduce five key concepts and a study classification system for identifying which mechanisms are relevant to any given analysis. We provide examples and discuss options for estimating parameters for bias analysis. This conceptual framework provides the \u2018links\u2019 between linkage error, information bias and selection bias, and lays the groundwork for quantitative bias analysis for linkage error. Linkage error can manifest as missing data, misclassification or measurement error, or erroneous inclusion or exclusion of people from an analysis. It can also cause splitting of one person\u2019s records into multiple units of observation, and merging of multiple units into one.Misclassification and measurement error can lead to information bias. Rates of misclassification and measurement error may be higher when links are meaningfully interpreted, such as when deriving vital status from linkage to a register of deaths.When inclusion or exclusion from an analysis rely on accurate linkage, linkage errors may lead to selection bias.When units of observation cannot be uniquely identified without linkage , linkage errors may lead to splitting and merging. Splitting and merging may lead to both information bias and selection bias.Considering only the links between two sets of records and the sampling frame for an analysis, there are 11 possible linkage structures that can help to identify the qualitative manifestations of linkage error, but linkage errors within each set may also need to be considered. ,With advances in computing technology and increasing use of secondary data for research, there has been rapid growth in analysis of linked data but little corresponding acknowledgement of the statistical problems introduced by linkage error: missed links between records relating to the same entity  and false links between records relating to different entities . This arRothman and colleaguesInformation bias arises from measurement error in quantitative variables or misclassification in categorical variables. One of the more straightforward impacts of linkage error is when a false link results in incorrect information being obtained from a record that belongs to a different entity. For example, if we link together reading and mathematics scores for two different children, we would introduce measurement error unless the two children happened to have the same scores. There are many situations in which missed links can also result in incorrect information being derived; this is especially likely when only a subset of records are expected to have links, and the presence or absence of a link is meaningfully interpreted, such as when we infer mortality from linkage to a register of deaths. In this case, it is not the data contained in the death record per se that provides information, but the existence of the link itself.When linkage is meaningfully interpreted, missed links and false links can both lead to misclassification. The misclassification, however, operates in opposite directions, so missed links and false links can offset each other\u2019s influence. For example, missed links to a register of deaths would cause false-negative misclassification of mortality, whereas false links could cause false-positive misclassification.When linkage is not meaningfully interpreted, missed links result in missing data and false links result in potential misclassification or measurement error. Note the caveat here; false links only lead to misclassification or measurement error when the information contained in the falsely linked records differs from the information that would have been derived from correctly linked records. For example, a link from one dead person to another person\u2019s death record would not result in misclassification of vital status, but it might result in measurement error in time to death. This potentially important caveat requires many of the statements in this section to be caged in uncertain terms  and its relevance to bias analysis will be discussed in the next section.Key concept 1: Links can be meaningfully interpreted to imply the value of some variable. When links are meaningfully interpreted, both missed links and false links can manifest as misclassification or measurement error in that variable, but in opposite directions.Selection bias occurs when the probability of inclusion in an analysis is correlated with one or more of the variables of interest.When linkage is not meaningfully interpreted and missed links lead to missing data, then how those missing data are handled determines the implications of linkage error. Invalid techniques for imputing missing data can induce information bias, and another common strategy for addressing missing data is exclusion . Exclusion of individuals with data that are missing introduces potential for selection bias when missing data from missed links are not missing completely at random (i.e. when the probability of missed links depends on one or more variable of interest).The third way that linkage error can affect inclusion in an analysis is more abstract; the double-counting that can occur when missed links cause one entity\u2019s records to be split into multiple apparent entities, and the undercounting that can arise when records relating to separate people are inappropriately merged because of a false link. Double-counting and undercounting can be operationalized as representing relative selection probabilities of greater than one or less than one, respectively.Key concept 2: When selection depends on the accuracy of linkage, linkage error may lead to selection bias. This can happen because linkage error leads: to misclassification or measurement error in selection criteria; to missing data in records that are subsequently excluded; or to splitting and merging.This splitting and merging of entities often involves some degree of both information bias and selection bias. For example, depending on whether they are linked, two hospital admission records may be counted as either one person admitted twice or as two people admitted once. Misclassification or measurement error may be implicated whenever variables of interest are derived from multiple records. In the hospital example, readmission statistics could be affected, but demographic characteristics that were constant across records or were derived from a single record would not be. Analyses involving variables derived from multiple records are therefore particularly susceptible to bias from merging and splitting.Merging and splitting is a concern whenever the target sample for an analysis is not uniquely identified in the data, prior to linkage. If a sample is to be drawn from a single, event-based file that must be \u2018internally linked\u2019 to enable analysis at the person level, then the units of analysis (people) can be affected by linkage error. Even when both files in a linkage contain only a single record for each entity, if the sampling frame includes people from either file then the sample cannot be uniquely identified until after linkage and the potential for merging and splitting remains. A missed link in these situations could result in somebody being counted twice (once in File A only and once in File B only) and a false link could result in two different people being counted once (as one person appearing in both files). The sample can only be uniquely identified prior to linkage when it is drawn from a single file that does not itself require internal linkage.Key concept 3: Unless the sample is uniquely identified prior to linkage, linkage error may lead to splitting and merging of entities (units of observation). Splitting and merging can be operationalized as a combination of varied probabilities of selection and misclassification or measurement error in variables that are meaningfully interpreted or otherwise derived from multiple records.Establishing the potential for merging and splitting requires careful consideration of the unit of observation. A set of hospital admission records, for example, may contain a uniquely identified sample if the unit of observation is admissions, but not if the unit is people, and not if the unit is sequenced events such as people\u2019s first admission (because first admissions cannot be identified until they have been linked to any other relevant admissions).These three key concepts provide three questions in identifying the manifestations of linkage error: (i) are links being meaningfully interpreted? (ii) is selection dependent on linkage? (iii) is there a possibility of splitting or merging? The answers to these are not always straightforward, especially in the case of establishing the potential for merging and splitting within an internally linked file, as discussed above. For links between multiple sets of records  we have found that it helps illustrate the sampling frame using a Venn diagram with shading in the region from which the analysis set (sample) is selected.Any two sets can intersect in three possible ways: (i) each set contains the same entities (their coverage overlaps perfectly); (ii) one set contains entities not included in the other (the latter is nested within the former); or (iii) each file contains entities not included in the other (their coverage intersects). Considering the different possible regions within these which could form the sampling frame for an analysis, we have identified 11 possible linkage structures . A missed link between records A and C may be of no consequence if there is an indirect link formed by links between A and B, and B and C. In essence, there are multiple possible ways for the same information to be derived from records A and C; either through a direct link between these records, or through an indirect link via record B.These caveats can all be parameterized in the same way, as the distribution of differences between the observed values derived from linked records, and the values that would have been derived from the (truly) matched records.Key concept 5: Linkage errors only have a meaningful impact on data quality when the information derived from the erroneously linked or unlinked records differs from the information that would have been derived from correctly matched records. Linkage error bias therefore depends on the proportion of each type of linkage error that results in incongruent information, and/or the distribution of differences in values between the (observed) linked records and (unobserved) matched records.Obviously, estimating this distribution of differences is problematic. However, it may often be reasonable or sufficient to assume that all linkage errors lead to meaningful differences . Furthermore, some of the techniques listed in We have identified three key concepts for determining the qualitative manifestations of linkage error, and two that relate to quantitative aspects that require measurement or estimation in bias analysis. Estimating and modelling every potentially relevant bias parameter will often be beyond the realm of feasibility; a balance must be struck between the requirement for accurate estimation, the availability of evidence to inform assumptions, the time required to collect that evidence and incorporate it, and the risk of ambiguity and human error that can be introduced by overly complex analysis.,Perhaps the biggest limitation of this conceptual framework is that it is rooted in what we (the authors) think of as \u2018deterministic analysis\u2019 of linked data; analysis that treats every pair of records as being either linked or not linked.We hope that this framework and classification system help to increase understanding of linkage error, and help researchers address bias in analysis of linked data. The next steps required are the development of generalizable formulae and software tools to make it easier to put these principles into practice."}
+{"text": "Coot is a tool widely used for model building, refinement, and validation of macromolecular structures. It has been extensively used for crystallography and, more recently, improvements have been introduced to aid in cryo\u2010EM model building and refinement, as cryo\u2010EM structures with resolution ranging 2.5\u20134 A are now routinely available. Model building into these maps can be time\u2010consuming and requires experience in both biochemistry and building into low\u2010resolution maps. To simplify and expedite the model building task, and minimize the needed expertise, new tools are being added in Coot. Some examples include morphing, Geman\u2010McClure restraints, full\u2010chain refinement, and Fourier\u2010model based residue\u2010type\u2010specific Ramachandran restraints. Here, we present the current state\u2010of\u2010the\u2010art in Coot usage. Originally, Coot was designed for interpretation of MX data, the focus being tools for moving and refining one or a small number of residues, or ligands.Coot have now been expanded to assist building of large macromolecular assemblies into such maps. Furthermore, modern computers now have multiple cores and these have been exploited to extend the range and speed of the Coot tools.Model building is an essential step in structural biology that facilitates interpretation of structural data obtained by different methods, including macromolecular crystallography (MX) and electron cryomicroscopy (cryo\u2010EM). Coot, include C\u2010alpha baton mode and main chain conversion, automatic finding of alpha helices, beta\u2010strands and ligands, placing helices and strands, generation of idealized DNA and RNA molecules, real\u2010space refinement, rigid\u2010body fit, rotate/translate zone, flip peptides, rotamer tools, and validation tools such as density\u2010fit analysis, rotamer analysis, and Ramachandran and Kleywegt plots.Coot GUI have now been redesigned to make the tools easier to find for novice users. Currently, the catalogue of tools is distributed into menu bars that refer to the type of task to be performed and is shown in alphabetical order. Also, the performance and speed of such tools have been optimized for large macromolecules and maps.Some of the tools that have been significantly used in Coot integrates additional restraints for nucleic acids and local distance restraints for cryo\u2010EM three\u2010dimensional (3D) reconstructions from other programs such as ProSMART and LIBG.Coot include those using a nonharmonic function 4 which augment previous RNA ToolsFaster refinement goes hand in hand with the addition of new restraint types. For instance, ensional D reconstCoot include representation, visualization, and validation of ligands. New features include improved chemical diagrams and 2D representation of geometry outliers for validation. Macromolecular model validation has been a mainstay of Coot functionalityOther improvements in 1.1Coot allow now to place full domains or chains and refine them. The difference is that whereas MR is performing a systematic search of rotations and translations, the tools in Coot for cryo\u2010EM model placement are a local translation and rotation search so that this process is started manually by placing the homolog structure near the center of the domain to be fitted using \u201cPlace molecule here.\u201d After the user has performed this operation, Coot tools can be used to fit the model into the map. Figure In cryo\u2010EM, de novo tracing of the main chains is often needed as the initial step towards structure interpretation. However, if the first operation for map interpretation is to fit the structure of a homolog or a previously obtained model into the map of interest, one would have no need for de novo tracing\u2014just as, in MX, one would not first try to solve a structure with heavy atom derivatives if molecular replacement (MR) was possible.1.2Nicholls and coworkersCoot, one can generate sharpened and blurred maps using different tools. For MX data, the Sharpen/Blur tool is interactive and is found in the Calculate menu. For cryo\u2010EM, we would not use the interactive Sharpen/Blur tool because the FFT takes too long for it to be interactive. In this case, a different Sharpen/Blur tool can be found in the cryo\u2010EM module. Additionally, the cryo\u2010EM module has a \u201cMulti\u2010Sharpen\u201d option, which runs Refmac5 to produce an mtz file that contains the coefficients for a number of sharpened and blurred maps. Simply by reopening the generated and saved mtz file, the user can select the preferred blurring/sharpening temperature factor. The module can be installed using Calculate \u2192 Modules \u2192 Cryo\u2010EM. When fitting a domain, we find that it is useful to use a large blur factor  before the fitting steps, as blurring allows a larger radius of convergence for the rigid body fit part of the algorithmIn 1.3Jiggle fit would then be carried out as the next step, typically on a chain or domain as described before.1.4Coot, the CCP4 program ProSMART would typically be used to generate this additional restraints set although other suites can be used to similar endsCoot. The use of these additional restraints, which tries to keep distance between atoms similar to those present in the starting model, is often what is needed at the first stages of refinement . These restraints help the atoms to move in a concerted manner. Typically, the application of restraints in Coot has involved the use of harmonic potentials. However, the application of these local distance restraints in Coot involves the \u201cGeman McClure\u201d (GM) robust estimator. Nicholls and coworkers explained the value of using GM restraints in protein refinement.Coot refinement has been improved to use multiple CPUs, now one can use longer distances (and hence use more restraints). Therefore, one might use distances of 6 or 7 \u00c5 in the local distance restraints generation , it is useful to add even longer\u2010range distance restraints to complement these restraints. These restraints might typically encode distance information for hydrogen bonds or conformation of corresponding structures from homologs. When working with n Figure d. It is 1.5Coot has been decoupled from the updating of the graphics by running it in its own thread. Consequently, the pressure to only refine small fragments, so that the graphics could update in a timely fashion, has been removed. Thus, more residues can be refined and their representation updated asynchronously rather than forced for every frame. The concomitant changes to the API, and the update of the calculations of the refinement to use multiple threads, considerably improves the ease of use of real\u2010space refinement, so that it is routine to refine residue selections as large as a domain or chain , Copy Fragment, Replace Fragment, and Merge Molecules are useful tools here. They can be found under \u201cEdit\u201d in the main menu bar. To illustrate typical operations, we are going to use an example where the \u201cmaster molecule\u201d has residues 50\u2013100, which need to be adjusted. One would copy out that fragment from the master molecule (Edit \u2192 Copy Fragment \u2192 Atom selection for fragment \u201c//A/50\u2013100\u201d), \u201cLast Only\u201d in the Display Manager will focus the attention on just that fragment. One can then operate on that fragment using different strategies, such as jiggle\u2010fit or real\u2010space refinement. Finally, the fragment will need to be merged back into the master molecule using \u201cEdit \u2192 Replace Fragment.\u201d This replaces the position of atoms in the master molecule with those from the fragment. \u201cMerge Molecules\u201d on the other hand will add the atoms of the fragment to the master molecule, with new chain identifiers being created if needed.Coot uses a proximity check to find the closest chain, and selects a new residue number that is suitably above those of the extant residues.There is an additional case of merging molecules: that of merging a ligand. It is typical in this case that a new chain identifier is not desired, but instead the chain identifier that matches the chain to which the ligand is begin attached should be used for the ligand. 1.7Coot, allowing quick generation of alternative hypotheses for the register of residues which the user can then inspect visually. Nudge residues can be used from the Cryo\u2010EM module . Each amino acid type  has its own set of rotamers which are tabulated in databases.Although this tool has been available in Coot, the same formalism for atom movement has been reworked to find high probability rotamers in low\u2010resolution maps. The previous rigid\u2010body\u2010fit\u2010based rotamer fitting tool in Coot allowed poor/impossible backbone geometry in low\u2010resolution maps, frequently resulted in distorted main chains . The score from the clash interactions and the density fit are combined for each hypothesis, and the best hypothesis is selected and replaces the current model if the score for that hypothesis is better than the score for the current positions of the atoms of the given residue. There is, as yet, no secondary structure dependence on the rotamer selection.The backrub motion introduces a change in \u03c4 (the N\u2010CA\u2010C angle) typically less than 2\u00b0, so while this change may be of some (negative) consequence, it is highly likely to be less than the beneficial changes in \u03c6, \u03c8, or \u03c7s of the fitted residue and the \u03c6 and \u03c8 of the neighboring residues. Any introduced \u03c4 angle strain can easily be reduced or removed by subsequent refinement .1.101.10.1Coot has the ability to generate A or B forms of RNA and DNA. These molecular fragments often provide useful templates for de novo model building in cryo\u2010EM 3D reconstructions. In addition to the standard molecular restraints ,1.11Coot.Asparagine (Asn)\u2010linked glycosylation is the most common type of N\u2010glycosylation of eukaryotic proteins, and it is also found in viruses, including HIV and Ebola.Coot to bring model building of carbohydrates to a stage where it can routinely produce high\u2010quality glycan models with cryo\u2010EM 3D reconstructions.It should be noted that the density of N\u2010linked carbohydrate in cryo\u2010EM reconstructions is often of lower quality that that from MX data. In cryo\u2010EM, we need to rely more on prior knowledge than optimization of the fit to density. Additional work is needed in 1.12Coot has had a means to rotate residues and other molecular fragments around rotatable bonds. More recently, a faster tool has been introduced that is particularly useful for ligands. Consider the case in which a ligand is placed in the active site of a protein, with a conformation that is not correct. The ligand has a benzene ring with a chlorine substituted at the meta position and the real/correct conformation needs the benzene ring of the ligand, and its substituents, to be rotated by 180\u00b0. Although the \u201cEdit Chi\u2010angles\u201d tool can do this, the manipulation is faster and more convenient with \u201cJED Flip,\u201d particularly with \u201cInteractive JED Flip,\u201d where the rotation is applied to the \u201cactive\u201d bond during refinement. JED Flip uses the torsion restraint to create a number of torsion angle deltas , the application of which is frequently the operation needed to correct the conformation of the ligand. If Interactive JED Flip is used, the ligand settles into the correct conformation with no additional intervention (see video). By default, Coot moves the smaller set of atoms on either side of the rotatable bond. (Infrequently) one might wish to rotate the larger fragment and this is achieved by \u201cReverse JED\u2010Flip\u201d (Shift G)  .1.13Coot has been extended to provide an interface to this functionality. Using the CCP4 Module , one can select an atom in each of two residues and define a bond order (default \u201csingle\u201d) which then constructs the input for Acedrg. Acedrg is run in the background and then Coot loads the dictionary produced by Acedrg so that it becomes available for Real\u2010Space Refinement.Acedrg1.14Coot allows the refinement of metals. Previous version of Coot did not interpret the LINK records for metals in macromolecules and hence selecting a metal atom in Real\u2010Space Refinement meant that there was only a nonbonded contact restraints between the metal and the metal ligand atoms. Now, Coot parses the LINK records and generates bond restraints for metal between nitrogen, oxygen, and sulfur metal ligands. Furthermore, with the addition of this bond restraint, the nonbonded contact restraint is no longer used. The target distances used for these bond restraints are element based derived from the metals in the Acedrg tables (full\u2010atom Acedrg atom types are not used so the restraints are not as accurate as they might be in the future).The new version of 1.151.15.1Coot by wrapping the functions of the RDKit . As well as de novo sketching, Lidia also supports the import of chemical structures from mol or mmcif files, a SMILES string, has a rather robust \u201cFetch\u201d tool which uses Wikipedia to convert potentially common molecule names to the International Nomenclature Name, and parses the drug box to import a representation of the ligand from DrugBank,https://www.chemspider.com/Chemical-Structure.1906.html), or PubChem.The \u201cLigand Display and Analysis\u201d (Lidia), the framework for 2D representation of ligands, has been previously described.Coot using the RDKit for the 2D layout of the chemical entity and adds rendering and representation of protein residues and interactions in the \u201cFlatland Ligand Environment View\u201d or FLEV mode (Ligand \u2192 FLEV this residue).Clarke and Labute1.16Coot graphic interface to proceed with model building and speed up the visualization process. Currently, as a result of multithreaded contouring, one can display larger areas of the working map as well as provide different view styles for the maps, that is, not only the traditional Standard Lines but also Solid/Transparent and Cut\u2010Glass representations. All of the above together facilitate map interpretation. These options are available under Display Control \u2192 Properties.In the past, small fragments of a map would be visualized in the 1.17Coot is using the map for navigation. When model building with Coot, one wants to add or move atoms to (or close to) the view rotation centre. The reason is that this way allows easier visualization of what is around a particular point if the point is at (or close to) the view rotation centre.Perhaps, the most convenient but overlooked feature of Coot determines the mapping into 3D space of the point under the cursor on both the front and back clipping planes, which gives us two 3D points in the model coordinates system. Coot then  draws a line between these two points and steps, in small increments, from the front 3D point to the back 3D point. As it does so, it queries the value of the active (fitting) map at each of those points and considers that value in relation to the contour level. When it finds a point above the contour level, it starts to record a density profile, and continues until it finds a point below the contour level. The weighted mean position of that profile then becomes the new rotation centre.One can do this quickly and easily navigate to the area of interest using the function blob_under_pointer_to_screen_centre bound to a key\u2010press . 1.18Validation of structures has been an important aspect of macromolecular model building for a number of years\u2014after it had been made apparent that it was possible to incorporate both gross and small errors in protein models.Coot go in this direction, and it is an on\u2010going topic of research.An important aspect of good macromolecular modeling tools is not only to detect problems in the structures but also to provide means to resolve them. The interactive validation tools of Coot currently incorporates an extensive validation menu to assess the model geometry and the fit of the models to the maps. The most practical quality indicators are the Geometry analysis, Difference Map Peaks, Density\u2010fit analysis, Rotamer analysis, and Ramachandran and Kleywegt plots.1.19Coot now uses \u201cThe (son of) Penultimate Rotamer Library\u201dThe Rotamer validation has been updated. 1.20Coot provides basic statistics for model atomic displacement parameters, called \u201ctemperature factors\u201d in the Coot interface\u2014an analysis of temperature factors can quickly direct the user to parts of the structure that have been erroneously modeled (or are more flexible than the majority of the structure). Interactive display of the temperature factor graph is available in the validation menu. The python functions to return the average and median temperature factors are mean_b_factor(imol) and median_b_factor(imol). The median temperature factor is often a more robust metric than the mean as it will not be affected by a small number of waters that have very high temperature factors.1.21Coot has been updated and improved in various ways. In general, the tool can be launched from the validation menu and will show the Ramachandran plot in a new window (Figure Dynarama. This way users can benefit from this validation tool and its features outside of Coot. The Ramachandran plot widget allows exporting of the plot together with the statistics in pdf and png format. In the future, the Ramachandran plot is envisaged to be even more interactive, for example, to update during the refinement, allow dragging, and flipping of residues within the plot, as well as contain gradient colors, in addition to the contour lines, to allow better visualization of the different regions.The Ramachandran plot tool in w Figure . A markew Figure c. The baw Figure b, where 1.22The idea of the ligand validation tool1.23Coot will progress in two major directions.The development of Although writing multithreaded code is difficult, the pay\u2010off is substantial. Larger atoms selections can be refined faster and with more restraints. Rotation/translation searches, torsion angle searches, and dynamic atom contact searches can be performed using multiple threads to reduce the wait\u2010time and increase interactivity.Secondly, a wholesale reworking of the dependencies will provide Python 3, more numerous and more fully featured modules, and an updated GUI which will eventually lead to graphics that are more interactive and better represent molecule shape of both the atoms models and maps.The combination of both the above will allow for interactive representation of rotamer probability, the Ramachandran plot, clashes, and other validation criteria.1.24https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/.At the time of writing, the Coot web page is at Coot web page. The source code repository is available at https://github.com/pemsley/coot.The software is licenced under the GNU GPL v3, GNU LGPL v3, and compatible licences and is available for free for academics and others. Links to source tar files and binary tar files is available from the The authors declare no conflicts of interest.Video 1 Fitting domains, using additional local distance restraints to refine a domain into a cryo\u2010EM map.Click here for additional data file.Video 2 Shuffle\u2010along/Nudge residues\u2013how the active residue selection and be shuffled along the change using locale distance restraints.Click here for additional data file.Video 3 Backrub Rotamers, a rotation illustrating the ways in which atoms can move to generate hypotheses in Backrub Rotamer fitting.Click here for additional data file.Video 4 RNA Fitting\u2010Domains: How to fit a fragment of RNA into a cryo\u2010EM reconstruction, with narration.Click here for additional data file.Video 5 JED\u2010Flip: Example of how a ligand can be quickly and easily rotated by chemically sensible angle differences to interactively examine the fit to density.Click here for additional data file."}
+{"text": "Since its post-World War II inception, the science of record linkage has grown exponentially and is used across industrial, governmental, and academic agencies. The academic fields that rely on record linkage are diverse, ranging from history to public health to demography. In this paper, we introduce the different types of data linkage and give a historical context to their development. We then introduce the three types of underlying models for probabilistic record linkage: Fellegi-Sunter-based methods, machine learning methods, and Bayesian methods. Practical considerations, such as data standardization and privacy concerns, are then discussed. Finally, recommendations are given for organizations developing or maintaining record linkage programs, with an emphasis on organizations measuring long-term complications of disasters, such as 9/11. From its humble beginnings in post-World War II public health research, the field of \u201crecord linkage\u201d\u2014that is, the matching of records for unique entities across one or more lists\u2014has exploded into a multi-field research focus see . As the Among the major 9/11 health cohorts, linkages are currently underway or have been conducted to cancer registries, the National Death Index, New York State hospitalizations, and various vital records. Moreover, several joint studies are being formulated to study pooled patient populations across cohorts. The reasons for this are both scientific and practical. As more data become available electronically and computational power improves, access to health data, at least from a technical point of view, has become easier. This is fortuitous as maintaining a large-scale research project over multiple decades among a trauma-exposed and aging population presents several challenges, chief among them attrition and reporting bias due to failing memories among respondents.This paper introduces the field of records linkage to the reader, assuming little previous experience with the topic. The goal is to more broadly inform the WTC community about this important topic and its implications for WTC research.Several disciplines have contributed to the data linkage literature. Unfortunately, one side effect has been a lack of consistent language surrounding record linkage research efforts. The result has been a variety of terms that are used for the same or similar concepts; Probabilistic record linkage is one example of multiple data combination techniques. In our experience, deterministic record linkage is either infeasible or only available for a subset of records for multiple reasons. In the case where a unique identifier is available, errors or missing values might prevent some proportion of records from being linked. More often, no unique identifier is available, and a simple, fixed rule will result in either too many links that are invalid  or true matches that remain unlinked . Probabilistic record linkage starts with data for one or more fields within each list and uses a probability model to determine the likelihood of a pair of records representing the same entity. For example, the name John Smith is a very common name, and therefore the likelihood that two records with the name John Smith really represent the same person is low. Jana Asher, in contrast, is a relatively rare name, so two records with the name Jana Asher are much more likely to represent the same person. Combined with a date of birth, a rare name can almost serve as a unique identifier.Data fusion, distinct from record linkage, involves the merging of records that represent different entities for the purpose of obtaining a better understanding of the concepts represented by the data. A simple example might help illustrate what data fusion accomplishes. Imagine that two distinct datasets each have spatial data for different non-overlapping coordinates, and some set of covariates that are different for the different datasets, such as temperature in one dataset and elevation in the other dataset. We can combine the datasets and use interpolation to estimate the temperature and elevation for all data points, creating a more complete set of information for the area. The interpolation makes this a less data-driven process than probabilistic record linkage, in that synthetic data are formed from a combination of the existing data and probability models.Finally, the most model driven of the techniques is called statistical matching. In statistical matching, records with similar characteristics across multiple covariates are matched together to compare values for a different set of variables. One application for statistical matching is to determine a treatment effect in the absence of a control within a randomized experimental design. Since randomization could not or did not happen, the alternative is to find a non-treated entity with characteristics as similar as possible to the treated entity, and pair those records for comparison. Another example is a demographic one; if a researcher wants to compare two variables that are on different datasets when there are not enough appropriate comparison fields for probabilistic record linkage, then statistical matching can be used to link the most appropriate records to each other.The remainder of this paper will be focused on probabilistic record linkage. The origins of record linkage as a field begin at the end of World War II; the original papers on record linkage related to family structure in the United States and elsewhere ,2,3 and Science [The first mention of computer-assisted record linkage in the literature occurred in 1959, when Harold Newcombe laid the groundwork for probabilistic record linkage in an article in Science ; around Science . In publScience . The ratThe breakthrough came from the research of Ivan Fellegi and Alan Sunter between 1967 and 1969 . Over thFrom about 1970 to 1999, most of the record linkage research was occurring at national statistics agencies. By the late 1980s, over 300 papers were being published annually, and the first books related to record linkage (both probabilistic and non-probabilistic) were being published. Much of the work had to do with finding optimal string comparators and improving the efficiency and accuracy of record linkage algorithms. In the meantime, computational power continued to grow, allowing larger and larger datasets to be linked in shorter and shorter time periods.By 2000, the computer science field had discovered record linkage and began to develop machine learning algorithms . Over thThe literature about record linkage has literally grown exponentially; a search of Google Scholar yielded over 5000 papers for 2018 alone see . AlthougFellegi and Sunter\u2019s model is based on a decision rule as to whether a pair of records\u2014these could be two different records from the same list, or a record from one list being compared to a record from another list\u2014are to be linked  or not. It was developed in the context of large governmental statistical agencies, and its original purpose was to link across records representing people. Typically, there are multiple fields that are directly compared for each pair of records under evaluation. For example, in m probability for the field, and the latter probability is called the u probability for the field.During the linkage process two specific probabilities must be estimated for each pair of fields. The first is the probability that the fields agree given that the underlying entity represented by the records is truly the same , and the second is the probability that they agree given that the underlying entities represented by the records are not the same . The former probability is called the m probabilities for the fields are typically close to, but not exactly, one. When the same entity generates two records , there is always a possibility that those records will not match due to human error or other random processes . For example, in The u probabilities for the fields vary quite a bit. In u probability depending on the frequency of the name in the population represented by the list(s) being compared. For example, if there are two lists being linked, and each of the lists represents the United States population, then the probability of the name \u201cJana\u201d is about 0.00015; the probability of the name Jane is about 0.001 [In contrast, the 2(m/u), which is called the agreement weight; if the field is determined to not agree then the weight is log2((1 \u2212 m)/(1 \u2212 u)), which is called the non-agreement weight and is always negative. The field weights (either agreement or non-agreement) are then summed to create an overall match weight for the pair of records. As can be seen in u probabilities lead to relatively smaller weights, which makes sense: Matching on gender, for example, does not make it much more likely the two records represent the same entity; however, matching on a rare last name does.Once the determination is made as to whether a field agrees or not, a weight is assigned to it. If the field is determined to agree, the weight is logIn the Felleg-Sunter process, once each pair of records has been assigned a match weight, the pairs are then put in order of their weights, with the largest weights coming first. At that point, two cut-offs can be set to separate the list of pairs into three sections. The top section\u2019s pairs are considered the \u201clinks\u201d; that is, the pairs of records that the process has determined  represent the same person. The bottom section\u2019s pairs are considered the \u201cnon-links\u201d; that is, the pairs of records that the process has determined do not represent the same person. The middle section will be reviewed, and a decision will be made individually for each pair as to whether it is a match. Note that the Fellegi-Sunter theory does not provide guidance for setting these thresholds, only that Type I error will be minimized for a fixed Type II error, or Type II error will be minimized for a fixed Type I error. Fellegi-Sunter does suggest that manual review of pairs over a range of weights can be used to determine above which weight pairs are almost certainly matches and below which weight pairs are almost certainly non-matches. More recently, record linkage software packages have included algorithms for determining optimal thresholds based on the record linkage weights.We have necessarily skimmed over a few details in this process. One is the method by which each pair of fields is compared. For example, names are often pre-processed to standardize spellings and convert nicknames to formal names, after which they are compared either exactly or through a string comparator. When a string comparator is used, it provides a distance measure such as the one developed by Matthew Jaro and William Winkler ; in shorThe other process not yet mentioned is the choosing of which pairs of records to compare. In record linkage processes with large datasets, it is usually computationally infeasible to compare every possible pair of records; for this reason, a blocking strategy is often used. Blocking is the process of restricting the comparisons to only pairs of records that agree on a set of specific fields. For example, if in a given blocking pass, both ZIP code and year of birth are used as the blocking variables, then only pairs of records with the exact same ZIP code and year of birth will be compared. This tremendously reduces the number of pairs of records to be compared; however, if there is an error in a record\u2019s ZIP code or year of birth, a true match might be missed. For that reason, in large record linkage projects, several blocking passes are used. For example, for a first pass, only records with identical ZIP codes and year of birth will be joined into pairs. In a second pass, only records with the same first and last names will be joined into pairs. When multiple blocking passes are used, they can be sequential (only records not linked in the first pass are linked in the second pass), or they can be overlapping , which will require further subsequent unduplication.The Fellegi-Sunter method remained the only well-known algorithm for probabilistic record linkage through the end of the 20th century. Advancements in the method had to do with processing speed, the use of expectation-maximization algorithms to determine match weights, creation of string comparators, and applications to new types of records. Eventually, practitioners began to realize that the Fellegi-Sunter algorithm was akin to a type of machine learning called na\u00efve Bayes.Basically, both the Fellegi-Sunter model and na\u00efve Bayes models rely on the concept of conditional independence; in the case of record linkage, this means that the agreement statuses for comparison variables are independent of each other under the condition of the true match status. However, this is not always the case. An example that readily demonstrates when comparisons are not independent that arises frequently in person-level linkage is for the variables first name and sex. For first name, the probability that a non-matched records pair  will agree (by chance) on first name will be quite low, less than 1%. The probability that a non-matched pair agree on sex is approximately 50%. However, in those cases where the non-matched records by chance agree on first name, the probability that sex agrees is much higher. That is, if we have two different people who have the first name \u201cMary\u201d, almost certainly they will agree on sex (an important exception is those who are transgender). Because the conditional probability of sex agreement differs from the unconditioned probability, the agreement status for sex is said to be non-independent of the agreement status for first name, and this fact has implications for the probability model used for evaluating the likelihood of matching.In machine learning, probabilistic record linkage can be replaced by one of several classification algorithms. In some cases, probabilistic record linkage becomes a supervised learning problem; that is, a training set of data is used to \u201cteach\u201d a classification algorithm, such as a decision trees, support vector machines, ensemble methods (such as random forests), or conditional random fields. The classification algorithm used for a specific problem is dependent on the type of data being linked; in most cases, the methodology requires that there be a large number of records in the datasets, as they must contain enough records to allow extraction of a sufficiently large training dataset. Decision trees have been used by business to unduplicate customer lists and build customer profiles that contain purchase histories . When thOnce a classification algorithm is selected, the training dataset is randomly selected from the larger collection of records; often, clerical review is used to determine which pairs of records represent true matches and which do not. Using the training data, the probabilities of a match given a particular agreement pattern across the fields is determined. For example, in k-means clustering. In k-means clustering, for each pair, a measure of similarity for each field is calculated. Unlike the match weights in the Fellegi-Sunter algorithm, these measures are set up so that 0 indicates a perfect match for the field, and higher numbers indicate the fields are less similar. A ceiling value of 1 can be set to mean that the fields completely disagree.An alternative option is an unsupervised method; that is, a method that does not rely on training data. An example is Multiple similarity measures have been used within unsupervised record linkage. For numeric data, similarity is fairly easy to measure. For text-based fields, the similarity measures are more complex. The Damerau-Levenstein distance, developed in the 1960s, is a single numeric measurement based on the comparison of two text strings for four common transcription errors: insertions, deletions, substitutions, and transpositions ,22. The For some record linkage tasks, semantic matching\u2014that is, matching across the meaning of two strings of text, or determining if two strings of text represent related concepts\u2014is the desired outcome. One class of similarity measures for semantic matching requires a known set of linked concepts against which the two strings of text can be compared; the similarity measure is based on how \u201cclose\u201d the two strings are in the map of known concepts. A different type of similarity measure is based on a large set of text documents in which the concurrence of the two strings of text is flagged; the similarity measure is then based on how frequently the two strings of text are found together. However, recent work has suggested that semantic matching is of highest quality when multiple similarity measures are aggregated to create a single measurement system .Note that when similarity measures are used, a binary decision for each field is no longer required. For example, in Once each of the pairs of records has an associated similarity measure vector, the distance between different pairs of records is measured. One common distance measurement is the Euclidean distance; this is determined by summing the squared distance\u2014that is, the difference in the field value between two record pairs\u2014across all the fields being compared. Record pairs that are \u201cclose\u201d to each other according to the distance measure are formed into clusters. Other possible distance measurements include the Manhattan distance, which replaces squaring the difference in field value between two record pairs with taking the absolute value of that difference, and the Hamming distance, which is used when similarity measure vectors contain only binary  values. In record linkage, typically three clusters are formed to match the three regions of match weights in the Fellegi-Sunter algorithm; one cluster represents the links, one the non-links, and one the possible links.Bayesian record linkage techniques are the most complex that have been developed to date and should be considered experimental. Bayesian techniques rely on probabilities of a match or non-match for specific agreement patterns that are either based on expert opinion or on previous projects. These prior probabilities are then combined with a new record linkage process involving new lists. At the end of the process, a posterior probability of match or non-match is determined for the record pairs, which allows the determination of links and non-links. Like machine learning processes, Bayesian record linkage can incorporate non-binary decisions as to whether a field agrees or disagrees for a given pair of records.m and u probabilities were assumed to follow a probability distribution of some type; for example, a uniform distribution with all possible probability values being equally likely, or a Beta distribution, with the parameters of the distribution set based on expert knowledge. The optimal (mean) values from the posterior distributions of the m and u probabilities were then used to create the match weights and complete the linkage process.Early efforts in Bayesian record linkage involved expansions of the Fellegi-Sunter algorithm. The m and u, and the use of the Dirichlet probability distribution for the prior distributions of m and u, which allowed the modeling of conditional dependence between those probabilities [Later work in Bayesian record linkage expanded the machine learning algorithms discussed earlier. Modifications included the use of training data to set the prior values for bilities . More rebilities .Although multiple record linkage models have been developed using Bayesian methods, in our experience, there is no software package available that allows a non-expert user\u2014that is, a person that has not learned Bayesian statistics and record linkage at a level expected of a person who has a Masters or PhD in statistics\u2014to complete Bayesian record linkage. Thus, for this type of procedure to be run successfully requires a statistician or data scientist who is trained in Bayesian computational methods. Further, it is our experience that while Bayesian record linkage seems promising, it is not so well developed as to warrant its use over the more familiar Fellegi\u2013Sunter-based processes.Record linkage research is occurring at phenomenal speed. A driver in the explosion of research around record linkage is the growth in the count and size of available datasets. Many recent advances in machine learning algorithms for record linkage have been motivated by the demand for more efficient and accurate web search tools. A related area is the need to be able to match across different languages as more countries are developing their own large data repositories. A final important area of record linkage research is based on the advent of repositories of non-traditional forms of data\u2014i.e., sound clips, visual representations, and multi-dimensional surfaces. Matching these types of data effectively and efficiently will require more sophisticated record linkage methodologies than are available today.A separate set of research questions is more practical in nature. In some contexts, researchers are being asked to obtain informed consent from all persons whose records will be matched for research purposes. In other contexts, researchers cannot access the large datasets that can be used for social and health-related research due to concerns around privacy and confidentiality. Current research topics related to these concerns revolve around privacy-preserving record linkage and understanding the bias introduced by the requirement for informed consent ,27. We tTheoretical models are the basis of record linkage methods; however, it is the practical considerations of record linkage that require the most effort to understand and problem solve. We will now review these practical considerations and make suggestions as to best practices.Due to the variety of ways in which records of human beings are transcribed, performing standardization prior to matching is almost always required. Organizations that frequently match across large datasets have standardization processes in place; for example, the U.S. Census Bureau will process data to standardize the sex field  and the format for date of birth, remove honorifics  and convert nicknames to formal names, and conduct address geocoding. However, 9/11 exposure and health data have been and continue to be collected by a variety of research teams both for research and clinical purposes, making data standardization a challenge. Commercial packages exist that will automate some of these processes. These packages range in prices and capabilities, and new tools are constantly being developed. For a recent example, please see smartystreets.com/pricing .If multiple organizations plan on sharing data, a common format for data entry is a time saving but not complete solution. Organizations can and do plan to use a common format for sex, date of birth, and honorifics; however, multiple human processes will continue to cause a certain level of error in the fields collected. For example, \u201cage heaping\u201d is a known problem in the demographic field; individuals have some probability of reporting their age to the nearest \u201c5\u201d or \u201c0\u201d instead of exactly . Individuals also vary in how they report information about themselves such as name; nicknames might be used in place of first names, and middle names may or may not be included.Missing data is a problem within any record linkage technique and can be due to a variety of factors. For example, participants may neglect to answer a question due to the sensitive nature of some topics, such as post-traumatic stress disorder symptoms. In addition, because most 9/11 research is longitudinal in nature, attrition has become a more salient problem over time. Even clinical data may be incomplete if a patient misses multiple follow-up visits or leaves a provider altogether.Incomplete data have been handled in several different ways. Some practitioners simply remove any records with missing data; however, this can cause quite a bit of bias in any estimation done with the linked data . Other pSome research has been done on alternative techniques for handling missing data. One possibility is to re-distribute the value of the match weight for the field containing the missing data to all other fields that are found to match, and the value of the non-match weight for the field containing the missing data to all other fields that are found to not match. In that way, the pair of records has similar \u201cweight\u201d to other records without the missing data. Another possibility is to impute the match status of the field containing a missing value based on the match status of the other fields being compared. Ong et al.  found thFinally, direct imputation of the missing values through a regression model or other modeling technique is a possibility, although it adds additional error into an already complex process .Measurement of error in record linkage processes is essential for providing an accurate measure of variance during estimation using linked data. The most common techniques for determining error rates include the use of a gold standard field , a clerical review process (which often is too costly and time consuming to consider), and model-based approaches. Resnick and Asher  propose Given the multiple potential record linkage techniques available, software for record linkage is not difficult to find. However, all benefit from a decent knowledge of record linkage techniques and the underlying statistical models to use. Recently Karr et al.  reviewedTypically, individuals with the data editing expertise to appropriately perform record linkage using the available free or low-cost software packages will be able to import data that starts in a variety of formats. In most cases, data stored in a commercial database can be exported into a comma delimited file (.csv); conversely, most software packages will be able to import comma delimited data.Data format. If different organizations use different data management systems, direct transmission of files without conversion into a commonly accepted format will cause issues. As mentioned before, most statistical and/or record linkage software can accept a comma delimited file as input.Data description. A separate file describing the data should be included; information about a dataset is typically called the \u201cmetadata\u201d. Metadata includes information on how the data were collected, a definition and value range for each variable in the dataset, and any restrictions on the data usage.Confidentiality agreement. All employees that will have access to the shared data should agree, preferably through signing a contract with the organization providing the data, to maintain the confidentiality of the data.Length of time data are available. If the data are only being shared for a limited time period, the parameters for that time period should be outlined prior to data transmission. In addition, requirements for the \u201cdisposal\u201d of the data once the time period is over should be agreed upon in advance.Institutional Review Board (IRB) requirements. In some cases, IRBs have determined that record linkage does not fall under human subjects protections; in other cases, IRBs have regulated record linkage projects. Any IRB restrictions that are already in place regarding the data to be shared must be understood by all parties engaging in the record linkage process.Health Insurance Portability and Accountability Act (HIPAA) requirements. In some cases, IRBs have required that informed consent be obtained from the individuals whose data will be used in the record linkage process. If data are being transferred between organizations, a new informed consent agreement might be required.Secure transmission of data. The protocol for data transmission should be agreed upon in advance and should have appropriate security protocols. Record linkage projects sometimes have stringent security requirements. For example, sensitive de-identified datasets sometimes require researchers to use a computer within a secured data center, separate from the Internet.If different organizations are sharing data files, a basic set of rules should be determined on how files will be transferred. Typically, the areas to consider are:The names of the individuals that complete the record linkage project.The purpose of the record linkage project; for what analysis will the linked data be used?The precedents of the record linkage project, if part of a longitudinal study, and where the documentation for previous iterations of the project can be found. Please note that even when record linkages are to be repeated at different time points with new data, each linkage should still have its own documentation.The names, file positions, and descriptions of the variables;The data collection process for the dataset;The organizational source (in house or outside organization) for the dataset;The date of acquisition of the dataset;The contact information for the person from which the dataset was obtained; andAny rules regarding the use and disposal of the dataset.The metadata for each file to be linked, including:The software that is used for the linkage.How many passes are performed when linking the data;If blocking is used, which variables are used as blocking factors during which passes;If parameters are set ahead of the process\u2014for example, if prior values for m and u probabilities are required\u2014what values are used for each pass.Any information as to how linked pairs, non-linked pairs and possible linked pairs are determined. For a standard Fellegi-Sunter process, this information includes the range of match weights for each of these groups; for a machine learning unsupervised clustering process, this information includes the mean value for each of these groups and the range of distances that determine which pairs are clustered into which groups.Some measurements of the error rate in the linkage process; ideally, these include a false-positive and false-negative rate.A definition for any new variables created during the linkage process.The methodology for the linkage, including:Any known limitations of the record linkage process, including issues with the data that might have made record linkage problematic, known methodological issues related to the algorithm used for the linkage, and specific issues that might have arisen during the record linkage project.The final disposition of the linked datasets, if they are not available indefinitely.In some cases, the linked data will be stripped of all identifiers, allowing the resulting dataset to be freely used without confidentiality constraints. If this is the case, the process of removal of identifying information should be outlined.Because record linkage is often performed by a limited set of individuals with the appropriate expertise, documentation of the linkage process is an essential part of the record linkage methodology. In other words, organizations should develop a pre-determined structure for a required report to be completed for every record linkage project. Such a report might include the following:Privacy and confidentiality are important considerations for all health data, particularly those surrounding stigmatized mental health conditions. For these reasons, data sharing is challenging between 9/11 cohorts. One option is a collection of techniques called privacy-preserving record linkage (PPRL) . First, There are multiple existing organizations that can serve as a third party during a linkage project. Data61, a digital research network based in Australia, developed a set of digital tools for PPRL called Anonlink . Data61\u2032It is possible to complete PPRL without paying a third party. There is a freely available PPRL package in the statistical software platform R ; howeverThere are multiple potential sources of bias in record linkage. Differential record linkage refers to the fact that data of lower quality typically result in less probability of linking data representing the same entity. If lower quality data were randomly distributed among the records being matched, this might not be a contentious issue; unfortunately, through studies like the ones completed by Lariscy ,39, we kAnother source of bias is introduced through pre-processing protocols and data standardization systems for record linkage; the majority of these have been developed in an English-centric linguistic cultural context. For this reason, they are not as useful in other language contexts. In their 2016 paper, del Pilar and Bail\u00f3n address this issue by creating phonetic encoding algorithms specific to Spanish names to allow lower quality Spanish name data to be matched . SimilarGender also plays a role. Data linkage often assumes gender is fixed but that is not the case for those who are transgender or non-binary. In addition, algorithms that match on last name put women at greater risk of not successfully matching because they often change their surnames at marriage.Record linkage processes that occur in multicultural contexts might encounter biases due to name standardization algorithms that are not appropriate for many of the naming conventions of the different subpopulations represented in the data. Practitioners must develop tools that account for naming conventions of those subpopulations; rules based on Hispanic and Asian naming conventions are the most likely to be needed.We have addressed multiple topics related to record linkage, including the three types of underlying models for the probabilistic aspects of record linkage, the basic methodologies by which these models are employed, practical considerations such as data standardization and data sharing issues, and potential sources of bias such as differential rates of linkage across minority populations.The success of a record linkage project, however, is dependent on the organizational structures that allow it to continue. In large data-driven organizations such as the U.S. Census Bureau, data governance structures are formalized through understood protocols for data sharing and a specific review committee for record linkage requests. Specific funding towards a team of record linkage experts is an annual line item in the organizational budget and may be a consideration as 9/11 research needs evolve.Record linkage cannot be accomplished without the appropriate in-house expertise. Expertise in information technology is not adequate; at minimum, an organization must have access to a data scientist or statistician that understands the probability models on which record linkage methods are built and can use statistical software packages to complete record linkage tasks. This individual must have enough technical expertise to read and understand the scientific literature related to record linkage as new methods are developed. Finally, they must be able to adapt the techniques being employed to complete the record linkage as data with different levels of quality are included in the process.In short, while record linkage can create avenues of research that might not otherwise be available, it is not an easy process to complete. Even the most expensive record linkage software requires a skilled practitioner and detail-oriented documentation of the steps taken for a specific record linkage project. Without institutional commitment to providing adequate resources and informed oversight for record linkage efforts, attempts to link data are likely to end in an unsatisfactory result."}
+{"text": "The Western Australia Data Linkage System (WADLS) is maintained and operated by the WA Data Linkage Branch (DLB) at the Western Australian Department of Health. DLB has pioneered a number of data linkage innovations, including the facilitation of genealogical research via the Family Connections system and streamlined data delivery via the Custodian Administered Research Extract Server. DLB\u2019s latest innovation is a new data linkage system called \u201cDLS3\u201d, which improves DLB\u2019s capability and capacity to handle the increasing volume and complexity of its routine operations. DLS3 was built entirely in-house and customised to meet the specific challenges that DLB has encountered throughout over twenty years of experience with a wide variety of linkages. This article describes the development and rollout of DLS3, including its design, architecture, benefits and limitations. Western Australia (WA) covers approximately a third (2.5M square kilometres) of Australia\u2019s total landmass and 10 per cent (2.6M people) of its population , the WADLS is the longest operating Australian linkage system and it is underpinned by strong privacy and security principles ,7. InitiDLB releases tailored, linked data extracts directly to Data Applicants, with the personal identifiers removed, for requests that are granted the applicable ethical, research and data governance approvals . The linWA lacks a population-wide personal unique identifier  that can be used for linkage. To overcome this challenge, DLB uses probabilistic linkage processes, where groups of records are compared using complex field matching algorithms. These algorithms compare a user-defined list of common fields, such as given name, surname, date of birth and address  and provide a score indicating their similarity. Most algorithms also incorporate user-defined parameters and frequency review to refine the score. These field-wise scores are combined to provide an overall match score for each pair of records under consideration, indicating the likelihood that the two records belong to the same individual .Each dataset linked by DLB has its own formatting, quality, completeness and other idiosyncrasies, which depend upon the context, standards and capabilities at the point of collection. Linkage is often best achieved via a customised approach to harness the strengths and minimise the quality issues associated with each dataset. Over time, these customised processes are maintained and enhanced to ensure the stability and suitability of ongoing linkage updates as the source dataset evolves.Historically, DLB\u2019s legacy linkage system used proprietary file-based linkage software (referred to as \u201cFLS\u201d hereafter) to perform the linkage process. The FLS was identified in isolation because its features were key to the legacy system and its limitations were difficult or impossible to avoid. The legacy system  grew organically to address the requirements of DLB.Linkage using this FLS involved the following steps, each customised by the user:Two datasets were chosen for comparisonblocking)Pairs of records meeting user-defined criteria were sub-setted (known as matching)These record pairs were evaluated using user-defined matching algorithms (weights assignment)Based on the outcomes of the matching algorithms, weights were assigned representing the likelihood that the two records belonged to the same person (threshold setting)Records were categorised as \u2018match\u2019, \u2018non-match\u2019 or \u2018potential match\u2019 based on user-defined weight thresholds  during the development and implementation stages. DLB\u2019s subject-matter experts collectively possess almost fifty years of data linkage experience (not including staff who have since moved on) and their detailed understanding of the idiosyncrasies and challenges that DLS3 would likely encounter ensured development of a system that was fit for purposeDLB designed DLS3 to comprise a number of fully integrated and function-specific components  that drive the key stages of the linkage process .DLS3 Services call upon a number of related Modules to perform functions that may span multiple Services. The two most important Modules are called \u2018Checking\u2019 and \u2018Storage\u2019, which are called by the \u2018Linkage\u2019 and \u2018Clerical Review\u2019 Services. The Checking Module runs a variety of quality assurance checks and flags suspicious potential links for further consideration. This includes threshold checks for potential matches that are assigned an \u2018intermediate weight\u2019 and require clerical review. The Storage Module stores the links in the WADLS links database, where they are managed and extracted as required.DLB is currently operating both DLS3 and some residual parts of the legacy data linkage system, with the latter undergoing phased retirement. Prior to each retirement phase, both systems run in parallel to confirm DLS3 is working correctly and to manage any data migration risks. The FLS has been retired and DLS3 is being used exclusively for cleaning, importation and data matching. DLB is currently finalising the clerical review, links storage and extraction functions; these are the final steps before all legacy tools can be retired.DLS3 has already yielded a number of key benefits. It creates considerable time and cost savings by running linkage strategies concurrently and delaying the clerical review stage until all potential links can be considered collectively. User input is reduced because successive stages are now integrated rather than requiring manual intervention. For example, the output of DLS3\u2019s cleaning and importation service flows directly into its linkage preparation service.Linkage quality is of considerable interest to data linkage centres and users of linked data ,13. It iDLB\u2019s approach to linkage quality focuses on understanding the nature of linkage errors, implementation of robust risk-mitigation strategies and a culture of continuous improvement . FollowiNumerous matching features have been implemented in DLS3 that were previously limited or impossible in the legacy FLS environment .DLB anticipates that DLS3 will continue delivering benefits as remaining modules are fully implemented and legacy tools are retired. DLS3 has been implemented using a phased replacement of the legacy system. The original design included some enhancements to the row-level unique identifiers  used to identify records submitted for linkage. Due to the requirement of maintaining compatibility with DLB\u2019s legacy FLS system (at least until it is completely replaced), it has been impossible to take advantage of enhancements in this area. DLB is confident solutions will be found to other limitations that may become apparent over time.DLS3, DLB has created an adaptable and sophisticated tool, informed by the DLB\u2019s extensive experience and understanding of all facets of data linkage. It delivers fast, high quality, scalable and cost-effective linkages to meet the current and emerging challenges of data linkage. As the demand for wide-reaching linkage infrastructure continues to grow, it strengthens DLB\u2019s position as a leader in data linkage innovation.This article has briefly described the development and rollout of its new data linkage system, DLS3, which was built entirely using the specialist skills and experience of the Data Linkage Branch in Western Australia. With This publication did not require ethical approval.TE and JA supervised the project. JT was lead developer on the project, supported by SH. TE drafted the original manuscript with input and critical feedback provided by JT, SH and JA."}
+{"text": "The Centre for Data Linkage (CDL) was established at Curtin University, Western Australia, to develop infrastructure to enable cross-jurisdictional record linkage in Australia. The CDL\u2019s operating model makes use of the \u2018separation principle\u2019, with content data typically provided to researchers directly by the data custodian; jurisdictional linkage where available are used within the linkage process. Along with conducting record linkage, the team has also invested in establishing a research programme in record linkage methodology and in developing modern record linkage software which can handle the size and complexity of today\u2019s workloads. The Centre has been instrumental in the development of practical methods for privacy-preserving record linkage, with this methodology now regularly used for real-world linkages. While the promise of a nation-wide linkage system in Australia has yet to be met, distributed models provide a potential solution. The Centre for Data Linkage (CDL) is a component of the national Population Health Research Network (PHRN) and was established in 2009 within Curtin University as part of the National Collaborative Research Infrastructure Strategy funded by the Australian Government. The focus was to develop and implement secure, state-of-the-art infrastructure to enable large scale and cross-sectoral data linkage for research in Australia (1). The Centre has continued to receive funding via the PHRN, supplemented with competitive research and consultancy funding.Australia\u2019s federated government system means that various datasets are gathered at different tiers of administration that operate independently from one another. Linkage units exist in many states across Australia, primarily linking local, state-based health datasets (secondary and tertiary care). The PHRN initiative aims to provide cross-jurisdictional linkage, i.e. linkage of data across nine different legal jurisdictions . The full potential of these data resources can only be realised by linking data between these jurisdictions to ensure complete population coverage.A federated government coupled with a complex authorizing landscape has made a move to an enduring national linkage map difficult to achieve in Australia in the first instance. The platform established by the CDL has been used for a number of large research projects requiring linkage of different state and national datasets, including those requiring the establishment of a long-term linkage map. It is anticipated that this project-based approach will evolve and mature into an ongoing enduring national linkage model.From the outset, the CDL focused on three key areas: i) carrying out record linkage for clients, ii) conducting research into record linkage methods, and iii) developing software and tools necessary for high quality record linkage. Each area is directly informed by and related to the others. Research questions are borne out of issues encountered during routine record linkage. Newly developed methodology published by the team is then implemented into the linkage software. The linkage software is then used operationally for routine linkage for clients. The CDL is very aware of the sensitivities associated with maintaining databases containing identifiable information, utilising the \u2018separation principle\u2019 to link data from multiple jurisdictions . This apUnder the model adopted by the CDL, the linkage team are supplied with demographic data from participating datasets. This information is used to generate a Linkage Map. The Linkage Map is central to the CDL linkage model and consists of 'pointers' to records in the contributing data collections; the Map identifies the same person within and between datasets. Although the creation of the Map requires access to individually identifiable demographic information, these data items are stored separately from the Linkage Map. For privacy reasons, the linkers do not have access to the content data - this remains under the full control of the relevant dataset's custodian. Content data is released by the original data custodian (not the CDL) to the researcher for approved research projects, either directly to the researcher or to a secure analytical research environment (i.e. a safe-haven).The development of a secure linkage infrastructure requires both secure linkage software to carry out the necessary functions, and a secure environment (including appropriate information governance) to host this software. In terms of an operating environment, the objectives of the CDL infrastructure are:To provide secure linkage systems and services to internal and external stakeholders, with adequate levels of availability;To provide an environment that is auditable and certifiable against the PHRN Information Governance Framework, and other industry standards;To provide a cost effective, low maintenance environment that can draw on shared services within a provider\u2019s managed environment .The infrastructure is designed to provide a platform for undertaking large linkage projects while meeting the requirement to provide a secure and controlled environment for working with sensitive data , 4.The infrastructure supports the following core functions:Provision of demographic information from data custodians to the CDL;Linkage of this data to create project specific linkage keys;Supply of keys back to the various data custodians in each jurisdiction;To future proof the secure data linkage facility, it was important to create scalable infrastructure that could accommodate both the increasing demand for linked data and the increasing volume and complexity of datasets to be linked.A challenge for the CDL was to translate information governance frameworks and standards into a set of rules, concepts and designs that could be implemented as a cost-effective technical solution . The modThe design process involved developing a set of guidance infrastructure architectures or \u2018Design and Implementation Guidelines\u2019 for a secure research computing environment to host the CDL infrastructure , 8. The The CDL also obtained human research ethics committee (HREC) approval to establish the core operations of the linkage system, that is, the capacity to receive demographic data to generate a Linkage Map and to release linkage keys to providers. In addition to this approval, other state-based HREC approvals were obtained to allow construction of a Linkage Map for specific projects so that state and territory data providers could release their demographic data to the CDL for linkage. Researchers wishing to use the linked data created through this process required further, additional ethical approvals.There are limited legislative barriers to record linkage in Australia. The overarching federal privacy legislation provides a mechanism for the release of data for health research with and without individual consent. Similar laws, with similar exemptions for research under condition, exist at the state level. However, there is also no obligation on data custodians to provide their data for linkage. A culture of risk aversion in government, particularly at the Commonwealth level, has existed in the past which has reduced access to government datasets for research . The proAn early environmental scan and software evaluation by the CDL suggested few if any available linkage systems could provide a robust enterprise-grade platform which could easily scale to the data sizes anticipated for linkage across Australia, and which could manage the complexity of ongoing enduring linkage for multiple research projects . As suchAt a basic level, the linkage software has been designed to undertake linkage across event-level datasets, based on fully configurable matching of demographic information using the standard Fellegi-Sunter probabilistic linkage approach. The software also performs data extraction to satisfy requests from jurisdictional data providers to supply encrypted project-specific identifiers for release to researchers .To ensure that the linkage system was \u2018fit for purpose\u2019, the software was developed to include linkage and management capabilities. The system was designed using a component approach which focused on system integration, interoperability and expansion capabilities to ensure future flexibility. The \u2018baseline\u2019 development criteria included the following requirements:Security was implemented in a role-based access control model. This method regulates access to the system based on the role of individual users. The system roles and their implementation were defined as part of the system architecture and are managed through standard operating procedures. User roles can be created, changed or withdrawn as the needs of the service change, without individually updating the privileges for every user. Administrative and monitoring functionality allows operators to manage linkage projects and data from different data providers. Linkage, quality assurance and data extraction processes are monitored through the user interface, and built-in audit trails track operations on all processes, data (records or transactions) and data custodian details.The system manages a range of projects from a simple \u2018one-off\u2019 project with a short life span through to enduring longitudinal datasets that are constructed and updated through the ongoing linkage of records over time. The ability to manage both types of projects ensures flexibility and versatility in linkage operations.All system components  have the capacity to handle large data volumes. The linkage projects carried out by the CDL regularly involve tens of millions of records and billions of matching transactions, and ensuring the software can manage these sizes is crucial.The system is designed to manage multiple projects without performance overheads. The user interface provides operators with the ability to create and manage projects (linkage and extraction), custodians and data. The system can manage multiple large projects without substantial or complex operator involvement. The system is also designed to manage the process of data extraction for specific research projects. Project extractions are controlled by the system and produce a project-specific linkage map for the researcher. The project keys generated in the Map are only relevant to an individual linkage project (even if the dataset appears in another linkage). This ensures no cross-over between projects or project teams.Unlike most linkage systems, the software has been designed to manage changes in data and links over time. The software can automatically process amended and deleted records as well as deal with new records. Unlike other designs, the system stores and processes all linkage transactions at the matching pair level. This allows the system to automatically detect and manage change to the linkage map as data is added (including new records and amendments to records). It also supports \u2018any point in time\u2019 referencing at the group or map level allowing operators to recreate the linkage structure for any records at any (previous) point in time.The linkage software uses a layered architecture built on the .NET framework and uses Microsoft SQL Server for data storage. The system is managed through a web interface which allows role-based access to linkage functions and features. Linkage tasks are run on a single processing node that allows compute resources to be vertically scaled as required. The linkage tools and services are installed within a specifically designed secure environment (ISO27001 certified) hosted at Curtin University.The development of software also provided synergies with one of the other core functions of the CDL; to conduct research into record linkage methods. The development of our own software has enabled improvements in record linkage methods to be directly translated into software and then used in a real-world production environment. A key example of this is the development and use of techniques for privacy-preserving record linkage.Privacy-preserving record linkage (PPRL) has been a major development in the record linkage arena internationally . These tTechniques for privacy-preserving linkage have been incorporated into CDL\u2019s linkage software and a number of pilot and/or proof of concept projects have tested and evaluated the capabilities of privacy-preserving linkage in a real-world context , 17.For these projects, the CDL has used the Bloom filter approach for privacy preserving record linkage. The process starts by encoding personally identifying information into Bloom filters (binary vectors). The method provides strong protection as the encoding process is irreversible and the encoded output is distorted to the extent that accidental recognition of an individual is impossible. The Bloom Filter encoding means that the matching can be carried out within the context of a traditional Fellegi-Sunter probabilistic linkage -16. ThesThe development of probabilistic linkage techniques that do not require the release of personal information but protect privacy through data encoding represents a significant breakthrough in data linkage methods. The application of these methods within operational linkage environments not only strengthens security but increases linked research opportunities as previously inaccessible datasets now become available for research. A recent key project for CDL has been the development of the Social Investment Data Resource (SIDR). The concept of the SIDR is to create an integrated and accessible resource containing de-identified, unit-level data about a cohort of young people who come into contact with the Western Australian criminal justice system. The West Australian (WA) criminal justice sector  has no routine way of linking up core datasets with and across organisations. Therefore, it is not possible to investigate the impact of any intervention program on any targeted group.Using the existing CDL architecture, the SIDR has been designed to be securely and strictly managed, providing secure access to authorised users through several mechanisms, including direct access and statistical analysis tools.The SIDR contains population-level data sourced from WA birth registrations, school enrolment information and other datasets from police, justice, child protection, housing and education. The linkage model requires the release of relevant name-identified data from key agencies  to CDL. The CDL matches these records, to determine the same individual across multiple datasets.To address legislative and privacy concerns, the SIDR linkage model has been designed to operate using a combination of clear text linkage and encoded data for privacy-preserving record linkage i.e. some of these linkages are conducted using personal identifiers, while privacy-preserving record linkage techniques are used in other circumstances.Significantly, this project also incorporates a bifurcated data linkage model, where data linkage responsibilities are shared between the CDL and the WA Department of Health Data Linkage Branch (DLB). The CDL has primary responsibility for linkage of non-health datasets and for enabling the build of the SIDR, while the DLB continues to undertake the linkage of all health-related data and to service research through the provision of value-added services (like family connections) , 19.The CDL has played a key role in Australian linkage infrastructure, and its linkage expertise is recognised nationally and internationally. Research into record linkage methods has resulted in over twenty peer-reviewed publications. The CDL has established a secure and efficient data linkage environment and developed innovative linkage software, utilized by CDL and a number of other organisations. The CDL initially focussed on large cross-jurisdictional and cross-sectoral linkage projects, which often involved tens of millions of records for a range of clients and research projects. More recently, the CDL has provided linkage services for health  and non-health  projects. The CDL team is relatively small with 8-10 people included in the various linkage activities.The CDL is also currently pioneering a distributed data linkage model with the DLB. Given the fragmented nature of health care service delivery in Australia, the impact of health funding, health service planning and health outcomes can only be achieved through efficient linkage/integration infrastructure. Experiences from other countries demonstrate the need to harness and harmonise the power and experience of linkage services and systems to improve the efficiency and quality within overall data linkage infrastructure. Given the numerous stakeholders and linkage services operating in Australia, the most promising path may not be to develop a single national linkage map managed by one organisation but to develop distributed models which utilize all available resources to achieve the same end goal.A challenge in Australia is to realise the potential of the infrastructure currently available across government and university sectors through compatible, sustainable and effective models which can maximise the capacity across all these systems. The methods and techniques around data linkage in Australia are well established, and new developments (exploiting advances in technology) have the potential to improve access, timeliness and efficiency.Challenges to realising the potential of data linkage in Australia remain. Increasing demand for data linkage services has put significant pressure on infrastructure to deliver in a timely fashion. The linkage community needs to focus on mechanisms which will ensure the timely delivery of data, particularly as the number, size and complexity of linkage research projects increase. Continuous improvement will also be essential. The infrastructure must continue to identify and implement new technologies to improve the efficiency of data linkage in Australia. Potential exists to improve the workflow and data flow between custodians, linkage units and researchers. Australia needs to ensure that data linkage infrastructure and technologies are interoperable and responsive to environmental changes around legislation, information technology, security and privacy.Although there has been considerable work to address the technical and methodological challenges associated with large-scale data linkage, the establishment of an enduring national linkage map has remained elusive. With linkage facilities in place across Australia, there are opportunities to leverage expertise and best practices. Development of interoperable systems would increase capacity and speed of data linkage processes. Partnerships between data linkage activities can drive innovation and efficiency within linkage systems.One solution may be through a distributed data linkage model which utilises existing data linkage services available through multiple linkage units to facilitate the access and linkage of multiple datasets. A distributed linkage approach would enable participating linkage units to continue maintaining their existing master linkage key systems, as well as operating in an integrated way to facilitate enduring linkages. It would utilise existing skills and experience, standards, governance arrangements and infrastructure in participating linkage nodes. The model would both standardise existing linkage approaches and provide a scalable platform to capture capability across multiple linkage nodes.A basic approach would involve the sharing across all linkage units of a common \u2018spine\u2019 file; a dataset or amalgamation of datasets which contains most of the population. Each linkage unit could then link their jurisdictions datasets to this spine independently. This would effectively result in a national linkage map, albeit one not held by any single party see . The modThe ultimate aim of any organisation dedicated to record linkage must be to improve access to and quality of linked data. Carrying out this mission will provide the community with a greater number of research outputs with greater confidence in their validity. The CDL has addressed this challenge in a number of ways. It has worked to carry out cross-jurisdictional and cross-sectoral linkage projects that would not have been possible otherwise. It has developed a research program dedicated to investigating best practice methods, and developing new methodologies for record linkage, with a strong focus on enhancing linkage quality and enabling data access. It has developed linkage software which meets the challenges of modern linkage units, which has been used not just to improve CDL\u2019s processes, but by outside linkage organisations as well.The research enabled by record linkage continues to improve lives through changes in health and social policy and clinical practice . The invNo ethical approval was sought for this publication."}
+{"text": "Penicillium italicum (blue mold) is one of citrus pathogens causing undesirable citrus fruit decay even at strictly-controlled low temperatures (<\u200910\u2009\u00b0C) during shipping and storage. P. italicum isolates with considerably high resistance to sterol demethylation inhibitor (DMI) fungicides have emerged; however, mechanism(s) underlying such DMI-resistance remains unclear. In contrast to available elucidation on anti-DMI mechanism for P. digitatum (green mold), how P. italicum DMI-resistance develops has not yet been clarified.P. italicum strains (highly resistant (Pi-R) versus highly sensitive (Pi-S) to DMI fungicides), with and without prochloraz treatment, to identify prochloraz-responsive genes facilitating DMI-resistance. After 6\u2009h prochloraz-treatment, comparative transcriptome profiling showed more differentially expressed genes (DEGs) in Pi-R than Pi-S. Functional enrichments identified 15 DEGs in the prochloraz-induced Pi-R transcriptome, simultaneously up-regulated in P. italicum resistance. These included ATP-binding cassette (ABC) transporter-encoding genes, major facilitator superfamily (MFS) transporter-encoding genes, ergosterol (ERG) anabolism component genes ERG2, ERG6 and EGR11 (CYP51A), mitogen-activated protein kinase (MAPK) signaling-inducer genes Mkk1 and Hog1, and Ca2+/calmodulin-dependent kinase (CaMK) signaling-inducer genes CaMK1 and CaMK2. Fragments Per Kilobase per Million mapped reads (FPKM) analysis of Pi-R transcrtiptome showed that prochloraz induced mRNA increase of additional 4 unigenes, including the other two ERG11 isoforms CYP51B and CYP51C and the remaining kinase-encoding genes  required for Slt2-MAPK signaling. The expression patterns of all the 19 prochloraz-responsive genes, obtained in our RNA-seq data sets, have been validated by quantitative real-time PCR (qRT-PCR). These lines of evidence in together draw a general portrait of anti-DMI mechanisms for P. italicum species. Intriguingly, some strategies adopted by the present Pi-R were not observed in the previously documented prochloraz-resistant P. digitatum transcrtiptomes. These included simultaneous induction of all major EGR11 isoforms (CYP51A/B/C), over-expression of ERG2 and ERG6 to modulate ergosterol anabolism, and concurrent mobilization of Slt2-MAPK and CaMK signaling processes to overcome fungicide-induced stresses.The present study prepared RNA-sequencing (RNA-seq) libraries for two P. italicum DMI-resistance mechanisms and revealed some diversity in anti-DMI strategies between P. italicum and P. digitatum species, contributing to our knowledge on P. italicum DMI-resistance mechanisms.The present findings provided transcriptomic evidence on  Penicillium digitatum (green mold) and P. italicum (blue mold) are well known as the predominant citrus pathogens causing postharvest diseases during fruits storing and transportation. The resulted economic losses are so great that aroused enormous attentions all over the world [Penicillium molds in the past decade, especially for P. digitatum isolates with high DMI-resistance [P. digitatum [P. italicum resistance induced by the DMI fungicides. It would be theoretically important to address molecular background of P. italicum isolates causing their DMI resistance.he world . The stehe world \u20136. Howevsistance , 7, consigitatum \u201313. HoweERG11-encoding proteins) can alter drug-target interactions and increase DMI-resistance for various fungal pathogens, as reported in the model yeast Saccharomyces cerevisiae [Candida albicans [Aspergillus fumigatus [Mycosphaerella graminicola [Monilinia fructicola [P. digitatum [CYP51s, recently, other genes encoding fungal ergosterol biosynthesis-related enzymes have been proposed to be potential targets, including ERG2 (encoding C\u2212\u20098 sterol isomerase) [ERG6 (encoding C\u2212\u200924 sterol methyltransferase) [ERG2 and ERG6 to cycloheximide resistance for S. cerevisiae has also been genetically emphasized [The mechanism of fungal DMI-resistance involves strategies targeting ergosterol-biosynthesis enzymes. The site mutations in CYP51s  \u201336 and Esferase) \u201340. The phasized .C. albicans [Mycosphaerella graminicola [P. digitatum strains [Tricholoma vaccinum [P. digitatum [Fungal DMI-resistance has also been ascribed to specific drug-transporter proteins that can reduce fungicide accumulation in fungal cells, including ATP-binding cassette (ABC) transporter family proteins, major facilitator superfamily (MFS) proteins, and multidrug and toxic compound extrusion (MATE) family proteins. ABC transporters have been functionally characterized in many fungal pathogens including green mold and verified to be up-regulated in their fungicide resistance \u201354. MFS albicans , MgMfsl minicola , and PdM strains , 58. Unl strains \u201361. To dvaccinum  and the igitatum .2+) signaling. The mitogen-activated protein (MAP) kinase signaling pathways, ubiquitously found in eukaryotes (from yeasts to various pathogenic fungi), comprise a set of cascaded protein kinases, MAP kinase kinase kinase (MAPKKK), MAP kinase kinase (MAPKK) and MAP kinase (MAPK), acting in series to modulate target protein activities [Alternaria alternata [P. digitatum [P. digitatum resistance to the fungicides iprodione and fludioxonil [Botrytis cinerea and functionally required for iprodione resistance [Fusarium graminearum resistance to fludioxonil [2+ signaling via Ca2+/calmodulin (CaM)-dependent kinases (CaMKs), usually linked with particular MAPK pathway(s), extensively participates in fungal responses to environmental stresses. The over-expression of CaMK2  in the yeast S. cerevisiae facilitated its resistance to some azole-fungicides  [Fungicide resistance is further associated with particular protein kinase signaling and calcium  . Recent onazole) \u201380.P. digitatum [A. fumigatus [Cercospora beticola [Fusarium culmorum [Candida glabrata [P. digitatum resistance to DMI-fungicide prochloraz through RNA-seq analysis [P. italicum resistance to such fungicides are poorly understood. Now we have isolated two P. italicum strains exhibiting desirably contrasting response to common DMI fungicides including prochloraz, i.e. Pi-R (highly resistant to prochloraz with EC50\u2009=\u200930.2\u2009\u00b1\u20091.5\u2009mg\u00b7L\u2212\u20091) versus Pi-S (highly sensitive to prochloraz with EC50\u2009=\u20090.007\u2009\u00b1\u20090.002\u2009mg\u00b7L\u2212\u20091). The purpose of this work was to compare transcriptomic profiles between these two P. italicum strains with and without prochloraz treatment, to identify differentially expressed genes (DEGs) involved in the azole-class fungicide resistance, and to provide theoretical cues to explain P. italicum anti-azole mechanism.RNA sequencing (RNA-seq) technology has become a powerful tool to profile transcriptomic response to reveal azole-resistance mechanism for some pathogenic fungi including prochloraz-resistant igitatum , voriconumigatus , tetracobeticola , tebuconculmorum , and fluglabrata . Our earanalysis . NeverthIn the present study, Pi-R and Pi-S were treated with or without DMI-fungicide prochloraz to prepare four RNA-seq samples, i.e., Pi-R-I, Pi-R-NI, Pi-S-I and Pi-S-NI. After Illumina sequencing, the four transcriptomic libraries contained 61,610,574, 70,012,472, 61,976,398 and 67,336,730 raw reads, respectively. By removing adaptor sequences and undesirable reads , 58,744,798, 66,490,626, 59,134,840 and 64,262,170 clean reads were generated from the four libraries with Q30\u2009>\u200990%, suggesting high quality for the present sequencing results. These clean reads were predominantly distributed in exon and intergenic regions  analysis was performed to visualize DEG profiles between Pi-R-I, Pi-R-NI, Pi-S-I and Pi-S-NI libraries Fig. . Pi-R anq-value 0.005  and an absolute value of log2(fold change)\u2009\u2265\u20091 were set as cut-off standard to identify DEGs between different libraries, including a) Pi-R-I vs Pi-R-NI, b) Pi-S-I vs Pi-S-NI, c) Pi-R-I vs Pi-S-I, and d) Pi-R-NI vs Pi-S-NI  958 DEGs between Pi-R-NI and Pi-S-NI (422 up-regulated and 536 down-regulated)  , including biological process (BP), cellular component (CC), and molecular function (MF). The number of total GO terms and its distribution in the three categories for each comparison are listed in Table q value \u22640.05), and the top 5 terms significantly enriched were oxidoreductase activity , oxidation-reduction process , single-organism metabolic process , catalytic activity , and single-organism process . In the comparison Pi-R-I vs Pi-S-I , and the top 5 terms significantly enriched were oxidoreductase activity , oxidation-reduction process , hydrolase activity , hydrolase activity , and transmembrane transport . Figure The DEGs were classified into three GO categories by the Blast2GO ), drug-target P450 gene ), steroid biosynthesis-related genes ) and MAPK/calcium signaling-related genes  and GO:0005509 ) were up-regulated in prochloraz-treated Pi-R, as compared to drug-untreated Pi-R or to drug-treated Pi-S. In contrast, most of these prochloraz-responsive DEGs, except for CYP51A, were down-regulated or unchanged in prochloraz-treated Pi-S, comparing to untreated Pi-S. GO enrichment also indicated lower transcript abundance of some of these prochloraz-responsive DEGs in Pi-R when compared with Pi-S under fungicide-free conditions  of the prochloraz-responsive DEGs mentioned above was summarized in Additional file Importantly, the up-regulated DEGs mapped to specific GO terms included a number of typical genes related to fungicide resistance. As summarized in Table q value\u2009=\u20090.013) and MAPK signaling pathway  (Table CYP51A (PITC_083360) in the comparisons Pi-R (I/NI) and Pi-S (I/NI), ERG2 (PITC_020620) in the comparisons Pi-R (I/NI) and Pi-S (I/NI), and ERG6 (PITC_014340) in the comparisons Pi-R (I/NI) and I (Pi-R/Pi-S); while the latter pathway included 1) up-regulated DEGs  in Pi-R-involved comparisons, i.e., Pi-R (I/NI) and I (Pi-R/Pi-S) and 2) down-regulated DEG  only in comparison Pi-S (I/NI). All the KEGG-enriched DEGs, as components of metabolic and/or signal-transduction pathway(s), were well coincident with the results of GO enrichment. In other words, the present GO-enriched DEGs, if involved in specific biological pathway(s), were exclusively KEGG-included, and certainly, pathway-irrelevant genes, e.g., drug-pump genes and drug-target genes, were KEGG-excluded, without exception.Further, KEGG enrichment was applied to identify pathways associating the prochloraz-responsive DEGs with resistance mechanisms. In the present four comparisons, KEGG analysis enriched prochloraz-responsive DEGs into only two pathways, i.e., steroid biosynthesis  Table : the forP. italicum response to DMI fungicides, included 1) drug-pump genes: ABC1 (PITC_032590), ABC2 (PITC_006400), MFS1 (PITC_098100), MFS2 (PITC_012240), MFS3 (PITC_056240), and MFS4 (PITC_091150); 2) ergosterol biosynthesis-related genes: CYP51A (PITC_083360), ERG2 (PITC_027000), and ERG6 (PITC_014340); 3) MAPK signaling-related genes: Mkk1 (PITC_088710) and Hog1 (PITC_062470); 4) Ca2+ signal transducer-related genes: CaMK1 (PITC_087700), CaMK2 (PITC_025800), EF-hand1 (PITC_033750), and EF-hand2 (PITC_036760). Additionally, FPKM-based unigene expression quantification combined with local Blast-based annotation revealed differential expression patterns for particular prochloraz-responsive unigenes in the present 4 comparisons, including CYP51B (PITC_064600), CYP51C (PITC_028940), Bck1 (PITC_061930) and Slt2 (PITC_008290)  and 2) cascaded association of Bck1 (encoding MAPKKK), Mkk1 (encoding MAPKK) and Slt2 (encoding MAPK) in Slt2-MAPK pathway, we also performed qRT-PCR validation for the 4 prochloraz-responsive unigenes that were not included in the present DEG list for comparison  erABC1 was strikingly increased in both Pi-R (I/NI) and I (Pi-R/Pi-S), by nearly 500- and 800-folds, respectively, while remarkably decreased in both Pi-S (I/NI) and NI (Pi-R/Pi-S); the similar (but not so strikingly) changing pattern was observed for the rest drug-pump genes including MFS1   was constructed from its parental strain Pi-R, exhibiting obviously lower prochloraz-resistance  as compared to the Pi-R wild-type  Fig. b, ergostPenicillium decay, but undesirably, a considerable number of resistant isolates including P. digitatum and P. italicum strains have developed [P. digitatum species by transcriptomic analysis [P. italicum species is still not clear, might due to rare opportunity to find highly DMI-resistant P. italicum strain(s). The EC50 values of P. italicum isolates towards DMI-fungicide(s) , published to date, were\u2009\u2264\u20090.92\u2009\u00b1\u20090.09\u2009mg\u00b7L\u2212\u20091, no more than moderate resistance level [P. italicum species [P. italicum strain (Pi-R) with extremely high resistance to some common DMI-fungicides including prochloraz  was also observed in the prochloraz-treated Pi-R rather than Pi-S  in Pi-R decreased the fungal resistance to prochloraz confirmed the involvement of particular drug-efflux pump in P. italicum anti-DMI mechanism  with close evolutionary association did differ in selecting drug-transporters to support their DMI resistance, and the mechanism(s) underlying need further research.Fungal resistance to azole-fungicides including a number of DMI-fungicides has been usually ascribed to over-expression of specific drug-efflux pumps such as ABC and MFS transporters , 58, 88.igitatum . The sim-S Table a, indicacriptome , as showMATEs, none was found to be differentially expressed in the prochloraz-induced Pi-R and Pi-S transcriptomes (Table MATE1\u20133) in P. digitatum resistance to prochloraz. The orthologous gene of PdMATE3 has been identified in the present Pi-R and Pi-S genomes, however, no mRNA transcripts of this gene was detected in the prochloraz-treated P. italicum strains. This might suggest transcriptional irrelevance of MATE transporter with P. italicum resistance to prochloraz. Unlike ABC and MFS transporters, MATE transporters are more associated with bacterial drug-resistance [MATE isogenes to DMI fungicides was only documented in prochloraz-resistant P. digitatum transcriptomes [P. italicum transcriptomes. Thus a potential debate on real function of MATE family member(s) in citrus Penicillium pathogens\u2019 resistance to DMI-fungicides would be an interesting study topic.More interestingly, regarding another class of drug-pump-encoding genes, i.e., es Table . In contsistance \u201362. To driptomes . But sucERG11s, the P450-dependent sterol 14\u03b1-demethylase (CYP51)-encoding genes , has been accepted as a primary strategy to develop fungal DMI-resistance [CYP51A in Pi-R (~\u20099-fold) was more than that in Pi-S (~\u20094.5-fold)  was observed only in prochloraz-induced Pi-R, rather than Pi-S  Fig. b, sugges-S Table b. This i targets , were upand ERG6 , 81, corScMkk1 and ScMkk2, have been characterized in the model yeast S. cerevisiae to function in CWI signaling-mediated fungicide resistance [Mkk1 (PITC_088710), the homolog of ScMkk1 in P. italicum species, was GO- and KEGG-enriched as up-regulated DEG in the prochloraz-treated Pi-R  and Slt2 (PITC_008290), the MAPKKK- and MAPK-encoding genes located upstream and downstream of Mkk1, respectively, were both up-regulated in prochloraz-treated Pi-R  and CaMK2 (PITC_025800), both up-regulated only in prochloraz-treated Pi-R  up-regulation of specific isogenes encoding ABC and MFS transporters, 2) simultaneous induction of all major EGR11 isoforms including CYP51A/B/C, 3) over-expression of ERG2 and ERG6 to modulate ergosterol anabolism, and 4) concurrent mobilization of Slt2-MAPK and CaMK signaling processes to adapt azole-induced stresses. Some differences in the choice of anti-DMI strategy between P. italicum and P. digitatum species were also discussed.In conclusion, the present work for the first time provided transcriptomic analysis of prochloraz-responsive gene expression profiles for two P. italicum strains Pi-R and Pi-S, used in this study, were isolated from rotten citrus fruits in local packinghouses (Yunnan Province) and orchards (Hainan Province), respectively. Pi-R exhibits dramatically high prochloraz-resistance (EC50\u2009=\u200930.2\u2009\u00b1\u20091.5\u2009mg\u00b7L\u2212\u20091), while Pi-S is prochloraz-sensitive (EC50\u2009=\u20090.007\u2009\u00b1\u20090.002\u2009mg\u00b7L\u2212\u20091)  as previously described [6 spores) was incubated with 200\u2009mL potato dextrose broth (PDB) medium for 2\u2009days at 28\u2009\u00b0C and 180\u2009rpm shaking, and the resulting mycelia were treated with or without prochloraz. In detail, prochloraz was added to the PDB medium at final concentration that was in agreement with EC50 value for each P. italicum strain . Prochloraz was pre-dissolved in 100\u2009\u03bcL DMSO, and the same volume of DMSO was added to 200\u2009mL PDB medium to prepare control samples. The prochloraz-induced and no-induced (control) samples were cultured at the same conditions (at 28\u2009\u00b0C and 180\u2009rpm shaking) for 6\u2009h before RNA extraction. The present study collected 4 samples in total for the following RNA manipulations, i.e. prochloraz-induced and no-induced Pi-R , and prochloraz-induced and no-induced Pi-S .escribed . AfterwaTotal RNA was extracted for the four fungal samples with three biological replicates, according to the method described before . The intP. italicum PHI-1 reference genome (GenBank accession number: JQGA01000000) [http://genome.jgi.doe.gov/Pendi1 /Pendi1.home.html), and the reference genome was indexed by Bowtie version 2.0.6.Raw reads stored in fastq format after the Illumina sequencing were first processed through in-house perl scripts to thoroughly remove low quality reads, as described before , to gene1000000)  using To1000000) . Prior tP-values were statistically corrected to assess the significance for the differences in transcript abundance according to Benjamini & Hochberg method [P-value \u22640.005 between two groups of comparison. The identified DEGs were hierarchically clustered by Cluster 3.0 [http://bioinfogp.cnb.csic.es/tools/venny/index.html. Further, DEGs were functionally enriched to Gene Ontology (GO) database (http://www. geneontology.org) using the GOseq R package based on Wallenius\u2019 non-central hyper-geometric distribution [http://www.genome.jp/kegg/), using the KOBAS software to test the significance of enriched DEGs in particular metabolic and signal transduction pathways [Gene expression level (transcript and/or unigene abundance) in each sample was estimated using HTSeq v0.6.1, according to FPKM analysis , which pg method . In the ster 3.0 , and theribution , and alspathways .P. italicum transcriptoms were selected for qRT-PCR validation, including 6 drug transporter genes, 5 ergosterol biosynthesis-related genes, 4 MAPK signaling pathway-related genes, and 4 Ca2+ signal transducer-related genes. Total RNA was extracted from the same P. italicum samples used for RNA-seq, according to Fungi RNA Kit user guide . First-strand cDNA synthesis was performed with PrimeScript\u2122RT reagent Kit with gDNA Eraser , according to the manufacturer\u2019s instructions, and the qRT-PCR was performed using a BIO-RAD CFX96 qPCR system with SYBR Green I fluorescent dye detection as previously described [\u03b2-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPD), were respectively applied as internal reference to calculate the relative mRNA abundance for the selected unigenes, according to the 2\u2013\u0394\u0394Ct method [t-test (n\u2009=\u20095) was applied in the SPSS Statistics 17.0 context to assess the significance of differences between the means (*p\u2009<\u20090.05 and **p\u2009<\u20090.01).Nineteen unigenes related to azole-drug resistance in the present escribed . The spet method  with 5 bmfs1 was derived from its parental strain Pi-R by introduction of mfs1- knockout cassette into the DMI-resistant P. italicum protoplasts that were prepared according to the method of Zhao et al. [hph) resistance gene (approximately 2.1\u2009kb) from the plasmid pTFCM  was inserted between the upstream and downstream flanking sequences of mfs1 gene  from the Pi-R genomic DNA. The knockout cassette was introduced into wild-type Pi-R protoplasts as described by Zhao et al. [Pimfs1 gene was replaced by the hph resistance gene.The mutant \u0394o et al. . Double-o et al. . In detao et al. , and thuAdditional file 1: Figure S1. PDA-based EC50 determination against prochloraz for two Penicillium italicum strains (Pi-R and Pi-S) with different response to the DMI prochloraz.Additional file 2: Table S1. EC50 values of Pi-R and Pi-S against the fungicides belonging to DMI, benzimidazole and phenylpyrrole classes.Additional file 3: Table S2. Primers used in the present qRT-PCR validation.Additional file 4: Figure S2. Genome mapping analysis for the clean reads from the present 4 RNA-seq libraries, i.e., Pi-R-I (a), Pi-R-NI (b), Pi-S-I (c), and Pi-S-NI (d).Additional file 5: Table S3. Summary of DEGs in the comparison between prochloraz-induced and no-induced Pi-R strains.Additional file 6: Table S4. Summary of DEGs in the comparison between prochloraz-induced and no-induced Pi-S strains.Additional file 7: Table S5. Summary of DEGs in the comparison between Pi-R and Pi-S strains under prochloraz-induced conditions.Additional file 8: Table S6. Summary of DEGs in the comparison between Pi-R and Pi-S strains without prochloraz induction.Additional file 9: Table S7. Summary of distribution (hit records) of 19 selected DEGs towards GO enrichments in the present 4 comparisons.Additional file 10: Table S8. FPKM values of the additional 4 prochloraz-responsive unigenes in the present comparisons.Additional file 11: Figure S3.GAPD-based qPCR validation of 19 prochloraz-responsive DEGs including drug transporter genes (a), ergosterol biosynthesis-related genes (b), MAPK signaling pathway genes (c), and Ca2+ signal transduction genes (d).Additional file 12: Figure S4. Comparison of prochloraz EC50 values between Pi-R (wild type) and its mfs1-knockout mutant (\u0394mfs1).Additional file 13: Table S9. Comparative analysis of prochloraz-responsive gene expression profiles between the P. italicum and P. digitatum transcriptomes."}
+{"text": "Alzheimer\u2019s and other dementias are chronic conditions that disproportionately impact some populations. The dementia continuum spans decades, so life-course approaches by state health departments (SHDs) are crucial. SHDs\u2019 implementation of the Healthy Brain Initiative Road Map (RM) represents policy/system/environmental actions to improve population-level dementia outcomes. METHODS. Alzheimer\u2019s Association uses online reporting by affiliate chapters to monitor SHDs\u2019 RM implementation. Association staff reviewed data and calculated frequencies by RM action and issue areas. Documentation from SHDs and national partners are supplementary data. RESULTS. Preliminary results from July-December 2019 indicate 34 SHDs were implementing 60 RM actions. Interventions clustered in risk reduction, early detection, workforce competencies, surveillance, and emergency preparedness. RM implementation lagged on access to community supports, attention to caregiving, and workforce supply analyses. A majority of states had multi-faceted initiatives. IMPLICATIONS. SHDs are deploying life-course approaches for dementia. SHD leaders must build towards a full response by expanding RM implementation."}
+{"text": "Grip strength declines with aging, is an indicator of overall health, and predicts mortality among older adults. Herein, we quantified the genetic contributions to grip strength among 4534 individuals, belonging to 574 families in the Long Life Family Study . Grip strength was measured using a handheld dynamometer, and the maximum value of two trials in the stronger hand was used. Quantitative trait linkage analysis was completed using pedigree-based maximum-likelihood methods with logarithm of the odds (LOD) scores >3.0 indicating genome-wide significance. Linkage analysis in the top 10% of families contributing to LOD scores was also performed to allow for heterogeneity among families (HLOD). All analyses were adjusted for age, sex, height and field center. Grip strength was lower per one year of older age , and overall: 24.3% of men and 19.3% of women had \u201clow\u201d grip strength according to European Working Group on Sarcopenia definitions. Grip strength was highly heritable . We identified a potentially novel locus for grip strength on chromosome 18p (LOD 3.18) with 26 families contributing to this linkage peak (HLOD = 10.94). Deep sequencing of the chromosome 18 region may yield fundamental insight on the biology of muscle weakness with aging, and may help identify novel therapeutic targets for treatment and prevention of this common condition."}
+{"text": "As control, 2% (w/v) CHX was mixed with 2% and 5.25% (w/v) NaOCl. Samples were subjected to the UHPLC-MS analysis. The mixture of ALX and NaOCl resulted in a yellowish precipitate formation, the amount of which depended on NaOCl concentration. Interaction of ALX and NaOCl resulted in the production of aliphatic amines. No PCA was formed when NaOCl was mixed with ALX. However, for the first time, we identified the possible products of the interaction. The interaction between NaOCl and ALX results in the formation of aliphatic amines; therefore, these compounds should not be mixed during endodontic treatment.Therapeutic success in endodontic treatment depends on successful infection control. Alexidine dihydrochloride (ALX) was recently proposed as a potential alternative to 2% chlorhexidine (CHX) as it possesses similar antimicrobial properties, expresses substantivity and does not produce p-chloroaniline (PCA) when mixed with sodium hypochlorite (NaOCl). However, the products released in this reaction have not been described to date. The aim of this study was to identify detected chemical compounds formed in the reaction of ALX and NaOCl with the ultra-high-performance liquid chromatography\u2013mass spectrophotometry (UHPLC-MS) method and assess whether precipitates and PCA are formed in this reaction. Solutions of ALX were mixed with the equivalent volume of 2% and 5.25% ( One of the main objectives of endodontic treatment is the eradication of infection from root canal systems by chemical and mechanical preparation. It has been widely proved that microbial reduction improves the prognosis of root canal treatment and therefore is the key to achieving endodontic success, as no apical periodontitis will develop without the presence of bacteria . It is aSodium hypochlorite (NaOCl), a basic solution used in irrigation protocol due to its unique tissue-dissolving capacities and broad antimicrobial spectrum, is not able to fulfill all criteria of effective chemical preparation, especially regarding smear layer removal and total eradication of microbiota. One-third to one-half of treated root canals remain infected when irrigated with 5.25% NaOCl only . It is tFor enhancing the bactericidal effect of NaOCl, 2% chlorhexidine (CHX) was advocated as an additional antimicrobial agent used in irrigation protocol . It is hw/v) ALX was equally effective in Enterococcus faecalis removal as 2% (w/v) CHX when tested on infected bovine dentin blocks [w/v) CHX, respectively [Alexidine dihydrochloride (ALX), a biguanide very similar to CHX, was first introduced to dentistry when tested as a mouth-rinse . It possn blocks . Furtherectively .w/v)) of ALX were mixed with a 4% (w/v) solution of NaOCl and analyzed with electrospray ionization mass spectrometry (ESI-MS) to determine whether PCA or precipitates were formed. As a result, the interaction of ALX and NaOCl did not produce PCA or precipitates; however, to date, no steps have been undertaken to identify chemical compounds formed in this reaction. The need for complete identification of products released is crucial for understanding the nature of this interaction and further assessment of the potential toxicity of these chemical compounds. The aim of this study was to determine and identify detected chemical compounds formed in the reaction of ALX and NaOCl with the ultra-high-performance liquid chromatography-mass spectrophotometry (UHPLC-MS) method.Kim et al. tested ALX as an alternative to CHX as a root canal irrigating solution . In thatw/v) NaOCl\u2013H2O, 1:99 (v/v)). The mixing of CHX and NaOCl resulted in the formation of a red-brown precipitate. The amount of precipitate varied depending on NaOCl concentration (with constant CHX concentration), but the precipitate was always present even if NaOCl concentration was highly diluted  during the analysis of the PCA standard mixture. The retention time of PCA was 0.56 min. The analysis of the precipitate mentioned above revealed the peak at the retention time of 0.56 min, corresponding to the ion of 128.0262 \u00b1 0.0050 m/z. The UHPLC-MS analysis of diluted supernatant showed no significant analytical signals, which may be attributed to significant ion suppression related to high salt concentration.Under the positive electrospray (ESI+) condition, a PCA molecule gives a characteristic ion: 128.0262 w/v) NaOCl\u2013H2O 1:99 (v/v), see When mixing ALX and NaOCl, a yellowish precipitate was formed. Similarly to the CHX\u2013NaOCl reaction, the amount of precipitate depended on the NaOCl concentration (with constant ALX concentration). Correspondingly, the precipitate appeared in each case, even for the lowest NaOCl concentration (5.25% (Because of the lack of an aromatic ring (or moieties that could react with NaOCl to form aromatic rings) in the ALX structure, no PCA is formed. However, ALX might be oxidized in a similar way to CHX, resulting in the formation of aliphatic amines. As was assumed, no peak characteristic of PCA was present either on a mass spectrum or on a chromatogram of any analyzed precipitate formed in an ALX\u2013NaOCl mixture. However, some other compounds that could be the products of the reaction between ALX and NaOCl were found. Their presence was predicted on the basis of possible reactions occurring in the mixture and confirmed by identifying these compounds on chromatograms  and corrThe relative reaction rates of the products depending on the concentration of NaOCl reagent have also been investigated and elaborated as the percentage of a particular peak area among investigated compounds  solution of chlorhexidine digluconate), CHLORAN 5.25% (5.25% solution of sodium hypochlorite) and CHLORAN 2% (2% (w/v) solution of sodium hypochlorite), were purchased from Chema-Elektromet . 4-Chloroaniline (p-chloroaniline) was supplied by Sigma-Aldrich . Ultrapure water was generated in-lab using a Milli-Q system from Merck Millipore .Methanol and acetonitrile (MS purity grade) were purchased from Sigma-Aldrich . Formic acid and ethanol were supplied by Merck . Alexidine dihydrochloride (> 98% purity grade) was purchased from Santa Cruz Biotechnology . Solutions used in endodontics protocols, namely Gluxodent  was prepared by dissolving analytical standard in a mixture of water and ethanol 4:1 (v/v). The stock solution of PCA (1 mg mL\u22121) was obtained by dissolving PCA in methanol. The standard working solution of ALX and the nonstandard working solution of CHX (500 ng mL\u22121) were prepared by diluting ALX stock solution or CHX commercial solution used in endodontics protocols in water\u2013acetonitrile 1:1 (v/v), respectively. Standard solutions of NaOCl were prepared by diluting 5.25% (w/v) NaOCl solution in water ). Moreover, a 2% (v/w) NaOCl commercial solution was used, without previous diluting. The standard solution of PCA (200 ng mL\u22121) was prepared by diluting stock solution in water and acetonitrile 1:1 (v/v). All solutions were stored in a refrigerator (+4 \u00b0C).The stock solution of ALX (4 mg mL2O\u2013ACN) 1:1 (v/v). Prepared samples were subjected to the UHPLC-MS analysis. Solutions of ALX and CHX were mixed with the equivalent volume (100 \u03bcL) of NaOCl standard solutions. A washing procedure was used to remove sodium ions, which can interfere with analytes and form adduct ions in the ion source of the mass spectrometer. Firstly, liquid supernatant from each sample was withdrawn and the precipitate was washed with cold water (+4 \u00b0C). Then, the sample was stirred and centrifuged . The washing procedure was repeated three times. Afterward, the precipitate was dissolved in 500 \u03bcL of methanol. Solutions made from a mixture of CHX and NaOCl and a mixture of ALX and NaOCl were 1000-fold and 50-fold diluted, respectively, in water-acetonitrile mixture (H2O\u2013ACN 1:1 (v/v) and subjected to the UHPLC-MS analysis.The liquid supernatant samples were 10,000-fold diluted in Hv/v) formic acid (phase A) and acetonitrile with 0.1% formic acid (phase B). In each analysis, 5 \u03bcL of sample was injected by an autosampler, and the separation was carried out in isocratic mode  within a Hypersil Gold Phenyl column  for 16 min. The flow rate of the mobile phase was 0.4 mL min\u22121. The column was thermostated at 25 \u00b0C. The electrospray ion source was operated under positive ionization conditions (ESI+). The other MS parameters were as follows: nebulizer pressure: 2.0 bar; dry gas flow: 5.5 L min\u22121; drying gas temperature: 200 \u00b0C; capillary voltage: \u2212500 V. The accurate mass of each ion was calibrated using sodium formate clusters. SmartFormula (Bruker) was used for the prediction of molecular formulas. The mass range was from 50 to 1500 m/z.The UHPLC-MS system consisted of the UltiMate 3000 RS liquid chromatography system  coupled with a tandem mass spectrometer with quadrupole and time-of-flight analyzers . A mobile phase consisted of water with 0.1% (The interaction of ALX and NaOCl results in the formation of aliphatic amines. This study showed that the reaction does not result in PCA formation. However, for the first time, we have shown that this interaction results in the formation of a yellowish precipitate, and we identified possible products of the reaction that can be potentially harmful. Therefore, ALX and NaOCl should not be mixed in endodontic irrigation protocol."}
+{"text": "The creation and evaluation of a national record linkage between substance misuse treatment, and inpatient hospitalisation data in England.A deterministic record linkage using personal identifiers to link the National Drug Treatment Monitoring System (NDTMS) curated by Public Health England (PHE), and Hospital Episode Statistics (HES) Admitted Patient Care curated by National Health Service (NHS) Digital.Adults accessing substance misuse treatment in England between 1 April 2018 and 31 March 2019 (n=268 251) were linked to inpatient hospitalisation records available since 1 April 1997.Using a gold-standard subset, linked using NHS number, we report the overall linkage sensitivity and precision. Predictors for linkage error were identified, and inverse probability weighting was used to interrogate any potential impact on the analysis of length of hospital stay.79.7% (n=213 814) people were linked to at least one HES record, with an estimated overall sensitivity of between 82.5% and 83.3%, and a precision of between 90.3% and 96.4%. Individuals were more likely to link if they were women, white and aged between 46 and 60. Linked individuals were more likely to have an average length of hospital stay \u22655 days if they were men, older, had no fixed residential address or had problematic opioid use. These associations did not change substantially after probability weighting, suggesting they were not affected by bias from linkage error.Linkage between substance misuse treatment and hospitalisation records offers a powerful new tool to evaluate the impact of treatment on substance related harm in England. While linkage error can produce misleading results, linkage bias appears to have little effect on the association between substance misuse treatment and length of hospital admission. As subsequent analyses are conducted, potential biases associated with the linkage process should be considered in the interpretation of any findings. This record linkage represents the first study of its kind to link centralised national level substance misuse treatment data and inpatient hospitalisation records.No single unique identifier, such as National Health Service number, is routinely collected within the National Drug Treatment Monitoring System and fewer personal identifiers are routinely collected than in other UK government held datasets.The limited availability of personal identifiers results in an increased risk of both false and missed matches, which could potentially affect the validity of any subsequently conducted analyses.Linkage error did not appear to lead to systematic bias and misestimation of sociodemographic and clinical factor associations with the average length of hospital stay.Routinely collected administrative data from the health and social care sector is increasingly used to both inform public health policy, and to generate research. While several initiatives across the UK have used national record linkages to further population-level understanding in specific disease areas,The number of people accessing specialist alcohol treatment has fallen by 19% between 2013 and 2017, while the number hospital admissions in which alcohol was recorded as a contributory factor has increased by 5% in the same timeframe.The National Drug Treatment Monitoring System (NDTMS) is the centralised database, collated and maintained by Public Health England (PHE), which receives monthly input from all local authority commissioned community drug and alcohol services in England.Although NDTMS has been previously linked with mortality data from the Office of National Statistics, and the Police National Computer,In this report we describe the process of record linkage and aim to evaluate the linkage quality and its potential impact on any subsequently conducted analyses. We believe this record linkage may result in the largest cross-sectional and longitudinal substance misuse database globally, and as such could become a resource which is able to support a large number of analytic outputs with the aim of improving the lives of those with substance use disorders.Patients accessing publicly funded specialist drug and alcohol treatment services in England provide written consent to share their information with NDTMS, and are informed that NDTMS records may be linked with data from specifically sanctioned UK government-held databases, including HES.The study benefited throughout from discussion with the South London and the Maudsley Biomedical Research Centre Data Linkage Service User and Carer Advisory Group, and the PHE Alcohol Treatment Expert Group which includes experts with lived experience. The former group represents a regular meeting of people whom have an interest in projects involving data linkage, and who have lived experience of mental health diagnoses, including substance use disorders. They receive on-going training on data matching processes, and hence can make recommendations on the acceptability of suggested data flows. The current proposal was presented in June 2018, and there was group-wide acknowledgement of the importance of the proposed linkage, based on personal experience of treatment experiences in drug and alcohol services. The group were content with the linkage methodology proposed, including the use of patient identifiers. Both groups will remain involved in subsequent analysis plans from any resultant linked data.Record linkage is the process of bringing together information pertaining to the same individual (or entity) from different databases. Linkage applies a set of criteria to determine whether or not records belong to the same individual, and aims to assess the true match status of each record pair: either a \u2018match\u2019, that is, records belong to the same individual, or a \u2018non-match\u2019, that is, records belong to different individuals. If record pairs are misclassified, error may be introduced as either \u2018false matches\u2019, that is, records from different individuals link erroneously or \u2018missed matches\u2019, that is, records from the same individual fail to link. Introduction of bias from linkage error, particularly if risk factors for important outcomes are associated with error rates, can impact the validity of findings derived from linked data.We selected all NDTMS records for adults accessing specialist drug or alcohol treatment in England between 1 April 2018 and 31 March 2019 as the test linkage population. The structure of the test linkage NDTMS data is such that one record represents one unique adult (n=268 251).10.1136/bmjopen-2020-043540.supp1Supplementary dataA Structured Query Language (SQL) algorithm was designed to facilitate NDTMS to HES APC linkage. Initial data cleaning in both datasets included the conversion of all missing or non-valid data to null values and the collapse of all postcodes relating to a no fixed abode (NFA) status into a single value. All NDTMS records contained a validly coded value for DOB and sex while 96.3% had a validly coded postcode, 94.7% a validly coded ethnicity and 18.4% a validly coded GP practice. NDTMS records that had missing or invalid postcodes  were excluded from linkage, as a combination of sex, postcode and DOB was the minimum\u2014but not necessarily sufficient\u2014data required to uniquely identify an individual. Of the remaining n=258 240 NDTMS records n=6878 (2.7%) shared the same combination of DOB, sex and postcode, of which n=164 (2.4%) did not have a valid entry for either ethnicity or GP practice.Matching was based on an exact match for each of the five variables described earlier, and conducted hierarchically in four stages as below:Stage 1: exact match on DOB, sex, postcode, ethnicity and GP practice.Stage 2: exact match on DOB, sex, postcode and GP practice.Stage 3: exact match on DOB, sex, postcode and ethnicity.Stage 4: exact match on DOB, sex and postcode.When records matched, they were removed from the dataset and not included in subsequent matching stages. As both databases are longitudinal, it was possible that several different values for postcode, and GP practice were recorded for each individual over time. Where more than one unique value was available the hierarchical algorithm attempted to link NDTMS records to HES APC records sequentially starting with the most recent value for each variable. All resulting records that linked with multiple records from the other dataset were removed and treated as non-links.A small subset of people in the full NDTMS sample (n=1328), who to date were taking part in the PHE individual placement and support trial,Using the \u2018gold-standard\u2019 sample the linkage rate was calculated as the percentage of NDTMS individuals linked to any HES APC record first by exact matching on only NHS number, and second using the four-stage deterministic algorithm described previously. The results were evaluated to determine the missed match rate, and the overall linkage precision, that is, the proportion of links that are true.Individuals linked using their NHS number were deemed to have been definitely hospitalised within their lifetime. Within this sample, individuals that were linked and not linked using the four-stage algorithm were compared with estimate rates of missed links. To allow for variation in patient characteristics and data quality between data providers, as well as between individuals, we used multilevel logistic regression with nesting of individuals within local authority commissioned treatment services and match status in NDTMS as the binary outcome . Model fit was examined using a likelihood ratio test comparing the multilevel model to a fixed-effects logistic model which did not account for nesting of individuals. We explored any association between match status and NDTMS sociodemographic , and clinical factors . For modelling purposes ethnicity was recoded into the binary categories of white and non-white,Challenges exist to assess the impact of linkage error when the outcome in question may not have been experienced by all people in the sample to be matched. When linking an individual\u2019s NDTMS records to HES APC it is difficult to know which matches have been missed as the HES database by design will only capture information about individuals who have been hospitalised. As such non-links could be due to an individual never having been admitted to hospital or being a missed match.For each unique linked individual, a binary outcome of their average length of hospital stay  was created to assess bias due to linkage error. This was chosen as it is clinically relevant, reflecting the current UK average length of hospital stay per person, and is recorded for all people within HES APC.While access to the linked dataset is only available within PHE, subject to approval, extracts of NDTMS are available to researchers through the Office of Data Release at PHE,The linkage was conducted using SQL Server Management Studio V.18.4. Additional analyses were conducted using STATA MP V.15.1, with the significance level set at 0.05.The overall matching for a unique person within the full NDTMS sample (n=268 251) to a HES APC hospitalisation record generated n=213 814 linked records, representing a linkage rate of 79.7%. The proportion linked according to the matching stages described earlier were: stage 1: 10.7%, stage 2: 5.7%, stage 3: 72.5% and stage 4: 11.1%.The overall matching for a unique person within NDTMS to a HES APC hospitalisation record using the \u2018gold-standard\u2019 subset of people with an NHS number available in NDTMS, generated n=1153 linked records using NHS number, representing a linkage rate of 86.6%. Using the four-stage algorithm within the \u2018gold-standard\u2019 population generated n=1053 linked records with a linkage rate of 79.3%. Although this was lower than the NHS number match rate this suggests that the majority of unlinked records were true non-links  and not missed matches. Of the n=1053 records linked using the four-stage algorithm, 102 were not linked by the gold standard. These included n=36 records that disagreed on NHS number and were therefore assumed to be false links, and 66 that had missing or invalid NHS numbers and could represent either false links or links missed by the gold-standard. These two possibilities suggest a precision of between 90.3% and 96.4%, respectively. Of the n=1153 records matched using the NHS number n=202 were not matched by the four-stage algorithm suggesting a sensitivity of between 82.5% and 83.3%, respectively.Weighting the probability of being linked to HES APC data using the four-stage algorithm demonstrated a correction of linkage bias within the \u2018gold-standard\u2019 sample, the results of which are summarised in The full linked sample had a total of 1 624 152 inpatient hospital admissions since HES database inception in April 1997 until January 2020, with a total time spent in hospital of 14 461 years, and an overall average length of hospital stay of 3 days. Using deterministic matching, a national longitudinal and cross-sectional dataset was built between NDTMS specialist community drug and alcohol treatment data and HES hospitalisation data in England, providing a linkage for 213 814 adults (79.7% of the full NDTMS cohort) to their inpatient hospital records. Using our linkage algorithm there were significant differences in the sociodemographic and clinical characteristics between the linked and non-linked samples, with individuals more likely to link if they were women, white, and aged between 46 and 60 years old. Using the linked data, we were able to demonstrate that individuals were more likely to have an increased average length of hospital stay if they were male, older, had no fixed residential address, and had problematic opioid use. These effects did not change substantially following inverse probability weighting, suggesting they were not driven by bias from linkage error.Very few studies have examined linkage error in the context of people with substance use disorders. Using our deterministic algorithm, n=54 437 (20.3%) of individuals were not linked to HES APC hospitalisation records. Linkage of the gold-standard sample suggests that approximately two-thirds of these are true non-links , and the remaining third are missed matches. When using the \u2018gold-standard\u2019 sample 86.6% of records matched using NHS number, as such 86.6% is likely to estimate the overall true match rate. We can thus infer that a roughly similar percentage of the n=10 011 NDTMS records with insufficient matching data should match and are therefore genuine missed-matches in the total sample (n=8670). Based on our linkage sensitivity these 8670 records constitute just under half of the total number of likely missed match records when using the four-stage algorithm. The sociodemographic and clinical characteristics of this cohort can be found in We found that older age groups were more likely to link which may reflect a greater availability of accurate personal identifiers in the records of this population as by living longer they have had greater potential exposure to drug and alcohol services compared with other age groups, and an increased number of hospitalisation records, and therefore potentially more values of matching variables. Previous research has suggested that individuals from black and ethnic minorities are more likely to have administrative records with inaccurately recorded dates of birth and higher levels of residential instability, which may be applicable to this sample, and partially account for the reduced likelihood in of linkage compared with white individuals.This represents the first study of its kind to link centralised national level substance misuse treatment data and inpatient hospitalisation records, and provides an example of how potential non-random loss between routinely collected administrative datasets can be adjusted for by weighting techniques.This linkage between substance misuse treatment and hospitalisation records offers a new powerful tool to evaluate the impact of specialist treatment on alcohol and drug related harm in England. Through its interrogation, and via additional sanctioned linkage to datasets from other government departments, for example, the Department for Work and Pensions, these data may hopefully be able to provide insight and knowledge to improve the lives of people with substance use disorders. While biases due to linkage error may produce misleading results in our sample, linkage biases appear to have little effect on the association between drug and alcohol treatment and length of hospital admission. However, without ongoing ability to probe information within the source data, potential linkage error could be introduced without future analysts being aware that there was need for it to be accounted for.In time, we hope this resource will generate a wide network of granular data and analytical expertise, which can be used to inform both commissioning and service provision to better meet the needs of people with substance use disorders in England. The immediate next steps are to evaluate the most common reasons for hospital admission within the cohort of people accessing drug and alcohol treatment and to assess the impact of engagement in, and successful completion of, drug and alcohol treatment on individual and national rates hospitalisation. It is important to note that as subsequent analyses of the resultant linked dataset are conducted, any potential bias associated with the linkage process should always be considered in the interpretation of any findings."}
+{"text": "This study aims to evaluate the ability of sodium thiosulfate (STS) to neutralize the adverse effect of sodium hypochlorite (NaOCl) on dentin micro-hardness.p value <\u20090.05 was considered significant.Fifty single-rooted teeth were longitudinally sectioned. The samples divided into a control and four sample groups (n\u2009=\u200920). All the samples were immersed in different solutions as follows, Control: Normal saline for 15\u00a0min, G1and G2: 2.5% NaOCl for 15\u00a0min, G3: 2.5% NaOCl for 15\u00a0min, followed by 5% STS for 10\u00a0min, G4: Normal saline for 15\u00a0min followed by 5% STS for 10\u00a0min. All groups except G1 incubated for one week before the test. The micro-hardness of samples was measured. Data were analyzed using the Kruskal\u2013Wallis test for pairwise comparisons. A p\u2009<\u20090.05). NaOCl alone (G2) or treated with STS (G3) resulted in a significant decrease in micro-hardness compared with the STS group (G4) (p\u2009<\u20090.05).All groups showed a significant decrease in the micro-hardness value compared with the control group. NaOCl for one week (G2) reduced the micro-hardness of dentine compared with samples, tested immediately after immersion in NaOCl (G1) (STS as a neutralizing agent could not prevent the dentin micro-hardness downturn caused by NaOCl. Irrigants and intra-canal medicaments such as NaOCl and calcium hydroxide may have some adverse effects on the physical and mechanical characteristics of dentin, result in reducing flexural strength, micro-hardness, and modulus of elasticity \u20135. NaOClSTS 5% is an antioxidant agent that recommends neutralizing the effect of NaOCl on dentin and improving the resin bonding properties . It reacThe effects of this neutralizing agent on the physical properties of dentin are unknown. Thus, this in- vitro study evaluated the effect of STS on the micro-hardness of dentin treated with and without NaOCl at different time intervals. This study hypothesizes that STS will reduce the effect of NaOCl on dentin micro-hardness by neutralizing its remnants on the dentin surface after one week.Fifty straight single-rooted teeth with relatively similar dimensions and morphology and closed apices were extracted for orthodontic or periodontal reasons collected with the patients\u00b4 informed consent. This study design was approved by the Ethics in Human Research Committee of Shiraz University of Medical Sciences (Ethics ID No. IR.SUMS.DENTAL.REC. 1398.138). Proximal view radiographs were taken to confirm the presence of a single patent canal. Teeth with root caries, cracks, curved canals, endodontic treatment, internal resorption, or calcification were excluded. Teeth were thoroughly cleaned of any soft tissue or calculus deposits and stored in isotonic saline solution at room temperature until the time of use. The crowns of all specimens were cut transversally at the coronal level of the roots with a double-faced diamond disc  at low speed with water coolant to ensure a uniform sample length of 14\u2009\u00b1\u20091\u00a0mm root length.Specimens were longitudinally sectioned in the buccolingual direction using a double-faced diamond disk at low speed, without passing through the canal space. A mallet and chisel were used to split the root. The root segments were horizontally embedded in auto polymerizing acrylic resin , leaving their dentin surface exposed. The dentin surface of the mounted specimens was ground flat and smooth with a series of ascending grades of carbide abrasive papers   under distilled water to remove any surface scratches and finally polished with a 0.1-Mm alumina suspension on a rotary felt disc  to obtain a smooth glassy mirror-like surface. The samples were divided randomly into one control and four experimental groups based on the immersion solution and incubation time:Control group: Normal saline for 15\u00a0min, G1 and G2: 2.5% NaOCl  for 15\u00a0min, G3: 2.5% NaOCl for 15\u00a0min irrigated with normal saline followed by 5% STS  for 10\u00a0min, G4: Normal saline for 15\u00a0min followed by 5% STS for 10\u00a0min. All groups except group 1 were incubated for 1\u00a0week in an incubator (37\u00a0\u00b0C with 100% humidity) before the micro-hardness test. The samples of group 1 were tested immediately after immersion in NaOCl.The micro-hardness measurements were taken either on the buccal or lingual side of each root. The sectioned root was divided equally into three-thirds representing the coronal, middle and apical thirds, and each area was tested separately. An indentation was made in the dentin surface approximately 200\u00a0\u00b5m from the canal-dentin interface for standardization Fig.\u00a0. The VicThe constant value of the equation was calculated from the specific geometry of the indenter, F is the applied load in grams and D is the diagonal of the indentation in \u03bcm .One specimen of each group was dehydrated, mounted and gold-sputtered, for evaluation under a scanning electron microscope  operated at 20KV. Photographs were taken from 3 points of each sample at 2000\u00d7 magnifications .Data were analyzed using the Kruskal\u2013Wallis test for pairwise comparisons. A p\u2009<\u20090.05). As shown in Fig.\u00a0p\u2009<\u20090.05).The micro-hardness medians (means\u2009\u00b1\u2009standard deviations) are shown in Table SEM micrographs showed longitudinal dentinal tubes with the patches of some smear layers in the control group. In the other specimens, a unique pattern of smear layer, which is totally covered the dentin was obvious, although this layer seems thicker and more homogenous in the samples, which were in contact with STS.p\u2009<\u20090.05). As shown in Fig.\u00a0p\u2009<\u20090.05).The micro-hardness medians (means\u2009\u00b1\u2009standard deviations) are shown in Table SEM micrographs showed longitudinal dentinal tubes with the patches of some smear layers in the control group. In the other specimens, a unique pattern of smear layer, which is totally covered the dentin was obvious, although this layer seems thicker and more homogenous in the samples, which were in contact with sodium thiosulfate.The results of the present study show that both 2.5% NaOCl and STS decreased the micro-hardness of dentin compared with the control group. The remnants of NaOCl after 1\u00a0week significantly reduced the micro-hardness of dentin compared with the samples treated with NaOCl for 15\u00a0min.Samples that were irrigated with NaOCl and then neutralized with STS, had significantly lower micro-hardness values than the samples irrigated with normal saline but were not different from the group immersed in NaOCl alone. Thus, STS not only reduced the effect of NaOCl on dentin micro-hardness but also may have had a synergistic effect on its weakening. Therefore, the hypothesis of this study was rejected.Different studies have shown that using NaOCl as an irrigating solution significantly reduces the micro-hardness value \u201333. MostThe interesting finding from this study is that the samples that were immersed for 15\u00a0min in NaOCl and irrigated with normal saline showed a significant reduction in micro-hardness after the one-week incubation. This finding indicates that the remnants of NaOCl and its oxidative products may remain active during this period leading to additional decreases in dentin micro-hardness. Concerning this subject, the use of an anti-oxidant agent like STS seems to be logical to reduce this effect. A recent study showed the recovery of resin bond strength to NaOCl treated dentin by using 5% STS for 10\u00a0min . STS canSEM micrographs also showed the same pattern of smear layers accumulation above the samples of NaOCl and STS Fig.\u00a0. This reThe reduction in dentine micro-hardness of all groups continued for one week even after irrigation the tested materials with normal saline. Neutralizing the remnants of NaOCl with STS did not prevent this adverse effect."}
+{"text": "We present a novel metric for measuring relative connection between parts of a city using geotagged Twitter data as a proxy for co-occurrence of city residents. We find that socioeconomic similarity is a significant predictor of this connectivity metric, which we call \u201clinkage strength\u201d: neighborhoods that are similar to one another in terms of residents\u2019 median income, education level, and (to a lesser extent) immigration history are more strongly connected in terms of the of people who spend time there, indicating some level of homophily in the way that individuals choose to move throughout a city\u2019s districts. Cities are defined by the flow of people through them\u2014the boundaries of metropolitan areas are drawn based on commuting patterns and on socioeconomic integration ; as peopWhile these person-based studies are valuable in analyzing social structure and identifying lack of interaction between, for example, different ethnic groups , there iFurther, studying location-connectivity as opposed to person-connectivity bypasses some of the obstacles that come with more traditional approaches. One such obstacle is that existing literature on segregation in activity spaces either relies on travel diaries and surveys , 12, or We propose a methodology to study connectivity between neighborhoods in Stockholm, Sweden, specifically as it relates to socioeconomic similarity: does the homophily in travel patterns identified in existing literature cause neighborhoods which are socioeconomically similar to one another to be more connected by the flow of people? First, we present a metric for calculating connectivity, which we call \u201clinkage strength\u201d, between any two neighborhoods in the city using data with high spatial but low temporal resolution. Our metric defines connectivity between two areas by co-occurrence of people in those areas\u2014co-occurrence has been used to measure connectivity between physical places in existing studies in other contexts , 14. OurThe paper is organized as follows. We introduce the methodology and datasets of our study in the Materials and Methods section. The outcome of the three regression analyses we perform are in the Results section. Finally, conclusions and future research directions are outlined in the Conclusions section.Our analysis requires two datasets: one describing movement of people between neighborhoods in Stockholm, and one describing socioeconomic dissimilarity between neighborhoods in Stockholm. In order to understand human mobility across the city, we analyze a set of geotagged tweets in the municipality of Stockholm between January 1, 2016 and April 30, 2019. By looking at successive geotagged tweets, we can understand users\u2019 general mobility spaces\u2014In order to understand the distribution of socioeconomic characteristics across Stockholm, we look at 2017 census data recorded at the level of Stockholm\u2019s 132 \u201cstadsdelar\u201d (singular: stadsdel)\u2014geographical units containing an average of around 6,000 residents. By aggregating tweet locations up to the stadsdel level, we are able to compare movement across neighborhoods to the socioeconomic characteristics of those neighborhoods; namely, we compare a connectivity metric calculated from Twitter data which we call \u201clinkage strength\u201d (described below) with dissimilarity between pairs of stadsdelar in three socio-economic features: income per person, the percentage of their population that was born in another country, and the percentage of their population that attended some amount of post secondary school .A and B, we use the following formula: Let UA be the set of users who tweet at least once in location A. Let xiA be the number of times user i tweets in location A. We define fA,B to be the connection, or linkage strength, between location A and location B created by Twitter users as follows:In this study, we define connection, or linkage strength, between two areas in terms of shared Twitter users. Users who tweet often in region A and in region B indicate some social or economic connection between the two regions (even if it occurs over the span of weeks) and some colocation of their residents. In order to quantify this type of connection between two locations xi,A times in location A and xi,B times in location B, they add min{xi,A, xi,B} units of linkage strength between A and B. Informally, users who spend time in both locations can be thought of as spreading resources or ideas between the two areas or forming personal connections between residents of the two areas. We take the minimum of xi,A and xi,B because it serves as a bound on the interaction created between the two neighborhoods. See In other words, if a user tweets We compare our linkage strength metric to that used in , 16, whiUsernames and text associated with the tweets are dropped from our dataset. User ids are hashed to new, random values in order to fully anonymize our data. Further, once linkage strength is calculated, tweets are no longer associated with individuals at all, and trajectories are untraceable in the data. Thus, we remove all identifying information from our data, and we do not add identifying data via home location estimation.A and B\u2014see We use a negative binomial regression to estimate the relationship between our explanatory variables (described below) and linkage strength between pairs of stadsdelar. Linkage strength is a count variable, but it is overdispersed with respect to a Poisson distribution ; negative binomial regression is appropriate for this kind of overdispersed count data . BecauseIn order to understand differences between stadsdelar in the impact of socioeconomic difference on movement patterns, we also estimate individual negative binomial regression models for each stadsdel. We use fewer explanatory variables\u2014the variables related to origin stadsdel (A) are no longer relevant, as we are looking at only pairs with the same origin stadsdel, and we remove several highly collinear variables, as they contain redundant information and we have fewer degrees of freedom in these smaller models. See We examine whether absolute difference in socioeconomic characteristics between two areas affects the linkage strength between them\u2014are neighborhoods more connected by flow of people to other neighborhoods \u201clike\u201d theirs, in terms of mean income, education levels, and immigrant makeup? We choose three socioeconomic variables for each stadsdel: mean income, percentage of the population with some post secondary education, and percentage of the population classified as \u201cfirst generation\u201d (those born outside of Sweden). These three characteristics are correlated with one another but distinct\u2014see We look at five control variables that can help to account for travel constraints and layout of the city: travel time between two locations, physical accessibility of each location in the city structure, number of points of interest in each location, population of each location, and expected linkage strength in a rank-distance null model of mobility.Travel times. We use the Google Maps API to calculate travel times via driving and public transit between two areas. As longer travel times indicate that areas are more expensive to travel between in terms of both time and money, we expect an inverse relationship between the travel time between two places and linkage strength between them. Analysis of this travel time data indicates heterogeneities in the strength of the transportation network between different neighborhoods\u2014see Accessibility. We use a labor accessibility index based on the SCAPER travel demand model [nd model , 18. ThePoints of Interest (POIs). We download information on points of interest in each stadsdel from OpenStreetMap. Points of interests include shops, restaurants, tourism sites, parks, among other types of places. As points of interest draw visitors to an area, they could help to explain movement around Stockholm. In order to specifically capture points of interest that would indicate attractiveness of an area, we filter out passive POIs which do not serve as a draw to an area, such as trash cans and surveillance cameras\u2014see Population. We use 2017 population counts from the Swedish census .Rank-distance null model. We estimate expected travel linkage strengths between areas of Stockholm using the rank-distance model in [A and B as inversely proportional to rankA(B), where rankA(B) is the number of points of interest closer to A than B is to A. In [model in . This moto A. In , Noulas We also include total Twitter activity and the individual socioeconomic characteristics of each stadsdel. In summary, the explanatory variables for the individual-stadsdel and all-stadsdel models are reported in Since our measure of linkage strength is symmetric, the characteristics of each location should contribute equally to it; for this reason, we fix the coefficients on socioeconomic characteristics, accessibility, points of interest, population, total amount of linkage strength and total number of tweets to be the same for both locations  by including their sums instead of their individual values. We also perform a log transform on accessibility, points of interest, population, and total number of tweets in order to capture the empirical relationship between those variables and our linkage strength metric.\u03b1 = .001, while foreign background has a signficant effect on linkage strength at \u03b1 = .1. For every standard deviation increase in socioeconomic difference, we see total linkage strength multiplied by the exponential of the given coefficient; thus, for every standard deviation increase in income difference between A and B , linkage strength between A and B is multiplied by e\u22120.097 = .91, leading to a 8.9% decrease in linkage strength between A and B. For every standard deviation increase in post-secondary education difference between A and B, we see a 10.0% decrease in linkage strength between A and B. For every standard deviation increase in immigrant makeup difference between A and B, we see a 3.4% decrease in linkage strength between A and B. Socioeconomic differences between neighborhoods thus produce statistically significant barriers to linkage strength throughout the city\u2014in the case of income and education, quite stark barriers; in the case of immigration status, relatively weaker barriers.The outcome of our regression analysis is detailed in Importantly, driving and transit time are also significant predictors of linkage strength, and have even larger effects than socioeconomic difference\u2014longer travel times by either mode of transportation are associated with significantly less linkage strength. This indicates that each serves a separate and significant role in connecting physical locations in the city. Strengthening physical infrastructure between parts of the city with low linkage strength could similarly serve to strengthen connectivity between dissimilar places: our results suggest that a decrease of one standard deviation in transit time (about fourteen minutes) is associated with about a 13.4% increase in linkage strength between two stadsdelar.We find that predictive effect of socioeconomic difference varies by stadsdel, and is even insignificant in some. In most stadsdelar where socioeconomic difference does have a significant predictive effect on linkage strength, that effect is negative, indicating that neighborhoods are more likely to be connected to other neighborhoods with a similar socioeconomic makeup, consistent with our full-city model. However, there is also a significantly positive effect in some stadsdelar\u2014some neighborhoods are significantly more likely to be connected to neighborhoods which are different from them, contrary to the overall trend in Stockholm see . Fig 6 sWe have identified significantly stronger linkage between neighborhoods of similar income, education levels, and immigrant makeup. This relationship persists even when controlling for factors induced by the structure and layout of the city, such as transit time between places and intervening opportunities, indicating some level of homophily in the way that individuals choose to move through neighborhoods. This lack of linkage strength between neighborhoods with different socioeconomic characteristics has important implications for social segregation in Stockholm. Researchers have already identified strong residential segregation between ethnic Swedes and immigrants to Sweden and between socioeconomic groups ; our resand foreign background dissimilarity coefficients in Stockholm, suggesting that they aren\u2019t well connected to the rest of the city in terms of linkage strength; our results suggest that encouraging new migrants to move elsewhere could potentially have the desired effect of faster integration into Sweden in that they may be more likely to be exposed to people from all across the city [Policymakers have already instituted various policies to try to ameliorate residential divides. For example, the Swedish Migration Agency discourages new immigrants to the city from living in certain socioeconomically challenged neighborhoods by withholding some state benefits if they choose to do so . The neithe city . Howeverdifferent from them by the flow of people. Using the individual-stadsdel models, we were able to identify which stadsdelar have the most significant homophily effect. These stadsdelar could potentially be areas of interest for Stockholm city planners as they plan for activities and spaces that will foster integration.In our individual-stadsdel models, we have found heterogeneities in the effect of socioeconomic dissimilarity on city connectivity across neighborhoods: consistent with the overall model, most significant socioeconomic difference coefficients in the income and education models were negative, but there were still some stadsdelar with significant, positive coefficients, indicating that they are more likley to be strongly connected to stadsdlar While the results are promising, it is important that we recognize potential biases in the Twitter data used in this study. Direct demographic information of Twitter users are not available, but language processing studies and formal surveys have found that Twitter\u2019s user population is, in general, younger, more educated, and wealthier than the general population , 31. FurOur distance calculations pose a further limitation to our analysis\u2014travel times were calculated using the geographic centroid of each stadsdel, which may not be representative of travel times between all points in the stadsdelar. Further research could help to illuminate exactly what biases arise from this, if any (see ).fA,B belongs to a cluster of observations associated with stadsdel A and a cluster of observations associated with stadsdel B. The natural statistical dependencies that will occur in data of this structure are known to cause deflated standard errors and thus over-rejection of the null hypothesis. We choose to account for this using clustered standard errors as recommended in [A and B. While strategies such as multilevel modeling have been shown to be even more effective at reducing over-rejection of the null hypothesis than cluster-adjusted standard errors, we believe that a single level model is sufficient and preferable in our context due to its simplicity and interpretability [Our data is cross-classified and multilevel in structure: each observation ended in , where ttability . It shoutability .It is worth exploring more deeply the causal mechanisms behind the lack of connection between dissimilar places that is demonstrated by our results. Specifically, analyzing the relationship between flow of people between places and infrastructure allowing for that flow  could have important implications for urban design, transportation planning, and the efficient orchestration of network slices. Using place-based measures like ours as opposed to person-based measures is especially amenable to this kind of analysis and planning, as they allow for the identification of weak spots in connectivity of the physical environment, as opposed to other measures of social segregation which look at lack of connectivity in social networks\u2014a metric agnostic of the physical environment.S1 AppendixWe discuss the cleaning process of our Twitter and point of interest data.(PDF)Click here for additional data file.S2 AppendixWe show that our model does not suffer significantly from bias related to residual spatial autocorrelation or multicollinearity.(PDF)Click here for additional data file.S3 AppendixWe describe the travel time data used in our model.(PDF)Click here for additional data file.S4 AppendixWe describe the labor accessibility index used in our model and show how it is distributed across the city of Stockholm.(PDF)Click here for additional data file."}
+{"text": "This study included 34 eligible studies and 1757 GCLSs. The clinicopathologic characteristics of GCLS were investigated from eligible studies, and the meta-analysis was performed. In addition, we compared the survival rates between GCLS and non-GCLS.  The estimated rate of GCLS was 0.062  0.040-0.097). GCLS was significantly correlated with the diffuse type of Lauren's classification, proximal tumor location, less-frequent lymphatic invasion, and lower pTNM stage. However, there was no significant difference in age, sex, tumor differentiation, vascular invasion, perineural invasion, pT stage, lymph node metastasis, and distant metastasis between GCLS and non-GCLS patients. EBV positive rates in GCLS and non-GCLS patients were 0.723 (95% CI 0.643-0.791) and 0.064 (95% CI 0.039-0.103), respectively. HER2 expression in GCLS was significantly lower than that in non-GCLS. GCLS patients had a more favorable prognosis than that of non-GCLS patients .  GCLS comprised 6.2% of overall GC and more frequent in the proximal portion of the stomach. Since GCLS was associated with better prognosis, the histologic finding can be useful for predicting the patient's prognosis. Gastric carcinoma (GC) includes various subtypes, such as tubular adenocarcinoma, poorly cohesive carcinoma, and GC with lymphoid stroma (GCLS) . Among tRelevant articles were obtained by searching the PubMed database through April 30, 2020. We used the following keywords: \u201cstomach or gastric\u201d and \u201cgastric carcinoma with lymphoid stroma or medullary carcinoma or lymphoepithelioma-like carcinoma.\u201d The titles and abstracts of all searched articles were screened for inclusion and exclusion. Included articles should have information on clinicopathological characteristics or prognosis in GCLS. However, nonoriginal articles, such as case reports and review articles, were excluded. In addition, those not written in English were not included in the present study. This protocol was reviewed and approved by the Institutional Review Board of Eulji University Hospital (EMC 2020-09-007).P value, and the O-E statistic (the difference between the number of observed and expected events) or its variance. If these data were unavailable, the HR was estimated using the total number of events, number of patients at risk in each group, and the log-rank statistic or its P value. Finally, if the only useful data were in the form of graphical representations of survival distributions, survival rates were extracted at specified times to reconstruct the HR estimate and its variance under the assumption that patients were censored at a constant rate during the time intervals [Data extracted from 34 eligible studies \u201335 incluntervals . The pubntervals . Data asQ and I2 statistics and expressed as P values. Additionally, sensitivity analysis was conducted to assess the heterogeneity of eligible studies and the impact of each study on the combined effects. Eligible studies included various populations having different tumor subtypes, tumor stages, and treatments. In addition, although the molecular and immunohistochemical tests were qualified, the methods were different between laboratories. Thus, in interpretations for estimated results, a random-effect model rather than a fixed-effect model was used. To assess publication bias, Begg's funnel plot and Egger's test were used; if it was significant, the fail-safe N and trim-fill tests were additionally used to confirm the degree of publication bias. The results were considered statistically significant at P < 0.05.The meta-analysis was performed using the Comprehensive Meta-Analysis software package . The prevalence of GCLSs among GC was investigated. Subgroup analyses based on the depth of the tumor, was performed. The clinicopathological characteristics of GCLS, such as age, sex, size, tumor differentiation, lymphovascular invasion, and pTNM stages, were compared with those of non-GCLS. EBV positivity from EBER in situ hybridization between GCLS and non-GCLS was compared. In addition, the differences of PD-L1, HER2, and p53 immunohistochemical expressions between GCLS and non-GCLS were investigated. Heterogeneity between the studies was checked by the n = 75), nonhuman studies (n = 17), and a language other than English (n = 7)  . Finally (n = 7) . These sP = 0.392 in a metaregression test). In addition, in sensitivity tests, there was no significant impact of each study on estimated prevalence rate.The estimated prevalence rate of GCLS among GCs was 0.062 (95% CI 0.040-0.097) . In earlNext, we compared the clinicopathological characteristics between GCLS and non-GCLS patients. Statistical significances was found in the tumor size, Lauren's classification, tumor location, lymphatic invasion, and pTNM stage using the metaregression tests . The tumP < 0.001 in the metaregression test). Microsatellite instability was found in 5.9% of GCLSs. PD-L1 immunohistochemical expression rates in GCLS occurred in 0.677 (95% CI 0.497-0.817) and 0.742 (95% CI 0.563-0.865) in tumor and immune cells, respectively. HER2 immunohistochemical expression rates were 0.026 (95% CI 0.005-0.120) and 0.632 (95% CI 0.403-0.813) in GCLS and non-GCLS, respectively.EBV positivity was found in 72.3% of GCLSs and 6.4% of non-GCLSs, respectively . There wPatients with GCLS had a better overall survival than those with non-GCLS . As shown in GCLSs comprise 1-7% of all GCs , 27. In Lim et al. reported treatment results of endoscopic resection for the early stage of GCLS (pT1/2) . HistoloMedullary carcinoma is also described in colorectal and breast cancers. The characteristic histologic finding of these carcinomas is poorly differentiated tumor cells with peritumoral lymphocytic infiltration. However, diagnostic criteria are slightly different between tumors. In breast cancer, medullary carcinoma is defined as (1) sheets of cells with indistinct cell borders  in greater than 75% of the tumor, (2) sharply circumscribed and pushing borders, and (3) moderate to poor differentiation . In coloThe characteristic finding of GCLS is peritumoral and tumor-infiltrating lymphocytes. In addition, cell nests of GCLS can be embedded in prominent lymphocytic infiltrates. Because studies against the immunotherapeutic effects of various cancers are recently increasing, the correlation between TILs and PD-L1 expression is useful to understand the treatment in GCLS. Because EBV positivity of GCLS was high, molecular characteristics of GCLS and EBVaGC may be overlapping. PD-L1 gene amplification was elevated in EBV-associated GCs . In the P < 0.001 in the metaregression test). The positive rates of EBV in non-GCLS varied from 0% to 19% among eligible studies [In GCLS, EBV positivity varied from 22.6% to 100% by reports , 27. We  studies , 32. How studies . Previou studies . HoweverThis study has some limitations. First, the analysis of MSI status in GCLS could not be performed due to insufficient information. Setia et al. reported that one case out of 17 GCLS showed a microsatellite instable (MSI) status . Second,"}
+{"text": "This study examined how users acquire spatial knowledge in an onscreen three-dimensional virtual environment when using overview maps. This experiment adopted a three (the size of overview maps) x two (the transparency of overview maps) between-subjects design. Three levels of the size of overview maps were evaluated, i.e., 1/2, 1/8, and 1/16 screen size. Comparisons between 20% transparent and 80% transparent were made. We asked 108 participants to complete spatial perception tasks and fill out questionnaires regarding their feelings. The results indicate the following: (1) The effects of the transparency of overview maps on users\u2019 spatial perception vary with the size of overview maps. The 80% transparent overview map is significantly more efficient than the 20% transparent overview map in the condition of 1/2 screen size. However, the result is opposite in the condition of 1/8 screen size. (2) Users like the 80% transparent overview map significantly better than the 20% transparent overview map in the condition of 1/2 screen size. (3) Concerning subjective evaluations of satisfaction, preference, and system usability, overview maps in the condition of 1/8 screen size are significantly better than those in the condition of 1/2 screen size. As three-dimensional virtual environment 3D VE) has become more widely used in recent years, the usability, interactivity, and immersion of VE interfaces are expected to be better. Compared to the real environment, spatial perception in VEs has been found much less accurate, especially in perceptual judgments of distance and size ,2,3. AddD VE has Various visual aids that provide spatial knowledge of the environment by graphical means  are usabThis is the first work to integrate the visual variables of size and transparency in one study of VE interfaces, making it possible to determine the most appropriate overview map design for user navigation.A design principle for better navigation performance: High level of transparency significantly improves efficiency for users with the overview map in the condition of 1/2 screen size. Low level of transparency significantly improves efficiency for users with the overview map in the condition of 1/8 screen size. For the 20% transparent overview map, 1/8 screen size and 1/16 screen size offer significantly higher efficiency than 1/2 screen size.The findings from this study suggest that reducing the size of overview maps could effectively improve users\u2019 subjective evaluations of the 3D VE user interfaces.In this paper, overview map design supporting navigation in a VE is studied. An experimental study examining the effects of the size and transparency of overview maps on users\u2019 spatial perception and subjective evaluations is conducted. The contribution of our work is composed as follows:Spatial cognition emerges from the interaction between an organism and environmental characteristics . Figure Healey  suggesteTransparency means the variations of color value and saturation , and it The above-mentioned studies have shown that the visual variables of size and transparency may play crucial roles in dealing with visibility issue, which is the most prevalent perception issue that can limit users\u2019 experience of a VE interface . The stuUsers\u2019 spatial perception can be evaluated by performing tasks based on the perceived spatial information about visible objects, which is particularly appropriate for interactive VEs . In the This study employed a between-subjects design involving two variables (the transparency of overview maps and the size of overview maps). Two discrete transparent conditions were tested, i.e., 20% transparent (mostly opaque) and 80% transparent (mostly transparent). The VE was experienced through a screen. The levels of the size of overview maps were 1/2 screen, 1/8 screen, and 1/16 screen. The dependent variables were task completion time and error count, evaluations of satisfaction, preference, and system usability.H1: The transparency of overview maps can make significant differences in users\u2019 spatial perception and their subjective evaluations of the 3D VE user interfaces.H2: The size of overview maps can make significant differences in users\u2019 spatial perception.H3: Users\u2019 subjective evaluations of the 3D VE user interfaces can be improved by reducing the size of overview maps.H4: A significant interaction effect exists between the size and transparency of overview maps.Four hypotheses guided the design of the experiment:Group with the 1/2 screen-size 80% transparent overview map: 13 females and 5 males;Group with the 1/8 screen-size 80% transparent overview map: 13 females and 5 males;Group with the 1/16 screen-size 80% transparent overview map: 12 females and 6 males;Group with the 1/2 screen-size 20% transparent overview map: 17 females and 1 male;Group with the 1/8 screen-size 20% transparent overview map: 15 females and 3 males;Group with the 1/16 screen-size 20% transparent overview map: 13 females and 5 males.One hundred and eight university students were recruited as volunteers to participate in the experiment via convenience sampling method, comprising 83 females and 25 males, ranging in age from 17 to 30 . Each group contained 18 participants, and each participant tested only one kind of overview map: Twenty-six people had never used overview map interfaces in VEs prior to participating in the experiment. Fifty-seven people had rarely used overview map interfaces in VEs. Twenty-five people had used the interfaces weekly. Ninety-nine percent of participants used smartphones or computers more than two hours per day . All participants used the experimental platform without any problem in basic operation. The study was designed in accordance with the latest version of the Declaration of Helsinki and was approved by the local ethical committee. All participants gave their informed consent for inclusion before they participated in the study.The prototype of VE is created with the 3DS Max software and the Unity 3D game engine. A two-story museum unfamiliar to the participants has been simulated. It consists of nine exhibition areas placed on the ground floor, including four Chinese painting exhibition areas, three introduction exhibition areas, a book exhibition area and a photographic exhibition area. Six exhibition areas are placed around the second floor, which is smaller than the ground floor. There are two Chinese painting exhibition areas, two book exhibition areas, a photographic exhibition area and a Chinese calligraphy exhibition area on the second floor. The overview map interfaces are created with the Photoshop software see . LandmarThe experiment was conducted on an iPad Air tablet computer using the iOS 9.3 operating system in an empty room. The VE interfaces were presented on the 9.7-inch screen with resolution of 2048 \u00d7 1536. Our study adopted the first-person perspective which is often used in virtual museums. Visual angles were changed by moving fingers across the screen. To reduce the number of variables affecting user performance, participants were allowed to move in the VE only by clicking the landmark icons on overview maps. Furthermore, there was no other navigation aid in the VE.Participants were required to fill out a survey questionnaire about gender, occupation, age, VE navigation experience, and familiarity with computers. Before starting the formal tasks, participants were introduced to the VE user interfaces and allowed to spend as much time as they needed until they felt familiar with the VE. The total duration of this part ranged between 1 and 5 min.By the method of randomized block design, the participants were randomly assigned to six groups to test one of the six VE user interfaces. The participants were informed that they should perform four spatial perception tasks in sequence as quickly and accurately as possible see . The firAfter all the tasks had been completed, participants were asked to complete a seven-point Likert scale to rate the overview maps in terms of subjective satisfaction (from 1 \u201cless satisfied\u201d to 7 \u201cvery satisfied\u201d) and preference (from 1 \u201cmost disliked\u201d to 7 \u201cmost liked\u201d). Participants were also required to fill out a SUS questionnaire see . Each stp = 0.059 > 0.05). No significant main effect of transparency  = 0.966, p = 0.328 > 0.05) was found. Significant interaction existed between the variables of size and transparency  = 6.556, p = 0.002 < 0.01).A 2 x 3 analysis of variance (ANOVA) was performed to analyze the collected data. The results generated from the descriptive statistics and two-way ANOVA of total task completion time are shown in In order to find out which factor is differentially effective at each level of a second factor, we selected the simple effects test . Table 4According to p = 0.471 > 0.05) or transparency  = 0.000, p = 1.000 > 0.05) was found. There existed no significant interaction effect between the variables of size and transparency  = 0.757, p = 0.471 > 0.05). Participants in each group made few mistakes.A 2 x 3 ANOVA was performed. The results generated from the descriptive statistics and two-way ANOVA of total errors occurred in all the tasks is shown in Satisfaction refers to users\u2019 experience of having positive feelings, such as confidence and control, in their performance of tasks. After completing the tasks, comparisons and analysis of subjective satisfaction were made using a 2 x 3 ANOVA. The descriptive statistics and two-way ANOVA of satisfaction are shown in p = 0.012 < 0.05). The results of post hoc comparison using the least significant difference (LSD) show a significant difference between the condition of 1/8 screen size and 1/2 screen size (p = 0.003 < 0.01). The satisfaction score of the overview map in the condition of 1/8 screen size  was significantly higher than that of the overview map in the condition of 1/2 screen size . No significant difference was found in the main effect of transparency  = 0.203, p = 0.653 > 0.05). There was no significant interaction effect between the variables of size and transparency  = 0.622, p = 0.539 > 0.05).The results regarding subjective satisfaction reveals a significant main effect of size  = 4.656, After the tasks were completed, participants were asked to rate how much they like the interface to recognize the most popular overview map. A 2 x 3 ANOVA was performed. The results generated from the descriptive statistics and two-way ANOVA of preference are shown in p = 0.008 < 0.01). The results of post hoc comparison using the LSD show a significant difference between the condition of 1/8 screen size and 1/2 screen size (p = 0.007<0.01). The condition of 1/16 screen size and 1/2 screen size also show a significant difference (p = 0.007 < 0.01). The preference scores of the overview map in the condition of 1/8 screen size  and 1/16 screen size  were significantly higher than that of the overview map in the condition of 1/2 screen size . No significant main effect of transparency  = 0.467, p = 0.496 > 0.05) was found. The interaction effect between the variables of size and transparency was significant  = 3.156, p = 0.047 < 0.05).The results regarding subjective preference reveal that there was a significant main effect of size  = 5.044, Based on the simple effect test results , the oveAccording to the interaction diagram illustrated in p = 0.029 < 0.05). The results of post hoc comparison using the LSD show a significant difference between the condition of 1/2 screen size and 1/8 screen size (p = 0.008 < 0.01). Participants using the overview map in the condition of 1/8 screen size  gave significantly higher SUS scores than those using the overview map in the condition of 1/2 screen size . It revealed no significant main effect of transparency  = 1.224, p = 0.271 > 0.05). No significant interaction effect was found between the variables of size and transparency  = 1.625, p = 0.202 > 0.05).A 2 \u00d7 3 ANOVA was performed. The generated results of the SUS questionnaire are shown in Besides the quantitative results, we also observed the process in which the participants interacted with the VE user interfaces, and we collected qualitative data from post interviews. The results are clearly positive: none of the participants had difficulties in using the experimental apparatus. All participants were satisfied with the simple operation of the system. Three participants who tried to zoom in on the overview map in the condition of 1/16 screen size commented that the contents of the overview map are a little hard to be recognized. None of the participants with overview maps in the condition of 1/8 screen size or 1/16 screen size tried to hide the overview map. Two participants reported that one of the difficulties in performing the tasks was the rapid change in visual angle.The analysis of total task completion time confirmed the fourth hypothesis that significant interaction effect exists between the size and transparency of overview maps. At 1/2 screen size, participants using the 80% transparent overview map performed significantly better than those using the 20% transparent overview map. At 1/8 screen size, participants using the 20% transparent overview map performed significantly better than those using the 80% transparent overview map. For the 20% transparent overview map, participants using overview maps in the condition of 1/16 screen size and 1/8 screen size performed significantly better than those using the overview map in the condition of 1/2 screen size. This might be because the 1/2 screen-size overview map and the 20% transparent overview map reduce the visibility and legibility of VE contents. Spatial perception tasks might be difficult when some relevant objects and locations are invisible. Smaller overview maps taking less screen space from the VE interface, and more transparent overview maps, allowing more underlying layer data to pass through them , can botConcerning subjective satisfaction, the results confirmed the third hypothesis that users\u2019 subjective evaluations of the 3D VE user interfaces can be improved by reducing the size of overview maps. The overview map in the condition of 1/8 screen size led to significantly higher user satisfaction compared to the condition of 1/2 screen size. This might be because the overview map in the condition of 1/8 screen size provides more details of the VE to make users feel easier to know the environment well. Another aspect that might explain the difference is that the overview map in the condition of 1/2 screen size, which is often deliberately hidden to avoid occlusion in the VE, makes the problem of focus switching worse [The results regarding subjective preference confirmed the third hypotheses as well. Users\u2019 subjective preference could be improved by reducing the size of overview maps. Users liked overview maps in the condition of 1/8 screen size and 1/16 screen size significantly better than those in the condition of 1/2 screen size, especially for the 20% transparent overview map. It might be that the overview map in the condition of 1/2 screen size masks more detailed spatial information that users may be interested in. Moreover, it is easier to switch attention between the layers of the VE and a small overview map. Similar to the results from total task completion time, the results regarding subjective preference also confirmed the fourth hypothesis that significant interaction effect exists between the size and transparency of overview maps. At 1/2 screen size, users liked the 80% transparent overview map significantly better than the 20% transparent overview map. Perhaps at 1/2 screen size, a high level of transparency effectively relieves the occlusion. This is consistent with previous studies that propose that making use of transparency influences user preference in navigation tasks , and a hThe results from the SUS questionnaire are consistent with those from users\u2019 subjective evaluations of satisfaction and preference, and also confirmed the third hypothesis that users\u2019 subjective evaluations of the 3D VE user interfaces can be improved by reducing the size of overview maps. The usability of the overview map in the condition of 1/8 screen size is considered significantly better than that of the overview map in the condition of 1/2 screen size. Perhaps this is due to the additional steps needed to display or hide the overview map in the condition of 1/2 screen size. It is difficult to switch attention between the layers of the detail view and a large overview map. The results are in accordance with previous studies suggesting that more mental and temporal demand is required to integrate the distinct views for understanding spatial relationships .Maps, a predominant form of navigation aid , need toThis study mainly explored how the size and transparency of overview maps affect navigation in a 3D VE. One hundred and eight participants spent an average of 15 min solving spatial perception tasks using an overview map. Our results suggest that significant interaction effect exists between the size and transparency of overview maps in users\u2019 spatial perception and subjective evaluations of the 3D VE user interfaces. At 1/2 screen size, users with the 80% transparent overview map have significantly higher efficiency and preference than those with the 20% transparent overview map. At 1/8 screen size, the 20% transparent overview map is significantly more efficient than the 80% transparent overview map. For the 20% transparent overview map, users with the overview maps in the condition of 1/16 screen size and 1/8 screen size have significantly higher efficiency and preference than those with the overview map in the condition of 1/2 screen size. Based on our work, we recommend that designers reduce the size of overview maps in navigating onscreen 3D VEs. The overview map in the condition of 1/8 screen size leads to significantly higher subjective evaluations of satisfaction, preference, and system usability than that in the condition of 1/2 screen size. Whether or not the results are consistent using immersive virtual devices and more natural navigation metaphors were not confirmed in this study. As a future work, we will investigate and extend our research to more complex tasks and more immersive VEs."}
+{"text": "Crassostrea gigas were chimeric, containing single nucleotide polymorphisms (SNPs) mapping to different linkage groups. Here, we merge 14 linkage maps constructed of SNPs generated from genotyping-by-sequencing (GBS) methods with five, previously constructed linkage maps, to create a compendium of nearly 69 thousand SNPs mapped with high confidence. We use this compendium to assess a recently available, chromosome-level assembly of the C. gigas genome, mapping SNPs in 275 of 301 contigs and comparing the ordering of these contigs, by linkage, to their assembly by Hi-C sequencing methods. We find that, while 26% of contigs contain chimeric blocks of SNPs, i.e., adjacent SNPs mapping to different linkage groups than the majority of SNPs in their contig, these apparent misassemblies amount to only 0.08% of the genome sequence. Furthermore, nearly 90% of 275 contigs mapped by linkage and sequencing are assembled identically; inconsistencies between the two assemblies for the remaining 10% of contigs appear to result from insufficient linkage information. Thus, our compilation of linkage maps strongly supports this chromosome-level assembly of the oyster genome. Finally, we use this assembly to estimate, for the first time in a Lophotrochozoan, genome-wide recombination rates and causes of variation in this fundamental process.Studies of linkage and linkage mapping have advanced genetic and biological knowledge for over 100 years. In addition to their growing role, today, in mapping phenotypes to genotypes, dense linkage maps can help to validate genome assemblies. Previously, we showed that 40% of scaffolds in the first genome assembly for the Pacific oyster  The physical, linear arrangement of genes in DNA molecules has profound implications at all levels of biological organization, from cell, to organism, to population. Thus, studies of linkage and linkage mapping have been fundamental for advancing genetic and biological knowledge for over 100 years . Today, Crassostrea gigas is a species of global commercial value and scientific interest, having been introduced from Asia to all continents but Antarctica for aquaculture (Mann 1979). Oyster cytogenetics has been fairly well studied. Cupped oysters of the genus Crassostrea have 10 pairs of chromosomes . The Pacific oyster omosomes . LongwelAllozymes furnished the first evidence of genetic linkage in bivalve molluscs and the first partial linkage or gene-centromere maps . The devC. gigas genome . The latter assembly  was accomplished, using third-generation DNA sequencing methods and Hi-C analysis, to associate 301 contigs with an N50 of nearly 3.2 Mb  methods enabled the generation of a large number of genetic markers for non-model organisms  in 2009. G0 families 23, 31, 47 and 92 were established using wild-caught parents by the Molluscan Broodstock Program (MBP) in 1996 \u2014F12, F20, F45, 2\u00d710, 51\u00d735, 23\u00d731, 23\u00d740, 31\u00d723, 40\u00d792, 47\u00d792, 92\u00d740, and 58\u00d719 (sire \u00d7 dam).  in 2015 .2 families and for family 58\u00d719, using GBS, and the follow-up bioinformatics analyses were guided by Genome Analysis Toolkit  (Except for the families genotyped by https://www.qiagen.com/us/shop/sample-technologies/dna/genomic-dna/dneasy-blood-and-tissue-kit/#resources), with minor modifications. We checked the quality of the extracted DNA by agarose gel electrophoresis, quantified DNA in each sample, following the protocol of Quant-iT PicoGreen dsDNA Assay Kit, and diluted or concentrated the sample DNA to a working concentration of 10 ng/\u00b5l. To make libraries for sequencing, we digested 100 ng of extracted genomic DNA in a final volume of 20 \u03bcL with the restriction enzyme ApoI (NEB # R0566L), in the buffer supplied by the manufacturer, at 50\u00b0 for 2 h, and then at 80\u00b0 for 20 min. We ligated common and barcoded adapters (designed with http://www.deenabio.com/services/gbs-adapters) to genomic DNA by incubating the mixture of digested genomic DNA, T4 DNA ligase (NEB # M0202L), H2O, and 10\u00d7 T4 DNA ligase reaction buffer at 22\u00b0 for 60 min, 65\u00b0 for 30 min, and 4\u00b0 for cooling (Table S2). We then pooled and cleaned the ligated products from different samples. We amplified the pooled products in 2 \u00b5l DNA template, 21 \u00b5l H2O, 25 \u00b5l NEB 2\u00d7 Taq Master mix (NEB # M0270S), and 2 \u00b5l Primer mix, using the PCR program, 5 min at 72\u00b0, 30 s at 98\u00b0, 14 cycles \u00d7 , 5 min at 72\u00b0, and holding at 4\u00b0 (Table S2). We then purified the amplified products by agarose gel electrophoresis, following protocols of QIAquick PCR Purification Kit, and extracted the DNA fragments, following the protocol of MinElute Gel Extraction Kit. Once libraries were constructed, we checked their size distributions on an Agilent Bioanalyzer and quantified their concentration using NanoDrop 2000 spectrophotometer. Sequencing libraries comprised 96 bar-coded samples, except for family 23\u00d731, which was sequenced in libraries containing 48 samples; this resulted in about 2\u00d7 greater sequencing depth and SNPs in family 23\u00d731 than in the other families (Table S3).GBS involved two steps, library preparation and sequencing. To construct libraries for sequencing, we first extracted DNA from all parents and progeny, following the DNeasy 96 Procotol, Purification of Total DNA from Animal Tissues , were assigned to the corresponding sample using a custom script in Python. The script also truncated reads having a full cut site or the beginning of the common adapter.We sent all libraries to the University of Southern California (USC) Genome & Cytometry Core for sequencing and sequenced each library in a single lane on an Illumina HiSeq instrument. We processed the GBS data generated from sequencing on the Linux clusters of the University of Southern California\u2019s Center for High-Performance Computing. Sequences that matched a barcode , followed by the 2 families, we aligned reads to the then-available v9 genome assembly, using the Burrows-Wheeler alignment tool , and processed alignments with the GATK software package v3.3.0 . The pedigrees of all well-genotyped progeny for the six, interrelated F2 families  were confirmed by relatedness analysis . To correct genotyping errors and fill missing genotype information, we first imputed uni-parentally segregating markers using the program Maskov according to i.e., a group of markers assigned to the same position on linkage maps) identified by JoinMap 4.1. Markers were grouped, using the independence LOD, which ranged from 2 to 26, across linkage groups and families.For family 51\u00d735 and the six, interrelated FnMap 4.1 , using t2r, the correlation coefficient for linear regression of marker rank orders on the ML map against marker rank orders on the RG map. When 2r was less than 0.95, we removed markers with large nearest-neighbor fits or markers with inconsistent positions between RG and ML maps. We considered RG and ML maps for a linkage group consistent when 2r reached at least 0.95; we then took the RG map as the final linkage map for that linkage group and brought back identical markers if their representative remained on the final RG map. Linkage groups were numbered according to the second-generation linkage map in We used both regression (RG) and maximum likelihood (ML) mapping methods to construct linkage maps. For the RG method, we used the Kosambi mapping function, with maximum recombination frequency of 0.4, minimum LOD of 1.0, and goodness-of-fit jump threshold for removing loci of 5.0. All other parameters were set to default values. We used only the first- or second-round regression linkage maps. For the ML method, which uses Haldane mapping units, we set chain length to 5,000, length of burn-in chain to 20,000, number of Monte Carlo EM cycles to 10, and chain length per Monte Carlo EM cycle to 5,000. All other parameters were set to default values. The parameter used to determine whether a locus fits well between its neighboring loci is nearest-neighbor fit (cM), with a larger value indicating a poor fit . We cons2 families and family 58\u00d719, we first mapped trimmed reads using BWA with default parameters , average spacing (between unique mapping positions), and genome coverage between the JM and LM3 maps of these six families, using Student\u2019s t-tests . We evaluated the reliability and consistency of different linkage mapping methods, by comparing rank orders of common markers on linkage maps constructed using RG method in JM, ML method in JM, and LM3 for the six, interrelated F2 families  , we evaluated the Chr_v1 genome assembly at the contig level. By comparing the ordering and orientation of the Chr_v1 contigs with that on the ALLMAPS-generated assembly, we assessed the Chr_v1 genome assembly at the chromosome level.By checking whether there is a block of contiguous SNPs on a contig assigned to linkage groups different than the consensus linkage group of the contig  between the two most distal SNPs on each linkage group by the physical distance between these SNPs on the corresponding chromosome of the Chr_v1 genome assembly. Next, we conducted a three-way ANOVA with family, sex, chromosome and two-way interactions among them as independent variables and RR as the dependent variable, to test whether these factors make a significant contribution to variation in recombination rate. Since we do not have replication of each family-by-sex-by-chromosome combination, the significance of the three-way interaction among family, sex and chromosome cannot be estimated and is thus excluded from ANOVA.To assess variation in recombination rate (RR), we used the ML maps for each parent of the six, interrelated F2 families using MareyMap  and ML  maps and physical positions of SNPs on the Chr_v1 genome assembly. We first removed outlier loci whose genetic distances did not increase monotonically with their physical positions, as defined by the instruction on MareyMap, because these outlier loci could arise from mapping errors on genetic or physical maps. We used the loess-based method, setting Span to 0.3 and Degree to 1. We removed loci with negative recombination rates and calculated recombination rates for the remaining set of loci. After standardizing RR values from ML maps to facilitate comparisons among families, we tested the null hypothesis that values below -1.28 and above 1.28, nominal 10th and 90th percentiles, were randomly distributed across the chromosome, using Pearson\u2019s chi-square test for complete spatial randomness as implemented by PROC SPP in SAS 9.4 . Analyses were done by family and by chromosome, using areas tightly defined by the length of chromosome mapped and the range in standardized RR values, nine quadrats per area, and a minimum of nine observations, so that the expected value in each quadrat was at least 1.0. We adjusted the probability threshold for significance at the \u03b1=5% level to 0.001 to account for multiple testing.To assess variation in recombination rate along each chromosome, we calculated recombination rates across the ten chromosomes of the six, interrelated FuscCg205, AY999703, on chr 2; ucdCg147, AF468549, on chr 3; ucdCg028, AF051178, on chr 5; ucdCg197, AF468595, also on chr 5; imbCg44, Y12085, on chr 7; and imbCg049, Y12086, on chr 8). Using the confidence limits for the gene-centromere distances from Lastly, in order to check whether there is reduced recombination around centromeres, we located the leftmost position of centromere-associated microsatellite markers detected by https://doi.org/10.25387/g3.13077470.Supplemental material available at figshare: 2 families. By comparing the genotype at common loci between duplicates of parents of the six, interrelated F2 families, we found that genotyping error rate was 2\u20133%. For the six, interrelated F2 families, numbers of SNPs generated by GBS, which passed filtering and genetic criteria, range from 4,615 to 13,885 (Table S3). On average, 65% of these markers  were input to JoinMap 4.1 (JM), of which nearly a third (32.5%) had identical genotypes. The number of markers placed on final RG maps ranges from 794 to 1,351 or from 16 to 34% of the markers input to JoinMap 4.1 for each family (Table S3).In total, we obtained genotypes for 12 parent oysters and 1,041 progeny (Table S1) for the six, interrelated F2 families range from 454.6 cM to 589.7 cM, with average marker spacings from 0.445 cM to 0.856 cM  between rank orders of markers on RG and ML maps are equal to or above 0.9999, as expected from the iterative method of map construction, while coefficients of correlations (2r) between rank orders of markers on RG and LM3 maps range from 0.9967 to 0.9991 , and between assignment to consensus linkage group and number of mapping families or level of family support . JM is \u223c1.72 times more likely to assign SNPs to the consensus linkage group than LM3, for SNPs mapped in only one family, and 2.56 times more likely, for SNPs mapped in more than one family . Using a decision tree , we clasC. gigas genome of 587,333,624 bases, accounting for 98.9% of the Chr_v1 genome assembly (Table S7B). Adding a default gap of 100 bases between contigs, ALLMAPS generates a genome totaling 587,361,424 bases, with chromosomes ranging from 39,883,917 to 78,822,797 bases in length (Table S7C). The remaining contigs, the total length of which account for only 1.1% of the Chr_v1 assembly, do not contain any high-confidence SNPs and, thus, are not placed by ALLMAPS. Comparing the sequence-based and linkage mapping-based assemblies, contig-by-contig, we find that the 275 contigs in common to both assemblies fall into five categories (Table S8): (1) 205 contigs (74.5%) assigned to the same chromosome and assembled in the same order by genetic and sequencing methods ; (2) 26 contigs (9.4%) assigned to the same chromosome but in different orders between the two assemblies; (3) 31 contigs (11.3%) assigned to the same chromosome, assembled in the same order, but reversed in orientation between the two assemblies; (4) nine contigs (3.3%) whose orientation in the ALLMAPS assembly is unknown; and (5) four contigs (1.4%) assigned to different chromosomes in the two assemblies. The 205 contigs that are assembled the same way by both assemblies, plus the 40 contigs assembled consistently but with reverse or unclear orientation, account for 89.1% of the 275 contigs in common (Table S8B). Of the 26 contigs that are placed in different orders, 18 are within three positions of aligning.ALLMAPS successfully places all high-confidence SNPs on 19 linkage maps from 12 families (Table S7A). Anchoring 288 contigs with these high-confidence SNPs, ALLMAPS assembles a chromosome-level 2 families, were analyzed by three-way ANOVA, with family, parent, and chromosome as the factors. The model is highly significant , yielding a grand mean RR = 1.97 cM/Mb (Table S9). As the two-way interaction of family \u00d7 chromosome is significant (P = 0.032), we test the significance of family and chromosome with this interaction term, finding that both main factors are significant . Neither of the two-way interactions involving sex is significant, though the one with family is marginal (P = 0.075); the main factor, sex, is highly significant when tested with the error term . Two families, 23\u00d731 and its reciprocal 31\u00d723, have significantly higher RR  than all other families (average RR = 1.72), which are not statistically different (vs. 1.74). Finally, a plot of the marginally significant family \u00d7 sex interaction shows that dams consistently have higher, though not necessarily significantly higher RR than sires across families and that the dams for the reciprocal 23\u00d731 and 31\u00d723 families appear to account for the high recombination rates of those families , across 10 chromosomes and the 12 parents of the six, interrelated Fifferent . Chromosifferent . Dams haIn addition to differences in recombination among families and chromosomes and between the sexes, we also observe variation in recombination rate along each chromosome . Random 2 families, we find all three maps are consistent with each other  with high-confidence SNPs contain blocks of two or more SNPs mapped in two or more families to a linkage group different than the consensus linkage group for their contig (Table S6). The total length of these \u201cchimeric\u201d blocks, however, amounts to only 0.08% of the total length of the assembly , and the chimeric blocks themselves are small, with a median length of only 33 bp . The lower proportion of chimeric contigs and the much smaller size of chimeric blocks suggest that the Chr_v1 assembly of the We assembled contigs into chromosomes, using ALLMAPS, and compared the order and orientation of 275 contigs in common with the Chr_v1 assembly. Overall, nearly 90% of these contigs are identically assembled by HiC analysis and ALLMAPS. Inconsistencies between the two assemblies for the remaining 10% of contigs may be largely driven by an insufficient number of high-confidence SNPs for accurate assembly based on linkage information. Identically assembled contigs contain 12 to 1,788, high-confidence SNPs, with a mean of 317 (Table S8B), while contigs with reversed or unclear orientation contain 1 to 403 SNPs, with a mean of 72. In contrast, 26 contigs that are ordered differently on the two assemblies contain 2 to 119 SNPs, with a mean of 36 (Table S8B); nevertheless, the median difference in order of these contigs is less than three places.The four contigs (1.4%) assigned to different chromosomes by HiC analysis and ALLMAPS contain only three to 14, high-confidence SNPs (Table S8B). Contig 237, which contains 14, high-confidence SNPs, contains 26 SNPs in total, 14 of which are assigned to LG2, while 12 are assigned to LG10; LG10 is consistent with the chromosome assignment by HiC analysis . While the 12 SNPs assigned to LG10 are excluded from ALLMAPS, because they do not pass the filtering criteria for high-confidence SNPs, we still suspect that contig 237 could either be part of chromosome 10 of the ALLMAPS-generated assembly or could comprise two pieces that should be assigned to chromosomes 2 and 10. Both possibilities may suggest a potential contig misassembly, but we cannot draw a firm conclusion with limited information at this point. For the remaining contigs , we do not find any solid evidence for these contigs being assigned to other linkage groups, but the small number of high-confidence SNPs on them may not provide sufficient information for ALLMAPS to generate a correct assembly (Table S8A).i.e., 30 out of 301 contigs). Linkage maps of larger families and more markers may be more helpful in assessing, and potentially enhancing this genome assembly.Altogether, Chr_v1 appears to be a reliable chromosome-level assembly of the Pacific oyster genome, which will make it invaluable for future studies. The assembly may still contain some small errors in assembly of contigs. Also, we note that we did not have enough high-confidence SNPs for checking \u223c10% of contigs  across a Lophotrochozoan genome. In addition, information from a set of interrelated Fcf. 1 hybrids, in a species with non-chromosomal and labile sex determination (Recombination rate varies significantly among families, among chromosomes, and between the sexes. Reciprocal families 23\u00d731 and 31\u00d723 have similar RRs that are significantly higher than those in the other four families, suggesting, perhaps, a heritable basis for variation in recombination rate that bears further investigation. Across family and sex, chromosomes 10 and 4 have the highest and the lowest RRs, respectively; the population and evolutionary consequences of this nearly twofold difference in RR merits further exploration as well. As reported previously (mination , suggestAlthough we expect to see lower recombination around centromeres , such a 2 families (e.g., hotspots at \u223c49 Mb on chromosome 2, in 23\u00d740, 31\u00d723, and 40\u00d792, \u223c53 Mb on chromosome 3, in 23\u00d740 and 31\u00d723, and \u223c40 Mb on chromosome 7, in 23\u00d740, 40\u00d792, and 47\u00d792; a coldspot at \u223c46 Mb on chromosome 6 in the reciprocal F2 hybrids 40\u00d792 and 92\u00d740; Finally, we find suggestive evidence for recombination hotspots and coldspots on all chromosomes but the first and across all six, interrelated Ffamilies . Some ho"}
+{"text": "AQP1 and negatively regulates AQP1 transcription. Thus, we discovered a novel function of ATF4 in controlling the process of TM remodelling in ET\u20101\u2010induced POAG through transcription suppression of AQP1. Our findings also detail a novel pathological mechanism and a potential therapeutic target for POAG.Primary open\u2010angle glaucoma (POAG) is the second leading cause of irreversible blindness worldwide. Increased endothelin\u20101 (ET\u20101) has been observed in aqueous humour (AH) of POAG patients, resulting in an increase in the out\u2010flow resistance of the AH. However, the underlining mechanisms remain elusive. Using established in vivo and in vitro POAG models, we demonstrated that water channel Aquaporin 1 (AQP1) is down\u2010regulated in trabecular meshwork (TM) cells upon ET\u20101 exposure, which causes a series of glaucomatous changes, including actin fibre reorganization, collagen production, extracellular matrix deposition and contractility alteration of TM cells. Ectopic expression of AQP1 can reverse ET\u20101\u2010induced TM tissue remodelling, which requires the presence of \u03b2\u2010catenin. More importantly,\u00a0we found that ET\u20101\u2010induced AQP1 suppression is mediated by ATF4, a transcription factor of the unfolded protein response, which binds to the promoter of  In the human eye, AQP1 is expressed in the iris and ciliary epithelium and mainly functions in AH production by transporting water out of the ciliary epithelium. Mice lacking AQP1 showed reduced aqueous fluid production and reduction of IOP.AQP1 gene and negatively regulates its transcription. We also address the hypothesis that during the early stages of glaucoma, increased humous levels of ET\u20101 mediates trabecular meshwork tissue remodelling, and we discovered a novel function of ATF4 in controlling the process of TM remodelling through transcription suppression of AQP1 in POAG.In this study, using in vivo and in vitro models, for the first time we demonstrated that water channel Aquaporin 1 (AQP1) expression is down\u2010regulated in trabecular meshwork cells, upon ET1 exposure, and that this contributes to a series of glaucomatous changes including actin fibre reorganization, collagen production, ECM deposition and contractility alteration of TM cells. This TM tissue remodelling process is mediated by the interaction between AQP1 and \u03b2\u2010catenin. More importantly, we found that ET\u20101\u2010induced suppression of AQP1 is mediated by ATF4, a transcription factor of the unfolded protein response that binds to the promoter of the 22.1Male NZW white rabbits aged 12\u00a0weeks were purchased from Guangdong Medical Laboratory Animal Center. Experimental animals were housed in individual cages and topical ocular ET\u20101 (2\u00a0\u03bcmol/L) or PBS eye drops were applied 3 times a day for 2\u00a0weeks and IOPs were recorded using opthalmotonometer weekly. Animals were killed at end of 2\u00a0weeks, and TM tissue were collected and fixed in 4% paraformaldehyde. Animal maintenance and experiment procedure were approved by the Laboratory Animal Ethics Committee of Shenzhen University.The primary human TM cell line was kindly provided by Dr Minbin Yu2.2TM tissues were immunostained using antibodies indicated in Table 2.3The HTMCs were seeded on coverslips in a 24\u2010well plate with a density of 8X104\u00a0cells/well. After corresponding treatment, HTMCs were washed with pre\u2010warmed PBS and fixed with 4% paraformaldehyde. The cells were permeabilized with 0.1% Triton X\u2010100 (Sigma\u2010Aldrich) and blocked with 5% goat serum (Life Technologies). For staining for collagen I, cells were incubated overnight with primary antibodies, including anti\u2010collagen I (Abcam), and the cells were washed and incubated with Alexa 488\u2010conjugated secondary antibodies (Thermo Fisher Scientific). The cells were probed with Alexa Fluor 594 phalloidin (Thermo Fisher Scientific). Afterwards, the coverslips were mounted with DAPI (Thermo Fisher Scientific). The pictures were taken by the LSM 510 (Zeiss) confocal microscope.2.4The supernatants were collected and collagen I and collagen III contents in the supernatants were measured using kits (R&D systems).2.5Collagen gel contraction assays were performed with a Cell Contraction Assay Kit  according to the manufacturer's instructions, with minor modifications by Dr Minbin Yu.2.6Total RNA was extracted using Trizol according to the manufacturer's instructions. The primers for target genes were obtained from the PrimerBank Database. QRT\u2010PCR was performed with the FastStart Universal SYBR Green Master reagent (Roche) and a Roche 480 real\u2010time PCR system. Target gene expression was calculated using the 2 (DDC[t]) method using b\u2010actin as the housekeeping gene. The results were presented as a relative value compared to the control group.5\u2032\u2010TGCCATCGGCCTCTCTGTA\u20103\u2032 (forward primer)5\u2032\u2010CAGGGTTAATCCCACAGCCA\u20103\u2032 (reverse primer)The primer sequences of AQP1 are as follows:2.7Treated and untreated HTMCs were rinsed with PBS and lysed in lysis buffer (Sigma) with 1X cocktail inhibitor (MERK USA). Cellular protein resolved by SDS\u2010PAGE was immunoblotted as previous study . Primary antibody for tested protein was listed as follows:Immunoreactive bands were revealed by ECL and visualized by the KODAK Image Station 4000MM PRO.2.85\u2032\u2010CCACGACCCTCTTTGTCTT \u20103\u2032;5\u2032\u2010GGAGGAGTATGACCTGGAT \u20103\u2032;5\u2032\u2010TTCTCCGAACGTGTCACGT \u20103\u2032;To knock\u2010down of AQP1 in HTMCs, the following siRNA sequences (sense strands) and scrambled control were purchased from Dharmacon :\u03b2\u2010catenin was knocked down with shRNA used in a previous study.To knock\u2010down of ATF4 in HTMCs, siRNA sense strands and scrambled negative control were purchased from Santa Cruz Biotechnology, CREB\u20102 siRNA(h):35\u00a0112).2.9Recombinant adenovirus coding AQP1 (Ad\u2010Flag\u2010AQP1) or for comparative control (Ad\u2010Flag) was constructed to express ZsGreen protein as a marker for the identification of infected cells.2.10P\u00a0<\u00a0.05).The pRL\u2010TK vector containing Renilla luciferase gene was cotransfected as an internal reference to correct the transfection efficiency. Luciferase activity of negative control pGL3\u2010Basic vector was used. The significance of luciferase activity was measured using a Dual\u2010Luciferase Reporter System (Promega). The activity differences were analysed using one\u2010way ANOVA test. The values were averaged from three independent replicates. Error bars represent SD (n\u00a0=\u00a03). Diverse letters aside by the column are defined as statistically significant difference . All data were expressed as mean\u00a0\u00b1\u00a0SEM. Statistical significance was analysed by one\u2010way ANOVA followed by a Student's 33.1Given that\u00a0ET\u20101 contributes to the pathogenesis of POAG, we adopted an ET\u20101 delivery strategy to establish models of POAG in vivo and in vitro. Topical administration of ET\u20101 to rabbit eyes for 2\u00a0weeks led to significant IOP elevation  was significantly attenuated in TM tissue of rabbits challenged with topical ocular ET\u20101 Figure A. Immuno3.3We then set out to determine whether AQP1 reduction was responsible for the pathological alteration of ET\u20101\u2010induced POAG. Knock\u2010down of AQP1 with si\u2010AQP1 in HTMCs led to a collapse of actin arcs and formation of thick actin bundles Figure A, altera3.4AQP1 has been reported to promote cell migration through its interaction with \u03b2\u2010catenin.3.5AQP1 promoter region (\u22122000\u00a0bp to +2500\u00a0bp)  (Figure AQP1 were fused to the luciferase reporter gene. Utilizing luciferase assay, we detected that the transcriptional activity of AQP1 is negatively regulated by ATF4, suggesting that upon ET\u20101 stimulation of HTMCs, ATF4 can bind directly to the AQP1 promoter region and negatively regulate AQP1 transcription (Figure We further investigated the possible mechanism underlying ET\u20101\u2010induced AQP1 suppression. We observed that ET\u20101 stimulation led to a time\u2010dependent inhibition of AQP1 at both protein and mRNA level Figure A,B. Give3.6In order to determine whether ATF4 is a requisite of ET\u20101\u2010triggered stiffness of HTMCs, we adopted a siRNA approach to knock\u2010down ATF4 Figure A,B, whic4Chronically elevated IOP is known to be the most important risk factor of POAG. Elevated IOP is implicated by disturbed AH homeostasis with increased resistance to drainage. Together with Schlemm's canal, TM facilitates the out\u2010flow of aqueous fluid and control of IOP in the eye. It has been observed that elevated IOP is significantly correlated with elevated ET\u20101 concentrations in AH of POAG patients.A relatively long process of gradually elevated IOP results in patients developing POAG. TM tissue remodelling is a complicated process that includes a series of intra\u2010trabecular meshwork cell responses to physical and chemical stress. ET\u20101, a small peptide with 21 amino acids, is synthesized and secreted by endothelial cells. In the eye, ET\u20101 can be synthesized at many locations, such as the vascular endothelium, retinal pigmented epithelium and non\u2010pigmented ciliary epithelium. Once secreted, it can then be circulated in the anterior and posterior aqueous fluid.Using rabbits as an animal model, we observed elevated IOP and AQP1 suppression in TM cells of eyes to which ET\u20101 was topically applied. AQP1 is expressed in the iris and ciliary epithelium and functions in aqueous fluid production and thus IOP regulation. More importantly, AQP1 is also expressed strongly in trabecular meshwork cells and regulates the cell volume.We noted that ET\u20101\u2010induced AQP1 suppression is highly associated with a reduction of \u03b2\u2010catenin, including stress fibre reorganization, collagen deposition and contractility alteration, which leads to a series of POAG characteristic features that affect HTMCs. Primary human trabecular meshwork cells used in the study were from one human patient due to donor limitation. However, our observations in the cell culture system correlate well with the results of the animal model. \u03b2\u2010catenin is an important regulator of the Wnt signalling pathway. Currently available knowledge related to the effects of AQP1/\u03b2\u2010catenin is mainly related to cytoskeletal remodelling,ET\u20101 has been reported to induce ER stressOur results show the harmful role of ATF4 in response to ET\u20101 in HTMCs. Interestingly, ET\u20101 can be synthesized and is secreted by human TM cells,Taken together, our results reveal a novel role of ATF4, where it functions in the process of POAG by negatively regulating the transcription of water channel AQP1 in HTMCs Figure A. This rNo potential conflicts of interest were disclosed.The authors contributed in the following way: YZ and YY conceived the study and designed the experiments. HZ performed the experiments with the help of YZ, XH, XS, XS, GL, XC, YY, JM, LC and XW. YZ, HZ and YY interpreted data and contributed to discussion. YZ and YY drafted the manuscript.\u00a0Click here for additional data file.\u00a0Click here for additional data file.\u00a0Click here for additional data file.\u00a0Click here for additional data file.\u00a0Click here for additional data file.\u00a0Click here for additional data file.\u00a0Click here for additional data file."}
+{"text": "To investigate the anti\u2010biofilm efficacy and working mechanism of several NaOCl concentrations on dual\u2010species biofilms of different architecture as well as the changes induced on the architecture of the remaining biofilms.Streptococcus oralis J22 and Actinomyces naeslundii T14V\u2010J1 were co\u2010cultured under different growth conditions on saliva\u2010coated hydroxyapatite discs. A constant\u2010depth film fermenter (CDFF) was used to grow steady\u2010state, four\u2010day mature biofilms (dense architecture). Biofilms were grown under static conditions for 4\u00a0days within a confined space (less dense architecture). Twenty microlitres of buffer, 2\u2010, 5\u2010, and 10% NaOCl were applied statically on the biofilms for 60\u00a0s. Biofilm disruption and dissolution, as well as bubble formation, were evaluated with optical coherence tomography (OCT). The viscoelastic profile of the biofilms post\u2010treatment was assessed with low load compression testing (LLCT). The bacteria/extracellular polysaccharide (EPS) content of the biofilms was examined through confocal laser scanning microscopy (CLSM). OCT, LLCT and CLSM data were analysed through one\u2010way analysis of variance (ANOVA) and Tukey\u2019s HSD post\u2010hoc test. Linear regression analysis was performed to test the correlation between bubble formation and NaOCl concentration. The level of significance was set at a\u00a0<\u00a00.05.The experimental hypothesis according to which enhanced biofilm disruption, dissolution and bubble formation were anticipated with increasing NaOCl concentration was generally confirmed in both biofilm types. Distinct differences between the two biofilm types were noted with regard to NaOCl anti\u2010biofilm efficiency as well as the effect that the several NaOCl concentrations had on the viscoelasticity profile and the bacteria/EPS content. Along with the bubble generation patterns observed, these led to the formulation of a concentration and biofilm structure\u2010dependent theory of biofilm removal.Biofilm architecture seems to be an additional determining factor of the penetration capacity of NaOCl, and consequently of its anti\u2010biofilm efficiency. Sodium hypochlorite (NaOCl) is the main irrigant of choice during root canal treatment, with concentrations employed ranging between 0.5% and 6% ; this is achieved by measuring the shifting that occurs at the greyscale level in pre\u2010 and post\u2010treatment greyscale images of biofilms acquired with the OCT  by means of confocal laser scanning microscopy (CLSM). The tertiary objective was to image real\u2010time by means of OCT the anti\u2010biofilm working action of the same concentrations of NaOCl during subtle flow.This study aimed at evaluating the anti\u2010biofilm efficacy of several NaOCl concentrations on dual\u2010species biofilms of different architecture. The primary objective was to assess by means of OCT the biofilm disruption and dissolution mediated by the static application of 2\u2010, 5\u2010, and 10% NaOCl on four\u2010day grown dual\u2010species biofilms comprised of clinical isolates of et al. et al. Streptococcus oralis J22 \u00a0and Actinomyces naeslundii T14V\u2010J1 (A. naeslundii)\u00a0were initially cultured in modified brain heart infusion broth (BHI)  . Next, the bacterial species were co\u2010cultured at concentrations of 6\u00a0\u00d7\u00a0108 cells\u00a0mL\u22121 for S. oralis and 2\u00a0\u00d7\u00a0108 cells\u00a0mL\u22121 for A. naeslundii for four days on saliva\u2010coated hydroxyapatite (HA) discs. This led to the formation of defined dual\u2010species biofilms in terms of thickness and structure  (control group), 2\u2010, 5\u2010, and 10% NaOCl . Before every experiment, a thiosulfate titration method was used to determine NaOCl concentration, and accordingly dilution with sterile demineralized water ensued. The treatment consisted of applying 20\u00a0\u03bcL solution statically (no flow) over the biofilms, followed by a 60\u00a0s interval, during which the biofilm samples were left undisturbed. Next, 20 \u03bcL sodium thiosulfate  was applied for NaOCl neutralization, and the samples were transferred in a volumetric jar with 20\u00a0mL adhesion buffer. The treated biofilms were scanned again with the OCT scanner under the same settings (post\u2010treatment scans). Quantification of changes on the biofilms was carried out by evaluating the scanned pre\u2010 and post\u2010treatment biofilm cross\u2010sections with an open\u2010source image analysis software . The distance in every column of pixels between the substrate and top of the biofilm  was calculated and compared for the pre\u2010 and post\u2010treatment images. To improve the accuracy of the data, different grayscale thresholds in each image were selected  and coherent layer  . Confocal laser scanning microscopy (CLSM) analysis of stained biofilm components, such as live/dead bacteria and extracellular polysaccharides (EPS), was also employed , and real\u2010time recording with the OCT scanner was performed for 60\u00a0s. Three biofilm samples per treatment group were used during three independent experiments.Furthermore, in order to gain more insight on the dynamics of the NaOCl bubble formation process over time, biofilm behaviour was registered in\u00a0real\u2010time by means of OCT during the subtle continuous flow of the several NaOCl concentrations over four\u2010day CDFF biofilms . Four\u2010day static biofilms had an extremely rapid and increased dissolution under the test conditions applied and were\u00a0therefore excluded from this type of experiment. CDFF biofilm\u2010carrying HA discs were inserted in a parallel plate flow chamber with the help of a custom\u2010made silicone mould; this ensured that the biofilm was always placed at the same level with regard to its vertical protrusion in the chamber, and parallel to the chamber surface and irrigant flow. Next, buffer (control), 2\u2010, 5\u2010 and 10% NaOCl were introduced at a low flow rate  and NaOCl concentration (predictor variable). Data are presented as mean and standard deviation (SD). The level of statistical significance was set at a\u00a0\u2264\u00a00.05.For each biofilm evaluation technique applied , 20 samples from each biofilm type were divided into four groups according to the treatment provided . Statistical analysis was performed using SPSS software . Normality of data was assessed through the\u00a0Shapiro\u2010Wilk test. One\u2010way analysis of variance (ANOVA) (or Welch\u2019s ANOVA) was carried out to detect the presence of significant differences among the various treatments employed for each biofilm type. Tukey\u2019s HSD (or Games\u2010Howell) P\u00a0<\u00a00.001), 2% NaOCl (P\u00a0<\u00a00.001) and 10% NaOCl (P\u00a0<\u00a00.001) , 2% NaOCl (P\u00a0=\u00a00.046) and 10% NaOCl (P\u00a0=\u00a00.005).Treatment with 5% NaOCl significantly increased biofilm disruption compared to the control  and 5% NaOCl (P\u00a0=\u00a00.018), while disrupting considerably more biofilm compared to 2% NaOCl  and 5% NaOCl (P\u00a0=\u00a00.003), while dissolving considerably more biofilm compared to 2% NaOCl.Treatment with 10% NaOCl significantly increased biofilm disruption compared to the control   Fig. b.Representative OCT scans of four\u2010day static biofilms pre\u2010 and post\u2010treatment are presented in Fig. P\u00a0=\u00a00.001), 5% NaOCl (P\u00a0<\u00a00.001) and 10% NaOCl (P\u00a0=\u00a00.002) , 5% NaOCl (P\u00a0=\u00a00.001) and 10% NaOCl (P\u00a0=\u00a00.007)  Fig. a and4 Maxwell element , 5% NaOCl (P\u00a0=\u00a00.001) and 10% NaOCl (P\u00a0=\u00a00.004)  Fig. a. The ma2 and E3 Maxwell elements in the four\u2010day CDFF biofilms (representing the bound water and EPS biofilm respectively) were not significantly affected, irrespective of the treatment applied  , as well as no significant differences among the several NaOCl concentrations , as well as no significant differences among the several NaOCl concentrations  were not significantly affected, irrespective of the treatment applied  and the control (P\u00a0<\u00a00.001). Also, treatment with 2% NaOCl resulted in a significantly higher per cent \u2018LIVE\u2019 bacteria compared to the control (P\u00a0=\u00a00.003) , while no significant differences were detected among the NaOCl groups , 2% NaOCl (P\u00a0<\u00a00.001) and 5% NaOCl (P\u00a0<\u00a00.001) , 2% NaOCl (P\u00a0<\u00a00.001) and 5% (P\u00a0<\u00a00.001)  Fig. . The linNbubbles\u00a0=\u00a04.6\u00a0\u00d7\u00a0CNaOCl \u2010 3.7, , where Nbubbles\u00a0=\u00a0amount of bubbles formed and CNaOCl\u00a0=\u00a0NaOCl concentration.P\u00a0=\u00a00.020), 2% NaOCl (P\u00a0=\u00a00.043) and 5% (P\u00a0=\u00a00.022). The linear regression analysis did not reveal any linear correlation between NaOCl concentration and the amount of bubbles formed.For the four\u2010day static biofilms, treatment with 10% NaOCl generated significantly more bubbles compared to control  concentrations, micro\u2010volume NaOCl solutions were applied statically over two structurally defined biofilms for a finite time interval (60\u00a0s). In the present study, NaOCl action was dependent solely on diffusion (and not convection). In that sense, factors that could alter the process of NaOCl diffusion into the bulk biofilm besides concentration, such as the biofilm structure, NaOCl reactivity with the biofilm matrix and time allowed for NaOCl diffusion (60\u00a0s) should be also taken into consideration.\u2010) and the extracellular polymeric substances of the biofilm matrix, such as proteins and polysaccharides  of NaOCl . By taking a closer look at the behaviour of the CDFF biofilms using OCT scans and snapshot images, an increase in the biofilm height Fig. a. AppareWith regard to the structurally less compacted four\u2010day static biofilms, the expected increased anti\u2010biofilm efficacy with increasing NaOCl concentration was only partially confirmed. In contrast to the dense CDFF biofilms, the increased anti\u2010biofilm capacity of the 10% NaOCl was clearly noticeable within the 60\u00a0s application interval, leading almost to complete biofilm disruption and dissolution. Moreover, the associated bubble count was significantly higher compared to the lower concentrations, indicating a stronger chemical effect resulting in gas\u2010associated bubble formation. Interestingly, 2\u2010 and 5% NaOCl concentrations did not demonstrate a significant difference in their disruptive and dissolving capacity, as opposed to their significant effect on the CDFF biofilms. Additionally, induction of bubble formation was barely noticeable and equally low in these groups.et al. The findings presented above suggest that biofilm structure drives the chemical interplay between the oxidizing reagent and the underlying biofilm and eventually the anti\u2010biofilm efficacy of NaOCl. Specifically, the four\u2010day static biofilms had a loose architecture compared to the four\u2010day CDFF biofilms as a result of the decreased bacterial density, increased EPS presence and significantly increased amount of \u2018free\u2019 water  and increase of the influence of the bacterial component (E4) noted for the 2% NaOCl\u2010treated CDFF biofilms. By increasing NaOCl concentration to 5%, penetration is increased and a chemical reaction with the deeper situated EPS occurs. This results in the formation of more bubbles in deeper layers  is illustrated Video . These lThe CLSM findings for the four\u2010day CDFF biofilm support the argument above and are consistent with the structural features of this biofilm type. Significant changes were detected only in the bacterial cell component of the biofilm (predominant biofilm component), while EPS remained mostly unaffected (less predominant biofilm component). Indeed, in the 10% NaOCl\u2010treated CDFF biofilms, per cent LIVE and DEAD bacteria were the highest and lowest respectively, compared to the other groups. This indicates that a chemical effect other than bacterial killing took place and is also in accordance with the biofilm detachment observed.et al. \u2010 and the EPS.In the four\u2010day static biofilms, the richer EPS content increases the reactivity sites with NaOCl, thereby limiting its penetration. Interestingly, for the 2\u2010 and 5% NaOCl, reactivity with the EPS seemed to be similarly low, as indicated by the lack of significant difference in biofilm disruption (OCT findings) and presence of EPS (CLSM findings) in the treated biofilms. The significant amount of water present in this biofilm type  comes in contact with an organic substrate (biofilm). Nevertheless, this is the first study linking bubble growth to biofilm removal and NaOCl concentration. Increasing NaOCl resulted in an increase in the amount of bubbles generated, irrespective of the biofilm type. While the generalized notion supporting that by increasing NaOCl concentration the chemical reactions with the underlying biofilm are potentiated and prolonged holds true, factors that influence bubble coalescence should not be overlooked. It has been demonstrated that the mechanism of bubble coalescence is strongly dependent on the presence of specific electrolytes and their concentration in aqueous solutions . The study of the viscoelastic properties combined with confocal laser scanning microscopy analysis of the remaining biofilms provided supplemental information in support of certain hypotheses about the interpretation of the findings presented. Lastly, a theory about the effect of the concentration of NaOCl on the stability of the bubbles generated was proposed based on bubble coalescence models.Dr. Busanello and Prof. Dr. So were financially supported by a CNPq scholarship, and a part of the study was financed by a Research Grant of the European Society of Endodontology (ESE). All other authors state explicitly that there are no conflicts of interest in connection with this article.Video S1. Real\u2010time rendering  showing a four\u2010day mature S. oralis J22/ A. naeslundii T14V\u2010J1 biofilm grown in a constant depth film fermenter during introduction of buffer at a flow rate 3.33 mL\u00a0min-1 in a parallel plate flow chamber (flow direction is from right to left). No biofilm removal or bubble formation is evident.Click here for additional data file.Video S2. Real\u2010time rendering  showing a four\u2010day mature S. oralis J22/ A. naeslundii T14V\u2010J1 biofilm grown in a constant depth film fermenter during introduction of 2% NaOCl at a flow rate 3.33 mL\u00a0min-1 in a parallel plate flow chamber (flow direction is from right to left).Click here for additional data file.Video S3. Real\u2010time rendering  showing a four\u2010day mature S. oralis J22/ A. naeslundii T14V\u2010J1 biofilm grown in a constant depth film fermenter during introduction of 5% NaOCl at a flow rate 3.33 mL\u00a0min-1 in a parallel plate flow chamber (flow direction is from right to left).Click here for additional data file.Video S4. Real\u2010time rendering  showing a four\u2010day mature S. oralis J22/ A. naeslundii T14V\u2010J1 biofilm grown in a constant depth film fermenter during introduction of 10% NaOCl at a flow rate 3.33 mL\u00a0min-1 in a parallel plate flow chamber (flow direction is from right to left).Click here for additional data file."}
+{"text": "Physical fitness (PF) is a multi-component construct and a biomarker of health. Worse PF is related to vulnerability and predicts worse academic achievements. Thus, assessing PF is important to monitor health in youth. This systematic review aimed to identify and inform physical education, health professionals and entities about existing PF batteries and field-tests that can be used in school settings. A comprehensive literature search was carried out in five electronic databases  to identify PF battery protocols that can be carried out in the school setting. Overall, 24 PF batteries were identified. Regarding the PF components assessed, only cardiorespiratory fitness and upper body strength were contemplated in all batteries. Middle-body strength and lower body strength were presented in most batteries . Agility (16 of 24) and body composition (16 of 24) were also considered in several batteries, although to a lesser extent. Flexibility (14 of 24) and speed (12 of 24) were the PF components less represented in the batteries. Among the 24 identified PF batteries, 81 PF tests assessing the different PF components were encountered. The advances in the PF field-based assessment in school settings and health in youth resulted in the amplification of the number of existing batteries. Considering the connection between PF and health and the opportunity that the school setting provides to assess fitness in children and adolescents, there is a need for standardization and a consensus of PF assessments in this specific setting. Physical fitness (PF) is a multi-component construct and a biomarker of health , 2. WorsAssessing PF reflects the impact of genetic and environmental factors on health-related PF components and consequently on health indicators . In lighPrevious systematic reviews identified a large number of test batteries available worldwide to test children's and adolescents' PF levels \u201318. ThesSo far no systematic review that provides a summary of all existing fitness test batteries for children and adolescents that can be carried out in the school setting under the specific circumstances of the school  has been carried out. Therefore, this systematic review aimed to identify and summarize the existing field-based health-related PF batteries that can be performed in children and adolescents to monitor and improve their health status.Data selection, collection, and analyses were performed following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement .*\u201d OR \u201cphysical performance\u201d OR \u201csport performance\u201d OR \u201cphysical condition\u201d OR \u201caerobic capacity\u201d OR \u201cmaximum oxygen consumption\u201d OR \u201cstrength\u201d OR \u201cflexibility\u201d OR \u201cmotor\u201d OR \u201cendurance\u201d OR \u201cspeed\u201d OR \u201cagility\u201d OR \u201cbalance\u201d OR \u201cbody composition\u201d OR \u201canthropometry\u201d OR \u201cbody mass index\u201d OR \u201cBMI\u201d OR \u201cskinfolds\u201d OR \u201cwaist circumference\u201d AND \u201cbatter*\u201d OR \u201cprotocol*\u201d OR \u201cassess*\u201d OR \u201cvalid*\u201d OR \u201creproduct*\u201d OR \u201cfeasab*\u201d OR \u201cmeasur*\u201d AND \u201cadolescent*\u201d OR \u201cchild*\u201d OR \u201cyoung*\u201d OR \u201cschool age\u201d OR \u201cschool-aged\u201d OR \u201cyouth\u201d. The keywords were selected and defined by consensus from all authors. Furthermore, the reference lists of individual studies that reported results or used PF batteries in their methodologies but did not present the protocol were searched for records containing those protocols. Records identified through this method were added as records identified through other sources.Five international databases  were searched for scientific articles published in peer-reviewed journals until the 30th of April 2020 containing PF battery protocols. In each database, a search was conducted taking into account a predefined combination of keywords. The combination of keywords used in each database was the following: \u201cfield-based test\u201d OR \u201cfitth of April 2020. Only records presenting PF batteries comprising field-based health-related PF tests for children and adolescents that could be performed in the school setting were included. Thus, inclusion criteria were the following: (1) presenting results on the identification, structure, validity, reliability or feasibility of PF batteries, or parts of it (including specific tests), assessing health-related PF components in children and adolescents; (2) containing PF batteries comprising field-based tests that can be performed in the school setting; (3) having a cross-sectional, prospective, observational, experimental, or narrative review study design; (4) being written in English, French, German, Spanish, or Portuguese. Records presenting findings on motor skills, other populations that were not children or adolescents, or not meeting all inclusion criteria were excluded.This systematic review includes scientific articles from peer-reviewed journals that contained PF battery protocols published until the 30The data extraction process was conducted based on PRISMA guidelines . After dEach identified PF battery was entered into a Microsoft Excel  spreadsheet, including information on author and year of publication; country; setting and age range of application; PF components assessed, and the PF tests used for each assessed component. The considered components of PF were body composition, CRF, upper body strength, lower body strength, middle-body strength, speed, agility, and flexibility. Also, a narrative synthesis was performed to describe each field-based health-related PF test in the identified PF batteries.n=5,838), 4,385 records were screened based on title and abstract, resulting in 4,154 records excluded. A total of 231 records were assessed for eligibility by full-text reads. Finally, 33 articles matched all inclusion criteria and were included in the qualitative synthesis. The flow chart of records selection is presented in A total of 10223 records  were identified. After removing duplicates (Most PF batteries (21 of 25) are exclusively for children and adolescents, while four of them are also extended to young adults  and adulRegarding the PF components assessed in the batteries, only the CRF and the upper body strength, endurance and power were contemplated in all PF batteries. Middle-body and lower body strength, endurance and power were presented in most of the PF batteries, 21 of 25 and 20 of 25, respectively. Other components as agility (17 of 25) and body composition (16 of 25) were also contemplated in most PF batteries, although to a lesser extent. Flexibility (14 of 25) and speed (13 of 25) were the PF components less represented in the batteries, notwithstanding they were present in at least 50% of the identified PF batteries.Among 25 identified PF batteries, a total of 87 PF tests, assessing the different PF components, were encountered. The PF component with the widest variety of different tests, that is, with 23, was CRF. It was followed by upper body strength, endurance and power with 21, speed with 10, middle-body strength and endurance with nine, body composition with eight, agility with seven, lower body endurance and power with five and flexibility with four different tests.This systematic review provides a summary of existent PF batteries from around the world containing field-based health-related tests that can be performed by children and adolescents and used to monitor health status. A total of 25 different PF batteries from European, American, Asian, and Oceanian countries were identified. This knowledge can be useful for selecting standardized and validated PF tests and batteries, adjusted for the school setting and considering different PF components, and simultaneously, allows direct comparison between peers of the same age from different geographic locations.Among children and adolescents, PF is associated with numerous health indicators, thus assessing PF has been suggested to be a reliable tool to monitor health in youth . FurtherBeing a multi-component construct, examining PF as a whole, using only one or two tests is a misconception, as different associations between PF components and health indicators are observed , 45. BecAssessing body composition is usually the result of different anthropometric measures and their relation, such as height, weight, or waist circumference, as well as methodologies to analyse the % of body fat, muscle mass, and hydration . The meaThe CRF is the most studied component of PF among children and adolescents , and notMuscular fitness, another important PF component, was also assessed in each of the PF batteries identified. However, different components of MF  were assessed across the batteries. Similar to CRF, MF is also associated with several health outcomes in youth , 46. A tA total of 25 PF batteries were identified in this systematic review and across them 87 different PF tests for body composition, CRF, and MF. A previous systematic review focused on PF tests indicated that the PACER (or 20-meter shuttle run), the handgrip strength and standing broad jump tests, the 4 \u00d7 10m shuttle run test, weight, BMI, skinfolds, circumferences, and % body fat estimated from skinfold thickness were the most reliable field-based PF tests for children and adolescents . In thisThis systematic review is not without some limitations. Firstly, the large number of articles and protocols for the same PF test may have resulted in an overlap of tests. Secondly, the terms selected to identify investigations and other documents describing the PF batteries, although highly thorough nevertheless may have excluded documents not matching the inclusion criteria. Also, the search was conducted in only five databases. Lastly, because of the different study designs and the integration of gray literature  the risk of bias and study quality assessment was unfeasible. Yet, most importantly, the major strength of this review is the ample number of articles reviewed and time interval search, which resulted in the identification of a rich set of PF batteries from around the globe.The advances in the PF field-based assessment on school settings and health in youth resulted in the amplification of the number of existing batteries. On the one hand, diversity allows choosing the battery that most fits the specific purpose and setting of the assessment. On the other hand, it somehow complicates the comparability of data from different contexts, countries, or regions. Therefore, considering the connection between PF and health and the opportunity that the school setting provides to assess fitness in children and adolescents, we highlight the need for standardization and a consensus of PF assessments in this specific setting. In the European Union, a unique and actualized European PF battery would allow comparisons between European children and adolescents from different countries, to contribute to adequate and specific education and health public policies in the future.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.AM and MP: conception and design and drafting the manuscript. DH-N, MP, JM, and FG: data acquisition. AM, SP, and BM: data analysis and interpretation. YD, AS, JM, DH-N, and AI: critical revision for intellectual content. DH-N and FG: administrative, technical or material support. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "Available and practical methods for privacy preserving linkage have shortcomings: methods utilising anonymous linkage codes provide limited accuracy while methods based on Bloom filters have proven vulnerable to frequency-based attacks.In this paper, we present and evaluate a novel protocol that aims to meld both the accuracy of the Bloom filter method with the privacy achievable through the anonymous linkage code methodology.The protocol involves creating multiple match-keys for each record, with the composition of each match-key depending on attributes of the underlying datasets being compared. The protocol was evaluated through de-duplication of four administrative datasets and two synthetic datasets; the \u2018answers\u2019 outlining which records belonged to the same individual were known for each dataset. The results were compared against results achieved with un-encoded linkage and other privacy preserving techniques on the same datasets.The multiple match-key protocol presented here achieved high quality across all datasets, performing better than record-level Bloom filters and the SLK, but worse than field-level Bloom filters.The presented method provides high linkage quality while avoiding the frequency based attacks that have been demonstrated against the Bloom filter approach. The method appears promising for real world use. Privacy preserving record linkage (PPRL) protocols involve determining which records from data collections describe the same individual where these records are encrypted or encoded so as to protect privacy. The challenge for these protocols is to allow for variations in data arising from missing, changed or incorrect identifiers  while at the same time ensuring that no information about the individuals within the dataset is revealed.honest-but-curious) model of security [PPRL techniques typically adopt a semi-honest  would result in those records being identified as belonging to different individuals. However, using too few identifiers can have the opposite effect, namely that separate individuals would be identified as the same person. This approach tries to use the optimum level of identifying information, allowing some error tolerance while correctly distinguishing between individuals .Cryptographic hash functions are used to convert the concatenated identifiers into a fixed length encoded form. These hash functions have several important properties that make them suitable for this purpose. They are deterministic, meaning the same input will produce the same encoded output. They have the property that a small change in the data input will change the hash value extensively so that the new hash value does not appear correlated with the old hash value. They are also one-way functions, meaning that it is not feasible to determine the original input data when given only the hash value, other than by hashing guesses of the possible input and checking these against the original hash value . To ensuThere are several variants of the anonymous linkage code approach. In Australia, the Statistical Linkage Key-581 (SLK) . AnotherHashed anonymous linkage keys combined with a secret key can provide strong privacy protection. Their weakness lies in ensuring a high level of linkage quality . Single This approach involves adding encoded personal identifiers into structures known as Bloom filters (a binary array); these Bloom filters are then compared. The encoding process uses a series of hash functions (again using the HMAC construction with a secret key) to map elements of the data field to positions within the Bloom filter. The encoding process allows string similarity metrics to be used, so that variations in spelling or typographical errors can be accommodated .There are two main variants of privacy preserving linkage using Bloom filters. The first is field based Bloom filters where every identifier  is encoded into its own Bloom filter. This allows the use of techniques typical of un-encoded record linkage, such as the use of field specific weights, and the ability to appropriately handle missing values, along with previously mentioned string similarity measures. Very high linkage quality has been achieved using this method . The secWhile Bloom filter methods have a greater tolerance for differences between records as compared to anonymous linkage codes, they also have weaker privacy protection. Both the field and record-level Bloom filter approaches have been shown to be vulnerable to frequency attacks -21. WhilSome of the earliest examples of privacy preserving record linkage involve encoding individual identifiers separately using hash functions. These encoded identifiers are then sent to the linkage unit who perform the linkage. Linkage can be carried out using standard deterministic or probabilistic methods; however, the encoding process does not allow string similarity comparisons to occur. To account for possible misspellings, techniques such as phonetic encoding can be used prior to the encoding process , 25.This method of linkage has been used in Germany and France for the linkage of cancer registries. The method appears to provide high linkage quality , 25; howIn this paper we present an alternate methodology to the approaches described above. The proposed method seeks to combine the best features of both approaches: namely, the privacy protection offered by the use of anonymous linkage code along with the linkage quality offered by the Bloom filter approach. An approach that could achieve linkage quality similar to the Bloom filter method without the associated privacy risks would be highly desirable .Unlike the Bloom filter approach, our method does not make use of approximate string matching. Rather it aims to achieve high quality linkage through utilising other important techniques from traditional (un-encoded) probabilistic linkage including the use of weights and methods for managing missing values.The proposed method extends the anonymous linkage code approach. For each record, a number of hashes are created, each from different sets of concatenated personal identifiers; we refer to these hashes as \u2018match-keys\u2019. Any pair of records with the same value for any particular match-key are identified as belonging to the same individual; that is, each match-key will directly identify an individual. Rather than use a predetermined set of match-keys, match-keys are generated based on parameters which describe the underlying characteristics of the data. These parameters are shared between the data custodians, and are used as input to the encoding process. Once encoded, data is sent to the linkage unit for linkage.The number of match-keys and the composition of each match-key are all determined as part of this privacy preserving approach. Importantly, this composition differs depending on the characteristics of the dataset(s) in question. These parameters are identified through utilizing methods from probabilistic record linkage.m and u probabilities. The m-probability is the likelihood that two records belonging to the same person have the same value for a particular field. The u-probability is the likelihood that two records belonging to different people have the same value for a particular field [Probabilistic linkage uses conditional probabilities to compute the likelihood of two records belonging to the same person . Recordsar field . These am and u probabilities for a particular dataset and for estimating the designated threshold [Numerous techniques exist for estimating hreshold . These ihreshold  and the hreshold .From the basic probabilistic model, it is possible to iterate through all possible combination of field state comparisons for a pair of records . We willm and u probabilities and an estimated threshold score, we can calculate the exact total score each combination of field comparisons would receive, and determine which would score above the threshold .The method as described creates match-keys for every field combination above the threshold. This can result in a large number of match-keys per record, creating large encoded datasets and increasing computational load. However, a great number of these created match-keys are redundant. For instance, if a match-key made up of encoded first name, surname and date of birth is considered identifying, then there is no need to also compute a match-key for first name, surname, date of birth and sex; no additional matches could possibly be found. In this way we can remove a large number of field combinations without affecting results. To identify the redundant field combinations is straightforward; given a field combination with a set of ree\u2019 see , any othPreliminary testing suggests this method can greatly reduce the number of match-keys required. A typical example of a linkage involving nine fields produced 402 field state combinations over the given threshold; after removal of unnecessary combinations, only 41 match-keys were required.In the next sections, we evaluate this simple method on a range of synthetic and real administrative datasets. We compare the results against those achieved with un-encoded linkage and against other privacy preserving techniques.De-duplication linkages were undertaken on a range of synthetic and real administrative datasets. Each dataset had either a truth-set available (for the synthetic datasets) or a gold standard benchmark with which to compare results . A range of different linkage methods were compared, including both un-encoded and privacy preserving methods. The un-encoded methods included probabilistic record linkage using approximate string matching and probabilistic linkage using exact matching only. Privacy preserving methods tested comprised field-level Bloom filters, record-level Bloom filters, the SLK-581 and the multiple match-key methodology. Parameters for each linkage method were calculated using the available truth-sets and gold standard benchmarks, with results reported at the threshold providing the optimal linkage quality (where F-measure was maximised). Parameters were shared across methodologies where possible. Results were compared using the precision and recall measures described below. Algorithms were implemented in Python 2.7 ; linkageSix separate datasets were included in the evaluation; two synthetic datasets and four real-world administrative datasets.The two synthetic datasets \u2018FER12\u2019 and \u2018BRO17\u2019 have been used in previously published research, and detailed information on the data generation process is available , 33. TheFour large-scale Australian health datasets were also used in this evaluation; these were hospital admission records from New South Wales (NSW) and South Australia (SA), and emergency department presentations from NSW and SA. Each dataset contained all records from the three years 2008-2010; only public hospital data was available in the South Australian datasets. Each dataset had previously been de-duplicated to a high quality by jurisdictional linkage units ; the links created by these units were used as the gold-standard benchmark against which our de-duplication results were compared. These linkage units utilised a variety of deduplication methods including intensive manual review of created links along with quality assurance procedures to analyse and review potential errors . The linEach dataset contained name information , sex, date of birth, and address information (street address and postcode). Fields used for linkage and the percentage of missing values within each dataset are described in Each dataset was de-duplicated using a range of linkage techniques; no linkages were conducted between any of the datasets. The same weights and blocking methods were used across linkage techniques, and multiple threshold scores were tested for each method . Agreement and disagreement weights were calculated directly from the available gold standard benchmark/truth-set. Two sets of blocks were used; Soundex of surname concatenated with sex, and full date of birth. This linkage strategy was based on a previously published \u2018default\u2019 strategy that has been regularly used in linkage evaluations , 37.Probabilistic linkage was carried out using un-encoded identifiers. All available variables were used in comparisons. Two probabilistic linkages were carried out; the first used the Jaro-Winkler string similarity metric  for alphField based Bloom filters were created according to a previously published methodology , 15. BloRecord based Bloom filters were created based on the cryptographic long-term key construction by Schnell , using tThe standard SLK-581 was also evaluated, created from the second and third letters of the individual\u2019s first name, the second third and fifth letters of their surname, along with full date of birth and sex .For the multiple match-key algorithm, weights were used to generate field state combinations. Linkage quality was calculated on all generated match-keys over the chosen threshold. The SHA-1 hash algorithm was used with output truncated to 90 bits per hash; this provided adequate security against collisions  while reducing file sizes.Linkage quality was measured using pairwise precision and recall, with the F-measure used as an overall metric of linkage quality. Results were reported at the threshold which maximised the F-measure.Linkage quality results for each tested linkage method across all six datasets are shown in As expected, un-encoded probabilistic record linkage using approximate string matching achieved the highest linkage quality across all datasets. Generally, the use of approximate string matching as compared to exact matching resulted in minor decreases in linkage quality; this decrease was larger for the synthetic datasets, likely due to their higher rates of error.In regards to privacy preserving techniques, field-level Bloom filters provided the highest linkage quality on all but one of the tested datasets. The multiple match-key method was the next best in terms of quality, with results only slightly below those for the field-level Bloom filters on most datasets. The record-level Bloom filters typically performed below that of the multiple match-key method, except for the SA Hospital dataset, where all three of these PPRL methods performed equally. The SLK method performed adequately on three of the four administrative datasets, however, results were lower than for all other tested methods. This method performed notably poorer for the NSW hospital dataset and both synthetic datasets due to the preponderance of missing values in these files.One notable outlier was the results from the BRO17 dataset, where the multiple match-key method outperformed all compared methods, including un-encoded methods. We attributed this to the fact that the multiple match-key method does not require blocking; the BRO17 dataset had high levels of missing values in all fields and the standard blocking strategy was likely not appropriate here.The number of hashes created in the multiple match-key method for each dataset varied from 14 (FER12 synthetic data) to 83 . Time taken for data encoding and linkage, and encoded file sizes (not reported but available from authors) were comparable to other evaluated methods.In general, the privacy preserving linkage methods evaluated here showed high linkage quality, providing continuing evidence of the viability of this method of record linkage. This was particularly apparent in datasets with few missing values or errors in identifiers, where all tested methods provided very high linkage quality.Based on these results, field-level Bloom filters are the privacy preserving method which provides the greatest linkage quality. The high quality returned from our linkages were consistent with those achieved previously . HoweverIn contrast, while the SLK method is simple to implement and can provide strong privacy protection if used appropriately , it does not appear suitable as an all-purpose privacy preserving linkage method, given the very poor linkage quality seen with some of the datasets. Although not tested here, we expect other anonymous linkage code methods to perform similarly to the SLK.The multiple match-key method introduced in this paper provided admirably high linkage quality. It was superior to the SLK method, which was the only evaluated privacy preserving method with similar privacy protections. The field level Bloom filter was the only privacy preserving method to produce higher linkage quality; this method has known deficits in terms of its privacy protections -21. It wA key consideration in assessing the viability of the multiple match-key privacy preserving method was determining the extent to which string similarity matching (which this method does not use) contributes to high linkage quality. Previous studies comparing results using string similarity matching to those without have found large decreases in error rates for some datasets . A numbeThe multiple match-key method presented here appears highly resistant to both dictionary and frequency attacks. Dictionary attacks are not possible through the use of a secret key in hashing (the HMAC construction) which is shared amongst data custodians and kept from the linkage unit. Frequency attacks also do not appear possible. Each particular match-key generated by this protocol is made up of a combination of fields that directly identifies an individual. If the same value of a match-key exists in two or more records, this means these records belong to the same person. As such any frequency analysis of match-keys will simply provide a list of which individuals who are found in the datasets most often, rather than provide any information on their identifiers.The hash-based encoding process used in this protocol means that similar input values do not result in similar match-keys, a feature of the Bloom filter approach which has allowed frequency attacks to occur. As the protocol does not create match-keys if one of their component fields is a missing value, it is also not possible to perform frequency attacks of match-keys on the subset of records where particular fields are missing. The use of inappropriate match-keys  would allow frequency attacks to occur. This could potentially occur through human or other error. Such a match-key is not advisable not just on privacy grounds but also on quality grounds, as it would of course also result in extremely poor linkage quality . In practice, this type of error would be easy to identify before data is encoded and sent to the linkage unit, and so is unlikely to occur.The use of more than one match-key provides one vector by which information about the individuals can be learnt. Information is leaked when comparing two records with some match-keys matching and others not-matching. For instance, if two records have the same match-keys for combinations that do not include surname, but different match-keys for combinations that do include surname, it is likely that the surnames differ between these matching records. This can reveal information about the record in question; for instance, as it is more common for women than men to change surname in their lifetime, we could guess that this record is more likely to be female than male. While the use of multiple match-keys can leak information, it does not appear able to re-identify an individual; rather, it suggests broad demographic groupings of which a record may be part. This privacy issue is not unique to the multiple match-key method but is inherent in all privacy preserving methods which use multiple encoded values. In situations where greater privacy considerations are required such that no information about an individual can be inferred, a single match-key (i.e. the SLK approach but using the HMAC construction) is the most viable option, despite its associated reduction in linkage quality.The privacy preserving method presented here achieves both high accuracy and appears to provide strong privacy protection. While the absence of approximate string matching in the method may present as a limitation, our results suggest that, in general, approximate string matching provides limited quality improvement. However, for certain datasets, approximate matching will be of greater importance, such as those with very few identifiers or large numbers of typographical errors, and in these situations, we expect the multiple match-key protocol will likely perform worse than other techniques such as record-level Bloom filters.The method proposed here is an extension of the anonymous linkage code concept to utilise more than one match-key. A similar method has been proposed by the ONS , althougFurther research is needed to investigate the performance of the multiple match-key method (as well as the other methods) in a real-world setting, where parameters must be estimated rather than calculated. Techniques for estimating weights and thresholds necessary for the multiple match-key methodology exist and have received evaluation in privacy preserving contexts . It shouIn this paper we describe and evaluate a new approach to PPRL. The results of our evaluation suggest this method can achieve very high quality results, while at the same time providing strong privacy protection.The differing privacy preserving protocols evaluated in this paper each have their own strengths and weaknesses, and will each be suitable in particular scenarios. The multiple match-key protocol does not achieve as high a quality as field-level Bloom filters but offers greater privacy protection. It provides slightly better linkage quality in most scenarios as compared with the record-level Bloom filter approach, while providing greater certainty regarding privacy. Finally, it provides greater linkage quality than that offered by a solitary match-key such as the SLK method. As such, we feel this protocol is an important and timely contribution to the current state of the art.This study received ethical approval under the Population Health Research Network\u2019s Proof of Concept project, which included approval for developing and refining linkage methodology. Approval was obtained from Curtin University Human Research Ethics Committee as well as from New South Wales Cancer Institute Human Research Ethics Committee and South Australian Department of Health and Aging Human Research Ethics Committee."}
+{"text": "Ji\u0159\u00ed (Georg) Proch\u00e1ska Fig.\u00a0 was bornSometimes, misfortunes paradoxically have a positive effect\u2014this was also the case in Proch\u00e1ska\u2019s life: touched by disease during typhus epidemics in 1771\u20131772, he was hospitalized in the Clinic run by Professor Haen, a famous Viennese physician, who noticed great potential in young Proch\u00e1ska and his desire for knowledge. Their meeting resulted in a surprising outcome\u2014after his recuperation, Proch\u00e1ska was given a job at the hospital, which provided him with sufficient financial resources to live and to study medicine. In 1774, he gained a position as a second assistant and, soon after, became first assistant to his mentor .Dissertatio Inauguralis Medica de Urinis. The subject of the doctorate corresponded to the interests of the era, still heavily influenced by the dogmas of Hippocrates\u2019 and Galen\u2019s theory of the four humors [In 1776, along with Haen\u2019s death, Proch\u00e1ska lost his position but he continued his work with another prominent specialist of the time\u2014professor of anatomy and ophthalmology, Josef Barth. He graduated under Barth\u2019s supervision and, on November 9, 1776, defended his doctoral dissertation, written in Latin, devoted to issues related to urine: r humors \u20134.De Carne Musculari Tractatus Anatomico-Physiologicus. In this dissertation (in Latin), he tried, in an interesting way, to explain the physiology of skeletal muscle contraction. Proch\u00e1ska hypothesized that contraction of a muscle is a consequence of the expansion of blood vessels, enabling an abundant supply of blood to the muscle tissue. According to his concept, increased blood flow to the muscle led to distortion of the muscle fibers, eventually provoking their contraction, and thus the contraction of the entire muscle [Proch\u00e1ska's research work resulted in a number of publications shortly after graduation. In 1778, he published a treatise on the anatomy and physiology of the circulatory system. In the same year, he published his first dissertation addressing neurological issues: e muscle . His conmagister of ophthalmology. In the same year, Proch\u00e1ska was offered a job at the University of Prague, where he attained the position of professor of anatomy and ophthalmology [On March 29, 1778, Proch\u00e1ska was promoted to almology . A few yAfter Professor Barth\u2019s resignation in 1791, Proch\u00e1ska returned to Vienna, taking up a position after his former mentor and promoter. As professor of anatomy, physiology and ophthalmology, he worked until his retirement in 1819. Proch\u00e1ska continued his research in Vienna, which resulted in further works in the field of anatomy and physiology, but he did not return to strictly neurological issues. Among these publications, the most important was a textbook on physiology, translated into several languages over the course of the nineteenth century. During his time in Vienna, Proch\u00e1ska created an imposing collection of anatomical preparations, later bought by the government for the enormous sum of 6000 florins. From 1805, he focused primarily on physiology, entrusting anatomical issues to his prosector, Michael Mayer. In addition to his scientific work, Proch\u00e1ska also ran a famous ophthalmology practice\u2014widely regarded as an outstanding Viennese specialist, he carried out over 3,000 cataract surgeries. At the same time, he was a man of great heart\u2014he treated and operated the poor for free [As well as his medical and university work, Proch\u00e1ska also participated in the activities of several scientific societies\u2014in Prague, Nancy and St. Petersburg. He was not only a good teacher, but also a great organizer\u2014for example, on the occasion of the visit of the Emperor Joseph II to Prague in 1783, Proch\u00e1ska gained funds to renovate the seat of the anatomy faculty at the local university [Not much is known about Proch\u00e1ska's private life. In 1779, he married Anna \u0160t\u011bp\u00e1nsk\u00e1, daughter of a Prague councilor, with whom he had a daughter, Emilia, in 1792. His wife passed away in 1810, at the age of 49. Proch\u00e1ska probably also had some artistic interests\u2014for example, it is recorded that, while on honeymoon, he was involved in painting and music. He died in Vienna, at about 7 O\u2019clock in the morning, on July 17, 1820, and was buried in St Mark\u2019s Cemetery , 10.Ji\u0159\u00ed Proch\u00e1ska was an ophthalmologist, physiologist, and anatomist but it is impossible to consider him a neurologist. Paradoxically, however, his contribution to the development of the emerging field of neurology is undeniable. His theory of the reflex, with its afferent and efferent branches and a decision-making center in between, became one of the foundations of clinical, experimental, and theoretical neurology in the century to follow."}
+{"text": "Colletotrichum lindemuthianum), Angular leaf spot (Pseudocercospora griseola) and Pythium root rot are important pathogens affecting common bean production in the tropics. A promising strategy to manage these diseases consists of combining several resistance (R) genes into one cultivar. The aim of the study was to determine genetic linkage between gene pairs, 2Co-4/Phg-2, on bean-chromosome Pv08 and Co-5/\u201cP.ult\u201d on-chromosome Pv07, to increase the efficiency of dual selection of resistance genes for major bean diseases, with molecular markers. The level of recombination was determined by tracking molecular markers for both BC3F6 and F2 generations. Recombination fraction r, among gene pairs, the likelihood of linkage, L(r), and logarithm of odds (LOD) scores were computed using the statistical relationship of likelihood which assumes a binomial distribution. The SCAR marker pair SAB3/PYAA19 for the gene pair Co-5/\u201cP.ult\u201d exhibited moderate linkage (r = 32 cM with a high LOD score of 9.2) for BC3F6 population, but relatively stronger linkage for the F2 population (r = 21 cM with a high LOD score of 18.7). However, the linkage among SCAR marker pair SH18/SN02, for the gene pair 2Co-4/Phg-2 was incomplete for BC3F6 population (r = 47 cM with a low LOD score of 0.16) as well as F2 population (r = 44 cM with a low LOD score of 0.7). Generally, the weak or incomplete genetic linkage between marker pairs studied showed that all the four genes mentioned earlier have to be tagged with a corresponding linked marker during selection. The approaches used in this study will contribute to two loci linkage mapping techniques in segregating plant populations.Anthracnose ( Diseases are critical production constraints for common beans in tropical countries, causing total crop failure when several pathogens attack susceptible bean genotypes under favorable conditions . Molecular markers and genetic maps accelerate identification of desirable homozygotes without need for progeny tests  marker pairs and their co-segregations could help to reduce sample sizes and time during marker assisted pyramiding (stacking together) of major genes in breeding programs for managing multiple diseases of common bean.Practically, a polymorphic marker must co-segregate with the gene of interest and so be present in the resistant progeny lines but absent in susceptible ones  score . On the other hand, the nonparametric linkage analysis is a model-free approach that studies the probability of an allele being identical by descent. Balding et al.  and StraThe recombination fraction estimate with the highest LOD score is considered the best estimate. The two-point LOD score between two loci, that is, a trait and a marker or marker-marker loci in this study was typically calculated over several recombination fractions between 0 and 1/2, and the recombination fraction that maximizes the likelihood (the maximum LOD score) is considered to be the best estimate of the recombination fraction  marker PYAA19 developed by Mahuku et al.  and species (ultimum) names. The gene symbol (\u201cP.ult\u201d) was thus put between quotation marks within the text and not italicised, unlike the other genes in this study  because it is not an official genetic symbol in common beans.In the study, the gene symbol \u201cP.ult\u201d is for u et al.  for seleThe findings will contribute to the efficiency of marker assisted pyramiding of disease resistance genes in common bean leading to simultaneous expression of more than one gene in a variety to develop durable resistance expression  to combine six disease resistance genes of common bean, namely 2, Co-5, Phg-2,Co-4 \u201cP.ult\u201d, I and bc-3  were identified among the 345 plants of BC3F6 population using genotypic electrophoresis gel profiles, harvested and seed used as parents to develop new crosses and populations . The F1 seeds from the two populations were harvested separatelty dried and planted in the screen house in the second season in 2015 to generate two populations of F2 plants which were monitored until maturity and harvested. The F2 seeds were planted in the screen house in sterile soils in raised wooden trays measuring 75 cm long \u00d7 45 cm wide and height of 13 cm during the third season to generate plants for DNA extraction.Five representative plants from progenies of the BC3F6 population in the field was collected from young leaves before flowering in eppendorf tubes, clearly labelled and transferred to the laboratory for extraction. DNA of each plant was isolated in the molecular laboratory facility at CIAT, Uganda, using the CTAB method according to Mahuku  and the sample left to incubate at room temperature for about 10 min in each wash, then the leaf disks were left to dry and ready for PCR reaction.Accu-Power PCR premix composed of DNA polymerase, dNTPs, reaction buffer, blue tracking dye and patented stabiliser. One microlitre of DNA for plants from BC3F6 population and leaf disk for the two F2 populations, 0.3 \u00b5l of forward and reverse primer, and 3.4 \u00b5l water was added to the premix to make a total reaction volume of 10 \u00b5l. The test sample tubes were placed in a thermocycler  for the PCR reaction cycles. Forward (F) and Reverse (R) primers of SCAR marker SBB14  was used to tag common bean lines with the 2Co-4 gene, SN02  for the Phg-2 gene, PYAA19  for \u201cP.ult\u201d and SAB3  for Co-5. The PCR products of markers used were separated on 1.2% agarose gel in 1X TBE at 140V for 30 min. The gel was then stained in 0.5ug/mL ethidium bromide for 20 min and the image was captured using the Syngene G: BOX gel documentation system .DNA samples for amplification through PCR were diluted to a factor of 1 in 30 \u00b5l solution and sorted out according to the field plan. The PCR reaction mix contained 5 \u00b5l of the 2,Co-4 SN02 for Phg-2, SAB3 for Co-5 and PYAA19 for \u201cP.ult\u201d) were computed for BC3F6 andF2 bean populationsto determine the number of recombinants and for further linkage analysis. This involved scoring polymorphic bands observed on electrophoresis gel pictures for each genotype using one for presence and zero for absence of bands. Recombinant genotypes were identified by counting band patterns in gel pictures in both BC3F6 and F2 populations.The SCAR marker frequencies  of obtaining the aforenetioned data set for recombination rates was computed using the following relationships, for example using data from BCThe LOD (log-odds) score is often used to assess the evidence for linkage and according to Balding et al.  is definL(data \u0406 r) = likelihood value at maximum estimate (MLE), while L(data \u0406 r = \u00bd) = likelihood value at maximum recombination fraction of 0.5.where r = recombination fraction. The two-point LOD score between two loci, that is, markermarker loci was calculated over several recombination fractions between 0 and 1/2, and the recombination fraction that maximizes the likelihood (the maximum LOD score) was considered to be the best estimate of the recombination fraction  and the rest of the genotypes (n= 183) were nonrecombinants  and LOD scores for testing linkage among gene pairs, mbinants . Similar2Co-4/Phg-2 are as shown in 2Co-4/Phg-2 was 0.47 showing weak linkage. However, the graph of the plot of likelihood (r) and recombination rates was maximum at 0.32 for gene pairs Co-5 and \u201cP.ult\u201d  and maximum recombination rates at 0.47 (the maximum likelihood estimate (MLE)) extracted from  \u201cP.ult\u201d . Similar \u201cP.ult\u201d  had the 2Co-4/Phg-2 and Co-5/\u201cP.ult\u201d from bean populations at F2 generation are shown in 2Co-4/Phg-2, 96 genotypes were recombinants  and the rest of the genotypes (n= 123) were non-recombinants  and LOD scores for testing linkage among gene pairs, mbinants . Similar2Co-4/Phg-2 is as shown in 2Co-4/Phg-2 was 0.44, showing weak linkage. However, the graph of the plot of likelihood (r) and recombination rates was maximum at 0.21 for gene pairs Co-5 and \u201cP.ult\u201d  and maximum recombination rates of 0.44 (the maximum likelihood estimate (MLE)) from  \u201cP.ult\u201d . Similar \u201cP.ult\u201d  had the 3F6 and F2 populations, respectively (2tabulated = 3.84) for a given gene pair in both BC3F6 and F2 populations. For gene pair, Co-42/Phg-2 the computed between the null hypothesis  and the alternative hypothesis . The marker loci for gene pairs 2Co-4/Phg-2 are thus not linked in BC3F6 and F2 bean populations studied. In contrast, for gene pairs, Co-5/\u201cP.ult\u201d, the computed chi value of 18.4 and 37.18 in BC3F6 and F2 populations, respectively (Co-5/\u201cP.ult\u201d are thus linked in BC3F6 and F2 bean populations studied.The summary of recombination fraction and likelihood data is shown in ectively  shows noectively  shows si2/Phg-2Co4 on bean chromosome Pv08 and Co-5/\u201cP.ult\u201d on chromosome Pv07. A strong genetic linkage among a pair of molecular markers located less than five centi Morgans (cM) apart on common bean chromosome implies that their two linked genes could be selected with only one marker to reduce genotyping costs.The objective of this study was to estimate recombination fractions and genetic linkage between gene pairs, 2/Phg-2Co-4 and Co-5/\u201cP.ult\u201d on common bean chromosomes Pv08 and Pv07, respectively.The recombination fraction summarized in 3F6 than F2 populationsfor gene pairs,2/Phg-2Co-4 (47cM vs 44 cM) and Co-5/\u201cP.ult\u201d (32 cM vs. 21 cM). This difference was attributed to the two generation studied with significant differences in levels of genetic variations.The results show incomplete genetic linkage between gene pairs, 2 populations derived their parents from from progenies of the BC3F6 population with single genes targerted. At BC3F6 the bean populations had possibly accumulated more recombinations than in F2s. Secondly, theprogeny lines in BC3F6 genotyped were derived from a four way cross comprising four parents used to develop the genetic pyramids  and angular leaf spot resistance (Phg-2) located on bean chromosome Pv08 co-segregate in bean populations due to genetic linkages was tested in this study. However, the weak genetic linkage between marker pairs studied shows that each of the four genes mentioned earlier have to be tagged with a corresponding linked markers during MAS. The study aimed to suggest strategies for reducing population size during gene pyramiding by finding a single marker locus (position of the chromosome) between genes or markers for simultaneous selection of resistance genes on the same bean chromosome(s), with linkages in coupling  and suggested overlap or very tight linkage of 4Co-1 and Phg-1 loci in the bean genome. The large genetic separation of SCAR markers SBB14 for 2Co-4 and SN02 for Phg-2 corroborates their physical positions in the Andean bean reference cultivar G19833 .In other studies on common bean, maximum linkage with no recombinants (0.0 cM) was reported for gene pairs, To define whether two markers are in linkage is to test whether the recombination fraction between these two markers is less than 0.5  proposed by Sun et al., (This study was based on dominant SCAR markers, which cannot differentiate homozygotes progenies from heterozygotes, we therefore recommend use of co-dominant markers to estimate linkage among gene pairs;  et al., . The fol2/Phg-2Co-4 on bean chromosome eight. The linkage was however relatively stronger among gene pair, Co-5/\u201cP.ult\u201d. There was difference in the value of recombination fraction between the BC3F6 and F2 population. This implies that selection for each of the resistance genes, 2, Phg-2, Co-5Co-4 and \u201cP.ult\u201d requires to be selected with their own SCAR marker due to lack of strong genetic linkages among these genes during marker assisted gene pyramiding targerting all the four genes in the same background.There was weak linkage among gene pair,"}
+{"text": "The two-minute walk test (2MWT) is a frequently used walking capacity test in persons with multiple sclerosis (pwMS). However, less is known about its relevance with regards to walking capacity during free-living walking performance. Therefore, the ecological validity of the 2MWT was tested by 1. computing free-living minutes with the same intensity (cadence) as during the 2MWT and 2. investigating the relationship between 2MWT cadence and minutes with the same cadence during free-living walking. 20 pwMS aged 44.2 \u00b1 12.2  score of 3.1 \u00b1 1.4) performed a 2MWT and wore an accelerometer for seven days. The number of pwMS reaching 100%, 90%, 80% or 70% of 2MWT cadence for at least one minute a day and minutes/day with at least 100%, 90%, 80% and 70% of 2MWT cadence during free-living walking was calculated. Six participants reached 100% of the 2MWT cadence for at least one minute/day during free-living walking. A total of 80% 2MWT cadence was the first intensity category that was reached by all participants during free-living walking. No significant correlation was found between cadence in the 2MWT and minutes in which this cadence was reached during free-living walking. Ecological validity with regard to walking intensity could not be confirmed in our study sample. Multiple Sclerosis (MS) is one of the most common autoimmune neurological diseases and one of the major causes of disability in young adults. Among the most prominent symptoms is disturbed gait. A total of 60% of persons with MS (pwMS) are not fully ambulatory 20 years after disease onset . FurtherThe goal of this study was to investigate the ecological validity of the 2MWT by comparing cadence (steps per minute) during the 2MWT to activity bouts with comparable cadence gathered from a seven-day accelerometer-based measurement of free-living physical activity. We assumed ecological validity of the 2MWT if (1) the 2MWT intensity (cadence) reflects intensity (minutes with the same cadence) of free-living walking performance, and (2) if higher 2MWT performance is associated with the higher walking capacity found during free-living walking . Taking into account that the 2MWT was actually not designed to reflect free-living walking intensity and is assessed under standardized conditions, we additionally performed an explorative analysis of free-living walking minutes with 90%, 80% and 70% of 2MWT cadence.Data were gathered as part of the\u201a MS bewegt\u2019 study . Participants were recruited from one inpatient and one outpatient clinic in Germany through the clinics\u2019 staff or via flyer. During baseline assessment, demographic data including age, gender, height, weight, disease course, duration of disease, Expanded Disability Status Scale (EDSS) score and information about the dominant side were collected and a 2MWT was performed. Patients were asked to wear an ActiGraph wGT3X-BT  accelerometer during the 2MWT and over a seven-day period at home. The ActiGraph was handed out, along with a wear-time diary, which was used to facilitate correct wear-time identification.The main inclusion criteria were age \u2265 18 years, MS diagnosis based on McDonald criteria, relapsing\u2013remitting, primary progressive or secondary progressive disease course, EDSS \u2264 6.5, no disease relapse during the last 30 days, and written consent. The main exclusion criteria were severe cardiovascular disease or cortisone therapy during the last 30 days.The 2MWT measures endurance and functional capacity in ambulation  and was During the test, participants were asked to cover as much distance as possible within a two-minute time frame. The length of pathway was 35m in the outpatient clinic and 50m in the inpatient clinic. Participants were instructed to turn around by walking around cones at the end of the pathway without stopping. PwMS were allowed to use walking aids if necessary and to slow down, stop or take a break if needed. During the test, participants wore a belt with an ActiGraph wGT3X-BT accelerometer on their preferred side on their left or right spina iliaca anterior superior. One tester walked next to the participant, recording distance by using a measuring wheel. Another tester counted steps with a pedometer and stopped time with a stopwatch.Participants were asked to wear the ActiGraph wGT3X-BT under the same instructions as in the 2MWT during waking hours for seven consecutive days at home, and to fill in a printed wear-time diary each day. ActiGraph data were included for further analysis if participants had at least four days with \u226510 h of wear-time, including one weekend day and one weekday according to the guidelines of Gabrys et al. (2015) .Accelerometry has been studied to be an objective method to examine community ambulation in pwMS . ActigraThe ActiLife software was configured the same way for the 2MWT and the free-living physical activity (PA) analysis. A sample rate of 100 Hz was used. Raw data (GT3X-files) were transformed into the proprietary AGD format with 60 s bouts using the low-frequency extension (LFE) filter, and then saved in CSV format for further analysis. Troiano algorithm was used for wear-time analysis and, additionally, wear-time diaries were analysed to ensure correct identification of wear and non-wear periods during the seven-day accelerometer measurement.p < 0.05.Mean, standard deviation and range were calculated for descriptive characterization of the sample. Steps of the 2MWT derived from the ActiLife software were divided by two to calculate average steps per minute (cadence). Afterwards, 90%, 80% and 70% of the 2MWT cadence were computed, and minutes with the corresponding cadence were extracted from the seven-day accelerometer measure in Microsoft Excel and described as average number of minutes per valid measurement day. For descriptive statistics, the number of participants reaching 100%, 90%, 80% and 70% of the 2MWT cadence for at least one minute per day was computed along with mean, standard deviation, minimum and maximum number of respective minutes. Scatter plots were made depicting minutes with at least 100%, 90%, 80% or 70% of 2MWT cadence during free-living walking in relation to the step cadence counted by the ActiGraph during the 2MWT. Spearman\u2019s rho was calculated to examine the association between the 2MWT performance and corresponding number of minutes during free-living walking. Analysis was carried out in SPSS Version 25.0 and significance was noted at All participants were properly informed about the study aims and procedures and gave written consent. The study was conducted in accordance with the Declaration of Helsinki and the protocol was approved by the ethical committee of the Baden-W\u00fcrttemberg Federal Chamber of Physicians .Descriptive statistics of the sample consisting of 20 pwMS are presented in Scatterplots visualizing the relationship of minutes walked with at least 100%, 90%, 80% and 70% of 2MWT cadence during free-living walking in relation to the 2MWT cadence measured in the clinic are presented in The 2MWT is a well-established and meaningful clinical measure of walking function. By examining its ecological validity, we aimed to explore possible new fields of application of the 2MWT results and its possibilities for informing the interpretation of patients\u2019 free-living walking performance by comparing it to free-living walking metrics. Only less than one third of the study population walked with 100% or more of the 2MWT cadence for at least one minute per day during free-living walking. A total of 80% of the 2MWT cadence was the threshold that was reached by all participants, suggesting that walking with intensities of 80% and less of total capacity is more common in free-living walking of pwMS. In addition, no significant correlation was detected between cadence in the 2MWT and corresponding minutes during free-living walking. This was also the case for 90%, 80% and 70% of 2MWT cadence. Based on this, people reaching higher cadences during the 2MWT were not found to also engage in more minutes of intense walking at their individual high-intensity level during free-living physical activity. Thus, participants showing a higher walking capacity during the 2MWT mostly do not make more use of their capacity during free-living walking. Consequently, based on our study sample and in terms of walking capacity, the 2MWT is not able to reflect free-living walking and the ecological validity with regard to walking intensity could not be confirmed. However, it should be noted that the 2MWT measures maximal walking capacity, which is only one influencing factor of free-living walking behavior. Previously, disability level, walking limitations, but also personal factors like physical-activity-related self-efficacy, self-regulation constructs and socio-demographic factors, were observed to be associated with physical activity . Also, tIn contrast to our results, moderate ecological validity of the 2MWT was found in previous studies. Gijbels et al. (2010) investigated the mean daily stride count and walking distance in the 2MWT and observed significant correlations in the total MS sample and in the subgroup of moderately disabled pwMS, but not in mildly disabled pwMS. The 2MWT was the most predictive variable for habitual walking performance measured through daily stride count in moderately disabled pwMS. Nevertheless, only half of the variances could be explained by the test, indicating that factors not captured by the test are influencing mean daily stride count. However, in their study, walking intensity was not taken into account, neglecting the purpose of the 2MWT to measure walking capacity .Stellmann et al. (2015) noted significant correlations of the 2MWT with different intensity-related parameters  from free-living physical activity and reported the 2MWT to have a moderate ecological validity. Unfortunately, steps per minute were not extracted to determine cadence, which means that their data are not directly comparable to our study data [Engelhard et al. (2018) examined the relationship between the maximum step rate  during free-living walking and the step rate during the 6MWT in pwMS and healthy controls. They found high correlations between the MSR and the 6MWT step rate and determined the MSR to be the best single parameter when measuring walking capacity in free-living walking, therefore being the real-world compliant to the 6MWT. Additionally, habitual walking step rate  together with the MSR were found to be even more efficient, since the MSR tended to overestimate 6MWT step rates and the HWSR had a trend of underestimating them. Engelhard et al. claimed that, in comparison to the MSR and HWSR, conventional habitual physical activity measures like average steps per day are poor measures of walking capacity since they are, in contrast to intensity-based measures, highly influenced by behavioral factors .Despite the correlations found in the above-mentioned studies, no significant correlations were discovered in our study. Our goal was to verify the ecological validity of the 2MWT not only by the calculation of linear associations between global measures, but by determining if the step rate performed during the 2MWT can be also observed during free-living walking. Furthermore, we examined the correlation of the 2MWT step rates and the number of minutes this step rate was reached during free-living walking. By doing so, we wanted to adhere to the original aim of the 2MWT to measure maximal walking capacity. Due to that, we did not compare the 2MWT to general free-living walking measures like steps per day, which also includes gait in low-intensity ranges. By observing 100%, 90% 80% and 70% of 2MWT cadence during free-living walking, we aimed to only target higher intensity walking behavior close to the maximal capacity found in the 2MWT. Thus, our approach to investigate the ecological validity of the 2MWT differed from the mentioned study approaches, explaining the lack of significant correlations in our study.When rating physical activity intensity, step rate thresholds are usually set on a cohort and not an individual level. For pwMS, Agiovlasitis and Motl (2014) determined step rate thresholds indicating moderate  and vigorous (between 118 and 140) physical activity .With the same moderate physical activity threshold of \u2265 82 steps, Neven et al. (2016) reported that 94% of mildly impaired and 36% of moderately impaired pwMS achieved two uninterrupted minutes at moderate intensity during walking in daily living. At low intensity (step rate < 82), all pwMS reached two and three minutes of uninterrupted walking. In general, people walked 14 \u00b1 14 min per day (1% of daytime) at moderate intensity . This isAs mentioned above, other influences, such as personal, environmental and behavioral factors or other symptoms , may be The study demonstrates some limitations which may have influenced the results. First, no standard procedure is available on how to assess wear time periods from the acceleration signals of the ActiGraph. The ActiLife software provides options to choose preset algorithms, and there is evidence about their accuracy depending on a number of parameters ,27. NeveSecond, we measured free-living walking in 60-s epochs. Therefore, the steps measured in one minute might have been accomplished within 40 s, followed by a 20 s break. This may dilute the actual walking intensity. To analyse data in shorter epochs of 10 s may be more accurate. However, since many measures are given as number per minute, we wanted to stick to that convention to facilitate calculations and comparability.Third, even if the ActiGraph was found to be a valid instrument for measuring steps during free-living walking in pwMS ,20, walkFourth, participants in this study walked, on average, 13,304 steps per day, which is unusually high for the MS population. For pwMS, a recent meta-analysis reports on average 5840 \u00b1 3096 steps per day . In thisFifth, we determined cadence to be an appropriate measure for walking intensity. However, the relationship between cadence and walking speed in this context could be biased by reduced walking function, especially reduced dynamic stability, in disabled pwMS ,34. An iLastly, we only provide a small study sample of 20 participants, which limits the generalizability of the results.Further studies with larger samples analysing the ecological validity of the 2MWT with regards to walking intensity are needed. Considering that no relation between the 2MWT and the walking performance in free-living walking with regard to walking capacity could be found, future research should further investigate determinants of free-living walking performance, including type of disease, symptoms and personal and environmental factors. Moreover, possible interactions of those factors with the extent to which walking capacity can be transferred into free-living walking behavior should be analysed. This, in turn, may help to individualise treatment and physical activity promotion interventions. Concerning accelerometer usage, the impact of different wear times and signal sensibility algorithms on the step outcome in populations with altered gait parameters or slow walking speed has to be evaluated.The 2MWT was not able to reflect free-living walking performance in terms of walking intensity. Thus, the ecological validity of the 2MWT with regard to walking intensity could not be confirmed in our study sample. Minutes with 100% of 2MWT cadence could only be found in less than a third of pwMS during free-living walking. A total of 80% of 2MWT cadence was reached by all participants, indicating that walking with intensities of 80% and less than total capacity is more common in free-living walking. Other aspects, like disease-related, personal, environmental, behavioral or symptomatic factors, may help to explain free-living walking performance. Future studies could further address the evaluation of determinants of daily living walking performance."}
+{"text": "Background. This study aimed to investigate the endodontic debridement efficacy of different sodium hypochlorite (NaOCl) irrigation regimens with and without ultrasonic agitation, followed by ethylenediaminetetraacetic acid (EDTA) via scanning electron microscopy (SEM) after using a rotary instrumentation system.Methods. Mandibular premolars (n=50) were randomly divided into five experimental groups (n=10) for root canal instrumentation with ProTaper Universal rotary system up to F3. The root canal system was treated with intracanal-heated NaOCl (100\u00b0C) or preheated NaOCl (55\u00b0C), followed by ultrasonic agitation and EDTA treatment. Samples irrigated with conventional needle irrigation (CNI) using normal saline solution were used as controls. Debridement efficacy was analyzed by SEM. A five-point scale was used to estimate the presence/absence of debris for each canal segment . The results were analyzed using one-way ANOVA and post hoc Tukey tests (P<0.05).Results. The experimental groups exhibited less debris compared to CNI with saline (P<0.05). The amount of debris decreased significantly for the group with NaOCl intracanal heating compared to extraoral heating. Ultrasonic agitation further enhanced the root canal debridement efficacy of NaOCl.Conclusion. In summary, intracanal heating of NaOCl with and without ultrasonic agitation followed by EDTA appears to be a promising method to flush debris from the root canal system. CNI is not, however, fully effective in delivering irrigants into the intricate areas of the root canal, such as the apical third, dentinal tubules, isthmus, and lateral canals, nor are they universally accepted.5 This is because the extent of flushing activity achieved by the CNI technique is only 0\u20122 mm from the needle tip depending on the depth of placement and the diameter of the needle, and canal cross-sectional shape and diameter.6Clinically, the success of root canal therapy\u2019s success relies heavily on the chemomechanical instrumentation and disinfection of the root canal system.5 Various methods have been studied to increase the potency of NaOCl and enable the use of low concentrations of NaOCl with decreased risk for toxicity or side effects.8 For example, NaOCl heating enhances the disinfecting and debridement properties due to an increase in the irrigation flow and reaction rate.10 Increases in irrigant temperatures are accomplished by preheating the irrigant extraorally or heating the NaOCl within the canal by utilizing ultrasonic equipment,8 or lasers.9 The heating of NaOCl often decreases its viscosity, enabling greater penetration, dissolving, and disinfection properties.10 Iandolo et al11 showed that NaOCl intracanal heating at 180\u00b0C significantly decreased debris compared to extraoral heating at 50\u00b0C. Besides, the ultrasonic agitation of intracanal-heated NaOCl resulted in improved debris removal relative to CNI and passive ultrasonic irrigation.Research firmly supports that sodium hypochlorite (NaOCl) solution is an ideal endodontic irrigant to flush debris from the root canal system.14 However, the heating of NaOCl solution above boiling point can cause heat transfer through the dentin, jeopardizing the surrounding tissues, alveolar bone, and periodontal ligament.16 Very few studies have evaluated the efficacy of NaOCl at its boiling temperature (96\u2012120\u00b0C).13 Therefore, this study aimed to evaluate the efficacy of NaOCl at a lower temperature (100\u00b0C), thereby reducing the above-mentioned risk and increasing the NaOCl efficacy. Furthermore, removing the debris from the root canal is more significant when EDTA and NaOCl are used in conjunction rather than alone.17 Limited data are available on the ultrasonic agitation of intracanal heating of NaOCl solution, followed by EDTA cleaning. Thus, we compared the root canal debridement efficacy of heated NaOCl with and without ultrasonic agitation, followed by EDTA. The null hypotheses were that extraoral heating of this irrigant does not significantly reduce the amount of debris compared with intracanal heating and that there is a significant difference between the percentage of remaining debris with and without ultrasonic agitation.Similarly, other studies have evaluated the efficacy of intracanal heating of NaOCl.Fifty mandibular premolars extracted for periodontal or orthodontic purposes were collected at the Maharashtra Institute of Dental Sciences and Research Centre with the donors\u2019 informed consent and Ethics Committee approval (Ref: MUHS/PH-T/E-1/2593/2019). The teeth with root resorption or open apex were excluded. The root segment was obtained by separating the crown of each tooth at the level of the cementoenamel junction. Two longitudinal grooves on the lingual or buccal surfaces of each root were created using a high-speed handpiece with a diamond bur to facilitate the vertical separation of the root segments.All the root canal instrumentation procedures were performed by a single trained operator. Root canal instrumentation was carried out with the ProTaper Universal system  in the sequence S1-S2-F1-F2-F3, at 200 rpm, and 3 N/cm torque. The root canal was irrigated during the procedure with 5 mL of 5% NaOCl. Intracanal heating of NaOCl was carried out at 100\u00b0C using Calamus Dual  with 30/04 carrier tips mounted 3 mm above the working length. Extraoral heating of NaOCl was carried out at 55\u00b0C using a kettle. The temperature was controlled using a thermometer. Ten activation cycles, each of 5 seconds, followed by 10 seconds of rest, were performed. After each activation cycle, the NaOCl was renewed, and the files were cleaned to remove debris to maintain effectiveness. The ultrasonic agitation was carried out using an ultrasonic activator . For control, CNI was carried out using normal saline solution with a 30-gauge side-vented needle. The five different protocols of agitation were as follows:Group A: Intracanal heating of 5% NaOCl to 100\u00b0C with ultrasonic agitation, followed by EDTA.Group B: Intracanal heating of 5% NaOCl to 100\u00b0C without ultrasonic agitation, followed by EDTA.Group C: Extraoral heating of 5% NaOCl to 55\u00b0C with ultrasonic agitation, followed by EDTA.Group D: Extraoral heating of 5% NaOCl to 55\u00b0C without ultrasonic agitation, followed by EDTA.Group E: Normal saline solution.18After root canal instrumentation, a stainless steel chisel was used to split the samples for scanning electron microscopy  imaging. Briefly, the samples were dried overnight, sputter-coated with gold , and photomicrographs  in threeHulsmann\u2019s scoring criteria for debris removal:Score 1: Clean root canal walls, with or without small debris particlesScore 2: Few debris agglomerationsScore 3: Debris agglomerations covering <50% of the root canal wallsScore 4: Debris covering >50% of the root canal wallsScore 5: Debris covering the complete or nearly complete root canal wallsStatistical analyses were performed with SPSS , and descriptive statistics were calculated for mean scores and minimum and maximum scores. One-way ANOVA and post hoc tests were used to compare the mean differences between individual groups.The comparisons of debris removal effects and mean value scores of the five debridement regimes are presented in 5 The effect of temperature on enhancing the efficacy of NaOCl has been documented in numerous studies.15 Preheating of NaOCl solution from 22\u00b0C to 45\u00b0C is one simple way to enhance its debris dissolution ability and antibacterial action. However, the NaOCl will be rapidly buffered in the root canal, minimizing its benefits.11 Previous studies have shown that intracanal heating of NaOCl solution substantially increases root canal debridement compared to preheated and not heated NaOCl solution.11 However, there are major differences in the literature regarding the debridement efficacy of NaOCl solution, arising from the use of various experimental conditions and mitigating variables that have affected the outcomes of some in vitro and ex vivo studies.19 As the NaOCl solution boils at temperatures between 96\u00b0C and 120\u00b0C, it is futile to use the heat carrier above the boiling point.20 In the present study, the 100\u00b0C intracanal heating of the root canal was efficient for successful irrigation and cleaning the endodontic space. The lower temperature used in this study can potentially prevent the surrounding periodontal complex,3 thereby providing safe and effective root canal debridement.The efficacy of NaOCl depends on the mechanical flush operation and chemical dissolving ability of the solution.21 The typical features of fluid agitation attempts are increasing the fluid\u2019s temperature, enhancing its chemical and biological activity. Presently, \u201cultrasonic agitation,\u201d which consists of activating irrigants by ultrasonic tips, is the most commonly used technique to enhance NaOCl efficacy.13 This technique permits the intensified stirring of the irrigant and formation of submicroscopic voids that create shear stress to disrupt debris and damage biofilms physically, thus resulting in a superior cleansing action.22 Interestingly, in the present study, there were no significant variations in debris removal between the ultrasonic agitation and no agitation groups with intracanal heating of NaOCl solution, followed by EDTA. These findings are close to those reported in a study by Mayer et al,21 showing that NaOCl and EDTA ultrasonic agitation did not reduce the scores of debris in straight root canals compared to the non-activated group.Significant attempts have been made to improve the capacity of NaOCl to remove and dissolve debris through physical fluid agitation using mechanical vibration, ultrasonic agitation, or pulsed lasers.24 These studies are mostly carried out on straight root canals, and various effects can be attributed to disparities in apical preparation, volume, and working times of irrigation and agitation. However, the present study indicated that the use of the EDTA following NaOCl\u2019s intracanal heating resulted in efficient cleaning of the root canal. Thus, the first hypothesis was confirmed: Intracanal heating of NaOCl solution enhanced debris removal. The second hypothesis, however, was not established as there was no significant difference in debris removal over the length of the root canal system, with and without the agitation of NaOCl solution. Although the results are satisfactory, further investigations are required to establish this strategy\u2019s effectiveness in complex root canal anatomies and its impact on the surrounding tissues.Nonetheless, this result contradicts many earlier trials where ultrasound agitation led to more successful debris removal than irrigation with a syringe and sonic agitation.For complete debridement of the canals, intracanal-heated NaOCl, followed by EDTA, was more effective than extraoral preheating followed by EDTA over the length of the root canal system. NaOCl solution at a temperature of 100\u00b0C was as effective as higher temperatures, thereby eliminating the need for heating the irrigants to high temperatures and safeguarding the periodontal complex.YD was responsible for the concept and experimental design, performed the experiments, and wrote the manuscript. RK and SG conceived the idea, hypothesis, and experiment design, and carried out supervision. SD, SG, and SB were responsible for assisting in the experiment and contributed to the discussion. ND was responsible for supervision analysis, interpretation of data, and wring the manuscript. All authors have read and approved the final manuscript.The authors acknowledge the help of K S Nagaraja Rao from Physics Department, Osmania University, Hyderabad, India for SEM imaging.This research received no external funding.The authors assert no conflicting interests concerning the authorship and/or publishing of this article.The use of human mandibular premolars and dental pulp stem cells (DPSC) for research was approved by the Institutional Ethics Committee of MIDSR Dental College, Latur (India)."}
+{"text": "Despite extensive studies on the formation of organic molecules in various extraterrestrial environments, it still remains under debate when, where, and how such molecules were abiotically formed. A key molecule to solve the problem, hexamethylenetetramine (HMT) has not been confirmed in extraterrestrial materials despite extensive laboratory experimental evidence that it can be produced in interstellar or cometary environments. Here we report the first detection of HMT and functionalized HMT species in the carbonaceous chondrites Murchison, Murray, and Tagish Lake. While the part-per-billion level concentration of HMT in Murchison and Tagish Lake is comparable to other related soluble organic molecules like amino acids, these compounds may have eluded detection in previous studies due to the loss of HMT during the extraction processes. HMT, which can yield important molecules for prebiotic chemistry such as formaldehyde and ammonia upon degradation, is a likely precursor of meteoritic organic compounds of astrochemical and astrophysical interest. This manuscript tackles the origin of organic molecules in carbonaceous meteorites. Identifying hexamethylenetetramine in three carbonaceous meteorites, the authors propose formation from ammonia and formaldehyde by photochemical and thermal reactions in the interstellar medium, followed by the incorporation into planetary systems. However, despite extensive studies on the formation of organic molecules in various extraterrestrial environments such as molecular clouds10, protosolar nebula12, and asteroids15, it still remains under debate when, where, and how such extraterrestrial molecules were abiotically formed.Presence of organic molecules in extraterrestrial environments has been widely accepted thanks to recent successes in the in situ detection of cometary molecules toward 67P/Churyumov-Gerasimenko6H12N4; monoisotopic mass of 140.1062\u2009Da), which is a polyheterocyclic organic molecule , ammonia (NH3), and methanol (CH3OH), HMT is in general a significant product (up to 60% by weight) in the total organic products20. Although the composition of products varies depending on the experimental conditions, HMT is generally abundant especially when methanol is used as an initial reactant20. Since methanol is abundant in interstellar ices3, the HMT formation is likely to take place in the interstellar medium (ISM) and become incorporated into solar system ices similar to other interstellar molecules22.A key molecule to solve the problems is hexamethylenetetramine  of interior samples of three carbonaceous chondrites, Murchison, Murray, and Tagish Lake, under mild conditions which utilized neither concentrated acidic solutions nor high temperatures for the extraction processes. The aqueous extracts were purified using cation-exchange chromatography and were then analysed using a high-resolution mass spectrometer (HRMS) coupled with a high-performance liquid chromatograph (HPLC)25. HMT was successfully detected from Murchison, Tagish Lake, and Murray meteorite extracts\u00a0at parts-per-billion levels.Since HMT is susceptible to degradation by 100\u2009\u00b0C waterm/z) of 141.1135\u2009\u00b1\u20090.0004, which corresponds to the protonated ion of HMT  formed by electrospray ionization (ESI), analysed by a HPLC equipped with an InertSustain PFP analytical column. One sharp peak was observed for each chromatogram at ~20.5\u2009min, which was consistent with HMT standard reagent , even under the different analytical conditions, the observed peak can be confidently assigned to HMT. The observed consistency in the fragmentation pattern of HMT by MS/MS experiments (see the \u201cMethods\u201d section) between the Murchison extract and the standard reagent further supports the above conclusion  m/z values well consistent with the HMT derivatives methyl-HMT (HMT-CH3), amino-HMT (HMT-NH2), hydroxy-HMT (HMT-OH), and hydroxymethyl-HMT (HMT-CH2OH),  and monohydroxy-monomethyl-HMT (HMT-OH(-CH3))  cannot be excluded. The absence of these species on the mass chromatograms for the HMT standard reagent , followed by HMT-CH2OH or its isomers (<0.6%), HMT-OH (0.2%), and HMT-NH2 (0.03%) , Fig.\u00a0 in the mace Fig.\u00a0 shows at19. Furthermore, the estimated relative abundances of these HMT-derivatives in the organic residues (orders of magnitudes less abundant than HMT)19 are in reasonable agreement with those of the meteoritic HMT-derivatives  compared to the elevated concentrations of HMT measured in the meteorite extracts argue that HMT is indigenous to the meteorites. In addition, the likely detection of several HMT-derivatives also bolsters this conclusion; unlike HMT itself, to our best knowledge, these HMT-derivatives are commercially unavailable and their presence in terrestrial environments has not been reported. However, these HMT-derivatives have been identified in organic residues produced by photolysis of interstellar ice analogues followed by warming to room temperatures, which mimics the processes of molecular evolution toward star formation26 and higher than that of sugars (<180\u2009ppb) and nucleobases (<~70\u2009ppb) in the Murchison meteorite6. In the Tagish Lake meteorite, the concentration of HMT (671\u2009\u00b1\u20099\u2009ppb) is also in the range of individual amino acid concentrations identified in acid hydrolysed water extracts of the Tagish Lake meteorite 7. While in Murray, the concentration of HMT (29\u2009\u00b1\u20099\u2009ppb) is lower than individual amino acid concentrations (51\u20132834\u2009ppb) in the same meteorite8. It is possible that differences in the Murchison/Tagish Lake and Murray parent body conditions  led to lower abundances or higher loss rates of HMT, which may partly be related to the formation of soluble organics. For example, Supplementary Fig.\u00a0m/z values corresponding to imidazole  and its alkyl-substituted homologues (up to seven carbon chains), which are proposed as the products after the hydrothermal degradation of HMT15. For Murchison and Murray, the presence of alkyl-imidazoles was strongly expected in their extracts; while, they were significantly depleted in Tagish Lake  is within the range of individual water-extractable and acid-produced amino acids (200\u20135000\u2009ppb) ppb is w28. However, the argument\u00a0that HMT is the origin of amino acids during workup is weakened by Murray, which has a similar abundance of amino acids to Murchison8, yet the HMT concentration was lower by about an order of magnitude than Murchison. Moreover, sample heterogeneity between different specimens of the same meteorite, which has been often invoked for explaining different quantitative results of some molecules including their different enantiomeric distributions in the same meteorites4, can also be invoked. On the other hand, it is likely that HMT is formed during our laboratory workup if both ammonia and formaldehyde are present in the aqueous extract29. Previous studies detected both molecules from carbonaceous meteorites after hydrothermal treatment and/or acid hydrolysis of meteorite powders at ~100\u2009\u00b0C or above32, implying that both free ammonia and formaldehyde are released from their acid-labile precursors after these treatments. Although it is not clear whether such precursors can contribute to the formation of HMT in aqueous solutions without acid and high-temperature treatment at room temperature, we expect that HMT formed as such does not constitute a significant fraction in the detected HMT abundance. Nevertheless, there are still a number of uncertainties on the origin of the difference in HMT abundance between three meteorites analysed in the present study (e.g. HMT abundance when each parent body is formed by accretion).Given the harsh extraction conditions of amino acid analyses, one possibility is that some of the HMT and its derivatives can form amino acids during routine amino acid extraction and workup. In fact, acid hydrolysis of HMT-containing organic mixtures yielded amino acids, and the role of HMT for amino acid formation has been investigated well in recent studies19. Though the typical interstellar values 33 are far higher than seen in any meteoritic compound. No levels of this extreme deuteration of HMT were visible. Yet, it is still possible that the HMT detected has an interstellar provenance and the ISM D was lost to exchange with comparatively D-poor parent body fluids. We have tested the D/H exchange in HMT upon heating with water and silicates to simulate possible variations in the deuteration level of meteoritic HMT through hydrothermal processes in asteroids. When fully deuterated HMT (C6D12N4) was heated with H2O under alkaline conditions (pH\u2009=\u200910) at 100\u2009\u00b0C, deuterium atoms in HMT were gradually replaced with hydrogen atoms in H2O, resulting in the formation of partly hydrogenated HMT like C6HD11N4 and C6H2D10N4 after several days , NH3, amines, amino acids, and nitrogen heterocycles37, many of which have been identified in carbonaceous meteorites after hydrothermal treatment at around 100\u2009\u00b0C or acid hydrolysis32. HMT that survived these desorption and degradation processes might be delivered to the Earth via meteorites and possibly interplanetary dust particles.Once HMT is incorporated into planetary systems and into a meteorite parent body, it has three likely fates: (1) physicochemical desorption from the surface of asteroids into the gas phase of the solar system, (2) decomposition, and (3) preservation. It is likely that desorption of HMT from asteroids could be induced either or both by external excitation energies  and by thermal processes, although these processes have not been studied experimentally so far. Laboratory studies strongly suggest that aqueous or thermal degradation of HMT on meteorite parent bodies has a potential to yield various kinds of molecules, such as formaldehyde 42, unless transformed into other (non-volatile) species by chemical reactions, both molecules are likely to be lost from grains during warming up phases toward star formation if the temperature of the grains exceeds the desorption temperature of both molecules. In contrast, since solid HMT does not desorb from grains even at 330\u2009K (refs. 18), it should have more opportunity to be incorporated into inner solar system bodies. Naturally, since HMT is in equilibrium with H2CO and NH3, it could also have been formed on meteorite parent bodies from both molecules if they are really present, which could keep the HMT concentration relatively constant. However, H2CO and NH3 have been identified in carbonaceous meteorites upon hydrothermal treatment at around 100\u2009\u00b0C or acid hydrolysis32; conversely these species may be from the decomposition of HMT on the parent body or during laboratory workup. As such, it will be challenging to constrain the location of HMT formation but its presence in the processed interstellar ice analogues20 can be a good indicator to explain its presence in meteorites. Hence, the presence of HMT in carbonaceous meteorites promises its pivotal role to carry interstellar prebiotic precursors to the inner solar system, which should contribute to the chemical evolution in the primordial stage on Earth.Among the various kinds of molecules which can form via hydrothermal degradation of HMT, both H44. The Murray (CM2) and the Tagish lake (C2 ungrouped) meteorites were both from meteorite trading companies with the certification. The exterior surfaces of these meteorite samples were independently washed by 0.1\u2009M HCl solution  with a soak (3\u2009min at ambient temperature) and gentle ultra-sonication  to peel the meteoritic surface layer, and the supernatant was removed. Then, the sample followed an organic solvent soak (3\u2009min at ambient temperature) by dichloromethane/methanol  with gentle ultra-sonication . After removing the supernatant, the chemically peeled samples were dried up by a vacuum freeze dryer  at ambient temperature. In a clean bench, the dried samples were gently powdered as fine as using a clean pestle and a clean mortar according to the previous work45 with the present blank test.The Murchison meteorite (CM2) was from a 10\u2009g chip taken from a 47.5\u2009g fragment originally from the Field Museum of Natural History, Chicago that had been stored at room temperature in a sealed glass desiccator for many years at the University of Chicago until it was opened in August 2015. The 10\u2009g chip was crushed and homogenized at the NASA Goddard Space Flight Center and a 2\u2009g portion of the powder was sent to Tohoku University. The sample quality (i.e. a degree of contamination) was previously evaluated for amino acids, suggesting very low levels of amino acid contamination based on their heavy carbon isotopic compositions and the detection of racemic alanine5 was used for this study. For further investigation of other reference carbonaceous meteorites, we conducted the water and solvent extraction for the fine powdered samples (2\u2009g for Murray and 0.5\u2009g of Tagish Lake) using ultra-sonication (10\u2009min with crushed ice in the sonic bath) with two bed-volume of ultra-pure water . After the solid/liquid separation by the centrifugation , the supernatant liquid phase was recovered; the water-extractable fraction procedures were repeated for three times. The fraction was then frozen and dried up by a vacuum freeze dryer  under ambient temperature. To remove inorganic salts and interfering organic matrix from the extracts, we isolated the HMT fraction using the cation exchange chromatography 46. The final elution containing HMTs was dried by a vacuum freeze dryer  under ambient temperature. The final fraction was dissolved in ~1\u2009mL of ultra-pure H2O and filtered by 0.20\u2009\u00b5m PTFE cartridge filter just before the HRMS. The pretreatment eliminates HPLC/ESI-Orbitrap-MS potential artefacts including a chromatographic retention shift47, ion suppression and ion-enhancement effect49. The recovery of HMT was measured using its standard reagent to be >90%. All glassware and the quartz wool were cleaned by heating in air at 450\u2009\u00b0C for 3\u2009hr.HMT and other water-extractable hydrophilic molecules  were recovered from ~2\u2009g of the Murchison powder and the cation desalting fraction as described in Furukawa et al.m/\u0394m\u2009=\u2009~140,000 at a mass-to-charge ratio (m/z) of 200 via an HPLC system  equipped with a reversed-phase separation column  at 40\u2009\u00b0C. The eluent programme for this HPLC setup is as follows: solvent A (H2O), solvent B (acetonitrile\u2009+\u20090.1% formic acid by volume)\u2009=\u200990:10 for the initial 5\u2009min, followed by a linear gradient of A:B\u2009=\u200950:50 at 20\u2009min, and it was kept at this ratio for 25\u2009min. The flow rate was 100\u2009\u03bcL\u2009min\u20131.The meteorite extract was introduced into an Orbitrap mass spectrometer  with a mass resolution of 19 at 30\u2009\u00b0C or an InertSustain Amide column  at 40\u2009\u00b0C in hydrophilic interaction (HILIC) chromatography mode to confirm that the detection of HMT does not depend on analytical columns , solvent B (acetonitrile\u2009+\u20090.1% formic acid)\u2009=\u2009100:0, followed by a linear gradient of A:B\u2009=\u200980:20 at t\u2009=\u200920\u2009min and it was kept at this ratio for 5\u2009min. The flow rate was 0.1\u2009mL\u2009min\u20131. The eluent programme for the HILIC mode analysis is as follows: at t\u2009=\u20090, solvent A (10\u2009mM ammonium formate plus 0.1% formic acid), solvent B (acetonitrile)\u2009=\u20091:99, followed by a linear gradient of A:B\u2009=\u200940:60 at t\u2009=\u200940\u2009min and it was kept at this ratio for 5\u2009min. The flow rate was 0.3\u2009mL\u2009min\u20131.The Murchison extract was also analysed using the same HPLC/HRMS equipped with other separation columns: a Hypercarb separation column m/z range of 50\u2013400 and a spray voltage of 3.5\u2009kV. The capillary temperature of the ion transfer was 300\u2009\u00b0C. The injected samples were vaporized at 300\u2009\u00b0C. We set up an inverse gradient programme to maintain the ionization efficiency during the ESI. To minimize analytical noise and the background signals in the LC and Orbitrap, we used high purity grade water and acetonitrile . Under these experimental conditions, the mass precision is always better than 3\u2009ppm for each chromatogram .The mass spectra were recorded in the positive ESI mode with a m/z 141.11\u2009\u00b1\u20090.2 were reacted with high-energy (30 in arbitrary unit) collision N2 gas to produce fragmental ions, in which the mass range of m/z 50\u2013160 was monitored using an Orbitrap MS with a mass resolution of ~140,000. The collisions of high-energy N2 with the protonated HMT ion (m/z 141.1135) gave two major fragmental ions; C5H10N3+ (m/z 112.0869) and C4H9N2+ (m/z 85.0761), as well as its non-fragmented parent ion  is a background signal on the LC condition. For the Tagish Lake and Murray meteorites, we were unable to perform MS/MS measurements due to the low concentration of HMT in the extracts.The MS/MS experiment was also performed using a hybrid quadrupole-Orbitrap mass spectrometer  with the identical HPLC and ionization conditions used for the full-scan analysis. The extracted positive ions 352 Fig.\u00a0. These m45 through the same extraction process to verify the potential impurity in the meteorite extracts. The mass chromatogram was shown in Supplementary Fig.\u00a05). The mass chromatogram was shown in the Supplementary Fig.\u00a0The solvent extraction blank analysis with ultra-sonication procedure was performed using 2\u2009g of combusted quartz sand6D12N4, CDN Isotopes; HMT-d12) was prepared to be 172\u2009mM and pH 10. About 100\u2009\u00b5L of the stock solution was transferred to a sample tube  made with pure Au whose one side had been tightly crimped by hand pliers. About 5\u2009mg of amorphous forsterite (~100\u2009nm in diameter) was also enclosed in the same sample tube as an analogue of asteroid minerals. After the headspace of the tube was purged with dry N2 gas, the other side was also crimped, and the sample tube was heated at 100\u2009\u00b0C for up to 31 days using an autoclave . We confirmed the weight of the sample tube did not change after heating, which indicated effectively no sample loss from the tube. The heated HMT solution was extracted from the sample tube, filtered by a hydrophilic PTFE membrane filter  to remove the silicate powders, and analysed by HRMS using a Thermo Scientific Exactive by flow injection. Analytical details have been reported in Oba et al. 50.Stock aqueous solution of fully deuterated HMT (CSupplementary InformationPeer Review File"}
+{"text": "To evaluate the effect of final irrigation of root canals with NaOCl solution at different temperatures on postoperative pain level and antimicrobial activity. 45 patients were randomly divided into three groups using a web program according to the irrigation selected: NaOCl 2\u00baC, NaOCl 25\u00baC and NaOCl 45\u00baC. First root canal samples were collected before treatment (S1). After chemo-mechanical preparation, final irrigation was performed with the selected irrigant  and second samples were collected (S2). Samples were subjected to quantitative real-time polymerase chain reaction to evaluate the levels of total bacteria. The root canal treatments were completed and the participants were given instructions to record postoperative pain levels at 24, 48 and 72 hours, 5 days and 1 week after treatment using a visual analog scale (VAS). The reduction in the number of total bacterial cell equivalents from S1 to S2 was statistically significant in all groups (p<0.001). The NaOCl 2\u02daC group reported significantly less postoperative pain than the NaOCl 45\u02daC group (p<0.05). Postoperative analgesic intake was significantly higher in the NaOCl 45\u02daC group than in the NaOCl 2\u02daC group (p<0.05). We conclude that final irrigation with NaOCl at different temperatures results in similar antibacterial effectiveness. Final irrigation with cold NaOCl (2\u02daC) is better than NaOCl 45\u02daC when comparing postoperative pain levels. Our randomized controlled clinical study aimed to evaluate the effect of final irrigation of root canals with NaOCl solution at different temperatures on postoperative pain level and antimicrobial activity. The null hypothesis was that the solution temperature does not change the solution antibacterial effectiveness or the postoperative pain level in patients presenting teeth with asymptomatic apical periodontitis.16 with a 80% power and a 0.05 alpha level. It was decided to enroll 15 patients per group to increase statistical power and to account for the loss of participants during the study. Clinical registration number is TCTR20200420005.Forty-five patients (26 women and 19 men) presenting incisor, canine or premolar teeth with radiographic and clinical evidence of asymptomatic apical periodontitis were included in our study. This study was approved by the ethics committee of the Faculty of Dentistry, Ataturk University (Opinion No. 2017-67) and an informed consent form was signed by all participants before the treatment. The sample size (n=13) was determined using a program , expecting a difference of 48% in proportions of positive cultures among groups,17 Exclusion criteria: patients that underwent treatment with antibiotics or NSAIDs within 1 month before the study; patients with any systemic disease; teeth that had received previous endodontic treatment; teeth with extensive destruction of the crown that prevented proper rubber dam isolation; and the presence of internal or external resorption. A web program (www.randomizer.org) was used to randomly assign the 45 participants into three groups (n=15). After randomization, the number of each group and of each patient were recorded. All the treatments were performed by one clinician (N.A.). Blinding the clinician to the groups was not feasible because of the recognizable temperature of the syringe. However, the patients were blinded to the groups.Teeth with necrotic pulps, clinically confirmed by pulp sensibility tests and the absence of bleeding on accessing the pulp chamber, no previous history of endodontic treatment, a pocket depth of <3 mm and having a periapical lesion with a periapical index score of 3, 4 or 5 ) were included.2O2 and 2.5% NaOCl for 30 s was used for disinfection of the crowns and surrounding structures (dam and clamp). After the disinfection protocol, the NaOCl was inactivated using 5% sodium thiosulfate. Access cavities were prepared using sterile round burs under cooling with sterile saline solution. The disinfection protocol described previously was performed again after completing access cavity preparation. To prevent the penetration of disinfectants into the pulp chamber and root canals, a sterile cotton pellet was placed on the floor of the pulp chamber. Sodium thiosulfate was used to neutralize NaOCl effectiveness and first sterility control samples were taken with sterile paper points from the coronal surface of the tooth, rubber dam, clamp and access cavity walls. Paper points were transferred to Eppendorf tubes containing a Tris-EDTA buffer  and then the samples were kept at -80\u00baC until bacterial presence was assessed with quantitative polymerase chain reaction (qPCR). The teeth with negative sterility control samples for bacterial presence in the qPCR assay were included in our study.Root canal samples were collected under strict aseptic technique. Before treatment, an oral rinse was performed with 0.12% chlorhexidine and before isolation of the teeth with the rubber dam, supra-gingival scaling was performed to remove plaque, followed by cleansing with pumice. Before the access cavity preparation, 30% HAfter working length determination with an electronic apex locator , the root canals were filled with sterile saline solution, not allowing them to overflow, and then a gentle filling motion was performed with a sterile #15 K-file . To obtain the first bacteriological sample (S1), paper points  were used to soak up the intracanal fluid in the root canal. The sampling procedure was repeated using three paper points and each paper point was left in the root canal for at least 60 seconds. Then the paper points were transferred to a tube containing a Tris-EDTA buffer. Any contact between the paper points and the cavity walls was avoided to prevent contamination during transfer of the paper points into the tubes.Root canals were prepared using Reciproc files  , according to the manufacturer recommendations. During preparation, root canal irrigation was performed using 1 mL of 1% NaOCl between three pecking motions of the file and final irrigation was performed with 5 mL of 17% EDTA followed by 5 mL of 1% NaOCl (at different temperatures). According to the final irrigation, the groups were divided as follows:NaOCl 2\u00baC: Syringes filled with 5 mL of 1% NaOCl were kept in a fridge at -2 \u00baC. Before the irrigation procedure, the syringe was removed from the fridge and when the temperature of the syringe, which was checked with a thermometer, increased to 2\u00baC, root canal irrigation was performed for 1 minute (the room temperature was 24.2\u00baC).NaOCl 25\u00baC: A CanalPro syringe heater  was used to heat a syringe filled with 5 mL of 1% NaOCl. The syringe was left in the device for 30 seconds to reach an average temperature of 26\u00b10.2\u00baC, because the device cannot be set to a specific temperature. Then the syringe was removed from the device and when the temperature of the syringe, which was checked with a thermometer, decreased to 25\u00baC, root canal irrigation was performed for 1 minute (the room temperature was 24.2\u00baC).\u00baC, root canal irrigation was performed for 1 minute (the room temperature was 24.2\u00baC).NaOCl 45\u00baC: A CanalPro syringe heater  was used to heat a syringe filled with 5 mL of 1% NaOCl. The syringe was left in the device for 20 minutes to reach an average temperature of 48\u00b10.2\u00baC, because the device cannot be set to a specific temperature. Then the syringe was removed from the device and when the temperature of the syringe, which was checked with a thermometer, decreased to 45After that, 2 mL of 0.5% sodium thiosulfate was used to inactivate the NaOCl and then the root canals were finally irrigated with distilled water. Second samples (S2) were taken as described above.Next, paper points were used to dry the root canals and the cold lateral compaction technique was used to obturate the root canals with gutta-percha cones and sealer . Permanent restorations were performed using flowable and nanohybrid composite resins . The participants were given instructions to record postoperative pain levels at 24, 48 and 72 hours, 5 days and 1 week after treatment using an 100 mm visual analog scale (VAS) as well as recording analgesic taken on the questionnaire. If a patient was referred to an unscheduled appointment, this was also recorded.\u00ae Green PCR Kits (Qiagen) on a Rotor-Gene real-time PCR instrument (Qiagen) in a total reaction volume of 20 mL universal 16S ribosomal RNA gene-based primers (Forward primer 5\u2019-ACTACGTGCCAGCAGCC-3\u2019 and reverse primer 5\u2019GGACTACCAGGGTATCTAATCC-3\u2019); qPCR reaction conditions were 95\u00b0C for 15 min, and 40 cycles of 95\u00b0C for 15 s, 55\u00b0C for 30 s and 72\u00b0C for 30 s. For each cycle, the accumulation of PCR products was detected by monitoring the increase in fluorescence of the reporter dye (double-stranded DNA-binding SYBR Green). All measurements were performed in triplicate for samples and standards. In all experiments, triplicates of appropriate negative controls containing no DNA template were subjected to the same procedures to exclude or detect any possible contamination or carryover.The total bacterial counts were evaluated using 16S ribosomal RNA gene-based qPCR with QuantiTect SYBR4 For intragroup analysis, the Mann-Whitney U test was used to compare reduction of counts of total bacteria between the two samples (S1 and S2), since the data were not normally distributed. The chi-square test was used to compare the number of root canals positive for bacteria in S1 and S2 among the NaOCl 2\u00baC, NaOCl 25\u00baC and NaOCl 45\u00baC groups. The number of bacteria in S1 and S2 samples and the reduction (%) in the number of total bacteria from S1 to S2 among the groups were compared using the Kruskal-Wallis test. Data were analyzed with GraphPad Prism Software  at a significance level of p=0.05.The Poisson regression model, which is the basic approach for modeling bacterial count data, was used as previously described.\u00ae SPSS\u00ae Statistics 20 software  at 5% significance level (p=0.05).The Kruskal-Wallis test was used to compare postoperative pain values among the groups, because the data were not normally distributed. Linear regression analyses were conducted to determine the confounding effects introduced by covariates . The chi-square test was used to analyze nominal data . The statistical analyses for postoperative pain values were performed using IBMSince all the sterility control samples yielded negative results for bacteria and there was no patient loss during follow up, 45 patients (26 women and 19 men) were included in our study. Each patient contributed with 1 tooth and there was no statistically significant difference among the groups in comparison of sex, age and tooth number distribution (p>0.05) .6, 5.18\u00d7106and 5.76\u00d7106 bacterial counts in S1 was decreased to a mean of 3.66\u00d7105, 2.7\u00d7105 and 3.31\u00d7105 bacterial counts in S2, respectively. The reduction in the number of total bacterial cell equivalents from S1 to S2 was statistically significant in all groups (p<0.001). The percentage of reduction was 94.7, 95 and 93.9 for the NaOCl 2\u02daC, 25\u02daC and 45\u02daC groups, respectively. There was no statistically significant difference among the groups in terms of percentage of reduction of total bacterial counts (p>0.05).The number of root canals positive for bacteria was also evaluated. Whereas all the S1 samples were positive for bacteria in all groups, 3 root canals from the NaOCl 2\u02daC group, 3 root canals from the NaOCl 25\u02daC group and 2 root canals from the NaOCl 45\u02daC group became negative for bacteria in S2 samples . The difLinear regression analyses revealed that postoperative pain level on day 1 was only influenced by the group (p<0.05). Sex, age and tooth number did not influence the postoperative pain level on day 1 (p>0.05). .The mean postoperative pain level was 6.67\u00b110.722, 16.87\u00b127.604 and 40.0\u00b150.709 for the NaOCl 2\u02daC, 25\u02daC and 45\u02daC groups, respectively. . Statist18 However, there was no statistically significant difference among the groups, when comparing NaOCl with different temperatures, in the removal of bacteria from root canals. Several studies investigated the antibacterial effect of NaOCl with different temperatures and conflicting results have been reported21. Sirtes, et al.11 (2005) observed a 100-fold increase in NaOCl antibacterial efficacy when the solution was heated from 20\u00b0C to 45\u00b0C. Similarly, Giardino, et al.20 (2016) demonstrated better NaOCl antibacterial efficacy at 45\u00b0C than at 20\u00b0C. However, Sirtes et al. used NaOCl solution with a concentration of 0.001% and they added bacteria in phosphate-buffered saline into the NaOCl solution. That is, they did not use extracted teeth or dentine slices in their investigation to mimic clinical conditions, which may have affected the results, because the presence of organic (tissue remnants and inflammatory exudate) and inorganic matter (dentine) can weaken the antibacterial effectiveness of the NaOCl solution23. Moreover, they incubated the bacteria in the NaOCl solution for 10 minutes. Giardino, et al.20 (2016) also assessed the NaOCl antibacterial efficacy after a 10-minute contact, which is a relatively longer contact time when compared with clinical conditions. Moreover, both studies were culture-based; which are less sensitive than the qPCR, which can detect as-yet-uncultivated bacteria.24 The methodological differences mentioned above could explain the difference in the findings of our study and the previous ones. Our findings could be explained by the strong antibacterial effect and the NaOCl concentration (1%) used in our study was efficient for killing sufficient bacteria in the root canal system. NaOCl can kill bacteria even at concentrations lower than 0.1%. Additionally, and consistent with our results, Carpio-Perochena, et al.19 (2015) compared NaOCl solutions with a concentration of 1% at different temperatures (22\u00b0C and 37\u00b0C) in terms of antibacterial efficacy and concluded that the temperature variation of the NaOCl is not relevant in killing or dissolving bacterial biofilms. Likewise, Gulsahi, et al.21 (2014) reported that there is no significant difference between the NaOCl at 25\u00b0C and 37\u00b0C in killing Enterococcus faecalis and Candida albicans for the same contact times. Therefore, it can be speculated that NaOCl with a concentration of 1% exerts a strong antibacterial efficacy that provides a substantial reduction in bacterial counts in the root canal system, regardless of the solution temperature.Our study compared the effect of NaOCl with different temperatures on elimination of bacteria from root canals and postoperative pain level. Since there was no significant difference among the groups in antibacterial effectiveness, but the temperature of the solution affected the postoperative pain level, the null hypothesis was partially rejected. According to the results of our study, chemo-mechanical preparation and final irrigation with EDTA + NaOCl was highly effective in significantly reducing the intracanal bacterial counts, irrespective of the NaOCl temperature. This is in agreement with previous studies, which reported statistically significant reduction of bacterial counts by chemo-mechanical preparation.25 Cryotherapy leads to reduced cellular metabolism by dropping local temperature, which causes reduced blood flow.26 Consequently, the effect of cryotherapy on limiting inflammation may explain the reduced postoperative pain value in NaOCl 2\u00b0C group.27 However, the most important finding of our study was that, although there was no statistically significant difference between the NaOCl 25\u00b0C and NaOCl 2\u00b0C groups, postoperative pain reached the highest level in the NaOCl 45\u00b0C group, with a significant difference when compared with the NaOCl 2\u00b0C group. This means that using preheated NaOCl for root canal irrigation results in a higher postoperative pain value. Additionally, in the NaOCl 45\u00b0C group, the need for analgesic intake was significantly higher than in the NaOCl 2\u00b0C group. There are no previous studies evaluating the effect of intracanal irrigation with preheated NaOCl on the level of postoperative pain, therefore, a direct comparison cannot be performed. However, the postoperative pain-enhancing effect of the preheated NaOCl can be explained by the fact that heat increases tissue temperature, which results in vasodilatation.28 Vasodilatation increases blood flow and allows leukocytes and plasma proteins to exit the circulation, which may cause an increase in inflammatory response, thus increasing postoperative pain.Our study also evaluated the effect of the NaOCl at different temperatures on the level of postoperative pain and showed that root canal irrigation with NaOCl at 45\u00b0C resulted in a significantly higher postoperative pain value than NaOCl at 2\u00b0C. This finding is in accordance with previous studies that reported less postoperative pain with the application of intracanal cryotherapy.12 (2015) showed that the solution temperature (45\u00b0C) decreased to body temperature (37\u00b0C) in 60 seconds when the solution was used for root canal irrigation in vivo. However, since the NaOCl exerts its antibacterial efficacy in seconds,13 60 seconds is enough to assess the antibacterial efficacy of the preheated NaOCl solution. Moreover, it can be claimed that a decrease in the temperature of the solution had no significant effect, since the preheated NaOCl group showed higher postoperative pain values than the NaOCl 2\u00b0C group. This means that the effect of the heat was clearly shown in our study.One of the limitations of our study is that the temperature of the solution in the root canal was not constant during the irrigation procedure. De Hemptinne, et al.We conclude that preheating NaOCl does not provide any extra antibacterial effect and results in a higher postoperative pain value than the cold NaOCl when used for final irrigation of root canals of teeth with asymptomatic apical periodontitis."}
+{"text": "Despite the potential of digital health interventions to improve the delivery of psychoeducation to people with mental health problems and their relatives, and substantial investment in their development, there is little evidence of successful implementation into clinical practice. We report the first implementation study of a digital health intervention: Relatives Education And Coping Toolkit (REACT), into routine mental healthcare. Our main aim was to identify critical factors affecting staff uptake and use of this online self-management tool for relatives of people with psychosis or bipolar.A mixed-methods, theory-driven , iterative multiple case study approach using qualitative analysis of interviews with staff and quantitative reporting of uptake. Carer researchers were part of the research team.In all, 281 staff and 159 relatives from Early Intervention teams across six catchment areas (cases) in England registered on REACT; 129 staff took part in qualitative interviews. Staff were positive about REACT helping services improve support and meet clinical targets. Implementation was hindered by: high staff caseloads and difficulties prioritising carers; perception of REACT implementation as research; technical difficulties using REACT; poor interoperability with trust computer systems and care pathways; lack of access to mobile technology and training; restricted forum populations; staff fears of risk, online trolling, and replacement by technology; and uncertainty around REACT\u2019s long-term availability.Digital health interventions, such as REACT, should be iteratively developed, evaluated, adapted and implemented, in partnership with the services they aim to support, and as part of a long term national strategy to co-develop integrated technology-enabled mental healthcare. Implementation strategies must instil a sense of ownership for staff and ensure they have adequate IT training, appropriate governance protocols for online working, and adequate mobile technologies. Wider contextual factors including adequate funding for mental health services and prioritisation of carer support, also need to be addressed for successful implementation of carer focussed digital interventions.ISCTRN 16267685.Study registration:  This is the first study to examine factors affecting staff uptake and use of a digital health intervention in UK mental health services.Mixed methods were used to understand staff engagement with the Relatives Education And Coping Toolkit (REACT) in Early Intervention teams across six locality based services, purposively sampled for geographical and ethnic diversity in order to maximise generalisability of findings.Findings highlight the need to embed technology development within clinical services; embrace an iterative long-term model of development, testing and adaptation; and provide adequate mobile technology, IT training and digital governance infrastructures to support new ways of working.Digital Health Interventions (DHIs) are increasingly being developed for people with severe mental health problems including psychosis and bipolar, to improve symptom monitoring , medicatHowever, evidence for successful implementation of DHIs in routine healthcare services is far more limited. Despite substantial investment, many DHIs are either not adopted by their intended users, are abandoned, fail to scale up locally or spread to other settings, or are not sustained over time , 10. We A recent systematic review of 26 studies reporting factors impacting on delivery of DHIs for people with psychosis or bipolar identified the following determinants of uptake: staff and service user attitudes; complexity of the user interface; staff / peer support to use the intervention; fit with existing service IT infrastructures; and costs to development and delivery . The majRelatives of people with psychosis or bipolar provide a large amount of unpaid care  but at hDHIs offer the potential for widespread dissemination of high quality, standardised care, made easily available, alongside a mechanism for uniting people online to share their experiences through peer support. Although DHI development costs can be substantial, ongoing delivery has the potential to be more cost effective in the long-term than face-to-face support and more accessible to those in rural areas and developing countries .The Relatives\u2019 Education And Coping Toolkit (REACT) is a supported self-management toolkit, providing accessible evidence-based information and support for relatives of people with psychosis or bipolar. Following evidence to support feasibility, acceptability, and effectiveness in reducing relatives\u2019 distress as a paper-based tool , REACT wWe used a theory-driven multiple case study design  integratNormalisation Process Theory (NPT) was used to guide data collection, analysis and interpretation. NPT focuses on the work done by staff to understand the processes by which a complex healthcare intervention is implemented, embedded, and integrated (or not) into practice . NPT hasThe study took place in Early Intervention (EI) teams across six National Health Service (NHS) trusts (cases) in England. Trusts provide healthcare to particular geographical areas, and within each trust there is an EI team which provides early intervention support to people with early signs of psychosis/bipolar. Cases were purposively sampled for geographical and ethnic diversity to maximise generalisability. To protect anonymity, a multi-layered taxonomy of birds and habitats was used for trusts and clinical teams  of relatives and staff advised on planning how REACT was introduced in each local context. IMPART leads were encouraged to customise their REACT site by adding a logo, times that the forum/direct messaging services were actively moderated and by whom, emergency contacts, and photos and biographies of the trust\u2019s REACT Supporters and IMPART leads. IMPART leads created REACT Supporter accounts for the staff moderating the forum and direct messaging, and clinician accounts for staff who could invite relatives to REACT by email.All trusts received pilot versions of REACT including an online manual from 19 September 2016 to 21 November 2016, during which minor edits were made. Each trust had at least one face-to-face training session, after the site went live, providing an overview of the importance of carer information and support; an outline of key components and how to use REACT; and aims of the IMPART study.Data were collected over 18\u2009months, first in two trusts (wave 1) in which key factors affecting implementation were identified, and in a further two (wave 2) following work done by the research team and stakeholders to design a revised implementation plan. This was repeated for the remaining two trusts (wave 3), with revisions culminating in a final list of recommendations based on understanding of key factors impacting on implementation across all six cases. Order of trusts was determined by pragmatic reasons related to the time taken for trust approvals.Staff engagement with the REACT toolkit was measured by the number of accounts staff created, and number of invitations sent to relatives over the data collection period. All data were recorded on the REACT website.Factors affecting implementation outcomes were explored through individual staff interviews; observations, document analysis; researcher reflective diaries, and Stakeholder Groups (SGs). Purposive sampling of participants was used to identify: IMPART leads (ILs), REACT supporters (RSs), team managers (TMs), and frontline clinical staff. The interview guide  was desiDescriptive summaries of implementation outcomes were calculated using quantitative measures of staff activity. Qualitative data sets collected from each case were analysed using framework analysis  supporteAll data were read and re-read by the Research Associates , and synthesised using the four main constructs of NPT  with guiIn wave 2 the implementation plan included the addition of: REACT booklets and business cards with details of how to access REACT for staff to give to relatives in face-to-face meetings; Staff office reminders with the REACT logo on including mugs and pens; automated email nudges for staff and relatives; a service planner to facilitate teams to allocate staff to key roles supporting REACT; and an auditing dashboard to show staff which relatives had been invited to use REACT. In wave 3, the plan evolved further to include a \u201cRequest Access Button\u201d for relatives who wanted to self-refer; staff induction packs for each role; a new \u201cREACT Champion\u201d role to promote REACT; a revised online \u201chow to\u201d manual for staff including a quiz to facilitate engagement; and printable PDF versions of REACT modules to offer as \u201ctasters\u201d for the website.Table\u00a0In all, 281 staff registered on REACT and between them, sent 355 invitations to 310 relatives, and 159 relatives registered. The highest number of clinician accounts were created at Lakes Trust (wave 3), least at Ocean (wave 2). Seashore Trust (wave 2) sent the most invitations to relatives, while Lakes Trust sent fewest. A minority of clinicians across all trusts sent invites, with the fewest being in Ocean, and most in Seashore. There was no evidence of an increase in implementation outcomes across the 3 waves, or as a result of the evolving implementation plan, though the timeframe of this analysis may have been too limited to adequately assess this. The aim of this study was to understand the factors determining these numbers, rather than to maximise them.Table\u00a0\u2026I think if REACT had been introduced as not research but just something we\u2019re going to do from now, that by the way we might evaluate, possibly it might have been seen as more of an infrastructural thing, this is what we will do, but yeah. The kiss of death is kind of true, but people switch off as soon as you go this is a person from X university and they\u2019re going to talk about this project. \u2026\u201d \u201cREACT had reasonable coherence for staff, in that they clearly understood what the toolkit was for, who it was aimed at, and the potential benefits for both staff .\u201cAn additional coherence problem was due to high staff turnover, disparately located teams, and preference for face-to-face training. Consequently, while staff understood the general purpose of REACT, many were not familiar with the detailed content and lacked confidence in promoting it to relatives.Mitigating strategies included delegating the role of inviting relatives to a small number of individuals (as in Marsh), and appointing a REACT champion to remind clinicians about REACT .\u201cIt shouldn\u2019t be psychology should it, REACT, it\u2019s just an information sharing but because it is held in psychology people are thinking oh it\u2019s another psychology strategy.\u201d .Coherence problems tracked through to cognitive participation. Staff agreed REACT could be of value to relatives, but only if delivered as part of a comprehensive care package, including face-to-face support. Opinions differed over who should have ownership over the delivery of REACT, depending on how the services were structured, and how REACT had been introduced. This impacted on buy-in from individual staff.\u201cI suppose it is a slight break with tradition isn\u2019t it, to use an internet-based source, because I suppose it depends how professionals are; our most valuable tool is ourselves and I don\u2019t know whether there\u2019s something that potentially is a bit daunting about handing that over to something else, i.e. technology\u201d .REACT was designed as an adjunct to a comprehensive service. However in trusts struggling to deliver services for relatives, staff feared relatives might become frustrated when they learnt from REACT about services they should have access to, but did not. Across professions, there were concerns about digital interventions replacing face-to-face therapies for psychosis, and particularly for older adults, who staff perceived as less likely to engage with DHIs.Commitment within clinical teams to integrate REACT into clinical practice was limited. REACT was rarely a regular item on any clinical meeting agendas. Tasks were usually allocated, not volunteered for, and training and supervision of key roles including the REACT Supporter role was lacking.REACT is just not the shark nearest the boat\u201d.\u201cI think there\u2019s an awful lot of new stuff happening right now, which makes it quite difficult for people to know what they should be focusing on [\u2026] you know one week we\u2019re saying this is the absolute priority, and then the next week we\u2019re saying, actually that\u2019s no longer absolute priority\u201d .For all trusts, the major challenges with implementation were located in the area of collective action, and how the introduction of REACT was managed through allocation of resources, and execution of protocols, policies, and procedures. Staff in all trusts were under great pressure, with high caseloads, competing priorities and, in some trusts (Woods and Seashore) high staff absence and turnover. All staff were working at (or beyond) capacity, lacking resources to deliver REACT. Staff felt forced to prioritise within priorities, leading to concentration of efforts on measureable outcomes (service user contacts rather than carer contacts), with financial incentives attached. As the Moor Trust IMPART lead put it during one of the SG meetings, \u201cIt was unclear to researchers or clinical staff who was responsible within trusts for the strategic direction of service development and who could facilitate the shift in priorities needed for staff to embrace novel interventions such as REACT.A further barrier to action was disjunction between the online nature of REACT (\u201cout of sight and out of mind\u201d), and staffs\u2019 primarily paper-based and community-located ways of working. This was exacerbated by lack of up-to-date mobile technology, and previous negative online experiences including a staff member having been \u201ctrolled\u201d  on a different online forum.\u201cAnd then we can go out then, I use my own personal phone \u2018cos obviously my work phone doesn\u2019t let me go on the internet, or I\u2019ll ask them to get their iPad out and I\u2019ll show them that way.\u201d.Staff who did try to use REACT, reported good interactional workability, promoting better consultations with relatives. However, some content was felt inconsistent with other aspects of the service (particularly the use of diagnostic terms), and the REACT dashboard used to create relatives\u2019 accounts was not sufficiently user friendly. The need for staff to sign into REACT using a unique username and password also caused difficulties, as staff were required to remember multiple versions of these for different online systems across the NHS. Many staff did not have access to health-service mobile technology and could not show relatives the REACT website during home-based consultations.Although most relatives had personal computers or tablets, staff did not feel it was appropriate to ask relatives to log in to their own desktop computers to be shown the site, particularly as these were often in the bedroom.\u201c\u2026\u2026it\u2019s absolutely no chance I\u2019m going to be putting my registration at risk to look over something that\u2019s,\u2026. but for the confidence, the security, the even if everybody.. somebody just says I need help right now doesn\u2019t get it within 20 minutes, a stereotype saying [?] off the bridge and then well they put a cry for help knowing REACT Champion was on duty and he didn\u2019t see it.\u201d .Finally, staff allocated to REACT Supporter roles did not feel confident or supported to moderate the online forum or respond to direct messages. Lack of clarity over who had clinical responsibility for REACT and absence of trust policies about managing risk online, resulted in poor relational integration (understanding of accountability and confidence in each other in delivering the practices required) and consequently lack of proactive engagement by REACT Supporters. Fear of being held responsible for risk was widespread across all trusts and in one (Lakes), led to some staff withdrawing support for REACT. No trusts seemed able to reassure staff or produce risk policies adapted for online communication.\u201cI think it\u2019s just not seeing any kind of outcomes coming from registering people and it looks like there\u2019s no activity on the site, I guess I mean people might be logging on, the few that have actually registered, but I guess there\u2019s no way of monitoring it, no way of knowing that people are actually finding it helpful or useful\u201d Where staff overcame these barriers and invited relatives, they were frustrated by the lack of feedback (reflexive monitoring) available from relatives about the site. Where staff sought feedback directly from relatives, this was generally positive about the module content, particularly hearing the stories of others. However, relatives were disappointed by the lack of forum activity. This was partly due the small population of relatives using the site, so no-one wanted to be the first; and partly due to lack of staff promotion of forum activity. As staff became aware of the lack of forum activity, they became less motivated to invite relatives, creating a vicious cycle.This is the first study that examined in detail the process of implementation of a DHI aimed at supporting relatives of people with psychosis or bipolar within national publicly funded mental health services. Staff engagement with REACT was facilitated by a good fit between the rationale and design of the toolkit, and the need for them to deliver support to carers as part of audited national clinical guidelines. The toolkit was easy to integrate alongside other services currently being offered, and staff could easily see the benefits of this approach in terms of accessibility to high quality information and support for relatives and staff. Positive feedback from relatives was a strong motivator of engagement. Barriers to implementation included high staff caseloads and difficulties prioritising supporting relatives; technical difficulties using REACT; poor interoperability with trust IT systems and care pathways; lack of access to mobile technology and IT training; restricted forum populations leading to low levels of use; staff fears of managing risk, online trolling, or replacement by technology; and uncertainty around REACT\u2019s long-term availability.Some of these factors are consistent with the findings of previous research into implementation of DHIs into physical healthcare settings , 41\u201343 aThe first is the significant impact of wider social context around mental health. Despite government initiatives to achieve parity of esteem for mental and physical health , mental The second is that digital confidence, competence, governance, and access to equipment were limited, suggesting that implementation of any new DHI would have been challenging. Managing risk was a high clinical priority, but risk management policies did not include online activity, leaving staff unclear of their responsibilities. Investment is needed to ensure staff have access to the mobile hardware they need, integrated IT platforms that support single login, and digital skills training. Governance issues around digital risk and responsibility urgently need to be addressed with clear staff policies.A third key barrier was staff perception that REACT was a research study, rather than a clinical initiative, despite extensive attempts to explain that the implementation focus of the work. This perception was compounded by the fact that the decision to adopt REACT was often made first by research leads not clinical teams. This impeded staff uptake, and was further compounded by lack of long-term funding for REACT, meaning there was no guarantee it would be available after the study. DHIs, such as REACT, need to be co-developed and iteratively evaluated, adapted and refined with extensive staff and service user input, as part of a long-term and resourced strategy to develop fully integrated technology-enabled services . The curBased on this study, we have outlined generalizable recommendations for successful implementation of DHIs, using REACT as an example  of data collection, restricting focus to the early stages of implementation and precluding an understanding of embedding, integration, or the impact of the evolving implementation plan; and the dual role of the research team in developing REACT whilst also collecting data to understand the process of implementation. Further research needs to test the generalisability of the factors identified as impacting on implementation of REACT in mental health services, to other DHIs and within other healthcare systems, and the effectiveness of the recommendations. Further research could also explore how individual differences among staff impact on levels of engagement, which has not been addressed here. This study has focussed only on uptake and use by staff, but equally important are the factors impacting on uptake and use by relatives who were offered REACT, and these will be reported elsewhere. There are many alternative theoretical frameworks that could have been used and of particular interest is the more recently proposed non-adoption, abandonment, scale-up, spread, and sustainability (NASSS) framework which has been specifically developed for DHIs . NASSS wIn the first implementation study of a DHI (in this study REACT) in the UK NHS mental health services, we identified many factors across staff coherence, cognitive participation, collective action and reflexive monitoring, that impact on the success of implementation and which are likely to be relevant to other DHIs in mental health. Some of these factors could be addressed by facilitating a model of DHI development that is strongly embedded in services, prioritises long-term intervention evolution and change, and manages staff expectations around uptake and effectiveness. However, wider contextual factors including inadequate funding for mental health services; lack of prioritisation of working with carers, and dissociation between research and clinical practice also need to be addressed."}
+{"text": "BackgroundApical periodontitis is caused by bacteria present in the root canal space. The removal of the infection is crucial to obtain healing. Canal irrigation is among one of the most important steps in eliminating bacteria. Sodium hypochlorite (NaOCl) is still the preferred irrigant due to its disinfecting and pulpal dissolution abilities. Heating NaOCl improves those abilities. However, the ability of intracanal heated NaOCl to kill bacteria has not yet been evaluated.ObjectivesThis study compared the disinfecting ability of different irrigation regimens using NaOCl with and without sonic and ultrasonic agitation, and with and without intracanal heating of NaOCl.MethodsThe canals of extracted mandibular premolars were prepared, sterilized and infected with\u00a0E. faecalis\u00a0for 28 days. The canals were then assigned to eight groups of 10 teeth depending on the NaOCl irrigation protocol. Group CONV: conventional irrigation with syringe and needle; Group END: NaOCl sonic agitation with EndoActivator; Group EDD: NaOCl sonic agitation\u00a0with EDDY; Group PUI: NaOCl passive ultrasonic agitation; Group H: intracanal heating of NaOCl; Groups END-H, EDD-H and PUI-H: NaOCl agitation\u00a0with EndoActivator, EDDY and passive ultrasound, respectively, followed by intracanal heating of NaOCl. The canals were sampled before (S1) and after (S2) the different irrigation protocols were performed, the colony-forming units were counted and the percentage of bacteria reduction was calculated for each group.ResultsThe number of bacteria decreased significantly for the different protocols (p < 0.001). The groups with NaOCl intracanal heating reduced bacteria significantly more than the other groups (p < 0.001). Five S2 samples in group H were free of bacteria. All of the S2 samples in the groups with NaOCl sonic and ultrasonic agitation followed by NaOCl heating were free of bacteria. Intracanal heating of NaOCl was more effective in killing bacteria than conventional irrigation, and sonic or passive ultrasonic agitation of NaOCl.ConclusionsIntracanal heating of NaOCl has the potential to be used as an adjunct to root canal irrigation in order to increase bacterial reduction in comparison to the conventional irrigation techniques involving sonic or ultrasonic agitation. Agitation of NaOCl followed by intracanal warming of the solution seems to be very promising in eliminating bacteria\u00a0from infected root canals. Apical periodontitis is mainly caused by bacteria present in the root canal space . One of Heating NaOCl increases its dissolving and disinfecting properties -7. WarmiCanal cleanliness and/or the presence of debris have not been shown to influence the repair of tissues . IndirecTooth selection and preparationEighty-eight single rooted mandibular premolars previously extracted for orthodontic reasons and stored in 0.9% saline solution were selected for this ex vivo study. None of the teeth had an endodontic treatment. The roots were fully developed. All teeth had only one canal, and did not show root fracture, resorptive defects or canal calcifications. The root surfaces were cleaned with periodontal curettes. The teeth were re-placed in a 0.9% saline solution until use.The access cavity was created. Patency was established using a #8 hand file  inserted in the canal until its tip was visible at the apical foramen. The crown was then reduced to standardize the length of the teeth. The working length (WL) was established a 17 mm, 1 mm shorter than the apical foramen. All canals were instrumented to working length with Reciproc 25 (0.25 mm at the tip)  followed by a Reciproc 50 (0.50 mm at the tip) (VDW GmbH). The instruments were used according to the manufacturer\u2019s instructions for use. The canals were irrigated during the preparation procedure with 6 ml of 5.25% NaOCl using a syringe and a NaviTip 31-ga endodontic irrigation needle with double sideport . The final irrigation protocol consisted of 3 ml of 17% ethylenediaminetetraacetic acid (EDTA) (Ultradent Products Inc.), 3 ml of distilled water and 3 ml of 5.25% NaOCl, each for 2 minutes. The NaOCl was inactivated with 3 ml 10% sodium biosulfate for 2 minutes. Sterile Reciproc 50 paper points (VDW GmbH) were used to dry the canals. The root surfaces and the apex of each tooth were sealed with cyanoacrylate. The teeth were mounted in silicone blocks. The specimens were distributed into eight groups of 11 specimens each. All the specimens were then sterilized at 121\u00b0C for 30 minutes.Control of sterilizationOne canal from each group was selected randomly to serve as a negative control and confirm sterilization. It was incubated and cultured in the same manner as described later for the root canal sampling procedure.Specimen contamination8\u00a0colony forming units per milliliter (CFU/mL).E. faecalis,\u00a0derived from ATCC 19433, was used and cultured aerobically on blood agar at 35\u00b0C for 48 h.\u00a0Inoculum was prepared in sterile BHI broth and turbidity was set to 0.5 McFarland corresponding to approximately 1.5 \u00d7 10Ten canals from each group were inoculated with 10 \u03bcL of the\u00a0E. faecalis\u00a0culture. The specimens were incubated for 28 days at 37\u00b0C. The inoculum was refreshed every day.Experimental groupsEach experimental group included 10 specimens. The eight groups were as follows:1-\u00a0Group CONV (n = 10): Each canal was irrigated with room temperature 5.25% NaOCl for 3 minutes using a syringe and a NaviTip 31-gauge endodontic irrigation needle with double sideport (Ultradent Products Inc.). A sterile stopper was placed on the needle to verify its insertion to 1 mm short of the working length. The needle was moved up and down over 3-5 mm during the irrigation. Each canal was rinsed with 9 mL of 5.25% NaOCl.2-\u00a0Group END (n = 10): Each canal was filled with room temperature 5.25% NaOCl. The EndoActivator sonic device  was used to agitate the solution in the canal with a specifically designed tip according to the manufacturer\u2019s recommendations . The tip3-\u00a0Group EDD (n = 10): Each canal was filled with room temperature 5.25% NaOCl. The EDDY sonic device (VDW GmbH) was used to agitate the solution in the canal with a specifically designed tip according to the manufacturer\u2019s recommendations . The tip4-\u00a0Group PUI (n = 10): Each canal was filled with room temperature 5.25% NaOCl. An ultrasonic tip with a noncutting end  mounted in a piezoelectric ultrasonic device  was inserted to 1 mm less than the WL and activated at the power setting of 4 for 20 seconds. Passive ultrasonic agitation (PUI) was repeated three times. The canal was flushed with 3 mL room temperature 5.25% NaOCl after each activation\u00a0cycle to refresh the solution .5-\u00a0Group H (n = 10):\u00a0Each canal was filled with room temperature 5.25% NaOCl. The solution was heated in the canal for 10 s using a Touch\u2019n Heat XF (size 0.30 mm and 0.04 mm/mm taper) electric heat carrier , attached to a System B unit  .\u00a0The tem6-\u00a0Group END-H (n = 10): Sonic agitation was performed three times as described for group END. After each time, NaOCl was aspirated, the canal was filled again with room temperature 5.25% NaOCl which was heated in the canal in the same manner as described for group H. Three cycles of heating were performed as for group H.7-\u00a0Group EDD-H (n = 10): Sonic agitation was performed three times as described for group EDDY. After each time, NaOCl was aspirated, the canal was filled again with room temperature 5.25% NaOCl which was heated in the canal in the same manner as described for group H. Three cycles of heating were performed as for group H.8-\u00a0Group PUI-H (n = 10): Passive ultrasonic agitation was performed three times as described for group PUI. After each time, NaOCl was aspirated, the canal was filled again with room temperature 5.25% NaOCl which was heated in the canal in the same manner as described for group H. Three cycles of heating were performed as for group H.At the end of each procedure, the NaOCl was then inactivated with 3 mL of 10% sodium biosulfate for 2 minutes.All solutions were delivered into the canal using a 3-mL syringe and a NaviTip 31-gauge endodontic irrigation needle with double sideport (Ultradent Products Inc.).Root canal sampling proceduresTen canals from each group were sampled.A first sample (S1) was taken from each canal prior to implementing the tested protocols. The canal was filled with a sterile saline solution. A #15 hand file (Maillefer) was used to loosen biofilm bacteria by scraping the root canal walls for 15 s. A sterile Reciproc 50 paper point was inserted in the canal to the working length. The paper point was left in the canal for 1 minute and placed in sterile BHI broth for 20 minutes. The solution was homogenized by vortex, serially diluted, plated on blood agar and incubated for 48 h at 37\u00b0C. Colony-forming units (CFUs) were counted.A second sample (S2) was taken from each canal after implementing the investigated irrigation protocol. The number of CFUs was determined as described for S1.Statistical analysisThe Statistical Package Software for Social Science  was used to perform the statistical analysis. The level of significance was set at\u00a0p\u00a0\u2264 0.05. Kolmogorov-Smirnov tests were conducted to evaluate the normality distribution of continuous variables. Wilcoxon tests were performed to compare colony-forming units before and after irrigation for the eight groups. The percentage of variation of CFU after irrigation was compared among the eight groups using Kruskal-Wallis tests, followed by Mann-Whitney tests.The results are shown in Table The number of CFUs decreased significantly between S1 and S2 for the different groups (p < 0.001).The percentage of bacteria reduction was very high for the CONV, END, EDD, PUI and H groups but significantly different from 100% (p < 0.05). The percentage of bacteria reduction was higher for END, EDD, PUI and H groups compared to group CONV (p < 0.001).\u00a0Group H performed better than groups END, EDD and PUI (p < 0.05).The CONV, END, EDD and PUI protocols were not able to completely remove bacteria in any of the 10 specimens. Five S2 samples were free of bacteria in Group H. All of the S2 samples in the groups in which the NaOCl was activated and heated were free of bacteria.The percentage of bacteria reduction was statistically significant between groups in which NaOCl was not heated and the other groups (p < 0.001). The percentage of bacteria reduction was not statistically significant among H, END-H, EDD-H and PUI-H (p = 0.166).This was the first study to evaluate ex vivo effect of intracanal heating of NaOCl on the elimination of bacteria.The methodology of the present study and the choice of\u00a0E. faecalis\u00a0were similar to other studies , 19. HowHeating NaOCl, and its sonic and ultrasonic agitation enhance its decomposition . TherefoIncreasing heating temperatures leads to a faster and greater antibacterial activity , 22-24. The results showed that the different irrigating protocols reduced significantly the bacteria counts. However, as expected and similar to previous studies, conventional irrigation without agitation (Group CONV) was the least effective compared to the other groups in reducing the number of bacteria , 26.In agreement with previous studies, sonic and ultrasonic agitation of NaOCl without heat were not successful in eliminating bacteria completely , 27. NaOHeating NaOCl reduced the number of bacteria significantly in in vitro studies , 23, 24.The disinfecting ability of sonic and ultrasonic agitation of NaOCl was similar when NaOCl was heated. All the S2 samples from the groups exposed to NaOCl agitation and intracanal heating of NaOCl were free of bacteria, in comparison to only five in heat-only group (group H). Although the difference in the percentage of bacteria reduction between the heat- and agitation-groups, and the heat-only group was not statistically significant, this finding could indicate the importance of sonic and ultrasonic agitation in association with heat application to render canals free of bacteria. The removal of the pulp tissue with sonic or ultrasonic activation of NaOCl prior to heat application would allow a better exposure of the bacteria to the heated NaOCl. A recent study showed that the ultrasonic agitation of intracanal heated NaOCl enhanced the penetration of NaOCl in the dentinal tubules and resulted in cleaner canals compared to PUI alone and conventional irrigation . In anotOne of the main objectives of the root canal treatment is to eliminate bacteria. Irrigation during and the end of the canal preparation plays a major role in the elimination of infection. Sodium hypochlorite is widely used as an irritant due to its antibacterial and pulp dissolution abilities. Warming of NaOCl inside of the canal enhanced pulp tissue dissolution and resulted in cleaner canals. However, the antibacterial ability of intracanal heated NaOCl has never been evaluated. The present study showed that intracanal warming of 5.25% NaOCl could be considered as an alternative to conventional irrigation protocols with sonic or ultrasonic agitation. The agitation of 5.25% NaOCl sonically or ultrasonically followed by intracanal heated NaOCl is very promising with regards to the removal of infection. Further evaluation is needed."}
+{"text": "M. tuberculosis. On a larger scale, it reinforces the importance of horizontal gene transfer in bacterial evolution and examines novel models and methods to provide a better understanding of the subtle effects of individual M. tuberculosis-specific virulence factors in infection settings that are relevant to the pathogen.This work sheds light on the role of the lipid 1-tuberculosinyladenosine in the evolution of an environmental ancestor to  Mycobacterium kansasii is an environmental nontuberculous mycobacterium that causes opportunistic tuberculosis-like disease. It is one of the most closely related species to the Mycobacterium tuberculosis complex. Using M. kansasii as a proxy for the M. kansasii-M. tuberculosis common ancestor, we asked whether introducing the M. tuberculosis-specific gene pair Rv3377c-Rv3378c into M. kansasii affects the course of experimental infection. Expression of these genes resulted in the production of an adenosine-linked lipid species, known as 1-tuberculosinyladenosine (1-TbAd), but did not alter growth in vitro under standard conditions. Production of 1-TbAd enhanced growth of M. kansasii under acidic conditions through a bacterial cell-intrinsic mechanism independent of controlling pH in the bulk extracellular and intracellular spaces. Production of 1-TbAd led to greater burden of M. kansasii in the lungs of C57BL/6 mice during the first 24\u2009h after infection, and ex vivo infections of alveolar macrophages recapitulated this phenotype within the same time frame. However, in long-term infections, production of 1-TbAd resulted in impaired bacterial survival in both C57BL/6 mice and \u2212/\u2212Ccr2 mice. We have demonstrated that M. kansasii is a valid surrogate of M. tuberculosis to study virulence factors acquired by the latter organism, yet shown the challenge inherent to studying the complex evolution of mycobacterial pathogenicity with isolated gene complementation. Mycobacterium tuberculosis virulence factors have been established by genetic knockout and complementation within the pathogen, producing evidence of an attenuation of virulence in ex vivo or in vivo experimental infections  (Rv3376-Rv3378c genomic island uniquely present in M. tuberculosis is known to encode a class II terpene cyclase (Rv3377c) and a tuberculosinyl transferase (Rv3378c). Together, the two enzymes are responsible for the conversion of geranylgeranyl pyrophosphate (GGPP) into the recently identified M. tuberculosis-specific lipid 1-tuberculosinyladenosine (1-TbAd), which is a potential diagnostic molecular marker for TB disease  events have happened during mycobacterial speciation and are associated with the stepwise emergence of pathogenic species \u201316. Fift\u2013(MKMTCA) . Althoug disease \u201320. 1-Tb\u2013N6-TbAd . While Grculosis , 19, it ow color .M. tuberculosis from phagosomal acidification inside macrophages  during long-term murine infection. This study demonstrates that we can use M. kansasii as a proxy of the MKMTCA to explore the complex evolution of M. tuberculosis. It also shows that gene acquisition likely provided advantages for specific contexts, including a possible and unexpected role in survival within alveolar macrophages during early stages of infection, despite tradeoffs or challenges under other circumstances requiring further evolution to overcome.We previously reported the important role of 1-TbAd in protecting rophages . In the M. tuberculosis-specific gene pair Rv3377c-Rv3378c into the M. kansasii genome within an integrative plasmid containing hygromycin resistance to produce M. kansasii::Rv3377-78c . As a control for subsequent experiments, an integrative \u201cempty vector\u201d (EV) was employed. After labeling with [14C]adenosine and lipid extraction using chloroform and methanol, radio-thin-layer chromatography (radio-TLC) was used to detect adenosine-linked lipids in M. kansasii::Rv3377-78c clones for comparison to M. tuberculosis strain H37Rv  (5%) staining showed that similar amounts of total lipids were spotted for each M. kansasii sample  and N6-TbAd (5.8\u2009min) were known  signals (m/z 540.354) matching the expected retention time and mass (m/z 540.3544) of the proton adducts of 1-TbAd. The extractions were performed at a range of pHs (4.5 to 7.4) since both the Dimroth reaction that generates N6-TbAd and the capture of lysosomotropic agents are sensitive to pH, as previously explained  (data not shown).High-performance liquid chromatography-mass spectrometry (HPLC-MS) was used to chemically identify the compounds produced by re known  to g. Whxplained , 22, 23.nditions  to g. Si3377-78c  and g. IM. kansasii, we assessed its influence on in vitro characteristics of the bacterial culture. M. kansasii::Rv3377-78c grew similarly to WT M. kansasii and M. kansasii::EV in Middlebrook 7H9 broth  and M. kansasii::Rv3377-78c (MKAN::Rv33778c) cultures were inoculated at equal OD600 into fresh pH-adjusted 7H9 (using HCl titration) and incubated at 37\u00b0C in a rolling incubator over 17 days. The starting pH of the cultures is indicated above each graph. OD600 was monitored every 2 to 3 days. The data are plotted separately for each of three independently growing cultures per strain. The data are representative of five independent experiments. Download FIG\u00a0S1, PDF file, 0.1 MB.1-TbAd production enhances growth at low pH. Copyright \u00a9 2020 Ghanem et al.2020Ghanem et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the M. tuberculosis estimated that 1-TbAd might naturally accumulate to micromolar concentrations in phagosomes, and 5 to 20\u2009\u03bcM 1-TbAd alters lysosomal pH and morphology in human macrophages  should not alter pH, but it would contact bacteria in high concentrations. As expected, the addition of synthetic 1-TbAd (pKa \u223c 8.5) and N6-TbAd (pKa \u223c 3.8) did not alter the pH of the 7H9 media  containing 1, 5, 10, or 20\u2009\u03bcM TbAd and monitored growth over 16\u2009days  measured by the addition of fluorescein and reading fluorescence against a standard curve. (b) Additional data for FIG\u00a0S2, PDF file, 0.2 MB.Chemical complementation with TbAd does not change 7H9 broth pH or enhance growth. (a) pH of 7H9 of Copyright \u00a9 2020 Ghanem et al.2020Ghanem et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the M. kansasii::EV or M. kansasii::Rv3377-78c was stained with carboxyfluorescein diacetate succinimidyl ester (CFSE) to measure their intracellular pH while monitoring their growth in different pH-adjusted 7H9 broth  overnight . The data are plotted as the median values. Download FIG\u00a0S3, PDF file, 0.02 MB.Copyright \u00a9 2020 Ghanem et al.2020Ghanem et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the M. kansasii::Rv3377-78c 1 day after aerosol infection compared with M. kansasii::EV (not shown). To test whether Rv3377c-Rv3378c was altering the dose administered or instead enhancing establishment or early growth, mice were aerosolized with M. kansasii::EV or M. kansasii::Rv3377-78c and their lungs were collected and homogenized shortly (4\u2009h) after infection and compared to bacterial counts 24\u2009h after infection . The top row shows the absolute CFU count data from independent experiments. The bottom row shows 24-h CFU/mean 4-h CFU ratio data from corresponding independent experiments. The data are plotted as the means \u00b1 SD. GraphPad Prism 8.1.2 was used to perform Welch\u2019s two-tailed unpaired t tests where statistical significance is indicated as follows: ns, not significant (P\u2009>\u20090.05); *, P < 0.05; **, P < 0.01; ****, P < 0.0001. Download FIG\u00a0S4, PDF file, 0.02 MB.Copyright \u00a9 2020 Ghanem et al.2020Ghanem et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the M. kansasii in the lungs by promoting survival in resident macrophages. First, using bone marrow-derived macrophages (BMDMs), we infected cells in culture with M. kansasii::EV or M. kansasii::Rv3377-78c and collected cell lysates at 4 and 24 h postinfection: the infections proceeded similarly with both bacteria unlike what we had observed in mouse lungs in vivo  Absolute CFU count data are plotted as the mean of technical replicates  \u00b1 SD, individually for three independent experiments. (Bottom) 24-h CFU/mean 4-h CFU ratio data are plotted for individual experiments and pooled (n\u2009=\u200915 per condition), shown as the mean \u00b1 SD. GraphPad Prism 8.1.2 was used to perform Welch\u2019s two-tailed unpaired t tests . Download FIG\u00a0S5, PDF file, 0.02 MB.Copyright \u00a9 2020 Ghanem et al.2020Ghanem et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the M. kansasii was directly introduced into the upper respiratory tract  to 0.27 (week 1), stabilizing at the latter proportion over time  Mice were sacrificed at 1 and 42 days postinfection to establish initial and persistent infectious dose, respectively. (b) Mice were weighed over the 42-day period to assess change in weight as a proxy for clinical status. Download FIG\u00a0S6, PDF file, 0.1 MB.High-dose infection with Copyright \u00a9 2020 Ghanem et al.2020Ghanem et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the \u2212/\u2212Ccr2 mice lose the ability to recruit non-tissue-resident immune cells and succumb to M. tuberculosis infection  in the mixed infection was valid, we compared hygromycin resistance of M. kansasii::EV  is an essential component for defense in the airways; nfection . We used3377-78c . Interespulation . With thasii::EV  and M. k3377-78c  after 4 l levels ; this isfections . Neitherfections . Thus, 110.1128/mBio.02645-20.8FIG\u00a0S7M. kansasii::EV and M. kansasii::Rv3377-78c retain hygromycin resistance in C57BL/6 mice. (a and b) Suspensions of M. kansasii::EV (MKAN::EV) (a) and M. kansasii::Rv3377-78c (MKAN::Rv33778c) (b) were used to infect WT C57Bl/6 mice. Lungs were isolated at 4 h and 4 weeks postaerosolization (n\u2009=\u20095 to 10 lung pairs per time point). CFUs were counted on 7H10 plates plus PANTA with or without 50\u2009\u03bcg/ml hygromycin (hyg50). (a and b) Mean pulmonary CFUs determined from plating with or without hyg (solid bars), and percent hyg resistance (+hyg/-hyg \u00d7 100%) (empty bars); points represent data from one mouse, and bars denote group mean. (c) Ratio of total pulmonary CFUs of 4 weeks over 4 h; GraphPad Prism 8.1.2 was used to perform Welch\u2019s two-tailed unpaired t tests where ns is not significant (P > 0.05). Download FIG\u00a0S7, PDF file, 0.04 MB.Copyright \u00a9 2020 Ghanem et al.2020Ghanem et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the M. kansasii to study the pathoevolution of M. tuberculosis. The less virulent nontuberculous mycobacterium (NTM) species is a suitable surrogate for the expression of M. tuberculosis-specific products, such as 1-TbAd, and can readily be used in vitro and for ex vivo and in vivo experimental infection models. We showed that 1-TbAd led to an improved survival during the first 24\u2009h of infection when tested in vivo, and ex vivo in alveolar macrophages, but the isolated addition of 1-TbAd to M. kansasii resulted in impaired persistence in different murine infection models.Our results indicate the feasibility of using M. kansasii::Rv3377-78c produced lipid species distinct from those seen in M. kansasii::EV. Our prior and current data indicated that transfer of TbAd biosynthesis genes to M. kansasii does not promote growth at neutral pH but confers increased growth in 7H9 media in the more acidic pH range (5.0 to 5.4)  . When adrophages , 22. In Rv3377-Rv3378c-dependent alkalization of 7H9 broth which might be explained by compartmentalization: whereas intracellular bacteria are bound in a small phagosomal compartment of 10\u221215 liter, growth in 7H9 media provides a much larger compartment for 1-TbAd to disperse in if it is physically shed from the bacterium. By a rough estimate, 1010 bacteria-worth of 1-TbAd would be required to change the pH of 1\u2009ml of 7H9 from 5.2 to 5.3, about 10,000 times the concentration of bacteria we inoculate  when 1-TbAd was generated through gene transfer and the action of enzymes in the bacterium. While not fully understood, these divergent outcomes whereby the compartment of origin controls the protective effect can be explained by the lysosomotropy model. Cytosolic 1-TbAd would be expected to shed its proton during membrane passage into the periplasm. The lack of pH control in the cytosol of M. kansasii::Rv3377-78c cultured in acidic media suggests that 1-TbAd does not act as a shield against proton flow into M. kansasii cytosol, which is the expected outcome if membrane penetration is required for the protective effect. These outcomes indicate that the consistently observed survival advantage could derive from 1-TbAd passage from the cytosolic membrane into the periplasm, mycolate membrane, or surface of M. kansasii. 1-TbAd may act on the bacterial population itself by targeting or protecting specific molecules during exposure to low pH, stopping damage from occurring.In phagocyte-free systems, we experimentally tested the hypothesis that the pH-dependent growth advantage of Rv3377c-Rv3378c-dependent metabolites, including 1-TbAd, do not have a direct growth-promoting effect. Our data also demonstrate that Rv3377c-Rv3378c-dependent metabolites protect against acid stress in vitro, using mechanisms that are independent of macrophage function, including lysosomes or activating receptors.It is noteworthy that genetic and chemical complementation provide different information about mycobacterial virulence factors, which in this case might result from the differential compartmentalization of the molecules. This result also argues that 1-TbAd must exert its protective effect at a specific location within the bacterial cell or cell wall. Exogenous 1-TbAd may simply not reach this specific location or not reach the location in an uncharged conjugate base state. Together with our genetic complementation data, it is clear that M. kansasii cell envelope composition; therefore, we can formally assign effects to Rv3377c and Rv3378c but cannot refute the possibility of an indirect pathway for the 1-TbAd effect. It is notable that the overall lipid profiles examined by TLC were not significantly altered by gene transfer. The Congo red retention assay - or lipopolysaccharide (LPS)-activated BMDMs for M. tuberculosis replication  as previously described  needles five times, 25-G needles five times, and 26-G needles three times, followed by low-speed centrifugation at 50 \u00d7 g for 5 min with passage through a 5-\u03bcm filter. To generate M. kansasii::Rv3377-78c, a 2.4-kb PCR fragment spanning Rv3377c-Rv3378c was generated using primers BamHI-Rv3377c-Rv3378c-F and HindIII-Rv3377c-Rv3378c-R (see hsp60 promoter and a selective apramycin resistance marker. The hsp60-Rv3377c-Rv3378c fragment was shuttled into the integrative vector pMV306 containing a hygromycin resistance cassette using XbaI and HindIII. All ligations were done using T4 DNA ligase (Fermentas). The resulting plasmid pMV306::Hsp60-Rv3377c-Rv3378c was verified by Sanger sequencing (Genome Qu\u00e9bec) to ensure the absence of frameshift or point mutations during the cloning process. An unaltered version of pMV306 with a hygromycin resistance cassette was used to create the empty vector (EV) control strain M. kansasii::EV. Following electroporation, M. kansasii::EV and M. kansasii::Rv3377-78c were grown in the presence of 100\u2009\u03bcg/ml hygromycin (Wisent).10.1128/mBio.02645-20.1TABLE\u00a0S1Table\u00a0S1, PDF file, 0.1 MB.Primer list. Download Copyright \u00a9 2020 Ghanem et al.2020Ghanem et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the M. kansasii::EV, M. kansasii::Rv3377-78c, and M. tuberculosis were grown to mid-log phase and subsequently incubated with 0.25 \u03bcCi/ml radiolabeled [8-14C]adenosine  for 14\u2009days. Polar lipid fractions were extracted using CHCl3\u2212CH3OH\u22120.3%NaCl (vol/vol/vol) (3\u2212CH3OH\u2212H2O 10:5:1 (vol/vol/vol) used as the mobile phase solvent. The radiolabeled signature was developed using Storm 840 PhosphorImager  to visualize adenosine-linked lipids in each lane. [8-14C]adenosine 1:100 was used as a no-lipid staining control. The plate was stained with 5% phosphomolybdic acid reagent (PMA) (Sigma) and heated briefly using an industrial blow dryer to visualize the total amounts of lipids loaded in each lane.vol/vol) , 37. ExtM. kansasii::Rv3377-78c, M. kansasii::EV, and M. kansasii parent strain were grown in 30\u2009ml of 7H9 media supplemented with albumin-dextrose-saline  to late log phase. Bacterial cell pellet and supernatant were separated by centrifugation at 5,000\u2009rpm for 5 min. The cell pellet was resuspended in 4\u2009ml of phosphate-buffered saline (PBS) at pH 7.4 and distributed equally into four 2-ml screw-cap tubes. The cells were pelleted by centrifugation and further resuspended in 1\u2009ml PBS at pH titrated to 7.4, 6.4, 5.5, and 4.5 with hydrochloric acid and incubated for 2\u2009h at 37\u00b0C. At the end of 2\u2009h, the cell pellet and the PBS supernatant were collected for lipid extraction. Ten volumes (3\u2009ml) of chloroform/methanol (C/M) at a ratio of 1:2 were added to the cell pellet and supernatant for extraction for 1\u2009h at room temperature. A second extraction under similar conditions was performed with 3\u2009ml of C/M at a ratio of 1:1. The extracted fractions were pooled and dried under a stream of nitrogen gas. Lipids from the 1-ml PBS supernatant were extracted using acidified ethyl acetate by adding 3\u2009\u03bcl of 6 N HCl and 1.4\u2009ml of ethyl acetate and mixing for 30 min in an Orbitron shaker. The mixture was centrifuged at 2,000\u2009rpm for 15 min to collect the upper organic phase and dried on glass under a stream of nitrogen gas at room temperature, and total lipids were weighed using an analytical balance. HPLC-MS separations were performed as described previously  for 2\u2009h , 38. Con600) was adjusted to 0.34, and 222\u2009\u03bcl of mid-log-phase declumped bacteria was added to 15\u2009ml of freshly prepared, pH-adjusted 7H9 in 150-ml roller bottles . Triplicate cultures were made per condition (strain and pH) and incubated at 37\u00b0C, rolling in the dark. OD600 was measured every 2 or 3\u2009days using two\u2009200-\u03bcl volumes of culture  and a Tecan Infinite M200 Pro plate reader. At days 8 and 17, 1\u2009ml was removed from each culture for centrifugation and recovery of supernatant, which was stored at 4\u00b0C until the extracellular pH was read using a micro pH combination electrode (AgCl) (Sigma-Aldrich), and Orion Star A111 meter (Thermo Scientific).For all pH experiments, liquid media were prepared as usual, and the pH was equilibrated to 4.0, 4.9, 5.0, 5.1, 5.2, 5.4, 6.0, 6.7, and 7.2 using 2 M HCl or NaOH. The optical density at 600 nm  control as indicated. The plates were incubated at 37\u00b0C in the dark, and OD600 was measured every 1 to 3 days with a Tecan Infinite M200 Pro plate reader.Synthetic 1-TbAd and eviously . Bacteri8 CFU was pelleted, the supernatant was completely removed, and the cells were resuspended in 0.3\u2009ml PBS containing 100\u2009\u03bcM carboxyfluorescein diacetate succinimidyl ester (CFDA-SE)  for 20 min at 37\u00b0C, shaking at 150 rpm in the dark. Bacteria were next diluted with 10\u2009ml of 7H9 and incubated for 4\u2009h at 37\u00b0C, rolling in the dark. A portion of bacteria was taken during this incubation for lysis in normal saline (0.9% NaCl) by beating with silica beads  to extract free protein-CFSE conjugate to generate a fluorescence-to-pH standard curve. After 4\u2009h, bacteria were washed and resuspended in normal saline to an OD600 of 0.4. Twenty microliters of bacteria in saline was subcultured into 180\u2009\u03bcl of freshly prepared, pH-adjusted 7H9 in 96-well plates . Lysed bacteria were plated similarly for the pH standard curve. Immediately, plates were placed in plate readers (Tecan Infinite M200 Pro) at 37\u00b0C, shaking and measuring fluorescence or OD600 every 30 min. Fluorescence at 528 nm was measured from 490-nm excitation (pH sensitive), and 520-nm fluorescence was measured from 450-nm excitation (pH insensitive). To calculate pH, 7H9 background was subtracted from all data first . Next, a standard curve of 490-nm excitation/450-nm excitation in relation to 7H9 pH was created from the CFSE-containing cell lysate. The 490/450 ratios calculated from the culture wells were applied to the standard curve to determine intracellular pH.Bacterial cultures were grown to mid-log phase and declumped as previously described. A total of 5\u2009\u00d7\u200910\u2212/\u2212Ccr2 mice (Jackson Laboratories) were used for experiments. Mice were approximately 6 to 16\u2009weeks of age upon infection over all experiments; different groups were age and sex matched. All protocols were approved by independent ethics oversight at the Research Institute of the McGill University Health Centre (RI-MUHC) and followed the guidelines of the Canadian Council on Animal Care (CCAC). C57BL/6 mice were infected through aerosolization  of bacterial cultures at an OD600 of 0.4 for 15 min as previously described (M. kansasii and M. kansasii::Rv3377-78c at an OD600 of 0.2. C57BL/6 \u2212/\u2212Ccr2 (C-C motif chemokine receptor 2 knockout) mice were infected through aerosolization of a low-dose mixed suspension at an OD600 of 1.0. Mouse lungs were harvested at 4 or 24\u2009h  and 14, 21, 28, 42, or 56\u2009days postinfection  into 1\u2009ml 7H9 and homogenized using an Omni Tissue Homogenizer TH  at high speed for 45\u2009s. Serial dilutions made in 7H9 liquid media from lung homogenates were plated on 7H10 plates containing polymyxin B, amphotericin B, nalixidic acid, trimethoprim, and azlocillin (PANTA) with or without\u200950\u2009\u03bcg/ml hygromycin. CFU were counted 2\u2009weeks after plating to determine bacterial burden.Male and female C57BL/6 and escribed . AlternaBone marrow was isolated from C57BL/6 murine tibiae and femora. Bone marrow-derived macrophage (BMDMs) were differentiated with recombinant macrophage colony-stimulating factor (M-CSF) (100 U/ml) (Peprotech) for a period of 7\u2009days as previously described , after w600 of 0.2 to 0.5, clumps were removed to ensure single-cell suspensions and adjusted in complete RPMI medium (without antibiotics) to an OD600 of 0.01. Macrophages were infected by replacing the medium with fresh medium containing bacterial suspension. After 4\u2009h of infection, the wells were gently washed three times with PBS to remove extracellular bacteria and fresh complete RPMI medium (without antibiotics) was added to each well. At indicated time points, the plates were spun down at 2,000 \u00d7 g for 5 min. Each well was subjected to PBS containing 1% Triton X-100 for 10 min at room temperature to induce macrophage lysis. Following serial dilution and plating, CFU were counted on 7H10 plates 2\u2009weeks postplating to determine bacterial burden.Macrophages were seeded into 96-well plates . Bacterial cultures were grown to an ODM. kansasii::Rv33778c/total  in competition assays to determine the comparative fitness of WT M. kansasii versus M. kansasii::Rv3377-78c (data point = CFU on 7H10 with hygromycin/CFU on 7H10).All calculations and statistical analyses were performed using Microsoft Excel or GraphPad Prism. Calculations included (i) the ratio of individual 24-h CFU values/mean 4-h CFU for murine lung and macrophage infection assays to determine bacterial proliferation (data point = 24-h CFU/mean 4-h CFU) and (ii) the proportion of"}
+{"text": "Thaumarchaeota are abundant constituents of dark ocean microbial communities, where their ability to couple ammonia oxidation and carbon fixation plays a critical role in nutrient dynamics. In this study, we describe an abundant group of putatively heterotrophic marine Thaumarchaeota (HMT) in the ocean with physiology distinct from those of their ammonia-oxidizing relatives. HMT lack the ability to oxidize ammonia and fix carbon via the 3-hydroxypropionate/4-hydroxybutyrate pathway but instead encode a form III-a RuBisCO and diverse PQQ-dependent dehydrogenases that are likely used to conserve energy in the dark ocean. Our work expands the scope of known diversity of Thaumarchaeota in the ocean and provides important insight into a widespread marine lineage.It has been known for many years that marine  Thaumarchaeota is a diverse archaeal phylum comprising numerous lineages that play key roles in global biogeochemical cycling, particularly in the ocean. To date, all genomically characterized marine thaumarchaea are reported to be chemolithoautotrophic ammonia oxidizers. In this study, we report a group of putatively heterotrophic marine thaumarchaea (HMT) with small genome sizes that is globally abundant in the mesopelagic, apparently lacking the ability to oxidize ammonia. We assembled five HMT genomes from metagenomic data and show that they form a deeply branching sister lineage to the ammonia-oxidizing archaea (AOA). We identify this group in metagenomes from mesopelagic waters in all major ocean basins, with abundances reaching up to 6% of that of AOA. Surprisingly, we predict the HMT have small genomes of \u223c1 Mbp, and our ancestral state reconstruction indicates this lineage has undergone substantial genome reduction compared to other related archaea. The genomic repertoire of HMT indicates a versatile metabolism for aerobic chemoorganoheterotrophy that includes a divergent form III-a RuBisCO, a 2M respiratory complex I that has been hypothesized to increase energetic efficiency, and a three-subunit heme-copper oxidase complex IV that is absent from AOA. We also identify 21 pyrroloquinoline quinone (PQQ)-dependent dehydrogenases that are predicted to supply reducing equivalents to the electron transport chain and are among the most highly expressed HMT genes, suggesting these enzymes play an important role in the physiology of this group. Our results suggest that heterotrophic members of the Thaumarchaeota are widespread in the ocean and potentially play key roles in global chemical transformations.The IMPORTANCE It has been known for many years that marine Thaumarchaeota are abundant constituents of dark ocean microbial communities, where their ability to couple ammonia oxidation and carbon fixation plays a critical role in nutrient dynamics. In this study, we describe an abundant group of putatively heterotrophic marine Thaumarchaeota (HMT) in the ocean with physiology distinct from those of their ammonia-oxidizing relatives. HMT lack the ability to oxidize ammonia and fix carbon via the 3-hydroxypropionate/4-hydroxybutyrate pathway but instead encode a form III-a RuBisCO and diverse PQQ-dependent dehydrogenases that are likely used to conserve energy in the dark ocean. Our work expands the scope of known diversity of Thaumarchaeota in the ocean and provides important insight into a widespread marine lineage. Crenarchaeota and Euryarchaeota were among the most well-studied archaeal phyla owing to the preponderance of cultivated representatives in these groups, but recent advances have led to the discovery of numerous additional phyla in this domain   , chemoliea (AOA) ; in thesea (AOA) , 16. Altronments \u201320.Thaumarchaeota that are ubiquitous in the dark ocean and potentially important contributors to carbon transformations in this globally important habitat.In this study, we characterize a group of heterotrophic marine thaumarchaea (HMT) with a small genome size that is broadly distributed in deep ocean waters across the globe. We show that although this group is a sister lineage to the AOA, it does not contain the molecular machinery for ammonia oxidation or the 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) cycle for carbon fixation but instead encodes multiple pathways comprising a putatively chemoorganoheterotrophic lifestyle, including numerous pyrroloquinoline quinone (PQQ)-dependent dehydrogenases and a divergent form III-a RuBisCO. Our work describes non-AOA members of the Nitrososphaerales within the class Nitrososphaeria, indicating their evolutionary relatedness to ammonia-oxidizing archaea (AOA). We also performed a phylogenetic analysis of the HMT MAGs together with reference genomes from the Thaumarchaeota and \u201cCandidatus Aigarchaeota\u201d using a whole-genome phylogeny approach, which suggests the placement of the HMT as a sister clade to the AOA (see We generated metagenome assembled genomes (MAGs) from 4 hadopelagic metagenomes from the Pacific (bioGEOTRACES samples) and 9 metagenomes from 750- to 5,000-m depth in the Atlantic (DeepDOM cruise). After screening and scaffolding the resulting MAGs, we retrieved 5 high-quality HMT MAGs with completeness of\u2009>75% and contamination of\u2009<2% , 22, we  AOA see . The ref AOA see , also fe AOA see  . The HMT clade is shown in blue. Black circles denote nodes with >80% bootstrap support, inferred using 1,000 ultrafast bootstraps in IQ-TREE. The collapsed node contains 147 genomes of ammonia-oxidizing archaea (AOA). Download FIG\u00a0S1, PDF file, 0.03 MB.Concatenated maximum-likelihood phylogeny based on 30 highly conserved marker genes including Copyright \u00a9 2020 Aylward and Santoro.2020Aylward and SantoroCreative Commons Attribution 4.0 International license.This content is distributed under the terms of the Thaumarchaeota, consistent with our 16S rRNA gene and concatenated marker protein phylogenies  and 13 metagenomes from the bioGEOTRACES samples and DeepDOM cruises  . For thi10.1128/mSystems.00415-20.6DATA SET\u00a0S1Data Set S1, XLSX file, 0.09 MB.Raw results from metagenome and metatranscriptome read-mapping analyses. Download Copyright \u00a9 2020 Aylward and Santoro.2020Aylward and SantoroCreative Commons Attribution 4.0 International license.This content is distributed under the terms of the To investigate the distribution of HMT across different depths in the water column, we assessed their relative abundance in metagenomes from Station ALOHA in the North Pacific Subtropical Gyre. We analyzed metagenomes sampled from 10 cruises of the Hawaii Ocean time series in 2010 and 2011 that correspond to depths of 25 m, 75 m, 125 m, 500 m, 770 m, and 1,000 m and were described previously . These r10.1128/mSystems.00415-20.3FIG\u00a0S3FIG\u00a0S3, PDF file, 0.07 MB.Depth profile comparisons of HMT (red) and AOA (blue) at Station ALOHA based on marker gene mapping (top) and whole-genome mapping (bottom). The line in the boxplot denotes the median value. Details regarding the mapping can be found in Materials and Methods. Raw data can be found in Copyright \u00a9 2020 Aylward and Santoro.2020Aylward and SantoroCreative Commons Attribution 4.0 International license.This content is distributed under the terms of the Thaumarchaeota but slightly larger than those of some DPANN archaea  and riboflavin metabolism , and genes for cobalamin biosynthesis. The absence of cobalamin biosynthesis is coincident with the acquisition of the vitamin B12-independent methionine synthesis pathway (metE) and is in sharp contrast to AOA, which have been postulated to be major producers of this coenzyme  between the HMT MAGs and a set of high-quality reference archaeal genomes  and DPANN lineages , HMT genrchaeota , with thrchaeota . Althougrchaeota , 47. TogHMT are putative aerobic chemoorganoheterotrophs, as evidenced by the presence of a full glycolysis pathway, the beta-oxidation pathway for fatty acid degradation, and a nearly complete tricarboxylic acid (TCA) cycle that can deliver reducing equivalents to a complete aerobic respiratory chain . No glyc10.1128/mSystems.00415-20.9DATA SET\u00a0S4Data Set S4, XLSX file, 0.06 MB.Curated metabolic reconstruction of HMT pathways. Download Copyright \u00a9 2020 Aylward and Santoro.2020Aylward and SantoroCreative Commons Attribution 4.0 International license.This content is distributed under the terms of the Four genes originally annotated as a pyruvate dehydrogenase complex also share homology with the branched-chain \u03b1-ketoacid dehydrogenase complex (BCKDC) and were annotated as such by the MaGe pipeline . This complex could allow energy conservation from branched-chain amino acids by converting them to fatty acids, which subsequently would be oxidized by the beta-oxidation pathway, generating reducing equivalents, acetyl-coenzyme A (CoA) and/or propionyl-CoA. Genes encoding both the membrane and binding components of an ABC transporter for branched amino acid transport are present in the MAGs, further facilitating these as energy sources. In addition to the transport of amino acids, the HMT genomes encode several zinc- and cobalt-containing metallopeptidases, including a leucyl aminopeptidase that could additionally furnish amino acids to this pathway. This metabolic module also apparently includes an electron transfer flavoprotein (ETF), known to serve as an electron carrier in beta-oxidation, and a membrane-bound ETF-quinol oxidase that could deliver electrons to the quinone pool .Thaumarchaeota. The MAGs encode a full complex I (NUO), but, like the AOA  that could accept electrons from the quinone pool. Like the AOA . Download FIG\u00a0S4, PDF file, 0.04 MB.Maximum-likelihood phylogeny of the ATPase subunits present in the HMT genomes and available Copyright \u00a9 2020 Aylward and Santoro.2020Aylward and SantoroCreative Commons Attribution 4.0 International license.This content is distributed under the terms of the c , which are rarely found in archaeal genomes. Individual MAGs encoded between 4 and 19 individual PQQ-dependent dehydrogenases, and we generated a nonredundant set of proteins from 7 HMT MAGs (the 5 we present here in addition to ASW8 and UBA57) that contained 21 total distinct PQQ-dehydrogenase variants . PQQ-depc \u201361 withoc \u2013. We hypoproteins . An analproteins , also abproteins .The divergent nature of the HMT quinoproteins makes substrate prediction for these enzymes extremely speculative. The most well-characterized of the PQQ-dependent enzymes are the membrane-bound glucose dehydrogenases (mGDH) and soluble methanol dehydrogenases (MDH). Both mGDH and alcohol dehydrogenases can act on a range of hexose and pentose sugars , with suPseudomonas sp. strain NBRC 111131 (accession no. WP_156358113.1), but even then the alignment had only 33.8% identity  in the HMT genomes is a notable feature of this group. We identified this gene in 4 of 5 HMT MAGs (missing only in HMT_AABW); it is also present in the ASW8 MAG, as previously reported (Thaumarchaeota (Beowolf and Dragon archaea)  , where tarchaea) , and thearchaea) . Howeverarchaea) , where tarchaea) .10.1128/mSystems.00415-20.5FIG\u00a0S5FIG\u00a0S5, PDF file, 0.4 MB.(A) Maximum-likelihood phylogeny of RuBisCo large subunits. Reference sequences and RuBisCO form classifications were obtained from Jaffe and Banfield   that is widespread in the global ocean. By analyzing MAGs from this group assembled from Atlantic and Pacific metagenomes, we show that they have small genomes and a predicted chemoorganoheterotrophic metabolism. Several unique features suggest adaptations to energy scarcity. The presence of numerous encoded PQQ-dependent dehydrogenases suggests the importance of oxidizing diverse carbon compounds and introducing reducing equivalents directly into the electron transport chain, which may be a critical component of their physiology in deep waters where energy is scarce. These PQQ dehydrogenases are among the most highly expressed genes in HMT and comprise up to \u223c3% of the total base pairs in their genomes, underscoring their likely importance. Further, HMT encode a highly expressed form III-a RuBisCO that potentially functions as part of a CORibosomal rRNA gene surveys have previously identified this group , 30, and32\u2013Thaumarchaeota has been focused on chemolithoautotrophic ammonia-oxidizing lineages, our findings lead to the surprising conclusion that chemoorganoheterotrophic thaumarchaea are also widespread in the global ocean. In addition to broadening our understanding of archaeal diversity in the ocean, this finding could have multiple implications for biogeochemical cycling in the deep ocean. First, archaeal lipid distributions are used as paleoproxies of past ocean temperature. Current interpretations of the isotopic composition of archaeal lipids from marine sediments  and an anomalous respiratory quotient .We constructed MAGs from metagenomic data generated from the DeepDOM cruise in the South Atlantic in April 2013 , 78 and We constructed MAGs from nine DeepDOM metagenomes using MetaBat v. 2.0 . We binnHaving assembled high-quality HMT MAGs from deepwater Atlantic metagenomes, we sought to identify this group in similar samples from the Pacific; therefore, we analyzed four deepwater metagenome sequences as part of the bioGEOTRACES cruises . For thehttps://github.com/tseemann/barrnap). We predicted signal peptides using the SignalP-5.0 server (http://phobius.sbc.su.se/) (http://raptorx.uchicago.edu/)  between MAGs using ANIcalculator v. 1  default. We pred0 server , transme.su.se/) , and tra.su.se/) . Annotat.su.se/)  on the M.su.se/)  as part go.edu/) . This maThaumarchaeota, we first evaluated the presence of these markers in a set of 23 complete Thaumarchaeota genomes that are currently available , which used a set of 145 marker genes (k__Archaea [UID2] marker set). For an alternative approach, we used a set of 162 highly conserved single-copy markers previously used to assess completeness in archaea , which wThaumarchaeota available on NCBI as of 1 December 2019. We added available \u201cCandidatus Aigarchaeota\u201d and \u201cCandidatus Geothermarchaeota\u201d genomes available on the IMG/M database  and Beowolf Thaumarchaeota identified in geothermal springs , but these genomes were included in this set nonetheless because they represent basal-branching Thaumarchaeota that are valuable for ancestral state reconstructions. The genomes in the high-quality set were chosen to represent as broad a phylogenetic breadth of Thaumarchaeota as possible and without overrepresenting any particular phylogenetic group, and to this end we manually curated this set to remove several AOA genomes if close relatives were still represented. A full list of the genomes included in these two sets can be found in We generated a second set of genomes for use in ancestral state reconstructions, which we refer to as the high-quality set. For this, we selected a subset of genomes from the full set described above. In general we only selected genomes with completeness\u2009of >90% and contamination of\u2009<2% (estimated using the lineage_wf function in CheckM v 1.0.18 with default parameters), with two exceptions: the Dragon https://github.com/faylward/markerfinder). This script uses a set of previously described Hidden Markov Models to identify highly conserved marker genes in genomic data . We selected a representative protein from each OG at random and used these for subsequent annotations. We used the hmmsearch command in HMMER3 to compare these proteins to EggNOG 4.5  and Pfam v. 31 (-cut_nc cutoffs) and the KEGG KAAS server to retrieve KO accession numbers, as described above for the genome annotations.We predicted proteins from each genome in the high-quality genome set using Prodigal v. 2.6.3  and subsThaumarchaeota to assess if the fosmids belonged to a closely related lineage. We did this by calculating pairwise best LAST hits of the proteins encoded in the MAGs and fosmids and averaging the percent identity of these hits . Full results can be found in We calculated the average amino acid identity (AAI) of the HMT MAGs with three fosmids that were previously sequenced from marine To quantify the abundance of the HMT group in metagenomes and HMT genes in metatranscriptomes, as well as for annotation purposes, we generated a nonredundant set of HMT proteins from 7 MAGs (the 5 MAGs generated in this study in addition to ASW8 and UBA57). This was done because these MAGs are highly similar (ANI of\u2009>95%); therefore, they have many similar or identical encoded proteins. Therefore, the consolidated nonredundant set of proteins from all HMT MAGs can be considered a representation of the pangenome of these closely related groups. For clustering the proteins, we used CD-HIT v. 4.6 (default parameters), with a combined file of all HMT proteins used as the input. Protein annotations were derived from the individual genome annotations . For read mapping, the proteins were then masked with tantan v. 13 (with the -p parameter) to prevent possible mapping to low-complexity sequences. Masked sequences were then formatted in a LAST database using the lastdb command from LAST v. 1060 (with the -p parameter). Subsequent mapping was done using the LASTAL command with the parameters -f BlastTab -u 2 -m 10 -Q 1 -F 15, which uses a translated mapping approach , as previously described , 123.To provide a comparison of HMT and AOA relative abundances, we also generated a nonredundant set of AOA proteins for comparison. For this, we used the same methods as those for the HMT MAGs, with proteins predicted from 147 reference AOA genomes used instead. These genomes were selected from our full genome set and represent genomes available in NCBI with estimated completeness of >50% and contamination of <5% , which we used for final comparisons. All values can be found in To estimate the global abundance and biogeography of HMT, we mapped raw reads from several metagenomic data sets onto the nonredundant set of HMT proteins. In addition to the DeepDOM and bioGEOTRACES metagenomes that we used for MAG construction , we also mapped reads from mesopelagic metagenomes from the Tara Oceans expedition and 89 metagenomes generated from the waters of Station ALOHA at depths ranging from 25 to 1,000 m  results and 2. FWe analyzed 10 metatranscriptomes collected during the same DeepDOM cruise in which the metagenomes were collected. These samples correspond to IMG/M accession numbers 3300011314, 3300011304, 3300011321, 3300011284, 3300011290, 3300011288, 3300011313, 3300011327, 3300011318, and 3300011316. Metatranscriptomes were processed using methods previously described . We mappThaumarchaeota tree using the \u201cace\u201d function in the \u201cape\u201d package in R . We estimated the presence of OGs in internal branches of the age in R , with OGPRJNA636088. The genomes of the 5 HMT MAGs are also available on the Aylward Lab FigShare account: https://figshare.com/articles/HMT_MAGs/12252731. Nucleic acid sequences for the MAGs, protein predictions, alignments for phylogenies, and other data products are available on the VTechData archival platform: https://data.lib.vt.edu/files/5712m673w.The MAGs described in this study have been deposited in DDBJ/ENA/GenBank and are associated with BioProject"}
+{"text": "TG/HDL-C ratio (THR) represents a single inherited surrogate predictor of hyperinsulinemia or insulin resistance that is associated with premature aging processes, risk of diabetes and increased mortality. To identify genetic loci for THR change over time (\u0394THR), we conducted a whole genome linkage scan among subjects of European ancestry who had complete data from two exams collected about seven years apart from the Long Life Family Study , a study with familial clustering of exceptional longevity in the US and Denmark. Subjects with diabetes or using medications for dyslipidemia were excluded from this analysis. \u0394THR was derived using growth curve modeling, and adjusted for age, sex, PCs, familial membership, and then log-transformed to approximate normality. Our linkage scan was built on haplotype-based IBD estimation with 0.5 cM average spacing. Heritability of \u0394THR was moderate (46%), and evidence for significant linkage (LOD>3) was identified on 3q28 (LOD=4.1). This locus harbors ADIPOQ among several other promising candidate genes. Interestingly, several studies previously reported suggestive evidence of linkage at this locus for relevant traits including adiponectin, dementia, AD and SBP. This linkage signal was not explained by significant GWAS SNPs for LPL or those under the peak (LOD attenuated to 3.7). In conclusion, we found a novel genetic locus on 3q28 for \u0394THR in subjects without diabetes selected for exceptional survival and healthy aging. Further query of sequence elements including rare functional and regulatory variants at this locus is underway which may reveal novel insights on insulin resistance mechanisms for aging."}
+{"text": "Novel, portable blood gas analyzers (BGAs) may serve as essential point-of-care tools in remote regions, during air travel or in ambulance services but they have not been extensively validated.We compared accuracy of a portable BGA to a validated stationary device.In healthy individuals and patients with chronic obstructive pulmonary disease participating in clinical field studies at different altitudes, arterial blood samples were obtained at rest and during exercise in a hospital at 760 m and in a high altitude clinic at 3100 m. Paired measurements by a portable BGA  and a stationary BGA  were performed to compute bias (mean difference) and limits of agreement (95% CI of bias).2 21.5\u221252.5 mmHg, and PaO2 45.5\u2212107.1 mmHg. Bias (95% CI) between devices were: pH 0.007 (\u22120.029 to 0.044), PaCO2 \u22120.3 mmHg (\u22124.8 to 4.2), and PaO2 \u22120.2 mmHg (\u22129.1 to 4.7). For pH, agreement between devices was improved by the equation to correct pH by portable BGA = \u22121.37 + pHmeasured \u00d7 1.19; bias after correction \u22120.007 (\u22120.023 to 0.009). The portable BGA was easily handled and worked reliably.Of 105 individuals, 248 arterial blood samples were analyzed, 108 at 760 m, 140 at 3100 m. Ranges of values measured by portable BGA were: pH 7.241\u22127.473, PaCOAccuracy of blood gas analysis by the portable BGA in comparison to the reference BGA was adequate for clinical use. Because of portability and ease of handling, portable BGA are valuable diagnostic tools for use in everyday practice as well as under challenging field conditions. Mountain travelers as well as airplane passengers experience hypobaric hypoxia that may induce hypoxia-related illness and changes in metabolic conditions. Patients with cardiovascular or pulmonary diseases are particularly susceptible. Unfortunately, there is a lack of information about these health risks, partly due to the heavy weight of conventional diagnostic instruments for biochemical analysis of blood, the required external electrical power supply, and related major logistical efforts and costs. To address these issues, novel, portable devices for the analysis of blood gases, electrolytes and other parameters have been developed.The EPOC device  is a battery-operated, portable blood gas analyzer (BGA) for use in challenging point-of-care settings such as in remote mountain regions, during air travel, or in emergency ambulance services . Near se2, and PaCO2) with clinically acceptable accuracy within ranges specified by the clinical laboratory improvement amendments (CLIAs)  . We perf (CLIAs)  comparedIn this study, arterial blood samples from participants in two randomized, placebo-controlled, double-blind, parallel trials were analyzed. The initial trials evaluated effects of preventive acetazolamide treatment (375 mg/day) on the incidence of acute mountain sickness (AMS) during a stay at 3100 m in patients with COPD and in age-matched healthy controls . The res1 40\u201380% predicted, pulse oximetry (SpO2) > 91%, and PaCO2 < 45 mmHg at 760 m. In trial 2, healthy individuals of the same age were admitted. The exclusion criteria were any acute disease or unstable health condition and allergy to sulfonamides.In trial 1, men and women with COPD living below 800 m were recruited. Inclusion criteria were an age of 18\u201375 years, COPD diagnosed according to GOLD, FEVArterial blood samples were collected once in the morning and over the course of the day before and toward the end of an exercise test, both at 760 and 3100 m. Each sample was analyzed immediately in triplicate, first by the Rapidpoint500, then by the EPOC and, finally, again by the Rapidpoint500. The value from the EPOC device was compared to the mean of the corresponding values by Rapidpoint500 to avoid bias from a time delay between measurements by the two devices. Changes in variables over the course of successive measurements in the same person were also analyzed. Over the course of 25 days, the repeatability of the EPOC and the Rapidpoint500 devices was additionally checked daily by comparison to two different calibration solutions with acidic and alkalotic pH ranges.2, PaO2) and eight other variables  were analyzed. The limits for the clinical acceptability are \u00b15 mmHg for PaCO2, \u00b13SD (\u00b18.0 mmHg) for PaO2, and \u00b10.04 for pH according to CLIA . Three main variables , integrated barcode scanner, and optional external printer. According to the manufacturer, EPOC can be operated at barometric pressure equivalents of altitudes up to 5100 m and temperature 15\u201330\u00b0C. For blood gas analysis, a droplet of blood is inserted into a small cavity of a test card containing sensors for the analysis. The calibration and analysis take approximately 7 min. Results are shown on the display and can be transmitted by wireless (bluetooth) connection to a printer and to a computer as needed. The EPOC test cards can be stored at room temperature (15\u201330\u00b0C).The Rapidpoint500 is a stationary BGA  with a turnaround time of 3\u20134 min. Cartridges for 250 or 500 measurements and waist are inserted into the device. Cartridges not inserted into the device require storage at 2\u20138\u00b0C. Operating conditions of the Rapidpoint500 are barometric pressure equivalents of altitudes up to 4572 m and temperature 15\u201330\u00b0C .P < 0.05 was assumed as statistically significant.Data are summarized as means \u00b1 SD. Agreement and precision are quantified according to Bland and Altman and CLSI guidelines, reporting mean differences between methods as bias, and 95% confidence interval as limits of agreement . Trends In 105 individuals, 248 radial artery blood samples were obtained for paired analyses by both devices, 108 at 760 m and 140 at 3100 m. 119 samples were collected at rest and 129 samples during exercise. The mean time between the first and second measurement in Rapidpoint500 was 3 min and 10 s (2\u201319 min). EPOC measurements were taken in-between.2, and 97% for PaO2. The 95% confidence interval for pH (\u22120.029 to 0.044) was only in the lower range within the clinical acceptability of \u00b10.04. The 95% CI for PaCO2 (\u22124.8 to 4.2 mmHg) was within the clinical acceptability range of \u00b15.0 mmHg. The 95% confidence interval for PaO2 (\u22129.1 to 4.7 mmHg) was only in the upper range within the clinical acceptability range of \u00b18 mmHg. The comparison of blood gases between EPOC and Rapidpoint500 are summarized in 2: PaO2corrected = 9.45 + PaO2measured \u00d7 0.90 (R2 = 0.94), corrected bias 0 mmHg (\u22126.2 to 6.2). A trend for overestimation of pH by EPOC in the low and underestimation in the high range could be corrected by the equation pHcorrected = \u22121.37 + pHmeasured \u00d7 1.19 (R2 = 0.91), corrected bias \u22120.007 (\u22120.023 to 0.009). No significant trend could be detected for PaCO2 (R2 of regression for PaCO2 = 0.7).Regression analysis of values measured by EPOC as dependent and values measured by Rapidpoint500 as independent variables provided the following equation to correct the PaOn = 50) over a period of 25 days  for the results in EPOC were for pH 0.2%, PaCO2 3.6%, and PaO2 7.2%. The ranges in Rapidpoint500 were pH 7.136\u20137.584, PaCO2 20.7\u201375.3 mmHg, and PaO2 61.7\u2013176.2 mmHg. The coefficients of variation for the Rapidpoint500 were pH 0.04%, PaCO2 1.8%, and PaO2 1.3%.To test repeatability of measurements, we performed repeated measurements of standard reference solutions with EPOC  and Rapidpoint500  for the pH and PaO2 but R2 = 0.87 for PaCO2 was slightly reduced with a significant bias and limits of agreement exceeding the recommended range. Unfortunately, a formal analysis of bias and precision of the measurements in absolute units was not presented which hampers a direct comparison to the current results. 2 \u22120.9 mmHg, and PaO2 6.4 mmHg. Among the two point-of-care devices, EPOC and i-STAT, they found bias\u2019s in pH 0.001, PaCO2 \u22122.3 mmHg, and PaO2 13.3 mmHg which were greater than the corresponding bias\u2019s among EPOC and Rapidpoint500 in the current study (PaCO2 \u22120.3 mmHg and PaO2 2.2 mmHg). In the same data set . Previous studies have evaluated the EPOC near sea level. data set , a lineaIn two studies, results of capillary blood analyzed with EPOC were compared to corresponding results of venous or arterial samples performed with standard devices in critically ill patients . The stuFurther studies performed with blood samples from animals (horses or dogs) are not discussed in detail here. These results showed a good agreement between EPOC and the reference analyzers .A major strength of our study was the use in various settings, i.e., at low and high altitude, in a large tertiary care center and in a remote high-altitude clinic, with a wide range of variables measured due to the testing at rest as well as during exercise. A limitation was that we compared the EPOC to only one type of stationary BGA even though the Rapidpoint500 is a well validated device. The storage of EPOC test cards at room temperature without dependency on a refrigerator was an important advantage for application of the device under field conditions. A limitation of the EPOC was a relatively long calibration time of 3 min after insertion of the test card. While this might be adequate for point-of-care assessments in an individual patient it might represent a limitation in a hospital setting with high-volume measurement sequences.The results of the current study show that EPOC allows accurate measurements of arterial blood gases in a challenging field setting. As the device was easy to use and robust it might serve as a valuable tool for point-of-care applications including at high altitude, during transports and in remote locations where electrical power is not available.The datasets presented in this article are not readily available because Data will be made available for research purposes within limits set by the ethics committee. Requests to access the datasets should be directed to KB.The studies involving human participants were reviewed and approved by Ethics Committee National Center of Cardiology and Internal Medicine, Bishkek, Kyrgyzstan. The patients/participants provided their written informed consent to participate in this study.KB, AB, and MF contributed to the conception and design of the study, data collection, analysis, interpretation, and drafting the article. LM, SS, MM, and MB contributed to data collection, analysis, and interpretation of data. TS, SU, and KB contributed to obtaining funding, to the conception and design of the study, acquisition, analysis, and interpretation of data. All authors critically revised the manuscript for important intellectual content, they approved the version to be published and all agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All authors contributed to the article and approved the submitted version.KB reports grants to his institution from the Swiss National Science Foundation. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "Mycobacterium kansasii is an important opportunistic pathogen of humans and has a close phylogenetic relationship with Mycobacterium tuberculosis. Seven subtypes (I\u2013VII) have been identified using molecular biology approaches, of which subtype I is the most frequent causative agent of human disease. To investigate the genotypes and pathogenic components of M. kansasii, we sequenced and compared the complete base-perfect genomes of different M. kansasii subtypes. Our findings support the proposition that M. kansasii \u201csubtypes\u201d I-VI, whose assemblies are currently available, should be considered as different species. Furthermore, we identified the exclusive presence of the espACD operon in M. kansasii subtype I, and we confirmed its role in the pathogenicity of M. kansasii in a cell infection model. The espACD operon is exclusively present in mycobacterial species that induce phagosomal rupture in host phagocytes and is known to be a major determinant of ESX1-mediated virulence in pathogenic mycobacteria. Comparative transcriptome analysis of the M. kansasii I-V strains identified genes potentially associated with virulence. Using a comparative genomics approach, we designed primers for PCR genotyping of M. kansasii subtypes I-V and tested their efficacy using clinically relevant strains of M. kansasii. Mycobacterium kansasii is the one of the most common causes of NTM disease in South America, South Africa and Europe  are increasingly being recognized as important opportunistic pathogens of humans. Reports have shown that more than 50 species of mycobacteria are associated with human diseases  . Phylogenetically, M. kansasii is closely related to M. tuberculosis , a group of non-RD1 loci . We confirm that all adult subjects provided informed consent, and a parent of the child participant provided informed consent on his behalf. Written informed consent was given.M. kansasii strains, designated KAUST-I to KAUST-V, that were originally isolated from either water or soil samples from five different European countries. Sixteen gDNA samples isolated from clinically relevant strains were also included in the study. These strains were collected from Radboud UMC Center of Infectious Diseases in the Netherlands. The isolates we collected were subtyped using the hsp65 gene as discussed before  were grown in Middlebrook 7H9  and EspACD- AflII (TTTTCTTAAGGTGGCCGCCCGTTTATGTAG). The DNA fragments were digested with HindIII and AflII and cloned into an AflII-restriction site containing a variant of pSMT3 digested with the same enzymes. The resulting plasmid, pSMT3-espACD-GFP, is a shuttle plasmid that is difficult to incorporate directly into M. kansasii. Therefore, we introduced the OriT region of pRAW  with an OD600nm = 0.8\u20131.0 were prepared. Simultaneously, THP-1 cells  = 5, or incubated with only medium. After 2 h of infection, the macrophages were washed with PBS and incubated with fresh medium without antibiotics. The cells were then treated with 1 mL of 1% Triton X-100 for 10 min at 37\u00b0C and collected at different time points after infection . Then, the obtained lysates were diluted to 1:10, 1:100, and 1:1,000, and 100 \u03bcL of each dilution was subsequently spread on Middlebrook 7H10 agar plates. The survival rate was evaluated as the percentage of colony forming units (CFUs) at different time points, taking the number of CFUs at time point \u201c0\u201d as the reference.Cultures of the different subtypes of TM Green Master Mix was used for the amplification, and the cycling conditions for these primers consisted of preheating at 95\u00b0C for 2 min, followed by 30 cycles of denaturation at 95\u00b0C for 30 s, annealing at 62\u00b0C for 30 s and extension at 72\u00b0C for 30 s. The final extension was performed at 72\u00b0C for 10 min after 30 cycles.The unique genes within each singleton pool were further investigated. We used BlastN against the non-redundant (nr) nucleotide database for the unique genes and refined the list of selected unique genes after comparison against the other species in the nr database. The unique genes were also confirmed by the binary alignment map (BAM) files generated by cross-mapping of Illumina reads to each subtype. The primer sets that target the unique genes are presented in TM) were added and treated six times by beating at maximum speed for 30 s. Then, the mixture was centrifuged, and the upper layer was incubated with 200 \u03bcL of chloroform. After centrifugation at 4\u00b0C at maximum speed for 20 min, an equal volume of isopropanol was added to the aqueous layer. The mixture was centrifuged at 4\u00b0C at full speed, and the supernatant was discarded. Then, 1.5 mL of 70% cold ethanol was added and centrifuged for 10 min. The ethanol was discarded, and the RNA was air-dried. The RNA was suspended in 30 \u03bcl of RNase-free water and incubated at 60\u00b0C until all of the RNA was dissolved. The RNA was then stored at \u221280\u00b0C before library preparation. For library preparation, DNA was removed using TurboTM DNase, and rRNA was removed using the InvitrogenTM Ribominus Kit. Strand-specific Illumina RNAseq libraries were prepared using the TruSeq kit following the manufacturer's manual, and the libraries were sequenced on a HiSeq2000 platform (IlluminaTM). The RNAseq reads obtained were first trimmed using the Trimomatic program  in the other four M. kansasii subtypes.Total RNA was extracted from 40 ml of exponential growth phase culture bacterial culture using the TRIzol protocol  sequencing methods to assemble genomes into single base-perfect contigs. All of the bacterial chromosomes of M. kansasii subtypes I-V were assembled into a single contig each, and the genome of M. kansasii subtype III included a large new 301,558 bp circular plasmid pMKIII01  is present exclusively in subtype I  and lipid metabolism   . In addi4, mbtC) . HoweverM. kansasii subtype I, twenty-one pathways for M. kansasii subtype II, nine pathways for M. kansasii subtype IV and 14 pathways for M. kansasii subtype V. Notably, metabolic pathways were significantly dominant in all subtypes.To determine the functional classifications and pathways of the DEGs associated with each subtype, all upregulated DEGs from each subtype were analyzed with EggNOG Mapper . Toxin/antitoxin (T/A) systems were previously only reported in M. tuberculosis while recent studies shows the expansion of the T/A systems from NTMs to MTBC  and one copy of a regulatory protein (rv1129c) that has been shown to be required for M. tuberculosis growth on cholesterol  that are present in subtype I, which was reported to be essential for endothelial-cell invasion and/or intracellular survival  genomes indicates that they may be important for virulence and pathogenicity.As expected, owing to the environmental niche of this bacteria, bspecies . Two copM. kansasii subtype III chromosome, whose G+C content is 66.22%. This may suggest that this plasmid was not originally from M. kansasii subtype III and has been transformed into the bacterial cells during the evolutionary processes. The new plasmid pMKIII01  than the pMKIII01  harbors hsp65 and ITS sequences, either requires restriction enzyme digestion steps or cannot distinguish the M. kansasii subtypes accurately ; Mycobacterium parakansasii (previously classified as Mycobacterium kansasii subtype III); Mycobacterium probekansasii (previously classified as Mycobacterium kansasii subtype IV); Mycobacterium novokansasii (previously classified as Mycobacterium kansasii subtype V); and Mycobacterium eurokansasii (previously classified as Mycobacterium kansasii subtype VI).Hence, we propose to consider M. kansasii subtype I DEGs is diverse and includes aminopeptidase, ADP-ribose pyrophosphatase and phenyloxazoline synthase , is mutually dependent  are outside the ESX-1 loci. They are homologous to rv3864~rv3867, located within the ESX-1 locus and required for ESX-1 secretion for virulence  under study accession No. PRJEB32175.The The studies involving human participants were reviewed and approved by The research protocol was approved by the Institutional Biosafety and Bioethics Committee of King Abdullah University of Science and Technology . The patients/participants provided their written informed consent to participate in this study.AP and AA conceived the study and obtained the funding and supervised the work. AP, QG, and AA designed the experiments. RU generated the strains for the complementation experiment. QG and FB-R performed the complementation experiment. YA, MA, and SA generated the Illumina data and helped in the analysis. QG generated the PacBio data, performed all the data analysis, and prepared the initial draft of the manuscript, followed by edits from AP, WB, AA, and JI. All authors have commented on various sections of the manuscript, which were finally curated and incorporated into the final version by QG, AP, and AA.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "Fragaria virginiana, was constructed using segregation data obtained from a pentaploid progeny population. This novel mapping population of size 178 was generated by crossing highly heterozygous F. virginiana hybrid \u201cLB48\u201d as a paternal parent with diploid (2n\u2009=\u20092x\u2009=\u200914) Fragaria vesca \u201cHawaii 4\u201d. The LB48 linkage map comprises 6055 markers genotyped on the Axiom\u00ae IStraw90 strawberry SNP array. The map consists of 28 linkage groups (LGs) organized into seven homoeology groups of four LGs each, and excludes a small 29th LG of undefined homoeology. One member of each homoeology group was assignable to an \u201cA\u201d subgenome associated with ancestral diploid Fragaria vesca, while no other subgenomes were defined. Despite an intriguing discrepancy within homoeology group VI, synteny comparisons with the previously published Fragaria\u2009\u00d7ananassa DA\u2009\u00d7\u2009MO linkage map revealed substantial agreement. Following initial map construction, examination of crossover distributions revealed that six of the total 5162  chromosomes making up the data set exhibited abnormally high crossover counts, ranging from 15 to 48 crossovers per chromosome, as compared with the overall mean of 0.66 crossovers per chromosome. Each of these six hyper-recombinant (HypR) chromosomes occurred in a different LG and in a different individual. When calculated upon exclusion of the six HypR chromosomes, the canonical  LB48 map had 1851 loci distributed over a total map length of 1873\u2009cM, while their inclusion increased the number of loci by 130, and the overall map length by 91\u2009cM. Discovery of these hyper-recombinant chromosomes points to the existence of a sporadically acting mechanism that, if identified and manipulable, could be usefully harnessed for multiple purposes by geneticists and breeders.The first high-resolution genetic linkage map of the ancestral octoploid (2n\u2009=\u20098x\u2009=\u200956) strawberry species,  Fragaria\u2009\u00d7ananassa, and its immediate 8x ancestors F. chiloensis and F. virginiana, is an ongoing challenge2. Genome assemblies in many diploid and polyploid plant species have been enhanced by anchoring to genetic linkage maps3, and/or by the choice of haploid or double-haploid individuals as the object of the sequencing effort6. Neither anchoring nor haploid approaches to genome assembly have yet been employed in an octoploid strawberry species.The assembly of reference genomes for polyploids, such as the octoploid (2n\u2009=\u20098x\u2009=\u200956) cultivated strawberry 7, and haploid or doubled-haploid clones have not been a readily available option in Fragaria. We have taken advantage of the fact that hybrid pentaploid progeny populations can be generated from certain diploid\u2009\u00d7\u2009octoploid crosses in Fragaria8. As allohaploids, these pentaploids offer potential advantages for genomic research, and can serve as \u201csurrogate haploids\u201d in relation to both linkage mapping and genome assembly. As a prelude to genome assembly and a basis for identifying gene\u2013trait associations in F. virginiana, we have used a pentaploid progeny population to construct a high-resolution F. virginiana genetic linkage map. We know of no prior study in which a pentaploid progeny population has been used for linkage mapping in Fragaria.The octoploid, cultivated strawberry is subject to inbreeding depressionF. vesca \u201cHawaii 4\u201d, an inbred hermaphrodite that universally serves as a model plant for Fragaria genetics/genomics, and for which a high-quality reference genome was available10. The resulting map defines the linkage groups to which a genome assembly could be anchored, and illuminates patterns of segregation distortion and genetic interference. Unexpectedly, our study has also revealed the presence of an intriguing genetic phenomenon, sporadic hyper-recombination (HypR), which if understood could be usefully harnessed in the service of plant breeding and genomic research.As the diploid crossing partner used to generate the pentaploid progeny population, we used 11. Most published octoploid strawberry linkage maps have been based on segregation analysis of the progenies of pseudo-testcrosses, relying on the heterozygosity of each crossing parent, and entailing the construction of separate male and female parental maps, which then may be integrated into a consensus map.As enumerated below, several octoploid strawberry linkage maps have been reported, generally consisting of the expected 28 linkage groups and displaying segregation patterns consistent with meiotic diploidization and disomic inheritance. However, the possibility of genomically localized polysomic inheritance patterns has also been suggestedF. \u00d7ananassa linkage maps, with initial emphasis on AFLPs and/or SSRs17, and more recently on high-throughput methods, including targeted sequence capture18 and SNPs21 genotyped on the Axiom IStraw90 strawberry SNP array21, or by other means23. Low-resolution SSR-based linkage maps have been constructed in F. virginiana25 and F. chiloensis25 for the purpose of mapping sex-determination loci.A variety of marker types have been utilized in the construction of 21. For the purpose of array design, SNP discovery was conducted in an octoploid germplasm panel consisting predominantly of cultivated strawberry (F. \u00d7ananassa) varieties, accounting for 85,663 of the loci targeted by the array. An additional 3751 loci were included based on identification in ancestral diploid F. iinumae as a basis for linkage map construction in that species26. Finally, 5648 single-nucleotide sites were chosen for interrogation using a non-discovery-based (\u201ccodon-based\u201d) approach21. Importantly, although the array design relied on the F. vesca Hawaii 4 Ver 1.1 reference genome assembly10, SNP discovery was not conducted in Hawaii 4 or any other F. vesca germplasm21. Our pentaploid project afforded opportunity to evaluate the performance of the array for genotyping in an octoploid other than F. \u00d7ananassa, and for which the array was not specifically designed.The first strawberry single-nucleotide polymorphism (SNP) genotyping array, the Axiom\u00ae IStraw90\u00ae array  was designed and advanced to commercial manufacture as an initiative of the USDA RosBREED Specialty Crops (SCRI) projectFragaria virginiana octoploid hybrid \u201cLB48\u201d (USDA germplasm accession PI 664374) was derived from a cross between two octoploid F. virginiana accessions: female clone \u201cL1\u201d (PI 660769) as the maternal parent and hermaphroditic clone \u201cBC6\u201d (PI 660767) as the paternal parent. L1 (subspecies virginiana) and BC6 (subspecies glauca) had been previously collected from the wild in New Hampshire and British Columbia, respectively . The pentaploid mapping population comprised 178 F1 hybrid seedlings generated from the cross between inbred, hermaphroditic diploid F. vesca \u201cHawaii 4\u201d, as the maternal parent, and highly heterozygous hermaphrodite LB48 as the paternal parent. On the basis of this parental pairing, the vast majority of marker segregation in the pentaploid progeny population was expected to be the product of heterozygosity in the octoploid parent, LB48, whereas Hawaii 4 served as a tester. Studied plants were clonally propagated and maintained at the Macfarlane Greenhouse facility of the New Hampshire Agricultural Experiment Station (NHAES) at the University of New Hampshire (UNH) in Durham, NH, USA.27 in order to evaluate the ploidies of the putative pentaploid hybrids. For this purpose, the 1C genome sizes of the parental (LB48) and grandparental (L1 and BC6) octoploids were estimated in comparison to diploid Hawaii 4.Flow cytometric analyses of propidium iodide-stained nuclei from freshly harvested young leaves were performed on a BD Accuri C6 instrument as described26. Briefly, young, partially expanded leaves were collected and refrigerated for no more than 5 days prior to DNA extraction. DNA was isolated using the E-Z 96 Plant DNA kit (Omega Bio-tek) followed by Proteinase K treatment as required by Affymetrix, Inc. DNA samples from 178 pentaploid progeny plants and two replicate sets of the grandparental (L1 and BC6), and parental (Hawaii 4 and LB48) individuals were sent to Affymetrix for genotyping on the Axiom\u00ae IStraw90\u00ae Fragaria whole-genome genotyping array (henceforth referred to as the IStraw90 array), and the Affymetrix GeneTitan\u00ae genotyping system was employed following the standard operation procedures . The resulting signal intensity files  were analyzed, and genotype calling was performed using the Affymetrix Axiom Analysis Suite software application. In the output data, markers were classified into the six standard Affymetrix conversion types (performance categories): PolyHighResolution (PHR), NoMinorHom (NMH), MonoHighResolution (MHR), CallRateBelowThreshold (CRBT), OffTargetVariant (OTV), and Other21.For the purpose of SNP genotyping, DNA samples were prepared by the method previously described by Mahoney et al.21. In this scheme, the \u201cminor\u201d allele \u201cB\u201d, would be detected as present versus absent in the progeny, resulting in two-category segregation with clearly separated clusters and robust genotype calling. If BB, the diploid parent would contribute the marker allele to all progeny, converting the assay from presence/absence to one of rare allele dosage, which creates problematic cluster compression in a polyploidy setting21. A three-category segregation pattern would result if both the diploid and octoploid parent were heterozygous for the same marker.Given the nature of the employed diploid (Hawaii 4)\u2009\u00d7\u2009octoploid (LB48) cross, the progeny of which were expected to segregate on the basis of heterozygosity in LB48, employed markers were required to display two-category segregation patterns corresponding to those expected of a testcross progeny in a octoploid setting. Given the existence of alternate alleles A and B, where B is the \u201cminor\u201d  allele of the marker in question, such a pattern would be expected if the diploid parent marker genotype was AA, and the octoploid parent marker genotype was determined by the number of homolog pairs carrying the marker locus: i.e., either (B/A)(A/A)(A/A)(A/A), or (B/A)(A/A)(A/A), or (B/A)(A/A), or (B/A), depending upon the effective ploidy of the marker site in questionn\u2009=\u2009missing data; a\u2009=\u2009haploid for one allele; and b\u2009=\u2009haploid for the alternate allele; while heterozygosity is not a permitted state in the haploid setting. The rubric for conversion of genotype calls from the Affymetrix format to that required for input into JoinMap is provided in Supplementary Table Linkage analysis was performed using JoinMap 4.1 , where the data set type was set as haploid (HAP). Input of genotyping data into JoinMap required conversion of Affymetrix genotyping codes to appropriate JoinMap codes. In the Affymetrix output data, four codes are used: \u22121\u2009=\u2009missing data; 0\u2009=\u2009homozygous or haploid for one allele; 2\u2009=\u2009homozygous or haploid for the alternate allele; and 1\u2009=\u2009heterozygous. In JoinMap, the relevant genotyping codes are Marker segregation resulting from heterozygosity in LB48 was nominally expected in a 1:1 presence:absence ratio, but the possibility of segregation distortion in at least some genomic regions was also anticipated, and so markers deviating from a 1:1 ratio were not excluded. Linkage clusters were calculated with a minimum logarithm of odds (LOD) score of 8.0. A series of increasingly refined linkage maps was produced using the maximum likelihood (ML) algorithm with default parameters. In order to temporarily reduce marker redundancy, if two or more markers shared the same set of progeny genotypes, initially all but one representative marker was automatically removed by JoinMap, then were later added back to the resultant linkage map using the \u201cAssign Identical Loci to Their Groups\u201d function.21, were identified and converted to missing data using in-house scripts. For this purpose, the singletons were identified as those interior  marker calls differing from both of the nearest flanking marker calls in the same individual. After singleton correction, map construction was performed again.After initial map construction, the majority of markers were in JoinMap linkage phase 0, so genotype calls of markers in the alternate phase 1 were converted by interchanging \u201ca\u201d and \u201cb\u201d genotype calls. After phase correction and map recalculation, the singleton markers, i.e., those putatively miscalled single markers that generated artifactual double crossovers28.In the resulting final map version, segregation distortion was detected via application of the chi-square goodness-of-fit test within JoinMap based on the 1:1 segregation ratio expected for each marker. In addition, crossover positions were identified and counted using an in-house script that detected genotypic shift (\u201ca\u201d to \u201cb\u201d or vice versa) between two adjacent marker calls, while ignoring missing data. The number of crossover points in each chromosome was then determined, and the distribution of crossovers was evaluated based upon expectations derived from the Poisson distribution10, upon which the array was designed21. Within each homoeology group, analysis of the subgenomic affinities of each chromosome to the ancestral F. vesca and F. iinumae genomes was performed using the approach previously employed19, whereby the array markers of the SNP\u2013SNP design category21 were categorized as displaying patterns of affinity of four possible types: affinity to both F. vesca and F. iinumae (YY); to F. vesca but not F. iinumae (YN); to F. iinumae but not F. vesca (NY); or to neither F. vesca nor F. iinumae (NN).Based upon its marker content, each linkage group was assigned to one of seven possible homoeology groups based upon the known positions of each IStraw90 array marker in the Hawaii 4 Ver 1.1 reference genomeF. \u00d7ananassa linkage map19 were compared as a means of identifying correspondences between F. virginiana linkage groups and those in the DA\u2009\u00d7\u2009MO map. The Circos29 diagrams were generated using the Circos program version 0.69\u20139.The map positions of markers shared between the canonical LB48 map and the previously published DA\u2009\u00d7\u2009MO 8. The octoploid/diploid ratios of FL2-A values ranged from 3.07 (for L1) to 3.16 (for BC6), while the 5xAJ/diploid ratio was 2.03. The FL2-A value of 95,660 for plant 5xAJ was approximately half of the sum of the Hawaii 4 and LB48 FL2-A values , as would be predicted for a Hawaii 4\u2009x\u2009LB48 pentaploid hybrid. The 99 additional progeny seedlings randomly selected for flow cytometric test using Hawaii 4 and LB48 as comparators all yielded 1C-values consistent with pentaploidy (data not shown).In their overall morphology, all progeny of the Hawaii 4\u2009\u00d7\u2009LB48 cross resembled the paternal parent (LB48) much more so than the morphologically distinct maternal parent (Hawaii 4), thereby providing an initial confirmation of progeny hybridity. All progeny plants displayed sexual sterility, as would be expected for odd-ploid hybrids. The flow cytometer output FL2-A readings (in parentheses) of nuclear fluorescence were obtained for diploid Hawaii 4 , LB48 , L1 , BC6 , and 100 progeny seedlings from the Hawaii 4\u2009x\u2009LB48 cross, including progeny plant 5xAJ , which had been previously confirmed as pentaploid via mitotic chromosome count21.After genotyping on the IStraw90 array, all parental and 178 pentaploid plant samples yielded the data that passed the quality control criteria. The average call rate for passing samples was 99%. In total, 138,099 SNPs were called, and were classified into the six standard Affymetrix conversion types (performance categories) based on criteria displayed by their respective cluster patterns  SNPs; and 760 belonged to one of the \u201chaploSNP\u201d categories .At the LOD score of 8.0, linkage clusters containing <20 markers were eliminated from further consideration, leaving 29 clusters, the smallest of which (LG29) had 72 markers while all others had numbers of markers ranging from 118 to 368 . Surprisingly, it was found that six individuals each harbored one chromosome (a different chromosome in each case) exhibiting an abnormally high number of crossovers, as further detailed below. Due to the atypical nature of these six hyper-recombinant (HypR) chromosomes, they were provisionally excluded from the data set by converting their genotype calls to missing data. Singleton correction was then performed, and the map was recalculated, resulting in the canonical LB48 linkage map that is shown in Fig. F. virginiana LB48, we chose to exclude the data from the six HypR chromosomes, and to exclude LG29, which was anomalous not only for its low number of markers but in other ways as subsequently described. Thus, the canonical LB48 map  linkage map from map Fig. . The summap Fig. . Over al21 on the seven pseudochromosomes (PCs) of the diploid Hawaii 4 Ver 1.1 reference genome10. Among these 28 LGs, best-percent matches ranged from 79% for LG02 up to 95% for LG06  design category (Supplementary Table F. vesca-derived) subgenome (Table F. iinumae-derived (\u201cB\u201d) subgenome or any other subgenomic category was not achievable using the employed approach. Thus, the LG numbers LG01 through LG28 were assigned in ascending sets of four, such that the lowest LG number in each set was assigned to the subgenome A member of the respective homoeology group  and hypothesized F. iinumae (b) subgenomes specified in the DA\u2009\u00d7\u2009MO map . At least one locus exhibited segregation distortion on 17 out of 28 canonical LGs  exhibited segregation distortion (X2 (1:1), 2% exhibiLGs Fig. , includiLGs Fig. . OtherwiLGs Fig. , while sLGs Fig. . Even moLGs Fig. .The marker segregation data for each linkage group is provided in Supplementary Table 28, where \u201cm\u201d is the mean event frequency, and \u201ci\u201d is the specific crossover count, in the present case ranging from 0 to 7. The Poisson frequency distribution was calculated as:The distribution of chromosomes exhibiting 0, 1, 2, 3, or more crossovers is expected to follow a Poisson distribution2\u2009=\u2009554, P\u2009<\u20090.001, df\u2009=\u20097). Specifically, the observed numbers of chromosomes with two or more crossovers were less than the expected numbers for all crossover count categories from 2 to 7 (Table Using the mean observed frequency (m) of 0.66 crossover points per chromosome Table , as deteIn total, 164 crossovers, or 4.8% of the total number of 3429 crossovers, occurred in the six exceptional HypR chromosomes, which comprised just 0.1% of the 5162 individual chromosomes in the total data set. Due to the atypically high level of crossing over in these six chromosomes, their respective marker data were excluded (by treating as missing data) from the calculation of the canonical LB48 linkage map, as previously described Fig. .Fig. 5CoInclusion of the segregation data from the six HypR chromosomes added a combined total of 91.04\u2009cM to the six linkage groups in question, and increased their combined number of loci by 130 Table . The incF. virginiana. The LB48 linkage map comprises 28 linkage groups distributed into seven homoeology groups of four members each, matching the expectations for a linkage map of a species with haploid chromosome number of 1n\u2009=\u20094x\u2009=\u200928, as is the case for Fragaria virginiana.Here, we report the use of a pentaploid (2n\u2009=\u20095x\u2009=\u200935) progeny population for the purpose of constructing a high-resolution linkage map, the canonical LB48 map, for the ancestral octoploid (2n\u2009=\u20098x\u2009=\u200956) strawberry species F. virginiana. Based as it is on segregation data from 178 gametes derived from parent LB48, the shortest theoretical distance between adjacent loci in the map is 0.56\u2009cM (=\u20091/178), as compared with the observed mean distance of 1.01\u2009cM. Any intervals shorter than the theoretical minimum, a few instances of which are evident in the \u201cPositions\u201d columns of Supplementary Table The LB48 map, comprising 6055 SNP markers distributed over 1851 loci and 1873\u2009cM of total map length, is the first high-resolution linkage map of the ancestral octoploid species Fragaria vesca30. The approach to subgenome identification taken here was patterned after that of Sargent et al.19, who defined the \u201cA\u201d subgenome linkage groups of the F. \u00d7ananassa DA\u2009\u00d7\u2009MO linkage map based upon the affinities of IStraw90 haploSNP (SNP\u2013SNP) marker tag sequences to the corresponding sites in the F. vesca \u201cHawaii 4\u201d Ver 1.1 reference genome. Similarly, these investigators19 tentatively identified a second F. \u00d7ananassa subgenome, designated \u201cb\u201d, that was putatively derived from the ancestral B genome diploid  Fragaria iinumae. This assignment was achieved by utilizing F. iinumae Illumina reads aligned to the corresponding SNP target sites in the Hawaii 4 Ver 1.1 genome assembly. In our present study, only the A-subgenome linkage groups of the LB48 map could be confidently identified based upon SNP\u2013SNP marker tag affinities.Within each homoeology group, the lowest LG number in an ascending series was assigned to the linkage group identified as corresponding to the \u201cA\u201d subgenome Table , which iF. \u00d7ananassa DA\u2009\u00d7\u2009MO linkage map19, both of which were based on SNP markers genotyped on the IStraw90 array. First, all 646 shared markers had the same homoeology group memberships in the LB48 map as they did in the DA\u2009\u00d7\u2009MO map, while the extents of synteny conservation and collinearity among members of each homoeology group was variable, but generally good. Interestingly, a clear correspondence was evident between each LB48 linkage group and one respective DA\u2009\u00d7\u2009MO linkage group, with two exception. In LG22, the LB48 linkage group with the most markers on the LB48 map, shared only three markers with the entire DA\u2009\u00d7\u2009MO map. Thus, LG22 has no counterpart on the DA\u2009\u00d7\u2009MO map. In contrast, LG24 displays correspondence at its \u201czero\u201d end to DA\u2009\u00d7\u2009MO LG6X2, and at its opposite end to DA\u2009\u00d7\u2009MO LG6b  from the expected 1:1 segregation ratio was seen in 1331 markers, or 22% of the 6055 markers comprising the LB48 map. Systematic bias toward alleles derived from grandparents L1 or BC6 was seen in parts of eight linkage groups for each, while in LG22 the segregation bias favored BC6 alleles at the top of the linkage group and L1 alleles at the bottom. Although it has been shown that the presence of segregation distortion is not an impediment to accurate map construction31, some investigators have chosen to exclude markers exhibiting segregation distortion from use in map construction32. If we had excluded markers exhibiting segregation distortion from our map construction pipeline, substantial regions of the map would have been lost, including: the top 32\u2009cM of LG10; the bottom 46\u2009cM of LG08; 52\u2009cM of LG17; and all but 3\u2009cM of LG14; as well as the entirety of LG29. Particularly when working with undomesticated germplasm and/or wide crosses, the automatic exclusion of markers exhibiting segregation distortion seems inadvisable, given the risk that genomic regions containing genes influencing gametophytic and/or zygotic viability or competitiveness may not be represented in the resulting map.Significant deviation  crossover categories is indicative of interference, whereby the presence of one crossover tends to suppress the occurrence of additional crossovers involving the same chromosome. However, the lower than expected count for the 0 crossover category and higher than expected count for the 1 crossover category is suggestive of a mechanism promoting the occurrence of a crossover. Thus, the overall pattern of disagreement between observed and expected crossover counts implies the existence of interacting mechanisms suppressing and promoting crossing over.In our data set Table , the meaTurning now to the HypR chromosomes, the Poisson analysis indicated that the occurrence of even one chromosome with 15 or more crossovers was vanishingly small, and that therefore the occurrence of six such chromosomes, with crossover numbers ranging from 15 to 48, cannot be attributed to chance. Importantly, the observed hyperelevation of recombination in just six out of 5162 chromosomes assayed is not incremental or genome-wide, but instead is drastic and narrowly focused. Thus, any proposed mechanism must offer an explanation not only for a hyperelevation in crossover number, but also its narrow yet seemingly arbitrary focus limited to one chromosome in each affected individual.If the causal factors underlying the observed instances of hyper-recombination can be identified, they may be amenable to manipulation to the benefit of plant breeders and geneticists. Inclusion of the HypR chromosome in linkage calculations in each case lengthened the respective linkage group, and added additional map loci. The latter resulted from the splitting of individual marker bins  of chromosomes from the LB48 paternal parent, and one haploid complement (1x\u2009=\u20097) from the F. vesca \u201cHawaii 4\u201d maternal parent. Despite the reported level of heterozygosity in Hawaii 437, our reliance upon polymorphisms between the paternal grandparents L1 and BC6 and the exclusion of markers exhibiting three-category (aa:ab:bb) segregation patterns sufficed to exclude any markers influenced by heterozygosity in Hawaii 4 from the final map. Accordingly, the LB48 map is a uniparental map, as distinct from most if not all octoploid Fragaria maps reported to date, which have been based on pseudo-testcrosses between two heterozygous parents, necessitating the initial construction and deconvolution of separate maternal and paternal maps.The LB48 map is the first linkage map in octoploid Fragaria. Marker genotype assignment is simplified in the pentaploid population, because all marker segregation patterns are of the two-category type (\u201ca\u201d versus \u201cb\u201d), and as noted above all segregation derives from the heterozygosity of just one parent. A benefit of two-category segregation is the simplification of the cluster analysis performed by the Affymetrix Axiom Analysis Suite software. A challenge associated with SNP genotyping in a polyploid is cluster compression, which arises because the dosage of the marker (or \u201cminor\u201d) allele, present in 0, 1, or 2 copies, must be ascertained against a background of a larger and variable number of \u201cmajor allele\u201d copies present in homoeologous chromosomes but not segregating21. The separation of genotype calls into just two clusters for PHR as well as NMH marker types , while the LB48 map has fewer markers (6055 versus 8407) and a proportionately lower locus density (0.99 versus 1.3 loci per cM). The IStraw90 array is populated primarily by markers discovered in an F. \u00d7ananassa germplasm panel21; however, the substantial number of markers available in our F. virginiana cross indicates that the IStraw90 array is also an effective genotyping platform in this ancestral octoploid species. For future studies of F. virginiana, it is noteworthy that only 2427 (or 39.6%) of the 6127 LB48 markers (including those on LG29) are represented on the derivative Axiom IStraw35 array38, whereas 4583 (or 54.5%) of the 8407 DA\u2009\u00d7\u2009MO markers are represented there. Thus, for future studies of marker/gene associations with traits of interest in F. virginiana, the IStraw90 array will likely be the preferred genotyping platform.Both the Fragaria research community. The LB48 map provides a new genomic resource for identifying marker\u2013trait associations and anchoring genome assemblies in F. virginiana, and by extension for the octoploid cultivated strawberry F. \u00d7ananassa. The features and properties of the LB48 map also provide insight into the consequences of conducting linkage analysis in a pentaploid mapping population, and into the performance of the IStraw90 strawberry SNP array as a basis for linkage mapping in F. virginiana, a strawberry species other than those for which the IStraw90 array21 and its derivative IStraw35 array38 were designed.In conclusion, the canonical LB48 linkage map provides a useful new genomic resource for the Finally, our data have unexpectedly revealed the existence of an intriguing and potentially useful genetic phenomenon, meiotic hyper-recombination (HypR). To our knowledge, this phenomenon has not previously been described in plant linkage mapping studies. However, the possibility may be considered that some previously published data sets may provide evidence of hyper-recombination in the absence of its recognition by the respective investigators. Alternately, just as some investigators have excluded markers with distorted segregation ratios from mapping data sets, some investigators may have discarded data from individuals exhibiting hyper-recombination or other departures from normal expectations. It will be of considerable interest to see whether our report encourages the recognition and reporting of other instances of hyper-recombination in plants and other eukaryotes.Supplementary Table S1 - Code Conversion RubricSupplementary Table S2 - Table Heading - Mapping DataSupplementary Table S2 - Mapping Data SpreadsheetSupplementary Table S3 - Map Comparison"}
+{"text": "However, ankle proprioception scores were significantly correlated with step length  and step cadence , and were significantly and negatively correlated with the stage of modified Hoehn and Yahr . The lack of relationship between ankle proprioceptive acuity and functional mobility in PD suggests that people with PD may be more limited by reduced sensorimotor integration or may rely more on other sensory input, rather than ankle proprioception, to achieve functional mobility, a finding consistent with sensory reweighting theory. In addition, poorer ankle proprioceptive acuity was associated with decreased step length and increased step cadence, suggesting that the shuffling gait observed in PD may be related to impaired ankle proprioception, which has important clinical implications for gait retraining in people with PD. Given that ankle proprioception was significantly and negatively correlated with the stage of modified Hoehn and Yahr, it may warrant being used as an objective biomarker to monitor the progression of PD.Previous research has found ankle proprioception to be impaired in people with Parkinson's disease (PD). However, the relationship between ankle proprioception and functional mobility in people with PD has not been fully investigated. The purpose of this study was to examine whether ankle proprioception is related to the functional mobility of people with PD. Forty-two participants with mild to moderate PD volunteered. Ankle proprioceptive acuity was measured in standing, by using active movement extent discrimination assessment (AMEDA). Functional mobility measures included the timed-up-and-go test (TUG), 30 s sit-to-stand test (30s-STS) and 10-meter walking test (10MWT). Step length and step cadence were recorded during the 10MWT. No significant correlation was found between ankle proprioceptive discrimination scores and any mobility performance measure in people with PD (\u22120.20<r<0.04, all  Parkinson's disease (PD) is the second most common neurodegenerative disease and may impair patients' functional mobility \u20133. FunctMotor impairments such as hypokinesia, bradykinesia and akinesia have already been recognized in people with PD , 10, howOne hypothesis is that impaired ankle proprioception would be significantly correlated with low functional mobility in people with PD , suggestIn order to test these hypotheses, we employed an ecologically valid, weight-bearing ankle proprioception test\u2013 the Active Movement Extent Discrimination Assessment (AMEDA), and a battery of functional mobility tests, including the timed-up-and-go test (TUG), 30 s sit-to-stand test (30s-STS) test and 10-meter walking test (10 MWT) to explore the relationship between ankle proprioception and functional mobility in people with PD. We hypothesized that ankle proprioception would be significantly correlated with functional mobility measurements and severity of the disease.The study involved 42 people with PD who were diagnosed by a neurological specialist according to the definition of the United Kingdom Disease Society . The stuParticipants completed the following tests in a randomized order: ankle proprioception testing (AMEDA), functional mobility tests including the timed-up-and-go test (TUG), 30 s sit-to-stand test (30s-STS) test and 10-meter walking test (10 MWT). The primary outcome measures are ankle proprioception and 10 MWT. Secondary outcome measures are TUG and 30s-STS. All tests were conducted during participants' optimally medicated phase . ParticiThe ankle AMEDA system was used to test the participant's proprioception at the ankle joint in functional standing  Figure . AccordiFor the TUG test , the parFor the 30s-STS , particiDuring the 10 MWT , particit-tests were conducted. The variables involved in independent t-tests were examined to determine that they were acceptably normally distributed.All data analyses were performed using IBM SPSS Statistics 22.0. For the demographic, ankle proprioception and functional mobility performance measures, descriptive statistics (mean \u00b1 SD) were calculated. Pearson and Spearman Correlations were carried out to examine the correlations between ankle proprioception and functional mobility test variables. Pearson's Correlation was used for continuous variables, including the AUC ankle proprioception score, TUG, the 10 MWT time, step length on the 10 MWT, and step cadence on the 10 MWT. Spearman's Correlation was calculated for the AUC ankle proprioception score with the stage of modified Hoehn and Yahr scores, 30s-STS and score from the NFOG-Q. To compare characteristics, severity of disease, ankle proprioception scores and all functional mobility performance scores between male and female participants, independent-samples p > 0.05). However, moderate correlations were observed between ankle proprioception and step length from the 10 MWT test , and between ankle proprioception and step cadence from the 10 MWT test . In addition, ankle proprioception had a significant correlation with the stage of modified Hoehn and Yahr .The demographic information, severity of disease, and results from the ankle proprioception and functional mobility tests are given in Impairments in proprioception and functional mobility are often seen in patients with PD, and these impairments are not commonly improved by medical treatments , 29, 30.Previous animal and human studies have established the influence of abnormal basal ganglia activity on proprioception in PD  and the One novel finding of the current study is that there was a significant correlation between ankle proprioception and step length and step cadence of the 10 WMT. Shortened step length and increased step cadence have been considered the most limiting factors in PD, and important factors that reflected as bradykinesia gait seen in PD , 38. TheHowever, the association between impaired ankle proprioception and altered gait in PD observed in the current study did not clarify the causal relationship between the two variables. It is possible that impaired ankle proprioception in people with PD may increase difficulty with optimal placing of the feet during walking, so that people with PD gradually develop the strategy of reducing step length and increasing step cadence. It is also possible that, due to fear of falling, people with PD reduce step length, which may subsequently reduce the range of motion at the ankle joint during walking. According to \u201cuse-it-or-lose-it\u201d theory, prolonged disuse of full range of movement of the ankle may consequently result in diminished ankle proprioception . If the In addition, the significant correlation observed between ankle proprioception and the stage of the modified Hoehn and Yahr indicated that the alteration of ankle proprioception may appear at the early stage of PD. Konczak et al.  also notThere are some limitations of the present study. Firstly, we did not record some information about PD patients' disease progression, including the duration of the disease, as well as the most effected side. Second, while we tested the patients when they were optimally medicated, we did not record the exact medication they took, and this may limit the implications of the findings in clinical practice. In addition, participants involved in the current study were individuals with mild to moderate PD, who had a relative high level of function. Therefore, it was unclear if the results obtained from this group would be similar to those with severe PD and low functional mobility. Besides, for performing the step count during the 10 MWT, we used a visual observation method, which might be less accurate than automatic step counting devices. Another limitation is that the TUG and 30s-STS have been used as functional mobility measures in the current study. Although these measures are clinically relevant and meaningful, they may not precisely reflect compensations and deviations in functional mobility. Future study is needed to use more objective measures to explore gait deficits in PD. Finally, the AMEDA approach to proprioception testing has the advantage of assessing proprioception in multi-joint movement, with muscular engagement. However, although this enhances the ecological compatibility of the test with functional mobility, it lacks the purity of isolated single joint proprioception testing, and might miss such deficits within this patient population.The lack of relationship between ankle proprioceptive acuity and functional mobility in PD suggests that proprioceptive acuity is not the main limiting factor in mobility in the current group of PD patients. Further study is required to establish whether this is compensated by other aspects of sensory input, rather than ankle proprioception, as this would be consistent with sensory reweighting. In addition, poorer ankle proprioceptive acuity was associated with decreased step length and increased step cadence, suggesting that the shuffling gait observed in PD may be related to impaired ankle proprioception, which has important clinical implications for gait retraining in people with PD. Given that ankle proprioception was significantly and negatively correlated with the stage of modified Hoehn and Yahr, it may warrant being used as an objective biomarker to monitor the progression of PD.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Ethics committee at the Shanghai University of Sport . The patients/participants provided their written informed consent to participate in this study.YW collected data and drafted the manuscript. JH participated in the study design, data analysis and helped to draft the manuscript. ZW and JZ helped to study design and data collection. JW, EP, GW, and RA made edits and comments to the manuscript. All authors conceived of the study, read and approved the final version of the manuscript, agreed with the order, and presentation of the authors.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "We discuss the latest developments in alternative battery systems based on sodium, magnesium, zinc and aluminum. In each case, we categorize the individual metals by the overarching cathode material type, focusing on the energy storage mechanism. Specifically, sodium-ion batteries are the closest in technology and chemistry to today\u2019s lithium-ion batteries. This lowers the technology transition barrier in the short term, but their low specific capacity creates a long-term problem. The lower reactivity of magnesium makes pure Mg metal anodes much safer than alkali ones. However, these are still reactive enough to be deactivated over time. Alloying magnesium with different metals can solve this problem. Combining this with different cathodes gives good specific capacities, but with a lower voltage . Zinc has the lowest theoretical specific capacity, but zinc metal anodes are so stable that they can be used without alterations. This results in comparable capacities to the other materials and can be immediately used in systems where weight is not a problem. Theoretically, aluminum is the most promising alternative, with its high specific capacity thanks to its three-electron redox reaction. However, the trade-off between stability and specific capacity is a problem. After analyzing each option separately, we compare them all via a political, economic, socio-cultural and technological (PEST) analysis. The review concludes with recommendations for future applications in the mobile and stationary power sectors. Our society depends on electricity, which powers our homes, jobs, hobbies, transportation and communication. We even have electricity on the go, which is essential as more and more people use mobile devices. Batteries have become a crucial component of our daily lives. Yet, unlike the power outlet at our homes that seemingly supplies an infinite amount of energy (as long as we pay the bills), batteries can only hold a limited amount of energy. So, how do these batteries store and release electrical energy? Indeed, the importance of this question is reflected in the awarding of the 2019 Nobel Prize in Chemistry to Stanley Whittingham, John Goodenough and Akira Yoshino for their research and development of lithium-ion batteries 2+ and results in fast intercalation kinetics. In the solvated ions ([Mg(DME)3]2+) co-intercalation, the DME molecules form a complex with Mg2+ ions. These complexes weaken the interaction between Mg2+ ions and the 2D host (MoS2) layers. This increases the intercalation kinetics of Mg2+ ions. Once inside the cathode lattice, the Mg2+ ion reacts with the MoS2. This reaction is the same as observed in metal oxides and metal selenides [4+ in the cathode is reduced by the electrons and not the Mg2+ ions, see Equation (9). In this reaction the Mo4+ is reduced to Mo2+, while the S2\u2212 remains unchanged. Co-intercalation between ether-solvents and Mg2+ ions may be a suitable route to overcome the sluggish intercalation kinetics in multivalent ion-based batteries. This co-intercalation was already shown to improve the kinetics for carbon-based cathodes as well [The mechanism for metal sulfide cathodes is similar to that of metal oxides . Howevermaterial . Here thelenides ,80. In t as well .(9)Mg2+2 cathode, magnesium anode and all-phenyl-complex electrolyte with Li as additive [2+ ion insertion by using a Mg/Li hybrid battery. The battery has a capacity of 146 mAh g\u22121 and a voltage of 2 V at a current density of 900 mA g\u22121. Furthermore, it shows high stability for 10,000 cycles.The best results for metal-sulfide based cathodes were obtained with a graphene wrapped VSadditive . This sy2+ ions into graphite layers is energetically unfavorable. However, when this problem is overcome, carbon-based anodes and cathodes can be used for Mg-ion batteries. The anode will function similar to that in sodium and lithium. The carbon cathode will use the possibility of the metallic magnesium anode. After the oxidation of the magnesium at the anode, Mg2+ ions (0.72 \u00c5) insert between the cathode carbon layers and are reduced [In general, the intercalation of Mg reduced ,84. The 2+ complexes were shown to co-intercalate into graphite [2+ ion is reduced and will form some metallic magnesium in the cathode material. To clarify the co-intercalation mechanism of graphite with Mg2+ ions, structural changes in the natural graphite were investigated through ex situ X-ray diffraction analysis at various states during charge and discharge. Comparing the experimental results with density functional theory (DFT) calculations, shows that Mg2+-diethylene glycol dimethyl ether (DEGDME) complexes are double stacked  is comparable to that of Li+ ions in graphite (1.8 \u00d7 10\u22129 cm2 s\u22121).Recently, Mggraphite . In the  stacked  in the f2+ ions associate with the reduced carbonyl groups of PDI-EDA, while the ClO4\u2212 anions can be bounded with the radical cations of the PTPAn cathode  as cathode, magnesium as anode and an all-phenyl-complex electrolyte, containing 1 M AlCl solvent . This sy4)2 and Mg bis(trifluoromethanesulfonimide), Mg(TFSI)2) [As previously mentioned, the cathode is not the only thing that has to be improved to make magnesium-ion based batteries viable. Metallic magnesium can react with all known electrolytes based on oxidatively stable solvents and salts, such as Mg(ClO(TFSI)2) . This re(TFSI)2) ,98, bism(TFSI)2)  and a bi(TFSI)2) . Out of 6S8 as cathode material. They propose their colloidal bismuth nanocrystals could serve as a new model anode and studied the Mg2+ ion intercalation mechanism of this material [2+ ions within Bi nanocrystals takes place through an alloying mechanism, leading to the simultaneous formation of \u03b1-Mg3Bi2 and \u03b2-Mg3Bi2, as in Equation (10). The reaction at the cathode is the same as for metal-sulfide cathodes, see Equation (11).The system that tested the bismuth nanocrystals as anode uses Momaterial . Specifi2Sn and Mg3Bi2. Since the alloying is reversible the Mg alloys can be used as an alternative for pure magnesium as anodes in Mg-ion batteries. This would allow for a wider range of usable electrolytes since the alloys do not appear to form a passivation layer.The other alloy materials were tested for their magnesium storage/release capabilities ,96,97,986S8 as cathode and 1 M solution of Mg(TFSI)2 in diglyme as electrolyte. However, these materials are incomparable as the bismuth nanocrystals are the only material tested as anode in a full cell set up. The other materials are only tested on their reversible magnetization/demagnetization mechanism. The system shows a stable capacity of 325 mAh g\u22121 over at least 150 cycles at a current density of 770 mA g\u22121 and delivers a voltage of ~1.2 V. This paper is also an example as it uses a benchmark cathode (Chevrel-phase Mo6S8) and a common electrolyte (Mg(TFSI)2 in diglyme) to test the electrochemical properties of their new anode material. If everyone in the field of battery chemistry was to use model systems while testing their material, understanding the added value of the new material would be significantly easier. Using magnesium metal alloys as anode material would be an interesting route to explore, since it negates the formation of a passivation layer on the anode, which is one of the major issues for magnesium anodes.The best results were obtained using Bi nanocrystals as anode, MoAs a whole, Mg-ion batteries are a promising alternative for Li-ion ones. Both cathodes and anodes have been researched extensively, leading to the discovery of magnesium-metal alloys as potential anodes. Using alloys would make Mg-ion batteries compatible with a variety of electrolytes that are unusable when using metallic magnesium. Both anode and cathode show high cycling stability and most materials show good rate capabilities. The biggest edge that lithium batteries have over Mg-ion batteries is the high voltage they deliver. The voltage of an average Mg-ion battery lies between 1\u20132 V, which is 2\u20133 times lower than that of a Li-ion one.\u22121) and its redox potential 2V10O25\u22c58H2O cathode. Here, the X-ray diffraction peaks showed the formation of Zn3(OH)2V2O7\u22c58H2O during discharge. The intensity of these peaks increased until the system was completely discharged and diminished during charging. Further evidence was found with X-ray photoelectron spectroscopy, which showed an increase in V4+ during discharge at the cost of V5+. This was reversed during charging and shows the highly reversible transformations between V5+ and V4+ during the cycling.As with the sodium and magnesium, the two main metal oxides cathodes for Zn-ion batteries are manganese and vanadium oxides. Yet their storage mechanisms differ: Vanadium oxide intercalates the Znmaterial ,108. Cle4\u22c53Zn(OH)2\u22c55H2O), which forms large flakes on the manganese oxide [3O4 nanoflowers. Both a MnO and ZnSO4\u22c53Zn(OH)2\u22c55H2O phase were observed after full discharge. Electrons from the anode are stored during the discharge via the reduction of Mn3+ (Mn3O4) to Mn2+ (MnO), see Equation (12). The surplus oxygen from the transition of Mn3O4 to MnO reacts with the ZnSO4 and H2O to form the ZnSO4\u22c53Zn(OH)2\u22c55H2O, see Equation (13). The resulting flakes are large enough to be observed with SEM 2//V6O13\u00b7nH2O battery [\u22121, at a current density of 5 A g\u22121. Furthermore, after 1000 cycles a capacity of 262 mAh g\u22121 is maintained. Overall, metal oxides are promising cathode material for Zn-ion batteries, owing to their high cycle stability (4000 cycles) and high rate-capabilities (20 A g\u22121) [\u22121. Furthermore, there is also a good understanding of the mechanism, making the rational design of cathode material significantly easier. If the capacity of these systems could be increased, they would become a competitive alternative to lithium-based batteries, especially for large grid energy storage.The possibilities of these materials are showcased with the Zn//Zn(CF battery . This sy0 A g\u22121) . They al4 as cathode [2+ ions with the VS4, as in Equation (14). However, recharging the material results in the partial conversion of VS4 to Zn3(OH)2V2O7\u22c52H2O (mind that vanadium is now V5+) and orthorhombic sulfur, see Equation (15). The authors note that more complicated conversions are likely to take place during discharge and do not yet fully elucidate the discharge mechanisms. One possibility is the formation of a sulfur cathode type battery. Although the mechanism is uncertain, good cycling stability for 170 cycles are observed. Furthermore, the Zn//Zn(CF3SO3)2//VS4 battery delivers a lower capacity (180 mAh g\u22121) than the metal oxide batteries, but has a high capacity retention.Metal sulfide cathodes for Zn-ion batteries can undergo multiple different charging mechanisms. Some follow the simple intercalation mechanism, with the reduction of the metal in the cathode ,112. Def cathode . With thOverall, the metal sulfides give reasonable capacities and high cycle stability and rate capacity. While unusual storage mechanisms are not observed for all materials, these can be a potential disadvantage (formation of unexpected phases) or advantage (high capacities) for these materials.+ ions incorporated in the material [3+/V4+ redox pair is essential for the battery, as demonstrated by the Na3V2(PO4)2F3 cathode. The Na+ ions are extracted during charging and some of the V3+ is oxidized to V4+, according to the X-ray photoelectron spectroscopy study, as shown in Equation (16). During discharge, instead of the Na-ions intercalating back into the material, Zn2+ ion inserts and forms Zn0.5Na2V2(PO4)2F3, see Equation (17). Alternatively, both Na+ and Zn2+ ions co-intercalate into Na3V2(PO4)3, behaving more like a Zn/Na hybrid battery. The hybrid system also results in a good initial discharge capacity of 101 mAh g\u22121 at a current density of 500 mA g\u22121 and delivers a voltage of 1.23 V at 50 mA g\u22121. It does lose a significant amount of this capacity (76 mAh g\u22121) after 200 cycles.Currently, polyanion Zn-ion batteries only use vanadium phosphates type materials as the cathodes. These follow a similar intercalation mechanism as the Na-ion polyanion batteries, even as far as having Namaterial ,114. AgaThese material are currently lacking compared to the metal-oxides and metal sulfide cathodes. They underperform in capacity, cycle stability and high rate-capabilities, while using similar elements. Maybe with enough time, these materials can perform better, but a serious breakthrough is necessary to overtake the metal-oxides and metal-sulfides.2+ ion storage mechanisms follows the rocking chair mechanism also observed in Li-ion batteries [2+ ion adsorption and desorption [+\u2013 groups are reduced to \u2013NH\u2013. At the same time, the =N\u2013 in polyaniline is reduced to \u2013N\u2212\u2013, which can bind the Zn2+ ions via electrostatic interactions [2+ ions that interacted with the negatively charged groups on the polyaniline being removed from the cathode material.The charge/discharge mechanism for carbon materials for Znatteries . Howeveratteries ,120. Forsorption . During ractions . After c2 and 3 M NH4Cl aqueous electrolyte [\u22121 at a current density of 8 A g\u22121, after 1000 cycles at this current density ~100% of the capacity is retained. Furthermore, the aqueous Zn-ion battery works well under bending, folding and twisting, making it an interesting material for flexible electronics. Carbon materials show excellent cycle stabilities as well as good rate capabilities. However, carbon suffers from low theoretical capacity, making the material not very appealing for small electronic devices. Carbon cathodes could be used for grid storage applications, where capacity is less crucial.The best results were obtained using a polyaniline cathode, zinc anode and 2 M ZnClctrolyte . The sysAs a whole, Zn-ion batteries are unlikely to replace Li-ion ones. They deliver a low voltage in the range of 1\u20132 V and have the lowest theoretical gravimetric capacity of all the described materials. However, aqueous Zn-ion batteries are among the safest battery systems and with the emerging flexible electronics, the need for flexible energy storage devices increases. For this application, Zn-ion batteries are a fitting choice as a lot of research has already been performed on flexible batteries ,125,126.\u22123) is about four times higher than lithium (2042 mAh cm\u22123) and its gravimetric capacity (2981 mAh g\u22121) is comparable to lithium (3861 mAh g\u22121) [Aluminum battery systems are a promising alternative for Li-ion batteries due to their low cost and high abundance . FurthermAh g\u22121) . The mosmAh g\u22121) ,131,132,mAh g\u22121) ,136,137,mAh g\u22121) ,139, metmAh g\u22121) ,140 metamAh g\u22121) ,142 and mAh g\u22121) . Here wexMyOz or even Al2O3 [3+ ion combines with AlCl4\u2212 ions to form Al2Cl7\u2212 ions, see Equation (18). Meanwhile, on the cathode side, a dissociation reaction of Al2Cl7\u2212 ions generating Al3+ and AlCl4\u2212 ions takes place. The released Al3+ ion simultaneously participates in the intercalation process in the cathode, resulting in AlxMyOz, as shown in Equation (19).The mechanism of the Al-ion storage mechanism depends on the cathode material. For metal oxides, the trivalent aluminum ions react with the metal oxide, forming Alen Al2O3 ,129,130.3+ ions released by the anode travel to the cathode material where they bind to the oxygen [5+ in the cathode framework being reduced to V4+ and V3+ by the electrons. However, this reaction has shown to be not completely reversible, leading to a loss in capacity due to accumulation of aluminum in the cathode material. ZnO was also tested as cathode in Al-ion batteries [3+ ions, a decrease in capacity was observed. This capacity fading was correlated to a decrease in crystallinity of their cathode material.Thus effectively, the Ale oxygen . This leatteries . Upon re2/C nanocomposite as cathode, aluminum as anode and AlCl3 in 1-ethyl-3-methylimidazolium chloride (1.3:1 mol) ionic liquid electrolyte [\u22121 at a current density of 50 mA g\u22121 and delivers a voltage of 1.95 V. Furthermore, the material shows no capacity fading after 20,000 cycles at a current density of 2 A g\u22121.The best results were obtained using SnOctrolyte . The sys3+ ions. In order to improve metal oxides as cathode materials for Al-ion batteries, the material must be stable upon repeated Al3+ ion intercalation/deintercalation. The material also needs to bind aluminum in a way where the reaction is reversible to improve the cycle stability and negate the accumulation of aluminum in the cathode material.Overall, metal oxide cathodes are promising material for Al-ion batteries. However, many materials are unstable or undergo irreversible reactions with Al4\u2212, rather than the Al3+ ions. During the charge of the battery, Al is deposited accompanied by the release of AlCl4\u2212 species on the metal anode and the intercalation of AlCl4\u2212 species on the graphite cathode; during the discharge of the battery, Al is dissolved with the formation of Al2Cl7\u2212 species in the anode and the AlCl4\u2212 ion is released at the cathode  as ionic liquid electrolyte [\u22121 at a current density of 10 A g\u22121. Furthermore, the systems retains nearly 100% capacity over 3000 cycles even at this high current density.The best results were obtained using nanosheet-bricked porous graphite as cathode, aluminum as anode and AlClctrolyte . The sysOverall, carbon materials are promising as cathodes in Al-ion batteries due to their high cycling stability and stability over a big range of current densities. However, the gravimetric capacity of carbon is significantly lower than that of other materials, so using carbon as cathode material makes it nigh impossible to obtain batteries with a high capacity.3S4 [2 [2 [2S4 [3+ ions, see Equation (18) [2+ is reduced to metallic tin after discharge, whereas the S2\u2212 was oxidized to S6+ during charge, as in Equation (20).Most metal sulfide-based cathodes  follow ion (18) . In the 3+ intercalation commonly observed for metal sulfides [7\u2212 ions into AlCl4\u2212 and Al3+ ions.The reaction shown in Equation (22) takes place during discharge. This is why the oxidation state of sulfur changes from 6+ to 2\u2212. The paper does not go into detail as to why their material shows chloroaluminate anion intercalation rather than the Alsulfides . It coulsulfides ,145. Anosulfides . For carsulfides ,147,148.3 in 1-ethyl-3-methylimidazolium chloride (1.3:1 mol) ionic liquid electrolyte. The system has a capacity of 406 mAh g\u22121 at a current density of 100 mA g\u22121, delivering a voltage of 2.4 V. After 500 cycles, capacity had decreased to 281 mAh g\u22121, which means the capacity decay rate is only 0.02% per cycle.The best results were obtained using the SnS cathode, aluminum anode and AlCl3+ in the cathode, which limits any increase in the charge/discharge current density [3+ ions to increase the performance at higher current densities.Overall, metal sulfides are promising materials for Al-ion batteries, delivering high initial discharge capacities and Coulombic efficiencies. However, the metal sulfide cathodes generally suffer from fast capacity decrease. Furthermore, the current densities for charge/discharge are still inferior to those for lithium-ion batteries. The rate performance of Al-ion batteries is influenced by the solid phase diffusion of Al density . To make+2 in the material does not change during charge/discharge, while the Se2\u2212 is oxidized to Se4+ or Se6+ [2\u2212 is oxidized. Upon discharge, the reactions can be described with Equations (20) and (21), with SnS replaced by CoSe in this example. In the second report the Co2+ is reduced to metallic cobalt, while the change in oxidation state of selenium is not shown. Here the reaction involves incorporation of Al3+ ions in the CoSe2 lattice, see Equation (21). The Co2+ is reduced during discharge while the Al3+ ions remains unchanged.Metal selenide cathode are electrochemically similar to sulfides. This makes sense as selenium is directly below sulfur in the periodic table. There are two articles describing the use of metal selenides as cathodes in Al-ion batteries. The intercalation mechanism is the same as described for the metal sulfide cathodes ,140. In  or Se6+ . The chl3 in 1-ethyl-3-methylimidazolium chloride (1.3:1 mol) ionic liquid as electrolyte. The system has a capacity of 427 mAh g\u22121 at a current density of 1 A g\u22121 and delivers a voltage of 2.1 V. However, the material shows fast capacity decay and the charge capacity is significantly higher than the discharge capacity, leading to poor coulombic efficiency.The best results were obtained using carbon encapsulated CoSe as cathode, aluminum foil as anode and AlClOverall, metal selenides are an interesting cathode materials for Al-ion batteries. However, current research is lacking and the materials that have been tested show rapid capacity decay. The metal selenides provide a high capacity and excellent rate capability. Metal selenides need to gain increased cycling stability and coulombic efficiency to become a viable option for cathode material. The high toxicity is also a factor.3+ ions as well as AlCl4\u2212 ions [3+ ions is the same as previously described, reduction of metal in the cathode material and binding of the Al3+ ions. However, for Cu3P as cathode material, a special mechanism is observed. In this mechanism the AlCl4\u2212 ions intercalate into the cathode material upon charging, oxidizing both copper and phosphor in the cathode material. Upon discharge, Cl\u2212 ions deintercalated from the material and partial reduction of P species occurs, while the oxidation states of Cu and Al remain unchanged.Metal phosphides/phosphites can show both intercalation of All4\u2212 ions ,142,143.\u22121 over at least 3000 cycles at a current density of 200 mA g\u22121. Overall, metal phosphides/phosphites can be a promising material for cathodes in Al-ion batteries owing to their high cycle stability. However, they suffer from rather low capacities. To become a viable option, the capacity must be increased considerably.The best results were obtained by using nickel phosphide as cathode. The system shows a stable capacity of 60.9 mAh gOverall, aluminum is a viable alternative for lithium for energy storage devices. Al-ion batteries show high capacities, high rate capabilities and aluminum is ~10 times cheaper than lithium . However\u22121; 3459 mAh cm\u22123) [Another alternative to Li-ion batteries that has received a lot of attention are the metal-sulfur (M-S) batteries . These systems promise a high theoretical capacity, thanks to the sulfur cathode (1672 mAh gAh cm\u22123) . HoweverAh cm\u22123) . This isThe energy storage mechanism for all metal-sulfur batteries follows a conversion type mechanism ,153,154.For aluminum a different reaction occurs as the reported systems use an ionic liquid electrolyte, leading to different redox chemistry, see Equations (18) and (23).\u22121 at current density of 1675 mA g\u22121 for 1500 cycles in a sodium-sulfur battery [There has not been much research into sodium-, magnesium-, zinc- and aluminum-sulfur batteries, since lithium-sulfur is already regarded as an alternative for Li-ion batteries. For the alternative metals, most of the research is towards the encapsulation of sulfur into porous cathode materials. For example, sulfur composite were prepared into sulfur loaded on a flexible carbon fiber cloth , sulfur  battery .62\u2212 and S42\u2212 species), and the final reduction product (Al2S3) have poor solubility in ionic liquid electrolytes [Sulfur batteries suffer from capacity fading due to sulfide dissolution and polysulfide shuttling . Howevertrolytes . This introlytes ,166, thetrolytes ,159 and trolytes ,158,166.PEST analysis, which takes into account Political, Economic, Socio-cultural and Technological aspects.The previous section focused on the theoretical and experimental capacities of the different non-lithium batteries. However, other parameters such as cost, safety, current supply, and total reserves, are as important for economic viability. Here we discuss these parameters and their consequences for the various battery types, using a point-based system for comparison. The parameters are based on the Availability is a key parameter for the viability of different batteries. Indeed, most of the other parameters, such as cost and political concerns, are influenced by international reserves. The points given in the final analysis are solely based on the world production and reserves. For lithium the reserves are estimated to be 53 million tons worldwide, with new reserves being found at an annual basis . The bigThe reserves of the substitute metals are much larger. Zinc has an estimated reserve of about 1.9 billion tons, with a 13.53 million ton production and 13.93 million ton consumption in 2017 . The mos2CO3. As lithium has the highest price, battery costs should reduce by changing to a different metal. The point distribution only takes the prices of the materials into account and does not contain any price variations associated with the modifications  necessary for non-lithium-based batteries.The cost of a battery comprises raw materials, labor and equipment costs . The rawSafety concerns govern any malfunction that can damage humans or their surroundings, for example explosions or combustion. However, toxicology effects are not addressed here since these deserve their own segment. Li-ion batteries are relatively safe and will only malfunction at high temperatures or when badly designed. However, lithium sulfur batteries are less safe, since the metallic lithium anode can violently react with both oxygen and water. The same principle holds for the Na-ion and Na-S batteries due to the similar reactivity of sodium and lithium. For aluminum, zinc and magnesium, both the M-ion and M-S battery contain metallic anodes, but these react less severe with oxygen and water.+ ions have the lowest mass per charge (6.94 gr/mol of charge), closely followed by the trivalent Al3+ ions (8.99 gr/mol of charge), divalent Mg2+ ions (12.16 gr/mol of charge), monovalent Na+ ions (22.99 gr/mol of charge) and the heaviest being divalent Zn2+ ions (32.69 gr/mol of charge). Another consideration is the lower battery weight when the generally metal containing cathodes are substituted by sulfur cathodes in M-S batteries. However, mass increase is only a problem for mobile applications. The lower the mass per charge, the higher the points given in this section.Changing lithium for another metal has a dramatic effect of the final weight of the battery. For sake of easy comparison, we assume that every battery will hold the same amount of charge unrelated to the used ions. Therefore, the mass per charge of the ions can be used to compare the different metals. LiThe toxicity of the batteries is quite difficult to define since the way of exposure can vary. Leaking of the battery can result in oral intake of any of the metals while an ignition or explosion of the battery can result in inhalation of metal particles. The danger of inhalation of metal particles do not vary much between the different metal-based batteries, thus we focus on the immediate danger in the oral uptake of the different metals. While Aluminum comprises about 5% of the earth\u2019s crust, it is not essential to humans and most flora and fauna. Therefore, the normal intake, 2\u20133 mg of aluminum, is low and is directly excreted from the body. Since the human body only has to handle small doses on a daily basis, a sudden high uptake of the metal will not be excreted and will cause toxic effects in our body . From thThe performance as a key factor has already been described in this review. We already discussed the maximum theoretical capacities of the different metals in this review. The more points in this area, the higher the theoretical capacity of the battery.A conclusion based on empirical data on the stability of the different battery types is difficult, because of the imbalance in research towards the different battery types. Therefore, we base the stability on the adherent problems of dendrite formation and polysulfide shuttling reported for the batteries. While these issues can be diminished by carefully designing anodes, cathodes and membranes, finding these solutions will diminish their short-term viability. All metal anode containing batteries suffer from dendrite formation, except the ones based on magnesium. Furthermore, sulfur batteries will suffer polysulfide shuttling which lowers their stability. Lower scores in this section are given to batteries which are more unstable anodes and cathodes.This review highlights the four main alternatives  for lithium in battery applications. The research on these materials is categorized in research towards the cathodes and anodes, and further classified into the different types of materials for these electrodes. Besides the practical aspects, e.g., specific capacity and cycle stability, we also focused on the different storage mechanisms for these materials. In general, metal-based cathodes  tend to give high specific capacities in any battery type. Alternatively, carbon cathodes (both as graphite and polymer type) yield lower capacities, although they do sometimes exhibit higher voltages.Since the chemistry of lithium and sodium is similar, most of the current Li-ion technology can be used for Na-ion batteries. If a sodium metal anode is used, the battery will be less safe, but not using these limits the achievable specific capacity. This is not the only problem of the anodes, with sodium and graphite being incompatible. Currently, Na-ion batteries suffer from a lower capacity and often \u201cpoor\u201d cycling stability, especially compared to lithium. This technology also scores low overall in the PEST analysis, with the exception of the cost, availability and current readiness. The biggest future for this type of batteries is in a transition period. The similarities, both in supplied voltage and industrial fabrication, requires a low investment to switch to this battery type. However, the low theoretical capacity will quickly require a different battery type.Magnesium-ion batteries are a promising alternative for Li-ion batteries. The formation of the passivated layer on the anode seems to be solved with magnesium-metal alloys. Their biggest drawback is their low voltage (2\u20133 times lower than lithium), requiring a complete overhaul of any battery carrying device. Additionally, the capacity of these batteries is currently low, even further diminished of one includes the supplied voltage. The high stability of these batteries will be a determining factor when the anodes and cathodes are improved for their capacitance.The low theoretic gravimetric capacity of zinc makes it an unlikely candidates for replacing Li-ion batteries in mobile applications. This is further diminished by their low voltage output and currently average specific capacity. However, aqueous Zn-ion batteries are among the safest battery systems, making them ideal for grid storage. Research should focus on the stability and capacity retention of these systems. Some have reported almost 100% retention, showing the possibilities for these types of batteries.Theoretically, aluminum is the best option for replacing lithium, with the highest theoretic gravimetric capacity. Furthermore, this battery type has an overall good score in the PEST analysis. The main drawback currently is the low capacity of the reported batteries, with also a visible trade-off between capacity and stability in literature. Current Al-ion batteries can be viable for grid storage, where capacity is less important than stability.In the more distant future, sulfur based batteries will replace Li-ion batteries. However, the type of metal that will be used is highly dependent on the battery technology at that time. These batteries have by far the highest capacity at high current densities and showcase more futuristic values. The biggest drawback (polysulfide shuttling) of these systems is already identified, improving the aim in the scientific research.We also identified a different problem within the battery research field. The lack of standardized testing conditions, such as standard cathodes, anodes, electrolyte and current densities, makes direct comparison between different systems difficult. To understand the contribution of new scientific work in this field, such standards are necessary."}
+{"text": "The fast and non-invasive detection of odors and volatile organic compounds (VOCs) by gas sensors and electronic noses is a growing field of interest, mostly due to a large scope of potential applications. Additional drivers for the expansion of the field include the development of alternative and sustainable sensing materials. The discovery that isolated cross-linked polymeric structures of suberin spontaneously self-assemble as a film inspired us to develop new sensing composite materials consisting of suberin and a liquid crystal (LC). Due to their stimuli-responsive and optically active nature, liquid crystals are interesting probes in gas sensing. Herein, we report the isolation and the chemical characterization of two suberin types (from cork and from potato peels) resorting to analyses of gas chromatography\u2013mass spectrometry (GC-MS), solution nuclear magnetic resonance (NMR), and X-ray photoelectron spectroscopy (XPS). The collected data highlighted their compositional and structural differences. Cork suberin showed a higher proportion of longer aliphatic constituents and is more esterified than potato suberin. Accordingly, when casted it formed films with larger surface irregularities and a higher C/O ratio. When either type of suberin was combined with the liquid crystal 5CB, the ensuing hybrid materials showed distinctive morphological and sensing properties towards a set of 12 VOCs . The optical responses generated by the materials are reversible and reproducible, showing stability for 3 weeks. The individual VOC-sensing responses of the two hybrid materials are discussed taking as basis the chemistry of each suberin type. A support vector machines (SVM) algorithm based on the features of the optical responses was implemented to assess the VOC identification ability of the materials, revealing that the two distinct suberin-based sensors complement each other, since they selectively identify distinct VOCs or VOC groups. It is expected that such new environmentally-friendly gas sensing materials derived from natural diversity can be combined in arrays to enlarge selectivity and sensing capacity.  It finds applications in a wide variety of fields, including food and beverage quality control , environSolanum tuberosum [O2CC5H11]) to selectively cleave acylglycerol bonds while conserving linear aliphatic ester bonds intact  grade), ethyl acetate, heptane, methanol were purchased from Fisher Scientific , acetic acid glacial (purity \u226599.7%) and ethanol (\u226599.8%) were obtained from Merck, and hexane from VWR . Solvents were of analytical grade and used as received.Cholinium hexanoate was synthesized and characterized as previously described . Hexanoi2.2Quercus suber L.) was obtained from Amorim & Irm\u00e3os SA . This powder, which is chemically similar to natural cork, does not have a proper size for agglomerate production or further uses, hence it is considered an industrial residue. The white potatoes  were purchased from Batatas Mirense, Lda.  and processed in the laboratory. Potato raw tubers were peeled; the peels were scrapped in boiled water to assure minimum pulp content, and dried at 50\u00a0\u00b0C until constant weight. Both plant sources were milled  and cleaned from extractives by sequential Soxhlet extraction with solvents of increasing polarity , as previously described [Industrial cork powder (from escribed . The ext2.3ca. 10\u00a0mg) were methylated and trimethylsilylated  prior to quantification by gas chromatography\u2013mass spectrometry (GC-MS) as described before [The hydrolyzable monomeric constituents of suberin samples  were acquired in DMSO-d6 using 5\u00a0mm diameter NMR tubes, at 60\u00a0\u00b0C. MestReNova, Version 11.04\u201318998  was used to process the raw data acquired in the Bruker spectrometer.Nuclear Magnetic Resonance (NMR) spectra were recorded using an Avance II\u00a0+\u00a0800\u00a0MHz  spectrometer, as previously described . All sub2.4p-value\u00a0=\u00a00.919, p-value\u00a0=\u00a00.907 for potato suberin and cork suberin, respectively. Therefore, the differences of each monomer between the two suberin samples were analyzed using a one-way Analysis of Variance (ANOVA). The contributions of each chemical class and of each monomer to the overall difference between both polymers were calculated using Principal Components Analyses (PCA) upon construction of a Pearson correlation matrix. The analyses were represented in biplots comprising the PCA and the Multidimensional Scaling (MDS) of all contributing points. The statistical analyses were performed using the software XL-STAT v.2014.5.03 (Addinsoft).The experimental replicas of the relative abundances (mg/g) obtained by GC-MS  displaye2.52 circular area in an untreated glass slide by drop-casting, and kept for 15\u00a0min in an incubator previously heated to 50\u00a0\u00b0C. To produce films of suberin without 5CB, the same procedure was followed except for the addition of 5CB.Suberin extracted from cork, and suberin extracted from potato peels were used to produce films of hybrid materials with birefringence and VOC-responsive properties. Briefly, suberin (either from cork or potato peels) was mixed with distilled water (2% w/v), sonicated for 2\u00a0h with cycles of 30\u00a0min  and left for 45\u00a0min under agitation (700\u00a0rpm) in an 80\u00a0\u00b0C bath. Afterwards, 5CB was added to the hot mixture (4.6% 5CB v/v) and vortexed for 10\u00a0min. Lastly, 5\u00a0\u03bcl of the final mixture was deposited onto a 20\u00a0mm2.6The surface topography of the casted materials (with and without 5CB) was observed by Scanning Electron Microscopy (SEM). SEM images were obtained on a Zeiss Auriga CrossBeam workstation equipped with a focused ion beam (FIB) column. The materials were previously coated with 20\u00a0\u03bcm of AuPd for better conductivity and placed on a carbon-aluminium support. Polarized Optical Microscopy (POM) was used to investigate the distribution of 5CB on the suberin matrix. POM images were taken with crossed (at 90\u00b0) polarizers and complemented with bright field (BF) microscopy images, using a Zeiss Axio Observer. Z1/7 microscope equipped with an Axiocam 503 colour camera and operated with ZEN 2.3 software for acquisition and processing of the images. The films surface composition was characterized by X-ray photoelectron spectroscopy (XPS). XPS was performed with a Kratos AXIS Supra spectrometer using a monochromated Al K\u03b1 source, running at 225\u00a0W. The detailed spectra were recorded with a pass energy of 5\u00a0eV. Charge neutralization with an electron flood gun was employed during the measurements, and all spectra were charge corrected to the C\u2013C, C\u2013H of the C 1s emission at 285\u00a0eV.2.7Glass slides with drop-casted hybrid films made of suberin and 5CB were used as VOC sensors in an in-house assembled e-nose. The sensing mechanism is based on the birefringence property of 5CB a\u2013c. Two The e-nose device  includesTo test the hybrid suberin films for responses to VOCs, the six independent sensor slots of the e-nose detection chamber were occupied with three cork suberin films and three potato peels suberin films. The films were, then, exposed sequentially to the headspace of 12 model solvents, which are structurally similar but from different chemical classes , during 21 consecutive cycles. Each cycle comprises 5\u00a0s of exposure to the VOC (exposure period) and 15\u00a0s of flushing with ambient air (recovery period). The solvents were previously heated to 37\u00a0\u00b0C for 15\u00a0min in a thermostatic water bath. The concentration of VOC in the sensor chamber was calculated between 12 and 15% (v/v), as explained in the detail in a previous publication of the group . Two ind2.8https://github.com/hgamboa/novainstrumentation). Then, the signals were divided in cycles and the cycles were normalized. In total there were approximately 100 cycles per VOC for each type of suberin film, which were used to train and validate an automatic VOC classifier. Data of cork suberin films was analyzed independently from the data of potato suberin films to allow studying the performance of the two types of sensors individually. For each type of suberin film, the cycles dataset was divided in training dataset (the 50 cycles per VOC from the first experiment) and validation dataset (the 50 cycles per VOC from the second experiment). Twelve features regarding the morphology of the waveform were extracted per cycle, as explained elsewhere [To process suberin film signals, data analysis tools based on Python libraries  were implemented. The signals were first filtered using the median filter  from SciPy library, and the smooth function (20 points sliding window) from novainstrumentation library . The most abundant monomers were systematically identified in the two suberin types, however the identification yield (estimated through peak area integration) was higher for cork suberin (63.12%) compared to potato suberin (38.18%)  . The limositions . The conThe monomeric pattern of cork suberin  largely In planta, suberin comprises a network of long-chain \u03b1,\u03c9-bifunctional acids esterified via glycerol units [ca. 4\u20135% (w/w) [ol units ,22. Therol units ,23, as iol units . Herein,5% (w/w) . Collect1H and HSQC spectra are comparable to those obtained before for similar samples, and the identification of the 1H and 13C chemical shifts for the constituent monomers of the two suberin types was based on those previously assigned through a combination of 1H\u20131H (COSY) and 1H\u201313C  correlation experiments [1H spectra of both suberin types and the glycerol CH-Acyl region of HSQC spectra are depicted in 2-X aliphatics, glycerol CH-acyl and aromatics were estimated through the integration of the 1H-spectrum as: 74%, 18%, 4% and 4%, respectively. For cork suberin these values were estimated as: 63%, 36%, 0.2% and 1%, consistent to those reported before [1H-spectrum  through changes in their molecular order, orientation and/or phase properties. A range of different methods can be used to monitor those changes, often utilizing the birefringence property. A common method is the measurement of the light intensity transmitted through a LC sensor placed between two crossed polarizers , some of which support that the biopolymer suffered structural rearrangements when mixed with 5CB. However, the uneven surface distribution of 5CB on the hybrid material of potato suberin, may limit the representativeness of the XPS measurements for these samples.In the hybrid materials formulation, the liquid crystal is in nearly twofold mass ratio excess relative to the suberin polymer . The surface elemental composition of the hybrid materials consisting of the 5CB with either suberin type was measured by XPS . In both3.3The hybrid materials containing 5CB and suberin of either type responded to VOC exposure. It was observed that VOC interaction with the hybrid materials causes a gradual reduction of the order of the 5CB molecules until isotropization. Subsequent exposure to ambient air facilitates the desorption of the VOCs from the film, and consequently the reorganisation of 5CB molecules. These changes in the liquid crystal order generate optical textures when the sample is observed using POM (Supplementary video). They also alter the light intensity transmitted through the film, which can be converted into a measurable signal detected by our custom e-nose ,8 Fig.\u00a0.Similarly to other biopolymer-based hybrid materials reported previously by our group ,8, the h3 moieties of suberin hydrocarbons could also occur. Similarly, polar yet aprotic VOCs  may preferentially interact with 5CB, whereas polar and protic VOCs  can interact with the protic hydroxyl groups present in \u03c9-hydroxyalkanoic acids that are abundant in both suberin types  ability of the two hybrid materials, their optical signals were analyzed independently. First, the complete signals were split into individual cycles, comprised of the combined 5\u00a0s exposure time and 15\u00a0s recovery time periods. Then, the values of 12 features representing the morphology of the optical signal waveform  were calper VOC for each type of hybrid material). This is called independent validation and allows for the evaluation of how the classifier performs when applied to unknown signals. Confusion matrices . The optical responses generated by the sensing materials are reversible and reproducible, showing stability upon storage at room conditions for three weeks. Future studies on the sensing performance  will allow a deeper understanding of these hybrid materials as sensors. Since the structural chemistry of suberin, which is found ubiquitously in plants, is plant species and tissue specific, a tremendous variety of distinct cross-linked suberin structures can be exploited. The employed extraction method can be tuned to further increase the diversity of the cross-linked suberin structures, mainly through control of the degree of esterification. Together this offers unlimited flexibility to produce sensors with distinctive sensitivity, in practice it would be possible to select the sensor according to the VOCs to detect. Finally, since the observed responses of the novel suberin-based sensors to the set of 12 different VOCs tested showed complementarity, the use of arrays of distinct suberin-based sensors is a promising alternative to their usage as key elements for future developments of the chemical sensing field, with potential broad applications, e.g. detection of spoiled fish 2\u201315% v/v.R\u00faben Rodrigues, Susana Palma, Vanessa Correia and Joana Pais: Investigation, validation, writing original draft; Marta Banza: Investigation, validation; Cl\u00e1udia Alves: Formal analysis; Jonas Deuermeier: Investigation; In\u00eas Padr\u00e3o, Celso Martins, Henrique Costa and Efthymia Ramou: Writing \u2013 review and editing; Cristina Silva Pereira and Ana Roque: Conceptualization, methodology, resources, supervision, Writing \u2013 review and editing.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper."}
+{"text": "The need for increased privacy protection in data linkage has driven the development of privacy-preserving record linkage (PPRL) techniques. A popular technique using Bloom filters with cryptographic analyses, modifications, and hashing variations to optimise privacy has been the focus of much research in this area. With few applications of Bloom filters within a probabilistic framework, there is limited information on whether approximate matches between Bloom filtered fields can improve linkage quality.In this study, we evaluate the effectiveness of three approximate comparison methods for Bloom filters within the context of the Fellegi-Sunter model of recording linkage: S\u00f8rensen\u2013Dice coefficient, Jaccard similarity and Hamming distance.Using synthetic datasets with introduced errors to simulate datasets with a range of data quality and a large real-world administrative health dataset, the research estimated partial weight curves for converting similarity scores (for each approximate comparison method) to partial weights at both field and dataset level. Deduplication linkages were run on each dataset using these partial weight curves. This was to compare the resulting quality of the approximate comparison techniques with linkages using simple cut-off similarity values and only exact matching.Linkages using approximate comparisons produced significantly better quality results than those using exact comparisons only. Field level partial weight curves for a specific dataset produced the best quality results. The S\u00f8rensen-Dice coefficient and Jaccard similarity produced the most consistent results across a spectrum of synthetic and real-world datasets.The use of Bloom filter similarity comparisons for probabilistic record linkage can produce linkage quality results which are comparable to Jaro-Winkler string similarities with unencrypted linkages. Probabilistic linkages using Bloom filters benefit significantly from the use of similarity comparisons, with partial weight curves producing the best results, even when not optimised for that particular dataset In recent years, record linkage centres have adopted many different models and linkage methods to ensure the protection of individual privacy as part of their operational processes. With growing demand for linked data, it has been critical for record linkage centres to implement methods which protect privacy, yet maximise the benefits that can be derived from data assets. As a result, research around privacy-preserving record linkage (PPRL) methods has become a pressing area of inquiry, with much focus on the use of Bloom filters -7. Much A Bloom filter is a probabilistic data structure that is used to approximate the equality of two sets; these similarity comparisons are extremely useful in record linkage allowing for typographic errors and variations in spelling. Bloom filters are implemented using an array of bits. Text values are first split into elements ; each element is added to the Bloom filter by applying one or more hash functions to it. The results of these hash functions determine which positions in the bit array are set to one.Typically, PPRL techniques that use Bloom filters are applied at either the field or record level. Field level Bloom filters encode each identifier into a separate Bloom filter . Record Probabilistic record linkage is preferred by many data linkage centres due to its proven track record of producing high quality linkage results from unencrypted identifiers -23. It hThere is little mention in the literature of Bloom filters being used in the context of probabilistic record linkage where the field similarity score is converted into a partial agreement weight during the calculation of a pair-wise score , 15, 28.Synthetic data was created using an amended version of the FEBRL data generator . DatasetWithin the \u2018corrupted\u2019 datasets, the fields containing errors were restricted to those that typically use a similarity comparison during record linkage (the \u2018similarity fields\u2019): first name, middle name, surname, address and suburb. The remaining fields were untouched. In the 1% error file, 1% of the designated fields were randomly selected to have their values corrupted, through the use of typographical errors, misspellings, truncation and replacement of values. The same procedure was used to generate a 5% error file, 10% error file and 20% error file.Real data was also used in our evaluation. An extract from the New South Wales (NSW) Emergency Department Data Collection was used to demonstrate the effectiveness of the partial agreement methods on real-world data . This daPrivacy-preserved versions of each dataset were created using field level Bloom filters for the \u2018similarity fields\u2019. These Bloom filters were constructed using the method first described by Schnell . Fields As per the Fellegi-Sunter approach, a single block, using the date of birth field value, was applied to reduce the comparison space. This field remained untouched during the corruption process and ensured full pairs completeness for our synthetic dataset linkages. The m- and u- probabilities for each linkage field within the datasets were estimated using known matches within the block. Known matches were identified using our generated key for the synthetic datasets, and the keys provided to us for the NSW administrative dataset (our \u2018truth sets\u2019). These probabilities were used for all linkages of all datasets.For the linkage of each dataset, the corresponding m- and u- probabilities were converted into agreement and disagreement weights as follows:Fields using exact comparisons used either the full agreement weight or the full disagreement weight. Fields using approximate comparisons used a value somewhere between these two weights. A missing field value on either side of the comparison resulted in a weight of zero. The weight values were summed across all fields to determine the total \u2018score\u2019 for each pairwise comparison.All pairs above a score of zero were recorded. Using the \u2018truth set\u2019 for each dataset, the number of missed matches  and incorrect matches  were calculated for each possible cut-off value above zero. False negatives and false positives were treated equally, the aim to minimise the sum of these misclassifications. Thus, the cut-off value with the smallest number of misclassifications was used as the best outcome  for that linkage. Records were grouped using transitive closure (\u2019merge\u2019 based) grouping, with all indirect links being honoured.For linkages of the privacy-preserved datasets, the S\u00f8rensen\u2013Dice coefficient, Jaccard similarity, and Hamming distance comparators were used to compare the similarity between Bloom filtered fields. S\u00f8rensen\u2013Dice coefficient and Jaccard similarity scores range from 0 to 1, where higher values represent greater similarity and a score of 1 represents identical values. Similarity is based on the set of bit positions set to one in each Bloom filter. Given two of these sets, A and B, similarities are calculated as follows:Hamming distance measures the difference between values. For Bloom filters, this is the number of bits that are different between each Bloom filter and resulting scores range from 0 to the length of the Bloom filter:The raw Hamming score is normalised by dividing all raw scores by the maximum raw score giving us a value between 0 and 1; lower scores represent greater similarity, and a score of 0 represents identical values.The method for modelling partial agreement required the distribution of matches and non-matches at defined similarity scores for each field in each dataset. This was achieved by performing a deduplication linkage on each dataset and recording matches and non-matches for each observed comparison. The study used the steps outlined by Winkler for estimating partial weights at specified similarity values :i=1,\u2026,N sub-intervals. We used N=20 resulting in sub-intervals at 0.05 increments.The similarity score range for all approximate comparison used is 0..1. This range was partitioned into j and each sub-interval For each field i\u03c4, was calculated as the probability of a match at interval i divided by the probability of a non-match at that interval:For each sub-interval \u03b4 is the comparator function, j\u03b3 is a comparison of the jth field,  is an arbitrary pair, M is the set of matches, and U the set of non-matches.Here i=1,\u2026,N, applying the normalised ratio vector to the field weight with the disagreement weight at 0 and the agreement weight at 1.The ratio vector \u03c4 is then used to create the partial weight curve for the complete set of sub-intervals In addition to partial weight curves, we used a simple cut-off value for the field similarity score to determine where the full agreement or full disagreement is applied. Cut-off values between 0.6 and 0.95 (in 0.05 increments) were used for all similarity fields. The cut-off value with a linkage result having the lowest number of misclassified pairs  was selected.We used the number of misclassified pairs as a measure of linkage quality. Baselines were created for each dataset by performing deduplication linkages using exact comparisons only. Deduplication linkages using Bloom filters with each of the approximate comparisons  were then compared to the baseline to measure the difference in linkage quality. Also, deduplication linkages using the Jaro-Winkler string comparison on unencrypted identifiers were undertaken to measure differences in linkage quality arising from the use of Bloom filters (the Jaro-Winkler comparator cannot be used directly on Bloom filtered data).The \u2018master\u2019 dataset of 1 million records contained multiple records belonging to the same individual. From this master dataset, a series of new datasets were created by removing or degrading the quality of particular fields. The partial weight curves were created for each field in each synthetic dataset shown in . DatasetThe results of the deduplication linkages for each synthetic dataset are shown in The performance of the similarity comparisons, when compared to the \u2018exact\u2019 comparisons, shows only a small reduction in linkage errors with the dataset containing 1% error. The benefit derived from partial agreements in data linkage appears minimal when the quality of the data is this high. However, a significant reduction in linkage error can be seen for the datasets containing at least 5% error across almost all similarity comparisons. The reduction in misclassified pairs for the dataset with 20% error, while high, is less than both the datasets with 5% and 10% error.While the results in The extract from the NSW Emergency Department Data Collection contained 4,304,458 records. Empty fields  were left as empty fields in the privacy-preserving version of the dataset.Dataset level weight curves were created for the NSW Emergency dataset using the same method used with the synthetic datasets. As with the synthetic datasets, deduplication linkages were undertaken using field level, dataset level and simple cut-off values. Also, a deduplication linkage was performed using the dataset level weight curves derived from the synthetic datasets.The results of all deduplication linkages on the NSW Emergency dataset are shown in The field level weight curves produced the best results, followed by both dataset level weight curves and the use of simple cut-off values. Similarly to the synthetic datasets, the field level and dataset level weight curves demonstrate a consistent improvement to recall while maintaining a high level of precision.Our results show that the use of Bloom filter similarity comparisons for probabilistic record linkage can produce linkage quality results comparable to the use of the Jaro-Winkler string similarity on unencrypted identifiers. With synthetic datasets, we found that the highest linkage quality was achieved using Hamming distance, producing fewer linkage errors (on the 10% error and 20% error datasets) than the Jaro-Winkler similarity on unencrypted identifiers. Regardless of the comparator used, all approximate comparisons improved the quality of the linkage, particularly as the level of error in the dataset increased. While the dataset with 20% error did not show the same proportional reduction (%) in misclassified pairs (as compared to datasets with only 5% and 10% error), the total number of misclassified pairs was vastly reduced. This \u2018dip\u2019 in reduction may be an artefact of the artificial error generation within the synthetic datasets, or it may be due to a limit on how much error can be accounted for using partial agreements.As expected, optimised partial weight curves for each field produced the best quality results. The dataset level weight curves, estimated as a single \u2018best-fit\u2019 curve for all fields, showed a well defined slope for each of the comparators, with only a small increase in the number of linkage errors for both the synthetic datasets and the NSW Emergency data. The synthetic datasets and the NSW Emergency datasets produced similar weight curves, so it was unsurprising the dataset level weight curves created from the synthetic data produced high quality results on the NSW Emergency data. The fact that these results were close suggests that it may be possible to estimate a generic curve (for each comparator) for use in the linkage of various types of data; however, further testing using a variety of real datasets is warranted.The S\u00f8rensen-Dice and Jaccard similarity comparators produced very similar linkage quality results across the range of datasets. The Hamming distance comparison appeared to produce the fewest errors for the synthetic datasets overall; however, its performance against the other comparators on the NSW Emergency data was inconsistent. This may be explained by Hamming\u2019s observed improved performance under higher degrees of error with the synthetic datasets. If the NSW Emergency data has a similar error rate to the 1% error dataset, Hamming distance\u2019s relative performance may also be similar.Field level and dataset level weight curves for all approximate comparators demonstrated improvement to recall while maintaining a high level of precision, a highly desirable outcome in many linkage settings. There is still a trade-off between missed matches and incorrect matches, however, and care must be taken in selecting an appropriate cut-off during linkage.A single cut-off value was shown to perform well in the context of determining agreement or disagreement in probabilistic linkage. The linkage quality using a cut-off value is lower than the linkage quality from an approximate weight curve , and the precision/recall trade-off is less desirable. However, the reduced level of error from an exact linkage is significant, and there appears to be some level of stability in the cut-off values themselves across our datasets. These results suggest that in the absence of being able to estimate a weight curve for a new dataset, whether it is due to size or complexity or time constraints, the use of a standard cut-off value is a viable alternative.There were several potential limitations to this study. This work uses previously linked real data as a benchmark. While this linked data is of very high quality, it may not be completely accurate. Bloom filtered comparisons on this particular linked data provided comparable results to Jaro-Winkler comparisons. However, this does not imply that these linkage methods are therefore equivalent in all aspects; specifically, ensuring high linkage quality with privacy preserving methods will always be far more difficult, given the limited ability to provide quality assurance or clerical review. Additionally, the synthetic datasets with introduced (manufactured) errors may not always capture the complexity of real datasets. Testing the performance of the Bloom filter comparisons against other kinds of datasets or \u2018gold standard\u2019 datasets would be a valuable exercise. However, such datasets are not always easy to find .Matching quality in probabilistic linkage benefits significantly from the use of similarity comparisons, with partial weight curves producing the best results. We have shown that this remains true even when the weight curve has not been optimised for the particular dataset being linked. This finding also applies to the comparison of Bloom filters within a probabilistic framework. Although determining the partial weight curves for producing optimal linkage quality typically requires the use of a truth set, our results show that adequate quality can be achieved through the use of weight curves derived from simulated datasets.All similarity comparisons produce significantly better results than \u2018exact\u2019 comparisons. Despite some of the challenges of working with Bloom filters and the range of comparators available, there is not a great difference between these comparators when used within a probabilistic framework. On the basis of these findings, our recommendation to linkage units is to choose the comparator that you are most comfortable with but to use a weight curve estimated for that particular comparator.Conversion of similarity scores to partial agreement weights is a quality optimisation available for all approximate comparisons (including Bloom filters) and is an essential element to maximising the pair-wise quality with the Fellegi-Sunter model of record linkage. Further work is required to determine how generalisable this option is, by analysing the weight curves with a broader variety of real-world datasets.Ethical approval for developing and refining linkage methodology was obtained from Curtin University Human Research Ethics Committee (HR 15/2010) as well as approval from New South Wales Cancer Institute Human Research Ethics Committee (HREC/10/CIPHS/37). Ethics approval included a waiver of consent based on the criteria in the national statement on ethical conduct in human research."}
+{"text": "Mycobacterium kansasii species comprises six subtypes that were recently classified into six closely related species; Mycobacterium kansasii (formerly M. kansasii subtype 1), Mycobacterium persicum (subtype 2), Mycobacterium pseudokansasii (subtype 3), Mycobacterium ostraviense (subtype 4), Mycobacterium innocens (subtype 5) and Mycobacterium attenuatum (subtype 6). Together with Mycobacterium gastri, they form the M. kansasii complex. M. kansasii is the most frequent and most pathogenic species of the complex. M. persicum is classically associated with diseases in immunosuppressed patients, and the other species are mostly colonizers, and are only very rarely reported in ill patients. Comparative genomics was used to assess the genetic determinants leading to the pathogenicity of members of the M. kansasii complex. The genomes of 51 isolates collected from patients with and without disease were sequenced and compared with 24 publicly available genomes. The pathogenicity of each isolate was determined based on the clinical records or public metadata. A comparative genomic analysis showed that all M. persicum, M. ostraviense, M innocens and M. gastri isolates lacked the ESX-1-associated EspACD locus that is thought to play a crucial role in the pathogenicity of M. tuberculosis and other non-tuberculous mycobacteria. Furthermore, M. kansasii was the only species exhibiting a 25-Kb-large genomic island encoding for 17 type-VII secretion system-associated proteins. Finally, a genome-wide association analysis revealed that two consecutive genes encoding a hemerythrin-like protein and a nitroreductase-like protein were significantly associated with pathogenicity. These two genes may be involved in the resistance to reactive oxygen and nitrogen species, a required mechanism for the intracellular survival of bacteria. Three non-pathogenic M. kansasii lacked these genes likely due to two distinct distributive conjugal transfers (DCTs) between M. attenuatum and M. kansasii, and one DCT between M. persicum and M. kansasii. To our knowledge, this is the first study linking DCT to reduced pathogenicity.The  Mycobacterium kansasii, a member of non-tuberculous mycobacteria (NTM), is an environmental bacterium that can cause pulmonary diseases mostly in patients with immunosuppression or underlying lung diseases. Although it depends on the geographical area, M. kansasii is usually classed as the second or third most common NTM isolated from patients . Bo. BoM. kaecretion . In addi strains . In addirulence) , was alsM. kansasii complex, a genome-wide association study was performed using treeWAS [To investigate the existence of genetic determinants supporting increased virulence in  treeWAS . This top < 0.00001) with the simultaneous score and the terminal score, respectively  and non-pathogenic (categories 4 and 5) phenotypes in the complete dataset was significant for four and two orthogroups  lacked the orthogroups identified by GWAS analysis (M. persicum for MK30 (25 kb) and MK52 (67 kb) and from M. attenuatum for MK20 (75 kb)  by a frameshift mutation in M. persicum  D. This ipersicum C. Anothetenuatum C.M. kansasii pathogenicity, we wanted to further assess the occurrence of distributive conjugal transfers (DCT) between all the members of the M. kansasii complex. For this purpose, coding sequences showing a higher average amino acid identity with another species than its own were considered to be probable recombinant regions, as highlighted in M. kansasii isolates . DCT signals were widespread in the M. kansasii complex, involving recombination of one gene up to large genomic fragments. Overall, M. kansasii presented larger regions of DCTs as compared with M. persicum, M. pseudokansasii and M. attenuatum. Conversely, several M. kansasii isolates presented fewer DCT signals in a similar fashion to the other species of the complex (Since DCT likely explained variations in  complex . In someM. kansasii complex (29.3%). Two conjugative plasmids were complete and circularized publicly available sequences (pMK12478 and pMK142). Interestingly, strain MK5 presented two different plasmids. All detected plasmids shared the same backbone structure as described by Ummels et al., although the approach used to identify plasmids likely biased their selection [traA/traI homolog but still shared type-VII and type-IV secretion systems homologs  (Conjugative plasmids were found in 22 out of the 75 strains of the election . Only fohomologs . As indi complex . Sizes r t-test) .p-value = 0.91, Chi-square). Furthermore, the presence/absence of single-copy orthogroups was assessed previously with the treeWAS analysis and returned no plasmid-encoded hit. Conjugative plasmids exhibited various gene functions within and between plasmids as shown by the diversity of COG categories found . Further studies using mutant and wild type strains will be needed to investigate the role of these genetic factors in the pathogenicity of the M. kansasii complex.Going along with this hypothesis, M. tuberculosis, variation in virulence were seen after functional modifications of master regulators of virulence, such as the PhoPR or the DosR regulon [M. kansasii species was significantly associated with pathogenicity. However, two genes, encoding hemerythrin-like and nitroreductase-like proteins, were significantly associated with pathogenic phenotypes  . Indeed,M. kansasii complex suggests the occurrence of HGTs most likely through conjugation among and within species. This is corroborated by the phylogenetic reconstruction based on core-plasmid genes that intertwines plasmids belonging to different species in the same clades. In the present dataset, the pathogenicity of the strains could not be correlated with the presence or absence of plasmids, or some of their genetic content, but we cannot exclude an eventual role in bacterial pathogenicity. Indeed, the incomplete nature of genome assemblies often renders difficult the identification of all contigs forming the plasmids, and the method used here could have biased our analysis towards the identification of previously known conjugative plasmids.The similarity of conjugative plasmids across different species of the M. kansasii complex, as well as among M. kansasii strains. DCTs, conjugative plasmid transfers and transduction were frequently observed and several evidence indicate that DCT was directly involved in gene losses affecting strain pathogenicity. While DCT was thought to be an important mechanism for HGT in mycobacteria [Overall, comparative analyses and genome-wide association studies on 74 genomes could identify several loci likely associated with the increased or decreased pathogenicity in species of the bacteria , it has,"}
+{"text": "AQP1-knockout (AQP1\u2212/\u2212) mice at different gestational days (GD). The expression and location of AQP1 and other AQPs in the placenta and foetal membranes of AQP1\u2212/\u2212 mice, AQP1-siRNA transfected WISH cells and oligohydramnios patients were also detected. Compared to control mice, AQP1\u2212/\u2212 mice exhibited reduced copulation plug and successful pregnancy rates, but these effects were accompanied by a larger AF volume and lower AF osmolality at late gestation. AQP9 expression was significantly decreased in the placenta and foetal membranes of AQP1\u2212/\u2212 mice, while AQP8 level was elevated in the foetal membranes of AQP1\u2212/\u2212 mice. Moreover, AQP9 expression was suppressed in WISH cells after AQP1 downregulation. Furthermore, AQP9 expression was associated with AQP1 level in the placenta and foetal membranes in oligohydramnios. AQP1 may play a critical role in regulating pregnancy outcome and maternal-foetal fluid homeostasis. Changes in AQP1 expression may lead to compensatory alterations in AQP8 and AQP9 expression in the placenta.To explore the effects of aquaporin (AQP) 1 on pregnancy outcome and the association between expression of AQP1 and other AQPs in the placenta and foetal membranes, the rate of copulatory plugs and pregnancy, amniotic fluid (AF) volume, osmolality and composition were determined in  The homeostasis of amniotic fluid (AF) exchange between matrix and foetus plays a vital role in a successful pregnancy, as either polyhydramnios (excess AF) or oligohydramnios (insufficient AF) may increase foetal morbidity and mortality during the perinatal stage  mice developed an increased AF volume and reduced AF osmolality  in both the placenta and foetal membranes of pregnant AQP1\u2212/\u2212 mice have not been investigated until now.Recently, the AQP1\u2212/\u2212 mouse model was generated to observe the effects of AQP1 depletion on the following gestation-related parameters on different gestational days: maternal pregnancy rate, foetal development and AF volume and osmolality. In addition, both the placenta and foetal membranes were collected to measure the mRNA and protein expression profiles of AQP3, AQP8 and AQP9 in mice after AQP1 depletion. Moreover, the correlation among the protein expression of AQP1 and other AQPs in the placenta and foetal membranes of patients with isolated oligohydramnios were also explored using an immunohistochemical method. Finally, after using small interfering RNA (siRNA) to interfere with AQP1 expression in human amnion epithelial WISH cells, the mRNA and protein expression levels of AQP3, AQP8 and AQP9 were examined.Therefore, a transgenic AQP1\u2212/\u2212 mice were established through intercrossing AQP1 heterozygous mice overnight  mice were donated by Prof. Yuanlin Song from Zhongshan Affiliated Hospital of Fudan University. AQP1\u2212/\u2212 and wild-type AQP1 (AQP1+/+) mice were mated separately. Gestational day 0.5 (0.5 GD) was defined as the day when a copulation plug was observed, with normal pregnant mice delivering foetuses at term on 19\u00a0GD to 20\u00a0GD. Meanwhile, the copulation plug and pregnancy rates were defined as the number of female mice with a copulation plug/number of total mice and the number of pregnancy/number of total mice, respectively. These two rates were recorded. Concept uses were collected from pregnant AQP1\u2212/\u2212 and AQP1+/+ mice at 9.5\u00a0GD (early pregnancy), 13.5\u00a0GD (middle pregnancy) and 16.5\u00a0GD (late pregnancy).All mice, which were 6\u20138\u00a0weeks old, were kept under a12-h light/dark cycle at a temperature of 23\u2009\u00b1\u20091\u00a0\u00b0C. A standard diet and deionized water were freely accessible to the mice. All animal experiments were carried out according to the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health. Protocols were approved by the Animal Care and Use Committee of Wenzhou Medical University.AQP1\u2212/\u2212 pregnant mice and five pregnant AQP1+/+ mice at each gestational day  were used for this study. At the appropriate gestational age, caesarean section was performed on the two groups of mice, and each gestational sac was carefully separated. The number of embryos per pregnant mouse and macroscopic atrophy were recorded at 9.5 GD, 13.5 GD and 16.5 GD. Each gestational sac was weighed and then ruptured. After the AF had been collected into an Eppendorf tube, the foetus, foetal membrane (including both the amnion and chorion) and placenta were weighed. These tissues were collected from foetuses on each gestational day (GD) and stored at \u2013\u00a080\u00a0\u00b0C for further study. The placental area was calculated after measuring the diameter by using the formula: S\u2009=\u2009\u03c0\u00a0\u00d7\u00a0(1/2\u00a0\u00d7\u00a0diameter)2. The AF mass was estimated by determining the difference in weight before and after-rupture.Five pregnant +, K+, Cl\u2212) and another ion (Ca2+).AF collected from each sac was centrifuged at 3000\u00a0rpm for 10\u00a0min to remove cellular debris. Subsequently, the AF from each litter of the same genotype was mixed and used to determine the AF osmolality and composition. The cryoscopic method was used to measure AF osmolality with automatic freezing point osmometer . Glucose and total protein in the AF were detected by the biuret method, and urea and creatinine levels were examined by enzymic methods. The electrode method was conducted with o-cresolphthalein complexone on an automatic biochemical analyser  to determine the concentrations of electrolytes  were purchased from the Type Culture Collection of the Chinese Academy of Medical Sciences . The cells were cultured in Dulbecco\u2019s modified eagle\u2019s medium (DMEM)  supplemented with 10% foetal bovine serum  and a100 \u00d7 Pen-Strep solution . The cells were incubated at 37\u00a0\u00b0C in a humidified atmosphere containing 5% COBoth AQP1 siRNA and control siRNA were purchased from GenePharma . The sequence of AQP1 siRNA was 5\u2032-GCU GUA CUC AUC UAC GAC UTT, and the antisense sequence was 5\u2032-AGU CGU AGA UGA GUA CAG CTT. The control siRNA sequence was 5\u2032-UGA CC UCA ACU ACA UGG UGT T, and the antisense sequence was 5\u2032-AAC CAU GUA GUU GAG GUC ATT. WISH cells were cultured to a confluence of 50% and subsequently transfected with either AQP1 siRNA or control siRNA at 40\u00a0nM using Lipofectamine 2000  in DMEM without foetal bovine serum following the manufacturer\u2019s protocol. The media were changed after 6\u00a0h of incubation and transfected cells were cultured for another 48\u00a0h for subsequent studies.2O at a volume of 10\u00a0\u03bcL, and reactions were carried out on a Light Cycler480 . The cycling parameters were as follows: 95\u00a0\u00b0C for 5\u00a0min, followed by 40 cycles of denaturation at 95\u00a0\u00b0C for 20\u00a0s, annealing at 58\u00a0\u00b0C for 1\u00a0min and extension at 72\u00a0\u00b0C for 20\u00a0s. Table \u25b3\u25b3Ct) method, and GAPDH was used as a normalization reference.Total RNA was extracted from the foetal membrane, placenta and WISH cells using TRIzol in accordance with the manufacturer\u2019s protocol. The absorbance of the RNA at 260\u00a0nm and 280\u00a0nm was quantified by spectrometry. From 1\u00a0\u03bcg of total RNA, cDNA was synthesized using a reverse transcription kit following the manufacturer\u2019s instructions . Each quantitative RT-PCR sample contained 5\u00a0\u03bcL of SYBRGreen Master Mix (TransStart TipTop), 1\u00a0\u03bcL of cDNA, 0.5\u00a0\u03bcL of primer pairs complementary to AQPs or GAPDH, and diethyl pyrocarbonate (DEPC) HPlacental and foetal membrane tissues and WISH cells were separately homogenized and lysed in radio immunoprecipitation assay (RIPA) buffer  supplemented with protease inhibitor cocktail . The lysate concentrations were determined by bicinchoninic acid (BCA) protein assay. Then, samples containing 40\u00a0\u03bcg of protein were separated by 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and the proteins were then transferred onto a polyvinylidene fluoride (PVDF) membrane . The membranes were blocked for 2\u00a0h with 5% skimmed milk in Tris-buffered saline with 0.1% Tween-20  at room temperature and then incubated at 4\u00a0\u00b0C overnight with a primary antibody. Membranes were incubated with the following primary antibodies at 4\u00a0\u00a0C overnight: monoclonal mouse anti-AQP1 antibody , polyclonal rabbit anti-AQP3 antibody , polyclonal rabbit anti-AQP8antibody , monoclonal mouse anti-AQP9 antibody , antibody against endogenous \u03b1-Tubulin was used as an internal control . The membranes were rinsed and probed with horseradish-peroxidase-conjugated secondary antibody  diluted 1:5000 for 2\u00a0h at room temperature. Finally, immunoreactive bands containing target proteins were visualized with enhanced chemiluminescence (ECL) substrate , and images were captured with an Amersham Imager 600 system .To detect correlations between the expression of AQP1 and other AQPs when AQP1 and other AQPs were decreased, a total of 30 patients with singleton pregnancies who received elective caesarean delivery were enrolled in this study; among these patients, 15 patients were identified as having oligohydramnios, while the other 15 had a normal AF volume. The gestational age of the pregnant women ranged from 37 and 40\u00a0weeks. Women were excluded when at least one of the following conditions was met: foetal abnormalities; a foetus small for the gestational age; premature rupture of the membranes; restricted foetal growth; treatment with several medications ; and diagnosis with complications that might influence the AF, such as hypertension, diabetes, cardiovascular disorders and autoimmune diseases. Ethical approval was received from the Ethical Committee of the Second Affiliated Hospital of Wenzhou Medical University. All subjects agreed to the study and provided written informed consent before starting the study.Before delivery, oligohydramnios was defined as an AF index (AFI)\u2009<\u20095\u00a0cm using ultrasound measurement, as a normal AFI ranges from to 8 to 18\u00a0cm  staining: monoclonal anti-AQP1 mouse antibody , polyclonal rabbit anti-AQP3 antibody , polyclonal rabbit anti-AQP8antibody  and monoclonal mouse anti-AQP9 antibody . The tissues were then incubated with secondary antibody for 20\u00a0min at room temperature, followed by incubation with diaminobenzidine as a chromogen for the appropriate duration. After washing with PBS for three times, the tissue sections were counterstained in haematoxylin, dehydrated, cleared and mounted in dibutyl phthalate polystyrene xylene. At least 10 representative staining fields were chosen under a microscope , and all immunopositive cells in these fields were analysed at random; the proportion of AQP-positive cells was determined for each case , placed in a 10\u00a0mM citrate solution (pH\u00a06.0) and heated at 100\u00a0\u00b0C in a microwave oven for antigen retrieval. Endogenous peroxidase activity was blocked with 3% HAQP1\u2212/\u2212 and AQP1+/+ mice were assessed by analysis of variance (ANOVA). Multiple comparisons were carried out by using a post hoc least significant difference (LSD) test. Correlation analysis of the expression of AQP1 and other AQPs was conducted using Pearson correlation analysis for normally distributed data; otherwise, the Spearman correlation test was used. A two-sided P\u2009<\u20090.05 indicated a statistically significant difference.SPSS 19.0  was used for statistical analysis. Differences in the copulation plug and pregnancy rates were analysed by chi-square test. Quantitative data are expressed as the mean \u00b1 standard deviation (SD). The statistical significance of differences in AF volume, osmolality and composition; feotal weight; amnion weight; placental area; and weight between the AQP1 depletion. We found that pregnant AQP1\u2212/\u2212 mice exhibited a reduced copulation plug rate  compared with that in pregnant AQP1+/+ mice , suggesting that AQP1 depletion led to a decrease in the copulation plug rate . The pregnancy success rate was higher in AQP1+/+ mice  than in wild-type controls . However, the numbers of macroscopic atrophic embryos per pregnancy recorded in AQP1\u2212/\u2212 mice and AQP1+/+ mice at each gestational age were no different. These results show that AQP1 deficiency reduces reproductive performance in female mice.First, we explored fertility in mice after AQP1\u2212/\u2212 and AQP1+/+ mice at 13.5 GD  concentrations was observed between the two groups at 13.5 GD , AQP1 mRNA expression was not detected in the placentas of AQP1\u2212/\u2212 mice but was observed in AQP1+/+ mice at both 13.5 GD and 16.5 GD . Moreover, among patients with a normal AFV, no significant difference in the expression of AQP1, AQP3, AQP8 or AQP9 was found in the placenta trophoblasts, amnion epithelial cells or chorion (Table P\u2009>\u20090.05). In patients with oligohydramnios, AQP9 expression was positively associated with AQP1 expression in both placental trophoblasts  and foetal membranes  .In this study, patients with oligohydramnios were chosen to determine whether the expression levels of AQP1 and other AQPs are correlated when AQP1 and other AQPs are decreased. The placenta and foetal membranes were immunostained with antibodies against AQP1, AQP3, AQP8 or AQP9 Fig.\u00a0. AQP1 anAQP8\u2212/\u2212 mice (Sha et al. AQP8\u2212/\u2212 mice displayed elevated fertility due to an increase in follicular maturation and ovulation via reduced granulosa cell apoptosis (Su et al. AQP4\u2212/\u2212 mice displayed reduced fertility and a lower pregnancy rate and litter size. This may be caused by the presence of smaller ovaries containing fewer antral follicles and corpora lutea and a decreased uterine response to gonadotropins in AQP4\u2212/\u2212 mice compared to wild-type mice (Sun et al. AQP1\u2212/\u2212 mice, demonstrating that AQP1 is involved in regulating the function of the female reproductive system. Nevertheless, the number of macroscopic atrophic embryos in AQP1\u2212/\u2212 mice and AQP1+/+mice did not differ at either 13.5 GD or 16.5 GD, suggesting that the AQP1 protein does not affect embryogenesis and development. Thus, further studies should be carried out to specifically elucidate the underlying molecular mechanism of reduced female reproductive performance due to the absence of AQP1 and cellular events responsible for the effect.AQPs are distributed throughout the male and female reproduction systems, where they facilitate gamete transport, fertilization and early embryo development (Skowronski et al. AQP1\u2212/\u2212 mice was dramatically higher than that of AQP1+/+ counterparts at 13.5 GD, and the embryonic weight of in AQP1\u2212/\u2212 mice was higher than that of AQP1+/+ mice at 16.5 GD. In support of the role of AQP1 in angiogenesis, the placentas of AQP1\u2212/\u2212 mice displayed an altered blood vessel structure, an increased number of trophoblast cells and nodules and enhanced nucleated red blood cell counts (Zheng et al. AQP1\u2212/\u2212 mice exhibit abnormal angiogenesis that results in hyperplasia and oedema. Indeed, an increased AFV and larger placentas were observed in AQP1\u2212/\u2212 mice at 16.5 GD, which might create a perfect environment for foetuses in utero and more nutrients to increase foetal weight. Our findings indicate that AQP1 has a vital function in placental and foetal growth.In our previous study, AQP1 was detected in placental vascular endothelial cells (Zhu et al. AQP1\u2212/\u2212 mice exhibit polyhydramnios (Zheng et al. AQP1\u2212/\u2212 displayed compressive deformation of the blood vessel structure and an increased number of syncytiotrophoblast nodules. Additionally, the permeability of trophoblast cells in AQP1\u2212/\u2212 mice was shown to be significantly decreased (Sha et al. AQP1\u2212/\u2212 mice serve as a model of polyhydramnios. Furthermore, polyhydramnios was observed at 16.5 GD rather than 13.5 GD, which is consistent with the occurrence of polyhydramnios in late gestation in humans (Brace AQP1-deficient mice showed impaired absorption of renal fluid and the inability to concentrate urine (Chou et al. AQP1\u2212/\u2212 mice at 16.5 GD; thus, we speculated that the observed polyhydramnios and decreased AF osmolality were associated with AQP1 (Mann et al. The foetuses of homozygous ns Brace . In the ns Brace . Althougns Brace . FurtherAQP1\u2212/\u2212 mice. Hence, upregulation of AQP8 in the foetal membranes after AQP1 depletion may lead to polyhydramnios through a compensatory mechanism, while the expression of AQP9 in the placenta and foetal membranes is decreased after AQP1 depletion. This phenomenon might be related to the different classifications and biological functions of AQPs. AQP1 and AQP3 play a vital role in passive water movement across the amnion (Damiano Specifically, AQP1, AQP3, AQP8 and AQP9, which play a vital role in maternal-foetal fluid exchange homeostasis, have been detected in the placenta and foetal membranes (Beall et al.  Damiano  and othe Damiano . AQP1 an Damiano , whereas Damiano . AQP3 mR Damiano .Furthermore, we explored the association between AQP1 and other AQPs in human WISH cells, and found that inhibition of AQP1 expression significantly reduced AQP9 expression. In our previous study (Zhu et al. In summary, while the water channel AQP1 may influence pregnancy outcome and participate in the regulation of AF volume and osmotic pressure, it does not affect embryogenesis and development. In addition, changes in AQP1 may lead to compensatory alterations in the expression of other AQPs in both the placenta and foetal membranes. Further studies should be carried out to clarify this compensatory mechanism in the future."}
+{"text": "AceDRG dictionaries corresponding to commonly occurring classes of missing linkages.Analysis of the Protein Data Bank revealed that over a third of entries contain covalent linkages without descriptions in the CCP4 Monomer Library (CCP4-ML). The CCP4-ML was updated with  AceDRG. As part of this effort, the CCP4-ML has also been extended using AceDRG link dictionaries for the aforementioned linkage types identified in this analysis. This will facilitate the identification of such linkage types in future modelling efforts, whilst concurrently easing the process involved in their application. The need for a universal standard for maintaining link records corresponding to covalent linkages, and references to the associated dictionaries used during modelling and refinement, following deposition to the PDB is emphasized. The importance of correctly modelling covalent linkages is demonstrated using a case study, which involves the covalent linkage of an inhibitor to the main protease in various viral species, including SARS-CoV-2. This example demonstrates the importance of properly modelling covalent linkages using a comprehensive restraint dictionary, as opposed to just using a single interatomic distance restraint or failing to model the covalent linkage at all.Covalent linkages between constituent blocks of macromolecules and ligands have been subject to inconsistent treatment during the model-building, refinement and deposition process. This may stem from a number of sources, including difficulties with initially detecting the covalent linkage, identifying the correct chemistry, obtaining an appropriate restraint dictionary and ensuring its correct application. The analysis presented herein assesses the extent of problems involving covalent linkages in the Protein Data Bank (PDB). Not only will this facilitate the remediation of existing models, but also, more importantly, it will inform and thus improve the quality of future linkages. By considering linkages of known type in the CCP4 Monomer Library (CCP4-ML), failure to model a covalent linkage is identified to result in inaccurate  interatomic distances. Scanning the PDB for proximal atom pairs that do not have a corresponding type in the CCP4-ML reveals a large number of commonly occurring types of unannotated potential linkages; in general, these may or may not be covalently linked. Manual consideration of the most commonly occurring cases identifies a number of genuine classes of covalent linkages. The recent expansion of the CCP4-ML is discussed, which has involved the addition of over 16\u2005000 and the replacement of over 11\u2005000 component dictionaries using  Hence, it is important to build accurate, reliable models of ligands that give confidence in the interpretation of the respective complex and its interactions. Whilst recent years have seen improvements in tools for ligand description, fitting, analysis and validation , as well as a number of known common post-translational modifications ; see Appendix Gemmi , as well as those that were not explicitly annotated as covalent linkages. Note that since the authors\u2019 original link records are automatically discarded upon deposition, we are unable to reliably distinguish post hoc between linkages that were modelled and those that were not modelled as being covalently linked during the structure-determination process. As a result of the analysis presented in Section 2AceDRG. In particular, extending the CCP4-ML to include descriptions for commonly occurring linkages  atoms involves efficient neighbour searching .Gemmi can also be used to generate and apply link records , including those between symmetry-related atoms. Indeed, Gemmi can add link records to a model for all linkages of known type that are found using the link-finding algorithm (or otherwise). In addition to their use in the analyses presented in the subsequent subsections, these functionalities of Gemmi have also been utilized within graphical interfaces to facilitate the practical application of covalent linkages ,s\u00a0=\u00a01.5; the dependency of our results on the chosen value of this parameter should be noted.All models in the PDB derived from X-ray diffraction experiments were analysed using REFMAC5 and thus do not require explicit link records.When searching for linkages of known type, the standard peptide and phosphodiester bonds  were excluded as these are handled automatically by Gemmi can report the graph geodesic distance (the number of edges in the shortest path) between atoms in the chemical graph of a macromolecule. For example, this allowed the exclusion of proximal atoms coordinated by metal ions , over half of these models have only one potentially missing link, whereas some PDB entries have a very large number of missing records  shows that only a single potential link instance appears unannotated for a third of these 43 linkage types; around a half have at most two instances. These might include rare linkage types involving rare components, as well as atom pairs that are modelled as being proximal despite not being truly covalently bonded. However, there are a number of known linkage types for which there are a large number of unannotated link instances in the PDB models. The ten most commonly occurring potentially missing linkage types are exhibited in Table 2The identified unannotated potential linkages comprise a total of 87 types of atom pairs for which there are linkage descriptions available in the CCP4-ML. These pertain to 43 unique linkage descriptions; some linkage descriptions can be used for multiple atom pairs . Fig. 1et al., 2016The interatomic distance (bond-length) distributions for these ten linkage types are shown in Fig. 22.4.Gemmi.We now consider atom pairs identified as potential linkages of unknown type. The identified \u2018potential linkages\u2019 may or may not actually correspond to atom pairs that can form covalent bonds. In the case of annotated linkages, the linkage type was deemed to be a valid linkage type by the PDB annotation pipeline, despite not having a corresponding entry present in the CCP4-ML. Information regarding the exact nature, source and treatment of the linkage is not available from the deposited model. Where such potential linkages are unannotated, it is possible that the atom pair should have been modelled and annotated as being covalently linked. Alternatively, it could be that the two atoms are not covalently linked, but rather have drifted together during refinement, ultimately becoming close enough to be identified as a potential linkage by the link-finding algorithm in Whether annotated or unannotated, the covalent linkages may or may not have been modelled and refined as such during structure determination. Restraints for a linkage of unknown type could have been defined in a custom dictionary, or otherwise just a simple link record could have been used in the absence of an appropriate dictionary (which is not a recommended treatment).et al., 2018REFMAC5. Since models of poor quality and/or derived using only low-resolution data tend to have high atomic positional uncertainty, those with nominal resolution lower than 3\u2005\u00c5 or an R factor greater than 30% were excluded (the R factor was used as Rfree was not available in too many cases). This resulted in a total of 129\u2005043 PDB model entries being included in our analysis.When analysing unannotated linkages, it became apparent that a large number of the proximal atoms detected as potentially linked by our algorithm were actually not covalently bonded, and rather were a consequence of the prevalence of inaccurate models in the PDB. In order to reduce the number of such false positives, when analysing linkages of unknown type we considered only the 137\u2005852 PDB entries for which there exists a corresponding pre-computed PDB-REDO entry . We also excluded atom pairs if one of the constituents was a water molecule (HOH) or an unknown component . In addition, since one of the primary objectives driving this investigation was to guide expansion of the CCP4-ML using AceDRG-derived link dictionaries, and AceDRG cannot currently generate dictionaries for metal-containing compounds, metals were excluded from the analysis of un\u00adannotated linkages of unknown type, as were H atoms and nonmetals with atomic number greater than 16. Specifically, potential linkages were only included if both constituent atoms had an element listed as one of B, C, F, N, O, P, S.Since high PDB-REDO was used to facilitate model-based and atom-based filtering, we used the coordinates corresponding to the original deposited PDB models when searching for potential linkages using Gemmi. Therefore, our results pertain to the status of models in the PDB and not the status of those remediated by PDB-REDO. A summary of the potential linkages remaining following filtering is shown in Table 3Whilst B factor resulted in a dramatic reduction in the number of identified potential linkages (a 45.3% decrease), especially for linkages that were not annotated (a 63.2% decrease); this is unsurprising due to the tendency of unreliable regions (with high B factors) to result in clashing atoms. Excluding potential linkages involving metals, water molecules and unknown components resulted in final set of 11\u2005188 atom pairs being identified as potentially missing linkages, which were found amongst 5739 PDB entries.It is evident that a large proportion of the models deposited in the PDB (38.3%) contain link records for which there is no corresponding description in the CCP4-ML. Filtering by Whilst the majority of the identified unknown potential linkage types were found annotated using a link record (4043 types), many of these types were found unannotated (3008 types). A number of these potential types (238) were found both annotated and unannotated amongst the PDB entries.i.e. identified proximal atom-pair types) are only found once amongst all model entries. There are comparatively few potential linkage types for which many individual instances are observed.The frequency distributions corresponding to the number of PDB model entries and the number of individual link instances for unannotated potential linkages are shown in Fig.\u00a03a). These are individually identified in Fig. 44rkm and 4rkn) and for which there are a large number of different types .The four PDB entries that exhibited the largest number of potential missing link instances can be seen as the rightmost observations in Fig. 34rkm (163 instances) and 4rkn (54 instances) correspond to the covalent linkage of cysteine and haem C , facilitated the identification of true classes of covalent linkages. Iterative exclusion of PDB entries that were found to contain many false-positive hits . Of the 11\u2005934 PDB model entries that contain unannotated metal-involving potential linkages, the vast majority (10\u2005694) were found to also contain other annotated metal-involving linkages. This reflects the inconsistent treatment/quality of metals within macromolecular models.Whilst the majority of metal-involving linkages are annotated using link records, around 11% are not  that were present in the CCD  the CCP4-ML contained over 13\u2005000 components, 73 linkages and 63 modifications. Many of these linkages and modifications involve the use of component wildcards; these may be applied to the linkage of any components that match the specified component type and atom nomenclature .As of late 2017 (Ai.e. not just NAG). The PDB contains examples of 14 such matching atom-pair types (with the most common being 289[C1]\u2013SER[OG]). However, there are also dictionary entries for MAN[C1]\u2013SER[OG] and XYS[C1]\u2013SER[OG] , which would take precedence over the more general pyranose[C1]\u2013SER[OG] linkage description.For example, the link description with identifier \u2018NAG-SER\u2019 can be applied to any pyranose[C1]\u2013SER[OG] glycosidic linkage (see Appendix trans/cis for peptide and \u03b1/\u03b2 for glycosidic linkages). More recently, substantial efforts have gone into updating and extending the CCP4-ML in line with developments in AceDRG for the creation of component and link dictionaries  the more detailed description of local atomic environments used by (ii) the utilization of a continually expanding wealth of high-resolution structural information in deriving geometric restraints: hundreds of thousands of small-molecule crystal structures.In cases where the CCP4-ML already contained a given component entry, the corresponding dictionary has been replaced with the new Such a detailed description of local geometric environments would not be possible were it not for the wealth of underlying prior information. This represents a substantial advancement compared with the approach used in the original derivation of the CCP4-ML component dictionaries.3.2.AceDRG, the maintenance of the CCP4-ML sometimes requires manual intervention. For instance, when new compounds with metals are added to the CCD, restraint files are constructed with LibCheck and manually curated. Another task performed manually is the revision of existing geometric targets in the light of new research, with an example being iron\u2013sulfur clusters  Checking the topology of the compound to see whether it matches one of the existing types, for example peptide.(ii) Checking the consistency of atomic nomenclature with other compounds in the category, for example a peptide residue should minimally contain the backbone atoms N, CA, C, O and OXT.etc.).(iii) If the topology and nomenclature of the compound are consistent with a specific category, then the appropriate type can be assigned  reflect the relative positions of electron clouds. It is known that the distances between the nuclei of H and parent atoms are longer than those between the centres of the electron clouds . Consequently, the H\u2014AceDRG-based updates described herein, the component dictionaries in the CCP4-ML are in the process of being extended to include additional information regarding H atoms derived from NMX diffraction data  in various coronavirus species, including SARS-CoV  in the Mpro inhibitor N3. This thioether bridge plays an important role in stabilizing the protein\u2013ligand interaction; all other attractive interactions between Mpro and N3 involve hydrogen bonds and hydrophobic contacts  is fundamental in the design of Michael acceptor thiol protease inhibitors  of the structures contained explicit link records, whilst in the other five this bond was incorrectly unannotated .Analysis of the 23 proximalPDB-REDO models re-refined using AceDRG dictionaries cannot be confirmed, as such information is not available.The interatomic distance distributions corresponding to these annotated and unannotated covalent linkages are shown in Fig. 64.2.AceDRG was used to generate a restraint dictionary for the CYS[SG]\u2013PJE[C20] covalent linkage , which is close to the \u2018ideal\u2019 distance between sulfur and sp2 C atoms. Note that this is lower than the AceDRG dictionary value , which is based on an sp3 C atom.a) indicates that something is wrong, and is a potential indicator of mismodelling . In this case, correctly modelling the covalent linkage  results in sensible geometry, a model that fits the density and no atomic clashes.As exemplified in Fig. 8ge Fig. 8b result4.3.6lu7; Jin et al., 2020pro. In the deposited model, the covalent linkage is annotated using a link record. In both the deposited model  and when using a link record but without providing a link dictionary , C20\u2013C21 is modelled as a double bond; this is reflected in refinement to an interatomic distance of 1.32\u2005\u00c5 in both cases. Re-refining the model using an AceDRG dictionary  results in C20\u2013C21 being modelled as a single bond, resulting in a more realistic interatomic distance of 1.51\u2005\u00c5 . This emphasizes the point that modelling a covalent linkage not only affects the two atoms involved in the linkage, but that the surrounding chemistry is also affected, highlighting the value of using comprehensive restraint dictionaries when modelling covalent link\u00adages.In the standalone PJE monomer there is a double bond between the C20 and C21 atoms . However, the C20\u2013C21 bond is changed to a single bond as part of the covalent linkage of CYS[SG] to PJE[C20]. Fig. 9el Fig. 9a and whry Fig. 9b, C20\u2013Cry Fig. 9c result6lu7, has come under scrutiny due to exhibiting certain pathologies  the B factors of all atoms of the PJE residue are set to a fixed value (20.0\u2005\u00c52). Unlike all other atoms in adjacent residues, it seems that the B factors of the PJE atoms were not refined prior to deposition.It should be noted that the quality of this particular example case, PDB entry et al., 2020Models such as this are currently being used in efforts to combat the COVID-19 pandemic .In all but one instance of the thioether bridge, the PJE[C20] atom had a larger B factors of atoms involved in unmodelled linkages . Concurrently, the B factor of PJE[C20]  in the N3 inhibitor will be higher than optimal due to not being appropriately restrained to the Mpro via the thioether bridge. This, combined with other incorrect assumptions regarding local chemistry (and of course incorrect atomic positioning) would negatively affect model interpretation.It is evident that there are large discrepancies between the e.g. van der Waals) forces are omitted from model refinement. Consequently, the atomic positions refine closer to their optimal values; this is reflected in more consistent B factors  a result of not modelling a covalent linkage. In this case, we focused on a particular atom pair (CYS[SG]\u2013PJE[C20]) that we knew was mismodelled to demonstrate how the original authors might have been alerted to such mis\u00admodelling had they looked at the B-factor divergence between those proximal atoms. However, it is not necessary to know which particular atoms are covalently bonded. Rather, comparing the B factors of any proximal buried nonbonded atoms can lead to the identification of modelling errors.It should be clarified that large B-factor restraints that are implicitly applied between covalently bonded atoms during refinement (by REFMAC5), B factors should become more consistent when they are modelled as covalently bonded  divergencesB-factor restraints were used for both refinements implies that the use of a detailed link dictionary does indeed result in a better model (i.e. one with more consistent B factors). In other cases, where the assumption of covalent linkage is incorrect, it would be expected that other pathologies indicating mismodelling would become apparent via other forms of local validation.Also, due to the by REFMAC, B facto5.Modelling covalent interactions between compounds requires special consideration during macromolecular model building and refinement. It is necessary to have complete chemical knowledge of the system, as well as a corresponding detailed restraint dictionary that describes extended local stereochemistry. Lack of automation and difficulties encountered during modelling have resulted in a large number of the covalent linkages being suboptimally modelled. In addition, failure to model covalent linkages at all has been a prevalent issue. Considering both annotated and unannotated potential linkages, we have investigated the extent of mismodelling of covalent linkages in macromolecular models. We have identified common types of missing linkages and subsequently extended the CCP4-ML with appropriate descriptions.In order to assess the general extent of problems involving covalent linkages in models in the PDB Section 2, many coi.e. proximal atom pairs that do not form covalent bonds, manual consideration of the most commonly occurring types identified a number of genuine classes of covalent linkages. AceDRG link dictionaries were generated for these classes . Where this is not the case, wwPDB annotators can change the CCD compound nomenclature in order to ensure consistency with other entries in the appropriate category. Such a remediation has recently been performed for carbohydrates; this involved the standardization of atomic nomenclature, which in turn allowed us to assign the types \u2018pyranose\u2019 and \u2018furanose\u2019 to many more compounds . This will greatly simplify the future modelling of glycosidic linkages. The prospect of a more general and comprehensive treatment of carbohydrates is on the horizon  were involved in dictionary generation, and thus the source of new and recently replaced CCP4-ML dictionaries can be traced. However, there is still the outstanding issue of how such information propagates to models deposited in the PDB. At present, component and link dictionaries are not deposited, and linkage identifiers that specify the exact dictionary, chemistry and restraints used during refinement are discarded upon deposition. This can have a direct effect on the subsequent interpretability and reproducibility of PDB models. For example, in Section 4.1In the CCP4-ML, we are now attempting to track the provenance of sources of prior knowledge; this is a relatively recent initiative. post hoc between linkages that were and were not modelled as being covalently linked during the structure-determination process. In some cases a deposited model might have been refined without an explicit linkage, but a link record was automatically added during deposition. Conversely, a model might have been refined under the assumption of a covalent linkage, and the link record subsequently discarded during deposition. In addition, since the connectivity annotation may be recalculated as a part of PDB model revision, the persistence of the existing link annotation is not guaranteed. Such inconsistencies between modelling assumptions during the structure-determination process and after deposition can lead to subsequent misinterpretation of the qualitative nature of macromolecular complexes.Furthermore, the fact that the original link records of the model authors, and indeed any connectivity annotations, are automatically discarded by the current wwPDB annotation pipeline is a major shortcoming of the deposition process. This meant that we were unable to reliably distinguish i.e. the automatic removal of link records, has been due to the heterogeneous qualitative nature of models deposited in the PDB; technical difficulties have been encountered when interpreting the information within submitted PDBx/mmCIF files that originate from different sources. This could be addressed by the adoption of a universal standard for the specification of connectivity records (including identifiers) and associated changes to wwPDB deposition recommendations/policy. Compliance with such standards would allow the connectivity annotation by the authors to be properly encapsulated, and noncompliance could be reported in the wwPDB validation report.The historical need for such a treatment by the wwPDB, AceDRG dictionaries, and different software suites may favour other sources of prior information (for example, traditional references such as Engh & Huber, 1991CCP4 suite, linkage identifiers are currently specified using LINKR records in PDB format and using the ccp4_link_id data item in PDBx/mmCIF format models (Nicholls et al., 2021Given that the CCP4-ML is now using We emphasize that it is important to properly model covalent linkages using a comprehensive restraint dictionary, as opposed to just using a single interatomic distance restraint, or indeed failure to model the covalent linkage at all. Addressing problems involving covalent linkages will facilitate future modelling efforts and ultimately improve the interpretation of structural data for biology and drug discovery."}
+{"text": "To evaluate a non-contrast respiratory- and electrocardiogram-gated 3D cardiovascular magnetic resonance angiography (CMRA) based on magnetization-prepared Dixon method  for the assessment of the thoracic vasculature in congenital heart disease (CHD) patients.70 patients with CHD  were retrospectively identified in this single-center study. REACT-CMRA was applied with respiratory- and cardiac-gating. Image quality (IQ) of REACT-CMRA was compared to standard non-gated multi-phase first-pass-CMRA and respiratory- and electrocardiogram-gated steady-state-CMRA. IQ of different vessels of interest  was independently assessed by two readers on a five-point Likert scale. Measurements of vessel diameters were performed in predefined anatomic landmarks . Both readers assessed artifacts and vascular abnormalities. Friedman test, chi-squared test, and Bland-Altman method were used for statistical analysis.Overall IQ score of REACT-CMRA was higher compared to first-pass-CMRA  and did not differ from steady-state-CMRA . Non-diagnostic IQ of the defined vessels of interest was observed less frequently on REACT-CMRA (1.7\u2009%) compared to steady-state-  or first-pass-CMRA . Close agreements in vessel diameter measurements were observed between REACT-CMRA and steady-state-CMRA : \u2212\u00a01.62\u20132.38\u00a0mm). REACT-CMRA showed high intra-  and interobserver  agreements regarding vessel diameter measurements. Fat-water separation artifacts were observed in 11/70 (16\u2009%) patients on REACT-CMRA but did not limit diagnostic utility. Six vascular abnormalities were detected on REACT-CMRA that were not seen on standard contrast-enhanced CMRA.Non-contrast-enhanced cardiac-gated REACT-CMRA offers a high diagnostic quality for assessment of the thoracic vasculature in CHD patients. Congenital heart disease (CHD) is the most common congenital disorder affecting about 0.8\u2009% of life births . AdvanceDue to its wide availability, noninvasiveness, and cost effectiveness, echocardiography is the first-line imaging modality in patients with CHD , 6. HoweBesides standard contrast-enhanced first-pass-CMRA with multiphase acquisition, which is performed without cardiac gating and during one breath hold, also contrast-enhanced respiratory- and electrocardiogram (ECG)-gated steady-state-CMRA\u2014acquired during a steady-state of contrast enhancement\u2014is used for thoracic vasculature imaging . In two Recently, a free-breathing flow-independent 3D relaxation-enhanced angiography without contrast and triggering (REACT) has been introduced, which utilizes two magnetization-preparation pulses and a 3D dual-echo Dixon method . This teThe purpose of this study was to evaluate the feasibility and performance of a non-contrast-enhanced REACT-CMRA compared to standard contrast-enhanced first-pass- and steady-state-CMRA in imaging the thoracic vasculature of patients with different types of CHD. The retrospective study was approved by the local institutional review board that waived\u00a0informed consent. 72 CHD patients who underwent CMR including non-contrast-enhanced and contrast-enhanced CMRA in our department between September 2018 and November 2020 were identified. There were no exclusion criteria regarding the type of CHD, pathologies, or previous surgical procedures/interventions. Two patients had to be excluded subsequently due to distinct motion and respiratory artifacts in all three CMRA sequences.All examinations were performed on a clinical whole-body 1.5\u2009T CMR system . For signal reception, a 32-channel torso coil with digital interface was used. The CMR protocol comprised ECG-gated bSSFP cine images in standard orientations , and phase-contrast velocity-encoded flow imaging in vessels of interest. The multi-phase first-pass-CMRA was performed during breath-hold after intravenous administration of gadobutrol  at a dose of 0.1\u2009mmol/kg body weight and a flow rate of 1.5\u2009ml/s, followed by a 20\u2009ml saline flush using the same injection rate. The single-phase steady-state-CMRA was acquired during injection of 0.1\u2009mmol/kg body weight of gadobutrol at a rate of 0.3\u2009ml/s, also followed by a 20\u2009ml saline flush, with ECG and respiratory navigator gating. Late gadolinium enhancement imaging in standard orientations was also performed. A detailed description of this protocol has been described previously .Non-contrast-enhanced REACT-CMRA was recently introduced as a relaxation-based flow-independent sequence by Yoneyama et al. . It is bAs a flow-independent technique, REACT-CMRA provides a simultaneous depiction of arterial and venous vessels. The reconstructed water-only images were used for analysis. REACT-CMRA was acquired before contrast injection. For imaging acceleration, parallel imaging with sensitivity encoding was used for all three CMRA techniques. All CMRA sequences were acquired in coronal orientation Fig.\u00a0. DetaileImage quality (IQ) of non-contrast-enhanced REACT-CMRA, contrast-enhanced first-pass, and steady-state-CMRA was qualitatively and quantitatively assessed by two readers with 4  and with 11\u2009years  of CMR experience. Both readers independently evaluated the images using dedicated software  in different sessions and on anonymized images. Both readers were blinded to the medical history in each case. The assessment of diagnostic value and the presence of susceptibility (e.g. related to stent implantation), flow (e.g. insufficiency jets), and fat-water artifacts  was performed in consensus by both readers. For evaluation of REACT-CMRA the water-only images were used.The ascending aorta (AAo), left pulmonary artery (LPA), left superior pulmonary vein (LSPV), coronary sinus (CS), and right coronary ostium (RCO) were defined as vessels of interest. Both readers separately rated IQ\u00a0based on a five-point Likert scale: (1) non-diagnostic, (2) poor , (3) intermediate , (4) good  excellent .AAo, LPA, and LSPV were defined for measurement of the vessel diameter. Measurements were conducted separately by both readers on previously defined positions according to proposed recommendations . For thiPrism  was used for statistical analysis. The Shapiro\u2013Wilk test was applied for the assessment of normal distribution. Continuous characteristics are presented as mean\u2009\u00b1\u2009standard deviation or as absolute frequency. Comparison of vessel measurements between the different CMRA sequences were compared by using one-way ANOVA followed by Tukey multiple comparison tests. Non-parametric Friedman test followed by Dunn test was used for multiple group comparison of image quality between the three applied CMRA techniques. Chi-squared test was used to compare the presence of non-diagnostic image quality. Bland\u2013Altman analysis was used to evaluate differences in vessel measurements between non-contrast-enhanced and contrast-enhanced CMRA and also to determine intra- and interobserver reliability of vessel diameter measurements. The level of statistical significance was set to P\u2009<\u20090.05.A total of 70 subjects  were included in this study. The most common types of CHD were coarctation of the aortic isthmus , tetralogy of Fallot , congenital aortic valve dysplasia , and dextro-transposition of the great arteries . The complete list of the underlying types of CHD is presented in Table\u00a0The observed mean total scan time was 1:54\u2009\u00b1\u20090:25\u2009min for multiphase first-pass-CMRA (including four phases), 6:06\u2009\u00b1\u20091:57\u2009min for steady-state-CMRA, and 6:21\u2009\u00b1\u20091:59\u2009min for REACT-CMRA\u00a0.IQ results of both readers are presented in Fig.\u00a0A non-diagnostic IQ was observed in 6 out of 350  evaluated vessels on REACT-CMRA, in 15/350  vessels on steady-state-CMRA (P\u2009=\u20090.046 versus REACT-CMRA), and in 73/350  vessels on first-pass-CMRA (P\u2009<\u20090.001 versus REACT-CMRA).AAo, LPA, and LSPV showed similar mean vessel diameters without significant differences between REACT- and steady-state- or first-pass-CMRA Table\u00a0. Bland\u2013AFor intra- and interobserver agreement, Bland\u2013Altman comparisons revealed closer 95\u2009% LOA for REACT- and steady-state-CMRA compared to first-pass-CMRA . REACT-CMRA improved the diagnostic value by more precise differentiation of even small vessels from directly adjacent structures due to high resolution and less blurring Fig.\u00a0. In totaSusceptibility artifacts were the most frequent encountered artifacts and affected each of the three CMRAs . They were mainly related to surgical or interventional procedures. Furthermore, flow artifacts were present in 10/70 patients (14\u2009%) on REACT- and steady-state-CMRA, respectively, but not on first-pass-CMRA. Fat-water separation artifacts were observed on REACT-CMRA in 11/70 cases (16\u2009%), which are specific to chemical shift encoding sequences and Dixon methods , 22. HowIn this study, we compared non-contrast-enhanced respiratory navigated and ECG-gated REACT-CMRA to conventional contrast-enhanced non-gated multi-phase first-pass-CMRA and contrast-enhanced respiratory navigated, ECG-gated steady-state-CMRA for assessment of the thoracic vasculature in a wide spectrum of CHD. We were able to show that the implementation of REACT-CMRA is feasible and yields precise vessel delineation, even in cases of complex cardiovascular anatomy as frequently observed in patients with CHD. The overall IQ of REACT-CMRA did not significantly differ from high-resolution contrast-enhanced steady-state-CMRA and was significantly higher compared to contrast-enhanced first-pass-CMRA. Vessel measurements of REACT-CMRA showed good intra- and interobserver agreement without difference compared to the standard contrast-enhanced sequences. Although artifacts were observed on REACT-CMRA, the overall artifact burden was low and did not limit diagnosis of the underlying disease. In some cases, REACT-CMRA showed additional diagnostic value compared to the established contrast-enhanced methods due to better background suppression and improved vessel delineation.CMRA techniques are an important component in almost every CMR protocol for initial diagnostic work-up or follow-up of children and adults with CHD. However, the application of a gadolinium-based contrast-agent is needed for the acquisition of standard CMRA sequences. Gadolinium-based contrast agents have a favorable tolerance and severe complications are rare since the introduction of new-generation agents. However, based on recent studies on gadolinium deposition in the brain, the restrained use of contrast agents is recommended due to uncertain long-term effects, especially in young patients , 25. MorRecently, REACT-CMRA was introduced by Yoneyama et al. . This neA non-diagnostic image quality level was seen in only 1.7\u2009% of all evaluated vessels on REACT-CMRA versus 4.3\u2009% on steady-state-CMRA and even 20.9\u2009% on first-pass-CMRA (most hereby affected vessel regions were CS and RCO). Moreover, a direct diagnostic benefit was yielded by the additional use of REACT-CMRA. The use of REACT-CMRA improved the detection of the proximal coronary arteries and showed additional diagnostic value in individual cases of complex cardiovascular conditions due to good blood-to-tissue contrast, high spatial resolution, and effective fat suppression of the epicardial fat. The mean scan time of REACT-CMRA was comparable to steady-state-CMRA and prolonged compared to first-pass-CMRA. However, the additional use of compressed sensing can be potentially used for accelerating image acquisition . The oveBased on our study results and clinical experience, we can recommend the use of contrast-free REACT-CMRA in patients with CHD. Besides its use as a valuable supplementary sequence to visualize complex anatomic structures, REACT-CMRA can be a great trade-off to assess the thoracic vasculature in children who require repetitive follow-up CMR examinations without the need of contrast agents. Other suggested areas of applications may be patients with pregnancy, severe renal dysfunction, connective tissue disease, or general follow-ups, e.g. for simple assessment of aortic or pulmonary artery diameters.It should be noted that unlike the standard time resolved first-pass-CMRA, REACT-CMRA\u00a0- like steady-state-CMRA - provides only static information about the thoracic vessels. However, since vascular stenoses in patients with CHD almost exclusively affect vessels close to the heart and corresponding information is obtained quantitatively by phase contrast flow measurements as standard, this limitation is negligible for the vast majority of cases. Fat-separation and flow-related artifacts may occur and should be known to avoid misinterpretation.Our study has limitations. First, the readers were not blinded to the CMRA sequences, which might have influenced observer bias. Second, due to a lack of standards, the image quality of non-contrast-enhanced techniques including REACT-CMRA may vary across institutions depending on the imaging parameter or even the CMR system. Third, no direct comparison to other non-contrast CMRA techniques like SSFP and bSSFP was made, as the current study focused on comparison to the clinical standard. A direct comparison to other non-contrast CMRA techniques is nevertheless useful and should be considered for future studies. Fourth, the comparison between non-ECG gated and ECG gated techniques is generally limited. Furthermore, digital subtraction angiography as the reference standard was not available. Fifth, there is a wide age range in the study cohort. Since non-contrast enhanced techniques are especially desirable in children a specific pediatric cohort of patients with CHD would be useful. Further studies are necessary to address these questions.REACT-CMRA enables contrast-free and reliable imaging of the entire thoracic vasculature in patients with CHD while providing higher image quality compared to the commonly used first-pass-CMRA and similar image quality compared to high-resolution contrast-enhanced steady-state-CMRA. REACT-CMRA is not only a useful alternative to standard contrast-enhanced CMRA but may also represent a decisive step toward contrast-free thoracic vasculature imaging in CHD patients."}
+{"text": "Drosophila melanogaster, population-level differences in chill tolerance among populations are not always found when a single trait is measured in the laboratory. We measured chill coma onset temperature, chill coma recovery time, and survival after chronic cold exposure in replicate lines derived from multiple paired African and European D. melanogaster populations. The populations in our study were previously found to differ in chronic cold survival ability, which is believed to have evolved independently in each population pair; however, they did not differ in chill coma onset temperature and chill coma recovery time in a manner that reflected their geographic origins, even though these traits are known to vary with origin latitude among Drosophila species and are among the most common metrics of thermal tolerance in insects. While it is common practice to measure only one chill tolerance trait when comparing chill tolerance among insect populations, our results emphasise the importance of measuring more than one thermal tolerance trait to minimize the risk of missing real adaptive variation in insect thermal tolerance.Species from colder climates tend to be more chill tolerant regardless of the chill tolerance trait measured, but for  While a minority of insects can endure freezing temperatures, most die at temperatures above freezing for reasons unrelated to ice formation. An insect\u2019s ability to survive and function at (relatively) mild cold temperatures is its chill tolerance, and this term can refer to a complex suite of traits: the temperature at which an individual enters a chill coma , its speed of recovery from chill coma when returned to benign conditions, its ability to withstand cold without injury or death, and its fitness (quantified in any number of ways) after cold exposure6.Thermal limits partly determine where insects can live: several measures of chill tolerance correlate strongly with latitudinal8 at the critical thermal minimum , and this is typically followed by muscle membrane depolarisation, and complete paralysis at the chill coma onset temperature (CCO)9.It is becoming increasingly clear that different measures of cold tolerance allow us to see the effects of cold on different organ systems, albeit indirectly. Chill coma onset, for example, involves neuromuscular signal transmission failure. Waves of spreading depolarisation first shut down the central nervous system10. At permissive temperatures, ion and water homeostasis are typically maintained through active ion pumping in the renal organs\u2014the Malpighian tubules and the rectum\u2014but active transport slows in the cold11. In the cold, active transport cannot compensate for passive ion leak, and as haemolymph Na+ and water leak down their own concentration gradients into the gut, K+ is concentrated in the remaining haemolymph12. High haemolymph [K+] causes muscle cell depolarisation, so it is thought that ability to recover K+ homeostasis and the degree to which homeostasis is lost in the cold determine an insect\u2019s chill coma recovery time14. Survival is also linked to the degree to which homeostasis is lost in the cold: high haemolymph [K+] is toxic and can lead to chilling injury and death by triggering cellular Ca2+ overload19.Insects need to restore ion balance upon rewarming because this balance is lost in the cold; insects susceptible to chilling progressively lose haemolymph ion and water homeostasis while they remain chilled21\u2014but in practice, they often do not. After cold acclimation or rapid cold hardening , multiple chill tolerance traits are often observed to improve 24. Correlations among related traits are also common in nature, and species and populations from colder regions tend to be more chill tolerant overall  than species or populations from warmer regions25. Among Drosophila species, CCO correlates strongly with two measures of survival , weakly with CCRT, and these traits also correlate with geographical distribution 2. Within a single species, Drosophila melanogaster, populations from colder regions are sometimes, but not always, more chill tolerant based on one or more of these traits. For example, higher latitude Australian populations are more chill tolerant based on CCO, CCRT and cold shock survival28. Similarly, in a large study of D. melanogaster populations from around the world, temperate populations were more chill tolerant based on CCRT (the only measure used) than tropical populations29; however, in a separate study of African and European D. melanogaster populations, critical thermal minimum was not related to latitude 30, and in Japan, there is minimal variation in cold shock survival and CCO among northerly/southerly populations within Drosophila species 31. Even focusing on a single population and a single trait can lead to conflicting evidence of variation in thermal performance. For example, survival following cold stress at a given temperature among 40 lines of the Drosophila Genetic Reference Panel (DGRP) does not always predict survival at another low temperature32.Since multiple organ systems are involved, chill tolerance traits can vary independentlyDrosophila species can be further complicated by not knowing how previously-tested population sets are related: CCO, at least, has a very strong phylogenetic signal33, which can cloud interpretation if not accounted for. One system for which phylogenetic relationships are known is the set of African and European populations of D. melanogaster used by Pool et al.34 to study the parallel evolution of chill tolerance traits. The system consists of paired, closely-related populations and a single outgroup . In each pair, one population is derived from flies caught in a relatively cold region, whereas the other is derived from flies caught in a relatively warm region  and confirmed clear differences in at least one measure of chill tolerance persist in these populations.Pool et al. (2017) used a chronic cold exposure assay (4 days at 4 \u00b0C) to demonstrate that populations from cold regions are more chill tolerant, and concluded that this tolerance likely evolved independently in each cold-region population. This system provides an opportunity to also examine whether and how other chill tolerance traits have evolved in cold vs. warm climates, without the concern that observed chill tolerance in separate regions might be inherited from a common ancestor rather than independently evolved. We thus set out to further characterise these populations using two of the most common chill tolerance measures\u2014chill coma onset temperature (CCO) and chill coma recovery time (CCRT). Because chill tolerance traits often do correlate both with each other and with climate of origin in natural systems, we expected that cold-climate populations would be more chill tolerant than their warm-climate counterparts based on one or both of these traits. To our surprise, the populations differed very little based on either the CCO or CCRT assays\u2014within or among pairs. To ensure that differences in chill tolerance had not simply been lost via lab adaptation, we further characterised survival during a 4 day exposure to 4 \u00b0C in French and Egyptian lines (the pair previously found to exhibit the greatest difference in recovery after chronic cold exposurePopulation was a significant predictor of CCO (p\u2009<\u20090.001), but this was driven entirely by the Zambian population (our outgroup), which had a significantly lower CCO than all other populations , even when CCRTs were adjusted based on room temperature  , once fluctuations in room temperature were taken into account (ambient temperature has a significant impact on chill coma recovery time (p\u2009<\u20090.001)). However, in the pairwise comparisons, CCRTs differed significantly only between Zambia and both the French (p\u2009=\u20090.01) and Egyptian (p\u2009=\u20090.01) populations; there were no significant differences within population pairs  and was largest after two days at 4 \u00b0C Fig. .Figure 5Drosophila melanogaster. Similar results have emerged from laboratory selection experiments (where selection pressure is controlled) and using the DRGP system to focus on genetic variation within a single D. melanogaster population21, but never, to our knowledge, in multiple wild-derived populations like those used here. We were surprised to find that chill tolerance traits vary independently even among populations that are known to differ significantly in one chill tolerance trait34\u2014in a way that matched the climatic conditions where their ancestors were collected. In some ways, this finding is not surprising: it is increasingly clear that the underlying mechanisms of several commonly measured chill tolerance traits differ9, and it is also possible to select on such traits separately21. However, it remains unclear why some wild-derived populations from colder regions display greater chill tolerance across a range of traits28, whereas others (e.g. the populations studied here) can only be differentiated if we measure the \u201cright\u201d trait. In other words, in order to draw meaningful conclusions about differences in stress tolerance (or the lack thereof), it is important to measure more than one trait20. Another surprise was the relatively low chill coma onset temperature (but unremarkable chill coma recovery time) of the Zambian population, derived from flies caught in the very warm ancestral range of this species. We do not have an explanation for this finding, but it may warrant further study.In this study, we confirmed that chill tolerance traits can vary independently in Drosophila species from cold climates do not evolve one chill tolerance trait and then stop2. Second, there may not have been enough evolutionary time for the populations to fully differentiate: D. melanogaster only left Sub-Saharan Africa some ten thousand years ago37, and it is possible that some chill tolerance traits may respond more quickly to the selection pressures of a cold climate than others. However, Australian populations have been shown to differ across multiple chill tolerance traits28\u2014and the species only reached that continent within the past few hundred years37. Finally, populations that do not appear to differ based on multiple chill tolerance traits may differ in their plastic responses to cold. Adult and developmental cold acclimation improve multiple chill tolerance traits 23. Plastic responses can be very strong and may be more ecologically important than differences in basal (i.e. non-plastic) chill tolerance27.There are several possibilities for why some cold climate populations might not evolve improvements in specific chill tolerance traits. First, it is possible that after evolving better chill tolerance in one trait, or other adaptations to a cold climate that we were unable to detect, these populations did not face the necessary selection pressure to drive improvements in other traits. Notably, however, at an interspecific level this does not appear to be the case: Drosophila species, including D. melanogaster, show evidence of acclimation by recovering the ability to stand shortly after falling into a chill coma38 or displaying improved cold shock resistance after exposure to 4 \u00b0C for several days39. Differences in chill tolerance plasticity have been studied on an intraspecific level before: Australian D. melanogaster populations, which have been shown to differ in multiple chill tolerance traits along a latitudinal gradient, do not differ in their response to either adult or developmental acclimation treatments40. However, a much larger study of D. melanogaster populations from around the world, including Africa and Europe, found that flies from temperate regions do indeed respond more strongly to a developmental acclimation treatment (i.e. their chill tolerance improved more) than flies from tropical regions29. That study measured only one trait, CCRT, and also found that flies from colder regions tended to have lower CCRTs than flies from warmer regions\u2014a pattern that we did not observe in our study populations\u2014but it demonstrates that intraspecific variation in the capacity for acclimation is possible.We note that the one trait that distinguished our populations, survival after 4 days at 4 \u00b0C, was one that may have permitted plasticity  during the assay. When held at temperatures slightly below the chill coma onset temperature, some min can depend on rearing temperature41, so adaptive variation in chill tolerance traits, or correlations among traits, within or among species could appear depending on rearing conditions. Such an approach may also offer an opportunity to explore potential trade-offs between basal and plastic cold tolerance, which have previously been explored in drosophilids43 .It remains to be shown whether plasticity is behind the discrepancies that we observed. We saw essentially no differentiation in two commonly- and rapidly-measured traits (CCO and CCRT), and clear differentiation in one trait  measured under conditions that would normally induce some form of acclimation. Nevertheless, we feel that this system, which contains populations that adapted separately to cold environments in Africa and Europe, would lend itself well to future studies of how different forms of plasticity affect different thermal tolerance traits. Within a single population, genetic correlations of CT46, and since we were able to confirm that the large difference in chill tolerance previously observed by Pool et al. (2017) between the French and Egyptian populations still exists. Therefore, we are confident that differences in chill tolerance seen in these populations reflect differences in their wild progenitors and are worthy of further study.An important caveat here is that we worked with laboratory-reared flies. However, past research has shown that chill tolerance traits are remarkably stable in lab reared flies over timeD. melanogaster populations tested here\u2014but the ability to survive a chronic, mild cold stress has. These findings support the notion that chill tolerance traits are mediated by different physiological mechanisms and highlight the importance of measuring more than one chill tolerance trait in wild-derived populations. A benefit of such paired population study systems is that they can allow us to see how chill tolerance and its underlying mechanisms can evolve under similar low temperature selection pressures.A cold environment can exert selection pressure on an insect and impact diverse chill tolerance traits in unexpected ways. To our surprise, traits that would allow higher activity in the cold (i.e. a low chill coma onset temperature) or faster recovery from chill coma have apparently not been selected for in the cold-climate African and European Drosophila melanogaster populations: three pairs of closely-related populations plus one outgroup (Zambia) ) consists of seven 7) consis34.To compare populations\u2019 cold tolerance, Pool et al. (2017) exposed flies to 4 \u00b0C for 96 h (4 days), returned them to room temperature, and then recorded the number that regained the ability to move or stand within 30 min. Within each population pair in that study, the population from the colder climate was significantly more cold tolerant than its warm-climate counterpart. This was especially the case for the France/Egypt pair47, so when flies were to be used in experiments, egg density was standardised to approximately 50 per vial. Sexually mature females (presumed to have mated) were isolated under CO2 on the day after emergence. CO2 exposure was brief (less than 10 min) and flies were then given a minimum of 48 h to recover48. All flies used in experiments were females between 5 and 7 days post-ecdysis.Flies were reared at 25 \u00b0C on Bloomington medium with a 12 h:12 h light:dark cycle. Larval density can impact cold tolerance49. To measure this, we used a temperature ramping assay51. Briefly, the flies  were placed individually in 3.7 mL glass screw-top bottles, which were clipped to a frame in a custom aquarium filled with a mix of ethylene glycol and water (1:1). The aquarium was connected to a 28 L programmable cooling bath  filled with the same ethylene glycol/water mixture. For the first 15 min of the experiment, the flies were held at their rearing temperature (25 \u00b0C), and then the temperature was ramped down at 0.1 \u00b0C/min. The temperature inside the aquarium was monitored with three type K thermocouples connected to a TC-08 recorder , and the average of the three thermocouples was used to estimate fly body temperature. To test whether flies were still capable of movement at temperatures approaching the chill coma onset temperature, the vials and metal frame were periodically tapped with a metal rod, and the CCO was recorded as the temperature when this failed to stimulate any response.A fly\u2019s chill coma onset temperature (CCO) is the temperature at which it becomes completely immobilized51. Room temperature was recorded and accounted for in subsequent analysis. We measured eight to ten flies in most (26) lines, but in six, we measured between eleven and fifteen. In order to ensure a similar number of replicates in all lines, we randomly sampled ten replicates from each of those six lines (using the sample function in R) for the statistical analysis.Chill coma recovery time (CCRT) was measured by holding flies individually in 3.7 mL glass screw-top vials at 0 \u00b0C (submerged in an ice-water slurry) for six hours, returning them to room temperature, and then measuring the time that it took for each one to stand on all six legs34, only two populations (one pair) were used in the survival experiment: France  and Egypt (4 lines). Flies were held in groups of 10 in vials  that were kept horizontal to reduce the risk of flies getting stuck in the food during exposure or recovery. The groups of flies were held at 4 \u00b0C for zero to four days , given 24 h to recover at 25 \u00b0C, and then evaluated for survival using a 5 point scale  modified from MacMillan et al.52.Because of the large number of flies needed to accurately measure survival and the number of lines in our study system, and because this trait has already been characterised across a large number of lines in this system53. To examine the effect of population (e.g. the population derived from flies collected in the South African highlands) on the chill coma onset temperature (CCO), we fit a general linear model with CCO as the response variable and line nested within population as predictor variables. In all analyses, normality of the residuals was confirmed through visual inspection. We then used Tukey\u2019s HSD (Honestly Significant Difference) test for pairwise comparisons among populations.All analyses were performed in R version 3.6.3Likewise, to examine the effect of population on chill coma recovery time (CCRT), we fit a general linear model with the log-transformed CCRT as the response variable (log transformation was necessary to ensure homogeneity of variance) with room temperature and line nested within population as predictor variables. Flies that did not recover during the 90 min observation time were given a CCRT of 90 min (17 out of 311 flies). Excluding these animals from the dataset entirely would artificially lower the mean, and we confirmed that doing so has no effect on our conclusions. Tukey\u2019s HSD test was then used for pairwise comparisons among populations.To test for a correlation between CCO and CCRT, we calculated the mean CCO and CCRT of each line and fit a general linear model with mean CCO as the response variable, and population and mean CCRT as predictor variables. Because room temperature may affect CCRT, we also ran this analysis after adjusting CCRTs for the effect of room temperature extracted from the general linear model above. This adjustment, however, had no effect on our conclusion, so we opted to show the uncorrected data.Mann\u2013Whitney tests were used to compare population survival scores  on each day of the chronic cold survival assay.Supplementary Information."}
+{"text": "Flexible batteries, which maintain their functions potently under various mechanical deformations, attract increasing interest due to potential applications in emerging portable and wearable electronics. Significant efforts have been devoted to material synthesis and structural designs to realize the mechanical flexibility of various batteries. Carbon nanotubes (CNTs) have a unique one-dimensional (1D) nanostructure and are convenient to further assemble into diverse macroscopic structures, such as 1D fibers, 2D films and 3D sponges/aerogels. Due to their outstanding mechanical and electrical properties, CNTs and CNT-based hybrid materials are superior building blocks for different components in flexible batteries. This review summarizes recent progress on the application of CNTs in developing flexible batteries, from closed-system to open-system batteries, with a focus on different structural designs of CNT-based material systems and their roles in various batteries. We also provide perspectives on the challenges and future research directions for realizing practical applications of CNT-based flexible batteries. Carbon nanotubes with diverse macroscopic structures and distinctive physical, chemical, and mechanical properties are promising candidate materials for flexible batteries. Rechargeable batteries, which store electrical energy by reversible Faradaic reactions, are dominant energy storage devices on the current market \u20133. They Flexible batteries need to undergo frequent mechanical deformations, such as bending, folding, twisting and stretching ,12. TheyThe flexibility of batteries may be achieved by either using intrinsically deformable materials or specially-designed structures Fig. . Various2 bonded carbon atoms, endowing individual CNTs with unique mechanical, physical and chemical properties [6 S m\u22121 for SWCNTs or over 105 S m\u22121 for MWCNTs at 300\u00a0K, thermal conductivity of 3500 Wm\u22121 K\u22121, Young's modulus of 1 TPa, tensile strength up to 300\u00a0GPa, a large specific surface area up to 1315 m2 g\u22121, and low mass density [Since Iijima's pioneering study in 1991 , carbon operties . For exa density . The out density . They haIn this review, we first discuss the unique merits that CNTs can offer for flexible batteries. Next, we highlight the recent progress of applying CNTs in various types of flexible batteries, including both closed-system and open-system batteries. Our discussion focuses on the material synthesis, fabrication of various battery components and structure design of batteries using either individual CNTs or their macroscopic assembles or composites. Last, we provide our perspectives on challenges and future research directions on using CNTs to realize flexible batteries in practical applications.Laser ablation, arc discharge and CVD methods have been extensively developed for the controllable preparation of CNTs. Because of the high energy input by a laser beam or an arc discharge, the prepared CNTs demonstrate a high degree of graphitization. Nevertheless, harsh vacuum conditions and continuous graphite target replacement are required . In the Due to their distinctive physical, chemical and mechanical properties Fig. , CNTs ha\u00a0000), which enables more efficient electron transfer compared to conventional conductive additives. Thus, they can serve as suitable conductive ingredients to form composite electrodes with less conductive electrode materials in flexible batteries.CNTs have high electrical conductivity with a large aspect ratio  and fast degradation under mechanical deformations resulting from weak interactions with electrode materials [The current collector is a crucial component in batteries that enables fast electron transfer and provides mechanical support for electrode materials. Conventional batteries use aluminum or copper foils as current collectors (10\u201320\u00a0\u03bcm in thickness). However, they are less suitable for flexible batteries because of their heavy weight . They were used as current collectors in flexible LIBs . Upon folding, graphite particles adhered well with the CNT collector without performance degradation. Figure For example, thin metal films (less than 200\u00a0nm) were deposited on super-aligned CNTs by electron-beam evaporation. The resulting composite films have low sheet resistances  was reported as a robust current collector for foldable LIBs [\u22122 was achieved. Impressively, the foldable LIB exhibited a high energy density of 170\u00a0Wh kg\u22121, which is approximately two times that of conventional LIBs based on metal current collectors.However, it should be noted that many studies reported that the mass loading of active electrode materials on CNT current collectors is only \u223c1\u00a0mg cmble LIBs . Since t\u22121) for LIBs, but it undergoes an excessive volumetric expansion (up to 400% during lithiation/delithiation cycling) [The feasibility of ion intercalation, together with excellent mechanical properties, makes CNTs attractive as anodes for flexible closed-system batteries. Early studies directly used CNT films as anodes in flexible LIBs. For instance, a free-standing CNT film was used as a flexible LIB anode without current collector and polymer . Howevercycling) . TherefoThere are two different approaches to forming flexible CNT-based composite anodes: surface deposition on the CNT outer surface or incorporation into the CNT matrix. The surface deposition is beneficial for enabling easy access to electrolytes. However, due to relatively weak interactions between CNTs and anode materials, such composite anodes often show relatively low electrical conductivity, and poor mechanical and electrochemical performances, especially under high mass loadings. In comparison, incorporating high capacity anode materials into the internal space of the porous CNT matrix provides more intimate interactions between CNTs and anode materials. Further, CNTs may also efficiently restrain the extrication and suppress the volume expansion of incorporated materials. Thus, this method is more widely used in current studies.et al. reported a CVD method to prepare flexible anodes by coating a uniform Si layer on CNT fabrics [et al. fabricated a stable and flexible film anode for SIBs by using a chemically bonded red phosphorus-CNT hybrid, and CNTs were cross-linked with a polymer binder, as depicted in Fig. For example, Evanoff  fabrics . CNT net2/CNT cathode for LIBs was prepared by a facile paper-making method with a high areal mass loading of 40\u00a0mg cm\u22122 [2 and a compressed thickness of 90\u00a0\u03bcm. LiCoO2 particles were uniformly embedded within the continuous 3D CNT matrix with desirable flexibility.Similar to flexible anodes, CNTs can play several beneficial roles in flexible cathodes: serving as robust substrates to provide the required mechanical flexibility, offering an electrically conductive network for less conductive cathode materials, and stabilizing some cathode materials. Many studies have focused on synthesizing robust CNT-based architectures that can host a high mass loading of active cathode materials without compromising their mechanical and electrochemical performance. For example, a flexible LiCoO\u00a0mg cm\u22122 . This fl\u22121) and energy density (2600\u00a0Wh kg\u22121) [\u221230 S cm\u22121 at 25\u00b0C), massive volume expansion (\u223c79%) and dissolution of polysulfides Li2Sn (3\u00a0\u2264\u00a0n\u00a0\u2264\u00a06) from the surfer cathode have limited the development of flexible Li-S batteries [Among a variety of closed-system batteries, Li-S batteries are attractive because they offer high theoretical capacity (1672\u00a0mA h gWh kg\u22121) . Howeveratteries ,47. Variatteries , papers atteries , spongesatteries  and foamatteries , have beatteries . Figure atteries . InteresAlthough many studies have reported various CNT-based current collectors and electrodes for flexible batteries, fewer efforts have investigated the assembly and performance of CNT-based flexible full cells. Due to the unique properties of CNTs, they can enable various types of flexible batteries with different cell configurations. Here, we highlight three kinds of cell configurations: thin-film batteries, cable batteries and stretchable batteries.et al. built a flexible quasi-solid-state thin-film SIB by using thin-film electrodes made of TiO2 nanosheets [2 and graphene oxide nanosheets. The resulting architecture can facilitate the electrolyte infiltration and ion intercalation. The assembled SIB, by coupling the TiO2/GO/CNT hybrid anode with a Prussian-blue-based cathode in polymer gel electrolyte, delivered cyclic stability under different deformative conditions , which showed enhanced electrical conductivity and high shape conformability by proper surface/interface engineering [The scalable production of CNT thin films is of considerable significance for realizing practical thin-film batteries. Assembly methods via CNT liquid dispersions usually have better scalability. The complex procedures and high temperature often limit the scalability of CVD methods. Much effort has been put into addressing this issue. For example, Wu ineering . Highly et al. fabricated a cable LIB with an ultrahigh-energy density of 215\u00a0mWh\u00a0cm\u22123 [2O3 nanoparticles [2O3 nanoparticles were distributed homogeneously on CNT surfaces without apparent agglomeration. A cable LIB was demonstrated using the Fe2O3/CNT anode coupled with a LiFePO4/CNT cathode.1D cable structures offer high mechanical flexibility. CNTs can be assembled into continuous 1D fibers by using wet or dry spinning techniques. Thus, cable batteries based on CNT fibers with desirable electrochemical and mechanical properties have been developed. For example, Liu mWh\u00a0cm\u22123 . CNT fibarticles . As illuet al. developed a coaxial cable LIB by sequentially scrolling two aligned CNT yarn electrodes onto a cotton fiber with gel electrolyte [\u22122. Other than cable LIBs, CNT fibers have also been used to build cable SIBs, Li-S batteries and Zn batteries [A unique advantage of cable batteries is that they can be further woven into flexible textiles to power lightweight and flexible wearable electronics. Peng ctrolyte . The aliatteries \u201363.Among various mechanical deformations, such as bending, twisting and stretching, it is more challenging to achieve stretchability in flexible batteries . However+ transfer pathways in PDMS. One study reported a porosity engineering method by phase separation of polymethylmethacrylate (PMMA) in PDMS and subsequent removal of PMMA to create Li+ transfer pathways [In the first method, CNTs are directly coated on elastic substrates, such as polymers or cotton textiles, to fabricate stretchable electrodes . Howeverpathways . The reset al. fabricated spring-like fiber electrodes by twisting aligned CNT films, demonstrating a high elongation of up to 300% [et al. used helical CNT fibers to fabricate a super-stretchy LIB with a strain of 600%, which was also produced from helical CNT fibers [The second method is to create free-standing elastic structures using CNTs as stretchable electrodes. For instance, Peng  to 300% . The res to 300% . HoweverT fibers . RecentlT fibers . The LIBT fibers ,73.Open-system batteries are typically composed of a metal anode, electrolyte, air cathode loaded with active catalysts and other additional components, such as current collectors and gas diffusion layers. The most common open-system batteries include LABs using non-aqueous electrolytes and ZABs containing aqueous electrolytes. In this section, we discuss recent progress in using CNTs to develop flexible open-system batteries. CNTs can be used in current collectors and anodes similar to those in closed-system batteries. As for cathodes, CNTs are usually employed as conductive supports for loading catalysts to accelerate the dynamics of ORR and OER processes. Thus, we will focus on the development of CNT-based air electrodes, which are unique to open-system batteries. Based on the morphology, synthesis methods and roles played by CNTs in air electrodes, CNT-based flexible air electrodes can be categorized into four types: (i) CNT powders added as active components or conductive additives supported on non-CNT flexible substrates, (ii) CNTs grown on non-CNT flexible substrates, (iii) free-standing CNT macroscopic structures assembled using CNTs, and (iv) free-standing macroscopic CNT substrates by direct synthesis. In the following subsections, we describe these four types of CNT-based flexible air electrodes, respectively.Many hybrid catalysts are comprised of CNTs as catalytically active components and/or conductive additives. They are synthesized by adding CNTs into metal salt precursors. The resulting hybrid catalysts in solid powders are then mixed with a binder solution to form a slurry, which is then drop-casted on non-CNT flexible substrates. The non-CNT flexible substrates provide mechanical flexibility, while the deposited catalysts deliver catalytic activities. The resulting flexible air electrodes can usually withstand simple bending and twisting mechanical deformations.et al. synthesized the perovskite lanthanum nickel oxide (LaNiO3)/ nitrogen-doped CNT (LaNiO3/NCNT) hybrid catalyst. The catalyst was then coated on a flexible substrate made of carbon cloth as an air electrode for flexible ZAB  [NCNTs are usually grown to provide additional catalytic activity. For example, Chen ble LABs . CNTs hable ZABs . Jiang eCNT/CNF) . A flexiet al. to act as air electrodes for ZABs [Although non-CNT flexible substrates provide necessary mechanical supports, they increase the total mass of electrodes, compromising the specific energy density of flexible batteries. Due to the strong \u03c0\u2013\u03c0 interactions among individual CNTs, they can be assembled into free-standing macroscopic structures and serve as air electrodes. Such macroscopic CNT structures provide continuous conductive paths and tunable pores for enabling electrolyte access. They can perform multiple functions in air electrodes, including current collectors, gas diffusion layers and substrates for catalysts. There are two approaches to creating such macroscopic CNT structures. One is to assemble them using individual CNTs. For example, CNT powders are dispersed in liquid solutions, and then flexible CNT films are produced by vacuum filtration , printinfor ZABs . Notablyet al. coated the porphyrin covalent organic framework on CNTs (CNT@POF) and then fabricated a free-standing film by filtration   [CNT networks grown by CVD are usually hydrophobic. Various surface functional groups, such as hydroxyl and carboxyl, have been introduced to CNT networks to improve catalytic activities and enhance interactions with other catalysts. For instance, Li D method . After eTs) Fig. g. The coTs) Fig. h. Benefi@N-OCNT) . The obtCNTs with excellent mechanical, electrical, structural and chemical properties have been shown to be superior materials for flexible batteries. CNT films and fibers can serve as lightweight flexible current collectors. Diverse CNT macroscopic assemblies, including 1D fibers, 2D films and 3D sponges and aerogels, have also found wide application as various flexible electrodes by incorporating active electrode materials or catalysts. CNT-based flexible batteries in different morphologies, including film, cable and stretchable batteries, have been demonstrated. The architecture of the batteries and the procedures of growth, assembling, functionalization and decoration of CNTs need to be optimized cooperatively to realize a satisfying performance. Although considerable advances have been achieved (Tables Hundreds of tons of CNTs have been used in conventional battery electrodes annually as conductive additives, which has driven the rapid commercialization of CNTs at a large industrial scale . The graEnhanced mechanical and electrical properties. The mechanical strength and electrical conductivity of one single CNT are at least an order of magnitude higher than that of traditional flexible substrates, such as metal meshes and carbon cloth. However, the properties of CNT assemblies are often not satisfactory; for example, the weak interactions and large contact resistances among individual CNTs. Welding adjacent CNTs together by metal nanoparticles or adding other conductive carbon nanoparticles to form cross-linked structures may be a practical solution. However, the precise control, scalability and cost of such methods have to be carefully studied. Furthermore, some novel CNT assemblies with unique architectures, such as wavy fibers/films or helical coils, may play important roles in stretchable components in flexible batteries.High-density CNT assemblies. Flexible batteries need to be very thin and light, thus they ought to have a high energy density. Free-standing CNT electrodes may avoid the use of metal current collectors, which can significantly reduce the mass of batteries. However, CNT assemblies often have a large surface area, low packing density and high porosity, which results in flexible batteries with low volumetric energy density. For closed-system batteries, electrodes with a large surface area favor the electrolyte decomposition and form a highly resistive SEI-like structure, resulting in a large first-cycle irreversible capacity. Besides, increasing the density of CNTs may also allow the loading of more active materials, which is beneficial in improving the overall energy density.Scalable manufacturing. Despite the fact that various CNT assemblies have been demonstrated in research labs by multiple methods, the lack of scalable manufacturing methods remains a key hurdle for the practical applications in flexible batteries. For liquid-dispersion-based assembly methods, the assembly of CNTs puts forward lots of technical demands for the precise control of length to diameter ratios and surface functional groups on pristine CNTs. Dry assembly methods currently still have a very high cost. Considering further requirements on CNT functionalization to load active materials, the methods still need significant improvements.Flexible CNTs can be combined with other inflexible materials, such as metal oxide and metal alloy particles, to obtain hybrid materials with desirable mechanical and electrochemical properties. To this end, the inert surface of pristine CNTs has to be functionalized by adding heteroatoms or oxygen-containing functional groups. The functionalized surface can offer strong interactions with active materials, thus ensuring the structural stability of resulting composite materials. However, the functionalization of CNTs often induces more defects, resulting in lower electrical conductivity and mechanical properties. The chemical stability of functionalized CNTs may also degrade, especially during long-term cycling in open-system batteries. For example, OER in metal-air batteries takes place in the strong oxidative condition, which easily leads to the oxidation of unstable carbon materials. Thus, precise control in the functionalization of CNTs is a big challenge.Besides, the functionalization method has to be able to modify CNTs with different properties at different locations precisely. For example, the uniform functionalization of CNTs is beneficial to load a large number of active electrode materials, thus increasing the overall energy density in closed-system batteries. For open-system batteries, since oxygen reactions occur at the three-phase interface, one side of the cathode needs to provide access to liquid electrolytes, and it supports catalysts, while the other side needs to be in contact with the air, and it prevents the leakage of electrolytes. Then the Janus structure, which is hydrophilic on one side and hydrophobic on the other, would be ideal. However, it is ambitious to just functionalize one side of the CNT films.Compared with conventional batteries, flexible batteries require performance evaluation related to both their electrochemical and mechanical properties. At present, it is still difficult to compare the results reported because diverse performance evaluation methods have been used. For example, the evaluation of electron flexibility requires a detailed description of multiple mechanical deformation conditions, such as bending, twisting and stretching. Such information is missing in many reports. For assembled flexible batteries, it is necessary to monitor their electrochemical performance under dynamic deformation conditions. After tests, electrode materials should be re-characterized to determine whether significant structural changes have taken place. Besides, when the energy density of flexible batteries is calculated, the total mass of all components should be considered rather than the mass of active electrode materials alone.Considering that the eventual target for flexible batteries is to power wearable electronics, more attention should be paid to the integrated system. It is necessary to optimize the configuration to make different functional parts of the integrated devices function individually and match well with each other. Additionally, continuous production techniques and comprehensive performance evaluation of the integrated system also need to be developed in the future.Although many challenges remain, recent advances have demonstrated the promising potential of CNT-based flexible batteries toward practical applications. Creating macroscopic CNT assemblies coupled with active catalysts with enhanced electrochemical and mechanical performances can push the performance limits of flexible batteries. The development of scalable production methods for CNT assemblies and flexible battery assembling techniques are vital in making flexible batteries competitive on the market. With ongoing research and development efforts, we believe that CNT-based flexible batteries will have a bright future."}
+{"text": "M. longissimus thoracis et lumborum on iridescence intensity and extent (=\u2009percentage of iridescent area) since interaction with light may be related to pH-induced alterations in microstructure. Muscles were injected with brines of different pH values, cooked, sliced perpendicular to muscle fiber direction, and visually evaluated by a panel of 20 experienced panelists.The objective of this study was to investigate the effect of pH change of cooked cured pork Muscles with lowest pH (5.38) showed the lowest iridescence score of 4.63 (p\u2009<\u20090.05). Iridescence was greatest in muscles with normal (5.78) and high pH , but did not differ significantly (p\u2009>\u20090.05). Iridescence was positively correlated (p\u2009<\u20090.01) with pH and water content, and negatively correlated (p\u2009<\u20090.01) with cooking loss. Hence, hydration state and light scattering from microstructure may be important factors that determine the degree of iridescence in cooked meat products. The shimmering, rainbow-like colors from iridescence are a well-known but still not fully understood meat color phenomenon. Iridescence can be a problem for the meat industry due to consumers concerns about the quality and safety of iridescent green-colored meat causing a rejection of those products , 2. SoluM. thoracis et lumborum, n\u2009=\u20095) were purchased from a local wholesaler . Each loin was divided into four equally sized pieces and randomly assigned to five different pH treatments: control, 0.2\u00a0M NaOH, 0.6\u00a0M NaOH, 1% lactic acid and 4% lactic acid. Injection brines were prepared by dissolving 15% (w/w) nitrite curing salt  in water (=\u2009control) and adding lactic acid or sodium hydroxide to obtain a brine with 4% lactic acid, respectively 0.6\u00a0M NaOH. These higher concentrated brines were then diluted with the control brine to obtain the 1% lactic acid, respectively 0.2\u00a0M NaOH brines. pH values were measured with a puncture type pH probe  connected to a pH-meter . The pH values of the brines were as follows: 7.37 (control), 12.07 (0.2\u00a0M NaOH), 12.13 (0.6\u00a0M NaOH), 2.23 (1% lactic acid), 1.57 (4% lactic acid). Muscles were injected  with 15% (w/w) brine, individually vacuum-packaged  and tumbled  for 2\u00a0h at 2\u00a0\u00b0C. After a resting period of 12\u00a0h (2\u00a0\u00b0C), the muscles were cooked in a cooking chamber  to a final core temperature of 70\u00a0\u00b0C . Cooking loss was calculated as the ratio of weight loss during cooking to the injected meat weight. Subsequently, muscles were cooled  and sliced  approximately 1\u00a0cm thick transversely to the longitudinal axis of the muscle fiber orientation. Each slice was individually vacuum-packaged to prevent drying and thereby reduction of surface iridescence and was evaluated visually by a trained sensory panel  for iridescence intensity  and extent . All panelists passed the Ishihara color test and were trained on the scale and the correct adjustment of sample rotation, observation and illumination angle to evaluate maximum iridescence. Iridescence score was calculated as the arithmetic mean of the intensity and extent and scores from the 20 panelists were averaged to give one score per slice. For proximate analysis, remaining muscles were finely comminuted . Water, ash and sodium chloride content were determined according to the official collection of methods of analysis  [Fresh pork loins  and OriginPro  software. Assumptions of normal distribution and homogeneity of variance were tested with Shapiro Wilk and Brown-Forsythe test. Data that met both assumptions were analyzed with one-way ANOVA (treatment as factor). Fisher\u2019s LSD post hoc test was calculated to test for differences between the treatments means. Data that did not meet the assumption of homoscedasticity were analyzed with a Welch ANOVA and Games-Howell post hoc test. Pearson correlation coefficient (r) was calculated to measure the dependence between iridescence and physicochemical properties. A significance level of \u03b1\u2009=\u20090.05 was used.All samples showed iridescence. The lowest iridescence score was observed in the 4% lactic acid treatment with the lowest ultimate pH Fig.\u00a0. StrongeThe purpose of this research was to investigate the influence of pH on meat iridescence in cured cooked pork meat. Iridescence was positively correlated with pH and water content, and negatively correlated with cooking loss. These findings support the hypothesis that iridescence is a special case of light scattering along the myofibers and that incoherent scattering from structural attributes suppresses iridescence. A major limitation of this study is the lack of information on the raw fresh muscle characteristics since it can be assumed that these parameters have a crucial impact on meat iridescence. Thus, it must be kept in mind that differences in iridescence might also arise from differences in the fresh muscle characteristics. In terms of possible solutions, there seems to be very limited approaches to reduce the potential problem of iridescence since lower hydration results in lower yields and reduced quality of processed meats."}
+{"text": "Suberin is a specialized cell wall modifying polymer comprising both phenolic-derived and fatty acid-derived monomers, which is deposited in below-ground dermal tissues  and above-ground periderm . Suberized cells are largely impermeable to water and provide a critical protective layer preventing water loss and pathogen infection. The deposition of suberin is part of the skin maturation process of important tuber crops such as potato and can affect storage longevity. Historically, the term \u201csuberin\u201d has been used to describe a polyester of largely aliphatic monomers , hydroxycinnamic acids, and glycerol. However, exhaustive alkaline hydrolysis, which removes esterified aliphatics and phenolics from suberized tissue, reveals a core poly(phenolic) macromolecule, the depolymerization of which yields phenolics not found in the aliphatic polyester. Time course analysis of suberin deposition, at both the transcriptional and metabolite levels, supports a temporal regulation of suberin deposition, with phenolics being polymerized into a poly(phenolic) domain in advance of the bulk of the poly that characterize suberized cells. In the present review, we summarize the literature describing suberin monomer biosynthesis and speculate on aspects of suberin assembly. In addition, we highlight recent advances in our understanding of how suberization may be regulated, including at the phytohormone, transcription factor, and protein scaffold levels. Plants have developed effective processes to facilitate their survival, including the production of secondary metabolites ,2. The bArabidopsis thaliana and the crop potato (Solanum tuberosum L.) have provided opportunities to elucidate the function of numerous suberin biosynthetic genes [\u03b2-ketoacyl-CoA synthase genes StKCS6 [AtKCS2, and AtKCS20 [StCYP86A33 [AtCYP86A1 [AtCYP86B1 [fatty acyl-CoA reductase genes, AtFAR1, AtFAR4, and AtFAR5, involved in the production of different chain-length primary fatty alcohols [AtGPAT5 [StFHT and AtASFT [The genomic resources of model organisms such as ic genes ,9. Molecs StKCS6 , AtKCS2, AtKCS20 ,12 encodCYP86A33 ,14, AtCYtCYP86A1 , and AtCtCYP86B1 ,17, threalcohols ,19, a gl[AtGPAT5 , aliphatd AtASFT ,23,24, ad AtASFT , AtABCG2d AtASFT . Since mIn the last two decades, advances in our understanding of suberin biosynthesis and deposition, and how these processes are controlled, have highlighted regulatory roles for phytohormones, transcription factors (TF), and Casparian strip membrane domain proteins (CASPs). This review provides an overview of the current state of knowledge of suberin monomer biosynthesis, the assembly of monomers into the suberin macromolecule, and the coordination of suberin deposition by phytohormones, TFs and CASPs. An increased fundamental understanding of the role of suberin in response to various stressors, and of the mechanisms that regulate the suberization process, may have important implications for crop improvement efforts, including enhanced tuber storage and resistance to drought stress and pathogen infection .13C-NMR [Suberin has been differentially described in the literature as a polyester-based aliphatic polymer with associated phenolics , and as a polymer comprising distinct poly and poly(phenolic) domains . These two views of suberin are based on the knowledge that suberized tissue contains both poly(phenolic) and poly polymers, but for which the structures are unresolved. The integrated model of suberin structure, originally proposed by Kolattukudy , and fur13C-NMR  and scan13C-NMR ) support13C-NMR . One dom13C-NMR ,33,34. T13C-NMR  and gene13C-NMR ,37 data 13C-NMR , and (3)13C-NMR . The mod13C-NMR  have que13C-NMR  more str13C-NMR  convinci13C-NMR . AltogetRegardless of which structural model accurately represents the structure of suberin, the poly(phenolics) consist of polymerized hydroxycinnamic acids and their derivatives, tyramine-derived hydroxycinnamic acid amides (at least in potato), and a small proportion of hydroxycinnamyl alcohols, i.e., monolignols  32,33,3,333,34. Over the last twenty years, many biosynthetic steps required for suberin production have been elucidated. Generally, characterization has focused on steps from two biosynthetic pathways implicated in phenolic and aliphatic monomer production required for the assembly of the respective poly(phenolic) and poly suberin domains, while novel aspects of linkage and assembly have also been recently elucidated. AtCYP86A1, encoding a CYP450 required to produce predominant aliphatic suberin monomers, was characterized in Arabidopsis using a cyp86a1/horst mutant [StCYP86A33 [cyp86a1/horst mutant [Total and relative suberin monomer composition varies between developmental stages, plant organs, and species, but is similar enough ,51 that t mutant , and subCYP86A33  includint mutant .The chemical composition of the phenolic domain of suberin has not been as thoroughly characterized as that of the aliphatic domain. In potato, which has been most studied in this regard, the SPPD is made up of hydroxycinnamic acids and their derivatives, including hydroxycinnamoyl amides and monolignols , all derFew studies address the involvement of phenylpropanoid metabolism during suberization. This is partly due to the highly localized and cell-specific deposition of suberin, making it technically challenging to distinguish suberin-specific biosynthesis from that of neighboring tissues. In roots for example, during normal growth and development, phenylpropanoid metabolism associated with CS formation can be overshadowed by the disproportionately greater number of phenolic monomers being synthesized and deposited in lignifying cells in the adjacent stele. Consequently, several distinct and complementary approaches are used to study phenylpropanoid metabolism during suberization: (1) histochemical analyses (primarily to track deposition), (2) molecular approaches using mutant analysis and/or fluorescent-tagged fusion protein expression, and (3) inducible systems such as wound-healing potato tubers. There are several recent and excellent reviews about phenylpropanoid metabolism, for example with emphasis on metabolome formation  and tranpara-hydroxylation by cinnamic acid 4-hydroxylase (C4H), yields 4-hydroxycinnamic acid, i.e., p-coumaric acid [p-coumaroyl-CoA has several possible metabolic fates. For example, p-coumaroyl-CoA can be shunted directly into the flavonoid branch of the phenylpropanoid pathway or serve as a precursor to other branch pathways involving side chain modification . An alternative metabolic fate, which, among other end products, channels carbon into suberin monomer formation, involves modification of the p-coumaric acid carbon skeleton into other common hydroxycinnamic acids in a series of hydroxylation and methyl-transfer reactions, beginning with the conversion of p-coumaroyl-CoA into p-coumaroyl-quinate/shikimate via hydroxycinnamoyl-CoA transferase (HCT) [p-coumaroyl-quinate/shikimate 3\u2032-hydroxylase (C3\u2032H) yields caffeoyl-quinate/shikimate [O-methyltransferase (CCoAOMT) yields the 4-hydroxy-3-methoxy-substituted hydroxycinnamate structure of ferulic acid [O-methyltransferase (COMT) [P-Coumaric acid, caffeic acid, ferulic acid, and sinapic acid (and their derivatives) are all found in the final suberin poly(phenolic) domain, albeit in a species-specific manner.Phenylalanine ammonia-lyase (PAL) catalyzes the first committed step of the phenylpropanoid pathway, converting the shikimate pathway-derived amino acid phenylalanine into cinnamate . The nexric acid . After cric acid , p-coumase (HCT) . Hydroxyhikimate , which ilic acid , which ilic acid , in conje (COMT)  in varioNicotiana tabacum L.) [In potato tuber suberin, hydroxycinnamoyl amides have been identified as part of the SPPD . These dacum L.) .Pinus taeda L. cells [Hydroxycinnamoyl-CoA thioesters can be converted into their corresponding monolignols by cinnamoyl-CoA reductase (CCR)  and cinnL. cells , the monL. cells .Historically, the SPPD of suberized tissues has been considered \u201clignin-like\u201d due to the presence of phenylpropanoid-derived monomers, including monolignols, cross-linked to the cell wall . However2O2 over 30 years ago [2O2 required for the peroxidase-mediated cross-linking of phenolics is generated by an NAD(P)H-dependent oxidase system is supported by evidence of reactive oxygen species production via oxidative bursts that occur upon tuber wounding [Solanum lycopersicum L.) as playing a role in the polymerization of lignin and phenolic suberin monomers in roots [Both the precise localization and mechanics of polymerization are critical for SPPD assembly. Here we focus on the process of polymerization and address recent progress in the coordination of where suberin is deposited in ears ago , in a prears ago ,93. In pears ago . The hypwounding ,63,64. Swounding . Phenoliwounding  and a diwounding . The invwounding . Conversin roots . These sThe SPAD consists primarily of modified fatty acids, primary alcohols, and glycerol . These aAcyl activation is typically an initial requirement for downstream fatty acid metabolism and is carried out by long-chain acyl-CoA synthetase (LACS) family enzymes prior to cutin monomer biosynthesis ,96. AlthChain elongation and oxidation represent two major fatty acid modification routes that yield the predominant aliphatic suberin monomers. Elongated chains can be reduced to form primary alcohols or decarboxylated to form alkanes, while a large proportion of oxidized fatty acids in the SPAD are short chains  that have undergone desaturation instead of elongation. However, some elongated fatty acids are also oxidized to yield VLC \u03c9-hydroxy and \u03b1,\u03c9-dicarboxylic acids and are found in the SPAD.AtKCS2/DAISY was first described by Franke et al. [daisy mutants produced root suberin that exhibited a concomitant decrease in C22 and C24 VLCFA-based constituent accumulation, with increased C16, C18, and C20 amounts. AtKCS20 was shown to be functionally redundant with AtKCS2, based on similar observations of C22 and C24 reductions in kcs20 mutants, and a more substantial alteration to root suberin aliphatics in kcs2 kcs20 double mutants [StKCS6 is involved in aliphatic suberin and wax monomer synthesis [StKCS6 silencing led to a drop in C28 and greater chain lengths, and led to accumulation of C26 and shorter chains, indicating that StKCS6 acts on C26 substrates, but might elongate shorter chains as well.Fatty acid elongation is carried out by endoplasmic reticulum membrane-localized fatty acid elongase (FAE) complexes made up of four enzymes . \u03b2-ketoae et al.  as a sal mutants . In potaynthesis . StKCS6 AtKCR1) that encodes an enzyme catalyzing the first reduction step by the fatty acid elongase complex to yield chain lengths greater than C18 for incorporation into different aliphatic polymers including the SPAD. There have been no potato homologs characterized to date.The KCS-generated \u03b2-ketoacyl-CoA is reduced by a \u03b2-ketoacyl-CoA reductase to a hydroxyacyl-CoA. Beaudoin et al.  describetrans-2,3-enoyl-CoA. In Arabidopsis, AtPASTICCINO2 (AtPAS2) encodes the third elongase complex enzyme [AtPAS2 characterization, it has demonstrated involvement in synthesizing VLCFA used as precursors for various lipidic compounds, including seed storage triacylglycerols, cuticular waxes, and sphingolipids.The next step involves a 3-hydroxyacyl-CoA dehydrogenase-mediated dehydration of 3-hydroxyacyl-CoA to yield x enzyme . While scer10 mutants. AtECR was characterized by Zheng et al. [The final enzyme in the fatty acid elongase complex is an enoyl CoA reductase (ECR) that reduces its substrate into the elongated chain with two additional carbons. A gene has been characterized only in Arabidopsis, based on g et al.  and showOxidation reactions yield modified fatty acids that comprise over half of the SPAD constituents in many plant species, including mid-chain epoxide and hydroxylated octadecanoates, and \u03c9-hydroxyalkanoic acids and their further oxidized \u03b1,\u03c9-dioic acid derivatives generated from saturated 16:0 to 24:0 chains as well as 18:1 unsaturated fatty acid. The presence of hydroxylated and dioic VLCFAs suggests that higher chain length products undergo elongation prior to oxidation . In potaAtcyp86a1/horst and Atcyp86b1/ralph mutants demonstrated the role of two monooxygenases with varying substrate specificities and functions, where the former yields shorter-chain \u03c9-hydroxy acids (\u2264C18) and the latter is responsible for the formation of very-long-chain C22-C24 \u03c9-hydroxylated fatty acids in root and seed suberin [AtCYP86A1 ortholog in potato, StCYP86A33, has been characterized in forward and reverse genetics studies, where its silencing led to a reduction in 18:1 and 20:0 \u03c9-hydroxy acids and \u03b1,\u03c9-dioic acids in tuber skin and a concomitant increase in 22:0 and 24:0 monomers [StCYP86A33 expression was found to complement the Arabidopsis cyp86a1/horst-1 mutant by re-establishing production of oxidized monomers [horst-1 mutants with either AtCYP86A1 or StCYP86A33 resulted in an increase in longer-chain distribution than the typically most abundant hydroxylated and dioic 18:1 monomers that is not consistent with RNAi-induced observations [Terminal carbon hydroxylations are carried out by cytochrome P450 enzymes belonging to the 86A, 86B, 94A, and 704B subfamilies , of whic suberin ,16. The monomers . StCYP86monomers . Complemrvations ,14. Thisrvations ,14,74.NtCYP94A5, StCYP94A26, is expressed in roots and wounded tubers.Oxidation of \u03c9-hydroxyhexadecanoic acid, into its corresponding \u03b1,\u03c9-dioic acid, was shown to be carried out by two NADP-dependent oxidoreductases in potato ,77, thouAtFAR1, AtFAR4, and AtFAR5. All are involved in primary alcohol biosynthesis in root suberin and appear to have different saturated chain length substrate specificities. In far1 mutants, C22 alcohols were reduced in quantity, far4 mutants showed decreased C20 fatty alcohols and far5 accumulated lower amounts of C18 alkanols, while heterologous expression in yeast demonstrated a range of chain length specificities from C18-C24 [Fatty acyl-CoA reductases (FARs) catalyze the conversion of fatty acids to primary alkanols for incorporation in the SPAD. Domergue et al.  used los C18-C24 ,104.AtCER1 and AtCER3 encode core components of a redox-dependent multi-enzyme complex that interacts with electron-transferring cytochrome b5 hemoproteins (CYTB5s) as cofactors to perform these alkane forming reactions after activation by long-chain acyl CoA synthase, AtLACS1 [Acyl-activated VLCFAs can also be routed towards the synthesis of waxes that make up a soluble  portion of the SPAD . Decarbo AtLACS1 ,80.The SPAD contains modified fatty acid monomers linked to glycerol, and wax components such as alkyl ferulates that represent the convergence of the main suberin-associated phenolic and aliphatic monomer biosynthetic pathways. Aliphatic monomers such as \u03c9-hydroxy acids and \u03b1,\u03c9-dioic acids are linked together by esterification to glycerol ,49. The sn-2 position of glycerol-3-phosphate represent a land plant-specific lineage of these enzymes [gpat5 loss-of-function mutants demonstrate substantial decreases in C20-C24 VLCFA and their \u03c9-hydroxy and dicarboxylic acid derivatives in suberin found in roots and seed coats, and overexpression of AtGPAT5 led to accumulation of sn-2 monoacylglycerols in the wax of Arabidopsis stems [AtGPAT7 is in the same clade as AtGPAT5, and its overexpression resulted in the accumulation of suberin monomers [Glycerol 3-phosphate acyltransferases (GPATs) catalyze the transfer of acyl-CoAs to glycerol, to yield monoacylglycerols. GPATs exhibit different regiospecificity, where GPATs capable of catalyzing acylation of the  enzymes . Arabidois stems . These fis stems . The woumonomers .Feruloyl-CoA transferases are involved in the conjugation of ferulic acid with modified fatty acid suberin monomers. Feruloyl transferases have been characterized in Arabidopsis and potato, where feruloyl-CoA acts as an acyl donor in the reaction with an \u03c9-hydroxy fatty acid acceptor to yield ferulate esters. The ferulate esters represent a point of convergence between the two major suberin-related biosynthetic pathways and also may act as a point of connection between suberin domains (see below), as they are proposed to promote linkage of the SPPD and SPAD, as well as between the SPPD and cell wall polysaccharides ,107,108.StFHT is a wound-inducible gene encoding a fatty alcohol/fatty \u03c9-hydroxyacid hydroycinnamoyl acyltransferase that catalyzes the conjugation of ferulate to \u03c9-hydroxyacids and primary alcohols in suberin, and primary alcohols in associated wax. StFHT may have a role in the synthesis of \u03c9-feruloyloxy fatty acids that serve as precursors for \u03c9-feruloyloxy fatty acid glycerol esters [In potato periderm, feruloyl transferase l esters ,24. In Al esters ,22.The transfer of suberin monomers, especially aliphatic monomers destined for the SPAD, are reliant in part on half-size ABC transporters and lipid transfer proteins . For exaabcg2, abcg6, and abcg20 loss-of-function mutant Arabidopsis lines to determine the role of these root and seed coat localized ABCG transporters. Aliphatic composition analysis of single mutants highlighted a role for each transporter in suberin production, although they did not have noticeable phenotypic changes. Triple mutants produced suberin with low levels of C20 and C22 saturated fatty acids, C22 alkanol, and C18:1 \u03c9-hydroxy acid and also displayed altered suberin organization and higher water permeability properties in roots and seed coats. The deposition of a suberin polymer in triple mutants, albeit altered, suggests that additional transporters are involved, and highlights the likeliness of substrate specificity along with some redundancy across transporters [Yadav et al.  used sinsporters . Lipid tsporters .reduced culm number 1 (rcn1) mutant plants [rcn1 mutant plants.The ABCG transporter required for root suberization in rice, RCN1/OsABCG5, was identified from loss-of-function t plants . RelativTo date, no definitive mechanism(s) has been described with respect to the assembly of SPAD monomers into a polyester macromolecule. In potato periderm suberin, two key acylglycerol building blocks provide the basis of the SPAD: glycerol-\u03b1,\u03c9-diacid-glycerol as the core block, and glycerol-\u03c9-hydroxy acid-ferulic acid to link the SPAD to the SPPD, see . Given tSlCD1/SlCUS1 expression in RNAi knockdowns and mutants led to a change in esterification of sn-1,3 and sn-2 positions of glycerol, demonstrating that SlCD1/SlCUS1 acts on primary and secondary hydroxyl groups of its cutin monomer substrates [GDSL-lipase/hydrolase family cutin synthases are acyltransferases that mediate the transesterification of major cutin monomers esterified to glycerol. Cutin synthases from tomato fruit (SlCD1/SlCUS1) and Arabidopsis flowers (AtLTL1/AtCUS1) were shown to catalyze a two-step enzymatic polymerization reaction in vitro using a predominant cutin monomer as substrate ,118,119.bstrates . Togethebstrates ,116. Duebstrates . While tbstrates . These mStFHT at this critical time post wounding [Both metabolic  and genewounding . Once thThe temporal deposition of suberin in wound-induced potato tubers may serve as a general model for suberin deposition in plant tissue. However, the coordinated deposition of phenolic and aliphatic monomers raises several questions, including how the process is regulated. There is also a limited amount of information about the poly(phenolic) composition of suberin polymers from species other than potato.The differential timing of SPPD and SPAD monomer synthesis and deposition suggests that the enzymes involved in these biosynthetic pathways are controlled by different modes of regulation. The mechanistic details of this differential regulation are not understood, but some aspects of the regulation of suberin biosynthesis have been described . For exaPhytohormones are involved in complex signaling networks to control physiological events, including the regulation of normal growth and development, and the coordination of abiotic and biotic stress responses. Accordingly, several phytohormones are known to mediate wound-induced signaling and gene expression, e.g., . In the Abscisic acid is a carotenoid-derived signaling molecule involved in many developmental and stress-related processes in plants, including responses to drought and plant-pathogen interactions ,126. AbsSoliday et al.  first poDe novo ABA biosynthesis is triggered by wounding tubers as shown by an increase in ABA accumulation and the induction of ABA metabolism at the transcriptional level ,128,129.StCYP86A33, StCYP86B12, StFAR3, and StKCS6 was delayed post-wounding by FD treatment, whereas exogenous ABA application (with or without FD treatment) enhanced transcript accumulation of these same aliphatic suberin associated genes. Similarly, insoluble aliphatic monomer accumulation was reduced in FD-treated tissues. In contrast, FD treatment had no apparent impact on the transcript accumulation of phenolic metabolism genes, while exogenous ABA and the combined FD + ABA treatments slightly increased the accumulation of some polar metabolites [PAL1 transcription during phenolic suberin biosynthesis, the overall impact on phenolic metabolism was less clear. Together, these findings suggest a role for ABA in the differential induction of phenolic and aliphatic metabolism during wound-induced suberization, at least in potato tubers [Actinidia deliciosa (Chev.) Liang and Ferguson) fruit tissue. Exogenous ABA application also led to increased content of phenols, flavonoids, alkanes, alkenes, alcohols, alkane acids, olefine acids, esters, glycerides, and tocopherols in wounded tissue. Han et al. [Several independent lines of evidence have established that de novo ABA biosynthesis is induced upon wounding and promotes the wound suberization processes via upregulation of suberin biosynthetic genes. For example, Woolfson et al.  used FD abolites . While Kabolites  demonstro tubers . In conto tubers  demonstrn et al.  concludeStFHT; [StCYP86A33 led to the identification of several ABA-linked response elements [StFHT promoter region contains numerous putative hormone- and stress-responsive cis-regulatory motifs, including those associated with ABA, jasmonic acid (JA) and salicylic acid (SA) [StFHT expression in wound-healing tubers [Overall, the involvement of ABA in wound-induced and native periderm suberization is evident, but knowledge of its targeted impact on recently characterized biosynthetic steps is limited. The identification of ABA-responsive promoter regions in suberin aliphatic biosynthetic genes, such as cytochrome P450s  and feruStFHT; ) provideelements . Regulatelements ,119. Thecid (SA) . Howeverg tubers .Agrobacterium-induced tumors [Agrobacterium-induced tumors in Arabidopsis accumulated high amounts of ABA, the ethylene precursor aminocyclopropyl carboxylic acid, osmoprotectants, and formed a suberized periderm-like protective layer. Analysis of gene expression in tumor tissue pointed to a distinct mechanism of drought acclimation in Agrobacterium-induced tumors, which differs from that of other tissues, such as leaves or roots. Several suberin related genes regulated by drought and/or ABA showed transcriptional activation in tumors, such as fatty acid \u03c9-hydroxylase (AtCYP86A1), phenylalanine-ammonia lyase (AtPAL1) and 4-coumarate-CoA ligase (At4CL2) as well as a suberin-associated peroxidase and lipid-transfer protein (AtLTP2) [In Arabidopsis, ABA plays a central role in suberin biosynthesis and deposition in drought-induced stress in d tumors . Specifi(AtLTP2) .GPAT5-driven GUS activity, Barberon et al. [GPAT5 localization via the transcriptional reporter line GPAT5::mCITRINE-SYP122. Using this reporter system, it was shown that ABA rapidly induces suberization and led to ectopic deposition in young root parts, as well as in the cortex [Abscisic acid is also involved in the developmental deposition of Arabidopsis root suberin, and likely plays a role in the suberization response to nutrient availability . Barberon et al.  demonstrn et al.  and GPATe cortex .StFHT contains many putative hormone-responsive motifs [StFHT expression [Other phytohormones, such as jasmonic acid (JA) and ethylene ,122, do e motifs  includinpression .Transcription factors (TFs) are proteins that bind to specific sequences of their target genes to control transcription, i.e., gene expression. Similar to the role of phytohormones, transcription factors are implicated in the regulation of plant growth and development and in various stress responses and can function within highly coordinated signaling pathways.Fifty-eight families of TFs, comprising approximately 320,370 members, have been described in 165 plant species . SeveralVitis vinifera VvMYB5a that controls the phenylpropanoid pathway in grapevine) [In the context of secondary metabolism, TFs have been shown to regulate entire pathways including multiple branches   or subgrapevine) . In the AtMYB41 in leaves of Nicotiana benthamiana resulted in the ectopic accumulation of transcripts for aliphatic suberin biosynthesis genes  and related metabolites (\u03c9-hydroxy fatty acid and dicarboxylic acids). Overexpression of AtMYB41 also led to increased phenylpropanoid and lignin biosynthetic gene transcripts and production of some metabolites like monolignols in leaves. Furthermore, the ectopic deposition of suberin in leaf epidermal and mesophyll cells resulting from overexpression of AtMYB41 resembled that typically observed in roots and native and wound potato tuber periderm [AtMYB92 in N. benthamiana resulted in a significant increase in the accumulation of suberin-related aliphatics [Several transcription factors have been described as regulators of suberin biosynthesis and deposition and are also important components of ABA signaling in response to abiotic stresses in plants. For example, Kosma et al.  confirmeperiderm . Similariphatics .MdMYB93 was identified by Legay et al. [Malus \u00d7 domestica) with high suberin deposition in fruit skins, via comparative transcriptomic analysis with non-russeted varieties. The authors demonstrated a correlation between MdMYB93 expression and putative apple homologs of AtCYP86A1, AtGPAT5, and AtCYP86B [N. benthamiana expression system to transiently overexpress MdMYB93 and measure its impacts on suberin related metabolism and transcriptome-wide gene expression. MdMYB93 expression in agroinfiltrated N. benthamiana leaves was accompanied by increased accumulation of suberin biosynthetic gene transcripts including those involved in cell wall development, lipid, and phenylpropanoid metabolism and ABCG family transporters. Overexpression of MdMYB93 also changed the composition of phenolic metabolites in N. benthamiana leaves [y et al.  in russeAtCYP86B . SubsequAtCYP86B  used a ha leaves , though Solanum lycopersicum) and russeted apple  fruit skins demonstrated the upregulation of suberin-related genes in suberized skin tissues [SlMYB93, apple MdMYB53, grape VvMYB107, potato StMYB93, rice OsMYB93, and Arabidopsis AtMYB107 and AtMYB9 [AtMYB107 and AtMYB9 in suberin biosynthesis and assembly through the analysis of loss-of-function mutants, in which they observed a significant reduction in suberin phenolic and aliphatic monomer components. The authors concluded that AtMYB9 and AtMYB107 coordinate the transcriptional regulation of phenolic and aliphatic monomer biosynthesis and deposition in suberizing tissue.Transcriptomic studies of suberization in wounded cuticle-deficient tomato  domain), very-long-chain-fatty-acid (VLCFA)-CoA, cuticular lipid components, root transport activities, hormone signaling, and cell wall regulation. SUB regulation of suberin biosynthesis and deposition appears to be specific to root endodermis, but does not affect CS formation [Cohen et al.  describeormation . The latormation , but doeormation  and soybormation .QsMYB1 is a MYB family member described from cork oak (Quercus suber) and shown to be involved in the regulation of phenylpropanoid and lignin pathways expressed in organs and tissues that undergo secondary growth [QsMYB1 expression is moderated by an alternative splicing mechanism that results in two different transcripts: QsMYB1.1 and QsMYB1.2. Each transcript was differentially expressed, depending on the nature of the abiotic stress applied to the tissue. For example, accumulation of QsMYB1.1 transcripts were downregulated slowly at high temperatures, while QsMYB1.2 was temporarily upregulated in response to drought stress [QsMYB1 across the cork oak genome, and determined that several genes were targeted by QsMYB1, including those encoding enzymes for monolignol and phenolic suberin component biosynthesis. Additionally, several members of the ABCG gene family and lipid-transfer proteins (LTPs) were regulated by QsMYB1, which signifies the important role of QsMYB1 in the regulation of lipid transport and suberin formation across the cellular membrane [y growth . QsMYB1 t stress . Capote t stress  developemembrane .AchnCYP86A1 [AchnFHT [AchnFAR [AchnCYP86A1 promoter to activate gene expression [N. benthamiana resulted in the accumulation of \u03c9-hydroxy acids, \u03b1,\u03c9-diacids, fatty acids and primary alcohols. The increase in primary alcohols was later shown to correlate with increased AchnFAR expression [AchnFHT promoter, while AchnMYB4 acted as a repressor [In kiwifruit, five TFs, AchnMYC2, AchnMYB4, AchnMYB41, AchnMYB107, and AchnABF2, which regulate expression of nCYP86A1 ,145, Ach[AchnFHT , and Ach[AchnFAR ,156 haveepressor . The autStCYP86A33, StKAR, StFHT, and StABCG11/StWBC11. Although TF binding activities were not investigated, the impact of StNAC103-silencing on these genes was consistent with an increase in their substrates, such as alkylferulate wax components, \u03c9-hydroxy acids, primary alcohols, and alkanes. The observed transcriptional repression activities of StNAC103 likely control suberin synthesis during wound-healing on a fine scale, and probably function to prevent premature suberization in certain root localizations in vivo [The NAC family constitutes one of the largest transcription factor families in land plants and is represented by 110 genes in potato . NAC tra in vivo . In Arab in vivo . A compa in vivo .StTHT [Phytophthora infestans)-resistant and -susceptible potato cultivars to elucidate phenylpropanoid metabolites linked to resistance, and identified the genes required for their biosynthesis, 4-coumarate:CoA ligase (St4CL) and tyramine hydroxycinnamoyl transferase (StTHT). StWRKY1 was shown to physically interact with target DNA, and its function was further validated using a gene silencing approach. StWRKY1 binds to the promoter region of its target phenylpropanoid biosynthetic genes, St4CL and StTHT, to activate transcription. Gene silencing of StWRKY1 caused a significant decrease in hydroxycinnamic acid amide abundance and a severe reduction in resistance to the late blight causing pathogen P. infestans [While a role for WRKY TFs in suberization has not been explicitly demonstrated, their ability to activate transcription of phenylpropanoid genes involved in SPPD monomer synthesis, like StTHT , offer iStTHT ,165,166.StTHT  charactenfestans . These rMYB9 [MYB107 [MYB41 [MYB39 [MYB93 [MYB39 and MYB93 are co-regulated by SHR and MYB36. They concluded that MYB39 could function as a potential downstream hub connecting the SHR regulatory network and ABA pathways to promote suberization [In the context of suberin biosynthesis, linkage, and deposition, a picture is emerging in which several TFs coordinately regulate suberin-specific gene expression under various developmental and stress conditions, in conjunction with specific phytohormones . Recent evidence supports the interaction between the TF-mediated developmental programs and stress hormone-mediated pathways in the regulation of Arabidopsis root endodermis suberization ,167. ForMYB9 , MYB107  [MYB107 ,170, MYB7 [MYB41 , MYB39 [1 [MYB39 ,171, and9 [MYB93 . Wang etrization . SimilarAchnMYB41, AchnMYB107, and AchnMYC2 expression was enhanced in response to ABA, whereas FD suppressed the expression of genes encoding these TFs [N. benthamiana leaves led to the upregulation of aliphatic suberin synthesis genes and an increase in the amounts of primary alcohols, \u03b1,\u03c9-diacids, \u03c9-hydroxyacids, and fatty acids [AchnCYP86A1 and AchnFAR and reduce accumulation of \u03c9-hydroxyacids and primary alcohols in wounded kiwifruit and in a N. benthamiana expression system [In kiwifruit, expression of several TFs has been linked to ABA ,155,156.n system .Arabidopsis thaliana, StMYB74 and StMYB102 in potato, MdMYB93 in apple, QsMYB1 in Quercus suber and AchnABF2, AchnMYB41, AchnMYB107, and AchnMYC2 in kiwifruit . Transport of nutrients to the stele takes place via two major pathways. The first is the apoplastic pathway, in which solutes diffuse freely through the apoplast. The second is the symplastic pathway, where solutes move cell-to-cell through the plasmodesmata . The endRoppolo et al.  identifiStCASP1-like/StCASP8 and StCASP1B2-like/StCASP9, are expressed during tuber periderm maturation  along with a cutin synthase-like GDSL lipase/esterase gene (StGDSL-like) [myb9 myb107 mutant seed coats [StCASP1-like/StCASP8 was expressed in the same temporal pattern as genes required for SPAD assembly and coincided with the upregulation of aliphatic monomer biosynthesis genes, along with the putative GDSL-type gene StGDSL.7 [StCASP1B2-like/StCASP9 and another putative GDSL-type gene, StGDSL.6, followed temporal expression patterns similar to aliphatic metabolism genes after wounding, and thus could be candidates for involvement in SPAD assembly [CASPs are found in many plant species ,182, wheSL-like) . It is tSL-like)  demonstrSL-like) . Such a ed coats . StCASP1StGDSL.7 . This suassembly .New candidate proteins involved in CS formation were identified during characterization of other AtMYB36-regulated genes  in ArabiCASPs are essential for the precise localization of the CS by forming a scaffold domain  within the plasma membrane that predicts the formation of the CS. Indeed, the Casparian strip domain provides a protein platform that allows localization of various enzymes like peroxidases, reactive-oxygen species-producing enzymes, transporters or combinations of these. Despite new understanding of the underlying mechanisms leading to CS initialization, many questions remain about the complex regulatory network underlying CS formation. Similarly, a more general role for CASPs in delineating sites for suberin deposition may exist, considering their wound-inducibility in potato tubers. Further studies on the identification and characterization of the CASPs and their associated proteins will offer more insights into the molecular mechanisms that determine the development, early differentiation, and CS formation in endodermal cells, and suberin deposition in general.Timely and effective suberization is vital to prevent water loss and infection, as well as healing wounds incurred during harvest and post-harvest handling and storage. This review provides insight into the molecular-genetic pathways leading to the biosynthesis and regulation of suberin deposition. There remains a need to elucidate the differential and temporal regulation of phenolic and aliphatic metabolic pathways required for suberin development. Recent investigations into the regulatory oversight of suberization have revealed the involvement of phytohormones including ABA, transcription factors that interact with ABA and biosynthetic genes, and novel candidates that may coordinate suberin domain assembly and linkage, such as CASPs. Collectively, these findings advance our fundamental understanding of what orchestrates the suberization process, and the potential to improve valuable agricultural products through the targeted engineering of crops with improved stress resistance traits, such as enhanced drought and pathogen resistance, or in identifying molecular markers to assist in the selection of crops with improved agronomic traits and post-harvest storability."}
+{"text": "Rhodotorula diobovata is an oleaginous and carotenogenic yeast, useful for diverse biotechnological applications. To understand the molecular basis of its potential applications, the genome was sequenced using the Illumina MiSeq and Ion Torrent platforms, assembled by AbySS, and annotated using the JGI annotation pipeline. The genome size, 21.1 MB, was similar to that of the biotechnological \u201cworkhorse\u201d, R. toruloides. Comparative analyses of the R. diobovata genome sequence with those of other Rhodotorula species, Yarrowia lipolytica, Phaffia rhodozyma, Lipomyces starkeyi, and Sporidiobolus salmonicolor, were conducted, with emphasis on the carotenoid and neutral lipid biosynthesis pathways. Amino acid sequence alignments of key enzymes in the lipid biosynthesis pathway revealed why the activity of malic enzyme and ATP-citrate lyase may be ambiguous in Y. lipolytica and L. starkeyi. Phylogenetic analysis showed a close relationship between R. diobovata and R. graminis WP1. Dot-plot analysis of the coding sequences of the genes crtYB and ME1 corroborated sequence homologies between sequences from R. diobovata and R. graminis. There was, however, nonsequential alignment between crtYB CDS sequences from R. diobovata and those from X. dendrorhous. This research presents the first genome analysis of R. diobovata with a focus on its biotechnological potential as a lipid and carotenoid producer.  Rhodotorula toruloides has been dubbed a \u201cworkhorse\u201d for biotechnological applications . . R. gramR. diobovata were also plotted against CDS of ME from Y. lipolytica (Y. lipolytica compared to other oleaginous yeasts. Dot-plots of CDS sequences of crtYB of R. diobovata versus R. graminis also showed a continuous match between the two sequences (R. diobovata versus X. dendrorhous, however, appear to have a series of sequence deletion events (Cytosolic and mitochondrial ME sequences of polytica c,d, whicequences a. Sequenn events b. The stRhodotorula diobovata, and revealed core differences between the amino acid sequences of R. diobovata proteins involved in the lipid and carotenoid biosynthesis pathways and those from other oleaginous yeasts. The unconventional mode of action of malic enzyme and ATP-citrate lyase in L. starkeyi and Y. lipolytica may be connected to their sequence organization. Dot-plot analysis of the coding sequences of malic enzyme and crtYB confirmed sequence homologies between R. diobovata and R. graminis sequences. There was, however, nonsequential alignment between crtYB CDS sequences from R. diobovata and those from X. dendrorhous. This research presents useful information for genetic engineering and the potential exploration of R. diobovata as a biotechnological \u201cworkhorse\u201d. This study presented the whole-genome sequencing of an oleaginous and carotenogenic basidiomycete,"}
+{"text": "While the short-term compatibility between NaOCl and 1-hydroxyethylidene-1,1-diphosphonic acid (HEDP) has been shown, it remains unclear whether ultrasonic activation of a combined NaOCl & HEDP solution immediately reduces the available chlorine and/or renders the NaOCl ineffective in dissolving organic tissue remnants. This was tested in three experiments: (1) direct activation in test tubes in an ultrasonic bath and then the activation by an ultrasonically oscillating tip (IrriSafe) in (2) an epoxy resin model containing a simulated isthmus filled with gelatin, and (3) extracted teeth with simulated resorption cavities filled with soft tissue. The control solutions were physiological saline and 2.5% NaOCl without HEDP. In (1), available chlorine after 30 s of ultrasonic activation (37 kHz) of test and control solution was assessed, as well as shrimp tissue weight loss in direct exposure. In (2) and (3), the ultrasonic tip was driven at 1/3 of full power using the respective unit, and areas of removed gelatin from the isthmus and tissue weight loss were used as the outcomes, respectively. Experiment (1) revealed no negative impact by HEDP on available chlorine (1), while all three experiments showed a highly significant ( Root canal debridement and disinfection performed in clinics can be performed according to various protocols . The corIrrigation with a pure NaOCl solution during and after root canal treatment leaves the root canal walls with a smear layer, and inorganic debris can accumulate in canal fins and ramifications ,11. TherHitherto, one aspect regarding the application of a combined NaOCl & HEDP irrigant for chemo-mechanical root canal treatment has not been investigated in detail. This is its ultrasonic activation, especially with regard to its effect on proteins and soft tissues . It woulIn this cascade of experiments, a combined NaOCl & HEDP solution prepared from a commercially available CE-marked etidronate salt  was scrutinized for its usefulness in conjunction with ultrasonic activation. The focus was on the targeted soft tissue-dissolving effect of the NaOCl, which is one of its main clinically desirable features ,25, and Firstly, the direct effects of ultrasonic energy on the immediate chemical stability and the soft tissue dissolving effect of the combined NaOCl & HEDP solution were studied in an ultrasonic water bath. Secondly, the ability of this combined solution to clean out gelatin from standardized simulated isthmus areas in epoxy resin models in conjunction with an ultrasonically activated endodontic instrument was tested. Thirdly, the same test was performed in extracted human single-rooted teeth containing simulated resorption cavities filled with soft tissue. The null hypothesis tested was that the addition of the etidronate powder under investigation to a 2.5% NaOCl solution did not alter the desired NaOCl effects in the above three experimental setups, especially in conjunction with ultrasonic activation.Physiological saline solution (0.9% NaCl), no activation vs. ultrasonic activation;2.5% NaOCl, no activation vs. ultrasonic activation;2.5% NaOCl containing 9% HEDP, no activation vs. ultrasonic activation.In this cascade of experiments, there were always two times three groups to control for the effect of NaOCl, HEDP, and ultrasonic activation in the different environments. These six groups used in all three experiments were:n = 12) were used in all the tests involving synthetic materials. However, based on the observed treatment effects and the ethical concerns when using an unnecessarily high number of extracted human teeth, the sample size in Experiment 3 was reduced to n = 10 . Based on this and to be uniform across the experiments, 12 samples per group (= 10 see .The sodium hypochlorite (NaOCl) solutions were prepared from a more concentrated stock solution with a content of above 4% NaOCl . NaOCl solutions were always prepared freshly on the days of the experiment by wt%/wt% dilution in deionized water. The etidronate powder was Dual Rinse HEDP (Medcem). One capsule (0.9 g) of powder was mixed with 10 mL of freshly prepared 2.5% NaOCl per application.The test and control solutions in this and all the following experiments were used at ambient temperature (23 \u00b0C). Their available chlorine was titrated using a 0.1 mol/L sodium thiosulfate solution  in a titration apparatus . Solution temperatures were assessed using a calibrated pocket thermometer with a slim steel probe . In this experiment, 1 mL of test or control solution was kept in a 2 mL safelock microcentrifugation tube . Tubes were suspended in an ultrasonic water bath  containing tap water at 38 \u00b0C. This water bath had an integrated water temperature control and an ultrasonic frequency of 37 kHz. The tubes were kept in vertical position in a rack  and activated or not (depending on group assignment) for 30 s.Experiment 3 was done under environmental conditions (23 \u00b0C and 40% relative humidity). Subsequently, tissue pieces were individually suspended in 1 mL of test or control solutions. The microcentrifugation tube containing the soft tissue was then suspended in the water bath as described above, and activated or not for 30 s. The overall exposure time of the shrimp tissue to test or control solutions (including positioning in the water bath and removing from it) was 1 min. Subsequently, the soft tissue pieces were washed in a 0.1 mol/L sodium thiosulfate solution  and NaCl 0.9%  blotted dry, and weighed again. The weight loss of the tissue samples after exposure to the test/control solutions and treatments was used as the outcome variable.To assess the effect of ultrasonic energy on the tissue dissolution efficacy of the solutions under investigation, pre-cooked cocktail shrimp meat  was used. Shrimp meat was cut to a standardized size, blotted dry, and pre-weighed in a precision balance . Weighing of the tissue pieces here and in Transparent models were prepared using a two-component epoxy resin that cures at room temperature . This model was an alteration of a previously published model to assess ultrasonic irrigation , with thIn the ultrasonically activated solutions, 2.5 mL of irrigant was administered for 30 s, followed by passive ultrasonic activation for 30 s . The ultImages were obtained using dental microscope OPMI PROergo  equipped with a digital camera . The removal of gelatin from an isthmus was calculated using ImageJ . First, the whole isthmus area was calculated and then the threshold for color was adjusted so that it reflected the remaining gelatin. The percent area cleaned of the gelatin was calculated using the \u201cAnalyze Particles\u201d tool. n = 12).After irrigation and imaging, the gelatin was cleaned from the models using hot water and a syringe. Subsequently, the clean models were filled again with hydrogel, randomized, and used for another cycle of irrigation. Each irrigation condition was repeated 11 times , and the canals were instrumented to size F3. Irrigation was performed using 5 mL of a 2.5% NaOCl solution for 1 min between each instrument change. Final rinse was performed with 5 mL of 17% EDTA  for 1 min to remove smear layer. The canals were then dried with sterile paper points (Dentsply Sirona). Longitudinal reference grooves of 0.5 mm depth were first made on the buccal and lingual surfaces of middle third of the root to serve as a guide, indicating the standard locations where simulated resorption cavities were to be made in the root canal. Each specimen was then embedded in Eppendorf tubes  using a putty impression material . Once the putty impression material was set, the roots were removed from the vials to enable assembly and reassembly of the specimens. Each root was then sectioned longitudinally into two halves using a diamond disc  under water cooling . The internal resorption cavity model was adopted from a published paper on root filling . All the cooling . Semicirn = 10) based on irrigation regimen as described above , dried, and then weighed using a precision balance in an airtight container . Both the root halves were then reassembled using a light curing resin barrier . Care was taken to maintain patency by placing a F3 protaper gutta percha point (Dentsply Sirona) between canal the two root sections. The apex of each root was closed with sticky wax to simulate a closed-end system. A plastic tube of 3 mm height was glued at the cementoenamel junction of each root which acted as a reservoir for irrigating solution. Each root was then be placed in their respective Eppendorf vials, and the samples were randomly divided into six groups . All the samples were weighed by a single investigator who was unaware of the test solutions used.All data presented here were normally distributed (Shapiro\u2013Wilk test). Chemical assessments were performed in triplicates and data are presented as means and standard deviations. Based on the error of measurement, the data is presented to one digit. For all other experiments the influence of the two independent variables \u201ctype of irrigant\u201d and \u201cultrasonic activation\u201d were computed using two-way analysis of variance (ANOVA). Subsequently, mean values were compared between all six groups by one-way ANOVA, followed by Tukey\u2019s highly significant difference (HSD) test. The alpha-type error was set to 5%.When solutions (23 \u00b0C) were placed in microcentrifuge tube into an ultrasonic bath (38 \u00b0C) and activated (test) or left non-activated for 30 s each, there was no difference in the available chlorine that was assessed immediately thereafter, indicating that the ultrasonic acitvation of the irrigant per se did not trigger a chemical reaction between the HEDP and the NaOCl  under thp > 0.001, each). Inter-individual comparisons between groups revealed that the ultrasonic treatment with NaOCl in the solution caused the statistically highest weight loss, while the presence of HEDP in the solution had no impact . Needle irrigation with saline was used as a negative control (p > 0.001). Similar results were observed when HEDP . However, mere irrigation with NaOCl and NaOCl & HEDP did dissolve some tissue (p > 0.05).The results in this experiment were comparable to those on soft tissue dissolution in the ultrasonic bath and also the removal of the gelatin from the simulated isthmus areas in riment 2 . Again, e tissue b, while e tissue a. Again,The current study showed in three independent experiments that the combination of a NaOCl solution with HEDP does not interfere with the possibility to activate the NaOCl ultrasonically. Moreover, this study confirmed the synergistic effect between NaOCl and ultrasound.This investigation is limited by the fact that it was a laboratory study with outcomes that may or may not be related to clinical treatment. Nevertheless, an attempt was made to use different modes of ultrasonic activation as well as different organic materials to investigate the dissolving NaOCl effects under ultrasonic activation. The first experiment was on the direct activation of irrigants in an ultrasonic water bath, where cavitation is the main driver. Sonochemical effects are related to the ultrasonic power used, the presence of bubbled gas, temperature, solvent composition, and reaction volume . These eExperiments 2 and 3) used epoxy resin models and extracted human teeth, respectively. This was to test an ultrasonic tip specifically designed for passive ultrasonic activation [Experiments 2 and 3 represented the standard clinical setting in terms of ultrasonic power recommended by the manufacturer.The two further experiments (tivation  in a simtivation , later ativation ,9,37. Ittivation . Cavitattivation . CavitatExperiment 2 [The observed synergistic effect of NaOCl and ultrasound is in conjunction with published data by other authors, who used Rhodamin B as an indicator , or a siriment 2 . The sliriment 2 , while tUnder the conditions of these experiments, the synergistic effect between ultrasonic activation of the irrigant and the NaOCl that was contained therein was confirmed, resulting in an enhanced effect on soft tissue and gelatin. The addition of HEDP from an etidronate salt did not hamper this effect. Consequently, none of the current findings speak against using a combined NaOCl & HEDP solution for ultrasonically activated root canal irrigation."}
+{"text": "Crotonylation is a kind of newly discovered acylation modification. Thousands of crotonylation sites have been identified in histone and non-histone proteins over the past decade. As a modification closely related to acetylation, crotonylation was reported to share many universal enzymes with acetylation. Crotonylated proteins have important roles in the regulation of various biological processes, such as gene expression, process of spermatogenesis, cell cycle, and also in the pathogenesis of different diseases, which range from depression to cancer. In this review, we summarize the research processes of crotonylation and discuss the advances of regulation mechanism of both histone and non-histone proteins crotonylation in difference physiological processes. Also, we focus on the alteration of the crotonylation under certain pathological conditions and its role in the pathogenesis of each disease. Scientists have focused on epigenetic mechanism study since the concept of Epigenetics was firstly proposed by Austrian developmental biologist Conrad Waddington in 1942 in Brucella. The data of LC-MS shown that a total of 5,559 crotonylation sites were identified on 1,525 different proteins, of which 331 sites on 265 proteins were significantly changed in BspF overexpression cells. So the authors speculated that BspF might influence the function of host proteins through its crotonylation to promote the intracellular propagation of BrucellaLysine crotonylation (Kcr) is one of histone lysine acylation modifications The research history of crotonylation is shown in Figure In 2015, Sabari et al. showed that the coactivator p300 had both acetyltransferase and crotonyltransferase activities and found that p300-catalyzed histone crotonylation directly stimulated transcription to regulate gene expression in vitroin vitroThe decrotonylases remove the covalent modification of lysine crotonylation. In 2012, histone deacetylase 3 (HDAC3) was firstly reported to own the activity of histone decrotonylase (HDCR) Certain specific domains are identified to participate in the process of transcriptional regulation induced by crotonylation. YEATS, Bromodomain and Double PHD finger (DPF) are three classes of domains that recognized acylation. In 2016, Andrews et al. found that transcription initiation factor TFIID subunit 14 (Taf14) was a reader of histone crotonylation The chemical formula and the regulators of crotonylation are shown in Figure in vivoIn addition to writers, erasers and readers, there are also other regulatory factors involved in the regulation of crotonylation. As a non-coding RNA, nuclear paraspeckle assembly transcript 1 (NEAT1) was associated with p300/CBP complex and its inhibition affected the location of H3K27 acetylation (H3K27Ac) and H3K27 crotonylation (H3K27Cr) to the transcription start site of many genes, including endocytosis related genes, which promotes the progression of disease The identified sites of histone crotonylation are exhibited in Figure Transcription is a crucial process in the expression of coding genes Rsad2, II6, Ifit1 in a YEATS domain-dependent manner in vitroMeanwhile, some readers of histone Kcr were proved to involve in gene expression and transcription. Li et al. discovered that AF9, a reader of histone Kcr, was involved in the recognition of p300-catalyzed crotonylation and gene expression process in the LPS-induced inflammatory response Figure . They shpeptidyl-prolyl cis-trans isomerase NIMA-interacting 4 (Pin4), coiled-coil domain-containing protein 160 (Ccdc160) and transcription elongation factor A protein-like 1  were greatly reduced in RS of CDYL transgenic mice, compared to that in wild-type mice. In addition, ChIP-qPCR assays detected the reduced levels of total histone Kcr and H2BK12cr on the promoter of related genes, showing that CDYL regulated the expression of sex chromosome-linked escaped genes through mainly influencing histone Kcr on the gene promoters to play an important role in spermatogenesis and thus male fertility Spermatogenesis is a highly conserved physiological process. The complicated process contains five steps: the proliferation of spermatogonia, spermatogonial differentiation into spermatocytes, spermatids produced by spermatocytes through meiosis, maturation of round spermatids, and the release of highly specialized mature spermatozoa Cell cycle contains four phases . Many intracellular and extracellular factors influence this process. During the cell cycle, certain protein has been dynamically regulated by modification, including ubiquitination, methylation, and phosphorylation. Serine phosphorylation, which most appears in the late of G2 stage and surges in H3 in the M stage, has been mostly studied Post-translational modification plays an important role in DNA damage Response (DDR) Zinc finger and SCAN domain-containing protein 4 (Zscan4) gene by decreasing the abundance of heterochromatic H3K9me3 and HP1\u03b1 at subtelomere. The induced expression of Zscan4 in turn slashed damage in telomere, and sustained length of telomere during chemically induced reprogramming Telomere is a small DNA-protein complex at the ends of the linear chromosomes of eukaryotic cell. Cap-like structure covers the ends of chromosomes to maintain the stability of the genome In mouse ES cells, histone decrotonylation was found to promote ES cell differentiation. Histone Kcr was elevated compared to differentiated cells The function of crotonylation on non-histone protein is paid more and more attention except for its roles on histone. Hundreds of crotonylated proteins and lysine residues have been identified using specific antibody enrichment followed by high-resolution mass spectrometry analysis. Bioinformatics analysis revealed that crotonylated proteins were particularly enriched for nuclear proteins involved in several physiological processes  in vitro in vitroCrotonylation has been reported to affect the localization of proteins. HP1\u03b1  belongs to the heterochromatin family and enriches in heterochromatin by binding to methylated histones in vitro; while for intracellular proteins, the way of degradation is ubiquitin proteasome pathway. Many researches have shown that protein acylation is closely related to proteasome-dependent protein degradation There are multiple pathways to break down protein. Lysosomal pathway is an approach for protein degradation entered cell The repair of DNA damage is a complex process that relies on particular pathways to remedy specific types of damage to DNA. Crotonylation also plays a key role in DNA repair. In 2020, Yu et al. had identified replication protein A 70 kDa DNA-binding subunit (RPA1) as one of the downstream substrates of CDYL Crotonylation is an important epigenetic modification form identified as a novel evolutionarily conserved histone PTM. Both histone and non-histone proteins were found to be crotonylated. Crotonylation is involved in various biological pathways that regulate diverse cellular functions ranging from gene expression to DNA damage. It is interesting to determine how their functions are mechanistically regulated by crotonylation in future.Kcr is a newly discovered post-translational modification, hence the associations between crotonylation and diseases are not fully clarified. More studies are needed to investigate the role of crotonylation in different diseases, which may provide new target for clinical therapy. The protein crotonylation associated diseases were summarized in Table Depression is a psychological disease and the pathogenesis of depression is perplexing, but it is currently believed that depression is mainly determined by related genes and environmental factors. Also, PTM was shown to involve in this process, including acetylation, methylation, phosphorylation, and so on. For example, H3K14ac levels in hippocampus of male C57/Bl6J mice showed a significant increase after ten continuous days of social defeat stress in vitro, thereby enhancing histone H3K4 crotonylation, H3K4 and H3K18 acetylation, and reducing H3K27 trimethylation + T cells, and histone crotonylation combined with other LRAs interfered with HIV latency. In the rhesus monkey-AIDS animal model infected with simian immunodeficiency virus (SIV), the expression of ACSS2 was highly induced in the intestine during acute primary SIV infection in vivo. Entering the chronic stage, the expression of ACSS2 decreased Human immunodeficiency virus (HIV) is a retrovirus which causes a multisystemic disease called acquired immunodeficiency syndrome (AIDS). HIV are divided into two types: HIV-1 and HIV-2, of which the former is more pathogenic. People who get infection has a long incubation period and its long latency is a main reason for its incurability. Researchers have found that the epigenetic regulation of histone played a key role in the process of HIV latency. NaCr is a strong latent reversal agent (LRAs) to increase the expression of acyl-CoA synthetase short-chain family member 2 (ASSC2) Various epigenetic mechanisms are involved in different kidney diseases, such as acute kidney injury (AKI), chronic kidney disease (CKD) and AKI-CKD conversion PGC-1\u03b1 and the decrotonylase SIRT3 in both TWEAK-stimulated tubular cells and in AKI kidney tissue PGC-1\u03b1 and SIRT3 and limited the expression of C-C motif ligand 2 (CCL2) in healthy kidneys and renal tubular cell. Consistent with these results, crotonate protected experimental mice from AKI by preventing the decrease of renal PGC-1\u03b1 and SIRT3 and increase of CCL2 in vivo and increasing histone crotonylation might have a protective effect from AKI.Acute kidney injury (AKI) refers to sudden (within 1-7d) and sustained (>24 h) declines in renal function, manifested by azotemia, water electrolyte and acid-base imbalance, and systemic symptoms. Post-translational histone modifications modulate gene expression under the circumstance of AKI. Recently, Olga et al. observed that histone H3k9cr was increased in folic-acid-induced AKI tissue and disclosed that inflammatory factors may participate in regulation of histone crotonylation in kidney tubular cells Immunoglobulin A nephropathy (IgAN) is one of the most common glomerular disease, characterized by IgA deposition, with or without the deposition of other immunoglobulins in the mesangial region. A proteomics analysis of crotonylation between healthy controls and IgAN patients was performed. By analyzing results in a bioinformatics manner, researchers focused on the characteristics of the crotonylayed proteins. Also, the functions of the differential crotonylayed proteins and the characteristics of the crotonylation sites in the amino acid sequence of proteins were analyzed. Integrated analysis revealed that crotonylated proteins were mainly involved in the humoral immune response in patients, especially in antigen processing and presentation Hemodialysis (HD) is one of the renal replacement treatments for patients with acute and chronic renal failure. Liquid chromatography tandem mass spectrometry (LC-MS/MS) coupled with highly sensitive immune-affinity purification was used to comparatively evaluate the crotonylation proteome of normal controls and maintenance hemodialysis patients Figure C. There natriuretic peptides B (Nppb), ultimately leading to the development of HCM Hypertrophic cardiomyopathy (HCM) is the most common genetic cardiovascular disease characterized by unexplained non dilated left ventricular hypertrophy (LVH). Recently, downregulation of short-chain enoyl-CoA hydratase (ECHS1) was showed in human hearts with hypertrophic cardiomyopathy. ECHS1 was reported to mediate histone crotonylation and contributed to cardiac homeostasis +/K+ transporting subunit alpha 1ATPase Na (ATP1A1). In Spitzoid tumor, the genes are lamin A/C (LMNA) and lamin B2 (LMNB2). In anaplastic large cell lymphoma, the genes related protein crotonylation are moesin (MSN), myosin heavy chain 9 (MYH9) and myosin heavy chain 10 (MYH10) Cancer is a major public health problem and owns high leading cause of death worldwideKcr is one of the non-acetyl lysine acylation modifications, which caused more and more concern. This article generalizes the related researches on crotonylation over decades by discussing the research processes of crotonylation and its involved physiological and pathological function. Particularly, we summarized histones and non-histones protein crotonylation in combination with specific modification sites, which shows that protein crotonylation is widely distributed in cells and it has significant roles in countless physiological processes. Lysine histone and non-histone proteins crotonylation is involved in many physiological processes from gene expression to protein stability. Also, the histone and non-histone proteins crotonylation associated diseases were discussed deeply here. It will be interesting to determine how their functions are mechanistically regulated by crotonylation in the near future. in vivo. The mechanism of crotonylation and its dynamic relationship with other acylation needs further exploration.Studies have shown that acetylation and crotonylation coexist in TSS or other regulatory elements of certain genes. This phenomenon suggests that there are intricate correlations between histone crotonylation and other types of histone modifications in the regulation of gene expressionUntil now, drugs that target histone crotonylation are still not available. Countless reasons contribute to its perplexity. Due to complex biochemical reactions of ester drugs metabolism in body, the lack of specific crotonylated enzyme is a main challenge, which limits further investigation of drugs targeting histone crotonylation. Despite the obstacles, studies on non-histone crotonylation made breakthroughs with the developments in high\u2010resolution LC\u2010MS/MS approaches. In 2017, Z Ju et al. designed the new coding schemes to predict protein crotonylation sites through databases"}
+{"text": "HMT) were investigated through physical methods and spectroscopically in dimethyl sulfoxide (DMSO) at ambient temperature. Evidently, HMT turned out as a sensor, selective and sensitive to silver ion (Ag+) only, among other cations, through colorimetric and fluorometric activities (observable by naked eye) and spectrally, both by UV-Vis and fluorescence spectroscopy. The resulting complex pedant (HMT-Ag) is highly responsive to the presence of fluoride ion (F\u2212) in aqueous soluble DMSO, evidenced by changes in absorption spectra as well as fluorescence quenching, upon addition of the respective ions. Consequently, spectral changes induced by the addition of these ions, were consistently concomitant with colour changes, from colourless to light brown (HMT-Ag) to dark brown (HMT-Ag-F) in daylight condition, while bright light blue colour (HMT) to dark blue brownish (HMT-Ag) under UV-light conditions. The experimental results were complimented by theoretical studies, which are well within agreement of one another.The photophysical properties of Hexamethylenetetramine ( Hexamethylenetetramine, Silver and Fluoride ion probe, Colorimetric sensor, fluorometric sensor. Over the years, its uses has been expanded to a wide variety of applications such as in the production of curing agents, nitrilo-triacetic acid, explosives, biocides and foodstuff industry; the practice which had since been discontinued due to its association with carcinogenicity and chronic toxicity . Th. ThHMT aO values , 39, 40. 1.53 eV . Evidentwith Ag+ a and b. is shown b, as sugHMT-Ag). Furthermore, the variation of molecular orbitals in HMT and its complex, are testament to the predicted interaction nature of the two species. In HMT, the concentration of the HOMO lies along the electron donor sources of nitrogen (N), while the LUMO lies more along the three carbon atoms of the chair-like conformation. In the complexed state (HMT-Ag), the concentration of the HOMO lies within the HMT ring, while the LUMO are predominantly localized on the silver atom . The variance in absorption spectra of HMT in different solvents is minimal, with only toluene displaying a slightly different HOMO-LUMO gap (5.88 eV). In addition, the frontier orbitals distribution were also investigated, in different solvents  interaction mode as indicated , more or less of its own size. Moreover, the chemical environment of the cavity mouth of HMT does play a vital role into the interaction between the two species. Guided by supramolecular phenomenon of self-assembly through weaker Van Der Waals force, HMT is only harmonious with Ag+ among all transition metal cations used, by displaying colorimetric activities once Ag+ is introduced. This has indeed confirmed the early studies, which succeeded in crystalizing HMT-Ag out of solutions, among all metals used. In rare studies, it has been reported that some group metals do form complexes bearing HMT as a ligand [In addition, the association of ndicated b. In soma ligand , 44, 45,3.7HMT-Ag pedant and the anions, as confirmed by colour changes above, spectroscopic analysis were conducted through the titrations of the complexed pedant (HMT-Ag) with F\u2212 in DMSO solution. Firstly, the molar titration of up to 10 equiv. of Ag+ to HTM, resulted in hyperchromic shift accordingly, till no noticeable spectral changes were observed anymore. However, the introduction of the molar amount of F\u2212 to HMT-Ag caused even more hyperchromic shift, simultaneously shadowed by subsequent colorimetric activities, signaling chemical interactions on the two entities , is evident that the coordination of Ag+ to HMT plays an intermediate role of inducing pedant functional recognition of fluoride ions. Thus, HMT-Ag complex can be classified as a colorometric and fluorogenic probe for fluoride ions in DMSO.Factually, absorption properties are closely associated to fluorescence behaviours in a particular molecular entity; the emission properties of  studies a. The mot of Ag+ b. The flh colour b inset. 3.9HMT-Ag as a fluoride sensor, by applying quantitative analysis using commercially obtained Colgate and a Mouthwash, all known to contain fluoride ions. The preparation of a toothpaste sample solution was prepared according to a known method, as 20 mg/ml in 1 ml of H2O. The Colgate aqueous solution was titrated against HMT-Ag (1 \u00d7 10\u22125 M in DMSO) in molar quantities as shown  of the University of Namibia, Namibia.This work was supported by the Data included in article/supplementary material/referenced in article.The authors declare no conflict of interest.No additional information is available for this paper."}
+{"text": "In the current review, we survey the current knowledge on genes/enzymes associated with the suberin biosynthetic pathway in plants, reflecting the outcomes of considerable research efforts in the last two decades. We discuss the function of these genes/enzymes with respect to suberin aromatic and aliphatic monomer biosynthesis, suberin monomer transport, and suberin pathway regulation. We also delineate the consequences of the altered expression/accumulation of these genes/enzymes in transgenic plants.Suberin is a natural biopolymer found in a variety of specialized tissues, including seed coat integuments, root endodermis, tree bark, potato tuber skin and the russeted and reticulated skin of fruits. The suberin polymer consists of polyaliphatic and polyphenolic domains. The former is made of very long chain fatty acids, primary alcohols and a glycerol backbone, while the latter consists of  The colonization of land by plants commenced c. 450 million years ago with the evolution of the first streptophyte algae lineage into terrestrial angiosperms . The mig16 and C18 \u03c9-hydroxylated fatty acids, and is immersed in and/or coated by very long chain fatty acid (VLCFA)-derived epicuticular waxes [20 , primary alcohols and a glycerol backbone [p-hydroxycinnamic acid byproducts that originate from the core phenylpropanoid pathway, with ferulic acid being the most abundant one [The cuticle layer is composed predominantly of the cutin polymer, made up of Cbackbone ,10 . Conversely, suberin lamellae can be found in specialized tissues, ranging from unicellular depositions in seed coat integuments a and rooA transmission electron microscopy analysis reveals the suberin polymer to be a polylamellate ultrastructure . Two typThe fine molecular structure of the suberin polymer has yet to be fully elucidated, most likely due to its complex polymeric nature. Correspondingly, most studies of suberin chemistry have relied on suberin extracts from different plant sources following hydrolysis and transesterification processes, which yield only monomers and/or small oligomers, not the complete chemical structure of the suberin polymer. Thus far, several hypothetical structures for the suberin polymer and its association with the cell wall have been raised . In addition to the aforementioned comprehensive reviews of the macromolecular structure of the suberin polymer, several recent reviews have outlined various aspects of the suberin pathway in plants. These include the possible cellular export and trafficking mechanisms of suberin ,21, the In the current review, we strive to provide an updated synopsis of the major efforts made in the last two decades to unravel novel genes/enzymes operating in the suberin pathway in plants. Our main focus is on the substantial advances made in the last couple of years. For the sake of simplicity, we survey the genes/enzymes in the suberin biosynthetic pathway according to their functional roles in: the formation of polyaliphatic monomers, the formation of suberin polyphenolic monomers, the transport of suberin monomers and the regulation of the suberin pathway. All genes/enzymes discussed in this review are presented in 16:0, C18:0 and C18:1 fatty acids in plastids by the fatty acid synthase (FAS) complex , where they are elongated into VLCFAs via the fatty acid elongation (FAE) complex (Suberin biosynthesis relies on the synthesis of C complex . Followi complex .\u03b2-KETOACYL-CoA SYNTHETASE (KCS) enzymes  KCS6 was suggested to be involved in the formation of suberin monomers with chain lengths >C28, as the silencing of this gene in the tuber skin periderm tissue explicitly reduced the levels of suberin-related VLCFAs with lengths above C28 [The first committed step in VLCFA biosynthesis is the condensation of C(2) units to acyl-CoA via the activity of  enzymes . Two ear suberin ,63. The  suberin  and that suberin . Further suberin . In addiatcyp86a1/horst mutant roots synthesized significantly lower levels of \u03c9-hydroxyacids, particularly those with chain lengths shorter than C20 [atcyp86B1/ralph mutants, including knockout lines and RNA interference-silenced lines, showed a strong reduction in the levels of C22 and C24 \u03c9-hydroxyacids and \u03b1,\u03c9-dicarboxylic acids [18:1 \u03c9-hydroxy and \u03b1,\u03c9-diacid levels, leading to a distorted, much thinner suberin lamella ultrastructure [Avicennia officinalis and its putative ortholog in Arabidopsis were shown to control root suberin biosynthesis. Heterologous expression of AoCYP94B1 in the Arabidopsis atcyp94b1 mutant and in wild-type rice (Oryza sativa) resulted in a higher accumulation of suberin content, eventually leading to increased tolerance against salt stress [atcyp94b3 mutant was rescued when it expressed AoCYP94B3 [Suberin-related VLCFAs are further oxygenated by members of the CYTOCHROME P450 OXIDASE protein family . Using rthan C20 . Similaric acids ,31. CYP8tructure . Recentlt stress . SimilaroCYP94B3 . 22, C20 and C18 primary alcohols. Heterologous expression of the three FARs in yeast established their activity as alcohol-forming enzymes with distinct but overlapping chain-length specificities ranging from C18 to C24 [far mutants reduced the levels of suberin primary alcohols by 70% to 80% in the polymeric and nonpolymeric fractions of roots of tissue-culture-grown plants. They also featured a reduction in suberin-associated root waxes of seven-week-old soil-grown plants and a decrease in the seed coat suberin polymer [Another archetypal class of suberin monomers is composed of primary alcohols. The formation of these components is executed by the reduction of carboxyl groups via the activity of FATTY ACYL-CoA REDUCTASE (FAR) enzymes . Using a8 to C24 . A subse polymer .atgpat5 mutant roots had 50% less aliphatic suberin, and their seed coats had seven-fold lower suberin-related dicarboxylic acid and \u03c9-hydroxyacid contents [20 and C22 \u03c9-hydroxyacids and dicarboxylic acids, typical of suberin [16 and C18:1 \u03c9-oxidized acyl-CoA components over ones with longer chains. GPAT5, however, acts on a wide range of long-chain \u03c9-oxidized and/or unsubstituted acyl-CoA components [atesb1 mutants display a two-fold increase in root suberin content [Glycerol is considered the backbone of the suberin polymer. Beisson et al.  showed tcontents . Subsequ suberin . Interesmponents . This bomponents . Another content . This pr content . Therefoquintgelp mutant lines led to an 85% decrease in the total suberin monomer content [Recently, a subset of five auxin-mediated GDSL-type esterase/lipase proteins (GELPs), GELP22, GELP38, GELP49, GELP51 and GELP96, were proposed to play a role in suberin polymerization in Arabidopsis roots . The expp-hydroxycinnamic acids such as cinnamic, coumaric and caffeic acids, with ferulic acid being the predominant one [ASFT is expressed in seed coat and root endodermis where suberization occurs [atasft mutants are almost completely devoid of ferulic acid in their polyphenolic domain, accompanied by substantial effects on the aliphatic suberin monomer levels [atasft mutants and similar to those of the wild type. An additional member of the BAHD family, namely, FATTY ALCOHOL:CAFFEOYL-CoA CAFFEOYL TRANSFERASE (FACT), was shown to catalyze the synthesis of alkyl hydroxycinnamate ester waxes, particularly alkyl caffeate esters  [Previous in silico and in vivo assays positioned many of the suberin monomer\u2019s biosynthetic enzymes in the endoplasmic reticulum (ER). Thus, it is predicted that the synthesis and modifications of aliphatic monomers occur in the ER. The core phenylpropanoid pathway that provides d et al.  projected et al. . In agre sativa) . atdso/atabcg11 mutants resulted in lower suberin content and suberin-related gene transcripts , which failed to develop long roots, apparently due to lower levels of C28 and C30 fatty acids and \u03c9-hydroxyacids [Regardless of which of these transport models will be revealed as the correct mode of suberin monomer transport, the apparent involvement of transporters, such as those belonging to the half-size ATP-BINDING CASSETTE (ABCG) and/or LIPID TRANSFER PROTEIN (LTP) superfamilies, is expected once suberin components reach the PM. Arabidopsis DSO/ABCG11 was the first transporter suggested to play a role in suberin monomer export. This was based on the observation that repressing DSO/ABCG11 expression in nscripts . Despitenscripts . Chemicaeability . Later, is seeds . Moreoveis seeds . A subse1 mutant . Lastly,oxyacids .atltpi4 mutant crown galls accumulated much less suberin, attributed to decreased synthesis of C18 VLCFAs. The ability of this transporter to act on VLCFAs typical to suberin was also demonstrated by expressing the protein in epidermis cells, which resulted in a significant rise in the levels of C24 and C26 VLCFAs [LTPG15, encoding GLYCOSYLPHOSPHATIDYLINOSITOL (GPI)-ANCHORED LIPID TRANSFER PROTEIN, was shown to be expressed in suberin-producing tissues of the root endodermis and seed coat . Howeve6 VLCFAs . Finallyeed coat . Seeds pic acids .Arabidopsis thaliana and Nicotiana benthamiana, seemingly by inducing the expression of suberin gene transcripts [atmyb1097 and atmyb9 mutant seeds displayed significant reductions in suberin content and altered levels of other seed-coat-associated metabolites [Actinidia chinensis Planch cv. Xuxiang) were shown to interact with the kiwifruit CYP86A1 promoter to activate the expression of its corresponding gene. Nicotiana benthamiana leaves expressing these two kiwifruit MYBs exhibited induced expression of suberin aliphatic genes and the accumulation of typical suberin components such as \u03c9-hydroxyacids, dicarboxylic acids and primary alcohols [Nicotiana benthamiana leaves induced the accumulation of suberin and the deposition of a suberin-like lamellar structure [atsub mutant roots accumulated less suberin content compared to wild-type roots [Nicotinana benthamiana leaves, it affected lipid homeostasis and yielded a 50-fold increase in suberin deposition [Quercus suber) MYB1 was shown to target genes involved in both suberin and lignin biosynthesis, transport and assembly, which are tightly associated with secondary growth and cork development [Malus x domestica) regulates suberin deposition during the formation of russeting on top of fruit skin during development [Solanum tuberosum) MYB102 and MYB74 genes were suggested as important regulators of wound suberization processes in potato tubers  internodes undergoing suberization during culm development or due to wounding, Figueiredo et al. [Nicotiana benthamiana leaves by activating the expression of suberin biosynthetic genes [Insights into the regulation of the suberin pathway were obtained only in the last decade. The first transcription factor identified as a positive regulator of suberin was Arabidopsis MYB41 . It was nscripts . Other mnscripts  isolatedabolites . Later, abolites . Interesalcohols . A distitructure . SUBERMApe roots . More repe roots  showed tpe roots . As in tposition . Apart felopment   protein facilities. For instance, Arabidopsis WRKY33 was recently identified as an upstream regulator of CYP94B1 oxidase, associated with suberin biosynthesis . In agre content . Lastly, content . atanac058 mutants displayed delayed root suberization, while the overexpression of ANAC058 caused ectopic deposition of suberin in roots [Unlike all the aforementioned transcriptional factors that positively regulate suberin biosynthesis, potato ANAC103 seems to negatively regulate it . Silenciin roots .The extensive study, over several decades, of the pathways underlying the formation of the cutin polymer, the major constituent of the cuticle, has led to the characterization of many genes and components involved in these processes. However, the first enzymes involved in suberin biosynthesis and deposition in plants were identified only in the last two decades, with many missing parts still remaining in the puzzle. In contrast to the cuticle, which covers all aerial parts of plants, the suberin lamella is hidden within the cells of specialized tissues, making it harder to investigate. Several key aspects of the suberin pathway remain elusive, particularly those involved in the polymerization of the suberin building blocks, the means of transport of suberin monomers to their deposition sites and the general chemical structure of the suberin polymer . Nonethe"}
+{"text": "Introduction. Primary hyperparathyroidism (PHPT) is a condition characterized by disorders of calcium\u2013phosphate metabolism and bone metabolism caused by pathological overproduction of parathyroid hormone (PTH). The diagnosis of overt PHPT is based on the presence of clinical symptoms and laboratory abnormalities typical of this condition: hypercalcemia, hypercalciuria and elevated iPTH levels. Imaging studies are not used for diagnostic purposes; they are performed to localize the parathyroid glands prior to potential surgical treatment. Technetium 99 m sestamibi scintigraphy (Tc99 m-MIBI) is the gold standard in the assessment of pathologically altered parathyroid glands. Other diagnostic options include cervical ultrasound (US), computed tomography (CT), magnetic resonance imaging (MRI) and positron emission tomography (PET). Parathyroid biopsy (P-FNAB) with iPTH washout concentration (iPTH-WC) assessment is still an underestimated method of preoperative parathyroid gland localization. Few studies have reported the utility of US-guided P-FNAB in preoperative assessment of parathyroid lesions. The aim of the study was to present our experience with 143 P-FNAB with iPTH-WC assessment. Material and methods. Laboratory results, US findings, P-FNAB complications and comparison with other imaging techniques were described and analyzed. Results. In 133 (93.0) patients, iPTH washout-to-serum ratio exceeded threshold level 0.5 and were classified as positive results. Median iPTH-WC in this group was 16,856 pg/mL, and the iPTH-WC to serum iPTH ratio was 158. There was no correlation between iPTH-WC and serum PTH, serum calcium, parathyroid gland volume and shape index. In the group of 46 operated patients, 44 demonstrated positive iPTH-WC results, which corresponds to a sensitivity of 95.6%. In Tc99-MIBI, radiotracer retention was found in 17 cases (in 24 MIBI performed), which corresponds to a sensitivity of 52.2%. P-FNAB did not cause any major side effects \u221292.5% of all patients had no or mild adverse events after this procedure. Conclusions. P-FNAB with iPTH-WC is a reliable method in parathyroid adenoma localization during PHPT. Its sensitivity for diagnosis of PHPT is much higher than that of Tc99-MIBI, and in some situations, P-FNAB with iPTH-WC may even replace that method. Furthermore, cost-effectiveness of iPTH-WC is at least similar to that of Tc99-MIBI. Complications of P-FNAB are mild and we can describe this method as a safe procedure. Primary hyperparathyroidism (PHPT) is a condition characterized by disorders of calcium\u2013phosphate metabolism and bone metabolism caused by pathological overproduction of parathyroid hormone (PTH) ,2. The cCervical ultrasonography (US) and Tc99 m-MIBI represent the first steps in parathyroid adenoma (PA) localization, with many centers using both methods in combination. This approach is very sensitive (90%) and highly accurate (97.2%) in PHPT, with greater experience resulting in more reliable results. Larger PAs (>1.8 cm) and higher preoperative ionized calcium levels (>1.49 mmol/L) are more reliably identified by sestamibi scan . HoweverThe diagnostic performance of US and Tc99 m-MIBI seems to be similar . US may The ultrasound-guided parathyroid fine-needle aspiration biopsy (P-FNAB) is a minimally invasive, safe and effective method for the diagnosis of parathyroid lesions. The procedure can be performed to localize a parathyroid tissue and distinguish it from surrounding structures. The use of preoperative P-FNAB can significantly increase the accuracy of parathyroid gland (PG) localization . HoweverFew studies have reported the utility of US-guided P-FNAB in preoperative assessment of parathyroid lesions. The aim of the study was to present our experience with 143 P-FNAB with iPTH-WC assessment, the safety assessment of FNAB of the parathyroid glands with iPTH-WC and an attempt to determine the cut-off value of iPTH-WC. Laboratory results, US findings, P-FNAB complications and comparison with other imaging techniques are described and analyzed.The study was conducted from 2017 to 2021 in the Department of Endocrinology and Internal Medicine in Gdansk. The study group consisted of 179 consecutive patients with suspicion of primary hyperparathyroidism PHPT who underwent P-FNAB with iPTH-WC assessment. Finally, examination results of 143 people with confirmed PHPT were included in the statistical analysis.-documented PTH-related hypercalcemia (laboratory assessment at the same time) with:-serum iPTH >69 pg/mL (laboratory reference ranges 11\u201369 pg/mL)-serum total calcium >10 mg% (laboratory reference ranges 8.9\u201310.0 mg/dL)-additionally, if serum total calcium was between 10.0 and 10.99 mg/dL\u2014at least two independent assessments on different occasions had to be documentedConfirmed PHPT:Age above 18 years old.Patients with suspicion of enlarged parathyroid gland(s) but without laboratory abnormalities typical of PHPT.Secondary or tertiary hyperparathyroidism.Age below 18 years old.Disinfection of the operating field;Biopsy of suspected structure (freehand technique) with a standard needles 25 G (0.5 \u00d7 25 mm or 0.5 \u00d7 40 mm) or 23 G (0.6 \u00d7 25 mm or 0.6 \u00d7 40 mm);Visualization of the tip of the needle in PG according to authors\u2019 own scale, QuOBo ;Aspiration of material\u2014usually small amount in the tip of the syringe;Topping up with 0.9% saline up to approximately 1 mL (if necessary);Transfer of the material (iPTH washout) to a test tube;Immediate transfer to the laboratory and iPTH-WC assessment;Simultaneous compression of biopsied field;USG control directly after P-FNAB and 15\u201330 min later.Before P-FNAB, peripheral blood for laboratory analysis  was obtained. These results were included in the statistical analysis. The next step was to perform an ultrasound with parathyroid localization. If the parathyroid gland was detected, P-FNAB was performed. The consecutive stages of FNAF included:Serum calcium concentration was determined by spectrophotometry using test kits from Abbott Laboratories. The coefficient of variation (CV) for intra-assay precision was <1.1%. iPTH concentration was determined by immunochemical method using the IMMULITE intact PTH kit by SIEMENS. The coefficient of variation (CV) for intra-assay precision was <5.7% (for mean iPTH range 72\u2013662 pg/mL). No CV for higher concentrations of iPTH were mentioned. High-Dose Hook Effect was not observed up to 500,000 pg/mL. Usually iPTH-WC above 250,000\u2013300,000 pg/mL were no further diluted. The highest iPTH-WC was 1,437,200 pg/mL. Positive results for iPTH-WC were considered when iPTH washout-to-serum ratio exceeded 0.5. This cut-off value was determined arbitrarily from the literature data and taking into account the unique methodology of sample dilution.Ultrasound examinations were performed by a sonographer with many years of experience in examining the thyroid and parathyroid gland, using the GE Loqiq 7 SE machine, with a linear 8\u201315 MHz probe. The examination was performed without any preparation of the patient, in the supine position, with the head tilted back. This is the position where the thyroid and parathyroids are best accessible. In order to achieve proper contact with the skin surface, a layer of standard ultrasound gel was applied to the test site.This scale, developed by the authors themselves, determines visibility of the needle tip in a suspected parathyroid gland. The scale ranges from 0  to 3 \u2014see details in Scales were created in February 2021 and have been regularly used since with both ranges from 0 to 3. Details of all scales are in https://doi.org/10.1002/(SICI)1097-0258(20000215)19:3%3C313::AID-SIM370%3E3.0.CO;2-K (27 December 2021)]. p-values of less than 0.05 were considered statistically significant. All analyses were performed using R (version 4.0.5) and R Studio (version 1.4.1103) software.Descriptive statistics were calculated for demographic characteristics and clinical features. Continuous variables were described as medians and 1st\u20133rd quartiles (Q1\u2013Q3). Categorical variables were analyzed using the two-sided Fisher\u2019s exact test and continuous variables using the two-sample Wilcoxon rank-sum (Mann\u2013Whitney) test. The study design allowed for investigation of diagnostic sensitivity of iPTH washout-to-serum ratio for confirmation of parathyroid tissue. An arbitrary cut-off value of 0.5 was chosen. Confidence limits for the sensitivity estimate were calculated based upon 10,000 resamples of the data set. A bootstrap method was used due to low number of false-negative results in the study sample [A total of 143 patients with PHPT who underwent P-FNAB with iPTH-WC assessment were included in the analysis. The median age was 61 years ; 127 (89%) of the patients were women and 16 (11%) were men. The median serum calcium concentration was 10.8 mg/dL (Q1\u2013Q3 10.4\u201311.5 mg/dL), phosphate 2.6 mg/dL (Q1\u2013Q3 2.2\u20132.8 mg/dL) and iPTH 116 pg/mL (Q1\u2013Q3 84\u2013167 pg/mL). US revealed a suspicious lesion of the left lower PG in 43 (30%), the left upper PG in 25 (18%), the right lower PG in 58 (41%) and the right upper in 14 (9.9%). In two cases (1.4%), the location of suspicious PGs was in the upper mediastinum. The suspicious PGs on ultrasound presented the largest dimension of 1.54 cm ; their median volume was 0.52 mL (Q1\u2013Q3 0.3\u20131.17 mL) and median shape index (ratio of longest to shortest dimension) was 0.49 (Q1\u2013Q3 0.4\u20130.6). Seventy-four (51.7%) patients underwent MIBI parathyroid scan. In 44 of these 74 patients (59.5%), the radiotracer retention site coincided with suspicious PG on ultrasound. There was no difference in laboratory and ultrasound characteristics between men and women or between younger and older patients.The median iPTH-WC was 13,373 pg/mL  and was several orders of magnitude higher than concurrent serum iPTH concentration with the median ratio of 108 (Q1\u2013Q3 27\u2013593). There was no correlation between iPTH-WC and serum PTH, serum calcium, PG volume and shape index.In 133 (93.0) patients, iPTH washout-to-serum ratio exceeded threshold level 0.5 and were classified as positive results. With the exception of iPTH-WC, there was no difference in demographic, laboratory and ultrasound characteristics between patients with positive and negative results , hyperplasia in 3 (6.5%) and carcinoma in 1 (2.2%) patient. Of the operated patients, 44 had positive iPTH-WC results which corresponds to a sensitivity of 95.6%. Twenty-four (52.2%) operated patients had MIBI scans performed. The radiotracer retention was found in 17 cases, which corresponds to a sensitivity of 52.2%. The bootstrap-derived 95% confidence intervals for these estimates were 89.1\u2013100% for iPTH-WC and 52.0\u201388.2% for MIBI scans.Post-FNAB PG volume measurements were available for 37 consecutive patients. The volume of PGs increased and the median volume after FNAB was 95% greater than the median volume before FNA. Median volume and Q1\u2013Q3 before and after P-FNAB was 0.44  and 0.86 mL , respectively.Measures of patients\u2019 compliance, quality of P-FNAB performance (QuoBo) and P-FNAB-related adverse effects were available only for a subset of patients. The results are shown in Embryology of PGs is complicated. The superior PGs are derived from the fourth branchial pouch and are closely related to thyroid lobes. They have a short road of embryological migration and their final localization is stable on the posterolateral surface of the middle to superior thyroid lobe. The inferior PGs are derived from the third branchial pouch. They have a long migration road and probability of ectopic localization is more likely. Typically, they are situated in posterolateral surface of the lower part of thyroid lobe and ectopically in mediastinum and thymus . LocalizParathyroids are small glands measuring approximately 2 \u00d7 4 \u00d7 5 mm. They have different shapes, but the oval (83%) and elongated (11%) shapes are the most common. Approximately 84% of population has typically four glands; this is crucial information from a practical point of view. Current ultrasound machines allow us to detect 2\u20133 mm structures, thus a healthy parathyroid is a potentially visible structure. Detecting a 2\u20135 mm parathyroid gland even with high levels of iPTH-WC does not confirm any parathyroid pathology . In our Inferior PGs receive end-arterial blood from the inferior thyroid artery (ITA). Superior PGs receive blood from ITA in approximately 80\u201385% and only in 15\u201320% from the superior thyroid artery. Sometimes visualization of ITA may be helpful in PGs detection. Risk of ITA injury during P-FNAB is low.The recurrent laryngeal nerve runs posteriorly to the inferior PGs, then crosses ITA and eventually passes the superior PGs running in front of it. In our material, no cases of recurrent laryngeal nerve or ITA injury were notedFrom a technical point of view, PGs visualization and PGs biopsy are two opposite poles of the same procedure. Patients should know that P-FNAB is usually slightly longer than thyroid biopsy. Difficulty visualizing the needle in PGs and different volume of dilution buffer are probably the two most common reasons for wide ranges of PTH-WC values in our and other studies. Below are the most important patient/doctor factors and some of the authors\u2019 tips to facilitate to the smooth and effective execution of P-FNAB .In our study, we performed biopsy in 143 patients with PHPT. Obviously, all biopsies were USG guided. Needle gauge were 23 or 25 and dilution buffer usually was 0.9% natrium chloride of approximately 1 mL. The number of needle passes ranged from one to three. This technique resembles other studies where needle gauge ranged from 21 to 27, the number of passes was usually one or two (maximum seven) and dilution buffer volume was from 0.5 to 1 mL of saline.Despite similar P-FNAB technique, proposed cut-off values were different. The first proposed approach is a fixed cut-off value, which is the same for every patient. The other is patient specific, where cut-off value depends on serum iPTH levels. The most common suggested fixed values of iPTH-WC were between 40 and 103 pg/mL ,23. HoweIt must be noted that assessed iPTH-WC is diluted approximately 2\u201310 times with saline. In our material, a few patients had iPTH level above 400 pg/mL; therefore, fixed cut-off iPTH-WC value of 40 pg/mL might not provide answers, whether it was PA or just venous blood diluted by saline. Furthermore, in our material, patients with tertiary hyperparathyroidism  have iPTH even above 4000 pg/mL, thus the cut-off value of 40 pg/mL was only 1% of serum iPTH. That is why we kindly recommend adopting patient\u2019s specific cut-off values. If iPTH-WC reaches similar concentration as serum iPTH, it means that \u201creal\u201d, not diluted, concentration is much higher. In our study (positive cases), iPTH-WC was much higher than iPTH, and the median iPTH-WC to serum iPTH ratio was 158. In our opinion, iPTH-WC equal to or higher than serum iPTH is a reasonable cut-off value as a proof that the visualized structure is an enlarged PG. Moreover, we suggest that lower iPTH-WC to serum iPTH ratio, i.e., between 0.5\u20131, should be interpreted as potential PG see .We developed a standardized security protocol in February 2021; therefore, it was used in part of the study group (40 cases). Nevertheless, no major complications were noted since the beginning of the study. According to the protocol, only three minor and three moderate complications were noted. Similar data were provided by Bancos et al. who described immediate complications in 5% of performed P-FNAB . Castellper se, thus sometimes the distinction as to what is the true cause of parathyromatosis may remain unresolved.Another theoretical potential risk of FNAB biopsy is tumor seeding along the needle tract (parathyromatosis), as parathyroid tissue can adhere to and grow in various settings, which explains the high success rate with autotransplantations. FNAB may lead to disruption of the lesion and seeding along the needle tract, causing separate secondary parathyroid lesions . KendricThe complication during surgery may be caused by the dense fibrotic reaction that may occur following FNAB, leading to increased adhesions to surrounding structures, such as the recurrent laryngeal nerve and resulting in less clear tumor borders and increased operative time.After P-FNAB, we observed that volume of biopsied PA was almost twice the previous size\u2014from 0.44 mL before to 0.86 mL after the procedure. It might be related to small bleeding inside PA. Interestingly, this phenomenon might be a helpful suggestion that biopsied structure was indeed a PA. Such relationship has never been described before.In our personal opinion, P-FNAB with iPTH-WC and Tc99 m-MIBI should not be considered as substitutes in every case. The main advantage of P-FNAB/iPTH-WC is its capability to detect small changes. Moreover, this examination has high specificity and positive predicted values . On the We suggest performing both of these procedures in young patients  with PHPT. Another group where iPTH-WC with Tc99-MIBI may be beneficial are patients with USG findings suggestive of many parathyroid-like lesions. In \u201cclassic\u201d cases of PHPT, P-FNAB with iPTH-WC may replace Tc99-MIBI.From a practical point of view, P-FNAB with iPTH is readily available, and crucial is the USG machine and the possibility of iPTH assessment. Unlike scintigraphy, no special workshop is necessary. In Poland, total costs  of Tc99 m-MIBI and P-FNAB with iPTH-WC are almost the same.Cytological examination of P-FNAB is difficult. Bancos et al. revealed than only 31% of P-FNAB were interpreted as parathyroid cells . AlthougMultiphase CT is becoming a viable first-line imaging option, as it has equal to superior sensitivity for PG localization when compared with scintigraphy and US imaging . ParathyA prospective study on 100 patients with PHPT compared the accuracy of 18 F-fluorocholine PET/CT with that of 99 mTc-MIBI or 99 mTc-tetrofosmin SPECT/CT in the preoperative detection of parathyroid adenoma ,35. The PVS is performed in patients with persistent or recurrent disease after previous parathyroid surgery, when repeated noninvasive imaging studies are negative or discordant . PVS, whP-FNAB with iPTH-WC is a reliable method in detection of PA during PHPT. Sensitivity during diagnosis of PHPT is much higher than Tc99-MIBI and in some situations may even replace that method. What is more, cost-effectiveness of iPTH-WC is at least similar to that of Tc99-MIBI. Complications of P-FNAB are mild and we can describe it as a safe procedure.Main limitations of this method are various cut-off values for true positive results. Moreover, this method is sensitive for P-FNAB technique with saline dilution volume or numbers of passes during biopsy. Regardless of these limitations, iPTH-WC equal to or higher than iPTH seems to be a reasonable cut-off value for positive results. Furthermore, iPTH washout-to-serum ratio from 0.5 to 1.0 should be considered as most likely positive and such cases should be considered individually .We recommend P-FNAB with iPTH-WC as an equivalent imaging method in PHPT."}
+{"text": "Privacy preserving record linkage (PPRL) methods using Bloom filters have shown promise for use in operational linkage settings. However real-world evaluations are required to confirm their suitability in practice.An extract of records from the Western Australian (WA) Hospital Morbidity Data Collection 2011\u20132015 and WA Death Registrations 2011\u20132015 were encoded to Bloom filters, and then linked using privacy-preserving methods. Results were compared to a traditional, un-encoded linkage of the same datasets using the same blocking criteria to enable direct investigation of the comparison step. The encoded linkage was carried out in a blinded setting, where there was no access to un-encoded data or a \u2018truth set\u2019.The PPRL method using Bloom filters provided similar linkage quality to the traditional un-encoded linkage, with 99.3% of \u2018groupings\u2019 identical between privacy preserving and clear-text linkage.The Bloom filter method appears suitable for use in situations where clear-text identifiers cannot be provided for linkage.The online version contains supplementary material available at 10.1186/s12874-022-01510-2. The task of privacy preserving record linkage (PPRL) involves identifying individuals from within and across datasets where these datasets have been encoded to ensure identifiers cannot be seen. Growing concerns about individual data privacy, along with an increasing demand for linked data from researchers has resulted in a burgeoning literature of new algorithmic approaches for providing PPRL . DespiteAn emerging method is one using Bloom filters within a probabilistic matching framework. The Bloom filter data structure is used to hold encoded personal identifiers . This meAn evaluation of this method by Randall et al . showed To address these challenges, we conducted a blinded evaluation where encoded administrative data was received and linked with no available knowledge of the \u2018answers\u2019, as in real-world scenarios.This project represented a collaboration between two linkage units (organisations who each regularly undertake linkage for clients) - the Centre for Data Linkage (CDL) at Curtin University , and WesThe overall approach and data flows are shown in Fig.\u00a0Ethical approval for this project was received from the Department of Health WA HREC (#2017/07). This approval included a waiver of consent.Two datasets were used in the study. The first was an extract of all hospital separations 2011\u20132015 from the Western Australian Hospital Morbidity Data Collection (morbidity). The second was an extract of all Western Australian Death Registrations  2011\u20132015. These datasets form part of the core data linkage system maintained by the WA-DLB, with updates to these collections regularly linked through their master system. There were 5,580,353 records in the morbidity extract and 68,955 records in the mortality extract. A summary description of the provided datasets is shown in Table\u00a0The data was encoded for privacy preserving linkage using field-based Bloom filters; each identifier was encoded into a separate Bloom filter, with a standard probabilistic record linkage method used on these encoded identifiers. This approach has been described previously in the literature ; previouLinXmart Simple Envelope Builder [The data was encoded at source by the WA-DLB, using the  Builder . This soNames, address and suburb were encoded into Bloom filters, while all other fields were encoded with a single hash value. The Bloom filter encoding used bigrams with no padding, a Bloom filter length of 512, with 30 hashes per bigram for names, 20 for address and 25 for suburb. The number of hashes used was lower for suburb and address fields as the average length of these fields was longer. All hashes used HMAC SHA-2. Only the first 20 characters of the input fields were used in creating the encoded data. Some basic pre-processing steps occurred as part of the encoding process - these included removing whitespace, converting all values to lower case, and removing non-alphanumeric characters.LinXmart software [The encoded data was linked using the software , using ssoftware .Prior to linkage, the encoded data was validated. This involved confirming the number of records and fields received, checking the frequency of each field against expected values, and cross-checking to ensure the encoding across the two files was the same. The existence of the same encoded value across datasets for a particular field was taken as evidence of the same encoding being used. During validation, two address values were identified as being more frequently occurring than expected. These addresses were assumed to be placeholders such as \u2018Unknown\u2019 or \u2018No Fixed Address\u2019. These were removed. Two postcode values were almost solely associated with these addresses. These were assumed to be \u2018dummy\u2019 postcodes (e.g. \u20189999\u2019) and also removed.The data files were then linked \u2013 both within and between dataset matches were sought. All available fields  were used in the comparison process. Fields encoded using Bloom filters were compared using the S\u00f8rensen\u2013Dice (Dice) coefficient. A default set of weights (m and u probabilities) were used; these had been developed and validated through project-based linkages previously carried out by the CDL. They are found in Additional file To reduce the number of pair comparisons, only a subset of records were compared. This reduction was achieved by placing the records in each file into blocks so that only record pairs that agree on certain fields were compared. The same blocking strategy was used for the privacy preserving and clear-text linkages; this ensured that all differences in results reflected differences between encoded and non-encoded record comparison, rather than due to records which were simply not compared by one party. The blocking strategy was defined by WA-DLB based on what is used routinely in its core linkage activity. These blocks have been trialed and validated over many years to reduce false positive links, which are problematic in an enduring multi-set system . The bloThe acceptance threshold for matches was set by manual examination of a random sample of record pairs at particular scores; the threshold value of 20 was chosen. While no personal identifiers could be seen during this process, it was still possible to carry out some level of manual review/quality assurance by looking at the pattern of identifiers that matched between pairs of encoded records. It was left for the linker to use their judgement based on linkage experience to identify patterns of identifiers likely to be/not be a match.After linkage, a number of quality assurance checks were carried out to investigate and potentially modify groups of records, such as those containing low-weighted pairs, those containing a large number of records, and those containing multiple death records. Record-pairs that appeared to contain male-female twins  were split, as were several groups with multiple death records. No other changes were made.DLS3 linkage software [The un-encoded data was linked by WA-DLB using their in-house software . This soThe matching strategy used by the WA-DLB was designed to link these particular datasets, taking into account their specific character and idiosyncrasies. Separate matching routines were used for morbidity-to-morbidity comparisons, as compared to morbidity-mortality record comparisons. The matching process is relatively complex, with different sets of comparisons and processing rules for each individual block  the linkage results found with PPRL and with clear-text linkage were exactly the same. These 68,478 mortality records linked to 10,191 hospital morbidity records.There were 68,955 mortality records in this study; the morbidity records that linked to each of these mortality records in both the clear-text and PPRL linkages were compared, with key results shown in Table There were 479 (0.7%) remaining mortality records for which differences were found between the PPRL and clear-text linkage. For 48 of these, the PPRL method linked additional morbidity records not found in clear-text linkage, while for 432 the clear-text linkage found additional morbidity records not found through the PPRL method .All differences were manually examined by the WA-DLB, who had access to the un-encoded personal identifiers.n\u00a0=\u00a042) were identified as being correct links. The reasons for the few incorrect PPRL links included the joining together of records belonging to twins, the joining together of a sibling pair, and the joining together of two unrelated individuals with similar names and dates of birth who resided at the same address (a hostel). An artefact of the incorrect joining of twins was the linking of multiple death records.Of the 48 mortality records where additional morbidity records were found through PPRL but not clear-text linkage, the majority  were identified as being correct links. A large portion  of these additional morbidity records were missed by the PPRL method due to discrepancies in the blocking approach used by the two linkage units; although efforts were made to ensure these were identical, differences were identified after linkage upon review, which meant that the PPRL effort did not bring together all of the records that were expected, and thus certain records did not have the opportunity to link. This discrepancy was due to operator error and differences in implementation between the two systems, rather than an inherent characteristic of the PPRL method. Other causes of the links missed by the PPRL method included differences in recording and parsing of address details, and inconsistently recorded first names. For addresses, these included comparisons involving addresses with and without particular key words like UNIT, LOT and FLAT, and comparisons involving acronyms or other codes identifying the individual had no fixed address. An additional challenge was comparing address that contained a place name to those that did not . For first names, the main challenge was identifying names with alternate spelling or diminutive forms (e.g. ELIZABETH and LIZ).Of the 432 mortality records where additional morbidity records were found through clear-text linkage, the majority of these  were identified as being incorrect links. These links were caused by incorrectly joining twins or cases of multiple death records incorrectly joining together, which were accepted due to WA-DLB\u2019s intentional omission of standard post-linkage checks. There were six mortality records which contained additional morbidity records from clear-text linkage, where some were correct and some were incorrect; these have been counted in both the correct and incorrect tallies above.Of the 432 mortality records where additional morbidity records were found through clear-text linkage, 11% (n\u00a0=\u00a0479), for approximately 20% (n\u00a0=\u00a097) the PPRL linkages were correct and for the remaining 80% (n\u00a0=\u00a0425) the clear-text linkages were correct. Of these last 425, a sizable proportion were missed by the PPRL linkage due to user error (discrepancies in the blocking approach), rather than inherent issues with the PPRL method.The results achieved by the PPRL method were highly comparable to those returned from clear-text linkage. Identical results were found for 99.3% of groups created by either method; of the remaining 0.7%  prior to encoding into Bloom filters. This may be an avenue for further improvements in PPRL linkage quality.While the evaluation here showed PPRL linkage quality to be essentially similar to that found with clear-text linkage, we do not expect this always to be the case. The nature of privacy preserving record linkage means it is more difficult to carry out quality assurance procedures and to identify instances where things have gone wrong. The resulting linkage quality may rely more heavily on the expertise of the linker than in clear-text linkage, where simple procedures such as manual review can easily identify processing errors. As such, the use of privacy preserving linkage techniques will always carry a greater risk of processing errors.High linkage quality is valued to ensure the integrity of any results derived from the analysis of linked data. There is limited understanding of exactly how linkage error can affect research results or of the level of linkage quality needed to ensure the validity of results , althougIn this evaluation, the encoded linkage strategy used the same blocking parameters as the clear-text linkage, which was provided by the clear-text linkers. This ensured all differences in results reflected differences in record-comparison between clear-text and encoded data. However, the relatively strict blocking criteria may have simplified the linkage for both parties, by reducing the available comparison space and therefore reducing the chance of false positives. It also had the disadvantage of not reflected real-world scenarios where blocking parameters are not provided.This evaluation focused on the linkage quality aspect of PPRL methods, but another important consideration are the privacy implications. Bloom filter methods are not impervious to attacks, with applications to both field/key , 13 and It is important that users understand the privacy aspects of the use of Bloom filters. In our model, Bloom filtered encodings are only to be released to a trusted third party, with significant legal and contractual safeguards and strong information governance in place. With these measures in place, Bloom filters are an important additional tool to reduce the risk of accidental of purposeful re-identification of individuals by the designated users who have access to them.These results demonstrate that PPRL methods can be used in real world applications. They can achieve very high linkage quality in real-world settings and should be considered as a solution, particularly where additional privacy protections are needed, or when data cannot be provided to linkage units in any other way. The nature of privacy preserving linkage means that there will always be a greater risk of processing error than with clear-text linkage and that care needs to be taken in the preparation and processing of encoded data.Additional file 1: Table 1. Match strategy used for PPRL. Table 2. Blocking strategy used for both PPRL and clear-text linkage."}
+{"text": "Campylobacter, being of particular importance. A strict cleaning procedure and use of an effective disinfection method for transport equipment are thus important to avoid introduction of Campylobacter to chicken and poultry farms, particularly during flock thinning. This study evaluated the efficacy of the disinfection procedure currently in use at one of the largest slaughter plants in Sweden and compared the effects with those of other disinfection methods. The evaluation was based on treatment ability to reduce the presence and amount of indicator bacteria belonging to the family Enterobacteriaceae and total aerobic bacteria. In 4 trials, sodium hypochlorite, peracetic acid, and drying with hot air, with or without sodium hypochlorite for final disinfection, were compared. The analysis was based on 40 cotton swab samples taken in each treatment, 20 after the soaking stage and 20 after the final disinfection step.Transport crates for poultry can contribute to the spread of pathogens, with those of public health interest, for example, Enterobacteriaceae. Since all crates treated with hot air were dry, transport conditions for the birds also improved, particularly in cold weather. A disadvantage is that this treatment is energy-consuming and would require substantial technical changes to the current cleaning process, increasing operating costs at the slaughter plant. However, considering the contribution of improved crate cleaning to overall hygiene control within the poultry supply chain and the beneficial effect on animal welfare, the costs may be justified.The results showed that use of a chemical disinfectant in combination with drying with hot air (dehumidifier) was the most effective treatment, with an average reduction of 3.4 log for total aerobic bacteria and 3.8 log for  Campylobacter infection for humans  used at the plant are composed of high-density polyethylene, with approximate dimensions 110 mm\u00a0\u00d7\u00a01163 mm\u00a0\u00d7\u00a01163\u00a0\u00d7\u00a0240 mm and fitted as drawers in steel frame . The cra\u25a0Prewashing is done using a set of spray nozzles, which apply cold water from above and below each crate.\u25a0To soften attached debris, the transport crates are then soaked for 90 s in an elongated tank filled with cold water with 0.5% (v/v) sodium hypochlorite  added as a detergent.\u25a0The main washing takes place in 3 consecutive washing modules using high-pressure nozzles spraying cold water on all surfaces of the crate.\u25a0The crates are dried and surplus water is removed by cold air jets applied in a drying module equipped with 5 large side-channel blowers, each 8.5 kW. Using high pressure and specially designed air blades, the crates are hit with air jets at a speed of approximately 235 km/hour.\u25a0Finally, the crates are disinfected with sodium hypochlorite (0.5% v/v) by a set of spray nozzles, which apply the solution from above and below to each passing transport crate for approximately 1 s.The cleaning system (Linco Food Systems) used for transport crates at the slaughter plant consists of the following 5 fully automated steps:A total of 4 different trials (A\u2013D) were carried out in the slaughter plant. Two of the trials (A and B) tested 2 chemical disinfectants commonly used by the food industry. In trial A (reference treatment), sodium hypochlorite  at 0.5% (v/v) was used, representing current practice at the slaughter plant. Trial B included the same automated cleaning step as trial A, but in the last step the crates were disinfected with 0.5% (v/v) peracetic acid .The other 2 trials (C and D) tested alternative treatments based on the principle of drying the crates with hot air. For this purpose, a test rig was designed and built adjacent to the slaughter plant. A dehumidifier  was installed on a steel container . One transportation module with 10 crates, cleaned according to the usual routine, was placed in the container. The crates were dried for 2 h, the maximum time available between cleaning and reloading transport crates on transportation trucks to maintain an even flow in the loading process. In trial C, the channel blowers were disconnected, and the transport crates were dried with hot air without using any chemical disinfectant in the last step of the cleaning process. In trial D, the combined effects of hot air and disinfectant were studied. The drying procedure in trial D was the same as for trial C and the disinfectant used was the same as for trial A, that is, sodium hypochlorite at 0.5% (v/v). Steps of the cleaning process that crates went through during each trial are shown in BPW) . One swab was used to sample the whole inside base by wiping the crate vertically with one side of the swab and horizontally with the other side.During each trial, the inner base of the crates was sampled with a sterile cotton swab measuring 10 cm\u00a0\u00d7\u00a010 cm , using a gloved hand. The swabs were moistened immediately before sampling, with 60 mL buffered peptone water  and after the final disinfection stage (referred to as \u2018postsample\u2019) for trial A and B, or the drying stage for trial C and D. Each cotton swab was then placed in a sterile plastic bag and 60 mL BPW were added. Each trial was carried out during 2 consecutive days. Ten pre- and 10 postsamples were taken during each day, on 3 different occasions on during each sampling day that is 20 pre- and 20 postsamples for each trial giving 80 pre- and 80 postsamples in total.All samples were transported in plastic insulated cooler boxes with frozen gel packs. The temperature was checked upon arrival and analyzed within 24 h. Samples with a minimum temperature of 0\u00b0C (not frozen) and at a maximum temperature of 4\u00b0C were accepted for analyses.PCA) (Oxoid in a Petri dish (9 cm diameter). After agar solidification, the plates were incubated at 30.0\u00b0C for 72 \u00b1 7 h. Plates with 25 to 250 colonies were selected for quantification, since these are considered to give the most accurate microbiological results.The samples were analyzed for total number of live, aerobic bacteria according to NMKL-method 86 . In brieEnterobacteriaceae was performed according to NMKL 144   in a Petri dish and left to solidify, and then an overlay of 5 mL VRBG was added and the plates were incubated at 37 \u00b1 1\u00b0C for 24 \u00b1 2 h. The numbers of suspected bacteria belonging to the Enterobacteriaceae were counted on plates with 15 to 150 colonies. Five colonies preliminarily identified as Enterobacteriaceae were cultured on blood agar and incubated at 37 \u00b1 1\u00b0C for 24 \u00b1 2 h. Presence of bacteria belonging to the Enterobacteriaceae was confirmed by oxidase test and the number of Enterobacteriaceae was expressed as log colony-forming units (CFU) per mL. The detection limit was log 1.0 CFU/mL.Analysis for bacteria belonging to the family NMKL 144 . The pre10-transformed. Standard deviations of bacterial reductions for each treatment were calculated. For the analysis, a linear mixed model was employed, and multiple comparisons were made by Tukey's HSD.The data obtained in the study were compiled and analysed using Excel and Before crate cleaning and disinfection, the total number of aerobic bacteria varied from 5.8 to 8.3 log CFU/mL, with a mean of 7.3, 7.1, 7.3, and 7.0 log CFU/mL in trial A, B, C and D, respectively . One samP < 0.001) and trials D and B (P\u00a0=\u00a00.01), but not between trials A and D .The greatest mean reduction in total aerobic bacteria was 3.4 log and was achieved in trial D . A signiEnterobacteriaceae varied from 2.0 to 6.2 log CFU/mL, with a mean of 4.5, 4.3, 4.8, and 4.1 log CFU/mL in trials A, B, C and D, respectively  in the average reduction seen for Enterobacteriaceae between trial D and all the other trials .Before crate cleaning and disinfection, the total number of ectively . Three s trial D . There wr trials . In factr trials . There wEnterobacteriaceae. The procedure of drying the crates alone without subsequent chemical disinfection  gave an average reduction of 1.5 log for total aerobic bacteria and 1.7 log for Enterobacteriaceae. Since no significant difference in Enterobacteriaceae was found between trial C and trials A and B, it appears that all these treatments were equally effective (or ineffective) in this instance. The significant difference in reduction between sampling days is hard to explain since the sampling procedure, the time of the delivery to the lab as well as the analyses were the similar for both sampling occasions. That might be an indication for either the cleaning system not operating consistently at all times, or that the outcome of cleaning procedures depends on the contamination level of the crates. Transport duration varied from half an hour up to 3.5 h. The longer transport duration the drier feces, which might be harder to remove from crates\u00b4 surface .The greatest reduction in bacteria on chicken transport crates in this study was achieved by applying hot forced air (dehumidifier) combined with a commonly used disinfectant (sodium hypochlorite), which gave an average reduction of 3.4 log for total aerobic bacteria and 3.8 log for There is no established standard for required level of cleanliness for transport crates, but it has been suggested that a 2 log reduction in microbial contamination is desirable and a 4 to 5 log reduction is clearly satisfactory . In our Campylobacter is sensitive to drying , leading us to conclude that they are equally effective, although the microbial reduction is not as high as desired in either case.Choosing the optimal disinfectant for transport crates is another issue. Sodium hypochlorite is commonly used because of its broad antibacterial spectrum, rapid bactericidal action, ease of use, stability in solution, relative nontoxicity to humans, low cost, and acceptable cleaning action . AnotherCampylobacter being introduced into chicken flocks during thinning. Furthermore, all transport crates subjected to hot air treatment  were fully dry, thus providing better transport conditions for the birds in cold weather conditions. However, using hot air on a large scale in practical conditions is energy-consuming and implementation would require substantial technical changes in the current cleaning process, inevitably increasing operating costs for the slaughter plant. Nevertheless, considering the contribution of improved crate cleaning to overall hygiene control within the poultry supply chain in general, especially with regard to Campylobacter, and the beneficial effect on animal welfare, the costs may be justified.Our results suggest that a system involving use of a chemical disinfectant in combination with drying with hot air has good potential for reducing the microbial load on transport crates for chickens and can thus mitigate the risk of"}
+{"text": "The RCPsych curriculum for core training in Psychiatry (2013) requires each Deanery to run regional MRCPsych teaching programme.The East Midlands School of Psychiatry run a local MRCPsych course aimed at all core psychiatry trainees in the deanery. Before the pandemic, the course took place between two venues \u2013 Nottingham and Leicester. During the pandemic, the course was delivered via Microsoft teams. We aimed to collect the feedback from trainees regarding the course to help shape the MRCPsych Course programme according to their training needs.We devised an online Microsoft forms questionnaire which included:Level of trainingNumber of exams passedRelevance of MRCPsych content to clinical practice and membership examUsefulness of mock exams, simulation scenarios and workshops towards clinical and exam practiceOverall experience of the courseWhich additional sessions they would like to be includedThe effect of COVID-19 on their ability to attend in MRCPsych programmeThese forms were sent to all the trainees in the region via email.Out of 44 trainees, 9 responded. 66.6% of the trainees who responded were CT1 and 33.3% CT2. 45% had passed Paper A and 55% had not passed any exams. 78% of them agreed and 11% strongly agreed that course was relevant to the clinical practice. 55.6% agreed that course was relevant to membership course. 44.4% agreed and 11% strongly agreed that mock exams were useful. 66.7% agreed and 11% strongly agreed that simulation case scenarios and workshops were useful for exam and clinical practice. 22.2% strongly agreed and 33.3% agreed that sessions were engaging and motivating. Overall experience of MRCPsych exam was rated as excellent (11%), good (55%), satisfactory (22%) and poor (11%).Suggestions to add additional sessions included antiracism in psychiatry, more mock exams, practical management of cases, to organise more interactive sessions on Microsoft teams, in-depth coverage of exam topics, to organise full day teaching sessions instead of half day.33.3% of trainees commented that COVID-19 had impacted on their ability to attend the exam as initially face to face sessions were cancelled till end of May 2020 and when started there were technical issues with the online platformConsider feedback received in modifying aspects of the MRCPsych courseTo share the results with trainers and course tutorsArrange relevant mock exam sessionsInclude the topics suggested by trainees and improve the experience of online learning by making it more interactiveLimitations: small sample size."}
+{"text": "Background and Objectives: The endodontic space is a complex area on both micro and macro levels; therefore, traditional irrigation techniques may not guarantee a complete cleaning of such a complicated tridimensional system. The presented ex vivo study aimed to evaluate root canal cleanliness, obtained through an equal volume of traditionally applied sodium hypochlorite (NaOCl), compared to ultrasonically activated NaOCl and ultrasonically activated NaOCl that had undergone intracanal heating NaOCl. Materials and Methods: A total of 60 freshly extracted human mandibular premolars underwent root sample length standardization (18 mm), root canal preparation and, based on the irrigation method employed, were randomly and equally assigned to three study groups, composed of root samples treated with ultrasonically activated NaOCl, ultrasonically activated NaOCl that had undergone intracanal heating and traditionally applied NaOCl. The root specimens were subsequently fixated with 4% buffered formalin solution and decalcified in Morse liquid. A total often 6-micron-thick serial cross-sections were obtained, dyed using hematoxylin and eosin and examined through an optical microscope at 40\u00d7, 100\u00d7, and 200\u00d7. Results: Ultrasonically activated NaOCl that had undergone intracanal heating showed a significantly smaller amount of debris compared to ultrasonically activated and traditionally applied NaOCl groups . Conclusions: Root canal cleanliness saw significant enhancements by ultrasonically activated NaOCl that had undergone intracanal heating. When teeth are diagnosed with inflamed vital pulps or infected with necrotic pulp tissues, the major aim of chemo-mechanical preparation is to target dissolving pulp tissue and dysrupt microbial biofilm. In particular, disinfection procedure should not only be confined to the major root canal space, but also reach the attached lateral canal system ,2,3,4. AConsequently, in view of subduing such shortcomings, endodontic research has focused, in recent years, on developing strategies to activate irrigation, to which end several techniques, including subsonic, sonic, ultrasonic, laser and manual dynamic ones, have been tested with no definitive results ,8,9. SucThe use of extra-orally heated canal irrigants, particularly sodium hypochlorite (NaOCl), is a well-known strategy for boosting pulp tissue solving effect ,11. HoweGiven these considerations, the current ex vivo study aimed to evaluate, at a microscopic level, in conservatively shaped root canals, root canal cleanliness obtained through an equal volume of traditionally applied NaOCl, compared to ultrasonically activated NaOCl and ultrasonically activated NaOCl that had undergone intracanal heating. The null hypothesis was that there was no significant difference among the three irrigation techniques applied.The present study was approved by the Institutional Review Board and the local Ethics Committee. Written informed consent forms were gathered from all of the participants.Sixty human mandibular premolars, freshly extracted as part of the orthodontic therapy plan, underwent periodontal tissues detachment, using a curette, immediately after tooth extraction, and were collected in separate vials, containing 5 mL of 10% formalin solution. The validity of the experimental design was expressed beforehand.The collected teeth were slitted at the level of the cemento-enamel junction to create root samples of a standardized length (18 mm). A K-file 10 mm size  was placed in each root canal until it was seen via the apex, and the working length was determined by deducting 0.5 mm from this measurement. Nickel-titanium rotary instruments, specifically 10/0.05 and 20/0.05 file instruments , were exclusively employed on the entire working length to deliberately establish a conservative shaping of the root canals. Throughout the entire canal shaping, irrigation was performed with a total of 5 mL of 3% NaOCl, via a 30G needle in a disposable syringe, renewed every minute, for each root canal. Subsequently, root canals were rinsed with sterile saline, further irrigated with 3 mL of 17% EDTA for 1 min to eliminate the smear layer and lastly rinsed with 3 mL of sterile saline.n = 20), composed of root samples treated with ultrasonically activated NaOCl (group A), ultrasonically activated NaOCl that had undergone intracanal heating (group B) and traditionally applied NaOCl (group C). The roots were tinted with nail varnish to avert irrigant extrusion. An equal volume of irrigant, 3% NaOCl , at 2 mm from the working length, in the three groups.The 60 specimens were randomly and equally assigned to three groups , NaOCl activation was performed through an ultrasonic file, connected to a cordless ultrasonic generator . The activation regimen comprehended eight cycles (20 s each), with 1 mL of irrigant freshened during each cycle. The ultrasonic tip was situated at 2 mm from the working length.In ultrasonically activated NaOCl that had undergone intracanal heating (group B), ultrasonic activation was conducted as previously described and preceded by intracanal heating. The internal heating was performed for 8 s using a System-B heat source XF-tip (30/0.04) at 180 \u00b0C  and was positioned at 3 mm from the working length.The traditionally applied NaOCl group (group C) specimens were irrigated with 8 mL of NaOCl through a disposable syringe and needle at a 2 mL/min pace.Thereafter, specimens were dried out and fixated, using 4% buffered formalin solution for 48 h and rinsed beneath running water for one hour. Then, samples were soaked in Morse liquid to reach decalcification for four weeks, with the solution being replenished every two days. Six micron-thick serial cross-sections were obtained from the roots, based on a previously applied protocol ,16. Ten Subsequently, obtained sections were examined under an optical microscope , at 40\u00d7, 100\u00d7, and 200\u00d7, using the dedicated Otpika Vision Lite software. Two independent, blinded, calibrated operators graded the quantity of pulp tissue debris in each section, based on the following criteria: grade 1\u2014detectable debris on 75\u2013100% of the area; grade 2\u2014debris on 50\u201374% of the entire area; grade 3\u2014debris on 25\u201349% of the entire area; grade 4\u2014debris <24% throughout the area. In case of disagreement between investigators, a third one evaluated the samples; final grading was accomplished by discussion among investigators.Frequency distribution of persisting pulp tissue and microbial biofilm debris was presented as lambda scores. Non-parametric tests were used for multiple comparisons among groups . Additionally, Mann\u2013Whitney test was employed for comparisons between pairs of groups.Ultrasonically activated NaOCl that had undergone intracanal heating (group B) manifested no or <25% debris for all of the sections (lambda 0.025), whereas the ultrasonically activated NaOCl group (group A) showed debris >25% in multiple sections and >50% in the remaining ones (lambda 0.0001). The traditionally applied NaOCl group (group C) revealed debris >50% in all of the sections.p value < 0.05) compared to ultrasonically activated irrigation alone and to syringe and needle irrigation (Ultrasonically activated irrigation preceded by intracanal heating resulted in significantly cleaner canals (rigation .The main purpose of endodontic treatment is the removal, as complete as possible, of damaged pulp tissues and microbial biofilm from the complex endodontic system ,15. ConsOrdinarily, bacteria can survive either as independent free-floating cells, in planktonic state, or as members of colonized surface-attached microbial communities, enclosed in a self-produced extracellular matrix that connects cells, overall identified as biofilm . BiofilmIt is well established that contemporary mechanical preparation strategies are not able to adequately reach root canal walls, thus leaving residual tissue and microbial residues inside the root canal system ,22,23,24Irrigation efficacy can be improved by employing activated irrigants; for instance, an easy way to activate NaOCl is its pre-heating to a temperature of 50 \u00b0C. Pre-heated NaOCl solution has been proved to have higher antimicrobial effect and tissue dissolving capabilities ,13. NeveThe endodontic space is a complex area, on both micro and macro levels; therefore, traditional irrigation techniques may not guarantee a complete cleaning of such a complicated tridimensional system. The irrigation technique employing ultrasonically activated NaOCl that has undergone intracanal heating may be considered easy to carry out in routine clinical procedures, as well as being economical, since it does not require specific equipment. Moreover, ultrasonically activated NaOCl that has undergone intracanal heating irrigation, through the reduction in NaOCl viscosity, and a consequent higher penetration into dentinal tubules, has been proved to enhance antimicrobial effect, as well as tissue dissolving capabilities ,12,13, aHowever, although, to the authors\u2019 knowledge, the present study is the first to assess from an hystological point of view these complex lateral macro- and micro-anatomies, following ultrasonically activated NaOCl that has undergone intracanal heating irrigation, especially combined to conservative shaping, and to compare root canal walls\u2019 cleanliness obtained via three different irrigation techniques, the small sample size means that if they are to strengthen such ex vivo observations, forthcoming investigations should compare the results obtained from the presented technique for a complex lateral canal system with those from other irrigant activation strategies, combined to conservative shaping, both in preclinical and in clinical settings."}
+{"text": "D324K overexpression significantly prevented RIPK1 cleavage and weakened the aggressiveness of AQP1-enriched TNBC cells. Overall, our findings clarify the underlying mechanism of cytoplasmic AQP1-driven TNBC progression and metastasis, in which RIPK1 exerts an essential role as a negative mediator and exhibits the potential as a therapeutic target for TNBC.The triple-negative breast carcinoma (TNBC) is the most aggressive subtype of breast cancer. In TNBC, Aquaporin 1 (AQP1), a water-transporting transmembrane protein, is aberrantly enriched in cytoplasm and causes tumor cell death evasion. However, the carcinogenetic bioactivities of cytoplasmic AQP1 cannot be attributed to the canonical \u201cosmotic engine model\u201d. In the present study, the receptor-interacting protein kinase 1 (RIPK1), a cell death regulator, was identified to negatively mediate AQP1-driven TNBC progression and metastasis. AQP1 overabundance and RIPK1 depletion occurred in TNBC, which were correlated with aggressive oncological features and poor prognosis. AQP1 bound with RIPK1, resulting in the inhibition of RIPK1/RIPK3/MLKL-mediated necroptosis and RIPK1/caspase-8/caspase-3-mediated apoptosis. Genetic inhibition of RIPK1 significantly exacerbated the pro-tumor effect of AQP1, while ectopic expression of RIPK1 notably blunted AQP1 signaling. Mechanistically, AQP1 binds to the D324 site of RIPK1, and facilitates RIPK1 cleavage and inactivation by excessively activating the caspase-8/RIPK1 negative feedback loop. RIPK1 The triple-negative phenotype is universally acknowledged to be responsible for the higher rates of relapse, metastasis, and mortality compared to other breast cancer subtypes. However, systemic treatment against TNBC remains limited with the conventional chemotherapy as the mainstay, because patients with triple-negative disease do not benefit from endocrine therapy or anti-HER2-targeted therapy, which have been widely applied in the setting of ER/PR-positive or HER2-positive breast cancers with improved prognosis3. Even worse is that some TNBC subsets fail to respond to chemotherapy4, necessitating an in-depth understanding of TNBC-specific signaling pathways and a de novo exploration of biomarker-sensitive therapeutic strategies.The triple-negative breast carcinoma (TNBC) is the most aggressive subtype of breast cancer, which is characterized by lack expression of estrogen receptor (ER) and progesterone receptor (PR), and non-amplification of human epidermal growth factor receptor 2 (HER2)7. AQP1 may behave as an oncogenic biomarker for numerous types of cancer that is able to sustain tumor pathogenesis through facilitating cell proliferation, migration, and angiogenesis11. Possible mechanisms may include the resistance to cell death that predisposes AQP1-expressing cells toward growth and metastasis14. Unfortunately, the exact effect of AQP1 on TNBC cell death evasion and the specific signaling pathway are still far from understood.Previous studies have identified Aquaporin 1 (AQP1), a water-transporting transmembrane protein, to be upregulated in TNBC and associated with the tumor development and progression21. Thus, the RIPK1 kinase has emerged as a promising therapeutic target for a wide spectrum of neurological, cardiovascular, renal, and hepatic disorders, and infectious and inflammatory diseases23. In addition, compelling evidence has documented that RIPK1 may be a bona fide biomarker and a novel target for cancer diagnosis and therapy by manipulating cellular demise27. However, the role of RIPK1 in breast cancer oncogenesis and development remains one of the major unsolved issues, which may lead to a better understanding of AQP1-related TNBC cell death escape.The receptor-interacting protein kinase 1 (RIPK1) is a central regulatory molecule in cell fate decision with dual bioactive properties as a coalescence of caspase-8-mediated apoptosis and a guardian of RIPK3-mediated necroptosisIn the current study, we report the aberrant expression of AQP1 and RIPK1 in TNBC that are associated with different prognoses. Further, we validate the interaction of AQP1 and RIPK1, and the suppressive effect of RIPK1 on AQP1-driven TNBC progression and metastasis. Finally, we identify the underlying mechanism of TNBC cell death resistance that AQP1 binds to the D324 site of RIPK1, and facilitates RIPK1 cleavage by promoting the caspase-8/RIPK1 negative feedback loop. Our results provide new insights into the essential role of RIPK1 as a negative mediator in AQP1-driven TNBC progression and metastasis, which might represent a potential therapeutic target for TNBC.28 was analyzed using the Oncomine database. Compared with the normal breast parenchyma, over ninefold upregulation of AQP1 in IDC was revealed with statistical significance . We next explored the subtype-specific abundance of AQP1 by deciphering the the Cancer Genome Atlas (TCGA), Genotype-Tissue Expression (GTEx) datasets (GSE1456/GSE6532/GSE7390). The result identified significantly higher expression level of AQP1 in TNBC than that in other subtypes .To determine the expression profile of AQP1 in invasive ductal breast carcinoma (IDC), the Finak\u2019s cohortp\u2009<\u20090.01). Intriguingly, AQP1 was observed with dominant cytoplasmic expression in TNBC cells instead of its common location on the cell membrane in normal breast parenchyma  and low-expression (n\u2009=\u200931) groups, according to the immunostaining score. As shown in Table p\u2009>\u20090.05); while the lymph node metastasis and radiotherapy occurred more frequently in the high-expression group (p\u2009<\u20090.01), suggesting that AQP1 abundance is strongly associated with TNBC progression. Meanwhile, to find out the association between AQP1 expression and the prognosis of TNBC, the invasive-disease-free survival rate was calculated in our cohort. The result demonstrated that TNBC patients with high tumor expression had less favorable survival compared with patients, whose tumors expressed a low level of AQP1 . Taken together, the upregulation of AQP1 plays an important role in promoting TNBC tumorigenesis and progression, and represents poor prognosis.To further confirm the subtype-specific expression pattern in big data analyses, tumor samples, and para-carcinoma normal tissues were harvested from 62 patients, who were pathologically diagnosed as unilateral TNBC at two tertiary medical centers. Consistent with our expectation, AQP1 abundance in TNBC was remarkably higher than normal control in both immunohistochemistry staining and Western blot analysis  but significant increase in RIPK1 expression intensity in IDC was observed in contrast to the normal breast tissue . However, we found RIPK1 was strikingly downregulated in TNBC compared with other breast carcinoma subtypes based on the analysis of the TCGA and GTEx datasets . Furthermore, a notable TNBC-specific reduction of RIPK1 abundance in our cohort was identified by immunohistochemistry staining and Western blot analysis . Meanwhile, the relative expression levels of caspase-8, RIPK3, and phospho-MLKL, known as critical execution molecules of programmed cell death, decreased drastically compared with normal breast tissues , indicating that the compromised RIPK1-mediated programmed cell death is associated with TNBC tumorigenesis.To investigate the RIPK1 expression in IDC, the Curtis\u2019s cohortp\u2009>\u20090.05); while negative axillary lymph node or earlier clinical tumor staging was more likely to be distributed in the high-expression group (p\u2009<\u20090.05), following the treatment decision of breast conservation and non-radiotherapy (p\u2009<\u20090.05). The invasive-disease-free survival analysis demonstrated enhanced TNBC-specific survival rate in patients with higher tumor expression of RIPK1 . Collectively, these findings imply that RIPK1 exerts an inhibitive role in TNBC development and a positive role in patient survival.Next, we examined the frequency of different RIPK1 expression levels in the current TNBC cohort. There was no statistically significant change pertaining to age, BMI, tumor size, and histological grade . To clarify this correlation, the co-immunoprecipitation and mass spectrum assays were performed in the clinical specimens. The results supported the existence of interaction between AQP1 and RIPK1 in TNBC , suggesting a tight link between enhanced AQP1 expression and TNBC cell growth. Meanwhile, AQP1 overexpression promoted TNBC cell migration ability, as demonstrated with notable elevation of wound healing percentage . The colony formation assays showed more migrated and invaded cells in AQP1-overexpressing groups than the cells in vector control group , indicating AQP1 raises the aggressiveness of TNBC cells, and may positively engage in TNBC progression and metastasis.To investigate the role of AQP1 in TNBC carcinogenesis and metastasis in vitro, the MDA-MB-231 and 4T1 cells stably overexpressing AQP1 were generated and verified by Western blot analysis Fig. . Both ofp\u2009<\u20090.01 for gain-of-function cell lines, p\u2009<\u20090.05 for loss-of-function cell lines). On the other hand, RIPK1-overexpressing cells exhibited significantly lower wound healing percentage in the scratch assay  and weaker migratory capacity in transwell insert-based cell migration assay with or without an extracellular matrix barrier , though they also possessed additional AQP1 expression. By contrast, AQP1-driven TNBC cell migration and invasion were dramatically exacerbated in the absence of RIPK1, displayed by accelerated wound healing  and improved cell penetration ability . To recapitulate, RIPK1 acts as a brake that arrests AQP1 signaling in TNBC cell proliferation, migration, and invasion in vitro.As increased AQP1 was accompanied by downregulation of RIPK1, it is hypothesized that RIPK1 dictates the oncogenic properties of AQP1 in TNBC. Thus the gain-of-function and loss-of-function approaches to overexpress and silence RIPK1 were employed to prove the assumption. As shown in Supplementary Fig. p\u2009<\u20090.01) at 14 days after transplantation than the vector control, and the trend lasted until the end of observation on the 32nd day. On the contrary, RIPK1 overexpression remarkably reduced the size of tumors to an extent even lower than the vector control from day 14 to day 32, though they were also AQP1 enriched . In addition, the final measurement of the tumors dissected from those mice identified a noted distinction among the three groups , suggesting the carcinogenetic activity of AQP1 in TNBC can be kept in check by RIPK1, which is consistent with the in vitro findings.To further validate the inhibitive effect of RIPK1 on AQP1-driven TNBC development, an orthotopic breast carcinoma mouse model was established by injecting transgenic 4T1 cells into the mammary fat pad of BALB/c mice Fig. . The volp\u2009<\u20090.01). However, the lung metastases declined strikingly in the double-transgenic group compared to that in AQP1-expressing group , demonstrating that the promotional effect of AQP1 on TNBC progression and metastasis in vivo was abolished by the upregulation of RIPK1. Moreover, 15-week consecutive observations revealed lower overall survival rate of mice injected with AQP1 transgenic cells than that of control mice . By contrast, the mice bearing double-gene-overexpressing cells exhibited considerably longer survival time than the mice in AQP1 group , indicating an improved prognosis of TNBC with high expression level of RIPK1. Overall, these findings proved that RIPK1 renders TNBC less susceptible to AQP1-triggered progression and lung metastasis in vivo.Next, the effect of RIPK1 on AQP1-induced spontaneous lung metastasis of TNBC were examined. 4T1 cells with AQP1 overexpression showed significant enhancement of the migratory activity in vivo compared with the vector control, represented by increased lung metastatic lesions , caspase-8-mediated cleavage (D324K), ubiquitination (K377R), and binding activity with RIPK3 (K530A and I539A). The mutants were immunoprecipated with AQP1-3\u00d7FLAG in HEK-293T cells. The results showed RIPK1 with D324K point mutant reduced the affinity of RIPK1 with AQP1 Fig. .Fig. 6AQ33, the amount of cleaved RIPK1 (cl-RIPK1) was tested in MDA-MB-231 cells stably overexpressing AQP1 and RIPK1D324K. In contrast to the vector control, AQP1 transgenic cells presented significantly higher cl-RIPK1/RIPK1 ratio . However, the activity of AQP1 in TNBC cell death evasion was blocked by the mutation of RIPK1 on D324 site, displayed by decreased cl-RIPK1/RIPK1 ratio, lowered absorbance at 450\u2009nm in CCK-8 assay, reduced wound healing percentage in scratch assay, and weakened cell penetration capacity in transwell insert-based cell migration assay . Moreover, compared with double-transgenic cells, RIPK1D324K overexpression alone presented less cl-RIPK1/RIPK1 ratio, but identical capacity of cell survival, migration, and invasion , suggesting that AQP1-driven TNBC progression and metastasis can be completely inhibited by loss of D324 site of RIPK1. It is also reflected by the fact that the viability and migration and invasion ability of AQP1-enriched cells were further mitigated in RIPK1D324K overexpression group, compared with that in wild-type RIPK1-transgenic group . Altogether these findings highlight the molecular mechanism of AQP1 in TNBC cell death resistance by overactivation of caspase-8-mediated RIPK1 cleavage  in our data, which may be attributed to the short follow-up interval or low patient numbers. Moreover, the abundance of RIPK1 is negatively correlated with that of AQP1 in our clinical cohorts, which inspired us to further explore the molecular relationship between them. The co-immunoprecipitation and mass spectra results confirmed that AQP1 binds to RIPK1 in TNBC. In addition, AQP1 and RIPK1 colocalize in the cytoplasm of TNBC tissue samples and cell lines, which paves the way to their mutual regulation. The in vitro results demonstrated that RIPK1 negatively regulates AQP1 signaling and TNBC cell proliferation, migration, and invasion. Besides, RIPK1 exhibited an inhibitive effect on the carcinogenic activity of AQP1 in vivo. The function of RIPK1 alone was not involved in the in vitro and in vivo study because the mechanism of AQP1-driven TNBC progression and metastasis is main aim of the current study, and RIPK1 was identified as a negative mediator. However, the regulatory role of AQP1 in TNBC-specific RIPK1 depletion remains unclear.RIPK1 represents an essential signaling node of cell death regulation in a variety of malignancies18. On the other hand, RIPK1 can bind to caspase-8 and FAS-associated death domain protein (FADD) to assemble the ripoptosome, resulting in the homodimerization and activation of caspase-8 to stimulate apoptosis21. Meanwhile, RIPK1 shares a key aspartate-specific residue (D324), which is the binding site of caspase-8 for RIPK1 cleavage, and plays an important role in termination of abnormal RIPK1 activation via the negative feedback mechanism38. The present study revealed both RIPK1/RIPK3/MLKL and RIPK1/caspase-8/caspase-3 pathways were inhibited in TNBC tumor samples and AQP1-expressing cell lines, which is in agreement with our presumption that both RIPK1-dependent necroptosis and apoptosis are attenuated by AQP1. Moreover, RIPK1D324K mutant failed to be immunoprecipitated by AQP1, demonstrating D324 is the binding site of AQP1. The caspase-8-mediated cleavage and inactivation of RIPK1 were exacerbated in AQP1-expressiong cells, whereas RIPK1D324K overexpression significantly prevented RIPK1 cleavage and weakened the aggressiveness of AQP1-enriched TNBC cells. Thereby it is reasonable that the combination of AQP1 with RIPK1 at D324 sequence is an obligatory step for TNBC to facilitate the recruitment of caspase-8, aggravate the cleavage of RIPK1, sequester RIPK1 in a quiescent form, and finally unleash tumor progression. It is argued that the deterioration of RIPK1 activity can promote caspase-8-FADD-induced apoptosis39, indicating that the cleavage and inactivation of RIPK1 may be unnecessary for AQP1-driven TNBC cell survival and migration. The claim, together with RIPK1-independent apoptosis, may just represent an idealistic paradigm when necroptosis is completely suppressed in cell cultures. Thus, RIPK1-dependent necroptosis and apoptosis have been identified as the main forms of cell death in the pathogenesis of multiple diseases40; however, in TNBC, the RIPK1-dependent cell death signals are arrested by AQP1 through excessively activating the caspase-8/RIPK1 negative feedback mechanism.Canonically, RIPK1 can recruit RIPK3, which further phosphorylates the mixed lineage kinase domain\u2013like pseudokinase (MLKL) to compose the necrosome, which is the core of the necroptosis machineryConsequently, the present work identifies a previously unrecognized pathway that RIPK1 binds to AQP1 and negatively mediates AQP1-driven TNBC progression and metastasis, and reciprocally, AQP1 adheres to and facilitates the cleavage of RIPK1 and suppresses the cell death signaling in TNBC. This initial foray will provide us a better understanding of the role of AQP1 in TNBC progression and create hope for potential therapeutic interventions  on the aggressive disease. However, further studies remain required to determine other components or adaptors of AQP1\u2013RIPK1 complex, the mechanism of caspase-8 recruitment by AQP1 after binding to RIPK1 and the intrinsic influence of the novel pathway on the microenvironment of TNBC.Mouse monoclonal antibodies against AQP1 (ab9566) and RIPK1 (ab72139) were purchased from Abcam . Mouse monoclonal antibodies against RIPK1 (610458), caspase-8 (9746), and RIPK3 (sc-374639) were bought from BD Biosciences , Cell Signaling Technology , and Santa Cruz Biotechnology , respectively. The rabbit monoclonal antibody against p-MLKL (S345) (ab196436), p-RIPK3 (S227) (ab209384) and p-RIPK3 (S232) (ab195117), and rabbit polyclonal antibody against RIPK1 (ab106393) and MLKL (ab194699) were purchased from Abcam. The rabbit polyclonal antibody against cleaved caspase-3 (9661) and caspase-3 (9662) were bought from Cell Signaling Technology. The rabbit monoclonal anti-\u03b2-actin antibody (AC026) and anti-\u03b1-tubulin antibody (AC013) were from Abclonal . The cell transfection regent (F231-02) was purchased from TransGen Biotech . The point mutation plasmids of RIPK1 were constructed using the HiFi HotStart DNA Polymerase .www.oncomine.org). TNBC datasets were downloaded from TCGA, GTEx, and the gene expression omnibus data repository (GEO)43. Additional information was extracted from the corresponding literatures and supplementary data. The expression levels of AQP1 and RIPK1 were normalized, log2 transformed, and compared using RMA and MAS version 5.0.The log2-transformed gene expression profiles of AQP1 and RIPK1 in invasive breast carcinoma were retrieved from the Finak\u2019s and Curtis\u2019s cohorts in the Oncomine database  or anti-RIPK1 (diluted 1:400) antibody at 4\u2009\u00b0C overnight. The sections were then incubated with biotinylated goat anti-rabbit immunoglobulin, and the reaction was visualized with the DAB complex followed by incubation with HRP\u2013streptavidin. The results were assessed by a single pathologist in a double-blind manner. The positivity was defined as the score \u22653 according to the previous scoring system, using staining intensity multiplied by staining area30. The percentage of positive cells per high-power field (HPF) was counted, and the average values of 5-HPF percentage counts were involved into the final calculation.The paraffin-embedded samples were cut into longitudinal sections of 5\u2009\u03bcm, which were stained by hematoxylin\u2013eosin and observed under a light microscope. The immunohistochemistry assessment for AQP1 and RIPK1 was conducted using standard techniques by streptavidin\u2013peroxidase method. The antigen retrieval was performed in sodium citrate  using an autoclave (120\u2009\u00b0C for 2\u2009minutes). After serial blocking with 3% HFor LC\u2013MS/MS analysis, we separated peptides using a Thermo-Dionex Ultimate 3000 HPLC system. The analytical column was a homemade fused silica capillary column  packed with C-18 resin . Each LC\u2013MS/MS run were searched against the AQP1 and RIPK1, using Proteome Discoverer  searching algorithm. Peptides assigned to a given protein group were considered as unique and more than two unique peptide matches was quantified.2 incubator at 37\u2009\u00b0C.The MDA-MB-231 cells  were obtained from the Cell Resource Center of Institute of Basic Medicine, Chinese Academy of Medical Sciences . The HEK-293T cells  and 4T1 cells  were purchased from the Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences . They were cultured in the Dulbecco\u2019s modified Eagles medium  supplemented with 10% fetal bovine serum , 100\u2009U/ml penicillinm, and 100\u2009\u03bcg/ml streptomycin  in a 5% COD324K-HA with a G418 selection marker were obtained, following the manufacturer\u2019s instructions . Lentiviruses were generated by co-transfection into HEK-293T cells with vectors or plasmids at 2\u2009\u00b5g/ml, pMD2.G envelope  and psPAX2  packaging vectors. The supernatants containing viral particles were collected to infect the TNBC cells at a multiplicity of infection of 25. Stable clones were screened out via puromycin or G418 selection at 1\u2009\u00b5g/ml.Stable overexpressing cell lines were established using a lentiviral system. The pCDH-CMV lentiviral vector expressing human AQP1 (NM_198098) ORF in conjunction with a tag of 3\u00d7FLAG and a puromycin selection marker was established, while the pCDH-RIPK1 (NM_003804)-HA and pCDH-RIPK127. Mouse RIPK1-siRNA duplexes (SR418761) and Scrambled Negative Control siRNA duplex (SR30004) were purchased from OriGene .To temporally knockdown the RIPK1 expression, TNBC cells at ~80% confluence were transfected with 200\u2009pmol control siRNA or siRNA targeting RIPK1, according to the manufacturer\u2019s instruction . The siRNA against human RIPK1 and Scrambled control siRNA were established, using previously described methodology27. In brief, the PCR-amplified products were digested with Dpn1 restriction enzyme  and transformed into TransT1 cells . The plasmid DNAs were extracted from different cell colonies and verified by sequencing. The plasmids were then transformed into HEK-293T cells together with the AQP1-overexpressing plasmid with a tag of 3\u00d7FLAG. The interaction of AQP1 and RIPK1 were further detected by co-immunoprecipitaion and subsequent Western blot analysis.Point mutation plasmids of RIPK1 were established, as previously describedtreatment group\u2009\u2212\u2009ODblank)/(ODstandard control\u2009\u2212\u2009ODblank)\u2009\u00d7\u2009100%.The TNBC cell proliferation was assessed by measuring the absorbance at 450\u2009nm wavelength using a CCK-8  under the direction of the manufacturer\u2019s instruction after incubation at 37\u2009\u00b0C for 1\u2009hour. The absorbance was detected every 24\u2009hours for 3 days. The cell viability was defined as  to calculate the wound healing percentage.The wound healing assay was applied to analyze the TNBC cell migration. Briefly, 5\u2009\u00d7\u2009105 TNBC cells in 100\u2009\u03bcl serum-free media were plated on the upper chambers, and 500\u2009\u03bcl of DMEM supplemented with 20% FBS was filled in the lower chambers. Then, the cells were allowed to migrate or invade for 20\u2009h, fixed with 4% formaldehyde, stained with 0.5% crystal violet, and rinsed by PBS. The chambers per condition were imaged in three independent tests with five random microscopic fields per chamber. The number of migrated or invaded cells were counted and analyzed by Image J software.In vitro migration and invasion assays were performed using a 24-well transwell insert . The upper chambers were precoated with Matrigel matrix  for the invasion assays. A total of 1\u2009\u00d7\u200910The tumors and cells were homogenized in the RIPA lysis buffer, containing protease inhibitor cocktail . Total protein was separated by sodium dodecyl sulfate\u2013polyacrylamide gel electrophoresis. Proteins were blotted to polyvinylidene difluoride membranes . Blots were incubated with primary antibodies (diluted 1:1000 except for \u03b2-actin antibody [diluted 1:3000] and \u03b1-tubulin antibody [diluted 1:5000]) at 4\u2009\u00b0C overnight, respectively. HRP-labeled secondary antibodies  were then added, and the blots were developed with the ECL plus kit (Amersham Biosciences). The images from the same experiments were scanned, and the gray values were processed and analyzed in parallel by Image J software.The tumor tissues or cells were prepared in the RIPA lysis buffer . The lysates were precleaned with protein G agarose beads  at 4\u2009\u00b0C for 4\u2009h. Control IgG, anti-RIPK1 antibody (diluted 1:200), anti-AQP1 antibody (diluted 1:400), or anti-FLAG antibody (diluted 1:200) was coupled to protein G agarose beads in lysis buffer for 4\u2009hours at 4\u2009\u00b0C. Then, the lysates were incubated with beads coupled with different antibodies or control IgG overnight at 4\u2009\u00b0C. The complexes were precipitated and collected for Western blot analysis.The colocalization of AQP1 and RIPK1 was examined in clinical specimens and TNBC cells. MDA-MB-231 and 4T1 cells stably overexpressing both AQP1 and RIPK1 were cultured in glass-bottom dishes . Cells were washed with PBS, fixed with 4% formaldehyde, and permeabilized by Triton X-100. The cells and tissue samples were incubated with anti-AQP1 (diluted 1:400) and anti-RIPK1 (diluted 1:200) antibodies at 4\u2009\u00b0C overnight, and then with goat anti-mouse antibody  and donkey anti-rabbit antibody  at room temperature for 1\u2009h. DAPI was used to stain the cell nuclei. The images of staining were captured by a Zeiss LSM800 confocal microscope.44. Briefly, the transgenic 4T1 cells (1\u2009\u00d7\u2009105/mouse) were orthotopically injected into the mammary fat pad of 7-week-old female BALB/c mice  using a 1-ml tuberculin syringe. The tumor length and width were measured with vernier calipers every 7 days in the first 2 weeks after implantation, and every 3 days in the remaining time until the end of observation. The tumor volume was defined using the formula V\u2009=\u2009(L\u2009\u00d7\u2009W\u2009\u00d7\u2009W)/2, where V represents tumor volume, L refers to tumor length, and W equals to tumor width.All animal care and studies were performed with the approval of the Ethics Committee of our institution and hospital. The orthotopic breast carcinoma mouse model was constructed, as previously describednd day after surgery. The tumors were obtained to precisely measure the diameters. The lungs were excised, fixed in formalin, paraffin embedded, and sectioned for hematoxylin\u2013eosin staining. The metastatic colonies of each lung sample section were photographed under a microscope at \u00d74 magnification. The metastatic lesion area from five random microscopic fields were measured using Image J software. In addition, the overall survival rate was estimated by Kaplan\u2013Meier curve and subsequent log-rank analysis in consecutive observations of 33 mice (11 per group) for 105 days.For spontaneous lung metastasis detection, six mice per group were sacrificed at 32t test, one-way analysis of variance with Bonferroni post hoc analysis and Pearson\u2019s correlation test, using GraphPad Prism version 8.01 and SPSS version 20.0. Survival curves were generated using the Kaplan\u2013Meier product-limit method and compared with the log-rank test. Significance was assumed with a p\u2009<\u20090.05.All results are presented as mean\u2009\u00b1\u2009SD (standard deviation). Statistical analysis was performed using two-tailed paired and unpaired Student\u2019s Further information on research design is available in the Supplementary InformationReporting Summary"}
+{"text": "Researchers and engineers can use the characteristic curves to evaluate the quality of the repurposed batteries. Furthermore, the profile datasets can be applied in the model-based engineering of repurposed batteries, e.g., fitting the variables of an empirical model or validating the results of a theoretical model.Owing to the popularization of electric vehicles worldwide and the development of renewable energy supply, Li-ion batteries are widely used from small-scale personal mobile products to large-scale energy storage systems. Recently, the number of retired power batteries has largely increased, causing environmental protection threats and waste of resources. Since most of the retired power batteries still possess about 80% of their initial capacity, their second use becomes a possible route to solve the emergent problem. Safety and performance are important when using these second-use repurposed batteries. Underwriters Laboratories (UL), a global safety certification company, published the standard for evaluating the safety and performance of repurposed batteries, i.e., UL 1974. In this work, the test procedures are designed according to UL 1974, and the charge and discharge profile datasets of the LiFePO Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.14495604 To overcome the temporary power shortage, many electrical energy storage technologies have been developed, such as pumped hydroelectric storage3, battery7, capacitor and supercapacitor10, compressed air energy storage13, flow battery16, fuel cell19, solar fuel23, superconducting magnetic energy storage27, flywheel28, and thermal energy storage29. Up to now, the pumped hydroelectric storage remains the main way for utility-scale electricity storage. This well-established technology has been commercially deployed since the 1890s3.The electrical energy storage system (EESS) is the capture of electrical energy produced at one time for use at a later time. The storage process involves converting electrical energy from forms that are difficult to store to forms that are more conveniently or economically storable, such as chemical, gravitational potential, elevated temperature, latent heat, and kinetic forms. The history of EESSs can be traced back to the early days of power generation, at the turn of the 20th century, where power stations were often shut down overnight, with lead-acid batteries supplying the residual loads on the direct current networks31. After several weeks, when the coal-fired Loy Yang power plant in Victoria failed, leading to a power shortage, the backup battery kicked in and delivered as much as 100\u2009MW into the national electricity grid in just 140\u2009ms33, responding even more quickly than the coal-fired backups that were supposed to provide emergency power. That shock absorber-type and emitter-type capacities help us to stop a blackout that would otherwise occur. The batteries are capable of providing inertia services and rapid frequency responses  to the grid36. Large-scale batteries begin to show their roles in supply electric networks since then.The development of renewable energy supply (mainly wind and solar photovoltaic) and electric vehicle (EV) industries advance the application of Li-ion batteries from small-scale 3\u2009C  products to large-scale battery energy storage systems (BESSs) and high-power mobile energy sources. The Li-ion battery exhibits the advantage of electrochemical energy storage, such as high power density, high energy density, very short response time, and suitable for various size scales (from 3\u2009C to utility usages). For example, the installation of the world\u2019s largest Li-ion battery  has been completed in South Australia in 201737, the Li-ion batteries are largely used, causing fundamental research, industrial development, as well as standard and policymaking in the field of Li-ion power batteries. Recently, the elimination of power batteries has largely increased, causing environmental protection threats and waste of resources. About 100\u2013120 GWh of EV batteries will be retired by 203037. Therefore, recycling and reutilization of such retired batteries have been promoted39. Some retired power batteries remain possessing about 80% of their initial capacity43. So they can be repurposed and utilized once again, for example, to serve the batteries in the stationary energy storage system47. Governments in various countries have acknowledged this emergent issue and prepared to launch their policies to deal with the recovery and reuse of repurposed batteries, such as coding principles, traceability management system, manufacturing factory guidelines, dismantling process guidelines, residual energy measurement, federal and state tax credits, rebates, and other financial support50.Accompanied by the vigorous promotion of commercialization and the popularization of electric vehicles worldwide52. In this work, the charge and discharge profiles of lithium iron phosphate repurposed batteries are measured based on UL 1974. The lithium iron phosphate battery (LiFePO4 battery) or lithium ferrophosphate battery (LFP battery), is a type of Li-ion battery using LiFePO4 as the cathode material and a graphitic carbon electrode with a metallic backing as the anode55. Although LFP batteries have a slightly lower energy density compared to other Li-ion cell chemistries due to their lower operating voltage, their special features, such as low cost, low toxicity, low self-discharge, high cycle life, high power, and high thermal stability, make them finds many roles in vehicle usage58, utility-scale stationary application61, and backup power64. The test procedures are designed according to UL 1974 and used to evaluate the safety and performance of the repurposed LFP batteries. The charge and discharge profile datasets provide researchers and engineers the characteristic curves to estimate the quality of repurposed batteries. Moreover, the profile datasets can be used in the model-based engineering of repurposed battery cells, e.g., fitting the variables of an empirical model or validating the results of a theoretical model.Safety and performance are important in using the repurposed batteries. Underwriters Laboratories (UL), a global safety certification company established in 1894, published the standard for evaluating the safety and performance of repurposed batteries in 2018, i.e., UL 197452 covers the sorting and grading processes of battery packs, modules, and cells as well as electrochemical capacitors that were originally configured and used for other purposes, such as EV propulsion67, vehicle auxiliary power70, and light electric rail applications73. Furthermore, the focused purposes intend for a repurposed application, such as for use in energy storage systems76 and other applications for battery packs, modules, cells, and electrochemical capacitors. This standard also covers application-specific requirements for repurposed battery systems and battery systems utilizing repurposed modules, cells, and other components. The UL 1974 standardIncoming open circuit voltage (OCV) measurements (Sec. 19.2 of UL 1974)Incoming high voltage isolation check (Sec. 19.3 of UL 1974)Capacity check (Sec. 19.4 of UL 1974)Internal resistance check (Sec. 19.5 of UL 1974)Check of BMS controls and protection components (Sec. 19.6 of UL 1974)Discharge/charge cycle test (Sec. 19.7 of UL 1974)Self-discharge (Sec. 19.8 of UL 1974)The battery module can be decomposed into cells and used components according to UL 1974. The used components of the battery systems, such as the battery enclosure, battery management system (BMS), thermal management systems, and other auxiliary systems, should not be considered for repurposing if they have already been used longer than the calendar expiration date specified by the original manufacturer. The cells preparing for repurposing will undergo the performance test for sorting. UL 1974 suggests that the following test procedures shall be conducted by the repurposed manufacturer as part of the routine analysis of the incoming battery assembly:The charge and discharge profile measurement according to Sec. 19 of UL 1974 is divided into two primary procedures. The first procedure with detailed steps containing Secs. 19.2 and 19.4 of UL 1974 are listed in Table\u00a0I per unit of nominal ampere hour capacity \u22121, 0.1\u2009h\u22121, and 0.2\u2009h\u22121, respectively. The charge current is gradually increased to avoid abnormal voltage raising. (The details of standard charge and discharge processes are stated in the following subsection).In the incoming open circuit voltage (OCV) measurements (P1S1 in Table\u00a0I over the full charge time tc (the full discharge time td), i.e., The capacity check of the battery cell according to the instructions of Sec. 19.4 of UL 1974 is designed as follows  is shown in P2S20\u2013P2S23 in Table\u00a0Vcut\u2009=\u20092.5 V and Vthres\u2009=\u20093.5 V), which is narrower than the safe working voltage range of new LFP battery cells (2\u20133.65\u2009V). The voltage range can be adjusted according to the manufacturer\u2019s design. In addition, the designed test procedures based on UL 1974 can be used for other types of Li-ion repurposed batteries.In this work, the voltage ranging from 2.5 to 3.5\u2009V is adopted for safe working of the repurposed LFP battery cells  The order of charge and discharge steps could be exchanged. (2) The values of the discharge cutoff voltage Direct current internal resistance (DCIR) of batteries indicates the resistance of current flowing through the battery. The value of DCIR is not fixed and varies depending on multiple factors, such as battery materials, type and concentration of electrolyte, temperature, as well as depth of discharge. The variation of DCIR has a great influence on battery discharge performance, especially for high power batteries. In general, the better the battery, the lower the internal resistance. Therefore, most battery manufacturers identify DCIR as a primary indicator for evaluating battery quality.79, IEC 61960-3 standard80, and ISO 12405-4 standard81. In UL 1974, the two-tier DC load method is adopted, offering an alternative method by applying two sequential discharge loads of different currents and time durations. The battery first discharges at a lower constant current I1 for t1 seconds, dropping to a voltage V1, and then discharges at a higher constant current I2 for t2 seconds, dropping to a voltage V2  Voltage and current during the discharge should be recorded at a rate not less than Some suggestions and comments from UL 1974: (1) The higher constant current is five times the lower one, i.e., The charge and discharge performance of the batteries were evaluated using the battery test system  as shown in Fig.\u00a0CR\u2009=\u20093\u20136\u2009h\u22121) for motor start and continuous medium power output (CR\u2009=\u20091\u20133\u2009h\u22121) for advancing the golf cart continuously. These battery modules have been used for 1\u20132 years, and then they reach the end-of-life (EOL).The first-life applications of these repurposed cells are power battery modules used in golf carts. The golf course is a relatively simple environment for design verification of the power battery. There are flat roads for continuous power output tests and some gentle slopes for the up and downhill tests. The power battery modules normally operate in two conditions: instant high power output . The detailed specification of the battery cells is available in the data repository82. An 18-digit code is used to mark the repurposed battery cell and the file folder of the dataset in the data repository, including the 2-digit vendor code, 1-digit battery type code, 2-digit specification code, 6-digit disassembling date code, and 7-digit serial number code  are included in the \u201cDatasets of repurposed battery cells\u201d folder of the data repository. There are 96 sets of data in total, without exclusion any of the cells in the modules for preserving the original distribution for further statistical or model analysis. On the other hand, some datasets of broken cells from the modules without repurposed value  are listed in the \u201cDatasets of broken battery cells\u201d folder. These datasets are valuable for researchers to realize the behavior of the broken cells without taking the risk  to do the test. Furthermore, the datasets provide some examples for researchers to recognize the abnormal behavior, and they can terminate the test while encountering similar behaviors, saving the experimental and engineering resources.82. The metadata description of each column in the dataset is shown in Table\u00a0The metadata description of each column in the dataset is exhibited in Table\u00a082) should exhibit the central tendency. For example, the DCIRs obtained by the two-tier DC load method at SOC\u2009=\u200985% and 20% locate around 0.0095 \u03a9 and 0.0165 \u03a9, respectively . The instrument is calibrated every year to guarantee the stability and precision of the measurements. For avoiding instability, an electric meter is used to randomly test the accuracy of voltage and current. From the statistical point of view, the key values obtained via test procedures 1 and 2 , four-wire sensing, or four-point probes method) is an electrical resistance (impedance) measuring technique that uses separate pairs of current-carrying and voltage-sensing electrodes  thermocouples attaching above the geometric center of the cell. In this study, one thermocouple is attached at the center of the cylindrical surface of the cell (as shown in Fig.\u00a0The test procedures 1 and 2 according to UL 1974 are designed for general-propose usage, i.e., the procedures could be used in testing LFP batteries and other types of secondary batteries. The profile datasets provided in this work can be used in the model-based engineering of repurposed battery cells: either to fit the variables of an empirical model or to validate the results of a theoretical model. The study involves no privacy or safety controls on public access to the data, i.e., everyone can access the data without limitations on data use."}
+{"text": "Mycobacterium kansasii infection is rare in kidney transplant recipients. The diagnosis may not be suspected readily due to non-specific clinical presentation. The diagnosis and treatment can be further delayed due to poor sensitivity of culture  and slow growth in culture media. Accurate and rapid diagnosis of disseminated M. kansasii infections in transplant recipients is important for antimicrobial management.Disseminated M. kansasii infections with unusual presentation in which rapid diagnosis was made using the Karius test (KT) are presented. The KT is a CLIA certified/CAP-accredited next-generation sequencing (NGS) plasma test that detects microbial cell-free DNA (mcfDNA). After mcfDNA is extracted and NGS performed, human reads are removed, and remaining sequences are aligned to a curated database of >1400 organisms. Organisms present above a statistical threshold are reported.Two cases of disseminated M. kansasii (on PAD 29) whereas plasma sent for KT on PAD 5 reported a positive test for M. kansasii at 284 molecules/microliter (MPM) in 4 days (on PAD 9). Case 2: A 59-year male kidney transplant recipient presented with generalized weakness, arthralgia, pericardial effusion, cytopenia, weight loss and intermittent fevers. Plasma sent for KT on PAD 12 was reported positive for M. kansasii at 1314 MPM in 3 days (on PAD 15). PET CT done simultaneously was consistent with an infection of an old AV graft in the left upper extremity. The AFB culture of the resected graft was confirmed as M. kansasii in 22 days on PAD 36. After the KT was available (before confirmation of M. kansasii on culture), the first patient underwent modification of empiric treatment and the second patient was started on specific treatment for M. kansasii.Case 1: A 31-year female kidney transplant recipient presented with a thyroglossal duct cyst, as well as swelling of her right metacarpophalangeal joint and left 3rd finger. AFB culture of the thyroglossal cyst aspiration done on post admission day (PAD) 2 took 27 days to be identified as M. kansasii casesTable of M. kansasii infectionRapid diagnosis of disseminated M kansasii infection much earlier than standard microbiology and thus helped in initiation and modification of pathogen directed treatment.Open-ended NGS plasma testing for mcfDNA identified disseminated All Authors: No reported disclosures"}
+{"text": "Background and Objectives: Neck and shoulder injuries commonly occur during boxing, and scapular dyskinesis is related to those injuries. This study investigated scapular dyskinesis with neck disability and shoulder malfunction in elite boxers. Materials and Methods: Seventy-two elite boxers participated in this study. Scapular dyskinesis was evaluated as normal, subtle, and obvious. Neck disability index (NDI), shoulder internal (IR), and external (ER) range of motion (ROM), isometric strength of IR and ER, and pectoralis minor length were measured and compared with the severity of scapular dyskinesis. Results: Thirty-eight boxers (52.7%) showed scapular dyskinesis. NDI score was significantly different . Isometric IR strength was significantly different . The length of the pectoralis minor was significantly different , and the dominant and non-dominant arm IR ROM was significantly different . Conclusions: The prevalence of scapular dyskinesis is high among elite boxers. Boxers with scapular dyskinesis presented shoulder malfunction as well as neck disability. Further investigation is necessary to examine the relationship between scapular dyskinesis and neck disability in boxers. Although boxing could lead to traumatic injuries, boxers sustain less frequent injuries compared to athletes of other contact sports ,2,3. Howp = 0.09) and obvious abnormality  of scapular dyskinesis among handball players. Kawasaki et al. in 2012, reported that scapular dyskinesis was associated with discomfort in the shoulder , and an asymptomatic shoulder with scapular dyskinesis revealed higher rate of discomfort within the shoulder during playing season (OR 3.6) among rugby players [Scapular dyskinesis is defined as abnormal static position and/or dynamic movement of the scapula . McClure players . In boxePreviously, researchers have reported factors that may lead to scapular dyskinesis. Factors identified with scapular dyskinesis include thoracic kyphosis and increased cervical lordosis, alteration in scapular muscle function, decreased flexibility of muscles around the scapula , glenohuScapular dyskinesis is common in overhead athletes . AlthougThis was a cross-sectional, single blinded study. We listed all elite boxing teams in the Republic of Korea and reached out to them using posters about the study to recruit participants. There are 263 registered elite male boxers at Korea Boxing Federation in the Republic of Korea; of these, 72 (144 shoulders) boxers volunteered to participate in this study. The inclusion criteria were as follows: elite boxers with four or more years of boxing experience. The exclusion criteria were amateur boxers, head or neck injury in the past three months or upper body surgery in the past one year. We required a sample size of 63 for an effect size 0.97, significance level of 0.05, and a power of 0.95 after calculating the sample size with G Power . We, however, recruited a larger sample size of boxers. An informed consent form was given prior to data collection. The CHA University\u2019s ethical review board approved this study (1044308-202010-HR-046-02).The demographic data of all participants, including age, height, weight, and boxing experience (years) were obtained. Next, the presence of scapular dyskinesis was evaluated using the scapular dyskinesis test (SDT). According to the SDT results, participants were divided into three groups . The grouping results were blinded from other measurements to prevent bias from other investigators. After SDT, the participants were asked to answer the neck disability index (NDI) questionnaire, following which, information was obtained on the internal and external shoulder rotational ROM, the length of pectoralis minor, and the isometric strength of internal and external shoulder rotation measurements. The independent variable was the grade of SDT . Dependent variables were other followed measurements. We compared the average of NDI scores, shoulder internal and external ROM and strength, and the length of the pectoralis minor to the severity of scapular dyskinesis to analyze the differences.The participants were asked to remove their shirts and hold the dumbbells in both their hands. The weight of the dumbbells was decided based on the weight of the subjects. Participants who weighed less than 68.1 kg were handed 1.4 kg (3 lb) dumbbells, and those who weighed more than 68.1 kg were given dumbbells that weighed 2.3 kg (5 lb). While standing upright, the participants were asked to flex their shoulders with their elbows straight in the thumbs up position until 180 degrees of flexion and returned to the starting position for 5 s, and this was repeated five times ,28. The The NDI includes 10 questions measuring neck pain-related disability . It is tThe participant\u2019s dominant and non-dominant shoulder IR and ER ROM were measured in the supine position. The dominant arm was decided to be the rear arm in a boxing stance . The shoMicro FET 2 hand-held dynamometer (HHD)  was used to measure shoulder isometric strength of IR and ER. The participant was in supine position with the shoulder in neutral position and the elbow flexed at 90 degrees. The investigator stabilized the upper arm, pushing down to the table. The HHD was located at 5 cm, proximal to the wrist on dorsal (ER) or ventral (IR) strength measure. The participant was asked to gradually increase the resistance to maximum. Two practice sessions followed by two final measurements were applied to measure the isometric strength of ER and IR . The relThe participant was in supine position and was allowed to rest to relax for one minute prior to data collection . The medt-test was used to compare IR and ER ROM of dominant and non-dominant arms. A normal distribution test was generated since the total sample size was 72 volunteers (with 144 shoulders). The level of significance was set at p < 0.05.SPSS 22.0  was used for statistical analyses. To compare the average of neck disability, shoulder rotational ROM and isometric strength, and the length of the pectoralis minor by the severity of scapular dyskinesis, a one-way ANOVA was used to analyze the data. The Shapiro\u2013Wilk test was used and the data were normally distributed. Bonferroni and LSD post-hoc tests were used to compare each group when the results were significantly different. A paired A total of 72 elite Korean boxers participated in this study. Thirty-eight boxers (52.7%) showed scapular dyskinesis in their shoulders. Thirty-four boxers (47.22%) were normal, thirty-three (45.8%) were subtle, and five (6.94%) showed obvious scapular dyskinesis in the dominant arm. Thirty-five boxers (48.51%) were normal, twenty-six (36.11%) were subtle, and eleven (15.27%) showed obvious scapular dyskinesis in the non-dominant arm .p = 0.025). However, the NDI score of the dominant arm with scapular dyskinesis was not significantly different between the normal, subtle, and obvious groups . We found no significant difference in IR ROM  and ER ROM . Although isometric IR strength was significantly different , isometric ER strength was not significantly different . The length of the pectoralis minor was significantly different between the normal and subtle groups , and normal and obvious (p = 0.001) scapular dyskinesis. There was a tendency of difference between the subtle and obvious groups (p = 0.054) and the length of the pectoralis minor.The associations between NDI, IR/ER ROM and strength, pectoralis minor length with scapular dyskinesis are presented in p = 0.001) (p = 0.915)There was a significant difference between the dominant and non-dominant arm\u2019s IR ROM  . There wThe purpose of this study was to investigate the incidence of scapular dyskinesis in boxers with neck disability and shoulder malfunction. We identified a high incidence of scapular dyskinesis  in boxers with neck disability and shoulder malfunction including decreased ROM, strength, and muscle length. The prevalence of scapular dyskinesis in overhead and non-overhead athletes was 61% and 33%, respectively . Overheap = 0.025). Although the NDI scores depicting obvious scapular dyskinesis presented mild disability in the neck, boxers with normal scapular dyskinesis showed no disability in their neck  [p = 0.029) [p = 0.013) [p = 0.002, p = 0.033) [We found that boxers with scapular dyskinesis in the non-dominant shoulder showed significantly higher NDI scores  . Scapulaability) . Neck paability) ,47. Acco= 0.029) ,48,49. T= 0.013) . Andres = 0.033) . Therefop = 0.001). Several previous studies have reported that overhead athletes presented with decreased IR ROM [p = 0.008) in baseball pitchers. In this regard, our results are similar to those from previous studies . Lenetsky et al. also reported decreased IR ROM in the dominant arm as compared to the non-dominant arm in boxers [IR ROM and ER ROM were not significantly different depending on scapular dyskinesis in boxers. However, IR ROM between the dominant and non-dominant arm was significantly different . Decreased rotator cuff strength is found in cases of scapular dyskinesis [p = 0.911) and the healthy population (p = 0.34) with and without scapular dyskinesis [We also found decreased shoulder IR isometric strength in cases with obvious scapular dyskinesis  [Shortened pectoralis minor could alter normal scapular motion and decrease the subacromial space . We foun= 0.001) . Our resWe recruited a large number of participants (N = 72) to investigate scapular dyskinesis-related malfunction in the neck and shoulder in boxers. However, there is no known gold standard test for scapular dyskinesis, and this could be the limitation of this study. A recent reliability study of SDT with the three grades  reported high inter-reliability (ICC = 0.86) (30). Kibler et al.  suggesteBoxers showed a 52.7% rate of incidence of scapular dyskinesis. The prevalence rate was lower than that seen with overhead athletes, but higher than that seen with non-overhead athletes. Boxers with scapular dyskinesis showed increased neck disability and decreased internal rotation ROM and strength, along with reduced pectoralis minor length. Scapular dyskinesis is identified in many shoulder injuries, and neck disability is also an important factor for the occurrence of scapular dyskinesis in boxers. Therefore, we recommend monitoring scapular dyskinesis in boxers to treat and prevent shoulder and neck injury. Further investigation is required to examine the relationship between scapular dyskinesis and neck and shoulder injury in boxers."}
+{"text": "The synthesis of four-bar linkage has been extensively researched, but for a long time, the problem of motion generation, path generation, and function generation have been studied separately, and their integration has not drawn much attention. This paper presents a numerical synthesis procedure for four-bar linkage that combines motion generation and function generation. The procedure is divided into two categories which are named as dependent combination and independent combination. Five feasible cases for dependent combination and two feasible cases for independent combination are analyzed. For each of feasible combinations, fully constrained vector loop equations of four-bar linkage are formulated in a complex plane. We present numerical examples to illustrate the synthesis procedure and determine the defect-free four-bar linkages. Linkage synthesis is to determine link dimensions of the linkage that achieves prescribed task positions ,2,3,4. TTo the best of the author\u2019s knowledge, the idea of hybrid task synthesis was proposed by Smaili and Diab  in 2006;Generally, there are three linkage synthesis methods which are graphical method, optimization method and numerical analytical method. The graphical method is to draw the linkage step by step under geometric constraints by the target positions on the linkage through the poles and rotation angles. Beyer  shows a The numerical analytical method is to formulate the kinematic constraint loop equations and solve for the appropriate link lengths and pivot locations. The research for numerical analytical method of linkage belongs to the area, numerical algebraic geometry, which was proposed by Sommese and Wampler  in 1996.In this paper, we present a numerical synthesis procedure for four-bar linkage that combines motion generation and function generation, which was not addressed before. The procedure is divided into two categories which are named as dependent combination and independent combination. Five feasible cases for dependent combination and two feasible cases for independent combination are analyzed. For each of feasible combinations, fully constrained vector loop equations of four-bar linkage are formulated in complex planes. In addition, we give numerical examples to illustrate the numerical procedure and determine the defect-free linkages. In what follows, we present how to perform the numerical procedure.F to the reference frame It is convenient to use vectors in complex plane to formulate constraint equations in planar kinematics . InsteadF, which isThe conjugate of Equation  denotes x-axis of the fixed frame F.The task of motion generation is to guide the coupler link of a four-bar linkage through prescribed points and orientations. In this paper, we define the prescribed point and orientation as motion task position (MTP) which is denoted as The conjugates of Equation  denote rAt MTP The conjugate of Equation  are(6)ONote that m denotes the maximum number of MTPs that a four-bar linkage can achieve during a movement. In Equation OThe unit vector n represents the maximum number of FTPs that a four-bar linkage can achieve. Note that the fixed pivots of the four-bar linkage must be specified in advance to measure the angles of input and output for function generation. Thus, in Equation (quations  and 11)T and T\u00afkquations  to elimiand T\u00afk,(Sk(B\u2212C)\u2212In m MTPs and n FTPs during a period of movement. The dependent combination means that there is at least a common task position (CTP) that the linkage moves through a MTP and a FTP simultaneously. On the contrary, there is no CTP during the movement, which is called independent combination.The task of mixed generation is also to determine the link dimensions of the four-bar linkage that achieves t denote the number of CTPs, and select one of CTPs as the first task position. For the rest of Let traints,O+Ql(A\u2212O)According to Equation , the conAccording to Equation , the conCombining Equations \u201316), th, th16), t, which are There are three cases for the value of When Equation  can be dEquation  and 16)t=1, Equat theory , the uppsmoothly ,25. The When Equation  are lineSubstituting Equation  into Eququations  and 16)O, O\u00af, C Equation .For case Equation  are applIt is easy to solve the equations to obtain the results of m MTPs and n FTPs without any CTP during the movement, namely The independence of mixed generation is a task to determine four-bar linkages that achieve m MTPs, we haveFor n FTPs, we haveFor x-axis of the fixed frame to vectors To decrease the number of unknowns, we establish the relationship between The rotation operations of input link Now, the relationship between Substituting Equation  into EquThe constraint equations for independent combination for mixed generation are combination of Equations  and 25)25). TherIn this section, we present numerical examples to illustrate the mixed constraint equations and determine non-defective four-bar linkages. For lack of space, we do not show numerical examples for all cases of mixed constraint equations. According to the number of unknowns in the nonlinear equations, we select one for maximum number of unknowns, the combination of For combination of In this example, the CTP is the fourth task position that the four-bar linkage passes through. substituting these values into Equations  and 16)16) to foBertine ,26 is apt number ,27 is 70The images of the four-bar linkage moves through each of prescribed task positions of For combination of Substituting the values into Equation  can obtaThe images of the four-bar linkage move through each of prescribed task positions of This paper presents a numerical procedure to synthesize four-bar linkage for mixed motion generation and function generation. The synthesis procedure is divided into two categories, dependent combination and independent combination. The feasible combinations are"}
+{"text": "Wearable power supply devices and systems are important necessities for the emerging textile electronic applications. Current energy supply devices usually need more space than the device they power, and are often based on rigid and bulky materials, making them difficult to wear. Fabric-based batteries without any rigid electrical components are therefore ideal candidates to solve the problem of powering these devices. Printing technologies have greater potential in manufacturing lightweight and low-cost batteries with high areal capacity and generating high voltages which are crucial for electronic textile (e-textile) applications. In this review, we present various printing techniques, and battery chemistries applied for smart fabrics, and give a comparison between them in terms of their potential to power the next generation of electronic textiles. Series combinations of many of these printed and distributed battery cells, using electrically conducting threads, have demonstrated their ability to power different electronic devices with a specific voltage and current requirements. Therefore, the present review summarizes the chemistries and material components of several flexible and textile-based batteries, and provides an outlook for the future development of fabric-based printed batteries for wearable and electronic textile applications with enhanced level of DC voltage and current for long periods of time.  However, many of these devices are based on conventional electronic circuit elements and substrates like printed circuit board and, therefore, stiff and inflexible, which restricts their widespread practical applications and also ease of wearability. The future growth of these wearable electronic systems depends on effective production and use of flexible and stretchable substrate materials in order to meet an increasing demand of intelligent systems and wearables ,2. TheseStudies regarding the potential application of textiles to store energy include supercapacitor ,16 and bIn this review, different flexible and textile-based batteries, as well as printing techniques applied for smart fabrics are discussed. The chemical and material components for each battery cells are analyzed to identify a fully flexible and sustainable power supply. The present review summarizes the current-state-of-the-art flexible and textile-based battery technologies, and highlights future research directions for developing fabric-based printed batteries with enhanced level of DC power to continuously power wearable and electronic textiles for long period of time.1.1.From the pre-industrial agricultural era to an exponentially developing information age, connected globally through networked technologies, a lot has been studied regarding the rapid changes and development of human being. Textiles arise in the first of these eras. Initially, different leaves were joined together by our predecessors to make their own styles of garment and apparel. Naturally available materials, including silk and cotton, were woven into more suitable and warm garment products. A wide range of man-made fiber materials including nylon, and Kevlar slowly came and largely improved our way of living over the last 100 years. Now with the machine age, the usual way of manufacturing the woven textile has grown to become the fabric of our lives. As a material from which is produced the interface between the outside environment and bare human body, textiles take an incomparable position in our society . The evoThe impact of electronic textiles on the economic development is expected to be extremely high according to IDTechEx report . With th1.2.There has been significant research interest in smart textiles and on the mechanism of powering electronic textiles which necessitates research into an energy source. Energy harvesting systems, including inductive coupling , thermoe1.3.This paper focuses on the critical review of different flexible and textile-based batteries, and gives a comparison between them in terms of their potential to power wearable and e-textile applications. Various printing techniques applied for smart fabrics are discussed and those applicable for thick-film deposition in order to obtain high-energy density textile battery will be identified. Regardless of the printing technology, proper adhesion of the printed layers with the substrate material is challenging to boost the scalability of printed batteries. Therefore, understanding the chemical and material components for each battery will be helpful for the future development of textile-based printed batteries with enhanced levels of DC power applicable for long periods of time.2.Printed batteries have received significant attention in research and industry, since they are ultra-thin, lightweight and environmentally safe while being tailorable. In order for the battery to be identified as printed-battery, the separate battery components which includes an electrolyte or separator, electrodes, and current collector, need to be manufactured using printing technologies. The design of electrodes and architecture of the battery are crucial in order to produce an efficient battery. The two most common printed battery designs are the stack or sandwich, and coplanar or parallel architectures. The stacking architecture, shown in In printed batteries, mostly inks are prepared for electrode materials as well as separator or electrolyte. For each component of the battery, these inks are made up of binders, solvents and compatible filler such as lithium-based material, based on specified properties of inks. Though printing technology provides complete freedom for the manufacturing of batteries over a large area and high-volume with minimal costs , developIn this section, the three most commonly used printing methods; dispenser printing, screen printing, and inkjet printing techniques are discussed. In all of these methods, developing fully functional ink for various battery components is critical, and also crucial in order to get printed-layers with appropriate and desirable properties. Moreover, the proper adhesion of layers onto the surface of substrates is also challenging for improving scalability of the printed battery. Each of these techniques have different ink properties and film thickness. Since areal capacity is related to the thickness of printed layers, thick film deposition methods are very attractive for high-energy batteries. Throughout this review, the term textile stands for those textiles or fabrics which are suitable for clothing purpose, and the two terms textile and fabrics are used synonymously.2.1.. Moreover, rapid prototyping technologies, which enable the printing of different electronic components simultaneously with the construction of a product, are now developing rapidly . In general, printing techniques can be classified as contact and non-contact techniques as in The incorporation of wearable technology looks set to remarkably enhance textiles in the near future. However, functionality alone is not enough for the world\u2019s current market where the market strategies are predominated by various fashions and styles. Due to the growth of digital technologies, which influences both the design as well as fabrication of the end product, new printing technologies involved in garment construction and printing of different patterns on textile surfaces are now possible . Moreove rapidly . In geneIn this paper, the three common printing techniques; dispenser printing , screen 2.1.1.In the last few years, many of the research works have focused on inkjet printing technology, which is a non-impact technology where the required image is produced on the surface of substrates using ink droplets. This technique is a promising method to fabricate polymer electronics and light-emitting diodes by depositing smart and functional materials. It consists of two operation modes: droplet on demand mode \u2013 which uses the piezoelectric actuating in order to produce ink droplets, and continuous printing mode \u2013 that uses magnetic field to produce ink droplets . This prInkjet printing of electronics started with printing of conductive layers on rigid materials, then later moved to printing of flexible substrate materials including papers and polyamides. Although this technology is popular in textile industries for the purpose of dyeing, obtaining higher pattern resolutions in the printing of electronic components on fabric substrates is still challenging. Some of the main concerns while conducting inkjet printing of electronic parts on textiles are due to the tight structure of textiles, diameter as well as natural porosity of fibers and yarns. Moreover, the printing inks should have to meet certain requirements, such as, lower viscosity, increased shelf-life, and lower surface tension. This technique has limited speed of printing and is commonly used to obtain thin-film layers; so, its practical application for printing thick battery electrodes is very low. However, due to its high pattern resolution, the technique is still more popular for designing and printing of small-sized batteries for various applications. Moreover, it is compatible with several rigid or flexible materials for depositing on paper or other substrates. However, this technology has several disadvantages when applied for smart fabrics , and usuwhere  (gcm\u22123) .2.1.2.Similar to that of inkjet and screen-printing technologies, dispenser printing is also applicable in the production of intelligent fabrics and wearables. Dispenser printing technology integrates the computerized printing technique of an inkjet printer and effective performances of screen-printing ink materials. This printing method is an additive process which provides capability of printing different inks and altering the pattern designs, easily and when required, which can be specified immediately on the computer. Moreover, as the dispenser printing inks are suitable for high-volume screen-printing techniques, products printed with dispenser printers can be taken for mass production in screen printers using similar inks, unlike the inkjet printing where inks need reformulation for mass production using screen printing technology. Dispenser printing is a non-contact printing technique for smart fabrics where it is possible to print directly from the computer designs like that of inkjet printing with the same thickness of layer as in the case of screen-printing. Electronic functional inks can be prepared at nano- or microscale level by using functional particles and combining with different chemicals such as binders, solvents and surfactants. The working principle of dispenser printing is based on a pneumatic system, which allows printing of the design directly from the computer software. Dispenser printing technology has been used for the production of non-woven conducting fabrics , and als2.1.3.Screen printing is an impact printing method where an ink is applied through a fine-mesh screen onto a surface, except in those areas blocked by the stencil to make them impermeable to the ink . A screeThis relation between ick film  depositiick film ,88. Whilick film ,90.Tabl3.[92]. Towards such an environment, lightweight, wearable and flexible electronic devices in various forms such as sensors integrated with textiles to monitor health would be widely adopted. Wearable electronic textiles, besides their feel being similar to regular fabrics, have additional embedded functions including sensing, processing, storage and communication , which enables them to be used in military activities, healthcare and medical textiles among others . So far, only the sensing functionality of these textiles, which provides an interface between different electronic components and the end-users, is an active area of research. Therefore, smart garments and wearables might be able to monitor different physiological variables and can be useful for several applications including healthcare monitoring. Future generation of these wearable systems would be thin and consistent to human body [96], so powering these devices without losing their mechanical property will be difficult [98] and at the same time a key, if done well, for successful commercialization of smart garments. The power supply for e-textiles can be performed through energy harvesting methods, such as photovoltaics, and also energy storing supercapacitors or batteries as given in Recent progresses in wireless technology, microelectronic systems, as well as internet accesses have helped electronic objects to be interconnected, so that they communicate with another, and provide users with sufficient information to make decisions as well as to improve their life ,92. Towanication , which eg others . So far,man body ,96, so pifficult ,98 and aOne method for powering wearable and textile electronic applications is by using the energy harvesting and wireless power transfer methods. However, many of the systems powered by the energy scavenging methods require the harvested energy to be stored until a sufficient amount of energy is available. Therefore, combining flexible energy harvesting methods with a textile-based energy storage provides the potential for sustainably and continuously power the future integrated electronic textiles and wearables. Compared to many of the harvesting techniques, the mechanical energy scavenging by using the piezoelectric or triboelectric nanogenerators is the most widely distributed harvesting method, available in all living environments including our body ,100. TheAn alternative to the implementation of energy harvesting techniques as an energy supply to wearable and textile electronics is to make use of the energy storage devices integrated with flexible and textile substrates. Energy storage devices include batteries, supercapacitors and fuel cells. Compared to both supercapacitors and fuel cells, batteries have a widely accepted market potential for applications requiring high energy storing capability. On the other hand, supercapacitors have high-power density; but, the problems of low-energy density and high self-discharging rate of supercapacitor still requires solution and hence limits the practical application of smart textile electronics. Knowing that the energy density is directly related to the working potential and capacitance of supercapacitor , the hybIn general, batteries have a widely accepted market potential for applications requiring high energy storing capability, than both microbial fuel cells and supercapacitors . There i3.1.Lithium-ion batteries (LIBs) are largely applicable in our everyday life. First commercially developed by Sony in the 1990\u2019s, these batteries are now available everywhere in consumer electronics. These batteries are the latest in the rechargeable battery technology, and the market potential for these batteries is growing quickly with enhanced investment levels across different value chains worldwide. Currently, the rigid lithium batteries produced in metallic foil materials are powerful and have a great influence on the battery technology for various applications due to their high-energy density and longer discharge lifetime. In the last ten years, a lot has been done to develop flexible lithium-ion batteries; however, because of limited capacity and high mass per unit volume of electrode materials, enhancing the energy density while keeping the flexibility, lightweight and charge/discharge cycle stability of battery is still challenging. These batteries are attractive battery chemistries which are useful for many different electronic devices and systems because of their high-energy density and proven charging or discharging cycle time. Alhough zinc-based alkaline batteries have shown high areal capacitance , most ofAnd, during discharging, it will be reversed; the lithium metal oxides, 4 and Li4Ti5O10 as its cathode and anode materials, respectively, and a solid poly (ethylene oxide) (PEO) electrolyte have been researched for electronic textile applications , where the batteries generated an open-circuit voltage of 1.3\u00a0V and 1.2\u00a0V, respectively. And to obtain enhanced durability and easy integration with wearable textile materials, liquid activated batteries on a textile substrate were also produced by using metallic electrode materials embedded with textiles. However, it is also reported that the use of metallic film on the design of the battery cell restricts the robustness as well as flexibility of the devices . Alternatively, a textile-based water activated battery was studied , where the battery demonstrated potential application of powering a light-emitting diode. This fabric-based battery, which needs water to be added for the activation process, was produced by using screen-printing of the silver, Ag, and aluminum, Al, electrode pairs. However, the fabrication process consists of many textile layers stacked up together to form the battery, which increases the weight of the device and is not comfortable to integrate with wearables. The battery contains three layers of a commercially available cotton fabric  inserted between two metallic electrode materials, which functions as anode and cathode as in 3, and aluminum chloride, AlCl3, compounds, and the other one is soaked into sodium nitrate, NaNO3, metal to be used as a salt bridge [120]. Thus, when water is added to a battery, the dry electrolytes will be hydrated and hence an electrochemical reaction will be produced. The general chemical reactions for the battery cells at the two electrodes are expressed as in (7) and (8), respectively.Most commercial lithium-ion batteries are made up of rigid metallic components, which restrict their ability to be integrated with textiles and other flexible materials. Also, flexible textile-based batteries using solid polymer electrolytic have been demonstrated promising results . Howeversearched  and showatteries . Howeverubstrate ,116, whe devices . Alterna studied , where tn fabric  insertedt bridge ,120. ThuA single battery cell produces a maximum potential of 1.3\u00a0V. Two of these printed battery cells combined in series before activation in 3.3.\u2212 consumed or produced, during discharge, is equal. These batteries have characteristics of long discharge cycle life, long storage time, and leakage proof. The most commonly used alkaline batteries applied for smart textiles and wearables consists of the zinc-manganese dioxide, Zn-MnO2, and the monovalent silver oxide-zinc, Ag2O-Zn, batteries.Alkaline chemistries are the predominant primary batteries which drives its energy from the zinc metal and manganese dioxide chemical reactions. In the basic environment, these batteries have a pH of approximately 14. The basic electrolytes for these battery chemistries are potassium hydroxide, KOH or sodium hydroxide, and NaOH, and hence it is named as alkaline battery. The amount of hydroxide ion, OH3.3.1.2 -Zn battery is not rechargeable; this is due to the fact that during plating the zinc \u2018dendrites\u2019 will be formed and also because of the corrosive nature of the electrolyte; this battery has not been widely applied for textile-based flexible electronic applications. This alkaline battery is environmentally safe and hence applicable for most of the flexible printed batteries. The printed, nontoxic, flexible and high-energy manganese dioxide-zinc-based battery was fabricated [The manganese dioxide \u2013 zinc battery is an alkaline battery, that contains the zinc anode, manganese dioxide cathode, and an electrolyte solution composed of potassium hydroxide, KOH. This battery chemistry has a higher gravimetric as well as volumetric energy density than the zinc-carbon chemistry, but it is more expensive. The MnObricated , where t2-Zn based printed alkaline battery chemistry has not been widely expanded.In this battery chemistry, the output voltage decreases with the depth of batter\u2019s discharge cycle life. One of the drawbacks for this battery because a constant voltage is desired for the circuit. Thus, the sloping open-circuit voltage with an increasing discharge time needs to be considered for manganese oxide-zinc based circuit applications; hence, the MnO3.3.2.2O-Zn, chemistry is one of the alkaline batteries which have received significant research attention because of their rechargeable chemistry and tolerance to high discharge current [2O, cathode and the zinc anode electrodes deposited onto polyester fabrics using a screen-printing technology. The chemistry of these batteries involves an oxidation-reduction reaction of the two electrode materials, where the monovalent silver oxide cathode and the zinc anode electrodes are reduced and oxidized as shown in (11) and (12), respectively.Among several commercially available flexible batteries, aqueous zinc chemistries were effective for the development of low-cost, high throughput products ,124. Due current ,130. The current . The suc current . The fab2O powder to prepare a conducting paste or ink that can be screen printed onto the fabrics. And a single printed battery cell in a 10\u00a0M NaOH conventional electrolyte generates a potential of 1.46\u00a0V, and approximately 80 \u00b5W of power. As a proof-of-concept, two of these screen-printed batteries onto textile fabrics combined in series as shown in The current collectors for this printed battery cell include fully flexible and electrically conducting copper (Cu), and nickel (Ni) threads ,134, whi4.2O-Zn, based printed battery demonstrated many advantages compared to lithium batteries as well as the MnO2-Zn based alkaline battery, to develop printed textile batteries. The energy density of the monovalent silver oxide-zinc battery is a bit smaller but still close to that of the lithium batteries. This is mainly due to the high output voltage as well as the large columbic capacity of the battery. However, this energy density could be enhanced by using the divalent silver-oxide (AgO) cathode, which provides greater operating voltage and higher energy density than the monovalent silver oxide-zinc battery. Currently, flexible batteries are constructed from active materials of very low thickness, due to which they have low capacity compared to traditional batteries. Due to its high theoretical capacity and low-cost, aqueous zinc-based batteries have been researched for the light-weight and flexible battery market. However, usually these batteries are not rechargeable and have high impedance; hence, they are limited for low-power, disposable electronics applications. The performance of batteries can be characterised by various parameters such as cycle life, and voltage/capacity of the battery  water or phosphate buffered saline (PBS), a single cell produces a potential of 1.3\u00a0V. As a proof, dual-cell printed batteries were combined in series, and powerup a 1.6\u00a0V light-emitting diode for about 30\u00a0minutes. In these types of batteries, instantaneous activation is required, and hence they offer a unique opportunity of generating power supply on-demand, so it simplifies the design as well as general operation of the devices. Repeated re-activation of the battery may result in a quicker voltage drops; hence this technique suffers from a non-uniformity and poor stability concerns. Moreover, the poor contact between the textiles and electrode materials contributes to a higher internal impedance; the higher the resistance, the lower the performance of the battery against high discharge currents. When the water is evaporated, the electrochemical reactions will come to an end, and the battery will be turned \u2018off\u2019 automatically, so they are considered as reserve batteries. Considering that the sensing measurements usually require a short period of time, these batteries can provide enough energy for many independent measurements over long durations. These batteries could be easily integrated into different textiles with optimum flexibility and indefinite shelf-life before activation, which is highly important for emergency applications.2O-Zn, based alkaline batteries printed on fabrics are presented here, which are safe and can be easily activated by ionically conducting liquids available on our body such as sweat. These screen-printed batteries are rechargeable unlike the manganese dioxide-zinc chemistry, MnO2-Zn, insensitive to air and water unlike the lithium-ion batteries, have good tolerance to high discharge current, and they behave like regular fabrics, which is crucial for electronic textiles. This is because the success and widespread acceptance of electronic textiles depends on their ability to be manufactured through existing textile technologies and use the textile area for storing as well as harvesting energy. These batteries were fabricated using a low-cost and high throughput screen-printing technology for enhanced flexibility in wearable and e-textile applications. A single printed battery produces a potential of 1.46\u00a0V, and further incorporation of engineering concepts into the battery cell enables voltage or current boost depending on the application requirements. As a proof, two of these screen-printed fabric-based batteries combined in series, using flexible electrical connections, could powerup a digital thermometer whose voltage and current requirements were 1.5\u00a0V and 12.5\u00a0\u00b5A, respectively. However, these monovalent silver oxide-zinc, Ag2O-Zn, batteries have high impedance, which results in low battery capacity and limited cycle life. Therefore, an increased focus needs to be placed on different battery chemistries and associated materials to overcome this problem and scale DC voltage and current for various e-textile applications.The monovalent silver oxide-zinc, Ag5.In the past few years, there has been significant research interest in the area of wearable and textile electronics as they remove the need for extra carriage of various devices. These wearable electronic devices have some important requirements such as flexibility, lightweightness and comfortability, among others. However, though there is progress in developing wearable and textile electronic devices, powering them still remains a challenging task. Usually, the current energy supply devices are constructed from rigid and bulky materials and often require more space than the devices they power. Therefore, energy supply for the next generation of flexible and smart textile electronic devices is key to the overall functionality of electronic textiles and wearables. Due to their high gravimetric and volumetric density as well as suitable power density for most applications, batteries are found to be the best alternatives to power electronic textiles. Printing technologies have a greater potential to fabricate light-weight and low-cost energy supply devices, by depositing different battery components with printable inks, that can be easily integrated with various textile electronic devices. Moreover, the printing processes are economical, can be customised easily, and also play a key role for high-volume roll-to-roll production of flexible batteries. Batteries can be printed on the fabrics and can also be adjusted according to the device\u2019s mechanical as well as power requirements. Different printed flexible power supply devices have been reported in the literature. However, the current flexible batteries are also constructed from materials of very low thickness, which again reduces the areal capacity of the batteries. Therefore, there is a need to develop flexible, lightweight textile-based batteries without any rigid electrical components to continuously and sustainably power electronic textile devices. In this review, different printing techniques, including screen printing, applied for electronic textiles and functional fabrics are discussed in detail. In all the printing techniques, the proper formulation of ink is a key to obtain sufficient electrical as well as mechanical properties of printed layer materials. Moreover, to enhance the scalability of printed batteries, proper adhesion of printed layer materials to the substrate is also very critical. Various battery chemistries and material components fabricated on textile substrates, such as lithium batteries, water-activated batteries and alkaline batteries, and their merits and demerits in terms of capacity or voltage, maintenance requirements, cycle life and safety are described. Each type of battery demonstrates their potential application to power different electronic devices of specific voltage and current requirements. Further incorporation of engineering concepts, using flexible electrical connections, to these batteries would help to boost the level of voltage or current depending on the application requirements. Therefore, batteries with the capability of generating high voltages are very important in e-textile applications, because the textile electronic circuits are generally designed to have a low-power consumption and a high input impedance; however, a lower impedance is desirable for component interconnections.2O-Zn, based textile battery, fabricated by depositing the zinc anode and monovalent silver oxide cathode onto textiles, behave like regular fabrics and can be directly integrated with different textile electronic devices. The battery, as manufactured, is in its dry form and later activated by bodily available liquids such as sweat. However, the battery showed limited cycle life and low capacity, which might be due to the poor electrical conductivity and high input impedance. Another species of silver oxide the so-called divalent silver oxide (AgO), offers comparably higher theoretical capacity, but it also has disadvantages of dual-voltage discharge curves and greater instability in alkaline solutions [In general, the success of printed flexible and textile batteries depends on the development of new active materials for all the battery components; the higher the energy density of active material, the more enhanced the areal capacity. Thus, achieving high-energy density and areal capacity of battery without any increment in the overall thickness of active layers is a very critical problem in the development of textile and flexible batteries. Among several commercially available flexible batteries, aqueous zinc-based chemistries have been researched for the lightweight and flexible battery market. This is mainly due to the fact that the zinc anode battery has high theoretical capacity and low-cost compared to the lithium-ion batteries, and also due to its safe battery chemistry. For the textile-based lithium batteries to be suitable for wearable and smart electronic textiles, they should not contain metallic current collectors and liquid electrolyte solutions. Although it is still a common challenge to design and develop waterproof power supply for wearable and textile electronic devices, the washability concern is more complicated in the case of lithium batteries because they contain several chemicals that are extremely sensitive to air and water. Water-activated batteries offer the advantage of generating power on-demand, so it simplifies the design process and reduces its size. However, these batteries suffer from poor stability due to repeated re-activation process. On the other hand, the monovalent silver oxide-zinc, Agolutions ,138. How"}
+{"text": "Acinetobacter are Gram-negative bacteria belonging to the sub-phyla Gammaproteobacteria, commonly associated with soils, animal feeds and water. Some members of the Acinetobacter have been implicated in hospital-acquired infections, with broad-spectrum antibiotic resistance. Here we report the whole-genome sequence of LC510, an Acinetobacter species isolated from deep within a pristine location of the Lechuguilla Cave. Pairwise nucleotide comparison to three type strains within the genus Acinetobacter assigned LC510 as an Acinetobacter pittii isolate. Scanning of the LC510 genome identified two genes coding for b-lactamase resistance, despite the fact that LC510 was isolated from a portion of the cave not previously visited by humans and protected from anthropogenic input. The ability to produce acyl-homoserine lactone (AHL) signal in culture medium, an observation that is consistent with the identification of the luxI and luxR homologues in its genome, suggests that cell-to-cell communication remains important in an isolated cave ecosystem. For exaRNA gene . In receRNA gene . In addinotation .Acinetobacter calcoaceticus that is associated with nosocomial infections. LC510 was isolated from a site deep within the Capitan Formation proximal to the region named \u2018Deep Secrets\u2019 at a depth below the surface of approximately 400\u2009m [Acinetobacter are found in soils, waters and occasionally animal feeds, some Acinetobacter species are known to infect humans with broad-spectrum antibiotic resistance and such environmental isolates may serve as a reservoir for additional resistance determinants [Of the 93 LC strains reported, LC510 stood out due to its initial species designation as rminants . In thisg for 10\u2009min. Genomic DNA (gDNA) extraction was performed on the pelleted cells using the QIAam DNA Mini kit  according to the manufacturer\u2019s instructions. The purified gDNA was quantified using the Qubit BR Assay  and normalized to 0.2\u2009ng \u00b5l\u22121 for Nextera XT library preparation . The constructed library was sequenced on an Illumina MiSeq (2\u00d7151\u2009bp run configuration) located at the Monash University Malaysia Genomics Facility that routinely sequences metazoan mitogenomes [Acinetobacter, or more broadly the family Moraxellaceae.The isolation, antibiotic characterization and 16S rRNA gene-based identification of strain LC510 have been described previously . For gDNogenomes  and occaogenomes  with no de novo assembly using Unicycler v0.4.7 (default setting with minimum contig length set to 500\u2009bp) [Acinetobacter pittii (WGS Project: BBST01), Acinetobacter lactucae (WGS Project: LRPE01) and A. calcoaceticus (WGS Project: AIEC01).Raw paired-end reads were adapter-trimmed using Trimmomatic v0.36  followed 500\u2009bp) . We then 500\u2009bp)  to calcuhttps://github.com/tseemann/abricate) to search for antibiotic resistance genes against the ResFinder database (minimum query length coverage and nucleotide identity of 90 %) [blaOXA proteins used muscle followed by maximum-likelihood tree construction with FastTree2 (1000) [Genome annotation used Prokka v1.13  and the of 90 %) . The ali2 (1000) .lacZ Agrobacterium tumefaciens reporter strain and X-gal as described previously [A single \u00bd strength tryptic soy agar plate colony of LC510 was inoculated into 50\u2009ml of sterile \u00bd strength tryptic soy broth and grown overnight at 30\u2009\u00b0C with shaking at 150\u2009r.p.m. An equal volume of ethyl acetate (EtOAc) was added to the culture followed by shaking at 50\u2009r.p.m. on an orbital shaker for 1 h. The EtOAc layer (upper layer) containing the extracted AHL was evaporated to dryness with a vacuum, concentrated and resuspended in fresh EtOAc to make a 20\u00d7 concentrated extract. Then, 25\u2009\u00b5l of the extract was spotted (2\u2009\u00b5l/transfer) onto a reverse-phase thin-layer chromatography silica gel 60 RP-18 sheet . In addition to the LC510 AHL extract, six synthetic AHL standards were also spotted in separate lanes for comparison. The chromatography was carried-out with a 70\u202f:\u202f30\u200a% methanol\u202f:\u202fwater mobile phase. The TLC was subsequently dried and overlaid with 1\u00d7 AB agar medium containing TraR-dependent eviously . After ohttps://pfam.xfam.org/family/Autoind_synth) that contains the probabilistic model used for the statistical inference of the LuxI-type family of autoinducer synthases [luxI and luxR homologue was visualized using Easyfig (blastn setting) [Proteins were scanned using HMMsearch v3.1b1 with an E-value cutoff of 1E-5 for the presence of Pfam profile PF00765 (ynthases . The gensetting) .50 length of 66.9\u2009kb) with a total length and GC content of 3 767 126\u2009bp and 38.63 %, respectively. Based on 16S rRNA gene identification, strain LC510 had previously been assigned to the species A. calcoaceticus  and blaADC (locus tag: YA64_000855). The blaoxa-213-like gene is commonly found among members of A. calcoaceticus and A. pittii  in strain M2 led to a substantial reduction in motility that could be rescued with the supplementation of its cognate AHL signal in the media [luxI mutant for LC510, using either transposon mutagenesis or homologous recombination [Given the demonstrated ability of several nd C8-OH . The luxi PHEA-2 . Such a annii M2 , hintinghe media . The prebination , followebination , will beA. pittii was presented in this study. The identification of two bla genes in the annotated genome of isolate LC510 that has no prior history of anthropogenic exposure supports previous work claiming that these genes contribute to the intrinsic antibiotic resistance in members of the species A. pittii. In addition, LC510 still retains the ability to engage in cell-to-cell communication in an isolated cave ecosystem, as evidenced by the presence of a luxI homologue in its genome and its ability to accumulate of N-acyl-homoserine lactones in culture medium.The whole-genome sequence of a Lechuguilla Cave isolate (LC510) belonging to the species"}
+{"text": "HMT+P brought the highest TDF content (15.3%), amylose content (31.24%), and RS content (15.71%), and the lowest amylopecyin content (30.02%) and RDS content (23.65%). HMT+M brought the highest slowly digestible starch (SDS) content (25.09%). The estimated glycemic index (eGI) was respectively reduced from 74.36 to 70.59, 65.87, 69.79, and 69.12 by HMT, HMT+P, HMT+M, and HMT+A. Moreover, a significant and consistent reduction in the heat enthalpy (\u0394H) of WQ was observed (p < 0.05), after four treatments. All these effects were caused by changes in the starch structure, as evidenced by the observed conjunction of protein and starch by a confocal laser scanning microscope (CLSM), the decrease in relative crystallinity, and transformation of starch crystal.The starch digestion processing of whole grain foods is associated with its health benefits in improving insulin resistance. This study modified the digestibility of whole quinoa flour (WQ) via heat-moisture treatment (HMT), HMT combined with pullulanase (HMT+P), HMT combined with microwave (HMT+M), and HMT combined with citric acids (HMT+A), respectively. Results showed that all the treatments significantly increased ( Global statistics indicate that diabetes mellitus (DM) has reached the epidemic level around the world, and one-tenth of people will suffer from this condition by 2030 . In receChenopodium quinoa Willd.) has recently attracted increasing attention due to its excellent nutritional compositions [Based on the modification effects on starch, HMT has gradually been used to modify the digestibility and physicochemical property of whole grains. Zheng et al. found HMT decreased the digestion rate of whole highland barley flour and enhanced its health benefits which include reduction in the body weight and serum glucose, improving oxidation resistance and altering the composition of gut microbiota in rats . Consistositions . Whole qositions . A serieositions ,18. Thusositions . If HMT Therefore, the major purpose of the present study is to investigate the modification effects of HMT and HMT combined with other treatments on WQ. The nutritional characteristics, digestibility, physicochemical properties, and granule morphology were respectively assessed.Three colored (red:black:white = 1:1:1) whole quinoa flour (WQ) produced in the year of 2019 were purchased from Sanjiang Fertile Soil Co., Ltd. . Total starch assay, glucose oxidase/peroxidase (GOPOD), dietary fiber, and \u03b2-glucan assay kits were provided by Megazyme International Ireland Ltd. . Pullulanase (EC 3.2.1.41), amyloglucosidase (EC 3.2.1.3), \u03b1-amylase (EC 3.2.1.1), pepsin (EC 3.4.23.1), and trypsin (EC 3.4.4.4) were provided by Sigma Chemical Co. . Other chemical reagents obtained the standards of the analysis grade.Hydrochloric acid-potassium chloride with pH = 2.0 was used to dissolve pepsin (10.0 mg/mL), then the miscible liquid was stored at 4 \u00b0C. Suitable amounts of \u03b1-amylase and amyloglucosidase were dissolved in 0.2 M sodium acetate buffer , and the concentrations were 290 and 60 U/mL, respectively.WQ was heat-moisture treated alone (HMT) or with pullulanase (HMT+P), microwave (HMT+M), and citric acids (HMT+A), respectively, according to the conditions obtained from previous optimal experiments (unpublished data). Briefly, the moisture content of WQ was adjusted to 30%, and then was sealed in a plastic bag. After equilibrium at 4 \u00b0C for 24 h, WQ were transferred to a steel pan and heated in an oven at 110 \u00b0C for 90 min, and then dried at 45 \u00b0C, and milled through an 80-mesh sieve. Then, for HMT+P treatment, HMT treated WQ was suspended in phosphate buffer solution  at 55 \u00b0C Pullulanase (40 U/g flour) was added to the slurry. After reaction for 8 h, the enzyme was inactivated by boiling water, and the pH value was adjusted to 6.8. The slurry was dried at 45 \u00b0C and milled through an 80-mesh sieve. For HMT+M treatment, HMT treated WQ was suspended in distilled water , and placed into a microwave oven . The treated sample was dried at 45 \u00b0C and milled through an 80-mesh sieve. For HMT+A, HMT treated WQ was suspended in citric acids solution , and kept in a 50 \u00b0C water bath for 45 min. Then, the pH value was adjusted to 6.8. The slurry was dried at 45 \u00b0C and milled through an 80-mesh sieve.The crude protein (method 46\u201312), crude fat (method 30\u201325), and ash (method 8\u201301) contents of samples were determined using AACC (2000) standard methods. Total dietary fibers (TDF) were determined using the dietary fiber kit  following the approved AOAC 985.29. Amylose/amylopectin content was determined using K-AMYL 12/16. AOAC 996.11 was used to determine the total starch content.2CO3 solution were added. After centrifugation, 3,5-dinitrosali-cyclic acid assay was used to determine the reducing sugar content of supernatant. Hydrolysisrate, RDS content, SDS content, and RS content was calculated as follows:20 was the reducing sugar content (mg) produced after 20 min of enzymatic hydrolysis; G120 was the reducing sugar content (mg) produced after 120 min of enzymatic hydrolysis.The method of Yu et al., 2017) was used to determine the extent of in vitro starch hydrolysis [17 was usThe GI were analyzed with a modified procedure established by Goni et al. . The staf is the final reaction time (180 min) and t0 is the initial reaction time (0 min).The area under the hydrolysis curve (AUC) can be calculated as follows:The hydrolysis index (HI) is the ratio of AUC between each sample and that of white bread (7300). The eGI was calculated using the equation:The experiments were carried out at least in triplicate to achieve re-producible results.A Differential Scanning Calorimeter  was used to analyze the samples\u2019 thermal properties and the result was described by the mothed of Huang, Dong, Zhang, Zhu, and Qu with some modifications . Six micThe method of Jan et al. was used to extract quinoa starch with some modifications . The quiA0 denotes the area under each peak, and At means the total area under the diffractograms.The morphology of the starch granules was examined by a scanning electron microscope  and confocal laser scanning microscopy  was respectively used to examine the morphology of the starch granules as described previously ,25. Sampp < 0.05.Data was expressed as the mean \u00b1 SD, included at least 3 replicates per sample. One-way Analysis of Variance (ANOVA) and Tukey\u2019s test were performed Statistical Product and Service Solutions (SPSS) version 22.0 . Statistical significance was set at p < 0.05), respectively, which was consistent with a previous report that the TDF content in whole wheat flour significantly increased after HMT [p < 0.05), and HMT increased the least, by 9.25% (p < 0.05). The results agreed with the results of Niu et al. who reported that HMT+M increased the amylose content in potato starch [p < 0.05), reduced by 3.12% and 44.53% for HMT and HMT+P, respectively. As previously reported, the increase in amylose content and the reduction in amylopectin content resulted from the breakage of some covalent linkages by excessive heating, and the degradation of the exterior linear chains of amylopectin during HMT [p < 0.05). Overall, HMT+P affected nutritional components most among the four HMT methods.The effects of different treatments on WQ nutrients are shown in fter HMT . This phfter HMT ,28. Addio starch . Additioring HMT ,31. The p < 0.05) from 2.89% (WQ) to 5.32% (HMT), 15.71% (HMT+P), 7.74% (HMT+M), and 12.49% (HMT+A), respectively. The results were consistent with Hung et al. who found that the RS content in rice was significantly increased by HMT [p < 0.05) the SDS content by 19.13%, 36.73%, and 20.27%, respectively. Asranudin et al. found that the SDS content increased to 4\u201316% in purple yam flour after heat-moisture treatment combined with bake treatment [The RS, SDS, and RDS content and eGI value of WQ under different treatments are shown in reatment .p < 0.05). The higher RS content and the lower RDS content led to the lower eGI value.In addition, different treatments reduced the eGI value to varying degrees: 74.36 (WQ) > 70.59 (HMT) > 69.79 (HMT+M) > 69.12 (HMT+A) > 65.87 (HMT+P). The results may be due to the changes of starch profile which reduced the starch digestibility. Among all the treatments, HMT+P had the greatest impact on WQ (0), peak gelatinization temperature (Tp), and conclusion gelatinization temperature (Tc), but lower \u0394H. The gelatinization enthalpy (\u0394H) is defined as the energy required for breaking the molecular interactions within the starch structure during gelatinization, mainly reflecting the dissolution of the double helix structure of starch [The thermal property of different samples is shown in f starch .p < 0.05) from 4.33 J/g to 1.05 J/g (HMT), 1.07 J/g (HMT+P), 0.53 J/g (HMT+M), and 0.64 J/g (HMT+A), respectively. Consistent with our results, the \u0394H of tartary buckwheat flour also reduced from 6.2 to 3.8 J/g after HMT [As shown, all treatments significantly reduced the \u0394H . The results agreed with Delinski et al. who reported that the relative crystallinity of amaranth starch decreased from 32.26% to 28.79% after HMT [HMT not only changed the crystal form of starch granules, but also reduced the relative crystallinity of starch granules. The relative crystallinity was decreased 6.77%, 13.55%, 33.86%, and 52.19% by HMT, HMT+P, HMT+M, and HMT+A, respectively (fter HMT . It was fter HMT . In addifter HMT . In summThe microstructure of WQ with different treatments is shown in Compared with HMT, HMT+P made the original particle structure and properties disappear, such as the rough and irregular surface and the larger particles. HMT+M made the granular structure disappear, the particles became larger, and the surface became rough compared to HMT. Han et al. believed that in the process of microwave radiation, the surface became rough easily due to heat effect from the inside to outside . CompareIn order to observe conjunction of protein and starch of different samples, a CLSM was used, and the result is shown in In conclusion, the nutritional composition, digestibility, and physicochemical property of WQ were modified by HMT, HMT+P, HMT+M, and HMT+A. HMT+P led to the highest TDF, RS content, and the lowest eGI value. All treatments significantly reduced the \u0394H of WQ. The conjunction of protein and starch was observed after four treatments, especially for HMT+M. HMT+A brought the most significant changes in the starch structure, as evidenced by the decrease in relative crystallinity and transformation of starch crystal. Overall, among the four treatments, HMT+P affected nutritional components most among the four HMT methods and HMT+A had the greatest effect on the physical and chemical properties of WQ. The results are expected to provide theoretical support for the development of functional food of quinoa."}
+{"text": "Mycobacterium kansasii can cause serious pulmonary disease. It belongs to a group of closely-related species of non-tuberculous mycobacteria known as the M. kansasii complex (MKC). Here, we report a population genomics analysis of 358 MKC isolates from worldwide water and clinical sources. We find that recombination, likely mediated by distributive conjugative transfer, has contributed to speciation and on-going diversification of the MKC. Our analyses support municipal water as a main source of MKC infections. Furthermore, nearly 80% of the MKC infections are due to closely-related M. kansasii strains, forming a main cluster that apparently originated in the 1900s and subsequently expanded globally. Bioinformatic analyses indicate that several genes involved in metabolism , ESX-I secretion, metal ion homeostasis and cell surface remodelling may have contributed to M. kansasii\u2019s success and its ongoing adaptation to the human host. Mycobacterium kansasii can cause serious pulmonary disease. Here, the authors present a population genomics analysis of 358 environmental and clinical isolates from around the world, supporting the idea that municipal water is a main source of infection, and shedding light into the pathogen\u2019s diversity and adaptation to the human host. Mycobacterium tuberculosis, diseases due to NTM have been an increasing concern in global health4, and in some developed countries, NTM is now responsible for more diseases than M. tuberculosis4. Mycobacterium kansasii is among the most pathogenic NTM and has the highest clinical relevance5. It is one of the last species to have diverged from a common ancestor before the appearance of the M. tuberculosis complex6 and is capable of causing aggressive and destructive pulmonary disease resembling tuberculosis7. In the mid-20th century, before the emergence of the HIV pandemic, M. kansasii was dominant among NTM diseases in several regions of United States, Europe, and Japan3. It is currently one of the most frequent causes of NTM pulmonary disease throughout the world  are environmental bacteria, but some species can cause opportunistic infections in humans. While they are not as pathogenic as rld Fig.\u00a0, with a M. kansasii infections are generally assumed to be acquired from environmental sources rather than by human-to-human transmission. Although municipal water distribution systems are believed to be the major reservoir for human M. kansasii infections14, water isolates are usually genetically distinct from clinical strains. Molecular typing has revealed that M. kansasii comprises at least six distinct subtypes that vary in prevalence and clinical relevance18. Very recently, based on genome-wide average nucleotide identity (gANI), it was proposed that the subtypes should more accurately be designated as closely related species20. The six subtypes were designated as M. kansasii (former subtype I), Mycobacterium persicum (II), Mycobacterium pseudokansasii (III), Mycobacterium ostraviense (IV), Mycobacterium innocens (V), and Mycobacterium attenuatum (VI), which together with Mycobacterium gastri, were recognized as the M. kansasii complex (MKC)20. M. kansasii (former subtype I) is responsible for the vast majority of infections due to the MKC species worldwide but is not often isolated from water sources18, and no definitive epidemiological link has ever been established between water reservoirs and clinical M. kansasii infections22. Instead, genotyping has shown that clinical strains of M. kansasii isolated from diverse geographic locations constitute a homogenous population18, suggesting potential human-to-human transmission of a successful clone. Potential transmission of M. kansasii between family members has been reported in several cases24. In addition, transmission has been recently revealed as a major route for the dissemination of dominant clones of Mycobacterium abscessus25, another NTM that can cause pulmonary infection.As with other NTM, M. kansasii has the highest clinical relevance among the MKC, as it has been associated with severe and even fatal disease in both immune-competent and immune-compromised patients, while the other MKC species are isolated only from immune-compromised patients or environmental sources17. Although M. kansasii causes more disease than the other MKC species, the genetic determinants of its pathogenic adaptation have not been addressed. In addition, clinical M. kansasii isolates can vary phenotypically, with strains showing either a smooth or rough colony morphology due to differences in cell wall hydrophobicity27. Similar to M. abscessus28, M. kansasii strains with the rough colony appear to be more virulent and can establish chronic systemic infections in mice29, but the genetic basis for the phenotypic differences has not been explained.Consistent with its clinical dominance, M. kansasii. The genomic analyses provided insights into its speciation, diversification, the sources of clinical infections, and possible genetic determinants associated with its ability to proliferation and cause disease in humans.In the current study, we analyzed the genomes of a worldwide collection of isolates to better define the global population structure of 20. Four species , each containing more than ten strains, were designated as the major species in the current study. A pairwise comparison of single nucleotide variants (SNV) among the strains of each of the four species revealed a median difference of 1888 to 3717 SNVs along the 2.12\u2009Mbp core genome .The core genome alignment consists of concatenated sequences of discontinuous sequence fragments in each strain, which does not fully represent the features of recombination, i.e., the genomic distribution and length of recombinant fragments. Therefore, recent recombination events were further explored by Gubbins analysis based on whole-genome alignments for each of the four major species. Evidence of recombination was found evenly distributed across the genomes of all four species Fig.\u00a0, with frsii Fig.\u00a0, Fig.\u00a03bAfter excluding the SNVs in the recombinant regions, we inferred phylogeny for each of the major MKC species to investigate the genomic differences between isolates based solely on non-recombinant mutations Fig.\u00a0, Fig.\u00a03aM. kansasii, was named the M. kansasii main cluster (MKMC). It contained 79.2% (244/308) of all the clinical isolates included in the current study. The MKMC contained 20 strains isolated from water sources, including one strain from the Czech Republic that clustered with clinical strains from neighboring Poland. The remaining 19 strains were isolated from an exposed cooling tower linked to a geothermal water source in Portland  during an outbreak of M. kansasii infections in the 1990s. In the maximum likelihood (ML) phylogeny, these water isolates clustered with eight strains isolated from patients in the town, including six patients who were part of the outbreak  predominantly belong to Lineage 2, while nearly all strains from USA and Canada (20/21) belong to Lineage 1. Strains isolated in Europe were the most diverse, constituting several branches in both lineages Fig.\u00a0, and BayD of \u22122.45 and \u22120.93 for individual core genes of the MKMC based on all SNVs or nonrecombinant SNVs respectively , a type VII secretion system associated with virulence in M. tuberculosis35. The genes are distributed in three genomic loci, one of which is comprised almost entirely of ESPs, the WhiB6 regulator, and a PPE protein associated with ESX-1  of all the MKMC isolates were used to identify convergent mutations or genes containing an unusually high number of mutations, which could be evidence of positive selectionzur mutations were identified in 38 clinical isolates, all of which were nonsynonymous or frameshifts  in an additional 35 clinical isolates, with several isolates carrying multiple unlinked mutations , an acyltransferase (papA3), and two glucosyltransferases (gtf1 and gtf2)  and a glucosyltransferase WbbL2), enzymes that are involved in lipooligosaccharide (LOS) synthesisf2) Fig.\u00a0. All thef2) Fig.\u00a0. Furtherrase papA, and two, enzymesl-lactate dehydrogenases  and a lipase , which are involved in lactate and lipid metabolism, respectively. The mutations were exclusively identified in clinical isolates, and an additional 14 clinical isolates were found to harbor fixed or unfixed mutations in these regions  were identified in codon 324 of mce1D, both resulting in the same amino acid substitution, suggesting they were likely gain-of-function mutations. Among the 12 isolates with a recombinant mce1D, five harbored the 970 G\u2009>\u2009C mutation.Convergent mutations that evolved at least three times were identified in the regions upstream of three genes encoding two M. kansasii yielded several insights into the population diversity, epidemiology, evolution, and host adaptation of this important pathogen. The phylogenomic analysis confirmed that previously defined M. kansasii subtypes represent closely related species, as has been recently proposed20. We found ample evidence of ongoing homologous recombination between the species, but no trace of recombination with any other mycobacterium species, further supporting the classification of these closely related species as the MKC20. Extensive recent and ancestral recombination events, likely driven by DCT, resulted in the mosaic genomes observed in the MKC species30, thereby demonstrating the importance of DCT in both the speciation and diversification of these species. Notably, there was evidence of recent recombination in the 16S\u201323S rRNA locus  is unique to M. kansasii and the other two (MKAN_RS11540 and MKAN_RS12085) including the ortholog of M. tuberculosis espA (MKAN_RS12085) are both present in some other MKC species. The fourth paralog is present in all MKC species except M. kansasii. Given the evolutionary complexity, the contribution of espA paralogs to the pathogenicity of M. kansasii is worthy to be further investigated.The comparative genomic analysis revealed genes associated with metabolism and virulence that were found only in the predominant M. kansasii predominantly belong to a homogenous cluster designated MKMC, and its clinical predominance but rare isolation from city water sources raises the possibility of human-to-human transmission. However, by investigating an outbreak of M. kansasii infections in Australia, we found genetic evidence that the patients were more likely to have acquired their infections independently from M. kansasii strains present in the city water system. This is consistent with previous suggestions that city water distribution systems constitute the principal reservoir for M. kansasii52. Our evolutionary analysis estimated that the MKMC originated in the early 1900s, possibly in Europe, although both the proposed date and geographic origin need to be confirmed by sequencing additional isolates from diverse global regions, especially in America and Africa. Considering the association of M. kansasii infections with urban areas, we speculate that the initial expansion of the MKMC was associated with the rapid urbanization of Europe since the 1900s53 and that it then spread and expanded with the urbanization of other global regions. Although it is unclear how the water-born MKMC could have achieved global dissemination during the past century, systems for storing potable water during long voyages could have played a role.Isolates of 54. We observed convergent mutations in a subunit (Mce1D) of a putative lipid/sterol transporter and also in the upstream region of a putative lipase involved in the lipid hydrolysis44, both of which may represent adaptations to a lipid-rich environment within the host. Besides lipids, host cell lactate was recently revealed as an important carbon source for bacterial growth within human macrophages55. More recently, convergent mutations in promoter and coding regions of lactate dehydrogenase gene lldD2 were extensively identified in M. tuberculosis. These mutations were thought to represent an adaptation to changes in host ecology and were associated with higher transmissibility56. We also identified mutations upstream of lldD1/2 in 14 MKMC clinical isolates, emphasizing the importance of lactate metabolism in host adaptation of mycobacteria. The convergent mutations in the regions upstream of lldD1/2 and the lipase gene likely resulted in upregulation of the corresponding enzymes, which could enhance the metabolic capabilities of M. kansasii within the host and facilitate its survival and replication.Several genes involved in metabolism and the stringent response appear to be under positive selections in the MKMC. Host cell lipids are a major carbon source for mycobacteria during infection and are critical for the survival of bacteria within the host58. The highest polymorphism was found in zur, which in M. tuberculosis encodes a transcriptional repressor that regulates the expression of genes involved in zinc and iron uptake and zinc mobilization60. Inactivation of Zur could increase the expression of genes that improve the ability of M. kansasii to compete with the host for the acquisition of zinc and iron  within the host39. The most-studied GBL signaling is the A-factor system associated with secondary metabolism and sporulation in Streptomyces63. Many of the mutation and recombination events in these two genes mapped to inner nodes of the ML phylogeny, suggesting that they could have been acquired before infecting humans, probably in city water distribution systems. While there is no solid evidence of mycobacterium sporulation, inactivation of these regulators could nevertheless modulate bacterial metabolism to facilitate the survival of M. kansasii in the urban water systems, where they would encounter low levels of nutrients and disinfectant residuals. LOS are polar glycolipids associated with cell wall hydrophilicity in several mycobacteria42. We found a high correlation between loss-of-function mutations in these genes and rough colony morphology in clinical isolates, consistent with previous findings in other mycobacteria42. The rough phenotype resulting from the absence of LOS has been associated with enhanced within-host survival and increased virulence in several mycobacteria, including M. kansasii and M. marinum64. In M. tuberculosis, a recent study demonstrated that the loss of LOS occurred in its Mycobacterium canettii-like ancestor and may have played a vital role in its evolution from an environmental mycobacterium to a professional pathogen43. Similarly, the mutations in the LOS synthesis genes of M. kansasii could also represent in-host selection for increased virulence and the ability to establish a persistent infection within the host.Seven genes associated with secondary metabolism showed potential positive selection, with nonsense and/or frameshift mutations likely causing loss-of-function. Among these are two TetR family regulators of GBL signaling and two genes involved in LOS biosynthesis. GBL signaling molecules are involved in the regulation of secondary metabolism and morphological development in actinomycetes65. Several mutations, including those in the LOS synthesis genes and the lldD2 promoter of the MKMC strains, mimic the ancestral adaptations of M. tuberculosis to a human host and suggest that M. kansasii may have the potential to evolve into a professional pathogen. The putative adaptive mutations we identified in the MKMC isolates all mapped to terminal phylogenetic nodes, suggesting that these putatively more human-adapted strains were not transmitted. However, considering the high similarity of pulmonary infections caused by M. kansasii and M. tuberculosis, we cannot exclude the possibility of aerosol transmission of M. kansasii, which would provide opportunities for multiple rounds of host adaptation. This should raise concern that some members of this species, particularly in the MKMC, may have the potential to evolve into highly adapted human pathogens.The extensive selection of mutations in clinical MKMC isolates represents a feature of opportunistic infections, where adaptive mutations are rapidly selected due to the shift from the environment to host nichesN\u2009=\u200922, from the national survey of drug-resistance during 2007\u20132008), Shanghai CDC , and The Third People\u2019s Hospital of Shenzhen  were included. Water isolates (N\u2009=\u200915) were obtained from public tap water across Shanghai in 2015, using a filtration method66. Briefly, 1\u2009l of water was passed through a membrane filter . The membrane was decontaminated by 15\u2009ml of 3% sodium dodecyl sulfate and 1% NaOH for 30\u2009min. The solution was neutralized with 40% phosphoric acid solution and then centrifuged for 15\u2009min at 2000 \u00d7 g. The sediment was then resuspended in about 500\u2009\u03bcl of the supernatant and plated on 7H10 plates. In Canada, clinical isolates  were obtained from the McGill University Health Centre mycobacteriology laboratory and identified as M. kansasii by the Laboratoire de Sante Publique du Quebec by 16S rRNA PCR and DNA sequencing. In Australia, clinical isolates  were referred to the mycobacterial reference laboratory at the Victorian Infectious Diseases Reference Laboratory (VIDRL) and identified by 16S rRNA PCR DNA sequencing. An addition of 28 clinical isolates, collected in 1990\u20131992 from global areas  and stored in VIDRL, were also included67. For clinical samples, specimens were cultured on L\u00f6wenstein Jensen (L\u2013J) slants, and multiple colonies that grew on the slants were scraped for DNA extraction. For water samples, specimens were primarily plated on 7H10 plates, from which a single colony was picked and subcultured on L\u2013J slants. Multiple colonies that grew on the slants were scraped for DNA extraction. Genomic DNA was sequenced on either an Illumina Hiseq 2000 or NextSeq 500 platform in single or paired-end mode with an expected depth of 100. Publicly available genomic sequences and short-read data were downloaded from the Assembly and SRA databases of NCBI respectively  and reverse primer16S-P2 (ACCGCGGCTGCTGGCAC) in these hospitals or by a local or national branch of the Center for Disease Control and Prevention (CDC). Clinical isolates collected by the China CDC (https://ncbi.github.io/sra-tools/). Sequencing reads were trimmed and filtered using Trimmomatic (v0.30)68 and draft genomes were assembled using SPAdes (v3.13.1)69 in the carful mode with reading correction, auto-sized k-mers, and mismatch corrections. The quality of assembly was evaluated using Quast (v5.02)70 and contigs of less than 200\u2009bp were filtered out. Pairwise genomic ANIs were calculated using fastANI (v1.1)71 with default parameters based on the assemblies.Public sequencing data were downloaded from NCBI and then converted into fastq files using the NCBI SRAtoolkit 73, and then homologous genes were clustered using the CD-Hit and MCL algorithms. To generate the core-genome alignment, the parameters were set to a minimum of 90% blastp identity, 100% coverage , and no paralog splitting . Sequences of individual core genes were aligned with MAFFT (v7.407)74 and then concatenated into a core genome alignment according to their genomic coordinates in the reference genome (NC_022663.1). RaxML (v8.2.12)75 was used to construct the ML phylogeny based on the core genome alignment with a GTR model and 1000 rapid bootstrap replications. iTOL (v5.7)76 was used for displaying and annotating phylogenies. SplitsTree (v4.14.5)77 was used to construct the phylogenetic network by the NeighborNet algorithm based on the core genome alignment. The Tajima\u2019s D statistic was calculated for individual core genes of the MKMC using PopGenome (v2.7.5)78 based on all and non-recombinant SNVs, respectively. For identification of M. kansasii specific genes, a minimum of 80% blastp identity with paralog splitting was set in Roary. Genes present in all M. kansasii isolates but absent in all isolates of any other MKC species were selected.Core genes for all MKC isolate included in this study were analyzed by Roary (v3.11.2)79. The core genome alignment was subjected to hierBAPS analysis with a uniform prior on the number of clusters. Genomic recombination was inferred using fastGEAR80 based on the core genome alignment with an integration number of 15 . The fastGEAR used BAPS to define the \u201cbest\u201d number of clusters and then detect \u201clineages\u201d that are genetically distinct in at least 50% of the alignment. fastGEAR detects both ancestral recombinations that affect all isolates in a lineage as well as recent recombination that affects a subset of isolates in a lineage. For each ancestral recombination, the larger lineage was assumed to be the donor (by default).Population structure was inferred using hierBASPM. kansasii, M. persicum, M. pseudokansasii, and M. attenuatum) were further explored with Gubbins (v2.3.4)81. The whole-genome alignment of each species was generated by an in-house pipeline based on Minimap2 (v2.17)82. Briefly, the contigs of individual strains were aligned to the reference genome by Minimap2 with the preset parameter \u201casm20\u201d for the alignment. By filtering secondary and short alignments (<200\u2009bp), the nucleotide corresponding to the reference genome at each site is determined to generate the \u201cpseudogenome\u201d for each isolate. Pseudogenomes of each species were concatenated to make a whole-genome alignment, which was subjected to Gubbins for recombination identification with default parameters. The donors of the recombination fragments were determined by a BLAST (v2.9.0)83 search against a local database containing all of the MKC genomes included in the current study and the representative reference genomes for other mycobacteria collected in the NCBI database. A cutoff value of identity was set to 99% to identify the probable donor strains. The outputs from Gubbins were viewed with Phandango (v1.1.0)84, or by an in-house python script that plots the recombinations with information including genomic coordinates and donor species.The recent recombinations in isolates of the major species /VarScan (v1.4.3) pipeline87. Variants were called at loci where the alternative base calls were supported by at least five reads that aligned to the reference in both forward and reverse directions. Variants in repeat regions, putative PE/PPE family genes, and transposable elements were excluded. Variants supported by \u226595% of the mapped reads were defined as fixed/homozygous mutations, otherwise, variants were defined as unfixed/heterozygous mutations. The homozygous SNVs in non-recombinant regions detected by both mapping- and assembly-based analysis were used to construct the ML phylogeny of the MKMC by RaxML based on the GTR model. We found several extraordinarily long terminal branches in the ML phylogeny for isolates from PRJ374853, which likely represent assembly errors, and the corresponding terminal branches were thus truncated to 0. Network (v5.0)88 was used to generate median-joining networks for the outbreak strains from Australia based on the concatenated SNV sequences.We applied mapping-based analysis to study the genetic variants among the MKMC strains. The trimmed reads were mapped to the reference genome ATCC 12478 by Bowtie2 (2.3.5)89. The BBM method inputs the posterior distribution of Bayesian inference to reconstruct the possible ancestral distributions of given nodes via a hierarchical Bayesian approach. The ML phylogeny of the MKMC constructed in the above section was used for the analysis. The strains were classified into six geographic regions based on where they had been isolated: East Asia, Australia, Europe, North America, South America, or South Africa. The Bayesian analysis was run with a fixed JC model for 5,000,000 cycles, 10 chains, a temperature parameter of 0.1, with sampling every 100 generations. Bayesian dating of the phylogeny was based on a subset of 121 strains of the MKMC with short-read data (to exclude assembly errors in publicly available genomes), clear dates of isolation, and genomes with proportions of recombinations <1.0%. The reference strain ATCC 12478, which was isolated in 1953, was also included. The temporal signal in the sequence alignments was investigated using TempEst (v1.5.3)90. As a complementary assessment of the temporal signal in the data, a date randomization test was performed on our datasets with the R package TipDatingBeast (v1.1-0)91. Sampling dates of the strains were randomly shuffled 20 times, and the randomized datasets were analyzed with BEAST (v1.8.0)92 using the same parameters as for the original datasets. If the 95% HPD intervals of root-height obtained from the original data do not overlap with the estimates obtained from the randomized datasets, a statistically significant temporal structure could be confirmed. BEAST was used to determine the timescale and the evolutionary rate of the MKMC using the tip-date calibration based on the whole-genome alignment. We used an uncorrelated lognormal distribution for the substitution rate and constant population size for the tree priors. The analysis was done in three chains of 5\u2009\u00d7\u2009107 generations sampled every 1,000 generations to assure independent convergence of the chains. Convergence was assessed using Tracer (v1.6)93 to ensure that all relevant parameters reached an effective population size of >100.The geographic origin of the MKMC was analyzed using the Bayesian Binary MCMC (BBM) method integrated into RASP (v4.0)38. To identify convergent mutations, non-recombinant homozygous mutations were analyzed against the maximum-likelihood phylogeny by TimeTree (v0.6.4)94. Homoplastic mutations independently evolved at least three times were identified as under potential positive selection. A circular plot was created using ClicO FS (v1.0)95 to display gene loci, recombination, and mutation densities of individual genes.Genes with convergent mutations or high numbers of non-recombinant mutations could have been subject to positive selection. A subset of 247 MKMC isolates with short reads data was used for the analysis. To identify genes with multi-diverse signatures, the non-recombinant homozygous mutations of all 247 MKMC isolates were used to calculate the mutation density (number of mutations per gene). Under the neutral evolution model, the number of substitutions per gene is expected to follow a Poisson process. The 95% confidence interval of mean predicted values from a Poisson distribution was estimated based on the Wald interval for the mean, and a significant deviation from the interval was taken as a signal of potential positive selectionFurther information on research design is available in the\u00a0Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Reporting Summary"}
+{"text": "Homo sapiens and Drosophila melanogaster and using Monte Carlo methods, we tested whether retrogenes exhibit significantly different linkage relationships than expected under a null assumption of uniform distribution in the genome. Overall, after excluding genes involved in the out-of-the-X pattern, no general pattern was found associating genetic linkage and retrogene survival. This demonstrates that selection on linkage may not represent an overarching force in retrogene survival. However, it remains possible that this type of selection still influences the survival of specific retrogenes.In retrogene evolution, the out-of-the-X pattern is the retroduplication of X-linked housekeeping genes to autosomes, hypothesized to be driven by meiotic sex chromosome inactivation during spermatogenesis. This pattern suggests that some retrogene survival is driven by selection on X-linkage. We asked if selection on linkage constitutes an important evolutionary force in retrogene survival, including for autosomal parents. Specifically, is there a correlation between retrogene survival and changes in linkage with parental gene networks? To answer this question, we compiled data on retrogenes in both  RMGDs epulation . Even nopulation . From anpulation , and conpulation . Other rpulation . Retrogepulation . While nOne aspect of retrogene survival that has been left relatively unexplored is the impact of linkage. Because RMGD creates new gene copies with different linkage relationships, selection on linkage may influence the fate of retrocopies. Survival of retrocopies may be a path for mediating selection on linkage, in contrast to a direct modification of recombination rate. An RMGD-based model of linkage modification would thereby complement the modifier allele models proposed by Nei in 1967 and built upon in later decades by Feldman, Barton, Otto, and others . An RMGDUtp14b renders males completely deficient in spermatogenesis  (D. melanogaster (accessed 1/17/21) (D. melanogaster: FB release 6). After removing retrocopies with missing information , a total of 4,426 retrocopies were found for humans, with 106 retrogenes. Retrocopies were paired with 1,431 parental genes with 809 unique network partners. In D. melanogaster, 82 retrocopies were found, 81 of which were retrogenes. These retrocopies were subsequently coupled with 64 parental genes with 109 unique network partners. Genetic distances were estimated using recombination maps for intrachromosomal parent-retrogene pairs; for interchromosomal pairs, distance was set to a default value of 0.5. (Retrogene data was collected from 3/21/21) . Regulat1/17/21) . Assembl of 0.5. .D. melanogaster dataset. As mentioned, the human data contains many more pseudoretrogenes than the D. melanogaster data; this difference is likely an artifact of data collection and prior research directions rather than an indication of the true rate of pseudogenization. Most of the data on human pseudogenes originates from D. melanogaster. Additionally, the density of regulatory networks differs between humans and D. melanogaster, with the latter exhibiting much denser and well-connected networks. Whether this is biological truth is a different question; however, for this study, we assume that the network data we have is an unbiased sample from the true relationships.There are a number of significant differences between the human and dplyr, rentrez, chromPlot, GenomicFeatures, MareyMap and reshape2 . All code and data used in this study are available via the author\u2019s GitHub repository (https://github.com/johnathanlo/retrogenes).Samples of random retrogenes are simulated by drawing genomic coordinates from the above distribution. Relevant statistics (described later in the Results) are then calculated. For each of our tests, this process was iterated 1,000 times to produce an approximate reference distribution for the relevant statistic. Test statistics were then calculated using the empirical data and compared against the reference distribution using a two-tailed test . The distance function is computed by applying recombination maps to the genomic coordinates of the parent/retrogene pair, with a default value of 0.5 for pairs on different chromosomes. The reference distribution was constructed as before by simulating a set of random parental genes and a corresponding set of random retrogenes and calculating the above statistic for 10,000 realizations. The test statistics were calculated from the data. No significant results were observed for either human or D. melanogaster . We define the mean over the population of parental genes asX3 using Monte Carlo methods as before, then compute the estimateTo obtain a distribution over the sample mean, we simulate the statistic xi assumed to be i.i.d. instances of X3 as defined above, and test at a significance level of \u03b1 = 0.05. The results show that lack of significance persists regardless of whether genetic distance is weighted by retrogenes or by parental genes  is the genetic distance between a and x as defined in the previous section; note dist  is a random variable, so X4 is random. Then we define the mean over the populationxi i.i.d. samples from X4 and test at a significance level of \u03b1 = 0.05. This test finds no significant deviation of the sample statistic from the expectation under the null for either humans or D. melanogaster  may mask signals from detection by our methods. Indeed, we know that in mammals and Drosophila, the out-of-the-X pattern is a definitive example of selection against linkage, while other studies have demonstrated that proximity may sometimes be selected for to derive the benefits of nearby regulatory regions or open chromatin formations  Distribution of retropseudogenes across the human genome. (B) Distribution of parental genes across the human genome.  Testing the out-of-the-X hypothesis in humans and Click here for additional data file."}
+{"text": "Photobacterium galatheae via mass spectrometry. Nerpa supports searching new genomes against thousands of known NRP structures, and novel molecular structures against tens of thousands of bacterial genomes. The availability of these tools can enhance our understanding of NRP synthesis and the function of their biosynthetic enzymes.Microbial natural products are a major source of bioactive compounds for drug discovery. Among these molecules, nonribosomal peptides (NRPs) represent a diverse class of natural products that include antibiotics, immunosuppressants, and anticancer agents. Recent breakthroughs in natural product discovery have revealed the chemical structure of several thousand NRPs. However, biosynthetic gene clusters (BGCs) encoding them are known only for a few hundred compounds. Here, we developed Nerpa, a computational method for the high-throughput discovery of novel BGCs responsible for producing known NRPs. After searching 13,399 representative bacterial genomes from the RefSeq repository against 8368 known NRPs, Nerpa linked 117 BGCs to their products. We further experimentally validated the predicted BGC of ngercheumicin from  Nonribosomal peptides (NRPs) are promising natural sources of antibiotics, immunosuppressants, anticancer agents, toxins, siderophores, pigments, and cytostatics . StartinIn contrast to regular ribosomal peptides encoded by short genes (about 1000 nucleotides long for prokaryotes), NRP production involves the coordinated action of giant biosynthetic gene clusters (BGCs) spanning hundreds of thousands of nucleotides. These BGCs encode multi-modular proteins, NRP synthetases (NRPSs), responsible for the assembly of NRP products. An NRPS BGC consists of one or more genes composed of NRPS modules wherein each module incorporates an amino acid into a final product . Each moThe cracking of nonribosomal code  enabled In non-collinear NRPS assembly lines, the order of genes in a BGC may deviate from their activation order due to rearrangement, skipping, stuttering, and iterative reuse of genes ,20. BesiIn a recent landmark study, MIBiG, a community-driven database of BGCs and their experimentally validated molecular products has been collected . As of AThe SeMPI web server addressed some of the antiSMASH limitations . It provGARLIC is a three-step approach to linking known NRPs and polyketides to their BGCs . First, http://cab.spbu.ru/software/nerpa (accessed on 30 September 2021).Here we present Nerpa, a method for screening genomes against NRP databases and linking predicted BGCs to their products. The tool works with non-collinear NRPS assembly lines and outperforms GARLIC in accuracy and efficiency. We demonstrate Nerpa performance by searching 13,399 bacterial genomes against 8368 NRPs. The run revealed known and novel BGC-NRP pairs, including a putative ngercheumicin BGC, experimentally validated via mass spectrometry. Nerpa is freely available as a command-line tool from Nerpa takes as input an NRP database and nucleotide sequences including complete genomes and draft assemblies . The pipmonomers), which include amino acids, lipid tails, and other types of residues. We distinguish between the monomers that originated from the decomposition of NRP structures (NRP monomers), and those predicted by genome mining (BGC monomers). Each monomer is typically identified by the core amino acid, its stereochemistry (D-/L-configuration), and whether it is methylated or not. BGC monomers additionally include specificity prediction scores. The alphabet of supported core amino acids is the same for both NRP and BGC monomers, and contains 58 residues Steps (i)\u2013(iii) operate with NRP building blocks  and revealed its vulnerability towards short BGCs with uncertain A domain specificities We downloaded all 13,399 reference and representative bacterial genomes from the NCBI RefSeq database  Supplem. Our tooNerpa identified numerous tentative connections between the RefSeq genomes and pNRPdb structures. We limited our analysis to the BGC-NRP pairs where the BGC genome is the best hit for the particular NRP structure and the NRP structure is also the best hit for the BGC. To be conservative, we kept only the pairs where the genome matches the genus of the original producer of the compound retrieved from the pNRPdb metadata. The resulting set links 117 BGCs to their putative products Microcystis aeruginosa, albeit without a link to the particular compound variant reported by Nerpa (<L-MeSer7>microcystin-LR). NCBI BLAST [Photobacterium galathea is novel since no analogs can be found in any database.BI BLAST  matched BI BLAST ,40. We mP. galathea S2753 [We cultured ea S2753  in diffeea S2753  as part ea S2753 , Derepliea S2753 , and Molea S2753  workflowPhotobacteria spp. [P. galathea mass spectra as ngercheumicins A and B and their derivatives with p values down to The ngercheumicin family comprises structurally similar variants A-B and F-I produced by ria spp. . Dereplientation . Dereplientation . The netP. galathea while the Nerpa alignment suggests its tentative biosynthetic pathway Our experiment validates the production of ngercheumicins by Breakthroughs in sequencing technologies enabled genome sequencing of thousands of NRP-producing organisms. Although the development of genome mining software, such as antiSMASH, facilitated high-throughput search for BGCs in these data, genes responsible for synthesis of most of the known NRPs still remain undiscovered. Currently, MIBiG, the largest community-curated BGC database, represents a minuscule fraction of all potentially known BGC-NRP pairs. While the expansion of MIBiG is of utmost importance for natural product research, it requires a lot of time-consuming manual work. Here, we demonstrate how a single push-of-a-button Nerpa run can be used for populating the MIBiG repository. First, our tool automatically finds known NRPS BGCs currently missing in MIBiG, such as ohmyungsamycin and stephensiolide. Second, Nerpa reveals novel BGC-NRP connections, which can further undergo experimental validation as shown for ngercheumicin.The small size of the available training data limits Nerpa accuracy. At the same time, our tool could be used for an iterative extension of the training set via collecting new trustable BGC-NRP pairs in a semi-automated fashion. This will allow both retraining of the current Nerpa parameters and development of a more sophisticated scoring. For instance, we may consider additional monomer modifications such as formylation. Furthermore, the enhanced training data will help to improve Nerpa\u2019s capability to analyse NRP-polyketide hybrids and even polyketides. Still, besides being primarily designed for NRPS BGCs, the current tool correctly linked microcystin-LR, an NRP-polyketide hybrid, to its gene cluster.We envision two main Nerpa applications in routine NRP research. Nerpa may match recently sequenced bacterial genomes against NRP databases to differentiate BGCs producing known versus novel compounds and thus prioritizing strains for the follow-up studies. Our tool may also screen recently elucidated NRP structures against large genomic databases to find their putative producers NRPS BGCs. The revealed BGCs could be useful for enhancing the NRP synthesis via heterologous expression and for searching or\u00a0bioengineering novel variants of the compound. To make the tool application fast and convenient, we provide all Nerpa-preprocessed chemical and genomics datasets used in this study. For further convenience, we are preparing a Nerpa web interface that will be integrated with the antiSMASH web services. We believe our software will benefit the community and facilitate the search and discovery of novel bacterial NRPs. Further development of genome mining software will help to extend the Nerpa functionality to other NRP producers such as fungi, plants, and sponges.NRP processing normally starts with a database of chemical structures in the isomeric SMILES format and consists of several steps explained in detail below . Non-isoNerpa converts the input structures into monomer graphs using rBAN . The monNerpa post-processes the rBAN output to infer monomer stereochemistry and recognize unidentified monomers. While rBAN ignores the stereochemistry, Nerpa inspects all chiral centers in the original chemical structure, allowing it to determine the stereoisomeric configuration (D-/L-) of the monomers. While rBAN performs the retro-biosynthesis of NRPs only, Nerpa supports NRP-polyketide hybrids . rBAN igWe classify edge annotations in the monomer graph into the backbone and tailoring classes. The backbone class consists of bond types that can be attributed to the activity of core NRPS modules. This class includes amide and double amide bonds and heterocycles such as thiazole, oxazole, and pyrimidine. All other bond types form the tailoring class. We also classify all graph nodes into Nerpa-supported and unsupported monomers. The former category contains monomers that can be predicted from a BGC c,d. The Nerpa starts linearization of a monomer graph by removing all tailoring bonds . Then thNerpa accepts genome sequences in the FASTA or GenBank format and processes them with the antiSMASH v5 genome mining pipeline . Users cNerpa converts each NRPS module into a BGC monomer. The tool considers A domains, which define the core amino acids, and M and E domains, which determine whether the amino acid is methylated and its stereochemistry. A module may also contain a dual function C/E domain  that is BGC monomer strip, or simply a strip, of an NRPS gene as a sequence of BGC monomers corresponding to the gene\u2019s modules. A strip may also include additional information, such as the presence of CS and TE domains in the gene  actually contains two clusters responsible for synthesis of syringomycin and syringopeptin . Erroneously merged BGCs complicate downstream analysis and deteriorate Nerpa results. To address this problem, we process each multi-gene BGC with two simple heuristics.First, Nerpa calculates the distances between adjacent NRPS genes in a BGC and if a distance exceeds a user-configurable threshold BGC monomer sequence or a set of plausible sequences reflecting the (unknown) NRPS assembly line of a BGC  BGC monomer strips. In addition to CS and TE domains discussed above, Nerpa also considers communication-mediating (COM) domains . COM domGiven a BGC, Nerpa first assumes the collinear NRPS assembly line and constructs the BGC monomer sequence accordingly. If the sequence is consistent, the processing is complete. If the inconsistency is caused solely by violation of conditions (i) or (ii), the tool puts the corresponding strip to the very beginning or ending of the sequence The Nerpa scoring module takes as an input one or several possible NRP and BGC monomer sequences e. We perLet ith monomer is m. A global alignment of A string Given an NRP monomer sequence We assume the independence of the aligned monomer pairs and rewrite Equation  as:(2)ScFor simplicity, we also assume that the matches of core amino acids, their methylations and stereochemistry affect the total probability independently. Therefore, Equation  is a sums to synthesize a core NRP monomer amino acid a may appear in an NRP by random chance, that is, how frequent a is comparing to all other NRP amino acids, Consider an alignment without indels. We compute the probability of a BGC module with substrate prediction m and e monomer components have at most three different states ..P(NRP\u2032|BThe Nerpa scoring relies on two major groups of parameters. The first group consists of f Norine  were useThe training of the remaining parameters requires known BGC-NRP alignments. For this purpose, we formed a dataset of 64 representative NRPS BGCs from MIBiG v2 . For eacBefore the training, the real specificity score scale We generated 100 equally-sized bootstrap samples from the training dataset #2 and used them for parameter learning. In the final step of the pipeline, Nerpa summarizes scoring results for all inputs e. First,Nerpa is implemented in C++ and Python v3. The NRP structure decomposition and linearization relies on rBAN , the RDK"}
+{"text": "Dear Editor,Osteosarcoma (OS) is the most common primary malignant tumour of bone with variable molecular biology and prognosis. This makes better patient stratification and precision treatment an urgent clinical need.In this study  [|log2 (fold change)|\u00a0>\u00a0.5 and adjust ] Figure . To our ] Figure  and B. E] Figure  and S1C,Meanwhile, transcriptomic analyses on 24 matched tumour and normal tissues collected in our hospital (hereafter referred to as Zhengzhou cohort) enriched similar Hallmark pathways such as protein secretion, UPR and MYC signalling Figure\u00a0 and E. CBased on this signature, we constructed a set of scoring systemFrom a translational standpoint, we were further interested in developing a prognostic signature consisting of a handful of genes with higher power. Based on the classification strategy above, we applied Kaplan\u2013Meier survival analyses coupled with univariate Cox regression to GSE21257 and TARGET cohorts and identified 21 candidate DEGs Table . Using G+ T cells, monocytes and M2 macrophages, and lower proportion of memory resting CD4+ T cells and M0 macrophages in the low\u2010risk subgroup (Figures\u00a0Additionally, we observed significant enrichment of multiple immune\u2010relevant gene signatures according to pathway enrichment analyses Figures\u00a0. In fact Figures\u00a0. Interes Figures\u00a0. Submap  Figures\u00a0, which wIn conclusion, our study underlines that UPR activation is a common molecular feature of OS, and offers a novel prognostic gene signature refined from this perspective with translational value Figure\u00a0.The authors declare no conflict of interest.SUPPORTING INFORMATIONClick here for additional data file.SUPPORTING INFORMATIONClick here for additional data file."}
+{"text": "In this paper, additive manufacturing was used in order to produce hose prototypes for peristaltic linear pneumatic actuators. In order to optimise the endurance of the actuator, we 3D printed different thermoplastic polyurethane elastomers with different shore hardness levels using ARBURG Plastic Freeforming technology. Furthermore, effects of the hose geometries on the lifetime of the actuator were investigated. Experimental evidence showed that the lifetime of the actuator was dependent on the combination of the hose design and on the material used to manufacture the hose. Moreover, experimental tests showed that the use of the Aurburg-Freeformer 3D printing technology led to a much higher hose endurance than the one reported by using the fused layer manufacturing technique. Interest in development of interactive robots capable of co-working and interacting with humans is gaining significant attention from different industries in recent years. Following this, pneumatic actuated systems could play a significant role in this context, as they are a simple solution to ensure safety requirements in cases of human\u2013robot contact and offer motion capabilities which are not achievable using more rigid actuation solutions ,2,3. In Among the recent reported actuation systems, peristaltic linear pneumatic actuators (PLPA) have been concluded to be a simple, cost-efficient alternative to other actuators ,15,16, wPLPA actuators are driven by pneumatic energy. They function on the basis of a moving body composed by two rollers that press a hose until it becomes sealed in the middle, creating two separate isolated chambers (see ). When aPLPA can be used to perform curved profiles, can be manufactured with low cost, and can also be built with very long strokes. These characteristics, along with a favourable friction behaviour for servo control applications, make them very attractive in comparison with conventional pneumatic actuators. More details on the friction characteristics of PLPA can be found in . HoweverIn order to explore the causes and potential solutions for this drawback, we conducted initial investigations using conventional hoses. These studies showed that the hoses typically fail due to crack formation in regions where the hoses fold ,18. RegaAs the next step, this study focused on the following points.Effect of the 3D printing technique on the PLPA endurance: ARBURG Plastic Freeforming 3D printing technique was used for manufacturing of the hose prototypes. This technique was chosen as it uses the droplet injection method, which appears to yield in more isotropic prototypes when compared to the FLM technique.Effect of the TPE material type (hardness) on the PLPA endurance: in this study, TPE materials with shore hardness levels of 60 A and 82 A were chosen for the printing material. TPE of 60 A is relatively more flexible when compared to 82 A. As such, circular hoses and the geometrically reinforced hoses were printed with both materials, and their corresponding endurances were tested in the PLPA setup.To this end, several hoses with designs similar to the ones used in  were pri3D printing experiments were conducted using two materials: material 1, TPE 60 A , and material 2, TPE 82 A . The mechanical properties of the used materials are listed in Table 2The experiments were conducted using ARBURG Plastic Freeforming (APF) technology with a freeformer 200-3X machine from ARBURG, Lossburg, Germany. The machine uses granulates as the raw material and plasticises them in a similar process to injection moulding. The molten polymer is discharged by the axial movement of the plasticising screw with a specific pressure. A nozzle closure system pulsed by a piezo actuator discharges up to 200 droplets of plastic per second with a diameter between 0.2 and 0.4 mm. Moreover, the precise positioning of the plastic droplets on previously calculated points is conducted by a movable three-axis build platform. This process is repeated in a layer-wise manner until the 3D object is manufactured. The machine has a build chamber volume of 154 mm \u00d7 134 mm \u00d7 230 mm. The build chamber can be heated up between 50 and 120 \u00b0C. The granulates are dried at 80 \u00b0C for 3\u20134 h before being guided to the print head. Pre-drying of the granulates avoids material processing problems that lead to bubble formation and thermal degradation due to moisture. A schematic view of the machine print principle is shown in \u25cbSpeed of axes when moving from point to point without discharge of material.Feed rate part carrier:\u25cbSpeed of the axes when discharging the filling of the part.Feed rate, continuous extrusion:\u25cbMaximum displacement speed of the axes when discharging the contour.Feed rate, discrete extrusion:\u25cbThis parameter describes the ratio of the width to the height of a droplet after it has been discharged.Drop aspect ratio:\u25cbThe material discharge is a nozzle flowrate parameter which determines the droplet volume.Material discharge:It should be noted that the discrete extrusion is used for printing the outside perimeters (contours) where a high geometrical accuracy of the printed part is desired. The continuous extrusion is used to print the infill of the printed parts in order to increase the printing speed.Initial trials were conducted for process parameter optimisation of TPE 60 A material. The first print attempts were performed using the available settings parameters of the APF software and material data sheet of TPE 60 A. In the design of the experiments, nozzle temperature and layer thickness were set to 200 \u00b0C and 0.2 mm, respectively. Further parameters such as build chamber temperature, extrusion speed, drop aspect ratio, and material discharge were varied. The examined parameters for the process optimisation of the TPE 60 A are listed in Afterwards, optimised process parameters for TPE 60 A material were used for printing the TPE 82 A material. The printing results have shown that TPE 82 A can be successfully printed using the optimised process parameters of TPE 60 A material. It should be noted that the printing angle for all hose geometries were set to 90\u00b0. Printing without support structures resulted in nonhomogeneous structures within the top layers. This nonhomogeneous structure was caused by the vibration of the flexible layers during the print as they came in contact with the nozzle.As such, all hoses were printed with internal support structures in order to minimise the vibration of the printed hoses caused by their flexible nature. The use of double acting pneumatic actuators requires the existence of two independent chambers. In the case of linear peristaltic actuators, these chambers are formed by the compression of the hose between two rollers. To avoid leakages, which would negatively influence the energetic efficiency of the actuator, the rollers must tightly compress the hose. In order to control the compression force, we used adjustment screws connected to a spring, as detailed in . The sprPrevious results in the literature showed that the configuration presented in The adjustment of the two spring washers was made in such a way that the leakage between chambers was lower than 5 slpm, regardless of the hose type. These leakages were determined using a mass flowmeter (Hastings HFM 201) and a pressure-reducing valve (Numatics Sentronic D), set for 3 bar (relative) working pressure. Further details can be found in .The motion of the carriage was obtained by connecting the actuator see  to a 5/3As mentioned in introduction, in this work, two hose designs were considered: designs A and B. Each design was printed using two different materials with different hardness levels. Material 1 is a soft and flexible (hardness 60 Shore A) material, while material 2 is a stiffer one (hardness 82 Shore A).Ps = 3 bar. The leakages between the PLPA chambers were measured at the beginning of the experiments and during the trials, before the hose failed. No significant differences in these measurements were found.Using the experimental setup presented in At the beginning of the trials, the hose compression force was estimated, as described in The PLPA endurance was tested for designs A and B and materials 1 and 2, as described in the previous sections. For each configuration\u2014A1, A2, B1 and B2\u2014three samples were tested. The average of the number of performed cycles for each hose is presented in Analysing Two main mechanical stress causes are present in the normal working operation of a PLPA: (i) the stress caused by internal pressure and (ii) the stress caused by the compression force of to the rollers. In previous studies using conventional circular-shaped hoses, it was found that all tested hoses failed due to cracks on the hose folded edges. This suggests that cause (ii) is the predominant one when compared with cause (i). This was the reason why design B was developed, in order to reinforce the hose folded edges. Although design B has the potential geometrical reinforcement advantage, it also has the potential disadvantage of leading to localised stress at the folded areas , which mOne possible justification to this fact is that softer materials tendentially lead to lower compression forces for similar sealing, as seen from the results presented in Another possible justification to this lies in the fact that the benefits of design B might somehow be compromised with material 1 as the softer material 1 does not allow the hose to maintain its shape when being pressurised. In fact, with the stiffer material 2, design B hoses tend to maintain its shape when being pressurised, in contrast to the printed hoses with the softer material 1. To illustrate this phenomenon, It can therefore be concluded that a compromise must be found between material and design in order to increase the hose longevity: in a softer material, effect (i) is predominant, and therefore design A might lead to higher endurance since it potentially leads to a more homogenous stress distribution. Furthermore, since the use of softer materials does not allow the hose to maintain design B shape, the benefits of this design might be compromised. In a stiffer material, effect (ii) appears to be predominant, and therefore design B with stiffer materials appears to lead to higher endurance. Further studies should therefore be made to explore materials with intermediate characteristics between materials A and B in an attempt to maximise longevity.Finally, it should be underlined that the endurance results obtained by hoses printed using the ARBURG Plastic Freeforming technology are significantly higher than the ones obtained with the fused layer manufacturing technique . This coIn this study, several hoses for PLPA prototypes were printed using the ARBURG Plastic Freeforming technology. Two types of TPEs were used for printing different designs . The 60 mm stroke hoses were experimentally tested for the 3D printed hoses in a PLPA setup. The experiments were conducted at 3 bar pressure, and the hose endurance was measured by running back and forth cycles.Results showed that printed circular hoses using TPE 60 A  underwent four times more life cycles when being compared to the circular hoses printed using TPE 82 A . On the other hand, printed hoses with geometrical reinforcement at the folding areas using TPE 82 A underwent five times more life cycles when being compared to similar designed hoses printed using TPE 60 A. It can therefore be concluded that the influence of the hose design on its endurance is dependent on the type of hose material. In fact, printed hoses with more flexible materials require less clamping forces, therefore contributing to increasing the hose endurance, but also being more affected by designs that introduce stress concentration factors. Finally, the obtained PLPA endurance by printed hoses using ARBURG Plastic Freeforming technology are significantly higher when being compared to the results obtained with fused layer manufacturing technique. As such, it can be concluded that the ARBURG Plastic Freeforming technique is more suitable for prototyping of PLPA actuators. Future studies will focus on the use of a PLPA for water hydraulics as it has a lower ecological footprint."}
+{"text": "Methods and Material. An in vitro experimental study that included eighty hemisected human root distributed into 8 groups: G1- distilled water; G2- 1% NaOCl/17% EDTA; G3- hypochlorous acid 0.025% HOCl, G4- 1% NaOCl/0.025% HOCl/17% EDTA, G5- 0.2\u2009g/100\u2009mL CS, G6- 1% NaOCl/0.2\u2009g/100\u2009mL CS, G7- CSnp, and G8- 1% NaOCl/CSnp. The wettability analysis calculated the contact angle (\u03b8) between a drop of a blood-like and root dentinal surface; topographic characterization with scanning electron microscopy (SEM) quantified the diameter and number of tubules per area; spectroscopy infrared analyses (IR-S) identified chemical changes in the inorganic (phosphate/carbonate) and organic phase (amide/methyl). Statistical analysis: a linear mixed model, Kruskal\u2013Wallis, and Holm\u2013Bonferroni correction (P\u2009<\u20090.05) were used.  To compare the effect of CS and CSnp on the wettability in root dentine with other irrigation protocols with an experimental P\u2009=\u20090.0001)) was found. A mean value of 67\u00b0\u00b1\u00b0for experimental groups (P\u2009=\u20090.07) was found, and we did not identify differences between them. The SEM identified greater tubular opening and erosion for G4 and greater dentinal permeability per area for NaOCl/CS. IR-S identified dentinal organic integrity with NaOCl-CS/CSnp compared to organic reduction promoted for NaOCl/EDTA.  Significantly higher wettability for G2 (27.1 (in vitro dentin determined an indirect association between the wettability and organic contents. The oxidative effect of NaOCl could be neutralized by CS-CSnp, and consequently, the wettability of the substrate decreases. This  The process of chemical conditioning the radicular dentin for orthograde or regenerative endodontics comes with tackling challenges such as disinfection, dilution of organic tissue, penetration capacity, and dentin chelation . When roDespite the irreversible ultrastructural alterations that deproteinate or demineralize the dentin matrix with the use of irrigants , sodium Wettability as physical property is quantified through a contact angle between a liquid and a substrate , 6, and In this perspective, 5.25% NaOCl and 17% EDTA solutions have been found to exhibit hydrophilic interactions with contact angles of 22\u00b0 and 55\u00b0, respectively, thus displaying adequate wettability for disinfection and adhesion of materials or cellular networks  on mean showed the largest tubular area of 5.1\u2009\u00b5m, marking a difference with the other analyzed groups  (P\u2009=\u20091) (P\u2009=\u20090.0006)  . Lastly,2e\u2009\u02d7\u200916) . For the (P\u2009=\u20091) . However\u20090.0006) .\u02d71), compared to distilled water as a negative control, confirming the chelating action of EDTA and CS in dispersion or CSnp  for the organic components (amide and methyl groups) was found to have decreased for [1%NaOCl\u2009+\u200917% EDTA], compared with the negative control, the HOCl, 0.2% CS, and CSnp, confirming the integrity of the organic component of dentin in the absence of NaOCl  and CSnp (63.2\u00b0) generated less surface energy on the dentinal substrate compared to conditioning with the [1% NaOCl\u2009+\u200917% EDTA] solution (27.1\u00b0). This difference allowed establishing its relation with the results observed in the topographic and chemical analyses after conditioning. Collagen and hydroxyapatite in dentin act antagonistically on contact with a liquid. The collagen present in the substrate possesses less capacity to break the molecular interactions, thus generating low surface energy. An opposite effect is created by the inorganic portion represented in the apatite crystals . AccordiHu et al.  observedC=O vibrations of the amide groups as part of the dentinal collagen is oxidized in [1% NaOCl\u2009+\u200917% EDTA]. On the contrary, with the presence of nonhydrolyzed collagen fraction for the CS and CSnp group, conditioned with NaOCl in equal concentration and time, corresponding to less oxidative activity on the organic component, by the action of the CS or CSnp, the wettability values identify this change.Therefore, if the organic fraction of dentin reduces the free energy on the surface of the substrate , then grTartari et al.  confirmeFor chelating agents, the wettability of the substrate is related to the surface roughness, which is significantly modified during demineralization . TherefoThe results of this research support that CS acts as a chelating agent ; howeverin vitro model to analyze the wettability of the dentinal substrate could be the first step to understand the role of chemical conditioning for cellular interaction.Wettability is a physical property that allows the interaction of the dentinal tissue with cellular materials or components , 7. ThisAn increase in chelation ability increases cell adhesion . In contin vitro model to evaluate the wettability prior to regenerative endodontics. The irrigating solutions modify the dentin wettability, and all wettability values are within the accepted therapeutic range. No indirect association between wettability and the integrity of the organic contents from the dentin was observed; therefore, the highest wettability achieved in the combination NaOCl\u2009+\u2009EDTA was related to the highest organic oxidation; on the contrary, the implementation of chelating agents such as CS and CSnp demonstrated apparent stability of the organic fraction of root dentin previously treated with NaOCl.The use of BLS allowed formulating an"}
+{"text": "Moreover, the activity of both PARP1 and PARP2 is suppressed upon their heteroPARylation. Taken together, our findings suggest that PARP2 can function differently in BER, promoting XRCC1-dependent repair (similarly to PARP1) or an alternative XRCC1-independent mechanism via hetero-oligomerization with PARP1.Poly(ADP-ribose) polymerase 2 (PARP2) participates in base excision repair (BER) alongside PARP1, but its functions are still under study. Here, we characterize binding affinities of PARP2 for other BER proteins  and oligomerization states of the homo- and hetero-associated complexes using fluorescence-based and light scattering techniques. To compare PARP2 and PARP1 in the efficiency of PAR synthesis, in the absence and presence of protein partners, the size of PARP2 PARylated in various reaction conditions was measured. Unlike PARP1, PARP2 forms more dynamic complexes with common protein partners, and their stability is effectively modulated by DNA intermediates. Apparent binding affinity constants determined for homo- and hetero-oligomerized PARP1 and PARP2 provide evidence that the major form of PARP2 at excessive PARP1 level is their heterocomplex. Autoregulation of PAR elongation at high PARP and NAD Base excision repair (BER) in mammalian cells is an efficient and complex process developed to detect and repair damaged bases and apurinic/apyrimidinic (AP) sites in DNA. Repair of single-strand breaks (SSB) involves enzymes and factors of the BER system and is generally considered a separate pathway of this process . Many prIn our previous study, we detected and characterized the interaction of PARP1 with functionally different key protein players of BER, AP endonuclease 1 (APE1), Pol\u03b2, and XRCC1, by using fluorescence-based and light-scattering techniques ,24. HereInteraction of PARP2 with the main BER proteins was studied by the fluorescent titration method using fluorophores with different spectral characteristics and hydrophobic properties. 5,6-Carboxyfluorescein- (FAM-), 5-Alexa Fluor 488- (AF-), sulfo-Cyanine 3- (Cy3-) and sulfo-Cyanine 5- (Cy5-) labelled proteins  were prepared using succinimidyl esters of respective fluorophores. The reaction was performed at pH 7.0 to label proteins preferentially at the N-terminal amino group . In each50) are apparent equilibrium dissociation constants of the complexes. The EC50 values determined for PARP2 complexes with Pol\u03b2 and XRCC1 are practically identical to each other and slightly (from 1.2- to 1.3-fold) exceed those characterizing self-association of PARP2 and its hetero-oligomerization with PARP1, indicating that PARP2 can interact with homologous and functionally different heterologous proteins with closely similar affinities. The affinity of PARP2 for APE1 was determined to be 3-fold higher than the affinity of PARP2 for Pol\u03b2.Initial studies were performed by using FAM-labelled PARP2 (or FAM-labelled partner protein) to determine relative affinities of PARP2 for other BER proteins under the experimental conditions developed previously for detection of various BER protein\u2013protein complexes, including those of PARP1 . Interac50 values determined for AF-PARP2\u2013PARP1 and Cy3-PARP1\u2013PARP2 pairs are practically identical to each other but substantially (~1.9-fold) lower than the respective value determined for the FAM-PARP2\u2013PARP1 pair  and 5 (Cy5), and the rhodamine dye Alexa Fluor 488  were additionally tested as fluorescent labels. The presence of sulfonate groups in the dye molecules improves solubility of their succinimidyl esters and decreases aggregation of labelled proteins. Furthermore, Cy3 and Cy5 are characterized by photostability, high extinction coefficients, and high quantum yields, enabling sensitive detection . The intRP1 pair . Thus, p partner , suggest partner C. The inda/Fd, where Fda and Fd are the AF-PARP2 fluorescence intensities measured in the presence of identical concentrations of Cy3-PARP1  or PARP1, respectively.Direct physical interaction of PARP2 with PARP1 was further confirmed by FRET, using AF-labelled PARP2 and Cy3-labelled PARP1 as a donor\u2013acceptor pair. The fluorescence was excited and monitored at the excitation and emission maxima of 5-Alexa Fluor 488. To correct for the fluorescence signal produced by the acceptor, unlabelled PARP2 was titrated with Cy3-labelled PARP1 in the control experiment. The corrected data were plotted to obtain the corrected binding curve (fitted to the four-parameter equation) B. The flThe influence of model DNAs mimicking intermediates of the BER process on the interaction of PARP2 with proteins was studied. The titration experiments for FAM-PARP2 with three various unlabelled proteins were performed in the absence and presence of the DNA duplex with a single-nucleotide gap (gap-DNA), a canonical substrate of Pol\u03b2, added at an excessive concentration . The binH). All three types of particle size distributions  were used for analysis. A volume-weighted distribution was applied as more relevant for estimation of the oligomeric state, because in this type of distribution, the contribution of each particle is related to the volume/mass of the particle that in accordance with Rayleigh approximation is proportional to R3. A typical volume-weighted size distribution profile obtained for PARP2 as an example is presented in H value for PARP2 slightly exceeded the theoretical value for the homodimer (5.0 vs. 4.67 nm) and was significantly lower than the respective value predicted for the homotetramer (5.0 vs. 6.28 nm)  technique for determination of hydrodynamic sizes of BER proteins and protein\u2013protein complexes to estimate their oligomeric state under true equilibrium conditions in solution was demonstrated for the first time in our previous work . Similar6.28 nm) . We founDLS measurements were performed for the equimolar mixtures of PARP2 with various BER proteins  characterized as the PARP2-binding partners. Values of the apparent equilibrium dissociation constants determined for the respective homo- and hetero-oligomeric complexes here  and prevH value determined from the volume-weighted size distribution for the binary mixture of PARP2 with Pol\u03b2 exceeded the respective value for the individual PARP2 (5.6 vs. 5.0) and was slightly lower than the theoretical value for the dimer-of-heterodimers (heterotetramer) (5.74) as a result of contribution of the homo-oligomerized small protein partner\u2013Pol\u03b2 (3.5)  (The Rl\u03b2 (3.5) . Thus, tvs. 5.6) . Based oH value determined from the volume-weighted size distribution for the binary mixture of PARP2 with XRCC1 (7.6) substantially exceeded the respective values for the PARP2 homo-oligomer (5.0) and XRCC1 homo-oligomer (5.7) (2 (6.44). The presence of self-associated proteins in the PARP2\u2013XRCC1 mixture detected by the cross-linking experiments  was comparable to the respective value for the PARP1 homo-oligomer (8.0) and exceeded the theoretical RH value for the heterotetramer (AB)2 (7.28) (The Rer (5.7)  and was eriments  evidentlllipsoid  can be r2 (7.28) . In the H value were performed every 3 min after initiation of the reaction by addition of NAD+ substrate, Mg2+ and gap-DNA to the protein sample. The magnesium ion coordinates intermolecular bonds with polyphosphate ligands [H values presented in H value measured before initiating the PARylation reaction was undertaken as a null point. Control measurements performed for the PARP2 mixture with activating DNA (in the absence and presence of other proteins), before addition of NAD+, revealed no change in the hydrodynamic radius due to the DNA presence. The RH value determined for PARylated PARP2 associate was 2.6 times lower than that for PAR-PARP1 . These results provide further evidence of the higher effectiveness of PARP1-catalysed PAR chain elongation. The RH value determined for the modified PARP2 after PARG treatment was only slightly higher than the RH value for the unmodified protein  exceeded to a similar extent the respective values measured for the two protein mixtures before the initiation of PAR synthesis (+) was not modulated by the small protein partners Pol\u03b2 and APE1. The volume-weighted size distribution profiles observed for the PARP2 binary mixtures with XRCC1 and PARP1 upon the PARylation reaction were presented in each case by two peaks , but were substantially lower than the RH value determined for the PARP2\u2013XRCC1 mixture before the PARylation reaction (7.6). Moreover, the RH value determined after the PAR degradation was lower than the theoretical value for the PARP2\u2013XRCC1 heterotetramer (5.8 vs. 6.44). Taken together, these results suggest that modification of PARP2 even with mono-ADP-ribose influences its interaction with XRCC1, modulating their oligomerization mode. The RH value measured upon the PARylation reaction for the PARP2\u2013PARP1 mixture (14.8 nm) was significantly lower than the respective values for the automodified PARP2 and PARP1 present alone (by 26-fold and 68-fold respectively). These data indicate mutual effects of PARPs on their PARylation and intermolecular association. The RH value determined after disruption of Mg2+-coordination bonds was 2.2-fold lower than the respective value for PARP1 alone, indicating strong inhibitory action of PARP2 on elongation and branching of PAR synthesized by PARP1.DLS measurements of the PARP2 hydrodynamic radius upon the PARylation reaction were performed in the presence of various protein partners at the equimolar concentration . The RH  or APE1 . The RH ynthesis . Combinewo peaks . The RH reaction . Peaks oprofiles , indicatFor visualization of the associates detected by DLS for PARylated PARP1/PARP2 and their mixtures with each other or BER protein-binding partners, we attempted to apply multicolour fluorescence detection by Celena S Digital Imaging System. Proteins labelled with distinct fluorophores were used to visualize them in protein mixtures. Images acquired with appropriate  fluorescence filters  were captured and superimposed to obtain a multifluorescent (merged) image. Images demonstrating presence of fluorescence intensity signals were obtained for protein associates formed upon PARylation reaction catalysed by PARP1 (combined with Cy3-PARP1) or PARP2 (combined with AF-PARP2), using an RFP filter for Cy3-PARP1 and a GFP filter for AF-PARP2 ; no sign2+-coordination bonds [2+ ions with PAR chains. Fluorescence signals for protein associates formed upon the PARylation reaction catalysed by the equimolar mixture of Cy3-PARP1 with AF-PARP2 were detected in images acquired with both RFP (for Cy3) and GFP (for AF) filters  of PARP2 complexes with APE1, Pol\u03b2, and XRCC1 determined in experiments with FAM-labelled proteins  is a multistep DNA repair process in mammalian cells for the processing of most common DNA lesions arising upon oxidative stress and ionizing radiation. The efficiency of BER depends on the coordinated action of enzymes that catalyse the sequential steps ,3. One oproteins  exceed b130 nM, ) and PAR0 nM,  an . This coTo determine the oligomeric states of PARP2 and particularly of its complexes with other BER proteins (never characterized before), we used the DLS technique. In our previous DLS study , we prop2+-coordination bonds with elongated and branched PAR polymers [+ used in these experiments fall within the range of intracellular concentrations of the enzyme and substrate [H value of PARP1 due to PARylation is significantly higher as compared to that for PARP2 (13.6 nm vs. 0.6 nm). Thus, for the reaction conditions used, PARP2 catalyses much less efficiently the elongation of PAR polymers than PARP1 does. These results may explain the predominant contribution of PARP1 to overall PAR synthesis in response to DNA damage in vivo [Using the DLS technique, we succeeded recently in detection of huge associates formed by PARylated PARP1, that are stabilized via intermolecular Mgpolymers . High coubstrate , suggestubstrate  can be pubstrate ,39. In t in vivo . In vitr in vivo ,22. In c in vivo  is stronH value of the intermolecular associate. In our previous DLS experiments performed for PARP1 under the same reaction conditions, no effect of XRCC1 on the size of PARylated PARP1 was detected. XRCC1 has been shown to regulate activity of both PARP1 and PARP2, assayed at a limited NAD+ concentration, but with different efficiency: the activity of PARP2 was significantly inhibited by the equimolar XRCC1 concentration, while at least a 4-fold XRCC1 excess was required to suppress the PARP1 activity [+ substrate is more effectively regulated by XRCC1.The DLS analysis of PARP2 PARylated in the presence of BER protein partners revealed strong inhibition of the automodification reaction by XRCC1 as shown by the 34-fold decrease in the Ractivity ,41. Our 2+-coordination bonds were smaller than the respective species detected for PARP1 alone . The purified proteins were dialyzed against a solution containing 50 mM Tris-HCl, pH 8.0, 200 mM NaCl, 5 mM DTT, and 40% glycerol and stored at \u221230 \u00b0C.Vectors for expression of human poly(ADP-ribose) polymerase 1 (PARP1), murine poly(ADP-ribose) polymerase 2 (PARP2), and bovine poly(ADP-ribose) glycohydrolase (PARG) were kindly provided by Dr. V. Schreiber ; human apurinic/apyrimidinic endonuclease 1 (APE1) and rat DNA polymerase \u03b2 (Pol\u03b2), by Dr. S. H. Wilson ; human X-ray repair cross-complementing protein 1 (XRCC1), by Dr. J. P. Radicella . PARP1 and PARP2 recombinant proteins were expressed in insect cells and purified as described previously , with soto refs. ,48,49. Hto refs. . The PARDNA oligonucleotides were synthesized and purified in the Laboratory of Biomedicinal Chemistry . Sequences of oligonucleotides were as follows: template\u20135\u2032-GGAAGACCCTGACGTTACCCAACTTAATCGCC-3\u2032, complementary oligonucleotide\u20135\u2032-GGCGATTAAGTTGGGTAACGTCAGGGTCTTCC-3\u2032, upstream primer 1\u20135\u2032-GGCGATTAAGTTGGG-3\u2032 and primer 2\u20135\u2032-GGCGATTAAGTTGGGT-3\u2032, downstream primer\u20135\u2032-pAACGTCAGGGTCTTCC-3\u2032. Double-stranded oligonucleotides (32-mer) containing a one-nucleotide gap (gap-DNA) or nick (nick-DNA) were prepared by annealing the upstream primer 1 or 2, respectively, and the downstream primer to the template oligonucleotide. A non-gapped 32-mer DNA (ds-DNA) was prepared by annealing the complementary oligonucleotide to the template. In each case, an equimolar mixture of the components was heated at 90 \u00b0C for 2 min and then slowly cooled down to room temperature.DLS measurements were performed using a Zetasizer Nano ZS  according to the method described in our previous work . Briefly2, 6 \u03bcM gap-DNA, 6 \u03bcM PARP2. Mixtures were equilibrated for 60 s prior to initiation of reaction by NAD+ addition to a 2.5 mM concentration. When indicated, samples contained 6 \u03bcM protein . After 15-min incubation, the reaction was stopped by the addition of EDTA to a 10 mM concentration. The RH value measurement was performed every 3 min during the reaction and immediately after EDTA addition. Then, the mixture was subjected to further treatment with 0.25 \u03bcM PARG for 18 h at 4 \u00b0C, and the RH value of the PARylated protein after enzymatic degradation was measured.Poly(ADP-ribosyl)ation (PARylation) reaction was carried out directly in a ZEN 2112 low-volume quartz cuvette used for DLS measurements. The reaction mixture (20 \u03bcL) contained 25 mM HEPES-NaOH, pH 7.5, 100 mM NaCl, 1 mM DTT, 10 mM MgClv/v) glycerol) using a 0.1\u22121.0 M gradient of NaCl concentration in buffer B. The labelled proteins were stored at \u221230 \u00b0C in a solution containing 50 mM HEPES, pH 8.0, 100 mM NaCl, 5 mM DTT, and 40% glycerol. The extent of protein labelling was quantified spectrophotometrically with a CLARIOstar microplate spectrometer , using the absorption coefficient of the protein based on the Expasy Protparam Data and the absorption coefficients for dyes taken from Lumiprobe protocols [32P-labelled NAD+ as described in The N-succinimidyl esters (SE) of 5,6-carboxyfluorescein , 5-Alexa Fluor 488 , sulfo-Cyanine 3  or sulfo-Cyanine 5  were dissolved in dimethylsulfoxide up to 10 mM concentration and used for protein labelling. The reaction mixture contained 100 mM MES, pH 7.0, 150 mM NaCl, 80\u2212100 \u03bcM protein , and 1.6\u22123-fold molar excess of the reagent over the protein. The reaction was carried out for 18 h at 4 \u00b0C in the dark and stopped by the addition of ethanolamine up to 2-fold excess over the reagent. Then, labelled proteins (except Cy3-PARP1) were dialyzed exhaustively against buffer A containing 100 mM HEPES, pH 8.0, 200 mM NaCl, and 10 mM DTT, to remove the free dye. Cy3-labelled PARP1 protein was purified from free dye by two successive chromatographies on HiTrap Capto S and Mono S columns  equilibrated with buffer B  and saturating concentrations, respectively; EC50 is the concentration of protein partner at which F \u2212 F0 = (F\u221e \u2212 F0)/2; n is the Hill coefficient. All experiments were performed at least three times.Fluorescence intensities of labelled proteins (used at the fixed concentration of 40 nM) were measured in the absence and presence of various concentrations of the potential partner (protein or DNA). In some cases, the experiments were performed in the presence of the second protein or DNA partner pre-added at sub-saturating concentration . The binding buffer contained 50 mM Hepes (pH 8.0), 100 mM NaCl, and 5 mM DTT. The measurements were performed in low-volume nontransparent polypropylene plates  using a microplate CLARIOstar spectrometer. The fluorescence intensity for 5,6-FAM and AF488 fluorophores was detected at the excitation wavelength of 482 nm and the emission wavelength of 530 nm. The excitation and emission wavelengths of 530 nm and 580 nm, respectively, were used for fluorescence intensity detection of Cy3-labelled proteins. In each independent experiment, fluorescence intensity was measured at each concentration of the protein/DNA partner in three repeats. The experimental data were processed quantitatively with the MARS Data analysis program . The binding curves were described by the equation:d) due to the presence of the acceptor (Fda):To detect protein\u2013protein interactions by the FRET approach, the fluorescence intensity of the AF-labelled PARP2 (donor-labelled probe) was measured in the absence and presence of various concentrations of the Cy3-labelled PARP1 (acceptor probe) as we detailed in our previous work . FRET ef0 value.In studying the effects of model DNA substrates on the PARP2 interaction with PARP1, XRCC1 or Pol\u03b2, the AF-PARP2/FAM-PARP2 (40 nM) was premixed with the respective DNA at an excessive concentration (400 nM), and the fluorescence intensity observed was taken as a starting F+, 1 \u03bcM gap-DNA, 50 mM Tris-HCl buffer (pH 8.0), 100 mM NaCl, 10 mM MgCl2, 1 \u03bcM PARP1 (an equimolar mixture of PARP1 and Cy3-PARP1) or 1 \u03bcM PARP2 (an equimolar mixture of PARP2 and AF-PARP2) in the absence or presence of 1 \u03bcM labelled protein . Mixtures were incubated for 30 min at 37 \u00b0C and then placed on ice to terminate the reaction. After addition of PEG 20K to a 0.8% concentration, the reaction mixture aliquot (6 \u03bcl) was placed between the microscope slide and 0.17 mm coverslip. Fluorescence images were captured using a Celena S Digital Imaging System with GFP, RFP, Cy5 filter cubes, and Plan Apochromat Fluor Oil 100X  objective.The PARylation reaction catalysed by PARP1/PARP2 was performed in a mixture (10 \u03bcl) containing 2 mM NAD"}
+{"text": "Meloidogyne spp. in sodium hypochlorite (NaOCl) is a helpful procedure to assess the population levels and to obtain inoculum. In this sense, laboratory and greenhouse experiments were done to evaluate the effect of the NaOCl concentration on the viability of M. enterolobii eggs. Additionally, the objective of this investigation was to corroborate the preferable treatments to count populations in cucumber galled roots or to obtain inoculum of M. enterolobii. It was shown that the effect of the NaOCl concentration on the viability of M. enterolobii eggs is potentially detrimental. The NaOCl concentration caused a higher hatching, which in turn, resulted in non-infective larvae. Therefore, the best treatments to obtain inoculum of eggs of M. enterolobii included the 0.75% NaOCl (with 8-min stirring), 0.5% NaOCl , and 0.3% NaOCl concentration . For a correct estimate of the egg population in roots, we show by several treatments that a concentration of 0.5% NaOCl  and 0.75% NaOCl (with 8-min stirring) give the highest results.Extraction of eggs of  Meloidogyne enterolobii Uang and Eisenback is a widely dispersed and highly pathogenic plant parasite. This RKN is capable for reproduction on most of vegetables, including cultivars with genetic resistance to other important RKN species such as M. incognita and M. javanica  javanica . In Mexicucumber . The yiecucumber . Currenterolobii . These merolobii . This teM. incognita from cucumber galled roots by grinding for 40\u2009sec in 20% NaOCl solution  that causes oxidative degradation of microbial cell structures upon contact, also, NaOCl damages vegetable tissues at concentrations above 200 ppm . In nemasolution . Simultasolution . Howeversolution . Similarsolution . Afterwagrinding .M. enterolobii will contribute to reduce variability in evaluations employing this technique. The objective of this investigation was to describe the effect of the NaOCl concentration on the viability of M. enterolobii eggs. Additionally, the objective of this investigation was to corroborate the preferable NaOCl-stirring treatments to inoculum of M. enterolobii.In recent years, different researchers have used the NaOCl method for different purposes, one example is to extract inoculum directly . AnotherCucumis sativus L. \u2018Espada\u2019) seeds were established in infested soil with a previously isolated local strain of M. enterolobii (Cucumber (erolobii . The plaA layer of Kimwipe tissue was set on nylon sieves in trays filled with sterilized tap water (Modified Baermann\u2019s Technique). Aliquots from each replication of extraction treatments containing 1,000\u2009\u00b1\u200910 eggs were spread on the Kimwipe tissue to allow the hatching and migration of J2. Nylon sieves with one-gram samples of galled tissue were used as a control. Trays were placed in a complete randomized design with six replications in the laboratory at 26\u00b0C for five days . Larvae Cucumber seedlings cv. Espada of two weeks after planting (WAP) were individually transplanted in 1.6-liter pots filled with steam-pasteurized (100\u00b0C\u2009\u00d7\u200945\u2009min) silty clay loam soil . Two holes of 4-cm deep and 1-cm wide were made in the soil surrounding each transplanted seedling with the use of a pipette. Then, each pot was inoculated with a 10-ml aliquot containing 1,500\u2009\u00b1\u200915 eggs from each replication of extraction treatments. Inoculation with 300\u2009\u00b1\u20096 J2 was used as control . Pots weTwo cucumber seedlings of three WAP were transplanted in 15-liter pots filled with soil as described above. Then, each pot was inoculated with a 20-ml aliquot containing 6,000\u2009\u00b1\u200930 eggs from each replication of extraction treatments. Inoculation with water or 1,200\u2009\u00b1\u200912 J2 were used as negative and positive controls . A rando\u03b1\u2009=\u20090.05) was used to determine main-effect means (SAS v.9.1) . In the same way, a Pearson\u2019s correlation coefficient test was used to establish relationships between the number of produced eggs and hatched J2. Additionally, these data were also analyzed by regression with numbers of hatched J2 as the independent variable (SigmaPlot v.14) .Prior to statistical analysis, data from both trials of each experiment were combined. Then, data from egg counts were ln transformed, and data from hatched J2 were first adjusted by subtracting the average amount of J2 obtained in the control and then \u221a transformed. Data were analyzed using the Proc GLM for a two-way ANOVA to determine the significance of both factors. A Fisher\u2019s least significant difference (LSD) test (p\u2009\u2264\u20090.05) when compared with stirring for 8 to 12\u2009min. At NaOCl concentration of 0.5% the level of extracted eggs was similar (p\u2009>\u20090.05) among stirring periods for 8 to 16\u2009min. However, at 0.75% NaOCl only the stirring period of 8\u2009min observed a similar (p\u2009>\u20090.05) extraction level when compared with the 0.5% NaOCl treatments. The lower (p\u2009\u2264\u20090.05) extraction level was observed with treatments of 0.3% NaOCl with stirring for 8 to 12\u2009min and 0.75 to 1.0% NaOCl with stirring for 12 to 20\u2009min (At low NaOCl concentration (0.3%) with stirring for 16 to 20\u2009min the extraction level was higher (o 20\u2009min .p\u2009\u2264\u20090.05) induction of hatching when compared with the remaining treatments (p\u2009\u2264\u20090.05) hatching level when compared with the rest of NaOCl treatments within each stirring period for 12 to 20\u2009min (The combinations of the NaOCl concentrations 0.75 and 1.0% with stirring periods for 16 to 20\u2009min resulted in a higher (eatments . This efo 20\u2009min .p\u2009\u2264\u20090.05) (data not shown). Importantly, although the weight of the cucumber root systems (concomitant variable) at 5 WAI varied from 10 to 22\u2009g among treatments (data not shown), the extraction level of M. enterolobii eggs was unaffected (p\u2009>\u20090.05). On the other hand, at 17 WAI the extraction of eggs from samples was useless to estimate the population levels from whole root systems. However, in the reproduction tests at 5 WAI, the treatment of 0.75% NaOCl with stirring for 8\u2009min observed a higher (p\u2009\u2264\u20090.05) reproduction level when compared with the treatments with 0.3% NaOCl with stirring for 8 and 20\u2009min (p\u2009\u2264\u20090.05) reproduction level when compared with all 1.0% NaOCl treatments and 0.75% NaOCl (12 to 20-min stirring). The 0.75% NaOCl with stirring for 8\u2009min resulted in a similar (p\u2009>\u20090.05) reproduction level compared to all 0.5% NaOCl treatments. On the other hand, the reproduction level with the 0.3 and 0.5% NaOCl treatments was similar (p\u2009>\u20090.05) within each stirring period. The 1.0% treatments showed the lower (p\u2009\u2264\u20090.05) reproduction levels within stirring periods for 8 to 16\u2009min. With the stirring period of 20\u2009min, the reproduction level observed was similar (p\u2009>\u20090.05) between treatments of 0.75 and 1.0% NaOCl (R\u2009=\u2009\u20120.86); i.e., the production of eggs decreased as the hatching increased  resulted in a higher count of 16,836 eggs per root system, when compared with eggs as inoculum  percentages of galling when compared with the rest of NaOCl treatments (p\u2009\u2264\u20090.05). Control treatment of 1,500\u2009\u00b1\u200915 J2 resulted in a similar galling level when compared to 6,000\u2009\u00b1\u200960 eggs from the 0.3% NaOCl treatment at 17 WAI (p\u2009>\u20090.05) . At 17 WAI, only the concentration of NaOCl was a significant factor in galling. Here, the treatment with 0.3% NaOCl resulted in higher (eatments . The 0.7\u2009>\u20090.05) .Meloidogyne spp., the galled roots can be directly treated with sodium hypochlorite via shaking, grinding or stirring to release the eggs from the gelatinous matrix attached to the root surface . The above means that despite the negative relation between hatching and egg production, a moderate induction of hatching resulted in a faster infection. In the same way, it is known that there is a relation between the number of RKN infecting roots and the size of galls . In our M. enterolobii eggs is potentially detrimental. However, it is achievable to obtain both a reliable estimation of egg population in roots and a viable inoculum. The combinations of 0.5% NaOCl with stirring for 8, 12, and 16\u2009min, and 0.75% NaOCl for 8\u2009min are preferable to extract the eggs of M. enterolobii. The NaOCl concentrations from 0.5 to 0.75% (with 8-min stirring) are more appropriate to induce a faster hatching of viable larvae. Finally, we conclude that the 0.3% NaOCl concentration is preferable to extract M. enterolobii eggs with more viability.We conclude that the effect of the NaOCl concentration on the viability of"}
+{"text": "Button battery ingestion accidents have been reported in multiple previous reports. However, ingestion of cylindrical-type batteries is significant less described in the literature. Cylindrical batteries can reportedly cause corrosive damage to the gastrointestinal mucosa after long-term retention, leading to ulceration and perforation. Here, we present a case of endoscopic removal of eight AA batteries that had been ingested and caused corrosive changes in the gastrointestinal mucosa. A 45-year-old man with mental retardation was brought to our hospital due to the suspicion of cylindrical battery ingestion. A plain abdominal x-ray revealed a total of eight cylindrical batteries. Esophagogastroduodenoscopy was performed approximately 24 hours after ingestion, and four AA batteries were removed using a polypectomy snare. The remaining four batteries were followed up and removed under colonoscopy after confirming that they had reached the rectum. Leaked components of retained cylindrical batteries can cause chemical mucosal damage in the gastrointestinal tract. Therefore, early extraction should be considered in case of cylindrical battery ingestion. On the other hand, when the cylindrical battery has passed the pyloric ring, conservative management with close monitoring is acceptable if there are no clinical symptoms. Additionally, a polypectomy snare is useful in the extraction of ingested cylindrical batteries. Button battery ingestion accidents have been reported in multiple previous reports. However, ingestion of cylindrical-type batteries has been significantly less often described in the current literature . TherefoIn this article, we report a case of endoscopic removal of eight AA batteries that were ingested and caused corrosive changes in the gastrointestinal mucosa, four from the stomach under esophagogastroduodenoscopy (EGD), and the remaining four that had passed beyond the pyloric ring were removed from the rectum under colonoscopy after follow-up, using a snare in both procedures.A 45-year-old man with mental retardation was brought to our emergency department from a psychiatric facility due to the suspicion that he had ingested cylindrical batteries\u00a0since all the cylindrical batteries in his room were missing. He had undergone colonoscopy at another hospital one week prior to the current presentation for further examination of positive fecal occult blood, at which time two AAA batteries had been removed. He was asymptomatic and his vital signs were normal. On physical examination, his abdomen was soft and non-tender, with no masses, and bowel sounds were normal. Laboratory data were unremarkable. A plain abdominal x-ray showed eight cylindrical structures in the left upper and lower abdomen, consistent with battery ingestion Figure .There was no evidence of perforation or obstruction. EGD was performed approximately 24 hours after the suspected ingestion, which revealed four AA batteries in the stomach. Moreover, there were scattered hemorrhagic erosions with black discoloration of the mucosa from the greater curvature of the middle part of the body of the stomach to the greater curvature of the fornix. The remaining four batteries were not visible on EGD up to the transverse part of the duodenum. The initial approach was to remove the AA batteries\u00a0using a Roth net, but this\u00a0was found to be difficult because the battery  was too large to be contained in the net (30 \u00d7 50 mm). Next, we used a polypectomy snare, which enabled easy extraction by holding the edge of the battery tightly with the snare and aligning the axis of the battery with the long axis of the esophagus Figures -2D.A total of four AA batteries were removed one at a time in this manner. The time required for the removal of a single AA battery was approximately five minutes. Since the patient was asymptomatic, the remaining four batteries, which had already moved deeper into the duodenum, were followed up with imaging examinations. During\u00a0follow-up, the patient fasted, and abdominal plain x-ray images were taken every 24 hours to monitor the movement of the batteries. No new clinical symptoms were observed during this period. On hospital day 3, all four batteries were confirmed to have reached the rectum. Since the size of the battery was large and it was expected to take time to pass the anus, we decided to perform foreign body retrieval under colonoscopy. Colonoscopy showed the four AA batteries in the lower rectum, with contents leaking from some of the batteries. All four batteries were removed using a polypectomy snare. After removal of the batteries, the rectum was examined and some easy-bleeding mucosa was identified Figures , 3B.The patient resumed eating on hospital day 4 and was subsequently discharged to a residential facility without any abdominal symptoms.\u00a0Plain abdominal x-ray 10 days after discharge showed no remaining batteries in his gastrointestinal tract.Our experience with this patient indicated the following clinical issues with such cases. Since cylindrical batteries cause chemical mucosal damage when retained in the stomach, their early removal is necessary. However, if the cylindrical battery moves beyond the pyloric ring into the small intestine, conservative management with close monitoring is also permissible. Additionally, polypectomy snares might be useful tools for the removal of ingested cylindrical batteries.Although many cases of accidental ingestion of button batteries have been previously reported, there are few reports of accidental ingestion of cylindrical batteries. Litovitz et al. reported that of 8,648 cases of battery ingestion from 1990 to 2008 in the United States, 8,161 (94.4%) involved a button battery and 487 (5.6%) were cylindrical batteries . Our patSince cylindrical batteries cause corrosive and toxic damage to the mucosa when they remain in the gastrointestinal tract, their early extraction is necessary. Guidelines issued by the American Society for Gastrointestinal Endoscopy recommend extraction when cylindrical batteries remain in the stomach for over 48 hours after ingestion . HoweverIn our case, a polypectomy snare was useful for the removal of the cylindrical batteries. Since cylindrical batteries are large and hard, and their surface is round and slippery, ingenuity is required for their removal. We summarize the case reports of cylindrical batteries that were removed by endoscopy in Table In all previous reports, the batteries were removed within three days of ingestion. A Roth net, polypectomy snare, and Dormia basket were used as removal devices. Among them, the basket and net were commonly used to extract small-sized batteries, including AAA batteries. On the other hand, among five cases with ingested AA batteries, similar to our case, a Roth net was used in three cases and a snare was used in two cases. Magnet catheters were used concomitantly in one of the four cases in which the battery was extracted with a snare. In the present case, a Roth net was first selected to retrieve the battery, but it was difficult to store the battery due to its large size. Hence, a polypectomy snare was used, which allowed easy removal of the batteries. Our experience suggests that endoscopic removal should be considered as early as possible if a cylindrical battery is retained in the stomach. In such cases, a polypectomy snare is especially useful for removing relatively large cylindrical batteries, because it can strongly grasp foreign objects with round and slippery surfaces.In conclusion, early endoscopic extraction of ingested cylindrical batteries should be considered if they remain in the stomach, due to the increased risk of ulceration and perforation. Contrary to current guidelines, a more aggressive approach should be adopted to remove the batteries, given their potential to cause mucosal damage. On the other hand, when a cylindrical battery has passed the pyloric ring, conservative management with close monitoring is acceptable if there are no clinical symptoms. Additionally, polypectomy snares are useful for the extraction of cylindrical batteries."}
+{"text": "Chosen reasons for FOTI included price followed by ease of use. In general, high price rated as the most perceived reason for not choosing a given NCDT followed by low practical applicability. Meanwhile, ease of use followed by relevant application ranked as the main reported reasons to choose an NCDTs.Early detection of caries lesions is key to a successful restorative dental treatment plan. The aim of this study was to investigate the preferences and attitude of graduate restorative dentistry residents (RDRs) regarding novel caries diagnostic technologies (NCDT) and to provide a brief overview of available technologies for both specialized and general dental practice. This cross-sectional study used an online questionnaire (17 questions) concerning RDRs\u2019 attitude, preferences, and insights regarding five available NCDTs. It was distributed among twenty RDRs at a local government dental school following a review session about NCDTs. Collected responses were analyzed statistically using one-way analysis of variance (ANOVA), chi-squared with Bonferroni correction, and Kruskal-Wallis tests at a 0.05 significance level. Sixty-five percent of RDRs reported an interest in NCDTs as a discussion topic and almost half of them were positive towards their use, however, sixty percent of respondents were hesitant to diagnose caries solely using NCDTs. Fiber-optic-transillumination (FOTI) systems were ranked the best overall and with regard to all the investigated criteria ( Dental caries is one of the most common wide-spread transmissible diseases affecting humanity ,2,3. It Restorative treatment of the detrimental effects of dental caries in the oral cavity by merely replacing lost dental hard tissues takes a major toll on healthcare services worldwide ,11,12. CThe dental practice is rapidly moving towards minimally invasive treatment modalities, which positively influence the development and introduction of several novel caries diagnostic technologies (NCDT) to facilitate early caries diagnosis leading to a possible end of the disease process to allow the utilization of effective remineralization preventive therapies ,15,30,31Despite the presence of numerous NCDTs on the market, their use amongst dentists is still limited . This miEthical approval was obtained from the research ethics committee of the Faculty of Dentistry in King Abdulaziz University, IRB protocol 031-03-17.This descriptive cross-sectional study was conducted using an online questionnaire that was formulated using Google Forms and was distributed amongst RDRs by sending them directly to their respective emails after an interactive theoretical session. The study\u2019s objectives and goals were explained explicitly to all RDRs prior to their participation and consent was obtained thereafter. Participation and enrollment of RDRs was voluntary and all responses were anonymized to confirm confidentiality. Participating RDRs were exposed to traditional and novel CDTs as part of their graduate education. Additionally, they had to individually conducted a literature review of currently available novel CDTs and their related information and updates. Prior to participation in the survey, RDRs attended a 3 h-long interactive session that included a restorative-dentistry-consultant-lead objective recap of the various CDTs as well as a resident-lead dynamic discussion of their previously prepared literature reviews emphasizing their conclusions and insights, as well as the CDTs\u2019 intricate details, including concepts, manipulation, indications, limitations, advantages, and disadvantages. Thereafter, a brief reflection regarding CDTs was prepared by each RDR. .The questionnaire\u2019s questions were formulated to assess attitude, opinion, and preference of restorative dentistry residents regarding NCDTs. They included questions concerning five commercially available NCDTs, namely: PTR and modulated LUM, FOTI/DIFOTI, QLF, LF, and NIT based devices. The questionnaire consisted of 17 questions in total; four multiple choice questions (MCQ), five Absolute Category Rating (ACR) scale questions that included five question items with the rating range from excellent to poor, seven questions using the 5-point Lickert scale and one free response format question. All questions were checked to establish both validity and reliability. The questionnaire was distributed amongst a group of restorative dentists with expertise in the field of caries diagnosis and the questions were adjusted accordingly. The 3 h interactive session prior to filling the questionnaire further ensured practicality and validity of the questionnaire. The average time needed to fill in the questionnaire was approximately 15 min.The questionnaire began with an inquiry of the RDR\u2019s demographic characteristics, then two Lickert scale questions addressed the participant\u2019s opinion regarding the presented topic and their opinion regarding the potential benefit of NCDT\u2019s use. Then the RDRs were asked to choose the preferred NCDT from the five previously presented categories. Afterwards, two MCQs assessed the respondents\u2019 most likely perceived reasons for choosing an NCDT device and the main perceived reasons negatively affecting their NCDT choice. This was followed by five Lickert scale questions that aimed at assessing the participating RDRs\u2019 attitude and interest in both NCDT\u2019s use and further education. The remaining questions  aimed at rating the five previously discussed NCDTs in respect to five parameters, namely: ease of understanding the principle, practicality , rice, clinical application , and patient\u2019s acceptance. Rating of each individual parameter for each NCDT was also included. The free response question including the RDR\u2019s personal reflections qualitatively assessed the respondents\u2019 opinions and insights regarding both presented NCDTs and other radiation-free caries diagnostic devices.p < 0.05). The received responses were tabulated and presented in the form of frequencies and percentages. Chi-squared statistical test was conducted to compare the five presented NCDTs, where each NCDT was rated according to five criteria; namely: ease of understanding, practicality , price, clinical application , and patient\u2019s acceptance.Statistical analysis of the results was conducted using SPSS computer software  at a significance level  was conducted to detect significant differences in the overall assessment of the diagnostic aids between the groups. This was followed by Bonferroni test for multiple comparison between the responses rating the five presented NCDTs.The Kruskal-Wallis inferential statistical test was conducted to rank the different NCDTs for each parameter with each parameter ranked individually to determine the preferred NCDT as reported by participating RDRs.All 20 RDRs that attended the revision session, agreed voluntarily to participate in the study and completed the questionnaire successfully resulting in a response rate of 100%. The participating RDRs population consisted of seven final year restorative dentistry residents, four residents in their third year of residency, six in their second year and only three first year residents.Regarding the RDRs opinion regarding NCDTs as a topic for further discussion, the majority of participating RDRs reported very high (13 (65%)) and high (4 (20%)) perceived relevance of NCDTs as a discussion topic. On the other hand, only two RDRs reported neutral responses, and one RDR reported a diminished perceived relevance of the topic. Regarding the RDRs opinion about the potential benefit of NCDTs in their own daily clinical practice, over half of them reported most beneficial (7 (35%)) and somewhat beneficial (4 (20%)), while a quarter of them were undecided on how beneficial the use of NCDT could be in their daily practice. When RDRs were asked about their preferred NCDT choice when taking all previously reviewed and discussed factors into consideration, an equal number of RDRs chose FOTI and NIT devices (8 RDRs), followed by PTR with modulated LUM and QLF (2 RDRs). None of the responding RDRs LF as their preferred device. Regarding the reasons for choosing a specific NCDT, the vast majority of RDRs chose ease of use and relevant application as their main reasons . On the other hand, when they were asked about their opinion regarding the main reason that would potentially cause an NCDT to be less valuable, almost half RDRs chose price as their main reason (45%). Three RDRs reported other reasons that were not included in the MCQ choices. They reported skepticism regarding the reproducibility, reliability, and specificity of an NCDT as other reasons to negatively influence their NCDT choice. Regarding the portion of the questionnaire aiming to assess the participating RDRs\u2019 attitude and interest in both NCDT\u2019s use and further education, the majority of RDRs reported a likely to very likely possibility (17 (85%)) of changing their current caries follow-up radiographic examination protocol if an NCDT was available in their clinic. However, less than half of them reported likely to very likely possibility of changing their current caries radiographic examination protocol when diagnosing new patients if an NCDT was available for use in their clinic. Furthermore, most RDRs were undecided when asked if they would completely rely on NCDTs to diagnose dental caries without intra-oral radiographs (12 (60%)). Despite this, almost half of the responding RDRs reported being completely against the exclusive use of NCDTs without radiographs during caries diagnostic procedures (8 (40%)), most of them reported a likely to very likely responses regarding the possible positive impact of NCDTs on the clinical outcome of their clinical practice (16 (80%)). Regarding the interest of the responding RDRs in NCDT-related further education, most of them reported likely very likely possibility of taking part in brief educational activities dedicated to improving their knowledge of their NCDT of choice (12 (60%)). p-value <0.001) as it received the highest number of excellent responses when counting all five criteria followed by LF . QLF had the lowest overall rank compared to the other NCDTs as it received more poor responses collectively, but the difference between QLF and PTR was statistically insignificant . Regarding the assessment of the five presented NCDTs, responses to the ACR questions portion of the questionnaire were analyzed using chi-squared test, where each NCDT was rated according to five criteria; namely: ease of understanding, practicality , price, clinical application , and patient\u2019s acceptance. According to RDR\u2019s responses in this section, FOTI ranked statistically significantly the best overall (chi squared p < 0.001). One-way comparison analysis of variance (ANOVA) showed that there is a statistically significant difference in the overall assessment regarding the afore mentioned criteria between the five NCDTs (p < 0.05). Other NCDTs were not statistically significantly different when compared to each other (p > 0.05).Following the ANOVA results, the results of the multiple comparisons between the investigated NCDTs using Bonferroni correction method are detailed in p < 0.001). The results for the NCDT ranking for each criterion and respective Kruskal-Wallis rank are demonstrated in Regarding the ranking of all NCDTs for each criterion, the sum of ranks for each NCDT according to each criterion was calculated, so that the NCDT with the lowest sum of ranks for each criterion would represent the best ranked NCDT. The null hypothesis of the Kruskal-Wallis statistical test comparing the mean ranks of the five NCDT groups was that the mean ranks of NCDTs were the same. The results showed that FOTI had the least sum of ranks across all investigated criteria, whereas QLF had the highest overall sum of ranks. Accordingly, FOTI was ranked the best reported NCDT and was statistically significantly different than the other NCDTs (The RDRs\u2019 responses to the open-ended question disclosing their insights and preferences about NCDTs were mainly expressing hopes of wide-spread practical utilization of novel technologies in caries diagnosis and monitoring, especially the more objective NCDTs as an aid to traditional visual-tactile and radiographic examination procedures in daily dental practice. Half of RDRs expressed their belief in that successful application of such technologies still depends on the professional interpretation of the examination results and one RDR expressed skepticism regarding the reproducibility of the NCDT diagnostic results and their efficiency in monitoring carious lesions over time. A couple of responding RDRs also expressed their fear of over treatment, due to the higher sensitivity of NCDTs leading to the detection of more incipient lesions and some false positives along the way. Almost all RDRs reported the usefulness of NCDTs as an adjunct mean in caries diagnosis in their daily practice, especially in cases where access to the surface under investigation is difficult, and/or radiographic examination is not possible or not desirable. All RDRs reported the need for further clinical research facilitating an evidence-based applicable easy utilization of NCDTs in restorative dental practice as well in general dental practice. They additionally expressed their concern regarding the high price of some the NCDTs that could render them difficult to be used by dentists across the various parts of the healthcare system in the country.The primary objective of the current investigation was to investigate the preferences and attitude of graduate restorative dentistry residents with regards to novel caries diagnostic technologies. Based on the significant differences found, the null hypothesis was rejected.Dental caries continues to be a prevalent disease affecting the global population . CurrentEffective preventive dentistry requires accurate early detection and effective monitoring of surface lesions ,17. AsseCaries diagnostic devices and techniques have gone through numerous developments over the last decade . This inDetection of caries lesions as accurately and as early as possible is the foundation of current dental practice ,32,53,64Numerous reports in the literature supports the beneficial use NCDTs as adjuncts to visual examination aided by intra-oral radiographs, where indicated, and not to completely replace it ,59,69,70Most RDRs in the current study generally preferred FOTI as a caries diagnosis adjunct above other NCDTs. FOTI has been widely utilized, preferred , and invEven through NIT based NCDTs with longer wavelengths have improved lesion detection specificity along with imaging capabilities and is less affected by stains than other NCDTs, thus minimizing the chance of false positive results ,60,69, iWithin the limitations of this study, it can be concluded that there is indeed an interest in the discussion, further education, and use of novel technologies by restorative dentists aiding in their caries diagnostic practices. When all factors are considered FOTI seems to be the preferred device amongst clinicians due to its affordability, ease of use, and practicality with possible additional uses in detection of other dental conditions such as cracks and fracture lines.There is a noticeable trend in dentistry these days towards preventive dental approaches through early detection and effective monitoring of incipient caries lesions, especially amongst restorative dentists. However, the complexity and high price of some of these technologies are major factors working against their effective use in daily practice. Thus, providing further education to dentists harboring an interest in NCDTs can help better generalize their proper use. Further investigation into the NCDTs use preferences and insights of dental practitioners specializing in other fields of dentistry such as pediatric dentistry and advanced general dentistry might help shed more light on the subject and aid NCDT developers in improving the availability of NCDTs for general dental use, which eventually may lead to improved dental healthcare services across all fields of preventive dentistry."}
+{"text": "The conditioning of the root canal wall during chemo-mechanical root canal treatment differentially affects the adhesion of root canal sealers. This investigation evaluated the impact of sodium hypochlorite (NaOCl) concentration as used in a root canal irrigation concept called continuous chelation, with 1-hydroxyethylidene-1,1-diphosphonic acid (HEDP) contained in the NaOCl solution that is applied. Fourier-transform infrared spectra of the dentinal wall were gathered. The consequential effects on push-out bond strength of an epoxy resin (AH Plus) versus a hydraulic CaSi sealer (BioRoot RCS) were assessed. Single-rooted extracted human teeth were used and irrigated with pure NaOCl at a concentration of 0% , 2.5%, or 5.25%. Dual Rinse HEDP (9%) was added to the solutions, or not added for further control. Pure NaOCl solutions caused a decrease in the amide III: phosphate ratios, which was counter-acted by the addition of HEDP. It was observed that the adhesion of the epoxy resin sealer under investigation was negatively affected by this NaOCl deproteinization of the canal wall in a dose-dependent manner, while the opposite was observed with the CaSi sealer. HEDP when used in conjunction with NaOCl was beneficial for the adhesion of both sealers. Instrumentation, disinfection and three dimensional obturation of the root canal system are the crucial factors for successful endodontic treatment. During mechanical instrumentation of the root canal, a smear layer is formed on the radicular dentin and clogs the orifices of the dentinal tubules . This smDual Rinse (9% HEDP)  is a medical device  based on this chemistry. It comes in a capsule containing 0.9 g of etidronate powder, which should be mixed immediately with fresh 10 mL of NaOCl solution of choice directly before treatment . Based oA root canal sealer plays an important role in the formation of a bond between root filling material and root canal dentin and also obtains a bacteria-tight seal of the root canal system . As the Dislodgement resistance also called push-out bond strength (POBS) is regarded as a pertinent prognostic factor to evaluate the association of a root canal sealer to the canal wall . It is kThe study protocol was approved by the Institutional Review board (IEC 768/2018) for the use of human teeth extracted according to individual treatment plans, which had nothing to do with this investigation. Soft tissue fragments and calcified debris on the specimens were removed using ultrasonic scalers. The specimens were stored in 0.2% sodium azide solution  at 4 \u00b0C until use.To study the influence of NaOCl concentration in continuous chelation, one capsule of Dual Rinse HEDP (Medcem) was freshly dissolved in 10 mL of physiological saline , 2.5% NaOCl, or 5.25% NaOCl before each experiment. These solutions without the HEDP were used as controls, resulting in six treatment groups.n = 12). However, because of the qualitative rather than quantitative nature of this data, the sample size in the FTIR experiment was reduced to n = 5.The number of samples included in this study was based on the amide III: phosphate ratios in dentin after 30 min of exposure  and the n = 5) based on irrigation solutions used . Because the etidronate powder  was directly dissolved in the NaOCl or saline solution as recommended by the manufacturer, there was only one solution per group. The specimens were placed inside microtubes containing 10 mL of either of these solutions for 30 min [\u22121 at 4 cm\u22121 resolutions by using 64 scans per measurement.Thirty root canal dentin slices with dimensions of 1 cm \u00d7 1 cm were obtained from the root canal of extracted human single rooted teeth. The dentin slices were obtained from the middle third of each root. The root canal dentin surface of each specimen was polished using silicon carbide abrasive paper of 120 grit  and alumina suspensions. After attaining a flat and smooth surface that favours the absorbance of infrared radiation, the specimens were immersed in distilled water in ultrasonic bath for 1 min to remove the residues from polishing. The specimens were dried with absorbent paper to avoid excessive dehydration to reproduce tissue characteristics found in the clinical environment. The compositional analysis of all specimens was performed using the FTIR spectrophotometer  with a diamond attenuated total reflection (ATR) set-up. The specimens were then positioned with the polished surface in contact with the diamond crystal of the ATR set-up and the initial FTIR spectra of each sample was recorded. Subsequently, the specimens were divided into six groups (r 30 min  in an orn = 12) based on the irrigation regimen (see above). Root canals were irrigated with 5 mL of irrigant after each instrument change during instrumentation, and the then again with 5 mL of that irrigant as a final rinse, followed by 5 mL of distilled water to remove chemical remnants from the canal system.A total of 144 single-rooted human teeth was selected for this experiment assessing six irrigants, 12 specimens per irrigant, and two sealers (one tooth per observation). Radiographs of the specimens were taken from buccal and mesial aspect to confirm a straight, single canal with mature apices and without any calcifications. The teeth were decoronated using a diamond disc  and working length was established by inserting a size 10 K file  into each root canal until it was just visible at the apical foramen  then subtracting 1 mm from the recorded length. Apices of all the teeth were sealed with sticky wax to prevent the flow of irrigants through the apex and to allow an effective reverse flow of the irrigant, thereby simulating a closed end system. The specimens were then randomly divided into two times six groups  based on sealers used, AH Plus (DeTrey) versus BioRoot RCS (Septodont).Root canals were biomechanically prepared using ProTaper rotary system  according to the manufacturer\u2019s instructions up to a size of F3. Then the taper of the canal was removed using Peeso reamer size 1\u20133 . Irrigation was performed using a 27-gauge side vented needle , which was inserted 1 mm short of the working length. Following the final irrigation regimen, the root canals were dried using paper points (Dentsply Sirona). Samples in each group were then subdivided into two groups  under continuous water cooling to obtain a slice of 2.0 \u00b1 0.1 mm thickness. Sealers were then mixed according to manufacturer\u2019s instructions and were filled into the root canals. Specimens were placed in 100% humidity for 48 h to ensure complete setting of the sealers.Every sectioned tooth sample which was filled with sealer was subjected to POBS measurement. The root canal diameter, as well as the thickness of each slice, was recorded using a digital calliper. Push out test was carried out using a universal testing machine . The force was applied in an apico-coronal direction at a crosshead speed of 1 mm/min using stainless steel plungers of 0.9 mm diameter positioned so that it will contact only the filling material. The maximum force (F) applied at bond failure was recorded in Newton.The POBS was calculated in MPa using the formula:The adhesion surface area was calculated by the following equation:p value < 0.05 was considered to be statistically significant (95% confidence level).Data was analysed using one-way analysis of variance (ANOVA) followed by Tukey\u2019s honest significant difference (HSD) test. The analysis was undertaken using SPSS software, version 20 . A Compositional analysis revealed that amide III: phosphate ratio decreased in all groups compared to immersion in the inert physiological saline solution, except for Dual Rinse HEDP in saline, in which the ratio increased . The maxIn carbonate: phosphate ratio, only the pure 5.25% NaOCl showed an effect that differed from the inert control solution .The raw data  revealedp < 0.05) except 5.25% + Dual Rinse HEDP group and Saline + Dual Rinse HEDP  between each other and also to the other groups. The maximum value of POBS was noted in 5.25% NaOCl + Dual Rinse HEDP (33.6 \u00b1 1.0 MPa), and the lowest bond strength was noted with saline + HEDP (3.1 \u00b1 0.7 MPa).When comparing the POBS values for BioRoot RCS, no significant differences were noted between 2.5% NaOCl + Dual Rinse HEDP and 5.25% NaOCl, as well as between Saline and Saline + HEDP . HoweverRejecting all null hypotheses, this study showed that the concentration of the NaOCl solution has a dose-dependent effect on the organic content of the root canal wall as assessed by FTIR, and that this effect also reveals itself in the adhesiveness of the two sealer types under investigation. Irrigant effects on sealer adhesiveness were not minor, as they resulted in a fold-factor increase or decrease in sealer adhesion compared to the control treatment utilizing a chemically inert physiological saline solution .This study is limited by its in vitro nature. However, since extracted human teeth were used, the results may still have clinical value. The dislodgment resistance of sealers in human root canals has been shown to be directly connected to their prevention of fluid leakage through the same . SealabiA further limitation of this work is that the dentin samples were treated with the experimental solutions for 30 min for FTIR analysis. This is because our pilot work demonstrated that this time of exposition was necessary to show clear-cut differences in amide III: phosphate ratios, which is in line with published information . HoweverThe FTIR results of the present study is in accordance with Tartari et al. ,39 who dIn the root canals filled with BioRoot RCS, POBS did not seem to be affected by the reduction in organic content at the dentin surface by 5.25% NaOCl. The statistically highest POBS, however, was seen with 5.25% NaOCl + Dual Rinse HEDP. This could be attributed to the continuous chelating action, which enabled the dissolution of organic components of dentin, as well conditioning of the inorganic part of dentin. This leads to the reduction of smear layer and smear plugs improving micromechanical retention and thereby yielding a higher bond strength to root canal dentin . Again, In conclusion, the combined use of NaOCl and Dual Rinse HEDP appears to result in a homogenous distribution of organic and inorganic components on the treated surface . The res"}
+{"text": "Introduction: Proprioceptive impairment is a common symptom after stroke. Clarifying how proprioception correlates with motor function after stroke may be helpful in optimizing proprioception-augmented movement training. Previous studies have shown inconsistent findings. A meta-analysis is an optimal method to explore the correlation and identify the factors contributing to these inconsistencies.Objective: To explore the correlation between proprioception and motor function after stroke through a meta-analysis, taking into account characteristics of the measurements used in these studies.Methods: We searched multiple databases until November 2021 for eligible studies that measured both proprioception and motor functions in persons with stroke and reported their correlation or data for correlation analysis. A meta-analysis of the correlations was performed. The subgroup analysis and meta-regression were further conducted to investigate potential factors contributing to the heterogeneity of correlation strength, based on the participants' characteristics, proprioception, and motor function measures.Results: In total, 28 studies comprising of 1,829 participants with stroke were included in the meta-analysis. The overall correlation between proprioception and motor function was significant , but there was heterogeneity across studies . The results of the subgroup analysis showed proprioception of the axial segment in weight-bearing conditions  and upper limb without weight-bearing  had a stronger correlation with motor function than proprioception of the lower limb without weight-bearing. The proprioception measured through ipsilateral matching  showed a stronger correlation with motor function than through contralateral matching. The International Classification of Functioning, Disability, and Health (ICF) domains of motor function, movement function , activity performance , and independence  showed a stronger correlation with proprioception than with other domains.Conclusion: There is a significant correlation between proprioception and motor dysfunction after stroke. The proprioception measured in the axial segment under weight-bearing conditions or measured with ipsilateral matching, and motor function, specifically in the ICF domains of movement function, activity performance, and independence showed a positive contribution to the association between proprioception and motor function. The correlation does not imply causation and might be underestimated by attributes of current tests for proprioception and motor function. Further studies are needed to clarify the cause-effect relationship. Proprioception is the sense of position and motion of one's own body parts and the force generated during movement , 2. It iProprioceptive deficits may present when damage occurs in the proprioceptive receptors , any parThe diverse settings of proprioception or motor function measurements could be the main cause of inconsistent findings on the correlation between proprioception and motor function after the stroke across studies. Owing to the complexity of the pathological processes of proprioceptive deficits, various methods have been developed to measure proprioceptive acuity , such asThis study aimed to investigate the correlation between proprioceptive impairment and motor deficits after stroke through a meta-analysis, taking the characteristics of measures into account. We hypothesized that proprioception and motor function are correlated in persons with stroke, and characteristics of proprioception or motor function might have different influences on the correlation.* OR position sense OR movement sense OR velocity sense OR force sense) AND (stroke OR hemiplegia OR cerebrovascular accident) AND  AND (Relation* OR correlation OR effect OR difference).The review was registered on the PROSPERO International System Evaluation Prospective Registration website (registration number: CRD42020184181) in May 2020 and was conducted according to the \u201cPreferred Reporting Project for Systematic Evaluation and Meta-Analysis\u201d (PRISMA). Databases, including Web of Science, CINAHL complete, SportDiscus, MEDLINE, and Academic Search Premier through EbscoHost, were searched until November 15, 2021. There was no limit regarding publication dates but restricted to English language articles. The search strategies related to stroke, proprioception, and motor function were used . The duplicates were then removed. Titles and abstracts were screened by three reviewers . When the abstract of an article suggested that it might meet the inclusion criteria, the full text was read to check its eligibility, and it was included if it fulfilled the selection criteria. The reference lists of the relevant articles were searched as additional sources. The corresponding author (XS) was consulted when there was a disagreement.Studies identified through the database search were evaluated by reviewers to ensure that the study met all of the following inclusion criteria:targeted at adults with stroke.included proprioception measures with a clear description and interpretation of the measurements and results.included motor function measures with a clear description and interpretation of the measurements and results.presented the results of the correlation between proprioception and motor function; the presented data of each function were sufficient to analyze their correlation.Studies were excluded when the following criteria were not met:The correlation was explored only after interventions.The description of proprioception or motor function measures was not sufficiently clear to interpret their correlation .Quality assessment of diagnostic accuracy studies scale-second version (QUADAS-2) was adopted to evaluate the methodological quality of the included studies. QUADAS-2 is the most recommended tool to date to evaluate the risk of bias in diagnostic accuracy studies  and assoTo ensure consistent assessments, a rating guideline of QUADAS-2 with detailed criteria was developed by reviewers with tailored signaling questions according to the research question of this study . Each sip. The correlation coefficients were extracted from the published data of most of the included studies or by analyzing the original published data of several studies using the Pearson correlation test (The extracted data consisted of the author (year), sample size, characteristics of participants, proprioception, and motor function measures, and correlation between proprioception and motor function in the form of correlation coefficients or ion test \u201322. The For proprioception measures, the following characteristics were extracted from the studies, including subtypes of proprioception tested, test region and position, matching side, matching movement, number of joint planes of task movements, and result data type.Proprioception generally has three subtypes which are position, motion, and force senses. Position sense refers to the ability to perceive the position of a joint or body part. Motion sense is the ability to identify the movement speed or direction of a joint or body part. Force sense is the ability to recognize the force of the muscles or joints , 2. The Proprioception is usually measured at limbs and trunks in weight-bearing or non-weight-bearing positions. For weight-bearing conditions, standing and sitting are common positions during which the trunk and lower limb are in the midline region of the body, thereby named as axial body segment. So, test regions and positions were categorized as \u201cthe upper limb or lower limb without weight bearing,\u201d and \u201cthe axial segment in weight-bearing position.\u201dMatching is the commonly-used task for measuring proprioception. It usually consists of two main steps, first generating a reference position or movement, then matching the reference position or movement. Based on the body part involved in the two steps of movement, the task has two kinds, \u201cipsilateral\u201d and \u201ccontralateral.\u201d Ipsilateral matching indicates that the body part  involved in reference-matching movement is the same as that in the reference-generating movement, while contralateral matching implies the limbs for reference generation and replication are on the opposite side. The movement of the matching task has two modes, \u201cactive\u201d or \u201cpassive.\u201d We described the mode of movement at each step of the matching task. For some tests, reference matching is just performed perceptually without movement, such as by participants reporting the position perceived during the first reference movement. The matching of these tests was described as \u201cipsilateral\u201d type and with \u201cperceptual\u201d mode.The joint plane includes frontal, sagittal, and transverse types. The number of joint planes was recorded as \u201csingle\u201d when one plane at one joint was involved, such as knee flexion-extension, and was otherwise recorded as \u201cmultiple.\u201dThe results of the proprioception test have three types of data: (1) continuous data refer to data that can be of any value, such as an error in distance or error in the degree of angle; (2) ordinal data refer to data with a set order or scale, such as the score of scales; and (3) categorical data refer to data reflecting types by classifying or grouping phenomena according to their properties; for instance, if a function is impaired or intact.For motor function, all the tests involving the muscle, movement functions at the body function level under the framework of the International Classification of Functioning, Disability, and Health (ICF), the performance or independence of general functional tasks and self-care with upper limbs involved, and mobility according to the ICF and participation level were regarded as relevant measures to extract . The chaA data extraction sheet was developed first, after which the work was independently performed by two researchers who further checked the accuracy of the extracted data.Comprehensive Meta-Analysis (CMA) software (version 3.0) was used for the meta-analysis. A CMA sheet was built, which consisted of study name, proprioception test name, motor function name, effect size data of correlation, and a series of moderators related to participant characteristics, and proprioception and motor function measures. The data value of the correlation between proprioception and motor function was standardized for meta-analysis according to the meaning of the test data value on corresponding functions. The correlation value was maintained when the paired proprioception and motor measures had both positive and negative indications for corresponding functions . For exaFor studies with more than one measure of proprioception or motor function presented, a priority order of either proprioception or motor function, which was determined by their frequency presented across the included studies, was used to select the most common measure of proprioception or motor function for meta-analysis to minimize the heterogeneity across studies.I2 statistic. If there was significant heterogeneity across studies, the random-effects model of the meta-analysis was used; otherwise, the fixed-effects model was used to analyze the correlation. For studies with significant heterogeneity, meta-regression tests or subgroup analyses were performed to explore the potential factors contributing to heterogeneity. Egger's test was used to evaluate the publication bias between the strength of the correlation and sample sizes. The strength of correlation was categorized into five levels according to the correlation coefficient (r): very weak (0.00\u20130.19), weak (0.20\u20130.39) , moderate (0.40\u20130.59), strong (0.60\u20130.79), and very strong (0.80\u20131.00)  tests were the most frequently adopted measures in the reviewed studies. Other details of the proprioception tests are presented in Half of the studies measured proprioception within a single joint plane, such as using the JPS test , 38, 39,For motor function, the FMA-UE section and MAS were the most common measures used in the reviewed studies. Other details of the motor function tests are presented in All the included studies were evaluated based on the modified QUADAS-2, the results of which are shown in r = 0.267, p < 0.05); however, there was significant heterogeneity across the studies   and combined position and motion sense  showed a comparable correlation with motor function (between-group difference: p > 0.05) (p = 0.055) occurred among the axial segment in weight-bearing conditions , upper limb , and lower limb without weight-bearing  (r = 0.412) showed a near-to-significantly stronger correlation with motor function than contralateral matching (r = 0.240) (between-group difference: p = 0.050) (p > 0.05) (p > 0.05) (p > 0.05) was found among the proprioception with continuous data, ordinal data, and categorical data results (The correlation between proprioception and motor function was significant ( < 0.05) . For sub > 0.05) . For pro < 0.05) . Proprio= 0.050) . Proprio > 0.05) . The pro > 0.05) . Compari results .p < 0.05). Muscle strength , movement function , activity performance , and activity independence  were significantly correlated with proprioception function, while other domains showed no significant correlation with proprioception function (p > 0.05) (p > 0.05)  . The res > 0.05) . Please p > 0.05).Upon further exploring the factors contributing to the heterogeneity of correlation between proprioception and motor function based on the characteristics of participants, including age, sex ratio, time after stroke onset, the ratio of persons with a right to those with left hemisphere stroke, and excluding persons with spatial neglect, we found that none of them showed a significant contribution to the strength of correlation (p < 0.05).There was a publication bias between the strength of the correlation and the sample sizes found by Egger's test (r = 0.267) exists between proprioception and motor function in patients with stroke . Based on the heterogeneity, we identified several factors contributing to the correlation strength from the characteristics of proprioception and motor function tests.This study first investigated the correlation between proprioception and motor function by considering their characteristics in a meta-analysis. The findings of this study verify our hypothesis that a significant but weak correlation   . Our metr = 0.174), much less than that in the axial segment with the trunk and lower limb involved in weight-bearing conditions (r = 0.443). We further found that similarly in non-weight-bearing conditions, proprioception in the upper limb showed a higher association with motor function (r = 0.292) than that in the lower limb. It is possibly because movements at the upper limb are usually executed without weight-bearing, in other words, proprioception tests for the upper limbs without weight-bearing have higher ecological validity than those for the lower limbs. However, for the phenomenon of weaker correlation for proprioception tested in the upper limbs without weight-bearing than that tested in the axial segment with weight-bearing, ecological validity related to weight-bearing is improper to explain, but other potential factors could exist, such as matching manner, which will be discussed in later paragraphs. Based on these findings, weight-bearing could be proposed as an important component of proprioception training for the lower limb or axial segment to boost the effect on improving motor function.Most proprioception tests targeted limbs in non-weight-bearing conditions in the included studies. This approach is supposed to ensure the purity of proprioception input; however, it weakens the ecological validity, especially for the lower limb, since most daily functional activities are performed in weight-bearing conditions . This nor = 0.412) moderately correlated with motor function, more strongly than proprioception measured using contralateral matching tasks (r = 0.240) . Thvia the gamma motor system (r = 0.479) than other modes, despite without a between-group difference in subgroup analysis which might be possibly due to a small number of studies adopting this mode. Besides, the test with multi-plane movement demonstrated a slightly stronger association with motor function (r = 0.315) than that with single-plane (r = 0.228), despite without a between-group difference in subgroup analysis. This finding could be explained by the ecological validity of the test since daily movements almost occur in multi-planes with multi-joints. Moreover, in most of the included studies, motor function was measured by a composite test or global performance with multi-joints involved, such as FMA and Berg Balance Scale (BBS), thus had higher formal similarity with multi-planes movement in proprioception tests, which could be another reason of the above finding. Thus, in the movement mode, a joint plane could be necessary to consider in designing proprioceptive training, giving priority to active mode, multi-planes, and multi-joints.For the reference generation and replication movement during matching tasks, different movement modes, such as passive-then-active, are suggested reducing the testing validity . The senr system , 44. DifNowadays, various technology-assisted proprioception evaluation systems have been developed for enhancing the precision of proprioception tests. Usually, these kinds of systems are used for biofeedback-augmented proprioceptive training. Despite the advanced functions and uses of the technology-assisted system, it cannot replace the clinical proprioception tests. Most clinical proprioception tests use ordinal or categorical data to record results that have less resolution , 32, 37 r = 0.338), activity performance (r = 0.239), and independence (r = 0.319) were more strongly correlated with proprioception than with muscle strength (r = 0.222), muscle tone (r = 0.116), environment-specific activity performance (r = 0.169), and participation (r = 0.000). These findings match the physiological mechanisms of motor control  than those with ordinal and categorical data , in spite of no significant between-group difference. The phenomenon implies that motor function measures with higher resolution might be more sensitive to detect the effect of proprioception-augmented movement training.Besides, the motor function measured with continuous data for the result showed a much stronger association with proprioception . The findings and conclusions in this study are those of the authors and do not necessarily represent the official position of the National Key R&D Program of China.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "VISPR is an interactive visualization and analysis framework for CRISPR screening experiments. However, it only supports the output of MAGeCK, and requires installation and manual configuration. Furthermore, VISPR is designed to run on a single computer, and data sharing between collaborators is challenging.To make the tool easily accessible to the community, we present VISPR-online, a web-based general application allowing users to visualize, explore, and share CRISPR screening data online with a few simple steps. VISPR-online provides an exploration of screening results and visualization of read count changes. Apart from MAGeCK, VISPR-online supports two more popular CRISPR screening analysis tools: BAGEL and JACKS. It provides an interactive environment for exploring gene essentiality, viewing guide RNA (gRNA) locations, and allowing users to resume and share screening results.http://vispr-online.weililab.org, while the source code is available at https://github.com/lemoncyb/VISPR-online.VISPR-online allows users to visualize, explore and share CRISPR screening data online. It is freely available at The online version contains supplementary material available at 10.1186/s12859-021-04275-5. VISualization of crisPR screens), an interactive visualization program as part of MAGeCK-VISPR. Although VISPR enables users to examine quality control (QC) metrics and pick genes of interest, it only supports output of MAGeCK. Users should install VISPR on a local computer, and manually modify a configuration file to run the program. Data sharing between different users is also challenging in VISPR, since VISPR is designated to run on a single computer. These restrictions limit its applications.CRISPR/Cas9, as a novel and powerful genome editing technology, mediated high-throughput screening enables systematic exploration of the functions of coding genes and non-coding elements in various contexts, including cancer, infectious disease, and development \u20136. We prhttp://vispr-online.weililab.org) or set up their own server from the VISPR-online source code  plot in Fig.\u00a0Besides, the distribution of The resulting view also provides some extra resources to explore the results. Individual genes can be viewed in Ensembl by clicking on the gene name. The GeneMANIA  can be eVISPR-online allows users to save data on the server and retrieve them later. As shown in Fig.\u00a0Although VISPR allows users to explore CRISPR screening results locally, but it requires manual installation and configuration. In this paper, VISPR-online is presented as an easily accessible and interactive screening visualization framework. Researchers can investigate their screening results with the web browser and easily share their findings with collaborators.The modularity of VISPR-online makes it easy to extend functionality. We will follow the latest research of screening analysis tools, update the number of species supported every year, and integrate more valuable functions to VISPR-online to perform new tasks.p values of selected samples are shown in CDF and histogram plots. Besides, VISPR-online provides session saving and retrieving functions. Accordingly, researchers can quickly resume their previous analysis process and share experimental discoveries with collaborators. VISPR-online is open-source, browser agnostic, and easy to install even on a laptop. More features will be added in the future versions of VISPR-online to further facilitate screening data analysis further.VISPR-online is a general interactive framework for CRISPR screening visualization. It supports most popular screening analysis tools, including MAGeCK, BAGEL, and JACKS, while its browser interface provides various visualization features. (1) positively and negatively selected genes are displayed in separated sortable tables. (2) gRNAs are displayed in their gene context. (3) read counts of all samples are presented in parallel coordinates. (4) http://vispr-online.weililab.org. Operating system(s): Platform independent. Programming language: Python 3, HTML, JavaScript. Other requirements: pypi packages . License: MIT https://opensource.org/licenses/MIT. Any restrictions to use by non-academics.Project name: VISPR-online. Project home page: Additional file 1. VISPR-online source code and sample data. Code and sample data used for test.Additional file 2. VISPR-online installation instructions and usages."}
+{"text": "As a new type of under-film drip irrigation, water-fertilizer integrated fertilizer application device in Northwest China, the hose pump has achieved excellent results in practical applications, but its pulsation has exhibited some adverse effects on the fertilization process. By analyzing the cause of pulsation and flow characteristics, we proposed a shell optimization method to reduce pulsation. We used a release time deformation curve as the shape curve of the outlet shell of the hose pump. Based on the fluid\u2013structure interaction analysis, we developed a numerical model of an optimized three roller hose pump and a conventional three roller hose pump for dynamic simulations. The simulation results showed the optimized hose pump flow pressure variation range was reduced by 26.92%, the average fluid flow velocity increased by about 10%, and mass flow rate improved by 8.84% over the conventional hose pump. We tested the optimized hose pump prototype and the conventional hose pump on the test bench. The test results showed that the pulsating pressure variation range of the optimized pump decreased by about 20%, and flow output increased by about 8.63%. These results suggest that shell shape optimization assist in the decrease of flow pulsation and contribute to further hose pump popularization. Water-fertilizer integrated fertilizer application devices include the venturi fertilizer applicator, differential pressure fertilization tank, and pump fertilization device2. Nowadays, pump fertilizer application devices are increasingly used due to their effective power characteristics and ease of precise control4. Hose pumps belong to the peristaltic pumps category, which is a kind of rotor type volumetric pump6. The hose pump exhibits effective self-priming performance and performs well in actual use, but the pulsation phenomenon occurs. The pulsation phenomenon has some influence on the pump body and also has adverse effects on the water-fertilizer integrated fertilizer application process9.In northwestern China, based on climatic conditions and planting characteristics, under-film drip irrigation with water and fertilizer integration is widely used in agriculture10 present a numerical study of the flow field in a novel \u201csoft\u201d acting peristaltic pump, and the results show the flow has generally a two-way pulsatile nature, moving forwards and backwards. Formato11 applied fluid\u2013structure interaction (FSI) modeling for predicting the fluid flow in a specific peristaltic pump; hyper-elastic material dynamics and turbulence flow dynamics were coupled to describe all the physics of the pump, and the applicability of FSI modeling for geometric optimization of pump housing was also discussed to prevent roller and hose parts pressure peaks. Zhou12 presented a generalized parametric model for the fluid field in a typical roller pump system and deduced analytical formulations of the moving boundary. Based on the model and formulations, the dynamic geometry and mesh of the fluid field can be updated automatically according to the time-dependent roller positions. The described method successfully simulates the pulsing flow generated by the pump. Elabbasi13 used the 3D FSI of COMSOL multi-physics to study the performance of a 180-degree rotating peristaltic pump with two metal rollers, accurately designed the flow parameters of a peristaltic pump, and carried out theoretical calculations and experimental verifications. Wang14 established a three-dimensional, two-way fluid structure coupling model of the hose pump to study its pressure fluctuation characteristics. The results show the roller pass frequency is the main frequency of the pressure fluctuation at the outlet of the hose pump.Some studies have been conducted on the flow characteristics, pulsation characteristics, and numerical simulations of hose pumps. Natarajan and Mokhtarzadeh-DehghanTo reduce the influence of the pulsation phenomenon, we first analyze the pulsation reason and flow characteristics of hose pumps then propose a hose pump shell shape optimization method. Based on fluid\u2013structure interaction (FSI) analysis, we developed the numerical model of the optimized three roller hose pump and the conventional three roller hose pump to dynamic simulations. To verify the practical application effect, we tested the optimized hose pump prototype and the conventional hose pump on the test bench.Take the conventional three roller hose pump as an example, as shown in Fig.\u00a015; it changes periodically because, when the drum is about to release the hose, the pressure acting on the pump hose disappears rapidly, the liquid fertilizer flow space in the pump tube increases rapidly, the negative pressure is generated inside the hose, the liquid fertilizer at the outlet is sucked back, and then the recovery deformation of the pump tube makes part of the liquid flow back to the recovery deformation zone of the pump tube.At the outlet position of the hose pump, the liquid fertilizer increases and decreases intermittently. The liquid in the pump tube does not flow at equal speed16. Figure\u00a0The pulsation phenomenon complicates the calculation of the hose pump flow rate. Liquid fertilizer viscosity, hose pump joints, pipe pressure, hose material properties, and hose pump speed have an effect on hose pump flowD represents the diameter of the circumferential pitch circle of the pump shell, in millimeters; and d represents the inner diameter of the hose, in centimeters.ThenTherefore, the volume of the liquid transported by the hose pump rotating a circle is as follows:n is the rotor speed, in rad/s.Ideally, assuming the hose is not deformed, thenIn the actual working process, the hose deformation will inevitably occur when it is squeezed by the roller. The actual flow rate of the pump will be as follows:Therefore, for the hose pump, without considering the volume occupied by the rollers compacting the pump pipe, the flow of the hose pump is independent of the number of rollers and other parameters, but it is related to the inner diameter of the pump pipe, the circumference diameter of the pump shell, and the rotation speed of the hose pump.19.We assume that the slower and more stable the hose pump roller releases the hose at the outlet, the smaller the pulsation of the medium output. The process of releasing the hose at the outlet by the pressure roller is studied in detail belowL, and the release rotation angle is\u00a0\u03b1,\u00a0the release time is t, and the internal diameter of the hose is d,\u00a0then when\u00a0\u03b1\u00a0from 0 turns to\u00a0\u03b1max , l will change from 0 to d.As shown in Fig.\u00a0n, the radius of the roller is r, the distance from the center of the pump body to the center of the roller is s,We assume that the rated speed of the hose pump is ThenAccording to the trigonometric theorem,t) is related to the distance from the center of the pump shell to the center of the roller, the inner diameter of the hose, and the velocity when determining the pump hose material. The function L\u2009=\u2009f(t) is independent of the radius of the roller.From the derivation of the mathematical equation, we can obtain the angle of rotation and the longitudinal distance of the process of releasing the hose from the roller. We found that the function L\u2009=\u2009f: a0\u2009=\u20096.458 ; a1\u2009=\u2009\u2212\u00a05.846 ; b1\u2009=\u2009\u2212\u00a03.348 ; w\u2009=\u200924.63 .Goodness of fit: SSE:9.03; R-square: 0.9988; Adjusted R-square: 0.9988; RMSE: 0.1747.General model Gauss 1:Coefficients (with 95% confidence bounds): a1\u2009=\u200913.71 ; b1\u2009=\u20090.1487 ; c1\u2009=\u20090.06959 .Goodness of fit: SSE:49.74; R-square:0.9932; Adjusted R-square: 0.9932; RMSE: 0.4092.Due to the high coefficient of determination of the Gaussian and Fourier curve fitting, both Gaussian and Fourier curves can be used as a function of the curve of the shell above the hose pump outlet. In practical applications, the shell processing forms such as casting or stamping can be selected for manufacturing and processing to achieve mass production.In this study\u2019s model, the radius of the rigid roller is 34.5\u00a0mm, the thickness of the hose is 15\u00a0mm, and the inner diameter is 25\u00a0mm. The distance from the center of the pump to the center of the roller is 94\u00a0mm. The geometric parameters of the model are brought into Eqs.\u00a0 and 8),,8),thenDue to the initial position delay, the Fourier function (9) in  was chosen as a function of the curve of the shell above the hose pump outlet.This article does not contain any studies with human participants or animals.All authors agree to publication of the manuscript.25. However, due to the large deformation of the hose, the convergence of fluid and structure is difficult, and the amount of computation is extensive. The pump could be considered a multi-body model and used for dynamic simulations in engineering applications28. This paper mainly studies the three roller hose pump commonly used in the fertilization process. To verify the feasibility and effectiveness of the optimization method, we performed simulations to compare the conventional hose pump and the optimized hose pump.To describe the model behavior of the hose pump accurately, the shell shape optimization of a hose pump requires an accurate model. There is a strong coupling relationship between pipe deformation and fluid flow, so fluid\u2013structure interaction (FSI) analysis is neededWe established two three-dimensional, two-way fluid\u2013structure coupling models of the hose pump, as shown in Fig.\u00a05 after mesh independent check. It is vital to carry out a grid independence test on the generated meshes. We set up three monitoring points at the outlet and used the flow velocity as the reference quantity for grid independence. We conducted grid independence tests with grid numbers of 2.0\u2009\u00d7\u2009105, 3.0\u2009\u00d7\u2009105,4.0\u2009\u00d7\u2009105 and 5.0\u2009\u00d7\u2009105. It is found that when the grid number was greater than 3.0\u2009\u00d7\u2009105, the difference in flow velocity at the monitoring points was small. Therefore, it is considered that the grid independence has been achieved when the grid number is 3.0\u2009\u00d7\u2009105. Therefore the models grid meets the requirements of calculation accuracy and speed.To accelerate the calculation speed and maintain the iteration accuracy, we transported the geometry models of the hose pumps into ICEM 19.0 for meshing. As shown in Fig.\u00a0k is the turbulent kinetic energy, The conservation of mass and momentum are the governing equations in the FSI simulations. It is assumed that the flow squeezing through the roller is incompressible. The three-dimensional time-averaged Navier\u2013Stokes equation (Reynolds equation) for incompressible turbulent flows. To simulate the actual flow of fluid in the rubber hose of the hose pump, we established turbulence modeling with realizable k-e. The realizable k-e model is defined as follows:We describe the fluid in an arbitrary Lagrangian\u2013Eulerian (ALE) framework. The ALE derivative induced by a deformation of the fluid domain, The deformed fluid domain is defined as 3\u00a0kg/m3. The volume of the hose is almost constant when it is extruded, so it is classified as a hyper-elastic material30. The inlet boundary condition type is mass-flow-inlet. The turbulence specification method at the inlet is intensity and hydraulic diameter. The turbulence specification method at the outlet is intensity and viscosity ratio. The finite volume method is used to discretize the transport equations. The coupling between the pressure and velocity of the model pump is solved by the SIMPLE algorithm.Without considering the dissipation factor, the interaction between the pipe and the roller is frictionless. The near-wall treatment is standard wall functions. For the fluid part, the standard slip-free condition is adopted, and the wall roughness of all flow fields is smooth. The working fluid is water at 22\u00a0\u00b0C, and the density is The speed of the hose pump was set to 65 r/min. The step control was defined by time, the time step was set to 0.002\u00a0s, and the end of step time was set to 2\u00a0min. Figure\u00a0We selected a cross section of the center of the hose to observe the pressure change. Figures\u00a0Figures\u00a0The mass flow rate does not vary with time, space temperature, and pressure. To verify the effect on the flow output of the hose pump, we compared the inlet and outlet mass flow rates of the conventional hose pump and the optimized hose pump. The net mass flow rate of the conventional hose pump was 0.2877146\u00a0kg/s, whereas the net mass flow rate of the optimized hose pump was 0.3131438\u00a0kg/s. The mass flow rate of the optimized hose pump was 8.84% higher than that of the conventional hose pump, indicating that the optimized hose pump\u2019s flow output performance was improved.To test the actual effect of the hose pump shell optimization, a prototype test was needed. We commissioned the shell, manufactured by a local company, as shown in Fig.\u00a0We conducted a test in a test stand system, as shown in Fig.\u00a0Because it was not possible to measure the pressure variation in the pump in real time, we observed the prototype test pulsating pressure variation in the outlet pipe. Figure\u00a0Figure\u00a0Gaussian function and Fourier function can well fit the curve of the roller releasing hose. The shell optimization method of using the extrusion curve as the shell shape curve of the outlet of the hose pump can be used to reduce pulsation.The three-dimensional two-way fluid\u2013structure coupling model of the hose pump can effectively simulate the fluid pressure and flow rate changes in the pump, which helps optimize the structure.The shell optimization hose pump pulsation pressure variation range can be reduced by about 20% and flow increased by about 8%, indicating that the structure optimization method of this paper is effective, which can provide reference for hose pump manufacturers.In this paper, we first analyzed the causes of pulsation and flow output characteristics of hose pumps then proposed a shell optimization method to reduce pulsation by using the squeeze curve as the outlet shell shape curve of the hose pump. We then conducted a simulation experiment to compare the flow variation of the optimized hose pump and traditional hose pump on the basis of fluid\u2013solid coupling simulation. Finally, we conducted a prototype comparison test. The following are some main findings and suggestions, which can be considered in the process of hose pump design and manufacture to improve the operational reliability of hose pumps."}
+{"text": "Male sexual orientation is a scientifically and socially important trait shown by family and twin studies to be influenced by environmental and complex genetic factors. Individual genome-wide linkage studies (GWLS) have been conducted, but not jointly analyzed. Two main datasets account for\u2009>\u200990% of the published GWLS concordant sibling pairs on the trait and are jointly analyzed here: MGSOSO  and HamThe online version contains supplementary material available at 10.1007/s10508-021-02035-3. Male homosexuality runs in families, and twin studies have shown that genetic contributions appear to account for a moderate proportion of the variation in male sexual orientation with heritability estimated at\u2009~\u200932%    line up very well with the original GWLS manuscripts\u2019 multipoint plots\u2013Fig.\u00a0Our primary analysis for this investigation was the joint analysis of multipoint linkage from the Hamer and MGSOSO datasets Below is the link to the electronic supplementary material."}
+{"text": "Saccharomyces cerevisiae as a model system, which exhibits no endogenous SULT activity nor possesses SULT-related genes. We observed that ectopic SULT4A1 expression in yeast displays similar subcellular localization as reported in mouse neurons and observed that SULT4A1 is associated with the outer mitochondria membrane. SULT4A1 expression stimulates colony formation and protects these cells from hydrogen peroxide and metabolism-associated oxidative stress. These SULT4A1-mediated phenotypes are dependent on extracellular sulfate that is converted in yeast to PAPS, the universal sulfonate donor for SULT activity. Thus, heterologous SULT4A1 expression in yeast is correctly distributed and functional, and SULT4A1 antioxidant activity is sulfate dependent supporting the concept that SULT4A1 has sulfate-associated activity.Sulfotransferase 4A1 (SULT4A1) is an orphan member of the cytosolic SULT superfamily that contains enzymes that catalyze the sulfonation of hydrophobic drugs and hormones. SULT4A1 has been assessed through all classical SULT approaches yet no SULT activity has been reported. To ascertain SULT4A1 function and activity, we utilized  SULTs are considered Phase 2 drug metabolizing enzymes that catalyze the sulfonation of hydrophobic drugs and hormones, which transforms these molecules into hydrophilic metabolites, to regulate their cellular activity and excretion2. SULT4A1 is a member of the cytosolic SULT superfamily based on sequence and protein structural homology with other SULTs3. The SULT4A1 amino acid sequence is highly conserved within vertebrates4 and no homologous SULT4A1 sequences have been reported in invertebrates. Moreover, the human SULT4A1 gene shows an unusually low rate of mutation and exhibits the lowest mutation frequency among all known human SULTs6. Most SULT isoforms are widely expressed in tissues; however, SULT4A1 protein is primarily detected in neurons of the central nervous system (CNS) of humans, rats, and mice12. The SULT4A1 CNS expression pattern and growing reports strongly suggest a critical function for SULT4A1 in neuronal development and function. Disruption of SULT4A1 expression contributes to neurodevelopmental syndromes; Sequence polymorphisms in the 5\u2019-UTR and haploinsufficiency of the SULT4A1 gene (deletions at loci 22q13.3) are associated with schizophrenia and Phelan-McDermid syndrome, an autism spectrum disorder17. The first direct SULT4A1 phenotypes came from studies utilizing zebrafish, revealing possible roles in phototransduction and excessive sedentary behavior during day(light)-time18. Subsequent generation of mouse sult4A1-knockout (KO) models showed a severe neurological phenotype that resulted in death 3 to 4\u00a0weeks after birth7. Although no activity has been described for SULT4A1, the protein in fact has an essential role in normal neuronal development19. SULT4A1, unlike other cytosolic SULTs, localizes to the cytosolic and mitochondrial subcellular fractions of mouse and human brains7. This suggests that SULT4A1 might have a supportive role in mitochondrial function. Indeed, Hossain et al. reported that ectopic expressed SULT4A1 has a direct regulatory role in mitochondria function and redox-homeostasis9. These observations could explain the critical regulatory role of SULT4A1 in neuronal cell populations that exhibit a remarkably high-energy demand and generate increased levels of oxidative stress in the form of reactive oxygen species. Due to the specific neuronal expression of SULT4A1, characterizing its activity in primary neurons and cultured neurons presents certain obstacles. Most importantly these models express additional SULTs that hinder identification of specific SULT4A1 sulfonation substrates20. We use the tractable eukaryotic single cell model organism Saccharomyces cerevisiae in our quest to ascertain SULT4A1 activity and function. Yeast is an established model organism to investigate function, activity, and post-translational modification of neuronal proteins and mitochondrial disease mechanisms23. Moreover, the yeast genome does not contain any homologous SULT sequences and yeast cells do not show any sulfonation activity24. Thus, SULT4A1 expression in yeast provides a clean model system including endogenously produced 3'-phosphoadenosine-5'-phosphosulfate (PAPS) from imported environmental sulfate for use in Met and Cys synthesis24. Herein, we report that ectopic SULT4A1 expression in yeast displays a similar subcellular cytosolic and mitochondrial distribution as mouse neurons and cultured neuronal cell models9. Moreover, we show that SULT4A1 is associated with the mitochondrial outer membrane. In addition, SULT4A1 expression protects yeast cells from hydrogen peroxide induced toxicity and metabolically generated oxidative stress. Strikingly, the SULT4A1 mediated protective and growth stimulating phenotypes are sulfate dependent, suggesting that SULT4A1 exhibits functional sulfate-activity.Sulfotransferase 4A1 (SULT4A1) was initially identified and cloned from human and rat brain two decades ago11. Yet, no SULT4A1 catalytic activity has been detected using many classical SULT approaches25. To ascertain the activity and function of SULT4A1, we utilized the yeast S. cerevisiae as a genetically tractable single cell organism that is a well-recognized model organism to study mitochondria and neuronal protein functions. Moreover, studying SULT4A1 in yeast has the advantage that yeast cells innately produce 3'-phosphoadenosine-5'-phosphosulfate (PAPS), solely for the biosynthesis of Met and Cys residues24. Since yeast does not exhibit SULT activity, we first needed to demonstrate that yeast can tolerate heterologous SULT4A1 expression and its potential cellular activity. To prevent potential adverse effects of ectopic SULT4A1, we used plasmid-borne galactose-inducible SULT4A1 expression. The GAL1 promotor is actively repressed when yeast is cultured in dextrose and becomes transcriptionally active when yeast is cultured in the presence of galactose. Other carbon sources are considered \u2018neutral\u2019 as they do not repress or induce transcription from the GAL1 promotor. Yeast cultures with and without SULT4A1 did not show any adverse effects when cultured in dextrose media (no expression) nor in galactose media (induced expression). Subsequent analysis of total cell extracts of galactose induced cultures of cells with and without SULT4A1 revealed that SULT4A1 forms a stable full-length protein  fractions. Exponentially growing cells, with or without SULT4A1, were induced with galactose for 36\u00a0h to induce robust SULT4A1 expression. We subsequently isolated the cytosolic and mitochondrial fractions from yeast spheroplast lysates via differential centrifugation and separation using a sucrose gradient. SULT4A1 was detected in cytosol and a weak signal was observed in the lysate of the mitochondrial fraction  SULT4A1 is associated and on which side of these membranes . To examine SULT4A1 association with the mitochondria outer membrane, we treated purified mitochondria with trypsin to proteolyze outer mitochondrial proteins and peptides before isolating total mitochondria lysates. Trypsin treatment degraded all SULT4A1 but not the VADC/porin1 loading control and SULT4A1 was detected in the control sample\u2014no trypsin Fig.\u00a0e. These 2O2-induced oxidative stress9. For yeast to be a functional model organism to decipher SULT4A1 activity and function, SULT4A1 should protect yeast cells from H2O2 induced oxidative toxicity. We first determined yeast sensitivity to H2O2 grown on galactose and dextrose. Independent of the carbon source, yeast cells start to show H2O2 sensitivity at concentrations\u2009>\u200910\u00a0mM under our growth conditions. Interestingly, the protective function of SULT4A1 expression in yeast is effective and ostensibly independent of H2O2 concentration  requirements via respiration/oxidative phosphorylation27. Yeast cells prefer to use the energetically less efficient fermentation process over the energy efficient oxidative phosphorylation pathways28. Yeast cells using fermentation have lower levels of metabolically produced ROS29. Lower ROS-levels reduces the amount of oxidative damage to lipids, proteins, and nucleic acids resulting in prolonged proliferation and potentially increased cell lifespan29. We examined SULT4A1\u2019s role in protecting cells against metabolically generated ROS by monitoring growth in liquid media supplemented with glycerol as non-fermentable carbon source, and the fermentable carbon sources, raffinose and galactose. Glycerol and raffinose, unlike galactose or dextrose, do not activate or repress transcription of the GAL1 promoter. We induced expression of SULT4A1 by co-expressing the chimeric Gal4-ER-VP16 transcription activator30. This chimeric transcription factor contains the Gal4 DNA binding domain that binds to its \u2018upstream activating sequence\u2019 in the GAL1 promoter, which forms an active transcription factor upon exposure to estradiol. Estradiol stimulates dimerization via the estrogen receptor dimerization domain (ER) while the VP16 transcription activation domain stimulates transcription. Thus, in the presence of estradiol this chimeric transcription factor stimulates transcription from the GAL1 promoter30. Yeast expressing SULT4A1 grow significantly better than cells without SULT4A1 with glycerol as a carbon source  present in the YNB mix. Omitting sulfate-salt from the media did not affect SULT4A1 expression levels indicating that these phenotypes are SULT4A1-dependent. These results suggest that the protective function of SULT4A1 to endogenous and exogenous generated oxidative damage is dependent on sulfate and by extension PAPS.Since no SULT activity for SULT4A1 has been reported, we studied the effect of omitting sulfate from the media on the SULT4A1 phenotypes described above. Omitting sulfate from the media prevents yeast from producing PAPS1 and is still an intriguing, unique isoform within the cytosolic SULT family. Although, SULT4A1 contains the highly conserved SULT-catalytic Lys-Lys-His residues, no sulfation activity by SULT4A1 for small  compounds has been reported25. A recent report suggested that SULT4A1 is able to sulfonate 1-napthol using the S. pombe \u2018enzyme\u2019 bag assay, yet this observation has not been reproduced by other laboratories32. Fission yeast and budding yeast do not possess any endogenous SULT-activity. SULT4A1 does appear to have multiple important functions in the central nervous system. In family studies, SULT4A1 shows transmission disequilibrium with the occurrence of schizophrenia33. SULT4A1 gene deletion has also been linked to Phelan-McDermid Syndrome (PMS), a generalized cognitive and developmental autism spectrum syndrome14. The first direct SULT4A1 phenotypes came from studies utilizing zebrafish that showed a role for SULT4A1 in expression of cone genes in phototransduction4, and regulation of activity levels during daylight18. Subsequent generation of homozygous \u039412 and \u039428 knockout mice, showed SULT4A1 selective expression to neurons with a cytosol and mitochondrial subcellular localization that is unique for cytosolic SULTs12. Moreover, SULT4A1 knockout pups of both sexes develop significant tremors, movement/balance issues, dysmorphic changes in snout and back, failure to thrive, bouts of apparent silent seizures, become immobile, and die or are euthanized between 21 and 24\u00a0days of age7. Subsequent studies showed that SULT4A1 protects cells against oxidative stress induced toxicity, supports mitochondria function and redox-homeostasis in mice cultured cortical neurons and SULT4A1 transduced SH-SY5Y cells9. Nevertheless, the catalytic activity of SULT4A1 remains an enigma25.SULT4A1 was identified and isolated 20\u00a0years ago6. These observations suggest that SULT4A1 function and activity is highly conserved and includes potential interfaces required for SULT4A1-protein interactions. As such we reasoned that baker\u2019s yeast might be a good model organism to ascertain SULT4A1 function and activity. Yeast does not contain any homologous SULT gene sequences and does not show any native SULT activity24. Yeast does natively produce PAPS (for Met and Cys biosynthesis), which is the sulfonate donor for all cytosolic SULTs24. Moreover, yeast is a tested model organism to study neuronal protein function and activity23. As such, we verified that expression of heterologous SULT4A1 produces a stable protein and does not induce toxicity in yeast. Moreover, SULT4A1 subcellular distribution is similar to that reported in mouse brain and neuronal cells9. We observed that SULT4A1 is localized in the subcellular cytosolic and mitochondrial fractions of yeast lysates and the artificially formed microsomal fraction (data not shown). These observations suggest that the ectopic expressed SULT4A1 in yeast is properly folded, localized to similar subcellular locations, and is functional. Further dissection of SULT4A1 association with the mitochondrial fraction showed SULT4A1 is associated with the mitochondrial membrane fraction and not with its soluble fractions. Membrane localization was suggested by Hossain et al. in their orthogonal projection of fluorescent immunostaining that showed partial colocalization with TOM70 in SULT4A1 transduced SH-SY5Y cells and intense mitochondrial localization of ectopic SULT4A19. To dissect SULT4A1 mitochondrial membrane localization, we treated the sucrose gradient purified mitochondrial fraction with trypsin. We observed that SULT4A1 is associated with the mitochondrial outer membrane and not located within the membrane like VDAC/Porin1 as this protein was not affected by trypsin treatment. This allows a more focused approach to ascertain how SULT4A1 is associated with the outer membrane and how it stimulates mitochondrial function, which seems to be a conserved event from yeast to vertebrate neurons.SULT4A1 is highly conserved among vertebrates and no homologous gene sequences are reported in invertebrates. Moreover, SULT4A1 shows the lowest number of SNPs in humans of all cytosolic SULTs2O2-induced stress/toxicity9. Ectopic expression of SULT4A1 protects yeast cells from H2O2 induced toxicity. In addition, SULT4A1 expression stimulates yeast colony formation and growth in liquid media under fermentative  and respiratory (mitochondria dependent generation of ATP) growth conditions. This SULT4A1 stimulated growth advantage in yeast could be related to similar events recently report by Culotta et al.34. These authors reported that SULT4A1 is involved in regulating neuronal branching and dendritic spine formation, which was significantly reduced in cells that were SULT4A1 deprived34. SULT4A1 provides a significant growth advantage under respiratory growth conditions that is potentially related to protection against increased levels of mitochondria generated reactive oxygen species (ROS) including H2O2 production. Yet, even under low mitochondrial produced ROS (fermentation) SULT4A1 granted a growth advantage to cells. Yeast fermentation reactions are similar to the Warburg effect known to provide advantageous growth conditions for cancer cells under low oxygen conditions35. However, this Warburg effect or aerobic glycolysis has also been reported to exists in specific areas of the brain where the glucose utilization exceeds the oxygen consumption and is associated with Amyloid beta resistance38. These yeast observations could be an indication that SULT4A1 might protect mitochondria and stimulate neuronal cell propagation during aerobic glycolysis that peaks during early childhood and becomes more restricted to specific areas during adulthood38. The question remains, how does SULT4A1 mitigate the effects of ROS and peroxide? Part of the answer is that SULT4A1 protective and growth stimulating effect is dependent on sulfate. We omitted the majority sulfate from the media under fermentative and respiratory growth conditions that diminished the growth advantage and protection to H2O2 induced stress/toxicity provided by SULT4A1 expression. Omitting sulfate from the media did not affect SULT4A1 protein levels, signifying that the observed phenotypes are SULT4A1 activity dependent. The residual protection of SULT4A1 to H2O2 is due to the low levels (~\u20090.5\u00a0g/L) of sulfate present in the commercial yeast nitrogen base without amino acids without ammonium sulfate mix (BD Difco) in the form of copper-, manganese-, zinc- and magnesium-sulfate salts. These results suggest that SULT4A1 possesses SULT activity. In yeast, sulfate is converted into the universal SULT sulfonate donor PAPS that in yeast is solely used as the sulfur donor for the synthesis of Met and Cys.Although, ectopic SULT4A1 is stably expressed and exhibits a similar subcellular localization as in neuronal cells, we needed to verify that this heterologous SULT4A1 protein functions in yeast as in neuronal cells. One testable function reported thus far is SULT4A1 protection from oxidative-HSaccharomyces cerevisiae is an appropriate model organism to investigate SULT4A1 function, activity, and its molecular mechanism of action.In Summary, the observations herein show that heterologous expression of SULT4A1 in yeast stimulates yeast growth under fermentative (aerobic glycolysis or Warburg effect) and respiratory growth conditions and protects cells from metabolically generated and exogenously induced oxidative stress/toxicity. All these SULT4A1 mediated phenotypes are sulfate-dependent, implying that they are dependent on SULT4A1 SULT activity. Additionally, ectopic SULT4A1 displays a similar subcellular distribution to the cytosol and the mitochondria as reported in neuronal cells, and that SULT4A1 is associated with the outer mitochondrial membrane. These observations suggest that SULT4A1 expression in yeast supports mitochondria function and regulates redox-homeostasis and protects against oxidative stress induced toxicity that are all dependent on sulfate. This implies that the interaction interfaces SULT4A1 used to associate with the mitochondrial outer membrane and cytosolic interaction partners are highly conserved from vertebrates to yeast. However, the question remains, how does SULT4A1 facilitate these sulfate-dependent phenotypes? Independent of what SULT4A1 activity is, its potential substrates and/or interaction partners exist in yeast and seemed to be conserved in vertebrate neurons. Thus, our observations reported here suggests that Saccharomyces cerevisiae strain MGY-250  was generated from FY-250  by gene replacement of the ura3-52 allele with LoxP-KANr-LoxP, followed by CRE-mediated recombination to yield ura3\u2206::LoxP39. The murine SULT4A1 open reading frame sequence was PCR amplified from PLVXmSULT4A1-Puro plasmid (generous gift from Dr. Andrabi9) with BamHI-XbaI ends  and cloned via directed gene replacement of TDP1 into YCpGAL1TDP1\u2022L (LEU2) vector resulting in pRS415GAL1mSULT4A1\u2022LEU2 (YCpGAL1SULT4A1\u2022L)39. Plasmid born mouse SULT4A1 was expressed from the galactose inducible (GAL1) promotor to prevent adaptation or cytotoxic effects of the heterologous SULT4A1 protein. To express SULT4A1 from the GAL1 promoter under growth conditions using non-inducible carbon sources (raffinose or glycerol), we co-expressed the chimeric transcription-activator GAL4-ER-VP16 from the pRS313ADH1HA-GAL4ERVP16-Flag\u2022HIS3 plasmid (generous gift from Dr. Kodadek30) and induce dimerization of the chimeric transcription factor with 1\u00a0\u03bcg/ml estradiol (E2) to obtain transactivation. All minimal media contains yeast nitrogen base without amino acids and ammonium sulfate (BD Difco), supplemented with essential amino acids mix without those used for autotroph selection, indicated carbon source, and with 5\u00a0g/L ammonium sulfate except when specially noted without sulfate. In all cases, gene deletions were confirmed by PCR followed by DNA sequencing and cloned alleles were verified by DNA sequencing. All experiments were independently repeated at least three times, and a representative experiment is depicted in \u201cCultures of yeast cells transformed with the indicated vectors were grown overnight at 30\u00a0\u00b0C in selective minimal media supplemented with 2% dextrose, diluted 1:100 in selective minimal media supplemented with 2% galactose, or with 2% raffinose or 3% glycerol and grown overnight at 30\u00a0\u00b0C. SLUT4A1 expression is induced with 1\u00a0\u03bcg/ml E2 in cultures supplemented with raffinose and glycerol. These overnight cultures were subsequently used for the following cell viability assays:595 of 0.25 in minimal selective media with the selected carbon source and grown until OD595\u2009~\u20090.6 to obtain exponentially growing cultures. For H2O2 toxicity spot test, these exponentially growing cultures were aliquoted and incubated for 1\u00a0h with the indicated concentration of H2O2 at 30\u00a0\u00b0C. Exponentially growing (treated) cultures were diluted to OD595 of 0.3 in TE buffer  and tenfold serially diluted, and 5\u00a0\u00b5l aliquots were spotted onto selective media plates containing indicated carbon source. Plates were incubated for 7\u00a0days at 30\u00a0\u00b0C with growth being recoded every day using a gel-doc system (SYNGENE G:Box). All images were processed via Adobe Photoshop 2021 to correct signal levels of the complete image before cropping the shown area and placed into Adobe Illustrator 2021 to generate final figures.(1) Spot test or colony formation assay. Overnight cultures were diluted to OD2O2 toxicity, two selective media plates with indicated carbon sources were each spread with 50\u00a0\u00b5l of the appropriate dilution of treated cultures (as described in Spot test) and incubated for 4\u00a0days at 30\u00a0\u00b0C and colonies were counted by hand. At least three independent assays were used, graphed, and analyzed using unpaired (two-tailed) t-test using Prism.(2) Quantitative colony formation assay. To quantify H595 of 0.05 in 5\u00a0ml selective media with the appropriate carbon source with estradiol when needed and grown at 30\u00a0\u00b0C. Every 24\u00a0h for 6\u00a0days the culture\u2019s OD595 were determined. Results of at least three independent assays were used, graphed and one way ANOVA followed by Tukey multiple comparison test of cell starting exponential growth phase using Prism.(3) Liquid growth curves. The overnight cultures were diluted to an ODyeast cell viability assay with the indicated carbon source and harvested on day 3 at an OD595 of 0.6\u20130.8, washed in cold sterile deionized water, and cell pellet was resuspended in 150 \u00b5L TEEG buffer  with 1% sodium deoxycholate, 1\u00a0mM phenylmethylsulfonyl fluoride (PMSF), protease inhibitor-EDTA free (Pierce), and 100 \u00b5L frozen (\u2212\u200920\u00a0\u00b0C) sterilized acid-washed glass beads. The samples were lysed at 4\u00a0\u00b0C in a bead beater, 10 cycles of 30\u00a0s on, 1\u00a0min off. The lysate was cleared from cell debris/glass beads and the supernatant fractions were extracted, and protein concentrations determined by Bradford assay. Lysate was boiled in SDS buffer for 10\u00a0min and stored at\u2009\u2212\u200920\u00a0\u00b0C or immediately used for immunoblotting/staining.Yeast cells transformed with the indicated vectors were grown as described in the yeast cell viability assay and exponentially growing galactose induced cells were harvested and resuspended in Zymolyase Buffer , flash frozen in liquid nitrogen and stored at\u2009\u2212\u200980\u00a0\u00b0C. Thawed cells were dosed with additional 1\u00a0mM PMSF and 200\u00a0\u00b5g/ml Zymolyase T-20 , incubated at 36\u00a0\u00b0C with gentle agitation for 1.5\u00a0h to generate spheroplasts. All following manipulations were done on ice or at 4\u00a0\u00b0C. Spheroplasts were harvested and resuspended in Subcell Frac Buffer  and lysed with 20 strokes in a glass Dounce homogenizer. The lysate was cleared of whole cells and large debris by repeated 200 RCF centrifugations and the crude mitochondria was pelleted from this cleared lysate. The supernate of this fraction was cleared by ultracentrifugation at 134,000 RCF for 1\u00a0h to generate the cytosolic fraction and microsomal (pellet) fraction. The crude mitochondria pellet was resuspended in 1\u00a0mL Subcell Frac Buffer and subjected to ultracentrifugation over a sucrose gradient (2\u00a0mL 32% sucrose layered over 1.5\u00a0mL 70% sucrose) at 134,000 RCF for 1\u00a0h. Pure mitochondria were recovered from the S70-band that formed between the 32 and 70% sucrose layers of the gradient. The pure mitochondria were washed in Subcell Frac Buffer then resuspended in Lysis Buffer . Mitochondria were lysed by vortexing hard 15 times for 15\u00a0s then sonicated 5 times 5\u00a0s at 20% output. Mitochondrial lysates were cleared by centrifugation at 17,000 RCF for 30\u00a0min and the supernatant fractions were collected. Bradford assays were conducted on all samples to determine protein concentrations and lysates were boiled in SDS buffer for 10\u00a0min and were stored at\u2009\u2212\u200920\u00a0\u00b0C or immediately used for immunoblotting/staining.Yeast cells transformed with the indicated vectors were grown as described in the Trypsin treatment of purified mitochondria: Sucrose gradient purified mitochondria were washed in Subcell Frac Buffer without PMSF and aliquoted into two samples. One sample was treated with 10\u00a0\u00b5g/ml trypsin and both samples were incubated for 30\u00a0min on ice with occasional gentle mixing. The trypsin was deactivated by the addition of 2\u00a0mM PMSF and the mitochondria were washed with Subcell Frac Buffer. Mitochondria were lysed as described in subcellular fractionation.Equal amounts of yeast lysate fractions were resolved on 4\u201314%  Tris\u2013Glycine SDS-PAGE gels and blotted onto a PVDF membrane (Bio-Rad) and immunostained with anti-SULT4A1  first followed by stripping and staining with anti-Histone H3 , anti-GAPDH , or anti-VDAC1/porin  antibodies. Blots were visualized with Clarity Western ECL substrate (Bio-Rad) chemiluminescence and imaged using a gel-doc system (SYNGENE G:Box). All images were processed via Adobe Photoshop 2021 to correct signal levels of the complete image before cropping the shown area and placed into Adobe Illustrator 2021 to generate final figures. All samples shown in the figure panels were resolved in same gel."}
+{"text": "Dear Editor,The regimen of nab\u2010paclitaxel and gemcitabine (AG) has been widely used as the first\u2010line\u00a0chemotherapy for advanced pancreatic cancer; the prolonged survival time is still less than 2 months.transK value in the tumor by Dynamic contrast enhancement magnetic resonance imaging (DCE\u2010MRI). Patients with pancreatic cancer treated with the three\u2010cycle AG regimen had significantly elevated transK , the rate constant (Kep), and initial area under the curve taken up to 60 s (iAUC60), were elevated in the AG group  treated with AG or gemcitabine alone, we first examined the  Figures  and 1B,  Figure . Clinica Figures , but not Figures . Using a Figures . Moreove Figures . We conf Figures  and S1F. Figures  and 1I,  Figures , and dec Figures  and\u00a01J.Interesting, VEGF promotes gemcitabine resistance in pancreatic cancer cells Figures\u00a0 and\u00a02B. A previous report stated that the levels of RRM1 were significantly reduced in myc\u2010depleted cells.One PDX model of human PDAC was also established. AG showed a stronger antitumor effect than gemcitabine alone Figure\u00a0. More imA 59\u2010year\u2010old male patient was diagnosed with PDAC confirmed by pathology. The CT and DCE\u2010MRI showed pancreatic body tumor and liver metastasis. After two cycles of AG treatment, both the primary and metastatic lesions were stable. In addition, he described that his back pain was significantly relieved. After two additional cycles of AG treatment, the primary tumor shrunk to some extent, while the liver metastatic tumor remained stable. However, new nodules (red arrows) in the right lung emerged Figures  and S3B.Overall, these observations suggest that there were more blood vessels and less stroma in the tumor tissue after 2\u20134 cycles AG treatment Figure , and thaThe authors have declared no conflict of interest.This study was conceived by Yi Qin, Xiaowu Xu, and Xianjun Yu. Zheng Zhang and Shunrong Ji designed the study. Zheng Zhang and Qifeng Zhuo performed the experiments. Zheng Zhang, Qiangsheng Hu, Wei Liu, and Yi Qin analyzed and interpreted the data. Wei Liu, Wenyan Xu, Guixiong Fan, Mengqi Liu, and Zeng Ye reviewed the manuscript. Zheng Zhang wrote the paper with comments from all authors. All authors read and approved the final manuscript.We have gained the informed consent of patients and approval from the Clinical Research Ethics Committee of Fudan University Shanghai Cancer Center. The reported patient gave consent for the publication of his case. All animal experimental procedures were performed in accordance with the protocols approved by the Institutional Animal Care and Research Advisory Committee of Fudan University Shanghai Cancer Center.All data generated or analyzed during this study are included in this published article and its supplementary information files or from the corresponding author upon reasonable request.Figure S1 Statistical analysis of the correlation between the gemcitabine\u2010based regimen and transK or VEGF in the gemcitabine group. (A\u2010D) Statistical analysis of transK, Ve, Kep, and iAUC60 in the gemcitabine group. (E) Human cytokine array analysis of the effect of VEGF stimulation on the expression of putative genes involved in gemcitabine sensitivity in the gemcitabine group. (F) Statistical analysis of the difference in VEGF in the gemcitabine group. (G) Representative IF image of patients treated by neoadjuvant therapy. The baseline is referring to the patient before therapy administration.Click here for additional data file.Figure S2 Silencing c\u2010Myc reverses the effect of VEGF on RRM1. (A\u2010D) The mRNA and protein levels of RRM1 were lowered by silencing c\u2010Myc, even with VEGF stimulation. (E and F) c\u2010Myc suppression or knockdown reversed the effects of VEGF on the IC50 values of gemcitabine of pancreatic cancer cells.Click here for additional data file.Figure S3 A real\u2010world case of a patient with pancreatic cancer treated with AG plus bevacizumab after progression in response to AG. (A and B) Treatment and disease progression for the presented case. (C) Changes in the VEGF levels of the patient during chemotherapy. (D) The graphical representation summarizes the pancreatic cancer tissues before and after AG treatment.Click here for additional data file.transK in PDAC.Table S1. Clinicopathological features and correlation of Table S2. Clinicopathological features and correlation of serum VEGF in PDAC.Table S3. Clinicopathological features and correlation of VEGF expression in PDAC.Click here for additional data file."}
+{"text": "Cassava plays a major role in improving food security and reducing malnutrition. The purpose of this study was to evaluate the influence of mechanical pressing coupled with ultrafiltration (UF) on the quality of different fractions of cassava leaves. Cassava leaves harvested from the greenhouse at the University of Hohenheim were passed through a mechanical screw press to extract the juice and separate the press cake. The juice was centrifuged and filtered to separate the sediment and clear supernatant. The clear supernatant was filtered using a 10 kDa UF system. The nutritional contents of the different fractions were analyzed at each processing step. The total phenolic content was significantly lower in the press cake that had a higher fiber and ash content. The juice and sediment fractions had higher crude protein and total phenolic content. Processing did not negatively affect the concentrations of essential amino acids except for tryptophan in the juice fraction. Non-protein nitrogen was mainly present in the UF permeate, illustrating the potential of UF for upgrading soluble protein fractions. The results indicated that the different fractions during processing could be a possible source of protein for food, feed , or fiber (press cake) for ruminant feed. Manihot esculenta Crantz), mainly cultivated for its root, is the world\u2019s seventh most important crop in terms of production and it is the main food crop in many countries within the tropics [\u22121DM) and minerals [Most of the global agricultural food production focuses on meeting energy requirements but the main cause of malnutrition is a shortage of proteins and vitamins in the diet . The use tropics . Cassava tropics ,5,6. The tropics . Conversminerals ,8,9,10 iminerals ,12. Seveminerals ,9,13,14.minerals .Despite the huge potential of cassava leaves, they have not been widely incorporated into the food system as an alternative protein source . The maiSeveral attempts have been made previously to extract protein from cassava leaves but the results indicated limitations in essential amino acids and protein recovery ,15,16,20White protein can be further purified from the leaf concentrate by combining different methods such as pH or heat precipitation, organic solvent precipitation, and flocculation . The comDetermination of the proximate composition and protein profiling during the processing of cassava leaves will provide insight into its further use as an alternative food crop . DiffereCassava leaves from six month-old plants grown in a greenhouse at the University of Hohenheim were harvested and taken to the laboratory. The leaves were ground in a kitchen chopper for 10 s at a speed of 10,200 rpm to reduce their size . The juice was extracted through a mechanical screw press with a 4 mm nozzle diameter and a screw speed of 18 rpm  . The lea2 filtration surface. To separate the retentate and permeate fractions, the processed clear cassava supernatant was passed through a membrane with 35 PSI transmembrane pressure (TMP), calculated asfP, rP, and pP are the feed pressure, retentate pressure, and permeate pressure, respectively, given in PSI.The molecular weight cut-off for the UF system was selected based on the SDS-PAGE result of the supernatant and previous reports of the cassava protein molecular weight of different varieties . UF was VCR) that relates to the concentration degree was calculated as follows [fV is the initial feed volume and rV is the final retentate volume, both in mm.For UF, the volume concentration ratio ( follows :(2)VCR=VnMF of the n = 6 fractions during processing was calculated according to Equation (3):nm is the mass of fraction n and inim is the initial mass of the sample.The mass fraction The mass loss of processing methods was calculated by the difference from the initial weight. MF during processing was reported on a fresh matter (FM) basis.Additionally, the samples were dried in an oven at 105 for 12 h  to calcun content of the six processing fractions was measured by the Kjeldahl method using the Kjeldahl analysis system  according to the manufacturer\u2019s guidelines. The CPn content was calculated with a conversion factor of 6.25. The results are expressed in g per 100 g of DM (g 100 g\u22121DM).The CP\u22121 was diluted in 3 mL 80% methanol, then mixed, and placed in a 60 \u00b0C water bath for 20 min. It was then centrifuged at 13,500 rpm for 10 min . The supernatant was transferred to a 10 mL volumetric flask where the residue was mixed again with 3 mL of 80% methanol and centrifuged. The supernatant was combined with the previous volume and was adjusted to 10 mL with 80% methanol. The extracted solution was kept at 4 \u00b0C until the analysis. The sample and standard were incubated for 2 h at room temperature in the dark using 80% methanol as a blank. The absorbance of the standards and the samples was measured using a UV-spectrophotometer  at 725 nm. The standard calibration curve was prepared with a gallic acid stock solution at 0.005 to 0.1 mg mL\u22121 .The total phenolic content (TPC) was determined by the Folin-Ciocalteu reagent method . A sampl mg mL\u22121 . The TPCThe amino acid profile was determined by an amino acid analyzer system  according to the methods described in the European Commission regulation, number 152/2009 .Proteins from the clarified supernatant, retentate, and permeate were investigated through sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) . A super\u22121DM).The neutral detergent fiber (NDF), acid detergent fiber (ADF), and acid detergent lignin (ADL) were measured before and after pressing by AOAC  using thp \u2264 0.05 using Tukey\u2019s honest significant difference test.One-way analysis of variance (ANOVA) was conducted for the data obtained from three independent replicates, except for the amino acid profile (two replicates), using the SAS statistical software package . The statistically significant difference of the means was defined as The fresh cassava leaves, press cake, juice, sediment, retentate, and permeate had an average DM content of 22.6, 11.0, 57.4, 32.7, 7.0, and 5.0%, respectively.After mechanical pressing, the press cake represented 22.0% of the initial mass on average and the juice 69.2%. This result was in line with previous results that also obtained 70% green juice from alfalfa leaves . The avep < 0.05) higher in the sediment (45.5 g 100 g\u22121DM) and lower in the permeate (13.9 g 100 g\u22121DM) fraction (\u22121DM) was high due to the young age of the plants used in the experiment [\u22121DM) had a significantly higher CP content than the press cake (28.4 g 100 g\u22121DM), which is similar to alfalfa pressing with a twin-screw extruder [\u22121DM based on the extraction method. Research conducted by Urribarr\u00ed et al. [\u22121DM) from the fresh leaves (18.6 g 100 g\u22121DM). Another study by Castellanos et al. [\u22121DM) and UF using 40 kDa membrane (43.9 g 100 g\u22121DM) was high compared to the fresh leaves (22.0 g 100 g\u22121DM). In the current study, a CP content of 34.7 g 100 g\u22121DM was concentrated in the retentate fraction. The different compositions of the individual fractions compared to other studies could be attributed to the different processing methods such as membrane cut-off or the variety and age of the plant [The CP content was significantly . The young age of the plant used in this experiment played a role in the low ash content (2.2 g 100 g\u22121DM) of the fresh leaves [\u22121DM) [Solanum africana (19.4 g 100 g\u22121DM), Amaranthus hybridus (22.3 g 100 g\u22121DM), Telfaria occidentalis (11.4 g 100 g\u22121DM), and Vernonia amygdalina (9.5 g 100 g\u22121DM) [\u22121DM) than in the sediment (16.8 g 100 g\u22121DM) [Atriplex lampa leaves (10 kDa membrane) that resulted in a reduction of the ash content from 40% to 23% [\u22121DM) after UF (40 kDa membrane) than in the fresh leaves (5.7 g 100 g\u22121DM). This deviation might be due to the difference in plant age, variety, and/or processing methods.The percentage of ash, an indication of the total mineral content of the fractions, varied from 1.3 g 100 gn et al.  for diffs [\u22121DM) , Solanum0 g\u22121DM) . During 0 g\u22121DM) , whereas0 g\u22121DM) . The low% to 23% . The cur% to 23%  that sta\u22121 which is similar to the current finding for fresh leaves. After pressing and centrifuging, most of the TPC moved to the juice and sediment fractions  in the permeate fraction , while a minimal loss was observed in the liquid fractions (juice and retentate) . The metfraction  is an inA large variety of bands ranging from 10\u2013200 kDa that show large (53 kDa) and small (12 kDa) RuBisCo subunits were generated in the retentate and supernatant fractions a. The sip < 0.05) higher in the press cake while the reverse was true for the juice fraction  resulted in highly nutritious fractions. UF was effective in concentrating cassava leaf protein in the retentate fraction without changing the protein profile of the leaf, although the total phenolic content (TPC) was high. The results indicated that cassava leaves, sediment, and the juice fraction could be potentially used as a source of protein with the limitations of a higher TPC. The press cake co-fraction could be a good source for ruminant feeds with a good fiber and a low phenolic content. The protein of the different leaf fractions established them as a possible source for complementing other conventional foods and feed. The amino acid profile of the different fractions apart from the permeate suggested the potential benefits of cassava leaves. These findings support the use of cassava leaves as a food security crop with balanced nutrients. Further studies should be conducted to address the cyanogenic potential and other anti-nutritional contents of the fractions. In addition, the optimization of the UF process, considering the transmembrane pressure, pH, volume concentration ratio, and temperature, will play an important role in the quality of the protein concentrate obtained from cassava leaves via pressing and UF."}
+{"text": "As a result, self\u2010powered electronic system could be constructed by integrating printed paper batteries with solar cells and light\u2010emitting diodes. The result highlights the feasibility of hydrogel reinforced paper for ubiquitous flexible and eco\u2010friendly electronics.Paper electronics offer an environmentally sustainable option for flexible and wearable systems and perfectly fit the available printing technologies for high manufacturing efficiency. As the heart of energy\u2010consuming devices, paper\u2010based batteries are required to be compatible with printing processes with high fidelity. Herein, hydrogel reinforced cellulose paper (HCP) is designed to serve as the separator and solid electrolyte for paper batteries. The HCP can sustain higher strain than pristine papers and are biodegradable in natural environment within four weeks. Zinc\u2010metal (Ni and Mn) batteries printed on the HCP present remarkable volumetric energy density of \u224826 mWh cm Hydrogel reinforced cellulose paper presents enhanced mechanical and conductivity properties that make them suitable as the separator and quasi\u2010solid electrolyte for paper batteries. The printed zinc batteries (Mn\u2013Zn and Ni\u2013Zn) are flexible, cuttable, and potentially integrable with other paper electronics. Burgeoning development of flexible electronics not only poses severe challenges to traditional manufacturing methods, but also raises further concerns on sustainability, in terms of the waste disposal and recyclability at the end of product life. Cellulose paper, derived from abundant natural biomass on earth, presents an ideal building block for developing flexible and environmentally sustainable electronics. Because of its portability, renewability and function tuneability, paper electronics could be the raising star of next\u2010generation functional devices, especially in the field of paper\u2010based flexible batteries, sensors, circuits, and diagnostic equipment. Imagine if these devices can be integrated on a piece of paper, it will greatly facilitate the application scenarios and enhance the commercial values. Meanwhile, the compatibility of paper to printing techniques allows facile ink printing of a wide variety of source materials, further flourishing the paper electronics.As fifth generation (5G) broadband has been set for commercial availability since 2020, full potential of Internet of Things (IoT) technology will be released by allowing the interconnection and communication of massive amount of IoT devices.1, 2 Compared with conventional lithium\u2010ion batteries, aqueous zinc\u2010ion batteries hold the advantages of low cost, high theoretical volumetric capacity (5854 Ah L\u20131), good rechargeability, and safe chemistry. Paper\u2010based zinc\u2010ion batteries are mainly shown in two categories. One is assembling active materials on the surface of cellulose fibers paper or directly fabricating freestanding active material films as paper\u2010like electrodes. This method hinders the printability of paper, making it almost impossible to use printing technology to fabricate batteries. The other type of paper zinc\u2010ion batteries employs the paper only as the substrate, in which the batteries are in\u2010plane printed on one side and present interdigitated configuration. Printing electrode inks on both sides is challenging because it may cause short of the battery due to the rough and permeable surface of paper. Accordingly, the printed micro\u2010batteries on paper deliver limited areal capacity and volumetric energy density (up to \u224810 mWh cm\u20133). The above limitations of energy storage components impede the integration and reliability of paper electronics.Among these printed paper devices, batteries are considered as the heart of paper electronics.19 Com but the electrode materials cannot be printed on the composite. In addition, the compatibility of the paper batteries with flexible circuits and devices allows the construction of a self\u2010powered paper system that integrates printed battery with circuit lines and solar cells. This work demonstrates the possibility of printed paper batteries and integration with flexible electronics toward the new era of paper electronics.Herein, we propose a new hydrogel reinforced cellulose paper (HCP) for printable zinc batteries and paper electronics. By combining the characteristics of cellulose paper and hydrogel, the composite paper presents favorable mechanical and conductivity properties that are suitable to its function as the separator and electrolyte for quasi\u2010solid zinc batteries. The anode and cathode materials are printed on the front and back of the paper, respectively. This is different from previous nano\u2010cellulose/hydrogel composites where the composites were employed only as the separator for energy devices,31 but2FigureTo fabricate the HCP, commercial cellulose filter paper (CP) was pre\u2010immersed in acrylamide solution with initiator and crosslinking agent. After heating under inert atmosphere, polyacrylamide (PAAM) hydrogel was polymerized among the fiber gaps in CP Figure1a. SincScanning electron microscopy (SEM) images in Figure\u00a0 After four weeks, HCP are completely degraded while the CP takes six weeks for full degradation. Hygroscopicity of hydrogel greatly facilitates the growth and reproduction of microorganism. For comparison, Celgard separator  and other three typical polymer separators (cellulose acetate (CA), polyethersulfone (PES) and polytetrafluoroethylene (PTFE)) retained their original shape after the same burial time , both CP and HCP became fractured after two weeks of burial Figure\u00a0. PAAM an33 AftIt is worth noting that the HCP only expands itself in longitudinal direction , and presents neglectable expansion in the lateral direction when it is immersed and swollen in electrolyte solution. This can be attributed to the anisotropic expansion of hydrogel which is constrained in the laminate stacking frame structure of cellulose fibers. This property guarantees the integrity of the printed electrode films during the activation of HCP electrolyte. As a proof of concept, radio\u2010frequency identification (RFID) antenna patterns were printed with a commercial printer on two pieces of HCP with the same size of 30\u00a0mm \u00d7 30\u00a0mm. One patterned HCP was swollen in liquid electrolyte. As shown in Figure\u00a0\u22121 from the impedance measurement , which can be rooted to the lower content of hydrogel in HCP. Compared with aqueous electrolyte in pure cellulose paper, the HCP electrolyte shows highly stable Zn plating/stripping behavior and smaller polarization ,37 whin Figure\u00a0. This enFigure2 and MnO2 were synthesized by a facile co\u2010precipitation method  powders with a certain amount of carbon black and PVDF. The carbon black enhances the electronic conductivity of the printed electrode, and the PVDF additive improves the bonding of electrodes and HCP substrate. The cathode and anode inks are printed on the front and back of HCP, respectively  according to discharge curves, respectively, which is equal to volumetric capacity of 20 and 27.5 mAh cm\u22123 (including the entire device volume). These values are significantly higher than previous flexible Ni\u2013Zn batteries (4.4 mAh cm\u22123) and Mn\u2013Zn batteries (3.3 mAh cm\u22123) under similar measured current densities. The plateaus in the charge and discharge curves correspond to the peaks in the cyclic voltammetry curves 2. When the current density is five times higher (from 1 to 5\u00a0mA cm\u22122), the capacity of Ni\u2013Zn battery slightly drops to \u22480.7 mAh cm\u22122, retaining 84% of the initial capacity. This rate capability is much better than that of Mn\u2013Zn batteries (34% capacity retention when current density increases from 1 to 5\u00a0mA cm\u22122). The resistance of the two battery systems were also investigated using impedance spectra . They are also much higher than the reported flexible NiCo\u2010Zn battery (8.0 mWh cm\u22123), Mn\u2013Zn battery (12.0 mWh cm\u22123), and Ni\u2013Fe battery (16.6 mWh cm\u22123). By evaluating the volumetric  energy and power densities, our printed paper batteries may represent the state\u2010of\u2010the\u2010art and supersede the commercial supercapacitors and lithium\u2010ion thin\u2010film batteries .40 The\u22122, the capacity of Ni\u2013Zn and Mn\u2013Zn batteries retained at \u22480.4 and 0.5 mAh cm\u22122, respectively. To prove the universality of the hydrogel reinforcement strategy, we also used commercial A4 copy paper for the fabrication of printed batteries  was employed and silver wires were printed on the same paper substrate to connect the electrodes to a red light\u2010emitting diode (LED). When the switch was turned on, the LED can emit light normally. As shown in FigureFor flexible energy devices composed of hydrogel electrolyte, safety, cuttability, and editability are their intrinsic features.58 To J\u2013V curve is shown in Figure The best destination for the paper batteries should be in paper electronics. The integration of our battery and other flexible electronics on paper will promote the electronic system into a compact, lightweight, and convenient configuration. In principle, it is possible to achieve the all\u2010in\u2010one paper electronic system by integrating built\u2010in flexible solar cells, batteries, circuitry, and energy\u2010consuming electronics. This concept is schematically illustrated in Figure\u00a0\u22122. The total energy released from the battery is \u22487.5 mAh , which is converted from light by the solar cell. Although we employed commercial solar cell in the experiment for conceptual demonstration, we are optimistic that with the fast advance in manufacturing technologies, more and more energy harvesting devices (such as solar cells and nanogenerators) can be printed on paper. Therefore, our printed zinc\u2010metal batteries may serve as reliable and low\u2010cost energy storage units in an all\u2010paper self\u2010powered system.To confirm the compatibility of the battery and solar cell on paper, the light charging performance of the integrated system was investigated. The battery was fully discharged first and stabilized for 10\u00a0min. Then the solar cell was illuminated by a standard solar simulator at one sun intensity Figure\u00a0, and thelar cells61 and3In summary, we have realized zinc\u2010based paper batteries by printing electrode inks on biodegradable hydrogel reinforced cellulose paper. The hydrogel paper serves as both electrolyte and separator for printed batteries, for which the hydrogel significantly improves the mechanical strength of the cellulose fibers and maintains the ion conductivity of the composite. As a result, the printed batteries are cuttable and flexible without the risk of short of contact failure. In addition, they exhibit high volumetric capacity and energy density which outperform other paper based or thin\u2010film energy storage units. The facile synthesis and high performance may push forward the advance of paper batteries for applications in printed electronics and integrated paper circuits.The authors declare no conflict of interest.Supporting InformationClick here for additional data file.Supplemental Video 1Click here for additional data file.Supplemental Video 2Click here for additional data file."}
+{"text": "A multi-platform open-source Python-based graphical user interface has been developed to provide access to automated classification and data management tools for biomolecular crystallization screening. Polo is a Python-based graphical user interface designed to streamline viewing and analysis of images to monitor crystal growth, with a specific target to enable users of the High-Throughput Crystallization Screening Center at Hauptman-Woodward Medical Research Institute (HWI) to efficiently inspect their crystallization experiments. Polo aims to increase efficiency, reducing time spent manually reviewing crystallization images, and to improve the potential of identifying positive crystallization conditions. Polo provides a streamlined one-click graphical interface for the Machine Recognition of Crystallization Outcomes (MARCO) convolutional neural network for automated image classification, as well as powerful tools to view and score crystallization images, to compare crystallization conditions, and to facilitate collaborative review of crystallization screening results. Crystallization images need not have been captured at HWI to utilize Polo\u2019s basic functionality. Polo is free to use and modify for both academic and commercial use under the terms of the copyleft GNU General Public License v3.0. The minimum specifications of any test machine were two processor cores and 4\u2005GB of RAM. Note that use of the MARCO model is CPU intensive and faster processors will result in significantly decreased classification times. It is recommended to use a display with a resolution of at least 1920 \u00d7 1080 to allow easy viewing of all interfaces. The Mac and Windows versions of Polo do not require the installation of additional software or programming languages, but Ubuntu users will need to install the unrar program to interact with rar archive files. Instructions to do so are available in the Polo manual and on the Polo website.3.Polo includes several interfaces for reviewing and classifying crystallization images, with or without use of the MARCO model.Allowing for easy use of the MARCO model alone, while improving accessibility of the algorithm, would not necessarily prove beneficial or increase adoption by average users without an interface that effectively conveys classification results, contextualizes them in the presence of all available imaging data, and allows for easy manual classification and review of model results. For this reason, 3.1.Polo interface have been based on protocols at the Crystallization Center at HWI, and an understanding of the experimental protocols and terms that have motivated Polo\u2019s design is necessary for developing an understanding of the interfaces. Typically, when a researcher sends a macromolecular sample for screening, it will be robotically dispensed onto a screening plate that is composed of hundreds of screening wells, each containing a unique chemical cocktail. As the experiment progresses in time, each well will be imaged, creating collections of images called runs; each represents the state of the experiment at a specific point in time. Runs are represented digitally as directories of images and may optionally contain metadata files describing the experimental conditions. Images are captured with standard bright-field (visible) microscopy. In addition, the Crystallization Center uses a Formulatrix Rock Imager 1000 with SONICC , which enables ultraviolet two-photon excited fluorescence (UV-TPEF) and second-harmonic generation (SHG) microscopies. UV-TPEF and SHG imaging modalities can be used to differentiate macromolecular crystals from precipitate and salt crystals and to identify sub\u00admicrometre-sized crystals  or by using remote file transfer protocol (FTP). Local files can be imported using either a file browser or drag-and-drop interfaces. For accessing and downloading image data from FTP servers, Polo provides a built-in FTP client built on Python\u2019s ftplib package . The FTP browser can be used to connect to any FTP server available over the internet and is not limited to connections served by HWI.After a run is imported, whether using FTP or a local file, it will be displayed in the Samples window which organizes runs by sample and imaging date. Runs whose sample cannot be determined will be imported under the name \u2018Non-HWI Run\u2019. Selecting a run from the list in the Samples window by double clicking will open it in all other interfaces. Individual runs within the sample can be classified with the MARCO model by selecting the run and then the Classify Selected Run button, or all runs within the sample can be classified by left-clicking the sample and selecting Classify All Runs.3.3.Polo\u2019s Slideshow Viewer enables viewing of experimental images whether they have been classified by the MARCO model or not. Image data are integrated with associated metadata, including human classifications, MARCO classifications and the associated confidence level, the well number, the modality used to capture the image, and the cocktail details that constitute the screening condition. Image metadata and cocktail information are displayed in the Image Details and Cocktail Details text browsers, respectively  Python package. Plots are accessible under the Plot tab and can be easily modified, resized or exported to png image files using the controls available in the Plot interface. The Plot interface includes ways to visualize human classification progress for the experiment, to compare human classifications against MARCO classifications and to assess the overall accuracy of the MARCO model against human classifications.3.5.3.Polo\u2019s Optimize interface provides a screen builder tool that enables users to generate these optimization screens automatically and export the results to HTML files for reference while in the laboratory. Optimization screens use the successful crystallization cocktail as the midpoint for generating the chemical gradients. Along with their screening-plate dimensions, the researcher enters the screening-plate well volume, the stock concentration of each reagent and the variance in concentration along each step in the gradient as a percentage. The Optimize interface then calculates the required dilutions for each well in the screening plate.Once successful crystallization conditions are identified, optimization is often required to produce diffraction-quality crystals. Optimization often occurs by varying concentrations of macromolecule and cocktail components in 24- to 96-well crystallization plates, where a precipitant is varied along each axis of the plate creating a gradient of conditions around the original positive hit condition. Manual calculations to determine the correct dilutions of precipitants for each well can be time consuming and error prone. 3.6.Polo provides the option to export image data, metadata and classifications to several file formats for use by both Polo and non-Polo users. These tools will be especially helpful in further analyses of crystallization metadata.3.6.1.Polo facilitates saving runs in the xtal file format. The xtal format is JSON-based and encodes image classifications, chemical conditions and image data in the same file. At the cost of large file size, xtal files enable high portability as images, classifications and chemical data are contained in a single file. These files can be shared amongst collaborators who are also using Polo. In addition, Polo enables export of chemical and classification data to mso files, which do not include encoded images and provide a lighter-weight solution for sharing image classifications and cocktail formulations. Detailed documentation on both file formats can be found at the Polo website . This eliminates the need to manually gather screening images and their metadata, empowering users to select screening images based on classification and to generate presentations that incorporate all images and associated metadata available in the Slideshow Viewer interface, including time-resolved and multi-spectra views . This currently limits some of Polo\u2019s functionality to non-HWI researchers as it is not feasible to exhaustively anticipate all file formats, naming protocols, data organization schemes etc. that may or may not be utilized by other crystallization screening experiments. Users with some experience in Python object-oriented programming should find the Polo source code flexible enough to create the necessary functionality required by their experimental and data management protocols .5.Polo is written in Python and uses the PyQt package from Riverside Computing as the graphical engine. All software included or used in Polo\u2019s implementation is open source and, at minimum, is free to use or modify provided that any derivative work also remains free and open source. A complete list of dependencies is available in Table S1 or on the Polo GitHub page (https://github.com/Hauptman-Woodward/Marco_Polo). Modification, improvement or customization of Polo\u2019s source code is encouraged. A self-installing distribution is available for Windows 10 and binary executables are available for Mac OS versions greater than or equal to High Sierra and Ubuntu Linux greater than or equal to 18.04. Current and previous releases of Polo can be downloaded from the GitHub releases page (https://github.com/Hauptman-Woodward/Marco_Polo/releases). Currently, root access is required to mark the Mac and Ubuntu distributions as executable. The Windows 10 distribution can be run without administrator privileges. In-depth installation instructions, video tutorials, sample data and source code documentation are available at the Polo website (https://hauptman-woodward.github.io/Marco_Polo/).6.Polo is an ongoing project under active development. In the future we plan to involve software developers and crystallographers from other organizations and crystal screening facilities to create a more universal application for structural biologists. We also hope to begin integrating tools for more in-depth chemical analysis of successful screening conditions. As the MARCO algorithm has been trained using bright-field images, the classifying function in Polo is only designed to perform using this type of image. We note the need to extend the classification capabilities to also include information from alternative imaging modalities, such as UV-TPEF and SHG. Polo in the current version provides the capability to view alternative imaging methods alongside the classified images, and we hope to extend these capabilities in the future. Potential contributors are encouraged to contact the authors, to suggest source code modifications and extensions, and to draft pull requests on Polo\u2019s GitHub site to aid in the further development of these tools.10.1107/S1600576721000108/ei5066sup1.pdfTable S1: Dependencies required. FIgs. S1 to S4. DOI:"}
+{"text": "An otherwise healthy 29-year-old man presented with multiple 1 to 3 mm purple papules present in the last 5 years on his glans and scrotum . He had Genital angiokeratomas are benign vascular lesions occurring most commonly on the scrotum or vulva.The differential diagnosis of angiokeratomas includes common nevus, blue nevus, primary melanoma, or cutaneous metastasis, angiomas, as well as others lesions with vascular patterns, such as basal cell carcinomas, and pyogenic granulomas. When angiokeratomas are diffusely present, evaluation for a lysosomal storage disease (LSDs)  and possible referral to a clinical geneticist should be considered .Penile angiokeratomas (PEAKERs) are an uncommon subtype of genital angiokeratomas . These b"}
+{"text": "The solar panels installed on a CubeSat are considered the main energy source of a nanosatellites. The deployment mechanism of a solar panel must be analyzed and tested extensively. Any suggested solar panel design should present a low vibrating free spinning deployment mechanism. This paper examines various types of solar panels to reach a conclusion of the efficient design when deployed on a 1U or 2U unit. However, calculations, analysis, simulations do not always give an extensive picture of how the satellite shall behave during deployment. Thus, testing in a microgravity environment gives a more accurate answer of how the satellite shall behave. In our work, various solar panels mechanisms are developed and eventually tested in microgravity. The first accordion structure for a 1U structure is tested in a microgravity environment through a parabolic flight with the National Research Council Falcon 20 aircraft. The results are recorded and analyzed to optimize the next design. The second design is based on a drag-sail mechanism for a 2U structure. The design is improved upon the first experiment results for the next parabolic flight. The simulated amount of power generated in orbit is also a main factor in our evaluation. The CubeSat project was launched by the University of Stanford and Cali Poly in 1999 to provide a pico-satellite standard with a lower cost and development time, increase space accessibility, and maintain frequent launches. The 1U CubeSat is a 10-cm cube up to 1.33 kg in weight [In this paper, two expandable designs were proposed to increase the number of additional solar panels on the 1U and 2U CubeSats and accordingly increase power generation. For the 1U CubeSat, an accordion origami concept is implemented, while for the 2U CubeSat, the drag-sail mechanism is used. Different tests and analyses are done on both designs to prove their practicability, such as structural analysis, dynamical analysis, power analysis, and microgravity experiment using a parabolic flight.In Sect.\u00a0The mechanical component concerns CubeSat\u2019s durability and strength as it is deployed into the outside space. During the process, the CubeSat mainly works in three forms of mechanical load: quasi-static, static, and dynamic loading. The work in  executedThe work represented in  includesIn , the worx-, y-, and z- separately. Results of equivalent stress (Von-Mises) distributions showed that the maximum stress that the primary and secondary structures experience is safe as it was small compared to the yield stress of Aluminum 6061-T6 and the total deflection was acceptable in comparison to the static deflection envelopes of the all components of the structures.The static response of their primary and secondary structure which is done using ANSYS software was shown in . This reNASA\u2019s Jet Propulsion engineers in Pasadena, California, think about how the principles of origami could be used for space-bound devices for easier deployment. Researchers say that origami can be useful in utilizing space solar power for earth-based purposes. Solar power acting like a power planet that wirelessly beams power down to Earth using microwaves. Panels used in space missions needed to be simple folds, collapsing like a fan or an accordion to simplify mechanical design for opening and closing . created A standard 1U Cubesat is proposed to have increased solar panels to increase the power generated . This moThe accordion can provide a large surface area from a small one with a simple mechanism. The accordion folds with be used to add the solar panels on it to increase the number of the solar panels of the cube-sat. The mechanism that will fold and unfold the accordion origami will be scissors mechanism which is used in elevators and hydraulic lifts. The accordion extendable CubeSat mechanism which is designed using SOLIDWORKS  is shownDrag sail is another type of origami folding technique that has a unique design to provide a large area of solar panels while opening. Drag-sail design consists of three main parts which are frame design, rotating mounting plate and Geneva cam represents unfold mechanism.As shown in Figs.\u00a0As shown in Figs.\u00a0During the microgravity envelope, the side forces or extra torques will lead to unstable panels deployment. Therefore, motion analysis is made to the grantee that the mechanisms of the extendable CubeSat designs will not lead to extra forces or torques and the velocities of the joints of the mechanism are symmetric. For the accordion mechanism, SOLIDWORKS motion analysis is used, as shown in Figs.\u00a0The motion analysis showed that every two corresponding links in the scissor mechanism have the same velocity. Figures\u00a0The analysis also showed that the scissors mechanism guarantees that the reactions of each two corresponding joints are the same which guarantees that there are neither side forces nor extra torques during panels deployment in the microgravity experiment. Figures\u00a0Drag-sail design should verify that the 4 m move with the same velocity to achieve successful folding for origami sail without any fail on opening.Center-to-center diameter Wheel diameter Driver pin diameter x axis.Studying Motion analysis for Geneva mechanism will be proceeded by giving the motor a certain mentclasspt{minimaAlso, the mechanism provides the same displacement of 4 m, because 4 m attached pins are connected with coupler with the same diameter from the coupler center. The velocity of the cam will be the same velocity of meter extension because of symmetric meters attached with coupler. As a result of velocity and transmission of the Geneva cam mechanism, the 4 m will provide symmetric velocity and displacement output for extension and retraction process .To evaluate the CubeSats performance during its presence in the space, the two proposed CubeSats models are imported to Systems Tool Kit (STK) program  which isSolar cells, which are placed on the solar panels, are used for power generation. AZUR space solar cells are chosen, which are space proved gallium arsenide solar cells, called GaAs triple-junction solar cells . The typmentclass2pt{minimThere are four different parameters that define the orbit which is the right ascension of ascending node (RAAN), eccentricity, inclination, and height. The mentioned orbit parameters values are shown in Table\u00a0The Accordion design is initially equal to approximately the standard 1U CubeSat when folded, but it can extend up to 50 cm using scissors mechanism, allowing more surface area of solar cells for power generation. The fully extended CubeSat is shown in Figs.\u00a0Simulating the Accordion CubeSat on STK while using the orbit details mentioned in Table\u00a0Here is the standard 1U CubeSat placed in the same orbit and simulated for the same duration for an accurate comparison. The simulation gives the graph in Fig.\u00a0Meanwhile, for the Drag-Sail design, it is initially equal to the standard 2U CubeSat when folded, but it can extend up to 500 Simulating the Drag-Sail CubeSat on STK while using the orbit details mentioned in Table\u00a0Here is the standard 2U CubeSat placed in the same orbit and simulated for the same duration for an accurate comparison. The simulation gives the graph in Fig.\u00a0The STK analysis results for the solar power generation of designs 1 and 2 which are the Accordion CubeSat and Drag-Sail CubeSat are shown in Figs.\u00a0For the parabolic flight, there were many constraints that were taken into consideration at the designing and implementation phases. The prototype size should be less than 30 The G-Force for the accordion design is an essential part in the project to ensure the safety of the cube while the deployment of the microgravity experiment. Without this test, all the experiment may fail. There are many methods for these tests; some can be done using analysis tool such as ANSYS and some can done using practical laboratories. The test for the accordion design is done in the laboratory. The G-Force test for the accordion design was implemented in German University in Cairo (GUC) crash test laboratory to the grantee that the cube is safe while deployment of the flight. Figure\u00a0G-Force test was performed on the drag-sail design to ensure that the cube will withstand the loading and boundary conditions during launching. The static structural analysis was done using ANSYS software . This anWhile Steel AISI 1020 was chosen for the 32 bolts in the design and the printed circuit board (PCB) which has a tensile yield strength of 294 MPa and a tensile ultimate strength of 394 MPa, steel AISI 1035 was chosen for the motor which has a tensile yield strength of 460 MPa and a tensile ultimate strength of 460 MPa, and AL 2024 was chosen for the controller Arduino which has a tensile yield strength of 324 MPa and a tensile ultimate strength of 324 MPa. Before the analysis, meshing using tetrahedron elements was performed on the structure which consists of 665,621 elements and 1,214,592 nodes, as shown in Fig.\u00a0X-, Y-, and Z-directions and a magnitude of 2G m/sIn the quasi-static analysis, the load which consists of three vectors in the Figure\u00a0Figure\u00a0X-, Y-, Z-directions simultaneously.Another configuration for the fixed supports was applied where the supports were placed on the four rails in the bottom only and the force was of magnitude 2G m/sFigure\u00a0X-, Y-, Z-directions simultaneously where the maximum deformation is a very small value Figure\u00a0Both configurations were proved to be safe for the CubeSat during launching process which means that the cube will not fail due to excess in stress or deformation.The flight includes 6 parabolas, each consisting of 20 s of reduced gravity. The flights were executed by the National Research Council (NRC) in Canada with the cooperation of Integrated Space Flight and Project PoSSUM. There was up to 3 min of level to be used for setting up experimental procedures. The material that was used for the prototype was acrylic. Laser cutting is used in the manufacturing process to convert the parts from SOLIDWORKS into a real prototype. The prototype is tested in a parabolic flight, as shown in Figs.\u00a0This study focused on increasing the power generated by the CubeSat which increases the functionality and processing capabilities, so it can be used in more challenging missions. Two expandable designs are proposed which are accordion CubeSat (1U) and drag-sail CubeSat (2U). Different analyses are applied to both CubeSats such as structural, dynamical, and power analysis. The accordion CubeSat is implemented and tested in the microgravity. Results show that both designs mechanism has neither side force nor extra torque that guarantees the cube stability in space. The generated power efficiency by the proposed CubeSats outperformed the standards, where the accordion CubeSat exceeded the standard 1U by 145.212%, while the drag-sail CubeSat exceeded the standard 2U by 477.867%. The microgravity experiment is done in a reduced-gravity aircraft during a parabolic flight and it shows that the accordion design is stable and can be used for future CubeSats. Future research directions include manufacturing and testing the drag-sail CubeSat to ensure its safety during flights and to be tested in microgravity, measuring the produced vibrations during the deployment of both CubeSats. Also, consider different orbital scenarios for the satellites."}
+{"text": "R. esculentum is a popular seafood in Asian countries and an economic marine fishery resource in China. However, the genetic linkage map and growth-related molecular markers are still lacking, hindering marker assisted selection (MAS) for genetic improvement of R. esculentum. Therefore, we firstly used 2b-restriction site-associated DNA (2b-RAD) method to sequence 152 R. esculentum specimens and obtained 9100 single nucleotide polymorphism (SNP) markers. A 1456.34\u00a0cM linkage map was constructed using 2508 SNP markers with an average interval of 0.58\u00a0cM. Then, six quantitative trait loci (QTLs) for umbrella diameter and body weight were detected by QTL analysis based on the new linkage map. The six QTLs are located on four linkage groups (LGs), LG4, LG13, LG14 and LG15, explaining 9.4% to 13.4% of the phenotypic variation. Finally, 27 candidate genes in QTLs regions of LG 14 and 15 were found associated with growth and one gene named RE13670  may play an important role in controlling the growth of R. esculentum. This study provides valuable information for investigating the growth mechanism and MAS breeding in R. esculentum. R. esculentum distributes in the northwest Pacific Ocean and is a popular seafood in Asian countries, especially in China2. R. esculentum is rich in protein and minerals while low in calories and fats, making it an ideal nutritive ingredient for developing oral formulations, functional food and nutricosmetics3. In addition, the collagen peptides from R. esculentum can accelerate the wound healing process of mice4 and have antihypertensive activity5, suggesting it can be applied in pharmaceutical industry. The edibility, nutritional value and medicinal properties of R. esculentum make it an economical fish resource and widely farmed in aquaculture systems of China2. However, overfishing decreased the population number of R. esculentum and caused the scarcity of natural resource6. To sustainable development and utilization of R. esculentum resources, it\u2019s necessary to investigate the genetic background and genetic improvement.Edible jellyfish 7. The advance of the 2b-RAD method attracts many researchers\u2019 attention and has accelerated the identification of SNP markers for constructing genetic linkage maps in farmed fishes, such as Carassius auratus8, Cyprinus carpio haematopterus9, Hypophthalmichthys nobilis10 and Hemibagrus wyckioides11. The average marker interval of these linkage maps was between 0.44 and 0.87\u00a0cM and helped mapping QTLs of interested growth traits for genetic breeding11. Based on the constructed linkage maps, many QTLs related to economic traits, such as body weight, body length, nutritional metabolisms and sex have been identified12. For example, one QTL related to body weight in Apostichopus japonicas, explaining 11.8% of the phenotypic variation7.Genetic linkage maps play an important role in studies of genome and geneticR. esculentum genome has been released13, the genetic linkage map and QTL for growth in R. esculentum have not been reported yet. In previous studies, the researchers only identified some markers in R. esculentum, such as microsatellite for detection of genetic diversity and conservation of germplasm resources14 and SNPs and simple sequence repeats (SSRs) for assisting MAS breeding15. To improve the growth of R. esculentum through MAS breeding, we identified SNP markers by 2b-RAD method and constructed the first genetic linkage map of R. esculentum. Furthermore, we detected some QTLs and genes related to growth traits of R. esculentum based on the linkage map. This study will provide some insights for investigating the growth mechanism and genetic research of R. esculentum in the future.Although the R. esculentum was close to 63%  ratio of 1.48. After filtering, 6674 SNP markers can be used for constructing the linkage map. Through the neighbor-joining tree and principal component analysis, we excluded the abnormal individuals and confirmed the parents and offspring as a whole for constructing the linkage map . For femR. esculentu13  was considered to play a key role in controlling the growth of R. esculentum . Of these candidate genes, 27 genes were overlapped, associating with both umbrella diameter and body weight Table . As we cum Table . Using ond Table .Table 4C16. SNP markers as one of the molecular markers were genotyped easily to construct the genetic linkage maps for guiding the genetic breeding of aquaculture animals17. Researchers have identified 1,034,708 SNPs in R. esculentum by transcriptome sequencing15, yet we only identified 9100 SNPs by 2b-RAD sequencing, much lower than that in the previous study15. The difference in SNP numbers between the two studies may be caused by the SNP analysis method. In the previous study, SNPs were detected by GATK2 software without reference genome while by RADtyping software according to R. esculentum genome in this study. Due to the simplicity and flexibility, the 2b-RAD method was extensively used for identifying SNPs and constructing high-density linkage maps for aquaculture animals19. By 2b-RAD method, Zhu et al. constructed the high-density linkage map of Pseudobagrus ussuriensis utilizing 7435 SNPs with a marker interval of 0.357\u00a0cM17. For R. esculentum, the marker interval of the linkage map is 0.58\u00a0cM at medium\u00a0density, higher than that of H. nobilis10, H. wyckioides11 and Channa argus19, lower than that of C. auratus8, P. ussuriensis17, Larimichthys crocea20. The difference of marker interval between R. esculentum and the other aquaculture animals may attribute to the SNP numbers used for constructing linkage maps. For P. ussuriensis17 and C. auratus8, 7435 and 8487 SNPs were used for constructing the linkage map, which is 1.96-fold and 2.38-fold higher than that of R. esculentum, respectively. However, the identified SNPs in R. esculentum are very important markers for genetic breeding and this is the first report of linkage map in R. esculentum.The molecular marker shows potential for investigating the growth of aquaculture animals in the genetic breeding industry11. Numerous QTLs about growth traits, such as body weight, body length, sex as well as disease resistance were identified based on the high-density linkage map25. For Nibea albiflora, 15 QTLs were detected associated with body weight, explaining 14.7\u201335.7% of the phenotypic variations21. For C. auratus at 2\u00a0months, eight QTLs in eight chromosomes were discovered associated with the body weight, explaining 10.1\u201313.2% of the phenotypic variations8. In this study, three QTLs distributed in three LGs were detected associated with body weight of R. esculentum and explained 9.4\u201310.5% of the phenotypic variations, following the result of C. auratus8. In addition, our studies showed that growth-related traits body weight and umbrella diameter in R. esculentum are positively correlated with the two close QTLs  may be the most promising according to their role in early growth and development of vertebrates8. EGF_CA domain is a calcium-binding EGF-like domain and needs calcium for performing biological function27. EGF_CA domain presented in extracellular  and membrane-bound27 and has three main roles, including protein\u2013protein interactions, as a spacer unit and structural stabilization28. Although the functional significance of EGF_CA domain in aquaculture animals is unclear, it is worth confirming if EGF_CA domain is associated with the growth of R. esculentum. With the release of genomic data of R. esculentum13 and the development of biotechnology, the gene function studies of R. esculentum will be improved for studying genetic breeding.Positional cloning of candidate genes with the help of QTL mapping may provide an efficient method for selective breeding in aquaculture animalsR. esculentum using the 2b-RAD method. Based on the linkage map, six QTLs were identified associated with the growth of R. esculentum and one candidate gene RE13670 containing EGF_CA domain in LG14 may play the key role in controlling the growth of R. esculentum. Although one full-sib family of R. esculentum is limited, the identified SNPs and genes for growth will accelerate the MAS breeding of R. esculentum.In this study, a total of 9100 SNP were identified and a high-density linkage map was constructed with a marker interval of 0.58\u00a0mM in of R. esculentum was constructed in Yingkou City, Liaoning province, China. We selected the two parents with a large difference in growth from the breeding pond and cultured their offspring in the nursery pond. The two parents and random 150 offspring at seven-month-old (juvenile jellyfish) were chosen for sequencing. The body weight and umbrella diameter of the offspring were measured . Secondly, the ligation reaction was conducted to add specific adaptors to the digested genomic DNA. Thirdly, the ligation products were amplified in MyCycler thermal cyclers (Bio-Rad). Fourthly, the PCR products were purified using a MinElute PCR Purification Kit and digested using SapI (New England Biolabs). Fifthly, the digested products were transferred to the tube containing magnetic beads for incubation and then transferred the supernatant to a new tube for ligation using T4 DNA ligase (New England Biolabs). After that, the ligation products were purified and barcodes were introduced by PCR using barcode-bearing primers. Finally, PCR products were purified and pooled for sequencing using the Illumina Novaseq 6000 PE150 sequencing platform.The 2b-RAD libraries of 30. The reference reads were extracted from R. esculentum genome (NCBI Genome ID: 56778) after electronic digestion using the BsaXI enzyme.The raw data of 2b-RAD sequencing were trimmed for getting the high-quality data, and then the high-quality data were aligned to reads. The reads with the BsaXI site were extracted and aligned to the reference reads using SOAP (version 2.21)31. The maximum-likelihood (ML) algorithm was used to detect homozygote or heterozygote in co-dominant markers. SNPs were filtered with the following criteria: (1) Segregating markers that could be genotyped over 80% of the progenies were retained; (2) Markers with a minor allele frequency (MAF) less than 0.01 were discarded; (3) Polymorphic loci that contain more than two alleles were excluded; (4) The aligned reads with more than two SNPs were discarded. Based on the detected SNPs, we constructed neighbor-joining tree using treebest (version 4.1)32 and performed principal component analysis using ADMIXTURE (version 1.3.0)33 for confirming the species as a full-sibling family. SNP markers were annotated using the software SnpEff (version 4.1)34.The aligned data were used to detect SNP by RADtyping software35. The male and female-specific linkage maps were constructed by software JoinMap (v5.0)36. The male-specific linkage map was constructed using paternal heterozygous genotype and maternal heterozygous and homozygous genotype. The female-specific linkage map was constructed using maternal heterozygous genotype and paternal heterozygous and homozygous genotype. The consensus genetic linkage map was constructed by merging male and female-specific linkage maps using the software MergeMap (http://138.23.178.42/mgmap/) 37.After filtering, the linkage group was divided with the LOD value of 2\u201315. Marker distances were calculated using Kosambi\u2019s mapping functionR. esculentum were performed by software MapQTL (v6.0)38. The interval mapping method was used for genome-wide QTL analysis and every one cM on each LG was scanned for searching the possible QTL. LOD threshold value at 95% level was calculated via 1000 permutation tests for each trait and QTL. LOD score of QTL that was greater than the LOD threshold value (2.5) at 95% level was declared significant.Based on the consensus linkage map, QTL mapping analyses of body weight and umbrella diameter in 39. We ascertained the candidate genes by combining their function annotation according to NR and KEGG database40 and analyzed results via online software NCBI-blast (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and conserved domain search service (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi).The genes located on the up-and down-stream 500\u00a0Kb distance of the associated genomic region of body weight and umbrella diameter were detectedSupplementary Information.Supplementary Figures.Supplementary Tables."}
+{"text": "The figures in this paper erroneously did not include the bars indicating significant differences between groups. The affected figures have been corrected online."}
+{"text": "Within the mammalian tissue, interstitial neutrophil migration can occur widely independent of \u03b22 integrins. This is in sharp contrast to neutrophil recruitment in zebrafish larvae (Danio rerio) where neutrophils originate from the caudal hematopoietic tissue and mainly migrate interstitially to sites of lesion upon the early onset of inflammation. However, neutrophils extravasate from the circulation to the inflamed tissue in zebrafish larvae at later-time points. Although zebrafish larvae are a widely accepted model system to analyze neutrophil trafficking in vivo, the functional impact of \u03b22\u00a0integrins for neutrophil trafficking during acute inflammation is completely unknown in this model. In this study, we generated zebrafish with a genetic deletion of CD18, the \u03b2 subunit of \u03b22 integrins, using CRISPR/Cas9 technology. Sequence alignments demonstrated a high similarity of the amino acid sequences between zebrafish and human CD18 especially in the functionally relevant I-like domain. In addition, the cytoplasmic domain of CD18 harbors two highly conserved NXXF motifs suggesting that zebrafish CD18 may share functional properties of human CD18. Accordingly, CD18 knock-out (KO) zebrafish larvae displayed the key symptoms of patients suffering from leukocyte adhesion deficiency (LAD) type I due to defects in ITGB2, the gene for CD18. Importantly, CD18 KO zebrafish larvae showed reduced neutrophil trafficking to sites of sterile inflammation despite the fact that an increased number of neutrophils was detectable in the circulation. By demonstrating the functional importance of CD18 for neutrophil trafficking in zebrafish larvae, our findings shed new light on neutrophil biology in vertebrates and introduce a new model organism for studying LAD type I.Neutrophils are key players in innate immunity and originate from the bone marrow of the adult mammalian organism. In mammals, mature neutrophils are released from the bone marrow into the peripheral blood where they circulate until their recruitment to sites of inflammation in a multistep adhesion cascade. Here, adhesion molecules of the \u03b2 Outside . Outsidammation , 17. In ntegrins .in vivo  during the primitive wave of hematopoiesis , 29. In When neutrophils of zebrafish larvae leave the CHT, they predominantly migrate throughout the larvae in the interstitial space . In comp2 integrins for neutrophil trafficking in zebrafish is rather elusive. In this study, we generated a model for LAD type I in zebrafish by genome editing using CRISPR/Cas9 technology to shed light on the role of CD18 for neutrophil trafficking in this model. We used sequence alignments to demonstrate that the amino acid sequences of zebrafish and human CD18 share a high similarity, supporting the putative significance of the zebrafish as a model to study integrin biology. Accordingly, CD18 knock-out (KO) zebrafish larvae recapitulated the main symptoms observed in LAD type I patients including reduced neutrophil trafficking to sites of sterile inflammation despite the fact that an increased number of neutrophils was detectable in the circulation using spinning disk confocal microscopy. Thus, CD18 KO zebrafish larvae may represent an interesting model to study LAD type I in vertebrates.To date, the putative role of \u03b2Sequence alignments of human, murine, and zebrafish CD18 and CD11b, using the longest isoforms from the UniProt database, were performed using Clustal Omega . Identit0 generation were raised and outcrossed with Tg zebrafish. The F1 generation was fin-clipped and specific mutations were identified by sequencing. For the gRNA1, we selected a mutant allele with a deletion of 2\u00a0bp , for the gRNA2 we selected a mutant allele with a deletion of 13\u00a0bp  and bred them to homozygosity. DNA isolation was performed with the PCRBIO Rapid Extract PCR Kit (PCR Biosystems) according to the manufacturer\u2019s instructions and PCR amplicons were sequenced to confirm the CD18 mutations.Two different CD18 KO zebrafish lines were generated by CRISPR/Cas9-induced genome editing. To this end, two different guide RNAs (gRNAs)  were designed using CHOPCHOP . gRNAs aFor PCR analysis, zebrafish larvae were euthanized by an overdose of tricaine  and frozen at -80\u00b0C. RNA isolation was performed with the RNeasy Mini kit (Qiagen) and cDNA synthesis with the Maxima First Strand cDNA Synthesis Kit for RT-qPCR with dsDNase (Thermo Fisher Scientific) according to the manufacturers\u2019 instructions. PCR was performed using the SsoAdvanced Universal SYBR Green Supermix (Bio-Rad Laboratories GmbH) and primer pairs against both WT and mutant CD18 for CD18 KO1  and against WT CD18 only  and primer pairs against both WT and mutant CD18 for CD18 KO2  and against WT CD18 only  in a peqSTAR PCR cycler (Peqlab/VWR). PCR products were separated in a 2% agarose gel and visualized with Midori Green (Nippon Genetics) in a gel documentation station (Peqlab).fli1:gfp;lyz:dsRed), Tg, and Tg, referred to as CD18 wild-type (WT), CD18 KO1, and CD18 KO2, respectively, were kept in E3 medium  at 28.5\u00a0\u00b0C and were analyzed at 3\u00a0dpf and 5\u00a0dpf. 1-Phenyl 2-thiourea  was added to the medium 24\u00a0hpf. Raising and housing of adult zebrafish as well as the experiments described were performed in accordance with animal protection standards of the Ludwig-Maximilians-Universit\u00e4t M\u00fcnchen and approved by the government of Upper Bavaria (Regierung von Oberbayern).Zebrafish embryos of the transgenic lines Tg and fixed in 4% paraformaldehyde in PBS at 4\u00a0\u00b0C over night . Larvae For live imaging of random neutrophil migration in the interstitial space as well as visualization of neutrophils in the circulation, zebrafish larvae were anaesthetized with 0.08\u00a0mg/ml tricaine (Pharmaq Ltd) in E3, mounted in 1.5% low melting agarose, and covered with 0.08\u00a0mg/ml tricaine in E3 for the duration of imaging . For neuTail fin transection assays were performed with zebrafish larvae at 3\u00a0dpf or 5\u00a0dpf anaesthetized with 0.08\u00a0mg/ml tricaine. The tip of the tail fin was cut with a clean scalpel and the larvae were either fixed immediately with 4% paraformaldehyde in PBS (0\u00a0h) or placed back in E3 at 28.5\u00a0\u00b0C and anaesthetized and fixed 1, 3 or 6\u00a0h after wounding . Imagingitgb2) of mammalian CD18. In addition to the itgb2 gene, the itgam gene (CD11b) has been described in zebrafish, while orthologues for the itgal gene (CD11a), the itgax gene (CD11c) and the itgad gene (CD11d) do not exist in this model according to Ensembl (https://www.ensembl.org) and National Center for Biotechnology Information (NCBI) databases (https://www.ncbi.nlm.nih.gov). This fact implies that the important role of LFA-1 for neutrophil trafficking in the mammalian system may have no equivalent in zebrafish and that Mac-1 may represent the important \u03b22 integrin in this model while LFA-1 is absent. To analyze this in more detail, we performed sequence alignments of the human, murine, and zebrafish amino acid sequences of CD18 . The CD18 KO1 and CD18 KO2 were generated using CRISPR/Cas9 genome editing in a transgenic zebrafish line expressing green fluorescence protein (GFP) under the ectively , 63, 64.The CD18 KO larvae developed normally up to 5\u00a0dpf and did not show any obvious phenotypical differences compared to CD18 WT larvae. To study the consequences of CD18 deficiency on neutrophils in this model, we fixed CD18 WT and CD18 KO larvae at 3\u00a0dpf and 5\u00a0dpf and imaged them using confocal fluorescence microscopy. Neutrophils could clearly be observed as individual, DsRed labeled cells in CD18 KO larvae as expected . Furthermore, expression of itgb2 is restricted to neutrophils, macrophages, and thymus according to the UCSC cell browser database  and Z03 (BW)].The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "Clinical outcome measures were 10 m walk test (10MWT), 6 min walk test (6mWT), lower extremity motor score (LEMS), proprioception, Berg Balance Scale (BBS), and Walking Index for Spinal Cord Injury (WISCI)-II. All participants were assessed within 48 h before and after the intervention.\u00a0All clinical outcomes were statistically improved after RAGT. Subgroup analysis according to the initial proprioception, WISCI-II in the normal group showed a statistically significant improvement compared to the abnormal group. Initial BBS and WISCI-II had a positive correlation with most of the final clinical outcomes. The final BBS had a strong positive correlation with the final 10MWT, 6mWT, and WISCI-II. Initial proprioception had a positive correlation with the final WISCI-II. The final proprioception also had a moderate positive correlation with 6mWT and BBS. This study\u2019s results suggest that the end-effector RAGT could promote proprioception, balance ability and walking ability. Postural control ability and proprioception also had a positive relationship with gait ability.The primary aim of this study was to reveal the effects of end-effector robot-assisted gait training (RAGT) on motor function, proprioception, balance, and gait ability in patients with incomplete spinal cord injury (SCI). The secondary aim was to determine the correlation between clinical outcomes. This study was a prospective and multi-center study. A total of 13 incomplete SCI patients who met inclusion criteria received 30 min of RAGT with Morning Walk The spinal cord plays an important role in connecting the brain and peripheral nerves. It transfers an ascending sensory signal from the periphery to the brain via the ascending tract  or descending motor signals from the brain to the periphery via the descending tract . If the spinal cord is injured by a traumatic or non-traumatic cause, the patient can have various degrees of motor and/or sensory impairment below the level of injury.For human gait, lower extremity muscle power, joint proprioception, visual balance, and cognition are needed. Balance ability can also be affected by joint proprioception and visual function. After spinal cord injury (SCI), patients experience various degrees of proprioceptive impairment. This is essential for locomotor recovery and skill learning after SCI ,3,4,5,6.Physicians should consider locomotor training for patients with incomplete SCI with some degree of motor function in their lower extremities. In the past, overground walking training with or without gait aid and/or physical assistance was the only intervention method. However, their therapeutic quality and duration are easily affected by the patients\u2019 motor functions. It is difficult to facilitate repetitive and physiological gait patterns by physiotherapists\u2019 physical assistance. Body-weight-supported treadmill training (BWSTT) is the next training method. A harness supports patients\u2019 body weight and provides more support than a gait aid. However, BWSTT still requires physical assistance from a physiotherapist to make the swing phase or support the stance phase ,8,9.\u00ae) with a treadmill base. RAGT allows the patient to experience physiological gait patterns repetitively and safely with body weight support by a harness ), proprioception (proprioception of the ISNCSCI at the ankle and knee), balance ability , and walking ability ). Proprioception evaluation of the ISNCSCI recommends using the great toe, ankle, and knee. According to the ISNCSCI, manual, proprioception (joint movement appreciation and position sense) is graded as absent, impaired, or normal. A grade of 0 (absent) indicates that the patient is unable to correctly report joint movement on large movement of the joint. A grade of 1 (impaired) indicates that the patient can consistently report joint movement with 8 of 10 correct answers only on large movement of the joint. A grade of 2  indicates that the patient can consistently report joint movement with 8 of 10 correct answers both small (approximately 10\u00b0 of motion) and large movement . Our purp-value was less than 0.05.After checking whether the data followed a normal distribution by Shapiro-Wilk test, we used non-parametric tests. Comparisons of pre- and post-RAGT effects in all participants were assessed using the Wilcoxon signed-rank test. Subgroup analysis was according to the initial proprioception status, the Wilcoxon signed-rank test was performed for within-group comparisons of RAGT effects and the Mann-Whitney U test was performed for between-group comparisons. We calculated the effect size at alpha = 0.05 with 80% power by using the G*Power version 3.1.9.6 software . Spearman\u2019s correlation was used to determine the correlation between the clinical outcome measures. SPSS ver. 25.0 software  was used for statistical analyses. Statistical significance was considered when the A total of 13 patients, including eight males and five females, participated in this study. Their median age was 52 years (age range 19\u201385 years), and the median time from onset was 48 days (range 19\u2013139 days). Twelve patients were injured by a traumatic cause and the other injured by a non-traumatic cause. Ten patients had tetraplegia with AIS D, one had paraplegia with AIS C, and one had tetraplegia with AIS D . After 2p = 0.002, 0.002, 0.003, 0.027, 0.001, and 0.001, respectively; After 20 sessions of RAGT, all outcome measures, including 10MWT, 6mWT, LEMS, proprioception of the ankle and knee, BBS, and WISCI-II, had significantly improved . After RAGT, only one participant could not perform the 10 MWT or 6mWT. He had the lowest WISCI-II level after RAGT among the participants, which was 2.Before RAGT, six participants had intact proprioception of the ankle and knee, and seven participants had decreased proprioception of the ankle or knee. As mentioned above, we hypothesized that the end-effector robot could assist proprioception and balance improvement by providing destabilization training. Therefore, we performed a subgroup analysis based on the initial proprioception status to compare the outcome measures.We divided the participants into two groups according to their initial proprioception of the ankle and knee. Participants with grade 2 of initial proprioception of the ankle and knee were classified into the normal group (n = 6). Participants with grade 0 or 1 of initial proprioception of the ankle and knee were classified into the abnormal group (n = 7).p = 0.028, 0.028, 0.028, and 0.027, respectively; p = 0.068, p = 0.027, 0.028, 0.018, 0.027, 0.018, and 0.018, respectively; In the normal group, the 10MWT, 6mWT, BBS, and WISCI-II were significantly improved  and strongly correlated with initial BBS  and WISCI-II . The final 6mWT was strongly correlated with the initial BBS  and WISCI-II . Final LEMS was moderately correlated with initial BBS  and strongly correlated with initial proprioception of the ankle and knee . Final proprioception of the ankle and knee was moderately correlated with initial LEMS  and initial WISCI-II . The final BBS score was strongly correlated with the initial BBS  and WISCI-II . The final WISCI-II was moderately correlated with the initial 10MWT , 6mWT , proprioception of the ankle and knee , BBS , and WISCI-II   .p = 0.039) and strongly correlated with the final 6mWT  and BBS . The final 6mWT was moderately correlated with final proprioception of the ankle and knee  and WISCI-II  and strongly correlated with the final BBS . Final proprioception of the ankle and knee was moderately correlated with the final BBS . The final BBS score was strongly correlated with the final WISCI-II score   .All clinical outcomes showed a significant improvement after 20 sessions of RAGT. End-effector RAGT improved gait speed (10MWT) and endurance (6mWT), LEMS, ankle and knee proprioception, balance ability (BBS), and ambulation capacity (WISCI-II). In previous studies on RAGT in SCI patients, the 6mWT, WISCI-II, and LEMS revealed improvements compared to the control group; however, the 10 MWT and BBS showed no significant improvement after the RAGT. ,20,21,22Before RAGT, two participants had WISCI-II level 8, and one participant had a WISCI-II level of 13. These three participants performed the 10MWT and 6mWT before RAGT. However, the others could not perform 10MWT and 6mWT before RAGT because their WISCI-II was less than 5. After 20 sessions of RAGT, 12 of the participants performed the 10MWT and 6mWT. The median values of improvement were 0.16 m/s and 52 m, respectively. Lam et al.  suggesteIn previous studies that showed no statistically significant improvement in 10 MWT, the participants were subacute or chronic SCI patients ,21,24,25To the best of our knowledge, this study is the first to use proprioception of the ankle and knee to evaluate end-effector RAGT effects in SCI patients. Only three articles have been published regarding end-effector RAGT in SCI patients ,17,19. OProprioception is an unconscious sensory perception of the body, joint position, and movement without visual feedback . DespiteIn this study, we divided participants into normal and abnormal groups, followed by a subgroup analysis according to the initial proprioception of the ankle and knee. As a result of the between-group comparison, WISCI-II in the normal group showed statistically significant improvement compared to the abnormal group. This result means that patients with normal proprioception before the intervention will have a more favorable improvement in gait ability than those who had initial abnormal proprioception. This supports the relationship between proprioception and gait ability.Postural control is also essential for the proprioception of gait . ProprioThe end-effector robot produced a gait cycle according to the footplate trajectory. Unlike the exoskeletal-type robot, only the patient\u2019s feet are attached to the robot\u2019s footplate, and the knee and hip joints move freely. These free movements of the hip and knee joints provide \u201cdestabilization training.\u201d In a previous study on end-effector RAGT in multiple sclerosis patients, postural stability (BBS) and balance confidence  improved as much as sensory integration balance training . In addiTherefore, we hypothesized that the end-effector RAGT could improve proprioception and balance ability. As a result, proprioception of the ankle and knee showed statistically significant improvement after RAGT in this study. In addition, BBS was also significantly improved, unlike a previous study that conducted RAGT with exoskeletal type in SCI patients . Thus, wThe other aim of this study was to determine which initial clinical outcome correlates with the final outcome and the correlations between final outcomes in patients with SCI. Initial BBS and WISCI-II scores were positively correlated with most of the final clinical outcome measures. The final BBS had a strong positive correlation with the final 10MWT, 6mWT, and WISCI-II. This means that postural control before intervention could play an essential role in the final favorable outcomes in gait. Postural control ability was positively correlated with gait ability.Initial proprioception had a positive correlation with the final WISCI-II. This is the same result as the subgroup analysis according to initial proprioception. In addition, final proprioception had a moderate positive correlation with the 6mWT and BBS. This result supports the relationship between proprioception and gait ability.The limitations of this study are the small sample size and the lack of a control group. A future randomized control study should be conducted to evaluate the effects of an end-effector RAGT on proprioception and balance ability in patients with SCI.This study is the first to assess proprioception in order to evaluate the effect of an end-effector RAGT in patients with SCI. In addition, we evaluated BBS as an outcome measure that lacks evidence of the effect of an end-effector RAGT in patients with SCI. The results of this study suggest that the end-effector RAGT could act as task-specific, repetitive, and desensitization training to promote proprioception, balance ability, and walking ability. In addition, postural control ability and proprioception were positively correlated with gait ability."}
+{"text": "Specific language impairment (SLI) is characterized by a delay in language acquisition despite a lack of other developmental delays or hearing loss. Genetics of SLI is poorly understood. The purpose of this study is to identify SLI genetic loci through family-based linkage mapping.We performed genome-wide parametric linkage analysis in six families segregating with SLI. An age-appropriate standardized omnibus language measure was used to categorically define the SLI phenotype.NOP9 (14q12) was reported previously. Linkage analysis identified a new SLI locus at 15q24.3-25.3 with the highest parametric LOD score of 3.06 in Family 315 under a recessive mode of inheritance. Suggestive evidence of linkage was also revealed at 4q31.23-q35.2 in Family 300, with the highest LOD score of 2.41. Genetic linkage was not identified in the other three families included in parametric linkage analysis.A suggestive linkage region replicated a previous region of interest with the highest logarithm of odds (LOD) score of 2.40 at 14q11.2-q13.3 in Family 489. A paternal parent-of-origin effect associated with SLI and language phenotypes on a nonsynonymous single nucleotide polymorphism (SNP) in These results are the first to report genome-wide suggestive linkage with a total language standard score on an age-appropriate omnibus language measure across a wide age range. Our findings confirm previous reports of a language-associated locus on chromosome 14q, report new SLI loci, and validate the pedigree-based parametric linkage analysis approach to mapping genes for SLI.https://doi.org/10.23641/asha.13203218 Most children acquire language without any formal instruction. Contrary to common expectations, language does not come easily to all children. Specific language impairment (SLI) is a language disorder that delays the acquisition of language skills in children who have no hearing loss or other developmental delays . The estFamily aggregation and twin studies provide evidence that the language acquisition difficulties children with SLI experience are inherited . Twin stAlthough SLI is known to aggregate in families , family-Linkage analysis is often the first step in family-based genetic investigation to narrow regions to identify causative genes and variations. The purpose of parametric linkage analysis is to identify coinheritance of chromosomal loci with a phenotype . LinkageMultiple genetic studies of SLI are reported, with mixed outcomes for linkage and association; consistently high heritability estimates and genetic loci are reported . SLI is Similarly, an investigation of Canadian families phenotyped as reading impaired (RI), language impaired, or clinically impaired showed linkage on chromosome 13q; subsequent follow-up investigation of American families replicated the linkage on 13q , 2002. PFounder populations and consanguineous families provide a unique opportunity for family-based analysis. The founder-based population of Robinson Crusoe Island, near Chile, has a high incidence (35%) of SLI , 2008. RN = 322), compiled at the University of Kansas (KU) Cohort, targeted the chromosomal loci 1p36, 3p12-q13, 6p22, and 15q21 . Previous behavioral investigations support a hypothesized relationship between SLI and RD, such that early language impairments are predicative of later reading impairments . An earlnd 15q21 . Sib-paind 15q21 . Linkagend 15q21 . This taTM4SF20, NFXL1, and SETBP1  and we acquired appropriate informed consent from all participants. Parents provided consent for all participants under 18 years of age. In total, 60 participants make up the six families, including six probands, 12 parents, 28 siblings, and 14 other relatives; 38 individuals are categorized as affected see . AffectiFamily 315 is the largest in the study; 18 individuals were available for single nucleotide polymorphism (SNP) genotyping and behavioral data was collected from 16 individuals. Genetic and language data were collected in Family 315 from three nuclear families (branches) descending from 315100. Branch 1 refers to the proband branch (descendants of 31510 and 31511), Branch 2 refers to the descendants of 31520 and 31521, and Branch 3 refers to the descendants of 31530 and 31531. Importantly, since the completion of genotyping in this study, additional behavioral information was collected in family members of Branch 2, including that one individual has a diagnosis of a rare syndromic condition, which presents with multiple severe phenotypes (not shown in https://www.asha.org/public/speech/disorders/childhood-apraxia-of-speech/), an uncommon motor speech disorder that makes it hard to speak, nor are there any in the full sample of participants. In addition, probands were monolingual native speakers of English and screened for nonstandard dialects. Family members (parents and siblings) of probands were also monolingual native speakers of English, without diagnosis of neurological conditions related to language, without hearing loss, and \u2264 55 years of age. Family members were not screened/exempt from participation for cognitive impairments or other developmental delays or autism at the initial time of assessment (based on parent report). Intelligible speech was required for validity of some of the phenotypes. Persons with hearing loss were not assessed/included in the study because the phenotype measures are not valid estimates of their language abilities.A full list of the phenotype measures collected as part of the ongoing longitudinal study of families in Rice's lab is described by Rice et al. in an earlier publication . ProbandSpeech articulation was assessed with the Goldman-Fristoe Test of Articulation\u2013Second Edition . All parThe omnibus language scores were obtained from a standardized language measure appropriate for the participant's age. Standardized omnibus language measures are designed to assess multiple dimensions of language, and age-standardized versions have been developed across a wide age range, following psychometric standards for reliability and validity. Although they do not measure all possible aspects of language acquisition, they are generally robust for broadly defined semantic and syntactic dimensions of language, across expressive versus receptive task demands. The omnibus language measures were chosen based on their high levels of reliability and validity, which is verified by monitoring of test\u2013retest reliability across times of measurement in this longitudinal study. We note any potential psychometric weaknesses work against our hypotheses of genetic risk in the families.The current study began following participants longitudinally in 1993 using an accelerated longitudinal design in which multiple age cohorts are sampled, such that children vary continuously in age at first assessment. The measures and their versions have remained the same throughout the course of the study in order to avoid confounds related to different items sets and different norming samples. It is essential to minimize this type of possible measurement error when comparing performance over time both within and across individuals. Training in the administration of the tasks includes reading the manuals, practice administration with adult trainers in the lab, observed administration with practice children in the lab, and followed by observed administration in the field with actual participants before being authorized to administer assessments independently. Routine intermittent live observations of actual data collection are conducted for monitoring reliability and validity of task administration, supplemented by viewing of video recordings of each session. Scoring of outcomes is also monitored and checked at various levels of processing, involving the examiners and other team members, on an ongoing basis. The measures were versions of two tests available at the outset of the study: for ages 4\u20136;11, the TOLD-2\u2013Primary Spoken Language Standard Score , and forN = 18) or Controls (N = 10). The oldest time of sequential annual assessments was for one participant in the SLI group, a total of 13 assessments with the final at 33.69 years; for the Control group, the oldest was 30.11, a total of 12 assessments. For a comparison between \u201cearly adulthood\u201d and \u201cadulthood,\u201d we calculated average omnibus standard scores per person in the age range 18\u201321 (\u201cearly adulthood\u201d) and 22+ years (\u201cadulthood\u201d). For the SLI group, the difference between these averages ranges from 2 to 6 standard score points (within standard error of measurement) for all but one participant who had a traumatic experience that coincided with two lower scores that bounced back into the usual level in subsequent assessments. For the Control group, the range in differences for the \u201cearly\u201d average versus the \u201clater\u201d average is 2\u20137, again within standard error of measurement, with one exceptional case of a period of three consecutive lower-than-average scores that later resolved to the previous levels. Overall, in our data, the general pattern of outcomes met our assumption that standard scores on the CELF-3 were unlikely to increase with age. Furthermore, we note that if language skills do improve in adulthood, then the use of younger age norms would inflate the adult standard scores, resulting in a lower likelihood of affection status, which would work against the study hypotheses. If such an inflation did occur, it is very small or nonexistent, as the average omnibus language standard score among proband parents in this study is 88.55 (SD = 15.16). Finally, recent analysis in this sample shows high test\u2013retest reliability, with correlations between occasions of measurement ranging from r = .715 (p < .0001) to r = .975 (p < .0001), offering further support for stability of measurement over time.We explored this expectation in our database, which includes participants with longitudinal assessments from childhood into adulthood. The sample was annually assessed beyond the ceiling age level. They entered as SLI . DNA was purified from the saliva samples using a standardized protocol with the Oragene prepIT\u2e30L2P kit. Extracted DNA was resuspended and stored in 1x Tris-EDTA (1X TE) buffer at \u221280\u00b0C for long-term storage.To obtain estimates of linkage, we performed familywise 2-point parametric linkage simulations using the FastSLink package in easyLINKAGE-Plus . LinkageSupplemental Table S1). Related phenotypes included in the search were performance on NWR tests, autism spectrum disorders (ASDs), CAS, and RD . The boundaries for all reported regions were determined based on the segment in which the majority of the markers surrounding the highest LOD score showed a positive LOD score.Suggestive and significant LOD score thresholds for complex traits have been proposed based on previous linkage simulations . Proposehttps://www.illumina.com/products/by-type/microarray-kits/infinium-qc.html). All DNA samples were diluted with 1X TE buffer to 50-ng/ul concentration, and 200 ng of DNA was used for SNP genotyping. Genome-wide SNP genotyping was outsourced to the Genetic Resources Core Facility at Johns Hopkins University School of Medicine. Genotype data were consistent with reported family relationships in all individuals . The array has 12,032 SNPs distributed across the autosomal chromosomes, though 38 were located within pseudo-autosomal regions. Additionally, 3,917 SNPs were located on the sex chromosomes  and mitochondrial DNA (141 markers).SNP genotyping was performed on the Illumina Infinium QC Array-24 that has 15,949 SNP markers equally spaced throughout the genome  were used in the Superlink-Online SNP 1.1 analysis. Superlink-Online SNP 1.1 has a cleaning tool, which removes markers that are uninformative or erroneous for various reasons . In totahttp://www.homozygositymapper.org; The linkage program MERLIN was used to draw haplotypes in Family 315 . Given tSupplemental Figure S1). In Family 387, under the recessive model of inheritance, two markers spanning chrX:89712929-90955058 showed single-point LOD scores equal to the maximum ELOD , Supplemental Table S3 (15q), and Supplemental Table S4 (14q). To provide additional context for the suggestive regions, the background linkage and the families with no significant linkage, the genome-wide linkage results for all six families are presented in Supplemental Figure S1. There were 10 additional markers in Family 315 with LOD scores greater than 2.0 . However, the markers, in the vicinity of the highest scoring markers, showed negative LOD scores .The highest calculated single-point LOD score was greater than 1.9 in the reported suggestive loci and matched the maximum ELOD in Family 300 and 489 see . The higSupplemental Figure S2). The highest single-point LOD scores of 1.3 and 0.55 were obtained in Branches 1 and 3, respectively, to chromosome 15q24.3-25 linkage region .The reduced single-point LOD score (compared to the maximum ELOD) in Family 315 led us to perform branch-wise linkage analysis, especially considering the presence of a rare syndromic condition (details of diagnosis not available) in Branch 2. We performed linkage analysis separately in Branch 1, Branch 2, and Branch 3 and all possible combinations of these branches  was used by the SLIC and linkage loci were reported . Their aSupplemental Table S1; Omnibus measures are also of interest as a phenotype because of the possible insight they can provide into the overlap with other related disorders that may share common causal pathways. For example, recall that early language impairments predict later reading impairments. Specifically, evidence from a longitudinal study of children with SLI shows that those with the poorest reading comprehension skills had significantly lower scores on both the Peabody Picture Vocabulary Test\u2013Fourth Edition and the CELF in eighth grade, as compared to those who had the poorest reading decoding skills . These rNOP9 overlapped with our three reported suggestive regions, though other regions with LOD scores greater than 1.2 in four families  are also noted if they coincided with previously reported regions or candidate genes associated with SLI, RD, NWR performance, ASD, and CAS . This overlap could help to narrow the region of interest in follow-up investigation of exome variations.Other challenges for gene discovery are at the level of variations in the size of gene regions of interest. The linkage region (~25 MB) in Family 300 is large, which could be due to a small family size. However, this could be a new SLI locus to be considered in the future with a larger data set. This locus may be a useful target for genome sequencing in this family showing a recessive inheritance patterns for language phenotypes. Additionally, some SNPs at 4q with the LOD score above 1.2 were observed in Families 387 and 430, indicating replication, although with less significance (see RBFOX2 gene (RNA binding Fox-1 homolog 2) associated with language and reading phenotypes  is especially noteworthy. A paternal parent-of-origin effect was identified previously at chromosome 14q12, specifically a nonsynonymous variant in the  489 see . The NOP 489 see . NOP9 enassembly . Efficieassembly . It is aassembly . Other gSupplemental Figure S2). Unlike with the region on chromosome 15q, no overlap was observed with other branches individually or in combination, indicating the homozygous region on 14q may not be of further interest in Family 315. Our haplotype analysis suggested a possibility of a distant common ancestor, but genome-wide homozygosity analysis negates it. The segregation of more than one haplotype in Branches 1 and 3 on chromosome 15q indicates that multiple alleles at this locus may be the cause of SLI in Family 315.We noted in the introduction that one of the advantages of pedigree analysis is that it can reduce trait variance of complex phenotypes at the behavioral level in order to better capture the genetic factors involved in the transmission of the trait . Yet, adRasGRF1 (Ras protein specific guanine nucleotide releasing factor). RasGRF1 is a regulator of the extracellular signal-regulated kinase signaling pathway via binding with a N-methyl-D-aspartate receptor subunit, specifically NR2B , Investigation , Methodology , Writing \u2013 original draft , Writing \u2013 review & editing . Kathleen Kelsey Earnest: Data curation , Supervision , Writing \u2013 review & editing . Shelley D. Smith: Data curation , Writing \u2013 review & editing . Mabel L. Rice: Conceptualization , Funding acquisition (Lead), Project administration , Resources , Supervision (Lead), Writing \u2013 review & editing . Muhammad Hashim Raza: Conceptualization , Investigation , Methodology (Lead), Project administration , Resources , Supervision (Lead), Visualization (Lead), Writing \u2013 original draft , Writing \u2013 review & editing .10.1044/2020_JSLHR-20-00102SMS1Supplemental Figure S1Click here for additional data file.10.1044/2020_JSLHR-20-00102SMS2Supplemental Figure S2Click here for additional data file.10.1044/2020_JSLHR-20-00102SMS3Supplemental Table S1Click here for additional data file.10.1044/2020_JSLHR-20-00102SMS4Supplemental Table S2Click here for additional data file.10.1044/2020_JSLHR-20-00102SMS5Supplemental Table S3Click here for additional data file.10.1044/2020_JSLHR-20-00102SMS6Supplemental Table S4Click here for additional data file.10.1044/2020_JSLHR-20-00102SMS7Supplemental Table S5Click here for additional data file."}
+{"text": "This cross-sectional study assesses whether physician group participation in Medicare bundled payments is associated with changes in costs or patient outcomes. Was participation in the Bundled Payments for Care Improvement (BPCI) initiative among physician group practices associated with changes in Medicare payments, patient selection, or clinical outcomes?In this cross-sectional study of 91 orthopedic groups in BPCI Model 2 and 169 propensity-matched controls, BPCI-participating practices decreased 90-day Medicare payments from $18\u2009257 to $15\u2009320 during the intervention, while control practices decreased payments from $17\u2009927 to $16\u2009170; 30-day and 90-day readmission rates decreased more among BPCI practices than controls, and 90-day healthy days at home increased.Group practice participation in BPCI for joint replacement was associated with reduced Medicare payments and improvements in clinical outcomes. Medicare\u2019s Bundled Payments for Care Improvement (BPCI) program, which ran from 2013 to 2018, was an important experiment in physician-focused alternative payment models. However, little is known about whether the program was associated with better quality or outcomes or lower costs.To determine whether participation in BPCI among physician group practices was associated with advantageous or deleterious changes in costs or patient outcomes.This cross-sectional study used 2013 to 2017 Medicare files and difference-in-differences (DID) models to compare the change over time in Medicare payments, patient selection, and clinical outcomes between 91 orthopedic groups in BPCI Model 2 and 169 propensity-matched controls for patients undergoing joint replacement. Analyses were performed between December 2019 and February 2021.Voluntary participation in BPCI.The primary outcome was 90-day Medicare payments; secondary outcomes were patient selection  and clinical outcomes .P\u2009<\u2009.001). Savings were driven by a decrease in postacute care spending. There were no differential changes in volume or comorbidities. The BPCI practices increased the proportion of patients discharged home compared with controls . There were no differential changes in 30-day or 90-day mortality rates or emergency department visits, but 30-day and 90-day readmission rates decreased more among BPCI practices than controls , and 90-day healthy days at home increased .There were 74\u2009343 patient episodes in the baseline period and 102\u2009790 during the intervention in BPCI practices, and 88\u2009147 patient episodes in the baseline period and 120\u2009253 during the intervention in control practices; 291\u2009214 of 461\u2009598 (63.1%) patients were women, and 419\u2009619 (90.9%) were White. At baseline, mean episode payments among BPCI-participating practices were $18\u2009257, which decreased to $15\u2009320 during the intervention, while control practices decreased from $17\u2009927 to $16\u2009170 (DID, \u2212$1180; 95% CI, \u2212$1565 to \u2212$795; Group practice participation in BPCI for joint replacement was associated with reduced Medicare payments and improvements in clinical outcomes. Medicare and other payers are increasingly moving toward alternative payment models in which clinicians are paid for the quality, rather than solely for the quantity, of care they provide. One example is the Bundled Payments for Care Improvement (BPCI) program, launched by Medicare in 2013. Hospitals, postacute care facilities, and physician group practices (PGPs) that joined assumed responsibility for quality and costs of care for a 30-day, 60-day, or 90-day episode that started with a hospital admission for 1 of 48 medical or surgical conditions.3 though according to a recent publication from the Center for Medicare & Medicaid Innovation,4 those improvements did not lead to savings for Medicare because they were offset by bonus payments paid out under the program. However, despite the fact that BPCI represented a large, national experiment in alternative payment mechanisms and enrolled hundreds of physician groups, little is known about whether the program was associated with better quality or outcomes or lower costs.Perhaps the most successful area for BPCI has been for major joint replacement of the lower extremity (MJRLE). Among hospital and postacute facility participants, studies have shown lower Medicare payments compared with concurrent controls,5Physician group practices could fare differently under BPCI than hospitals or postacute facilities. They may be more nimble in changing postdischarge follow-up to prevent rehospitalizations, and they may have preexisting relationships with patients that help them determine cost-effective postacute care pathways. Additionally, because their revenue is driven by professional fees rather than facility fees, they may benefit less from rehospitalizations. Studies of the largest alternative payment model under Medicare, the Medicare Shared Savings Program, showed that accountable care organizations led by PGPs were more likely to succeed.6 but was subsequently put on hold during the COVID-19 pandemic. Understanding patterns in BPCI could be important to informing program design and monitoring strategies under BPCI-Advanced.As the Centers for Medicare & Medicaid Services (CMS) and other payers continue to seek payment models that reduce costs and improve quality, understanding the association of PGP participation in BPCI with costs and outcomes is important. Furthermore, in October 2018, Medicare launched a follow-on program to BPCI, called BPCI-Advanced, that was joined by more than 500 PGPs7 and was the most commonly selected condition by PGP participants in BPCI.In this study, we aimed to fill this gap by examining changes in payments, patient selection, and clinical outcomes for patients of PGPs participating in BPCI. We focused on MJRLE because it is the most common type of surgery performed among Medicare beneficiaries8 in which participants assumed accountability for all Medicare payments  during an episode triggered by a hospital admission and continuing for 30, 60, or 90 days postdischarge. The PGPs were paid fee-for-service rates, but payments were retrospectively reconciled against targets on a quarterly basis. Target prices were set based on each practice\u2019s unique historical utilization, minus a discount of 2% to 3% depending on the episode length chosen by the participating entity.There were 4 participation models in BPCI, but 79% of PGPs enrolled in Model 2,STROBE) reporting guideline.This study was approved by the Office of Human Research Administration at the Harvard School of Public Health. The requirement for informed consent was waived because the data were deidentified. This study followed the Strengthening the Reporting of Observational Studies in Epidemiology  numbers for physicians affiliated with each practice in each year. When NPIs were affiliated with more than 1 taxpayer identification number (TIN) , we used data from the Medicare Carrier file to assign them to the TIN with which they billed the plurality of their claims for MJRLE. For both BPCI and potential control TINs, we linked these data at the TIN level to public data from Physician Compare for practice characteristics, such as location and size, and at the county level to the Area Resource File for market characteristics, such as median income. Market share was calculated as the proportion of all admissions in a county for MJRLE by each PGP. Market competitiveness was calculated using the Herfindahl-Hirschman Index.1 Using propensity scores based on PGP and market characteristics, each BPCI PGP was matched without replacement with up to 3 control orthopedic PGPs within the same region and the same baseline volume tertile . Automated matching was restricted to PGPs with a log odds propensity score absolute difference below 0.5. We then hand-matched 20 practices  by removing the within-region match requirement and selecting the remaining potential control within the same volume group with the closest number of surgeons in the practice (the dominant factor in the propensity model). Any practice or market characteristic with a standardized mean difference (SMD) of 0.2 or higher after matching was included in the regression models described below as a covariate.To select controls, we used a modification of an approach used in a prior study of BPCI.We used 100% Medicare inpatient files from January 1, 2013, to September 30, 2017, to identify index admissions for MJRLE for which either the discharging clinician or the operating clinician belonged to a participating PGP or to a control PGP, per CMS\u2019s BPCI inclusion rules. We included only beneficiaries who were continuously enrolled in Parts A and B during their episode of care and excluded those with end-stage kidney disease.We considered baseline to be January 1, 2013, through September 30, 2013, for all PGPs and controls. The intervention period varied, because PGPs could join BPCI on a rolling basis, and was set for each BPCI PGP and its matched controls as the time from 3 months postenrollment through the end of September 2017. For the 91 BPCI practices analyzed here, the mean intervention period was 22.9 months, or just over 7 quarters.1 For each admission for MJRLE, standardized Medicare-allowed episode payments  for the initial hospital stay plus 90 days following discharge were calculated using 100% inpatient, skilled nursing facility (SNF), home health agency, and durable medical equipment claims. We refer to these as Medicare Part A payments and consider this our primary outcome. For the roughly 20% of patients for whom we had Part B outpatient and physician claims , we also calculated 90-day standardized Part B payments. Total payments were Winsorized at the 95th percentile of national episode payments annually per CMS specifications and adjusted for inflation to prices in 2017.Our primary outcome was the change in Medicare Part A payments per episode. Because few PGPs chose 30-day or 60-day episodes, we analyzed 90-day episodes for all participants, as has been done previously.Secondary outcomes included changes in patient selection, measured by the mean Medicare Chronic Conditions Warehouse comorbidity score; per-PGP quarterly volume; changes in key individual components of payment and Part B payments; and changes in 30-day and 90-day readmissions, 30-day and 90-day mortality, healthy days at home, and the proportion of patients discharged home, having any SNF stays, and using any home health services.Market and PGP characteristics were compared between BPCI PGPs and their matched controls using SMDs. Our primary approach was an \u201cintention to treat\u201d approach, in which we included all participating PGPs regardless of whether they ultimately dropped out of the program, which they could do without penalty at any time. Our models are described in greater detail in the eMethods in the P value less than 0.05 was considered statistically significant. Secondary end points and subgroup analyses should be considered exploratory. Analyses were performed using SAS, version 9.4 (SAS Institute).For our primary outcome, the change in Medicare Part A payments per episode, a 2-tailed There were 91 orthopedic PGPs that participated in BPCI Model 2 and 2951 that did not. Of these, the BPCI PGPs were successfully matched to 169 controls . There wP\u2009<\u2009.001) (Raw Medicare payments were well matched between BPCI and control PGPs in the baseline period but diverged as PGPs joined the BPCI program over the course of 2014 . At base\u2009<\u2009.001) . There wP\u2009=\u2009.18) . The BPCI PGPs also had a relative increase in healthy days at home  . There wIn exploratory subgroup analyses, among patients who had MJRLE with fracture, there were relative decreases at BPCI PGPs vs controls in 30-day mortality  (eTable 7 in the We found that PGP participation in BPCI for MJRLE was associated with significant reductions in overall Medicare payments, with no deleterious changes in clinical outcomes. Savings were largely driven by reductions in SNF payments, both in patients with and without fractures undergoing MJRLE .The intent of BPCI was to incentivize clinicians to reduce unnecessary services and prevent avoidable readmissions. Results of the current study suggest that orthopedic surgery practices that joined BPCI were able to reduce SNF use following MJRLE without increasing readmissions. While SNF spending dropped in both subgroups, the proportion of patients discharged to SNF did not decline in the subset of patients undergoing MJRLE for fracture, who were older and sicker than their elective surgery counterparts. This suggests that reductions in SNF use were concentrated among the healthiest patients, where such care may not have been necessary. However, long-term functional status data are needed to determine whether reductions in SNF use might be associated with worse long-term outcomes in mobility or pain.We found a small relative decrease in the proportion of patients undergoing surgery with BPCI PGPs who qualified for Medicare on the basis of a disability. There were no other changes that might suggest broad risk avoidance, including in comorbidity scores, the proportion of patients of minority race/ethnicity, or the proportion dually enrolled in Medicaid. Claims data cannot discern the appropriateness of any of the procedures examined, but we did not see changes in volume that suggest patient shifting as the major source of savings under the program.3 although direct comparisons are not feasible owing to the many differences between the patients seen in each setting. For example, the patients enrolled in BPCI via PGPs were markedly less likely to have a fracture and were younger and less sick than prior reports of BPCI patients in hospitals or at postacute care facilities.10 Physician group practices may have advantages in achieving savings, including long-term relationships with patients that might lead to greater comfort sending patients home rather than to SNF care, or to better-aligned financial incentives, because few PGPs have ownership relationships with postacute facilities, unlike hospitals. We did not see an increase in outpatient spending to suggest that PGPs added clinic visits to achieve reductions in SNF care, although it is possible that nonbilled services, such as phone calls, home visits, or remote monitoring, may have been provided.The findings we document among PGPs participating in BPCI are similar or larger than those documented among hospitals and SNFs participating in the program,12 Additionally, there have been national and single-center studies showing savings for MJRLE among hospitals participating in BPCI Model 2.15 This study lends further support to the notion that episode-based payments for joint replacement can save money without adversely affecting patients.To our knowledge, there have been no prior studies of PGP performance in BPCI published in the peer-reviewed literature. There have been 3 annual federal evaluations of BPCI; despite differences in methodology from the approach we used, they also found lower costs for patients cared for under BPCI.4 noting a lack of net savings under BPCI did not break their findings out by hospital vs physician group or postacute care facility participation. We used a short preintervention baseline period; during this period, PGPs were surely aware they were nearing the beginning of the intervention and may have been preparing to redesign care. However, the finding that pretrends were similar between intervention and control PGPs is reassuring. We had a limited follow-up period, and longer-term follow-up may be necessary to evaluate how care evolves.16 Finally, we do not know how PGPs achieved the changes that we observed; this is an important area for future mixed-methods and qualitative research.There are limitations to the study findings. The BPCI is a voluntary program; generalizability to mandatory models is uncertain. We focused on orthopedic PGPs joining the MJRLE bundle because it was the most commonly selected, with adequate volume for analysis, and because identifying control practices is easier when examining a single specialty. These findings may not generalize to other types of practices or bundles. Our lists of participants were obtained from Medicare and not verified by PGPs, which could introduce error. While we included NPIs assigned to each PGP based on annual CMS data files, it is possible that individual clinicians changed practice affiliation midyear, which would lead to misclassification. As Medicare has not released data on target pricing nor on PGPs\u2019 savings or losses under the program, we could only evaluate BPCI\u2019s impact on patients and their outcomes, and not its overall financial impact for PGPs or to the federal government. The Center for Medicare & Medicaid Innovation publicationIn this cross-sectional study of US Medicare data, PGP participation in BPCI for MJRLE was associated with reductions in Medicare payments and improvements in clinical outcomes. It is unclear if these results would generalize outside this voluntary model."}
+{"text": "CCP4 is reviewed and the method of link-dictionary generation used by AceDRG is described. An overview of the various protocols available for the modelling and application of covalent linkages within the CCP4 suite is presented, providing instructive guidelines with a focus on practical application.The mechanism for modelling covalent linkages in  CCP4 suite are considered. The mechanism used for modelling covalent linkages is reviewed: the use of dictionaries for describing changes to stereochemistry as a result of the covalent linkage and the application of link-annotation records to structural models to ensure the correct treatment of individual instances of covalent linkages. Previously, linkage descriptions were lacking in quality compared with those of contemporary component dictionaries. Consequently, AceDRG has been adapted for the generation of link dictionaries of the same quality as for individual components. The approach adopted by AceDRG for the generation of link dictionaries is outlined, which includes associated modifications to the linked components. A number of tools to facilitate the practical modelling of covalent linkages available within the CCP4 suite are described, including a new restraint-dictionary accumulator, the Make Covalent Link tool and AceDRG interface in Coot, the 3D graphical editor JLigand and the mechanisms for dealing with covalent linkages in the CCP4i2 and CCP4 Cloud environments. These integrated solutions streamline and ease the covalent-linkage modelling workflow, seamlessly transferring relevant information between programs. Current recommended practice is elucidated by means of instructive practical examples. By summarizing the different approaches to modelling linkages that are available within the CCP4 suite, limitations and potential pitfalls that may be encountered are highlighted in order to raise awareness, with the intention of improving the quality of future modelled covalent linkages in macromolecular complexes.In this contribution, the current protocols for modelling covalent linkages within the  In addition to requiring knowledge of the particular atoms that are covalently bound, it is necessary to have a complete chemical description of the system  entries  that specifies for a particular atom pair within the model to be treated as covalently bound. Also, a separate link-description dictionary is required which specifies the chemical connectivity and geometric restraints associated with a particular linkage (including references to any required modifications to the bonded compounds). Whilst not technically a strict requirement, such dictionaries are highly recommended in order to avoid poor resultant model geometry; thus, they should be considered as a requirement in modern application.In this section, we shall reflect on the usage of link records and restraint dictionaries for describing covalent linkages, according to implementations within the REFMAC5 without the need for link records. This holds true for any peptide bond between two monomers categorized as \u2018peptide\u2019 (any amino-acid residue with standard backbone-atom naming) in the CCP4-ML; the equivalent applies to nucleotides with the group name \u2018DNA\u2019 or \u2018RNA\u2019. There are 509 amino-acid and 270 nucleotide components in the CCP4-ML that are linked automatically. Indeed, any linkages that have descriptions in the CCP4-ML are automatically created and applied during refinement by REFMAC5 if the potentially linked atoms have the same chain identifierLink records are only needed for nonstandard bonds. For example, they are not required for peptide or phosphodiester linkages between adjacent residues, which are defined in the CCP4-ML. It should be noted that peptide bonds involving a noncanonical amino acid such as selenomethionine (MSE) or phosphothreonine (TPO) are also recognized by et al.  that uniquely references the full link description, which may be found in the CCP4-ML or in a custom dictionary. Any information regarding link identifiers is not currently used by the OneDep system at the point of deposition . This \u2018link distance\u2019 is typically ignored during refinement , although exactly how this information is interpreted and utilized is implementation-specific; this is a common cause of confusion.REFMAC5, if a LINK record is specified in the absence of a corresponding dictionary entry to describe that covalent linkage, then only a single covalent-bond restraint is applied between the two atoms. If the atom types are present in the CCP4-ML, with a corresponding restraint representing their bonding, then that restraint is used. Determining the appropriate stereochemistry, and thus the appropriate restraint, can be difficult, especially in the absence of explicitly modelled H atoms; this may potentially result in inappropriate restraints. If a matching restraint is not available in the CCP4-ML  then a restraint is generated with a target value equal to the \u2018link distance\u2019 reported in the LINK record. If the link distance is absent then REFMAC5 calculates a default target value based on the covalent radii of the atoms.In et al., 2021Either way, if a restraint dictionary is not available then only a single interatomic distance restraint is used to represent the covalent linkage. This means that other geometric properties (for example inter-component angles) that represent the local structural configuration are not restrained. However, such restraints are recommended in order to ensure that, for example, the relative orientation of the linked components is reasonable. In addition, modifications to the internal restraints for each of the involved components are not applied; the effect of this can be dramatic, especially when the covalent linkage results in chemical changes within the components . Consequently, compared with the use of a detailed dictionary, this typically results in an atomic model of suboptimal quality  is left up to the downstream refinement software. Consequently, 2.4.et al., 2004et al., 1991etc.) are identified by a unique component identifier, which in current practical usage is treated as synonymous with \u2018residue name\u2019, \u2018monomer id\u2019 and \u2019three-letter code\u2019.Restraint dictionaries are used to describe the connectivity and geometry of molecular components (Vagin (i) The description of the covalent linkage itself: references to the components and atoms to be linked and the qualitative nature of the bond, along with associated distance, angle, torsion and planar restraints.(ii) Descriptions of the modifications that need to be applied to each of the dictionaries of the linked components in association with the particular covalent linkage, including any changes to the atomic composition (for example removing atoms), connectivity, chemical properties and geometric restraints of the individual components.In addition to those for the individual components, dictionaries describing all modelled covalent linkages between components are also required. Whilst analogous in format to component dictionaries, these \u2018link dictionaries\u2019 are distinct in terms of content. They comprise two facets.REFMAC5 selects the first matching link entry. Consequently, it is important that the connectivity annotation record within the model references the correct identifier for the corresponding link dictionary; it is worth being mindful of such considerations when using link dictionaries.Both link records and modification records are assigned their own identifiers, which must be unique and self-consistent in order to avoid ambiguity; link descriptions cross-reference particular component modifications by their identifiers. Note that there may be multiple modifications that could be applied to a given component, and there may be multiple link types that use the same modification. Indeed, there is a separate link description for each chemical linkage type. There may theoretically be multiple link descriptions corresponding to the bonding of a given atom pair between two particular residues that correspond to different chemistries; for example, differing bond orders of the covalent linkage (and implied changes to protonation) and/or differing modifications to be applied to the chemical composition/properties of either of the linked components. In the case of such ambiguities, Note that it may be necessary to reuse component and link dictionaries both within and between models; a given model may exhibit multiple instances of the same covalent linkage, and different models may exhibit the same local chemistry. For example, there are 4469 instances of the \u03b1-1,3-glycosidic linkage, which is the covalent bond between the O3 and C1 atoms of pyranose components, amongst 1740 PDB entries (up to 36 link instances per model). Another example is the covalent linkage between LYS[NZ] and PLP[C4A] , of which there are 1598 instances modelled amongst 792 PDB entries (up to 12 link instances per model).et al., 2021In order to facilitate reusability, component/link dictionaries are usually located in separate files from the model. Pre-computed dictionaries corresponding to many of the most commonly occurring components and link types, including the \u03b1-1,3-glycosidic linkage, are distributed as part of the CCP4-ML. The CCP4-ML has recently seen substantial expansion, including the addition of link dictionaries for commonly occurring covalent linkages, including LYS[NZ]\u2013PLP[C4A]  may be physically located in separate files or accumulated into an aggregate dictionary. Due to the format compatibility of PDBx/mmCIF model and restraint dictionaries, any dictionary information used during refinement may be additionally encapsulated when using the PDBx/mmCIF model format (for the purposes of completeness and tracking the provenance of utilized prior knowledge).REFMAC5 only allows a single custom dictionary to be provided as input, it is necessary for dictionaries to be accumulated prior to model refinement. Where multiple dictionaries are used, it is necessary to ensure that they do not conflict in order to avoid potential ambiguity and error. In response to this need, a new tool to facilitate dictionary accumulation is now available in CCP4, which performs validation in order to ensure the compatibility of dictionary entries and includes the ability to automatically reassign modification identifiers where necessary.Gemmi library for structural biology  dictionaries from a simple chemical description, as well as the generation of initial coordinates corresponding to a low-energy conformer. AceDRG has recently been extended to allow the generation of link dictionaries, using the same fundamental procedural principles as for component-dictionary generation.In this section, we shall discuss the approach to link-dictionary generation implemented in AceDRG considers the composite component complex as a whole and generates a dictionary for this complex as if it were a single monomer. The end result is that the linkage is modelled as if it were a natural part of one larger hypothetical molecule, and thus the resultant link dictionary contains geometric restraints derived from detailed information regarding the local chemical and structural environment (up to the third order).The introduction of a covalent bond between two monomers affects the internal chemistry and geometry of each of the two components. Consequently, instead of attempting to treat the two monomers and the link independently, JLigand for the creation of link dictionaries using LibCheck  Construction of an initial composite component: a hypothetical molecule comprising the two components to be connected by a covalent linkage (Section 3.1(ii) Derivation of a detailed stereochemical description of the composite component, including information about bond lengths, angles, torsions, chiral centres, rings and planar groups (Section 3.2(iii) Qualitative and quantitative comparison of the geometric descriptions of the individual and composite components. Differences between these descriptions are included in the output link dictionary . Given such a chemical specification, AceDRG firstly sanitizes the valences of the linked atoms to report any possible gross errors such as valency violations. This sanitization involves adding/deleting bonded H atoms to/from the linked atoms in order to achieve the required valency. If the valency must be reduced but there are no bonded H atoms, AceDRG will adjust the formal charges of the atoms as necessary. Where multiple valences are possible, for example for sulfur and boron, the option that would involve minimal modification is selected. Once all necessary modifications have been applied and validated, the bonding pattern of the composite compound is constructed and the whole composite compound is sanitized.3.2.AceDRG generates a stereochemical dictionary using the procedure described by Long et al. ; these are the recommended routes when using CCP4 Cloud. AceDRG can also be executed from a command-line interface, as well as via the Make Covalent Link task in CCP4i2. Both Coot and the Make Covalent Link task can add link records to a model; the latter of these can scan a given model for matching instances of a linkage and apply link records accordingly (maintaining the appropriate identifiers). In cases where there are multiple custom restraint dictionaries , they must be accumulated into a single aggregate dictionary, ensuring internal consistency and uniqueness of nomenclature and identifiers. This aggregate dictionary, along with any required dictionaries from the CCP4-ML, is used by Coot and REFMAC5 during the iterative model-building and refinement process. The final model deposited in the wwPDB contains link records, but without link identifiers.Fig. 24.1.1ajs; Rhee et al., 1997et al. . In such cases, it is important to follow the typical convention in order to avoid extra work upon deposition of the final model in the wwPDB. Replacing a residue by its modified counterpart can be performed efficiently with the \u2018replace residue\u2019 tool in  al. 2012 and Tabl al. 2021.The composite component complex approach may also be required in difficult cases, such as when there are multiple linkages between the same two components or when a link dictionary involves more than two components (a use case not currently supported by modern dictionary generators).ProSMART , they should not be used to describe excessive changes to internal component chemistry. Indeed, it is important to ensure that both components to be linked are modelled using appropriate monomer descriptions before attempting to model the covalent linkage between them.Note also that the use of the linkage mechanism should be restricted to describing the result of chemical reactions in which two components become covalently bound: this has a clear biological interpretation. Other geometric restraints that involve multiple residues, for example hydrogen-bond restraints from 4.2.REFMAC5 and Coot automatically detect and apply linkages to a model based on the proximity of atoms. When multiple link dictionaries are available that match a given atom pair (in the CCP4-ML and/or a custom restraint dictionary), REFMAC5/Coot must decide which dictionary to use. In such cases, the dictionary with the most detailed matching specialization will be selected; exact matches are preferred over wildcard entries, and conformational analysis may be performed in cases where there are multiple exact matches. However, note that any such potential ambiguities are avoided if the model contains connectivity annotation records that specify exactly which link dictionary should be used for each particular instance  that set the correct link identifier in the coordinate files based on an extendable dictionary of 48 common pyranose\u2013pyranose linkages before being passed to REFMAC5  and as part of an RNA polymer (identifier A). As long as the correct residue name is used, REFMAC5 and Coot will use the correct linkage restraints without the need to add specific link-record annotation.As part of the process of the correct application of covalent linkages and the efficient use of existing descriptions in the CCP4-ML, an important step is ensuring the use of the correct residue nomenclature. Even when two monomers seem to be identical, it is always important to use the one with the correct identifier, et al., 19774ub6  ligand.In some cases special care is required when selecting the appropriate component identifier for a particular compound. Haem groups are an example of this  produces and adds a standard LINK record to a model . This is found in the CCP4 module . The CCP4 module contains a menu item Make Link via AceDRG which opens a dialogue that asks for the following.(i) The bond order corresponding to the covalent linkage (default: single).(ii) Which non-H atoms should be deleted (if any).(iii) Which bond orders change as a result of this new linkage (if any).An interface to the link-dictionary generation functionality has recently been added to Coot executes AceDRG to produce the required link dictionary, which is then imported into Coot so that it is available for subsequent real-space refinement. On successful reading of a link dictionary, Coot provides visual feedback by representing the new linkage as a dotted line between the linked atoms.The user then clicks on the two atoms to be linked. 4.6.JLigand was originally designed as a graphical interface for LibCheck, allowing users to visually create and edit chemical graphs for ligands and produce component and link dictionaries, as well as generate regularized coordinate models. JLigand is now able to use AceDRG for component- and link-dictionary generation; AceDRG is recommended over LibCheck. However, LibCheck can still be used as a contingency in cases that AceDRG cannot presently handle .JLigand is closely integrated with Coot: following the selection of two atoms in Coot, JLigand is launched displaying the two components to be linked. JLigand can then be used to specify the details of the covalent linkage . The link dictionary is then generated and communicated back to Coot, at which point Coot generates and applies the corresponding link record to the model. JLigand provides a more interactive graphical alternative to the AceDRG interface of Coot.JLigand uses a mechanism similar to AceDRG when generating link dictionaries  project management . Descriptions for the two components to be linked are required: these may be automatically imported from the CCP4-ML using the relevant three-letter code or from a custom component dictionary. Where required, such dictionaries can be created separately using AceDRG via the Make Ligand task. The interface automatically inspects the component dictionaries in order to determine the lists of atoms within the two components. After selecting the atoms to be linked, and specifying the linkage bond order, the user may also select to optionally delete atoms, change bond orders and change formal charges within each of the components.Recently, the This task may be used in isolation in order to make an abstract link description, or it can be used in conjunction with a particular model. In the latter case, the model is searched for all potential instances of the specified linkage type, according to proximity criteria. The user may then select whether to automatically apply link records for all identified potential instances of the linkage, or to add just one link record for a specific instance.Make Covalent Link task utilizes the Gemmi library . Thus, any component and link dictionaries generated during, for example, one Coot job are naturally accessible and used in any subsequent REFMAC5 and Coot jobs.In a particular revision, the dictionary of restraints includes accumulated descriptions of ligands and linkages created in any Coot task in CCP4 Cloud performs different actions depending on the presence of a dictionary for that linkage type. If a link description is not present then Coot inserts a standard LINK record into the output coordinate file  and asparagine (ASN) was not modelled using a link record (noting that 32\u2005927 such linkages are modelled amongst 6505 PDB entries). The N-linked glycosylation involves the removal of an O atom (O1) from NAG and the addition of a single bond between NAG[C1] and ASN[ND2]. Fig. 4AceDRG). Bond, angle, torsion and chirality restraints in the vicinity of the linkage are updated . Planarity restraints within both components are removed, and a new planar group involving both components is added . In the example model the covalent linkage is not modelled, and thus the interatomic distance between the linked atoms is unrealistically long (2.28\u2005\u00c5) due to repulsive forces during refinement. Re-refining the model using the AceDRG link dictionary results in an interatomic distance of 1.51\u2005\u00c5, which is closer to the target value of 1.431\u2005\u00c5 . Whilst here we exemplify the manual modelling of a covalent linkage, note that Coot contains automated tools to facilitate the building of N-linked glycans building of N-linked glycans . Planarity restraints are removed, and a new planar group involving both components is added . In the example model, the interatomic distance between the linked atoms is 1.0\u2005\u00c5 .Fig. 55.3.AceDRG link dictionaries facilitates the accurate modelling of a methionine\u2013tyrosine\u2013tryptophan (MET\u2013TYR\u2013TRP) cross-link. The first linkage is a single bond between MET[SD] and TYR[CE1], and the second is a single bond between TYR[CE2] and TRP[CH2].et al., 2005AceDRG link dictionary includes a description of this chemical modification.Fig. 6AceDRG link dictionary. In the absence of a link dictionary, the target values for models with link records derive from the CCP4-ML . It is evident that there is a greater discrepancy between the default and AceDRG target values for MET\u2013TYR than for TYR\u2013TRP. This indicates that compared with the simple default covalent radii-based target values, the more detailed description of local stereochemistry adopted by AceDRG results in little difference to the linkage bond length for TYR\u2013TRP but in a substantial difference in the case of MET\u2013TYR . The latter exemplifies the utility of the more detailed and accurate description of stereochemistry provided by AceDRG.Table 11sj2 . Fig. 6e) shows the model with PDB entry 5jhy refined against higher resolution data (1.4\u2005\u00c5) using the same AceDRG link dictionaries.Whilst the changes to coordinates resulting from the use of link dictionaries may be subtle, especially in cases where the data are of sufficiently high resolution to clearly indicate the position of each atom, the use of a more detailed dictionary nevertheless results in models that are more consistent with previous observations/prior knowledge (d), indicating that the covalent linkage may not have been modelled in the original deposition. Re-refining the model with link records but without a link dictionary results in interatomic distances that are closer to the target values (the TYR-TRP linkage distance is affected more than MET-TYR), yet there is still a large discrepancy between the model and (default) dictionary values for both linkages. However, re-refinement using the AceDRG dictionary results in inter\u00adatomic distances that are much more consistent with the AceDRG target values.As can be seen in Table 1This highlights the importance of correctly modelling covalent linkages using comprehensive restraint dictionaries. Whilst the resultant effect on the coordinate parameters may be subtle, this treatment may be important for the subsequent interpretation and detailed analysis of interactions and strain. Here, we have focused purely on the interatomic distance corresponding to the covalent linkage itself, although in practice it may also be useful to analyse the behaviour of other geometric features in the linked components when determining an appropriate modelling strategy.6.AceDRG, and have provided an overview of the various practical routes available for the modelling and application of covalent linkages within the CCP4 suite.In this contribution, we have reviewed the mechanism for describing covalent linkages: the use of link-annotation records to specify the existence of link instances within a model, along with an appropriate restraint dictionary for each type of covalent linkage. We have described the process of link-dictionary generation using i.e. modifications of the chemical composition of components and restraints describing intra-component stereochemistry). Such changes can have an effect on model geometry and thus subsequent interpretation, and so it is always advisable to use modelling assumptions (and restraints) that most accurately reflect the understanding of the chemistry within the crystal structure.It is important to model covalent linkages using a sufficiently detailed link dictionary, which, in addition to containing inter-component stereochemical restraints, also reflects any changes to the individual components as a consequence of the reaction  must be made manually, and thus care is needed when deciding linkage chemistry. Often, the MX data quality/resolution is insufficient to unambiguously determine appropriate chemistry, although inspecting discrepancies between model and density maps can provide diagnostic information by indicating potential errors. Referring to literature detailing the nature of a particular chemical reaction can aid this, noting that different environmental conditions can result in different chemistries (for example protonation states may vary with pH). In some cases complementary experiments and referring to higher resolution analogues may aid such decisions.AceDRG can successfully be used to generate link dictionaries for the majority of covalent linkages, there are a number of scenarios that are currently unsupported; for example, when a covalent linkage (or the dictionary description) involves atoms from more than two components: there is presently no formal mechanism for dealing with this scenario in mmCIF restraint dictionaries. Notably, AceDRG cannot presently create dictionaries involving many metal-containing compounds . Metals pose additional challenges, such as determining the coordination and analysing/describing environmental interactions. The ability to routinely and robustly create restraint dictionaries for metal-containing compounds is a future prospect. Also, care should be exercised in cases where a compound is involved in multiple covalent linkages.Whilst CCP4 , software developers from different suites (who facilitate the process) and the wwPDB (who ensure the appropriate encapsulation of relevant information during deposition). Ensuring that all parties cooperate using a cohesive unified framework is a challenge. However, doing so is important in order to aid the quality and future interpretation of deposited models.10.1107/S2059798321001753/ir5021sup1.pdfSupplementary Figures. DOI: Click here for additional data file.10.1107/S2059798321001753/ir5021sup2.gzLink dictionaries generated by AceDRG, corresponding to the examples presented in Section 5. DOI:"}
+{"text": "This article presents a dataset on political connections, Sharia, and abnormal returns surrounding the M&A announcement of listed firms on Indonesia Stock Exchange (IDX) during the period 2010\u20132016. The dataset provides both short-run and long-run abnormal returns. Using an event study methodology, I calculate cumulative abnormal returns (CAR) as short-run abnormal returns and buy-and-hold abnormal returns (BHAR) as long-run abnormal returns. This dataset may be useful for researchers who study political connections, Sharia, and M&A performance. The data presented in this article are related to the research article entitled \u201cPolitical connections, Sharia and M&A performance: Evidence from Indonesia\u201d   Specifications TableValue of the Data\u2022The dataset provides short-run and long-run abnormal returns of firms that carried out M&A in the Indonesian market between 2010 and 2016, especially for firms with political connections and Sharia shares. I determine the date of the M&A announcement as the date on which the deal was publicly announced. This dataset is useful for investigating the short-run and long-run performance of M&A in Indonesia.\u2022The dataset contains firm-level data such as political connections, Sharia shares, abnormal returns, and various financial data . Consequently, the dataset is particularly useful for those studying M&A performance (both short-run and long-run) and the role of political connections and Sharia compliance.\u2022The dataset can also be compared with other datasets that apply different Sharia indexes, political connections' criteria, and measurements of abnormal returns to study similar phenomena.1The unbalanced panel comprises 48 M&A deals from 40 non-financial public firms in Indonesia covering the period 2010 to 2016.Short-run abnormal returns\u2013measured by CAR  for the full sample are presented in 2As mentioned in the previous section, my dataset contains political connections, Sharia compliance, CAR, BHAR, and various financial data . 2.1Following Faccio 2.2A firm is considered as Sharia compliant if it has Sharia shares, i.e., is a constituent of the Indonesia Sharia Stock Index (ISSI).22.3I rely on an event study methodology to measure both short-run and long-run abnormal returns surrounding M&A announcement.2.3.1itR is s the daily return for firm I on day t, and mtR is the daily return for market index m on day t. The estimation window is defined from 180 days to 30 days  prior to the M&A announcement. Then, using the basic market model, the abnormal return of security i for period t is:First, I employ a basic market model to estimate the alpha and beta of the sample firm:I use three days before to three days after the M&A announcement  and four days before to four days after the M&A announcement  as the event window.2.3.2BHAR as follows:i,tR is the realized return of security i on day t. I use market return on day t, which I denote as benchmarkR. The mean market-adjusted BHAR is defined as:Following Barber & Lyon I use twenty-four months after the M&A announcement (BHAR [24]) and thirty-six months after the M&A announcement (BHAR [36]) as the event window.2.4This dataset also includes firm-level financial data on size, leverage, ROA, risk, and growth. Firm size is calculated as the natural logarithm of total assets. I calculate leverage by the sum of total short-term and total long-term debt divided by total assets. ROA is measured as the ratio of net income divided by total assets. I measure risk by the daily stock return standard deviation  before the M&A announcement. Finally, growth is calculated as the growth rate of total assets over the last fiscal year.The author declares that this work does not involve the use of human subjects.Budi Wahyono: Conceptualization, Formal analysis, Investigation, Data curation, Writing \u2013 original draft, Writing \u2013 review & editing.The author declares that he has no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper."}
+{"text": "Record Linkage has versatile applications in real-world data analysis contexts, where several datasets need to be linked on the record level in the absence of any exact identifier connecting related records. An example are medical databases of patients, spread across institutions, that have to be linked on personally identifiable entries like name, date of birth or ZIP code. At the same time, privacy laws may prohibit the exchange of this personally identifiable information (PII) across institutional boundaries, ruling out the outsourcing of the record linkage task to a trusted third party. We propose to employ privacy-preserving record linkage (PPRL) techniques that prevent, to various degrees, the leakage of PII while still allowing for the linkage of related records.Mainzelliste as the data source. Our solution does not rely on any trusted third party and all PII is guaranteed to not leak under common cryptographic security assumptions. Benchmarks show the feasibility of our approach in realistic networking settings: linkage of a patient record against a database of 10\u00a0000 records can be done in 48\u2009s over a heavily delayed (100\u2009ms) network connection, or 3.9\u2009s with a low-latency connection.We develop a framework for fault-tolerant PPRL using secure multi-party computation with the medical record keeping software https://github.com/medicalinformatics/SecureEpilinker subject to the AGPLv3 license. The source code of the modified Mainzelliste is available at https://github.com/medicalinformatics/MainzellisteSEL.The source code of the sMPC node is freely available on Github at Bioinformatics online. New correlations between diseases and medical indications require combining data usually originating from different sources, such as genomic data, laboratory values or clinical data. In particular for rare diseases, individual treatment facilities will generally not have enough cases to be able to draw statistically significant conclusions. For this purpose, it is essential that data stored at different locations for a given patient are correctly linked (referred to as \u2018record linkage\u2019). At the same time, it is necessary to safeguard the patient\u2019s privacy and manage the data according to applicable data protection laws . In part) the identity data  and (ii) the medical data (MDAT). In this article, we will only be considering record linkage using information from the IDAT. One cannot always assume that patient data will be always complete and free of errors. Nonetheless, we would like to be able to match patient data across datasets wherever possible. In 1969, Fellegi and Sunter published the first mathematical description of record linkage, the process of pairwise comparison of records from two sets of records to find the pairs that likely represent identical entities   used forHowever, even when using Bloom filters, identity data\u2014albeit in encrypted form\u2014is still being stored in a central location and can, in principle, be misused for unauthorized reidentification. In fact, many Bloom filter-based solutions are vulnerable to frequency and cryptanalysis attacks . AlthougAn optimal record linkage process would completely avoid storing IDAT\u2014in any form\u2014outside of the original treatment facilities and, thus, render such attacks impossible. Ideally, none of the record linkage parties would obtain any new information from the linkage process. This is the promise of Secure Multi-Party Computation (sMPC). This technique is based on the principle that in a computation performed across multiple parties, each participating party only knows their own input and the result of the given computation  a linkage service. We developed a close integration into Mainzelliste, to deploy a holistic ID management and linkage solution based on an open-source software that is already in wide use.In this article, we describe the design and implementation of Mainzelliste Secure EpiLinker (MainSEL), a variant of sMPC integrated as an extension into Mainzelliste. A record linkage setup using MainSEL is comprised of a 1.1While being studied for over fifty years, record linkage algorithms and techniques gained increased traction and interest in the last decade. In comparison to the classic publications of record linkage  the focuexact-only matching of databases.A number of techniques use Bloom filters and hash-based message authentication codes (HMACs) to provide privacy in the linkage process  or the i1.1.1More recently, exchange groups in the record linkage configuration.They use a total of four Bloom filters, in which (fragments of) different fields are combined. Expert knowledge of probable errors is encoded in the choice of fragments and fields. In contrast, our solution takes a much more general approach by implementing the full field-tested EpiLink algorithm . Our method also resolves ties between several probabilistically matching records by determining the highest match score before evaluating the score threshold. For this, we implemented a novel quotient-ordering circuit that is able to calculate the maximum of many quotients, with its index, in sMPC. Their solution cannot resolve ties between multiple matches. Additionally, our solution compares fields like the date of birth or zip code with exact equality and combines the individual field comparisons in a weighted sum to give the final score, before evaluating the score threshold, whereas, as mentioned above, their solution only performs a threshold comparison on individual field comparisons.Furthermore, our solution performs the whole post-processing of linkage information still in sMPC, whereas Lazrig et al.\u2019s solution reveals per Bloom filter whether it matched, which leaks information  and in particular contains Lazrig et al.\u2019s method as a special simplistic case: Circuit Hence our solution is more generally applicable and delivers a higher quality of matches , are not composable with sMPC security guarantees. In fact, the security notion of DP is contrary to the sMPC security goals in the case of PPRL, as sMPC aims to reveal the correct, exact result of the ideal function and DP aims to reveal only noisy, i.e. (boundedly) approximate results. Even hybrid systems using differentially private blocking mechanisms as a pre-processing for the sMPC record linkage  for increased clarity of the description.In this article, the function 2.1Trusted Third Party (TTP), often called a linkage unit. Since we use secure multi-party computation, we do not require any trusted third party.Record linkage describes the task of linking records from different data sources that belong to the same entity. In general, this may include two or more data sources. We deal with the task of peer-to-peer, that is, two-party record linkage. In this setting, classic solutions off-load the record linkage to a match-cardinality) or linkage of the datasets for scientific evaluations. The MainSEL software implements the field comparison, match classification and the match-cardinality application. In Section 2.4 we introduce a linkage service that enables the secure use of the linked datasets in arbitrary follow-up applications. Note that this service is not a TTP.Privacy-Preserving Record Linkage (PPRL) generally advances in several steps: data pre-processing, blocking/filtering, field comparisons/similarity and match classification. This is then followed by some application of the record linkage classification, e.g. the counting of matches  between two records that attains values between 0 and 1. The database record with the highest similarity score is then compared to a threshold parameter  Given a record M records. For each kx, we determine the best matching record and check whether the score reaches the threshold. The count of those matches now determines the above introduced match-cardinality, which we denote with Let In summary, we care about the following two functionalities: 2.1.1To determine the similarity score of two records, we implemented the same algorithm as used by the Mainzelliste software, which is inspired by the EpiLink software . This leS of two records x and y is a normalized weighted sum of field similarities, yielding a score between 0 and 1: The similarity score x and y have ix and iy, each, for I is the field index set. ix and jy are non-empty and 0 otherwise. Similarity of fields of index i are determined using the functions ie and if are the error rate and average frequency of values, respectively. Those values are statistically derived once for a set of fields and then fixed, see The two records Two fields are determined to match if their score is above a certain threshold. Weighting each field\u2019s impact on the final score differently improves the score\u2019s ability to veraciously categorize matches.s and w for the numerator and denominator, which we also call the field-weight and weight component of a  score, because we often need to work with them individually, especially when describing the sMPC solution. The actual division We introduced the definitions Tie-solving order. We often need to determine the maximum of a set of quotients, for which we introduce a special order. On quotients s1, w1) and , we define the tie-solving order as \u00a0Exchange groups. In real record linkage scenarios, linkage quality can be improved by grouping some fields into so-called exchange groups, like first, sur- and birth name. Such fields may be accidentally swapped when entered. The score G, its symmetric group. It has size Let G\u2019s sub-score for permutation \u03c3 is now defined as G must be of the same comparison type and that GS for a group G is now determined as the maximum of all sub-scores for all permutations, using the tie-solving order A group x and y now becomes \u00a0Gs and Gw are the numerator and denominator of the group scores GS, as defined in Let 2.1.2i, we use either simple equality or Dice-coefficients of Bloom filters  is applied to string fields like first and surname where fault tolerance is desired. Strings x are converted into a Bloom filter Hamming-weight, that is, the number of set bits of a bit vector and X and Y. Write x and S\u00f8rensen-Dice-coefficient . While the foundations of this subfield of cryptography were laid in the 1980s : Informally, a distributed protocol is said to semi-honest or honest-but-curious attacker model. In this setting, protocols aim to be secure against an attacker who correctly follows the protocol, but additionally tries to learn as much as possible about the other parties\u2019 inputs and outputs. While stronger security models  exist, the semi-honest model is a good fit in a setting as ours, where the parties are regulated by law and known in advance.Throughout this article, we will focus on the sizes (i.e. the number of records to be linked) need to be known in advance. However, in cases where this information is still considered sensitive, the parties can pad their databases with dummy elements to any reasonable upper bound on the size, thus hiding the actual size, at the expense of increased computational complexity.While the inputs of the parties participating in an sMPC protocol remain secure, the input Summing up ABY\u2019s security assumptions cf. , the wea2.3In order to calculate the main functionalities When expressing algorithms in a circuit, it cannot have dynamic control flow, because otherwise information of intermediary results would leak. Thus, all branches have to be evaluated and all loops have to be unrolled. For efficiency reasons, all unrolled loops are executed in parallel and all sums are calculated as balanced binary-trees to minimize the circuit depth.Because we lack dynamic control flow, we decided to not apply blocking mechanisms, as is usually done in record linkage pipelines. Blocking describes the pre-filtering of records such that less records need to be fully compared. We also have to evaluate all field similarities, even if either field is empty (in which case this pair of fields does not contribute to the score).x by Aarhus and the records C 1.  \u00a0cf. ,C 2. determines the highest score and its index C 3. match bittests for a match by calculating the \u00a0Given a record s and w in parallel, use them for (C2) and (C3) and never actually calculate the divisions M records k and then simply sum the match bits.It is more efficient to calculate the field-weight- and weight-sums 2.3.1x as a circuit input or the circuit implementation of function x. Sans-serif font is used for circuit variables, typewriter for circuit functions/algorithms. We define A, B and Y (This adheres to the notation of ABY.), respectively, and denote the spaces of values of bit-length l in those protocols as l and protocol Y. It is mainly relevant to the discussion in Section 2.3.5. The superscript bit-length l or subscript protocol p are sometimes omitted for brevity.To describe the individual circuit components, we introduce the notation 2.3.2weight precision and similarity or Dice precision, which is the same for all fields We start by introducing the fixed-point presentations of weights and field similarities, which are the only two real number variables in the similarity score. Let The field similarity measures T is multiplied with Similarly, the real-valued threshold The real weights This leads to the highest possible precision because the weights occupy the full range of 2.3.3no operation if the same protocol is chosen for As has been shown \u2013(C3). Inputs are only mentioned in the circuit sub-component where they are used, thus omitted in superordinate components. Private inputs by Aarhus and Berlin are stated as semicolon-delimited pairs. A high-level overview of the circuit layout is shown with Circuit \u2013(C3). Inj for Berlin \u2019s input individual field of a single record Now follows the description of the circuit implementation of Circuit ol \u03b1 cf. . It uses Circuit  for the ores cf. .Similarity circuits. The field similarity i has Dice similarity type, we use the field entry\u2019s Bloom filter as the input to the circuit, i is denoted by Note that both circuits output the similarity as values in protocol Maximum quotient circuit. Remember that a group\u2019s sub-score is the quotient ores cf.  and is dNote that the index is not needed for the calculation of a group weight, but will later be used when calculating the maximum over all scores to determine the best match in Circuit Now the actual 2.3.5L for arithmetic circuit components and the resulting fixed-point precisions n weights of bit-length n weights of length n field-weights of length L of space It follows a discussion about the chosen bit-length L\u2009=\u200932 and n\u2009=\u20098 fields, such that We compared the fixed-point score calculation as implemented in our circuits to the same calculation done in double floating point precision on a large number of random inputs. The observed deviations are < 1% for 2.4In this section we describe the MainSEL record linkage system\u2019s design. It is comprised of the Mainzelliste as the data source and SEL as the sMPC compute unit. Both components communicate with each other via JSON REST interfaces. We illustrate the systems\u2019 communication interface, the record linkage and ID management workflow and possible additional modes of operation.2.4.1The sequence of communication is divided into two phases: the initialization phase and the linkage phase. During local initialization the connection to the local data source and the structure of the records, as well as their weights, are configured. Then an arbitrary number of remote targets can be configured. Every call between each of the parties is authenticated via a pre-shared key and executed over a secure channel, e.g. TLS-secured .The initialization phase is completed when the configurations between the local SEL and the (multiple) remote SEL s, as well as between the local SEL and the linkage service, are tested. The test assures connectivity and compatible algorithm configurations.The linkage phase see  starts wWith these requirements satisfied, the actual sMPC is executed between the local and the remote SEL [step (3)]. At the end of the computation, each side holds one share of the index of the best match, as well as shares of the match bits. These shares are then sent to the linkage service. Additionally, the remote SEL sends their encrypted IDs to the linkage service [step (4)]. It combines the shares and the best matching IDs are de- and re-encrypted. The information whether a match occurred is stored together with the linkage ID (LID) in encrypted form. This LID is transmitted to the local SEL, which sends it to the given callback address for storage or evaluation in the Mainzelliste [step (5)].2.4.2The usage of the record linkage process results is a privacy concern in itself. To avoid re-identification by two colluding actors on both sides, the returned LID must not reveal any information about the matching result. However, this very information is the basis for the detection of duplicates and the assignment of pseudonyms.Linkage Service, a component only concerned with generating and encrypting LIDs. This component does not constitute a trusted third party, as it has no functionality in the linkage process and does never receive any private information. It only holds a secret key for every party for re-keying the generated LIDs and generates random IDs. This setup is used to prevent collusion of adversaries in both locations.Confidential pseudonymization is achieved by introducing the To prepare for confidential LID management, one data source contacts the Linkage service to generate random IDs for all its records. Those random IDs are encrypted with the corresponding party\u2019s secret key. After receiving the linkage result as well as the list of IDs from the server, the linkage service decrypts the LIDs. If the linkage results in a match, a matching bit is concatenated to the decrypted ID and this string is re-encrypted with the client\u2019s secret key. If the records do not match, a new random ID is generated and encrypted with the client key. This procedure ensures that every LID looks like a random string and even two actors on both sides are not able to examine or compare IDs to learn matching records.To identify the matching records, a process that requires the patients\u2019 consent for the data exchange from both parties is executed which grants the linkage service permission to decrypt the LIDs and distribute them in plain text. This information allows the identification of matches as well as the quality of matches.In this scenario, only the linkage service is allowed to generate LIDs. Otherwise the security of this procedure would be compromised. To check the validity of signatures, MainSEL uses a randomly chosen but sufficiently long zero padding of the plain text LIDs. This enables the linkage service to verify the validity of LIDs and that they belong to the correct party.LID Generation without a LS. The described outsourcing of ID management is desirable for regulatory reasons, but not required from a cryptographic protocol perspective. The same functionality could be realized within the sMPC circuit, for example in the following way: both parties input an additional randomness per record. If 2.4.3As described in the introduction of Section 2.1, we can easily extent the This mode of operation is relevant for a number of real world applications, especially in the research and treatment planning of rare diseases. Patients with rare diseases are regularly recorded in multiple hospitals and research facilities, often with differing or uncertain diagnoses. This leads to a high amount of duplicate records in joint cohort studies. The current legal process for finding those duplicates includes all legal requirements required for transferring and processing the complete identifying dataset. This process is unreasonably complex for the feasibility analysis stage of a study, where e.g. cohort sizes are determined.3This section provides benchmarking results for our implementation of the sMPC circuit as set forth in Section 2.3 and describes the experimental setup. The interpretation of the reported benchmarks is discussed in Section 4.3.1As we implement the established, well understood record linkage algorithm of the Mainzelliste software , only the size and structure of the circuit need to be known, but the input data can be set later. More specifically, in this phase the parties perform base OTs and OT-extension and exchange multiplication triples (Arithmetic Sharing) or Yao keys. Details can be found in the description of the ABY framework , a full cross-linkage of two medium-sized databases with 10 000 patients each would take 78\u2009h for the setup and 17\u2009h for the online phase, or approximately 4\u2009days in total. In the high latency (100\u2009ms) networking setup C, it would take almost 17\u2009days. We expect to drastically reduce this time in future work by adding record linkage blocking techniques to our procedure, which, for classical and Bloom filter-based record linkage, have already been implemented in recent versions of Mainzelliste. However, this setup would only need to run once initially, when two parties enter the secure record linkage system. Once the system is online and linked, securely linking a newly admitted patient to an existing database of size 10 000 would take 6.1\u2009s online time for circuit variant A. In network environment C, it would take 48\u2009s for variant initial linking phase, while during normal operation the complexity becomes In circuit variant The optimal configuration of our system depends on the requirements of the scenario. For most environments, the optimization of the online times is sensible, as the setup phase can run between timing critical processes. For all cases other than having small databases and very high latencies, using variant This work is easily generalizable to augmented patient data. If, for example, the IDAT fields used in this work were augmented by equality-check MDAT values, runtimes would not be impacted heavily. As displayed in Our results are in alignment with A. We can also conclude that machines with more computational power would unfortunately not lead to significant improvements in runtime.The studied network environments reveal widely known bottlenecks of sMPC. Firstly, we can identify the network communication as the computation\u2019s main impediment cf. . By eithThe legal question whether the transmitted data is \u2018personal data\u2019 is not answered yet in the European Union. Past decisions of the European Court of Justice and the German Federal Court of Justice lead to our understanding that record linkage without the patients consent might be legally permitted, as encrypted data is only personal data for parties having access to the encryption key as well as third parties having the legal right to demand disclosure of the key . In the 5In this work, we presented a novel method to perform privacy preserving record linkage with no information leakage, guaranteed by the utilization of provably secure multi-party computation. Most importantly, in the environment relevant for medical research in the foreseeable future (semi-honest setting and the absence of quantum computers), record linkage via MainSEL ensures that no record linkage party learns anything apart from the intended record linkage result\u2014not even in an indirect (e.g. Bloom-filtered) form. Our implementation includes integration interfaces, optimizations and operation-ready deployment methods.) the secure record linkage algorithms and (ii) the interfaces and application.Due to carefully designed cryptographic protocols, as well as a novel high-level approach to generate optimized integer division circuits, our solution provides reasonable runtimes for linking mid-sized to large data sources as well as in an online mode for large and very large data sources. Albeit the promising results, this work opens up possibilities for further optimization and research in the following two categories:  is future research.two-party computation framework (ABY), so using MainSEL to fully link k databases of size N, each, requires the naive pairwise matching of multi-party successor to ABY will be explored in the future.Note that we implemented a pair-wise record linkage algorithm using a secure Locality Sensitive Hashing based techniques, are shown to be incompatible to those strong privacy guarantees , or even software library, with moderate effort.Independent of those areas of improvement, further regulatory and legal work is a necessary condition to allow practical usage of secure record linkage. With this work we hope to contribute to this process by providing practical benchmarks and technology details. In our opinion secure record linkage can contribute to a more privacy-preserving, better auditable and less bureaucratic digitized medicine.We would like to stress, that even if secure record linkage is computationally intensive, many application scenarios become legally or intent-wise possible only through the privacy guarantees of our solution.This work was supported by the German Federal Ministry of Education and Research (BMBF) through the HiGHmed Consortium [01ZZ1802G]; and the German Research Foundation (DFG) through the MAGIC project [LA 3859/1-1] and the Research Training Group GRK 1651 (SOAMED).Conflict of Interest: none declared.btaa764_Supplementary_DataClick here for additional data file."}
+{"text": "This ex vivo study aimed to measure the performance of an electronic apex locator (EAL) in the presence of sodium hypochlorite irrigants with different concentrations. Two EALs (Root ZX Mini and Locapex 6) were used to locate the apical foramen in 10 extracted single-rooted teeth in the presence of 0.5%, 2.5% and 5% sodium hypochlorite. Visual working lengths were also determined using #10 K-file under a microscope before the electronic measurements were made. The performance of both EALs was compared for the electronic working lengths determined under the different concentrations of sodium hypochlorite. A multiple-way ANOVA and PLSD Fisher\u2019s test with an \u03b1 risk fixed at 5% were conducted. There were no statistical differences in the working lengths determined by both EALs between the three groups with different concentrations of sodium hypochlorite and their visual control measurements. When a \u00b1 0.5 mm margin was applied, the Root ZX Mini and the Locapex 6 presented 88% and 83% accuracy, respectively. Sodium hypochlorite concentration in irrigants does not affect the accuracy and reliability of either the Root ZX Mini or the Locapex 6. Electronic apex locators are reliable with any concentration of sodium hypochlorite irrigants. Error-free determination of working length (WL) is instrumental in ensuring the integrity of the periapical area during an endodontic procedure. The working length is defined as the distance between a coronal reference point and the apical point, consensually defined by the apical constriction, at which canal preparation and obturation should terminate ,2. TradiThe shortcomings of the radiographic technique are easily overcome using an electronic apex locator (EAL). The EALs have been presented as valid instruments for identifying the apical constriction and determining working length as an alternative to the radiographic method . The newRoot ZX developed by Kobayashi and Suda  and manuHowever, the reliability of EALs has been questionable in the presence of irrigants in the root canal space. In particular, the presence of electroconductive solutions such as sodium hypochlorite (NaOCl) present inside the canal significantly reduced the impedance and, therefore, resulted in shorter measurements, whereas more extended measurements were detected in the lower electroconductive solution . HoweverTherefore, this ex vivo study aimed to assess the impact of three different concentrations of NaOCl on the accuracy of two electronic apex locators: Locapex 6  and Root ZX Mini (Morita Co.). The null hypothesis was that there were no differences among the three different concentrations of NaOCl and between the EALs.The protocol of this study using extracted teeth was approved by the institutional review board of Institute of Dental Sciences, Siksha \u2018O\u2019 Anusandhan (SOA/IDS/IRB 2021/9-I). Thirty single-rooted caries-free teeth, which were freshly extracted due to periodontal or orthodontic reasons with mature apices, were collected from the Department of Oral surgery. Twenty teeth from the collected teeth samples, which had either apical resorptions, metallic restorations, prosthetic or endodontic treatments, were excluded from the study. Ten teeth were finally selected. Incisal edge or cusps of the included teeth were flattened with a 12 mm cylindrical diamond burr  with a high-speed handpiece  to create a stable and reliable coronal reference point. The access cavity in each selected specimen was made with a 10 mm spherical diamond bur . An initial #8 K-file  was introduced to explore the canal and determine the initial visual-WL (VL) by advancing the file until the tip was tangential to the major foramen under a stereomicroscope  at 16\u00d7 magnification. The rubber stop was then adjusted to the coronal reference point and the WL (VL) was measured. The value was recorded using an endodontic ruler and microscope magnification, with 0.1 mm precision. Two EALs were used in this study: Locapex 6 (LPX6) and Root ZX Mini (MRZX) . The MRZTooth specimens and labial clips for each EAL were embedded in a plastic container filled with an unset mixture of alginate . The alginate was mixed according to the manufacturer\u2019s instruction and was left to set . The irrigating solutions were freshly prepared and divided into three groups according to the concentration of NaOCl: An original concentration of 9.6% NaOCl  was diluted with sterile water for Irrigant 1 (IR1) with 0.5% NaOCl, Irrigant 2 (IR2) with 2.5% NaOCl and Irrigant 3 (IR3) with 5% NaOCl. The irrigation solutions were then used in order of lowest to highest concentration. When testing the different concentrations of NaOCl, the previously applied irrigation solution was aspirated, and canal was dried with paper point  to avoid concentration modifications. The root canal was flushed with 1 ml of irrigant, delivered using a side slotted and vented needle . After irrigation, the pulp chamber was gently air-sprayed and coronal part of the canal was partially dried with optimal sized paper points. For each specimen tooth, the electronic WLs were measured in IR1, IR2 and IR3 recorded using both EALs, LPX6 and MRZX. Additional irrigation using 0.5 mL irrigants and drying process using air spray and paper points was repeated between each electronic WL measurement. The size #10 K-file connected to file holder was advanced till the \u201cAPEX\u201d signal flashed on the screen for the LPX6 and the first red bar next to the \u201cAPEX\u201d signal flashed in the MRZX. The rubber stop was adjusted to the coronal reference point, the file was taken out and the WL was confirmed using an endodontic ruler under a dental operating microscope  at 10\u00d7 magnification. Two measurements per EAL were recorded for each tooth in each group. The same procedure was repeated for all groups IR1, IR2 and IR3. The statistical analysis was performed with StatView 5.0 . VL and WL measured using LPX6 and MRZX were compared, regardless of NaOCl concentration, using a multiple-way ANOVA test and PLSD Fisher test to assess the comparability of both methods . The Fisher test was then used to study the influence of NaOCl concentration on each EAL separately. For all tests, the \u03b1 risk was set at 5%.p > 0.05). The WL measured visually (VL) and electronically (MRZX and LPX6) were not statistically different .The test subjects  were deemed comparable and formed a homogenous group. The PLSD Fisher test showed that there was no statistical difference between the VL and EAL measurements  proportionally decreased with NaOCl concentration  . This fiAnother explanation might be linked to the experimental model, which could affect the conductive properties of EALs. In a few studies, teeth were either immersed in 0.9% NaCl or a gelatine model . At the The \u00b10.5 mm margin of error is defined as being the strictest clinical tolerance and clinically acceptable . The accSimilar to another study , the apiAn endodontic ruler is a routinely and most common WL transfer measuring device in dental practice , althougVarious ways to simulate in vivo conditions to determine the working length include 1% agar, gelatine, alginate, and flower sponge soaked in 0.9% saline and alginate models . HoweverUnder the limitations of this study, both MRZX and LPX6 were found to be reliable with 90% accuracy regardless of the concentration of NaOCl within an error margin of \u00b10.7 mm interval. Although this correlates with previous studies, there is the necessity for further research with additional parameters such as preflaring or EAL integrated endodontic motors used in NaOCl, heated NaOCl or other irrigating solutions."}
+{"text": "In response to calls for research to improve human-machine teaming (HMT), we present a \u201cperspective\u201d paper that explores techniques from computer science that can enhance machine agents for human-machine teams. As part of this paper, we (1) summarize the state of the science on critical team competencies identified for effective HMT, (2) discuss technological gaps preventing machines from fully realizing these competencies, and (3) identify ways that emerging artificial intelligence (AI) capabilities may address these gaps and enhance performance in HMT. We extend beyond extant literature by incorporating recent technologies and techniques and describing their potential for contributing to the advancement of HMT. Human-machine teaming (HMT)Effective HMT is contingent upon the success of complex interactions between human and machine agents, and between these agents and their environment . HoweverIn this perspective, we (1) briefly summarize the state of the science on critical team competencies identified for effective HMT, (2) highlight gaps preventing machines from fully realizing these competencies, and (3) identify emerging artificial intelligence (AI) capabilities that show promise for enhancing these competencies in machine agent teammates. Our goal is to show how HMT can integrate cutting edge advancements from computer science to improve capabilities of machines to function as teammates.Psychologists and engineers have long explored the use of machines to augment and improve human task performance . In earlAs machines gained intelligence and the ability to adapt in their interactions with humans, researchers e.g.,  developeIn the last decade, the conversation has shifted from machines as tools to machines as teammates . The inttransportable teamwork competencies, are applicable in any effective team, regardless of the team or task environment  pertaining to its abilities to afford an operator\u2019s comprehension about an intelligent agent\u2019s intent, performance, future plans, and reasoning process\u201d  adaptability  has argued that to move beyond knowledge-based AI methods (first wave) and statistical machine learning AI methods (second wave), we need approaches that can integrate both first and second wave methods to support contextual understanding and adaptation , will also enable machines to determine when and how best to communicate with teammates, further enhancing the trust and ability that affords machine teammate-likeness. Finally, the creation of causal and counterfactual inference capabilities unique to third wave AI will allow machines to be truly adaptive teammates that possess the ability to recognize and reason about the underlying factors that produce changes in the HMT and environment.The capabilities afforded by recent technological advancements show promise for allowing machines to possess the transportable teamwork competencies identified as universally critical to teams . For exaWhile these new technological approaches show promise, more work is needed to refine them to the level required for effective teamwork. Current AI research focuses on developing specific learning and performance capabilities and often does not incorporate findings or insights from the teaming and HMT literature. For example, consider OpenAI Five, a team of five trained neural-network models that can coordinate together to beat a team of five top human champions at Dota 2, a multiplayer online battle arena game . While tContrastingly, if the OpenAI Five possessed the capabilities outlined in this perspective piece , then they should better support communication, coordination, and adaptation with humans. As this example shows, more work is needed to better understand how insights from human teaming and HMT might be integrated into the development of these emerging machines. Increasing collaboration between computer scientists and HMT researchers in examining these insights would be beneficial. With machines operating at new levels of sophistication, true HMT may become possible at a larger scale than seen before.KS, LB, CM, RW, and ES contributed to the writing and content of this paper. All authors contributed to the article and approved the submitted version.RW was employed by company Soar Technology, Inc.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "Although 234 SINEs have been identified so far, only 23 are from insect species (SINEbase: Plutella xylostella, among which PxSE1, PxSE2 and PxSE3 were tRNA-derived SINEs, PxSE4 and PxSE5 were 5S RNA-derived SINEs. A total of 18 related SINEs were further identified in 13 lepidopteran insects and a baculovirus. The 3\u2032-tail of PxSE5 shares highly identity with that of LINE retrotransposon, PxLINE1. The analysis of relative age distribution profiles revealed that PxSE1 is a relatively young retrotransposon in the genome of P. xylostella and was generated by recent explosive amplification. Integration pattern analysis showed that SINEs in P. xylostella prefer to insert into or accumulate in introns and regions 5\u2009kb downstream of genes. In particular, the PxSE1-like element, SlNPVSE1, in Spodoptera litura nucleopolyhedrovirus II genome is highly identical to SfSE1 in Spodoptera frugiperda, SlittSE1 in Spodoptera littoralis, and SlituSE1 in Spodoptera litura, suggesting the occurrence of horizontal transfer.Here, five SINEs were identified from the genome of Lepidopteran insect genomes harbor a diversity of SINEs. The retrotransposition activity and copy number of these SINEs varies considerably between host lineages and SINE lineages. Host-parasite interactions facilitate the horizontal transfer of SINE between baculovirus and its lepidopteran hosts.The online version contains supplementary material available at 10.1186/s12864-021-07543-z. B1 and Alu, are originated from 7SL RNAs, other eukaryotes primarily harbor tRNA-like SINEs [snRNA) (SINEU) and the 3\u2032-end of the large ribosomal subunit (LSU or 28S rDNA) (SINE28) have been identified in avian, crocodilian and mammalian genomes, respectively [) during retrotransposition [Short interspersed nuclear elements (SINEs) are Class I transposable elements (TEs) that propagate by a copy-and-paste mechanism , 2. SINEke SINEs , and SINke SINEs , 6. Receectively . The chaposition .Alu elements have been identified in the human genome [ZmSINE3 were detected in Zea mays [As non-autonomous retrotransposon, the replication rate and survival of a SINE is dependent on the partner LINE activity, and its genomic copy number varies greatly between families and host species. For example, as high as 1 million copies of n genome , whereasZea mays . On the Zea mays . InteresZea mays , 14, sugAlu SINE inserted into human pluripotency-associated transcript\u00a05 (HPAT5) regulated related microRNAs through its let-7 binding site, which is essential for inner cell mass formation during early embryonic development [Transposable elements play an important role in the epigenetic regulation of the genome and generation of genomic novelty. A growing body of evidence has recently accumulated indicating that SINEs have a deep impact on genome organization and gene structure by generating regulatory elements for gene expression , 16, exoelopment .Plutella xylostella (L.), which is one of the most damaging insect pests of cruciferous vegetables around the world. We investigated the structures and insertion regions of these SINEs. The distribution of these SINEs in other lepidopteran insect species was also surveyed.While SINEs have been well characterized in human , other mPxSE1, was identified by homology search in DBM genome database . The PxSE1 is 263\u2009bp long, includes GT dinucleotide repeats at 3\u2032-tail and a 72-bp tRNA-related region at the 5\u2032-end with 64% identity to 72-bp tRNAArg of Drosophila melanogaster, which contains box A and box B of the RNA Pol III promoter , poly(T) or simple sequence repeats at 3\u2032-end. The average divergence varied from 0.035 to 0.13 , which was located within ORF27 encoding an unknown protein.BLAST searches were performed to detect PmSE1 in Papilio machaon and PzSE1 in Papilio zelicaon, whereas MsSE1 in M. sexta and CfSE1 in Choristoneura fumiferana showed the highest evolutionary divergence (0.436)  was observed between HaSE3 as a query [PxSE4 and PxSE5, in DBM  with its 43-bp 3\u2032-end being 84% identical to that of PxSE5 , encoded L1_EN (Endonuclease domain of the non-LTR retrotransposon LINE-1) and RT domain, and was terminated by ATGT tetranucleotide repeats in the short 3\u2032 untranslated region (3\u2032 UTR)  from WGS was inserted as a 1686\u2009bp fragment, which shared 71.8% identity with mariner-8_BM from B. mori [PxLINE1.1 were also found in the other 7 lepidopteran insect genomes  and expressed sequence tags (EST) database using BLASTN. Three elements, LaSE2, CsSE2 and ObSE2, with high identities to PxSE4 were found in genomes of Lerema accius, C. suppressalis and Operophtera brumata, respectively  of PxSE5 have more than 95% identity with its consensus sequence  of PxSE1, 2478 out of 5056 copies (49%) of PxSE2, 2470 out of 5158 copies (48%) of PxSE3, 2265 out of 4415 copies (51%) of PxSE4 and 902 out of 1952 copies (46%) of PxSE5 were found in introns  inserted into CDS of a gene encoding nitrogen permease regulator 3-like protein. The 21-bp fragment at 5\u2032-end of PxSE2.2 contributed 7 amino acids to the N-terminus of the protein  was found to share 95% identity to the sequence (WNNL01000005.1:\u00a0248783\u2013248238) of Spodoptera exigua genome  except the highly identical heads. Similarly, two 5S rRNA-derived SINEs, PxSE4 and PxSE5, also shared 98.7% identity in 159\u2009bp region of their bodies. Previous studies have found that the conserved bodies of SINE mainly include the V-domain, CORE-domain, Deu-domain, Nin-domain, Ceph-domain, Inv-domain, Pln-domain, Snail-domain, and Meta-domain [PxSEs are different from these known domains. A hypothesis has proposed that nonautonomous LINEs that have only 5\u2032 and 3\u2032 regions of original LINEs can be a source of enigmatic middle body of SINEs [ObSE2 and 5S rRNA-derived PxSE4, ObSE2 is a tRNA-derived SINE.Up to now, more than 234 SINEs have been isolated from the genomes of human, mammals, reptiles, fishes, mollusks, fungus, green plants, and insects [a-domain \u201343. HoweP. xylostella, and seven LINEs in each of the lepidopteran insects were identified with 3\u2032-end similar to that of PxSE5 and SfSE1, suggesting that the LINE identified in this study is an ancient retrotransposon and might widely exist in Lepidoptera insects. However, the 5\u2032 regions among SfSE1, PxSE5 and PxLINE1 shared a large divergence, indicating these SINEs exploded after the exchange of 3\u2032-tails. Moreover, the distinct 3\u2032-end in other PxSEs suggested that these SINEs might be mobilized by other LINEs that were not identified yet.SINEs can be composed of 5\u2032 and 3\u2032 regions of nonautonomous LINEs, and their 3\u2032 tails will also exchange with other LINEs under the pressure of natural selection to facilitate rapid amplification . The taiP. xylostella, the copy numbers of SINEs of tRNA origin is relatively higher than that of 5S rRNA origin. In particular, the copy numbers of PxSE5 is only 1952. Previously, it was speculated that the type 1 promoter in 5S rRNAs is more dependent on upstream signals than the type 2 promoter in tRNAs, resulting in the Pol III promoter in a retroposed 5S rRNA copy presumably remains silent or is expressed at a low level [MsSE1 and MsSE2 in M. sexta and SfSE1 in S. frugiperda were 7513, 16,157 and 11,117, respectively, whereas only 4521 copies of ObSE1 and 863 copies of ObSE2 were found in O. brumata. The genome sizes of M. sexta and S. frugiperda are around 400\u2009Mb, while O. brumata has larger genome size of 618\u2009Mb. Hence, SINE copy number may not correlate with genome size. Some factors of 3\u2032-tail, such as poly(A) tail or short direct repeats length, sequence conservation and distance to the transcriptional terminator, may affect the retroposition efficiency of the SINE families [The copy numbers of SINEs varies among different families and species. In families , 47. In PxSE1 showed that it most likely is a relatively young retransposon in the genome of P. xylostella and was generated by recent explosive amplification. The scattered distribution of PxSE5 copies also suggests that it is older than other SINEs.Based on the divergence of the copies from the consensus sequence, the relative age distribution of identified SINEs was analyzed. Scattered age profiles were found in most SINEs among all species or within the genus, suggesting that the activity and accumulation of these SINEs are dynamic processes that can vary considerably between host lineages and SINE lineages. Especially, the highly identity and concentrated PxSINEs inserted into introns accounted for 44\u201351%, only 2\u20138 copies were inserted into exons, indicating that PxSINEs prefer to insert or accumulate in introns of genic regions. However, the proportions of different SINEs located within introns of Solanaceae range from 15 to 54% [Zoysia japonica and maize [PxSEs into introns may provide signals for alternative splicing and polyadenylation, which may be a reflection of the host response to an ever-changing environment.The ability of TEs to replicate and move in the genome affects the genomic structure, gene expression, and the divergence and evolution of host species \u201351. The 5 to 54%  and 96% nd maize . In micend maize . We specImportantly, we also noticed that only 25 copies of SINEs inserted into the genic exonsof DBM, of which 13 copies were found in 3\u2032 UTR. In eukaryotic cells, some proteins (such as PUF protein) can bind to regulatory elements in the 3\u2032 UTR of mRNAs and control mRNA stability, translation and localization . The genSmaI-cor SINE between coregonid and common ancestor of salmonid , Sauria SINE between reptiles and mammals [HaSE2 SINE between Aphis gossypii and Lepidoptera insects [PxSE3 families and host species in this study. Interestingly, SlNPVSE1, a SINE copy inserted into the baculovirus, shared more than 90% identity to the consensus sequence of SfSE1, SlittSE1 and SlituSE1  DNA can also replicate in five non-permissive cell lines including SF21AEII, CLS-79, SpLi-221, hi-5 and BmN4 [PxSE1 between baculovirus and Lepidoptera insects. This is not surprising, because population genomics supported baculoviruses as vectors of horizontal transfer of insect transposons [Helitron transposon Hel-2 and Tc1-like transposon TCp3.2 between insects and associated baculoviruses has been detected [Sauria SINE from reptiles to mammals [PxSE1-like elements mediated by baculoviruses.Increasing evidence showed that HTT is a common phenomenon. So far, no less than 5689 HTT events have been recorded . However mammals , HaSE2 S insects . The lon insects , 60, as . exigua . S. exigand BmN4 , indicatnsposons . Similardetected , 65. Recdetected , 67 and detected . Additio mammals . Hence, P. xylostella, among which PxSE1 is a relatively young retrotransposon and was generated by recent explosive amplification. Homology searches revealed scattered distribution of these elements in other Lepidopteran insects with variable copy numbers. The preference of PxSINEs to insert or accumulate in introns of genic regions indicated that P. xylostella SINE families contribute to structural variation in introns. The identification of PxSE1-like elements in the baculovirus and related lepidopteran host insects provides evidence of horizontal transfer facilitated by host-parasite interactions. These data may have implications for understanding the evolution and HT mechanisms of SINEs.In this study, we identified three tRNA-derived SINEs and two 5S RNA-derived SINEs in the genome of P. xylostella WGS was downloaded from NCBI [The 235 publicly available insect databases of WGS assemblies including 33 Lepidoptera insects, EST, nucleotide (Nr/Nt), and TSA from National Center for Biotechnology Information (NCBI)  were used in this study  [The association of DBM SINEs with annotated genes were investigated using custom Perl script from MapGene2Chrom (2c_v1.0) . The int2c_v1.0) . The numwww.psc.edu/biomed/genedoc). The phylogeny of full consensus sequences of SINE families was built by MEGA 7.0 using Maximum Likehood with K2\u2009+\u2009G model [SINE\u2019s tRNA-like structure was checked with tRNAscan-SE , using m\u2009G model . The rel\u2009G model .Additional file 1: Figure S1. Characteristic of PxSE1 in P. xylostella. (A) the sequence of PxSE1. The pink nucleotides are TSD sequence, gray background present A box and B box structure, green background is 3\u2032tail sequence. (B) the homology search of PxSE1 in Repbase database.Additional file 2: Figure S2. The consensus sequence of tRNA and 5S rRNA related SINE transposons in insect genomes. Nucleotides in red font are 3\u2032 tail sequences.Additional file 3: Figure S3. Multiple sequence alignment the consensus sequence of PxSE1 (A), PxSE2 (B), PxSE3 (C), PxSE4 (D), PxSE5 (E) and their empty site sequences. The nucleotides of TSD are indicated with the red words. The nucleotides of 3\u2032 tail sequence are indicated with the gray background.Additional file 4: Figure S4. Multiple sequence alignment and evolutionary divergence estimation between the consensus sequences of SINEs. The number of base differences per site from between sequences are shown. All ambiguous positions were removed for each sequence pair. Evolutionary analyses were conducted in MEGA7.0.Additional file 5: Figure S5. The 8 copies with high identity to PxLINE1.1 in P. xylostella (A) and the multiple sequence alignment of one copy (AHIO01028576.1: 13049_14357) and Mariner-8_BM (B).Additional file 6: Figure S6. Alignment of potential LINE transposons in 8 lepidopteran insects genome. PxLINE1 in P. xylostella , TaLINE1 in Tuta absoluta (SNMR 01038797.1: 8533\u201311852), McLINE1 in Melitaea cinxia (APLT01012314.1: 14517\u201316103), CcLINE1 in Conopomorpha cramerella (SJJU01072145.1: 61771\u201365266), GmLINE1 in Galleria mellonella (NHTH01000021.1: 4230671\u20134228043), ArLINE1 in Adela reaumurella (WYDE01048472.1: 2507\u2013535), AhLINE1 in Adoxophyes honmai (BHDV01006067.1: 48096\u201349728), DpLINE1 in Dendrolimus punctatus (JAABVI010000027.1: 8196917\u20138193378).Additional file 7: Figure S7. The origin analysis of ObSE2. (A) the alignment of 75-bp fragment at 5\u2032-end of ObSE2 and 72-bp tRNA-related region of D. melanogaster. (B) the schematic representation of structure of ObSE2.Additional file 8: Table S1. The copies of PxSE1 in the genome of P. xylostella.Additional file 9: Figure S8. Examples for the relative age distribution of SINE families in other species based on the identity to the species-specific consensus. The abscissa showed that the identity between each consensus sequence and the copies. The ordinate showed that the copy numbers of sequence with the same identity.Additional file 10: Figure S9. The typical integration pattern of SINEs within genome of P. xylostella. (A) (B) and (C) are schematic diagrams of several copies inserted into the introns of LOC105382892, LOC105381513 and LOC105383359, respectively. (D) Statistics number of different SINE families inserted into the same gene.Additional file 11: Figure S10. Paralogous empty sites of PxSE1 (A) in P. xylostella and SfSE1 (B) in S. frugiperda. The nucleotides of TSD are indicated with the red background. The nucleotides of 3\u2032 tail sequence are indicated with the gray background.Additional file 12: Table S2. The databases of NCBI used for Blast searches, including 8 WGS databases, 3 EST databases and 2 TSA databases as well as the Nr/nt database."}
+{"text": "Bilal U, Alazraqui M, Caiaffa WT, et al. Inequalities in life expectancy in six large Latin American cities from the SALURBAL study: an ecological analysis. Lancet Planet Health 2019; 3: e503\u201310\u2014In this Article, due to a linkage error with the Panama City data, life expectancy data, P90\u2013P10 gaps, and associations with education have been corrected for Panama City in the Summary, table 2, the Results, where appropriate in the Discussion, in figures 1 and 2, and in the appendix. Additionally, support from the SALURBAL investigators has been added to the Acknowledgments section. These corrections have been made as of Jan 3, 2020."}
+{"text": "Arabidopsis thaliana, we tested the biochemical and physiological responses to water deficiency and NaCl treatment in mutants that are differentially affected in suberin composition and lamellae structure. Chronic drought stress increased suberin and suberin-associated waxes in wild-type plants. Suberin-deficient mutants were not more susceptible than the wild-type to the chronic drought stress imposed in this study. Nonetheless, the cyp86a1-1 cyp86b1-1 mutant, which had a severely altered suberin composition and lamellae structure, exhibited increased water loss through the root periderm. Cyp86a1-1 cyp86b1-1 also recorded lower relative water content in leaves. The abcg2-1 abcg6-1 abcg20-1 mutant, which has altered suberin composition and lamellae, was very sensitive to NaCl treatment. Furthermore, cyp86a1-1 cyp86b1-1 recorded a significant drop in the leaf K/Na ratio, indicating salt sensitivity. The far1-2 far4-1 far5-1 mutant, which did not show structural defects in the suberin lamellae, had similar responses to drought and NaCl treatments as the wild-type. Our results provide evidence that the suberin amount and lamellae structure are key features in the barrier function of suberin in reducing water loss and reducing sodium uptake through roots for better performance under drought and salt stresses.Suberin is a cell-wall-associated hetero-polymer deposited in specific plant tissues. The precise role of its composition and lamellae structure in protecting plants against abiotic stresses is unclear. In  Resistance to drought and high salinity have been the main foci in crop improvement efforts due to reductions in crop productivity caused by climate change . DroughtArabidopsis thaliana, the poly-aliphatic domain contains long-chain and very-long-chain fatty acids, \u03c9-hydroxy fatty acids, \u03b1, \u03c9-dicarboxylic fatty acids, and primary fatty alcohols, as well as glycerol and ferulic acid [Being sessile, plants have evolved multiple acclimation and adaptation mechanisms to respond to environmental stresses. Analysis of these protective mechanisms will contribute to our knowledge of plant tolerance to stress conditions. Plants have various hydrophobic barriers that provide protection against water loss. Suberin is one such cell-wall-associated barrier located in certain tissues, such as the root endodermis and periderm . Suberinlic acid . The moslic acid . The sublic acid ,9. In Arlic acid , particulic acid ,9.In Arabidopsis, root suberin deposition changes through plant development ,12,13. DOryza sativa) revealed the induction of suberin deposition under salt stress [Avicennia\u2009officinalis roots [CYP86A1, which encodes a fatty acid \u03c9-hydroxylase involved in suberin monomer biosynthesis, results in higher root hydraulic conductivity than the wild type [esb1 has ectopic suberin deposition in roots. Compared to the wild type, esb1 has higher water-use efficiency (WUE) and a lower transpiration rate associated with enhanced suberin deposition in roots [Suberin deposition can also be modulated in response to different environmental stresses. North and Nobel  reportedt stress  and oxygt stress . Additiot stress . An incrt stress . Suberinis roots , by wateis roots ,22, and is roots ,24. Franis roots  reportedild type ,27. The in roots . Conversin roots ,30. In ain roots .abcg2-1 abcg6-1 abcg20-1), suberin biosynthesis (far1-2 far4-1 far5-1 and cyp86a1-1 cyp86b1-1), or regulation of suberin synthesis (myb92-1 myb93-1). Compared to the wild type, roots of the triple abcg2-1 abcg6-1 abcg20-1 mutant roots have a distorted suberin lamellae structure and major reductions in alkyl hydroxycinnamates of suberin-associated waxes [abcg2-1 abcg6-1 abcg20-1 is 70% higher than the wild type in roots of 10-day-old seedlings and three times higher than the wild type in roots from 7-week-old plants [abcg2-1 abcg6-1 abcg20-1 is reduced in 20:0 and 22:0 fatty acids and 22:0 fatty alcohol [ABCG triple mutant was reported to be more permeable to water and salts [FAR1, FAR4, and FAR5 cause reductions in 22:0-OH, 20:0-OH, and 18:0-OH, respectively, and triple far1 far4 far5 mutants have a 70% overall reduction in primary alcohol content in the suberin polymer  and a greater than 90% reduction in alkyl hydroxycinnamates in the suberin-associated waxes [CYP86A1 and CYP86B1 result in strong reductions in C16 and C18 [cyp86a1-1 cyp86b1-1 has not been previously reported and our data showed that it is reduced in all chain lengths of DCAs and \u03c9-OH FA monomers and thus is expected to have a major perturbation of suberin structure and function. Additionally, the double mutant myb92-1 myb93-1 was included in this study because our preliminary studies showed that compared to the single mutants (myb92-1 and myb93-1), the double mutant has reduced amounts of all suberin monomers. Further, apple MYB93 is thought to play a major role in the regulation of suberin deposition in russeted apple skins [Nicotiana benthamiana [Although the above-described studies imply a relationship between suberization and physiological parameters in plants that relate to water and solute uptake, there is still a gap in our understanding of the role of suberin composition and ultrastructure in tolerance to drought or salinity. We hypothesized that Arabidopsis mutants altered in suberin composition and ultrastructure would have altered water relations and altered solute uptake in roots. Mutants selected in this study were affected in suberin monomer transport . Earlier time points were not included in our experiment because these have been previously reported [A time-course of poly-aliphatic suberin production in roots was analyzed using 4- to 7-week-old wild-type Arabidopsis plants to better understand how developmental suberin biosynthesis, which is that produced normally under control growth conditions, is regulated under non-drought conditions  plants in the same time period. Our preliminary investigations revealed that introducing drought stress in sand-grown Arabidopsis when plants were younger than 2-weeks old  resulted in high seedling-death rates. In addition, introducing drought stress for only one week did not result in a significant difference in suberin production between control and drought-stressed plants. However, when the length of the drought treatment was extended to 2 weeks, there was a significant difference in suberin production between control and drought-stressed plants. Therefore, the comparisons in suberin production and leaf biomass between control and drought-stressed plants were carried out in 4-week-old plants and older. Plants were 2-weeks old (after sowing) when the drought stress was introduced. Drought stress reduced the leaf biomass of wild-type plants after 2, 3, and 4 weeks a. Total far1-2 far4-1 far5-1, cyp86a1-1 cyp86b1-1, and myb92-1 myb93-1, respectively, it was reduced by only 27% in abcg2-1 abcg6-1 abcg20-1, which had the lowest total dry mass under control conditions. Wilting symptoms were not displayed in any of the mutants, but compared to non-drought conditions, drought significantly reduced the leaf relative water content (RWC) in all genotypes except for cyp86a1-1 cyp86b1-1  of all genotypes investigated, but with some significant differences among them a. While yp86b1-1 b. Cyp86agenotype .cyp86a1-1 cyp86b1-1 and myb92-1 myb93-1, had not been chemically and structurally characterized. Therefore, we investigated their suberin composition and lamellae structure. Of note, a recent study reported the suberin composition of myb41 myb53 myb92 myb93, a quadruple mutant that shows an overall ~ 78% decrease in suberin monomers compared to the wild type [At the time this experiment was conducted, two of the Arabidopsis mutants selected for our study, namely ild type .myb92-1 and myb93-1, the double mutant myb92-1 myb93-1 had a lower total suberin content (p = 0.000). Compared to WT, myb92-1, myb93-1, and the double mutant myb92-1 myb93-1 showed 22%, 11%, and 40% reduction in the total suberin content, respectively  increase in total suberin content after drought stress, resulting from increases in FAs, DCAs, and primary fatty alcohols . However, in both wild-type and cyp86a1-1 cyp86b1-1 plants, there was no significant difference in root water loss between the control and drought treatment . Therefore, in the next experiment we used plants that were grown only under control (non-drought) conditions to investigate if total suberin content helps to reduce water loss through roots  in cyp86a1-1 cyp86b1-1   . The PCAreatment . Cyp86a1abcg20-1 . After N scatter . Except trations a\u2013c. Abcgreatment a. The cht stress . Analyse\u2013Wallis) . Taken tIn this study, we report changes in Arabidopsis root suberin accumulation in response to chronic water deficiency and high NaCl in wild-type plants. In addition, to better understand how altered suberin amount, composition, and lamellae structure in roots regulate water and ion transport, the stress responses of selected mutants with different degrees of suberin deficiency were investigated. The results show that (1) chronic drought stress increases the rate of suberin biosynthesis and the total root suberin-associated wax content in wild-type roots; (2) suberin production exhibits plasticity under different soil water contents; (3) chronic drought stress, but not increased NaCl, induces suberin in all tested genotypes; and (4) mutants with suberin deficiency and distorted lamellae experienced increased water loss through the root periderm as well as ion imbalances upon NaCl treatment relative to the wild type.Previous studies have examined changes in developmental suberin deposition under non-stress conditions ,11 in Arcyp86a1-1 cyp86b1-1 mutant, which also presented a loss of lamellae ultrastructure. In contrast, a 33% decrease in root suberin content in the myb92-1 myb93-1 double mutant was not associated with a deformed lamellae structure. These two mutants had differences in their monomer compositions, but our analysis cannot determine which specific type(s) of monomers, if any, are critical to forming the lamellar structure observed by TEM. On the other hand, the abcg2-1 abcg6-1 abcg20-1 mutant has many defects in suberin composition, but its total suberin content is higher than that of the wild type, and the mutant fails to form suberin lamellae [far1-2 far4-1 far5-1 mutant shows changes in suberin monomer composition, specifically near the elimination of primary fatty alcohols, but it has normal total suberin content and lamellae structure [MYB41 and GPAT5 promoters drive the expression of reporter genes in cortical cells under ABA and NaCl stresses [We tested various mutants differentially altered solely in suberin composition or altered in both composition and lamellae structure to evaluate whether the analyzed mutants have different vulnerabilities to stresses. We observed a 60% decrease in root suberin content in the double lamellae . The fartructure . Thus, itructure  also emptructure . In addistresses ,42. Howecyp86a1-1 cyp86b1-1 double mutant presented the lowest leaf relative water content, implying it is the most water-stressed genotype. This mutant showed a high rate of water loss through the root periderm and severely decreased root suberin content with a distorted ultrastructure. Conversely, esb1-2, a mutant with high levels of poly-aliphatic suberin ectopically deposited in roots [esb1-2 was associated with higher WUE [The root periderm acts as the first line of defense against the underground environment. Suberin is a protective biopolymer found in the periderm and endodermis of roots, yet its specific contribution to the stress response function of the root periderm has not been elucidated. Under control conditions, the in roots  and a lain roots , had a lin roots . A lowerin roots . Enhancegher WUE ,37 that gher WUE .far1-2 far2-1 far5-1 was similar to the wild type, which indicates that primary alcohols that are part of the suberin polyester and root suberin-associated waxes  do not play a major role in resistance to chronic drought stress. Vishwanath et al. [far1-2 far2-1 far5-1 are more affected than the suberin polymer. Despite the defects in root suberin-associated waxes, the physiological parameters tested in this study did not indicate any chronic drought susceptibility in far1-2 far2-1 far5-1.The drought stress response observed in h et al.  showed tcyp86a1 mutant were compared [Cyp86a1 exhibited a salt-sensitive phenotype, with 5-week-old hydroponic-grown cyp86a1 plants displaying more withered leaves and down-curling of leaf tips after a 50 mM NaCl treatment [cyp86a1 leaf membranes suffered more serious injuries by NaCl treatment [cyp86a1 cyp86b1 double mutant. Additionally, there was an increase in Na accumulation and a reduction in K in shoots in the cyp86a1-1 single mutant [Abcg2-1 abcg6-1 abcg20-1 recorded a very high Na content and a very low K content even under control conditions, indicating increased movement of Na to the shoot, while the far1-2 far4-1 far5-1 mutant was not affected by NaCl treatment. Thus, the ability of a plant to tolerate salinity can depend on how much it limits Na transport to the shoot, while maintaining K content [cyp86a1-1 cyp86b1-1 and abcg2-1 abcg6-1 abcg20-1 mutants have reduced tolerance to salt stress due to high Na and low K in shoot tissues. In contrast, increased suberin in the esb1-2 mutant reduced solute leakage in roots [abcg2-1 abcg6-1 abcg20-1 suffered from ion imbalance due to uncontrolled uptake of Na, which resulted in high mortality by NaCl treatment. The cyp86a1-1 cyp86b1-1 and myb92-1 myb93-1 mutants recorded significantly decreased leaf K/Na ratios relative to the wild type, but these two mutants appeared to have suffered less from ion imbalance than that of abcg2-1 abcg6-1 abcg20-1.Recently, the effects of NaCl on the hydroponically grown wild type and the compared . Cyp86a1reatment . Additioreatment . Similare mutant . Abcg2-1 content . Therefoin roots . Krishnain roots  also detin roots . High Nain roots ,47. Mainin roots . A decrein roots . Additioin roots ,50,51. Tcyp86a1-1 cyp86b1-1 and myb92-1 myb93-1 had increased suberin, while these genotypes had decreased suberin under NaCl stress, indicating different suberin regulatory mechanisms in different stresses. These findings provide evidence for the regulated deposition of suberin in the root periderm as a mechanism to protect against abiotic stresses and contribute to future research targeting enhanced stress resistance in crops.In conclusion, our results indicate that the chemical composition and ultrastructure of suberin help to prevent water loss and limit uncontrolled Na uptake under high-salinity conditions in Arabidopsis. The results also indicate that suberin synthesis shows plasticity at different water levels and prevents root dehydration under chronic drought stress, which is important to maintain water balance in a plant. Additionally, our study showed that under drought stress, Arabidopsis thaliana wild type (Col-0 ecotype) and double and triple T-DNA insertion lines of suberin mutants were either generated by the authors or obtained from the Arabidopsis Biological Resource Center (ABRC). Homozygous T-DNA insertion lines were confirmed by PCR genotyping using primers listed in v/v) was calculated based on the volume of the cup. Thirty pots from each genotype were kept at 20% (v/v) water content by adding the required amount of water daily. The growth chamber conditions were 21\u201322 \u00b0C, 40\u201360% humidity, and long-day conditions (16 h light/8 h dark cycle). Two to three seeds were sown initially and kept covered with transparent plastic domes for one week and thinned out to one healthy seedling after one week. To induce the drought stress, watering was withdrawn in 20 pots from each genotype on the 10th day after sowing. Three days after water withdrawal, the soil moisture level reached 5% (v/v) and was maintained at that level throughout the experiment for drought-stressed plants. To analyze the regulation of suberin production under re-watering conditions, two weeks after drought stress was introduced, 10 replicate pots were watered again to reach 20% (v/v). For the salt-stress assay, 20 pots were maintained at 20% water. To induce the salt stress in wild-type and mutant plants, 0.1 g of NaCl was added to 10 replicate pots of each genotype for three consecutive days starting on the 10th day after sowing. The NaCl concentration in the soil solution reached 100 mM on the 14th day (2 weeks after sowing). Since preliminary studies revealed a high rate of salt-induced mortality in the abcg2-1 abcg6-1 abcg20-1 mutant, 50 mM NaCl was instead used for this mutant by adding 0.15 g of NaCl on the 10th day after sowing of that mutant.At four weeks of age, a fully developed young leaf from each plant was collected for drought-stressed and control treatments to determine the leaf relative water content (RWC). The leaves were stored in a re-sealable plastic zipper bag, and the fresh masses (FM) were determined immediately. These leaves were then hydrated overnight by placing them between moist paper towels for 24 h, after which the saturated mass of the fully turgid leaves (SM) was measured . Leaves n-tetracosane , 1-pentadecanol (15:0-OH), heptadecanoic acid (17:0), and tridecyl (13:0)-ferulate, which served as internal standards. Extracts were evaporated under nitrogen and derivatized with 100 \u03bcL of N,O-bis-(trimethylsilyl)-trifluoroacetamide (BSTFA) plus 100 \u03bcL of pyridine at 110 \u00b0C. The derivatized samples were allowed to cool and the solvent evaporated under nitrogen. The samples were re-suspended in heptane:toluene (1:1 v/v) for analysis by gas chromatography (GC) following the method described in [Five-week-old wild-type tap roots were used in this study because it has been found that suberin-associated wax deposition mostly takes place after 4 weeks . Each trribed in .v/v), chloroform:methanol , chloroform:methanol , and 100% methanol as described in [Roots were immersed in 4 mL of hot isopropanol and incubated for 30 min at 85 \u00b0C. The samples were delipidated for 24 h each with chloroform:methanol  at 450 W at \u226437 \u00b0C, held under vacuum for 1 min, 40 s at 100% power, and held under vacuum for another 3 min. Samples were rinsed with water and then placed in the microwave under vacuum (>20 mm Hg) at 250 W at \u226440 \u00b0C for 1 min 40 s at 100% power. Samples were then dehydrated via an ethanol series of 30, 50, 70, 80, 90, 95%, and twice with 100%, followed by two times in acetone. At each dehydration step, the samples were microwaved at 250 W at \u226437 \u00b0C for 40 s at 100% power. Infiltration was performed three times in Spurr low-viscosity resin  in the microwave at 450 W at \u226443 \u00b0C for 2 min at 100% power. Samples were polymerized in capsules in the microwave at 750 W at \u226460 \u00b0C for 19 min at 100% power, at \u226470 \u00b0C for 12 min at 100% power, at \u226480 \u00b0C for 12 min at 100% power, and at \u2264100 \u00b0C for 45 min at 100% power. Embedded tissues were sectioned to 100 nm thickness using a diamond knife and a Reichert Ultracut E ultramicrotome  and collected on copper grids that were coated with formvar and carbon . Grids were treated with 10% hydrogen peroxide for 10 min and stained with 10% uranyl acetate in methanol for 8 min, and then Reynold\u2019s lead citrate for 10 min [Microwave processing of the samples was adapted from ,57. Sampr 10 min . The samesb1-2, was used in this experiment [The method described in  was usedperiment . For eac3 on a hotplate at 110 \u00b0C for 8 h. The solution was diluted into deionized water gravimetrically to 10 g and analyzed by microwave plasma-mass spectrometry (MP-MS) (Agilent 4200 MPES) and inductively coupled plasma-mass spectrometry (ICP-MS) (Agilent 8800QQQ Triple Quadrupole ICP-MS). The elements Ca, K, Mg, and Na were measured by MP-ES, and the elements Li, B, Al, Si, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Mo, and Cd were measured by ICP-MS. Reproducibility due to sample heterogeneity and measurement uncertainty was evaluated using 10 mg replicate portions corresponding to the same homogenized plant specimen.Leaf tissue samples were oven dried at 85 \u00b0C for 48 h in 7 mL perfluoroalkoxy (PFA) vials until the sample weight reached a constant weight such that the samples were completely dehydrated. The dried leaves were ground to a fine powder using a mortar and pestle, and approximately 10 mg of each sample was weighed in a PFA vial, lightly closed, and digested with 0.5 mL of 15 M HNOt-test was conducted to determine if there was a significant difference between the means of two groups (either between control and treatment or wild type and mutant). Principal Component Analysis (PCA) was carried out to investigate the segregation of the leaf ionomic phenotype of the wild type and mutants using data obtained by ICP-MS.Statistical analyses were performed using SYSTAT Version 13 . Prior to analyses, if necessary, log transformation of data was carried out to attain a normal distribution. To evaluate whether drought or NaCl treatment had any effect on the measured traits, ANOVA tests were performed. Significant interactions between treatment (drought or NaCl) and genotypes enabled us to identify the percent changes in tested parameters among different genotypes. The Kruskal\u2013Wallis test was conducted when assumptions of normality were not met. Student\u2019s"}
+{"text": "Andrias davidianus is an important amphibian species in China because of its increasing economic value, protection status and special evolutionary position from aquatic to terrestrial animal. Its large genome presents challenges to genetic research. Genetic linkage mapping is an important tool for genome assembly and determination of phenotype-related loci.The Chinese giant salamander A. davidianus. The genetic length of LGs ranged from 17.61\u2009cM (LG30) to 280.81\u2009cM (LG1), with a mean inter-locus distance ranging from 0.11(LG3) to 0.48\u2009cM (LG26). The total genetic map length was 2643.10\u2009cM with an average inter-locus distance of 0.24\u2009cM. Three sex-related loci and four sex-related markers were found on LG6 and LG23, respectively.In this study, we constructed a high-density genetic linkage map using ddRAD sequencing technology to obtain SNP genotyping data of members from an full-sib family which sex had been determined. A total of 10,896 markers were grouped and oriented into 30 linkage groups, representing 30 chromosomes of We constructed the first High-density genetic linkage map and identified three sex-related loci in the Chinese giant salamander. Current results are expected to be a useful tool for future genomic studies aiming at the marker-assisted breeding of the species.The online version contains supplementary material available at 10.1186/s12864-021-07550-0. Andrias davidianus, historically widely distributed in China, is the world\u2019s largest extant amphibian. Due to its habitat destruction and hunting by man, it has been classified as endangered by the International Union for Conservation of Nature and Nature Resources since the 1980s [A. davidianus focused on the immune mechanisms [The Chinese giant salamander he 1980s , 3. Becahe 1980s . It is cchanisms \u20138, and ochanisms .Salmo salar [Cyprinus carpio [Paralichthys olivaceus [Megalobrama amblycephala [Larimichthys polyactis [Oreochromis niloticus [Ictalurus punctatus [Lates calcarifer [Rana temporaria [Hyla arborea [Lissotriton newts [Previous studies reported that the Chinese giant salamander has a huge genome (about 50 Gb) which was a big challenge to assemble , and to mo salar , 14, Cyps carpio , Paraliclivaceus , Megalobycephala , Larimicolyactis , 19, Oreiloticus , Ictalurunctatus , and Latlcarifer , all of mporaria , Hyla ar arborea , and Lison newts . HoweverA. davidianus without a genome, the CIM method was effective approach. In the previous study, we explored the female-specific marker to identify the genetic sex of the salamander which was important to detect the sex reversal salamander treated by high temperature and sex hormone in the study of sex determination mechanism [A. davidianus full-sib family to construct a high-density linkage map based on the ddRAD sequencing. Using the constructed linkage map. We identified three sex-related loci in different linkage groups. These results provide an effective genetic tool for A. davidianus genome assembly and marker-assisted breeding.Quantitative trait locus (QTL) mapping is an effective means to relate phenotype to the genome. In previous study, QTL mapping has been used in various species for different traits location including loci related to ecomic phenotype  and resiechanism , 33. we echanism , 35. StuNinety-seven RAD-seq libraries from two parents and 95 offspring were constructed and sequenced on an Illumina HiSeq X platform to generate raw reads. Based on the data trimming, 10.49 billion reads comprising \u223c1516.07 Gb of sequencing data were individually partitioned into RAD tags according to their multiplex identifiers (GenBank accession No. SRP155453). Finally, female and male parent datasets which contained 210,000,000 and 190,000,000 clean reads, respectively, were correspondingly partitioned into 1,887,649 and 6,378,461 RAD tags. A total of 1458.71 Gb of data  were produced and divided into 302,909,245 RAD tags  from the 95 offspring, for individual SNP discovery  per 41\u2009bp sequence was used to identify the SNP of the parents contained the RAD tags. A panel of 10,896 high-fidelity SNPs in both parents was identified, and alleles for each marker were assigned to their respective parent donor. The SNPs were analyzed across the 95 offspring with genotyping data and classified into three categories using an in-house script: maternal heterozygous (3343 SNPs), paternal heterozygous (6066 SNPs), and heterozygous in both (1487 SNPs) Table S.A. davidianus. In both the maternal and paternal map, a total of 10,896 segregating SNPs were successfully classified into 30 linkage groups which was consistent with the karyotype 2n\u2009=\u200960  to 280.81\u2009cM (LG1), and a mean inter-locus distance ranged from 0.11 (LG3) to 0.48\u2009cM (LG26). The maximum density of SNP marker in linkage group was LG3 containing 878 effective loci and minimum density was LG30 containing 105 effective loci  and LG11(qs-3). The likelihood of odd (LOD) of the loci were 5.8, 4.3, and 3.1, which explained 29.37, 12.00, and 6.78% of the phenotypic variation, respectively. Genotyping results of SNP 97.nn_np_297384, 99.nn_np_294732, 99.nn_np_278109, and 90.hk_hk_96246 in the sex-related loci were correlated with phenotype by the single-marker analysis of the WinQTL cart 2.5 . These results indicated the LD differ in maternal and paternal linkage groups.Thirty linkage groups were constructed with the average marker-interval of 0.24\u2009cM, which was the highest density linkage map in amphibians. In common frog, 107 SSR markers was distributed in 15 linkage groups with total length 1698.8\u2009cM . In LissA. davidianus sex-related QTL. Three loci were identified on the maternal map LG6  and LG11 (qs-3). The genetic distance of the QTL locus ranged from 0.1\u2009cM to 3.5\u2009cM suggested a narrow regions in the linkage group. This may provide valuable information for sex-related gene detection. While the same method was used to identify the loci on the maternal and paternal maps, all observed sex-related loci were on the maternal map. It is possible that some key sex-related markers were homozygous on the paternal map. Sex chromosomes have a greater number of repeat sequences, making the assembly of sex chromosomes and the construction of a sex linkage group more difficult, and sex is readily influenced by factors other than chromosome [A. davidianus. We analyzed sex related markers in this region and found correlation coefficient which ranged from 57.81 to 68.18% between phenotype and genotype in eight maternal SNP marker. Possibly due to low sequencing depth, some key SNPs in sex-related loci were not mapped on the linkage group. This region was considered as sex-related loci. So many SNPs in this region was related with sex too [A. davidianus through expanded single-linkage data in future. Further research requires expanded knowledge of the A. davidianus genome.The high-density linkage map provided the means of fine mapping romosome . Some seromosome , and 175romosome . In the A.davidianus. The genetic length of LGs ranged from 17.61\u2009cM (LG30) to 280.81\u2009cM (LG1), with a mean inter-locus distance ranging from 0.11(LG3) to 0.48\u2009cM (LG26). The total genetic map length was 2643.10\u2009cM with an average inter-locus distance of 0.24\u2009cM. At last, we found three sex related loci on the maternal map LG6  and LG11(qs-3). Genotyping results of four SNP markers  in the sex-related loci were significantly correlated with phenotype which indicated the four markers were sex-related markers. We constructed a High-density genetic linkage map and mapped 3 sex-related loci, which provides the linkage map is a useful tool for genomic study and provides a genetic basis for marker-assisted breeding of Chinese giant salamander.We constructed a high-density genetic linkage map using ddRAD sequencing technology to obtain SNP genotyping data from one full-sib family. A total of 10,896 markers were grouped and oriented into 30 linkage groups (LG), representing 30 chromosomes of A. davidianus including two parents (8 year old) and 95 salamander larvae  from one F3 full-sib family were collected from Zhejiang Yongqiang Chinese Giant Salamander Limited Company . The salamanders were soaked in 4\u2009L MS222 with the concentration of 0.5\u2009g/L for 10mins to anaesthetize. After all the salamanders were unconscious, the vertebra was broken from the neck, then the skin of the neck was removed, and muscle tissue was collected. All operations were carried out as per Yangyze River Fisheries Research Institute Care Committee (no. 2013001). The muscle tissue was collected from each salamander using the tissue DNA extracting kit  to extract genomic DNA following manufacturer\u2019s instruction. The concentration and quality of DNA were detected by Agilent 2100 Bioanalyzer  and 1% agarose gel electrophoresis. Genetic sex of each offspring was identified using a sex-specific marker explored by our lab [Total of 97 apparently healthy  our lab .The RAD library construction, sample indexing, and pooling followed the natural population , 45. TheP\u2009<\u20090.001. The linkage map was constructed using joinmap and Lep-Map. Joinmap was used to divide markers into groups using a grouping tree function in CP model with LOD\u2009=\u20098.0 [RAD-based SNPs were first tested against the expected segregation ratio at the beginning of the linkage analysis. Markers were filtered out by a chi-square test for separation ratio at the intersection sites with OD\u2009=\u20098.0  and Lep-OD\u2009=\u20098.0 . KosambiCIM method of WinQTL Cart 2.5 software was used to map the sex-related loci on the male and female linkage maps respectively . Model sAdditional file 1: Table S1. Original data of each sample for RAD sequencingAdditional file 2: Table S2. Genotyping information and location of each SNP on the 30 LGsAdditional file 3: Table S3 Distribution of each SNP on the 30 LGs"}
+{"text": "Transient technology seeks the development of materials, devices, or systems that undergo controlled degradation processes after a stable operation period, leaving behind harmless residues. To enable externally powered fully transient devices operating for longer periods compared to passive devices, transient batteries are needed. Albeit transient batteries are initially intended for biomedical applications, they represent an effective solution to circumvent the current contaminant leakage into the environment. Transient technology enables a more efficient recycling as it enhances material retrieval rates, limiting both human and environmental exposures to the hazardous pollutants present in conventional batteries. Little efforts are focused to catalog and understand the degradation characteristics of transient batteries. As the energy field is a property\u2010driven science, not only electrochemical performance but also their degradation behavior plays a pivotal role in defining the specific end\u2010use applications. The state\u2010of\u2010the\u2010art transient batteries are critically reviewed with special emphasis on the degradation mechanisms, transiency time, and biocompatibility of the released degradation products. The potential of transient batteries to change the current paradigm that considers batteries as harmful waste is highlighted. Overall, transient batteries are ready for takeoff and hold a promising future to be a frontrunner in the uptake of circular economy concepts. A critical discussion from the circular economy point of view about the degradation mechanisms, transiency time, and biocompatibility of transient batteries is provided. Although degradation processes may be random, to allow easy control of the transiency through external stimuli, the degradation of transient materials should preferably be activated by triggers such as pH, light, temperature, or the presence of specific gases/liquids. The time required for complete degradation can range from a few minutes, up to 45 days. Transient technology is rapidly gaining ground for biomedical applications, environmental sensors, or information\u2010sensitive hardware systems where they prevent access to data after application. Examples in the biomedicine include implanted medical devices (IMDs), skin\u2010patchable monitoring, wound healing systems, soft\u2010tissue sensing, or electroactive controlled release of drugs. In comparison with the chronic implants which are aimed to stay in the body permanently, transient medical devices can be implanted to diagnose/treat a disease and afterward disappear with no need of additional surgery for device retrieval, reducing potential risks, costs, and chronic inflammation caused by permanent devices. Such temporary devices are disintegrated, dissolved, resorbed, or degraded in a programmed fashion under relatively mild conditions at their end\u2010of\u2010life (EOL).Transient technology is a growing research area where materials, devices, or systems are able to undergo controlled degradation processes which ultimately result in their dissolution into the environment, leaving behind minimal or even nontraceable products after a period of stable operation.1 SimiFigure\u00a0 The device is usually present in the form of a 2D foil, where the active components are supported onto a mechanically flexible sheet, typically polymeric in nature. The material compositions are chosen based on both the performance and ability to degrade/dissolve with negligible associated harmful effects. Upon the application of an external trigger, a series of chemical reactions occur, and the device begins to degrade/dissolve. After a given time, all the components disappear, releasing degradation products into the medium. These products arise from the breakdown of the component materials into their respective constituents. While metals usually degrade into ionic species, polymers have the ability to degrade into soluble monomer units or oligomers (chains comprising few repeating units). As some of the biodegradation products may result toxic when exceeding certain concentrations, special care should be paid when designing transient devices. WEEE generation and management seriously jeopardize sustainability and it is considered as one of the foremost environmental issues in different countries. Additionally, the multicomponent character of WEEE comprising noble, ferrous/nonferrous metals, plastic, glass, and toxic chemicals, together with their mechanically/chemically durable nature seriously threatens human and environmental safety. In this scenario, many countries have implemented different policy approaches to face WEEE, where European Community Directive 2012/19/EU and RoHS Directive 2011/65/EU  are the flagship policies.Because of the new consumer trends, faster obsolescence, and the advent of the Internet\u2010of\u2010Things, the quantity of household and industrial electronics being consumed has continuously risen over the last years. This unprecedented increase in the use of electronic devices is leading to enormous amounts of disposed waste electrical and electronic equipment (WEEE) once electronic devices are discarded.16 WEE As a result, some materials can be only recycled up to 5 times before their quality decreases to the point where it can no longer be used. Moreover, despite the huge economical investment into the development and promotion of recycling, not all the regions worldwide have proper waste management policies and infrastructures. Also, collecting recyclable materials can be a real issue as they are not disposed correctly by consumers or there may be a lack of enough collection points. As a result, current recycling activities are not able to keep up with the pace of the global growth of WEEE, and only 17.4% of the generated WEEE in 2019 was properly collected and recycled. The whereabouts of the remaining surplus raises serious environmental concerns as a large fraction of such untraced material is probably mixed with other waste streams and directly disposed into oceans or landfills, seriously threatening the balance of our ecosystem. This is even more exacerbated in regions such as Africa, Oceania, or the Americas, which have WEEE recycling rates of 0.9%, 8.8%, and 9.4%, respectively. If new policies and technologies directed to face the large quantity of untraced WEEE are not properly applied, this dilemma could be aggravated in the near future as the global WEEE production is expected to increase from 7.3\u00a0kg per capita in 2019 to 9\u00a0kg by 2030.Recycling of electronic goods can be pursued as an alternative to the increasingly pressing issue of WEEE accumulation in the environment. However, due to the applied mechanical and thermal treatments during recycling together with the accumulation of a wide variety of elements, recycling often results in downcycling, yielding goods with poorer functionalities in comparison with the original material.19 As  That way, transient materials are incorporated into the earth's biogeochemical cycles through biodegradation, balancing the overall carbon cycle. When intended to be recycled, transient batteries can facilitate the process as the readily degradable character of the battery encasing avoids the first manual disassembly step. The chemical recycling process to extract nonmetal fractions is also simplified as straightforward processes involving aqueous extraction can be applied to many natural polymer electrolytes (as they present easily tunable solubility properties). Additionally, biopolymers such as lignin have been proven to efficiently recover Co2+ from a leaching solution through adsorption, avoiding the need of hydro\u2010 or pyrometallurgical approaches involving harsh experimental conditions. Interestingly, these materials can be then carbonized to yield electrodes that can be reused, providing novel cues toward upcycling. Overall, transiency offers new possibilities for recycling as the dissolution/degradation of a given component facilitates the recovery of other materials, reaching retrieval rates as high as 96%. Not only recycling strategies but also material scarcity and safety play a pivotal role toward sustainable battery design. Transient batteries contain cathodes based on nontoxic and relatively earth\u2010abundant elements such as zinc, molybdenum, or naturally occurring bioactive compounds such as melanin. This is in sharp contrast to the widespread use of critical raw materials such as cobalt in the energy storage field, which present serious issues in the supply chain. Therefore, transient batteries represent an opportunity to shift from the use of scarce materials to greener solutions, which is an urgent task as the global demand of cobalt is expected to increase by 60 times by 2030 considering the key role of batteries to store renewable energy and the accelerated electric vehicle adoption during the COVID\u201019 pandemic.Considering that the electrochemical energy storage systems represent the most hazardous components of WEEE, new strategies are required to manage discarded batteries. The application of biodegradable materials into transient electronic devices could bypass the environmental burdens associated with the often complex, expensive, and labor\u2010intensive collecting, mechanical sorting, and recycling of WEEE as they can be simply degraded in the environment with no harmful effects.14 Tha Due to tight dimension constraints (IMDs such as pacemakers have a preferred maximum volume of 1 cm3), the implementation of transient devices for biomedical applications has been limited. In principle, wireless power transfer technology can be applied in small transient IMDs as a means to transmit electrical energy through electromagnetic fields. Different IMDs have been powered operating in the megahertz region, reaching input powers of a few Watts. However, power transfer efficiency is below 50% (even below 3% in many cases), transmitter\u2013receiver distance is usually limited to 100\u00a0mm, and more importantly, the surrounding tissue of the IMD being powered may suffer undesired heating issues. Therefore, IMDs with directly connected batteries are preferred.Most of the efforts carried out till date have been devoted to transient electronics , providing good examples of the potential of transient technology.29 Due3) and a power density high enough to drive a cardiac pacemaker. This is in contrast to transient batteries that are not aimed for biomedical applications, where the less stringent dimension requirements allow battery volumes up to 512 mm3 (16\u00a0\u00d7 16 \u00d7 2\u00a0mm).The miniaturization of IMDs has been delayed because of the challenge to reduce the battery size, where a bulky and durable packaging is needed to avoid the contact of the toxic battery components with the body. In order to enable fully autonomous transient devices that do not rely on external power sources, the development of transient energy storage systems plays a pivotal role. Miniaturization of transient batteries, for example, could enable self\u2010powered bioimplants as shown by Wallace and co\u2010workers, who reported a bioimplantable magnesium\u2010based transient battery with a thickness of just 300\u00a0\u00b5m  in transient energy storage systems was investigated by Fu et\u00a0al., who reported a transient LIB with a capacity of 3 mAh cm\u22122 and a working voltage above 2.0\u00a0V, which was able to degrade under alkaline conditions in aqueous potassium hydroxide solution.Electrochemical energy storage systems in general, and batteries in particular, either primary (nonrechargeable) or secondary (rechargeable), are especially attractive as they outperform other energy storage systems in terms of energy density and energy conversion efficiency.35 To  As schematically summarized in Figure\u00a0 The separator, on the contrary, is not considered as an active part but serves to physically isolate both electrodes while enabling ionic conduction between them. The separator is usually found in the form of a porous solid membrane soaked with a liquid electrolyte, or as a gel. Current collectors enable the flow of the electrical current from the positive to the negative terminal through the device that is being powered. Finally, an external encapsulation is required to provide a protecting coating that avoids parasitic corrosion or damage of the other battery components.The fundamental operation of transient batteries is similar to durable batteries and relies on the conversion of chemical energy to electrical energy.36 As  sodium, aluminum, magnesium, or zinc\u2010ion chemistries are usually composed of chemically stable carbon nanomaterials/metals/metal oxides as electrodes and current collectors, nondegradable polymers as separators, organic liquids as electrolytes, and the full cell is protected by a metallic casing for high safety assurance. In this context, the potential environmental and human health effects of secondary batteries were studied using life cycle impact assessment, concluding that such batteries can be classified as hazardous due to their excessive contents of lead, cobalt, nickel, copper, chromium, thallium, and flammable electrolytes. Therefore, limiting both human and environmental exposures to these hazardous pollutants should be a priority, particularly in those regions of the world that lack adequate infrastructure for battery waste collection, sorting, and recycling.Conventional secondary batteries based on lithium,39 sod Another advantage of transient batteries is that they can be degraded \u201con\u2010site\u201d with no harmful effects, avoiding the need for often overly tedious, time\u2010consuming, and expensive waste collection and processing. As the choice of materials fulfilling these stringent requirements is mostly limited to degradable and nontoxic chemistries, most of the transient batteries reported so far suffer from low open\u2010circuit voltage (VOC), low power densities, and short lifetimes, failing to provide enough power for practical applications.Transient batteries relying on green materials may offer a promising alternative. This requires a major paradigm shift in the design of batteries. While conventional batteries are designed for being long\u2010lasting, degradable batteries are just the opposite. Active materials , the separator/electrolyte pair, and current collectors need to be rethought so they can be degraded once a predetermined external trigger is applied. Additionally, battery packaging/casing should be redesigned as its task of permanently protecting the battery cell from external influences is no longer given. Instead of simply isolating the anode, cathode, separator, and electrolyte from the surrounding medium, the encasing should allow interaction with the surrounding environment  when necessary. Interestingly, when batteries are intended for recycling, the degradation of the battery components can even help to recover the materials more efficiently.25 Ano By contrast, batteries intended for environmental applications are mainly aimed at reducing the contaminant accumulation in the environment from conventional batteries, so a partial disintegration is acceptable. In this context, a quick transiency (from seconds to few minutes) is preferred for batteries powering secured hardware to prevent unauthorized access, while batteries for consumer electronics survive for a longer period of time in the environment. However, to avoid waste accumulation into marine and land environments, these transiency times should ideally be below 1 year. To further compare such requirements, which in turn define the material selection and design, the different degradation and electrochemical characteristics of conventional batteries and transient batteries are listed in Table\u00a0It is clear that material choices and design considerations for transient batteries are sharply distinct from those required for conventional batteries . Depending on the intended application, full battery degradation may or may not be necessary. In the case of batteries powering biomedical devices such as cardiac pacemakers or electrocardiogram signal detectors, all the components should be completely degraded in few days into monomers (for polymeric parts) or ions  that could be absorbed or excreted by the body with no toxic response. The complete degradation is an essential requirement as the accumulation of certain battery components may result in undesired biological responses .46 By  Even if biodegradable/compostable materials cannot be considered as the ultimate remedy, such materials can support the transition to a circular economy as they create harmless secondary products , thus merging efficient resource utilization, value creation, and economic growth.Because of their degradability, transient batteries can provide new approaches toward a \u201ccircular economy,\u201d which establishes a framework for an economy that is restorative and regenerative by design (Ellen MacArthur Foundation). Although the circular economy is still an area open to debate, it is generally accepted that \u201cthe circular economy seeks to break away from the linear economy characterized by \u201cmake, use, dispose\u201d in favor of a more circular model based on \u201creuse, recycle, or biodegrade\u201d (Bio\u2010based Industries Consortium).\u201d Similarly, in the words of the European Compost Network, \u201cRecycling biodegradable wastes and resource efficiency lie at the heart of the circular economy.\u201d50 EveFigure\u00a0 but may not be desirable at all for devices intended to perform stimulation functions for short\u2010term usage, such as tissue regeneration or wound healing (a few weeks). In fact, to effectively remove the device once it has served its purpose, fast in vivo transiency rates are needed for other biomedical applications such as electrocardiogram signal detectors, which usually operate for just a few days. Apart from the biomedical applications, transient batteries can be also applied to reduce the environmental burdens caused by conventional electrochemical energy storage systems. In those ex vivo applications, the transiency characteristics are mainly driven by the time that the device should function. In this context, degradation under controlled composting aerobic conditions at 58\u00a0\u00b0C, 50% of humidity, and pH of 6.5\u20138  20200:2015 for plastic materials) is typically fast. By contrast, many compostable polymers are not degradable (or their kinetics are extremely slow) in marine , fresh water , or even landfill  environments.Apart from the electrochemical performance, one of the current bottlenecks for efficient transient batteries is their relatively slow transiency rates originating from the sluggish chemical reactions occurring between the battery constituents and the environment . However, as summarized in Figure\u00a052 butMoreover, addressing the potential impact of the transient batteries during their degradation under regulated conditions  and unregulated conditions such as in rivers or marine environments is of pivotal relevance toward the full understanding of how transient batteries could provide an environmentally respectful solution to our society. In this sense, there is an urgent need for a comprehensive understanding of the degradation mechanisms involving transient batteries as well as cataloging their degradation characteristics. Recognizing how transient batteries degrade and which are the resulting degradation products may help researchers to design and apply appropriate materials for transiency. few efforts have been devoted to addressing the transient energy storage devices. In particular, no works have summarized the degradation characteristics of transient batteries, which is of prime interest to exploit the full potential of transient devices. Accordingly, this work critically reviews the progress made on transient batteries, covering key aspects of their degradation in terms of the governing mechanisms, degradation kinetics, and composition of the products. The effect of the degradation products of the anode, cathode, separator, electrolyte, current collectors, and encapsulation on both the human body and environment are comprehensively addressed. Their assembly and electrochemical performance in terms of energy density, specific capacity, working voltage, Coulombic efficiency (CE), and lifetime are analyzed. Along with the biomedical applications, the potential of transient technology for implementation into a circular economy perspective is highlighted. Finally, we provide an outlook on the current challenges and future requirements for transient batteries.Although several works have reviewed the recent progress made on transient electronics,54 few2 It should be noted that not all the battery components need to be biodegradable, as fading by mere dissolution or disintegration is also enough to provide the transiency. During biodegradation processes, the starting material is transformed into H2O, CO2, CH4, and biomass, thus closing the loop back to nature.Depending on the application field, different definitions of biodegradability have been provided in the literature. From a transient electronics perspective, biodegradable can be assigned to a material that is able to decompose in a physiological environment  into constituents of lower molecular weight after a specified period of time.55 It Figure\u00a0 to 45 days  as shown in Figure\u00a0 To investigate the possible toxicity of the degradation products, other works conducted in vivo degradation studies  and current collectors. So far, transiency times for batteries ranging from 1\u00a0min Figure\u00a04a2]2Figurs Figure\u00a0\u2010left[8s Figure\u00a0.[12]2.1 However, natural biopolymers have also gained attention and are now applied in batteries as highly electrolyte\u2010wettable and thermally resistant separators, as binders in electrodes sustaining highly reversible electrochemical reactions, or as multifunctional membranes inhibiting the loss of electroactive species during cycling. These features contribute to an increased ionic conductivity, enhanced battery safety, improved cycling performance, and enhanced battery life span.Thanks to their physicochemical properties, low cost, and ease of processing, polymers are commonly applied as packaging and separator materials in transient batteries. Current batteries mainly rely on petroleum\u2010derived polymers as separator and electrode binder materials.57 HowFigure\u00a0 Naturally occurring polymers are further classified into plant\u2010derived polysaccharides  and animal\u2010derived materials . Natural polymers offer excellent properties such as biocompatibility, nontoxicity, easy availability, and affordability. Disadvantages of natural polymers include a marked batch\u2010to\u2010batch variation due to different sources of origin, a strong immunogenic response associated with their bioactivity, and they require complex and expensive purification procedures. Synthetic polymers, on the other hand, display predictable properties, batch\u2010to\u2010batch uniformity, and they can be tailored to provide the desired physicomechanical properties for specific applications.As schematically summarized in Figure\u00a059 NatAmong synthetic polymers, polyesters, polyanhydrides, and polycarbonates are the most widely applied degradable polymers in transient batteries. Some of the synthetic polymers commonly found in transient devices can be extracted from biomass as well. Obtaining synthetic polymers from biomass can contribute toward sustainability and ultimately toward the circular economy, thus fulfilling one of the goals of transience technology. One of the most prominent polymers in this sense is polylactide (PLA), which can be entirely produced from lactic acid (LA), a product extracted from the fermentation of agricultural products.64 The degradation depends on environmental conditions  and physicochemical properties of the polymer . Overall, four main biomaterial degradation mechanisms are found: hydrolytic degradation, enzymatic degradation, oxidative degradation, and physical degradation. Generally, naturally derived polymers are prone to undergo enzymatic or oxidative degradation, whereas synthetic biodegradable polymers are susceptible to hydrolytic degradation.Polymer degradation occurs when the average length of the main chain is reduced through the cleavage of chemical bonds.65 The Either way, the packaging is the first material suffering transiency as it shields the battery from the external environment. The transient properties of the packaging are directly related to the morphological, structural, and chemical features of the encapsulation. The thickness of the polymeric packaging, its molecular weight, and crystallinity degree are some of the key characteristics that can be easily modulated to achieve tailored transiency. As a matter of fact, whether surface or bulk erosion occurs is mainly related to the chemical nature of the polymers themselves, e.g., while PLA or polyglycolic acid (PGA) show a bulk degradation process, poly(ortho esters) are inherently surface\u2010eroding polymers. However, the geometry and shape of the polymeric components  can influence such degradation mechanism by determining the contact surface area and the prospective water diffusion kinetics into the bulk. Generally, thick films are degraded following a surface erosion mechanism, while a shift from surface to bulk erosion can occur provided the thickness drops below a critical thickness value. Moreover, in the case of bulk erosion, the thicker the sample, the faster the degradation as products autocatalyze degradation reactions when accumulated within the interior of the sample. In surface erosion, larger surface\u2010to\u2010volume ratios accelerate degradation reactions by exposing additional polymer chains to chain scission reactions. Additionally, larger molecular weights delay degradation kinetics as a result of the less available chain ends to undergo catalytic reactions, while polymers having large amorphous regions are more sensitive to hydration and in consequence to hydrolysis, boosting their transiency.Based on triggers such as water, light, temperature, or pH changes, fully transient electronics (full dissolution), and partially transient electronics  have been reported so far.54 Eit Regarding the separators, thicknesses from 50 to 300\u00a0\u00b5m are commonly observed in transient batteries as they offer a compromise between resistance against dendrites and ion diffusion resistance. The reaction kinetics of hydrolytic and enzymatic degradation as well as environmental or biological effects of the released products are also summarized in the next section.Hereunder, the most commonly found degradation mechanisms of polymers applied either as packaging materials or separators in transient batteries are discussed. The most common thicknesses of polymeric packaging are in the range of 40\u2013150\u00a0\u00b5m (to effectively protect the battery from surrounding medium), although packaging thicknesses reaching up to 500\u00a0\u00b5m have also been reported.2 Rega2.1.1 Different factors can yield hydrolyzable bonds. The formal charge of the reacting carbon markedly affects the reactivity of the polymer toward hydrolysis. During hydrolysis, the oxygen atom of H2O attacks the positively charged carbon atoms of the macromolecules via a 2nd order nucleophilic substitution reaction, and therefore chemical groups with a charge value above 0.3 electron charges are hydrolytically active. This is of particular relevance in the case of esters, amides, carbonates, carbamates, ureas, anhydrides, and orthoesters, which are vulnerable to hydrolysis as a result of their charge value >0.3.\u00a0Similarly, conjugated structures influence the reaction kinetics as their presence stabilizes the chemical groups, hindering bond scission. Additionally, steric effects can make hydrolyzable bonds less accessible to cleavage.Hydrolytic degradation is the main degradation mechanism of synthetic polymers (polyesters mostly) and is based on a water\u2010induced random scission of susceptible bonds.61 DifFigure\u00a0Mw is markedly reduced. Such Mw reduction yields soluble chains which can be expelled into the surrounding medium. This degradation process is characterized by a simultaneous Mw reduction and mass loss throughout the whole specimen. On the contrary, surface erosion consists of a controlled layer\u2010by\u2010layer degradation , resulting in a linear and controlled mass loss and Mw decreases over time. Water diffusion determines whether bulk degradation or surface erosion will occur. If diffusion is faster than the hydrolysis of surface chains, a bulk erosion process takes place. The polymer is thus saturated by the degrading medium , and a nonlinear mass loss occurs over time. During this process, degradation products (oligomers) containing hydroxyl and carboxylic acid groups are accumulated within the polymer matrix and consequently, the reaction is autocatalyzed. Conversely, when the hydrolysis of surface chains is fast, surface erosion occurs. As autocatalytic effects are suppressed due to the unrestricted diffusion of degradation products away from the polymer matrix, this process is generally expressed as a linear loss in mass over time. These two mechanisms are not independent and a combination of both may also occur.The determination of how the degradation of polymeric materials proceeds and controlling chain\u2010scission events is important for a rational design of transient batteries. As schematically shown in  PLA is the most prominent polyester which has found application in transient batteries as both encapsulating and separator material. For instance, a thick PLA film was used as the encasement for an electrochemical cell, together with a PLA spacer, to print the electrode pair and confine the NaCl/poly(\u03b5\u2010caprolactone) (PCL) composite inside the cell. PLA showed good tensile strength and remarkable compatibility with PCL. As shown in Table\u00a0 Subsequently, autocatalysis starts due to the increase in acidic conditions. The formed lactic acid oligomers diffuse through the material and dissolve in water. Diffusion is assisted by the plasticization effect of water, which increases the free volume. In this case, degradation happens faster in the bulk of the sample than on the outer layer. Amorphous PLA loses 50% of its original weight in 8 days at 70\u00a0\u00b0C and pH value of 5.4, with alkaline media and elevated temperatures favoring its degradation process. The as\u2010generated degradation products include LA, CO2, and H2O, which can be metabolized in the body or can be ejected through urine and breath, making PLA a suitable material for transient batteries aimed at biomedical applications. However, it should be taken into account that LA may cause severe inflammation of surrounding tissues due to its low pKa value (the logarithmic acid dissociation constant) of 3.08, highlighting that a complete understanding of the transiency products is crucial.Among synthetic polymers, polyesters, wherein repeating units are bonded via ester linkages, show ideal properties to be applied in transient batteries. Their high susceptibility to nucleophilic attack by hydroxide ions make them suitable alternative materials to traditional petroleum\u2010based nonbiodegradable polymers.69 PLAco\u2010glycolic acid) (PLGA), which is a copolymer of PLA and PGA, is one of the most remarkable examples. PLGA was used as an encapsulation material to develop a fully biodegradable primary magnesium\u2013molybdenum trioxide (Mg\u2013MoO3) battery system. Using Mg and MoO3 as the anode and cathode, respectively, and an alginate\u2010based hydrogel electrolyte, a battery lifetime of 13 days was achieved, which is larger than most of the reported transient batteries that last for a maximum of 4 days. Accordingly, this system seems appropriate for therapeutic stimulation functions that operate for a few weeks. Moreover, full in vivo and in vitro biodegradability was achieved. The degradation kinetics of PLGA films in phosphate\u2010buffered saline (PBS) solution revealed that as soon as the films were immersed in the liquid environment, water diffused throughout the samples to yield bulk chain scission events. As a result, PLGA was degraded into its LA and glycolic acid (GA) units, releasing acidic molecules into the solution which caused a pH decrease. These oligomers are eventually broken down to yield CO2 and H2O. The LA\u2010to\u2010GA ratio determined the degradation kinetics of PLGA. With increasing LA content, the degradation kinetics slowed down due to the hydrophobicity of LA and higher glass transition temperature which increased chain stiffness and reduced the susceptibility of ester groups to hydrolysis. A PLGA with a 50:50 LA\u2010to\u2010GA ratio showed a 20% weight loss after 10 days in PBS . PLGA shows excellent biocompatibility properties due to the easy assimilation and transformation of LA and GA by Krebs cycle.As some polymers do not display the mechanical or chemical characteristics required for transient batteries, copolymerization has been pursued as a fruitful strategy to upgrade their functional properties. Poly revealed that PCL barely loses 3.3% of its weight after 90 days.Taking advantage of the hydrolytic degradation of PCL without any toxic effects,78 a b Both in vivo and in vitro studies indicate that PGS undergoes surface degradation, which avoids a sudden release of degradation products and makes this polymer especially useful for biomedical applications. A PGS implant in the subcutaneous area of Sprague\u2010Dawley rats degrades fully within 60 days, while only 17.6% of its weight is lost in 60 days in PBS at 37\u00a0\u00b0C. As PGS is composed of naturally occurring glycerol and sebacic acid monomers, the human body can easily metabolize the degradation products. Besides, no catalysts or additives are necessary during PGS synthesis, avoiding possible toxic effects when intended for biomedical applications.Poly(glycerol sebacate) (PGS) is a simple glycerol\u2010ester\u2010based polymer that was applied as a substrate in degradable electronic devices. PGS\u2013cinnamate together with silver nanowires was applied as electrode material of an ingestible current source.11 Bot Polyanhydrides have a hydrophobic main chain linked by easily hydrolyzable anhydride groups which undergo degradation through a surface erosion process. Polyanhydrides together with PLGA were applied in a primary Mg\u2013MoO3 transient battery as coatings capable of being fully degraded within 48 h in moisturized environments. Polyanhydrides degrade in vitro and in vivo into their corresponding acids with no biologically adverse effects, demonstrating their high potential for biocompatible energy storage devices.In addition to polyesters, other classes of synthetic polymers have been applied in transient batteries. Water\u2010activated primary batteries with a Mg anode and Fe, W, or Mo cathodes were packed using polyanhydride.74 Pol2O5) cathode, a Li metal anode, a nonwoven PVP nanofiber separator, aluminum and copper current collectors, and a sodium alginate encasement. The highly porous structure of the PVP separator enables a rapid dissolution once the aqueous trigger is applied. Similarly, a fully degradable battery composed of a Sn\u2010doped V2O5 cathode, a Li metal anode, a PVP separator, and a PVA encapsulation was designed by Wang et\u00a0al. As PVA is highly soluble in water, the authors improved the stability of the battery by coating the PVA encapsulation with a thin polycarbonate layer, which is a water\u2010resistant polymer that rapidly dissolves when exposed to alkaline media. Therefore, when the battery was exposed to an aqueous potassium hydroxide (KOH) solution, a dissolution time for the whole transient battery of only 8\u00a0min was achieved. Based on a spray coating process, Fu et\u00a0al. also applied a waterproof polycarbonate layer onto the outer surfaces of the PVA encapsulation to develop a transient lithium pouch cell capable of withstanding corrosion by the surrounding aqueous environment.Water\u2010soluble polymers such as polyvinylpyrrolidone (PVP) and polyvinyl alcohol (PVA) are other frequently applied synthetic polymers in transient batteries. A transient battery capable of dissolving in water in solely 10\u00a0min was obtained based on a vanadium oxide , which is a salt in the liquid state at temperatures below 100\u00a0\u00b0C, forming a polyelectrolyte. A SF\u2013choline nitrate (SF\u2013[Ch][NO3]) polyelectrolyte was reported in a fully biodegradable thin\u2010film Mg battery by Jia et\u00a0al. As a result of the combination of SF with the biocompatible IL, the ion\u2010conducting membrane was degraded in a buffered protease XIV solution after 24 h with 89% weight loss. The presence of the IL favored the formation of the amorphous structure of SF, allowing a high transiency rate. Moreover, the good biocompatibility of SF makes this material an interesting platform to develop transient devices.Among animal\u2010derived biopolymers, silk fibroin  and chitosan (a linear polysaccharide extracted from crustaceans) have been applied in transient batteries, thanks to their facile processability, mechanical strength, and versatile functionalization, all of which are advantageous in comparison with other animal\u2010derived biopolymers.86 Biod\u2010glucosamine and N\u2010acetylglucosamine molecules connected by \u03b2\u2010(1\u21924) linkages, has been applied as a host material to obtain ionically conducting membranes. For example, chitosan was combined with [Ch][NO3] to obtain a biocompatible Mg\u2013air battery for implantable applications. Chitosan provides good mechanical support and dimensional stability, while [Ch][NO3] supplies charge carriers and acts as a plasticizer. As a result, a mechanically flexible and highly ionically conducting material was obtained. Although different enzymes can degrade the \u03b2\u2010(1\u21924) linkages, lysozyme is the most commonly found enzyme for chitosan degradation. However, special care should be paid to the chitosan deacetylation degree (DD) as DDs above 95% are not degradable by lysozymes. Gelatin is another biodegradable animal\u2010derived polymer with huge potential in transient devices. Gelatin is a collagen\u2010derived polymer and it has been extensively applied in food and pharmaceutical industries because of its good film\u2010forming properties, low price, nontoxicity, and biodegradable character. As a matter of fact, an edible and biodegradable electrochemical power source packaged within a gelatin capsule to facilitate oral delivery was reported. When the device is hydrated, the gelatin capsule is dissolved and allows hydration of the electrodes, which are deployed to contact each other and initiate the discharge of the electrochemical sodium ion cell .Chitosan, a linear polysaccharide formed by ications.32 Chi Cellulose is best degraded when exposed to cellulases, which hydrolyze \u03b2\u2010(1\u21924) glycosidic linkages to obtain glucose molecules. Sodium alginate, a polysaccharide that can be extracted from brown algae, was applied as a water\u2010soluble encapsulating material for a rechargeable lithium\u2010based transient battery capable of delivering high voltage and capacity. Despite its high solubility in water, sodium alginate shows good stability in conventional organic electrolytes, making this material suitable for transient batteries containing organic liquid electrolytes. When exposed to enzymatic environments, lyases cleave sodium alginate chains via a \u03b2\u2010elimination mechanism, resulting in biocompatible oligosaccharides.Cellulose and alginate are the most common plant\u2010derived polymers in transient batteries. Cellulose, which can be extracted from wood or cotton, has been used as a platform material to fabricate porous separators for a LIB capable of undergoing transiency in 30\u00a0min (when exposed to water) through a combination of dispersion of insoluble and dissolution of soluble components.56 Cel2.2The current collector is a critical component of batteries as it provides mechanical support to the electrode materials (cathode/anode) and collects electrons from them. In conventional batteries, the selection of active materials and current collectors is mainly based on their electrochemical stability and performance. These requirements become more stringent when it comes to transient batteries as these materials should not only show high electrochemical performance but also adequate degradability in a suitable fluidic solution. Moreover, for applications in the human body, these materials should exhibit biocompatibility and generate degradable products with minimum deleterious effects. Regarding nonbiological applications, the materials are expected to show stable performance during their operation period, and then disappear in their surroundings at controlled rates without releasing toxic products.Figure\u00a0 Typically used biodegradable metals in transient batteries include Mg, Mg\u2010based alloys, iron (Fe), molybdenum (Mo), zinc (Zn), and tungsten (W). All these metals except W are essential metallic elements for the human body with significant physiological roles. Metal oxides such as MoO3 and manganese dioxide (MnO2) were applied as cathode materials for biodegradable batteries due to their high solubility in aqueous solution, biocompatibility at controlled levels, and edibility. Metal oxides have been widely applied as electrode materials in conventional batteries , zinc\u2010ion batteries, etc.) due to their large theoretical capacities, capacity retention, and cyclability. Generally, metal oxides such as titanium, manganese, or zinc oxides are chemically and thermally stable, can be obtained through large\u2010scale manufacturing approaches, are abundant, affordable, and their working voltage and energy density can be tuned by morphology and chemistry design .Biodegradable metals, metal oxides, and organic\u2010based materials are the most commonly employed electrode materials for transient batteries designed to power IMDs  and aluminum (Al) for current collectors. These materials are chosen based on their electrochemical stability in organic electrolytes and fast dissolution behavior in alkaline solution formed by the reaction of Li metal with water. Among nonmetallic electrodes, eumelanins, a subclass of melanin pigments, and quinone redox species, were explored as anode materials for aqueous batteries. These biologically derived materials are well\u2010known to exhibit excellent in vitro and in vivo biocompatibility along with biodegradability via free radical degradation mechanism.Gold (Au) nanoparticles as electrode materials offer bioinertness and catalytic properties.8 AlteA complete understanding of the degradation mechanisms, kinetics, and electrochemical performance of the abovementioned materials is essential for developing more advanced and high performing transient batteries. The degradation behavior of materials depends on a multitude of factors ranging from solution chemistry to the redox environment. Additionally, it is critical to assess the impact of generated products on the immediate surroundings, either within the human body or in natural environments. In the following section, these key points involving the dissolution mechanism, kinetics, and potential biological or environmental impact of the active materials and current collectors proposed for transient batteries will be discussed.2.2.1 As a well\u2010known biodegradable metal, Mg is routinely found in structural implants and is the preferred choice as anode material in transient primary batteries due to its high theoretical specific charge capacity (2200 mAh g\u22121), high physiological tolerance (300 mg per day), biocompatibility, and appreciable negative electrode potential (\u22122.3\u00a0V vs standard hydrogen electrode). Mg degrades in aqueous solution to produce hydrogen gas and magnesium hydroxide (Mg(OH)2), a biocompatible corrosion product shown to enhance bone growth in vivo. The degradation behavior of Mg is influenced by a variety of factors including the composition of degrading media, temperature, and the shape/form in which Mg is conformed in the batteries. Different shapes of Mg used in transient batteries include thick Mg films micropatterned by subtractive etching of Mg foil, electrodeposited Mg microstructures from organic solvents, and foil format. They all differ from each other in terms of crystal orientation, electrical resistivity, surface morphology and grain size that eventually dictate their degradation behavior. Tsang et\u00a0al. studied the degradation behavior of electroplated Mg in PBS  and revealed its lower corrosion resistance in comparison to commercial Mg foil, most likely due to its larger grain size and high surface roughness. The same group used electroplated Mg as anode for PCL\u2010encapsulated Mg\u2013Fe biodegradable batteries and demonstrated its complete dissolution within 20 days in PBS solution , agitated at 50\u00a0rpm to mimic body conditions. Thick foil format of Mg as anode and current collector was utilized in fully transient Mg\u2013Mo and Mg\u2013MoO3 battery systems. In Mg\u2013Mo primary batteries, the dissolution of Mg and Mo foils together with a polyanhydride encasing took place slowly in PBS solution at 37\u00a0\u00b0C for the initial 11 days and then completely disappeared in the next 8 days as the temperature of the solution was increased to 85\u00a0\u00b0C. Conversely, the degradation of Mg\u2013MoO3 proceeded faster as the Mg foil, the sodium alginate hydrogel, and the MoO3\u2013PLGA layer completely disappeared in PBS solution  within 9 days. These different transiency times are related to the different dissolution rates of Mg and Mo foils, reported to be 1\u201310 and 0.02 \u00b5m per day, respectively.Most of the efforts reported to date have been devoted to the development of nonrechargeable (primary) batteries. Therefore, if not stated otherwise, the batteries reported here are intended for single use. The general mode of degradation of biodegradable metals is through a corrosion process. As summarized in Figure\u00a0103 As A dissolution rate of 0.05\u20130.5 \u00b5m h\u22121 was observed for a Mg thin film in SBFs , while a AZ31 thin film dissolved at a rate of 0.02\u20130.1 \u00b5m h\u22121 due to its improved corrosion resistance. Khan et\u00a0al. developed a Mg\u2013Zn anode system for transient Mg primary batteries via combinatorial magnetron cosputtering. The rationale behind their study was to identify the optimum combination in the Mg\u2013Zn system that would provide higher electrochemical performance and longer lifetime. The corrosion resistance of Mg was found to be improved with increasing Zn concentration; however, no degradation experiments were reported in the paper. A new biodegradable anode consisting of Mg\u2013Zn (3\u00a0wt%)\u2013Zr (0.8\u00a0wt%) (MZZ) alloy coated with biocompatible \u03b2\u2010tricalcium phosphate nanorods (\u03b2\u2010TCP) was also developed. The degradation rate of MZZ and \u03b2\u2010TCP\u2010coated MZZ alloy was monitored by measuring the weight loss in both samples after long\u2010term immersion in SBF at 37\u00a0\u00b0C. The average corrosion rate of \u03b2\u2010TCP\u2010coated MZZ alloy (0.365 \u00b5m h\u22121) was found to be slower than that of MZZ alloy (0.62 \u00b5m h\u22121), thus confirming the protective nature of the coating. Structural analysis showed the presence of many corrosion pits on the surface of MZZ alloy, while \u03b2\u2010TCP\u2010coated MZZ alloy maintained its structural integrity without any dramatic change. Tsang et\u00a0al. minimized the parasitic corrosion of Mg anode by passivating its surface with biodegradable polymers, i.e., PCL and PGS for microelectromechanical\u2010system\u2010enabled biodegradable batteries. However, no dissolution experiments were conducted to show the effect of these polymers on the overall dissolution behavior of Mg under physiological conditions.Despite the attractive properties of Mg as anode, its rapid corrosion in aqueous solution and high self\u2010discharge rates have limited its application. To obtain longer degradation times and better performance for transient Mg\u2010based batteries, alloying of Mg with biocompatible metals and surface coating methods were developed. AZ31 Mg alloy containing 3% Al and 1% Zn by weight was used as anode material in primary Mg batteries, increasing battery lifetime by 6 times compared to pure Mg.106 A  The dissolution rate of Fe foil in PBS solution  is reported to be 0.0034 \u00b5m h\u22121, which is much slower than the corresponding thin film in Hank's solution . The difference in dissolution rates of metal foil and thin film is attributed to their morphological differences and different compositions of the two solutions. Preliminary in vivo tests in the native descending aorta of pigs  demonstrated excellent biocompatibility of pure Fe in the form of stents with no significant neointimal proliferation, no pronounced inflammatory response, and no organ toxicity. Like Fe, Mo foil also has a slower dissolution rate of 0.00083 \u00b5m h\u22121 in PBS solution . But unlike iron, the anodic oxidation of Mo in nearly neutral electrolytes yields a soluble product, mainly a mixed\u2010valence oxide containing Mo(IV), Mo(V), and Mo(VI). The exact ratio between different valence states depends on the pH of the degradation medium, and the solubility of this mixed\u2010valence oxide determines the overall degradation kinetics of Mo. Au nanoparticles (NPs) deposited on biodegradable silk film were applied as a bioinert catalyst toward oxygen reduction reaction for an encapsulated Mg\u2013air battery. The biodegradation process of the battery was conducted in buffered protease solution at 37\u00a0\u00b0C wherein Au NPs physically fragmented in the solution due to the dissolution of the supporting substrate. These NPs were reported to be biocompatible and under optimized enzyme treatment, can be eliminated from the body through renal excretion, phagocytosis, and/or endocytosis.Among the cathode materials for transient batteries, Fe is most widely explored due to its advantageous mechanical and electrochemical properties. Degradation of Fe in biofluids and distilled water occurs very slowly and in a nonuniform manner due to the formation of insoluble corrosion products (oxides) that accumulate on the surface, preventing further corrosion.107 Th2.2.23 is a layered material that is being explored for numerous applications including medical devices, lithium\u2010ion battery cathodes, chemotherapy agents, and heterogeneous catalysts. Huang et\u00a0al. first reported the potential of MoO3 as the cathode for a fully biodegradable primary Mg\u2013MoO3 battery. A thick slurry of MoO3 powder mixed with a biodegradable polymer, PLGA, was cast on top of a Mo foil to obtain a well\u2010connected 3D network structure. This 3D network structure promoted battery performance due to the increase in effective surface area as well as increased conductivity of the MoO3 layer. The transiency of the entire battery was observed in PBS solution at 37\u00a0\u00b0C. The dissolution behavior of MoO3 was found to be controlled by the encapsulation layers, representing an advantage to obtain a desired release rate of Mo in the degradation medium. Being able to control the molybdenum concentration in the solution/electrolyte is essential to achieve maximum cell viability. A concentration of 7\u00a0mol% MoO3 is compatible with human immortalized keratinocyte (HaCaT) cell line, while higher concentrations showed slightly reduced cell viability. The in vitro results indicated that the MoO3/PLGA film did not present any toxic effects on L\u2010929 mouse fibroblast cells and exhibited excellent biocompatibility. The presence of MoO3 indeed resulted in increased growth ability of the L\u2010929 cells. The understanding of the dissolution kinetics of MoO3 is critical in determining its fate in the environment and within the human body. The dissolution of MoO3 is a slow hydrolysis process, resulting in the formation of molybdate anions as the dominant degradation product following the reaction as shown in Equation\u00a0 was reported. An increase in dissolution rate was observed with increasing pH (maximum at pH 9.25\u00a0\u00b1 0.2) and temperature (0.0001 min\u22121 at 0\u00a0\u00b0C compared to 1.09 min\u22121 at 40\u00a0\u00b0C). Below pH 2, MoO3 is shown to be stable against hydrolysis. Recent work demonstrated the dissolution kinetics of MoO3 nanoribbons in six different degrading media including Nanopure water (pH 7.0), U.S. Environmental Protection Agency moderately hard water (pH 7.8), phosphate\u2010buffered saline (pH 7.4), Roswell Park Memorial Institute Medium , simulated lung fluid , and phagolysosomal simulant fluid . The nanoribbons showed complete dissolution in buffered media, i.e., RPMI, PBS, and SLF in which most of the dissolution generated H+ ions , which is within the tolerance limit of the human body (70\u201388 \u00b5g per day). Ag nanowires eventually oxidize, corrode, and resorb by the body without any in vitro toxicity. 8\u00a0mg of MnO2 was used for optimum performance, while the recommended daily allowance of MnO2 is 11\u00a0mg for adults. Higher doses (100\u2013250 \u00b5g mL\u22121) of MnO2 particles (size 1\u20132\u00a0\u00b5m) have elicited adverse responses such as lactate dehydrogenase leakage during in vitro toxicity evaluation in rat liver cells. Regarding the dissolution behavior, MnO2 is highly insoluble in water, undergoing reductive dissolution with the release of soluble Mn2+ ions in the presence of electron donors. The reductive dissolution of MnO2 is a surface\u2010controlled process and the reduction rate shows strong pH dependence with increasing solubility under acidic conditions. Chen et\u00a0al. demonstrated a unique breakup nature of MnO2 nanosheets under mildly acidic conditions and employed this dissolution behavior for drug delivery and ultrasensitive pH\u2010responsive magnetic resonance imaging applications. The dissolution rates of Mn oxides also show a positive correlation with specific surface area and reduction potential. In environmental settings, Mn oxides readily undergo cation exchange reactions and are thus applied for adsorptive removal of heavy metal pollutants. This property of Mn oxides can be utilized in transient batteries for environmental resorption as the release of these oxides at controlled rates during the degradation process would benefit the environment in the remediation of contaminants in soil and water treatment applications.Another interesting metal oxide that has found its way into transient sodium\u2010ion batteries is MnOpacitors.120 Mn The transient rechargeable LIB consisted of V2O5 as cathode and Li metal as anode along with a biodegradable separator and thin films of Cu and Al deposited onto a sodium alginate substrate as current collectors. V2O5 is a layered material that has attracted great attention as cathode material for LIBs due to its high theoretical capacity of 294 mAh g\u22121, low price, and abundant sources. Its choice as the cathode material is mainly ascribed to its dissolution behavior in alkali solution as given by the following reactions in lithium hydroxide (LiOH) solutionFu et\u00a0al. reported the first rechargeable transient battery based on mature Li\u2010ion technology as an extension of transience technology to more advanced batteries.2 The 2+ and VO(OH)+) is favored. During the battery dissolution process, basic pH was attained after Li metal, serving as anode for transient LIBs, reacted with water to form LiOH solution. This basic environment also triggered the dissolution of the Al current collector to produce LiAl(OH)4 as the degradation product. Cu metal did not dissolve at high pH values, but it was found to disintegrate into smaller pieces due to the dissolution of its supporting substrate, sodium alginate, in water. Although this battery presented a novel degradation mechanism, there are several possibilities to further improve the electrochemical performance. To achieve high areal energy density, a transient LIB was fabricated using LiAl alloy as anode, and an origami\u2010inspired high\u2010capacity V2O5 as cathode. Just like Li metal, LiAl alloy also exhibited fast dissolution behavior in 1.5 m KOH solution with a transience time of only 2\u00a0min. The origami\u2010inspired cathode not only provided high areal energy density due to its folded design but also facilitated an increased rate of transience. Further improvement in the electrochemical performance of pure V2O5\u2010cathode\u2010based transient batteries was achieved by doping tin into V2O5 nanofibers, forming a porous freestanding electrode with a 3D network. The Sn\u2010doped cathode offered a high areal capacity of \u22482 mAh cm\u22122 and upon immersion into concentrated alkali stimuli, V2O5 fully dissolved into a soluble salt, and the Sn ions formed water\u2010soluble SnO32\u2212. Although V2O5 is extensively found in transient secondary batteries, it is worth mentioning that many biological response studies demonstrated notable toxicity of this oxide. As a matter of fact, micrometer\u2010sized V2O5 was included in the Environmental Protection Agency (EPA) \u201cP\u2010list\u201d of acutely hazardous chemicals. Bulk V2O5 has been reported to be genotoxic, destroyed liver architecture in male guinea pigs, and occupational exposure of workers to vanadium oxide resulted in rhinitis, bronchitis, and pneumonitis. The unregulated release of such oxides into the environment during the degradation process of batteries could lead to toxic effects. More recently, a transient primary LIB was developed using biodegradable PVA, cellulose, and active materials such as lithium cobalt oxide (LiCoO2) and lithium titanate (Li4Ti5O12). The transient behavior of this battery is based on a very interesting approach using chemical dissolution of soluble components such as PVA, cellulose and physical redispersion of insoluble materials, i.e., active materials and carbon black. The detailed degradation behavior of these polymers has been explained in the previous section.In alkaline solution, vanadium exists predominantly in +5 oxidation state, whereas at acidic pH, +4 valence state as vanadyl cations , the insoluble dark\u2010brown pigments, were tested in aqueous NIBs due to their unique physical and chemical properties including reversible cation binding abilities. The full cell was fabricated by pairing eumelanin anode with the \u03bb\u2010MnO2 cathode and this full cell provided a lifetime of 5 h when operated at a discharge current of 10 \u00b5A, much longer than the conventional batteries used in ingestible devices. Although no degradation studies were reported in this work, other literature studies provide information on biocompatibility and biodegradation behavior of melanins. In vitro and in vivo biocompatibility of thin melanin films by examining Schwann cell attachment and growth, as well as neurite extension in PC12 cells, were delineated by Bettinger et\u00a0al. The results pointed to enhanced Schwann cell growth and neurite extension in PC12 cells by melanin thin films, thus confirming its potential as a biodegradable material. Moreover, melanin implants placed near peripheral nerve tissue in Sprague\u2010Dawley rats were observed to be degradable in vivo with only small fragments remaining after 8 weeks. Chemical degradation of melanin pigments by oxidation with permanganate or hydrogen peroxide, breakdown with hydrogen iodide, and ultraviolet\u2010induced photodegradation have also been thoroughly studied.As a promising alternative to conventional inorganic materials, organic compounds have been studied as electrode materials for biodegradable energy storage devices because of their intrinsic advantages like easy fabrication, mechanical flexibility, structural diversity, and acceptable theoretical capacity.75 Mel However, it should be taken into account that in spite of their biodegradability, degradation products from quinones show acute toxic response in aquatic organisms and can cause cancer in humans.Quinones, cyclic compounds containing two carbonyl groups in an unsaturated six\u2010membered ring structure, can be potentially used as electrodes in degradable redox flow batteries, thanks to their good solubility, suitable redox potential, scalability, biodegradability, and low cost.138 Ho3Table\u00a0Figure\u00a0\u22121, open\u2010circuit voltage (VOC) of 0.4\u20130.7\u00a0V, and a lifetime of 24 h (limited by the depletion of the active Mg) was reported in PBS liquid electrolyte. The low output voltage delivered by the battery was increased by connecting the cells in series to obtain a stable voltage of 1.5\u20131.6\u00a0V for up to 6 h, which is enough to power a light\u2010emitting diode (LED) and a wireless radio circuit. To extend battery lifetime and improve VOC, Jia et\u00a0al. replaced the liquid electrolyte with a chitosan\u2013choline nitrate gel polymer electrolyte (GPE). As shown in Figure\u00a0VOC of 1.33\u00a0V (middle point of the discharge curve) for 160 h when cycled at a current density of 10 \u00b5A cm\u22122, which is 40\u00a0mV above the VOC of the batteries with the liquid electrolyte. However, at higher current density, the obtained voltage was 60\u00a0mV lower than that of liquid electrolyte due to slower ion mobility inside the GPE. Nonetheless, the biocompatible ionic liquid\u2013biopolymer electrolyte enabled a volumetric power density of 3.9 W L\u22121 for the Mg\u2013air battery, which is adequate to power certain IMDs such as pacemakers or biomonitoring systems.In addition to the degradation mechanism and kinetics, the ability to store energy is of paramount relevance for transient batteries to reach commercialization stage. In this section, we summarize the electrochemical performance of primary and secondary transient batteries . As a result, the extent of hydrogen evolution originating from the reduction of water upon corrosion is reduced. Further, the PCL dip\u2010coated Mg half\u2010cells showed a 70% increase in specific capacity at 280 \u00b5A cm\u22122 in comparison to the noncoated samples, delivering a maximum specific capacity of 930 mAh g\u22121 at 330 \u00b5A cm\u22122. For full cells, the PCL\u2010coated Mg\u2013Fe batteries managed to achieve a stable discharge voltage at current densities as high as 400 \u00b5A cm\u22122 and featured a high energy density of 694 Wh kg\u22121, which is two orders of magnitude higher than that for Mg\u2013Mo batteries. As both thickness and permeability of polymeric coatings influence the mass transfer resistance, the effect of varying the thickness of PCL and PGS on the electrochemical performance of the batteries was studied. The PGS\u2010coated batteries achieved longer discharge lifetimes than uncoated ones, showing the highest capacity and CE of 0.7 mA h\u22121 and 13.5%, respectively (film thickness of 10\u00a0\u00b5m). As PGS thickness increases, lower average potential and less stable discharge profiles were obtained due to the increased charge transfer resistance and accumulation of reaction products at the PGS\u2013Mg interface. PCL\u2010coated batteries showed similar thickness dependence on the battery performance. In addition to the encapsulation layer, a solid electrolyte for Mg\u2013Fe batteries comprising PCL and NaCl was fabricated to maintain the stable electrochemical environment inside the cell, making the battery cell immune to the continuously changing surrounding environment. Operating voltages of 0.45 and 0.95\u00a0V were obtained for discharge rates of 100 and 12.5 \u00b5A cm\u22122, respectively. In another work, a Mg\u2013Fe battery featuring electroplated Mg as anode exhibited a capacity and power of 1.2 mAh and 36 \u03bcW at a current of 55 \u00b5A, respectively. A similar surface coating strategy was pursued with \u03b2\u2010tricalcium phosphate nanorods on a biodegradable Mg alloy (MZZ). At a current density of 100 \u00b5A cm\u22122, the battery with \u03b2\u2010TCP\u2013MZZ alloy showed a plateau voltage of 1.05\u00a0V for 1800 h in comparison to only 625 h for the noncoated MZZ alloy . The full battery delivered a capacity of 2.2 mAh cm\u22122 with a plateau voltage of 1.03\u00a0V at a current density of 5 \u00b5A cm\u22122. Interestingly, as indicated by the discharge curves in Figure\u00a0Jia et\u00a0al. demonstrated a silk\u2010based compact Mg battery using an anode composed of AZ31 Mg alloy, Au NPs deposited onto a crystallized silk film as a cathode, and SF\u2013choline nitrate as a polymer electrolyte.8 The 3 battery with a VOC of 1.6\u00a0V, battery lifetime of 50 h delivering 1.5\u00a0V (0.6\u00a0V for 250 h), and areal energy density of 6.5 mWh cm\u22122 was developed according to the requirements for implantable electronics. The output voltage achieved for a single Mg\u2013MoO3 cell could power a LED in PBS solution for up to 16 h . Such a primary battery was able to power a water\u2010monitoring device, although it suffered from a low Coulombic efficiency of 13.3%. The same quinone chemistry has been recently applied in a paper\u2010based flow battery. Efficiencies as high as 98% were achieved by tuning the size of electrode and flow rate of the battery. The primary battery featured a lifetime of about 30\u00a0min and a cell energy density of 3.1 Wh L\u22121 cm\u22122 for an electrode length of 20\u00a0mm.A biocompatible NIB based on organic electrodes, i.e., melanin\u2010based anode together with  The battery presented a working voltage of 2.8\u00a0V and was able to deliver an energy of 0.29 mWh (energy density of 480 Wh kg\u22121). As shown in Figure\u00a0\u22121 and CE of 99%. A step forward in this direction came from the development of an origami V2O5\u2010cathode\u2010based LIB, which showed a stable cycling performance for up to 20 cycles  and a high working voltage above 2.0\u00a0V, comparable to conventional LIBs. To further enhance the battery performance, metal ion doping was used to synthesize a Sn\u2010doped V2O5 cathode. This battery provided 0.27 mAh cm\u22122 capacity at a current density as high as 17.76 mA cm\u22122. The transient battery with the Sn\u2010doped V2O5 cathode had a capacity of 2 mAh cm\u22122 at an areal current density of 0.3 mA cm\u22122 compared to 1.2 mAh cm\u22122 for the pure V2O5 cathode. The high performance of the doped cathode was ascribed to its better electronic conductivity and electrochemical reversibility. Finally, a transient LIB battery based on a LiCoO2 (LCO) cathode and a Li4Ti5O12 (LTO) anode generated a total specific capacity of 2.27 mAh g\u22121 and a CE of 12.5% when discharged at 20 \u00b5A cm\u22122. The battery was able to power a 1\u00a0V calculator for \u224815\u00a0min. However, the transient LIB presented a relatively poor electrochemical performance due to nonuniform interfaces and poor connections between the battery components.Regarding secondary batteries, the first transient LIB was shown in 2015 by Fu et\u00a0al. through a combination of cut\u2010and\u2010stack and shadow mask metal deposition techniques.2 The s Figure\u00a0.[3]  For example, batteries delivering 30\u2013100 \u03bcW are sufficient for pacemakers and cardiac defibrillators , while neurostimulators (operation time of a few months) demand power in the range of a few microwatts to milliwatts, and 100\u20131000 \u03bcW are necessary to power drug pumps (lifetime of a few hours). However, further research is needed to boost the capacities and operation times of transient batteries so that they can power more sophisticated devices similar to conventional batteries, e.g., cochlear implants , retinal stimulators (250\u00a0mW), actuators (\u224850\u2013100\u00a0mW), or portable electronic devices (0.5\u201312 W), which usually operate in the potential range of 1.5\u20133.0\u00a0V.The power provided by transient batteries reported so far is enough to support ultralow power IMDs and biosensors, which require output voltages from 0.3 to 1.5\u00a0V.143 Fo4Figure\u00a0\u22122, specific energy densities up to 4.70 mWh cm\u22122, and working voltages above 2.0\u00a0V have been obtained so far, many of the reported transient batteries suffer from relatively low power density and short lifetime.While substantial efforts have been devoted to the development of transient electronics in general, transient batteries remain relatively unexplored. It is thus essential that researchers also focus their attention on transient energy storage devices, where technology interacts with nature to leave no permanent footprint. lifetime.12 On the other hand, synthetic biodegradable materials such as polyesters present easy tunability of their degradation profile. However, their biocompatibility should be examined from the raw materials to the final product considering all processing steps. Interestingly, besides their biodegradability, many of those polymers are also biosourced, meaning that they are obtained from renewable resources . Polymers that undergo selective depolymerization back to their initial constituent's feedstock may provide novel approaches for the valorization of transient batteries. This may reduce the environmental footprint related to the intensive use of fossil\u2010based raw materials while limiting greenhouse gas emissions.Given the diverse fields of potential applications, transient batteries must meet many requirements, ranging from negligible toxicity of the degradation products, when intended for biomedicine, to a rapid transiency for security devices. To date, water\u2010soluble polymers such as PVA, biodegradable polymers such as PLA, PCL, polyanhydride, or biodegradable and renewable polymers including silk fibroin, cellulose, gelatin, chitosan, and sodium alginate have been applied in transient batteries. Plant\u2010based polysaccharides  and animal\u2010based polymers  are enzymatically degradable, although their batch\u2010to\u2010batch variability makes their degradation slightly unpredictable.151 On body fluids such as gastric juice, urine, saliva, blood, or sweat have been directly applied as electrolytes. As schematically shown in Figure\u00a0 Because of its abundant polar \u2014OH groups, cellulose is usually applied as the separator, which upon contact with fluids acts as battery electrolyte. Generally, these batteries cannot be recharged as they stop functioning as soon as the body fluids acting as electrolytes get exhausted. One of the earliest attempts using body\u2010fluid\u2010based electrolytes instead of the conventional flammable organic or corrosive electrolytes was reported by Lee in 2005, who built a human\u2010urine\u2010activated microbattery composed of a magnesium anode and a copper\u2010chloride\u2010doped paper cathode. When the urine is dropped onto the paper between the Mg and CuCl layers, the CuCl reacts to form MgCl2 and Cu, delivering a total power of 1.5\u00a0mW with a maximum operating voltage of 1.4\u00a0V, which is enough to drive urine screening on\u2010board biosensors. The battery could be reactivated again upon urine soaking. More recently, a sweat\u2010activated primary battery was assembled using a Mg anode, a Ag/AgCl cathode, and a cellulosic separator encased within an elastomeric microfluidic system with multiple outlets to expel excess sweat, allow fresh sweat to enter, and permit the release of hydrogen gas as a product. With ionic conductivity values ranging from 1 to 10 mS cm\u22121, the sweat, containing naturally excreted dissolved salts, provides the adequate aqueous electrolytic conditions for closing the circuit. However, the separator of this battery required an impregnation step with NaCl to ensure enough ionic conductivity due to changes of the sweat composition. A maximum operating voltage of 1.8\u00a0V was obtained, resulting in a specific capacity of 67 Ah kg\u22121 and capable of operating for 5 h. As a representative example of a transient battery powering an in vivo device, a gastric\u2010fluid\u2010activated biocompatible battery comprising a Zn anode and a Cu cathode was reported. During operation, Zn undergoes galvanic oxidation and the inert Cu cathode returns the electrons to the solution. The cell delivered an average power of 0.23 \u03bcW mm\u22122 for 6.1 days to support a temperature sensing and wireless communication system located within the gastrointestinal tract of pigs.To avoid toxicity issues arising from electrolyte leakage of batteries using organic electrolytes,154 bo As shown in Figure\u00a00.44MnO2\u00a0cathode, a NaTi2(PO4)3@C anode, and a microporous polyacrylonitrile separator were used. The battery showed a specific capacity of 41 and 40 mAh g\u22121 at a current density of 0.2 A g\u22121 for normal saline or cell\u2010culture medium, respectively  or a cell\u2010culture medium mimicking the fluid present around cells in the human body .159 As+ concentration in sweat depends on the sweat rate, thus influencing the ionic conductivity and the battery performance. Therefore, it is not at all trivial to design a transient battery that can ensure a constant electrochemical environment inside the cell, especially if the battery is located within a constantly changing human body. ILs, nonvolatile room\u2010temperature molten salts, represent other sustainable electrolyte choices as they are highly conducting and present good biocompatibility.Even though the use of internal biofluids as battery electrolytes is an interesting strategy, it should be considered that the constantly changing nature of biofluids can have undesirable effects on the resulting electrochemical performance. The pH, chemical composition, and viscosity of gastric fluids can vary throughout the day, while the Naeat rate,160 th2O to form LiOH) metals/metal oxides, or biologically derived compounds  are found to be suitable as electrodes because of their capability to reversibly bind/host different ions. Alloy composition and microstructure design may help to obtain electrodes with limited parasitic corrosion. Although pure metals and metal oxides are the most common materials to develop batteries, primary batteries with continuous operation for 100\u00a0min using solely organic materials are possible.Additionally, bioresorbable (magnesium), biocompatible , or dissoluble 2 meta With an output voltage and power of 3.0\u00a0V and 2.8\u00a0mW, respectively, the battery could be disposed in an organic waste container. After 60 days, 54\u00a0\u00b1 4% of the battery weight was biotically degraded under standardized anaerobic conditions (the worst\u2010case scenario for biodegradation).In comparison to the many efforts carried out to develop transient batteries for the biomedical field, scarce attempts have been made to develop high\u2010power biodegradable batteries for environmental applications. In this regard, Esquivel et\u00a0al. developed a biodegradable flow battery based on cellulose, carbon paper, beeswax, and organic redox species.141 Wi As conventional batteries usually require a high\u2010tech treatment for effective recycling, transient batteries can facilitate material recovery, enabling cheaper and more efficient recycling processes. In a complementary manner, transient batteries can provide a suitable solution to the accumulation of dangerous electronic waste that is dumped inadequately, significantly improving environment and people's health in the future.Intense endeavors are now focusing on the reuse, remanufacturing, and recycling of batteries to expand their useful life as a strategy to reduce the potential harmful effects of batteries on human health and the environment.165 As Due to their inherent degradable character, transient batteries have a particular potential to progress on several SDGs. Transient batteries may contribute to the \u201cSDG 7: Affordable and clean energy\u201d by making widely available the access to more sustainable energy storage systems. Lowering our dependence on the use of nonrenewable resources that have a destructive impact on the biosphere, transient batteries have the potential to achieve \u201cSDG 12: Responsible consumption and production.\u201d Finally, the use of transient batteries may protect the environment against the disposal of durable or toxic materials, protecting \u201cLife below water\u201d and \u201cLife on land,\u201d SDGs 14 and 15, respectively.Bearing this in mind, it is evident that transient batteries have a huge potential to facilitate the ecological transition by introducing a new model based on circularity. In this context, in 2015, the United Nations General Assembly established 17 Sustainable Development Goals (SDGs) which aim to promote prosperity while protecting the planet.166 Du\u03b2\u2010sheet structure. Another aspect worthy of noticing is that the chemistry of the battery components  could be tailored in such a way that the battery operates normally until the introduction of specific triggers such as water, pH change, exposure to light, or temperature initiate the degradation process. In any case, one should take into account that focusing exclusively on the transiency may lead to inferior battery performance. Therefore, researchers must find an adequate balance in the trade\u2010off between device\u00b4s electrochemical performance and transient behavior.In this review, we highlight that the transiency depends on both the nature of the material itself  and the environmental conditions of the medium . Therefore, if one can foresee the degradation conditions of the battery, it should be possible to precisely engineer the battery design to obtain a specific degradation time frame. Controlling the geometry of battery components  may allow a fine\u2010tuning of the kinetics of the battery transiency. For example, longer\u2010lasting transient batteries are achieved by increasing encapsulation thickness or by decreasing the exposed surface area (no porosity). The chemistry of the device also plays a major role in the transiency, as chemical cross\u2010linking of the encapsulation/separator or surface modification  may lower the susceptibility of the material to undergo chain\u2010cleavage reactions. Moreover, the crystal structure of the materials itself can be tuned to obtain tailored degradation behavior, as the degradation rate in the aqueous environment of biopolymers such as silk is dependent by several orders of magnitude on its crystalline tructure.88 Ano2, lithium iron phosphate, etc.) as these compounds will be found together in an actual device. It should be taken into account that not all biodegradable materials undergo degradation processes in all environments, i.e., some may biodegrade in waste processing facilities, while others may do better in soils. Moreover, a biodegradable material does not per se mean that it is biosourced, compostable, or renewable. To date, the transiency of batteries has mainly been proven under simple in vitro tests based on hydrolytic degradation, either in deionized water or in simulated biofluids like PBS. However, to get a better picture, it is essential to evaluate their degradation not only in PBS, an in vitro surrogate of physiological fluid at 37\u00a0\u00b0C, but also in different body fluids such as gastric acid, saliva, blood, or urine. It is important to note that in vivo degradation behavior can be very different from in vitro behavior. For some polymers such as poly(trimethylene carbonate) or poly(\u03b5\u2010caprolactone), in vivo degradation occurs much faster than in vitro degradation due to the action of enzymes. The presence of complex compounds such as proteins, but even simple ions can lead to unforeseen results comparing with in vitro studies. For example, calcium ions can accelerate the dissolution rates of silicon nanomembranes, while proteins show just the opposite effect. Therefore, in vivo degradation analyses offer the most compelling approach to assess the degradation behavior and potential risks of transient batteries. It is also worth mentioning that although the biocompatibility of different inorganic materials used in transient batteries has been evaluated through cell toxicity tests and animal models, their environmental impact has not been investigated yet. To examine the potential environmental impact, future research works on transient batteries should focus their efforts on the analysis of the biodegradation process according to harmonized standards such as ISO 14855\u20101:2012, ASTM D5338, ASTM D6868, ASTM D7044, ASTM D8029, or CEN EN 13432:2000. However, as transient batteries might be used in a wide variety of environments, even the benefit of standardized tests can be somewhat limited.Despite significant progress, a lot of work remains to be carried out to better understand the life cycle  of materials that are being developed for transient batteries, in particular those for the anodes and cathodes. This applies not only to pure elements  but also to their chemical compounds  are required. A demonstration of fungal biodegradation of transient batteries so that they can be applied as forestry fertilizers should also be developed. These results may shed light on the design and fabrication of transient batteries for biomedical and environmental applications.Additionally, in vivo degradation studies of transient batteries may further deepen the understanding on how different battery components interact/behave in real biological solutions or what maximum dosage of materials is allowed to prevent threat to the human body. Those studies have revealed, for example, that hydrogen gas formation during battery degradation can lead to undesirable tissue necrosis and blood clotting (in vitro studies may just show that the battery is degradable). The degradation of a battery in PBS was compared with in vivo degradation in the subcutaneous area of Sprague\u2010Dawley rats during 4 weeks.12 Bat which can be used for agricultural or cosmetic purposes. Particularly interesting chemistries are zinc\u2010ion batteries, which contain a high capacity zinc metal as anode and safe mildly acidic or neutral water\u2010based electrolytes (pH 3\u20137). Zinc is cheap, highly abundant, and represents an essential element for cells as it acts as an intracellular secondary messenger in several cellular processes. As a matter of fact, a zinc anode has been combined with a Pd cathode to fabricate a gastric battery for a wireless endoscopy application, highlighting the potential of zinc\u2010ion batteries for implantable applications. Mg batteries represent another interesting option, which, using body fluids as electrolytes, can yield a theoretical voltage of 3.09\u00a0V and an energy density of 2840 Wh kg\u22121. In any case, it should be noted that pure magnesium suffers from quick corrosion in aqueous solutions, so more resistant Mg alloys such as AZ31  are desired.Ideally, transient batteries are envisaged as an additional opportunity to provide novel functionalities to the degrading medium. This can be achieved by incorporating additives into transient devices that can be then released in a controlled fashion during the EOL of the battery, enriching the environment. For example, silk fibroin releases amino acids during its degradation,89 whiAlthough the debate about whether recyclable or biodegradable is the best for the environment is ongoing, without any doubt, biodegradable transient batteries represent a step forward in developing more sustainable alternatives to standard batteries. Transient batteries provide us an opportunity to change the current paradigm which considers batteries as harmful waste, to use them as nurture for the environment. Transient batteries hold a promising future to be a frontrunner in the uptake of circular economy concepts, opening new and highly innovative application fields.The authors declare no conflict of interest."}
+{"text": "SQUAMOSA promoter binding protein-like (SPL or SBP box) genes, making it difficult to accurately distinguish their roles in regulatory networks that affect numerous biological functions. Here, we collected data about miR156 and miR529 family members from representative land plants and performed sequence comparisons, phylogenetic analysis, small RNA sequencing, and parallel analysis of RNA ends (PARE) analysis to dissect their evolutionary and functional differences. Although miR156 and miR529 are highly similar, there are differences in their mismatch-sensitive regions, which are essential for target recognition. In land plants, miR156 precursors are conserved mainly within the hairpin region, whereas miR529 precursors are conserved outside the hairpin region, including both the 5\u2019 and 3\u2019 arms. Phylogenetic analysis showed that MIR156 and MIR529 evolved independently, through divergent evolutionary patterns. The two genes also exhibit different expression patterns, with MIR529 preferentially expressed in reproductive tissues and MIR156 in other tissues. PARE analysis revealed that miR156 and miR529 possess specific targets in addition to common targets in maize, pointing to functional differences between them. Based on our findings, we developed a method for the rapid identification of miR529 and miR156 family members and uncovered the evolutionary divergence of these families, providing insights into their different regulatory roles in plant growth and development.MicroRNA156 (miR156) and miR529 have high sequence similarity and recognize overlapping sites in the same target genes,  MicroRNAs (miRNAs) are 21\u201324 nucleotide (nt) long noncoding RNAs that regulate many physiological and developmental processes in plants and animals . In planPlant miRNAs recognize nearly complementary sequences that are generally located within the coding sequences of their targets . Thus, wSQUAMOSA promoter binding protein-like (SPL or SBP box) genes, which encode transcription factors that share a common SBP domain [SPL is a broadly investigated regulatory module in plants. By regulating SPL expression, miR156 is involved in regulating many biological processes, including flowering time, branching/tillering, and environmental stress responses [The sequences of miR156 and miR529 family members are highly similar, and both target P domain ,14. ManyP domain ,17,18. mesponses ,19,20. Cideal plant architecture1 (IPA1), which has pleiotropic effects on rice plant architecture [SPL genes targeted by miR156 and miR529 in three plant species (moss (Physcomitrella patens), rice (Oryza sativa), and maize (Zea mays)) revealed that miR529 targets comprise a subset of miR156 targets and that the targets regulated by miR156 alone and by both miR156 and miR529 are under different levels of selection pressure [MIR529 genes have a higher average loss rate than MIR156 genes after identical duplication events [SPL family genes have retained miR529 target sites [Arabidopsis thaliana plants overexpressing a miR529 precursor from a monocot that naturally lacks miR529 displayed phenotypes similar to that of an spl mutant, indicating that the retained target sites of miR529 were still functional [MIR529 and MIR156 genes.miR156 is preferentially expressed in rice seedlings, whereas miR529 is mainly expressed in panicles, and both miRNAs confer complex spatiotemporal regulation of their target gene itecture . A studypressure . A few sn events . Interesn events ,25,26,27et sites . Transgenctional . HoweverA. thaliana miR161 family members miR161.1 and miR161.2 [MIRNAs were also found to undergo alternative processing, such as MIR319C in melon (Cucumis melo L.) [Some annotated pre-miR156s (such as maize Zma-miR156j) also contain a 21-nt sequence that can be annotated as miR529 in the stem region just behind the mature miR156 sequence. Perhaps these annotated pre-miR156s can also generate miR529 by alternative processing. In some cases, different miRNA family members are indeed formed by alternative processing, such as the rice miR444 family members miR444a.1 and miR444a.2 [miR161.2 . Some anmelo L.)  and whetMIR529 from MIR156 based on their sequence features. We also predicted miR156 and miR529 family members from 50 land plants for which genome sequences were available to investigate their evolutionary relationship. MIR156 is phylogenetically distant from MIR529, and they exhibit different expression patterns and possess specific target genes in addition to common ones. This study illustrates the evolutionary differences between miR156 and miR529 and provides insights into their regulatory roles in plants from an evolutionary perspective.In the present study, we developed a rapid method to distinguish P. patens), a gymnosperm (Picea abies), and four angiosperm species  [To explore the relationship between miR529 and miR156, we downloaded their precursor and mature sequences from miRbase 22  and selebicolor) ,38,39,40Aquilegia coerulea) and Far-miR529 from tall fescue (Festuca arundinacea)  with only a 3-nt difference from Zma-miR529 was found in the Zma-miR156j precursor just behind the mature miR156 sequence B. This odinacea) . Since tP. abies, the mature sequences of Pab-miR156i, Pab-miR156p, Pab-miR156u, and Pab-miR156q lie in the 3\u2019 end of the precursor sequence. This pattern differs from that of most mature miR156 sequences, which commonly lie in the 5\u2019 arm, so we omitted these four precursors from further analysis. In most miRNA precursors, the sequences from miRNA-5p to miRNA-3p are highly conserved. Thus, we used this sequence, which we named miRNA-5p/loop/miRNA-3p, for phylogenetic analysis. miR529-5p/loop/miR529-3p and miR156-5p/miR156-3p were divided into two distinct clades with strong bootstrap support , using Phytozome v.12.1 , while approximately 10 nt following miR529-3p were relatively conserved (40\u201370%). Therefore, the additional conserved sequences of MIR529 genes are located outside the miR529-5p/loop/miR529-3p region  for which genome sequences were available [To explore the evolutionary history of miR529 and miR156, we identified predicted vailable ,46,47,48Spirodela polyrhiza, which is considered to be a basal monocot) during evolution. Notably, miR529 is almost completely absent in core eudicots, but it still exists in most monocots. miR529 family members were detected in only a few eudicots: Nelumbo nucifera , Vitis vinifera, Ficus carica, and Cannabis sativa. Only three orders of Lycophytes remain on Earth: Lycopodiales, Selaginellales, and Isoetales [miR529 was discovered in broyophytes and is regarded as the earliest-diverging clade of land plants ,49. miR5soetales . Based osoetales .Marchantia polymorpha, but these sequences are not capable of forming stem-loop structures  and three monocots . The National Center for Biotechnology Information Gene Expression Omnibus (NCBI GEO) identifiers of the sRNA-seq datasets are listed in To examine the functional relationship between miR156 and miR529, we used the model plant maize as an example to study the expression patterns of these miRNAs by analyzing sRNA-seq data  from vegSPL genes are common targets of both miR156 and miR529. Using PARE analysis, we detected a series of SPL transcripts cleaved by both miR156 and miR529. The cleavage site of miR529 in the target mRNA is located 4 nt before the cleavage site of miR156. Based on the nucleotide counts at the cleavage site, we generated target plots (T-plots) to illustrate the cleavage status of each target gene  and SBP29 (Zm00001d021573), are cleaved only by miR156 (COX19 family (CHCH domain family) [OsSPL13 (LOC_Os07g32170) and BdSBP13 (Bradi1g26720) were identified as miR156 targets in O. sativa and B. distachyon, respectively (COX19 orthologs were identified in O. sativa (Os03g068550) and B. distachyon (Bradi1g12230), but no miR529 cleavage site was identified in these genes, suggesting that these genes might not be targeted by miR529 and that COX19 might be a maize-specific miR529 target.In addition to common targets, we identified a few miR156- and miR529-specific targets. T-plot analysis showed that two y miR156 C,D. miR1 family) , are cleectively . COX19 oSPL genes in members of the liverwort family are regulated by miR529 but also contain binding sites for miR156 [M. polymorpha, miR156 target sites were detected in the SPL genes of this plant. PARE was used to predict miR529 targets in M. polymorpha [MYB family genes are miR529 targets in M. polymorpha [r miR156 . Althouglymorpha , and thelymorpha , but theS. bicolor, P. patens, and M. polymorpha [SPL genes. Thus, most studies to date have focused on the miR156-SPL module, while the miR529-SPL regulatory relationship has to some extent been ignored. In the current study, we discovered that miR156 and miR529 exhibit different expression patterns in different tissues and developmental stages, indicating that the miR529-SPL module has a unique biological function. It is therefore important to distinguish between these two miRNA families and clarify their evolutionary relationship.miR529 is an ancient miRNA family that was first reported in rice  and whoslymorpha ,38,52. DSPL genes as well [The MS region is an important marker of differentiating miRNAs in plants. Notably, we determined that the MS regions of miR156 and miR529 are not identical: their sequences differ by three nucleotides, GAC, located at positions 2\u20134. However, this difference cannot be used to distinguish miR529s from miR156s because some pre-miR156s contain miR529-like sequences; thus, we investigated whether such miR156s and miR529s are alternatively processed from a single pre-miRNA. We collected available data for miR156s and miR529s from plant species in which miRNAs were identified by sRNA-seq and found no evidence of miR529-like sequences being produced from pre-miR156s. This finding implies that miR529s are not the result of alternative processing of miR156 precursor sequences. Consistent with this notion, no obvious evolutionary relationship was identified between pre-miR529s and pre-miR156s. Recently, another miRNA-miR535 was found to have sequence similarity to miR156 and miR529, and it has been reported to target MIR529 sequence has greater similarity to MIR156 than to MIR529 genes, suggesting that it was mistakenly annotated as miR529 in miRbase.By analyzing the 40 bp-miR529-5p/loop/miR529-3p-40 bp sequences, we found that UGAC/CGAC, the first four nucleotides of miR156, never appear in miR529s, and therefore, this characteristic can be used to rapidly distinguish miR156s from miR529s. The first four nucleotides of Aqc-miR529 in miRbase are UGAC, and the Aqc-M. polymorpha, one of the most ancient land plants. Among green algae, we were not able to determine whether miR529 is present in Zygnematophyta and Coleochaetophyta, which are considered sister groups to land plants, due to the lack of reference genomes for these clades. However, miR529 is absent in other green algae, including Chlamydomonas reinhardtii, Dunaliella salina, Volvox carteri, Coccomyxa subellipsoidea, Micromonas sp. RCC299, and Ostreococcus lucimarinus. We could not determine with certainty whether miR529 appeared in green algae, but we suspect that miR529 was important in the early history of land plants.Our analyses showed that miR529 has frequently been lost in plant species during evolution. We traced the origin of miR529 to early land plants before the emergence of SPL genes are common targets of miR156 and miR529, they could be regulated by the two miRNAs during different developmental stages or in different tissues. Indeed, some SPL genes are targeted by miR156 in shoots and miR529 in tassels.The high degree of similarity between miR156 and miR529 raises the issue of whether they participate in common or diversified regulatory pathways. We detected different expression patterns for miR529 compared to miR156 in maize. miR529 levels are high in tassels and relatively low in other tissues, while miR156 is mainly present during the vegetative stage. As SPL genes are targeted by both miR156 and miR529: some are miR156-specific or miR529-specific targets. Some SPL genes  in rice are thought to contain only miR156 target sites [SPL13 orthologs Zm00001d006451 and Zm00001d021573 in maize are cleaved only by miR156, as revealed in our PARE analysis. We also detected miR529-specific targets in maize, but these targets are not conserved in rice. PARE analysis revealed that MYB genes are targets of miR529 in M. polymorpha [SPL module, the two miRNAs might have evolved specific targets and undergone functional diversification to better coordinate plant growth and development.However, not all et sites . The SPLP. patens, Osa-miR529a-b from rice) and miR156s  were used as queries to the search genome sequences by BLAST analysis in the Phytozome v.12.1 , FernBase , ConGenIE , and NCBI databases  [Mature sequences of miR156 and miR529 were retrieved from miRBase 22 . To idenst 2021) ,58,59,60st 2021) : if the https://www.zhaolab.org/psRNATarget/, accessed on 15 August 2021) was used to predict putative targets of miRNAs as follows (last accessed on 15 August 2021). (a) Target prediction: the miRNA was submitted to the \u201csubmit small RNAs\u201d section and \u201ccDNA library\u201d was selected with default parameters; (b) cleavage site prediction: both the miRNA and target mRNA were submitted to the \u201csubmit small RNAs and targets\u201d section with default parameters. The mismatch between the miRNA and target was required to be less than 3 nt at the 5\u2019 end and less than 6 nt at the 3\u2019 end of the miRNA [The psRNATarget server   according to the manufacturer\u2019s instructions. A NanoDrop 2000 spectrophotometer  was used to determine the quality of the extracted RNA, and only RNA of high integrity was used for subsequent analysis.Maize cultivar \u2018B73\u2019 were planted in a green chamber at 25 \u00b0C with 65% relative humidity under a 14-h/10-h light/dark cycle. The stalks from maize seedlings and tassels from maize at R1 stage were collected as samples. Total RNA was extracted from Parallel analyses of RNA end (PARE) libraries (GEO access: GSE175630) were constructed as previously described  and sequAdapter-free sequences of sRNA-seq data  were dowE-value < 0.05 and cleavage category = 0 (T-plot).Raw sequences were filtered to remove low-quality reads, and adapter sequences (TGGAATTCTCGGG) were removed using an in-house perl script. The remaining sequences were analyzed using CleaveLand 4.3  with -n O. sativa and Brachypodiumdistachyon. The results were analyzed and corrected by manual inspection, and only the longest isoforms were retained as representative protein sequences for each identified homologous gene.The protein sequences of the miR156 and miR529 target genes were downloaded from MaizeGDB 2018  and usedhttp://tcoffee.crg.cat/apps/tcoffee/do:regular, accessed on 15 August 2021) [Multiple sequence alignments of 40 bp-miR156-5p/loop/miR156-3p-40 bp (the genomic sequences from 40 bp upstream of miR156-5p to 40 bp downstream of miR156-3p) and 40-bp-miR529-5p/loop/miR529-3p-40 bp (the genomic sequences from 40-bp upstream of miR529-5p to 40-bp downstream of miR529-3p) sequences from different plant species were performed using the command-line version of T-Coffee  (st 2021) . Mafft_mst 2021) ,58. The https://www.ebi.ac.uk/Tools/msa/mafft/, accessed on 15 August 2021) with default settings, the gap opening penalty is 1.53 and the gap extension penalty is 0.123. Phylogenetic reconstruction was performed by the neighbor-joining (NJ) method with the default model, and phylogenetic trees with 1000 bootstrap replicates were drawn by Mega 7.0 [miR529-5p/loop/miR529-3p and miR156-5p/loop/miR156-3p were aligned using the MAFFT method with Clustal W2 online software (Mega 7.0 ."}
+{"text": "To study the structure-performance relationship, a series of nanostructured Fe-Cu binary oxides (FCBOs) were prepared by varying synthesis conditions. The obtained binary oxides were well characterized using X-ray diffraction (XRD), transmission electron microscope (TEM), Brunner-Emmet-Teller (BET), magnetic and Zeta potential measurement techniques. Both As(V) and As(III) sorption on the FCBOs were evaluated by batch tests. Results show that the surface structure and crystallinity of FCBOs are greatly dependent on preparation conditions. The crystallinity of FCBOs gradually increases as the synthesis pH value increasing from 9.0 to 13.0, from amorphous phase to well-crystalline one. Simultaneously, the morphology change of FCBOs from irregular agglomerate to relatively uniform polyhedron has been observed. The sorption of arsenic is greatly influenced by the crystallinity and structure of FCBOs, decreasing with increasing degree of crystallinity. The amorphous FCBO has higher surface hydroxyl density than well-crystalline one, which might be the reason of higher sorption performance. As(V) is sorbed by the FCBOs via formation of inner-sphere surface complexes and As(III) is sorbed through formation of both inner- and outer-sphere surface complexes. This investigation provides new insights into structure-performance relationship of the FCBO system, which are beneficial to develop new and efficient sorbents. They were directly used and no further purification was done. As (V) and As (III) stock solutions were prepared with deionized water using Na2HAsO4.7H2O and NaAsO2, respectively. Arsenic working solutions were freshly prepared by diluting stock solutions with deionized water. Glass vessels were used as reactors. Before use, reactors were firstly cleaned using 1% HNO3 solution and then washed several times with deionized water.Analytical grade chemicals including FeCl3.6H2O) and 5.0\u00a0g copper (II) sulfate pentahydrate (CuSO4.5H2O) were dissolved in 400\u00a0ml deionized water. Under vigorous mechanical-stirring, NaOH solution (3\u00a0M) was added dropwise to raise the pH of mixture to a predetermined value (9.0 or 11.0 or 12.0 or 13.0). The formed suspensions were continuously stirred for 0.5\u00a0h, aged at 100\u00b0C for 6\u00a0h using a hot water bath. After cooling, the prepared suspensions were washed several times with distilled water. Afterwards, they were treated by filtration and dried at 55\u00b0C for about 24\u00a0h. The dried FCBOs were crushed into fine powders (0.5\u201350\u00a0\u00b5m) and stored in a desiccator. According to the synthesis pH value, these FCBOs are denoted as FC1, FC2, FC3, and FC4, respectively.A series of FCBOs with a Fe/Cu molar ratio of 2:1 were prepared at different pHs, according to a slightly modified method described by Ms) and magnetization remanence (Mr) measure of particles\u2019 magnetism, was determined using vibrating sample magnetometer at room temperature . Specific surface area was determined by nitrogen adsorption (BET-method) using a surface area analyzer . A zeta potential analyzer  was used to analyze zeta potential of the FCBOs.X-ray diffraction analyses were performed on a Rigaku D/Max-3A diffractometer using Ni-filtered copper K\u03b1 one radiation . The morphology of FCBOs was analyzed using a transmission electron microscope . Specific saturation magnetization . After 4\u00a0h shaking at 130\u00a0rpm with the temperature maintained at 25\u00b0C, the mixture solution was passed through a 0.45\u00a0\u03bcm membrane. The filtrate was titrated using the HCl solution and residual NaOH in it was neutralized until pH up to 7.0, then the amount of surface hydroxyl can be calculated based on the amount of NaOH consumed.The density of surface hydroxyl sites was determined by a surface titration method . Fe-Cu bA series of batch experiments were performed to investigate the sorption of arsenic on FCBOs. A certain amount of FCBO was put into 100\u00a0ml glass vessels containing 50\u00a0ml arsenic solution of different concentrations. The vessels were then oscillated on a shaker at 170\u00a0rpm for 24\u00a0h. After reaction, all samples collected were filtrated using 0.45\u00a0\u00b5m membrane and then were analyzed for arsenic. More detailed description of sorption experiments can be seen in the Supplementary Material.3, and stored in acid-washed glass vessels. Arsenic concentration was determined using an inductively coupled plasma mass spectrometry machine .Prior to arsenic analysis, the aqueous samples were diluted to a concentration below 100\u00a0\u03bcg/L, acidified with concentrated HNO2) (JCPDS 80-0656); the peaks at 31.7 and 32.3\u00b0 are attributed to the hydrated copper hydroxide (Cu(OH)2\u00b7nH2O) (JCPDS: 42-0638); the peak at 35.8\u00b0 might be ascribed to both copper oxide (CuO) (JCPDS: 45-0937) and 2-line ferrihydrite formation (2O4) (JCPDS: 34-0425). With a further increase in synthesis pH value from 12.0 to 13.0 (FC4), the intensity of these peaks increases, indicating a greater crystallinity.X-ray diffraction patterns of the as-prepared FCBOs are presented in ormation ; the pea2O4 particles are dominant under this condition. These results agree with those of XRD analysis.Transmission electron micrographs (TEMs) of the FCBOs are shown in S-shape type, while those of FC1 and FC2 exhibit a nonhysteresis straight line, indicating that they are paramagnetic or superparamagnetic. The parameters of magnetic properties are summarized in Ms) increases with increasing synthesis pH value. For FC1 and FC2, they demonstrate very weak magnetism and the value of saturation magnetization is less than 0.4 emu/g. As the synthesis pH increases from 11.0 to 12.0, the saturation magnetization of FC3 rises sharply to 19.5 emu/g, which is far higher than that of FC2 and is very close to the CuFe2O4 nanoparticle (20.6 emu/g) synthesized via citrate-nitrate combustion method . Under weak acidic conditions, the surface of the FCBO is positively charged because of protonation and H2AsO4\u2212 is dominant species in aqueous solution, which is beneficial for electrostatic attraction between the surface of FCBO and the aqueous H2AsO4\u2212. With an increase in solution pH, the surface of FCBO becomes less positively charged and even negatively charged. At the same time, HAsO42- (a more negatively charged As (V) species) becomes to be dominant. Therefore, the attraction between the surface of FCBO and As (V) species weakens and as a consequence, As (V) sorption decreases.The influence of solution pH on arsenic sorption was investigated and the results are shown in ka1 of the acid. The pka1 of H3AsO3 is 9.2. The reduction in As (III) sorption at pH above 9.1 may be ascribed to the Coulombic repulsion between the negative surface of FCBOs (pHpzc = 7.3-9.0) and negatively charged As (III), whereas the predominant form of As (III) species is H2AsO3\u2212.Compared to As (V), the influence of solution pH on As (III) sorption is markedly different. Its sorption enhances gradually as solution pH increases and a maximum sorption occurs at about pH 9.1. Afterwards, further increase in pH decreases the sorption of As (III). Similar phenomena have been reported for the As (III) sorption by other binary metal oxides . General2 molecule, which is smaller than the arsenic molecule. Partial surfaces of the FCBOs are inaccessible to arsenic molecule. Additionally, the As(V) and As(III) maximal sorption capacities of Fe-Cu binary oxide synthesized at pH 7.5 are 82.7 and 122.3\u00a0mg/g, respectively  were employed to fit the isotherm data. The fitting results and obtained parameters are shown in R2). However, the Freundlich model is more suitable to describe the adsorption of As(III) on Fe-Cu binary oxides except for FC4, according to the correlation coefficients. The As (V) adsorption is likely a monolayer adsorption because the Langmuir model supposes that the adsorption process is a monolayer adsorption. While As (III) adsorption is a multilayer adsorption since the Freundlich model presumes that adsorption occurs on the heterogeneous surface and follows multilayer adsorption.For As (V), the Langmuir model is more favorable for fitting the data, giving higher correlation coefficients (R2) of linear regression for As (V) and As(III) is 0.971 and 0.925, respectively. It should be noted that the intercept is negative, indicating that not all surface hydroxyl groups are efficient for arsenic, especially for FCBO with high crystallinity. This could be explained as follows. The space structure of arsenic species  is remarkably larger than that of hydrogen ions, which was used to determine the amounts of surface hydroxyl groups. Therefore, partial hydroxyl groups on the surfaces could not be available for the arsenic molecules. To some extent, the arsenic sorption capacity of FCBO could be evaluated by the amounts of surface hydroxyl groups.The surface of metal oxides in water is easily hydroxylated, due to the dissociation of chemisorbed water molecules, and the formed surface hydroxyl groups are responsible for anions adsorption from water by the exchange with hydroxide ions . To revepzc (pH at point of zero charge) of the virgin FC1, FC2, FC3 and FC4 were about 9.0, 8.8, 8.1 and 7.3, respectively. Evidently, the pHpzc of the FCBOs decreases with increasing in crystallinity. This could be explained as follows. The pHpzc values of CuO and Cu(OH)2 are commonly over 9.2  and the pHpzc of As(V)-adsorbed FC1 is about 6.7. Apparently, As (V) is specifically adsorbed by the FC1, since the specific sorption of anions leads to a shift of the pHpzc of adsorbent to a lower pH value (pzc of FC1 has been found after reaction with As(III). Commonly, the adsorption of uncharged As(III) species can not result a significant shift in pHpzc of adsorbents  adsorbed on the FC1 was oxidized to As(V) by the dissolved oxygen because the experiments were conducted in an open system and the present CuO content might catalyze this reaction. For the FC2, FC3 and FC4, similar phenomena have been observed.The zeta potentials of synthesized Fe-Cu binary oxides before and after reaction with arsenic were measured. As presented in over 9.2 , and the 7.6\u20138.7 . As a mid values . For thepH value . Howeversorbents . The sli\u22121 may belong to the vibration of O-H stretching and the peak at 1,631\u00a0cm\u22121 may be ascribed to the deformation vibration of water molecules, implying that the surface of FC1 sorbed water molecules through physical adsorption; the peak at 1,116\u00a0cm\u22121 may be assigned to the vibration of SO42-  of As-O-M groups  is specifically sorbed by the FCBOs via formation of inner-sphere surface complexes, while As (III) is sorbed through formation of both inner- and outer-sphere surface complexes.A series of Fe-Cu binary oxides were prepared under different solution pH values. The crystallinity and saturation magnetization of prepared Fe-Cu binary oxide increased with an increase in synthetic pH value. Simultaneously, the morphology of FCBO changed gradually from irregular agglomerate to relatively uniform polyhedron. The adsorption of arsenic on FCBOs is remarkably affected by the surface structure and crystallinity, decreasing as the degree of crystallinity increases. Surface hydroxyl density of FCBOs is an important parameter to evaluate its arsenic adsorption ability. Nevertheless, the adsorption ability may be overestimated if only this parameter is used. As (V) is sorbed by the FCBOs via formation of inner-sphere surface complexes and As (III) is sorbed through formation of both inner- and outer-sphere surface complexes. This investigation provides new insights into structure-performance relationship of FCBOs system, which are beneficial to develop new and efficient sorbents. However, the characterization of FCBOs is still not insufficient in this study and more powerful techniques such as X-Ray Absorption Fine Structure (XAFS) are needed to reveal further the structure-performance relationship of FCBOs system in future study."}
+{"text": "Linking Brazilian databases demands the development of algorithms and processes to deal with various challenges including the large size of the databases, the low number and poor quality of personal identifiers available to be compared , and some characteristics of Brazilian names that make the linkage process prone to errors. This study aims to describe and evaluate the quality of the processes used to create an individual-linked database for data-intensive research on the impacts on health indicators of the expansion of primary care in Rio de Janeiro City, Brazil.We created an individual-level dataset linking social benefits recipients, primary health care, hospital admission and mortality data. The databases were pre-processed, and we adopted a multiple approach strategy combining deterministic and probabilistic record linkage techniques, and an extensive clerical review of the potential matches. Relying on manual review as the gold standard, we estimated the false match  proportion of each approach  and the missed match proportion  of the clerical review approach. To assess the sensitivity  to identifying social benefits recipients\u2019 deaths, we used their vital status registered on the primary care database as the gold standard.In all linkage processes, the deterministic approach identified most of the matches. However, the proportion of matches identified in each approach varied. The false match proportion was around 1% or less in almost all approaches. The missed match proportion in the clerical review approach of all linkage processes were under 3%. We estimated a recall of 93.6% (95% CI 92.8\u201394.3) for the linkage between social benefits recipients and mortality data.The adoption of a linkage strategy combining pre-processing routines, deterministic, and probabilistic strategies, as well as an extensive clerical review approach minimized linkage errors in the context of suboptimal data quality. A variety of administrative data is available for analysis in Brazil, including live births, mortality, outpatient clinics, and publicly funded hospital care. Health information is produced at the various administration levels  using the same systems and under the same standards, yielding National Databases .Record linkage has been used in specific projects conducted by the Ministry of Health, State and Municipal Health Secretariats, as well as university researchers. Databases with personal identifiers are assigned to the latter, after approval by a research ethics committee. To access the databases, the researchers must meet many requirements aimed to ensure privacy and data security .Linking Brazilian databases demands the development of algorithms and processes to deal with various challenges including the large size of the databases as well as the low number and poor quality of personal identifiers available to be compared , 4. In aDespite the increased popularity of record linkage in Brazil, only few initiatives linked various health databases , 6, 7 toA reform of the public health system in Rio de Janeiro City started in 2009. By then, the coverage of primary health care (PHC) was 3.5%, reaching 55% in 2015 , giving This study aims to describe and evaluate the quality of processes used to create an individual-linked database for data-intensive research on the impacts on health indicators of the expansion of primary care in Rio de Janeiro, Brazil.Table\u00a0The Social Benefits National Registry  is the database where people who want to receive welfare and social benefits from the Brazilian government are registered. These benefits include the cash-transfer program (Programa Bolsa Fam\u00edlia\u2014PBF), the low-cost energy social program , and a continuous pension benefit for the elderly and handicapped (Benef\u00edcio de Presta\u00e7\u00e3o Continuada\u2014BPC) , 12. TheThe Electronic Medical Registry (EMR) was implemented to be the main clinical, administrative and epidemiological data management tool of primary care. It was designed to allow integration with the Brazilian Primary Care Information System (SIAB\u2014Sistema de Informa\u00e7\u00e3o da Aten\u00e7\u00e3o B\u00e1sica), nowadays replaced by the e-SUS , 14. TheThe Hospital Admissions Information System (SIH) is an administrative database for the authorization of hospital admissions, including payments and auditing in the public health system , which oThe Brazilian Mortalilty Information System (SIM) started in 1975 in order to unify the various Death Declaration Forms being used in the states. The SIM database records individual deaths certificates, including description of the causes of death and the population profile . The codWe used PostgreSQL  and OpenWe linked the CadU database to the FHR, SIH, and SIM databases, one at a time. In all of these processes, we combined deterministic and probabilistic linkage, plus clerical review approaches. We also linked FHR to EMR records, however, performing only the deterministic procedure. A pilot study showed a minimal gain as well as a high cost in terms of the number of candidate record pairs that needed to be manually reviewed when we added the other approaches. The reasons for this low performance are the absence of the mother\u2019s name attribute in EMR data, and the greater efficiency of the deterministic approach, since the common personal identifiers of FHR and EMR datasets are generated by the same computerized system using a shared table. The electronic medical records were stored in ten different files, each containing data from health facilities located in the same region of the city. Due to the large size of these files, we linked each of them to the FHR database separately.We adopted a sequential strategy, sending to the probabilistic approach only the records for which a match was not identified in the deterministic phase Fig.\u00a0. LikewisWe carried out the deterministic linkage using PostgreSQL . The rulWe used OpenReclink  for the We used the Levenshtein edit distance to compare names, which measures the minimum number of edits  required to change one name string into the other , and an We post-processed all the record pairs classified as matches (first to sixth blocking pass) or potential matches (seventh blocking pass) using PostgreSQL . All recEight reviewers manually assessed the candidate record pairs classified as potential matches. Each reviewer was assigned a batch of non-overlapping candidate pairs. The reviewers were trained and evaluated by one research expert in clerical review, who was also responsible for their supervision. They assessed the same attributes used in the probabilistic process, along with the address. We let the reviewers decide each attribute's agreement, and the final resolution of the candidate record pair (match or non-match) without using any set of detailed criteria. We only oriented the use of few general rules for record pairs classification, which were developed empirically based on the experience gained in previous projects, as follows: (a) if the individual\u2019s name is rare, then the record pair should be classified as a true match, even in the presence of disagreements in one or more other attributes; (b) if the individual\u2019s name is common, then the record pair should be classified as a true match only if all other attributes agreed; (c) the individual\u2019s name is not common neither rare, the record pair should be classified as a true match if the date of birth and either the mother\u2019s name or the address agreed Fig.\u00a0. The namThe training consisted of a 3-h session when the rules were presented along with real examples. After that, the reviewer had to classify correctly at least 90% of 200 pairs of records to be approved.The final phase was the merge of the matched record pairs generated in each approach and the identification and elimination of record pairs wrongly assigned as matches. We manually reviewed any duplicated record pairs of the same individual in a one-to-one match situation . Likewise, we sent to manual review five or more repeated records of the same individual in the CadU versus SIH linkage process (one-to-many case). Finally, we removed all personal identifiers, keeping only the new unique identifiers created in the pre-processing phase.We rely on manual review as the gold standard to evaluate the linkage quality. Two reviewers who did not participate in the initial clerical review process evaluated the samples of records pairs. The reviewers were aware of the status of the record pair assigned in the different approaches , and they could either agree or disagree. In the case of disagreement, the supervisor decided the final status (match or non-match). Hence, pairs of records automatically classified as matches in the deterministic or the probabilistic approaches were manually reviewed for the first time. In contrast, the pairs of records classified as match or non-match in the clerical review approach were reviewed a second time by a different reviewer.N\u2009=\u2009744). We used this sample size to estimate the odds ratios for potential factors associated with linkage errors that we intend to evaluate in a future analysis. Likewise, we drew a simple random sample from record pairs classified as non-matches in the clerical review approach.We drew from each approach, without replacement, simple random samples from record pairs classified as matches  of each approach  and the missed match proportion  of the clerical review approach. For the linkage between the CadU and the SIM databases, we determined, in addition, the recall proportion using as the gold-standard the information about the vital status registered on the FHR. First, we selected all record pairs from the linkage between CadU and FHR with a date of death between 1999 and 2016 (N\u2009=\u20094179). In doing that, in the CadU database, we were able to add the information about each individual's vital status registered in the FHR database. Then, we evaluated how many of the individuals identified as deceased in the FHR (the gold standard) were also identified as deceased through the linkage between CadU and SIM. We calculated the recall proportion for the entire population and according to using the FHS services (yes/no). It was the only situation where we combined information from three databases .Each member of a family registered with FHS teams, at least in theory, should be recorded in the FHR database. Nevertheless, 297,280 individuals recorded in the CadU database, who were in a family with a FHS registered individual, did not have a match record in the FHR database. This find could be due to missing data in the FHR database or linkage error. To clarify this question, we drew a sample of 744 records from these CadU records and extensively manually searched them in the FHR database.Approval for this study was obtained from the Brazilian National Commission for Ethics in Research \u2014number 2.689.528.Table\u00a0In all linkage processes, the deterministic approach identified most of the matches. The linkage of the CadU database to the FHR database identified the highest proportion of matches deterministically. In contrast, the linkage of the CadU database to the SIH database presented the lowest percentage of matches detected through the deterministic approach and the highest percentage identified through clerical review Fig.\u00a0. That liWe estimated the false match and the missed match proportions using the manual review as the gold standard. The false match proportion was around 1% or less in almost all approaches except for the clerical review in the linkage between the CadU and SIH databases (3.89%) and the CadU and SIM databases 2.55%)  for the linkage between the CadU and SIM databases, based on the information about the vital status registered on the FHR. The recall proportion did not vary significantly according to the use of the FHS services: yes\u2009=\u200992.8% ; no\u2009=\u200991.6% .Finally, analyzing a sample of the 297,280 individuals recorded in the CadU database, who were in a family with an ESF registered individual and did not have a match record in the FHR database, we found that 89.7% of them (N\u2009=\u2009667/744) were missing in the FHR database.Even under suboptimal conditions, we managed to create an individual-linked database for data-intensive research with low linkage error rates by adopting a linkage strategy that combined multiple approaches. Our strategy is in line with a recent guideline for linking data for health service, prepared for the Agency for Healthcare Research and Quality (AHRQ) . It recoWe created the individual-linked database to evaluate the impacts on health indicators of the expansion of primary care in Rio de Janeiro, Brazil. We used CadU as the study population and linked it to FHR/EMR datasets to evaluate primary care exposure. To evaluate hospitalizations and mortality, we linked CadU to SIH and SIM databases, respectively.We used the same general strategy to link the CadU database to the FHS, hospitalization (SIH), and mortality data (SIM). However, the proportion of matches identified in each approach varied. The linkage between CadU and FHR databases found more than 90% of the matches through the deterministic approach, while the linkage between CadU and SIH databases identified three quarters. We implemented the deterministic approach using simple rules aiming to minimize false match errors . The rulThe number and quality of personal identifiers also influence the probabilistic approach , which mThe clerical review is the most labor-intensive and time consuming process in record linkage . When hiPre-processing was the second approach in terms of time and resource consuming in our study. Data cleaning is considered an essential step for improving record linkage in the scenario of poor data quality . HoweverWe tailored all the approaches to minimize false match errors. Unlike missed match errors, false match errors are positively correlated with the size of the databases to be linked . Moore eTo evaluate the impacts on health indicators of the expansion of primary care, we used record linkage to classify both the exposure to primary care and the outcomes  . TherefoOne limitation of our study was the use of the manual review as the gold standard for estimating the false match and the missed match proportions. However, to minimize errors due to the inherent subjectivity of manual classification, the supervisor decided the final status (match or non-match) whenever the reviewer of the validation sample assigned a discordant class from the initial classification. The linkage strategy adopted was complex, making it difficult to obtain a representative group of records classified as non-matches. Hence a further limitation was the lack of assessment of the recall measures for almost all linkage processes, except for the linkage between the CadU and the SIM databases. For this linkage, the gold standard was the information about the vital status registered on the FHR. Therefore, the analysis was restricted to the individuals registered in the CadU database who were found in the FHR database. However, we believe that the results observed for this particular subset of the CadU individuals may be generalized to the whole CadU population, as selection bias is unlikely. We carried out the linkage between the CadU and the SIM databases without knowing which individuals were also registered in the FHR database. Also, we estimated that about 90% of the individuals recorded in the CadU database, who were in a family with a FHS registered individual and did not have a match record in the FHR database, were missing in the FHR database. This result suggests that significant linkage errors are less likely to explain missed matches in the linkage of the CadU to the FHR databases. Finally, the reviewers of the linkage quality evaluation were aware of the record pairs status assigned in the initial review process, which might have contributed to overestimate the accuracy measures.Newcombe , a pioneIn conclusion, the adoption of a linkage strategy combining pre-processing routines, deterministic, and probabilistic strategies, as well as an extensive clerical review approach, minimized linkage errors in the context of suboptimal data quality. Although we reported our experience of linking Brazilian databases, we believe that the processes we developed to deal with various challenges can help Population Data Science researchers worldwide."}
+{"text": "Genome-wide, clusters of multiple SNPs in the 10\u20136 to 10\u20138p-value range were found at chromosomes 5p13, 5q31, 7q32, 8p22, and 10q23, highlighting glutamate-related genes. This is the first reported GWLS and genome-wide association study on CGN. Further increasing genetic knowledge about CGN and its relationships to male sexual orientation should help advance our understanding of the biology of these associated traits.Male sexual orientation is influenced by environmental and complex genetic factors. Childhood gender nonconformity (CGN) is one of the strongest correlates of homosexuality with substantial familiality. We studied brothers in families with two or more homosexual brothers , who self-recalled their CGN. To map loci for CGN, we conducted a genome-wide linkage scan (GWLS) using SNP genotypes. The strongest linkage peaks, each with significant or suggestive two-point LOD scores and multipoint LOD score support, were on chromosomes 5q31 (maximum two-point LOD\u2009=\u20094.45), 6q12 (maximum two-point LOD\u2009=\u20093.64), 7q33 (maximum two-point LOD\u2009=\u20093.09), and 8q24 (maximum two-point LOD\u2009=\u20093.67), with the latter not overlapping with previously reported strongest linkage region for male sexual orientation on pericentromeric chromosome 8. Family-based association analyses were used to identify associated variants in the linkage regions, with a cluster of SNPs (minimum association The online version contains supplementary material available at 10.1007/s10508-021-02146-x. Male sexual orientation is moderately heritable (30\u2009~\u200940% heritability) and appears multifactorial, with evidence of multiple genetic and environmental contributions via family, twin, and segregation analyses , prior to genetic analyses below, we transformed CGN scores via sqrt(2\u2009+\u2009CGN) to normalize them .We studied a set of families each with two or more homosexual brothers (409 concordant sibling pairs in 384 families) collected largely from community festivals for a linkage study on male sexual orientation and detailed previously , and (2) removal of samples . Following the QC filter application in the larger dataset  using PLINK v1.9  and rigorous quality control (QC) steps were previously detailed . In the GWLS, we detected suggestive two-point linkage (LOD\u2009\u2265\u20092.2) , one (rs7841264) at 8q24 was intronic in CASC8 (cancer susceptibility 8), and the other four  were intergenic. However, we note that linkage signals are imprecise  and thus, larger regions containing additional genes are implicated. Our multipoint nonparametric GWLS results . For our GWAS QC, we achieved a \u03bb1000\u2009=\u20091.046 . The top regions (Supplementary Table 1) were on chromosomes 5p13 , 5q31 , 7q32 , 8p22 , and 10q23 . There are a number of genes of potential relevance to CGN in and around these regions, as described below. Regional association plots for the top linkage regions are displayed in Supplementary Figs. 2 (chromosome 5), 3 (chromosome 6), 4 (chromosome 7), and 5 (chromosome 8). Of note, the linkage peak on chromosome 5q31 also contains a cluster of associated  SNPs . In addition, we display a regional association plot for chromosome 10q23 , which though not in a top linkage region did show genome-wide significant association for 9 SNPs.Our GWAS Fig.\u00a0 showed s\u20136\u2009<\u2009p\u2009<\u200910\u20138p value) SNPs from the GWAS, 2 of which are genome-wide significant associations, thus with both linkage and association positional evidence. However, none of the genes in the immediate region of this cluster have obvious putative connections to CGN.In this first GWLS on CGN in males, we found genome-wide significant linkage with multipoint support for several linkage regions, most notably at chromosomes 5q31 and 8q24 Fig.\u00a0. This wa\u20136\u2009<\u2009p\u2009<\u200910\u20138, including two loci  breaching genome-wide significance  for association with CGN and detected several additional regions . These regions contain a number of genes of putative relevance to the trait, some of which we highlight next. At the 5p13 SNP cluster, the nearest gene is SLC1A3, a brain expressed glutamate transporter which has been implicated in some behavioral phenotypes, e.g., attention deficit hyperactivity disorder, mood disorders, cortico-limbic connectivity during affective regulation  in the mouse, N-Methyl-D-aspartate receptor -dependent synaptic plasticity in the hippocampus was decreased, and these mice displayed mild social interaction deficits, increased self-grooming, and modest anxiety-like behaviors, which were reversed by pharmacological NMDAR activation  with SNPs reaching genome-wide significance , contributory genetic variants generally have individually small effects, leading to challenges in generating replicable findings. Other limitations include the current study being on a predominantly European ancestry sample and only on males, using retrospective recall of CGN rather than prospective ratings, and not including a replication sample. Replication and extension efforts are somewhat hampered in that relevant survey questions are often not included in large biobank samples such as for CGN; however, there are more sexuality data-points becoming available in some instances . Additional and larger studies in the future should provide further insight into genetic contributions to CGN and also to its relationship with sexual orientation.Supplementary file1 (DOCX 38555 kb)Below is the link to the electronic supplementary material."}
+{"text": "BRCA1 and BRCA2), which focuses on the identification of genetic factors modifying cancer risk of BRCA1 and BRCA2 mutation carriers, and GENEPSO (prospective cohort of BRCAx mutation carriers), which focuses on environmental and lifestyle risk factors.Linking independent sources of data describing the same individuals enable innovative epidemiological and health studies but require a robust record linkage approach. We describe a hybrid record linkage process to link databases from two independent ongoing French national studies, GEMO  and supervised machine learning (ML). This approach (named \u201cPRL\u2009+\u2009ML\u201d) combined together the candidate matches identified by both approaches. We built the ML model using the gold standard on a first version of the two databases as a training dataset. This gold standard was obtained from PRL-derived matches verified by an exhaustive manual review. ResultsThe Random Forest (RF) algorithm showed a highest recall (0.985) among six widely used ML algorithms: RF, Bagged trees, AdaBoost, Support Vector Machine, Neural Network.Therefore, RF was selected to build the ML model since our goal was to identify the maximum number of true matches. Our combined linkage PRL\u2009+\u2009ML showed a higher recall (range 0.988\u20130.992) than either PRL (range 0.916\u20130.991) or ML (0.981) alone. It identified 1995 individuals participating in both GEMO (6375 participants) and GENEPSO (4925 participants).Our hybrid linkage process represents an efficient tool for linking GEMO and GENEPSO. It may be generalizable to other epidemiological studies involving other databases and registries.The online version contains supplementary material available at 10.1186/s12874-021-01299-6. Record linkage is a process that allows to identify records appearing in different databases and referring to the same entity  , but whiLinkage methods are usually classified as either deterministic or probabilistic , 3, 4. DBRCA1 and BRCA2)  and y\u2009=\u2009. d is the number of matching variables; in our study, d\u2009=\u200910. The space of comparison is the Cartesian product X\u2009\u00d7\u2009Y which contains of all possible record pairs . All matching variables are discrete numerical values except MUT_HGVS which is a string. A similarity vector s\u2009=\u2009 is then computed as si\u2009=\u2009 where xi, yi are the i-th matching variables and  is a measure of similarity given by the Jaro-Winkler similarity simJW for the string matching variable (MUT_HGVS) and by the binary similarity simB (i.e. exact agreement) for the others  is computed as a weighted sum of the similarity vector S:w\u2009=\u2009 is the vector of weights. Weights are computed using the EpiLink approach [fi denotes the average frequency of values taken by the variable and ei the estimated error rate. We assumed ei\u2009=\u20090.01 for all matching variable [The probability of matching for record pair  were tolerated here. After blocking, the imputation of missing data could be then performed. The missing data in similarity for MUT_HGVS (numeric) were imputed by Bayesian linear regression and those for other categorical matching variables were imputed by logistic regression.The labeled record pairs were randomly partitioned into two sets: the training dataset (60%) on which we trained ML models, and the test dataset (40%) on which we evaluated the predictive performance of the trained models. ML models were built by using the similarity vector of the six variables . We employed six broadly used ML algorithms  . We compS from Eq. S > t) and non-matches  \u2264 t). We manually reviewed not only these potential matches [Step 1  based on the gold standard of true matches from step 1.Once the best performing ML algorithm and the optimal linkage method was chosen, it is necessary to train a final ML model for the ML or PRL\u2009+\u2009ML linkage method to predict the new true matches on the updated dataset. To achieve this, after the blocking step and the imputation of missing data on the whole record pairs (from step 1), we trained a ML model on a larger subset than any \u201cdataset A\u201d used in the cross-validations step  by contacting the recruiting center or the laboratory that performed the genetic test.The role of the manual review is to verify whether the candidate matches identified by the linkage method are indeed true matches. In our study, the manual review was conducted by verifying the HGVS nomenclatures of Up to September 2016, 4688 individuals had been enrolled in GEMO and 3339 in GENEPSO,. After data pre-processing, 15,653,232 record pairs were built as the Cartesian product of the two databases in dataset 1. The PRL score of each record pair was computed from Eq. We applied 5-fold cross-validation on the 15,653,195 record pairs that were labeled in the previous step. Within this cross-validation, we call A the training dataset  and B the test dataset . After blocking, each dataset A was randomly partitioned into a set Atrain containing 60% of the record pairs and a set Atest containing the remaining 40% of the record pairs Table . The aveIn the 5-fold cross-validation step, the averaged performance of three linkage methods  was assessed on dataset B Figs. c and 3. In conclusion, the PRL approach was very sensitive to the threshold and did not perform better than RF, except for the measure of recall at threshold 0.6, which, naturally, comes as the cost of a lower precision. Conversely, RF had a high precision but a modest sensitivity. PRL\u2009+\u2009RF had very high recall, and had a precision similar to that of PRL. Since the goal of our study was to minimize the number of FN, we chose the combined linkage method PRL\u2009+\u2009RF with the less conservative threshold of 0.6, which achieved the highest recall.In order to train a final RF model Fig. d for PRLBRCA1/2 mutation carriers had been enrolled in GEMO and 1586 in GENEPSO. These updated GEMO and GENEPSO samples constitute dataset 2. The combined linkage method PRL\u2009+\u2009RF was applied on this dataset to identify the new true matches (Fig. As of December 2019, 1687 new hes Fig. a. Besidehes Fig. b.Fig. 4Finally, 738 of the 1315 candidate pairs suggested by the combined linkage method were true new matches. This is consistent with the precision achieved with a threshold of 0.6 on dataset 1 Fig. . This coBRCA1/2 mutation carriers from 1693 families that had been enrolled in both studies.To summarize, in December 2019, GEMO included 6375 participants and GENEPSO included 4925 participants, and our hybrid record linkage identified 1995 PRL has a lower computational cost but the linkage quality is impacted by the choice of the threshold on the likelihood score. Lower thresholds lead to more FP whereas higher thresholds lead to more FN. The ML approach reaches higher precision, requesting fewer manual reviews. However, the blocking step can lead to FN if the data contain errors in blocking variables. We found that the PRL\u2009+\u2009ML combined method, having the highest recall compared to either of the two methods alone, improves linkage by identifying more true matches, but at the cost of additional manual reviews.In a context where manual review cost is to be capped and missing true matches is tolerated, the ML approach, which has a much higher precision to the expense of a lower recall, is an interesting option. Another possibility, which we expect from our results on dataset 1 Fig.  to reachWe expect linkage performance to be related to the number of matching variables. Had more matching variables been shared between GEMO and GENEPSO, the most discriminating matching variables could have been identified using feature selection algorithms, resulting in a lower computational cost. Here, with 10 matching variables, such a strategy was not necessary. On the other hand, if too few matching variables had been available, one could expect ML models to have lower performance, giving the advantage to PRL.Previously, Elfeky et al.  describeIn this study, the two databases are limited in size. However, larger databases may be challenging for record linkage. In this case, the traditional blocking technique that we employed here is a first step towards reducing computational complexity. In addition, partitioning the data into a larger number of smaller blocks and processing them in parallel using our hybrid record linkage process could be used to maintain a reasonable computational time. Besides, In order to decrease the burden of manual review, we could aim to achieve a high precision instead of having high recall by choosing a higher PRL score threshold. Thus, the manual review could serve for linkage method tuning.BRCA1/2 mutation carriers. PRL and ML were combined to classify the record pairs into matches and non-matches, and the ML model was built on a training set labeled by using PRL followed by manual review.In this paper, we propose a hybrid record linkage process which involves both PRL and ML approaches Fig.\u00a0. The hybGEMO and GENEPSO are ongoing studies and their respective databases are continuously updated. About 730 new subjects are included each year in GEMO, and 590 in GENEPSO. Hence, we will apply our hybrid approach on the updated versions of the two databases on a regularly basis, so as to identify new matches and increase the statistical power of research projects involving linked participants. Our hybrid record linkage process was driven by the need of a specific epidemiological question and may be generalizable to other epidemiological or translational studies involving other databases and registries.Additional file 1:Table S1. Confusion matrix. Table S2. Score distribution for all record pairs comparisons between GEMO and GENEPSO in dataset 1. Table S3. Size of each dataset A after blocking. Table S4. List of matches identified by either PRL or RF. Table S5. Performance of the unsupervised machine learning models."}
+{"text": "Dear Editor,in vivo and in vitro by configuring cellular metabolic profile.The NLRP3 inflammasome is an intracellular surveillance multimolecule platform that senses broad ranges of pathogen\u2010derived, environmental, and endogenous stress\u2010induced factors,in situ ASC speck formation both in primary and ASC\u2010GFP expressing immortalized macrophages. (Figure\u00a0in vitro (Figures\u00a0In order to discover novel NLRP3 inflammasome inhibitors, we treated primary macrophages with various natural products upon NLRP3 inflammasome activation. Out of more than 40 compounds tested, we determined xanthone as a potential target Figures\u00a0 and\u00a01B. . Figure\u00a0. Consist Figures\u00a0 and\u00a01I,  Figures\u00a0.Mitochondria dysfunction and potassium efflux are two most common triggers of NLRP3 inflammasome activations.Being the metabolic hub of cell, mitochondria function and dynamics are closely related to cellular metabolic profile.In order to investigate how xanthone regain mitochondrial integration, we employed both 4D proteomics and untargeted metabolomics analysis Figure\u00a0. We illuin vivo. Intraperitoneal injection of xanthone could significantly decrease sera IL\u20101\u03b2 level, improve survival rate, with no change of TNF\u03b1 level in LPS\u2010induced sepsis mice (Figures\u00a0We finally asked whether xanthone could suppress NLRP3 inflammasome  Figures\u00a0. In addi Figures\u00a0 and\u00a04E.  Figures\u00a0 and\u00a04G,  Figures\u00a0. Further Figures\u00a0 and S5B.in vivo data showed the beneficial effects of xanthone in both LPS\u2010induced systemic sepsis and topical keratitis. Together, our data showed that xanthone could serve as a promising inhibitor to treat excessive NLRP3\u2010related diseases.In summary, based on unbiased screening, we newly identified xanthone as a NLRP3 inflammasome inhibitor. Mechanistically, we showed xanthone metabolically rewired macrophages to obtain mitochondria fitness upon NLRP3 inflammasome activation Figure\u00a0. Our in The authors declare no conflict of interests.Supporting InformationClick here for additional data file."}
+{"text": "Leopard signs were obtained from in 70 out of 223 grids surveyed, with a na\u00efve leopard occupancy of 0.31. The model\u2010averaged leopard occupancy was estimated to be 0.5732 (SE 0.0082) with a replication\u2010level detection probability of 0.2554 (SE 0.1142). The top model shows the additive effect of wild boar, ruggedness, presence of livestock, and human population density positively affecting the leopard occupancy. The detection probability of leopard was higher outside the protected areas, less in the high NDVI  areas, and higher in the areas with livestock presence. The presence of wild boar was strong predictor of leopard occupancy followed by the presence of livestock, ruggedness, and human population density. Leopard occupancy was higher in west Chure (0.70\u00a0\u00b1\u00a0SE 0.047) having five protected areas compared with east Chure (0.46\u00a0\u00b1\u00a0SE 0.043) with no protected areas. Protected areas and prey species had positive influence on leopard occupancy in west Chure range. Similarly in the east Chure, the leopard occupancy increased with prey, NDVI, and terrain ruggedness. Enhanced law enforcement and mass awareness activities are necessary to reduce poaching/killing of wild ungulates and leopards in the Chure range to increase leopard occupancy. In addition, maintaining the sufficient natural prey base can contribute to minimize the livestock depredation and hence decrease the human\u2013leopard conflict in the Chure range.Conservation of large carnivores such as leopards requires large and interconnected habitats. Despite the wide geographic range of the leopard globally, only 17% of their habitat is within protected areas. Leopards are widely distributed in Nepal, but their population status and occupancy are poorly understood. We carried out the sign\u2010based leopard occupancy survey across the entire Chure range  was 0.5732 (SE 0.0082) with a detection probability of 0.2554 (SE 0.1142). The top model included wild boar, ruggedness, presence of livestock, and human population density as covariates. Further, maintaining a sufficient natural prey base can contribute to minimize the livestock depredation and hence decrease the human\u2013leopard conflict in the Chure range.The model\u2010averaged leopard occupancy in the Chure range (~19,000\u00a0km Chure is the young mountain range consisting of fragile sedimentary rocks such as mudstones, shale, sandstones, siltstones, and conglomerates , Asian elephant (Elephas maximus), leopard, gaur (Bos gaurus), sloth bear (Melursus ursinus), pangolins (Manis crussicaudata and M. pentadactyla), and hyena (Hyaena hyaena). Ungulates such as wild boar (Sus scrofa), barking deer , sambar (Rusa unicolar), chital (Axis axis), and three primates rhesus monkey (Macaca mulata), Assamese monkey (Macaca assamensis), and Terai gray langur (Semnopithecus hector) serve as prey species for a range of carnivores including the leopards.A large part of the Chure range (>70%) is forested and is the potential habitat for various wildlife such as leopards. The range consists of 23.4% of the forests nationally and 3.5% of other woodland covers of Nepal  for easy organization of the survey. Each block was further divided into grids of size 10\u00a0\u00d7\u00a010\u00a0km2 and surveyed in two to three shifts successively. We chose 10\u00a0\u00d7\u00a010\u00a0km2 grid size because it was larger than the home range size of leopards, that is, 6\u201390\u00a0km2 in lowland Nepal and similar habitats  with over 5\u00a0years of field experience in wildlife research conducted the survey in the field. The survey team was trained on survey protocols and wildlife sign identification before starting the survey to ensure the quality of the data. Out of 322 grids cells in the entire Chure range, 223 were surveyed which falls in the forested areas. The rest of the grids (n\u00a0=\u00a0109), which either fall entirely outside of the forests or was inaccessible due to undulating steep rugged terrain, were omitted from our study. Each grid was further divided into 16 subgrids of 2.5\u00a0\u00d7\u00a02.5\u00a0km2  for the uniformity to search the presence of leopard sign and associated covariates influencing their occupancy and detection. The survey was conducted between 2016 and 2018. We could not cover the entire Chure range in a single year due to the large area and limited human resources available. We carried out the survey in the same season (postmonsoon) to avoid the potential bias from surveys in different years.The Chure range was divided into 4 blocks  and \u201c\u03b81\u201d = Pr (leopard presence in a replicate/grid occupied and was present in the previous replicate). We also checked the performance of the standard occupancy model  to obtain the true occupancy of leopards of Chure range  across the grid influence the leopard occupancy positively and negatively, respectively . We separated the wild boar (W) from other prey species because many studies reported leopards avoiding the wild boar , human disturbance , and livestock presence (R), and human population density (PD). If a grid falls more than half inside the national park or buffer zone, it was coded as \u201c1\u201d and \u201c0\u201d if it falls outside. The human population density (PD) was obtained from the Gridded Population of the World Version 4  for each grid was calculated using 90\u00a0m ASTER DEM  as a covariate that affects the detection probability. Before adding the covariates in our analysis, we tested the Spearman correlation coefficient (r) using PAST version (4.0)  and livestock were highly correlated , vegetation cover measured as NDVI\u2014Normalized Difference Vegetation Index (N), terrain ruggedness index (Appendix\u00a0. The dat\u03b80 and \u03b81) . We used a constant model for replicate\u2010level occupancy parameters (R), vegetation cover (N), and livestock (L) using the global model for occupancy. Then, the suitable model structure of Pt was kept constant and \u03a8^ was varied for the top covariate model structure on grid\u2010level occupancy. We modeled covariate stepwise such that if it improved the model fit, then was retained to combine with other covariates in multivariate models that we considered significant from our a priori model building. We applied combination of covariates as additive effects in the model and eliminated models that failed to converge. We identified top competitive models that fit the data well with delta AIC\u00a0<\u00a02. The competitive models were averaged based on model weights , and other parameters. We applied the parametric bootstrapping to the untransformed \u03b2 parameter from the top models via simulating 1,000 random deviate to obtain the standard deviation of the mean .We also could not ignore the possibilities that some of the covariates or other unknown factors influencing the leopard presence contribute to variation in the leopard abundance and hence influence the replicate\u2010level detectability (Pt). To address this, our occupancy model focused on identifying the suitable covariate model structure for Pt from sample effort (Samp_Eff), management type (IO), ruggedness . In the east, there was no spatial correlation in detection, while we checked the performance of the standard occupancy model . They were present in half (49%) of the grids where leopards were detected. Other prey species combined  were present in 111 grids (52%). Lopping and encroachment were recorded on 97 grids (45%) whereas livestock sign was detected in 117 grids (55%).L, leopard detection increased with increase in livestock presence, opposed our prediction) to be the top detection model  was assessed.We fit 26 (15 detection and 11 occupancy) a priori alternative model that described expected covariates combination effecting leopard's occupancy and detection. Our result showed the model containing the additive effect of management regime , vegetation cover , and livestock presence (\u03a8^) of leopard in Chure range obtained after model averaging , and livestock  in Chure was 0.5732 (SE 0.0082) with the detection probability 0.2554  potential available habitat of the Chure range. Further, we estimated the grid\u2010specific occupancy (\u03a8^) and variation of leopards across the Chure range  as a covariate for detection (pe) (Appendix\u00a0pw), the additive effect of management regime , vegetation index , and livestock presence  to be the top detection model , obtained after model averaging (w\u00a0=\u00a00.30), was prey species  and vegetation index . The model\u2010averaged \u03a8^e was 0.46  out of 7,100\u00a0km2 surveyed. Similarly, for the west Chure range, the top model for leopard occupancy (\u03a8^w), obtained after model averaging (w\u00a0=\u00a00.69), was management regime , tiger presence , and prey species . The model\u2010averaged \u03a8^w was 0.70  out of 15,300\u00a0km2 surveyed  of Nepal. We found the spatial replicate model performed better than the standard occupancy model. Our result showed that more than half of the Chure range was occupied by leopards. Leopard occupancy was higher in the west Chure (0.7) compared with East (0.5). The additive effects of the covariates on the top model influencing the leopard occupancy were the presence of wild boar , human population density (positive with human density\u2014opposed to our assumption), terrain ruggedness , and the presence of livestock (positive\u2014opposed to our assumption). Similarly, the additive effect of the covariates on the top model influencing the detection probability of the leopard was management regime (higher outside the protected areas\u2014opposed our prediction), vegetation cover (less in the densely vegetated areas\u2014as predicted), and livestock presence (higher in the areas with the presence of livestock\u2014opposed our prediction).This is the first comprehensive survey of leopard occupancy covering the entire Chure range  in the west Chure range in Terai Arc Landscape  in the east and Shuklaphanta National Park (ShNP) was higher ((0.70 (SE 0.047))) compared with east Chure range ((0.46 (SE 0.043))). There are five national parks with source populations of leopards in west Chure range. Leopards are highly adaptable in terms of foraging strategy and flexible for habitat selection in the rugged Chure area , showed co\u2010occurrence with tigers . Other studies have also documented the high density of leopard co\u2010occurring with tigers within protected areas through spatial and diet partitioning  has a positive influence on leopard occupancy of western Chure range. A study on relative abundance of ungulate species (including cattle) in Terai Arc Landscape based on pellet count (pellet groups per 10\u2010m2 plots) documented relatively lower abundance in forest outside the protected areas  than tigers (2.3 to 2.9 per 100 km2)  which touches a small portion of the Chure range in the northwest ) in the east Chure range compared with the west Chure range (\u03a8^w= 0.70 (SE 0.047)). Hence, this study of leopard occupancy distribution helps wildlife managers and policymakers to guide for identifying locations to focus on leopard conservation in the Chure range.The tiger\u2010focused conservation activities in protected areas in the west have increased their number nearly twice since 2010  as covariates, but their influence in the model was weak. We believe the rarity of prey other than wild boar in the Chure range is the reason for such results in contrast to our expectation of strong relation between predators (leopard) and prey , and livestock (L) positively influenced both the leopard occupancy  and the detection . We suggest that maintaining a sufficient natural prey base can contribute to minimize the livestock depredation and hence decrease the human\u2013leopard conflict in the Chure range.Leopards are specialized solitary hunters primarily hunting wild ungulates, but also kill livestock if opportunity arises ; Data curation ; Formal analysis ; Investigation ; Methodology ; Supervision ; Validation ; Writing\u2010review & editing . Saneer Lamichhane: Conceptualization ; Data curation ; Formal analysis ; Investigation ; Methodology ; Writing\u2010original draft . Rajan Regmi: Conceptualization ; Writing\u2010review & editing . Milan Dhungana: Conceptualization ; Writing\u2010review & editing . Shyam Kumar Thapa: Data curation ; Writing\u2010review & editing . Anil Prasai: Data curation . Aashish Gurung: Data curation ; Writing\u2010review & editing . Santosh Bhattarai: Data curation ; Writing\u2010review & editing . Rajan Prasad Paudel: Data curation . Naresh Subedi: Conceptualization ; Data curation ; Formal analysis ; Funding acquisition ; Investigation ; Project administration ; Supervision ; Validation ; Writing\u2010review & editing ."}
+{"text": "Here we demonstrate that HPF1 can stimulate the DNA-dependent and DNA-independent autoPARylation of PARP1 and PARP2 as well as the heteroPARylation of histones in the complex with nucleosome. The stimulatory action is detected in a defined range of HPF1 and NAD+ concentrations at which no HPF1-dependent enhancement in the hydrolytic NAD+ consumption occurs. PARP2, comparing with PARP1, is more efficiently stimulated by HPF1 in the autoPARylation reaction and is more active in the heteroPARylation of histones than in the automodification, suggesting a specific role of PARP2 in the ADP-ribosylation-dependent modulation of chromatin structure. Possible role of the dual function of HPF1 in the maintaining PARP activity is discussed.Poly(ADP-ribosyl)ation catalyzed by poly(ADP-ribose) polymerases (PARPs) is one of the immediate cellular responses to DNA damage. The histone PARylation factor 1 (HPF1) discovered recently to form a joint active site with PARP1 and PARP2 was shown to limit the PARylation activity of PARPs and stimulate their NAD Kurgina et al. conduct in vitro characterization of the effect of HPF1 on the catalytic output of PARP1 and PARP2 under several experimental conditions. The authors report that the DNAdependent and DNA-independent autoPARylation of PARP1 and PARP2, as well as the heteroPARylation of histones in complex with the nucleosome are stimulated by HPF1 in a certain range of HPF1 and NAD\u2009+\u2009concentrations. Poly(ADP-ribose) (PAR) composed of linear and/or branched repeats of ADP-ribose is synthesized by enzymes called poly(ADP-ribose) polymerases (PARPs) that utilize NADon) Fig.\u00a0 or targe.\u00a02 Fig.\u00a0.Fig. 1Di4. PARP2 was also discovered as an enzyme that catalyzes the synthesis of PAR5. The role of PARP2 and its cooperation with PARP1 is under intensive investigation10. In human cells, the majority of PARP activity is exerted by PARP1 (around 90%) and by PARP2 (10\u201215%)7. It is known that neither PARP1 nor PARP2 is required for viability in mice, but parp1\u2212/\u2212parp2\u2212/\u2212 double knockouts are embryonic lethal with considerable genomic instability11. PARP2 shares significant homology with PARP1 in the catalytic domain structure, but differs in the domain architecture due to the absence of zinc fingers and BRCT domain7. Roles of PARP1 and PARP2 in base excision repair and single-strand break repair were intensively studied14. Overlapping functions of PARP1 and PARP2 in regulation of these processes were shown by using model DNA duplexes16 as well as nucleosomes18. PARP2 is comparable with or even more efficient than PARP1 in binding DNA breaks, but has a lower affinity for intact DNA and AP sites19. Difference in the affinities of PARP1 and PARP2 for various DNA structures in the nucleosome context was lately revealed18. PARP2 synthesizes shorter PAR chains than PARP1 does and also inhibits PARP1-catalyzed PAR synthesis via hetero-oligomerization of the two PARPs10. It has been shown that removal of histone H3 is largely suppressed in PARP2-deficient cells20. Thus, PARP1 and PARP2 can perform both overlapping and specific functions in maintaining genome stability, and these enzymes cooperate in the regulation of cellular processes.The best studied PARP family member, PARP1, is the abundant nuclear protein involved in multiple cellular processes, among them, DNA repair regulation. PARP1 serves as a sensor of the DNA lesions usually induced by ionizing irradiation and oxidative stress and signals to recruit the appropriate proteins to the sites of damage22. HPF1 was shown to complete the active site of these enzymes via complex formation, and the role of the joint active site was suggested to switch the PARylation specificity to serine residues23. HPF1 plays an essential role in the PARP1- and PARP2-catalyzed PARylation of histones25. It was proposed that HPF1 binds only the DNA-activated form of PARP1/PARP2 because the autoinhibitory helical domain (HD) when bound to the ADP-ribosyl transferase (ART) subdomain of the catalytic (CAT) domain prevents complex formation of HPF1 with PARP1/PARP229. The conformational change of HD induced by DNA/nucleosome binding promotes the interaction of PARP1/PARP2 with HPF130. It is interesting that shortening of the synthesized PAR chains was detected in the presence of HPF122. The authors explained this effect by shielding of amino acid residues important for PAR chain elongation  in the complex with HPF1. In addition to affecting the substrate specificity and the length of polymer chain in the ADP-ribosylation reaction, HPF1 was shown to enhance NAD+-hydrolysis catalyzed by PARP131. Recently it was shown that initiation and elongation steps of ADP-ribosylation are distinctly regulated by HPF1, ARH3, and PARG hydrolases, and that the HPF1-dependent initial attachment of ADP-ribose to Ser residue can be elongated by PARP1 alone32.Recently the new histone PARylation factor (HPF1) modulating activity of PARP1 and PARP2 was discovered21. Rudolph and coauthors from Luger\u2019s group showed that HPF1 differently modulates affinity of some PARP inhibitors for the PARP1-nucleosome complex, but this effect does not extend to PARP233. Therefore, the comparative study of the mechanism of HPF1-dependent modulation of the activities of PARP1 and PARP2 is of particular interest.PARP1 and PARP2 are known as therapeutic targets for the treatment of malignant tumors and other diseases. Several inhibitors of PARP are already using as anticancer drugs. Cells with knockout of HPF1 revealed high sensitivity to PARP inhibitors+-hydrolase activity of PARPs was detected. PARP2 was revealed to catalyze more efficiently modification of histones than the automodification.In the present study, we compared effects produced by HPF1 on the PARP1 and PARP2 activities in the autoPARylation reaction performed in the presence of DNA or nucleosome, as well as in the heteroPARylation of histones. HPF1 was found to stimulate the automodification of both PARP1 and PARP2 in a concentration-dependent manner, with the effects detected for PARP2 exceeding those for PARP1. Under optimal HPF1 concentration (produced the highest stimulatory action) no enhancement of NAD32P]NAD+  constructed from this DNA were used. The products of PARylation reaction were analyzed by SDS-PAGE separation and quantification, using ATP . NAD+, NH2OH, reagents for electrophoresis and basic components of buffers were purchased from Sigma\u2013Aldrich (USA).Recombinant wild-type human PARP1 and murine PARP2 were expressed and purified as described in detail previously49 were performed as described previously47. Briefly, DNA was obtained by PCR ; nucleosome was reconstructed by dialysis of DNA-histones mixture against a gradient of NaCl from 2\u2009M to 10\u2009mM. The homogeneity of the nucleosome sample was analyzed by the electrophoretic mobility shift assay on a 4% nondenaturing PAG.The sequences of the oligonucleotides for model DNA were as follows: template\u20145\u2032-GGAAGACCCTGACGTTCCCAACTTTATCGCC-3\u2032, downstream primer\u20145\u2032-pAACGTCAGGGTCTTCC-3\u2032, upstream primer\u20145\u2032-GGCGATAAAGTTGGG-3\u2032. The model 31\u2009bp DNA containing nick was prepared by annealing the upstream primer and the downstream primer to the template. The mixture was heated at 90\u2009\u00b0C and stepwise cooled to 40\u2009\u00b0C, incubated for 15\u2009min and cooled to 4\u2009\u00b0C. The effectiveness of hybridization was checked by PAGE in nondenaturing conditions. The amplification of Widom603 147\u2009bp DNA and subsequent reconstitution of nucleosomes using the Widom603 sequenceE. coli cells, a plasmid was constructed. The HPF1-coding sequence was amplified by PCR using specific primers and total HeLa cDNA. The resulting PCR product was annealed with the linearized pLate31 vector . The amplified DNA plasmid was characterized by the Sanger sequencing method at the SB RAS Genomics Core Facility . Next, E. coli Rosetta (DE3) cells were transformed with the pLate31-HPF1 plasmid. The transformed cells were incubated in a Studier autoinduction system in a 1\u2009l of culture. The growth was carried out for 18\u2009h at 18\u2009\u00b0C. Further, cells were harvested and lysed, and the HPF1 protein was purified by sequential chromatographies on the Ni-NTA agarose column , MonoQ 5/50 column , and Superdex 16/600 column . The protein concentration was determined spectrophotometrically, using the adsorption coefficient of the protein based on the Expasy Protparam Data.To obtain recombinant human HPF1 by expression in 2, 1\u2009\u00b5M [32P]NAD+ , 250\u2009nM DNA (nucleosome), 500\u2009nM PARP1 (PARP2), and 0.06\u201216\u2009\u03bcM HPF1. When indicated, the reaction mixture contained no DNA. The reaction was initiated by adding [32P]NAD+ to a protein-DNA mixture preassembled on ice. After incubating the mixtures at 37\u2009\u00b0C for 15\u2009min for PARP1 and 45\u2009min for PARP2, the reactions were terminated by the addition of SDS-PAGE sample buffer and heating for 3\u2009min at 95\u2009\u00b0C. Where indicated, reactions were treated with 1\u2009M hydroxylamine  for 1\u2009h at 37\u2009\u00b0C before addition of loading buffer. The reaction products were separated by 10% (20%) SDS-PAGE (a ratio between acrylamide and bis-acrylamide of 99:1); bands of proteins labelled with [32P]ADP-ribose were analyzed by using the Typhoon imaging system  and Quantity One Basic software (Bio-Rad). The radiolabelled signals of modified proteins were quantified as follows: the total (raw) signal of the smeared band of modified protein (indicated for each protein in the autoradiograms) was quantified and the same-size background signal of gel in the respective lane was subtracted from the raw signal. The quantitative data presented in histograms were obtained in at least three independent experiments.Catalyzed by PARP1 and PARP2 autopoly(ADP-ribosyl)ation and covalent labelling of histones were carried out in a standard 10\u2009\u03bcl reaction mixture containing 50\u2009mM Tris-HCl, pH 8.0, 50\u2009mM NaCl, 5\u2009mM MgCl2, 1\u2009\u00b5M or 10\u2009\u00b5M [32P]NAD+, 250\u2009nM nucleosome, 500\u2009nM PARP1 (PARP2), and 1\u2009\u00b5M (for PARP1) or 0.5\u2009\u03bcM (for PARP2) HPF1. Reactions were initiated by addition NAD+ (5\u2009\u00b5l) and stopped by dispensing 5\u2009\u00b5l aliquot of the mixture to the SDS-PAGE sample buffer, using Multipette E3 dispenser (Eppendorf) for the repetitive dispensing every 10\u2009seconds (until first 60\u2009s of the reaction); the aliquots of longer incubation were dispensed manually.PARP1/PARP2-catalyzed reaction was carried out in a 70\u2009\u03bcl reaction mixture containing 50\u2009mM Tris-HCl, pH 8.0, 50\u2009mM NaCl, 5\u2009mM MgCl+ hydrolytic consumption catalyzed by PARP1 and PARP2 was carried out in a standard 10\u2009\u03bcl reaction mixture containing 50\u2009mM Tris-HCl, pH 8.0, 50\u2009mM NaCl, 5\u2009mM MgCl2, 1\u2009\u00b5M [32P]NAD+, 250\u2009nM nucleosome, 500\u2009nM PARP1 (PARP2), and two different concentration of HPF1: 1\u2009\u00b5M (for PARP1) or 0.5\u2009\u03bcM (for PARP2), and 16\u2009\u03bcM. After incubating the mixtures at 37\u2009\u00b0C for 15\u2009min for PARP1 and 45\u2009min for PARP2, the reactions were terminated by the addition of SDS-PAGE sample buffer and heating for 3\u2009min at 95\u2009\u00b0C. The reaction products  were separated by 20% SDS-PAGE (a ratio between acrylamide and bis-acrylamide of 99:1) and their yields was analysed by phosphorimaging and quantified as described in the subsection \u201cTesting of PARP activity in the poly(ADP-ribose) synthesis\u201d. The quantitative data presented in histograms were obtained in three independent experiments.The reaction of NADt-test was used for the statistical analysis. Significant levels are: *p\u2009<\u20090.05; **p\u2009<\u20090.01; ***p\u2009<\u20090.001.All experiments were repeated three times. Data are presented as mean values\u2009\u00b1\u2009SD. The Further information on research design is available in the\u00a0Transparent Peer Review FileSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Reporting Summary"}
+{"text": "The rapidly growing field of miniaturized smart electronics has forced us to search for compatible microscale power sources with reliable electrochemical performance, various form factors, manufacturing scalability, and safety . Among tNatl. Sci. Rev. by Wu. et al. [2 planar micro-batteries as a breakthrough approach. The Zn//MnO2 planar micro-batteries, which were based on interdigital patterns of Zn ink as an anode and MnO2 ink as a cathode, with high-conducting graphene ink as a metal-free current collector, showed outstanding electrochemical performance, aesthetic diversity, mechanical flexibility, and modularization.A recent study published in . et al.  reported2 micro-batteries were fabricated by a low-cost and scalable screen-printing technique as illustrated in Fig. 2 micro-batteries with various complex-shaped planar geometries, resulting in the fabrication of multiple parallel interdigitated micro-batteries via in-series/in-parallel connections  of conventional lithium thin-film batteries. The Zn//MnO2 micro-batteries also provided long-term cyclability, high capacity retention of 83.9% after 1300\u00a0cycles at a current density of 5 C, which far exceeds those of stacked Zn//MnO2 batteries reported to date. Furthermore, the Zn//MnO2 planar micro-batteries exhibited exceptional flexibility without capacity loss under serious deformation and high voltage/high capacity through facile serial and parallel connection of bipolar cells. The serial or parallel Zn//MnO2 planar micro-batteries were assembled with unit cells one by one, which were packaged by dropping electrolyte onto the project area of interdigital microelectrodes.The Zn//MnOons Fig.\u00a0b, indivions Fig.\u00a0c, flexibons Fig.\u00a0d, and flons Fig.\u00a0e and linons Fig.\u00a0f. The pltes Fig.\u00a0g. They d2 micro-batteries with in-plane geometry presented in this study hold great promise as a high-performance, safe, flexible, and shape-versatile printed microscale power source that can be directly integrated with various miniaturized electronics. This study will be of broad interest to scientists and engineers involved in nanotechnology, chemistry, material science, and energy storage, and contributes to enriching development perspectives and directions of planar microscale power sources for potential use in future microelectronics. Research directions on printable batteries are currently focused on (i) synthesis of highly conducting and stable battery component inks with tunable rheological properties associated with electrochemical performance, (ii) design of battery shapes and configurations with fully printable techniques, (iii) development of industrially scalable printing techniques, and (iv) monolithic/seamless integration of printable batteries with electronic devices [The low-cost, environmentally benign Zn//MnO devices ."}
+{"text": "Point clouds were obtained after laser scanning of the concrete panel, SMW, and sheet pile which is most widely used in retaining structures. The surface condition of the point cloud affects the displacement calculation, and hence both local roughness and global curvature of each point cloud were analyzed using the different sizes of the kernel. The curvature of the three retaining structures was also analyzed by the azimuth angle. In this paper, artificial displacements are generated for the point clouds of 100%, 80%, 60%, 40%, and 20% of the retaining structures, and displacement and analysis errors were calculated using the C2C, C2M, and M3C2 methods. C2C method is affected by the resolution of the point cloud, and the C2M method underestimates the displacement by the location of the points in the curvature of the retaining structures. M3C2 method had the lowest error, and the optimized M3C2 parameters for analyzing the displacement were presented. Retaining structures have been used in various excavation sites to prevent the failure of soils. Precast concrete panels are widely used when reinforcing methods need to be applied after excavation is completed. The lateral movement of the ground can be suppressed by being used with reinforcement methods, such as anchors and soil nailing in concrete panels ,5,6,7,8.Various monitoring techniques have been applied to control the displacement in excavation sites. Monitoring techniques, such as inclinometers, extensometers and total stations have been used to measure vertical and horizontal displacements . In ordePoint clouds are obtained by laser scanning, since numerous points discontinuously represent a structure, the effect of resolution causes a major error in the monitoring of results. The Cloud to Could (C2C) comparison method, which directly compares points and points, is most affected by resolution. In order to minimize the effect of the resolution, techniques, such as Cloud to Mesh (C2M), which analyzes the distance after representing the point cloud as a mesh, have been developed . Since tIn this paper, laser scanning was performed to collect the point clouds of concrete panels, SMW, and sheet piles, which are widely used as retaining structures. The concrete panel was constructed with an anchor as shown in Since the three retaining structures are scanned by a laser scanner in this paper they have different surface features, the displacement of the point cloud can be affected by surface conditions during the analysis. The laser scanner used in this paper is a Leica P40 and Topcon GLS-2000. The range accuracy of the Leica P40 is 1.2 mm + 10 ppm, and the maximum resolution is 0.8 mm at 10 m. The accuracy of the single point of Topcon GLS-2000 is 3.5 mm, and the maximum resolution is 3.1 mm at 10 m.The field conditions and laser scanning settings: In order to perform equivalent analysis of each point cloud, pre-treatment of the point clouds are required. The point clouds of each retaining structure are extracted in a size of 3.2 m in width and 1.45 m in length. Since the number of points in the extracted point clouds was all different, the number of points in the sheet pile and SMW were equally selected by random extraction according to the point cloud of the concrete panel which has the lowest number of points. R is roughness calculated for points within a kernel radius of j, \u0394Z is the difference in elevation from a measurement point and the least-squares plane is calculated by the nearest neighboring measurement points and n is the number of measurement points within a kernel radius. The displacement calculation of the point cloud is influenced by the roughness and curvature of the point cloud . TherefoIn this paper, the roughness was analyzed by increasing the radius of the kernel to 0.1 m, 0.25 m, 0.5 m, 0.75 m, and 1.0 m to evaluate not only the local roughness but also the global curvature of the retaining structures see . Small kThe results of the kernel analysis of sheet piles are shown in In order to compare the roughness behavior of the three retaining structures, the roughness histograms were compared as shown in In order to analyze the curvature of the three retaining structures, the azimuth angles of each point were evaluated. An azimuth angle close to 0 means that it coincides with the directionality of the entire surface, and an increase or decrease of the azimuth angle means the curvature increases. In this paper, displacement was calculated using C2C, C2M, and M3C2  methods. In order to apply the C2M and M3C2 methods, the point cloud shown in z-axis except for one point cloud extracted from group A as a reference. The shifted distances are 0 mm, 2.5 mm, 5 mm, 7.5 mm, and 10 mm  has to be performed before M3C2 displacement is calculated. A Principal Component Analysis was performed on the neighbors of point i within a sphere of radius D/2 and chose the Dopt scale at which the third component is the smallest. It is ensured that a minimum of 10 points is used to compute the normal at Dopt. Otherwise, the scale has to be selected at a larger size as soon as this requirement is met . PCA anaThe displacement of the retaining structure has to be strictly controlled during excavation and hence it is important to monitor the entire retaining structure. Although laser scanning can monitor the entire retaining structure in three dimensions, errors may occur depending on the condition of the surface and the displacement calculation method. Therefore, in this paper, the roughness and curvature analysis of the surface of the three retaining structures was conducted by the point clouds of concrete panels, SMWs, and sheet piles. The analysis error was also calculated when the C2C, C2M, and M3C2 methods were applied to the displacement calculation of each retaining structure. The detailed conclusion of this paper is as follows.(1)in order to understand the roughness and curvature features of the point cloud in the three retaining structures, a roughness histogram analysis using the kernel was performed. In the result of the kernel radius of 0.1 m, the concrete panel had the highest local roughness, and it was found to be 4.2 times and 14.8 times rougher than the SMW and sheet piles, respectively. In the 0.5 m radius of the kernel where global curvature can be found, the sheet pile showed the largest value. Although SMW is also affected by the mixture of randomly protruding concrete and ground, the global curvature of the concrete panel is not reflected as roughness at a radius of 0.5 m compared to the other retaining structures. Therefore, in the displacement analysis, the effect of the global curvature of the sheet pile should be considered, and the influence of the randomly protruding area should also be considered for the SMW.(2)the curvature of the retaining structure was analyzed with the histogram of azimuth angle, and in the concrete panel, the azimuth angles of the points are evenly distributed over the entire angle due to the zigzag-shaped surface. In the SMW, the azimuth angles of the points were intensively distributed around \u221210 \u00b0 and 75 \u00b0. In the sheet pile, the points are concentrated around 0\u00b0 azimuth and the rest are distributed around 47\u00b0 and 137\u00b0 azimuth. In the azimuth analysis results, sheet piles are affected by global curvature, concrete panels are affected by local curvature, and SMW is affected by random curvature.(3)the displacement and analysis errors of C2C, C2M, and M3C2 were calculated by shifting the 100%, 80%, 60%, 40%, and 20% point clouds of each retaining structure by 0 mm, 2.5 mm, 5.0 mm, 7.5 mm, 10.0 mm. The displacement calculated by C2C showed the largest error in all retaining structures, and it is confirmed that the error was determined by the resolution of the point cloud.(4)a curved area existed in all three retaining structures, and the C2M displacement was underestimated because the negative C2M displacement was calculated according to the direction of the point in the curved section. Therefore, when analyzing the displacement of the retaining structure by C2M analysis, the influence of negative C2M displacement has to be removed.(5)M3C2 analysis had the highest accuracy in displacement calculation among the three analysis methods for retaining structures. PCA analysis is essential in order to get high M3C2 displacement accuracy of retaining structures with curvature and roughness. The optimized D for the M3C2 analysis of concrete panel, SMW, and sheet piles introduced in this paper were 0.142 m, 0.146 m, and 0.200 m, respectively."}
+{"text": "Our aim was to determine if the effect of heating up NaOCl at body temperature (BT) contributed to an improvement of the efficacy of XPF. Sixty-two single-canal human roots previously instrumented were infected with E. faecalis inoculum at 0.5 McFarland and incubated at 37 \u00b0C for two weeks. Twelve specimens were randomly selected as positive control, and the remaining fifty were divided into five experimental groups (n = 10). The first two were irrigated with 2.5 vs. 5.25% NaOCl at room temperature (RT), activated with PUI, and the other three were irrigated with XPF. Of these three, two were irrigated using 2.5 vs. 5.25% NaOCl at RT and one was irrigated with 5.25% NaOCl at BT. Our results showed that NaOCl was effective in biofilm removal for all experimental groups (p > 0.05), especially in the groups irrigated with 5.25% NaOCl at room temperature (RT) activated with PUI and the group treated with 5.25% NaOCl at BT with XPF. These groups were the most successful ones (p < 0.001). NaOCl, activated with XPF, was as effective as PUI in biofilm removal from the apical third of the canal when it was used at higher concentration and heated up. This study indicates that XPF only reached the efficacy of PUI when NaOCl was heated up.The objectives of the present study were to assess the antibacterial effectiveness of two sodium hypochlorite (NaOCl) concentrations (2.5% and 5.25%) activated by means of two techniques, passive ultrasonic irrigation (PUI) and XP-endo Enterococcus faecalis, which is capable of invading the root canal and forming biofilm alone, even in adverse conditions [The prognosis of endodontically treated teeth with apical periodontitis is highly dependent on the removal of biofilm from inaccessible areas of the root canal system . One of nditions . For thinditions ,4.Additionally, one of the factors which may hinder root canal disinfection is the complexity of the root anatomy, such as multiple macroscopic canals and thousands of microscopic dentinal tubules covering the canal walls ,6,7. ThiWhereas endodontic files facilitate the cleaning of macroscopic areas, irrigating solutions may help to reduce the number of microorganisms in the whole root canal system, including in dentinal tubules. However, some irrigating agents which have proved effective during in vitro tests are less efficient in vivo as a consequence of the great influence that environmental conditions have on both the mass and structure of biofilm in the root canal ,11,12.At present the irrigant most commonly used during endodontic treatment is NaOCl due to its antibacterial and proteolytic properties. However, it is essential to activate it during the procedure in order to impel it through the whole root canal system and to force it to penetrate into the dentinal tubules. The use of ultrasound with passive technique (PUI) after instrumentation is one of the methods which best achieves this aim ,14.\u00ae Finisher (XPF), offer a competitive alternative to ultrasounds. XPF is an ISO 25/.00 rotary instrument made of nickel\u2013titanium MaxWire\u00ae alloy , which at body temperature becomes more malleable. This effect allows the file to contract and expand following canal irregularities. In this way, when this file is used after instrumentation the turbulence created by its free movement not only improves wall cleaning but also increases the penetration of irrigating agents into the dentinal tubules [Nevertheless, newly designed irrigation systems such as XP-endo tubules ,16,17.E. faecalis bacterial biofilm on human roots by using NaOCl at two different concentrations, 5.25% (the highest biocompatible one) and 2.5% (the one most commonly used) [For these reasons, both systems were selected to carry out this in vitro study. Our aim was to compare their efficacy on the elimination of mature ly used) .In the case of XPF, an additional group was included, which heating the 5.25% NaOCl up to BT to ensure that the file was able to turn into the martensitic phase and allow the irrigant to reach the deepest areas of the canal ,20.There are few studies that use NaOCl heated up at BT when using XPF as an irrigant activation technique and, at the same time, compare it with PUI for biofilm removal. Taking into account that one of the main causes of endodontic failure on necrotic teeth is the persistence of biofilm within the most apical canal sections, we consider that this newly designed instrument could be a good alternative to ultrasound, and have tested it with different temperature and NaOCl concentrations.About 80 extracted human teeth were collected from an anonymous pool of teeth of San Pablo CEU University in order to ensure the 62 valid teeth after radiological assessment required for our study. For this reason, informed consent is not available. The approval for the study was obtained from the Research Ethics Committee of San Pablo CEU University (certificate 340/19/14), who determined that it complies with the essential ethical requirements demanded in this area (Annex 1).\u00ae Dental One Brasseler Boulevard Savannah, GA 31419) to standardize the root length (about 15 mm).Crowns were sectioned by using a diamond disc  and mesio\u2013distal (M\u2013D) projections to assure that the 62 selected teeth had one single canal and were free of any type of pathology or open apex.n = 10), depending on NaOCl concentration, activation technique, and irrigant temperature. The first group was irrigated with 5.25% NaOCl at RT activated with PUI, the second one with 2.5% NaOCl at RT and PUI, the third one with 5.25% NaOCl at RT and XPF, the fourth one with 2.5% NaOCl solution at RT and XPF, and the last one with 5.25% NaOCl at BT and XPF.Twelve specimens were randomly selected as positive control and the remaining fifty were assigned into the five different experimental groups  in order to confirm the presence of biofilm in the canal walls after the bacteria inoculation and colonization.\u00ae and WaveOne\u00ae Gold Primary files (Dentsply Maillefer), using distilled water as an irrigator. Finally, the roots were sealed with nail varnish in order to emulate the periodontium.Concerning root canal preparation, the working length (WL) was determined at 1 mm from the apical foramen (AF) by using a stainless-steel K-file size #10. Apical patency was performed using a K-flexofile (Dentsply Maillefer) size #15. The instrumentation was followed by the use of ProgliderEnterococcus faecalis was obtained from the CECT (Spanish Type Culture Collection). It was cultured on Slanetz\u2013Bartley agar plates with 1% sodium azide, incubated at 38 \u00b0C for 24 h and subjected to several successive passes for stabilization. Fresh cultures were prepared two days before the inoculation. The inoculum was obtained by diluting colonies in sterile physiological saline until reaching 0.5 McFarland concentration.Each specimen was introduced in a sterilized Eppendorf tube with 500 mL of brain heart infusion culture (BHI) and autoclaved at 120 \u00b0C and 1.5 atm for 20 min.During the tests carried out to determine the final protocol, contamination in neither the BHI culture nor in the dentin extracted from the canals was observed in any the teeth autoclaved as mentioned above after being incubated for two weeks.E. faecalis was pipetted into all root canals, working in a laminar flow cabinet. This procedure not only facilitated the entry of bacteria into the canal, but also aided their penetration into the dentinal tubules. After two weeks, two samples were longitudinally sectioned, making a small groove with a 0.19 mm diamond disc and splitting them into two slices with the aid of a steel blade. Immediately afterwards, both slices were covered with an ionic liquid1-butyl-3-methylimidazolium bis-(trifluorometha-nesulfonyl) (Sigma\u2013Aldrich 711713) and left to dry for 15 min [Immediately afterwards, 50 \u03bcL of r 15 min . The excr 15 min .\u00ae, Dentsply Tulsa Dental) placed 2 mm from the AF. At the end of each treatment, the NaOCl was neutralized with a 5% sodium thiosulfate solution, using the same volume as the previous NaOCl replenishment and applying it with the same technique to avoid post-treatment NaOCl activity, following the procedure carried out by Radcliffe et al. in their experimental work [During each complete procedure, all of the specimens were irrigated with the same volume of NaOCl (4.5 mL) and for an equal retention time (1 min), using a sterile syringe and a 30 G side-vented needle  operated with an ultrasonic device  set at power 5. The irrigant was activated in three twenty-second cycles (flow rate 0.075 cm3/s), placing the tip 1 mm from the AF and using 1.5 mL NaOCl for each replenishment.PUI activation was performed with an IrriSafe3/s). The irrigant was activated in two thirty-second cycles, placing the tip 1 mm from the AF. In the case of the group irrigated with 5.25% NaOCl at BT, a test tube containing the irrigant was immersed in a bottle warmer with water heated up to 37 \u00b0C. We confirmed the right NaOCl temperature by means of a laser thermometer during the whole procedure.The irrigant activation with XPF was performed with a VDW Silver motor, set at 1 Ncm and 800 rpm, with a gentle in-and-out motion following the manufacturer\u2019s recommendations  and usin\u00ae XA file (Dentsply Maillefer) inserted through the AF up to a depth of 4 mm. This material was weighed to standardize the samples. Next, dentin chips from each specimen were diluted in 1 mL of physiological saline buffer and vortexed for 1 min to facilitate bacteria detachment from dentin.After finishing each disinfection treatment, about 20 mg of dentinal chips were extracted from the apical third of the canal by using a sterile Protaper Next50 \u03bcL from the previous suspensions was pipetted and diluted tenfold, and 100 \u03bcL from each dilution was seeded on a Slanetz\u2013Bartley agar plate with 1% sodium azide. These plates were incubated at 37 \u00b0C for three days. The colony-forming units (CFU) obtained from each sample allowed to quantify the number of microorganisms remaining in the apical third after each treatment.p value is lower than 0.05.As the data obtained after CFU counting were overdispersed in relation to the normal model, two non-parametric tests were used: Kruskal\u2013Wallis, to check if there was any significant difference among the groups, and Wilcoxon\u2013Mann\u2013Whitney to analyze the differences between pairs of groups, taking into account that a difference is considered statistically relevant whenever In order to visualize the formation of biofilm, dental roots were inoculated with bacteria as indicated in the materials and methods section. After two weeks of incubation, the roots were longitudinally sectioned and the root canals were analyzed by scanning electron microscopy (SEM) .p < 0.05)   . At the = 0.168) .E. faecalis biofilm from the apical third using two different activation systems (XPF and PUI) with the aim of determining the most effective procedure.This study has analyzed the efficacy of NaOCl at two different concentrations (5.25% vs. 2.5%) in the removal of mature PUI was used in three twenty-second cycles, placing the tip 1 mm from the AF. A higher number of cycles would not have contributed to a significant reduction of the organic matter or debris, as shown by Duque, Van der Sluis, and Macedo ,26,27 inHowever, although XPF was also inserted 1 mm from the AF, instead of using it for one minute at a time it was used in two thirty-second sequences. This was done with the aim of achieving a balance between a higher efficiency due to the NaOCl replenishment and a lower malleability of the file as the consequence of a lower frictional heating  .Other researchers, such as Bao et al.  and SasaThe final findings revealed that the samples from the group irrigated with 5.25% NaOCl at RT activated with PUI and the ones irrigated with 5.25% NaOCl at BT activated with XPF showed a higher reduction in the bacteria count, as no CFU were detected in the samples from the first one and in the majority of the samples from the second one, decreasing the efficiency when XPF was used at RT.The 300-fold CFU reduction observed between the group of samples irrigated with 5.25% NaOCl at BT activated with XPF and the one treated with 5.25% NaOCl at RT activated with XPF could not have been explained only by the NaOCl temperature effect, estimated by Sirtes G et al. [This fact was also confirmed in the study published by Pacheco-Yanes et al.  with theWith regard to the efficiency of both techniques on biofilm removal, our findings contradict those of Bao et al. , who in Concerning NaOCl concentration, both systems (PUI and XPF) showed higher efficiency with 5.25% NaOCl, concurring with the results of other studies ,33 whichOur research has shown a partial bacteria reduction in the groups irrigated at the lower concentration (2.5%), being more effective in the group activated with PUI. However, this slightly significant difference between PUI and XPF could not have been relevant in a clinical endodontic treatment when NaOCl is used at RT as, according to the in vivo study carried out by De Hemptinne et al. . In thisFor this reason, further in vivo studies would help to clarify if this lower NaOCl concentration would be enough to guarantee the success of the endodontic treatment on infected teeth.Within the limitations of this study, it was concluded that the activation of 5.25% NaOCl at RT with PUI was more effective in bacteria reduction and biofilm destruction than the one achieved with XPF in the same conditions. However, when 5.25% NaOCl was heated up at BT and was activated with XPF, both systems showed similar efficacy. On the other hand, the activation of 2.5% NaOCl was partially efficient when using either PUI or XPF.In order to try to overcome the limitations of this study, it would be interesting to perform further long-term in vivo studies in infected teeth with chronic apical periodontitis, comparing the effectiveness of XPF to activate NaOCl at BT vs. RT, and assessing the efficacy of the treatment by means of micro-CT with follow-up evaluation."}
+{"text": "EUROCAT is a European network of population-based congenital anomaly (CA) registries. Twenty-one registries agreed to participate in the EUROlinkCAT study to determine if reliable information on the survival of children born with a major CA between 1995 and 2014 can be obtained through linkage to national vital statistics or mortality records. Live birth children with a CA could be linked using personal identifiers to either their national vital statistics  or to mortality records only, depending on the data available within each region. In total, 18 of 21 registries with data on 192,862 children born with congenital anomalies participated in the study. One registry was unable to get ethical approval to participate and linkage was not possible for two registries due to local reasons. Eleven registries linked to vital statistics and seven registries linked to mortality records only; one of the latter only had identification numbers for 78% of cases, hence it was excluded from further analysis. For registries linking to vital statistics: six linked over 95% of their cases for all years and five were unable to link at least 85% of all live born CA children in the earlier years of the study. No estimate of linkage success could be calculated for registries linking to mortality records. Irrespective of linkage method, deaths that occurred during the first week of life were over three times less likely to be linked compared to deaths occurring after the first week of life. Linkage to vital statistics can provide accurate estimates of survival of children with CAs in some European countries. Bias arises when linkage is not successful, as early neonatal deaths were less likely to be linked. Linkage to mortality records only cannot be recommended, as linkage quality, and hence bias, cannot be assessed. Congenital anomalies are structural anomalies and genetic syndromes that occur during development of the embryo and are a leading cause of perinatal and infant mortality in Europe . Around Death certificates are a reliable source of information on the number of deaths, as all deaths must be registered. However, although the primary cause of death such as infection is listed, a US study found that CAs are often not listed as an underlying cause of death . This meOne aim of the EUROlinkCAT study is to investigate the survival of children with specific CAs for the first 10 years of their lives by linking livebirths with CAs in EUROCAT registries to mortality records from various administrative sources. This study reports on the quality and accuracy of linkage to national vital statistics or mortality records in order to provide information for future researchers considering conducting similar studies in other population groups.www.eurocat-network.eu) were invited to participate in the HORIZON 2020-funded EUROlinkCAT study. Initially, 20 registries from 12 countries agreed to try to link all livebirths with a CA in their region to mortality records up to their 10th birthday  which include diagnoses of CAs  vital statistics containing civil registrations data such as birth and death registrations, where each liveborn baby would be expected to have a record; and (ii) mortality records containing only death registrations. Registries linking to vital statistics databases are able to determine the proportion of successful and unsuccessful matches; i.e. if a EUROCAT case is identified in vital statistics, a match has occurred; if a EUROCAT case is not identified in the vital statistics, a match has not occurred. However, when linking to mortality records the number of successful and unsuccessful matches cannot be quantified, as if a EUROCAT case is not identified in the mortality records, it is likely to be because the child is still alive, but it may also be because the linkage failed (a missed match).The method of linkage was generally electronic and determined by the institution providing the mortality data, who also specified the linkage identifiers see . Some reLinkage errors occur when an individual is matched to another person\u2019s record  or fails to be matched with their record (missed match). Researchers from Ulster University (UU) worked with registries to standardise their data to a common data model (CDM), details of which are given in an earlier paper (Protocol paper submitted). The use of a CDM enabled a central linkage quality syntax script to be developed by the St George\u2019s, University of London (SGUL) team which were distributed to all registries to evaluate the accuracy of the linkage by comparing characteristics of matched and not matched records in order to identify any factors leading to missed matches. For example, deaths within the first day of life may be less likely to be linked if a unique ID was not allocated at birth. The institutions performing the linkage were asked to specify for each matched case if the match was considered \u201cstrong\u201d (i.e. confidence in matching coded as excellent or good) or \u201cweak\u201d (i.e. confidence in matching coded as fair or poor), with guidance provided based on the combination of identifiers used. Some of the linking institutions used their own local definitions, usually based on a scoring system, as to what constituted a \u2018strong\u2019 or \u2018weak\u2019 match.The EUROCAT registries have ethics permissions and procedures for routine surveillance, data collection and transmission of anonymised data to a central database, according to national guidelines. Local registries follow national legislation as to whether parental consent is needed for registration of babies with anomalies . A commoFor registries that linked to vital statistics, the odds of linkage occurring were examined by fitting univariate logistic regression models to all EUROCAT cases being linked to vital statistics with linkage failure as the outcome and each of the specific factors measured in EUROCAT as the independent variable. For registries linking only to mortality records, the odds of known deaths in the EUROCAT data being identified in the mortality records were examined by fitting univariate logistic regression models to all known deaths amongst EUROCAT cases with linkage failure as the outcome and specific factors measured in EUROCAT as the independent variables.The values for maternal age, gestational length, number of babies in the pregnancy, infant sex, and birth weight in the EUROCAT data were compared with those in the linked data. Maternal age was judged to agree if the values differed by 1 year or less, birth weight was judged to agree if the values differed by <100 g and gestational length was judged to agree if the values differed by less than 1 week.Five countries had limitations on the release of aggregate data and analytic results if the numbers of births involved are very small. The Northern Netherlands released data if all exported results were rounded to the nearest five. Rounding all frequencies ensures that original numbers cannot be inferred. For Denmark, a few named researchers at SGUL and UU were allowed access to the aggregate data for the purpose of collating and including in pooled-analysis, on condition that it was securely stored and processed, that any individual results involving fewer than five people were not released; and that personal identification was not possible from any released results. The SAIL databank  provided data to the CRR with the requirement that aggregate data on fewer than five people were not released, and could not be calculated from any information in the public domain. The registry from Antwerp, Belgium could not release any information on three or fewer cases. NHS Digital (England) allows small numbers to be published if the analysis is national, otherwise numbers below eight need to be suppressed.Out of 21 registries who agreed to participate in the study and to link their data, one registry from \u00cele de la R\u00e9union was unable to obtain ethics permissions to perform the linkage. Five English registries received approval to link their data 3 years after the initial application to do so; at the time of writing only three registries have completed linkage and their results are reported in this paper. Seven registries linked using only deterministic methods. Six registries used a combination of deterministic and probabilistic methods i.e. they linked cases first using deterministic methods, and then resorted to probabilistic methods for unlinked cases. Two registries used probabilistic methods only. Three registries linked all cases manually to mortality records . Zagreb could only obtain identifiers for 78% of cases, born between 2011 and 2014 hence the registry was excluded from survival analysis due to the potential for bias. Ukraine reviewed all their cases manually and Basque Country reviewed their cases in the first few years of data collection due to concerns about too few mortality records being linked.The registries were asked to classify the strength of the linkage. The linking institutions for the eleven registries that linked their CA data to vital statistics classified all their matches as strong, with the exception of the UK English registries, where strong matches accounted for between 92% - 99% of all matches.st December 2015 due to adoption or to leaving the region or country covered by the vital statistics database. Ten of the eleven registries that linked to vital statistics had information on loss to follow-up, seven with national coverage . The Emilia Romagna registry did not have loss to follow-up information. The proportion of births lost to follow-up was under 2% for five registries, 2.6% for Denmark: Funen, 3.6%-6.7% for the UK English registries and 9.8% for Wales.For four registries , the proportion of known deaths occurring in the unlinked cases was higher than the proportion of deaths in the linked cases .In registries that linked to vital statistics, characteristics of the live births recorded in the CA registries can be compared to live births that were linked and those that were not to determine if linkage success is associated with any specific risk factors. For registries that linked to mortality records no such comparison is possible. However, EUROCAT registries report survival for the first week of life and many also have survival in the first year of life. Therefore, the characteristics of live births known to have resulted in a death but not linked can be compared to those live births who were linked to the mortality records. This will give an indication of any factors associated with linkage success, but the estimates will be much more imprecise as the sample sizes are much smaller and there is bias as the EUROCAT registries are more likely to have a death recorded if it occurs within the first week of life.We report the accuracy and completeness of record linkage when linking CA registry data to national vital statistics or mortality records in 18 registries in 13 European countries to examine survival of children born with a CA over a 20-year period from 1995 to 2014. For registries linking to vital statistics, the accuracy of the linkage was assessed over time and was shown to be excellent for Finland, Norway, and Denmark: Funen and good for Paris, Wales, and the Northern Netherlands, with very few children having incomplete follow-up periods. Although the linkage improved over time for the two Italian and three UK English registries, they were unable to link at least 85% of all live born cases in the early years. As a result, Italian and English data for the early years will be excluded from future analyses, as it was not sufficiently accurate. In contrast, it was extremely difficult to assess the accuracy of the linkage for registries that only linked to mortality records.For both types of linkage there was an indication that live births resulting in deaths within the first week of life were less likely to be linked. Preterm births and those with low birthweights were also less likely to be linked, possibly as these are risk factors for neonatal deaths. A low proportion of deaths occurring in the first week of life compared to the first year of life, particularly if below 40%, may be an indication of unsuccessful matching, regardless of the type of linkage. For Saxony-Anhalt, another indication that some deaths may be unlinked was that the survival, particularly of anomalies associated with high fatality rates, was significantly higher than that of any other registry .There are several reasons why early deaths, particularly those occurring during the first hours and days of life, were less likely to be matched. Firstly, assigning national ID numbers can take several days and may not be completed before the death certificates are completed. Secondly, if the child dies within minutes of birth they may also be incorrectly classified as a stillbirth or even a spontaneous abortion  and hence may not receive an ID number. Thirdly, a birth in a maternity unit immediately transferred to a neonatal intensive care unit, possibly in another region, where the child dies may not be linked. Studies have shown that those who die in the first week are less likely to receive a death certificate than those who die later. Also, extremely preterm newborn babies are less likely to get either birth or death certificates compared to full-term newborn babies, even in high-income countries ,16.Overall, only five registries distinguished between strong and weak links because for most other registries a successful match required exact agreement on several identifiers, such that all matches were by definition strong. Of these two registries linked to mortality records and three are the UK English registries linking to the same Vital Statistics. Linkages defined as \u201cweak\u201d in one registry were reclassified as \u201cnot linked\u201d. One registry classified all their links as weak due to permission not being given to use a unique national ID for matching. The UK English linkage score measures the strength of match to a hospital admissions database but all matched individuals have already been successfully traced through the personal demographics service. In the context of this study, a measure of linkage strength did not appear to be useful.If a child with a CA was linked, the linked data, if present, were found to be accurate in most registries for maternal age, gestational length , multiple birth status, infant sex, and birth weight. For governance reasons, Wales is only able to provide week of birth, which explains the lower accuracy found between the Welsh EUROCAT and linked variables for gestational age. In nine registries, more than 20% of information was missing for at least one variable in the linked mortality data. With the exception of infant sex, the other linked data for the UK English registries  were missing more than 20% overall. Valencian Region was excluded from this analysis as their mortality records held no information on these variables. In all registries, the accuracy and completeness improved over time.Studies involving data from the Nordic countries, where unique national ID numbers are used to identify individuals in their national databases, have obtained the high levels of linkage observed in this study. Comparing the linkage results from this EUROlinkCAT study with those from other countries is difficult as many have not reported any information about the accuracy of the linkage . Some stOther studies have examined the survival of children with CAs by linking to mortality records . In a stFuture studies planning identification of mortality during and after the neonatal period via linkage with mortality records should take into account that linkage to vital statistics is the method of choice. Linkage to mortality records alone does not enable an accurate assessment of linkage quality to be performed. There was evidence that poor linkage could bias survival estimates as those deaths occurring in the first week of life were less likely to be linked. Therefore, the accuracy and completeness of information must be considered when determining the inclusion of data into an analysis.S1 File(DOCX)Click here for additional data file."}
+{"text": "Dulong (Drung people) are one of the ethnic minorities of China, consisting of a small population living in remote and mountainous regions with limited facilities. Over the years, the Dulong have maintained their livelihood by collecting wild medicinal and edible plants. Therefore, through their experience and understanding, they had accumulated sufficient traditional knowledge about local plant resources. Since ancient times, wild edible plants have been essential to the food security of the Dulong people. However, there is almost no comprehensive report available on WEPs consumed by the Dulong people. The objectives of this study were\u00a0to: (1) make a systematic study of WEPs used by Dulong people, (2) record traditional knowledge related to WEPs, (3) analyze multiple uses of WEPs, and (4) evaluate species with significant cultural significance to Dulong people.Ethnobotanical survey including free listing, semi-structured interviews, key informant interviews and participatory observations was conducted in Dulongjiang Township, Gongshan County, Yunnan Province, Southwest China. A total of 127 informants were selected using the snowball method and information about WEPs, including vernacular name, food categories, parts used, mode of consumption, collection season, and other local uses were collected. The RFC and CFSI were calculated to identify the most culturally significant WEPs. One-way analysis of variance was performed to evaluate whether the four reference variables  significantly influenced the number of plant species mentioned by the respondents.A total of 148 species of WEPs consumed by the Dulong people belonging to 58 families were collected, including wild vegetables (71), wild fruits (52), staple food substitutes (15), spices (7), nuts (4), tea substitute (2), liquor making materials (3) oils and fats (3), and culinary coagulants (1). WEPs are used in a number of different ways, including as fuelwood, feed, and medicine. Food substitute plants accounted for the majority of the top 27 wild food plants identified by RFC and CFSI. It was observed that farmers have more knowledge of WEPs, and moderate education level informants reported less WEPs used.Maianthemum atropurpureum, Caryota obtusa, Cardiocrinum giganteum, and Angiopteris esculenta with economic potential can be developed to provide a source of income for the residents. More studies of the nutritional value, chemical composition, and biological activities of WEPs are needed. The demands and development of local communities can be realized under the premise of protecting WEPs and the associated traditional knowledge. More attention should be paid to the value of WEP and underutilized plants during future rural development.The WEPs used by the Dulong people are diverse and abundant in the Dulongjiang region. In the future, WEPs such as The online version contains supplementary material available at 10.1186/s13002-022-00501-3. China is classified among the countries with the richest biodiversity of plants in the world and has a wide variety of wild edible plants (WEPs) with abundant reserves and a wide distribution . WEPs reWith the development of the social economy and the modernization of agricultural science and technology, human utilization of WEPs has never decreased . CollectCurrently, conducting ethnobotanical surveys of wild edible plant resources has attracted the interest of many ethnobotanists and has become the focus of research \u201324. Therpinyin, one of the smallest ethnic groups in China and containing only 6930 people, is mainly concentrated in the Dulongjiang area of northwest Yunnan. Dulong people live near water and choose forests as habitats. In the long process of interaction with the living environment, many WEPs were consumed, and traditional ecological knowledge about them has been accumulated due to unique topography, rainy stereoscopic weather, closed traffic conditions, and abundant natural resources  were selected using quantitative indices (CFSI). The higher the CFSI value, the more important the role this plant plays in the diet . The corEleven food substitute species were evaluated and selected based on FC and CFSI. In the past, the Dulong people had poor living conditions and often faced a lack of clothing and food, so many plants were used as a substitute for food. These grain substitute plants have been gradually introduced into their home gardens for cultivation in the previous wild state.Caryota obtusa is a secondary protected plant species distributed mainly distributed in its Maku village, and the cultivation and management of it have a stronger position in the development history of the Dulong people. After processing one mature tree, about 100\u2013300 kg of sago starch can be obtained. Therefore, as an important food substitute plant, it helped Dulong people manage the resource shortage food gap during the period of resource shortages and played an important role in maintaining the livelihood security of the Drung and the nutritional value of a balanced diet. During their farm work, Dulong people will remove weeds for C. obtusa or cultivate some seedlings near their houses from the mountains. Generally, when there are major events, such as building a house or celebrating a marriage, C. obtusa will be planted in the home gardens in case of unexpected needs. To adapt to the harsh environment, a special cultivation and management mode for C. obtusa was generated by Dulong people. This traditional knowledge is related not only to the local biophysical environment but also to the traditional culture, which helped to maintain the population of C. obtusa and guaranteed regional food security. There are several ecological concepts in the mode of managing and utilizing C. obtusa by Dulong people, which provides a reference method for the cultivation and protection of C. obtusa resources.To illustrate the homogeneity of WEPs between different places, the JI was used to compare our study with eight previous investigations in China and neighboring countries , 67\u201371. Chenopodium album, used by all 5 ethnic groups  used by Dulong and other three ethnic groups.To explore WEPs differences in the use of WEPs between the Dulong and several other ethnic groups in northwest Yunnan, China, we used a Venn diagram to visualize WEPs used by the five ethnic groups . There is only one plant species, namely ups Fig.\u00a0. There aRibes alpestre, Rubus ellipticus, Rubus niveus, Capsella bursa-pastoris, Zanthoxylum bungeanum, Galinsoga parviflora, Taraxacum mongolicum, Houttuynia cordata, Pyrus pashia, Plantago asiatica, Mentha canadensis, Fagopyrum dibotrys, and Debregeasia orientalis) used by Dulong and other two ethnic groups. Dulong, Tibetan, and Monba showed higher similarities of consuming WEPs, which may be related to their living habits and types of surrounding plants.There are 13 species  and 40.94% (52) of the total, respectively. Previous studies have shown that gender is a critical variable that influences the distribution of local knowledge, and women often have more traditional knowledge because they are usually unemployed in rural areas and dedicate themselves to household and subsistence activities , 72. KhaThe average number of species is 6.1 mentioned by respondents under 19\u00a0years of age, 18.0 by the respondents between 20 and 39, 22.9 by respondents between 40 and 59, and 29.9 by respondents over 60. There is no homogeneity of variance in the one-way analysis of variance (<\u20090.05) . Maybe it is because uneducated people are more dependent on agricultural activities, while educated people generally choose non-agricultural jobs. With the development of the economy and the improvement of living conditions, many people are willing to choose non-agricultural activities, which is one of the reasons for the loss of knowledge retention and transmission of WEPs  and had more traditional knowledge than other groups , 14.5 by respondents with other occupations. There is a significant correlation with other occupations Fig.\u00a0a. An impFor the remoteness of the village, the average number of species mentioned by the respondents in the vicinity of the village is 18.7 and 21.8 by the respondents in the far village. However, there are no significant differences between the far village and the nearby village in the Dulongjiang region Table .The Dulong people have invaluable knowledge of WEPs, the use of which is generated under a specific cultural and ecological background. Due to the unique topography, climatic conditions, and extremely high biodiversity of the Dulongjiang region, it has generated abundant wild edible plant resources, which is its ecological foundation . AlthougRecently, with the improvement of living standards, the requirements for dietary balance and variety of foods have gradually increased, and wild food plants have created unprecedented opportunities, especially in the domestication of WEPs, the selection of excellent varieties, the development of WEPs with economic potential and WEPs for both medicine and food. Some similarities exist between medicine and food, and many plants are both edible and medicinal. Plants in local food cultures are inseparable from traditional therapeutic systems . These mAmomum tsaoko in the Dulongjiang region to increase income and promote economic development significantly impacts the local understory vegetation. Moreover, not only are plant resources threatened, but traditional knowledge related to these resources is also gradually being lost (Additional file At the same time, WEPs also face many challenges with economic development and improvements in transportation. Many studies from various regions have found that sociocultural factors are the main drivers of reduced consumption of WEPs , 74. OthIt seems to have happened within one overnight that Dulong people completed the transition from traditional livelihoods of gathering, fishing, and hunting to the poverty alleviation of entire tribes, and the traditional knowledge of WEPs is bound to be impacted. With local residents' incomes rising, many people are reluctant to collect WEPs, and the younger generation is not very interested in them, which is the main reason for the loss of traditional knowledge of WEPs.In summary, 148 WEPs and associated traditional knowledge used by Dulong people were recorded. Multiple uses of these WEPs were analyzed and the most culturally significant WEPs of the Dulong people were identified by quantitative methods. In the future, wild vegetables and fruits with economic potential can be developed to be a source of income for residents. The excellent traits of WEPs can be preserved and exploited by cross-breeding new varieties. More detailed analysis of the nutritional value, chemical composition, and biological activities of WEPs is expected to be performed. The needs of Dulong people and the development of the local community can be realized under the premise of protecting WEPs and related traditional knowledge.Additional file 1.Appendix A: Table 1 CFSI data. Appendix B: Table 2 One-way ANOVA data."}
+{"text": "Arabidopsis thaliana and rice (Oryza sativa), and the results show that our tool competes favorably against existing SINE annotation tools. The simplicity and effectiveness of this tool would potentially be useful for generating more accurate SINE annotations for other plant species. The pipeline is freely available at https://github.com/yangli557/AnnoSINE.Short interspersed nuclear elements (SINEs) are a widespread type of small transposable element (TE). With increasing evidence for their impact on gene function and genome evolution in plants, accurate genome-scale SINE annotation becomes a fundamental step for studying the regulatory roles of SINEs and their relationship with other components in the genomes. Despite the overall promising progress made in TE annotation, SINE annotation remains a major challenge. Unlike some other TEs, SINEs are short and heterogeneous, and they usually lack well-conserved sequence or structural features. Thus, current SINE annotation tools have either low sensitivity or high false discovery rates. Given the demand and challenges, we aimed to provide a more accurate and efficient SINE annotation tool for plant genomes. The pipeline starts with maximizing the pool of SINE candidates via profile hidden Markov model-based homology search and de novo SINE search using structural features. Then, it excludes the false positives by integrating all known features of SINEs and the features of other types of TEs that can often be misannotated as SINEs. As a result, the pipeline substantially improves the tradeoff between sensitivity and accuracy, with both values close to or over 90%. We tested our tool in  By integrating all known features of short interspersed nuclear elements (SINEs), AnnoSINE is a tool for both accurate and fast genome-scale SINE annotation in plant genomes. Zea mays) genome (Transposable elements (TEs), which are repetitive and mobile DNA sequences, are very abundant in most eukaryotic genomes. For example, TEs constitute \u223c50% of the human genome  and \u02c380%) genome . Thus, TSolanum pennellii also suggested a complex interplay between TEs and stress-related genes , and pepper (Capsicum annuum) can differ greatly, from about 500 to 20,000 , tomato (Solanum lycopersicum), and Nicotiana benthamiana, which diverged 24 million years ago  of SINEs and libraries containing sequences of verified intact SINEs. In order to quantify the performance of AnnoSINE, we tested it in rice (O.\u2002sativa) and A.\u2002thaliana with the standard SINE library . When we tested our method, we used species masking, meaning that the training data (SINEs) do not contain any SINE from the test species. Thus, the cross-species SINE annotation experiments emulate SINE annotation in a newly assembled genome.The input to In Step 3 of our method, we used BLAST to determine the copy numbers of each SINE. We applied the constraints on the alignment length  in A.\u2002thaliana and rice. RepeatModeler can output the SINE consensus sequences by identifying repetitive sequences and conducting TE classification. We thus directly use the consensus SINE sequences to annotate all SINEs in the genome via RepeatMasker.Our method is compared with four tools that can output SINE annotations, including SINE-Finder, SINE Scan, SINE Base, and RepeatModeler. The performance comparison is shown in Below we summarize the performance of different tools.A.\u2002thaliana and 80% in rice). However, it also has a high false discovery rate .SINE-Finder has high sensitivity  at the cost of sensitivity.SINE Scan reduced the FDR substantially .SINE Base improved the tradeoff between sensitivity and precision. Both the base level and element-level accuracy can reach 99% for rice. The base level and element-level accuracies are \u223c87% and 94% for A.\u2002thaliana. In addition, its sensitivity is \u223c35% lower than SINE Base in rice.The performance of RepeatModeler is slightly worse than SINE Base in AnnoSINE1 not only has the highest sensitivity at the base level and element level but also has better precision (\u02c390%) for A.\u2002thaliana. AnnoSINE2 also has good performance. Although its sensitivity is 10% lower than SINE-Finder, its precision is 91% higher than SINE-Finder in A.\u2002thaliana. For rice, both AnnoSINE1 and AnnoSINE2 have the best sensitivity (\u02c395%) and precision (80-90%) at the base level, indicating that our tool can generate more accurate SINE boundaries. AnnoSINE, which integrates AnnoSINE1 and AnnoSINE2, has the highest sensitivity and the best F1 score in all experiments.In comparison, AnnoSINE and SINE Scan are not associated with any FP at the seed level, suggesting the high precision of the two programs. However, when we apply RepeatMasker for annotating SINEs at the genome-scale, a small portion (3%\u20135%) of apparent FPs are generated using the seed sequences as the library. It is likely due to the fact that the seed sequences in the standard library and the test library are not exactly the same, causing a low level of discrepancy in the final annotation output.We quantify the seed-level performance by examining the overlaps between the standard library and the library generated via other tools. The performance comparison is shown in It is worth noting that although RepeatModeler has high sensitivity (87%) and precision (99%) at the seed level for rice, its accuracy and sensitivity dropped substantially at the genome scale (sensitivity <30%). The decreased performance is mainly caused by two problems. First, the RepeatModeler\u2019s seed sequences contain two consensus sequences over 1\u2009kb in length. Although only a small region of the two long sequences is similar to SINE, the entire consensus sequence is annotated as a SINE sequence by RepeatModeler. Thus, the non-SINE portions of the consensus sequences lead to some FP annotations in the genome. In addition, there are 30 seeds (in the standard library) that are missed by RepeatModeler. Because those 30 seeds represent the abundant SINE families in the genome, many FNs are generated during the genome-wide annotation. As a result, the sensitivity is \u02c230% at the genome-wide level.AnnoSINE. As expected, the running time depends on the genome size and proportion of SINE elements in the genome. Our pipeline can achieve good performance with reasonable running time.We also benchmarked the efficiency of SINE annotation tools on the MacBook Air platform with a 1.6\u2009GHz Intel Core i5 processor and an 8\u2009GB 2133 MHz LPDDR3 memory. AnnoSINE, which integrates all known features of SINEs for boosting SINE annotation. First, we apply the HMM for homology-based search to increase sensitivity. Then we compliment the homology-based approach with a structure-based search to maximize the pool of SINE candidates. Most importantly, we carefully examine and categorize the types of false positives within the SINE candidates and design corresponding filters for those false positives, which allows our tool to achieve both high sensitivity and precision.SINEs are short and highly heterogeneous, making SINE annotation more challenging than annotating other TEs. Although there are existing tools for annotating SINEs, they have various limitations. For example, SINE-finder has a very high false-positive rate (\u02c390%), whereas SINE Scan has very low sensitivity. SINE Base is entirely dependent on the known SINE library to implement homologous sequence identification, and it is unlikely to perform well for novel SINEs that are not contained in the known library. The current version leads to low sensitivity in rice. To generate more accurate SINE annotations, we developed AnnoSINE has the best performance among all tested tools, there is still room to optimize its performance. With more and more SINEs available, one future direction is to apply deep learning-based methods for SINE annotation. But this method requires a large amount of training data to prevent overfitting. In addition, deep learning models are not as accurate as pHMM-based models in identifying accurate loci of SINEs in genomes. In this work, we made several attempts to use deep learning models for SINE annotation. First, we implemented three well-studied deep learning models to distinguish SINEs from non-SINEs using the position-specific copy number profiles. Convolutional neural network-bidirectional long short-term memory (CNN-BiLSTM) has the same performance as the customized screening method in A.\u2002thaliana, while its performance is slightly worse in rice. As there are many parameters to train for the deep learning models, we suspect that inferior performance is mainly attributed to the limited training data. Second, we also formulated the SINE annotation problem as a learning problem using BiLSTM. In particular, the learning model can label each base as \u201cinside SINE\u201d or \u201coutside SINE\u201d and thus can use the output labels directly for genome-scale annotation. However, the final performance is much worse than the boundaries obtained by pHMM-based homology search. Thus, although an automatic pipeline based on learning can save users from adjusting the parameters, the limited training data at the current stage does not support its utility for this application. With the increased availability of sequenced genomes and their SINE annotations, deep learning-based methods may obtain some unique advantages in not only SINE annotation, but general TE annotation.Although AnnoSINE, that takes genomic sequences as input and outputs a nonredundant SINE library, redundant library, and genome-scale SINE annotation. The benchmark experiments against several other existing tools show that AnnoSINE demonstrates a substantially better tradeoff between sensitivity and precision at both the base- and element-level evaluations. As the current SINE annotations in most plant genomes are far from ideal, we hope that this tool can make contributions in providing fast and accurate SINE annotations for more plant genomes in different taxa groups.SINEs are a group of highly diverse TEs with degenerate sequence and structural characteristics, making genome-scale SINE annotation difficult. In order to deliver better SINE annotation for plant genomes, we developed a SINE annotation tool, AnnoSINE identifies SINE candidates using two methods in the first step. A homology-based search is utilized to find SINE candidates if the query genome is closely related to the genomes with known SINE annotations. If the query genome is distant from those genomes, structure-based de novo SINE search programs can be applied to identify putative SINE elements. To distinguish the two pipelines, we name them AnnoSINE1 and AnnoSINE2, which represent homology- and structure-based methods for SINE candidate generation, respectively.Our tool With the increased availability of SINE annotation covering different plant taxa, the homology-based search would generate sensitive SINE identification in related plants, particularly those in the same family.We apply profile HMM (pHMM)-based homology search because it is more sensitive in finding remote homologs than pairwise sequence alignment tools such as BLAST . We firsOnce the pHMMs are built for all the families, our tool conducts a homology search for any query genome or assembled sequence using nhmmer in HMMER3. The default E-value is 1.0, but the users can adjust the threshold for a more stringent or lenient homology search.In order to identify potential SINEs that lack substantial sequence similarities with known SINEs, we use the structure-based de novo SINE search tool to generate the candidates. We chose SINE-Finder, the most sensitive de novo SINE search tool for identifying putative SINE sequences using structural-based features . AlthougStep 1 can produce a large number of SINE candidates. However, both homology search and de novo structure-based search may return false candidates. To further screen for bona fide SINEs, we examine TSDs around each candidate SINE, which is an essential feature of SINEs . HoweverTSD-based screening cannot remove all false candidates. In addition, homology or structure-based tools may not yield accurate SINE boundaries in the genome. For example, HMM could be constructed from SINEs that are shorter than the SINEs in the input genome. Thus, homology search can underestimate the length of actual SINEs. For example, although Arabidopsis and Brassica share many SINEs with decent sequence similarities, their length distribution has lineage-specific patterns. The SINEs of Arabidopsis tend to be longer than their homologs in Brassica, as shown by the length distribution profiles of SINEs in Specifically, we extend both 5\u2032- and 3\u2032-ends of each SINE candidate for 100\u2009bp, where the 5\u2032- and 3\u2032-ends are determined by Step 1. As shown in All the returned SINE copies from BLAST search are used for producing an MSA, which is constructed using the query as the template. The MSA will be converted into a position-specific copy number profile, where each position contains the number of aligned bases. Considering that the SINE copies should be highly similar, we refine the SINE boundaries using the copy number profile.Let the number of sequences that can be aligned with the candidate SINE be We developed two methods to distinguish SINEs from non-SINEs using the position-specific copy number profiles. As shown in Helitrons. We can identify it by the presence of a gap in the alignment, which corresponds to positions with repeat number <We observed three types of repeat family structure profiles that are not associated with true SINEs. We sketched them in In contrast to method 1, which uses customized parameters to distinguish the copy number profiles of SINEs and non-SINEs, method 2 uses a learning-based automatic pipeline for the classification of SINEs. By using the position-specific copy number profiles of SINEs and non-SINEs as the positive and negative training data, we implemented three binary classification models: CNN, BiLSTM, and CNN-BiLSTM. Of the three models, the hybrid CNN-BiLSTM has the best performance in distinguishing SINEs from non-SINEs as shown in After we annotate SINEs, we classify them into superfamilies by examining the origin of SINE heads . At the Tandem repeats in DNA are duplicated or similar copies of two or more continuous nt patterns . SINEs mMITEs are prevalent in plants . With siThe nonredundant library is generated by searching and removing the redundant seed sequences. We applied CD-HIT with the identity cutoff of 80% to group similar SINEs into one cluster . For eachttps://www.repeatmasker.org/; After constructing the seed library, we use RepeatMasker (https://ftp.ncbi.nlm.nih.gov/genomes/genbank/plant/). We downloaded all plant genome annotation files in the general feature format that contain SINE annotation and used them as our training data. Of the 62 plant genomes with SINE annotations, 60 are from flowering plants. They cover multiple families, including Amborellaceae, Brassicaceae, Fabaceae, Solanaceae, Myrtaceae, Rosaceae, Euphorbiaceae, Arecaceae, Salicaceae, and Poaceae. In order to test the performance of SINE annotation on \u201cnew\u201d species, we conduct cross-species experiments by removing the test species from the training data. Thus, there is no overlap in the SINE sequences between the training (for pHMM construction) and testing datasets.In order to build the pHMMs in Step 1, we need to retrieve some known SINEs as the training data for model construction. There are many plant genomes with TE annotations at NCBI\u2019s Genbank , and sequences that are shorter than 800\u2009bp were examined. A few new SINE families were identified and added to the standard library. To build a more comprehensive library, all the related intact SINE elements in the genome (<5\u2009bp truncated at each end), with \u02c320% divergence (based on RepeatMasker\u2019s output) to the element in the library were collected, and redundancy was reduced as described in Step 7. Thereafter, the sequences of nonredundant distant relatives were added to the library to form the final standard library for subsequent analysis  CNN. (B) BiLSTM. Performance comparison of learning-based methods. The values of TP, FP, and FN of different SINE annotation tools at the element level (see Methods about the definition of element level). The values of TP, FP, and FP of different SINE annotation tools for seed level evaluation. Standard SINE library for rice. Standard SINE library for A.\u2002thaliana.kiab524_Supplementary_DataClick here for additional data file."}
+{"text": "Although several studies have shown that release of water channel proteins, aquaporin 1 (AQP1) and AQP2 in urinary extracellular vesicles (uEV\u2010AQP1 and \u2010AQP2), were altered in experimental kidney injury models, their release in human chronic kidney disease (CKD) has been largely unexplored. The aim of the present study was to clarify whether the release of uEV\u2010AQP1 and \u2010AQP2 is altered in patients with CKD. Urine samples were collected from 15\u00a0healthy volunteers  and 62 CKD patients who were categorized into six glomerular filtration rate (GFR) categories  in between 2005 and 2016 at Miyazaki Prefectural Miyazaki Hospital, Japan. uEV\u2010proteins were evaluated by immunoblot analysis. The release of AQP1 and AQP2 were significantly decreased in patients with both CKD G4 and G5, in comparison with the normal group. The area under the receiver operating characteristic (ROC) curve (AUC) values for AQP1 and AQP2 in patients with CKD G4 and G5 were 0.926 and 0.881, respectively. On the other hand, the AUC values in patients with CKD G1\u2010G3 were 0.512 for AQP1 and 0.680 for AQP2. Multiple logistic regression analysis showed that AQP1 and AQP2 in combination were useful for detecting CKD G4 and G5, with a higher AUC value of 0.945. These results suggest that the release of uEV\u2010AQP1 and \u2010AQP2 was decreased in patients with CKD G4 and G5, and these proteins might be helpful to detect advanced CKD. Patients with CKD frequently show fluid overload  is a significant public health concern worldwide , AQP2, sodium\u2010potassium\u2010chloride co\u2010transporter 2, and sodium\u2010chloride cotransporter, suggesting that these renal proteins in uEVs could provide information on renal disease states  that include exosomes  and microvesicles have been identified in various type of biological fluids such as serum, urine, milk, and saliva, and a focus of intense translational research to identify novel biomarkers  are altered in experimental kidney injury models such as gentamicin  protein, apoptosis\u2010linked gene 2\u2010interacting protein X (Alix), and Tamm\u2010Horsfall protein (THP), all of which are reported to be possible uEV marker proteins . Urinary osmolality was measured using an automatic osmometer .2.3g at 4\u00b0C for 15\u00a0min and the supernatant was centrifuged at 17,000\u00a0g at 4\u00b0C for 30\u00a0min. Thereafter, the supernatant was mixed with a protease inhibitor mixture  followed by ultracentrifugation at 200,000\u00a0g  at RT for 1\u00a0h using a thickwall polycarbonate tube  and the MLA\u201055 rotor . The resulting pellet (a fraction rich in EVs) was suspended in a solution containing a protease inhibitor mixture. The suspension was then mixed with 4\u00d7 sample buffer  and subsequently, the sample was incubated at 37\u00b0C for 30\u00a0min. The final samples were stored at \u221280\u00b0C until the use.First midstream urine of the day was collected in the morning from each CKD patient. The procedure for isolation of uEVs was performed as described previously , anti\u2010AQP2 antibody , anti\u2010TSG101 antibody , anti\u2010Alix antibody , and anti\u2010THP antibody , anti\u2010rabbit IgG , anti\u2010mouse IgG , and anti\u2010goat IgG . The antibody\u2010antigen reaction was visualized by using a Super Signal chemiluminescence detection system (Thermo Fisher Scientific) and the detected signal was quantified using the software package Win Roof software V5.7  or ImageQuant TL software .We always loaded one constant control sample comprising a mixture of the samples from three healthy individuals in the same gel, and the resulting band intensity was expressed as a percentage of the constant control band intensity.2.5http://boxplot.tyerslab.com) . Statistical significance was accepted for all tests at p <\u00a00.05. Receiver operating characteristic (ROC) curves were generated and the sensitivity and the specificity of the cut\u2010off values were calculated using the StatFlex software package . The results were also confirmed using Easy R , 0%, 15.6%), \u2010AQP2 , and \u2010TSG101  were significantly lower than those in the normal group , respectively. In patients with CKD G4, the levels of uEV\u2010AQP1 and \u2010AQP2 were also significantly decreased to 23.5%  and to 34.2% , respectively, but no significant change for uEV\u2010TSG101  was observed. The levels of uEV\u2010Alix and \u2010THP did not differ significantly among the groups.We also checked the relationship between urine osmolality and uEV\u2010AQP1 or \u2010AQP2. No significant correlations were obtained .Next, ROC curve analysis was performed to examine the diagnostic accuracy of these uEV\u2010proteins for the patients. Examination of uEV\u2010AQP1 or \u2010AQP2 in the 19 patients with CKD G4 and G5, and those in the 15 normal healthy volunteers yielded a ROC AUC of 0.951 for AQP1 , 0.888 to 1) and 0.884 for AQP2 (95% CI: 0.757\u20131) Figure . AQP1\u00a0haWe also performed ROC curve analysis for the patients with CKD G1 to G3. This yielded AUC values for AQP1, AQP2, TSG101, Alix, and THP of 0.512 (95% CI: 0.362\u20130.662), 0.680 (95% CI: 0.546\u20130.814), 0.703 (95% CI: 0.532\u20130.875), 0.623 (95% CI: 0.463\u20130.784), and 0.521 (95% CI: 0.359\u20130.683), respectively . The predicted probabilities (P) for uEV\u2010AQP1 and \u2010AQP2 in combination were calculated by equations of 1 / (1\u00a0+\u00a0e\u2212x) and x\u00a0=\u00a05.66277\u00a0\u2212\u00a00.0591 * (AQP1) \u2013 0.0210 * (AQP2). The summarized data are shown in Figure As the AUC values for uEV\u2010AQP1, \u2010AQP2, and \u2010TSG101 for patients with CKD G4 and G5 were high (>0.8)  machinery is thought to be important for the biogenesis of multivesicular endosomes, containing intracellular vesicles that become exosomes. The ESCRT machinery comprises four protein complexes: ESCRT\u20100, \u2010I, \u2010II, and \u2010III. TSG101 is a component of ESCRT\u2010I and Alix is an adaptor protein in the ESCRT machinery , the recommended protein intake for patients with CKD G4 and G5\u00a0has been <0.8\u00a0g/kg/day (KDIGO Board members, This study had several limitations. A larger research population would have been desirable, especially as only six of the participants had CKD G4, making it more difficult to obtain the precise AUC value and significance relative to the normal group. We did not study the level of renal protein expression in CKD patients, which meant we were unable to investigate the mechanism underlying the decreased release of uEV\u2010AQP1 and \u2010AQP2. Establishment of a more accurate measurement method would be essential for determining the cut\u2010off values for uEV\u2010AQP1 and \u2010AQP2, since immunoblot analysis is a semi\u2010quantitative approach. Furthermore, in our study the relationship between release of uEV\u2010AQPs and proteinuria or treatment history of patients could not be examined, and these points should be examined by increasing the number of cases in the future.In conclusion, this study has demonstrated that reduction of uEV\u2010AQP1 and \u2010AQP2 was associated with advanced CKD. ROC analysis revealed that uEV\u2010AQP1 and \u2010AQP2 reflected CKD progression of G4 and above, and particularly, the use of both uEV\u2010proteins in combination yielded better results than the use of either protein alone. Overall, these findings suggest that uEV\u2010AQP1 and \u2010AQP2\u00a0may be applicable as novel biomarkers for diagnosis of advanced CKD.We have no financial interest to disclose.S.O.\u2010H., N.Y.\u2010I., and M.I. conceived and designed the research; S.O.\u2010H., N.Y.\u2010I., and M.I. performed experiments; S.O.\u2010H., H.S., Y.S., and M.I. analyzed the data; S.O.\u2010H., N.Y.\u2010I., Y.S., and M.I. interpreted the experimental results; S.O.\u2010H., H.S., and M.I. prepared figures; S.O.\u2010H. drafted manuscript; S.O.\u2010H. and M.I. edited and revised manuscript; S.O.\u2010H., N.Y.\u2010I., H.S., Y.S., and M.I. approved final version of manuscript.Fig S1Click here for additional data file.Fig S2\u2010S6Click here for additional data file.Table S1Click here for additional data file."}
+{"text": "We present the first international initiative to compile and share information on pro pluvia rogation ceremonies, which is a well-studied proxy of agricultural drought. Currently, the database has more than 3500 dates of celebration of rogation ceremonies, providing information\u00a0for 153 locations across 11 countries spanning the period from 1333 to 1949. This product provides data for better understanding of the pre-instrumental drought variability, validating natural proxies and model simulations, and multi-proxy rainfall reconstructions, amongst other climatic exercises. The database is freely available and can be easily accessed and visualized via  Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.14372093 These impacts span a wide variety of socioeconomic sectors, including agriculture3, energy4, and tourism5, among others. The environmental impacts of drought have also been well-documented6, as evidenced by forest decay and mortality7, forest fires8, changes in biodiversity9, etc. In the literature, much effort has been made to quantify, by means of drought indices, the spatial and temporal variability and changes of drought during the last 50 or 100 years11. Nonetheless, the relatively short-period of these comprehensive assessments does not allow for capturing some key characteristics and processes, such as trends and variability of drought in the pre-industrial era or the atmospheric mechanisms controlling this variability at multidecadal or longer time scales.Drought is one of the most important natural hazards, with adverse impacts on both natural and human environments13. They were celebrated in different cultures and regions, in supplication to gods for changing the environmental or social risks brought to their communities. Although Catholics have been the most studied rogations with climatic perspectives, similar ceremonies have been found in many religions and societies14. A typical example in Islam is the Salat al istisqa\u2019, in which sunnah is encouraged to pray for rain.The rogation ceremonies for rain are a highly accurate documentary proxy of the occurrence of past agricultural droughts15. The rogations had a well-defined bureaucratic process with letters and money transfer among institutions that have left paper records in various archives, primarily municipal and ecclesiastical, but also in archives of agricultural guilds, from which the date of the celebrations can be retrieved. The process of pro pluvia rogations  began when farmers noticed a lack of rainfall for crop development or cattle feeding. The farmer guilds sent a formal request to the local government to celebrate a PPR. The local government accepted this request, especially when crop failure was plausible, forwarding the request to the ecclesiastic authorities. Then, the ecclesiastic council decided on celebrating (or not) the ceremony and defined which liturgical act was required. If the decision was positive, the ecclesiastic council responded to the local government with the date and the proposed liturgy. Finally, the local government announced the rogation celebration16. In normal conditions, the cost of the celebration was paid by the local government a few days after the celebration. However, delays in these payments were documented frequently, as the ecclesiastical authorities claimed payments to local government months after the celebration of the rogation.St. Mamertus, Bishop of Vienne, established the Roman Catholic rogations at the end of the fifth century21, France23, Ecuador24, Mexico27, Italy28 and Portugal29): to generate precipitation30 or atmospheric circulation modes reconstructions (e.g. North Atlantic Oscillation (NAO)31, El Ni\u00f1o-Southern Oscillation (ENSO)32); to validate natural proxies34, or to understand the social and ecological impacts of droughts35. All these works concur that PPRs are an accurate drought proxy, with extraordinary date precision. However, the rescue of PPR dates is a highly time-consuming task, given that it requires reading from hundreds of thousands of pages to extract relevant information to construct a PPR series dating back to three or four centuries. A key challenge of constructing PPR data is the capability of cities, villages, or towns to preserve their historical documentation and accordingly build series without gaps. In general, continuous series can be obtained for the last 500 years; the series suffered from data discontinuity before 1500.PPRs have been used in the last decades to understand drought variability in the pre-instrumental period across different countries (e.g. SpainPro pluvia ROgation database (INPRO), which incorporates information from more than 3500 PPRs spanning 153 different locations across the globe. The availability of this dataset for the research community will increase the reuse of PPRs in different climatic or social studies. Moreover, this initiative is open to any contributions of new research and we encourage any researcher to share their PPRs through the IMPRO initiative.Due to the demonstrated utility of this proxy, as well as the significant effort made by many researchers to rescue rogation ceremonies, a specific international repository for this proxy may be of particular importance for the research community. This repository can assure data quality and the reuse of this invaluable climatic information for different applications. For this reason, this work presents the INternational A wide range of documentary sources can be used to retrieve information about rogations Fig.\u00a0. Each doOfficial records of the institutions directly involved in the celebration of PPRs: Categorizes whether the identified virus is mosquito-borne or mosquito specific virusThese records include ecclesiastical authorities, government institutions, and agricultural guilds. The bureaucratic process required to celebrate a rogation ceremony was documented in the official record books of these institutions, generating dossiers with the letters and payments crossed among different institutions. Unfortunately, the archives of the agricultural guilds are frequently lost. As such, we have focused only on governmental and ecclesiastical archives.Official records books (chapter acts): They are the minutes of the assemblies of the local government or ecclesiastical authorities. Ordinary assemblies were normally celebrated on a weekly basis, but extraordinary assemblies were carried out more frequently to cope with emerging circumstances that required rapid reaction. Chapter acts reflect the decisions made by authorities over centuries. They have been frequently available in paper format. This increases the cost of consultation, and can make the task overwhelming, in some cases due to the limited opening hours of the archives preserving these acts. However, other collections have been digitized. These sources typically provide daily resolution, the motivations of the rogations are well defined, and the documentation can be complete and continuous for the last 400 or 500 years.Rogation dossiers: they are preserved in ecclesiastical and local authorities archives. They contain the original letters that these authorities exchanged about the celebration of rogation ceremonies, from the request of agricultural corporations to payment of different services . This documentation, when preserved, provides extraordinary detail about the development of liturgical acts, cost of ceremonies, and any discrepancy among ecclesiastical and municipal authorities. In some way, the official records described in the previous paragraph are a summary of these dossiers. Unfortunately, rogation dossiers are available only in a paper format, being mostly lost or uncatalogued. This makes their query a challenging task. The preserved dossiers were generated mainly from PPRs corresponding to extraordinary liturgical acts.Private diaries: As PPR celebrations were rare and important events that disturbed ordinary life, the precise dates of some of these ceremonies were recorded in private diaries. Nevertheless, these diaries usually cover a short period of time (few decades) and miss some minor rogations.th century onwards. In the last decades, significant efforts have been made to digitize historical newspapers . These historical sources allow for a digital and remote consultation of a great number of newspapers without the need of travelling to newspaper archives.Newspapers: Newspapers inform about important events that occurred in their area of interest. Rogation ceremonies were recorded frequently with their date, cause, and liturgical acts. Newspapers occasionally published rogations after the celebration, while on other occasions they published celebration announcements several days in advance. Indeed, this documentary source is not useful for generating continuous records at a specific location. Rather, it is more important for securing records in small towns or villages where other primary sources were lost. Newspapers were issued daily or weekly, first handwritten and printed from the 17Iconographic testimonies: Engravings, paintings or photographs of pro pluvia rogations celebrations provide information about the ceremonies, such as participants, liturgy, streets decoration. Nevertheless, they are frequently restricted to celebrations with extraordinary liturgy.In the last decades, some scientific works have provided information about PPRs. Commonly, these articles are based mainly on primary sources, which have been cited properly. Daily resolution is not always provided. This is the most reliable secondary source given that they were produced by scientists after careful data quality testing.Annals of cities, chronicles and similar books: References to rogation ceremonies can be found in books that recover the history of a city or a region. These are usually based on different sources that can be cited properly or not. This makes the traceability of rogations uncertain. The dates of rogations are not given on a daily basis in these sources. Rather, they often refer to these events at the monthly, seasonal, and annual scales. Importantly, only rogations associated with liturgical acts, such as processions or pilgrimages, are primarily documented.Monographs about religious images or relics: these monographs provide information on the dates of the most important rogation ceremonies. However, dates of rogations are commonly provided at a coarser temporal scale. In addition, lists of rogations are mostly not exhaustive. Albeit with these shortcomings, these sources can provide valuable information about very old PPRs whose primary sources were lost.1614, expenses36, area occupied by the text in chapter acts37) has not been compiled. This is simply because this information is not always available. Moreover, it can be more subjective and/or less comparable among different locations.For each document, we have compiled the date of PPR with the best available resolution. This is the most objective information and it is provided by all records with a resolution ranging from daily to annual. Other information used in some rogations indices  Fig.\u00a0. The ear39. It is a unique .csv file. Each row provides information about the celebration of one PPR, including the date of the celebration, location (with the geographic coordinates), and documentary source from which it was retrieved.The database is in figshare repositorynovenas (nine days) and triduo (three days)). In such cases, we provide the date of the first day. In our database, there are four columns related to the date: \u201cyear\u201d, \u201cseason\u201d, \u201cmonth\u201d, and \u201cday\u201d. We filled in these columns depending on the information provided by the document. We found that 48% of the rogation records had daily resolution, compared to 44%  and 4% for monthly, and annual scales.Date of celebration: When possible, we provide the exact date of celebration of each rogation on a daily scale. The date recorded in the database is the date when PPR was celebrated. This is not a trivial question, because we had a bureaucratic period of around one week for each rogation (i.e. from the first time that famers requested celebration to the exact date of celebration). Some liturgical acts lasted some days (e.g. Location: We provide the name of the city or town where the PPR was celebrated and an approximate latitude and longitude in the World Geodetic System 1984 (WGS84). The coordinates were approximated to the center of the locations. When two or more rogations are held in the same location , we treat them all as one location, with the same name and geographical coordinates.Type: All the retrieved PPR were referred to drought. We have flagged with an asterisk those celebrations in which the motive is not clearly specified in the document, but the researcher considers that it is caused by drought.Documentary source: There are three fields to describe the documentary source: i) type of source ; ii) details of the source from which data were retrieved; and iii) a reliability categorization of the source .For secondary sources that do not cite properly their primary documentary source or were directly based on secondary sources.For secondary sources based on primary sources, which were cited correctly. These sources were produced by specialized researchers, with a possibility to verify the information from their primary source.Only for primary sources with direct evidence.The validation of our dataset depends largely on the reliability of the documentary sources. For this reason, we calculated a reliability classification, with values ranging from low (1) to high (3) for each record:Our findings reveal that 16%, 68%, and 16% of the PPRs had high, moderate, and low reliability, respectively. However, it should be noted that the level of reliability can be improved, following the consultation of new documents.Another important question in the quality of the dataset is record duplication. When the same PPR date was recorded in more than one source, the record with a higher value in the reliability index was retained, while the other was deleted. When both records had the same reliability index, we merged the records, providing details of both sources for the PPR record. Duplicated records were defined as two PPRs, belonging to the same location, with less than three days of difference. However, this definition extended also to identical dates provided at different temporal resolutions . Figure\u00a039 and for an easy consultation of the dataset, we have created a public web viewer, available at http://inpro.unizar.es/. This viewer allows direct inquiries for any time span between 1333 and 1949. For each location, we provided information about the total number of rogations for the selected period (centre of the circle) and the percentage of rogations in the different seasons: spring , summer , fall , winter , and no seasonal detail (black). When clicking on a location, information about the rogations for that location during the selected time period  is displayed. Also, the complete database can be downloaded. The viewer is a useful tool for a quick screening of drought conditions in a particular location or period. The viewer is a useful tool for a quick screening of drought conditions in a particular location or period.In addition to the figshare repository6. As such, PPRs had a noticeable seasonality, associated with the requirements of soil humidity of the major crops or pastures dominating in each region for a particular time. This notion must be carefully taken into account when comparing information among regions or seasons. This makes the use of this proxy data more complicated on the annual scale.It is important to note that the PPRs are proxies of agricultural droughts and are thus associated with a loss of humidity in soils, which is controlled largely by a lack of precipitationThe quality and homogeneity of the rescued series depend largely on the consulted documentary sources. Accordingly, long, continuous, and reliable series of rogation ceremonies can be very useful to promote our understanding of drought variability in a particular location and for individual dates. Also, the short series of rogation ceremonies can be appropriate to understand the extension or intensity of drought events detected by long rogation series or natural proxies, or to identify extreme droughts."}
+{"text": "Aquaporin 1 (AQP1) is one of thirteen known mammalian aquaporins. Its main function is the transport of water across cell membranes. Lately, a role of AQP has been attributed to other physiological and pathological functions including cell migration and peripheral pain perception. AQP1 has been found in several parts of the enteric nervous system, e.g., in the rat ileum and in the ovine duodenum. Its function in the intestine appears to be multifaceted and is still not completely understood. The aim of the study was to analyze the distribution and localization of AQP1 in the entire intestinal tract of mice. AQP1 expression was correlated with the hypoxic expression profile of the various intestinal segments, intestinal wall thickness and edema, as well as other aspects of colon function including the ability of mice to concentrate stools and their microbiome composition. AQP1 was found in a specific pattern in the serosa, the mucosa, and the enteric nervous system throughout the gastrointestinal tract. The highest amount of AQP1 in the gastrointestinal tract was found in the small intestine. AQP1 expression correlated with the expression profiles of hypoxia-dependent proteins such as HIF-1\u03b1 and PGK1. Loss of AQP1 through knockout of AQP1 in these mice led to a reduced amount of bacteroidetes and firmicutes but an increased amount of the rest of the phyla, especially deferribacteres, proteobacteria, and verrucomicrobia. Although AQP-KO mice retained gastrointestinal function, distinct changes regarding the anatomy of the intestinal wall including intestinal wall thickness and edema were observed. Loss of AQP1 might interfere with the ability of the mice to concentrate their stool and it is associated with a significantly different composition of the of the bacterial stool microbiome. Aquaporins (AQP) are a group of water channels that have been found in many organs and tissues in different species, including humans, animals, plants, and lower organisms ,2,3. ThiAQP1 has foremost been found in the kidney and in the lungs; its primary and best described function is the transport of water ,9,10,11.The hypoxia-inducible factor 1 alpha (HIF-1\u03b1) is known as a key regulator of cellular response to hypoxia and, therefore, plays an important role in the regulation of oxygen homoeostasis. Under hypoxic conditions, HIF-1\u03b1 is upregulated and activates cellular responses to low oxygen levels ,28. ReceThe intestinal microbiota have been increasingly investigated in the past years. It has been shown that many diseases present with a shift in the bacterial population of the intestinal tract, as the gut microbiota play an important role in drug metabolism, prevention of pathogenic bacteria, and immune system maturation ,38. In tTaken together, it appears that the current knowledge on the localization and function of AQP1 in the intestines and the enteric nervous system in mammals remains incomplete. Although the functional impact of AQP1 expression in the intestinal tracts remains unclear, it is possible that AQP1 may inflict on the neuronal function of the enteric nervous system, both under physiological and pathophysiological conditions.This study analyzes the localization of AQP1 in the entire intestinal tract of mice. AQP1 expression correlated with the hypoxic expression profile of the various intestinal segments, as well as with aspects of colon function including the ability of mice to concentrate stools and their microbiome composition.In the first part of the study, we characterized the expression of AQP1 throughout the gastrointestinal tract, the correlating expression of AQP1-regulating and hypoxia-inducible factors, and the resulting changes of the anatomical structure of the intestinal wall in the AQP1-KO animals.In order to characterize the distribution of AQP1 along the intestinal tract, we stained all segments of the intestinal tract by H.E. staining, by staining AQP1 immunohistochemically, and by co-staining AQP1, calretinin, and S100B by immunofluorescence staining. AQP1 has been proven to be positive in the endothelial cells of blood vessels, which we confirmed throughout and which served as an internal positive control in all stainings. We found AQP1 to be positive to some extent in the mucosa throughout all segments of the intestinal tract, although markedly more in the small intestine, as well as in the esophagus. AQP1 expression in the serosa layer was most prominent in the esophagus. Not surprisingly, the submucosal glands were highly positive for AQP1. Furthermore, AQP1 positivity was found primarily in the structures of the enteric nervous system. While the esophagus did not show much expression of AQP1 either in the myenteric or in the submucosal plexus, AQP1 expression was present in the submucosal plexus and the myenteric plexus in all other intestinal organs. However, the submucosal plexus was more prominent in the segments of the small intestine compared to the colon. In the enteric nervous system, AQP1 localizes around the calretinin positive ganglia and colocalizes with some S100B positive nervous fibers. p-value < 0.007). RNA expression of AQP1-KO mice was confirmed to be negative . In all organs, the amount of HIF-1\u03b1 in the AQP1-KO mice was higher than in the WT mice with a similar distribution pattern in the jejunum, ileum, duodenum, colon, cecum, stomach, and esophagus (p < 0.0001 and < 0.0002).Based on the knowledge that AQP1 expression is regulated by HIF-1\u03b1 expression, e.g., under hypoxic conditions, we examined HIF-1\u03b1 mRNA expression in WT and AQP1-KO mice. HIF-1\u03b1 mRNA analysis in the wildtype mice shows the highest amount of HIF-1\u03b1 in the jejunum, followed by a declining amount of HIF-1\u03b1 in the ileum, the colon, the cecum, the duodenum, the stomach, and the esophagus. The expression between the small intestine and the rest of the organs showed a significant elevation (sophagus C. The dip < 0.02). In the AQP1-KO mice the amount of PGK1 was, with the exception of the stomach and the ileum, higher than in the WT mice. This increase was significant in the duodenum and the jejunum (p < 0.0001 and < 0.007).We further investigated the mRNA expression pattern of PGK1, as its expression is also upregulated by HIF-1\u03b1 and can indirectly give a further indication of HIF-1\u03b1 regulation in the absence of AQP1. RNA analysis for PGK1 showed the highest level in the WT mice in the ileum followed by the jejunum, the stomach, the esophagus, the duodenum, and the cecum D. In accp < 0.0001), the duodenum, the jejunum (p < 0.00001), the ileum (p < 0.002), and the cecum. However, in the esophagus and the colon (p < 0.006), the intestinal wall AQP1-KO was thinner compared to the WT mice.A remarkable difference in the intestinal wall thickness was visually obvious between WT and AQP1-KO mice, especially in the colon, and therefore characterized in more detail B. Measurp < 0.012), the jejunum (p < 0.049), and the cecum (p < 0.0004). In the esophagus (p < 0.039) and the colon (p < 0.007) the muscular layer was significantly thinner in the AQP1-KO compared to the WT mice.The measurements for the muscular layer are summarized in p < 0.000001) and the ileum (p < 0.002). The mucosal layer was thinner in the esophagus and the colon of AQP1-KO compared to the WT mice. This difference was significant in the colon (p < 0.0007).With the exception of the esophagus, the mucosa represented the largest part of the intestinal wall in the gastrointestinal tract of WT and AQP1-KO mice E. The muWe observed an increase in the wet-to-dry ratio in all intestinal segments of the AQP1-KO mice, indicating an increased intramural water content in the intestine of AQP1-KO mice F. These In the first part of the study, we found three distinct locations of AQP1 expression in the serosa, the mucosa, and the enteric nervous system, each pointing to a different function of AQP1 in each anatomical aspect or segment. The presence of HIF-1\u03b1 mRNA expression, as well as the mRNA expression of its downstream target PGK1, which were activated as key regulators in response to hypoxia, correlated with the AQP1 mRNA expression pattern in the intestinal segments. A similar pattern was also found for AQP1 protein expression in the immunostaining. Most interestingly, the anatomical changes that we could relate to the AQP1-KO, involved a significantly increased thickness of the mucosal layer in the parts of the small intestine with the highest physiological AQP1 expression and a significantly reduced thickness of the intestinal wall of the colon regarding the mucosal as well as the muscular layers. These findings, together with the expression of AQP1 in close proximity to ganglia and fibers of the enteric nervous system of submucous and myenteric plexus, suggest a functional consequence resulting from the lack of AQP1. Therefore, we investigated some aspects of colon function, including the ability of WT and AQP1-KO mice to concentrate stools, biophysical properties, and the microbiome of their stool.Dry weight of stools was measured for all WT and AQP1-KO mice. Although the results did not show significant differences between the groups, the ability to concentrate stools varied between the WT and AQP1-KO group. Overall, WT mice seemed to be more prone to liquidize their stool. This could be a sign that the passing of stool is normal or facilitated in WT mice, while AQP1-KO might have greater difficulty defecating. This could be a consequence of an impaired water homeostasis of the AQP1-KO mice, or it could have a more complex background.p-value of 0.013.In order to test if the lack of AQP1 in the intestine resulted in changes of the microbiome, the stool of all mice was pooled in groups of WT and AQP1-KO mice and isothermal microcalorimetry was performed. A distinct difference in heat profile of the stool was observed with the AQP1-KO mice showing a decreased heat production rate and decreased total heat B. Metabop < 0.009). The relative abundance in the bacteroidetes  and the firmicutes  were lower in the AQP1-KO compared to the WT mice. Overall, the absolute abundance in the AQP1-KO was higher  than that of the WT mice .To analyze the differences between bacterial composition in the WT and AQP1-KO mice, the microbiome was further investigated on the phylum level E. The reIn the second part we show that the loss of AQP1 in the intestinal tract resulted in changes of anatomical structure of the intestinal wall, which might decrease the ability of the colon to concentrate stool and result in distinct changes of the microbiome.AQP1 expression was prominent in three distinct locations within the gastrointestinal tract of mice, in the serosa, the mucosa, and the enteric nervous system. Each location pointed towards a different possible function of AQP1 in each anatomical aspect or segment. Overall, AQP1 was found in all parts of the gastrointestinal tract but it was more prominent in some regions in certain locations than in other.Expression of AQP1 in the serosa was most prominent in the esophagus, the only gastrointestinal organ that is not covered by a peritoneal sheath. One reason for a prominent AQP1 expression in this part could be an enhanced necessity for AQP1 in the esophageal to act towards water homeostasis. The presumed main function of AQP1 here would be that of a water channel. The expression of AQP1 in the mucosal layer presumably mainly fulfills a water channel function.In the jejunum and ileum, the expression of AQP1 in the mucosa was especially high . This coThe third location, the expression of AQP1 in the cells of the enteric nervous system, was observed throughout the gastrointestinal tract. AQP1 was expressed in the submucosal as well as in the myenteric plexus of the enteric nervous system, suggesting a mechanism by which AQP1 contributes to neuronal function.Throughout the gastrointestinal tract, the expression of AQP1 mRNA also correlated with the mRNA expression of the hypoxia-dependent factors HIF-1\u03b1 and PGK1 . A similLoss of AQP1 in the gastrointestinal tract did not lead to a complete loss of gastrointestinal function. Mice did not show growth deficits or increased occurrence of gastrointestinal disease. However, it led to distinct changes regarding the anatomy of the intestinal wall.Looking at the organs with the highest physiological AQP1 expression, jejunum and ileum, the loss of AQP1 led to a significant increase in the intestinal wall thickness. This suggests that here the main function of AQP1 could be to maintain water homeostasis. One reason for this could be a swelling of the mucosa that occurs in the absence of AQP1. Water could be retained within the intestinal wall leading to a thickening, especially of the mucosal layer of stomach, jejunum, and ileum, probably the segments with the highest water fluctuation. Another explanation could lie in a hypertrophy of the mucosa. In that case, mice would aim to regulate water homeostasis in the absence of AQP1 by an increasing mucosal cell number. It is likely to assume a combination of both.In our wet-to-dry measurements, we observed an increase in the wet-to-dry ratio in AQP1-KO mice in all segments of the intestinal tract. This indicates a disturbance of volume regulation in the absence of AQP1 and is in line with results from a previous study on the lens epithelial water permeability in AQP1-KO mice. Here, an approximately 3% greater basal water content was found in the lens of AQP1-KO mice compared to that of WT mice . It has In the literature there are several references, mostly on traumatic ischemia models, linking increased AQP1 expression with edema. In a rodent testicular ischemia\u2013reperfusion model the increase in AQP1 expression was closely linked to testicular edema after reperfusion . SimilarIn our samples we observed AQP1 expression in WT mice most prominently in the mucosa and in cells of the enteric nervous system. Lack of AQP1 in AQP1-KO mice might therefore not only lead to edema, but also to a disturbance of the enteric nervous system and, possibly as a consequence of both changes, to alterations of smooth muscular contractility.If we look at the colon, in which coordinated muscle activity is most important for passing stools, we found a significant increase in water content in the AQP1-KO mice compared to the WT mice. Hypothesizing that the intestinal edema leads to a reduced contractility of the intestinal muscle, we congruently observed a thinning of the entire intestinal wall including muscle and mucosal layer. Thus, a correlation between intestinal wall edema and wall thickness could be observed. Reduced contractility of the muscle layers could lead to changes of the stool passage time in the colon and thus lead to changes in the stool microbiome.If we look at the colon in particular, we found anatomical changes that we could relate to the AQP1-KO. They included a significantly reduced thickness of the intestinal wall of the colon affecting both the mucosal as well as the muscular layers. As we saw AQP1 expression throughout the intestinal tract in the submucosal as well as in the myenteric plexus of enteric nervous system, there might be a mechanism in which AQP1 contributes to changes in neuronal function resulting in reduced muscular and mucosal thickness in the colon. It has been described that the nerve fibers of the myenteric plexus in particular control the muscle tone and contractions of the intestine and that the submucosal plexus has its main function in controlling the secretion of the mucosa . Gao at How the water channel function, the ion channel function, or a possible gas channel function of AQP1 protein impact these processes in detail remains the focus of further research.Our findings also hint that the ability of the mice to concentrate stool might be compromised, indicating once more an important role of AQP1 in water homeostasis by its water channel function. Our data furthermore showed that the loss of AQP1 expression was associated with a changed composition of microbiota in the stool.Heat production as measured by isothermal microcalorimetry is a measure of the overall metabolic activity, in this case, of bacteria. Our measurements suggested a different bacterial composition of WT and AQP1-KO stool, which could be confirmed by detailed microbiome analysis. Detecting differences in the composition of stool bacteria by measuring heat production, as was proven by the microbiome analysis, could provide the base for a fast and fairly simple differentiation of bacterial composition of stool samples in the future.Alpha diversity of WT and AQP-KO mice do not seem to differ much. Since both indices (Shannon- and Simpson-index) consider richness and abundance of the samples, results must be interpreted with caution. Beta-diversity in our experiment showed a clear shift of the phyla. Earlier studies have already shown that the phylum of bacteroidetes and firmicutes are the two largest phyla in the gut of the mice as well as of humans ,62. In a+/+, 4 KO AQP1\u2212/\u2212 and 1 HT AQP1\u2212/+) and the intestines provided by from the Department of Physiology of the Hannover Medical School . The AQP1-KO mice had been generated from breeding pairs of heterozygous AQP\u2212/+ mice kindly provided by Dr. Alan S. Verkman . Animals were anaesthetized and sacrificed in accordance with the German Tierschutzgesetz \u00a74 2015/222. Organs, specifically the esophagus, stomach, duodenum, jejunum, ileum, cecum, and colon were immediately removed. These organs were separately snap frozen and cryo-conserved. Each organ was embedded in OCT  according to a modification of the \u201cSwiss role\u201d described by Meier-Ruge [Ten mice were bred from a C57BL/6 background  when IHC staining was positive for AQP1, and heterozygous DNA expression was confirmed. Results of this mouse were excluded from all analysis except for the pooled stool analysis.One of the five originally as AQP1-KO classified mice was reclassified as heterozygous . The kit was used according to the R&D System\u2019s protocol, and a rabbit primary antibody against AQP1  was used at a dilution of 1:400. The samples were than counterstained with hematoxylin , mounted with aquatex  and covered up with a Cover-Slip. Every staining was accompanied with a negative control using antibody diluent .Immunofluorescence staining was performed as a triple staining with antibodies against AQP1, calretinin, and S100B. The staining was performed according to a standardized protocol, using the rabbit anti-AQP1 antibody  at a dilution of 1:400, the chicken anti-calretinin antibody  at a dilution of 1:200 and the guinea pig anti-S100B antibody  at a dilution of 1:400. As secondary antibodies, anti-rabbit A488 , anti-chicken A647 , and anti-guinea pig A555  were used at a dilution of 1:2000. Tissue slices were mounted with DAPI . For every sample an additional negative control without the primary antibodies, was carried out.The immunohistochemical and the immunofluorescence stainings were analyzed using an Olympus BX63 microscope with the accompanying CellSens Software (Olympus). To evaluate the intensity of AQP1 expression in the immunohistochemical staining two independent examiners scored representative specimen sections of each mouse and each organ. A scoring system with a range from 0 to 3  was applied. Results of both examiners were correlated. In the few cases of incongruity, the sections were reexamined, and a joined scoring was reached. Furthermore, the immunohistochemical pictures were analyzed with ImageJ-win32 to measure the color intensity of AQP1 for each picture in the wildtype mice. For the measurement a threshold was set by HSB 195\u2013255/30\u2013255/0\u2013200 as a reference to our immunohistochemical staining and the color intensity was measured with this border. The results were depicted as the sum of pixels in each picture according to these settings.n = 4) and WT mice (n = 5) were measured. A total of 4\u20136 measurements were taken from each intestinal segment per mouse.Furthermore, the thickness of the anatomical layers  of the different parts of the intestine was measured using ImageJ-win64. All intestinal samples of AQP-KO mice /dry weight.\u2212\u0394CT method \u00d7 1000.Tissue slices of 50\u00b5m were shredded and lysed in Buffer RLT Plus (QIAGEN). RNA isolation was subsequently performed using the RNeasy Plus Mini Kit  according to the manual. RNA was eluted in 30 \u00b5L or 14 \u00b5L nuclease-free water, respectively. RNA concentration was determined using a Colibri Microvolume Spectrometer (BioConcept AG). cDNA synthesis was performed using the GoScript\u2122 Reverse Transcription System  from up to 200 ng RNA per reaction. A Biometra T-Personal Thermal Cycler was used. Quantitative PCR was performed using the FastStart Universal SYBR Green Master (Rox) . AQP1, HIF-1\u03b1, and PGK1 specific primers  were used for the amplification of cDNA. Per reaction, 5 \u00b5L Master Mix was mixed with primers forward and reversed (0.5 \u00b5M), 1 \u00b5L of cDNA template or water in NTC, and water to a final volume of 10 \u00b5L. PCR reactions were performed in triplicates in MicroAmp\u2122 Optical 384-Well Reaction Plates  in a ViiA 7 Real-Time PCR System (Applied Biosystems) using the associated software. Data were analyzed using the 2For microcalorimetric measurements a differential nanocalorimeter  was used. Stool samples were weighted to ensure that samples of similar weight were processed and to allow for further standardization. They were transferred into 3 mL calorimetric glass vials. Vials were then sealed and inserted according to the manufacturer\u2019s instructions. Sterile glass vials filled with the same weight of sterile saline solution served as reference samples, acting as inert thermal references. Following thermal equilibration, measurements were recorded with the thermostat set at 37 \u00b0C. The microcalorimeter data were sampled at a frequency of 1 data point per second and further resampled to obtain an effective sampling rate of 1 data point every 300 s over >250 h . Data were stored by the TAM assistant software and exported as a CSV file that could be edited in commonly used spreadsheet software. As we did not perform this experiment with biological replicates, statistical analysis was not possible.Before removal of the organ, stools were sampled from all individually caged 10 mice immediately after defecation and put into weighing bottles in Hannover. After determining the wet weights, the stool samples were dried in a drying chamber at 105 \u00b0C for 24 h and weighed again to obtain the stools\u2019 dry weight.t-test if data were normally distributed and the Mann\u2013Whitney test if not. p-values less or equal to 0.05 were considered statistically significant. Graphs were created utilizing Graph Prism 9 software or R, using the R-packages \u201cggplot2\u201d [Data were analyzed using Microsoft Excel, R  and Grapggplot2\u201d  and \u201cggpggplot2\u201d .Bacterial RNA was isolated from the stool samples of each mouse separately. RNA was analyzed though a service by Microsynth AG Balgach Switzerland according to their protocols. Different bacterial RNA were characterized and their relative abundances measured by the company.The biodiversity in the gut microbiota samples was assessed using the Shannon and the Simpson index . The comAQP1 is found in a specific pattern in the serosa, the mucosa, and the enteric nervous system throughout the gastrointestinal tract. Although AQP-KO mice retained gastrointestinal function, distinct changes regarding the anatomy of the intestinal wall were observed. Loss of AQP1 might interfere with the ability of the mice to concentrate their stool and is associated with a significantly different bacterial expression pattern of the stool microbiome."}
+{"text": "In this article the wrong figures appeared due to a database error\u00a0as figures 1 and 2; the updated Figs."}
+{"text": "It was because of our mistakes, we mixed the figures used in our manuscript, and after it was published, we attention that our figures were in the wrong place. Thus, we provide below the new figures with corrected information."}
+{"text": "Calcaneocuboid coverage decreased in plantar and medial regions  and increased in the lateral region . Significant subluxation occurs across the medial regions of the talar head and the plantar medial regions of the calcaneocuboid joint. Coverage and distance mapping provide a baseline for understanding Chopart joint changes in PCFD under full weightbearing conditions.A key element of the peritalar subluxation (PTS) seen in progressive collapsing foot deformity (PCFD) occurs through the transverse tarsal joint complex. However, the normal and pathological relations of these joints are not well understood. The objective of this study to compare Chopart articular coverages between PCFD patients and controls using weight-bearing computed tomography (WBCT). In this retrospective case control study, 20 patients with PCFD and 20 matched controls were evaluated. Distance and coverage mapping techniques were used to evaluate the talonavicular and calcaneocuboid interfaces. Principal axes were used to divide the talar head into 6 regions  and the calcaneocuboid interface into 4 regions. Repeated selections were performed to evaluate reliability of joint interface identification. Surface selections had high reliability with an ICC\u2009>\u20090.99. Talar head coverage decreases in plantarmedial and dorsalmedial (\u2212\u200979%,  Through these structures, most of the pathological features associated with PCFD occur2. Abduction of the midfoot, medial arch collapse, and forefoot varus may have substantial or minor contributions from the talonavicular and the calcaneocuboid joint5. As PTS occurs in PCFD, structures distal to an initially fixed talus are expected to deviate dorsolaterally, contributing substantially to the described deformities7. Prior work attempted to assess these behaviors using various methods like simulated weight-bearing computed tomography (WBCT) and fluoroscopy, finding abduction and eversion but conflicting results regarding plantarflexion through these joints10.The transverse tarsal joint complex (Chopart articulations) is a key element of the peritalar subluxation (PTS) seen in progressive collapsing foot deformity (PCFD)12. Using two-dimensional tools in coronal plane imaging, the amount of subluxation at the posterior and middle facets was found to be correlated with PCFD diagnosis and severity14. Dibbern et al. performed an objective three-dimensional (3D) WBCT analysis of the subtalar joint in PCFD using 3D distance maps (DMs) and introducing the concept of coverage mapping (CM)15. They showed that subluxation of the calcaneus underneath the talus was more prominent in the middle facet than in the posterior facet of the subtalar joint, while simultaneously identifying decreases in interbone distance in the sinus tarsi and subfibular regions, explaining lateral impingements in PCFD17.The recent use of WBCT to evaluate PTS has produced important data to help understand this pathological functioning9. Information provided by the 3D mapping specifically related to plantarflexion and subluxation may be of particular value in diagnosing, staging, and estimating treatment impacts in PCFD. Therefore, the objective of this study was to compare distance and coverage map differences between loaded Chopart joints of PCFD and control patients using full weightbearing CT. We hypothesized that a significant amount of decreased articular coverage, indicative of subluxation, would be present in the talonavicular and calcaneocuboid joints in PCFD compared to controls. We further hypothesized that medial widening and lateral narrowing of intra-articular distances would be observed in the talonavicular and calcaneocuboid joints consistent with subluxation. Finally, we sought to understand whether present gold standard methods for selection of articular surfaces are reliable for use in understanding joint interaction.These CM and DM techniques may help improve understanding of bone positioning and interactions through the Chopart complex in PCFD as previous research has not directly assessed the articular interfaces of the talonavicular and calcaneocuboid joints under physiological load18.This retrospective case control study obtained University of Iowa\u2019s institutional review board approval (IRB# 201904825). It complied with the both the Health Insurance Portability and Accountability Act (HIPAA) and the Declaration of Helsinki. Informed consent was obtained from all subjects. This manuscript follows the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE case\u2013control) guidelines 18.19. Patients were excluded if they were found to have a rigid deformity at physical examination, any prior PCFD surgery or metallic implants deterring visualization of the first and fifth rays. Class E deformities  were also excluded19.The first 20 patients with a PCFD were selected from a randomized list to have undergone a WBCT at our institution between 2014 and 2021. Adults (over 18\u00a0years old) with clinical diagnosis of PCFD were included. Patients presenting with a stage 1 (flexible) class A, B, C, D, or a combination of classes were admitted20.A matched control group of 20 feet was selected from adult volunteers that underwent WBCT. Individuals were excluded if they had any hindfoot complaint (current and prior), signs of any deformity or arthritis  noticeable during imaging assessment. A foot and ankle offset (FAO) bellow 5.2% was required for this group of patientsWBCT acquisition was conducted with patients instructed to bear weight in a natural, upright standing position with feet approximately at shoulder width to distribute weight evenly between their two lower limbs. Studies were performed with a cone-beam computed tomography (CT) scanner .15.The 3D boundaries of the talus, calcaneus, navicular, and cuboid were extracted from WBCT images using an automated segmentation protocol . Resulting surfaces were exported as triangulated surface models to Geomagic Design X (3D Systems). Articular facets were selected in Geomagic Design X in two separate trials by the same reader21. The cuboid facet of the calcaneus was similarly divided into quadrants: medial, superior, lateral, and inferior. Measurements performed in articular areas were defined using the distance along the normal direction of vectors projected from the subchondral bone of the hindfoot  to their respective midfoot counterparts (the posterior facets of the navicular and cuboid).Distance measurements were performed along the talar head (articular surface for the navicular) and the articular surface for the cuboid on the calcaneus. Detailed regional analysis was conducted by dividing the talar head into six subregions Fig.\u00a0 using th22. Other previous studies have used thresholds of four millimeters in joints in the foot23. We wanted to ensure adequate characterization of coverage in the joint; the most prudent way to do so was to raise the coverage threshold by one millimeter. Since the method of distance measurement was based on the normal vectors, most regions that were uncovered were defined as such due to the lack of intersection of the normal vector with the opposing bone, not due to being greater than the five-millimeter threshold.Coverage percent was calculated by finding both the total area of each division and the total area of the division that was covered. The area covered divided by the total area of the division yields the coverage percent for that division. We defined coverage as anywhere on the joint that had a joint space width (JSW) of less than five millimeters. We chose a threshold of five millimeters in order to capture all possible parts of the joint that could be considered covered. Lintz et al., reports distances of approximately two millimeters in the talonavicular joint15. Pink was chosen to highlight uncoverage of articular regions, as a result of the overall 3D deformity in PCFD, that were either completely uncovered or had distances greater than 5\u00a0mm. Blue indicated coverage of the joint with less than 5\u00a0mm distance between bones  facetnes Fig.\u00a0.Figure 224. Finally, a repeatability study was performed to quantify variability in articular selections on the calcaneus and talar head. Models were recreated and the joint area was selected on each bone.The talonavicular coverage angle (TNCA) was obtained using the segmentation and the automatic angle tool based on two-dimensional (2D) projections of 3D axesP values for data sets that were normally distributed were calculated using a two-tailed Student\u2019s T-test; P values for data sets that were not normally distributed were calculated using the Mann\u2013Whitney-Wilcoxon U test. Pearson correlation was utilized for correlation between variables. Intraclass correlation coefficients were computed to evaluate interrater selections of the articular areas.Statistical calculations were performed in MATLAB and Excel using Visual Basic for Applications. Normality was determined using the Shapiro\u2013Wilk Test. This work was conducted under University of Iowa\u2019s institutional review board approval (IRB# 201904825). Subjects signed an informed consent prior to inclusion.The author affirms that this manuscript is an accurate, honest, and transparent account of the study reported; that no important aspects of the study were omitted; and that any discrepancies from the study as planned  were carefully explained.P\u2009=\u20090.80), sex (P\u2009=\u20091.00), and BMI distributions (P\u2009=\u20090.40)  and dorsal medial regions . Coverage also decreased in PCFD for both the dorsal middle  and plantar middle (\u2212\u200926% p\u2009=\u20090.003) regions. Corresponding increases in coverage were seen in both the dorsal lateral  and plantar lateral  regions of the PCFD group. Overall coverage of the talar heads was similar among groups (p\u2009=\u20090.22). CMs for every joint are shown in Fig.\u00a0Significant decreases in coverage were seen in all middle and medial regions of the talar head of PCFD patients in comparison to controls Fig.\u00a0. The larp\u2009=\u20090.002) but not in the dorsal  region of PCFD patients. A significant decrease in coverage was observed in both the plantar  and medial  regions. Changes in overall coverage of the calcaneocuboid facet were not significant (p\u2009=\u20090.649) when comparing the studied groups. TNCA had a mean of 31.52\u00b0 (SD\u2009+\u2009\u2212\u20096.78) in controls and 45.13 (SD\u2009+\u2009\u2212\u20097.08) in PCFD patients (p\u2009<\u20090.001). Talonavicular coverage was negatively correlated (r\u2009=\u20090.75) and influenced (R2\u2009=\u20090.57) by the TNCA . Parallelly, the lateral regions on the talar head experienced an increase in coverage when compared to the controls . The changes in coverage on the plantar and dorsal subregions of the talar head were similar eliminating pure plantarflexion as the cause of this subluxation. This may indicate a tendency toward medial abduction and external rotation of the navicular as the root cause of these changes in coverage. These findings are in line with work by Louie et al., who was also able to identify a medial to lateral shift in coverage (p\u2009<\u20090.0001)8. However, Louie et al. found more pronounced plantar uncoverage than our cohort, potentially explained by differences in imaging acquisition (simulated weight-bearing CT and WBCT)8. Kitaoka et al. using cadaveric analysis, demonstrated a shift for a more central and dorsal contact distribution in PCFD patients5. Further, Malakoutikhah et al. observed a decrease in overall contact pressure and subluxation of the talonavicular joint when their finite model was collapsed10. These could contribute to the understanding that the deformity has a complex out of plane rotational component rather than a simple sagittal and axial movement26. Phan et al., using dual fluoroscopy, were able to observe similar behavior at the talonavicular joints of flatfoot patients, demonstrating increasing abduction  and external rotational  in comparison to controls9.In patients with PCFD, subluxation occurred on both the talar head and the calcaneocuboid facet. Significant subluxation was noted on the medial side of the talar head in patients with PCFD, specially on its plantar aspects  and medial  subregions in patients with PFCD. Increases in coverage on the lateral and dorsal subregions occur, but only the lateral increase in coverage was significant . Comparable comportment was observed by Phan et al. at the calcaneocuboid joints of flatfoot with higher external rotation movement . The fact that PTS produces instability at the subtalar joint and the calcaneus also moves around the talus may explain why coverage changes were not as large on the calcaneal cuboid joint. Similarly, Wang et al. noticed lesser movement at the calcaneocuboid joint in comparison to the talonavicular and subtalar joints27. The study demonstrated 3.93\u00b0, 5.04\u00b0 and 5.97\u00b0 of dorsiflexion; 5.82\u00b0, 8.21\u00b0, and 15.46\u00b0 of eversion; and 9.75\u00b0, 7.6\u00b0, and 4.99\u00b0 of external rotation in normal feet during midstance in CC, talonavicular, and subtalar joints, respectively27.On the calcaneocuboid facet, significant subluxation occurred at the plantar  in our study. This is similar to what Louie et al. reported, finding similar overall coverage of the talus (62% vs. 56%) and navicular (98% vs. 92%) when comparing symptomatic flatfoot and neutral aligned subjects through CM. This is likely due to PTS increasing coverage and contact in some areas and decreasing by similar amounts in others thus providing a mean neutral value23. Another argument, raised by Louie et al., is that some of the PCFD can be secondary to a pediatric flatfoot and present dysplastic alterations to bone and joint that could create abnormal cartilaginous relations in subluxed areas29. The last possibility is that even in full weightbearing conditions, early PCFD may not experience true subluxation through the Chopart articulation. In this scenario hindfoot PTS, forefoot deformity, and ligamentous laxity cause changes through the midfoot that are within the compliance of highly mobile articulations.Differences in overall coverage were not observed in either joint when comparing PCFD and control patients  and increases superomedially  and inferomedialy  talonavicular regions30. Similar to our study, no changes in distance were observed by this study at the calcaneocuboid joint30.This explanation is supported by the absence of differences seen among PCFD and controls in the overall and regional distance mapping (ps\u2009>\u20090.224). Since our sample is composed of flexible (stage 1) middle-age PCFD patients (mean 49.5\u00a0years old), early signs of joint degeneration (narrowing) would not be expected. In contrast to the subtalar joint where forces applied to the region are primarily vertical, making impingement  a valuable marker of topography, the tarsal joints present perpendicular to gravity resulting in increased potential for shearing and decreased potential for static extraarticular impingement2, respectively. Compared to the average area of the articular surfaces, the mean of differences between each trial averaged 9.4\u2009\u00b1\u200938.1\u00a0mm2. The average difference between the two selections was at most 10% of the overall area. These differences are negligible relative to the magnitude of differences seen in the overall talonavicular and calcaneocuboid joints; they are likely to average out over a population. However, increased reliability may be important to consider when looking at subregional analyses of individual cases where local variance in selection may impact results more dramatically. Therefore, automated methods are desirable to increase reliability before considering these results in the context of individual cases.To account for potential differences in coverage derived from variance in selection, we evaluated the current gold standard of manual selection at two time points. Surface selections had a high reliability with an ICC greater than 0.99. The mean areas of the for the cuboid and talar head articulations were 433.7 and 947.6\u00a0mmThis study has several limitations. As a retrospective study, it could not evaluate the linear progression of the disease. Additionally, patients were not followed over time to identify changes secondary to PCFD. The study\u2019s findings cannot be applied to class E  and stage 2 (rigid) subjects which may involve later stage deformity. The matched control group consisted of a heterogeneous group of healthy volunteers. Although we observed statistically significant differences, previous sample calculations or power analysis were not performed. This could have underestimated the changes and contributed to similarities in overall coverage and distance mapping. Patient functional assessment was not executed, preventing correlation between symptoms and imaging findings. Finally, the use of WBCT and 3D coverage and distance mapping are still not widely accessible, decreasing the study\u2019s reproducibility.In conclusion, our study results show that significant subluxation occurs on the medial region of the talar head and the plantar medial regions of the calcaneocuboid joint. To our knowledge, this is the first study to assess subluxation across the entire Chopart joint under full weightbearing conditions. These results provide a baseline to understand changes occurring at the transverse joint of PCFD patients, providing data that might help in disease management. Coverage and distance mapping provide objective information that could lead to earlier diagnosis and better assessment treatment impact when reestablishing joint interfaces. Future research is needed to increase reliability through automation and continue the search for more complete understanding of physiological bone congruence and changes associated with collapse and to halt articular degeneration in PCFD.Supplementary Information."}
+{"text": "Multicentre-observational study.The 6-minute walk test (6mWT) is an established assessment of walking function in individuals with spinal cord injury (SCI). However, walking 6\u2009min can be demanding for severely impaired individuals. The 2-minute walk test (2mWT) could be an appropriate alternative that has already been validated in other neurological disorders. The aim of this study was to assess construct validity and test-rest reliability of the 2mWT in individuals with SCI. In addition, the influence of walking performance on sensitivity to change of the 2mWT was assessed.Swiss Paraplegic Center Nottwil, Switzerland; Balgrist University Hospital, Z\u00fcrich, Switzerland.Fifty individuals (aged 18\u201379) with SCI  were assessed on two test days separated by 1 to 7 days. The first assessment consisted of a 2mWT familiarization, followed by a 2mWT and 10-meter walk test (10MWT) ) in randomized order. The second assessment consisted of 2mWT and 6mWT in randomized order. Tests were separated by at least 30\u2009min of rest.r\u2009=\u20090.980, p\u2009<\u20090.001). The 2mWT correlated very strongly with the 6mWT  and the 10MWT , and moderately with the WISCI II . Sensitivity to change was slightly affected by walking performance.The interclass correlation coefficient between the 2mWT assessed on the first and second test day was excellent (The 2mWT is a valid and reliable alternative to the 6mWT to measure walking function in individuals with SCI.NCT04555759. Almost half of all spinal cord injuries (SCI) are functionally incomplete , 2, meanOne of the most established assessments of walking function in individuals with SCI is the 6-minute walk test (6mWT) which measures the distance an individual is able to walk within 6\u2009min. The 6mWT is widely used to measure endurance, fatigability, and cardiovascular fitness , showingVariability in gait performance is expected to be greater in individuals with a lower walking function ; for exar\u2009=\u20090.8\u20130.99), and (5) the distance walked per minute will decrease over time in the 6mWT but not the 2mWT.We hypothesize that (1) the 2mWT will show a very strong correlation of 0.8\u20130.99 with the 6mWT, (2) the 2mWT will show a good to excellent test-retest reliability, ICC higher than 0.8, (3) the SEM and the MDC are influenced by the walking function, (4) the walking function has no influence on the correlation between the 2mWT and the 6mWT  in <60\u2009s), and ability to walk without physical assistance. We excluded individuals who walked <60 meters in 6\u2009min due to a potential high variability of walking performance as this may occur in individuals with poor walking function . The excThis study was approved by the Ethical Committee of the Canton of Zurich and the Ethics Committee for Northwest/Central Switzerland (BASEC 2020-01473) and was conducted in accordance with Good Clinical Practice guidelines and the Declaration of Helsinki. Prior to enrollment, written informed consent was obtained from all participants. Individuals were invited to participate on two test days, separated by 1\u20137 days. A maximum of 7 days has been chosen because it can be assumed that the walking function remains stable during this time period. On the first day, a familiarization run of the 2mWT was performed. After a break of at least 30\u2009min, the 2mWT and the 10MWT were performed in randomized order, again separated by at least 30\u2009min of rest. The WISCI II was applied and scored based on the performance in the 10MWT. The second testing day consisted of 2mWT and 6mWT, performed in randomized order and separated by a 30\u2009min break.Examiners from both centers were trained all together in performing the assessments to optimize the standardized collection of all outcome measures.The walk tests were performed according to the Guidelines of the American Thoracic Society , except Any braces and/or habitual assistive devices were permitted but needed to be kept consistent on both testing days. Subjects were instructed to walk as far as possible, but safely, during the respective test time of 2 or 6\u2009min. Standardized instructions were read out to the participants. Every minute during the test, they were informed about the remaining time and were encouraged to keep up the good work. No other communication occurred during the test.Subjects were allowed to take rest breaks if needed, but time continued to run during the break.r\u2009=\u20090.86) [The 6mWT has shown very good construct validity (10MWT: \u2009=\u20090.86)  in indivThe 10MWT was performed with a flying start, i.e., subjects were instructed to walk a total distance of 14 meters including two meters to accelerate and two meters to decelerate. Time was measured for the middle ten meters and rounded to the next tenth of a second.Participants performed a total of four 10MWT runs: two at their preferred walking speed (10MWT self), and two at their maximal walking speed (10MWT max), i.e., as fast as possible, but still safe. The average of the two runs each was taken as the final result for each variant.r\u2009=\u20090.89; 6mWT: r\u2009=\u20090.95) [The 10MWT has shown very good construct validity (TUG: \u2009=\u20090.95)  and exce\u2009=\u20090.95) ) in indiThe WISCI II is an ordinal scale to assess walking capacity . It captFor the present study, the WISCI II was scored based on the performance of the 10MWT.The WISCI II has shown good construct validity  .The sample size of 50 was chosen based on the COSMIN guidelines .There was no missing data as, in accordance with the study protocol, the three drop-outs have been replaced. No further cleaning was necessary since all datasets were complete and plausible. Data were analyzed using RStudio for Mac, version 1.3.1093. Descriptive data are reported using mean\u2009\u00b1\u2009standard deviation or median (range).Data were tested for normality using the Shapiro\u2013Wilk test. If data were normally distributed, construct validity was tested using the Pearson correlation. Otherwise, a Spearman rank correlation was applied.R) were used for the interpretation of the strength of the association: 0.00\u20130.19: \u201cvery weak\u201d, 0.20\u20130.39: \u201cweak\u201d, 0.40\u20130.59: \u201cmoderate\u201d, 0.60\u20130.79: \u201cstrong\u201d and 0.80\u20131.0: \u201cvery strong\u201d.According to the guide from Evans et al. , the folBased on studies in other neurological populations \u201316, a \u201cvTest-retest reliability was determined by calculating the interclass correlation coefficient (ICC)  between the 2mWT from the first and the second test day . To inteIn addition, a Bland\u2013Altman plot was created to estimate the absolute agreement of the two measurements of the 2mWT.The SEM SD\u00d71\u2212ICC  and the M\u00d71.96\u00d72  were calt-test was used to compare the distances walked in the 2mWT in the first and the 2nd\u2009min. Moreover, a Friedman test was applied to compare the minute intervals of the 6mWT with paired post-hoc Wilcoxon Tests (Bonferroni corrected), if applicable. A significance level of p\u2009<\u20090.05 was selected.A paired Although the maximal time window for the two test days was narrow with 7 days it cannot be excluded that individuals with a sub-acute SCI could show a spontaneous improvement in walking performance within this time window. Therefore, the study sample was divided into an acute/sub-acute (0\u20136 months after injury) and a chronic (>6 months after injury) SCI group.To investigate whether SEM and MDC are influenced by the walking performance, the study sample was separated into slow and fast walkers. Community ambulation is often measured at crosswalks, where speed is the primary concern . In the Two subgroup analysis were performed: (1) the influence of time since injury on construct validity and test-retest reliability and (2) the influence of walking performance on SEM and MDC was analyzed, each by creating two subgroups.Fifty-three individuals with SCI were recruited for this study. Three individuals had to be excluded, two due to medical reasons (after screening) and one was not able to complete the 6mWT due to pain during walking. As specified in the protocol, these drop-outs have been replaced, resulting in the a priori targeted sample of 50. Descriptive details about the participant characteristics can be found in Table\u00a0The 2mWT showed very strong correlations with the 6mWT . The Bland\u2013Altman plot . The SEM and the MDC are presented in Table\u00a0The walking distance of the 2mWT on the first and second test day showed a very high ICC .The mean distance walked for the 2mWT was 114.1\u2009\u00b1\u200953.3 meters on the first day and 118.8\u2009\u00b1\u200954.0 meters on the second day. For the 6mWT, the average walking distance was 338.2\u2009\u00b1\u2009165.8 meters. Walking distances during 1-min intervals of the 2mWT were not different (Day 1: p\u2009<\u20090.001), with all intervals being significantly different from the first minute except for the sixth minute , since the velocity in the 6mWT is lower than in the 2mWT.In the 6mWT, minute intervals differed significantly . Correlation coefficients of the 2mWT with the other assessments can be found in Table\u00a0The fast walkers walked on average more than twice as far as the slow walkers in the 2mWT  and the 6mWT . The correlation between the 2mWT and the 6mWT remained, even when splitting into subgroups (p\u2009<\u20090.001)). Results of the different gait tests divided into the two subgroups are presented in Table\u00a0In addition, test-retest reliability was preserved for each subgroup   Fig.\u00a0.The calculation of SEM and MDC for the subgroups showed that these values are influenced by gait performance. The values for the slow walkers are slightly higher than for the fast walkers  one block (~80 meters) 1 year post injury . Hence, The main findings of the present study are that in individuals with SCI (1) the walking distance assessed in the 2mWT and the 6mWT are highly correlated, (2) the 2mWT has an excellent test-retest reliability, (3) SEM and MDC are different for fast and slow walkers, (4) the walking function has only a minor effect on the correlation between the 2mWT and the 6mWT, and (5) the 2mWT cannot capture exhaustion.It has been shown that the 2mWT correlates strongly with the 6mWT in individuals with neuromuscular disease , multiplIt should be noted that generally only final distances  are documented for these tests, e.g., during clinical routine, but also most of the research reports. If this is the case, the 2mWT is a valid alternative to the 6mWT to describe walking function in individuals with SCI, given the very high correlation coefficients for the overall patient sample, but also for the slow and fast walker subgroups separately.The subgroup analysis further revealed that the test-retest reliability of the 2mWT is slightly lower in the slow walkers since they tend to show more day to day variability in their walking performance. The slightly lower ICC of the slow walkers is also reflected in a slightly increased SEM and MDC. This underlines that it is important to calculate SEM and MDC separately for slow and fast walkers in functional tests.In this study, we investigated test-retest reliability by comparing the distance of 2mWTs that were performed 1\u20137 days apart. Previous studies have shown that there is a learning effect in repeated walking tests . To prevWe excluded individuals with a walking distance <60 meters in 6\u2009min. This is a limitation of the study since our results may not apply to individuals with more severely impaired walking function.Moreover, we used a hallway length of 35 meters instead of the 30 meters suggested in the Guidelines of the American Thoracic Society . HoweverIn the present study we did not consider all aspects of responsiveness  but the proper assessment of responsiveness would be critical in order to use the 2mWT to measure the potential impact of interventions on walking function over time in individuals with SCI. We can see from our data that the 2mWT will be more responsive in detecting walking function improvements in fast walkers, as they show less day to day variability and therefore smaller SEM and MDC than in slow walkers. Further research is required to determine all aspects of responsiveness of the 2mWT in measuring change over time or effects of interventions in individuals with SCI.Reliable, valid, and practicable assessments of walking function that are easy to use are of great importance, in the clinic and in research. This study demonstrated good to very good construct validity and excellent test-retest reliability of the 2mWT in individuals with SCI. Based on these findings, the 2mWT can be suggested as a suitable alternative to the 6mWT in individuals with SCI comparable to the investigated group. Its short application time may allow clinicians and researchers to measure walking function efficiently.Supplementary Table S1"}
+{"text": "Dear Editor,1We reported a new role of epithelial cell adhesion molecule (EpCAM) on maintaining the longevity of intestinal epithelial cells (IECs) via regulating the TSC1/mTORC1/Sirtuins pathway. EpCAM has been used as a cell surface marker of various cancer tissues and many kinds of stem cells,\u2013/\u2013 mice were first compared. Similar to previously reported,\u2013/\u2013 pups showed no bodyweight gain, serious diarrhoea and damaged intestines  and EpCAM Figures\u00a0, S1A\u2013C.  Figures\u00a0, S1F. Th\u2013/\u2013 mice compared to the WT controls  and telomerase RNA component (TERC), components of Telomerase, Figures\u00a0, S2A\u2013C.  Figures\u00a0, S2D. 53n Figure . TERT ans Figure . Howevers Figure . These r\u2013/\u2013 Caco\u20102 cells , marked by the increase of \u03b3H2AX,s Figure . These r\u2013/\u2013 Caco\u20102 cells (Figure \u2013/\u2013 pups, the expression of Sirtuins significantly increased in the small intestines of them (Figures\u00a0Tert in the intestines of mutant pups (Figures\u00a0\u2013/\u2013 mice.To explore the mechanisms of EpCAM deficiency on accelerating the aging of IECs, members of Sirtuin familys Figure . After a Figures\u00a0, S6B. MD Figures\u00a0, S6C. \u03b3H Figures\u00a0, S6D. Th\u2013/\u2013 pups (Figures\u00a0\u2013/\u2013 Caco\u20102 cells, and the ratios for p\u2010mTOR/mTOR and p\u2010S6/S6 remarkably increased in EpCAM\u2013/\u2013 cells (Figures \u2013/\u2013 IECs. Rapamycin was administrated to EpCAM\u2013/\u2013 pups to reduce the hyperactivation of mTORC1 in IECs of them (Figures\u00a0\u2013/\u2013 pups was slightly improved after administration of rapamycin (Figure\u00a0Tert and Sirtuins and reduced the level of \u03b3H2AX in EpCAM\u2013/\u2013 Caco\u20102 cells (Figures \u2013/\u2013 pups (Figures\u00a0To study signal pathways related to the premature aging of IECs caused by EpCAM deficiency, the mTORC1 pathway Figures\u00a0, S7A. TS Figures , S8A. Th Figures\u00a0, S7B. Thn Figure\u00a0. The pron Figure\u00a0, S7C. Thn Figure\u00a0, S7C. Ra Figures , S8B\u2013D.  Figures\u00a0, S7D, inIn summary, EpCAM deficiency causes the premature aging of IECs via TSC1/mTORC1/sirtuins pathway Figure\u00a0. TherefoThe authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.Figure S1 EpCAM was successfully knockout in the intestinal epithelial cells both in vivo and in vitroFigure S2 EpCAM deficiency affected the compositions of telomerase and telomeres in the intestinal epithelial cells both in vivo and in vitroFigure S3 EpCAM deficiency caused accumulation of unrepaired DNA double\u2010strand breaks in caco\u20102 cellsFigure S4 EpCAM deficiency affected the expression of members of sirtuin family in the intestines of E18.5 embryonic mice and caco\u20102 cellsFigure S5. Hyperactivation of mTORC1 induced the premature aging of EpCAM\u2010/\u2010 Caco\u20102 cellsFigure S6 Quantitative analysis of the western blot results in Figure 3Figure S7 Quantitative analysis of the western blot results in Figure 4Figure S8 Quantitative analysis of the western blot results in Figure S5Table S1 Sequences of primers used for qPCRTable S2 The primary and secondary antibodies used for western blotClick here for additional data file."}
+{"text": "Formulaic language is a general term for ready-made structures in a language. It usually has fixed grammatical structure, stable language expression meaning and specific use context. The use of formulaic language can coordinate sentence generation in the process of writing and communication, and can significantly improve the idiomaticity and logic of machine translation, intelligent question answering and so on. New formulaic language is generated almost every day, and how to accurately identify them is a topic worthy of research. To this end, this article proposes a formulaic language identification model based on GCN fusing associated information. The innovation is that each sentence is constructed into a graph in which the nodes are part-of-speech features and semantic features of the words in the sentence and the edges between nodes are constructed according to mutual information and dependency syntactic relation. On this basis, the graph convolutional neural network is adopted to extract the associated information between words to mine deeper grammatical features. Therefore, it can improve the accuracy of formulaic language identification. The experimental results show that the model in this article is superior to the classical formulaic language identification model in terms of accuracy, recall and F1-score. It lays a foundation for the follow-up research of formulaic language identification tasks. Formulaic language identification, also known as multi-word expression identification, refers to finding formulaic language that meets a certain definition from a large amount of corpus, and is also an essential task in natural language processing. The use of formulaic language can not only coordinate sentence generation in the process of writing and communication, improve the accuracy and fluency of language expression, but also have important theoretical and practical significance for computer-assisted language teaching and machine translation\u00a0 between words and basic features into the Graph Convolutional Network (GCN) for feature representation. The obtained features can effectively represent the grammatical structure of formulaic language so that the relationship between words can be fully utilized. Finally, the high-order neighbor information between words can be captured. \u2022The high-order neighbor information is input to the CRF layer for decoding so that the problem of formulaic language recognition is regarded as a sequence labeling problem. This method can obtain the label category of each character, achieve the purpose of recognizing formulaic language, and provide a new idea for solving formulaic language recognition problems. \u2022We designed three groups of comparative experiments, and the experimental results proved the effectiveness of the proposed model. This model can recognize formulaic language in the text, which lays a foundation for expanding formulaic language corpus in machine translation.The rest of this article is organized as follows. Section 2 reviews related work. Section 3 describes a formulaic language identification model based on GCN fusing associated information, including basic feature extraction module, feature extraction module fusing associated information and label representation module. Section 4 verifies the effectiveness of the model through experimental analysis. Section 5 summarizes the work of this article and tells the shortcomings and future research directions.At present, the research on formulaic language is either descriptive research based on the corpus or confirmatory research for language acquisition. There are very little researches related to the recognition of formulaic language. In fact, formulaic language recognition is the basis of applied research on formulaic language\u00a0. Howevert-value, dispersion rate, criticality, etc.\u00a0, Support Vector Machine (SVM), Conditional Random Field (CRF), Maximum Entropy (ME) and so on are used by researchers to recognize formulaic language.\u00a0etc.\u00a0 proposedWith the development of deep learning,\u00a0The recognition method based on statistics can effectively recognize formulaic language with high frequency of co-occurrence. However, its main problem is that it does not consider the meaning of multi-word combinations and only takes frequency or correlation as the basis of discrimination, so the accuracy of recognition is low. The rule-based recognition method can accurately identify the formulaic language whose form is consistent with the rules defined in the model, which has the problem that the rules are not comprehensive enough. With the development of machine learning, some scholars try to use classifiers such as RF and SVM to recognize formulaic language through classification technology. However, it is necessary to extract features that can represent samples\u00a0. The morTo sum up, aiming at the problem of formulaic language recognition, this article proposes a formulaic language identification model based on GCN fusing associated information. This model represents the feature vector through word embedding technology, integrates the associated information that can represent the characteristics of formulaic language, and uses GCN to obtain deeper semantic features. The features obtained in this way are closer to the formulaic language than the features extracted manually in machine learning. Finally, considering the dependency between tags, CRF is used to decode tags to achieve the purpose of recognizing formulaic language. This model not only takes into account the frequency of word co-occurrence, but also considers the dependencies of words in sentences, and achieves good results in formulaic language recognition.The model in this article is mainly divided into three parts: basic feature extraction module, feature extraction module fusing associated information and label representation module. Its overall structure is shown in In the process of natural language processing, the computer cannot directly use text data. The text data needs to be expressed as a feature vector, and then the feature vector is used as the model\u2019s input. This article uses the word embedding vector generated by the embedding layer in Torch as the part-of-speech feature, the feature vector trained by GloVe word vector technology as the semantic feature, and the late fusion of the part-of-speech and semantic features as the basic features of the model.The most significant difference between formulaic language and general multi-word expression is that the structure of formulaic language is mostly fixed, which is often in the form of Verb-noun or subject\u2013predicate\u2013object. Therefore, part-of-speech features are regarded as one of the features to identify formulaic language. First, we use the Stanford part-of-speech tagger to analyze the text. The examples of part-of-speech analysis results are shown in In addition, for text processing, characters, words, phrases more reflect the lexical information of the text rather than its semantic information, so they can not accurately express the content of the text. For formulaic language, it is a multi-word unit with high frequency and has complete structure, meaning and function, so semantic feature is also an essential feature of formulaic language. This article uses the feature vector trained by GloVe to represent the semantic features of formulaic language.GloVe\u2019s full name is Global Vectors for Word Representation. It is a word representation tool based on global word frequency statistics. It can express a word as a vector composed of real numbers. These vectors capture some semantic characteristics between words\u00a0. Its impX is constructed according to the corpus. Each element Xij in the matrix represents the number of times that word i and context word j appear together in a context window of a specific size. Generally speaking, the minimum unit of this number is 1, but GloVe doesn\u2019t think so: according to the distance d between two words in the context window, it proposes a decreasing weighting function: decay\u00a0=\u00a01/d to calculate the weight. That is, the farther the distance, the smaller the weight of the two words in the total count.(1) A co-occurrence matrix (2) Construct the approximate relationship between Word Vector and Co-occurrence Matrix, as shown in bi and Among them, the (3) Construct the loss function, as shown in Among them, xrepresents the number of co-occurrences, and xmax represents the maximum number of co-occurrences.Among them, Because LSTM is good at capturing the long-distance and long-term dependence of sentence context information, it can better avoid the problem of gradient disappearance and gradient explosion and has higher computational efficiency. Nevertheless, LSTM cannot capture the two-way information of sentences. For the task of formulaic language identification, if the forward information and backward information of sentences are added, the model can learn more semantic information when processing text\u00a0. Therefosi\u00a0=\u00a0, input it into the Bi-LSTM network, we can get the hidden layer representation si. Each unit obtains the current hidden vector ht according to the calculation of the previous hidden vector ht\u22121 and the current input vector xt, and its operation is defined as follows:For sentence it, ft, ct, ot, ht are the state of input gate, forget gate, cell state, output gate and hidden layer when the t-th text is input; W is the parameter of the model; b is the bias vector; \u03c3 is the Sigmoid function; tanh is the hyperbolic tangent function.In the formula: Bi-LSTM model is composed of forward LSTM and reverse LSTM models. The LSTM network of each layer outputs a hidden state information respectively, and the parameters of the model are updated by back propagation. The structure of Bi-LSTM model is shown in t represents the input of the network at time t, the LSTM in the box is the standard LSTM model, t, and t.\u2295 indicates splicing operation. The output representation of Bi-LSTM at time t is defined as t is directly spliced by the forward output and the reverse output.In Feature fusion includes early fusion and late fusion. Early fusion is to fuse multi-layer features first and then train the model on the fused features . In this article, early fusion is used as a comparative experiment. First, the part-of-speech features and semantic features are fused, and then the fused features are input into Bi-LSTM. The generated results are used as the basic feature vector. The structure diagram is shown in Compared with early fusion, late fusion first trains the model with a single feature and then fuses the training results of multiple models. The advantage of late fusion method is that it can flexibly select the model\u2019s results and improve the fault tolerance of the system. The amount of calculation of fusion information is reduced and the real-time performance of the system is improved\u00a0. In thisThe basic feature extraction module uses the word embedding model to train in large-scale text. It can obtain word vectors rich in text semantic features and part-of-speech features, but the syntactic structure information in the text is ignored. As the basis of language understanding, the syntactic structure can effectively represent the grammatical structure of the text and reveal the relationship between the components of the text. For formulaic language, it is a multi-word unit with high frequency of co-occurrence, and several words with high correlations may form formulaic language. Therefore, it is very important to select the features that can represent the relationship between words for identifying formulaic language. Based on this, this article uses the mutual information between words and the dependency syntactic relation of sentences as the associated information of formulaic language.X and Y is defined as: Mutual information measures the correlation between two random variables, that is, the amount of information about another random variable contained in one random variable\u00a0. The mutp is the joint probability distribution function of X and Y, p(x) and p(y) are the edge probability distribution functions of X and Y, respectively. If we want to measure the correlation degree of any two words x and y in a data set, we can calculate it as follows: where p(x) and p(y) are the probability of independent occurrence of x and y in the data set. It can be obtained by directly counting the word frequency and dividing it by the total number of words; p is the probability that x and y appear in the data set at the same time. Directly count the number of times they appear at the same time, and then divide it by the number of all unordered pairs. Using mutual information to calculate the relationship between binary words, the higher the mutual information, the higher the correlation between x and y, and the greater the possibility of forming formulaic language.where etc. The dependency syntactic relation of the sentence \u201cevaluations play an invalid role in X.\u201d is shown in Dependency syntax reveals the dependency relationship and collocation relationship between words in a sentence. One dependency relationship connects two words, one is the core word and the other is the modifier. This relationship is related to the semantic relationship of the sentence. The dependency relationship between words in a sentence includes subject-predicate relationship, verb-object relationship, inter-object relationship, In the formulaic language identification using dependency syntactic relation, the existing studies mostly use the dependency syntax in the text to construct rules, extract features, and then input them into the classifier to recognize formulaic language through classification. Although this method can achieve certain results, the nonlinear semantic relationship between components in sentences has not been learned and utilized. Spatially, the relationship between words can be represented by a graph through mutual information and dependency parsing. The GCN is used to process the correlation information, so that the nonlinear semantic relationship between words in the sentence can be extracted.aij in its adjacency matrix A\u025bRN\u2217Nrepresents the mutual information value between the ith node and the jth node in the graph. For the GCN with dependency syntactic relation as input, firstly, the dependency syntax of sentences is analyzed, with words as nodes and the dependency between words as the representation of edges. The element aij in its adjacency matrix A\u025bRN\u2217Nrepresents the dependency between the ith node and the jth node in the graph. If there is a dependency between the two nodes, the aij is 1, otherwise it is 0. For example, in the example of dependency syntactic parsing \u201cevaluations play an invaluable role in X.\u201d, the adjacency matrix A constructed based on dependency syntactic parsing is shown in In the feature representation based on GCN, the mutual information and dependency syntactic relation between words are used to determine the word connection relationship, and the basic features of words are represented as nodes. For the GCN with mutual information as input, firstly, the corpus is used as the data set to calculate the mutual information value between words, the word is used as the node, the mutual information value between nodes is used as the representation of the edge. The element C, that is, the feature vector dimension of each node Xi is C, the channels output by GCN is F, that is, the feature vector dimension of each node Zi is F, and finally the label Yi of the node is predicted by the feature of the node.In order to directly conduct deep learning modeling on graph data, the specific method adopts a variant model of convolution neural network-Graph Convolution Neural Network proposed by\u00a0G\u00a0=\u00a0, V is a vertex set containing N nodes, E is an edge set including self-loop edges . The feature information of graph G\u00a0=\u00a0can be represented by Laplace matrix, as shown in Specifically, given a graph Or use the symmetric normalized Laplasse matrix, as shown in A is the adjacency matrix of the graph, IN is the n-order identity matrix; D\u00a0=\u00a0diag(d) is the degree matrix of the vertex, as shown in where Based on the Fourier transform of graph, the graph convolution formula can be expressed as x is the basic feature vector of the node; g is convolution kernel; U is the eigenvector matrix of Laplace matrix L.In the formula, In order to reduce the amount of calculation, scholars used Chebyshev polynomials to simplify the graph convolution formula in 2017. Finally, the layered propagation formula of graph convolution can be expressed as \u03c3 is the activation function; W is the weight matrix to be trained.In the formula, Conditional Random Field (CRF) is a statistical-based learning model first proposed by G\u00a0=\u00a0, Y\u00a0=\u00a0Yv|v\u00a0\u2208\u00a0V is a set of random variables Yv indexed by node V in G. For the conditions that X has been given, if each random variable Yv obeys the Markov property, the probability of Yv can be expressed as X,\u00a0Y) is established as a conditional random field. For an undirected graph u\u00a0\u223c\u00a0v represents that node u and node v are adjacent in the undirected graph G.Among them, X, and X is used as a precondition. P(y|x) is defined as a joint probability distribution, and the maximum value among them is selected as the corresponding sequence label\u00a0 The difference between Experiment 1 and Experiment 2 lies in the comparison between Bi-LSTM and CNN. The experimental results show that Bi-LSTM is much better than CNN in feature extraction. Because the identification of formulaic language is a typical sequence labeling problem, Bi-LSTM can capture the long-distance and long-term dependence of sentence context information and can capture the two-way information of sentences. However, CNN cannot capture the long-distance dependence information well, so it is better to use Bi-LSTM in formulaic language recognition. It is worth noting that the Recall in the result of using CNN to extract features is relatively high, indicating that it can recognize more formulaic languages. However, it can also recognize many non-formulaic languages, so the Precision is not very high.(2) The difference between Experiment 1 and Experiment 3 is that the way of feature fusion is different. Experiment 1 adopts early fusion and Experiment 3 adopts late fusion. The experimental results show that the late fusion method has higher Precision and the early fusion method has higher Recall. The main reason is that the early fusion method can recognize more results when recognizing formulaic languages, but it can also recognize many non-formulaic languages, so its Precision is low. The features of the late fusion method are more accurate, and the recognition results are not as much as those of the early fusion, but the formulaic languages can be recognized more accurately. According to the F1-scores of the two methods, the effect of late fusion is better than that of early fusion.(3) Experiment 4 adds CNN layer based on Experiment 3, but the results after adding CNN are not as good as the previous results. The main reason is that CNN captures local correlations and each layer of CNN has a fixed span. Naturally, this layer can only model limited distance information. In Experiment 4, Bi-LSTM has obtained the long-distance dependency of the context, and then CNN is added after Bi-LSTM to deeply abstract the features, some correct formulaic languages are filtered out, resulting in a significant decline in Precision and Recall.(4) Experiment 5 adds feature extraction based on GCN fusing dependency syntactic parsing on the basis of Experiment 3. The experimental results showed that after adding dependency syntactic features, the Recall and Precision are reduced. Because dependency syntactic relation mainly focuses on the dependency between two words in a sentence, it is easy to cause that the extracted word string does not belong to formulaic language, so the recognition effect is not good.Experiment 6 adds feature extraction based on GCN fusing mutual information on the basis of Experiment 3. The experimental results show that after adding the mutual information features, the Recall increases, indicating that the number of correctly recognized formulaic language is more. However, due to the reduction of Precision, F1-socre is not improved. At the same time, compared with experiment 5, it shows that mutual information features can better represent the characteristics of formulaic language than dependency syntactic features.(5) Experiment 7 (the model proposed in this article) inputs the dependency syntactic features and mutual information features into GCN for feature extraction. The experimental results show that although the independent dependency syntax features (Experiment 5) do not show good results, after combining the two, the Precision. Recall have increased significantly. The reason is that the features of dependency syntactic parsing and mutual information complement each other in the identification of formulaic language to achieve efficient extraction of formulaic language. It also shows that dependency syntax features and mutual information features are important features to measure formulaic language.(6) From the train_time and test_time in In summary, through ablation experiments, the results verify the effect of formulaic language identification model based on GCN fusing associated information. That is, through the late fused part-of-speech features and semantic features as the basic features, dependency syntactic relation and mutual information as the associated information, the combined model can reduce the errors of a single model and enhance their advantages.Because the model in this article involves two GCNs, one is GCN based on dependency syntactic parsing, and the other is GCN based on mutual information. Therefore, when selecting the number of layers of GCN, we use Experiment 5 and Experiment 6 in ablation experiment to do two groups of comparative experiments and select the optimal number of network layers by comparing the experimental results.(1) Experiment 5 is a GCN structure based on dependency syntactic parsing. Experiments are carried out by setting 1, 2, 3, 4 and 5-layer graph convolution respectively. The experimental results are shown in (2) Experiment 6 is a GCN structure based on mutual information. Experiments are carried out by setting 1, 2, 3, 4 and 5-layer graph convolution respectively. The experimental results are shown in Analysis of experimental results:It is generally believed that increasing the number of network layers can reduce the error and improve the accuracy, but it will also complicate the network, thus increasing the training time of the network and the tendency of over fitting. Therefore, the number of network layers required by different features is different. In this article, dependency syntactic parsing only uses 0 or 1 to describe the relationship between two words, which is not good enough for the interpretation of formulaic language, so it needs three-layer graph convolution to extract better features. Mutual information can better represent the characteristics of formulaic language, so two-layer graph convolution can extract better features.From the above analysis, it can be seen that in formulaic language identification model based on GCN fusing associated information, the layers of the two GCNs are set to 2 and 3 layers respectively.In order to verify the effectiveness of the model proposed in this article, the CNN_Bi-LSTM_CRF model in literature\u00a0 and the  \u2022CNN_Bi-LSTM_CRF: This article performs the task of named entity recognition. Because formulaic language recognition and named entity recognition are similar, and this model performs well in the field of named entity recognition, this model is used as a comparative experiment. The word vector is trained with word2vec, and the word vector of the text data obtained after word2vec training is spliced to generate the word vector matrix, which is then used as the input of CNN convolution layer. The CNN module extracts the spatial feature information of the text through convolution and the collection of vector matrix. Then, the results are input into Bi-LSTM for forward and backward training. Finally, the vector with sentence feature information is put into CRF for decoding and prediction to obtain the final sequence. \u2022Bi-LSTM_CRF: This article describes the Deep-BGT system that participated in the PARSEME shared task 2018 to automatic identification of verbal multi-word expressions (VMWEs). Author uses Bi-LSTM model with a CRF layer on top. The input layer includes word vectors generated by fastText word embedding technology, POS and dependencies. Each input vector is represented as splicing these three features, similar to early fusion technology. Because formulaic language is also a kind of multi-word expression, the combination of Bi-LSTM and CRF is the mainstream method in the field of multi-word expression recognition, so this model is used as a comparative experiment.The experimental results of the above two models and the model proposed in this article on formulaic language recognition are shown in Analysis of experimental results:(1) As the CNN_Bi-LSTM_CRF model is the primary method in named entity recognition, the experimental results show that this model does not perform well in the task of formulaic language recognition. It can be seen that in different tasks, although the two tasks are very similar, we should also start from the essence of the object in the task, mine the characteristics that can represent the research object, and design a unique model. At the same time, through the CNN_Bi-LSTM_CRF model and Experiments 2 and 4 in the previous section, it can be found that the effect will not get better after adding CNN to the model. Hence, CNN is not suitable for formulaic language identification task.(2) Bi-LSTM_CRF model is used to identify multi-word expressions. Compared with Experiment 1 in the previous section, the difference lies in the different input features. The input features of Experiment 1 are part-of-speech features and GloVe word embedding features, while Bi-LSTM_CRF model inputs part-of-speech features, fastText word embedding features and dependency syntactic relation. The experimental results show that the F1 -score of Bi-LSTM_CRF model is higher. However, the Recall of Experiment 1 is higher, indicating that dependency syntactic relation is conducive to recognizing formulaic language.At the same time, in comparison with the model in this article, the main difference is that the Bi-LSTM_CRF model only constructs the dependency syntactic relation into a simple feature vector, splices it with other features and then trains them. The model in this article constructs the graph structure through the syntactic dependency tree and then extracts the features through GCN. The advantage of GCN is that it can gather the information on all edges and points. Thus, the boundary ambiguity between words is eliminated, and any two non-adjacent nodes in the graph are each other\u2019s second-order neighbors. They can receive each other\u2019s non-local information through two-node updates. The aggregated features in this method can more accurately represent formulaic language, so the effect of identifying formulaic language is better.In order to make an in-depth analysis of the shortcomings of this model, we investigated the examples of formulaic language recognition errors under the best experimental results and selected three representative sentences for analysis. The specific contents are shown in It can be seen from the table that the reason for the recognition error in the first example is that the word \u2018since\u2019 is not recognized. The reason for the recognition error in the second example is that the word \u2018to\u2019 is recognized. The reason for the recognition error in the third example is that \u2018restore to\u2019 is also regarded as a formulaic language. For the first two examples, in fact, the model\u2019s positioning in the sentence is very accurate. However, due to the uncertainty of manual labeling, fixed evaluation indicators and other factors, it can only be considered that this word string recognition is wrong. If the recognition effect is viewed from the perspective of word coverage, it must be higher than the existing evaluation indicators.In the third example, although \u2018restore to\u2019 is a non-formulaic language, it contains many characteristics of formulaic language, resulting in the model mistakenly considering it as formulaic language. This kind of word string usually contains many characteristics of formulaic language, such as Verb + preposition and other structures. However, it may not have a specific function and meaning in the fields of scientific and technological literature writing, so they are not formulaic languages. This situation is a problem we should think about and solve in the future.In this article, a formulaic language identification model based on GCN fusing associated information is proposed. The part-of-speech features and semantic features fused through late fusion are taken as the basic features. Then the two associated information of mutual information and dependency syntactic relation are input into GCN together with the basic features for feature representation. This combined representation can capture the syntactic and semantic structure of multi semantic web. It can also conduct a more in-depth downstream semantic analysis. Finally, we input the feature vector output from GCN layer to CRF layer for decoding, obtain the label category of each character, and get the formulaic language. Several groups of comparative experiments show that compared with the existing models, the proposed model can improve the accuracy of formulaic language identification and verify the model\u2019s effectiveness. In addition, it should be noted that the formulaic language identification model proposed in this article can obtain strong identification performance with only a small proportion of labeled text. For the identification of other tasks, it has a certain universal reference value. However, this model still has some shortcomings in analyzing the fuzziness and fuzzy boundary of some formulaic languages. The word strings described by facts in sentences cannot be understood and learned by the model, which needs further consideration in future research.10.7717/peerj-cs.984/supp-1Supplemental Information 1Click here for additional data file."}
+{"text": "Blattella germanica. All described SINEs have tRNA promoters, and the start of their transcription begins 11 bp upstream of an \u201cA\u201d box of these promoters. The number of copies of the described SINEs in the B. germanica genome ranges from several copies to more than a thousand copies in a SINE-specific manner. Some of the described SINEs and their degenerate copies can be localized both in the introns of genes and loci known as piRNA clusters. piRNAs originating from piRNA clusters are shown to be mapped to seven of the nine types of SINEs described, including copies of SINEs localized in gene introns. We speculate that SINEs, localized in the introns of certain genes, may regulate the level of expression of these genes by a PIWI-related molecular mechanism.In recent decades, experimental data has accumulated indicating that short interspersed nuclear elements (SINEs) can play a significant functional role in the regulation of gene expression in the host genome. In addition, molecular markers based on SINE insertion polymorphisms have been developed and are widely used for genetic differentiation of populations of eukaryotic organisms. Using routine bioinformatics analysis and publicly available genomic DNA and small RNA-seq data, we first described nine SINEs in the genome of the German cockroach,  Short interspersed nuclear elements (SINEs) are nonautonomous retrotransposons transcribed by RNA polymerase III. The conversion of SINE RNA into DNA and the subsequent process of integration into different locations of the genome are controlled by the molecular machinery of autonomous retrotransposons \u20134. SimilTypical SINEs are 150\u2013600 bp in length with a composite structure consisting of three parts: \"head\", \"body\" and \"tail\", sequentially localized starting from the 5\u2019- end of the TE. \u201cThe head\u201d is a fragment of one of the types of RNAs transcribed by RNA polymerase III: tRNA, 5S, or 7SL. Usually, SINEs contain tRNA fragments , 9. SomeThe main structural and functional component of \"the body\" of SINEs is the relatively extended region responsible for the binding of the protein product of the \"partner\" autonomous retrotransposons with the SINE RNA and subsequent reverse transcription of this RNA and SINE integration into the genome. Note that it is far from always possible to trace the similarity of the nucleotide sequence between SINE and its autonomous \"partner\" . Moreove\"The tail\" of typical SINEs represents repetitive microsatellite motifs or poly (A) sequences. The molecular mechanism of the formation of these sequences remains largely unclear to date.During the integration of retrotransposons, sequential cutting occurs first at the bottom and then at the upper strand of the target site. Depending on the particular retrotransposon, the second strand break can occur downstream, upstream, or in line with the bottom strand nick, resulting in target site duplications (TSDs), target site deletions or blunt insertions, respectively . Since tWhereas transposable elements (including SINEs) have been considered selfish or junk DNA \u201331, receSince the target-site specificity of the integration of SINEs is dictated by the molecular machine of their autonomous partners, it is logical to assume that the distribution of autonomous and nonautonomous retrotransposons in the genome will be similar. However, the retrotransposons in the genome have been shown to occupy distinct parts of the genome, and their regional densities are negatively correlated with each other . SINEs aOf particular interest, from our point of view, are studies of the role of a recently discovered class of small RNAs in the regulation of the activity of TEs and the epigenetic modification of the integration sites of TEs mediated by these small RNAs. piRNAs (PIWI-interacting RNAs) are the largest class of small noncoding RNAs of 26 to 31 nucleotides in length expressed in germinal and somatic cells; they are found in complexes with proteins of the PIWI family, for which they were named . piRNAs piRNAs have been shown to regulate transpositional activity through RNA decay  and/or through DNA methylation and histone modification . DNA methylation and histone modification are known to be able to lead to repressive heterochromatin formation and change the dynamics of expression of a gene localized in this region \u201349. It wBlattella germanica, has been increasingly considered a model object for studying the molecular genetic organization of eukaryotes and may serve as a suitable reference model for studying the molecular biology of insects [In recent decades, the German cockroach,  insects \u201356.B. germanica. We examined a representative sample of approximately a thousand genes previously annotated in the genome of the German cockroach and identified the genes containing SINE copies in the introns. Degenerate copies of seven of the nine described SINEs were shown to be localized in piRNA clusters, and the corresponding piRNAs are mapped to copies of SINEs localized in gene introns. We consider the results obtained as the first step toward studying the possible regulatory function of SINEs of B. germanica.In this study, we first described the structural and functional organization of SINEs of Finally, since the number of SINEs in the genome, as a rule, is large enough and their integration occurs into random sites of the genome, the pattern of integrated copies of these TEs can be considered a polymorphic molecular genetic marker that allows solving the problems of population genetics, in particular, determining the genetic distances between populations of living organisms.B. germanica was reported [https://www.ncbi.nlm.nih.gov/Traces/wgs/PYGN01).Recently, the 2-Gb genome of reported , which gB. germanica genome, we used two methodological approaches: the first approach was based on an automatic search using the previously published program SINE_Scan 1.1.1 [To identify SINEs in the an 1.1.1 ; the sechttps://github.com/maohlzj/SINE_Scan) was used with the default parameters  was used with the default parameters . The \"Assemble\" command of this software was run with the following parameters of \"Assembly\" settings tab: Algorithm\u2014Local alignments, Min. percent identity = 90, Min. overlap length = 150, with the following visual analysis of the sequences in \"Contigs\" folder. The candidates for SINE should have no less than three aligned sequences; the beginning of tRNA should have a shift no more than five nucleotides in length; overlapping sequences should have a length no less than 150 nucleotides  [-25 were used for the following analysis.For each of the sequences considered a candidate for SINE, sequences were found in the genome of blast+/)  were useAt the final stage, based on the comparison of similar nucleotide sequences corresponding to each of the SINE types, consensus sequences were predicted using the Jalview V.2 program .https://www.girinst.org/censor/index.php, and 2) regular online Blastn search, which allowed to compare the sequences we analyzed with all the sequences presented in the GenBank database (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome)To identify evolutionarily conserved sequences within the SINEs we described, we used two approaches: 1) the CENSOR program , 62, avahttps://sines.eimb.ru).The duplications of the integration sites were predicted online by TSD (target site duplication) Search program (B. germanica were determined using the DotPlot ability in the UGENE 34.0 software package [The number of copies of each of the SINE types and the coordinates of each specific copy in the genome of  package . The folhttps://www.ncbi.nlm.nih.gov/gds/?term=GEO%20GSE87031) was used for analysis. To detect the piRNAs localized in piRNA clusters, we used previously described methodological approaches [A publicly available small RNA-seq database  to SINEs consensus sequences and genes of  program .In our study, sequences referred to SINE had to meet the following criteria: 1) had a length of at least 150 bp; 2) contained a sequence corresponding to the RNA polymerase III promoter; 3) were represented in the genome by at least three copies; and 4) were flanked by target site duplications.B. germanica (Sbg1 \u2013Sbg9). The lengths of the identified SINEs were Sbg1\u2013306 bp, Sbg2\u2013313 bp, Sbg3\u2013613 bp, Sbg4\u2013456 bp, Sbg5\u2013582 bp, Sbg6\u2013359 bp, Sbg7\u2013564 bp, Sbg8\u2013355 bp, and Sbg9\u2013658 bp.We identified nine SINEs of To identify SINEs, we used two methodological approaches see \u201c\u201d. We firIn B. germanica that have at least 90% similarity both with each other and with the consensus sequences corresponding to Sbg7 or Sbg8. The consensus sequences of Sbg7 and Sbg8 were shown to have one nucleotide substitution each , the tRNAscan 2.0.3 program  predicte \u2013Sbg6, t in Figs . Note thB. germanica have one nucleotide substitution within the \u201cA\u201d or/and \u201cB\u201d boxes  [https://sines.eimb.ru) [Reticulitermes lucifugus, a closely related species of B. germanica, these distances are different for SINEs Talua, Talub, Taluc and Talud   and SINEeimb.ru)  databaseB. germanica SINEs was investigated by comparing their nucleotide sequences with previously described TEs of various species presented in the following databases: RepBase [https://www.ncbi.nlm.nih.gov) using the program CENSOR [The structure of \u201cthe body\u201d of  RepBase , SINE BA RepBase , and NCBm CENSOR , 62 and B. germanica, sequences similar to the sequences of autonomous retrotransposons, potential partners of the studied SINEs, were also not identified. The exceptions are Sbg8 and Sbg9, which contain the same sequences as the sequences of the Locusta migratoria RTE retrotransposon  in this structural part of retrotransposons. Interestingly, each of the Sbg1, Sbg4, Sbg6, Sbg8, and Sbg9 copies identified in the B. germanica genome contains perfect microsatellite motifs: \u201caccttt\u201d, \u201ctggaa\u201d, \u201cca\u201d, \u201cttag\u201d, and \u201cttag\u201d, respectively. However, only a few copies of Sbg5 contained the motif \u201ctcaga\u201d. Sbg3 copies contain different repeat variants, apparently representing different variants of duplications of short nucleotide sequences of the parent SINE copy  sequences, whereas all other opy Figs .The molecular mechanisms responsible for changes in the lengths of STRs have been the subject of study over the past several decades since the discovery of these structural formations in eukaryotic genomes. The main mechanism leading to an increase/decrease in the number of repeats in the STR locus is now generally accepted to be slipped strand mispairing that occurs during genomic DNA replication . The numB. germanica had duplications of the integration site, represented by direct repeats 10\u201315 bp long, flanking the retrotransposons  to 1849 (Sbg4) copies with 90% or more similarity to consensus sequences. The number of \"degenerate\" copies, reflecting the evolutionary age of a particular SINE type, also varies widely depending on the SINE type . Fig 2B SINEs can be subdivided into distinct subfamilies by specific diagnostic nucleotide changes. Older subfamilies are generally very abundant, while younger subfamilies have fewer copies , 4. NoteSbg9 deserves special consideration. This TE is a dimeric SINE formed by the combination of two SINEs: Sbg1 and Sbg8. Apparently, among the SINEs described by us, Sbg9 is the most evolutionarily young, formed relatively recently, and, probably, for this reason it is represented in the genome by such a small number of copies. Note that dimeric SINEs are quite widespread in the described genomes of living organisms [for review, see B. germanica, may have not only general scientific value but also applied value. B. germanica is a synanthropic species of organisms that can live both in human residential premises and in public institutions and on livestock farms. The spectrum of the negative influence of these insects on human life is unusually wide. A particular danger is the ability of these insects to be carriers of pathogenic microorganisms and cause severe allergic diseases in humans [B. germanica populations and the possibility of determining the migration flows of this insect species are of particular importance. Currently, several types of molecular genetic markers are used to solve the problems of population genetics of B. germanica: polymorphism of the ribosomal RNA gene cluster [From our point of view, the SINEs described in the genome of the German cockroach, n humans \u201375. In t cluster ; polymor cluster \u201379; patt cluster . DespiteB. germanica. Over a thousand copies of some types of SINEs were shown to be present in the genome of B. germanica. We can assume that, as has been shown for other species of organisms studied in this regard, the SINEs described by us can be used to analyze the polymorphism of the integration sites of these TEs and, as a consequence, to develop a new type of molecular genetic marker that allows differentiation of populations of B. germanica. However, it is obvious that the resolution of the proposed markers must be verified experimentally.SINEs are represented in the genomes by many copies, and the transposition of young copies occurs constantly at a certain level. Based on these properties, we can assume that SINEs can be considered unique informative molecular genetic markers that make it possible to differentiate populations of eukaryotic organisms. Indeed, at present, this type of marker is actively used for these purposes [for example, see B. germanica?piRNAs play an important role in the control of the transpositional activity of mobile elements \u201349. Do pB. germanica. B. germanica showed that two of the nine SINEs described by us (Sbg3 and Sbg7) do not contain nucleotide sequences that are complementary to piRNAs selected in the above-described way; at the same time, from 46 (Sbg2) to 2290 (Sbg4) piRNA reads were mapped to the consensus sequences of the remaining SINEs. The results obtained indicate that the transposition activity of at least some of the SINEs described by us could be regulated by piRNAs.To address this question, we checked if piRNAs sequences would map to the SINEs of As noted above, piRNAs can regulate the transpositional activity of TEs not only through RNA decay but also through DNA methylation and histone modification of TE sites. DNA methylation and histone modification lead to a change in chromatin conformation and cause a change in the dynamics of transcription of a specific TE \u201349. ObviB. germanica genes, and, if so, are piRNAs, potentially capable of causing local changes in chromatin conformation, mapped to these SINE copies?Are SINEs (or their degenerate copies) localized in the introns of B. germanica genome, for which the potential functional activity has been determined and for which the exon-intron structure has been described. A total of 801 genes were identified. Mapping of piRNAs Click here for additional data file.S2 File(PDF)Click here for additional data file.S1 Script(PY)Click here for additional data file.S1 FigAreas of DNA sequences corresponding to SINEs are highlighted in blue. Gray background\u2013DNA sequences corresponding to the SINE environment and having a low level of similarity. Vertical lines in red, green, black, and bright blue indicate single nucleotide substitutions.(PDF)Click here for additional data file.S2 FigpiRNAs localized in piRNA clusters were mapped to the described SINE sequences; then, this piRNA fraction was retrieved, and the accumulated pool of piRNAs (9261 reads) was used for subsequent analysis.(PDF)Click here for additional data file.S3 Fig(A)\u2013The consensus nucleotide sequence of the corresponding SINE with the designation of the tRNA structure. The tRNA nucleotide sequences are highlighted in gray; \u201cA\u201d and \u201cB\u201d boxes are highlighted in blue; nucleotides other than canonical nucleotides are highlighted in yellow background and red font (explanation in the text). (B)\u2013Distribution of the corresponding SINE copies in the B. germanica genome. Blue vertical lines indicate the positions of SINE localized in direct orientation, red vertical lines\u2014in inverted orientation. (C)\u2013The result of the alignment of the consensus sequences and the sequences of twelve similar to the consensus sequence SINE copies presented in the genome, along with the nearest environment. Direct repeats flanking the retrotransposon are highlighted with a green background. Poly(A) sequences and short microsatellite repeats are highlighted in yellow, blue and pink backgrounds. Variable nucleotides are highlighted in red fonts. (D)\u2013Alignment of Sbg1, Sbg8 and Sbg9, demonstrating that Sbg9 was formed by combining Sbg1 and Sbg8.(PDF)Click here for additional data file.S4 FigThe consensus nucleotide sequences of the corresponding SINEs with the designation of the tRNA structure are shown. The tRNA nucleotide sequences are highlighted in gray; \u201cA\u201d and \u201cB\u201d boxes are highlighted in yellow; nucleotides other than canonical are highlighted in red font. The distances from the start of transcription of SINEs to the corresponding \u201cA\u201d boxes are highlighted in pink.(PDF)Click here for additional data file.S5 Fig(A)\u2013Masked Sbg8 sequence and the local sequence alignment of Sbg8 and RTE retrotransposons of Locusta migratoria. (B)\u2013Sequence of RTE retrotransposon of L. migratoria. The sequence fragment similar to the 3\u2019-end of Sbg8 is highlighted in blue.(PDF)Click here for additional data file.S6 FigReads mapped in direct orientation are highlighted in blue, and reads mapped in reverse complement orientation are green.(PDF)Click here for additional data file.S1 TableF and R represent forward and reverse complement orientations, respectively.(PDF)Click here for additional data file."}
+{"text": "We examine the effect of immobilization configurationto show that the complex grafted in a semirigid configuration catalyticallyoutperforms the rigid, flexible configurations and even the homogeneouscounterparts. Using the semirigid catalyst, we are able to obtaina TON of up to 800 and a TOF of up to 37 h\u20131 under1 atm CO2. The catalyst is shown to be recyclable withonly minor leaching and no change to product selectivity. We furtherexamine a range of epoxides with varying electron-withdrawing/donatingproperties. This work highlights the benefit arising from the constrainingeffect of a solid surface, akin to the role of hydrogen bonds in enzymecatalysts, and the importance of correctly balancing it.The conversion ofCO If anthropogenicCO2 can be efficiently and sustainably converted to startingchemicals and materials, it will serve as a pivotal point for thedramatic global climate changes the world is experiencing.1 The coupling of CO2 with epoxidesto cyclic carbonates is one of the promising routes for CO2 utilization with a 100% atom-economy.2 Cyclic carbonates are an important class of chemicals which havemany applications such as green solvents, precursors for pharmaceuticals,fine chemicals, electrolytes in lithium-ion batteries, and precursorsfor biodegradable polymer synthesis.4 Industrially,the main cyclic carbonate products are propylene carbonate and ethylenecarbonate.5 As far as we know, the mostactive reported catalysts for the CO2\u2014epoxide couplingreactions to cyclic carbonates are homogeneous metal complexesbased on Al(III), Co(II/III), or Cr(III) sites at temperatures rangingfrom 100 to 150 \u00b0C and CO2 pressures greater than10 atm.9 Interestingly, bimetallic aluminium complexes11 and dinuclear iron(III) complexes having a reduced Robson macrocycleligand system,12 in the presence of anionic cocatalyst, were reported to be active for the synthesis ofcyclic carbonates at atmospheric CO2 pressure. For example,the work of North et al. nicely demonstrates the conversion of variousterminal epoxides into cyclic carbonates at 1 atm CO2 pressurewith a reasonable turnover number (TON) up to 320 and a turnover frequency(TOF) value of nearly 13 h\u20131 using bimetallic aluminiumcomplexes grafted on silica.17 With these dinuclear catalysts, the cycloaddition reaction was shownto be promoted through a dual activation mechanism via cooperativeinteractions of the two metal centers.20One of the greatestenvironmental challenges of today\u2019ssociety and in the coming decades is the mitigation and utilizationof anthropogenic carbon dioxide  catalyst (LFe2Cl3ClO4)  1 using tSupporting Information. The molecular structureof the di-iron(III) complex (LFe2Cl4) is as reported by Williamsand co-workers and is shown in 12 The modifieddi-iron(III) complex (LFe2Cl3ClO4) that was used as a precursor for grafting is shown in Figure S1). Usingseveral reported grafting protocols,29 the modified complex was immobilized through interactionwith either surface hydroxy or amino groups on nonporous high-surface-areasilica to obtain the active solid catalysts. The di\u2013iron complexgrafting modes are shown in 2 surface and hence relatively flexible(LFe2-NH/SiO2). This provides the complex with a higher configurationalmobility potentially reducing steric effects for catalysis and substrateaccess; (b) immobilized directly through Si\u2013OH groups to establishtight binding to the SiO2 surface. Given the short lengthof the two Si\u2013O\u2013Fe bonds that hold the complex closeto the surface, the configurational freedom of the complex is significantlyhindered and is therefore considered to be rigid (LFe2-O/SiO2); (c) immobilized through an amine and a surface silanol givingthe complex a semirigid mode (LFe2-O-NH2/SiO2). This essentially is an intermediate statebetween flexible and rigid forms. Analysis of the materials usingthermogravimetric analysis\u2013mass spectrometry (TGA\u2013MS)provided information as to the grafting loading and thermal stability. The MS data indicatethat mass loss in the range of 200\u2013600 \u00b0C is due to thedecomposition and combustion of the di\u2013iron complex.30 Cross-referencing this analysis with the Fecontent by inductively coupled plasma-optical emission spectrometry(ICP\u2013OES) (Table S2), the loadingof the complex in all of the catalysts was found to be similar to\u223c13% wt for LFe2-NH/SiO2, \u223c18% wt for LFe2-O/SiO2, and \u223c15% wt for LFe2-O-NH2/SiO2. Interestingly, the TGA data also showthat the onset temperature for decomposition increased in the followingorder: LFe2-O/SiO2 (224 \u00b0C), LFe2-O-NH2/SiO2 (243 \u00b0C), and then LFe2-NH2/SiO2 (252 \u00b0C). These resultsare consistent with a decrease in the degree of interaction of thegrafted molecule with the solid support, which has been shown to promotethermal decomposition.32The full synthesis details for the modified reduced Robson-typemacrocycle coordinated di-iron(III) catalyst shown in \u20131 inthe Fourier transform infrared (FTIR) spectra between the free di-ironmacrocycle  and the graftedmaterials, we clearly see significant shifts in the N\u2013H bandsfollowing grafting .34 In addition, the intensity of the band at 955 cm\u20131,33 pertaining to the Si\u2013OH bendingvibration, was found to decrease following immobilization of the complexas LFe2-O-NH2/SiO2  or LFe2-O/SiO2 . For LFe2-NH/SiO2 , the Si\u2013OH band at \u223c955 cm\u20131 only partly diminished compared to the primary amine bands at 3370and 3305 cm\u20131 in the APS . These results indicate successful binding of the LFe2Cl3ClO4 complex to the silicasupport. Analysis of the immobilized and free catalysts using diffusereflectance (DR)-UV-vis spectroscopy showed bands at \u223c200,235, and \u223c280 nm, which are related to \u03c0\u2013\u03c0*excitation .35 An additional band, related to n\u2212\u03c0*excitation of the macrocycle ligand, was found at \u223c320 nm.37 Notably, in the case of APS-supported catalysts(LFe2-NH/SiO2 and LFe2-O-NH2/SiO2), the spectra exhibited broad absorption bandstypical of grafted chiral Fe(III)-salen complexes on amine-functionalizedsurfaces.36 Consistent with the immobilizationof the di\u2013iron complex to the SiO2 support, we foundthat the UV\u2013vis bands were slightly shifted compared to thebands of the free di\u2013iron complex. In addition, the ligand-to-metalcharge transfer transitions in all solid catalysts were centered at\u223c440 nm, whereas the band for the free complex is significantlyred-shifted to \u223c580 nm. This serves as a strong indicationthat the di-iron metal centers are strongly affected by the interactionwith the SiO2 support.37Comparing the characteristicN\u2013H vibration bands of thecomplex in the range of 1480\u20131616 cm3/2 and Fe 2p1/2 are obtained in the regions of 710 and 723 eV along witha weak satellite peak at 718.9 eV, confirming the existence of theFe3+ oxidation state in all immobilized catalysts . Interpretation of the intensity ofthe O 1s signal was only possible for the free complex where the signalfrom the SiO2 support was not present . What was noticeable is that all immobilized catalystsshowed a similar main BE at \u223c532.6 shifted by \u22120.4 eVfrom the 533.0 eV O 1s BE measured for the physical mixture of thecomplex with bare SiO2 . This seeming shift may result from the consumption of a high bindingenergy component, namely SiOH, during the grafting process. Consistentwith the IR data, the diminished SiOH component is the most prominentfor the tight binding complex. The most insightful information wasgained from the N 1s XPS spectrum  (synthesissolvent) coordinated at the di-iron centers. The presence of coordinatedTHF species was confirmed by the analysis of the Fe, N, Cl, and Oatomic composition from XPS; see schematic and detailed explanationsin Tables S1\u2013S3. Fitting of the LFe2-O/SiO2 N 1s signal showed that only the second peak upshiftedby \u223c0.5 eV as compared to the LFe2Cl4 sample, indicatingthe coordination of the di\u2013iron complex to an electron-donatingsurface, that is, to Si\u2013OH groups (Table S1).Based on the atomic concentration of Fe and Cl and N as well as theN 1s peak area ratio (\u223c3:1), we concluded that the complexwas grafted either via two Si\u2013OH or via one Si\u2013OH andthat the two forms occurred with a 3:1 abundance (Tables S1 and S2). For the LFe2-O-NH2/SiO2 catalyst, the measured N 1s peak area ratiowas found to be 3:2, which closely matches with the binding throughboth Si\u2013OH and grafted NH2. The atomic concentrationof Fe indicates that about 3/4 (0.73 mmol) of the NH2 remainedunbound (Tables S1 and S2). For the LFe2-NH/SiO2 catalyst, the N 1s first peak appears at a low bindingenergy of 399.5 eV, which corresponds to the value seen for the LFe2Cl4 free complex , which confirms exclusive grafting through the NH2 groupsin contrast to the case of LFe2-O-NH2/SiO2.To further probe the properties of the di-iron complex and theimmobilization mode, we performed X-ray photoelectron spectroscopy(XPS). The binding energies (BEs) of Fe 2patalysts S8. Interatalysts S8. Interatalysts S8. Inter40 To examine the coordination, we recorded the electronparamagnetic resonance (EPR) spectra at 15 K for all catalysts  ferric ions (hs-FeIII). On the other hand, therigid LFe2-O/SiO2 and semirigid LFe2-O-NH2/SiO2 catalysts displayed two hs FeIII, species, one with g = 4.3 and a second one with g\u22a5 = 6.7 and g|| = 2, indicating the presence of two different Fe III environments.However, the semirigid LFe2-O-NH2/SiO2 catalyst showed a strong EPR signal at g = 4.3 and a less dominant signal at g\u22a5 = 6.7 and g|| = 2, which is a superpositionof the first two catalysts, indicating the presence of the two bindingmodes. These observations are consistent with the conclusions drawnfrom the XPS data. In sum, the above results clearly show that thedifferent grafting protocols resulted in the formation of differentbinding modes. Presumably, the tight binding of the complex to thesurface in LFe2-O/SiO2 makes for a relatively rigid structure.The grafting through the amine only in LFe2-NH/SiO2 createsloose binding rendering the complex more flexible, whereas bindingthrough both amine and hydroxy generates a semirigid complex.It can be expected that the differentimmobilization modes, depictedin atalysts 2 (right)2 with 2 equivalentsof bis(triphenylphosphino)iminium chloride ([PPN]Cl). As reportedand verified here, under the current reaction conditions, PPNCl wasinactive on its own.11 In addition, allcatalysts were found to be inactive without the addition of PPNCl.PPNCl is a bulky cationic initiator, which acts to labialize the metal-nucleophilebond of either the Cl or carbonate to promote the rate-limiting, epoxidering-opening step or to facilitate the ring closure (backbiting) step.11 A proposed mechanism with respect to the currentactive catalyst is shown in Figures S10 and S11. The catalysts were initially evaluated for CO2 conversionusing the relatively bulky cyclohexene oxide (CHO) as the couplingagent, which is less active for ring-opening as compared to otherepoxides due to its bicyclic nature. The selectivity to the cycliccarbonate was generally >90%, and the isolated cyclic carbonateyieldwas used to determine TON and TOF. The catalysis results at 1 atmof CO2 show that the semirigid LFe2-O-NH2/SiO2 catalyst was more active (TOF of 10.8 h\u20131) as compared to the rigid LFe2-O/SiO2 (TOF =8.3 h\u20131) and the flexible LFe2-NH/SiO2 (TOF= 5.6 h\u20131) catalysts (S4). Thisfinding is consistent with what is known for enzyme-inspired syntheticcatalysts, which show enhanced performance when able to balance betweenthe flexibility needed for correct catalyst alignment and the rigidityof the support required for stability of the active sites34 and accessibility of the substrate.42 Notably, the semirigid LFe2-O-NH2/SiO2 catalyst exhibited higher activity as comparedto the homogeneous counterparts, whereas the rigid and flexible showedsimilar or diminished performance .Reaction testing of these catalysts was conducted in neat epoxideat 80 \u00b0C and under 1 or 15 atm of COatalysts 1 and S4.1H NMR showed that forall catalysts, the methyne protons had a chemical shift of 4.64 ppm(versus 3.90 ppm for the trans-CHC), which meansthat the cis-cyclohexene carbonate (cis-CHC) was exclusively produced .11 Only few reported homogeneous catalystsshow the formation of the cis-CHC product by thecoupling with CO2.44 Further testing was done for the semirigid LFe2-O-NH2/SiO2 catalyst with various epoxidesubstrates . We furtherfound that the epoxides having stronger electron-withdrawing groupsgave higher TOF in contrast to electron-rich epoxides. This trendcan be observed by the obtained yield, TON and TOF, which decreasedin the following order: phenyl glycidyl ether (PGE) > styrene oxide(SO) > tert-butyl glycidyl ether (TBGE) > CHO.Ascan be expected, upon increasing the CO2 pressure to 15atm at 80 \u00b0C, the yield and TOF for cyclic carbonate productionsignificantly increased giving TOF values close to 37 (Table S4). As shown in LFe2-O-NH2/SiO2 catalyst could be reused, following a washingstep, at least 6 times without significant loss in TON or change toselectivity, Reaction productanalysis using produced S12.11 Onproduced S12.11 On2 immobilized di\u2013ironcomplex. The combined results by XPS, EPR, TGA\u2013MS, DRIFTS,and DR-UV-vis spectroscopy show three distinct immobilization modes,namely, flexible, semirigid, and rigid. Catalytic testing demonstratesthat the catalyst in the semirigid mode (LFe2-O-NH2/SiO2) outperformed both the flexible (LFe2-NH/SiO2) and rigid (LFe2-O/SiO2) modes and the homogeneouscatalyst in the conversion of CO2 and epoxides to cycliccarbonates. The semirigid catalyst mode was shown to have markedlyhigh activity, exhibiting high TON values in the range of 100\u2013800and TOF values close to 37 h\u20131 for cyclic carbonatessynthesis at 1 atm CO2. This is remarkable given that otherexamples in the literature including microporous organic network systems,47 MOFs,48 and SiO2 supported di-aluminum complexes17 have lower TON at 1 atm CO2 or requirethe presence of tetraalkylammonium salts and CO2 pressuresgreater than 10 atm to reach similar TON values (Tables S5 and S6in Supporting Information). We furtherdemonstrate that the semirigid catalyst was reused up to six timeswithout significant loss in TON or change in selectivity. As far aswe know, this is the only example of a recyclable heterogeneouslycatalyzed cis-cyclohexene carbonate synthesis at1 atm CO2.In conclusion,the current work describes the synthesis, characterization,and catalytic testing of a modified SiO"}
+{"text": "Objective\u2003This study evaluated the dynamic cyclic fatigue resistance of the XP-Endo Shaper (XPS), associated with chlorhexidine digluconate (CHX) or sodium hypochlorite (NaOCl) in two different formulations: gel (G) or liquid (L).Materials and Methods\u2003Sixty XPS were used in an artificial stainless-steel canal, and the files were fully immersed in the irrigating solution throughout the experiment until the fracture. The files were divided into six groups (n\u2009=\u200910) based on the irrigation solution used: NaOCl(L), NaOCl(G), CHX(L), CHX(G), natrosol gel (NAT) (control), and lubricating oil (LO) (control). The artificial canal was manufactured 1.5\u2009mm wide, 20\u2009mm long, and, 3.5\u2009mm deep with a straight cervical segment measuring 14.29\u2009mm; an apical segment of 4.71\u2009mm with 3\u2009mm radius; and 90\u2009degrees of curvature apical 1\u2009mm long straight segment. Resistance to cyclic fatigue was determined by recording the number of cycles to fracture (NCF).Results\u2003The CHX(G), CHX(L), and OIL (LO) groups showed no significant difference between them and presented longer time to fracture (p\u2009>\u20090.05). NaOCl(L) shows the lowest NCF without significant differences between NaOCl(G) and NAT. The NCF of the NaOCl(G) was statistically similar to the CHX(L) and statistically lower than the CHX(G) and OIL groups. NAT did not present a statistical difference of the NaOCl(L), NaOCl(G), and presented a significantly lower NCF than the CHX(G) (p\u2009<\u20090.01).Conclusion\u2003The use of CHX(G) resulted in increased cyclic fatigue resistance of the XPS instruments compared to NaOCl or LO. The development of new nickel-titanium (NiTi) alloy led to a higher success rate of root canal treatment, reducing clinical time and instrument fracture.Enterococcus faecalisbiofilm, but this effect is less than that of NaOCl liquid.However, endodontic files are unable to reach all root canal walls,Chlorhexidine digluconate (CHX) is an option for endodontic irrigants that could replace hypochlorite with some limitation.Despite technological innovations in endodontics, fractures from mechanized NiTi instruments still occur in two ways: torsional or cyclic fracture.n\u2009=\u20093), the significance level of 5%, and power of 90%, the sample calculation indicated the need for 60 files (n\u2009=\u200910) in the present study.The sample size and power for statistical testing were calculated by analyzing variance using the software G*Power 3.1.9.4. For the effect size of 0.571, obtained from a pilot study (n\u2009=\u200910) based on the irrigating solution, CHX(L) chlorhexidine liquid 2% ; CHX(G) chlorhexidine gel 2% ; NaOCl(L) sodium hypochlorite liquid 5,25% ; NaOCl(G) sodium hypochlorite gel 3% ; NAT natrosol gel  (control group), LO lubricating oil WD-40  (control group).Sixty 25-mm long XPS instruments were equally assigned to six groups (The files were inspected for deformities at high magnification (13.6\u2009\u00d7\u2009) , and none of them was discarded. Noncorrosive stainless-steel blocks against NaOCl or CHX were used to test resistance to dynamic cyclic fracture. The artificial canal was 1.5\u2009mm wide, 20\u2009mm long, and 3.5\u2009mm depth, with a straight cervical segment of 14.29\u2009mm, a long curved apical segment of 4.71\u2009mm with a radius of 3\u2009mm and a curvature of 90\u2009degrees, and a long straight apical segment of 1\u2009mm.These dimensions allow the file to rotate freely within the artificial canal, both angularly and in a dynamic motion. The canal was covered with an acrylic plate to prevent instrument slippage, visualize the instrument during its action, and keep the irrigating solution within the simulated canal. The interface between the metallic block and the acrylic plate around the metallic canal was sealed with silicon  to prevent leakage and keep the simulated canal full of irrigating solution. The irrigating solution was inserted into the simulated canal by using a 3-mL syringe (Ultradent Products Inc \u2013 EUA) and needle Navitip 30G 21mm long (Ultradent Products Inc \u2013 EUA).The stainless-steel block with the artificial canal was positioned vertically on a heating plate  at a constant temperature (37\u00b0C\u2009\u00b1\u20091\u00b0C), measured by a laser thermometer pointed at the simulated canal , so the substance temperature into the root canal was accurately measured.The contra-angle handpiece was fixed to the mechanical system that enables a dynamic axial movement of the file inside the simulated canal. The mechanical motion system consists of a linear guide, coupled with a savox sc-12 56t69 engine  that performs back and forth movements, controlled by an electronic device that controls the speed amplitude of the axial movement. The files were placed inside the simulated canal coupled to a 6:1 reduction handpiece , driven by a VDW Silver Reciproc motor . Cyclic fatigue tests were performed by rotating the instruments in continuous rotation at 800\u2009rpm and torque of 1\u2009Ncm.The instruments were inserted 20\u2009mm into the canal, fitted with a silicone stop to register this length. A back-and-forth axial movement at a speed of 3.0\u2009mm/s and amplitude of 3.0\u2009mm were applied to the instruments to simulate clinical pecking motion. The continuous rotation of the file's movement occurred until the fracture could be visually observed. The file movement within the simulated canal was recorded with iPhone 6s , using 4K recording technology. The movie was analyzed in Microsoft Movie Maker , and the time at which the instrument began to rotate until the moment of the fracture was registered in seconds. The number of cycles to fracture (NCF) was calculated by using the following formula: NCF\u2009=\u2009time (seconds)\u2009\u00d7\u2009revolution per minute/60.The fragment lengths were measured by using a 150-mm digital caliper (accuracy of\u2009\u00b1\u20090.03\u2009mm/0.001). The maximum point of stress in both artificial canals was also analyzed.A descriptive analysis of the NCF was performed according to the irrigant type (CHX and NaOCl) in its different formulations (G or L) and according to the control groups (LO and NAT). The normality and homoscedasticity were confirmed by the Shapiro\u2013Wilk and Levene tests, respectively. Tukey's test was used to compare the averages for the NCF according to the irrigant type (groups\u2009=\u20096). The significance level was set at 5%.p\u2009=\u20090.066;The CHX(G), CHX(L), and OIL (LO) groups showed no significant difference between them and presented longer time and NCF. NaOCl(L) shows the NCF without significant differences between NaOCl(G) and NAT. The NCF of the NaOCl(G) was statistically similar to the CHX(L) and statistically lower than the CHX(G) and OIL groups. NAT did not present a statistical difference of the NaOCl(L) and NaOCl(G), and presented a significantly NCF than CHX(G). The irrigating agents did not significantly influence the length of the file fragment , whereas previous studies evaluated at room temperature.Despite NaOCl is potentially irritating for periapical tissues, especially in high concentrations, it is the substance most widely used for root canal irrigation in endodontics because of its effective antimicrobial activity and ability to dissolve organic tissues.On the other hand, CHX (L and G formulation) was used in the present study because both CHX and NaOCl were equally effective in reducing endodontic infection, despite their different molecular mechanisms.p\u2009<\u20090.01). Therefore, the null hypothesis was rejected, corroborating with another study.In this study, OIL(LO), CHX(L), and CHX(G) did not differ significantly from each other; however, the NCF were statistically higher compared to NaOCl(L)  provided the endodontic file the most significant resistance to fracture in this experimental model similar to CHX(L). Chlorhexidine gluconate is a cationic biguanide used as an intracanal irrigant in a liquidIn the NaOCl(G) group, the time to fracture and the NCF was similar to the one verified with the use of CHX(L) and NAT but did not reach the time until fracture and NCF verified with the OIL and CHX(G). When exposed to a NaOCl, even in a G formulation, the lower fracture resistance could be attributed to the induced corrosive zones, which are likely to reduce the resistance to cyclic fatigue of the instrument.Immersion of instruments in NaOCl before cyclic fatigue testing for 3 to 5\u2009minutes did not affect the cyclic fatigue of NiTi endodontic instruments.The mean lengths of fractured segments were recorded to evaluate the maximum concentration area of the compression and tensile stresses of the tested files inside the canal curvature. There was no significant difference between the groups, regardless of the broken fragments' lengths (4.5\u2009\u00b1\u20090.5\u2009mm). This matches the file's location inside the curvature; the point of maximum stress was similar in each circumstance, suggesting standardization of the experiment.Based on the current study's findings, it is possible to conclude that CHX (G) and CHX (L) increased the time to fracture and the NCF of XPS files. In addition, XPS instruments were significantly more resistant to cyclic fatigue when irrigated with CHX(G) than with NaOCl(G), NaOCl(L), or CHX(L). However, the circumstances tested in these dynamic models are very different from those present in clinical practice. Therefore, further studies are needed to evaluate other irrigating solutions and their influence on XPS instruments' cyclic fatigue."}
+{"text": "As modern society experiences rapid changes, the unpredictability of the labor market is increasing. University students preparing to join the workforce may experience increased anxiety and stress due to the heightened uncertainty regarding their career plans. Regulating such negative emotions and adjusting to the changing circumstances may influence their career development. Thus, the current study aimed to investigate the relationship between cognitive emotion regulation (CER) \u2014 specifically adaptive CER and maladaptive CER \u2014 and career decision-making self-efficacy (CDMSE), with career adaptability (CA) as a mediating factor. The path analysis model consisting of adaptive CER, maladaptive CER, CA, and CDMSE was tested with 357 Korean university students who were facing the school-to-work transition. The results of the study were as follows. First, adaptive CER was positively related to CA and CDMSE, while maladaptive CER was negatively related to CA only. Second, CA and CDMSE were positively related. Third, CA partially mediated the relationship between adaptive CER and CDMSE and fully mediated the relationship between maladaptive CER and CDMSE. Based on these results, theoretical and practical implications are proposed, and the limitations of the study are discussed. Modern society is changing rapidly and becoming more complex, and various factors such as the great recession around the world, increased inequalities caused by polarized jobs, and the advancement of technology, including automation and artificial intelligence, are affecting the labor market . The proDue to such various changes, the labor market is becoming increasingly unpredictable as new opportunities arise and traditional jobs disappear. In such fluid and unstable times, university students who are preparing to join the workforce can no longer expect a linear development in their careers . They shRecent studies have focused on career adaptability (CA) as an important factor that needs to be enhanced in times of unpredictability, especially during the pandemic . CA, oneadaptivity or adaptive readiness refers to a rather stable personality trait of flexibility and willingness to adapt; adaptability indicates the psychosocial resources individuals have to cope with various career-related challenges and tasks; adapting or adaptive responses refers to behaviors that function to accomplish developmental tasks in the changing context; and adaptation is the goodness of fit resulting from adaptive readiness, resources, and responses , which has been addressed as one of the adaptive responses in the career construction model of adaptation . CDMSE rUsing the career construction model of adaptation, the current study also focuses on cognitive emotion regulation (CER) as adaptive readiness to reflect the impact of students\u2019 regulation of negative emotions on CA and CDMSE. The role of emotions in career development has long been emphasized, as they are closely related to work experiences and career-related choices , 2004. SIn sum, the purpose of the current study is to find supporting evidence for the relationships among CER \u2014 specifically adaptive CER and maladaptive CER \u2014 CA, and CDMSE in the framework of the career construction model of adaptation using a path analysis model. Also, the mediating effect of CA between either adaptive or maladaptive CER and CDMSE is examined using mediation analysis. Examining the relationships among the variables can provide supporting evidence to confirm the model of adaptation in the Korean context as well as insight for counselors and educators to assist university students in a successful transition to the world of work in an era of uncertainty.Adaptivity refers to adaptive readiness and is defined as a rather stable and context-general personality trait of flexibility and willingness to adapt when faced with undefined career-related problems . CognitiAdaptability is a psychosocial resource needed for individuals to manage various career-related challenges, tasks, and transitions, and it consists of four resources: concern, control, curiosity, and confidence . SavickaAdapting refers to adaptive beliefs and behaviors that are implemented to accomplish career tasks in the changing context, and the adaptive responses include career planning, career exploration, career decision-making difficulties, and occupational self-efficacy . In the Adaptation is the final dimension in the career construction model of adaptation. It indicates the goodness of fit resulting from adaptive readiness, resources, and responses . VariablCognitive emotion regulation is examined as adaptivity because emotions play a critical role in career development , 2004. AHowever, previous studies on CDMSE have mostly focused on the role of emotional intelligence which included emotion regulation as one of its sub-factors. Moreover, the results regarding the relationship between emotion regulation, as a sub-factor of emotional intelligence, and CDMSE were inconsistent. That is, regulation of emotions did not predict CDMSE in While emotion regulation is a complex process involving biological, social, behavioral, and cognitive elements, Hypothesis 1: Adaptive cognitive emotion regulation is positively related to CDMSE.Hypothesis 2: Maladaptive cognitive emotion regulation is negatively related to CDMSE.According to the career construction model of adaptation, CA refers to coping resources that are predicted by stable personality traits or individual differences such as cognitive flexibility and willingness to adapt . Based oStudies that examine the relationship between emotion regulation and CA seem scarce. Most of the studies have investigated the relationship between CA and emotional intelligence, with emotion regulation as a sub-factor, and have found that emotional intelligence positively predicts CA . It has Hypothesis 3: Adaptive cognitive emotion regulation is positively related to career adaptability.Hypothesis 4: Maladaptive cognitive emotion regulation is negatively related to career adaptability.Career decision-making self-efficacy and CA have been examined as relating variables in numerous studies. In most of the previous studies, CDMSE was examined as a mediating variable leading to CA. For instance, However, the career construction model of adaptation illustrates the mechanism by which CA operates in the process of adaptation . That isHypothesis 5: Career adaptability is positively related to CDMSE.Various studies investigated the mediating role of CA in relation to other variables in the framework of the career construction model of adaptation. In the Korean context, Hypothesis 6: Career adaptability mediates the relationship between adaptive cognitive emotion regulation and CDMSE.Hypothesis 7: Career adaptability mediates the relationship between maladaptive cognitive emotion regulation and CDMSE.SD = 1.23), and 75.9% of them responded that they were actively seeking a job at the time of participating in the survey. Since four participants did not provide their majors, they were excluded from analysis. The resulting number of participants included in the final analysis was 357, and all participants responded to all survey questions without any missing information.Initially, a total of 361 undergraduate students facing a school-to-work transition in Korea voluntarily consented to participate in this study. Before collecting data, the ethical considerations of the study were evaluated and approved by the Institutional Review Board of Sejong University, South Korea . Data were collected in December 2020 by sending an online survey link to university students who were enrolled in a nationwide panel of a data collecting institute. The participants had been informed of the purpose of the study, possible beneficial and harmful outcomes of participation, and that they could stop participating in the study at any time without any disadvantage. Using a screening question, only the students in the 3rd and 4th year in a 4-year university were included because they are generally more actively involved in job-search-related activities in South Korea compared to students in the 1st and 2nd year in university. It took approximately 15 min for the participants to complete the online survey. The gender composition of the participants was 54.8% female and 45.2% male, and the majority of the participants were in their 4th year in university (65.4%). The majors of the participants were liberal arts and social sciences (31.6%), natural sciences and engineering (29.1%), economics and business (16.6%), medicine and pharmacology (10.5%), arts and kinesiology (7.2%), and undefined (1.1%). The mean age of the participants was 24.08 (The Cognitive Emotion Regulation Questionnaire (CERQ), originally developed by Career adaptability was measured using the Career Adapt-Ability Scale (CAAS) originally developed by Based on previous studies that examined university students\u2019 career-related variables, we controlled for some of the demographic factors, such as gender, major, and job-searching status for the final outcome (CDMSE) of the model. First, we included gender as a control variable because previous studies reported an inconsistent relationship between gender and the level of CDMSE e.g., . Second,jamovi 2.5.5 was used to examine the descriptive statistics of the data to inspect possible outliers. Then, we examined the bivariate correlations among the studied variables: adaptive CER, maladaptive CER, CA, and CDMSE. We also conducted Plus8.In the screening phase, jamovi 2.5.5 for Harman\u2019s single factor test.By the nature of the study design  the relationship among the studied variables might be inflated or deflated due to common method variance (CMV) , 2012. TRMSEA), the comparative fit index (CFI), and the standardized root mean squared residual (SRMR) rather than the chi-square (\u03c72) fit statistic due to its poor performance with a large sample  model with 18 items for adaptive CER and the second-order factor model with the five factors as indicators for higher-order factor . Second, we tested the four-correlated factor  model with 16 items for maladaptive CER while investigating the adequacy of the second-order factor model posing a general factor for the four sub-factors. Third, we tested the four-correlated factor  model with 24 items for CA while testing the second-order factor model with the four factors as indicators. Finally, we tested the four-correlated factor  model with the 13 items for CDMSE and the second-order factor model with the four factors governed by a general factor, namely CDMSE. The appropriateness of a CFA model was evaluated based on the alternative fit indices, such as the root mean square of approximation , 2.73 (SD = 0.63), 3.76 (SD = 0.58), and 3.46 (SD = 0.55), respectively. The level of maladaptive CER was slightly lower than adaptive CER, which means that the participants used adaptive CER more than maladaptive. The skewness of the variables ranged from \u20130.09 to \u20130.70, indicating all variables were somewhat negatively skewed, but all values did not exceed the acceptable range (<| 2|) for retaining the univariate normality assumption (p < 0.001), indicating that multivariate normality was rejected.We inspected the data based on the descriptive statistics and found that no outliers existed. The descriptive statistics of the main variables  are displayed in sumption . The kurr = 0.58; p < 0.01) and CDMSE . Yet, the correlation between adaptive CER and maladaptive CER was not statistically significant . Although no prior study directly examined the correlation between adaptive CER and maladaptive CER, the study by r = \u20130.17; p < 0.001) and CDMSE . Third, the correlation between CA and CDMSE was positive and very high .Since the multivariate normality was not sustainable for the main variables, we refer to Spearman\u2019s correlation. We conducted Harman\u2019s single factor test to examine the method effect due to the design of the current study . The result of Harman\u2019s single factor test indicated that only 25.4% of the variance of the 71 items was accounted for by the single factor assumed to be the effect of the common method. Hence, we garnered that the problem of CMV was not prominent in our study based on the criterion (>50%) in p < 0.001), 368.54 (p < 0.001), 930.09 (p < 0.001), and 262.03 (p < 0.001), respectively, we employed the robust Maximum Likelihood estimation method which was recommended for non-normal data  = 210.839, p < 0.001; RMSEA = 0.044; CFI = 0.955; SRMR = 0.049] and second-order factor model  were acceptable. For maladaptive CER, both the four-correlated factor model  and second-order factor model  were adequate after imposing one correlation between unique factors of 9th and 17th items. This is because the correlation between these two items exhibited the largest modification index, and the items, which belong to the same sub-factor \u2018blaming others,\u2019 had similar contents: \u201cI feel that others are responsible for what has happened\u201d and \u201cI feel that basically cause lies with others.\u201d For CA, both the four-correlated factor model  and second-order factor model  reasonably fitted to the data. For CDMSE, both the four-correlated model  and second-order factor model  were appropriate. Since the second-order factor model of every scale was acceptable, using the total score for the path analysis model was empirically legitimate. More detailed information regarding the tested CFA models and standardized parameter estimates can be found in the 2(df= 12) = 16.56, p = 0.17; RMSEA = 0.033; CFI = 0.991; SRMR = 0.056] as shown in R2s of CDMSE and CA were 0.63 and 0.40, respectively, indicating that approximately 63% of the variance of CDMSE was explained by adaptive and maladaptive CER, CA, and control variables , and 40% of the variance of CA was explained by adaptive and maladaptive CER.To test the hypothesized model  of adaptSE = 0.04; p < 0.05), supporting Hypothesis 1. However, \u03b2 from maladaptive CER to CDMSE was not significant, indicating that Hypothesis 2 was not supported. \u03b2 from adaptive CER to CA was 0.61 , indicating a strong positive relationship, while \u03b2 from maladaptive CER to CA was \u20130.14 , supporting both Hypotheses 3 and 4. Since \u03b2 from CA to CDMSE was 0.74 , Hypothesis 5 was supported, which indicates that CA was positively related to CDMSE after controlling for gender, job-searching status, and majors. As presented in Both SE = 0.05; p < 0.001) with a 95% BS-CI of , which supported Hypothesis 6 that CA would mediate the relationship between adaptive CER and CDMSE. Since the direct effect from adaptive CER to CDMSE was statistically significant, we can consider that CA partially mediated the relationship between adaptive CER and CDMSE, and that the total effect of adaptive CER to CDMSE was positive and significant . The standardized indirect effect of maladaptive CER through CA to CDMSE was \u20130.11  with a 95% BS-CI of , indicating that Hypothesis 7 was supported. The non-significant direct effect from maladaptive CER on CDMSE combined with the significant bivariate correlation between them allowed us to interpret that CA fully mediated the relationship between maladaptive CER and CDMSE. However, the total effect of maladaptive CER to CDMSE was not significant , which means that the overall effect of maladaptive CER to CDMSE was offset.We used a 95% bias-correct bootstrapping confidence interval with 2000 bootstrapping samples to test the mediation effect of CA between adaptive CER and CDMSE and that between maladaptive CER and CDMSE. The current study found supporting evidence for the adequacy of the career construction model of adaptation  with adaRegarding the relationship between CER and CDMSE, adaptive CER was positively associated with CDMSE, supporting Hypothesis 1, although the magnitude of the relationship was not large. The role of emotion in the process of career decision-making has been addressed by many studies, most of which have focused on emotional intelligence in relation to career decision-making-related variables. Specifically, studies have found that emotional intelligence had a positive effect on CDMSE . Howeverr = 0.77) offset the negative and small correlation between maladaptive CER and CDMSE (r = \u20130.14). That is, CA fully mediated the relationship between maladaptive CER and CDMSE, and more discussions will ensue.On the other hand, the results showed that maladaptive CER had no significant relationship with CDMSE, which did not support Hypothesis 2. The significant relationship between maladaptive CER and CDMSE disappeared after CA was taken into consideration. It seemed that the strong positive correlation between CA and CDMSE , we found that CA partially mediated the relationship between adaptive CER and CDMSE. This result is comparable to various studies that have investigated the mediating role of CA. For instance, Meanwhile, CA had a mediating effect between maladaptive CER and CDMSE, as postulated in Hypothesis 7. As a whole, however, the effect of maladaptive CER to CDMSE was suppressed based on the non-significant total effect. According to Students\u2019 gender, major, and job-searching status were selected as control variables for CDMSE, but none of the chosen control variables had a significant relationship with CDMSE. Most of all, gender showed no significant relationship with CDMSE. Previous studies showed inconsistent results. Specifically, Moreover, students\u2019 major and job-searching status had no significant relationship with CDMSE. Although no prior study seemed to have directly investigated the effects of major and job-searching status on CDMSE, we added these control variables because major was related to career decision , and jobThe theoretical and practical implications of the findings of the current study are discussed as follows.The findings of this study have theoretical significance in that they provide supporting evidence for The results also have practical implications for university counselors and educators working with students in the school-to-work transition in today\u2019s world of rapid advancement and increased uncertainty. First, the impact of emotion regulation on career development should be acknowledged. Counselors and educators should address students\u2019 emotional responses to the ever-changing world of work and examine cognitive strategies that students can use to regulate their emotions. As Next, interventions are needed for students with maladaptive CER. Previous studies propose that interventions should aim to challenge maladaptive CER and enhance the use of more adaptive CER strategies . More adAlso, considering its mediating effect, the role of CA should be emphasized. CA is malleable and can be gained through training . StudiesSince CA indicates psychosocial resources one can draw upon to manage various career-related tasks and challenges, fostering university students\u2019 CA would not only help students make a successful transition into the workforce, but also help them adjust to changes in their workplace. Studies have found that CA is associated with various organizational outcomes, such as career satisfaction , higher Finally, the aforementioned practical implications, namely intervening to assist students in using more adaptive CER and enhancing CA, are related to CDMSE. CDMSE refers to self-efficacy which is not simply related to making decisions about what jobs to select, but to gather the necessary information and making sound decisions for one\u2019s career. Hence, gaining an understanding of CDMSE for university students, who are about to join the world of work, may provide some insight at the organizational level as well. In a study involving professionals in the electronic media industry, CDMSE was found to be positively related to career optimism . In anotThe present study has several limitations. First, the study was conducted with cross-sectional data to examine the relationships between the variables. To find evidence for causal relationships, longitudinal and experimental studies may be needed. Identifying causal relationships among the variables may provide more rigorous support for the career construction model of adaptation, as well as insight for devising more comprehensive interventions for students.Second, we tested the CMV using Harman\u2019s single factor test, but the method is known for its insensitivity and inability to handle the CMV. Future studies that use a single-administration self-report survey design should use more robust methods, such as a marker variable , to deteThird, the study used the total scores of the variables to examine their relationships, but to have a more comprehensive understanding of relationships among the examined variables, the sub-factors should be considered in the future. As proposed, interventions to challenge maladaptive CER and enhance CA would be needed to assist university students in their transition to the workforce, and understanding the functions of the variables\u2019 sub-factors would help design specific interventions or training programs.Fourth, we used only 13 items out of the 25 items in the original CDMSE scale since some item contents did not apply to our study. Therefore, we should be careful to interpret the results related to CDMSE, as they might not be directly comparable to the other studies in which the whole item set was used. Furthermore, in future studies, the reliability and validity of CDMSE should be reevaluated for university students who face a school-to-work transition.Finally, the generalizability of the results may be limited since the sample of the study consisted only of Korean university students. The circumstances of the labor market and domestic economy, as well as the situations of university students, may look very different in other countries. To extend the generalizability of the results, future study is needed to encompass participants from other cultures and countries.Despite the limitations, this study contributes to the existing literature by identifying the relationships among adaptive and maladaptive CER, CA, and CDMSE, confirming the career construction model of adaptation. Based on the results of the study, practical interventions can be designed and implemented to assist university students in their transition period in the context of this unpredictable, changing world.10.17632/cmrrccbmdh.1).The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: Mendeley Data (doi: The study involving human participants were reviewed and approved by Institutional Review Board of Sejong University. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.AL and EJ equally contributed to the conceptualization of the study. AL was mainly responsible for writing the introduction, literature review, and discussion sections. EJ took charge of conducting formal data analysis and writing the \u201cMaterials and methods\u201d and \u201cResults\u201d sections. Both authors contributed to the article and approved the submitted version."}
+{"text": "In-hospital falls are a serious threat to patient security and fall risk assessment (FRA) is important to identify high-risk patients. Although sensor-based FRA (SFRA) can provide objective FRA, its clinical use is very limited and research to identify meaningful SFRA methods is required. This study aimed to investigate whether examples of SFRA methods might be relevant for FRA at an orthopedic clinic. Situations where SFRA might assist FRA were identified in a focus group interview with clinical staff. Thereafter, SFRA methods were identified in a literature review of SFRA methods developed for older adults. These were screened for potential relevance in the previously identified situations. Ten SFRA methods were considered potentially relevant in the identified FRA situations. The ten SFRA methods were presented to staff at the orthopedic clinic, and they provided their views on the SFRA methods by filling out a questionnaire. Clinical staff saw that several SFRA tasks could be clinically relevant and feasible, but also identified time constraints as a major barrier for clinical use of SFRA. The study indicates that SFRA methods developed for community-dwelling older adults may be relevant also for hospital inpatients and that effectiveness and efficiency are important for clinical use of SFRA. Fall accidents are a major threat to the health of older adults, resulting in injuries and even premature death. The frequency of falls increases with age and frailty level . ApproxiApproximately 10% of all falls occurring among older adults cause severe injury, most commonly fractures . The numAlthough fall prevention has been relatively well-studied among older adults living in the community, research in care facilities and hospitals is more limited. A Cochrane Review on fall prevention interventions for older adults  includedThere is a major need for clinical tools that are easy-to-use and generate objective, accurate, and quantitative risk assessment in clinical settings ,31. ThisThe overall aim of the study presented in this article was to investigate whether examples of SFRA methods might be relevant for FRA at an orthopedic clinic. The study\u2019s research questions were:RQ1: In what situations do staff at an acute ward for inpatient orthopedic care assess older patients\u2019 fall risk? Can SFRA support the staff in any of these situations?RQ2: Which SFRA methods suiting the clinical FRA situations and the clinic\u2019s procedures can be identified from scientific literature?RQ3: What are the staff\u2019s views on the value of SFRA and its utilization for the clinic?To address the three research questions, the study adopted a qualitative design with an inductive approach  and fourSub-study 1 was a focus group aiming at identifying situations where SFRA might assist the FRA currently performed at an orthopedic clinic. Two situations were selected through the focus group interview and written communication between researchers and the clinic.Sub-study 2, published in [ished in , was a sSub-study 3 was a comparison between the identified SFRA methods  from sub-study 2 and the two selected clinical FRA situations from sub-study 1. This sub-study identified SFRA methods which might be relevant for the two selected clinical FRA situations.Sub-study 4 investigated the clinical staff\u2019s views of whether and how the SFRA methods identified in sub-study 3 might contribute to their clinical work with FRA in older adults.Sub-study 1 and 4 were conducted at an orthopedic clinic at a hospital located in a medium-sized Swedish city with approximately 150,000 inhabitants. The clinic performs out-patient and inpatient care , and pre-and post-operative rehabilitation.For sub-study 1, the participants worked on the acute inpatient ward and were recruited by their managers. They all received written information from their manager and the researchers before providing a written consent to participate.For sub-study 4, the participants were clinical staff who attended a research seminar held at the orthopedic clinic. They also received oral and written information, through a Mentimeter presentation, prior to providing their written consent.The participants were asked to complete study specific questionnaires collecting background information. The quantitative data on the participants\u2019 background information was analyzed by descriptive statistics.Data on clinical FRA situations where SFRA might be relevant was collected in a focus group interview with clinical staff. The focus group interview aimed to address RQ1.The interview, which was approximately 90 min, was led by a moderator (first author). Two other researchers (second author and a research engineer) supported the interview in the role of assessors, taking notes and asking complementary questions. All researchers had an engineering perspective, and the authors had experience in collecting user feedback from healthcare staff. To clarify and add information to the interview responses, the participant responsible for the research at the orthopedic clinic also contributed with additional questions to the staff. The interview was audio recorded and transcribed verbatim. In the introduction, the participants were informed that the purpose of the interview was to explore how the staff performed FRA of older patients. As an opening question, they were asked to describe the group of patients aged \u226565 where FRA is relevant. Thereafter, a semi-structured interview guide, containing questions about how fall risk is currently assessed during a patient stay within the ward, and how they thought technology could support their FRA, was used.The verbatim transcript was analyzed by all three researchers to identify situations where the staff performed FRA of older adults. They identified a list of FRA situations. The brief description of each FRA situation was produced by the moderator who extracted and summarized information on environment, situation, participants, and aim per FRA situation from the assessors\u2019 notes. Thereafter, joint discussions between the researchers on whether or not complementary information from the transcripts should be added were held.From the identified situations, the researchers selected two FRA situations for further exploration. The selection was performed based on the following criteria: (1) all selected situations should contain FRAs based on motions, i.e., not only questionnaires; and (2) taken together, the selected situations should include different types of FRAs and involve different professions. The FRA situations were selected during a joint discussion between all three researchers until consensus was reached. The results of the analysis were sent to the focus group participants, the staff\u2019s team leader and the clinic\u2019s operational developer for feedback via the person being responsible for research at the clinic. The results were also presented at a staff meeting, in which staff was given the opportunity to provide additional feedback. The feedback was integrated into the brief descriptions of the identified FRA situations.The aim was to identify SFRA methods that had been evaluated with older adults and to contribute with information on the characteristics of each method and the evaluation methodology used. The systematic literature review, previously published in , was perThe studies\u2019 populations of interest, investigated test results, comparator test results, and outcomes  were def(1)The SFRA method used assessment tasks considered to be relevant for performing a FRA in the following situations: walking to the bathroom, the transitions sit-to-stand and stand-to-sit, putting on slippers, walking in stairs, getting into and out of bed, and activities in daily living (ADL)s .(2)The SFRA method used one or two sensors since a higher number of sensors was not considered feasible for the clinical setting.To address RQ2, all studies included in the systematic literature review of SFRA , i.e., sThe aim of sub-study 4 was to address RQ3. Data were collected during a research seminar directed towards staff from the entire orthopedic clinic. The seminar included presentations of three research projects of which the overarching study presented in this article was one . The outhttps://www.menti.com (accessed on 1 February 2023) by entering a password.The presentation  started The first two questions were used to collect written consent to participate and to allow the researchers to use the information provided according to the described data handling. Thereafter, the participants answered six background questions related to their profession, workplace, number of years in the profession, current FRA procedures, use of technology for FRA, and interest in using technology for FRA.The presentation continued with the provision of brief information on methods used and results obtained in sub-studies 1\u20133. For sub-study 3, a table presenting the ten selected studies in terms of publication record, FRA method, study population, number of sensors, sensor position(s), and SFRA outcomes was presented. Thereafter, the participants answered ten questions related to each identified SFRA method\u2019s value for the FRA situations and the clinic\u2019s operations.Thereafter, information on the four SFRA evaluation studies involving patients that were identified in sub-study 2 was provided. Three of these were not selected in sub-study 3 due to not classifying individuals. The participants answered two questions related to the relevance of the FRA methods used in the four studies involving patients. The sequences of questions related to SFRA and FRA methods included two slides explaining the FRA methods. The aim of these slides was to provide information on the methods in case the participants lacked knowledge of them.For each question, the researchers monitored the number of answers entered in order to allow all participants to answer before proceeding to the next question or information slide. A technical issue with Mentimeter was that also participants entering the code after the written consent questions had been asked were able to answer all remaining questions, i.e., without providing consent to participate and permitting the researchers to use the provided information. Therefore, the answers from these people were omitted in the analysis. The lack of a possibility to provide written consent in later stages of the questionnaire is a limitation in the Mentimeter questionnaire tool.The analysis of the data depended on the nature of the question. Multiple-choice questions were analyzed by summarizing the number of answers per response alternative. Free-text questions were analyzed by coding the content of each answer, clustering codes according to similarity, and summarizing the number of answers per cluster. The number of responses per question was also counted. For certain clusters, quotes illustrating the participants\u2019 views were also extracted. The first author made a first analysis which was reviewed by the second author. Differences in views on how to interpret responses were discussed until consensus was reached.The study addressed the following three research questions:RQ1: In what situations do staff at an acute ward for inpatient orthopedic care assess older patients\u2019 fall risk? Can SFRA support the staff in any of these situations?RQ2: Which SFRA methods suiting the clinical FRA situations and the clinic\u2019s procedures can be identified from scientific literature?RQ3: What are the staff\u2019s views on the value of SFRA and its utilization for the clinic?Five individuals participated in the focus group interview. One was responsible for the research at the clinic and the others represented different categories of health personnel including assistant nurse, nurse, occupational therapist, and physiotherapist. The assistant nurse and the nurse worked on an acute inpatient ward while the physiotherapist and the occupational therapist worked both in inpatient and outpatient care. As for experience in the field, three individuals had <5 years working with patients \u226565 years of age, and one had 5\u201310 years of experience working in a similar setting. All four reported assessing fall risk in older adults several times per day and voiced an interest to use technology in FRA. They further expressed that the technology could contribute to fall prevention as well as to recognize some risks.Six clinical FRA situations were identified from the focus group interview . The sitBased on results published in 33 articles, the systematic review of SFRA literature, previously published in , identifFifteen out of the 33 articles in sub-study 2  evaluateThe ten selected articles are presented in The study included 13 participants. As shown in The participants described that the patients\u2019 fall risk is assessed in various settings some of which included meetings with outpatients, as well as during inpatient enrollment, post-surgery rehabilitation planning, etc. They described that they observe and collect information both from the patients and their caregivers/relatives . They meThe remainder of this section presents the participants\u2019 views on SFRA in terms of: relevance and feasibility of the assessment tasks , the parSome participants were familiar with \u22651 of the FRA methods used in the SFRA approach selected in sub-study 3. As shown in The FRA methods found to be most relevant in clinical work were activities in daily life followed by gait in daily life and gait tests. Interestingly, only one respondent perceived that TUG was relevant in clinical work. Moreover, all FRA methods were found feasible for the clinical work of 3\u20135 participants.Sub-study 2 identified three evaluations of SFRA methods\u2019 abilities to discriminate between groups of patients with different fall risk levels ,52,53. DThe participants stated that they would like to obtain information on their patients\u2019 fall risk, both in general but also in specific activities/situations from SFRA .The participants\u2019 willingness to dedicate time to SFRA-related work, i.e., to mount and remove sensors, and to review SFRA results, varied . The maxFour out of eight participants envisioned time constraints as a barrier for using SFRA in their clinical work see . One of The participants anticipated both positive and negative outcomes from using SFRA in their clinical setting see . A majorThis study aimed to investigate whether examples of SFRA methods might be relevant for FRA in an orthopedic clinic. The study addressed the three research questions:RQ1: In what situations do staff at an acute ward for inpatient orthopedic care assess older patients\u2019 fall risk? Can SFRA support the staff in any of these situations?RQ2: Which SFRA methods suiting the clinical FRA situations and the clinic\u2019s procedures can be identified from scientific literature?RQ3: What are the staff\u2019s views on the value of SFRA and its utilization for the clinic?The major results of the study in relation to RQ1\u2013RQ3 are the following:RQ1: In the acute inpatient orthopedic ward, FRA is performed in several situations during a hospital stay. The FRA is mainly performed through the staff\u2019s observations while structured FRA is performed during patient enrollment and post-surgery. The patient\u2019s ability to safely perform daily activities and move is at focus. Several professions  contribute to clinical FRA.Although Cameron et al.  identifiRQ2: There is evidence in scientific literature that SFRA can discriminate between groups of older adults with different levels of fall risk and classify older adults according to risk levels. However, many SFRA evaluations involve older adults living independently and base the FRA on clinical tests  performed under observation. Nevertheless, it was possible to identify ten examples of evaluated SFRA methods which classified older adults according to fall risk, used 1\u20132 sensors, and included assessment tasks reflecting activities and movements of daily life . These examples support Shany et al.\u2019s  conclusiRQ3: In the entire orthopedic clinic (including both in- and outpatient care), FRA is mainly performed by observations and the staff\u2019s clinical experience. In addition, medical records, patients\u2019 own descriptions, patient characteristics, physiological measurements, and structured questionnaires are used as complementary sources of information. FRA is mostly performed without technology and several healthcare professions contribute.The staff working with in- and outpatient orthopedic care find that SFRA tasks reflecting activities and movements of daily life are relevant and feasible for their clinical work. More specifically, FRA based on gait tests, and gait and ADL were found clinically relevant by a larger number of staff compared with the TUG and standing balance tests. Despite not being familiar with the elbow flexion test, some staff perceived that the test might be relevant for FRA after a brief introduction to the concept.The staff wanted SFRA to contribute with information on a patient\u2019s fall risk, both in general and in specific situations or activities. Context-specific fall risk information was also found being highly useful by older adults wanting to better understand their fall risk . Some stThe staff expressed concerns (such as questioning whether the patients would accept the technology and anticipated non-effective SFRA as costly and a waste of resources). They also expressed potential values  related to SFRA. Both the concerns and potential values may be related to the perceived usefulness of SFRA, which according to UTAUT  is a detAlthough SFRA has mainly been evaluated among older adults living independently and by collecting sensor data from assessment tasks performed under observation, this study has shown that SFRA includes FRA methods that may be relevant in orthopedic clinics. In this study, clinical staff from in- and outpatient orthopedics care expressed that, in order to be clinically relevant, SFRA must be effective in reducing falls, reliable, smooth, and easy-to-use. These views are supported by the UTAUT model."}
+{"text": "The concept of utilizing a large temperature difference (>20\u2009\u00b0C) between the surface and deep seawater to generate electricity, known as the ocean thermal energy conversion (OTEC), provides a renewable solution to fueling our future. However, it remains poorly assessed how the OTEC resources will respond to future climate change. Here, we find that the global OTEC power potential is projected to increase by 46% around the end of this century under a high carbon emission scenario, compared to its present-day level. The augmented OTEC power potential due to the rising sea surface temperature is partially offset by the deep ocean warming. The offsetting effect is more evident in the Atlantic Ocean than Pacific and Indian Oceans. This is mainly attributed to the weakening of mesoscale eddy-induced upward heat transport, suggesting an important role of mesoscale eddies in regulating the response of thermal stratification and OTEC power potential to greenhouse warming. Ocean thermal energy conversion (OTEC) resources provide a renewable solution to fuel our future. Here the authors show a significant increase of OTEC resources under greenhouse warming with the increasing rate regulated by oceanic eddies. Global warming and ocean acidification caused by the increased concentration of CO2 exert many different adverse effects on the ecosystems and human society3. Therefore, the replacement of fossil energy with decarbonized sources is essential for tackling these crises5. Unlike many other renewable technologies based on intermittent energy sources such as winds and sunlight7, the ocean thermal energy conversion (OTEC) is capable of steadily providing humanity with vast amounts of electrical power8 associated with a moderate levelized cost of energy (140\u2013157 USD MWh\u22121)7 and competitive by-products9. It has been estimated that the total electrical power generated by OTEC  across the global ocean could reach up to 8\u201310 terawatts (TW)10, with only 1.9 TW of electricity generation by fossil fuels during 2020 in contrast11, indicating an abundant energy potential.Fossil fuels as energy sources have been on heavy dependence since pre-industrial times, resulting in massive emissions of greenhouse gases, especially carbon dioxide  depends on the squared temperature difference \u2206T between surface and deep (1000\u2009m) seawater . During the past half century, more than 90% of the anthropogenic heat surplus accumulated in the oceans via the heat flux at the sea surface, of which two thirds is absorbed in the upper-700\u2009m water column12. Accordingly, the global mean sea surface temperature (SST) has increased by an amount (0.78\u2009\u00b0C)13 larger than the deep ocean counterpart (0.06\u2009\u00b0C averaged within 700\u20132000\u2009m) since 1960s14, leading to enhanced thermal stratification15. In the future, the strengthening of thermal stratification is likely to continue due to greenhouse warming16, implying enriched OTEC resources. Despite this simple response of thermal stratification to greenhouse warming in terms of the global average, the geographic pattern of anthropogenic thermal stratification change is complicated15 and strongly affected by the heat transport of oceanic flows20. On the one hand, the SST changes caused by local sea surface heat flux changes can be advected elsewhere by oceanic flows like a passive tracer, particularly into the deep ocean via the ventilation processes19. On the other hand, changes in surface wind and buoyancy forcing under greenhouse warming drive changes in oceanic flows that redistribute the heat in the ocean and further affect the efficiency of ocean uptake of anthropogenic heat surplus via the redistribution feedback20. Yet existing knowledge of OTEC power potential change in a warming climate is mainly derived from a one-dimensional (1-D) model21 with an oversimplified representation of heat transport by oceanic flows22. This causes large uncertainties in the projected OTEC power potential change by the 1-D model.The OTEC power potential density 23 have an oceanic resolution (~1\u00b0) too coarse to resolve the ocean thermal structure in the coastal ocean where deploying OTEC plants is much more feasible than in the open ocean24. Moreover, even in the open ocean, coarse-resolution CGCMs have an evident bias in the simulated ocean thermal stratification26 partially due to deficiencies in representing effects of smaller-scale processes such as oceanic mesoscale eddies29, casting doubt on their validity in projecting the trends of ocean thermal stratification and OTEC power potential in the future.Coupled global climate models (CGCMs) provide a more reliable projection of ocean thermal stratification change under greenhouse warming compared to the 1-D model. However, so far most of the CGCMs participating in the Coupled Model Intercomparison Project Phase 6 (CMIP6)30 (referred to as CESM-H) (see \u2018CESM-H simulation\u2019 in Methods section) to assess the change of OTEC power potential and its underlying dynamics under the high carbon emission scenario. It should be noted that the assessment in this study does not consider the feedback effect on ocean thermal stratification caused by the effluent discharge when utilizing OTEC31. Nevertheless, it has been demonstrated22 that such feedback is negligible for a moderate cold-water intake rate such as 5\u2009m year\u22121 used for the computation of OTEC power potential in this study . As shown below, CESM-H shows good skills in reproducing the OTEC power potential in the past half century, providing us confidence in its reliability in projecting the future change of OTEC power potential.In this study, we use an unprecedented long-term high-resolution simulation based on the Community Earth System Model\u22121 during 1955\u20132021, not significantly different from the observed value 1.80\u00a0\u00b1\u00a00.22 TW century\u22121. In contrast, the time-mean global OTEC power potential for the CMIP6 CGCM ensemble mean is 7.21\u00a0\u00b1\u00a00.30 TW during 1955\u20132021, indicating a smaller time-mean \u2206T or equivalently weaker thermal stratification in the CMIP6 CGCMs than the observation and CESM-H  during 1955\u20132021 similar to those in the observation and CESM-H, in accordance with the similarity among the linear trends of \u2206T in the observation, CESM-H and CMIP6 CGCM ensemble mean  observation (see \u2018Observation products\u2019 in Methods section) and compared to an ensemble of coarse-resolution ~1\u00b0) CGCMs in CMIP6 , is 127.4\u2009\u00b1\u20090.3 million km2 in the observation, closer to 116.6\u2009\u00b1\u20091.0 million km2 in the CESM-H than 99.9\u2009\u00b1\u20093.2 million km2 in the CMIP6 CGCM ensemble mean.We next examine the present-day (1992\u20132021) spatial distribution of net Fig.\u00a0 that is ean Fig.\u00a0. However24. The EEZ covers 49.7% of the OTEC region and accounts for 52.0% of the global OTEC power potential in the observation  is slightly higher than the observed one. Similar to the case of global OTEC power potential, the time-mean OTEC power potential over the EEZ in the CMIP6 CGCM ensemble mean (3.71\u2009\u00b1\u20090.13 TW) biases low by 23.8% (20.9%) compared to the observation (CESM-H) due to its smaller time-mean \u2206T  is not statistically different from those in the observation and CESM-H.At present and in the foreseen future, deployment of OTEC plants in the exclusive economic zone (EEZ) would be more practical and cost-effectiveion Fig.\u00a0, with thion Fig.\u00a0. The tim\u00a0\u22121 Fig.\u00a0. The CES \u2206T Fig.\u00a0. HoweverBased on the above comparisons, we conclude that the CESM-H generally provides a reliable simulation of OTEC power potential during 1955\u20132021. Specifically, it simulates a linear trend of OTEC power potential similar to those in the observation and CMIP6 CGCM ensemble mean but outperforms the CMIP6 CGCM ensemble mean in the simulated time-mean OTEC power potential. This lends support to using the CESM-H for projecting the future OTEC power potential change by the end of this century.Pnet around the end of this century is similar to the present one but its magnitude becomes systematically larger  is more than twice 1.99\u2009\u00b1\u20090.59 TW century\u22121 during 1955\u20132021  is about twice 0.97\u2009\u00b1\u20090.20 TW century\u22121 during 1955\u20132021  ger Fig.\u00a0 owing to021 Fig.\u00a0. The tim021 Fig.\u00a0. The timT and Pnet  but decreases southeastward to ~15\u2009kW\u2009km\u22122 near the Yucatan Channel. This heterogeneous change of Pnet mimics that of SST change under greenhouse warming  and Gulf of Mexico (GOM), the two representative marginal seas of the Pacific and Atlantic Oceans, respectively. The SCS located at lower latitudes has generally higher SST than the GOM, resulting in larger values of present-day \u0394lly Fig.\u00a0. The chaPnet depends on the value of \u2206T, sea surface warming acts to increase Pnet but deep ocean warming has the opposite effect. Although the surface ocean is projected to warm faster than the deep ocean in terms of the global average16  and heat sink by mesoscale eddies (\u22123.22\u2009\u00b1\u20090.09\u2009W\u2009m\u22122), with the turbulent vertical mixing (\u22120.03\u2009\u00b1\u20090.01\u2009W\u2009m\u22122) and OHC tendency (0.05\u2009\u00b1\u20090.14\u2009W\u2009m\u22122) more than an order of magnitude smaller. The cooling by mesoscale eddies is largely attributed to their induced upward heat transport at 800\u2009m (\u22122.97\u2009\u00b1\u20090.10\u2009W\u2009m\u22122) as a result of baroclinic instability39.To uncover the underlying dynamics of strong warming at 1000\u2009m in the Atlantic Ocean, an OHC budget analysis is performed for the 800\u22121200\u2009m water column over the Atlantic OTEC region  becomes one of the dominant terms . They account for 74.1% of the OHC tendency anomaly, whereas this fraction value is reduced to -3.4% and 29.3% for mean flows and turbulent vertical mixing, respectively. The effect of mesoscale eddies is mostly ascribed to the reduction of upward eddy heat transport at 800\u2009m. Its value decreases from 2.97\u2009\u00b1\u20090.10 W\u2009m\u22122 during 1992\u20132021 to 1.76\u2009\u00b1\u20090.04\u2009W\u2009m\u22122 during 2071\u20132100. Such a decrease implies a weakened baroclinic instability under greenhouse warming which might be partially due to the enhanced stratification41 that reduces the available potential energy stored in mean flows through flattening the isopycnals42. It should be noted that the reduced upward heat transport of mesoscale eddies also occurs in the Pacific and Indian Oceans. However, the upward eddy heat transport in the Pacific and Indian Oceans has a much shallower vertical structure than that in the Atlantic Ocean , the OHC tendency anomaly , it does not necessarily mean that the OTEC power potentials in the CESM-H and coarse-resolution CMIP6 CGCMs have consistent responses to greenhouse warming. In fact, with the rising greenhouse gas emission in the future as in the high carbon emission scenario, the difference in the projected trends of OTEC power potential between the CESM-H and coarse-resolution CMIP6 CGCMs during 2022-2100 is qualitatively similar to its historical counterpart but quantitatively becomes sufficiently large to be statistically significant  forcing during 2006\u20132100. Comprehensive descriptions of CESM-H can be found in a recent overview paper30.The CESM-H simulation is performed based on CESM version 1.3. It has a nominal 0.25\u00b0 and 0.1\u00b0 horizontal resolution for the atmosphere and ocean components, respectively. There are 30 vertical levels in the atmosphere and 62 levels in the ocean. The simulation consists of two components. The first one is the 500-year-long pre-industrial control (PI-CTRL) simulation with the climate forcings fixed to the 1850 conditions. The second one is the 250-year-long historical and future transient simulation (HF-TNST) during the 1850\u20132100 period. The HF-TNST simulation is branched off from the 25044. This error is two orders of magnitude smaller than the \u2206T threshold (20\u2009\u00b0C) for OTEC and does not affect the secular change of OTEC power potential.Monthly averaged potential temperature and diagnostic outputs of heat transport by resolved flows are saved during 1878-2100. In this study, we use the potential temperature as a proxy for temperature. This may introduce a small error on the order of 0.1\u2009\u00b0C for the temperature at 1000\u2009m30. This may bias the simulated secular change of OTEC power potential in HF-TNST caused by greenhouse warming. To minimize such bias, we subtract the trend of temperature at 1000\u2009m during the years 350\u2013500 in PI-CTRL from that during 1950\u20132100 in HF-TNST.After 250 year\u2019s spin-up, the model drift in the deep ocean (1000\u2009m) temperature becomes small but still noticeable45 is used in this study to evaluate the OTEC resources during 1955\u20132021. The monthly temperature data are on 1\u00b0\u2009\u00d7\u20091\u00b0 regular grids and have 26 levels between 0\u20132000\u2009m.An observational global OHC dataset provided by Japan Meteorological Agency (JMA)47 per unit area, formulated as31:wcw\u2009=\u20095\u2009m year\u22121 is the deep cold seawater intake rate, \u03c1\u2009=\u20091026\u2009kg\u2009m\u22123 is a reference density of seawater, cp\u2009=\u20094000\u2009J (kg\u00b7K)\u22121 is the seawater specific heat capacity, \u03b5tg\u2009=\u20090.75 is the turbo-generator combined efficiency, \u03b3 is the flow rate ratio of the surface warm water intake over the deep cold water fixed as 1.5, T is the absolute temperature of the warm water intake and \u2206T is the temperature difference between surface and deep ocean (1000\u2009m) seawater intakes48. The first term on the right-hand side of Eq. 52 but is more than an order of magnitude smaller than the intense wind-driven upwelling O(1 m day\u22121) in the eastern boundary upwelling systems53.It should be noted that the cold water used in the system is pumped up from the deep ocean and will not be discharged back to its initial depth, but directly to the surface, leading to a reduction in the SST, \u2206\u03b8(t) as:54 is used to compute the s.e. of OLS estimators To compute the standard error (s.e.) of statistics discussed in the main text such as the time-mean value and slope of the linear trend, we model the time series of some quantity N\u2009=\u200936 is the ensemble number of coarse-resolution CMIP6 CGCMs, the subscript i corresponds the i-th CGCM, and the braces represent the average across the CGCMs. To derive Eqs.  is the three-dimensional oceanic flow, Km is the turbulent vertical diffusivity, \u2207 = , the overbar denotes the three-month average, the prime denotes the perturbation from the three-month average, and \u2329\u2026\u232a denotes the horizontal average. The horizontal mixing by subgrid-scale processes is dropped, as its effect is negligible compared to other terms when averaged over a sufficiently large area considered here, i.e., the Atlantic OTEC region , the heat flux convergence of mesoscale eddies (Qeddy), and turbulent vertical mixing (Qmix). The Qmean is computed based on the monthly model output of u and T, while the Qeddy is derived from subtracting Qmean from the model diagnostic output of uT. The Qmix is computed as the residue.The term on the left-hand side of Eq.  is the OSupplementary InformationPeer Review File"}
+{"text": "The Demographic Index (DI) comprises of five linked administrative datasets, used for population estimation. Current linkage methods are not ideal to utilise the power of this asset. Using the 2021 England and Wales Census, we are developing an innovative composite linkage method to fully utilise the power of the DI.Using non-greedy deterministic and probabilistic linkage methods, we will link the DI to the Census at a composite level where we believe links exist \u2013 i.e., linking a Census cluster (consisting of linked Census and Census Coverage Survey (CCS) records) with a DI cluster (consisting of linked records from the data sources used to make the DI). We will then conduct a pairwise linkage of records from these linked clusters to link individual source records to the Census. We will utilise clerical review to resolve uncertain and conflicting links and to inform the quality of our linkage.We anticipate producing a high-quality linkage that will inform how the coverage of the DI compares to Census (through the composite-level linkage) and the quality of the DI itself (through the pairwise-level linkage). We have developed a clerical matching system that can display composite-level linkage, i.e., candidate cluster-pairs. We will tailor our clerical review and quality assessment to records that fall within carefully chosen postcode areas, to ensure all hard-to-count groups and geographical areas are sampled. Working with large datasets is a challenge we are overcoming by using distributed computing and search space reduction.The 2021 Census has been previously linked to the CCS with high accuracy; these records are considered intrinsically linked.To assess national population estimates\u2019 quality and the policy decisions based upon them, we are linking a key composite population-level dataset to the 2021 England and Wales Census. The presentation will showcase the methods we are developing and how we are ensuring the highest quality possible."}
+{"text": "Background: As a systematic fungicide, prochloraz is often used to control banana freckle disease, and it is significant to assess the safety and risk of prochloraz. Methods: The dissipation kinetics and distribution of prochloraz in bananas were measured by high-performance liquid chromatography (HPLC). Results: The results showed that the fortified recoveries in bananas were 83.01\u201399.12%, and the relative standard deviations (RSDs) were 2.45\u20137.84%. The half-life of prochloraz in banana peel (3.93\u20135.60 d) was significantly lower than it was in whole banana (8.25\u201310.80 d) and banana pulp (10.35\u201312.84 d). The terminal residue of prochloraz in banana fruits was below the maximum residue level  at pre-harvest intervals (PHI) of 21 d. Moreover, the residue of prochloraz in banana peel was always 1.06\u20137.71 times greater than it was in banana pulp. The dietary risk assessment results indicated that the prochloraz residue in bananas at PHI of 21 d was safe for representative populations. (4) Conclusions: We found that a 26.7% prochloraz emulsion oil in water (EW) diluted 1000-fold and sprayed three times under field conditions was safe and reliable, providing a reference for the safe application of prochloraz in bananas. Musa acuminata), the most important fleshy fruit around the world, is widely planted in China [Banana  ethyl-imidazole-1-carboxamide) is a systemic imidazole fungicide and is widely used on banana for controlling freckle disease due to its high efficiency . It can Prochloraz is a low-toxicity pesticide; however, its metabolite 2, 4, 6-trichlorophenol is characterized by stable structure and poor biodegradability, which can easily cause serious environmental pollution and is suspected to be a strong carcinogen . TherefoThe objectives of this study were (a) to determine the dissipation dynamics of prochloraz residue on banana fruits under field conditions; (b) to analyze the prochloraz\u2019s residual distribution and permeability of banana fruits; (c) to evaluate the exposure risk of prochloraz on banana fruits to representative populations in China. We aimed to provide a reference for the safe application of 26.7% prochloraz emulsion oil in water (EW) in bananas.18, and graphitized carbon black (GCB) were obtained from the Agela Technologies Incorporated Company . The stock standard solution of prochloraz and 2, 4, 6-trichlorophenol was prepared at 100 mg/kg by methanol (HPLC-grade) and stored in a refrigerator at \u221220 \u00b1 1 \u00b0C.Prochloraz standard  and 2, 4, 6-trichlorophenol standard  were obtained from Aladdin . A 26.7% prochloraz emulsion oil in water (EW) was supplied by the Jinan Yinong Chemical Limited Company . Ethyl acetate, acetonitrile , petroleum ether, formic acid, anhydrous sodium sulfate, pyridine hydrochloride, and anhydrous NaCl were obtained from the Yongda Chemical Reagent Factory . Acetonitrile and methanol (chromatographic-grade) were supplied by the Oceanpak Company . N-propyl ethylenediamine (PSA), ODS-CThe field experiment was conducted in Guangzhou , Yuxi , and Lingao  in 2021. The production soil in three test location was red loam, with 4.1% organic matter and a pH at 5.5 in Guangzhou, 3.4% organic matter and a pH at 5.7 in Yuxi, 3.5% organic matter and a pH at 4.5 in Lingao. The banana varieties planted in Guangzhou, Yuxi, and Lingao were the Guangdong banana no. 2, Zhongjiao no. 9, and Brazil banana, respectively. Each treatment (location) had 6 plots with a buffer zone of 1 m, and each plot had six trees. At each location, three plots were randomly selected to have prochloraz sprayed, and the remaining three plots served as the control area. We diluted 26.7% prochloraz (EW) 1000-fold (maximum recommended dosage) with water and sprayed the mixture on whole banana plants three times at 7 days after the previous spray. The water consumption was 900 L/ha. In the control areas, the bananas were not sprayed with prochloraz throughout the whole developmental stage. In order to research the dissipation dynamics of prochloraz and the terminal residues in different parts of the banana under field conditions, representative banana samples were acquired at random from every point at 0 (2 h), 1, 3, 5, 7, 14, 21, 28, and 35 d after the last spray. All banana samples were homogenized and stored in the refrigerator at \u221220 \u00b1 1 \u00b0C.18, 0.050 g GCB, and 0.050 g PSA were added for purification. Next, 1500.0 \u03bcL of methanol (chromatographic grade) was added until complete dissolution. The final step was to filter the supernatant with a 0.22 \u03bcm filter membrane and analyze the samples using HPLC.The banana fruit samples were peeled with a knife to separate the peel and pulp, and then each was put in a centrifuge tube. We added 5.0 g samples of a whole banana, the peel, and the pulp to a disposable centrifuge tube. We then added 25.0 mL acetonitrile to the disposable centrifuge tube and extracted samples by ultrasonication for 1 h. Next, 5.0 g NaCl was added to the centrifuge tube and centrifuged at 4000 r/min for 5 min using the centrifugal machine. We placed 25 mL supernatant solution into a flask. The supernatant of the acetonitrile layer was aspirated and mixed with 2.0 g of anhydrous sodium sulfate before the pressure was reduced. We then concentrated the mixture to 3.0 mL and transferred it to a 20 mL stopper tube to be dried with nitrogen. Next, 1.0 g of pyridine hydrochloride was added into the stopper tube and hydrolyzed in an oil bath at 220\u2013240 \u00b0C for 1.5 h; after allowing the mixture to cool naturally, we took the tube out and cleaned it twice with 10.0 mL of water, transferred the tube to the separation funnel, extracted the transferred reactant with 40 mL \u00d7 2 ethyl acetate, and discarded the inorganic phase. We combined the mixture with the organic phase, dehydrated it using anhydrous sodium sulfate, and then rotated it to evaporate the solution until it was dry. Next, the cleaning solution was extracted to a centrifuge tube, and 0.050 g C18  and with water: acetonitrile (35:65), as the mobile phase, consisted at a flow rate of 1 mL/min. The sample size, detection wavelength, and column temperature were 35 \u00b0C, 290 nm, and 10 \u03bcL, respectively.A Shimadzu LC-20A HPLC  was used to analyze the 2, 4, 6-trichlorophenol. The chromatographic column was analyzed with an Agilent Eclipse XDB-CMethod validation studies were tested for the whole banana fruit, banana peel, and banana pulp, including the linearity, limit of detection (LOD), limit of quantitation (LOQ), and fortified recovery, to evaluate their accuracy and precision. The LOQ was defined as the minimum amount of the measured substance in the sample that could be quantitatively determined with certain accuracy, identified by multiple additive recovery tests . The LODFor fruit consumption groups of different ages and genders, we calculated the dietary intake risk for prochloraz in bananas based on the groups\u2019 specific body mass and fruit intake, combined with the terminal residue data in our study . The dieochloraz . The acuochloraz :ESTI = Lnce dose .If %ADI (%ARfD) > 100%, this indicates an unacceptable risk of chronic (acute) dietary intake of prochloraz from bananas; the greater that the %ADI (%ARfD) is, the greater the risk is. Conversely, the chronic (acute) dietary intake risk of prochloraz from bananas is acceptable if %ADI (%ARfD) \u2264 100% .0 is the initial residual concentration of prochloraz, Ct is the residual concentration of prochloraz at the time of t, t1/2 denotes the half-life of prochloraz, and k is the constant of the dissipation degradation rate [The dissipation dynamics and half-life of prochloraz were calculated using Equations (6) and (7), respectively :Ct = C0eion rate .The data were sorted using Excel, and a statistical analysis was performed using analysis of variance (ANOVA) with the SPSS 17.0 Statistical Package for Social Sciences  . The valThe content of prochloraz in each part of banana was determined by measuring the content of 2, 4, 6-trichlorophenol and converting it according to Equation (1). The standard linear regression formula and correlation coefficient for 2, 4, 6-trichlorophenol are y = 15499 x + 25.825 and 0.9999, respectively, indicating that 2, 4, 6-trichlorophenol standard kept a good linear relation in the concentration range of 0.05\u201310 mg/L. As shown in p < 0.05). Furthermore, the prochloraz half-life in the banana peel from Yunnan was significantly different from that of the prochloraz half-life in the banana peel and pulp collected in Guangdong and Hainan (p < 0.05).As shown in The prochloraz terminal residues in different parts of the banana fruit from the three southern provinces of China were determined at 21, 28, and 35 d. As shown in The prochloraz residues in the banana peel and pulp from the three southern provinces of China were measured, and their ratios were computed to measure the prochloraz permeability and distribution in the banana peel and pulp. The results indicated that prochloraz has great diffusivity in banana fruits A. The prDietary intake risk is related to food intake and body weight, which varies in different populations. As shown in Contamination of crops by pesticide residues is an ongoing challenge, and the rate of dissipation after the application of pesticides is an important factor and useful tool for evaluating residues\u2019 behavior . To eval"}
+{"text": "Dual-inhibitors of PARP1 and PARP2 are promising anti-cancer drugs. In addition to blocking PARP1&2 enzymatic activity, PARP inhibitors also extend the lifetime of DNA damage-induced PARP1&2 foci, termed trapping. Trapping is important for the therapeutic effects of PARP inhibitors. Using live-cell imaging, we found that PARP inhibitors cause persistent PARP2 foci by switching the mode of PARP2 recruitment from a predominantly PARP1- and PAR-dependent rapid exchange to a WGR domain-mediated stalling of PARP2 on DNA. Specifically, PARP1-deletion markedly reduces but does not abolish PARP2 foci. The residual PARP2 foci in PARP1-deficient cells are DNA-dependent and abrogated by the R140A mutation in the WGR domain. Yet, PARP2-R140A forms normal foci in PARP1-proficient cells. In PARP1-deficient cells, PARP inhibitors - niraparib, talazoparib, and, to a lesser extent, olaparib - enhance PARP2 foci by preventing PARP2 exchange. This trapping of PARP2 is independent of auto-PARylation and is abolished by the R140A mutation in the WGR domain and the H415A mutation in the catalytic domain. Taken together, we found that PARP inhibitors trap PARP2 by physically stalling PARP2 on DNA via the WGR-DNA interaction while suppressing the PARP1- and PAR-dependent rapid exchange of PARP2. Our results showed that PARP1 exchanges rapidly at the site of DNA damage with or without PARP inhibitors , clinical PARP inhibitors cannot allosterically lock purified PARP1 on model DNA substrates . The successful targeting was confirmed by Southern blotting with a 3\u2032 probe . PCR was carried out under the following conditions: 94\u00baC for 5 min\u00a0followed by 32 cycles 94\u00baC 30 s, 62.5\u00baC 30 s, 72\u00baC 30 s. The product corresponding to the wild-type allele is \u223c280 bp, the conditional allele is \u223c370 bp, and the deleted allele is \u223c600 bp.The ditional  and Rosaditional . Loss of in mice . To genel allele  by placi3\u2032 probe . Upon Sc3\u2032 probe . Two ind3\u2032 probe . The folPARP1 knockout (KO), PARP2 KO, and PARP1/2 double knockout (DKO) were generously provided by Dr\u00a0Keith W. Caldecott at the University of Sussex  were isolated (as single clones) from 4-hydroxytamoxifen  treated Cre-ERT2/+Parp1C/CRosa26a, Cre-ERT2/+Parp2C/CRosa26\u00a0and Cre-ERT2/+Parp1C/CParp2C/CRosa26 iMEFs. The successful deletion was verified by PCR and western blotting for Parp1 and Parp2 proteins  cells of wild type (WT), f Sussex  and cultproteins . Primaryproteins .The PARP inhibitors olaparib , niraparib , and talazoparib  were dissolved in dimethyl sulfoxide (DMSO) and used at 1 \u03bcM final concentration. Anti-PARP1 antibody  was used at 1:5000. Anti-PARP2 antibody  was used at 1:2000. Anti-XRCC1 antibody  was used at 1:5000. Anti-PAR antibody  was used at 1:1000. Anti-Histone H3 antibody  was used at 1:5000. Anti-\u03b1-Tubulin antibody  was used at 1:5000. Anti-\u03b2-Actin antibody  was used at 1:10\u00a0000. 8-MOP   was added 10 min\u00a0before micro-irradiation at a final concentration of 100 \u03bcM when used.88-570 (large) was generated by replacing the coding sequence of \u0394NTR-PARP2 with the coding sequence of 88\u2013570aa of human PARP2. All mutations were validated via Sanger sequencing.The DsRed-mono-C1-XRCC1 and pEGFP-C1-PARP2 plasmids were generously provided by Dr\u00a0Li Lan at Massachusetts General Hospital  and Dr\u00a0XRPE-1 cells were treated with or without 1 \u03bcM niraparib for 1 h\u00a0in the presence of 0.1 mg/ml MMS. Cells were then harvested and lysed in lysis buffer containing 100 mM KCl, 2.5 mM MgCl2, 5 mM EDTA, 50 mM HEPES, 3 mM dithiothreitol, 10% glycerol, 0.5% Triton X-100, and protease inhibitor cocktail  for 45 min on ice with tapping every 15 min. Lysates were centrifuged at \u223c16 000 g for 15\u00a0min at 4\u00b0C. Supernatants containing soluble proteins were collected and the pellet containing chromatin bound proteins was washed twice with lysis buffer, then subjected to sonication for 10 min. The chromatin and soluble extracts were mix with SDS-PAGE loading buffer and incubated at 95\u00b0C for 7 min. Then samples were subjected to Western Blotting with indicated antibodies.\u00ae-Multi\u00a0+\u00a0Microplate Multimode Reader  and plotted as a dose-response curve using GraphPad Prism. To express Empty-IRES-hCD8 and PARP2RA-IRES-hCD8 in the PARP1/2 DKO RPE-1 cells, the cassettes were packed into retrovirus and positively infected cells were purified with anti-hCD8 MACS beads as describe before \u00a0. The absorbance of formazan product and the reference were measured at 550 and 650 nm, respectively on a GloMaxe before .4 RPE-1 cells or MEFs were seeded onto each 35 mm diameter glass-bottom plates on day 1. On day 2, the cells were transfected with plasmids encoding fluorescence protein-tagged PARP2 and/or XRCC1 via Lipofectamine 2000  or Lonza 4D-Nucleofector\u2122 X according to manufacturer instructions. Live-cell imaging was performed 24 h\u00a0after transfection with a Nikon Ti Eclipse inverted microscope  equipped with the A1 RMP confocal microscope system and Lu-N3 Laser Units . Only cells with moderate yet reliable expression (\u223c200\u20131000 a.u.) of the GFP- or RFP- tagged protein were imaged. Laser micro-irradiation and timelapse imaging were carried out using the NIS Element High Content Analysis software (Nikon Inc.) and a 405 nm laser (energy level \u223c500 uW for a \u223c0.8 \u03bcm diameter region).Live-cell imaging analyses were performed as described before  with mint1/2 is defined as the time needed for the fluorescence level to reach 50% of the maximum recovered intensity  after bleaching and the fluorescence intensity in the bleached area before bleaching, while ntensity .hPARP2 and mPARP2 were expressed and purified as previously described . CirculaPARP2 was serially diluted in binding buffer (50 mM Tris\u2013HCl (pH 8.0), 150 mM NaCl, and 0.01% IGEPAL) from 4 \u03bcM to 90 nM in a volume of 10 \u03bcl of a 384-well plate (Corning 3575). Fluorescently labeled pNick  was added to each well and incubated for 30 min to ensure complete association. All concentrations cited are final concentrations in a volume of 20 \u03bcl. Fluorescence polarization (FP) using excitation at 482 nm (bandwidth 16 nm), dichroic filter at 496 nm, and emission at 530 nm (bandwidth 40 nm) was monitored from the top of the plate using a BMG Labtech CLARIOstar plate reader.+ (63 \u03bcM\u20132 mM). All concentrations cited are final concentrations in a volume of 30 \u03bcl. Control wells lacked NAD+. Fluorescence polarization (FP) was monitored as in the DNA binding assays, but in a time-dependent mode with 7 s intervals over the course of 45 min. Assays in the presence of HPF1 were performed using the injector mode and 1 s read intervals on the CLARIOstar plate reader in the presence of 200 \u03bcM NAD+\u00a0and 2 \u03bcM HPF1 over the time course of 4 min.PARP2  was diluted in binding buffer in the presence of fluorescein-labeled pNick (7.5 nM) across seven wells of a 384-well plate (Corning 3575) in a volume of 20 ul and pre-incubated for 30 min to ensure complete association. Dissociation of labeled DNA was initiated by the addition of 10 \u03bcl of varying concentrations of NADParp1 KO, Parp2 KO\u00a0and Parp1/2 DKO iMEFs. XRCC1 is recruited to the DNA damage foci by directly binding to PAR (Parp2 KO) does not statistically significantly affect GFP-Parp2 foci intensity, loss of endogenous Parp1 (in Parp1 KO and Parp1/2 DKO cells) decreases Parp2 foci intensity by \u223c2/3  cells than in PARP1 KO isogenic control cells  intervals for a total of 5 min\u00a0(300 s)  Figure  and\u00a0B. U) Figure . Consist) Figure , nirapar) Figure \u2013C. This s Figure . Similars Figure . Taken tParp1/2 DKO iMEF cells. Strikingly, niraparib increased PARP2 foci intensity >3-fold, reaching and exceeding the level in control Parp1-proficient cells  and efficiently  in DMSO treated controls  (t1/2\u00a0=\u00a01.3 s) is very similar to those bleached at 180s after irradiation  and talazoparib  and relatively moderately delayed by olaparib     reportedadiation . In cont) Figure  and\u00a0B. T) Figure , suggestntensity . Moreoves Figure  and delas Figure . As expeKO cells . CollectPARP1/2 DKO cells, we generated GFP-PARP2-E545A and GFP-PARP2-H415A with alanine substitutions in the conserved H-Y-E catalytic triad of PARP2 , with PARP2-H415A  even slower than PARP2-E545A  , but did not measurably affect the exchange of PARP-H415A   Figure . Neverth) Figure . Togethe) Figure .PARP2 KO RPE-1 cells and intensified the weak \u0394NTR-PARP2 foci in the PARP1/2 DKO cells  both with and without niraparib, suggesting the additional 17 aa deletion in the unstructured NTR link region might compromise DNA binding   Figure \u2013H. The kPARP2-WT , indicatPARP2-WT \u2013G, suggePARP2-WT  and ectoKO cells . The finKO cells  where niKO cells . As a reKO cells .in vivo and in the presence of PARP inhibitors remains elusive. Using quantitative live-cell imaging, we showed that PARP2 can be recruited to DNA damage sites through both a PARP1-dependent (predominantly) and a PARP1-independent mechanism. Loss of endogenous PARP1 and its PARylation activity markedly attenuate\u00a0PARP2 foci   ,52,53, win vivo (PARP1/2 DKO cells are brighter than those in PARP1 proficient (i.e. PARP2 KO) cells, consistent with a model where endogenous PARP1 competes with the WGR domain of PARP2 for DNA binding. Despite the ability for NTR to bind to DNA and PAR, we, like others (PARP1/2 DKO cells than the PARP2-\u0394NTR (\u0394aa1\u201370), highlighting a role for aa 70\u201387 of the unstructured NTR region for DNA binding. Two isoforms of PARP2 were noted in UniProt (Entry: Q9UGN5). Isoform 2 was used here and in prior studies  cells are consistently lower than those in PARP1/2 DKO cells, suggesting that endogenous PARP1 might compete with PARP2-DNA interaction (Nearly a decade has passed since the Pommier group first reported that PARP inhibitors trap PARP2 on chromatin after DNA damage\u00a0, but theOS cells . Among tP Figure . PARP2 iP Figure . The maxeraction . In thiseraction . Despiteeraction  and\u00a0H, teraction \u2013G. Niraperaction . These oeraction , the HA eraction , potentieraction . AlternaUsing live-cell imaging and FRAP, our study revealed the mechanism underlying PARP2 trapping that is distinct from PARP1. In the case of PARP1, PARP inhibitors delay DNA repair without affecting PARP1 exchange, to cause continuous recruitment and exchange of PARP1 and persistent foci  Figure . In the gkac188_Supplemental_FileClick here for additional data file."}
+{"text": "The focus of the questionnaire was the following: expectations and motivations for conference participation, the importance of conferences for the scientific qualification process, interaction, and communication among colleagues, and individual pay-off of the conference visit. A pilot study was conducted including 10 orthopaedic surgeons for testing comprehensibility, ease of reading, and acceptability of questionnaires. The questionnaires were kept confidential and collected with strict anonymity from all the participants. A total of 197 completed response sheets, were considered for final analysis. Out of the total participants, 29.9% (n=59) were faculties and 70.1% (n=138) were delegates. Forty-four point two percent  delegates and 71.2% (n=42) faculties were frequent conference-goers.There is a lack of enough evidence in determining the actual usefulness of conferences and continuing medical education (CME) programs among clinicians with active practice ,2. This According to the delegates, the main motivation behind attending scientific conferences was to present their work. Sixty point one percent  strongly agreed, and another 23.2% (n=32) agreed to this response. (n=83) strongly agreed, and another 23.2% (n=32) agreed to this response. Seventy-four point seven  of delegates strongly agreed/agreed that they were directed by their teaching faculty to attend the conference. The majority of the delegates were unsure whether attending conferences was added to their curriculum vitae (CV) . The academic content was the most important factor according to 89.1% (n=123) delegates. Seventy-one percent  of delegates strongly agreed/agreed that venue and location were important for the conference. Conference kits, quality of the food and beverages, and cultural programs were strongly agreed/agreed upon as important by only 14.5%, 74.7%, and 76.1% of participants respectively. Interestingly, 60.1% (n=83) of delegates reported that presenting a paper was more important than publishing their research. Overall, 54.3% (n=75) of delegates agreed that conferences are truly academic events.According to the faculties, the primary motivation behind attending conferences was also to present their research work with 55.9% (n=33) strongly agreeing while 20.3% (n=12) agreeing with this response. Adding to their CV for future endeavors was strongly agreed/agreed upon by 59.3% (n=35) faculty. Only 37.3% (n=14) strongly agreed/ agreed that they attend conferences to oblige their friends while a majority 45.8% (n=27) remained neutral to the response. Eighty-six point four percent  and 89.7% (n=53) strongly agreed/ agreed that they attend a conference to make professional connections and to visit the local place respectively. The majority of them were motivated by the talks given by other faculties, and most of them picked up a research topic from the earlier conferences attended by them. Most of them agreed that they have changed their teaching/clinical practice from the knowledge gained from previous conferences. According to 52.5% (n=31) of the attending faculties, the conference is more of an academic event, while 61% (n=36) agreed that publishing a scientific work remains more important than only presenting it at these meetings.Comparing the responses of both delegates and faculties, the main motivation behind their attendance was, identified to present their research work (p= 0.529) and the quality of the conference was found to depend mainly on academic content (p=0.203). Responses of frequent conference-going delegates and faculties were also compared and all questions had similar responses except, most of them did not attend the full conference. Publishing research work was significantly noted to be more important (p=0.006) among the faculties than only presenting the research work, at a scientific conference. The strengths of the study are the anonymity of responses, two different well-structured questionnaires catering to the perceptions of both delegates and faculties, and a national-level medical conference. Further studies with a larger number of participants for validating this questionnaire-based survey may be considered in the future. An online survey engaging a larger number of participants would have been better for an overall analysis of the actual academic utility of scientific conferences.These conferences help in developing interest in research works and in picking up topics for future research activities. Previous studies too highlight that the attending faculties and delegates were greatly motivated by the talks and presentations given by other faculties and it helped them to update their knowledge for improving the healthcare delivery system -5. To so"}
+{"text": "The probe TPE-kana 1 showed strong affinity towards bovine serum albumin (BSA) compared to its other biological competitors. The recognition of BSA have been investigated employing UV\u2013Vis absorption and fluorescence emission spectroscopy. The significant color change of TPE-kana 1 with BSA can be observed by necked eye, where the role of AIE-active TPE molecule is handle in both optical and colorimetric changes. The quenching of fluorescence of TPE-kana 1 with BSA was characterized by fluorescence spectroscopy, with 71.16% of quenching efficiency. Moreover, the Stern\u2013Volmer quenching constant was calculated and found to be 2.46\u2009\u00d7\u2009107\u00a0M\u22121. Probe TPE-kana 1 showed detection limit of 2.87\u00a0nM (nM) towards BSA with binding constant 7.56\u2009\u00d7\u2009107\u00a0M. A molecular docking study is also performed to investigate the detail interactions between TPE-kana 1 with the sites of BSA via non-covalent i.e., H-bonding, \u03c0-cation interactions, \u03c0-donor hydrogen bonds and \u03c0-\u03c0 interactions. The lowest binding energy conformation was found at\u2009\u2212\u200910.42\u00a0kcal/mol.A novel tetraphenylethylene (TPE) functionalized aminoglycoside antibiotic kanamycin (TPE-kana  Most of these methods showed changes in their photophysical properties due to chemical reactions or physical interactions with proteins. Among the reported methods, the fluorescence probe methods provide an attractive advantage such as high selectivity and excellent sensitivity, rapid response and simplicity towards proteins. Bovine serum albumin (BSA) is one of the most widely investigated proteins from the serum albumin series of proteins due to its structural similarity with human serum albumin (HSA)10. Literature search revealed that different fluorescence reagents have been employed for the detection of BSA16 Most of these fluorescent materials showed aggregation caused quenching (ACQ) effect, which in turn led drastic reductions in their fluorescence emission peaks. To overcome ACQ effect, researchers utilized aggregation induced emission (AIE) materials for BSA biomolecule detection21.In recent years, discovery of AIE-active biosensors for bioanalytes (proteins) sensing is attracted much attention. Researchers have developed several methods for sensitive and selective detection of proteins19. Another important factor to mention, that the protein albumin exhibited structurally selective two main binding sites such as site-I and site-II, from which binding site-I mainly occurs through hydrophobic interactions, whereas binding in site-II occurs through not only hydrophobic interactions but also hydrogen bonding, and electrostatic interactions involved21. According to recent reports probes with high selectivity and sensitivity towards serum albumin and exhibiting specific binding at only site II21. Therefore, there is a need to develop a new, highly efficient chemosensors for the selective detection of proteins are on great demand.The development of AIE materials to detect high sensitivity is highly challenging due to the significant role of proteins and other biomolecules in the living system22. Various amide conjugated biomaterials have been reported for different biological applications such material design, sensing of biomolecules and biomedical applications26. The molecular modelling and functionalization of aminoglycosides through covalent and non-covalent interactions have been used for the fabrications of various types of biomaterials such as hydrogels, nanoparticles, chemosensor, biointerfaces, amphiphiles and microstructures30. In our recent work, we have also shown that the self-assembled flower-like microstructure due to coordination of primary amino and hydroxyl group of kanamycin with Cu2+ ions and furthermore we have shown that flower shown to be having high surface areas, thus, used as a photocatalyst for the degradation of organic dyes31.On other hand, aminoglycoside antibiotics have possessed unique structurally features with number of amino and hydroxyl group. The presence of multiple amino groups on kanamycin allows it to modify through amidation reactions. The amino group at 6\u2032 position of kanamycin is more reactive and undergoes site selective amidation, compared to other amino group due to steric hindrance factor1), a strong fluorescent chromophore for selective detection of BSA via supramolecular interactions. The detection of BSA using AIE-active TPE-kana 1 in aqueous solution was found to be most powerful tool due to its easy preparation, rapid response, excellent sensitivity, high selectivity, and non-destructive nature, as the responses of TPE-kana 1 towards various other biological competitors are insignificant compared to the BSA.In this work, we described the synthesis aniline 4 was obtained by reducing compound 3 using hydrazine hydrate. The compound 4 was then heated with succinic anhydride in toluene yielded 4-oxo-4-phenyl)amino)butanoic acid 5, the compound 5 was then upon amide coupling with kanamycin antibiotic resulted in the formation of target compound TPE-kana 1 (70% yield). All the compounds were confirmed by 1H NMR spectroscopy and the final target molecule was characterized by IR, NMR, HRMS sophisticated techniques and purity was confirmed by high-pressure liquid chromatography (HPLC), and also optical rotation as illustrated in ESI Fig. 1 attributed to amide > C=O stretching of \u2013CONH and peaks at 1589, 1521 are attributed to \u2013N\u2013H bending modes of secondary amide. The 1H NMR spectrum of TPE-Kana 1 was recorded in deuterated methanol solvent. The chemical shift (\u03b4) values in the region from 7.71 to 7.67\u00a0ppm and 7.29 to 7.26\u00a0ppm correspond to two H-bonded amide protons. The formation of compound TPE-kana 1 was further confirmed by the HRMS: (m/z) calculated 912.4025 found 912.4036 [M]+, 980.2810 [M + 3Na]+. The purity of TPE-Kana 1 was analyzed by HPLC and shown to be 95% purity of TPE-kana 1. The specific rotation for the TPE-kana 1 was calculated using a polarimeter, the obtained specific rotation is \u03b123D = \u2212 1.4929 .In the present study, kanamycin, an aminoglycoside antibiotic functionalized with highly fluorescent 1,1,2,2-tetraphenylethene (TPE) molecule (emits white light), was successfully synthesized. Fluorescent TPE-functionalized kanamycin TPE-kana 1 in water were investigated using UV\u2013Vis absorption and fluorescence spectroscopy  and primary amino (\u2013NH2) group of kanamycin functionalized TPE as a AIE active moiety. The presence of multiple -OH and amine \u2013NH2 group of kanamycin and TPE group leads to such different absorption and emission behavior of TPE-kana 1. The multiple \u2013OH and amine \u2013NH2 group kanamycin could provide utmost opportunities for the formation of both non-covalent interactions at different pH which may leads to construction of versatile biomaterial framework. Moreover, this different behavior of TPE-kana 1 at different pH was also characterized using scanning electron microscopy (SEM) which shows almost similar mode of aggregations at pH-4 and 9.2, a globular particular aggregates like structure of TPE-kana 1 was observed. Whereas at pH-7 the TPE-kana 1 produces leaf-like structures  dependent changes of TPE-kana opy Fig. . In UV\u2013Vres Fig.  and in pres Fig. .1, we investigate whether the kanamycin antibiotic affect the AIE behavior  of TPE-kana 1 we studied UV\u2013Vis absorption and fluorescence emission behavior of TPE-kana 1 in different proportion of THF-water mixtures. TPE is a strong AIE active compound, exhibits AIE behavior upon aggregation, and shows strong emission of light. The naked eye detection study showed that upon increasing the water ratio up to 99% the significant increase in the fluorescence was observed under 365\u00a0nm UV-light Fig.\u00a01 were investigated using UV\u2009\u2212\u2009Vis absorption and fluorescence emission spectra in the THF/H2O solvent mixture with different water fractions (fw\u2009=\u20090 to 99%) , the SEM image is shown in ESI Fig. After successful synthesis of TPE-kana 1 (5\u2009\u00d7\u200910\u22125\u00a0mol/L) was prepared by dissolving in DMSO solvent. The stock solution from DMSO was used to prepare solution of TPE-kana 1 in distilled water. A series of biomolecules (4 equiv.) such as BSA, heparin, ascorbic acid, sodium pyrophosphate, sodium oxalate, sodium citrate, adenosine triphosphate, glutamic acid, aspartic acid, sodium acetate, and glucose were added to TPE-kana 1 in water. The sensing performance of TPE-kana 1 was then monitored by the color change in the UV light (365\u00a0nm) and is depicted in Fig.\u00a01 did not show such color change in presence of other biomolecules, as shown in Fig.\u00a01 as a fluorescent sensor can be used for selective detection of BSA in solution.The stock solution of the TPE-kana UV light 65\u00a0nm andUV light 65\u00a0nm and1 in distilled water using UV\u2013Vis absorption, fluorescence spectroscopy and also studied competitive experiments to figure out the selectivity of TPE-kana 1 toward BSA. The theoretical docking studies employed to investigate interaction of TPE-kana 1 with BSA either site I and/or site II or within them.Furthermore, we have examined sensing capability of TPE-kana 1 towards biomolecules. TPE-kana 1 shows selectivity towards BSA protein only as compared with other biomolecules/proteins as shown in Fig.\u00a01 (5\u2009\u00d7\u200910\u22125\u00a0M) exhibit absorption maxima centred at 306\u00a0nm, and which upon BSA addition a new absorption band appeared near 280\u00a0nm wavelength with the enhanced intensity and broaden peak at 306\u00a0nm and no such effect occurred with various other biological competitors as shown in Fig.\u00a01 with BSA not only through intermolecular hydrogen-bonding between the amino- and hydroxyl-functional group of TPE-kana 1 with BSA but also packing of the TPE-kana 1 in the pocket of BSA, which leads to change in the optical properties of TPE-kana 1. For a detailed understanding, titration of TPE-kana 1 with BSA (0\u2013120\u00a0nm) in distilled water (D/W) was evaluated, which was monitored at absorbance band i.e., 280\u00a0nm and 306\u00a0nm band as shown in Fig.\u00a01 shown to be excellent candidate towards selective sensing of BSA.The UV\u2013Vis absorption spectroscopy was employed to investigate the sensing response of TPE-kana 1 (stock solution was prepared in DMSO) through detection in distilled water at room temperature. The fluorescence spectra of TPE-kana 1 (5 \u00d7 10\u22125\u00a0M) exhibited typical emission band at 476\u00a0nm upon excitation at \u03bbex\u2009=\u2009310\u00a0nm as shown in Fig.\u00a01 estimated 0.42 in D/W at room temperature. The selectivity TPE-kana 1 towards BSA with the addition of a series of anions and biomolecules was monitored in the changes at 476\u00a0nm emission band. It clearly shows no significant changes with the addition other competitors except BSA, in particularly upon addition of BSA the emission intensity of TPE-kana 1 at 476\u00a0nm dramatically decreased with appearance of a new emission band at 357\u00a0nm. Further, upon gradual incremental addition of BSA (0\u2013120\u00a0nM) over the solution of TPE-kana 1 in D/W, the fluorescence emission gradual decrease 476\u00a0nm and enhanced emission at 375\u00a0nm, Fig.\u00a01: BSA was found to be decreased to 0.17 as compared with TPE-kana 1 (\u03a6\u2009=\u20090.47). The emission intensity at 476\u00a0nm considerably decreased from 422 to 107 as the concentration of BSA increased from 0 to 120\u00a0nM and further additional of BSA does not show any more changes may be due to the detection limit. This clearly suggest the interaction of TPE-kana 1 with BSA protein over other biological competitors almost silent, because of hydrophobic interactions along with H-bonding between \u2013OH and \u2013NH2 group of kana moiety and amino acids of BSA followed by \u03c0\u2013\u03c0 interactions between tryptophan amino acid of BSA and TPE unit of TPE-kana 1. We believe that the strong H-bonding between kanamycin of TPE-kana 1 bind within the molecular cleft of BSA, which can be clearly seen in changes under UV\u2013Vis absorption  was calculated using equation [(I0\u2009\u2212\u2009I)/I0]\u2009\u00d7\u2009100%, where I0 and I are the fluorescence intensities before and after addition of the BSA. After the addition of 120\u00a0nM of BSA solution the initial emission intensity of TPE-kana 1 was quenched by approximately 71.16%, the quenching efficiency (\u03b7) was calculated to be about 71.16%.The initial fluorescence intensity of TPE-kana 1 for BSA detection, the fluorescence response of TPE-kana 1 with several anions (120\u00a0nM) as well as biological competitors (120\u00a0nM) of BSA such as heparin, ascorbic acid, sodium pyrophosphate, sodium oxalate, sodium citrate, adenosine triphosphate, glutamic acid, aspartic acid, sodium acetate, glucose was investigated. TPE-kana 1 did not show a considerable response neither to the anions and not to biological competitors to BSA as illustrated in Fig.\u00a0To evaluate the selectivity study of TPE-kana sv) was calculated by employing fluorescence emission intensity (I0/I) as a function of increasing BSA concentration [Q] by the following relation; I0/I\u2009=\u2009TPE-kana 1\u2009+\u2009Ksv [Q], where I0 and I are the emission intensities of TPE-kana 1, before and after addition of BSA, respectively, Ksv is the quenching constant (M\u22121), and [Q] is the molar concentration of BSA. The Stern\u2013Volmer plot of TPE-kana 1 with BSA is showed in Fig.\u00a0sv value obtained for BSA was 2.46\u2009\u00d7\u2009107\u00a0M\u22121. This indicated that the BSA exhibited exclusive quenching ability towards fluorescent TPE-kana 1 in water.The Stern\u2013Volmer quenching constant (K1 with BSA in distilled water was carried out by adding increasing concentrations of BSA solution (0\u2013120\u00a0nM) and the emission intensity as a function of BSA added was then plotted, Fig.\u00a0To evaluate the detection limit, emission titration of TPE-kana \u03c3/m.Detection limit\u2009=\u20093 m is the slope between emission at 476\u00a0nm and concentration of BSA. The detection limit for BSA was shown to be 2.87\u00a0nM (R2\u2009=\u20090.907) in distilled water showing the fluorescence intensity of TPE-kana 1 at 476\u00a0nm (\u03bbex\u2009=\u2009310\u00a0nm) as a function of BSA concentration. Hence the detection limit of TPE-kana 1 in distilled water was found to be 2.87\u00a0nM which is higher or comparable with other molecules used for detection of BSA (Table Where \u03c3 is the standard deviation of the emission of the free sensor and SA Table .a) between 1 and BSA. The linear relationship of absorption intensity as a function of 1/[BSA] was found to be 7.56\u2009\u00d7\u2009107\u00a0M with R2\u2009=\u20090.9968. This suggest that the TPE-kana 1 to have strong binding affinity towards BSA. Furthermore, SEM images clearly shows strong interaction between TPE-kana 1 with BSA, which produces highly packed coagulated microstructure , Lys294 (2.21\u00a0\u00c5), Arg217 (1.67\u00a0\u00c5) as shown in Fig.\u00a01 as shown in Fig.\u00a01 forms conventional hydrogen bonding, carbon-hydrogen, van der Waals, and \u03c0-type of interactions play the major role for the binding pocket residue of BSA protein.To evaluate binding mode of TPE-kana 1 as a molecular probe for selective and sensitive detection of BSA. TPE-kana 1 showed significant AIE properties in THF:water solvent systems. Furthermore, TPE-kana 1 exhibited BSA induced decreased fluorescence emission intensity in distilled water compared to other examined anions and biological competitors in aqueous medium. The sensing of BSA was well characterized by using UV\u2013Vis absorption and fluorescence emission spectroscopy, the quenching of fluorescence observed with BSA. Moreover, the quenching efficiency was calculated and obtained was 71.16%. The Stern Volmer quenching constant was calculated and found to be 2.46\u2009\u00d7\u2009107\u00a0M\u22121. The limit of detection of TPE-kana 1 towards BSA was found to be 2.87\u00a0nM. The binding constant was calculated and found to be 7.56\u2009\u00d7\u2009107\u00a0M and R2\u2009=\u20090.9968. The molecular docking study revealed the significant binding affinity of TPE-kana 1 for BSA with the lowest binding energy conformation was found at\u2009\u2212\u200910.42\u00a0kcal/mol. These results clearly indicated that TPE-kana 1 may use as a promising recognition tool for the BSA in solution as well as biomedical waste.In summary, we have successfully designed and synthesized TPE-functionalized kanamycin antibiotic TPE-kana 3 and 4 were synthesized using reported literature procedure and compound 5 and TPE-kana 1 were synthesized using following procedure  as an internal standard and CDCl3-d and MeOD-d4 as deuterated solvents. Thermofisher exactive orbitrap MALDI-TOF measurements were used for HRMS, a Schimadzu Biotech Axima performance spectroscopic instrument. UV\u2013Vis absorption spectra were recorded using a UV\u2013Vis 1800 Shimadzu spectrophotometer and fluorescence emission was measured on an Agilent, Carry Eclipse spectrofluorophotometer.Compounds ure Fig. . All rea1 was prepared in DMSO and was stored in a cold and dark place. The stock solution was used for the experiments after appropriate dilution in distilled water (D/W). The concentration of TPE-kana 1 was confirmed by absorbance at 306\u00a0nm for the TPE fluorophore UV\u2013Vis absorption spectra (250\u2013550\u00a0nm) of the samples in a 10\u00a0mm path length cuvette was measured using a UV\u2013Vis 1800 Shimadzu spectrophotometer fluorescence emission spectra of TPE-kana 1 in a 10\u00a0mm path length cuvette were measured using an Agilent, Carry Eclipse Spectro fluorophotometer with excitation at 310\u00a0nm. All absorbance and fluorescence measurements were carried out in 100% aqueous solutions at room temperature.A stock solution (1\u00a0mM) of TPE-kana UV\u2013Vis absorption and fluorescence emission measurements: UV\u2013Vis absorption and fluorescence emission of the TPE-kana 1 upon the addition of 4 equiv. of BSA and other biological competitors. The 2\u00a0mL TPE-kana 1 solution (5\u2009\u00d7\u200910\u22125\u00a0mol/L) in distilled water (D/W) was placed in the quartz cell, and the absorption and emission spectra were recorded with the addition of BSA and its other competitor\u2019s different anions (20\u2009\u00d7\u200910\u22123\u00a0M). The absorption and emission spectra were completely recorded at room temperature.UV\u2013vis absorption and fluorescence emission titration of the TPE-kana 1 upon the addition of BSA: The 2\u00a0mL TPE-kana 1 solution (5\u2009\u00d7\u200910\u22125\u00a0mol/L) in D/W was placed in the quartz cell and the fraction of BSA solution (0\u2013120\u00a0nM) was added, and the corresponding absorption and emission spectra were recorded at room temperature.1 (5\u2009\u00d7\u200910\u22125\u00a0mol/L) was used to prepare solution for necked eye experiments by dissolving it in D/W. Then BSA and its other competitors such as heparin, ascorbic acid, sodium pyrophosphate, sodium oxalate, sodium citrate, adenosine triphosphate, Glutamic acid, aspartic acid, sodium acetate, and glucose were added, the photograph were taken under 365\u00a0nm UV light.The DMSO stock solution of the TPE-kana 1 with BSA was carried out by adding increasing concentrations of BSA solution (0\u2013120\u00a0nM) and the emission intensity as a function of BSA added was then plotted.To determine the detection limit, emission titration of TPE-kana 1 with BSA, we employed molecular docking calculations using AutoDock4.2.6 software (https://autodock.scripps.edu/)35. The crystal structure of BSA was retrieved from the protein database (source code: 4OR0.pdb). Here, chain A was considered for the molecular docking study. The three-dimensional atomic co-ordinate of TPE-kana 1 was generated using the Discovery Studio Visualizer35. For the docking study, a grid box size of 80\u2009\u00d7\u200980\u2009\u00d7\u200980 with a spacing of 0.375\u00a0\u00c5 was built around the active site of BSA which is present in between the domain IIA and IIIA. Next, we employed a local docking protocol, to explore a binding mode of TPE-kana 1, similar to an earlier study34. Here, we keep BSA as rigid and TPE-kana 1 as a flexible molecule. The output docking conformations were generated by applying the Lamarckian Genetic Algorithm (LGA). These output conformations were further clustered using an all-atom RMSD with a cut-off of 4\u00a0\u00c5. The clusters were further analyzed based upon binding, van der Waals, and electrostatic energy, etc. The lowest binding energy conformation of TPE-kana 1 was further analyzed for the bonding interactions using PyMol (DeLano 2002) and Discovery Studio visualizer35, respectively.To investigate the binding mode of TPE-kana High-pressure liquid chromatography (HPLC) Column: INERTSIL-ODS C18, Solvent: ACN: Water, 9:1, Flow rate: 1\u00a0mL/min, Run time: 30\u00a0min.Supplementary Information.Supplementary Legends."}
+{"text": "B4GALT7 or B3GALT6 genes both deranging the biosynthesis of the glycosaminoglycan linkage region of chondroitin/dermatan sulfate and heparan sulfate proteoglycans. In this study, we have analyzed the linkage regions of urinary chondroitin sulfate proteoglycans of three siblings, diagnosed with spEDS and carrying biallelic pathogenic variants of the B3GALT6 gene. Proteoglycans were digested with trypsin, glycopeptides enriched on anion\u2010exchange columns, depolymerized with chondroitinase ABC, and analyzed by nLC\u2010MS/MS. In urine of the unaffected mother, the dominating glycopeptide of bikunin/protein AMBP appeared as only one dominating (99.9%) peak with the canonical tetrasaccharide linkage region modification. In contrast, the samples of the three affected siblings contained two different glycopeptide peaks, corresponding to the canonical tetrasaccharide and to the non\u2010canonical trisaccharide linkage region modifications in individual ratios of 61/38, 73/27, and 59/41. We propose that the relative distribution of glycosaminoglycan linkage regions of urinary bikunin glycopeptides may serve as a phenotypic biomarker in a diagnostic test but also as a biomarker to follow the effect of future therapies in affected individuals.The spondylodysplastic type of Ehlers\u2013Danlos syndrome (spEDS) is caused by genetic defects in the  B3GALT6 gene and diagnosed with spondylodysplastic Ehlers\u2013Danlos syndrome, which we propose may serve as a biomarker for this disease.We have identified a >100\u2010fold relative increase in the non\u2010canonical trisaccharide versus the canonical tetrasaccharide linkage region of tryptic bikunin glycopeptides, prepared from urine of three siblings with identical biallelic variants of the 1CHST14 or DSE genes, and spondylodysplastic EDS (spEDS) is caused by pathogenic variants in the B4GALT7 or B3GALT6 genes. The latter two genes are coding for two unique galactosyltransferases  that work in sequence adding two galactose  residues to what is to become the canonical tetrasaccharide \u201clinkage region,\u201d common to all glycosaminoglycans of chondroitin/dermatan (CS/DS) and heparan sulfate (HS) proteoglycans, which links the glycosaminoglycan chains to the core proteins  constitute a very heterogeneous group of heritable connective tissue disorders, mainly presenting with skin hyperextensibility, joint hypermobility, and general tissue fragility.Proteoglycans (PGs) are composed of a core protein to which one or multiple glycosaminoglycan chains (GAGs) are bound.O\u2010) linked to Ser\u2010215 of bikunin  in the urine of healthy human individuals.b3galt6 knock\u2010out zebrafish showing a typical dysplastic phenotype resembling the human spEDS\u2010B3GALT6.B3GALT6 gene, and showed a typical spEDS phenotype.B3GALT6, complementary to the clinical signs and genetic analyses.The structure of the tetrasaccharide linkage region, common to CSPGs/DSPGs and HSPGs, was settled more than 50\u2009years ago by the two groups of Roden and Lindahl et al.22.1B3GALT6 gene (NM_080605.3) and their unaffected mother  deletion variant and the B3GALT6:c.953C>T, p.(Pro318Leu) missense variant. Informed consent was obtained from the patients and/or their participating parents. This study was approved by the Ethics Committee of the Ghent University Hospital .The individuals participating in this study include three previously reported siblings with biallelic pathogenic variants in the 2.2g, for 10\u00a0min at room temperature. The supernatants were collected, frozen, and kept at \u221220\u00b0C until analysis. Linkage region containing glycopeptides was prepared from urinary proteoglycans using the protocol described inThe time of urine sampling was at ages 29\u2009years , 33\u2009years (PIV:2), 36\u2009years (PIV:1), and 54\u2009years (mother), respectively. Morning urine was sampled and immediately centrifuged at 2000\u2009\u00d7\u200920, and from this 3 \u03bcl were injected onto an Acclaim Pepmap C18 precolumn  and passed to the analytical column (350\u2009\u00d7\u20090.075\u2009mm I.D.) packed in\u2010house with 3\u00a0\u03bcm ReproSil\u2010Pur C18\u2010AQ particles (Dr. Maisch). The gradient was 10\u201350% B\u2010solvent  in A\u2010solvent (0.2% formic acid in dH2O) over 60\u2009min at a flow rate of 300\u2009nl/min, and then 50%\u2013100% B over 5\u00a0min, with a final hold at 100% B for 10\u00a0min. MS scans were done in positive ionization mode at 120\u2009000 resolution (at m/z 200): an automatic gain control (AGC) target value of 106 and a mass range of m/z 600\u20132000. The MS/MS (MS2) spectra were collected in the data\u2010dependent mode with a duty cycle time of 3\u00a0s and a dynamic exclusion of 10\u00a0s. The most abundant 2(+) \u2013 7(+) charged precursor ions for each MS scan were fragmented by higher\u2010energy collision dissociation (HCD) at normalized collision energy (NCE) levels of 20%, 30%, and 35%. The isolation window was set to 2.5\u00a0m/z units, the MS2 mass range was m/z 100\u20132000 at 30\u2009000 resolution, and MS2 data were collected in profile mode.Glycoproteomic analyses were performed as technical triplicates on an Orbitrap Fusion Tribrid instrument and the Easy\u2010nLC 1200 system (Thermo Scientific). Samples were dissolved in 20\u2009\u03bcl 3% acetonitrile and 0.2% formic acid in dHData analysis was performed with Mascot distiller (Matrix Science) and manual verification and complementary fragmentation analysis of the glycopeptide spectra using the Qual browser of the Xcalibur software. This glycoproteomic technique allows for identification of the type of GAG chains, their linkage regions, the identity of the original proteoglycan, and their sites for glycosylation.3In the control urine of the mother, we identified GAG\u2010modified glycopeptides of 16 different CSPGs  or a disulfated pentasaccharide  in affected children. In a recent publication, the Bruneel group reviewed the status of known proteoglycan inherited biosynthesis defects and the analytical tools available for diagnosis.The almost complete dominance (99.9%) of the canonical hexasaccharide modification in the mother control sample fits well with what is well known for CSPGs and what we have described before for bikunin.b3galt6 KO model, having an EDS\u2010like phenotype, we found the non\u2010canonical linkage region only on biglycan and aggrecan, whereas in the wild\u2010type zebrafish, the canonical linkage region of CSPGs was found not only on biglycan and aggrecan but also on epiphycan, osteopontin, and syndecan\u20103 (as well as on four different HSPGs).Interestingly, in the urine sample of the individual PIV:1, the non\u2010canonical form of the linkage region also appeared on a glycopeptide of neuropeptide W Figure\u00a0, withoutIn conclusion, the glycoproteomic approach used here may become a suitable diagnostic method for clinical purposes but may also be helpful in deciphering the biological complexity of deranged GAG biosynthesis and the importance of linkage region modifications and core protein structures for the biosynthesis of both CSPGs and HSPGs.The work was supported by grants from the Swedish Research Council (2017\u201000955), the Swedish state under the agreement between the Swedish government and the county councils, the ALF agreement (ALFGBG_721971) and Research Foundation Flanders .Mahnaz Nikpour, Fredrik Noborn, Jonas Nilsson, Tim Van Damme, Olivier Kaye, Delfien Syx, Fransiska Malfait, and G\u00f6ran Larson all declare that they have no conflict of interest.All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation  and with the Helsinki Declaration of 1975, as revised in 2000. Informed consent was obtained from the patients and/or parents participating in this study. The study was approved by the Ethics Committee of the Ghent University Hospital .No animal subjects were used in this study.Appendix S1. Supporting InformationClick here for additional data file."}
+{"text": "Pseudostellaria heterophylla is a traditional Chinese herbal medicine, which has been cultivated for hundreds of years. Viral diseases of P. heterophylla occur widely and limit the yield and quality of this medicinal plant. In this study, five leaf samples of P. heterophylla with typical viral symptoms were collected from four main producing regions that are distributed in Fujian, Guizhou, and Anhui Provinces in China and analyzed by next-generation sequencing. Comprehensive bioinformatics analyses revealed that nine viruses in five genera Carlavirus, Potyvirus, Fabavirus, Cucumovirus, and Amalgavirus infected P. heterophylla. Among these viruses, three novel and two known carlaviruses, tentatively designated Pseudostellaria heterophylla carlavirus 1, 2, and 3 , Jasmine virus C isolate Ph (Ph-JVC) and Stevia carlavirus 1 isolate Ph (Ph-StCV1), respectively, were first identified in P. heterophylla. PhCV1-3 share a similar genomic organization and clear sequence homology with members in the genus Carlavirus and could potentially be classified as new species of this genus. One novel amalgavirus, tentatively designated P. heterophylla amalgavirus 1 (PhAV1), was first identified in P. heterophylla. It had a typical genomic organization of the genus Amalgavirus. In PhAV1, the +\u20091 programmed ribosomal frameshifting, which is prevalent in most amalgaviruses, was identified and used in the expression of RNA-dependent RNA polymerase (RdRp). Combined with a phylogenetic analysis, PhAV1 could potentially be classified as new species of the genus Amalgavirus. In addition, multiple Broad bean wilt virus 2 (BBWV2) variants, Turnip mosaic virus (TuMV), and Cucumber mosaic virus (CMV), which have been reported in P. heterophylla, were also detected in this study. The distribution of PhCV1-3, Ph-JVC, Ph-StCV1, TuMV, BBWV2, and CMV in four production regions in Fujian, Guizhou, and Anhui Provinces was determined. This study increased our understanding of P. heterophylla virome and provided valuable information for the development of a molecular diagnostic technique and control of viral diseases in P. heterophylla. Pseudostellaria heterophylla (Miq.) Pax is also known as \u201cTai-zi-shen\u201d or \u201cHai-er-shen\u201d in China. It is a member of the family Caryophyllaceae and one of the most popular traditional Chinese herbal medicines. It can be used to treat spleen deficiency, anorexia, weakness after illness, and spontaneous perspiration symptoms , Zherong County (in Fujian Province), and Xuancheng City  display symptoms, such as foliar mottles, mosaics, ringspots, and leaf malformations, as well as the dwarfing of plants. To date, TuMV , BBWV2 , CMV , and Tobacco mosaic virus  are the four viruses that have been reported to infect P. heterophylla (ven 100% . Such serophylla .Carlavirus of the family Betaflexiviridae comprises 61 confirmed species (ICTV).Bemisia tabaci; The genus Amalgaviridae is a recently reported family of double-stranded RNA viruses that is an amalgam of the families Partitiviridae and Totiviridae, which encode proteins phylogenetically related to former, but their structure is more closely related to the latter .1 Putative amalgaviruses have also been reported in fungi and bryophytes , a rapidly developing technique for viral detection and diagnosis that is suitable for a variety of plants, animals, and fungi  Isolation kit  and treated with an RNA Clean XP Kit  and an RNase-Free DNase Set  according to the manufacturer\u2019s instructions. The quality and quantity of total RNA were measured using a NanoDrop spectrophotometer (Thermo Fisher Scientific) and Agilent2100 . After the removal of ribosomal RNA using a Ribo-Zero Magnetic Kit , the libraries were built using a TruSeq RNA Sample Prep Kit . Barcoded libraries were paired-end sequenced on an Illumina HiSeq X platform according to the manufacturer\u2019s instructions.de novo using CLC Genomics Workbench 6.0.4  according to the scaffolding contig algorithm. The second assembly was then conducted using CAP3 software.E-value\u2009<\u20091e\u22125.Total RNA was extracted from the leaf tissue using a mirVanaP. heterophylla viruses were obtained by overlapping reverse transcription (RT)-PCR and rapid amplification of cDNA ends-PCR (RACE-PCR). The primers used in RT-PCR and RACE-PCR were designed based on the viral contig sequences using Primer Premier 6  or the neighbor-joining method (BBWV2) with 1,000 bootstrap replicates in MEGA X .P. heterophylla leaf samples were collected from the four different main production regions of P. heterophylla in June of 2020 and 2021, including one sample in Shibing County (SB) and one sample in Danzhai (DZ) County in Guizhou Province, two samples in Zherong County (ZR-HB and ZR-GX) in Fujian Province and one sample in Xuancheng city (XC) in Anhui Province, in China . These results confirmed the presence of both viruses in P. heterophylla. One BBWV2 isolate was identified in libraries DZ and ZR-HB and designated BBWV2-DZ and -ZR-HB, respectively. Two BBWV2 isolates were identified in libraries XC and ZR-GX and designated BBWV2-XC1, -XC2, \u2212ZR-GX1 and -ZR-GX2, respectively. Pairwise comparisons showed that the polyprotein encoded by RNA1 shared 73.41 to 98.15% nt sequence identities and 82.71 to 99.09% amino acid (aa) sequence identities with each other  of the viral-related reads in the ZR-HB, ZR-GX, and DZ libraries . Three n3TX3NTX22GDD , was identified near the C-terminus of the RdRp domain of these three carlaviruses , Potato virus S , and Tagetes carlavirus 1  with nt identities of 69.95% (55% query coverage), 69.75% (19% query coverage), and 71.15% (87% query coverage), respectively. Pairwise comparisons between PhCV1, PhCV2, PhCV3, Ph-JVC, Ph-StCV1, and 15 of the most similar carlaviruses were performed based on the nt and aa sequences of Rep and CP using MAFFT program and SDT software. The Reps of PhCV1, PhCV2, and PhCV3 shared 53.50\u201361.30%, 52.80\u201354.90%, and 53.70\u201366.90% nt sequence identities and 38.50\u201356.40%, 36.70\u201341.90%, and 39.20\u201372.20% aa sequence identities with Reps in the other aligned carlaviruses , S6. TheApple stem pitting virus  was used as an outgroup. The phylogenetic tree based on Rep revealed that PhCV1 clusters closely with Chrysanthemum virus B (CVB); PhCV2 clusters closely with Butterbur mosaic virus (ButMV), and PhCV3 clusters closely with TaCV1  was found in the ORF1 of PhAV1 , which is predicted to encode RdRp with 1,054 aa residues, with an estimated molecular weight of 120.4\u2009kDa. A BLASTP analysis showed that the CP of PhAV1 had the highest degree of identity (30.73% aa sequence) with that of Phalaenopsis equestris amalgavirus 1 , and RdRp of PhAV1 had the most identity (51.01% aa sequence) with that of Cucumis melo amalgavirus 1 , the only species of the genus Zybavirus that infects fungi  and stevia (Stevia rebaudiana), respectively. Based on informatic analyses of the genomic features and phylogeny, PhCV1, PhCV2, and PhCV3 were proposed to be new members of the genus Carlavirus, and PhAV1 was proposed to be a new member of the genus Amalgavirus. These data indicate that a rich diversity of viruses infect P. heterophylla.rophylla . To our n nature . Therefot ranges . JVC andP. heterophylla growing regions was detected by RT-PCR . Of the 12 samples collected in Anhui Province, 11 were simultaneously infected with CMV, BBWV2, and one or more carlavirus(es). A total of 20 samples were collected in Guizhou Province. One virus was detected in 10 samples, two in two samples, four in three samples, and no viruses were detected in five samples. This showed that severe viral infections in P. heterophylla were present in all three provinces, particularly in Fujian and Anhui Provinces. BBWV2 was the most common virus in the three provinces with an incidence of 100% in Fujian Province, 83.33% in Anhui Province, and 50.0% in Guizhou Province  in the ZR-HB, ZR-GX, and DZ libraries  and two known (Ph-JVC and Ph-StCV1) carlaviruses were identified in ibraries ; Table 1aviruses . The symgenicity . The N-tgenicity . The CRPgenicity . The assP. heterophylla by exploring its transcriptome. BLASTP and phylogenetic analyses based on the aa sequences of PhAV1 RdRp showed that PhAV1 clusters in the amalgaviruses clade. It displayed <51.01% aa sequence identity with the other amalgaviruses, indicating that PhAV1 could be a new species of the genus Amalgavirus. The +1 PRF is used in the expression of many genes of viruses, such as amalgaviruses, fijiviruses, and the influenza A virus  of +1 PRF was identified in the ORF1 of PhAV1. This could result in the direction (+1) of the frameshifting that is involved in the expression of RdRp.Most of the putative amalgaviruses that have been recently identified were discovered by the analyses of a transcriptome dataset. Similarly, we identified PhAV1 in  A virus . The shiP. heterophylla, and the RNA1 of isolates ZR-GX1 and XC1, RNA2 of isolates ZR-GX1 and XC2 shared a low degree of nt similarity (RNA1\u2009<\u200975.66% and RNA2\u2009<\u200974.19%) with other isolates in P. heterophylla and other isolates in the NCBI database (P. heterophylla.\u201cBBWV2 exhibits a high genetic variation with sequence variants that are continuously distributed in a wide sequence space compared with most plant viruses. This sequence distribution (genetic structure) is not associated with geographic locations, suggesting long-distance gene flow, and it has been primarily shaped by negative selection and recombination .\u201d Six isdatabase . AlthougP. heterophylla. It is also the first report of the infection of P. heterophylla by JVC, StCV1, and multiple BBWV2 variants. The findings of this study provide useful information for the development of a molecular diagnostic technique to establish a more effective P. heterophylla viral disease control strategy.In conclusion, this study is the first report of the complete nucleotide sequences of new viruses, including PhCV1, PhCV2, PhCV3, and PhAV1, that infect https://www.ncbi.nlm.nih.gov/genbank/, ON241319, ON241320, ON241321, ON241322, ON241323, ON241324, ON241325, ON241326, ON241327, ON241328, ON241329, ON241330, ON241331, ON241332, ON241333, ON241334, ON241335, ON241336.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: RW and WD contributed to conception and design of the study, collected the samples and conducted the experiments. RW, YL, and SL performed the statistical analysis. RW, KG, YL, and SL discussed the results, drafted and revised the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the Key project at central government level: The ability establishment of sustainable use for valuable Chinese medicine resources (2060302), CAMS Innovation Fund for Medical Sciences (CIFMS) (2021-I2M-1-032), and the National Natural Science Foundation of China (81873095).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "Unfortunately, we switched the labels for \"colour\" and \"motion\" in Figures\u00a04 and 6 as well as Figures\u00a0S4 and S5, which provoked a discrepancy with the findings presented in the original report. The figures have now been corrected online. We apologize for any confusion this may have caused."}
+{"text": "The physical and chemical properties and sustained release properties were determined by a transmission electron microscope (TEM), field emission transmission electron microscope (FETEM), atomic force microscope (AFM), laser particle size analyzer, Fourier transform infrared spectrometer, and contact angle tester. The results indicated that the prochloraz nanocapsules were spherical, the average particle size was about 100 nm, and the encapsulation efficiency and loading rates were 86% and 30%, respectively. The nanocapsules tended to expand in acidic solutions, and this promoted the release of prochloraz more quickly, which could be verified by the biological test of anthrax. At the same time, the prochloraz nanocapsules can protect the pesticide from sunlight. Therefore, this work provides a promising approach to improve the utilization efficiency and prolong the duration of pesticides, which might have a huge potential application prospect.In this work, prochloraz pH-responsive nanocapsules were developed by the Pickering emulsion polymerization method with isophorone diisocyanate (IPDI) as the reaction monomer and nano Fe 3O4 particle-branched polyethyleneimine (PEI).In this work, prochloraz pH-responsive nanocapsules were developed by the Pickering emulsion polymerization method with isophorone diisocyanate (IPDI) and nano Fe It is widely used to control eyespot disease and powdery mildew on cereals, and it is also effective against a broad spectrum of fungal diseases affecting fruits and vegetables.1\u20133 It has been reported that the half-life of prochloraz after photolysis in an aqueous solution is 10 days. The dissipation half-lives in soil range between 5 and 37 days under field conditions. It is therefore necessary to develop a novel formulation to increase the stability of prochloraz in the environment.Prochloraz [4 Consequently, it is urgent to develop new approaches to reduce the loss and improve the utilization efficiency of pesticides. A promising method to resolve this problem involves the construction of controlled-release pesticide systems, which will fulfill the demand of prolonging the duration and enhancing the utilization efficiency of the pesticides.5\u20139Pesticides are considered as the most effective way to control weeds, pests, and diseases in modern agriculture for the promotion of grain yields. However, traditional pesticides tend to enter the environment easily through runoff, volatilization, and leaching, leading to serious environmental issues and even hazards to human beings.10,11 Accordingly, various kinds of nanomaterials have been used as carriers in these controlled-release pesticide systems such as polymers,12,13 inorganic nanomaterials,14 and nanocomposites.15 There are some chemical preparation methods for synthesizing nanocapsules, such as in situ polymerization, suspension polymerization, interfacial polymerization, and emulsion polymerization. Pickering emulsion polymerization possesses many advantages including the reduction of foaming, lower toxicity and lower cost, and it has been extensively studied. Solid particles as surfactants and fillers provide a direction to prepare stable Pickering emulsions, such as nano-SiO2 particles and nanocomposite latex particles. Magnetic nanoparticles have been widely studied because of their fascinating magnetic separation properties and wide range of potential applications in pigments, medicine, biomedical and bioengineering fields, etc.16\u201319Recently, controlled-release pesticides are attracting more and more attention all over the world.At the same time, a pesticide needs to be released quickly in environmental conditions (such as pH and temperature) when a plant disease occurs and in the presence of insect pests. Thus, environmental-responsive pesticide capsules should be designed to meet the requirements for controlling pests. Many diseased plants can secrete oxalic acid and other compounds during disease development, resulting in a slightly acidic environment. At this point, we designed a capsule that can be rapidly released under acidic conditions. At the same time, this capsule can maintain the drug's stability and its effectiveness to achieve its quick action and timely prevention and control of diseases.3O4 as the reaction monomer and emulsifier, respectively. Meanwhile, the release kinetics, light stability, and efficacy of the prochloraz nanocapsules against the anthracnose pathogen are studied further.In our study, the anthracnose pathogen, a widely present fungus, was used as the target, and 5% prochloraz pH-responsive nanocapsules were prepared by the Pickering emulsion polymerization method with isophorone diisocyanate (IPDI) and polyethyleneimine (PEI)-modified nano-Fe2.2.1.2\u00b74H2O, FeCl3\u00b76H2O and NaOH were analytical chemicals purchased from Chengdu Kelong Chemical Reagent Factory . Acetonitrile and methanol were HPLC grade and purchased from J. T. Baker (USA).The model pesticide prochloraz (purity 98%) was supplied by Hubei Jusheng Technology Co., Ltd. . IPDI (purity 99%) was purchased from Jining Hongming Chemical Reagent Co., Ltd . PEI (purity 99%) was purchased from Shanghai Tengzhun Biotechnology Co., Ltd. Tristyrylphenol ethoxylate (600#) was purchased from Zhongyue Chemical Technology Co., Ltd . Xylene, FeCl2.2.3O4 was prepared by chemical co-precipitation.20 First, 4.4 g FeCl2\u00b74H2O was dissolved in 10 mL deionized water and filtered by a membrane; 5.2 g FeCl3\u00b76H2O was dissolved in 100 mL deionized water. Then, the FeCl3 solution was added into the flask, stirred and heated to 70 \u00b0C; 7 mL FeCl2 solution was added into the flask, and 12 mL concentrated ammonia water with 25% mass fraction was added rapidly under intense stirring. After 1 hour, a black sol-like liquid was obtained, separated by a magnet, and washed several times with methanol and water, and the samples were freeze-dried. Then, 1 g PEI was dissolved in 100 mL deionized water, and 1 g nano-Fe3O4 was added to the solution and stirred to form a brown sol-like liquid. The samples were separated by magnets, washed several times with deionized water, and freeze-dried to obtain PEI-modified nano-Fe3O4. The physical and chemical properties of PEI-modified nano-Fe3O4 were characterized by TEM and Fourier transform infrared spectroscopy.In this study, magnetic nano-Fe2.3.3O4 (0.1 g) was added to 14 g deionized water containing 100 \u03bcL acetic acid and stirred to form an aqueous phase. Then, 1 g of prochloraz, 0.4 g of 600# and 2 g of IPDI were dissolved in 1 g of xylene to form the oil phase. After that, the oil phase was added into the water phase dropwise under stirring. Prochloraz nanocapsules were obtained after stirring for 6 h for full reaction of IPDI and PEI. The physical and chemical properties and sustained release properties of prochloraz nanocapsules were characterized by TEM, FETEM, laser particle size analyzer, Fourier transform infrared spectroscopy, HPLC and contact angle tester.PEI-nano-Fe2.4.\u22121, temperature: 40 \u00b0C, detection wavelength: 220 nm, injection volume: 10 \u03bcL. The encapsulation efficiency and drug loading rate of nanocapsules were calculated by the following formulas.The prochloraz nanocapsules were washed and centrifuged, and the precipitated capsules were dried to form powder capsules. Dry prochloraz nanocapsules were weighed and placed in the test tube. An appropriate amount of methanol was added and prochloraz was completely dissolved in methanol by ultrasonic dispersion. Then, the samples were centrifuged at a high speed and the supernatant was taken to prepare the samples after filtering. The concentration of prochloraz was determined by HPLC. The chromatographic conditions are as follows: mobile phase: methanol and water, volume ratio: 80\u2009:\u200920, flow rate: 1 mL min2.5.At is the residue amount of prochloraz at time of t, and A0 is the initial amount of prochloraz.Prochloraz nanocapsules (5 mL) and EC were added to a Petri dish and placed under an incandescent lamp. Methanol was added to the Petri dish after 3, 5 and 7 days. The active ingredients were extracted repeatedly by ultrasound and detected by liquid chromatography. Then, the degradation ratio (DR) of prochloraz in the nanocapsules and EC was calculated as follows:2.6.21 and cultured for 144 hours at 25 \u00b0C so as to increase the strain reserve. The nanocapsules were diluted at different concentrations and mixed into the sterilized melt PDA medium. The PDA medium with different concentrations of prochloraz was prepared by pouring into a 9 cm diameter dish and cooling. Sterilized water was added to the PDA medium as the CK. Each sample was repeated three times in the experiment. After PDA was solidified, the bacterial samples were punched with a 6 mm diameter perforator and then inserted into the PDA medium. After 144 hours of culture, the colony diameters were determined by the cross-over method. The inhibition rate is calculated by using the formula and the toxicity curve and the median effect concentration (EC50) was obtained. Certain amounts of the diluted prochloraz nanocapsule solution and emulsifiable concentrate solution were added to the melt PDA medium with pH 5.5, 7 and 8.5. The concentration of prochloraz in PDA was EC50. Each sample was repeated three times in the experiment. The anthracnose pathogen was cultured by the same method, and the inhibition efficiency was calculated after 7 days.The tested strain is anthracnose pathogen, which was cultured in the College of Plant Protection, Southwest University. Citrus anthracnose fungus was inoculated on sterilized potato dextrose agar (PDA)3.3.1.3O4. From the figure, we can see that nano-Fe3O4 is evenly distributed and the average particle sizes are in the range of 5\u201310 nm. Comparing 3O4 has better dispersibility. This also indicated that PEI successfully modified nano-Fe3O4.3.2.3O4 on the surface of the capsules. The particle size is about 80\u2013130 nm, which can also be proven in \u22121 stands for N\u2013H of the polyurea stretching vibration. The absorption peaks at 1240\u20131458 cm\u22121 are due to the C PBM data was replaced with SVG by xgml2pxml:<glyph-data id=\"z.dbd\" format=\"PBM\" resolution=\"300\" x-size=\"8\" y-size=\"10\" xml:space=\"preserve\">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns=\"http://www.w3.org/2000/svg\" version=\"1.0\" width=\"13.200000pt\" height=\"16.000000pt\" viewBox=\"0 0 13.200000 16.000000\" preserveAspectRatio=\"xMidYMid meet\"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform=\"translate scale\" fill=\"currentColor\" stroke=\"none\"><path d=\"M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z\"/></g></svg>O, CN, and CC stretching vibrations and N\u2013H and C\u2013H flexural vibrations. These peaks prove that IPDI and PEI-nano-Fe3O4 react to form polyurea. For prochloraz, the main characteristic infrared absorption peaks were attributed to the CO stretching (1637 cm\u22121), C\u2013N stretching (1567 cm\u22121) and C\u2013H of pyridine flexural vibration . All of the characteristic absorption peaks can be found for the prochloraz nanocapsules, which indicate that prochloraz is successfully encapsulated in the nanocapsules.3.3.\u22121 and their contact angles were measured. We found that the contact angles of EC, EW and nanocapsules were 45.52\u00b0, 40.58\u00b0, and 25.28\u00b0, respectively. The contact angles of the nanocapsules were significantly smaller than those of EC and EW, indicating that the nanocapsules had better wettability. At the same time, we analyzed the relationship between the adhesion work and time under different dosage forms, as shown in The contact angles of the prochloraz emulsion concentrate (EC) (A), emulsion in water (EW) (B) and nanocapsules (C) on cowpea leaves are shown in 3.4.3.4.1.3O4, which bind with H+ to produce a repulsive force and enlarge the capsule gap. At the same time, due to the negative zeta potential of the capsule as a whole, the increase in H+ will also destroy the potential balance and make the capsule unstable. The release mechanism of the prochloraz nanocapsules is shown in Certain amounts of the prochloraz nanocapsules were packed in dialysis bags (MW: 3500). The release behavior and stability of the prochloraz nanocapsules were studied by changing the pH under the condition that the stirring speed was 100 rpm and the temperature was kept constant (25 \u00b0C). 3.4.2.Certain amounts of prochloraz nanocapsules were packed in dialysis bags (MW: 3500). The release behavior and stability of the prochloraz nanocapsules were studied by changing the temperature under the condition that the stirring speed was 100 rpm and the pH was kept constant (pH = 7). 3.4.3.22\u201324 It can be seen from The origin software was used to fit the simulation equation of the cumulative release rate under different pH values. The zero order equation, first order equation, Higuchi equation and Peppas equation were chosen as the fitting models.n is an index, which reflects the release mechanism: Fickian diffusion (n < 0.43), non-Fickian or anomalous diffusion (0.43 < n < 0.85), and case II transport (n > 0.85).25 The results showed that the diffusion index n was between 0.43 and 0.85 at different pH values; thus, the release of nanocapsules belonged to non-Fickian or anomalous diffusion.Further results for fitting with the Peppas equation are shown in 3.5.Owing to the low light stability, prochloraz tends to degrade under incandescent light irradiation, resulting in low utilization efficiency. In order to obtain light stability, the prochloraz degradation ratios of nanocapsules and EC were investigated under incandescent light irradiation. As shown in 3.6.50 to be about 0.1 mg L\u22121. Then, we carried out the inhibition test of the nanocapsules and EC for the fungus. The results are shown in In the indoor bioassay experiment, we first set up a series of concentration gradients to fit the toxicity curve of prochloraz to the anthracnose pathogen and calculated ECFrom the chart, we find that the inhibition rate of EC shows a downward trend and the inhibition rate of the nanocapsules at the 7th day is significantly higher than that of EC, which may be ascribed to the photolysis of prochloraz in EC during the culture process; also, the prochloraz nanocapsules can effectively alleviate the photolysis of prochloraz. The inhibition rates at different pH values  were 48.8%, 47.7%, and 45.3% on the 7th day, respectively. The results showed that the inhibition rate of nanocapsules under acidic conditions was higher than that under alkaline conditions on the 7th day. This may be because the prochloraz nanocapsules are released faster under acidic conditions.4.In this work, prochloraz pH-responsive nanocapsules were developed by the Pickering emulsion polymerization method. The release rate of the prochloraz nanocapsules is faster under acidic conditions and the release of the nanocapsules belongs to non-Fickian or anomalous diffusion. The nanocapsules have excellent magnetic separation performance (more than 50%). In addition, they can prevent the photolysis of prochloraz. The results of an indoor bioassay also indicated that the inhibition rate of 5% prochloraz nanocapsules was significantly higher than that of EC on the 7th day.There are no conflicts to declare."}
+{"text": "FLOWERING LOCUS C (FLC). Accordingly, we investigated the effect of proline on flowering time in more detail by analyzing the relative expression of the main flowering time genes in p5cs1 p5cs2/P5CS2 proline-deficient mutants and found a significant upregulation of FLC expression. Moreover, proline-deficient mutants exhibited an adult vegetative phase shorter than wild-type samples, with a trichome distribution reminiscent of plants with high FLC expression. In addition, the vernalization-induced downregulation of FLC abolished the flowering delay of p5cs1 p5cs2/P5CS2, and mutants homozygous for p5cs1 and flc-7 and heterozygous for P5CS2 flowered as early as the flc-7 parental mutant, indicating that FLC acts downstream of P5CS1/P5CS2 and is necessary for proline-modulated flowering. The overall data indicate that the effects of proline on flowering time are mediated by FLC.The recent finding that proline-induced root elongation is mediated by reactive oxygen species (ROS) prompted us to re-evaluate other developmental processes modulated by proline, such as flowering time. By controlling the cellular redox status and the ROS distribution, proline could potentially affect the expression of transcriptional factors subjected to epigenetic regulation, such as  Moreove and FT1 .stn7 (state transition 7), for example, Dietzel et al., (2015) [stn7 mutants exposed to high-light treatments. Furthermore, in wild-type plants, redox signals generated from the chloroplast were found to enhance the activity of histone acetyl-transferase (HAT) and histone deacetylase (HDAC), two key enzymes of epigenetic regulation. Moreover, Rai et al., (2018) [Lablab purpureus, the increase in O2\u2022\u2212 and H2O2 caused by high-temperature stress can be relieved by the combined application of salicylic acid (SA) and sodium nitroprusside (SNP)\u2014a nitric oxide (NO) donor. Notably, the supplementation of SNP and NO to temperature-stressed plants led to increased activity of antioxidant enzymes and altered profiles of DNA methylation and demethylation.The involvement of FLC in proline-mediated flowering may suggest a link between redox regulation and the epigenetic regulation of flowering. Indeed, recent studies suggest that the redox status of the cell affects the enzymes involved in plant epigenetic modifications, such as DNA methylation and histone protein acetylation, leading, in turn, to chromatin remodeling and differential gene expression . In a co, (2015)  found no, (2018)  reported+/NAD(P)H ratio in cytosol and mitochondria, as well as the generation of ROS [The relationship between the redox environment and proline is complex. Proline metabolism is affected by the redox status  but alson of ROS ,51, beha1-pyrroline-5-carboxylate reductase (P5CR). The reactions are coupled with the oxidation of NAD(P)H to NAD(P)+ and are subjected to feedback inhibition by proline and other cofactors [+/NAD(P)H ratio in the cytosol, proline synthesis feeds the pentose phosphate pathway [2O2 [1-pyrroline-5-carboxylate dehydrogenase (P5CDH). This process requires FADH and NAD+ reduction and feeds the electron transport chain to produce ATP. When the electrons generated by proline oxidation exceed the capability of the electron transport chain, however, O2\u2022\u2212 and H2O2 are also produced as by-products of mitochondrial respiration.During proline synthesis in the cytosol, glutamate is reduced to proline by the sequential action of the P5CS and \u0394ofactors . In addi pathway , and in way [2O2 . During A delicate equilibrium between ATP and ROS production is maintained by multiple regulatory systems, by modulating the relative activities of ProDH and P5CDH and directing proline catabolism toward glutamate production or the proline\u2013P5C cycle. In this cycle, the P5C generated by proline oxidation in the mitochondrion is not further oxidized to glutamate by P5CDH but re-enters in the cytosol, where it is reduced back to proline by P5CR. This seemingly futile cycle allows the transfer of reducing equivalents into the mitochondrion and the generation of ROS and may behave as a master regulator of redox balance and ROS signaling.FLC, however, suggests that proline may affect the epigenetic regulation of FLC expression by modifying the redox status of the cell.The classical models of gene induction involve binding either transcription factors or small regulative molecules to the promoters of target genes to regulate their transcriptional activity. Unfortunately, this model of action does not seem not to apply to proline, to the best of our knowledge. The interesting finding that proline-deficient mutants have high levels of p5cs1 p5cs2/P5CS2 mutants and showed, through molecular, physiological, and genetic experiments, that the effects on the flowering time of proline metabolism depend on FLC. Although this finding cannot explain how proline can modulate FLC expression, in view of the results of this study, it may suggest a possible mechanism of epigenetic regulation, which deserves further investigation. However, considering the multiple development pathways in which proline seems to be involved, we cannot rule out, at present, that proline-mediated FLC regulation may occur through different mechanisms acting at a transcriptional or post-transcriptional level.In this communication article, we investigated the molecular basis of the late flowering in Arabidopsis mutants used in this work were of Columbia-0 (Col-0) ecotype and were grown in a growth chamber at 24/21 \u00b0C with a light intensity of 300 \u00b5E\u00b7m\u22122\u00b7s\u22121 under 16 h light and 8 h dark per day (under either 16 h light and 8 h dark (long days) or 8 h light and 16 h dark (short days)). Seeds were stratified for three days at 4 \u00b0C, surface-sterilized, germinated on MS1/2, and potted a week after. p5cs1 p5cs2/P5CS2 mutants were generated by crossing a homozygous p5cs1 mutant  obtained from the Salk collection, with a heterozygous p5cs2 mutant (GABI_452G01) obtained from the GABI-Kat collection [p5cs2 mutants are embryo-lethal, and homozygous lines cannot be produced [p5cs1 p5cs2/P5CS2 quasi-double mutants was first described by Mattioli et al., (2009) [All the p5cs2 mutant allele and carry out segregation analysis, heterozygous p5cs2/P5CS2 single mutants and p5cs1 p5cs2/P5CS2 quasi-double mutants were germinated on MS1/2 plates supplemented with 6 \u03bcg/mL sulfadiazine (sul). Vernalization experiments were performed by keeping seeds in the dark at 4 \u00b0C for three months. We performed the flowering time analysis by counting the number of rosette leaves present at the appearance of the first flower bud [To maintain the ower bud ,55. We chttps://www.ncbi.nlm.nih.gov/tools/primer-blast/, accessed on 15 August 2022) and are reported in ACTINE8 (ACT). All the analyses were performed in triplicate on, at least, three independent samples. Statistical analysis was performed on raw Ct values. Total RNA for RT-qPCR was extracted from leaves or apical shoots using a NucleoSpin RNA Plant  according to the manufacturer\u2019s instructions. To prevent mRNA decay, we dissected leaves and apices in a cold room and threw them in liquid nitrogen immediately after dissection. RNA quality (A260/A280 ratio > 2) and quantity was assessed with a NanoDrop 1000 . All samples were taken when we saw the first floral bud in the wild-type population. Reverse transcription was performed from 1 \u03bcg of total RNA using a QuantiTect Reverse Transcription kit  as recommended by the manufacturer. Plant material was collected from plants close to flower transition, with still no visible signs of flowering. To enrich tissues in SAM, apices were dissected under a Zeiss Stemi SV6 stereo-microscope.Molecular techniques were performed according to standard protocols. For PCR analysis, genomic DNA was extracted with a modified CTAB method, according to Stewart and Via (1993) . Real-tiA T-DNA insertion mutant in a Col-0 background was identified in the GABI-KAT collection  and obtap5cs1 p5cs2/P5CS2 mutants cannot transmit pollen grains bearing the p5cs2 mutant allele [p5cs1 p5cs2/P5CS2 and flc-7 were made using the former as female and the latter as male. Seeds from outcrossed siliques were grown under sulfadiazine selection, and resistant plantlets were checked by using PCR to identify individuals heterozygous for all the mutations. Seeds from self-fertilized F2 plants were grown under sulfadiazine selection, and resistant plantlets were checked by using PCR to identify individuals heterozygous for p5cs2 and homozygous for p5cs1 and flc-7.Since t allele , genetic"}
+{"text": "Nigrospora nonsegmented RNA virus 1 (NoNRV1) has been reported previously in the fungus Nigrospora oryzae, but its biological effects on its host are unknown. In this work, we isolated a strain 9-1 of N. oryzae from a chrysanthemum leaf and identified NoNRV1 infection in the isolated strain. The genome sequence of NoNRV1 identified here is highly homologous to that of the isolate HN-21 of NoNRV1 previously reported; thus, we tentatively designated the newly identified NoNRV1 as NoNRV1-ZJ. Drug treatment with Ribavirin successfully removed NoNRV1-ZJ from the strain 9-1, which provided us with an ideal control to determine the biological impacts of NoNRV1 infection on host fungi. By comparing the virus-carrying (9-1) and virus-cured (9-1C) strains, our results indicated that infection with NoNRV1 promoted the pigmentation of the host cells, while it had no discernable effects on host growth on potato dextrose agar plates when subjected to osmotic or oxidative stress. Interestingly, we observed inhibitory impacts of virus infection on the thermotolerance of N. oryzae and the pathogenicity of the host fungus in cotton leaves. Collectively, our work provides clear evidence of the biological relevance of NoNRV1 infection in N. oryzae, including pigmentation, hypovirulence, and thermotolerance. Cryphonectria hypovirus 1 (CHV1), which causes hypovirulence in chestnut blight fungus Cryphonectria parasitica and has been used as a biocontrol agent [Curvularia thermal tolerance virus (CThTV)-infected C. protuberata is required for the growth of a panic grass, Dichanthelium lanuginosum, in soils with temperatures >50 \u00b0C [Mycoviruses, also known as fungal viruses, are widespread in major fungal species, including mushroom, yeasts, oomycetes, and filamentous fungi. The majority of mycoviruses have a genome composed of one or more double-stranded RNA (dsRNA) segments . Recentlol agent ,18,19. Is >50 \u00b0C . Transcrs >50 \u00b0C , which ws >50 \u00b0C ,23.C. parasitica [Biological pigments are present ubiquitously in nature and have complicated chemical compositions and variable colorization properties. If not all, many fungi produce pigments which serve various biological functions, including harnessing solar energy for metabolic use and protection against ionizing radiation . Some myrasitica . The brorasitica . Fungi arasitica ,31. Melarasitica . A previrasitica \u2014suggestirasitica .Nigrospora oryzae is a widespread endophyte or weak pathogenic fungus that infects a broad variety of crops, including maize (Zea mays) [Oryza sativa) [Sorghum sp.) [Gossypium hirsutum) [N. oryzae was released [N. oryzae, including N. oryzae victorivirus 1 [N. oryzae victorivirus 2 [N. oryzae mitovirus 1/2 [N. oryzae fusarivirus 1 [N. oryzae partitivirus 1 [N. oryzae nonsegmented RNA virus 1 (NoNRV1) [N. oryzae an ideal microorganism for the study of mycovirus\u2013host interactions. NoNRV1 is an unclassified virus and has a dsRNA genome of 2857 bp encoding two separated open reading frames (ORFs). ORF1 encodes a protein of unknown function, and ORF2 is responsible for encoding a putative RNA-dependent RNA polymerase (RdRP) [N. oryzae isolated from a chrysanthemum plant, analyzed its genome sequence and phylogenesis, and tested its biological effects on the fungal host. ea mays) , rice (O sativa) , sorghumhum sp.) , cotton irsutum) , and othirsutum) ,40,41,42released  which wiivirus 1 , N. oryzivirus 2 , N. oryzirus 1/2 , N. oryzivirus 1 , N. oryzivirus 1 , and N. (NoNRV1) , which we (RdRP) . That isN. oryzae strain 9-1 was isolated from the surface-disinfected leaf on potato dextrose agar (PDA) plates at 28 \u00b0C in darkness. The isolated fungus was maintained on PDA plates, and its hyphal morphology and spore structure were observed using a Nikon light microscopy . To identify the isolated strain, the internal transcribed region (ITS) fragment was amplified and sequenced according to the reported procedure [A chrysanthemum leaf with mild mosaic symptoms was collected in 2018 at Xinsha Island, Zhejiang Province, China. rocedure . The obtN. oryzae strain 9-1 according to the methodology reported previously with a slight modification [TM III reverse transcriptase , according to the manufacturer\u2019s instructions. The full-length cDNA was obtained by a regular PCR using Primer B and Q5 High-Fidelity DNA Polymerase (NEB). The resultant cDNA was cloned into the vector pLB-simple  for sequencing. DsRNA fragments were isolated from fication , CF-11 cfication . BrieflyAs a nucleoside analog, the drug Ribavirin was reported to be efficient to cure specific mycoviruses . To remoTo assess the efficacy of curing NoNRV1-ZJ from its host 9-1 by Ribavirin treatment, both positive and negative strands of the virus were detected by RNA gel blotting assays, as described previously . BrieflyTotal RNAs were extracted from the mycelia of 9-1 or 9-1C using TRIzol (Thermo-Fisher), according to the manufacturer\u2019s instructions. One microgram of total RNA was used as template for synthesis of first-strand cDNA using 6mer random primer and reverse transcriptase SSIII (Thermo-Fisher). Two microliters of the first-strand cDNA samples were used as templates for PCR reactions using ORF1-specific primers  and Q5 DNA polymerase (NEB). PCR reactions was run in a thermocycler (Eppendorf) with 98 \u00b0C for 30 sec, 35 cycles of 98 \u00b0C for 10 s, 56 \u00b0C for 15 s, and 72 \u00b0C for 15 s, followed by an extension of 72 \u00b0C for 5 min. The resultant PCR products were separated on 1% agarose gel and visualized under UV light after ethidium bromide staining.N. oryzae was assayed as described previously [Virulence of eviously , with mieviously . The virSearches for homologous sequences in GenBank were carried out using BLAST . ConservNigrospora species [N. oryzae isolates , Ustilaginoidea virens nonsegmented virus 1 , Purpureocillium lilacinum nonsegmented virus 1 , Phytophthora cactorumusti-like virus 1 , Conidiobolus non-segmented RNA virus 1 , and a proposed partitivirus species (MN033127). ORF2 located downstream of ORF1 encodes an approximately 83 kDa, putative RdRP . Sequence analyses showed that the dsRNA molecule is composed of 2857 bp with a G+C composition of 57.72%. A BLAST search for the whole dsRNA sequence showed that it has a 99% identity with the previous isolate HN-21 of NoNRV1 , which ieviously , the poseviously . In addiive RdRP A, with aRdRPs have a conserved catalytic structure, which contains a set of seven motifs A-G . These sThe 5\u2032 untranslated region (5\u2032 UTR) and 3\u02b9 UTR of the NoNRV1-ZJ genome are 34 nt and 56 nt in length, respectively. The 5\u2032 UTR sequence was predicted to form two short hairpins with a \u2206G value of \u20133.90 kcal/mol D. The 3\u2032To determine the relationship of NoNRV1-ZJ with other mycoviruses, we constructed both phylogenetic trees based on the ORF1 or ORF2 proteins of selected viruses. It is worth noting that only seven viruses were included in the phylogenetic analysis of ORF1 because no other mycoviruses reported so far have an equivalent ORF1. These seven viruses are unassigned, non-segmented mycoviruses, with the exception of a partitivirus species isolate H1. The phylogenetic tree showed that NoNRV1 forms a clade with PlNV-1, UvNV-1, UvNV-2, and the partitivirus species A. IntereN. oryzae, an attempt was made to remove the virus in the isolate 9-1 by culturing the fungus on Ribavirin-containing PDA plates with five successive passages. Afterwards, total RNAs were extracted from untreated (9-1) and Ribavirin-treated (9-1C) mycelia and used for detection of the positive and minus strands of the viral genome by RNA gel blot analysis. Here, the total RNA extracted from cucumber mosaic virus (CMV)-infected plants was used as an RNA ladder after hybridization with a CMV-specific probe. In the 9-1 sample, both positive and negative strands of NoNRV1-ZJ were detected markedly at the position slightly below CMV RNA2 (3038 nt), which is consistent with the genome size of NoNRV1-ZJ  or oxidative (H2O2) chemicals. Each chemical at a high concentration showed obvious inhibition of the growth of both fungal cultures, and 9-1 had no growth advantages over 9-1C on the chemical-containing plates , and alleviates host virulence in cotton leaves. Our work provides phenotypic evidence that uncovers the biological impacts of NoNRV1 infection on fungal hosts.In this work, we determined the infection of NoNRV1 in the fungus UGAA or AUAUG  at the ORF1/ORF2 junction that has been illustrated well in the Helminthosporium victoriae virus 190S [The NoNRV1 genome encodes two separated ORFs. It is a mystery how the 3\u2032 proximal ORF2 is translated. Non-canonical translational strategies, including translation stop/restart, and ribosomal \u22121 or +1 frameshifting have been identified as the molecular mechanisms used by some mycoviruses to translate 3\u2032 proximal ORFs ,75,76,77rus 190S ,67,74. Irus 190S . Thus, wC. protuberata with infection by CThTV [N. oryzae mycelia in the presence of NoNRV1 is the outcome of increased melanin. Firstly, melanin is important for fungi to protect themselves from adverse stresses [Here, we reported the interesting phenomenon that NoNRV1 infection promoted pigmentation in host hyphae on solid media B. A simiby CThTV . CThTV iby CThTV ,80. Howestresses , while Nstresses . HoweverPestalotiopsis theae chrysovirus-1, PtCV1; Sclerotinia sclerotiorum hypovirulenceassociated DNA virus 1, SsHADV-1) were reported to be hypovirulence-inducing agents which convert their pathogenic fungal hosts into non-pathogenic, even beneficial endophytes [N. oryzae pathogenicity in cotton leaves  on its fungal host at a high temperature (32 \u00b0C). This is similar to our finding that NoNRV1 infection made its host completely lost thermotolerance at 37 \u00b0C (N. oryzae isolate (9-1C) grew as well at 37 \u00b0C as did at 28 \u00b0C, suggesting that N. oryzae itself is extremely thermotolerant. Exposure to high temperature is a useful way to cure mycoviruses in their hosts [The relationship between mycovirus and fungal heat-resistance is complicated. Previously, CThTV has been reported to enhance fungal host heat-tolerance , which cat 37 \u00b0C B. Strikiir hosts ,89. Thus"}
+{"text": "This paper describes the creation of a comprehensive, linked Australian population spine including name and address history. This spine was developed using four national datasets and linked to multiple State and Territory datasets. This enabled the creation of linkage maps which could then be used to produce de-identified linked datasets.Initially the spine was created using identifiers from Medicare Consumer Directory (MCD), Social Services data (DOMINO) and National Death Index (NDI). The COVID-19 vaccination program covering almost the entire Australian population provided the opportunity to add the Australian Immunisation Registry (AIR).Probabilistic linkage was used to link MCD and DOMINO with linkage rates of 97.3%. MCD and NDI data were also linked probabilistically. Most AIR identifiers shared IDs with MCD and were linked deterministically with the remainder linked probabilistically.Based on the linkage results, unique identifiers were created for everyone appearing in at least one of the four datasets. Only unlinked records with very incomplete information in NDI and AIR were excluded. All unique combinations of names and addresses for each individual were added to the combined spine. This allowed us to cover the data gaps of each dataset and create a comprehensive history not possible when using a single data source.Linkage maps were created between all contributing data sources. State and Territory datasets were also linked to the spine using probabilistic linkage. These linkages were then reused for multiple projects.Linkage to the combined spine increased linkage efficiency  as well as increased linkage accuracy and a reduction in the transfer of personal information to provide a better service to the Australian research community."}
+{"text": "Ceratitis capitata (Diptera: Tephritidae), is an invasive pest, that is currently expanding its geographic distribution from the Mediterranean coasts to more temperate areas of Europe. Given that low temperature is a primary determinant of insect species\u2019 range boundaries especially in the Northern Hemisphere with pronounced seasonality, we used chill coma recovery time for assessing latitudinal clines in basal chill tolerance of C. capitata adults. We selected six populations obtained from areas with broad climatic variability based on the main bioclimatic variables of temperature and precipitation, spanning a latitudinal range of about 19\u00b0 from Middle East to Central Europe. Adults were exposed to 0\u00a0\u00b0C for 4\u00a0h, and time to regain the typical standing position of a fly at 25\u00a0\u00b0C were recorded. The post-stress survival after a period of 8\u00a0days was also recorded. Results revealed that adults from Israel and Austria were less chill tolerant than those from Greece, resulting in curvilinear trends with latitude. Analysis of macroclimatic conditions revealed combined effects of latitude (as a proxy of photoperiod) and macroclimatic conditions on chill coma recovery time. Nonetheless, there was not a deleterious effect on post-recovery survival, except for flies obtained from the northern most point . Overall, it seems that evolutionary patterns of basal chill coma recovery time of C. capitata adults are driven mainly by local climatic variability.The Mediterranean fruit fly,  Species with tropical or subtropical origin have been established in colder temperate regions, wherein they had to adapt to seasonal thermal variation and extreme weather events4. Hence, latitudinal clines in cold tolerance have been shaped in response to extreme winter minima overwinter survival6. Otherwise, evolutionary changes in the seasonal timing of life-history events, such as diapause termination, are expected to protect post-diapause adults from low temperatures that fall below critical thermal limits for activity during their active growth and reproduction periods7. In addition, chill-susceptible adults with increased chill tolerance can be protected from chilling injuries, which may have negative impact on fitness-related traits8. Performance at sub-lethal temperatures is therefore considered as a key limiting factor for population resistance and resilience at higher latitudes9. As a result, chill tolerance is a growing topic of research in an attempt to understand the species\u2019 distribution limits with climatic variability, particularly for invasive insect species11.In the Northern Hemisphere, the northward expansion of many terrestrial insects has been driven by climate warming and/or human-mediated transportation and trade13. Prolonged exposure to low temperature causes a loss of ion balance and hemolymph hyperkalemia. High haemolymph [K+] can lead to chilling injuries, the effects of which cascade across tissues and may cause cell death (apoptosis) in the neuromuscular system; this process limits the ability of insects to recover, stand or fly, even if ion balance is restored. Recovery to warmer conditions involves both the rapid recovery of the temperature-induced depolarization and the energetically costly restoration of ion (and sometimes water) homeostasis, including upregulation of genes for repairing chilling injuries. Hence, the ability to quickly re-establishing homeostasis after cold stress directly affects the adaptation to low temperatures14. Sensitivity to such cold stress differ among population/insect species, and determines how fast individuals can conclude their foraging activities in order to feed, mate and/or escape from predators after a cold night and/or a frost event. Therefore, there are multiple potential fitness benefits from a fast recovery, suggesting that chill coma recovery can be a trait under selection15. To date, most efforts in understanding chill tolerance have focused on Drosophila spp. and/or Drosophila populations from tropical habitats that exhibit longer chill coma recovery time than those from temperate environments17, implying that chill coma recovery time can be a useful metric for disentangling inter- and intra-specific variation in chill tolerance10.One of the commonly measured chill tolerance trait is chill coma recovery time (CCRT), which refers to the time needed under benign conditions to recover neuromuscular function following a period of chill coma induced by temperatures that are commonly below the critical thermal limit for activityPorcellio laevis18, the winter ant Prenolepis imparis19 and the temperate-zone butterfly Lycaena tityrus20 with the high-latitude/altitude populations showing an increased resistance to cold, in line with the climatic variability hypothesis21. Colder environments are expected to harbor populations with higher chill tolerance in line with local climatic  variability and particularly the great variation in thermal minima across environmental gradients22. Chill coma recovery time was found to be correlated with daily minimum temperature in the Australian endemic species Drosophila serrata5, the minimum temperature at the coldest month for temperate and tropical Drosophila melanogaster populations from the coastal eastern Australia6, and both the annual mean temperature and annual mean minimum temperature for the common woodlouse, P. laevis from Chile18. In addition, Poikela et al. (2021) reported combined effects of latitude and bioclimatic variables on chill coma recovery time of Drosophila flavomontana adults by asserting that latitude is a proxy of photoperiod that serve as a reliable cue for seasonal temperature changes in the Northern Hemisphere. Chill coma recovery time of adults have evolved in response to latitudinal varying photoperiod but they are also associated with macroclimatic conditions of low-altitude coastal areas, wherein chill tolerance increases. Therefore, it seems that there is no \u2018gold standard\u2019 choice of environmental parameter for the relationship between insect chill tolerance and distribution limits23. A general assumption holds that latitude provides a better description of the geographical distribution while bioclimatic variables are key predictors of the thermal stress that limits performance and species\u2019 persistence24.At the intraspecific level, the main selective pressures driving local adaptation in chill coma recovery time are inferred either from geographical proxies of environmental variation, such as latitude or altitude, or from a range of bioclimatic indicators of local climatic variability related mainly to temperature and precipitation. Linear latitudinal or altitudinal clines in chill coma recovery time have been observed for a few non-drosophilids, including the common woodlouse Ceratitis capitata (Wiedemann) (Diptera: Teprhitidae), is a highly damaging phytophagous pest with more than 300 host plant species, including cultivated trees of Prunus spp., Citrus spp. and Pyrus spp.25. It is an invasive pest, originated from eastern Sub-Saharan Africa, which has been dispersed in almost all tropical and sub-tropical regions of the world26. In the Northern Hemisphere, medfly has long been established in the Mediterranean Basin and Middle East but it is only recently that expanded its geographic distribution up to central Europe27. Since 2010, medfly adults have been captured in fruit-producing regions of Vienna, where seems to have been established 27. Even though medfly distribution is mainly attributed to anthropogenic activities based on transportation and/or trade, it is suggested that cold tolerance of C. capitata adults have jointly supported northward expansion of the species by facilitating population resistance to cold stress33. Thus far, evolutionary patterns of cold tolerance for C. capitata adults have been addressed only for some southern African populations in terms of critical thermal minima32.The Mediterranean fruit fly (medfly), C. capitata adults, previous studies revealed that they are able to recover after a single short frost event in approximately 20\u00a0min (e.g. 0\u00a0\u00b0C for 1-4\u00a0h)34. At the intrapopulation level, flies with slower recovery time had reduced life expectancy, higher initial mortality rate, and worse climbing performance than their counterparts with faster recovery30. Nevertheless, only C. capitata flies that were reared for multiple generations under constant laboratory conditions have thus far been used for estimating chill coma recovery time. Eventhough lab-adaptation can result in rapid evolutionary changes in stress-related traits of insects35 , any domestication effects on basal chill tolerance of C. capitata adults remain unexplored. Our preliminary data revealed slower recovery with artificial rearing under constant laboratory conditions for medfly adults from Greece than the wild flies  from populations located at environmentally heterogenous habitats in order to be the most representative of the climatic variability faced at C. capitata habitats in the temperate zone across the Northern Hemisphere. Given the heterogeneity of climate based on K\u00f6ppen-Geiger climate classification for the six fly collecting sites37, we initially quantified local climatic variability by performing a principal component analysis on the main bioclimatic variables of temperature and precipitation ). Then, we predicted that flies from the high-latitude, colder site in Central Europe will have lower chill coma recovery time than flies from the low-latitude, warmer site in Middle East, being in line with linear latitudinal clines reported previously for non-drosophilids species20. In an attempt to address the complex nature of the chill coma recovery time and distinguish whether latitudinal clines in chill tolerance of medfly adults have evolved in response to changes in photoperiod, macroclimatic conditions or their combination38, we assessed the effects of both local climatic conditions (based on bioclimatic variables) and latitude (as a proxy of photoperiod) on chill coma recovery time, accordingly to Poikela et al. (2021). Then, we assessed how chill coma recovery time, a non-lethal trait, accounts for geographic variation in post-recovery fitness, by estimating survival of both sexes for a period of 8\u00a0days under benign conditions. We predicted that populations with faster recovery will show higher survival than those with delayed recovery, in accordance with the previously reported intrapopulation variability in chill coma recovery time30. Considering that chill coma recovery time might be an important metric of performance under climatic variability, this study aims to provide insights regarding the recent northward expansion of medfly populations and a better understanding of population resistance after short frost events for making sound pest management decisions.Here, we used chill coma recovery time as a chill tolerance metric for assessing evolutionary patterns of The temperature-precipitation background of the six sites was characterized by principal component analysis (PCA) on 19 bioclimatic variables ) (BIO2), and temperature of annual range (BIO7) as well as precipitation of the wettest month BIO13), precipitation of the wettest quarter (BIO16) and precipitation of the coldest quarter (BIO19) . Pairwise comparisons revealed that chill coma recovery time was longer for flies from Yotvata than those from the four Greek populations. Marginal differences in chill coma recovery time were recorded for flies from Campos and Heraklion .Average chill coma recovery time ranged from 22.1\u2009\u00b1\u20091.4\u00a0min (Yotvata) to 16.2\u2009\u00b1\u20090.5\u00a0min (Volos) for the six ons Fig.\u00a0. Cox Region Fig.\u00a0. Flies fR2\u2009=\u20090.86, p\u2009=\u20090.053) between chill coma recovery time and latitude, indicating increased chill tolerance between 30 and 40oN latitude, which represent the Greek populations, or otherwise the Mediterranean Basin  and climatic variables, included latitude, PC1 and their interaction as explanatory factors (see Supplementary Table ts Table .Table 1Lp\u2009<\u20090.001)  Fig.\u00a0. Fifty p01) Fig.\u00a0. Recoverp\u2009<\u20090.001) and sex (p\u2009=\u20090.011) were significant predictors of the post-recovery survival (see Supplementary Table Survivorship of the recovered flies after remaining 8\u00a0days at benign conditions ranged from 52.5% (Vienna) to 97.5% (Heraklion) among the six populations .Pairwise comparisons revealed that post-recovery survival of flies from the northernmost population of Vienna was lower than those of flies from all Greek populations, except from Volos. Post-recovery survival was marginally lower for flies from Vienna than that from Yotvata, which was marginally lower than those from Heraklion. Survivorship of flies from Heraklion was marginally higher than those from Volos Fig.\u00a0. SurvivoC. capitata adults from six populations in the temperate zone across the Northern Hemisphere using a common-garden experimental approach. Despite geographic variation in chill coma recovery time, no linear latitudinal trends were found. Instead, a curvilinear relationship between chill coma recovery time and latitude was shaped, with a faster recovery for flies residing in the Mediterranean Basin. In an attempt to link the above latitudinal clines to macroclimatic conditions by using 19 bioclimatic variables, we found combined effects of regional climatic variability with latitude (as a proxy of photoperiod) on chill tolerance, underlying the complex nature of chill coma recovery time. Post stress survival was high for the recovered adults from all populations but Vienna. Survivorship of females was higher than those of males. It is seems, therefore, that chill coma recovery time of C. capitata adults is mainly driven by the local climatic variability of their habitas across the Northern Hemisphere.This study examined the evolutionary responses in basal chill tolerance of 6. Drosophila species overwinter as adults and faster recovery from a cold stress provides fitness benefits during the cold season by increasing overwinter survival18. On the other hand, latitudinal trends of chill coma recovery time were absent across Australian populations of Bactrocera tryoni36 and Bombus vosnesenskii workers from Western United States39. In this study, C. capitata adults that reside in the Mediterranean Basin (35\u201340\u00b0) exhibited faster recovery than those from southern and northernmost populations, indicating a curvilinear relationship between chill coma recovery time and latitude across the Northern Hemisphere. One possible explanation for the low chill tolerance of Vienna adults is that populations near high-latitude range edges, especially if they are recent expansions, are likely to have phenotypes far from their local optimum because of higher genetic load40. Consistent with this expectation, populations from low-latitude range edges are also likely to perform more poorly than central populations across test sites, supporting the slower recovery of Yotvata flies as well. Moreover, recovery curves of adults from Yotvata and Vienna populations significantly differ from those from the Greek populations, validating a less steep, slower recovery with higher variability among individuals within populations. Even though the above patterns could be indicative of chilling injuries that results in lower fitness30, the increase in trait variance also increases the opportunity for selection under climatic stressful conditions41.Several studies have reported linear latitudinal clines in chill coma recovery time, particularly for drosophilidsMyrmica rubra, inhabiting sub-Arctic regions in the Northern Hemisphere, resulting from a connection with climates experienced by ancestral populations11. Given that gene flow is common among Greek populations42, ideally we could have controlled for population relatedness for excluding any phylogeographical connections, but genetic data were not currently available for all of the tested populations. On the other hand, the measurements of chill coma recovery time of Australian D. melanogaster populations revealed local adaptation to climatic selection along a latitudinal cline despite strong gene flow43. Thus, gene flow can either hinder or promote adaptation at range edges depending on the balance between the costs of migration and genetic drift44. Overall, considering the complicated interplay of selection, gene flow, and drift that affect evolutionary potential at range edges40, genetic studies are needed for elucidating the net effects of evolutionary forces on C. capitata populations that are resided in the Northern Hemisphere.Curvilinear trends in chill coma recovery time were also demonstrated for the invasive widespread ant species 18, albeit exceptions exist39. Latitude, which is not a real environmental variable, can serve as a proxy of photoperiod that is a more reliable cue for seasonal temperature changes than environmental temperature itself23. Presuming that chill coma recovery time is ecologically relevant to climatic variability15, an attempt was made to link chill coma recovery time of C. capitata adults with the macroclimatic conditions of the six sites. Model revealed that chill coma recovery time was significantly associated with PC1 and an interaction between latitude (as proxy of photoperiod) and PC1. On the one hand, the combinations of extreme temperature and precipitation (PC1) based on the annual mean temperature, the mean temperature of winter and summer seasons and the precipitation of the warmest season, which shape the climatic profile of the two climatic edge populations (namely the populations located at cold-climate Vienna and warm-climate Yotvata area), were associated with low chill tolerance. Temperature extremes, such as the minimum temperature of the coldest month that serves as proxy for the winter cold thresholds in each site, were also associated with chill tolerance in C. capitata flies, in line with drosophilids6. On the other hand, latitude found to be associated with the above bioclimatic variables (PC1) for estimating chill tolerance of C. capitata adults as well. This is probably due to site-specific differences in the seasonal availability of host fruits and the occurrence of frost events as well as differences in the overwintering capacity of C. capitata adults that regulate the duration of their flight period.Bioclimatic indicators of each collection site can be a reliable indicator of climatic variability to address geographical variation in thermal selection45. The day of the last spring frost as well as the phenological events have been advanced47, increasing the risk of exposure of the most vulnerable stage of insect life cycle and tree phenology to subsequent spring frosts. For example, the recent frost events of April 2016 and April 2017 caused crop losses in apple production in Austria48, which is the main fruit host of C. capitata in this area. In addition, late spring frost events are more severe to coastal areas compared with continental areas47. Moreover, C. capitata adults are more vulnerable to frost events (LLT50: 0\u00a0\u00b0C for 8\u00a0h)33 than immature stages 49, resulting in geographical variation in overwintering capacity of medfly adults among the six site. Specifically, C. capitata overwinters as larvae (particularly 1st and 2nd instars) within fruits in Thessaloniki51 and Campos (Chios)52, while it overwinters in all stages in Crete due to mild winters53. Similarly, adults are captured all year round along the coastal plain and the Jordan Valley in central region of Israel54. In the area of Campos (Chios), adult flight period expand from June to January with peak captures from August to November52. In Volos, adult captures increases from May to November, but some adults may be captured until January . In Thessaloniki, no adults are detected from December to the end of June, with increasing capture rates in autumn25. By contrast, the flight period is narrowed in Vienna, with most adult captures throughout August and September27. As a result, cold winters with low minimum temperatures are associated with adult absence from the coldest sites during the winter, which prevent them from being exposed to frost events during the coldest season when photoperiod is short 50. In contrast, frost events are more often in autumn and winter for flies from latitudes around 30\u00b0 (Yotvata) than in spring, as it is the case for flies in the Mediterranean Basin45. As a result, flies from the warmest area are on wings during seasons with short photoperiod when it is more likely to be exposed to frost events than during warmer seasons with long photoperiod. It is therefore, suggested to further assess the photoperiodic cues jointly to thermal cues for minimizing the chance of missing ecologically relevant patterns of basal chill tolerance in C. capitata flies.In Europe, relatively most frost events (when the daily minimum temperature drop below 0\u00a0\u00b0C) are expected in spring, particularly for populations around 40\u00b0 latitudeper se55. Nevertheless, latent chilling injury, which refers to cold-induced damage days after the stress56, has been rarely investigated and then often with contradictory results58. A recent meta-analysis revealed that survival is significantly decreased after extreme weather events, as opposed to reproduction and abundance9. Accordingly, post stress survival can be a useful proxy of population resistance after frost events for C. capitata flies. In this study, flies from all populations but Vienna demonstrated compensatory mechanisms during cold stress in order to reduce deleterious effects on survival. Even though both sexes need to adjust their physiology in order to survive a frost event8, the sex-related differences in post stress survival of C. capitata flies indicate that females are likely to shift a part of the investments into reproduction during the post-stress period, incurring survival costs. Nevertheless, all females that managed to recover were mated and reproductively mature and therefore, remaining alive for a period of 8\u00a0days after being exposed to chilling stress, gives them the opportunity to resume reproduction activities, and potentially increases population resistance. To this end, further studies are needed for determing the ability of the recovered females to reproduce effectively, by measuring their fecundity and fertility. Despite this limitation, this study contributes towards improving our understanding of how frost events during the adult life can affect long-term fitness of C. capitata flies and whether carry-over effects of frost events differ among C. capitata populations. The above knowledge could be useful both for predicting its distribution limits across the Northern Hemishere and for making sound pest management decisions in each area after a frost event. To this end, field validation of the results is a prerequisite for sustainable pest management decisions since laboratory may not provide transferable outcomes for pest management of C. capitata59.The impact of sub-lethal stress on insect individuals may be of greater ecological importance than the ability to survive temperature extreme C. capitata, revealing no linear latitudinal clines in the basal chill tolerance for populations residing in the temperate zone across the Northern Hemisphere. In the future, the use of more sampling sites, either within the same climate zone or from another climate zone met through the currently distribution range of C. capitata in the Northern Hemiphere, is highly recommended for excluding the possibility of alternative results under different sampling schemes. Moreover, a single frost event seems not to limit fitness of C. capitata flies in the Northern Hemisphere, but population resistence for flies from Vienna came under question. However, it is worth noting that chill coma recovery time is a plastic trait for C. capitata flies29, and it is therefore likely that flies from the relatively less chill tolerant populations to compensate their low basal chill tolerance with high cold acclimation capacity, as it is the case for other insects60. In this sense, the geographic patterns of developmental plasticity and adult acclimation on chill coma recovery time of C. capitata adults need to be addressed. It is also a need for further studies on seasonal variation in chill tolerance for multiple (\u2265\u20093\u00a0years), at least for sites where C. capitata adults are on-winds all year around, as previous study reveals that chill coma recovery time of a natural population of D. melanogaster respond adaptively to seasonal shifts in temperature that are characteristic of temperate regions61. Last but not least, plasticity patterns of chill tolerance can be ideally combined with studies on their mechanistic base, for making sound predictions of the impact of climatic variability on population persistence and distribution8.Overall, this study is the first to address geographical patterns of chill coma recovery time of the adults of the widespread invasive pest 20 , with the average temperature of the warmest month being below 22\u00a0\u00b0C, the coldest averaging above 0\u00a0\u00b0C, and at least four months with average above 10\u00a0\u00b0C. There are no strict seasonal patterns of precipitation. All Greek populations but Thessaloniki have a typical hot summer Mediterranean climate (Csa), with at least one month\u2019s average temperature above 22\u00a0\u00b0C, four months above 10\u00a0\u00b0C, and the coldest above 0\u00a0\u00b0C. Winter is the wettest period while the driest month of summer receives less than 40\u00a0mm. The climate in Thessaloniki is classified as cold semi-arid (Bsk), and it is characterized by cold, relative wet winters and hot dry summers. Israel lies in a transition zone between the hot and arid southern part of West Asia and the relatively cooler and wetter northern Mediterranean region. Yotvata belongs to one of the three dryland zones in southern part that is characterized by an extremely hyper-arid climate (Bwh)62. Summers are hot and totally dry, following by mild winters with low average annual rainfall that greatly varies from year to year. Hence, Yotvata population represents a climatic edge population in the temperate zone of the Northern Hemisphere while Vienna could be characterized as a climatic edge population located at the highest latitude of the current distribution.We used six populations that were originated across the temperate zone of the Northern Hemisphere. Populations were obtained from three countries: Austria (Vienna), Greece  and Israel (Yotvata: Arabah). Population sampling sites spanning from\u2009~\u200929\u00b0 to 48\u00b0N latitude with up to 157\u00a0m altitude in order to avoid altitudinal clines in chill tolerance20 Fig.\u00a0. Accordi0) , AT-AGES and Agricultural Research Organization (ARO) for Greek, Austrian and Israeli populations respectively. Flies were allocated at 4 cages in each generation, and after rearing for 1\u20132 generations in fruits under standard laboratory conditions, pupae from Austria (F2) and Israel (F1) were delivered by a courier agent to UTH. Upon emergence, adults kept in wooden (30\u2009\u00d7\u200930\u2009\u00d7\u200930\u00a0cm), wire-screened cages provided with water and a standard adult diet . All cages were held at similar low densities  and females were allowed to oviposit on 5-cm-diameter hollow, plastic hemispheres of red color (domes) that were artificially punctured with 40\u201350 evenly distributed holes on their surface. Each dome was fitted in a 5-cm-diameter hole made on the cover of a 5.5-cm-diameter plastic petri dish. Water was placed in the base of the petri dish in order to maintain humidity levels (beneath the dome) adequate enough for female oviposition. A plastic cup containing 0.5\u00a0ml of orange juice was placed in the base of the petri dish to stimulate oviposition.Pupae were retrieved from field infested fruits  from the six sampling sites, from late summer to early winter based on the local availability of infested host fruits. Collected fruits were transferred to laboratory, placed in plastic containers on a layer of sterilized sand and remained under standard conditions  until pupae collection. The collected pupae from different sites were used to raise separate, site-specific populations/colonies. Wild adults  for avoiding maternal, trans-generational or other epigenetic effects of field populations as well as laboratory adaptation issues that may raise under prolonged rearing conditions63. The above common garden approach will provide evidence that observed phenotypic differences are not environmentally induced and help to identify the role of local selective factors64.All flies were reared for three to four generations in the UTH laboratory conditions before being used for the chill coma recovery assays. Specifically, we used flies reared up to FC. capitata lab-adapted adults at 0\u00a0\u00b0C for 4\u00a0h is sufficient to induce chill coma and cause variability in recovery time among individuals within a population31, we predicted that recovery from the same cold stress will provide a clear-cut discrimination among the six C. capitata populations. In addition, we used 10-day-old adults to control for any potential age-related differences in chill tolerance, which can markedly influence trait assessments65.Based on previous results found that an exposure of 13. There were no dead flies observed during chill coma recovery time assays (0\u00a0\u00b0C for 4\u00a0h) or censored flies .To determine chill coma recovery time (CCRT), we used 20 males and 20 females for each population. Upon emergence, adults from each population were placed into Plexiglas cages (20\u2009\u00d7\u200920x20cm) with ad libitum access to adult diet food and water. On adult day 10, groups of 8\u201310 mixed-sex adults of the same population were transferred into empty 35-mL glass vials with a cotton wool stopper. Vials were immersed in an ice-water slurry at 0\u00a0\u00b0C for 4\u00a0h (in the dark) in a Styrofoam cooler box placed at room temperature (25\u00a0\u00b0C). The temperature within vials were checked by placing an analog thermometer into an empty glass vial immersed into the ice-water slurry. Following chill coma, flies were immediately placed individually in petri dish (5\u00a0cm in diameter) in a supine position (using a paintbrush), and their recovery was monitored for one hour under laboratory conditions . Petri dishes were sealed with a transparent plastic lid to prohibit escape of recovered flies. A fly was scored as recovered when it was able to right itself and stand on its legs in a normal posture or fly, without any interference or stimulation from the observer. The time period needed at 25\u00a0\u00b0C until reach the upright position was termed \u201cchill coma recovery time\u201dFor each population, flies that recovered from chill coma assays were transferred back into their Plexiglas cages . All flies had ad libitum access to adult diet and water and remained under standard laboratory conditions  for the next 8\u00a0days. Dead males and females were recorded daily. Post-recovery survival of males and females were calculated as the percentage of the recovered males and females that remained alive 8\u00a0days after the chill coma assay.66. To characterize macroclimatic conditions of the six collection sites, we extracted 19 bioclimatic variables (Bioclim1-19) related to temperature and precipitation from WorldClim database  using latitudinal and longitudinal coordinates for each site 67  model selection was used to distinguish among a set of possible linear regression models describing the relationship of latitude (as a proxy of photoperiod), PC1, PC2 on chill coma recovery time. BIC uses a stronger penalty for including additional variables to the model. The general linear model was used for the parameter estimation of the best-fit model with meaningful biological meaning. The relationship of chill coma recovery time with latitude was initially examined using a linear regression model but the model fit was inadequate (R2\u2009=\u20090.009) so a second-degree polynomial model was adopted. A Cox proportional hazards regression  was applied to model recovery times adjusting for population, sex, and their interactions. Kaplan\u2013Meier curves and pairwise log-rank tests (package emmeans71) were used for the comparison of populations\u2019 recovery rates. A logistic regression model was used to examine the effect of population and sex on post-recovery survival. Statistically non-significant interactions were removed by the model. Benjamini-Hochberg (B-H) correction was used to adjust for multiple comparisons in both Cox proportional hazards and logistic regression models. P-values less than 0.05 were considered statistically significant.Statistical analysis was conducted using R version 4.1.1 Supplementary Information."}
+{"text": "To describe the record linkage system that is currently implemented at the Provincial Health Data Centre (PHDC) in the Western Cape, South AfricaTo assess its output to date with respect to types of matches and duplicates trendsTo describe the errors affecting patient matchingWe apply a stepwise deterministic record linkage approach to link patient data that are routinely collected from health information systems in the Western Cape province of South Africa. Variables used in the linkage process include South African National Identity number (RSA ID), date of birth, year of birth, month of birth, day of birth, residential address and contact information. Matching records are established from sequentially running the data through multiple passes formed by various combinations of linkage variables. Descriptive analyses are used to estimate the extent of mismatches and duplication in the provincial patient master index (PMI).The proportion of duplicates dropped from approximately 16.8% in December 2015 to 9.6% in October 2020, indicating improved data linkage over time.Duplicates mainly arise from spelling errors, and surname and first names carry most of the errors, with different first names and surname for the same individual in approximately 22% of duplicates.Linkage is also affected by completeness, with less than 30 % completeness for the South African national identity (RSA ID) number which is mainly because RSA ID is not mandatory when seeking healthcare.Linkage improvement could be due to improved registration practices. Further improvements are possible by repeating data linkage where patients register before creating a new patient record following a failed search. This could use the PHDC linkage approach whilst leveraging all data in addition to search terms used by the clerk."}
+{"text": "Dear Editor,1 Therefore, precisely managing cellular ROS levels, meaning to keep redox homeostasis properly, stands as an aim for health and longevity. Actually, this aim is challenging, as it asks not only to restrain excessive ROS accumulation at the right time and right place but also to guarantee proper ROS levels for fitting physiological requirements. Traditional ways for eliminating ROS put more attention on the efficiency and identified a variety of drugs and enzymes with antioxidant properties, known as enzymatic and non-enzymatic ROS scavengers, respectively. However, how to accurately manage the integrative effect of ROS scavengers in vivo remains a problem. Actually, the effectiveness of non-enzymatic antioxidants in vivo is controversial, explained mostly by the uncertainty to reach a given organ with the appropriate concentration or to work there persistently.2 As to enzymatic ROS scavengers, although their overexpression in a given place can be achieved, this mode is often unwieldy or harmful to intrinsic physiologic functions in cases.3 For this reason, we attempt to develop new ways for in vivo antioxidation, for establishing a high-quality balance between efficiency and safety. As the consequence, we innovated a gene expression system that can limit the excessive ROS accumulation but keep the physiological level of ROS.The moderate level of reactive oxygen species (ROS) contributes to cellular functions such as proliferation, differentiation, and infection resistance, but the excessive level of ROS causes oxidative damage which underlies the basic mechanism for aging and geriatric diseases.This enzymatic scavenger expression system is named as SeHed (Stress-evoked Hydrogen peroxide elimination device), featuring by its stress-evoked response and the moderate capacity for mitochondrial hydrogen peroxide elimination. Popularly speaking, this system works like an automatic fire extinguisher that can put out the burst of ROS under intensive stress conditions. Structurally, SeHed is composed of a stress-sensing promoter portion and a mitochondria-targeted catalase encoding portion Fig. .Fig. 1Th4 the performance of this promoter was tested by luciferase reporter assay and GFP reporter assay. As expected, it sensitively responded to H2O2 and TNF\u03b1 stimuli , acting for sensing and responding to external or internal stresses that result in detrimental ROS accumulation. We chose this promoter based on the bioinformatic analysis to an oxidative stress-induced senescence system, in which the NF-\u03baB pathway was the most responsive signal working for pro-senescent gene expression Fig. . After smCat), which was a modified version of human catalase, featuring the deletion of peroxisomal localization signal and the addition of mitochondrial localization signal at the N-terminal  did Fig. , S12a\u2013d.6 SeHed possesses a special feature for cellular ROS elimination, which means it is adept to eliminate H2O2 in time, in mitochondria, and in a moderate extent. This makes it facilitating to keep the efficiency and safety of anti-oxidant treatment in a balanced status. SeHed also supports the concept that the relief of the detrimental ROS accumulation and the maintenance of physiological necessary ROS are equally important for antioxidant therapy and for healthy improvement (Fig. In summary, based on the concept of precise anti-oxidation,ent Fig. . We hopeSupplementary_MaterialsSupplementary_MaterialsEthics declarationsSupplementary Figures 1Supplementary Figures 2Supplementary Figures 3Supplementary Figures 4Supplementary Figures 5Supplementary Figures 6Supplementary Figures 7Supplementary Figures 8Supplementary Figures 9Supplementary Figures 10Supplementary Figures 11Supplementary Figures 12"}
+{"text": "Population Data BC (PopData) is an agency in British Columbia, Canada, that routinely performs linkages of various administrative and researcher-collected data to a population spine. We developed a linkage report template in order to increase transparency of linkage process and outcome for end users and data providers.PopData performs probabilistic and deterministic data linkage using an in-house software. A literature review identified existing guidelines and examples of linkage reporting. A survey collected input from a wide range of end users about their interest in receiving linkage reports and specific information that is important to their work. A draft template was developed by PopData\u2019s linkage experts and data scientists which then was reviewed by PopData staff and external partners. Privacy requirements, mode of delivery, readability to the intended audience and operational feasibility were carefully considered.The resulting template built on our existing internal linkage summaries. The report follows a framework suggested in the literature with three key components: 1) information on the data source and linkage fields, 2) data pre-processing and linkage methodology, and 3) linkage results, presented in tables and figures, including overall linkage rates, detail on matched fields, and the distribution of linkage weights of linked and unliked pairs. In addition, an appendix describes the linkage methods and population spine in detail, and supplementary notes will comment on unique issues related to the data, when those are applicable. Educational materials to aid understanding of linkage methodologies and reporting are also under development.Linked data are increasingly used in research, making it important to provide information on linkage process and performance to the research community. Rigorous and standardized linkage reports produced by data centres can facilitate evaluation of the impact of linkage performance on research findings and enable transparent reporting in peer-reviewed research."}
+{"text": "Batteries and supercapacitors have become important elements of everyday life, thanks to the extensive effort contributed to their development. It is interesting to look back at the historical development of batteries and supercapacitors during the last two centuries since the invention of the first battery\u2014called voltaic pile\u2014by Alessandro Volta in 1800 and the first patent on electrochemical double layer capacitors by H. I. Becker in 1957 . A surve2 and Li/S), as well as sodium ion and dual-ion insertion batteries.It often happens that improving the performance of an energy storage system becomes increasingly difficult as the system approaches the practically-attainable power/energy density which is usually 50%\u201360% of the theoretical limit. This along with the insufficient cycling stability especially in the case of batteries constitute two of the most important challenges in today\u2019s energy storage research. Therefore, a marked further development of batteries and supercapacitors definitely requires both fundamental and applied research covering various aspects such as the underlying electrochemical/electronic processes, structure-performance relationships, new storage mechanisms, as well as material discovery and optimization. Accordingly, in the last 2\u00a0decades several new research directions have been followed that may be all together called \u201cpost-lithium ion batteries.\u201d The most important ones are probably solid state batteries, flow batteries, lithium metal batteries  through metal ion doping can significantly influence the electrochemical performance is presented here by Last but not least, as Research Topic Editors, we would like to thank all authors, peer-reviewers, and the Frontiers\u2019 team for their valuable contributions to this Research Topic, and hope the readers will appreciate the reported findings as much as we did."}
+{"text": "This study evaluated the effect of two natural antioxidants on the compromised bond strength of a resin\u2010modified glass ionomer (RMGI) to the sodium hypochlorite (NaOCl)\u2010affected pulp chamber dentin.n\u2009=\u200912), according to the solutions used for immersion: (1) Control, distilled water; (2) NaOCl, 5.25% NaOCl for 20\u2009min;\u00a0(3) NaOCl/Ethylenediaminetetraacetic acid (EDTA); 5.25% NaOCl for 20\u2009min\u2009+\u200917% EDTA for 1\u2009min;\u00a0(4) NaOCl/TA, 5.25% NaOCl\u2009+\u200910% tanic acid (TA) for 5\u2009min;\u00a0(5) NaOCl/EDTA/TA, 5.25% NaOCl\u2009+\u200917% EDTA\u2009+\u200910% TA for 5\u2009min;\u00a0(6) NaOCl/PA, 5.25% NaOCl+ 10% proanthocyanidin for 5\u2009min;\u00a0and (7) NaOCl/EDTA/PA, 5.25% NaOCl+ 17% EDTA\u2009+\u200910% PA for 5\u2009min. The RMGI was bonded on the treated dentin using a Tygon tube. After 24\u2009h of storage, microshear bond strength (\u00b5SBS) was tested. Data in MPa were submitted to one\u2010way analysis of variance and Tamhane test.Forty\u2010two sound third molars were split into halves. The exposed pulp chamber dentin was ground to provide the flat dentin surfaces and divided into seven groups (p\u2009<\u2009.05);\u00a0and in the NaOCl/EDTA/TA group, the increased bond strength was at the level of the control group (p\u2009>\u2009.05). NaOCl/PA and NaOCl/EDTA/PA and NaOCl groups had comparable \u00b5SBS.NaOCl significantly decreased the \u00b5SBS; NaOCl/EDTA and NaOCl/TA significantly increased the \u00b5SBS, higher than the control group (TA could be suggested to provide effective bonding of RMGI and immediate sealing of the pulp chamber dentin after NaOCl irrigation. Forty\u2010two freshly, caries\u2010free third molars were collected and stored in 0.5% chloramine T at 4\u00b0C until use. Each tooth was cut mesiodistally to expose the dentin surface of the pulp chamber. Each tooth incision was mounted in self\u2010curing acrylic resin, exposing the pulp chamber dentin. The dentin surface of the pulp chamber was ground with 600\u2010grit silicone paper under running water for 30\u2009s before any treatments. All the materials used in the study are listed in Table\u00a0The 84 samples were then randomly divided into 7 groups of 12 each according to the different solutions used. (1) Control group: the samples were immersed in distilled water for 20\u2009min;\u00a0(2) NaOCl group: the samples were immersed in 5.25% NaOCl for 20\u2009min and then washed with running water for 1\u2009min;\u00a0(3) NaOCl/EDTA group: after immersion in 5.25% NaOCl and washing, the samples were immersed in 17% EDTA  for 1\u2009min and then washed with running water for 1\u2009min;\u00a0(4) NaOCl/TA group: after treatment with 5.25% NaOCl, the samples were immersed in 10% TA for 5\u2009min; the solution was prepared using 10\u2009gr of TA in the form of powder  dissolved in 100\u2009ml of distilled water\u00a0and then washed with water for 1\u2009min;\u00a0(5) NaOCl/EDTA/TA group: after treatment with NaOCl and EDTA, the samples were immersed in 10% TA for 5\u2009min and then washed with water for 1\u2009min;\u00a0(6) NaOCl/PA group: after treatment with 5.25% NaOCl, the samples were immersed in 10% PA for 5\u2009min; the solution was prepared using 10\u2009gr of grape seed extract in the form of powder  dissolved in 100\u2009ml of distilled water\u00a0and then washed for 1\u2009min; and (7) NaOCl/EDTA/PA group: after preparation with NaOCl and EDTA, the samples were immersed in 10% PA for 5\u2009min and then washed for 1\u2009min.2, BlueLEX, Monitex) for 20\u2009s. The bonded samples were kept in a humid environment for 24\u2009h at 37\u00b0C. A wire of 0.2\u2009mm diameter was inserted around the interface of the RMGI/dentin. The interface, wire loop, and load cell center were aligned as straight as possible. The microshear (\u00b5SBS) bond strength test was performed using a universal testing machine  at a speed of 0.5\u2009mm/min. The debonded specimens were observed under a stereomicroscope at \u00d740. Fracture modes of the specimens were classified as follows: A: adhesive failure at the interface, B: cohesive failure in RMGI, and C: mixed failure, a combination of adhesive and cohesive failure. At first, the normal distribution of the data was analyzed using the Kolmogorov\u2013Smirnov test and confirmed; then, the data were analyzed by one\u2010way analysis of variance and Tamhane post hoc statistical tests (p\u2009<\u2009.05).After preparing the samples, RMGI  was mixed according to the manufacturer's instructions and packed in cylindrical molds (Tygon tubes) with a diameter of 1\u2009mm and a height of 1\u2009mm, which was placed on dentin of the pulp chamber. The RMGI was then cured with a light\u2010curing unit (1200\u2009mW/cm3p\u2009<\u2009.001). The Tamhane analysis test was applied to compare the groups. NaOCl significantly decreased the bond strength (p\u2009=\u2009.006). Compared to the control group, NaOCl/EDTA and NaOCl/TA treatment yielded a significantly higher bond strength . However, the increased bond strength in NaOCl/EDTA/TA was not significant (p\u2009>\u2009.05). NaOCl/PA and NaOCl/EDTA/PA with similar results did not significantly affect the reduced bond strength of the NaOCl group (p\u2009>\u2009.05). Mixed failure was the main fracture mode in all groups, except for NaOCl, NaOCl/PA, and NaOCl/EDTA/PA groups in which adhesive failure was predominant  was bonded immediately. Accordingly, NaOCl led to a decrease in the \u00b5SBS of FLC. This negative effect has been previously reported for some adhesives to the pulp chamber dentin (Cecchin et al.,\u00a0In this regard, no study was reported on the interaction of pulp chamber dentin and RMGI with endodontic irrigants. The adverse effect reported in Sekhar and colleagues study was attributed to the dissolution of collagen, leading to the impedance of ionic bonding of RMGI to the dentin collagen and hybrid layer formation (Sekhar et al.,\u00a0RMGIs are self\u2010bonded to the dentin structure through two mechanisms: ionic interaction between anions of polyalkenoic acid chains and calcium ions of hydroxyapatite around collagen and micromechanical interlocking, hybrid layer and resin tag formation into surface roughness and porosity resulted from the self\u2010etching ability of RMGI (Imbery et al.,\u00a0The results of the current study demonstrated that applying EDTA, TA, and EDTA followed by TA could remarkably improve the SBS of FLC. According to previous studies, EDTA increased the bonding effectiveness of RMGI to the smear layer\u2010covered dentin (Fagundes et al.,\u00a0The two bonding mechanisms of RMGI could contribute to the bond strength of RMGI. However, its chemical bonding is essential to provide bonding durability (Saad et al.,\u00a0The null hypothesis tested in this in vitro study has to be partially rejected since TA has been revealed to exert a considerably beneficial effect on the bonding ability of the RMGI to the pulp chamber dentin, whereas PA had no effect.The present study was performed on the smear layer\u2010covered dentin produced by grinding. This grinding is necessary to provide a flat dentin surface for bond strength testing. However, in the intraoral situation, RMGI was bonded directly on the pulp chamber dentin with no dominant smear layer because it is not prepared during ET (Schellenberg et al.,\u00a0The present research was a short\u2010term bond strength study. Further long\u2010term research with different antioxidant/irrigant\u00a0agents and various application times is suggested to be conducted to evaluate the longevity of the bonding of RMGIs.In the current vitro study, we have only evaluated the short\u2010term effect of the two antioxidants on the bond strength of RMGI to NaOCl\u2010affected dentin. As the aging process may influence the durability of the bond, long\u2010term investigations with other antioxidant agents by using different application times are suggested. Furthermore, in vivo studies should be conducted before the advisement of antioxidants in clinical procedures.5The adverse effect of NaOCl irrigation on the bonding ability of the RMGI to the pulp chamber dentin could be completely recovered by a 5\u2010min\u00a0application of TA solution. PA was not capable of inducing any reversing effect on the compromised bond strength.All authors contributed to the conception, design, data acquisition and interpretation, and statistical analysis\u00a0and drafted and critically revised the manuscript. All authors gave their final approval and agree to be accountable for all aspects of the work.The authors declare no conflict of interest."}
+{"text": "Words of estimative probability (WEPs), such as \u2018possible\u2019 and \u2018a good chance\u2019, provide an efficient means for expressing probability under uncertainty. Current semantic theories assume that WEPs denote crisp thresholds on the probability scale, but experimental data indicate that their use is characterised by gradience and focality. Here, we implement and compare computational models of the use of WEPs to explain novel production data. We find that, among models incorporating cognitive limitations and assumptions about goal-directed speech, a model that implements a threshold-based semantics explains the data equally well as a model that semantically encodes patterns of gradience and focality. We further validate the model by distinguishing between participants with more or fewer autistic traits, as measured with the Autism Spectrum Quotient test. These traits include communicative difficulties. We show that these difficulties are reflected in the rationality parameter of the model, which modulates the probability that the speaker selects the pragmatically optimal message. Our ability to express probability is of great importance in daily and scientific life. Sometimes, we can use precise numbers when referring to probabilities; for example, we might say that the probability of a fair coin landing on heads is 50%. But very often, we do not\u2014or cannot\u2014know the exact probability of a particular event. In those cases, we might prefer to use what Kent  called wBecause of their central importance, the meaning and use of WEPs has been studied extensively across many disciplinary boundaries \u2019 stands for the probability of an event x:(1)\u2003x\u301b = [P(x) > P(not-x)]a.\u2003\u301athere is a good chance that x\u301b = [P(x) > 0]\u2003\u2003b.\u2003\u301ait is possible that x\u301b = [P(x) < P(not-x)]\u2003\u2003c.\u2003\u301ait is unlikely that The first view holds that sentences containing WEPs have crisp prototype-based approach is often couched within the framework of fuzzy logic. Whereas the truth-conditional view assumes that sentences with WEPs are always either true or false, fuzzy logic argues that they can be true or false to varying degrees  . These traits include difficulties with pragmatic communication  whether patterns of gradience and focality in the use of WEPs can be explained on the basis of a threshold-based semantics, and (ii) whether participants with more autistic traits are less likely to select the pragmatically optimal message than participants with fewer autistic traits.interpret WEPs , we recruited 255 participants on Mechanical Turk.We used a relatively open-ended sentence frame rather than one that steered participants towards using WEPs from a specific part of speech. Our motivation for this decision was that we wanted to see which WEPs naturally come to mind, and to accommodate different response preferences observed in previous studies . By contrast, monotone decreasing WEPs like \u2018impossible\u2019 license inferences from sets to subsets, as shown in (3). Non-monotone WEPs like \u2018possible but not certain\u2019 license neither type of inference.(2)\u2003It is possible that he ordered salmon.\u2003\u2003\u2192 It is possible that he ordered fish.(3)\u2003It is impossible that he ordered fish.\u2003\u2003\u2192 It is impossible that he ordered salmon.The threshold-based approach argues that WEPs denote thresholds on the probability scale. The type of threshold associated with a WEP depends on its Monotone increasing WEPs place a lower bound on the probability scale; monotone decreasing ones an upper bound. Non-monotone WEPs often place both a lower and upper bound on the probability scale.We determined the monotonicity of the WEPs in our sample by consulting our intuitions about the validity of arguments such as (2) and (3). Consequently, the following WEPs were classified as monotone decreasing: \u2018not likely\u2019, \u2018not very likely\u2019, \u2018unlikely\u2019, \u2018very unlikely\u2019, and \u2018impossible\u2019. All other WEPs were classified as monotone increasing.TH. This lexicon associates each message m with a threshold \u03b8, so that the truth value of m in state t is:Based on the foregoing, we may define a threshold-based lexicon \ud835\udd0ffuzzy logic, which argues that the truth value of a sentence can take any value in the  interval , or messages with a higher truth value over messages with a lower truth value (\ud835\udd0fPT).Given a lexicon, we may define two types of speakers in order to connect the hypothesised semantics to the data from the production experiment: a salience. Some WEPs come to mind more easily than others, as evidenced by their fluctuating production frequencies. To model effects of differential salience, we pair each message m with a salience value PSal(m), which is treated as a free variable.We further assume that the available messages vary in their Slit as follows:m in a state of affairs t is proportional to (i) the salience of m, and (ii) the truth value of m in t.Thus, we may define a literal speaker rationally, i.e., they try to optimise the probability that their audience arrives at the correct interpretation   of perceiving the actual state of affairs t as t\u2032. Since the visual displays in the production experiment were upper-bounded, PCf(t\u2032|t) is defined as the product of the probability PANS(t\u2032|t) of maintaining an approximate representation of the number t as t\u2032 and the inverse probability PANS(100 \u2212 t|100 \u2212 t\u2032). These probabilities, in turn, are specified as follows:To model the accuracy of participants\u2019 estimates, we define the confusion probability w stands for Weber\u2019s fraction, which represents the accuracy of participants\u2019 estimates. To parametrise w, we carried out an experiment (Exp. 2) in which we presented 50 participants with the same types of displays used in the production experiment  is a speaker production rule, either literal or pragmatic, the production probabilities under approximate perception of the actual state are:We added the numerosity estimation module to our speaker models. If Taken together, we may distinguish four speaker models, varying the lexicon between threshold-based and prototype-based, and the speaker type between literal and pragmatic.To illustrate model performance, For proper statistical model comparison, we look at how well each model is able to predict the test dataset by calculating the expected log pointwise predictive density using the \u2018elpd\u2019 function from the R package \u2018loo\u2019 , with participants at the median assigned to the high-AQ group. The average AQ of the high-AQ group was 26 (range: 22\u201336); of the low-AQ group 14 (range: 3\u201321). Due to computational limitations, we could not incorporate AQ as a continuous measure; hence, this analysis is inevitably coarse-grained.To put these AQ values in perspective, Baron-Cohen et al.  suggest For this analysis, we focus on the pragmatic threshold-based model. Using STAN, we obtained samples from the posterior distribution over free parameter values conditioned on the data from the production experiment. We fit the model using the combined training and test datasets. Crucially, we fit different lambda parameters for the datasets from high-AQ and low-AQ participants.By fitting the model on the entire dataset, rather than on the datasets from high-AQ and low-AQ participants separately, we ensure that all parameters except for the lambda parameter remain constant across both groups of participants, so that differences in the lambda parameter cannot be interpreted as statistical \u201cspandrels\u201d compensating for differences in other parameters. Reassuringly, the same pattern of results emerges if the model is in fact fit on both datasets separately.t-test indicated that this difference was significant (t(31520) = 206, p < .001). This analysis suggests that participants with a high AQ were less likely to select the pragmatically optimal message than participants with a low AQ.People associate WEPs with gradient and focalised ranges on the probability scale. It has sometimes been concluded that these patterns of use must be reflected in the underlying semantics of WEPs  is said to be characterised, in part, by a pragmatic deficit. Hence, we intuited that this deficit might be reflected in the model parameters, specifically in a parameter that modulates the degree of rationality. We indeed find that participants with more autistic traits\u2014as measured using the Autism Spectrum Quotient test\u2014were estimated to have a significantly lower rationality parameter than participants with fewer autistic traits.scalar inferences, such as the inference from \u2018some\u2019 to \u2018not all\u2019  and nominal  ones. The pragmatic model assumes that these WEPs compete with each other to the same degree. At the same time, we observed that about 40% of the participants consistently produced either adjectival or nominal WEPs. Consequently, adjectival WEPs were more likely to co-occur with other adjectival WEPs than with nominal ones, and vice versa, suggesting that expressions from the same part of speech compete with each other more strongly than with expressions from different parts of speech. An interesting direction for future research is to encode such differential levels of \u201calternativeness\u201d into the model.The communication of probability is of great importance in high-risk areas such as healthcare .All authors formulated the project, and designed and conducted the experiments. MF developed the probabilistic model. BvT and MF implemented and tested the model. All authors wrote the paper.1\u2003semantic rather than pragmatic meaning.A note on terminology: we use the term \u2018meaning\u2019 to narrowly refer to the conventional content of an expression rather than what someone who uses that expression conveys, i.e., to refer to 2\u2003Supporting Information contain an appendix with more details about the production experiment, as well as anonymised data and analysis files.The 3\u2003Supporting Information contain an appendix with more details about the numerosity estimation experiment, as well as anonymised data and analysis files.The 4\u2003Note, in passing, that if we construe prototype-based meanings as reflections of patterns in language use, it seems redundant to assume that they enter into a further pragmatic reasoning process. Nevertheless, for completeness, we also consider the possibility of a pragmatic speaker using prototype-based meanings."}
+{"text": "As the most aggressive tumor, the outcome of pancreatic cancer (PACA) has not improved observably over the last decade. Anatomy-based TNM staging does not exactly identify treatment-sensitive patients, and an ideal biomarker is urgently needed for precision medicine. Based on expression files of 1280 patients from 10 multicenter cohorts, we screened 32 consensus prognostic genes. Ten machine-learning algorithms were transformed into 76 combinations, of which we selected the optimal algorithm to construct an artificial intelligence-derived prognostic signature (AIDPS) according to the average C-index in the nine testing cohorts. The results of the training cohort, nine testing cohorts, Meta-Cohort, and three external validation cohorts (290 patients) consistently indicated that AIDPS could accurately predict the prognosis of PACA. After incorporating several vital clinicopathological features and 86 published signatures, AIDPS exhibited robust and dramatically superior predictive capability. Moreover, in other prevalent digestive system tumors, the nine-gene AIDPS could still accurately stratify the prognosis. Of note, our AIDPS had important clinical implications for PACA, and patients with low AIDPS owned a dismal prognosis, higher genomic alterations, and denser immune cell infiltrates as well as were more sensitive to immunotherapy. Meanwhile, the high AIDPS group possessed observably prolonged survival, and panobinostat may be a potential agent for patients with high AIDPS. Overall, our study provides an attractive tool to further guide the clinical management and individualized treatment of PACA. BRCA-mutated PACA patients. However, a recent study confirmed that although the PARP inhibitor olaparib extended progression-free survival of patients (3.8 months vs. 7.4 months), the overall survival (OS) was not significantly improved  has a 5-year survival rate of only 11% and ranks fourth among tumor-related deaths in the United States . Due to improved . Reassurimproved . Thus, iBRCA1/2 mutations, NTRK fusion, DNA mismatch repair deficiency (dMMR), and microsatellite instability-high (MSI-H) have been gradually brought into clinical guideline , we constructed and multicenter validated a nine-gene artificial intelligence-derived prognostic signature (AIDPS) via 76 machine-learning algorithm combinations. In 13 multicenter cohorts, AIDPS exhibited robust performance in predicting OS, relapse-free survival (RFS), immunotherapy, and drug efficacy. After incorporating several vital clinicopathological features and 86 published signatures of PACA, our AIDPS also demonstrated stable and dramatically superior predictive capability. In addition, in other common digestive system tumors such as liver hepatocellular carcinoma (LIHC), stomach adenocarcinoma (STAD), colon adenocarcinoma (COAD), and rectum adenocarcinoma (READ), the AIDPS could still accurately stratify the prognosis. Overall, our study provides an important reference for achieving early diagnosis, prognostic evaluation, stratified management, individualized treatment, and follow-up of PACA in clinical practice.Our workflow is outlined in To evaluate the prognostic performance of AIDPS, we categorized PACA patients into high and low AIDPS groups according to the median value. The Kaplan\u2013Meier curve for OS and RFS demonstrated the high AIDPS group possessed significantly longer survival in the PACA-AU-Array training cohort  curves. The calibration curves of both the PACA-AU-Array training cohort and Meta-Cohort showed that AIDPS had good prediction performance . The areKRAS, TP53 or CDKN2A mutations  was applied to elucidate the potential functional pathways of AIDPS. As illustrated in rognosis .TP53, CDKN2A, and oncogene KRAS were more frequently mutated in the low AIDPS group than high AIDPS group, whereas the opposite was true for SMAD4, TTN, and RNF43 (BRCA1/2 mutations-related) was enriched in the high AIDPS group, while mutational signature 1 (age-related) was more dominant in low AIDPS group.To investigate genomic heterogeneity between the high and low AIDPS groups, we performed a comprehensive analysis of mutations and copy number alteration CNA, . As shownd RNF43 . Next, bMYC within 8q24.21, and the distinct deletion of the tumor suppressor genes CDKN2A, CDKN2B, and SMAD4 within 9p21.3 and 18q21.2  expression between the two groups. According to single-sample gene set enrichment analysis (ssGSEA), we found that the low AIDPS group exhibited a relatively higher infiltration abundance of immune cell types, including activated CD4+ T cells, CD56dim natural killer cells, central memory CD8+ T cells, gamma delta T cells, and type 2T helper cells  web tool, the low AIDPS group resulted in significantly lower TIDE scores and higher immunotherapy response rates . The resPAK1 activity was required for gemcitabine resistance in PACA, and that PAK1 inhibition enhanced the efficacy of gemcitabine. Consistent with this study, our results revealed that patients with low PAK1 expression possessed distinctly lower estimated AUC values, suggesting greater sensitivity to gemcitabine (As illustrated in citabine . Afterwacitabine .https://clue.io/) tool to identify candidate compounds for PACA. After taking the intersection with the results obtained by CTRP and PRISM, we ended up with two candidate compounds: ATPase inhibitor ouabain and histone deacetylase (HDAC) inhibitor panobinostat. Among them, panobinostat with a \u201398.11 CMap score was highly sensitive in PACA patients, suggesting that it could become a potential therapeutic agent for PACA patients in the high AIDPS group  was more sensitive to the ATPase inhibitor ouabain and HDAC inhibitor panobinostat. Therefore, in clinical settings, our AIDPS may become a reliable platform to further serve individualized decision-making in PACA.Over the past 20 years, the incidence of PACA has increased by 0.5\u20131.0% per year, but the 5-year survival rate only improved from 5.26% to 10%, without a significant breakthrough . The lacIn the era of precision medicine, anatomy-based TNM staging is far from meeting the needs of clinicians for ideal biomarkers that could accurately evaluate the prognosis and predict the efficacy of PACA patients . RecentlOverfitting is one of the troublesome problems encountered by artificial intelligence and machine learning in biomedical model development, with several models fitting well in the training cohort but poorly in other external validation cohorts . After mKRAS, TP53, or CDKN2A mutations, our AIDPS showed significantly improved accuracy. After stratifying PACA patients into the high and low AIDPS groups, we demonstrated that there was no outstanding difference in age, gender, and TNM stage, but the low AIDPS group had more advanced grades, which also contributed to its worse prognosis to some extent. Furthermore, given the excellent performance of AIDPS in PACA, we additionally measured its performance in four common digestive system tumors LIHC, STAD, COAD, and READ and found that AIDPS could accurately stratify patients. These findings indicated that AIDPS constructed in PACA, as a biomarker, has broad prospects for generalization to other tumors.In addition, compared with several common clinical and molecular characteristics such as TNM stage, grade, MAP3K8 and PCDH7, and lower methylation levels of PCDHB1 and SPAG6 in the high AIDPS, all corresponded to obviously prolonged OS, suggesting that methylation modification might play an indispensable role in its better prognosis.GSEA functional enrichment analysis was applied to elucidate the underlying biological mechanisms of AIDPS. The low AIDPS group was mostly enriched in immune and proliferation-related pathways, which partly explained its more advanced grades and worse prognosis. In addition, many recent studies have reported the emerging promise of epigenetic alterations especially DNA methylation in the early diagnosis and prognostic follow-up of PACA . TherefoTP53, CDKN2A, and oncogene KRAS. Numerous studies have revealed that TP53, CDKN2A, and KRAS mutations promoted invasion, metastasis, and immune escape in PACA patients, resulting in worse prognosis  We systematically collected 13 large multicenter cohorts and selected the algorithm with the largest average C-index in the 9 testing cohorts to construct our AIDPS. (2) Unlike current prognostic models for a certain pathway, our AIDPS was based on 15,288 intersection genes from training and nine testing cohorts, which avoided the omission of other indispensable biological process in the initiation and progression of PACA. (3) In order to prevent the inappropriate modeling methods due to personal preference, we combined 10 recognized machine-learning algorithms into 76 combinations and selected the best model based on their accuracy. While we have tried to be as rigorous and comprehensive as possible in our research, some limitations should be noted. First, although we collected 13 independent multicenter cohorts, further validation in prospective study was warranted. Second, in spite of the nine genes included in AIDPS having appeared in numerous prognostic signatures of PACA, which indicated their consistent prognostic value. Their roles in PACA remain to be elucidated, and more functional experimental validation is required in the future. Finally, further clinical trials are necessary to affirm the therapeutic effect of panobinostat in PACA patients with high AIDPS.In conclusion, based on 32 CPGs from training cohort, nine testing cohorts, and three external validation cohorts, we constructed and validated a consensus prognostic signature (termed AIDPS) via 76 machine-learning algorithm combinations. After incorporating several vital clinicopathological features and 86 published signatures, AIDPS also exhibited robust and dramatically superior predictive capability. Of note, our AIDPS has important clinical implications for the clinical management and individualized treatment of PACA, and patients with low AIDPS are more sensitive to immunotherapy, while panobinostat may be a potential agent for patients with high AIDPS. In addition, in other prevalent digestive system tumors, the nine-gene AIDPS could still accurately stratify the prognosis, suggesting a strong possibility of extrapolation. Overall, our study provides an attractive tool for prognostic evaluation, risk stratification, and individualized treatment of PACA patients in clinical practice.http://portal.gdc.cancer.gov/), International Cancer Genome Consortium , ArrayExpress (https://www.ebi.ac.uk/arrayexpress/), and Gene Expression Omnibus  public databases according to the following procedure: (1) more than 40 samples with survival information; (2) at least 15,000 clearly annotated genes; and (3) patients with primary tumors and no other treatments were given before resection. Finally, we enrolled 1570 samples from 13 cohorts, TCGA-PAAD (n = 176), ICGC-PACA-AU-Seq , ICGC-PACA-AU-Array , ICGC-PACA-CA , E-MTAB-6134 (n = 288), GSE62452 (n = 65), GSE28735 (n = 42), GSE78229 (n = 49), GSE79668 (n = 51), GSE85916 (n = 79), GSE21501 (n = 102), GSE57495 (n = 63), and GSE71729 (n = 125). The FPKM data in the TCGA-PAAD was downloaded from UCSC Xena database (https://xenabrowser.net/datapages/) and further converted into log2 (TPM + 1) format. The RNA-Seq data of ICGC were downloaded from its portal and converted into log2 (TPM + 1) format. The normalized exp-Array data from ICGC, ArrayExpress, and GEO were generated directly from their portal. Detailed clinical and pathological information of these 13 cohorts is presented in We collected datasets from The Cancer Genome Atlas  for the next study according to the following criteria: p<0.05 and all hazard ratios (HRs) consistently >1 or <1 in more than 8/10 cohorts.To construct a consensus prognosis model for PACA, we performed our previous workflow . (1) FirPatients in the training cohort, nine testing cohorts, Meta-Cohort (obtained by removing batch effects and repetitions from training cohort and nine testing cohorts), and three external validation cohorts  were categorized into high and low AIDPS groups according to the median value. The prognostic value of AIDPS was evaluated by Kaplan\u2013Meier curve and multivariate Cox regression analysis. The calibration curve and receiver-operator characteristic (ROC) curve were plotted to assess the predictive accuracy of AIDPS.With the attention paid to the stratified management and precise treatment of PACA, many prognostic signatures have been constructed, including m6A-related lncRNA signature, metabolic reprogramming-related signature, and SMAD4-driven immune signature, etc. . To compWe compared the differences in several pivotal clinical traits such as age, gender, TNM stage, and grade between the high and low AIDPS groups. In addition, to explore the application value of AIDPS in other prevalent gastrointestinal tumors, we acquired the mRNA expression and survival data of LIHC, STAD, COAD, and READ in the same way as TCGA-PAAD, and further performed Kaplan\u2013Meier analysis.DESeq2 package, all genes were ranked in descending order according to log2FoldChange (log2FC). Next, we identified GO and KEGG enriched pathways by the clusterProfiler package and further selected the top five pathways in normalized enrichment score (NES) for visualization.GSEA was applied to identify specific functional pathways in the high and low AIDPS groups. After differential analysis using the maftools package. (2) As described in deconstructSigs package to extract the mutational signatures for each PACA patients, and selected mutational signature 1 (age-related), mutational signature 2 (APOBEC activity-related), mutational signature 3 (BRCA1/2 mutations-related), and mutational signature 6 (dMMR-related) with higher frequency of occurrence in PACA for visualization (http://firebrowse.org/). We finally selected regions with broad-level CNA frequency >20% and several genes located within chromosomes 8q24.21, 9p21.3, and 18q21.2 for display.To investigate the genomic alteration landscape in the high and low AIDPS groups, we performed a comprehensive analysis of mutation and CNA data in the TCGA-PAAD. (1) After obtaining the raw mutation file, we calculated the TMB of each sample and visualized the top 15 genes through the lization . (3) Rec;Following the pipeline developed in previous studies . (5) Finally, we identified potential drugs for patients in the high AIDPS group by the intersection of the compounds obtained from (3) and (4).As shown in https://clue.io/) is a publicly available web tool for discovering candidate compounds that may target AIDPS-related pathways based on gene expression signature  < 0.05 was considered statistically significant.All data cleaning, analysis, and result visualization for this research were performed in R 4.1.2. Continuous variables were analyzed by Wilcoxon rank-sum test or Student\u2019s https://github.com/Zaoqu-Liu/AIDPS, copy archived at swh:1:rev:f9d929456fa9f0cd7721cea6a5e2a1412ffae4a7; All data generated during this study are included in the manuscript and supporting files. The basic script for our AIDPS model is available on the GitHub website  that performed best on the task, the authors were able to identify a reduced set of features  which, when measured in the patient, allow for fairly accurate prediction of patient survival and prognosis. Importantly, three new external data sets GSE21501, GSE57495, GSE71729 were used in the validation. public reviews designed to be posted alongside the preprint for the benefit of readers; ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by Caigang Liu as the Senior Editor. The following individual involved in the review of your submission has agreed to reveal their identity: Anthony C C Coolen (Reviewer #2).Thank you for submitting your article \"Comprehensive machine-learning survival framework develop a consensus model in large scale multi-center cohorts for pancreatic cancer\" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:1. The authors should repeat their complete pipeline after randomizing within each of their ten data sets the clinical outcomes (i.e. shuffle the time-to-event outcomes). If one would obtain again a signature with comparable prognostic features, then one can be sure that what is reported here is largely a manifestation of overfitting due to dimension mismatch, and hence of no interest.2. The authors should provide a better understanding of why agreement/optimization amongst a large number of combined ML methods was chosen as a way to drive feature selection. Comparing to existing methods is one thing, currently, there isn't really a clear comparison here to other feature selection approaches.3. The authors should provide a thorough explanation of the biological relevance of the 9 genes in relation to the mutational load/copy number changes/ immunological infiltration found in low AIDPS patients.Reviewer #1 (Recommendations for the authors):Things I think would strengthen the paper:1. A better understanding of why agreement/optimization amongst a large number of combined ML methods was chosen as a way to drive feature selection. Comparing to existing methods is one thing, but there isn't really a clear comparison here to other feature selection approaches, nor much discussion of why this one was chosen. Is this meant to be a reproducible approach, for other problems, or is the point just to get the AIDPS features? Why is this approach to feature selection preferred/superior.2. Right now the current pipeline seems highly unwieldy because of all the models considered. Is this meant to be a general and reproducible approach? if so, the authors need to demonstrate more clearly that this same approach can be done by others with ease.3. Everything in the subsequent analyses comparing high and low AIDPS groups hinges on the assumed accuracy and generality of the model. Once you have 9 genes, though, you should be able to study those features using a range of methods, some of which are picked to have high interpretability. I am sorry if I missed this, but at the moment it is not clear to me what best an ML method can do is using the 9 genes, and whether there is an interpretable method that works well but might imply a non-trivial relationship amongst the genes that matters to the outcome. This seems more informative than studying the statistics of high and low AIDPS groupsReviewer #2 (Recommendations for the authors):To rescue their claims one could suggest to the authors the following simple but clean and fair test. The authors could repeat their complete pipeline after randomizing within each of their ten data sets the clinical outcomes (i.e. shuffle the time-to-event outcomes). If that exercise is found to make the signal disappear , then their present results should be studied further as possibly relevant. If, on the contrary, one would obtain again a signature with comparable prognostic features, then one can be sure that what is reported here is largely a manifestation of overfitting due to dimension mismatch, and hence of no interest.Reviewer #3 (Recommendation for the authors):From the biological point of view, currently, there was little explanation of the biological relevance of the 9 genes in relation to the mutational load/copy number changes/ immunological infiltration found in low AIDPS patients. Please discuss these in sufficient detail.eLife. Your revised article has been evaluated by Caigang Liu (Senior Editor) and a Reviewing Editor.Thank you for resubmitting your work entitled \"Comprehensive machine-learning survival framework develop a consensus model in large scale multi-center cohorts for pancreatic cancer\" for further consideration by The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:1. The improvements to figure 7  meant to address Reviewer #1's request seem to all focus on treating the aidps features separately. Please show the accuracy when these aidps features are considered together as a nine dimensional feature vector in one model.2. For reviewer #2, please remove the newly added material called `performance of AIDPS in randomly split cohorts', as this adds nothing relevant (as it s again testing on discovery data). Please take out all mentioning of `validation' or `validated' from the manuscript, other than when discussing the tests on the three new data sets GSE21501, GSE57495, GSE71729; only the latter can be called validation sets. Please compare the performance of existing signatures to that of the authors' own signature on the three new data sets GSE21501, GSE57495, GSE71729.The main reason for this suggestion is that the revised manuscript still tests their own signature on discovery data but that of the competitors on unseen data, which is not a fair comparison. Unless the authors remedy this they should (i) remove all mentioning of superior performance of their signature (since the comparison did not involve a level paying field) and (ii) remove all mentioning of `validation' other than in relation to the three new data sets.3. Please include (4. Current research progress on these nine AIDPS genes) in the rebuttal, which is a summary of what is known about the biological function of the nine genes, as an Appendix or Supplementary information in the manuscript.Reviewer #1 (Recommendations for the authors):The authors have largely addressed the concerns raised in my previous review. As best I can tell, they have not yet shown how a simple ML model (such as linear regression or xgboost) performs on the same task when only fed with the 9 aidps features simultaneously. The improvements to figure 7 meant to address my request seem to all focus on treating the aidps features separately, so it useful to know they perform poorly when separate\u2026 what about when they are considered together as a nine dimensional feature vector in one model?Reviewer #2 (Recommendations for the authors):In my first report I put forward two points. First, that the authors used the same data sets both in the discovery stage and the validation stage. Second, that they compared the performance of competitor signatures to theirs by using these same data. This has the following consequences: 1. They could not claim that their signature had been validated, because what they called validation data were in fact the discovery data . 2. The comparison between their signature and the existing ones was not fair . Therefore, all claims on their signature having been validated and on it being superior to existing signatures were not supported by the available evidence. I also suggested that they could test for overfitting impact by repeating their previous analyses step by step with outcome-randomized versions of the original data. This should make all true signals disappear and is hence a clean and fair test.In the revised version of their manuscript the authors did not carry out the requested re-analysis with outcome-randomized data. Instead they (a) sampled several new data sets from their original data (without outcome randomization), and (b) tested their signature on three independent new data sets. Exercise (a) seems pointless; of course the signature would again be able to predict, since one is testing it once more on samples from the discovery set. Exercise (b), although not what was requested by this referee, is more useful. Here the authors report again significant performance of their signature, with AUC values in the range 0.68-0.70, lending weight to the suggestion that their signature makes sense.I conclude from the authors' reply and from the new version of the paper that point 1. above has been partly addressed, but not point 2. If the authors, as it seems, do not wish to carry out the requested analysis with outcome randomization, then they have the following options:Option A;\u2013 Remove the newly added material called `performance of AIDPS in randomly split cohorts', this adds nothing relevant .\u2013 Take out all mentioning of `validation' or `validated' from the manuscript, other than when discussing the tests on the three new data sets GSE21501, GSE57495, GSE71729; only the latter can be called validation sets.\u2013 Remove all performance comparisons between the new signature and the existing ones (since this comparison is presently not fair) and all conclusions based on these comparisons.Option B:\u2013 Remove the newly added material called `performance of AIDPS in randomly split cohorts', this adds nothing relevant .\u2013 Take out all mentioning of `validation' or `validated' from the manuscript, other than when discussing the tests on the three new data sets GSE21501, GSE57495, GSE71729; only the latter can be called validation sets.\u2013 Compare the performance of existing signatures to that of their own signature on the three new data sets GSE21501, GSE57495, GSE71729; this comparison would be fair.I would personally strongly suggest option B, since otherwise the paper would in my view be too weak in terms of the amount of justified conclusions to warrant publication (but that is a decision for the editor to make). In addition, I would still hope that the authors choose to comply with my original request for application of the full pipeline to outcome-randomized discovery data. This would clarify objectively, for the authors' own benefit, the extent to which their original tests and conclusions may have been affected by overfitting.Reviewer #3 (Recommendations for the authors):The authors have addressed my comments. I find the following v useful as a summary of what is known about the biological function of the nine genes and wonder if it can be incorporated as an Appendix or Supplementary information:\"The nine AIDPS genes were rarely studied in tumors, most of which focused on their roles in biological phenotypes such as tumorigenesis and progression, and few studies investigated their roles and guiding significance in PACA immune microenvironment and treatment. Specifically, we found that SELENBP1 owned lower expression in the low AIDPS group with poor prognosis was associated with better tumor prognosis, and its decreased expression resulted in poor prognosis of many tumors such as lung adenocarcinoma. Meanwhile, DCBLD2, PRR11, EREG, ADM and TGM2, which were overexpressed in the low AIDPS group, played an essential role in the proliferation, invasion and metastasis of many tumors such as PACA, hepatocellular carcinoma and colorectal cancer, and lead to their chemotherapy resistance and were potential targets for enhanced efficacy. However, the functional roles and mechanisms of UNC13D and CDCA4 in tumors are not well understood, and more high-quality studies are needed in the future. These findings are consistent with our study, and the specific functional characteristics are summarized as follows:1. SELENBP1, encoding a novel human methanethiol oxidase, whose expression is significantly decreased in the progression of Barrett's esophagus to adenocarcinoma and can affect chemotherapy sensitivity (PMID: 20332323). This is consistent with our results that SELENBP1 expression was decreased in the low AIDPS group with worse prognosis .2. PLCB4 encodes a phospholipase, and activating PLCB4 mutation was recently identified in primary leptomeningeal melanocytic tumors (PMID: 28499758).3. DCBLD2 is upregulated in glioblastoma and head and neck cancer, and phosphorylation of DCBLD2 Y750 activates AKT pathway which in turn enhances EGFR-driven tumorigenesis (PMID: 25061874). DCBLD2 has also been found to play a crucial role in the invasion, progression, and metastasis of neuroendocrine tumors (PMID: 18827820). These results are consistent with an increased expression of DCBLD2 in the low AIDPS group with a dismal prognosis.4. Proline-rich protein 11 (PRR11) is a novel tumor-related gene, which has been found can promote breast cancer growth and anti-estrogen resistance through the PI3K pathway (PMID: 33127913). In addition, it has also been demonstrated that PRR11 affects autophagy and promotes proliferation in non-small cell lung cancer (NSCLC) through the Akt/mTOR pathway , and recent studies have also found that PRR11 can promote the tumorigenesis and progression of renal clear cell carcinoma (ccRCC) by regulating E2F1 stability (PMID: 34499617). Overall, these results are consistent with higher expression of PRR11 in the low AIDPS group with a worse prognosis.5. UNC13D mutation play an essential role in familial hemophagocytic lymphohistiocytosis (FHL), but its role in tumors remains unknown and needs to be further explored .6. EREG, as an EGFR ligand epithelial regulatory protein, plays a fundamental role in the growth and proliferation of breast and colorectal cancers  and is associated with tamoxifen resistance in breast cancer (PMID: 30967627) and efficacy of panitumumab and adjuvant chemotherapy in colorectal cancer .7. Adrenomedullin (ADM) encodes a pre-prohormone that is cleaved into two bioactive peptides, which can dilate blood vessels, regulate hormone secretion, and promoting angiogenesis. Many studies have confirmed that ADM plays a major role in the proliferation, invasion, angiogenesis, and metastasis of PACA, hepatocellular carcinoma, prostate cancer, melanoma, ovarian cancer, and endometrial cancer and may become a novel therapeutic target .8. In addition, currently one of the few studies support the oncogenic role of cell division cycle associated protein 4 (CDCA4) in tumors and nuclear factors induced as E2F transcription factor families that regulate E2F-dependent transcriptional activation and cell proliferation, but their potential mechanism, especially effect on PACA tumorigenesis and progression require further investigation .9. Transglutaminase 2 (TGM2), a promoter of stemness and radiotherapy resistance in glioma (PMID: 33483373), has been confirmed in many studies to promote colorectal cancer progression via P53 and Wnt/\u03b2-catenin signaling pathways , as well as is associated with tumor-promoting inflammation in gastric cancer (PMID: 32467608). These results are consistent with the TGM2 expression dramatically increased in the low AIDPS group with worse prognosis, suggesting that TGM2 may play an important role in the development and progression of PACA.\" Essential revisions:1. The authors should repeat their complete pipeline after randomizing within each of their ten data sets the clinical outcomes (i.e. shuffle the time-to-event outcomes). If one would obtain again a signature with comparable prognostic features, then one can be sure that what is reported here is largely a manifestation of overfitting due to dimension mismatch, and hence of no interest.Thank you and reviewer 2 for your insightful recommendations. In response to this problem, we supplement the contents of Figure 3\u2014figure supplement 4-5, and detailed explanations are given in lines 584-637 of this author\u2019s response. The results from 22 randomized split validation cohorts and three external test cohorts consistently confirmed that AIDPS can robustly predict the prognosis of pancreatic cancer (PACA) patients. Thanks again for your advice, which we are sure makes our validation of AIDPS more rigorous, reliable and meaningful.2. The authors should provide a better understanding of why agreement/optimization amongst a large number of combined ML methods was chosen as a way to drive feature selection. Comparing to existing methods is one thing, currently, there isn't really a clear comparison here to other feature selection approaches.Thank you and reviewer 1 for your questions. As to why we choose agreement/optimization as our feature selection method, we conducted in-depth thinking and careful investigation again, and made a detailed and rigorous discussion. Specific responses are presented in the recommendation section of reviewer 1 in lines 120-213.3. The authors should provide a thorough explanation of the biological relevance of the 9 genes in relation to the mutational load/copy number changes/ immunological infiltration found in low AIDPS patients.Thank you and reviewer 3 for your recommendations. As you mentioned, our study explored the genomic landscape such as mutation and copy number alteration (CNA) of the 9-gene AIDPS and their impact on the tumor microenvironment (TME), focusing on its guiding value for decision-making and clinical management in PACA patients, while ignoring the biological functions of these nine genes as independent individuals. To compensate for this defect, we supplemented two parts, Figure 7\u2014figure supplement 2-3, to explore the biological functions of AIDPS and its nine genes related to mutation, CNA, and immune infiltration in four aspects. A detailed response to this section can be found in the recommendation section of reviewer 3 in lines 644-799.Reviewer #1 (Recommendations for the authors):Things I think would strengthen the paper:1. A better understanding of why agreement/optimization amongst a large number of combined ML methods was chosen as a way to drive feature selection. Comparing to existing methods is one thing, but there isn't really a clear comparison here to other feature selection approaches, nor much discussion of why this one was chosen. Is this meant to be a reproducible approach, for other problems, or is the point just to get the AIDPS features? Why is this approach to feature selection preferred/superior.With advancements in high-throughput sequencing techniques and computational biology, numerous predictive signatures have been proposed according to various machine-learning approaches. However, two questions worth considering are why a particular algorithm should be selected and which solution is the optimal one. The selection of algorithms by researchers may rely largely on their own preferences and bias. Thus, to generate a consensus signature, we collected 10 prevalent algorithms and then combined them into 76 combinations. As to why we use agreement/optimization as a feature selection method, we will next answer from the following five aspects:1. Why perform feature selection?For clinical prediction model development, the 10 events per variable (10 EPV) rule recommends that feature variables should be included according to the number of positive events, which in general requires more than 10-fold higher number of positive events than the independent variable included . According to the 10 EPV rule, 1280 samples from our ten cohorts contained 772 positive events (OS of 1), which means that the independent variables included in our study at the beginning should be less than 77. However, given that there are 15,288 intersection genes in the ten cohorts, we need to conduct the first stage of variable screening for the initial covariates that are severely mismatched with the sample size. There are two commonly used variable screening methods: one is differential expression analysis of tumor and normal samples, and it is believed that differentially expressed variables may play a crucial role in the tumorigenesis and progression , and the other is univariate Cox regression analysis of survival variables, and variables with independent prognostic significance are selected for subsequent analysis  ; and even some studies combine the two methods . Because the ten cohorts lacked normal samples , univariate Cox regression analysis was used for variable screening in this study.2. Why choose consistent prognostic genes (agreement)?It is well-known that the widespread tumor heterogeneity caused by different genetic background, pathological type, differentiation state and even living environment poses a great challenge to treatment and is also an urgent problem to be solved in precision medicine. Because of spatiotemporal heterogeneity, the same gene may have different prognostic performance in different cohorts, for example, A1BG was a protective factor in the TCGA-PAAD and GSE79668, and had no significant prognostic value in most cohorts, but it was indeed a risk factor in the GSE71729. Therefore, in order to obtain more stable prognostic genes, drawing on previous experience , we obtained 32 consensus prognostic genes in the first stage for subsequent machine-learning modeling according to the screening criteria of consistent prognostic significance in at least 8/10 cohorts.3. Why build machine-learning algorithm combinations?Based on the 32 consensus prognostic genes obtained from the appeal step, we selected the PACA-AU-Array with larger sample size as the training set to construct the consensus model. However, according to the 10 EPV rule, the PACA-AU-Array  allowed up to 16 variables to be included for subsequent model construction. To solve this problem, among the ten machine-learning algorithms we collected, RSF, LASSO, CoxBoost and Stepwise Cox have the function of dimensionality reduction and variable screening. Therefore, we combined them with other algorithms to make the model more simplified and more conducive to clinical translation.4. Why choose the largest average C-index (optimization)?The pros and cons of a model lies in whether it can maintain good predictive ability in the validation sets, that is, it has strong generalization ability. In existing studies, the concordance index  and the time-dependent ROC curve  are the most common and widely recognized indicators for evaluating the predictive performance of machine-learning survival models. Among them, C-index can be used to judge the accuracy and discrimination ability for global prediction, and the performance is relatively stable. While the area under the timeROC curve (AUC) depends on the time node, reflecting the prediction accuracy at a certain moment, and the overall performance is not very stable. Therefore, we chose C-index to evaluate the predictive performance of the 76 algorithm combinations.In addition, the aim of our study was to obtain a predictive model with strong generalization ability that could maintain excellent performance in each validation cohort and have a good likelihood of clinical translation. Some models, such as random survival forest (RSF) model, performed extremely well on the PACA-AU-Array training set (C-index = 0.914), but it performed very poorly on the nine validation sets . This is mainly because some algorithms are easy to overfit in the training set. In this situation, we thus cannot say that RSF is the optimal model. Therefore, in order to avoid the impact of model overfitting as much as possible, following the previous workflow , we chose the largest average C-index of nine validation sets as our feature selection method.5. From the results, how is the effect of our feature selection approach?In summary, based on the feature selection criteria of consistent prognostic value (agreement) and the largest average C-index (optimization), we combined CoxBoost and Survival-SVM to construct a 9-gene AIDPS. In nine validation sets, in addition to owning an average C-index of 0.675, AIDPS also exhibited robust and superior predictive capability  in another recognized indicators, timeROC, and outperformed the existing 86 gene expression signatures. Meanwhile, the composition of 9-gene AIDPS is relatively simple, and it is more likely to construct a multigene panel for clinical translation. In addition, as recommended by reviewer 2, we randomly split the Meta-Cohort into 22 validation cohorts (from Cohort_100 to Cohort_1150) and again thoroughly collected three external test cohorts, including GSE21501 (n = 102), GSE57495 (n = 63) and GSE71729 (n = 125). The univariate Cox regression analysis, Kaplan-Meier survival curves, AUCs of timeROC curves, C-index, and calibration curves consistently demonstrated robust and good predictive performance of AIDPS . Therefore, based on the above results, we can preliminarily illustrate that AIDPS generated by our machine-learning survival framework has great potential for clinical application in PACA patients with insidious onset and poor prognosis.2. Right now the current pipeline seems highly unwieldy because of all the models considered. Is this meant to be a general and reproducible approach? if so, the authors need to demonstrate more clearly that this same approach can be done by others with ease.Thank you for your questions. In existing predictive models, researchers mostly chose the modelling algorithms based on their knowledge limitations and preferences, and it is worth thinking about which method is more suitable or which solution is optimal. Therefore, to construct a consensus model with robust predictive performance and stronger generalization ability and extrapolation possibilities, we collected ten classical machine-learning algorithms, among which RSF, LASSO, CoxBoost and Stepwise Cox have the functions of dimensionality reducing and variable screening. Given that our training set has only 161 positive events and a maximum of 16 variables can be accommodated for subsequent model construction according to the 10 EPV rule, we combined these four algorithms with other algorithms to generate 76 combinations to make the model more simplified and more conductive to clinical translation. This does make our pipeline seem unwieldy, but at present this method has better performance and application prospects, and the same pipeline has also been applied to the following high-level studies:(1) In February 2022, our team published an article entitled: \u201cMachine learning-based integration develops an immune-derived lncRNA signature for improving outcomes in colorectal cancer\u201d in Nature Communications (PMID: 35145098). In this study, based on 43 immune-related lncRNAs with consistent prognostic significance obtained by univariate Cox regression analysis, we combined ten machine-learning algorithms into 101 algorithm combinations and demonstrated that the model constructed by Lasso combined with stepwise Cox (direction = both) possessed the best predictive performance in a training set and six validation sets (average C-index of 0.696). In addition, we confirmed that the high-risk group was sensitive to fluorouracil-based adjuvant chemotherapy, whereas the low-risk group showed more abundant immune cells infiltration and was more sensitive to pembrolizumab.(2) In January 2022, our team published another article entitled: \u201cIntegrative analysis from multi-center studies identities a consensus machine learning-derived lncRNA signature for stage II/III colorectal cancer\u201d in EBioMedicine (PMID: 34922323). In this study, based on 27 stable recurrence-related lncRNAs, we combined ten machine-learning algorithms into 76 algorithm combinations and demonstrated that CMDLncS generated by Lasso combined with stepwise Cox could accurately detect recurrence in patients with II/III colorectal cancer (average C-index of 0.777). In addition, patients in the high CMDLncS group were likely to benefit from fluorouracil-based adjuvant chemotherapy, whereas the low CMDLncS group showed greater sensitivity to bevacizumab.(3) Recently, Nan Zhang et al., from Xiangya Hospital of Central South University, published the other article entitled \u201cMachine learning-based identification of tumor-infiltrating immune cell-associated lncRNAs for improving outcomes and immunotherapy responses in patients with low-grade glioma\u201d in Theranostics (PMID: 35966587). The authors identified 136 tumor-infiltrating immune cell-associated lncRNA (TIIClncRNA) of low-grade glioma (LGG) by comprehensive analyzing the sequencing data of purified immune cells, LGG cell lines, and bulk LGG tissues. After that, based on 46 prognostic TIIClncRNAs obtained by univariate Cox regression analysis, the authors combined ten machine-learning algorithms into 101 algorithm combinations, and picked out TIIClnc signature that derived by combining RSF and CoxBoost algorithms using the largest mean C-index (0.744) in the TCGA training set, Xiangya in-house validating set, and two external validating sets. In addition, the authors found that TIIClnc signature was significantly associated with the expression of CD8, PD-1, and PD-L1, as well as immune characteristics such as microsatellite instability, tumor mutation burden, interferon \u03b3. Results from multiple datasets demonstrate that TIIClnc signature accurately predicts superior immunotherapy response across cancer types.In addition, the complete flow and essential scripts of our pipeline has also been published in the GitHub website (https://github.com/Zaoqu-Liu/IRLS). To our knowledge, some people have been applying our pipeline in solid tumors such as melanoma, bladder cancer, hepatocellular carcinoma, gastric cancer and have achieved good results. For PACA, our AIDPS model has also been uploaded to the corresponding catalogue (https://github.com/Zaoqu-Liu/AIDPS), and the researchers entered the expression matrix of these nine AIDPS genes to obtain patient-specific risk score. Thanks again for your questions, which let us think again about the extensibility and generalizability of our pipeline.3. Everything in the subsequent analyses comparing high and low AIDPS groups hinges on the assumed accuracy and generality of the model. Once you have 9 genes, though, you should be able to study those features using a range of methods, some of which are picked to have high interpretability. I am sorry if I missed this, but at the moment it is not clear to me what best an ML method can do is using the 9 genes, and whether there is an interpretable method that works well but might imply a non-trivial relationship amongst the genes that matters to the outcome. This seems more informative than studying the statistics of high and low AIDPS groupsThanks for your questions and suggestions. As you said, once we have these nine AIDPS genes, we can conduct a series of studies. Therefore, we supplemented Figure 7\u2014figure supplement 1-3 to further explore the relationship between AIDPS and nine AIDPS genes and prognosis, immune cells infiltration and immune checkpoint molecules (ICMs) expression. Next, we will explain our findings in following four sections:1. Prognostic performance of AIDPS and nine AIDPS genesOur previous results consistently indicated that AIDPS could accurately predict the prognosis of PACA patients . According to your advice, based on large multi-center data from the training set, nine validation sets, and three newly collected external test sets, we performed an integrated analysis of survival variables using AIDPS and nine AIDPS genes as continuous variables. As shown in Figure 7\u2014figure supplement 1A, AIDPS was an independent protective factor in all cohorts . Correspondingly, because the 32 consensus prognostic genes used to construct AIDPS with consistent prognostic value in at least 8/10 cohorts, nine AIDPS genes had relatively consistent prognostic performance in the ten cohorts . However, in the three external test sets, GSE21501, GSE57495 and GSE71729, the performance of nine AIDPS genes was hardly satisfactory. Overall, compared with the nine independent AIDPS genes, our AIDPS constructed by CoxBoost and Survival-SVM brings significant performance improvement, and its simple composition and robust performance also have greater possibility of clinical translation and extrapolation.2. Correlation between AIDPS and its nine genes at expression levelTo describe the biological relevance between AIDPS and its nine genes, we performed Pearson correlation analysis referring to previous studies . As shown in Figure 7\u2014figure supplement 2A, SELENBP1 and PLCB4 had strong positive correlations with AIDPS (R = 0.46 and 0.57), while DCBLD2, PRR11, UNC13D, EREG, ADM, CDCA4, and TGM2 had very strong negative correlations with AIDPS (R from -0.57 to -0.75) in the whole TCGA-PAAD. Moreover, there was a positive correlation between the two genes positively correlated with AIDPS (R = 0.23). Similarly, there were also positive correlation between the seven genes negatively correlated with AIDPS (R from 0.19 to 0.59), while there were moderate negative correlation between the two genes positively correlated AIDPS and the seven genes negatively correlated AIDPS (R from 0 to -0.55). Based on the rationale of weighted gene co-expression network analysis : genes with similar expression patterns may be co-regulated, functionally correlated or in the same pathway. The strong correlation within the nine genes explains the rationality of our AIDPS from the perspective of biological function to some extent.In addition, to investigate the changes of the nine AIDPS genes with AIDPS changes, we performed Pearson correlation analysis again in the high and low AIDPS TCGA-PAAD, respectively, and the low AIDPS group exhibited a consistent trend with the whole TCGA-PAAD . Interestingly, the overall correlation was lower in the high AIDPS group and numerous genes showed different or even opposite trends to the whole and the low AIDPS group , suggesting that AIDPS may have stronger biological relevance and potential function in PACA patients with low AIDPS .3. Relationship between nine AIDPS genes and immune molecules and immune cellsGiven the obvious impact of AIDPS in the TME and its guiding significance for immunotherapy, we compared the relationship of AIDPS and its nine genes with 27 ICMs and 28 immune cells in the whole, high AIDPS, and low AIDPS TCGA-PAAD. The results again corroborate our previous finding that the low AIDPS group had higher immune cells infiltration and ICMs expression . In the whole TCGA-PAAD, AIDPS showed a prominent negative correlation with ICMs and immune cells, and SELENBP1 and PLCB4, which were positively correlated with AIDPS, also exhibited a corresponding trend. However, DCBLD2, PRR11, UNC13D, EREG, and TGM2, which were negatively correlated with AIDPS, had a significant positive correlation with ICMs and immune cells . The high AIDPS group also showed the similar results as the whole cohort . Interestingly, in the low AIDPS group, AIDPS showed an observably positive correlation with ICMs and immune cells different from the whole and high AIDPS cohort. Meanwhile, PLCB4, which was positively correlated with AIDPS, showed a similar trend, while DCBLD2, PRR11, UNC13D, EREG, ADM, CDCA4 and TGM2, which were negatively correlated with AIDPS, were negatively correlated with ICMs and immune cells .4. Current research progress on these nine AIDPS genesAfter careful literature research, we found that SELENBP1 that owned lower expression in the low AIDPS group with poor prognosis was associated with better tumor prognosis, and its decreased expression led to poor prognosis of many tumors such as lung adenocarcinoma. Meanwhile, DCBLD2, PRR11, EREG, ADM and TGM2, which were overexpressed in the low AIDPS group, played a vital role in the proliferation, invasion and metastasis of many tumors such as PACA, hepatocellular carcinoma and colorectal cancer, and lead to their chemotherapy resistance and were potential targets for enhanced efficacy. However, the functional roles and mechanisms of UNC13D and CDCA4 in tumors are not well understood, and more high-quality studies are needed in the future. These findings are consistent with our study. The specific functional characteristics of these nine AIDPS genes are summarized as follows:(1) SELENBP1, encoding a novel human methanethiol oxidase, whose expression is significantly decreased in the progression of Barrett\u2019s esophagus to adenocarcinoma and can affect chemotherapy sensitivity (PMID: 20332323). This is consistent with our results that SELENBP1 expression was decreased in the low AIDPS group with worse prognosis .(2) PLCB4 encodes a phospholipase, and activating PLCB4 mutation was recently identified in primary leptomeningeal melanocytic tumors (PMID: 28499758).(3) DCBLD2 is upregulated in glioblastoma and head and neck cancer, and phosphorylation of DCBLD2 Y750 activates AKT pathway which in turn enhances EGFR-driven tumorigenesis (PMID: 25061874). DCBLD2 has also been found to play a crucial role in the invasion, progression, and metastasis of neuroendocrine tumors (PMID: 18827820). These results are consistent with an increased expression of DCBLD2 in the low AIDPS group with a dismal prognosis.(4) Proline-rich protein 11 (PRR11) is a novel tumor-related gene, which has been found can promote breast cancer growth and anti-estrogen resistance through the PI3K pathway (PMID: 33127913). In addition, it has also been demonstrated that PRR11 affects autophagy and promotes proliferation in non-small cell lung cancer (NSCLC) through the Akt/mTOR pathway , and recent studies have also found that PRR11 can promote the tumorigenesis and progression of renal clear cell carcinoma (ccRCC) by regulating E2F1 stability (PMID: 34499617). Overall, these results are consistent with higher expression of PRR11 in the low AIDPS group with a worse prognosis.(5) UNC13D mutation play an essential role in familial hemophagocytic lymphohistiocytosis (FHL), but its role in tumors remains unknown and needs to be further explored .(6) EREG, as an EGFR ligand epithelial regulatory protein, plays a fundamental role in the growth and proliferation of breast and colorectal cancers  and is associated with tamoxifen resistance in breast cancer (PMID: 30967627) and efficacy of panitumumab and adjuvant chemotherapy in colorectal cancer .(7) Adrenomedullin (ADM) encodes a pre-prohormone that is cleaved into two bioactive peptides, which can dilate blood vessels, regulate hormone secretion, and promoting angiogenesis. Many studies have confirmed that ADM plays a major role in the proliferation, invasion, angiogenesis, and metastasis of PACA, hepatocellular carcinoma, prostate cancer, melanoma, ovarian cancer, and endometrial cancer and may become a novel therapeutic target .(8) In addition, currently one of the few studies support the oncogenic role of cell division cycle associated protein 4 (CDCA4) in tumors and nuclear factors induced as E2F transcription factor families that regulate E2F-dependent transcriptional activation and cell proliferation, but their potential mechanism, especially effect on PACA tumorigenesis and progression require further investigation .(9) Transglutaminase 2 (TGM2), a promoter of stemness and radiotherapy resistance in glioma (PMID: 33483373), has been confirmed in many studies to promote colorectal cancer progression via P53 and Wnt/\u03b2-catenin signaling pathways , as well as is associated with tumor-promoting inflammation in gastric cancer (PMID: 32467608). These results are consistent with the TGM2 expression dramatically increased in the low AIDPS group with worse prognosis, suggesting that TGM2 may play an important role in the development and progression of PACA.In summary, our study demonstrated that AIDPS outperformed nine AIDPS genes alone in prediction performance, so we chose AIDPS instead of the nine genes alone for subsequent analysis. The biological relevance studies exhibited that AIDPS and its nine genes had inconsistent or even opposite performances in the high and low AIDPS groups, suggesting that the high and low AIDPS groups owned relatively strong biological heterogeneity. In addition, our literature survey found that these nine AIDPS genes were rarely studied in tumors, and most of these focus on their roles in biological phenotypes such as tumorigenesis and progression, and few studies investigated their roles and guiding significance in PACA immune microenvironment and treatment. Meanwhile, our study showed that the high and low AIDPS groups had observably different prognosis, functional characteristics, immune infiltration, genomic variant landscape, and treatment response. Moreover, stratifying PACA patients into high and low AIDPS groups for individualized treatment and clinical management is also in line with the concept of precise treatment. Therefore, in this study, we focused on the high and low AIDPS groups. Relevant contents have been added and marked in red in the corresponding section of the revised manuscript. Thanks again for your questions and suggestions, and we are sure that your suggestions have enriched and refined our research.Reviewer #2 (Recommendations for the authors):To rescue their claims one could suggest to the authors the following simple but clean and fair test. The authors could repeat their complete pipeline after randomizing within each of their ten data sets the clinical outcomes (i.e. shuffle the time-to-event outcomes). If that exercise is found to make the signal disappear , then their present results should be studied further as possibly relevant. If, on the contrary, one would obtain again a signature with comparable prognostic features, then one can be sure that what is reported here is largely a manifestation of overfitting due to dimension mismatch, and hence of no interest.Thank you for your advice. During the construction of AIDPS, in order to eliminate the heterogeneity between different cohorts as much as possible and obtain relatively stable prognostic genes in PACA, we performed univariate Cox regression analysis and identified 32 consensus prognostic genes according to the following criteria of consistent prognostic significance in at least 8/10 cohorts. Afterwards, we selected the PACA-AU-Array with larger sample size as the training set and validated the model using the remaining nine cohorts and Meta-Cohort, and confirmed that AIDPS has robust and good predictive performance. In doing so, as you are concerned, there is indeed the possibility of applying validation sets to train the model and resulting in a lack of rigor in the validation results. But we did this to make more adequate use of the 10 PACA cohorts to obtain a robust model. In a sense, if we also use the genes of a specific biological pathway for modeling according to popular practice, then our model will be like the collected 86 signatures and is hard to generalize in other cohorts or populations, and unable to further clinical translation and serve the clinical management and precision treatment of PACA patients. To rescue our claims, as you recommended, we supplemented the contents of Figure 3\u2014figure supplement 4-5 to further test the performance of AIDPS. The details are as follows:1. Performance of AIDPS in randomly split cohortsFollowing your recommendations, we used the sample function of R software to perform non-return random sampling from Meta-Cohort synthesized from 10 PACA cohorts, and 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, and 1150 samples were successively selected to form a new cohort. Kaplan-Meier survival analysis, univariate Cox regression analysis, timeROC, and C-index were used to measure the performance of AIDPS in these cohorts. The Figure 3\u2014figure supplement 4 is presented from left to right as follows: the results of Kaplan-Meier survival analysis showed that the survival curves of patients in the high and low AIDPS groups were remarkably separated ; univariate Cox regression analysis also exhibited that AIDPS was an independent protective factor in these cohorts (P <0.001 in the Cohort_100 and <0.0001 in the other cohorts); the timeROC curves for 1-, 2-, 3-, 4-, and 5-years OS also showed that AIDPS owned AUCs greater than 0.7 in the vast majority of cohorts, and at least greater than 0.65 in the relatively poor Cohort_100, Cohort_200, and Cohort_300; plus C-index greater than 0.65 in all cohorts, suggesting that our AIDPS can accurately predict OS in PACA patients.2. Performance of AIDPS in three additional independent test cohortsFurthermore, to more objectively test the performance of AIDPS, in addition to the training and validation sets mentioned earlier, we again screened and searched for three external test cohorts, GSE21501 (n = 102), GSE57495 (n = 63), and GSE71729 (n = 125). The results of univariate Cox regression analysis showed that AIDPS was an independent protective factor in all three cohorts . Kaplan-Meier survival analysis also exhibited that patients in the low AIDPS group owned a significantly shorter OS . The AUCs of 1-, 2-, and 3-year OS were 0.677, 0.681, and 0.761 in the GSE21501; 0.682, 0.728, and 0.747 in the GSE57495; 0.676, 0.693, and 0.714 in the GSE71729 . In addition, the calibration curves of these three external test cohorts also confirmed the good predictive performance of AIDPS .Overall, the results of the above randomized split validation cohorts and three external test cohorts consistently confirmed that AIDPS can robustly predict the prognosis of PACA patients. Thanks again for your suggestions, and we are sure that your suggestions makes our verification more rigorous, reliable and meaningful.Reviewer #3 (Recommendation for the authors):From the biological point of view, currently, there was little explanation of the biological relevance of the 9 genes in relation to the mutational load/copy number changes/ immunological infiltration found in low AIDPS patients. Please discuss these in sufficient detail.Thank you for your recommendations. As you mentioned, our study explored the genomic landscape such as mutation and copy number alteration (CNA) of the 9-gene AIDPS and their impact on the tumor microenvironment (TME), focusing on its guiding value for decision-making and clinical management in PACA patients, while ignoring the biological functions of these nine genes as independent individuals. To compensate for this defect, we supplemented the correlation analysis between AIDPS and nine AIDPS genes in the whole, high AIDPS, and low AIDPS TCGA-PAAD. Considering the outstanding different TME between the high and low AIDPS groups, we added a correlation analysis of AIDPS and its nine genes with 27 immune checkpoint molecules (ICMs) and 28 immune cells obtained by ssGSEA in the whole, high AIDPS, and low AIDPS TCGA-PAAD. In addition, to describe the overall relationship between AIDPS and its nine genes and mutation/CNA in PACA patients, we added expression information of these genes in the process of Figure 6A to generate Figure 7\u2014figure supplement 3. Next, we will explore the biological relevance between AIDPS and its nine genes with mutation, CNA, and immune infiltration from the following four aspects:1. Correlation between AIDPS and its nine genes at expression levelTo describe the biological relevance between AIDPS and its nine genes, we first performed Pearson correlation analysis referring to previous studies . As shown in Figure 7\u2014figure supplement 2A, SELENBP1 and PLCB4 had strong positive correlations with AIDPS (R = 0.46 and 0.57), while DCBLD2, PRR11, UNC13D, EREG, ADM, CDCA4, and TGM2 had very strong negative correlations with AIDPS (R from -0.57 to -0.75) in the whole TCGA-PAAD. Moreover, there was a positive correlation between the two genes positively correlated with AIDPS (R = 0.23). Similarly, there were also positive correlation between the seven genes negatively correlated with AIDPS (R from 0.19 to 0.59), while there were moderate negative correlation between the two genes positively correlated AIDPS and the seven genes negatively correlated AIDPS (R from 0 to -0.55). Based on the rationale of weighted gene co-expression network analysis : genes with similar expression patterns may be co-regulated, functionally correlated or in the same pathway. The strong correlation within the nine genes explains the rationality of our AIDPS from the perspective of biological function to some extent.In addition, to investigate the changes of the nine AIDPS genes with AIDPS changes, we performed Pearson correlation analysis again in the high and low AIDPS TCGA-PAAD, respectively, and the low AIDPS group exhibited a consistent trend with the whole TCGA-PAAD . Interestingly, the overall correlation was lower in the high AIDPS group and numerous genes showed different or even opposite trends to the whole and the low AIDPS group , suggesting that AIDPS may have stronger biological relevance and potential function in PACA patients with low AIDPS .2. Relationship between nine AIDPS genes and immune molecules and immune cellsGiven the obvious impact of AIDPS in the TME and its guiding significance for immunotherapy, we compared the relationship of AIDPS and its nine genes with 27 ICMs and 28 immune cells in the whole, high AIDPS, and low AIDPS TCGA-PAAD. The results again corroborate our previous finding that the low AIDPS group had superior immune cells infiltration and ICMs expression . In the whole TCGA-PAAD, AIDPS showed a prominent negative correlation with ICMs and immune cells, and SELENBP1 and PLCB4, which were positively correlated with AIDPS, also exhibited a corresponding trend. However, DCBLD2, PRR11, UNC13D, EREG, and TGM2, which were negatively correlated with AIDPS, had a significant positive correlation with ICMs and immune cells . The high AIDPS group also showed the similar results as the whole cohort . Interestingly, the tread in the low AIDPS group was contrary, AIDPS and PLCB4 were observably positively correlated with ICMs and immune cells. Meanwhile, DCBLD2, PRR11, UNC13D, EREG, ADM, CDCA4 and TGM2, which were negatively correlated with AIDPS, were negatively correlated with ICMs and immune cells .The disparate result of AIDPS and its nine genes in the high and low AIDPS groups indicated that these AIDPS genes may be closely related to the immune regulatory pathway, which may play a crucial role in anti-tumor immune and immunotherapy. This also provides a rationale to some extent that AIDPS can accurately predict immunotherapy response in PACA patients.3. Relevance of nine AIDPS genes with mutation and copy number alterationOur previous study found that the low AIDPS group owned outstandingly higher genomic alterations such as tumor mutation burden (TMB) and CNA load, which could provide more neoantigens for anti-tumor immune and immunotherapy and lead to better immunotherapeutic efficacy . As shown in Figure 7\u2014figure supplement 3, the proportion of top 15 gene mutation and CNA loci (frequency >20%) were obviously higher in the low AIDPS group. In addition, we also found that SELENBP1 and PLCB4, which were positively correlated with AIDPS, were significantly lower in the low AIDPS group, suggesting that they were significantly negatively correlated with TMB and CNA burden. Correspondingly, DCBLD2, PRR11, UNC13D, EREG, ADM, CDCA4, and TGM2, which were negatively correlated with AIDPS, was remarkably increased in the low AIDPS group, hinting they were significantly positively correlated with TMB and CNA burden .4. Current research progress on these nine AIDPS genesAfter careful literature research, we found that the nine AIDPS genes were rarely studied in tumors, most of which focused on their roles in biological phenotypes such as tumorigenesis and progression, and few studies investigated their roles and guiding significance in PACA immune microenvironment and treatment. Specifically, we found that SELENBP1 owned lower expression in the low AIDPS group with poor prognosis was associated with better tumor prognosis, and its decreased expression resulted in poor prognosis of many tumors such as lung adenocarcinoma. Meanwhile, DCBLD2, PRR11, EREG, ADM and TGM2, which were overexpressed in the low AIDPS group, played an essential role in the proliferation, invasion and metastasis of many tumors such as PACA, hepatocellular carcinoma and colorectal cancer, and lead to their chemotherapy resistance and were potential targets for enhanced efficacy. However, the functional roles and mechanisms of UNC13D and CDCA4 in tumors are not well understood, and more high-quality studies are needed in the future. These findings are consistent with our study, and the specific functional characteristics are summarized as follows:(1) SELENBP1, encoding a novel human methanethiol oxidase, whose expression is significantly decreased in the progression of Barrett\u2019s esophagus to adenocarcinoma and can affect chemotherapy sensitivity (PMID: 20332323). This is consistent with our results that SELENBP1 expression was decreased in the low AIDPS group with worse prognosis .(2) PLCB4 encodes a phospholipase, and activating PLCB4 mutation was recently identified in primary leptomeningeal melanocytic tumors (PMID: 28499758).(3) DCBLD2 is upregulated in glioblastoma and head and neck cancer, and phosphorylation of DCBLD2 Y750 activates AKT pathway which in turn enhances EGFR-driven tumorigenesis (PMID: 25061874). DCBLD2 has also been found to play a crucial role in the invasion, progression, and metastasis of neuroendocrine tumors (PMID: 18827820). These results are consistent with an increased expression of DCBLD2 in the low AIDPS group with a dismal prognosis.(4) Proline-rich protein 11 (PRR11) is a novel tumor-related gene, which has been found can promote breast cancer growth and anti-estrogen resistance through the PI3K pathway (PMID: 33127913). In addition, it has also been demonstrated that PRR11 affects autophagy and promotes proliferation in non-small cell lung cancer (NSCLC) through the Akt/mTOR pathway , and recent studies have also found that PRR11 can promote the tumorigenesis and progression of renal clear cell carcinoma (ccRCC) by regulating E2F1 stability (PMID: 34499617). Overall, these results are consistent with higher expression of PRR11 in the low AIDPS group with a worse prognosis.(5) UNC13D mutation play an essential role in familial hemophagocytic lymphohistiocytosis (FHL), but its role in tumors remains unknown and needs to be further explored .(6) EREG, as an EGFR ligand epithelial regulatory protein, plays a fundamental role in the growth and proliferation of breast and colorectal cancers  and is associated with tamoxifen resistance in breast cancer (PMID: 30967627) and efficacy of panitumumab and adjuvant chemotherapy in colorectal cancer .(7) Adrenomedullin (ADM) encodes a pre-prohormone that is cleaved into two bioactive peptides, which can dilate blood vessels, regulate hormone secretion, and promoting angiogenesis. Many studies have confirmed that ADM plays a major role in the proliferation, invasion, angiogenesis, and metastasis of PACA, hepatocellular carcinoma, prostate cancer, melanoma, ovarian cancer, and endometrial cancer and may become a novel therapeutic target .(8) In addition, currently one of the few studies support the oncogenic role of cell division cycle associated protein 4 (CDCA4) in tumors and nuclear factors induced as E2F transcription factor families that regulate E2F-dependent transcriptional activation and cell proliferation, but their potential mechanism, especially effect on PACA tumorigenesis and progression require further investigation .(9) Transglutaminase 2 (TGM2), a promoter of stemness and radiotherapy resistance in glioma (PMID: 33483373), has been confirmed in many studies to promote colorectal cancer progression via P53 and Wnt/\u03b2-catenin signaling pathways , as well as is associated with tumor-promoting inflammation in gastric cancer (PMID: 32467608). These results are consistent with the TGM2 expression dramatically increased in the low AIDPS group with worse prognosis, suggesting that TGM2 may play an important role in the development and progression of PACA.In conclusion, previous studies and our analysis results consistently indicate that the nine AIDPS genes are correlated with mutation, CNA, and TME in PACA. This also essentially supports the important impact of AIDPS on the prognosis and immune microenvironment, as well as its role in the clinical management and precision treatment of PACA. The relevant contents have been added and marked in red in the corresponding section of the revised manuscript. Thanks again for your valuable comments, and we are sure that your hard work has made our study more rigorous and meaningful.The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:1. The improvements to figure 7  meant to address Reviewer #1's request seem to all focus on treating the aidps features separately. Please show the accuracy when these aidps features are considered together as a nine dimensional feature vector in one model.Thank you and Reviewer #1 for your reminder. As you mentioned, in this revision, we supplemented the content of Figure 4\u2014figure supplement 1D, focusing on exploring the performance of this 9 AIDPS genes in other machine-learning algorithms. The results exhibited that our AIDPS model constructed by survival-SVM is the best choice for 9 AIDPS genes obtained after dimensionality reduction by CoxBoost from 32 consensus prognostic genes, which is also consistent with our previous pipeline results . Specific responses are presented in the recommendations section of Reviewer #1 in lines 69-88.2. For reviewer #2, please remove the newly added material called `performance of AIDPS in randomly split cohorts', as this adds nothing relevant (as it s again testing on discovery data). Please take out all mentioning of `validation' or `validated' from the manuscript, other than when discussing the tests on the three new data sets GSE21501, GSE57495, GSE71729; only the latter can be called validation sets. Please compare the performance of existing signatures to that of the authors' own signature on the three new data sets GSE21501, GSE57495, GSE71729.The main reason for this suggestion is that the revised manuscript still tests their own signature on discovery data but that of the competitors on unseen data, which is not a fair comparison. Unless the authors remedy this they should (i) remove all mentioning of superior performance of their signature (since the comparison did not involve a level paying field) and (ii) remove all mentioning of `validation' other than in relation to the three new data sets.Thank you and Reviewer #2 for your suggestions. Following the Option B of Reviewer #2\u2019s recommendations, we have removed all contents related to the \u201crandomly split cohorts\u201d added in the first revision. In addition, in our latest manuscript, we refer to the PACA-AU-Array as the training cohort, the remaining nine cohorts involved in screening consensus prognostic genes as the testing cohorts, while the three external datasets GSE21501, GSE57495, and GSE71729 are called the validation cohorts. Moreover, in this revised manuscript, we checked our wording again and used the \u201cvalidated\u201d and \u201cvalidation\u201d only in these three validation cohorts. Furthermore, to remedy our claims, we compared the performance of our AIDPS with 86 published signatures in the three new validation cohorts GSE21501, GSE57495 and GSE71729, and again demonstrated our AIDPS stability and superior performance . A detailed response to this section can be found in the recommendations section of Reviewer #2 in lines 151-190.3. Please include (4. Current research progress on these nine AIDPS genes) in the rebuttal, which is a summary of what is known about the biological function of the nine genes, as an Appendix or Supplementary information in the manuscript.Thank you and Reviewer #3 for your suggestions, and we have placed the section \u201cthe current research progress of these nine AIDPS genes\u201d in Appendix 1 and uploaded it with this revised manuscript.Reviewer #1 (Recommendations for the authors):The authors have largely addressed the concerns raised in my previous review. As best I can tell, they have not yet shown how a simple ML model (such as linear regression or xgboost) performs on the same task when only fed with the 9 aidps features simultaneously. The improvements to figure 7 meant to address my request seem to all focus on treating the aidps features separately, so it useful to know they perform poorly when separate\u2026 what about when they are considered together as a nine dimensional feature vector in one model?Thank you for your recommendations. As you mentioned, in the last revision, we confirmed that nine AIDPS genes alone do not predict the prognosis of PACA patients, ignoring their performance as a nine-dimensional feature vector. In this revision, we supplemented the content of Figure 4\u2014figure supplement 1D, focusing on exploring the performance of this 9 AIDPS genes in other machine-learning algorithms. It is worth pointing out that since these nine AIDPS genes are already dimensionality reduced from 32 consensus prognostic genes by CoxBoost, machine-learning algorithm combinations are no longer considered here. Therefore, we used the expression files of these nine AIDPS genes in the PACA-AU-Array training cohort to construct 18 models via 10 common machine-learning algorithms  and interrogated their performance in the remaining 12 multi-center cohorts. The results are shown as follows:As expected, among all 18 models, the model built by survival-SVM, namely our 9-gene AIDPS, achieved a maximum mean C-index of 0.666 in the remaining 12 multi-center cohorts. That is, for the 9 genes obtained from 32 consensus prognostic genes after dimensionality reduction by CoxBoost, these results reconfirmed our previous pipeline results , and the AIDPS model constructed by survival-SVM was the best choice. Thanks again for your reminder, we are sure that your suggestions make our study more rigorous and meaningful.Reviewer #2 (Recommendations for the authors):In my first report I put forward two points. First, that the authors used the same data sets both in the discovery stage and the validation stage. Second, that they compared the performance of competitor signatures to theirs by using these same data. This has the following consequences: 1. They could not claim that their signature had been validated, because what they called validation data were in fact the discovery data . 2. The comparison between their signature and the existing ones was not fair . Therefore, all claims on their signature having been validated and on it being superior to existing signatures were not supported by the available evidence. I also suggested that they could test for overfitting impact by repeating their previous analyses step by step with outcome-randomized versions of the original data. This should make all true signals disappear and is hence a clean and fair test.In the revised version of their manuscript the authors did not carry out the requested re-analysis with outcome-randomized data. Instead they (a) sampled several new data sets from their original data (without outcome randomization), and (b) tested their signature on three independent new data sets. Exercise (a) seems pointless; of course the signature would again be able to predict, since one is testing it once more on samples from the discovery set. Exercise (b), although not what was requested by this referee, is more useful. Here the authors report again significant performance of their signature, with AUC values in the range 0.68-0.70, lending weight to the suggestion that their signature makes sense.I conclude from the authors' reply and from the new version of the paper that point 1. above has been partly addressed, but not point 2. If the authors, as it seems, do not wish to carry out the requested analysis with outcome randomization, then they have the following options:Option A;\u2013 Remove the newly added material called `performance of AIDPS in randomly split cohorts', this adds nothing relevant .\u2013 Take out all mentioning of `validation' or `validated' from the manuscript, other than when discussing the tests on the three new data sets GSE21501, GSE57495, GSE71729; only the latter can be called validation sets.\u2013 Remove all performance comparisons between the new signature and the existing ones (since this comparison is presently not fair) and all conclusions based on these comparisons.Option B:\u2013 Remove the newly added material called `performance of AIDPS in randomly split cohorts', this adds nothing relevant .\u2013 Take out all mentioning of `validation' or `validated' from the manuscript, other than when discussing the tests on the three new data sets GSE21501, GSE57495, GSE71729; only the latter can be called validation sets.\u2013 Compare the performance of existing signatures to that of their own signature on the three new data sets GSE21501, GSE57495, GSE71729; this comparison would be fair.I would personally strongly suggest option B, since otherwise the paper would in my view be too weak in terms of the amount of justified conclusions to warrant publication (but that is a decision for the editor to make). In addition, I would still hope that the authors choose to comply with my original request for application of the full pipeline to outcome-randomized discovery data. This would clarify objectively, for the authors' own benefit, the extent to which their original tests and conclusions may have been affected by overfitting.Thank you for your thoughtful suggestions. Following the Option B, we have removed all contents related to the \u201crandomly split cohorts\u201d added in the first revision. In addition, in our latest manuscript, we refer to the PACA-AU-Array as the training cohort, the remaining nine cohorts involved in screening consensus prognostic genes including TCGA-PAAD, PACA-AU-Seq, PACA-CA-Seq, E-MTAB-6134, GSE62452, GSE28735, GSE78229, GSE79668, and GSE85916 cohorts as the testing cohorts, while the three external datasets GSE21501, GSE57495, and GSE71729 are called the validation cohorts. Moreover, in this revised manuscript, we checked our wording again and used the \u201cvalidated\u201d and \u201cvalidation\u201d only in these three validation cohorts. Furthermore, to remedy our claims, we compared the performance of our AIDPS with 86 published signatures in the three new validation cohorts GSE21501, GSE57495 and GSE71729, and again demonstrated our AIDPS stability and superior performance. The results are exhibited in Figure 4\u2014figure supplement 1:As shown in Figure 4\u2014figure supplement 1A-C, our AIDPS has robust predictive performance, ranking fourth in the GSE57495 cohort as well as fifth in the GSE21501 and GSE71729 cohorts, which is superior to almost all existing signatures. Notably, although the 6-gene signature of Stratford JK was significantly better than AIDPS in the GSE21501 and GSE71729 cohorts, it was constructed in the GSE21501 cohort and performed very poorly in other cohorts, with C-index even less than 0.6 in the GSE57495, TCGA-PAAD, PACA-AU-Seq, et al. . The 15-gene signature of Chen DT had observably superior performance in his own training cohort GSE57495, but it was unsatisfactory in the GSE21501, GSE71729, PACA-CA-Seq, and other cohorts . Similarly, Kim J\u2019s 5-gene signature performed well in the training cohort GSE71729 as well as GSE21501 and PACA-AU-Seq, but very poorly in most other cohorts such as GSE85916, GSE57495, PACA-CA-Seq, and E-MTAB-6134 . In addition, as shown in Overall, using the PACA-AU-Array as the training cohort, nine PACA cohorts as the testing cohorts, the three external datasets GSE21501, GSE57495, and GSE71729 as the validation cohorts, the results of all cohorts confirmed that our AIDPS could better predict the prognosis of PACA patients. Thank you again for your questions and we are sure that your suggestions make our validation more rigorous, credible and persuasive.Reviewer #3 (Recommendations for the authors):The authors have addressed my comments. I find the following v useful as a summary of what is known about the biological function of the nine genes and wonder if it can be incorporated as an Appendix or Supplementary information:Thank you for your suggestions, and we have placed the section \u201cthe current research progress of these nine AIDPS genes\u201d in Appendix 1 and uploaded it with this revised manuscript."}
+{"text": "Arabidopsis (GASA) family genes play critical roles in plant growth, development, and stress responses. However, the biological functions of GASA proteins in lettuce have yet to be thoroughly investigated.Lettuce is one of the most extensively farmed vegetables in the world, and it prefers cool growing conditions. High temperatures promote premature bolt formation, reducing quality and yield. The gibberellic acid-stimulated GASAs were identified in lettuce including, three groups of LsGASA proteins based on the phylogenetic analysis. Except for one, all GASA proteins included a conserved GASA domain with 12 cysteine residues. Cis-element analysis showed that LsGASAs were closely associated with light, phytohormones, and stress resistance. Five segmental and three tandem duplication events were observed in the LsGASA family based on duplication analysis. GASA synteny analysis among lettuce, Arabidopsis, tobacco, and rice revealed that LsGASA5 is highly collinear with all species. Six of the 20 LsGASA showed increased expression patterns at specific time points in the shoot apical meristem when subjected to heat stress. According to gene expression analysis, the majority of GASA were highly expressed in flowers compared to other organs, and six GASA exhibited highly increased expression levels in response to NaCl, abscisic acid, and gibberellin treatment. Furthermore, LsGASA proteins are predominantly found in the plasma membrane and/or the cytosol.Using genome-wide analysis, 20 LsGASA genes for their diversity and biological functions. Moreover, our results will be useful for further studies on the function of lettuce GASA in abiotic stress- and heat-induced bolting signaling.This study provides a comprehensive characterization of The online version contains supplementary material available at 10.1186/s12870-023-04101-5. GASA/GAST gene family is found in a variety of plant species and includes a signal peptide at the N-terminus and a conserved domain with 12 cysteine residues known as the GASA domain (PF02704) at the C-terminus, [GAST1 (GA-stimulated transcript\u00a01) was first characterized in tomato (Solanum lycopersicum L.) [GASAs have been identified in several species, including 19 in tomato (Solanum lycopersicum L.) [Arabidopsis  [Oryza sativa L.) [Theobroma cacao L.) [Triticum aestivum L.) [Vitis vinifera L.) [Nicotiana tabacum L.) [The icum L.) , 15 in Ain PF0270 at the Civum L.) , 14 in gacum L.) .GASA is involved in phytohormone responses, including gibberellin (GA), abscisic acid (ABA), auxins (IAA), brassinosteroids (BR), and salicylic acid (SA) [GASA4 and GASA6 are downregulated by JA, ABA, and SA in Arabidopsis but abundantly expressed by GA, brassinosteroids, auxins, and cytokinins [GASA1 expression analysis in wheat revealed that ACC, ABA, and MeJA were responsible for its induction [GASA and DELLAs, which are negative regulators of GA signaling, have been discovered which imply that GASA significantly contributes to GA signaling [GsGASA1 is involved in suppressing root development through the accumulation of DELLA proteins under cold stress [GASA influences resistance to abiotic stress [Arabidopsis specimens that overexpress FsGASA4 (Fagus sylvatica) and TaGASR1 are more resistant to oxidation, and salt during seed germination and seedling growth [GASA proteins are essential for many biological processes and play a key role in plant development, such as stem elongation , flowericid (SA) , 15. Fornduction . Severalignaling . GsGASA1d stress . Other sc stress , 20. Forg growth  and heatg growth .Lettuce is a cool\u00a0season crop with an optimal growth temperature range of 15\u201325\u00a0\u00b0C that is mostly used in salads. In recent years, the consumption of lettuce has increased significantly owing to its health benefits, such as allelopathic activity and a variety of bioactive phytochemical nutrients including anthocyanins, phenolic acids, and carotenoids , 23. HarFLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1), which play important roles in the flowering molecular pathway, were upregulated by heat treatment and delayed bolting and flowering were observed after the knockdown of soc1 and ft by RNAi from lettuce [Environmental factors such as heat stress restrict plant growth and productivity . The tra lettuce , 28. BolGASAs are important regulators of plant development, heat-induced bolting with GASA expression has not yet been studied in lettuce. In this study, we identified a novel GASA family in the lettuce genome and examined their gene structure, conserved protein motifs, cis-acting elements, chromosomal localization, and synteny. The expression of LsGASA in the apical meristem tissues in response to heat stress was investigated. In addition, transcript levels of heat induced GASA were analyzed in response to abiotic stress and hormones, and subcellular localization was observed.Elucidation of the molecular regulatory network of heat-induced bolting in lettuce might be crucial for the development of heat-resistant lettuce since lettuce bolting time affects yield, quality, and consumer preference. Although GASAs were renamed LsGASA1 to LsGASA20 according to their chromosomal locations and distributed across lettuce genome loci 1, 2, 3, 4, 8, and 9  and four genes  showed pairwise synteny with NtGASA and AtGASA, respectively. Furthermore, LsGASA5, LsGASA12, and LsGASA14 showed pairwise synteny with the OsGASA family, with LsGASA5 being syntenic with the both OsGASA.To deduce the evolutionary relationship of ice Fig.\u00a0. NumerouLsGASA3/4, LsGASA12/13, and LsGASA15/16). On the other hand, segmental duplications were detected on three gene pairs, including LsGASA1/11, LsGASA6/8, and LsGASA9/10 , auxin-responsive elements (TGAelement and AuxRR-core), gibberellin-responsive elements , and MeJA response elements (TGACG-motif and CGTCA-motif). Except for GASA19, all other GASAs contained at least one sequence of hormone-responsive cis-acting elements. LsGASAs contain various stress-related elements, including ARE, DRE core, GC motif, LTR, MBS, MYB, MYC, and TC-rich repeats. Most GASA contains 35 MYB, 70 MYC, and 48 ARE elements in their promoters. All GASA contained ARE elements. In the promoter of LsGASAs, light-responsive elements  showed the largest number compared to hormone-responsive and stress-responsive elements. Among the various elements, all GASA contained Box\u00a04 elements that were associated with light-responsive elements  that displayed increased expression in the SAM of lettuce under heat treatment. These results suggest that the six LsGASAs may be involved in heat-induced bolting mechanisms.For further study, we selected six LsGASAs across an array of lettuce tissue samples, we analyzed the expression of the six GASAs in leaves, roots, stems, seeds, and flowers  in SAM were highly increased under heat treatment , which are heat-induced genes in SAM, were investigated for their expression patterns in different organs and response to abiotic stress and hormones. Tissue-specific expression revealed that LsGASAs were highly expressed in the roots and flowers. Six LsGASAs exhibited diverse expression patterns in response to different abiotic stressors and hormone treatments. In addition, the subcellular localization of the six LsGASAs was mostly in the plasma membrane, cytoplasm, or both. Overall, this study provides fundamental information on the LsGASA family and its responses to various abiotic stresses. This study will allow for a comprehensive investigation into the functional roles of each LsGASA; these findings will then allow for future studies into the characterization of mechanisms that underlie heat-induced bolting in lettuce.In this study, we identified 20 LsGASA, the hidden Markov model (HMM) profiles of the GASA domain (PF02704) from the Pfam database (https://pfam.xfam.org/) were used as a query, and the putative GASA protein sequences were identified using HMMER v 3.0 [https://plants.ensembl.org/index.html) with a predefined threshold of E\u2009<\u20091e-5. The selected proteins containing GASA domains were confirmed using InterProScan (https://www.ebi.ac.uk/interpro/search/sequence-search). The ExPASy ProtParam (https://web.expasy.org/protparam/) tool was used to estimate the physical and chemical characteristics of all identified GASA proteins, including their isoelectric point, molecular weight, and grand average of hydropathy.To identify ER v 3.0  searchinLsGASAs was determined using the lettuce genome database. The MapChart program was used to graphically map lettuce chromosomes [LsGASA were aligned using ClustalW and KaKs_calculator 3.0 [LsGASA pairs. The divergence time (T) was calculated using the following eq. [x\u2009=\u20096.56\u2009\u00d7\u200910\u2212\u20099.The chromosomal localization of omosomes . The CDSator 3.0  was usedwing eq. :1\\documeArabidopsis genome. GFF files were obtained from EnsemblPlants (https://plants.ensembl.org/index.html) for lettuce and Arabidopsis, and from the Sol Genomics Network (https://solgenomics.net/organism/Nicotiana_tabacum/genome) for tobacco. The genome sequences and GFF files of lettuce and other species were used as input files for the One-Step MCScanX tool in TBtools. Consequently, the output files, Ctl, simplified GFF, and collinearity file were used for the dual synteny plot in TBtools for synteny visualization.TBtools  was usedhttps://gsds.gao-lab.org/) was used to display the exon-intron structures of the LsGASA.A phylogenetic tree was constructed using the neighbor-joining method with 1000 bootstrap replicates using MEGA X software . Multiplhttp://meme-suite.org/), and the subcellular localization of LsGASA proteins was predicted using WoLF PSORT (https://wolfpsort.hgc.jp/) and Plant-mPLoc server (https://rostlab.org/services/nlsdb/). Furthermore, the cis-acting elements of the promoters up to 1500\u2009bp upstream of the start codon of all LsGASAs were predicted using PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/).The conserved motifs of LsGASA proteins were predicted using MEME Suite  and then cloned into the pMDC43 vector using LR Clonase . Recombinant vectors containing the GFP expression cassette were examined for transient expression using lettuce protoplast transfection. The subcellular localization of LsGASAs was observed using a confocal laser scanning microscope .To examine the subcellular localization of LsGASAs, the CDS of D. atrakce  was used in this study. Germplasm was provided by the National Agrobiodiversity Center, Korea, with permission for use in this experiment. D. atrakce was grown in 32 cell trays and placed in a growth chamber  set at 22/20\u00a0\u00b0C (16\u2009h/8\u2009h), 60% relative humidity, and 18,000\u2009lx light intensity. Plants were exposed to abiotic stress and hormone treatment 25 d after planting. To examine the responses of the LsGASAs to abiotic stresses and phytohormone applications, 25-day-old seedlings were subjected to diverse treatments such as heat stress , 20% polyethylene glycol (PEG 6000), 250\u2009mM sodium chloride (NaCl), low temperature (4\u00a0\u00b0C), 150 \u03bc\u039c of GA3, 150 \u03bc\u039c of paclobutrazol (PAC) and 100\u2009\u03bcM of ABA, and the treated lettuce seedling shoot apical meristem (SAM) and leaves were harvested at time intervals of 2, 6, 12, 24, 48 and 72\u2009h. All the obtained samples were immediately stored at \u2212\u200980\u00a0\u00b0C until further use.The lettuce cultivar Detenicka atrakce\u2019 was fixed with 4% paraformaldehyde, followed by vacuum for 20\u2009min. An ethanol series of 30, 50, 70, 95, and 100% was used to dehydrate the fixed samples. The dehydrated samples were cleaned with tert-butyl alcohol  series . The cleaned samples were infiltrated and embedded in Paraplast Plus (Sigma-Aldrich) at 58\u00a0\u00b0C. Embedded samples were cut into 8-\u03bcm thick sections using a microtome (Leica RM2255). For observation, sectioned samples were stained with 0.05% toluidine blue O (Sigma-Aldrich) in citrate buffer (pH\u20094) and coverslips were applied using Canada balsam . Finally, slides were observed under an upright microscope .SAM of heat-stressed \u2018https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi? LINK_LOC=BlastHome)  and the extract was treated with TRIzol reagent . A Power cDNA synthesis kit  was used to synthesize first-strand circular DNA (cDNA) from 1\u2009\u03bcg total RNA according to the manufacturer\u2019s instructions. Polymerase chain reaction (PCR) was used to confirm the specificity of gene-specific primers designed using Primer-BLAST  under heat stress conditions using ImageJ program.Additional file 5: Fig. S3. Expression analysis of six LsGASA in different tissues . Error bars represent the standard error of the mean.Additional file 6: Table S3. List of specific primers used in the study."}
+{"text": "The addition of etidronic acid (HEDP) to sodium hypochlorite (NaOCl) could increase the antibiofilm potency of the irrigant, whilst maintaining the benefits of continuous chelation. Studies conducted so far have shown that mixing HEDP with NaOCl solutions of relatively low concentration does not compromise the antibiofilm efficacy of the irrigant. However, the working lifespan of NaOCl may decrease resulting in a reduction of its antibiofilm efficacy over time (efficiency). In this regard, continuous irrigant replenishment needs to be examined. This study investigated the response of a dual\u2010species biofilm when challenged with 2% and 5% NaOCl mixed with HEDP for a prolonged timespan and under steady laminar flow.Streptococcus oralis J22 and Actinomyces naeslundii T14V\u2010J1 were grown on human dentine discs in a constant depth film fermenter (CDFF) for 96\u00a0h. Biofilms were treated with 2% and 5% NaOCl, alone or mixed with HEDP. Irrigants were applied under steady laminar flow for 8\u00a0min. Biofilm response was evaluated by means of optical coherence tomography (OCT). Biofilm removal, biofilm disruption, rate of biofilm loss and disruption as well as bubble formation were assessed. One\u2010way anova, Wilcoxon's signed\u2010rank test and Kruskal\u2013Wallis H test were performed for statistical analysis of the data. The level of significance was set at a \u2264.05.Dual\u2010species biofilms comprised of Increasing NaOCl concentration resulted in increased biofilm removal and disruption, higher rate of biofilm loss and disruption and increased bubble formation. Mixing HEDP with NaOCl caused a delay in the antibiofilm action of the latter, without compromising its antibiofilm efficacy.NaOCl concentration dictates the biofilm response irrespective of the presence of HEDP. The addition of HEDP resulted in a delay in the antibiofilm action of NaOCl. This delay affects the efficiency, but not the efficacy of the irrigant over time. OCT provides quantitative measurements of biofilm removal, whilst revealing the biofilm structure at the mesoscale level  from NaOCl  is effective against biofilms as it both kills the biofilm microorganisms and breaks down the biofilm polymeric matrix  is invariably included in root canal irrigation regimens aiming at enhanced cleanliness and disinfection of the root canal system , that are statically applied on mono\u2010species biofilms. Despite their undisputed value, direct translation of their findings is difficult, as clinical parameters such as irrigant flow and replenishment ought to be taken into account. In addition, the strength of mono\u2010species biofilms to hydrodynamic shear stresses is expected to differ considerably from multi\u2010species biofilms  2021\u00a0guidelines    (pre\u2010cultures). S.\u00a0oralis were cultured in an aerobic incubator, at 37\u00b0C, for 24\u00a0h and A.\u00a0naeslundii in an anaerobic incubator, at 37\u00b0C, for 48\u00a0h. Following, pre\u2010cultures were mixed with 190\u00a0ml of fresh BHI and incubated aerobically for S.\u00a0oralis and anaerobically for A.\u00a0naeslundii for another 16\u00a0h (main cultures). Next, bacteria were harvested by means of centrifugation (6500\u00a0g), with two washing steps of the bacterial pellets with sterile adhesion buffer  in between  , phenylmethylsulfonyl fluoride was added to a final concentration of 1\u00a0mM as a protease inhibitor. Afterwards, the solution was centrifuged again, dialysed  against demineralized water and freeze\u2010dried for storage. The lyophilized saliva was dissolved in 30\u00a0ml of adhesion buffer (1.5\u00a0g/L), stirred for 2\u00a0h and centrifuged at 6500 g\u00a0rpm, 10\u00b0C for 5\u00a0min. The dentine discs were exposed to the reconstituted saliva under static conditions, at 4\u00b0C, for 14\u00a0h. It has to be noted that the lyophilized saliva is not submitted to any sterilization process. Saliva lyophilization does not guarantee sterilization. Nevertheless, prior to lyophilization saliva is centrifuged twice to remove any micro\u2010sized debris, including bacterial cells. This decreases considerably the bacterial load. After salivary protein adsorption, the substrate is inoculated with a large number of bacterial cells, which will eventually predominate over any salivary bacterial cells present on the surface. Thus, we expect none  interference to the formation of the dual\u2010species CDFF biofilms . After placing the saliva\u2010coated dentine discs on each platform, the height was set at 250\u00a0\u03bcm distance between the disc and the rim of the holder, thus allowing for the development of biofilms of standardized thickness (250\u00a0\u03bcm). Two hundred millilitres of the dual\u2010species bacterial suspension were used to inoculate the CDFF. Inoculation was performed dropwise, at a rate of 1.67\u00a0ml/min, whilst the CDFF table was slowly rotating. After inoculation, table rotation was stopped to allow for further bacterial adhesion onto the dentine discs. Finally, rotation was resumed and the biofilms were grown under continuous supply of modified BHI at a rate of 45\u00a0ml/h, at 37\u00b0C, for 96\u00a0h. Before proceeding to biofilm treatment with the irrigants, the thickness of each sample was measured with the aid of OCT. Only samples reaching the thickness of 250\u00a0\u03bcm (pre\u2010set height in the CDFF holders) were used in the experiment.To ensure reproducible and standardized development of bacterial cell\u2010dense biofilms, a constant depth film fermenter (CDFF) was used , 5% NaOCl and 5% NaOCl combined with HEDP. Prior to the experiments, iodometric titration was carried out to determine the concentration of the stock NaOCl solution (Sigma\u2010Aldrich) and the desired NaOCl concentrations were prepared by diluting stock NaOCl with demineralized water; the NaOCl solutions used in the experiments had a pH 12. Finally, for the preparation of the NaOCl/HEDP irrigant solutions, 4.5\u00a0g of DualRinse HEDP was mixed with 50\u00a0ml of either 2% or 5% NaOCl for 2\u00a0min. After mixing, the solution was drawn back into a 50\u2010ml polypropylene syringe with a Luer Lock opening and used immediately.Four irrigant solutions were used to challenge the biofilm, namely, 2% NaOCl, 2% NaOCl combined with HEDP . Three independent experiments were carried out, during which 3 independent biofilm\u2010carrying dentine discs from each irrigant group were treated with the corresponding irrigant . Sample size was determined based on findings from preliminary investigations and data previously published , letting only the biofilm on top of the dentin disc to be exposed to the bulk of the irrigant. Irrigant was passed through the PPFC using a peristaltic pump at a flow rate of 0.05\u00a0ml/s. During irrigation, 2D real time, cross\u2010sectional recordings were acquired by means of optical coherence tomography . Recordings were taken in 2\u00a0separate time intervals, named Phases I and II. In Phase I, the short\u2010term effect of the irrigant on the biofilm was recorded (0\u2013180\u00a0s exposure). In Phase II, the long\u2010term effect of the irrigant on the biofilm was recorded (300\u2013480\u00a0s exposure). The time intervals were chosen based on preliminary experiments on biofilms exposed to the plain NaOCl solutions showing a high NaOCl activity at the first 180\u00a0s that gradually decreased, until it plateaued after 480\u00a0s. Real\u2010time imaging was performed at 25 frames/s , by setting the field of view at a span of 5\u00a0mm and the refraction index at 1.33. Each acquired OCT biofilm image represents a series of consecutive xz\u2010plane images taken along the diameter of the circular sample , that is a series of axial intensity profiles containing depth\u2010resolved structural information along the longest distance from one end of the circular biofilm\u2010carrying dentine disc to the other (5\u00a0mm)  were submitted to treatment with each irrigant (Ndisrupted layer) and a layer exhibiting higher greyscale pixel intensity . The total number of pixels measured after background noise subtraction accounted for the total biofilm present. The number of pixels measured within the disrupted and coherent layer, which resulted after background noise subtraction and applying a multilevel thresholding, accounted for the disrupted and coherent biofilm present, respectively. The outcome measures used to assess the biofilm response to the different irrigant solutions were the following:xt:0t\u00a0=\u00a00\u00a0s is defined as the time point at which interaction between the irrigant and the biofilm takes place, namely, immediately after the introduction of the irrigant solution in the parallel plate flow chamber.Percentage total biofilm at each measurement point xt:Percentage disrupted biofilm layer at each measurement point 0\u00a0=\u00a00\u00a0s (baseline measurement).Rate percentage total biofilm loss (%/s), indicated by the percentage total biofilm loss over time, normalized against the starting point t0\u00a0=\u00a00\u00a0s (baseline measurement).Rate percentage\u2010disrupted biofilm forming (%/s), indicated by the percentage disrupted biofilm present over time, normalized against the starting point tAmount of bubbles formed during Phase I (0\u2013180\u00a0s) and Phase II (300\u2013480\u00a0s) (OCT slide\u2010by\u2010slide imaging). A bubble was counted as an \u2018event\u2019 when it could be visualized from the moment of its initiation until its rupture (before the end of the observation period) or when it was visible throughout the observation period.The open source image processing package Fiji was used to analyse the cross\u2010sectional images acquired during the two phases of the OCT recordings. The image stacks, consisting of 3800 images with a resolution of 1000\u00a0\u00d7\u00a0376\u00a0pixels, were reduced to 380\u00a0measurements. This yielded a data point for every 0.4\u00a0s. A multilevel Otsu threshold was used to segment biofilm from the background  or anova for normally distributed data and Wilcoxon signed\u2010rank tests or Kruskal\u2013Wallis H for non\u2010normally distributed data was performed. The level of statistical significance was set at a \u2264.05. Rejection of a null hypothesis, namely when p\u00a0\u2264\u00a0.05, was followed by pairwise comparisons .Statistical analysis was performed using R statistical package (version 3.6.3). Measurement points of the OCT recording were binned into 30\u00a0s intervals. Data normality were assessed with the Shapiro\u2013Wilk test. One\u2010way repeated measures analysis of variance . Within the first 180\u00a0s, both 5% NaOCl and 5% NaOCl/HEDP had disrupted a significant amount of biofilm, which was not the case for the 2% NaOCl and 2% NaOCl/HEDP. For the rest of the observation period, no further disruption was caused by the 5% NaOCl and 5% NaOCl/HEDP. Two percent NaOCl started causing significant biofilm disruption after 300\u00a0s, whilst 2% NaOCl/HEDP elicited a significant biofilm disruption only towards the end of the observation period (480\u00a0s) , keeping the same significant disruptive potential for 300\u00a0s, compared to 2% NaOCl and 2% NaOCl/HEDP. At the end of the observation period (480\u00a0s), 2% NaOCl/HEDP had caused the least biofilm disruption, without any significant difference compared to other irrigants (Table During the first 180\u00a0s (Phase I), 5% NaOCl decreased the percentage total biofilm the fastest, at a rate significantly higher than for 2% NaOCl and 2% NaOCl/HEDP and considerably higher than 5% NaOCl/HEDP. Five percent NaOCl/HEDP decreased the percentage total biofilm faster only when compared to 2% NaOCl during Phase I. During the last 180\u00a0s (Phase II), all irrigant solutions decreased the percentage total biofilm faster compared to 2% NaOCl/HEDP Figure .During the first 180\u00a0s (Phase I), 5% NaOCl and 5% NaOCl/HEDP induced biofilm disruption in a significantly higher rate compared to 2% NaOCl and 2% NaOCl/HEDP Figure . During During the first 180\u00a0s (Phase I), exposure of biofilms to 5% NaOCl and 5% NaOCl/HEDP led to a significantly higher bubble count compared to 2% NaOCl and 2% NaOCl/HEDP Table . A consiThe response of a bacterial dense dual\u2010species biofilm to relatively low and high NaOCl concentrations, either with or without the addition of HEDP, under laminar irrigant flow, was analysed. We showed that NaOCl concentration, irrespective of the addition of HEDP, was the driving factor of biofilm disruption and removal, enhancing efficacy and boosting the early efficiency of the irrigant. Remarkably, a slight volumetric increase (swelling) of the biofilm was observed in the first minutes following application of 2% NaOCl, with or without HEDP. On the other hand, 5% NaOCl solutions, with or without HEDP caused a significant biofilm disruption immediately after coming in contact with the biofilms. Mixing NaOCl with HEDP resulted in a delayed antibiofilm effect of the irrigant, a finding that was more prominent in the 5% NaOCl solutions. Finally, a distinct pattern of bubble formation was evident between the 2% and 5% NaOCl groups. Biofilms exposed to 5% NaOCl demonstrated a rapid growth of numerous large bubbles compared to the small\u2010sized and considerably less bubbles formed within the biofilms exposed to 2% NaOCl.in vitro models measure biofilm removal after \u2018one\u2010off\u2019 NaOCl application, thus neglecting the merit of irrigant refreshment and volume that are vital part of endodontic irrigation as this is clinically practised. In our model, a steady laminar flow was generated in a parallel plate flow chamber. The flow allowed for a more efficient transport of the NaOCl by the convective motion of the fluid  affect biofilms can be compensated to a large extent by mechanical instrumentation, irrigant agitation or the use of intracanal medicaments. Especially for mechanical debridement, its role in reducing the bacterial load is well\u2010established , and secondly to the biofilm viscoelastic properties  within the first 180\u00a0s, whilst slowing down thereafter. Five percent NaOCl solutions contain a higher quantity of reactive OClDuring the last 180\u00a0s, 2% NaOCl without HEDP showed an increase in the biofilm removal rate, approaching the rate of the higher concentrated solutions. At first, this supports the idea that the assumingly compromised antibiofilm efficiency of the lower concentrated NaOCl solutions can be compensated by more frequent exchange, a larger volume and longer exposure times (Alves et al., \u2212 within the first hour after mixing, which limits the working lifespan of the solution (Biel et al., \u2212 is consumed when NaOCl comes in contact with biofilms (de Beer et al., Previous studies have shown that combining 5% NaOCl with HEDP result in some loss of the free available OClNaOCl reacts with the proteinaceous and polysaccharidic content of the biofilm matrix (Hawkins et al., When 2% NaOCl is applied, the lower reactivity between the irrigant and the biofilm does not seem to allow for large bubbles to form. Also, taking into account that these bubbles are formed in the upper biofilm layers due to the limited penetration of the 2% NaOCl compared to the 5% NaOCl, their buoyancy is initially low. Nonetheless, small bubbles also cause disruption in the biofilm structure. This disruption is associated with the structural re\u2010arrangement occurring naturally as a result of the volumetric expansion caused by the formed bubble. Consequently, the biofilm coherence is reduced, albeit below the critical failure level, thus similarly to what happens to a hydrogel as it swells (Macedo et al., Contrary to our working hypothesis, the continuous presence of a chemically \u2018inert\u2019 chelator did not have a synergistic effect on the antibiofilm capacity of NaOCl against CDFF biofilms. Within the limitations of this study, NaOCl concentration seems to be the driving factor that determines biofilm response (Petridis et al., HEDP slows down the efficiency of NaOCl in terms of biofilm removal/disruption, but leads to similar results when biofilms are exposed to NaOCl/HEDP combined solutions for longer periods.NaOCl concentration affects the rate of biofilm disruption and removal.Bubble formation resulting from the reaction between NaOCl and the biofilm contributes to disruption of the biofilm structure and the biofilm volumetric expansion associated with the less concentrated NaOCl solutions.Bubble formation parameters, such as growth rate and final size, are dependent on the concentration of the irrigant solution.Optical coherence tomography is a valuable imaging tool for real\u2010time monitoring of interactions between reactive solutions and biofilms.Based on the findings and within the limitations of this study, the following conclusions can be drawn:The authors deny any conflicts of interest related to this study.All study protocols were approved by the Institutional Review Board of the University Medical Center Groningen and judged as not falling under the scope of the Medical\u2010Scientific Act for research with humans.Mariana Macial Batista Borges: Study conception and design, data collection, data analysis and interpretation, writing and revising the paper, final approval to the submitted version. Ren\u00e9 J.B. Dijkstra: Study conception and design, data collection, data analysis, writing and revising the paper, final approval to the submitted version. Flaviana Bombarda de Andrade: Study conception, revising the paper, final approval to the submitted version. Marco Antonio Hungaro Duarte: Revising the paper, final approval to the submitted version. Michel Versluis: Data interpretation, revising the paper, final approval to the submitted version. Lucas W.M. van der Sluis: Study concept and design, data interpretation, writing and revising the paper, final approval to the submitted version. Xenos Petridis: Study concept and design, data analysis and interpretation, writing and revising the paper, final approval to the submitted version.Appendix S1Click here for additional data file.Video S1Click here for additional data file.Supplementary MaterialClick here for additional data file."}
+{"text": "To develop a step-by-step process for probabilistic linkage of national clinical and administrative datasets without personal information, providing guidance on selecting variables for linkage, estimating match weights, and choosing the probabilistic linkage threshold. To validate this process against deterministic linkage using patient identifiers.We undertook probabilistic linkage without personal information using electronic health records from the National Bowel Cancer Audit (NBOCA) and Hospital Episode Statistics (HES) databases for bowel cancer patients undergoing emergency surgery in England. We selected linkage variables based on completeness, and ability to discriminate between matches and non-matches, assessed using a novel score derived from m-probabilities and u-probabilities. Taking deterministic linkage using patient identifiers as the reference-standard, we calculated sensitivity and specificity of probabilistic linkage, plotted a Receiver Operating Characteristic curve across alternative thresholds of match weights, and compared patient characteristics and estimates from fitted regression models between linkage methods.When considering the ability to discriminate between matches and non-matches, patient and administrative variables tended to discriminate better than clinical variables. 81.4% of NBOCA records were linked to HES using probabilistic linkage, versus 82.8% using deterministic linkage. Most NBOCA records were linked to HES using both methods . Probabilistic linkage had over 96% sensitivity and 90% specificity compared to deterministic linkage using patient identifiers. Patients that linked deterministically, but not probabilistically, were younger and more likely to have emergency admission, but otherwise had similar characteristics. Regression models for mortality and length of hospital stay according to patient and tumour characteristics were not sensitive to the linkage approach.Probabilistic linkage without personal information can be used as an alternative to deterministic linkage using patient identifiers, or as a method for enhancing deterministic linkage. It allows analysts outside highly secure data environments to undertake linkage while minimising costs and delays, protecting data security, and maintaining linkage quality."}
+{"text": "Anxiously attached individuals tend to report stronger parasocial relationships withtheir favorite media figures than people with other attachment orientations. Researchershave suggested that these individuals may be inclined to see their favorite media figuresas safe and secure attachment figures. The purpose of the current study was to evaluatethis possibility by assessing the qualities of people\u2019s favorite media figures,particularly within a television context. A sample of 200 online participants filled outan attachment measure, reported their favorite television figure, and rated severalaspects of the television figure\u2019s personality. It was expected that anxiously attachedindividuals would be drawn to figures that are high in warmth, emotional stability, andsensitivity. Instead, results showed that these individuals preferred figures with greateranxious and insecure characteristics. These results suggest that anxiously attachedindividuals may not see their favorite media figures as safe and secure attachment figuresas previously theorized. Exploratory analyses failed to show significant effects for thesecond attachment dimension, attachment avoidance, or for the interaction between anxietyand avoidance. People often form intimate bonds with celebrities and fictional characters they encounterin the media .AlthougAttachment theory arose from the work of British psychiatrist and psychoanalyst John workingmodels, mental representations of the self and close others that shape a person\u2019sexpectations regarding their close relationships  in their real-life attachment figures.Hypothesis 1: Individuals with high levels of attachment anxiety havefavorite media figures that are high in warmth, emotional stability, and sensitivity.One might wonder whether individuals with high levels of attachment avoidance are drawn tomedia figures that possess certain qualities. Unlike anxiously attached individuals, peoplewith high levels of attachment avoidance do not appear to have particularly strongparasocial relationships with their favorite media figures . It seemResearch Question 1: Do individuals with high levels of attachmentavoidance have favorite media figures that possess certain qualities?Studies in this area tend to consider attachment anxiety and attachment avoidanceseparately, but it should be noted that these two dimensions can interact with one anotherto create variations in attachment orientations. For instance, some people report highlevels of attachment anxiety and low levels of attachment avoidance, whereas other peopleshow the opposite pattern. Still others report high levels or low levels of both dimensions.It is currently unclear whether these dimensions interact to affect media figurepreferences, and if so, what the specific pattern of results might be. Although it isexpected that high levels of attachment anxiety will be related to a preference for safe andsecure media figures, the uncertainty surrounding the avoidance dimension makes it difficultto predict how it might interact with anxiety to impact media figure preferences.Research Question 2: Is the interaction between attachment anxiety andattachment avoidance related to favorite media figure qualities?The purpose of the current study was to learn more about how attachment dimensions arerelated to media figure preferences. This study focused on television figures specifically,as past research in this area has concentrated primarily on this type of media e.g., .N\u2009=\u200922), if they provided suspicious or incomprehensible answers  (N\u2009=\u200925), if they selected a media figure that did notappear on television (N\u2009=\u200927), or if they were statistical outliers thatprovided extreme or unusual scores (N\u2009=\u200914). This left a final sample of200 participants. The sample consisted of 89 males and 111 females. Participants had amean age of 44.06\u2009years (SD\u2009=\u200913.60). The sample was largely White (79%),with the rest of the sample identifying as Black or of African descent (8%), Asian orPacific Islander (7.5%), or another race or ethnicity (5.5%). Participants were offered amonetary incentive of 50 cents American for their participation in the study.Participants were recruited through the crowdsourcing Internet marketplace AmazonMechanical Turk (MTurk). Participation was restricted to residents of the United Statesand Canada who had a successful completion rate of 97% or above for their MTurk tasks. Atotal of 288 individuals completed the study. Participants were dropped from the analysisif they failed attention questions embedded within the questionnaire to 7(strongly agree). Responses to the items were averaged to obtaincomposite scores for each subscale. Cronbach\u2019s alpha was .96 for the anxiety subscale and.93 for the avoidance subscale.Television figure qualities. The qualities of participants\u2019 favoritetelevision figures were assessed with a 16-item instrument loosely derived from theSixteen Personality Factor (16PF) Questionnaire from strongly disagree) to 7 (stronglyagree).Participants were redirected from MTurk to the Qualtrics online survey platform, whichhosted the study materials. After completing an informed consent form, participants filledout the demographic questionnaire and the attachment scale. Next, they were asked to writedown the name of their favorite television figure, either a fictional character  or a nonfictional personality . They were also asked to write down the name of thetelevision program that their favorite television figure appears on. Finally, they filledout the instrument assessing their favorite television figure\u2019s qualities. Once datacollection was complete, data were entered into SPSS Statistics (Version 26.0) forstatistical analysis.Game ofThrones (N\u2009=\u20096), Sheldon Cooper (played by Jim Parsons) fromThe Big Bang Theory (N\u2009=\u20094), Rachel Green (played byJennifer Aniston) from Friends (N\u2009=\u20094), and Jean-Luc Picard (played byPatrick Stewart) from Star Trek: The Next Generation(N\u2009=\u20094). Overall, fictional characters (N\u2009=\u2009147) were morecommon than nonfictional personalities (N\u2009=\u200953).Participants selected a wide variety of television figures as their favorites. The mostfrequently selected figures were Jon Snow (played by Kit Harington) from Pearson correlations were used to assess the bivariate relationships between attachmentanxiety and each of the television figure descriptors, as well as attachment avoidance andthe television figure descriptors. Due to the number of statistical tests being performedwithin each set of correlations, the alpha level of .05 was adjusted using theHolm-Bonferroni method . The Holr(198)\u2009=\u2009.33,p\u2009<\u2009.001, and tension, r(198)\u2009=\u2009.27,p\u2009<\u2009.001. There were also significant negative associations betweenattachment anxiety and two television figure descriptors: reasoning,r(198)\u2009=\u2009\u2212.25, p\u2009<\u2009.001, and self-reliance,r(198)\u2009=\u2009\u2212.21, p\u2009=\u2009.003. In general, participants withhigher levels of attachment anxiety reported favorite television figures that are moreapprehensive and tense, with poorer reasoning and lower levels of self-reliance. Nosignificant associations between attachment avoidance and the television figure descriptorswere found.Results showed significant positive associations between attachment anxiety and twotelevision figure descriptors: apprehension, pr(197)\u2009=\u2009.33, p\u2009<\u2009.001; tension,pr(197)\u2009=\u2009.27, p\u2009<\u2009.001; reasoning,pr(197)\u2009=\u2009\u2212.25, p\u2009<\u2009.001; and self-reliance,pr(197)\u2009=\u2009\u2212.21, p\u2009=\u2009.002. No significant associationsbetween attachment avoidance and the television figure descriptors were found.An exploratory analysis was performed to assess whether the correlational results persistedafter accounting for the fictional or nonfictional nature of the television figures. Forthis analysis, the correlation analysis was repeated using partial correlations withfictional/nonfictional status as the control variable. Once again, results showed thatattachment anxiety was significantly related to apprehension,F\u2009=\u20092.44,p\u2009<\u2009.001 (Wilks\u2019s \u03bb\u2009=\u2009.56). However, peel off testing showed that themodel was no longer significant after removing the first function,F\u2009=\u20091.25, p\u2009=\u2009.176. In other words, the firstfunction accounted for the significant relationship between the two sets of variables.A canonical correlation analysis was used to gain a more comprehensive understanding of howthe attachment dimensions were related to the television figure descriptors. Canonicalcorrelation is a dimension reduction technique that assesses the relationship between twosets of variables. Attachment anxiety, attachment avoidance, and the interaction betweenanxiety and avoidance formed one set of variables. The television figure descriptors formedthe second set of variables. The canonical correlation analysis yielded three functions withcanonical correlations of .57, .33, and .28, respectively. The full model across all threefunctions was statistically significant, Past research has shown that anxiously attached individuals tend to have strongerparasocial relationships than people with other attachment orientations. Researchers havesuggested that anxiously attached individuals see their favorite media figures as surrogateattachment figures that offer a greater degree of safety and security than their real-lifeattachment figures. The results of the current study cast doubt on this notion. Participantswith higher levels of attachment anxiety tended to report favorite television figures thatthemselves exhibited higher levels of anxiety and insecurity. It seems unlikely thatanxiously attached individuals would derive feelings of safety and security from suchfigures, particularly when there are alternative television figures (perhaps even on thesame program) that could more effectively provide such feelings. Previous studies from theparasocial relationship literature have shown that people are often drawn to media figuresthat remind them of themselves . This fiTo account for the link between attachment anxiety and parasocial relationship strength,researchers may need to consider other perspectives. For instance, it is possible thatanxiously attached individuals do form attachment bonds with their favorite media figures,but the nature of these bonds might not be as secure as previously thought. In this case,their media attachments would mirror their real-life attachments to a certain degree.Researchers should also consider the possibility that anxiously attached individuals do notsee their favorite media figures as attachment figures at all, as there are other potentialreasons why these individuals might develop strong parasocial connections. For instance,studies have shown that anxiously attached individuals tend to have a higher need forbelonging and affiliation than people with other attachment orientations . PerhapsAlthough the statistical analysis for this study revealed a number of significant resultsfor attachment anxiety, no comparable results were found for attachment avoidance or theinteraction between anxiety and avoidance. This outcome is not particularly surprising, aspast research from the parasocial relationship literature has failed to show a consistentlink between attachment avoidance and people\u2019s bonds with television figures. People withhigh levels of attachment avoidance clearly use television and have favorite televisionfigures. However, attachment avoidance may not be related to preferences for specific typesof television figures, regardless of whether it is considered in isolation or in conjunctionwith attachment anxiety . Attachment anxiety by itself seems to be the best indicator of preferred televisionfigure qualities.There were some important limitations to this research. Ratings of television figurequalities were used to assess whether anxiously attached individuals see their favoritetelevision figures as safe and secure attachment figures. However, cognitive, affective,and behavioral responses to these figures would provide an alternative and potentiallymore valid means to test this possibility. Although anxiously attached individuals aredrawn to relatively anxious and insecure television figures, it is possible (unlikelythough it may seem) that they derive a sense of attachment security from such figures.Future researchers should assess people\u2019s responses to media figures to better determinehow anxiously attached individuals view these figures. Researchers should also consideralternate, nonattachment reasons for why anxiously attached individuals might be drawn tosuch figures, such as the satisfaction of belongingness needs.The qualities of participants\u2019 favorite television figures were assessed using singleitems representing each of Cattell\u2019s 16 personality factors. These collective items couldbe administered fairly quickly (the full version of Cattell\u2019s 16PF Questionnaire contains185 items in its current form) while also providing a nuanced view of people\u2019s favoritetelevision figures. However, these single items likely lacked the sensitivity andreliability of multi-item measures. Several studies have shown that the performance ofsingle-item measures is often comparable to that of multi-item measures e.g., . RegardlThe results of this study challenge the assertion that anxiously attached individuals seetheir favorite media figures as surrogate attachment figures that compensate fordeficiencies in real-life attachment security. Although unexpected, these results open someinteresting avenues for future research in this area."}
+{"text": "The aim of this study was to develop and validate a semi-automated segmentation approach that identifies the round window niche (RWN) and round window membrane (RWM) for use in the development of patient individualized round window niche implants (RNI) to treat inner ear disorders. Twenty cone beam computed tomography (CBCT) datasets of unilateral temporal bones of patients were included in the study. Defined anatomical landmarks such as the RWM were used to develop a customized 3D Slicer\u2122 plugin for semi-automated segmentation of the RWN. Two otolaryngologists (User 1 and User 2) segmented the datasets manually and semi-automatically using the developed software. Both methods were compared in-silico regarding the resulting RWM area and RWN volume. Finally, the developed software was validated ex-vivo in N = 3 body donor implantation tests with additively manufactured RNI. The independently segmented temporal bones of the different Users showed a strong consistency in the volume of the RWN and the area of the RWM. The volume of the semi-automated RWN segmentations were 48 \u00b1 11% smaller on average than the manual segmentations and the area of the RWM of the semi-automated segmentations was 21 \u00b1 17% smaller on average than the manual segmentation. All additively manufactured implants, based on the semi-automated segmentation method could be implanted successfully in a pressure-tight fit into the RWN. The implants based on the manual segmentations failed to fit into the RWN and this suggests that the larger manual segmentations were over-segmentations. This study presents a semi-automated approach for segmenting the RWN and RWM in temporal bone CBCT scans that is efficient, fast, accurate, and not dependent on trained users. In addition, the manual segmentation, often positioned as the gold-standard, actually failed to pass the implantation validation. The incidence of inner ear disorders\u2014e.g., idiopathic sudden sensorineural hearing loss (ISSHL) and Meniere\u2019s disease (MD)\u2014in the population of industrialized countries, is estimated at 5\u201320 per 100,000 people annually for ISSHL and 513 per 100,000 people annually for MD ,5,6. ISSThe temporal bone is a major part of the lateral skull base that contains critical structures including the middle ear, the inner ear, cranial nerves, and numerous vessels . The onlTo achieve a sustained drug delivery to the inner ear, the substance has to be supplied continuously at the RWM resulting in continued diffusion to the inner ear. Thereby, a high concentration of active ingredient would be achieved locally, while the systemic burden on the organism remains low. Side effects for the patient can be significantly reduced. A new approach that offers the potential for sustained inner ear local drug delivery is an additively manufactured, patient individualized, drug-loaded implant that fits precisely into the RWN. In order to manufacture such an individualized implant, a three-dimensional (3D) representation of the patient specific RWN is constructed based on image segmentation of a computed tomography (CT) or cone beam computed tomography (CBCT) scan of the temporal bone. Manual segmentation\u2014manual slice-by-slice identification and outlining of the relevant anatomy of the RWN in CBCT scans of temporal bones using a computer software\u2014is time consuming and requires considerable effort by trained technicians or clinicians ,21. ConsCurrent segmentation approaches of medical images represent the structures of interest by identifying image voxels based on their intensity level variations, or Hounsfield values (HV) .The process of auto-segmentation of the inner ear is facilitated by the fact that the cochlea is a fluid filled structure mainly surrounded by radio dense hard bone , providiTo date, several software tools that can enhance and accelerate the segmentation of structures in the temporal bone have been developed  but noneIn our prior study an otolaryngologist used her anatomical knowledge in addition to the image intensity and manually segmented the anatomy of 50 RWN and found variations in volume and shape of the RWM and RWN . HoweverTo verify the accuracy of the developed semi-automated approach a comparison of 20 clinical cone beam computed tomography datasets of unilateral temporal bones was performed by semi-automated segmentation using a customized 3D Slicer\u2122 plugin with a previous manual segmentation of these datasets.The applicability of the developed software was verified in three body donor implantation tests. The respective region of interest (ROI) was imaged, the developed semi-automated segmentation approach was used to generate a RWN reconstruction, a RNI was built by additive manufacturing and the implantation feasibility and fitting accuracy were evaluated in the respective donor and compared to a RNI made for the same RWN based on manual segmentation.In order to develop an individualized RNI we wrote a software tool that assists the user to create a suitable 3D model that is based on a CBCT volume image. The software was validated by additively manufacturing RNIs and performing implantation tests to determine implantability and therefore the suitability of the software for clinical use. To assess the time saved for the users, we compare the developed software to the manual segmentation procedure.Twenty anonymized unilateral temporal bones CBCT datasets of patients were included in the study. The protocol for this retrospective study for using the patient\u2019s data was approved by the responsible ethics committee approved (Project identification code 3699-2017). The selected patients were included based on no history of oto-surgical manipulation, no diseased or malformed cochleae. A clinical 3D ACCUITOMO 170 Digital CBCT scanner  was used for scanning the patients. Resulting CBCT volumes with an exposure time of 30.8 s and a computed tomography dose index of 8.04 mGy and were reconstructed in an isometric voxel size of 0.08 mm \u00d7 0.08 mm \u00d7 0.08 mm and exported as Digital Imaging and Communications in Medicine (DICOM) data using i-Dixel software version 1.68  . The semLike in our earlier work , for eacAs depicted in In the following paragraphs, each step within the development of the software is described in detail. In short, the boundary between the inner ear and the middle ear, i.e., the RWM, was defined by applying an oval cut-out of a saddle shaped surface with four control points (step 1). The bony area was identified by thresholding the CBCT intensities within the ROI (step 2). After the user determines the boundary of the RWN towards the middle ear, the RWN, which will be filled by the implant-body, is completely defined (step 3). The user can add and adjust a handle to the implant that can be used by the surgeon to hold the implant with forceps step 4; e. Since To perform these steps, a cubic ROI with an edge length of 5 mm, centered at the estimated position of the RWN is cropped from the CBCT scan c. When u1. The round window membrane cannot be identified in clinical CBCT images. At best, in really good images, it is possible to identify a slight contrast between the air in the RWN and the liquid in the cochlea to determine the boundary between middle and inner ear. To overcome this, a saddle shaped surface model of the RWM, which is represented by a bilinear interpolation of four points, is implemented see b and allThe initial location of these four points relative to the center of the RWM is based on a mean position that has previously been determined in high resolution \u00b5CT scans. In \u00b5CT datasets of temporal bone specimens that were scanned with a voxel size of 16 \u00b5m \u00d7 16 \u00b5m \u00d7 16 \u00b5m, we placed four fiducials in a way that the bilinear interpolation fitted the anatomical structure to a high degree. The mean positions were obtained by repeating this for six RWMs and averaging the positions within our CCS. The user can place the mean RWM model in the clinical scan and manipulate the points in 3D in order to fit the structure of the individual RWM and cochlea.2. To threshold the bone around the RWN, fitting the peaks for bone, soft tissue, and air in the intensity-histogram of the ROI provides an initial estimation for the threshold value for bone. This value can further be refined by the user. In case that the CBCT image has a high noise level, a slight Gaussian blurring with a kernel width typically in the order of one voxel can be applied by the user to obtain a smooth surface of the implant.3. Since the RWN is a half-open structure without a clear border towards the middle ear cavity, the extend towards the middle ear is somewhat arbitrary. The user can refine the extent to which the niche is filled by dragging a slider in the user interface (UI) . To undeThe user can not only influence the \u201cwater level\u201d, i.e., the RWN filling, interactively in the UI  but also4. A handle for the implant is created on the surface of the implant facing the middle ear e. The shhttp://www.slicer.org, accessed on 12 January 2022)  [Manual segmentation: An experienced otolaryngologist, highly specialized in segmentation of the temporal bone, performed the manual segmentation of the 20 CBCT datasets using 3D Slicer\u2122 version 4.11  . The RWNMA, USA) . In shorMA, USA) .Semi-automated segmentation: Two otolaryngologists, one experienced and one at the beginning of her residency, performed individually, after a brief explanation of the new software, the semi-automated segmentation as described above on each of the 20 CBCT scans.Step (1) Removing voxels inside the RWN model.Step (2) Removing voxels classified as bone.Step (3) Removing voxels that are above the \u201cspill-over\u201d filling level.Data analysis: Aiming to compare the segmentation methods, we focus on the volume of the RWN calculated by counting the voxels of the implant, before adding the handle and multiplying it with the voxel volume. We calculated the Dice similarity coefficients (DSC) and Jaccard indices (J). In order to better understand where the differences between the manual and semi-automated segmentation arise from, we remove voxels from the manual segmentations that would not be classified as implant by applying the steps 1\u20133 described in the text above:In addition, we compared the area of the RWM calculated based on the number of voxels making up the contact surface between niche and scala tympani.The results of the semi-automated segmentation were compared to the manual segmentation of the same 20 CBCT scans.To evaluate the implantation feasibility and fitting accuracy of RNI generated from manual segmentations in comparison to RNI generated from the semi-automated segmentation, three previously anonymized formalin-fixed human temporal bones\u2014with in total three RWN\u2014were implanted. The use of human temporal bones was approved by the responsible ethical committee and registered under the number 9236_BO_K_2020. An experienced otorhinolaryngologist performed all manual and semi-automated segmentations of the RNI as described above and implanted and compared the 3D printed individualized RNI (see below). The implantation trials were blinded as the otorhinolaryngologist did not know which RNI\u2014manually segmented or semi-automatically segmented RNI- were handed for the corresponding temporal bone to avoid bias.For the development of the corresponding RNI, a mobile intraoperative CBCT scanner ) was utilized for image acquisition. All images were captured with an isometric voxel size of 0.3 mm \u00d7 0.3 mm \u00d7 0.3 mm. All the segmentations of these CBCT images were done by the same experienced otorhinolaryngologist who performed the manual and semi-automated segmentations of the 20 temporal bones mentioned above.After the RNI were printed (see the description of the printing process below) the fitting accuracy of the manually segmented and semi-automatically segmented RNI was evaluated and compared based on surgical visual judgment and tactile feedback regarding correct representation of the surgical approach, the general handling of RNI with the handle and the fitting accuracy of the RNI. The insertion was done by an experienced otorhinolaryngology surgeon (User 1) and a conventional transmeatal approach through the external ear canal was performed to visually assess the RWN using a surgical microscope ). For the insertion of the RNI standard surgical forceps were used.\u00ae Manufacturers Series , equipped with a low temperature printing head operated by pneumatic pressures of 5 bar and an UV Curing Head at 365 nm. Medical grade UV silicone  with its silicone catalyst  in a ratio of 50:1 was prepared using the Speedmixer\u2122 DAC 150.1 FVZ  for two minutes operated by 3500 rpm. The silicone was loaded into the low temperature head attached with a 200 \u03bcm dispensing needle tip  and printed at 27 \u00b0C at a movement speed of 2 mm/s. The silicone was crosslinked layer-by-layer using the UV-light head of the printer.After the manual and semi-automated segmentation, a Standard Tesselation Language (STL) file of each digital model was generated as a routine function in 3D Slicer\u2122 and used for 3D printing. The STL file was loaded into the Perfactory RP software version 3.2.3594  and was sliced into 160 \u03bcm slices . The resulting file was imported to EnvisionTEC Visual Machines software version 2.10.130r12, where the model was assigned an infill with a fiber spacing of 0.2 mm and a 90\u00b0 layer-to-layer rotation, and a single contour outline. The RNI were 3D printed using a 3D-BioplotterComparing the volume of the RWN of the semi-automated segmentation of the two users with the manual segmentation , one canThe manual segmentation for labeling the RWN took around 30 min for each unilateral temporal bone dataset, whereas using the semi-automated application took only three to five minutes and that included the creation of the RNI STL file for 3D printing.p = 1.7 \u00d7 10\u22125), the volume of the semi-automated RWN segmentations were 48 \u00b1 11% smaller than the manual segmentations. This difference in volume between semi-automated and manual segmentation is quantified by calculating the DSC the Jaccard index as shown in The bars of the manual and automated segmentations show a clear correlation  removes 16 \u00b1 11% of the voxels , cropping inside the RWN (towards the cochlea) removes 19 \u00b1 7% of the manual segmentation, applying the threshold for bone removes 21 \u00b1 8% of the voxels. When all the rules are applied on the manual segmentation, the remaining segmentations have 7 \u00b1 6% less volume compared to the semi-automated segmentations.Using three temporal bones, the otorhinolaryngologist considered the general handling prior to the insertion of all RNI  including their handle with the forceps as feasible. Direct visual contact with the tip of the instrument and with the RNI could be sufficiently obtained throughout the handling towards the RWN using a binocular microscope. The handle on the surface of all RNI enabled appropriate handling with the forceps and the arrowed side of the handle provided information about the orientation in which the RNI should be implanted. During visualization of the RWN in one temporal bone obstructions were visible in the RWN which were removed before insertion of the RNI.After the handling, image documentation and rating of the implantations of both RNI\u2014manually segmented and semi-automatically segmented\u2014in each temporal bone the RNI were unblinded.The manually segmented RNI did not fit into the corresponding RWN in all three temporal bones. The volume of the RWN seemed to be too large to pass the border of the bony edges of the RWN. Several attempts to press the manually segmented RNI into the corresponding RWN failed .The otorhinolaryngologist rated the fitting of all semi-automatically segmented RNI as good, with all implants sitting pressure-tight in the RWN, allowing visualization of all bony edges of the RWN without room for wobbling in the RWN .Assembly time for the insertion of the semi-automated segmented RNI was less than ten seconds in all three implantations and the total time from the beginning of the transmeatal approach to final positioning of the RNI in the RWN was less than 10 min. Traditional studies on temporal bones mainly involved cadaveric dissections and histopathologic analysis , but witThis is the first study to use a semi-automated approach for segmentation of the RWN anatomy. Our group focused on developing this approach since it is an important step in the process of establishing 3D printed individualized implants for RWN based drug delivery to the inner ear. The advantages of our semi-automated approach of RWN segmentation are fourfold: (1) it requires only little manual input, (2) yields segmentation results surpassing those created by trained experts because it avoids over-segmentation as demonstrated in the ex-vivo validation, (3) delivers results considerably faster than manual. (4) Non-expert users can produce better results  with the help of the software than the experienced surgeons performing the manual segmentation. The semi-automated method avoids over-segmentation mainly because it is not influenced by the windowing (brightness/contrast) setting of the DICOM viewer, which makes it more reliable.Additive manufacturing of drug loaded individualized implants that can be positioned in the individually shaped RWN may overcome the uncontrolled delivery of drugs to the inner ear. Additive manufacturing, also referred to as 3D printing, enables to create implants adjusted to the individual anatomical needs of a patient ,41,42. TThe first step in developing additively manufactured implants requires images obtained from a comprehensive CT or CBCT scan of the region to be implanted. These images are used to produce a computer-aided design drawing, STL file format respectively, of the object to be manufactured . Even thU1, SemiU2) and the J  .The volume of the manually segmented RWN was 48 \u00b1 11% bigger than the volume of the corresponding RWN using the semi-automated approach and this also explains the relatively small DSC and J between the semi-automated approach and the manual segmentations . CroppinExcluding overlapping areas and removing voxels from the RWN and RWM analysis, as shown in Limitations of our study include that a small number of users tested the semi-automated approach and could make suggestions for suitable iterations and only anatomically normal temporal bones were segmented. There is also a need to investigate how the segmentation algorithms performs in relation to abnormal anatomy of the temporal bone such as vestibulocochlear malformations . In addiWhile there is much progress in deep learning and related methods for fully automated segmentations, we did not find any literature specifically for the RWN. A prerequisite for deep learning is a large high quality training data set. As it turns out to be a difficult manual task and just training a deep learning network would likely just replicate the human over-segmentations. Therefore, we hope that our semi-automated tool can help to collect enough high quality and human reviewed segmentations to train a deep learning network in the future.The aim of the study was to write a plug in software for a semi-automated segmentation of the RWN to ease the segmentation of the region for a more efficient workflow of additively manufactured individualized drug delivering RNI. We did not aim to evaluate whether semi-automated segmentation is better or worse than manual segmentation but aimed to validate our approach using manually generated data sets. But in the ex-vivo experiments we figured out that the manually segmented RNI did not fit into the RWN but the RNI based on our written software did nicely fit into the respective individual niche. Therefore, we can state that the developed plug allows a segmentation which is so close to the real anatomical condition that based on this software 3D printed RNI fit precisely into the individual RWN."}
+{"text": "Population covering educational attainment registers have been proven helpful for planning and research concerning educational efforts. Regular linking of different databases is needed to build and update such a register. Without unique national identification numbers, record linkage must be based on quasi-identifiers such as names, date of birth and sex. High-quality record linkage require the unique identification of persons. Therefore, available identifiers should be sufficient for unique identification despite missing identifiers for some cases. Redundant identifiers can achieve this goal. However, the data protection principle of data minimization, as recommended in the European General Data Protection Regulation, aims to avoid additional data if possible for the given purpose. Therefore, a ministry commissioned a simulation study to inform legislators on the minimum set of identifiers needed for a national register. A microsimulation of the population consisting of nearly 20 million people was implemented to generate data on accumulating changes and errors in identifiers over ten simulated years. The simulation covered, for example, international migration, regional mobility, marriages, school careers and mortality. Each event triggered changes of identifiers according to specified error probability models. The resulting data were linked by different record-linkage procedures. Linkage quality and linkage bias dependent on the available identifiers were assessed. We report on the design of the simulation study, the linkage results and recommendations for the minimum set of identifiers. The results may be helpful for the design of other population covering registers."}
+{"text": "However, in outbred forest trees, it is almost impossible to generate the F2 population because of their high heterozygosity and long generation times. We proposed a novel strategy to construct an integrated genetic linkage map that contained both parental recombination information, with restriction-site-associated DNA sequencing (RADSeq) data in an F1 hybrid population of Populus deltoides and Populus simonii. We selected a large number of specific RAD tags to construct the linkage map, each of which contained two SNPs, one heterozygous only in the female parent and the other heterozygous only in the male. Consequently, the integrated map contained a total of 1154 RAD tags and 19 linkage groups, with a total length of 5255.49 cM and an average genetic distance of 4.63 cM. Meanwhile, the two parent-specific linkage maps were also constructed with SNPs that were heterozygous in one parent and homozygous in the other. We found that the integrated linkage map was more consensus with the genomic sequences of P. simonii and P. deltoides. Additionally, the likelihood of the marker order in each linkage group of the integrated map was greater than that in both parental maps. The integrated linkage map was more accurate than the parent-specific linkage maps constructed in the same F1 hybrid population, providing a powerful genetic resource for identifying the quantitative trait loci (QTLs) with dominant effects, assembling genomic sequences, and performing comparative genomics in related Populus species. More importantly, this novel strategy can be used in other outbred species to build an integrated linkage map.The genetic linkage maps of the traditional F Without any doubt, accurate linkage maps are more powerful for locating QTLs and anchoring chromosome-scale sequences [Genetic linkage maps display the linear orders of groups of DNA markers with genetic distances between them, playing a crucial role in identifying the quantitative trait loci (QTLs), assembling genomic sequences, and performing comparative genomics ,2,3,4. Fed lines . Mather dividual . Thus, wequences ,7. In adequences ,9.2 populations as in the inbred lines, due to their long generation times and high heterozygosity [1 hybrid population was usually established by crossing two diverged individuals as a mapping population, and the so-called \u201cpseudo-testcross\u201d strategy was applied for linkage mapping, leading to two independent parental linkage maps [1 hybrid population [2 maps in inbred lines have rarely been reported to date. The main reason is that the overwhelming majority of molecular markers segregate in the ratio of 1:1 in the F1 progeny, at which one parent is heterozygous and the other homozygous, but there were no sufficient markers segregating as bridges in both parents for generating a complete linkage map [2 linkage maps in inbred lines.However, for outbred species, especially in forest trees, it is almost impossible to derive the Fzygosity ,11. Therage maps ,13,14,15pulation ,17,18,19kage map . Meanwhikage map ,21,22,231 hybrid population of P. deltoides and P. simonii. In our previous studies [P. simonii [P. deltoides and P. simonii. Such a linkage map differed from those constructed with the \u201cpseudo-testcross\u201d strategy in forest trees, providing full genetic information for locating quantitative trait loci (QTLs), assembling genomic sequences, and performing comparative genomics. Furthermore, the proposed strategy could be applied to other outbred species for constructing an integrated linkage map with an F1 hybrid population.With the advances in next-generation sequencing technologies, a large number of single nucleotide polymorphisms (SNPs) can be obtained in a fast and cheap way across a hybrid population for genetic mapping. These SNPs provide opportunities for mining special markers to perform various genetic studies. In the current study, we proposed a novel strategy to construct an integrated linkage map that contained both parental recombinant information between the adjacent markers in an F studies ,24, a la simonii . We chos1 hybrid population for constructing the parental integrated linkage map. The F1 population was derived by crossing a female P. deltoides and a male P. simonii in the springs of 2009 and 2011, as described in our previous studies [http://www.ncbi.nlm.nih.gov/Traces/sra, accessed on 1 May 2022).We randomly selected 257 trees in an F studies . The selP. simonii [mem command; (2) each SAM file was converted into a BAM file and then was sorted and indexed with SAMtools; (3) each sorted BAM file was converted into a BCF file using BCFtools with its parameters set as follows: \u201cbcftools mpileup -Obuzv -a AD,INFO/AD \u2013f\u201d; (4) for each parent, a VCF file was obtained from its BCF file with the command as \u201cbcftools call-m -v -f gq\u201d; (5) the SNPs of each parent were extracted from their VCF files and merged into a site list file, in which the allele read coverage depth (DP) of each genotype at each SNP site was \u22653, and the corresponding genotyping quality (GQ) was >30; (6) for each progeny, a VCF file was derived from its BCF file according to the site list file obtained above, with the command as \u201cbcftools call-m -v -f gq; (7) the SNP genotypes for all individuals were extracted from their VCF files such that the allele DP \u2265 3 and GQ > 30, finally leading to a SNP dataset.First, the PE read data of each tree was filtered to produce high-quality (HQ) data with the NGS QC toolkit (v2.3.3) . Next, w simonii  to call  simonii , SAMtool simonii . The whop < 0.01) or had \u226520% missing genotypes, it was removed from the dataset.A chi-square test was performed for each SNP to check whether it follows the Mendelian segregation ratio. If an SNP seriously deviated from the Mendelian ratio .As a novel strategy, we selected the RAD tags (<1000 bp) that contain two SNPs to construct an integrated linkage map of  simonii A. These  simonii B,C. Firssoftware . Apparennto \u201cab\u201d . It is esoftware . Third, software  and FsLiaa\u00d7ab with JoinMap. In order to investigate the performance of the three linkage maps, we compared their collinearities with a reference sequence. Additionally, for a linkage group, the linear orders of the three linkage maps were compared by calculating the likelihoods with the transformed and merged genotype data using MapMaker.After the above procedures, the female linkage map and an integrated linkage map were generated. Meanwhile, the male linkage map was obtained by just using the SNPs of 1 hybrid population by aligning the HQ reads to the reference genome of P. simonii. Like the results in our previous studies [ab\u00d7aa and aa\u00d7ab, amounting to 29,466 and 18,863, respectively. From the two types of 1:1 segregated SNPs, a total of 1154 RAD tags were obtained for constructing an integrated linkage between the two parents. Each RAD tag had two SNPs within a range of <1 Kb, one of which was segregated in the type of ab\u00d7aa and the other in aa\u00d7ab  thresholds ranging from 5 to 17 and thus perfectly matched the karyotype of Populus  of all the RAD tags  [2 population from inbred lines, the two haplotypes of an individual can be discriminated from its genotype with the RAD tags. For this reason, the probability of an assumed QTL genotype conditional on the flanking RAD tags could be easily derived, and thus the likelihood of the QTL effects could be established. After that, the calculation for the parameters can be implemented through the expectation\u2013maximization (EM) algorithm [Populus species.In theory, the integrated linkage map provides a powerful genetic resource for identifying QTLs, with additive and dominant effects in the Fing (IM)  and comping (IM) . Unlike lgorithm . We expeP. deltoides and P. simonii with next-generation sequencing data. However, the conventional linkage mapping methods for such a hybrid population led to two parent-specific linkage maps, each lacking the full recombinant information of both parents. The integrated linkage map proved to be more accurate than the two parent-specific linkage maps, providing a powerful genetic resource for identifying quantitative trait loci (QTLs) with dominant effects, assembling genomic sequences, and performing comparative genomics in Populus. Most importantly, this novel strategy can be used in other outbred species for constructing an integrated genetic linkage map.In this study, a novel strategy was proposed and successfully applied to construct an integrated linkage map for"}
+{"text": "HIF1A-silenced cells, the mRNA and protein expression levels of AQP1 remained unaltered. In the permeability experiments, a statistically significant volume increase was observed at the 360 s time-point in the LPS-exposed HPMECs, while LPS-exposed HIF1A-silenced HPMECs did not exhibit cell swelling, implying a dysfunctional AQP1. AQP1 did not seem to affect cell apoptosis yet could interfere with endothelial migration and/or proliferation. Based on our results, it seems that HIF1A silencing negatively affects AQP1 mRNA and protein expression, as well as AQP1 function, in the setting of lung injury.Aquaporin-1 (AQP1), a water channel, and the hypoxia-inducible factor 1\u03b1 (HIF1A) are implicated in acute lung injury responses, modulating among others pulmonary vascular leakage. We hypothesized that the AQP1 and HIF1A systems interact, affecting mRNA, protein levels and function of AQP1 in human pulmonary microvascular endothelial cells (HPMECs) exposed to lipopolysaccharide (LPS). Moreover, the role of AQP1 in apoptosis and wound healing progression was examined. Both AQP1 mRNA and protein expression levels were higher in HPMECs exposed to LPS compared to untreated HPMECs. However, in the LPS-exposed  Diffuse endothelial injury, intense promotion of the coagulation system, increased capillary permeability, hyaline membrane formation, and hypoxemia comprise the main pathological characteristics of acute respiratory distress syndrome (ARDS) ,2. ARDS When injury is present in the lungs, the damage caused to the endothelium results in a diffuse inflammatory response and edema formation in the alveolar space. To eliminate the expansion of inflammation, endothelial cells become active, and alter their shape and function to facilitate the innate and adaptive immunity responses . EndotheAquaporin 1 (AQP1), a water channel, is expressed in the majority of endothelial cells and epithelial barriers in healthy humans . In the On the other hand, the degree of hypoxemia is the main parameter for the ARDS severity classification . HypoxiaHIF1A affects AQP1 mRNA and protein expression levels, as well as the water permeability function of AQP1. Additionally, we investigated whether, in our model, AQP1 is implicated in other biological processes, including cell apoptosis and wound healing, apart from its principal role as a water channel.In this study, we aimed to investigate the interaction between AQP1 and HIF1A in lipopolysaccharide (LPS)-exposed human pulmonary microvascular endothelial cells (HPMECs). We examined how the silencing of AQP1 mRNA expression levels were measured in untreated HPMECs (ctr), HPMECs exposed to LPS for 24 h, and HIF1A-silenced HPMECs either exposed or not to LPS. The changes in AQP1 mRNA expression levels are presented in AQP1 mRNA levels in HPMECs exposed to LPS for 24 h were elevated 175-fold compared to untreated HPMECs (p < 0.001). However, in the HIF1A-silenced HPMECs, exposure to LPS did not affect AQP1 mRNA expression (p > 0.05).p < 0.01). The AQP1 protein levels in the HIF1A-silenced HPMECs exposed to LPS were not significantly increased compared to untreated HPMECs [1.68 (0.83\u20131.74) vs. 0.80 (0.79\u20130.96), respectively, p > 0.05; We also examined the protein expression levels of AQP1. The AQP1 relative protein expression is presented in HIF1A silencing promote functional changes in HPMECs by interfering with AQP1 activity. The results of the permeability assay are presented in p < 0.0001), indicating an increase in AQP1 function. On the contrary in the LPS-exposed HIF1A-silenced HPMECs there was no increase in cell volume at the 360 s time-point . When HPMECs were exposed to HgCl2 (0.3 mmoL/L for 5 min), an AQP1 blocker, the cells exhibited cell volume shrinkage compared to untreated HPMECs , as expected.AQP1, as mentioned above, is a water channel and among others, its main function is water movement. Therefore, we examined whether LPS exposure and AQP1-silenced HPMECs, HPMECs exposed to either the AQP1 inducer, phorbol 12-myristate 13-acetate (PMA), or its inhibitor HgCl2, and in HIF1A-silenced HPMECs exposed to LPS. As presented in We next aimed to examine whether dysregulated AQP1 expression affects apoptosis in HPMECs. Therefore, we measured the activity of caspase 3 in untreated HPMECs, HPMECs exposed to LPS, HIF1A silencing interfere with the postulated role of AQP1 in wound healing. The migration rate was calculated as the percentage of wound closure over time. Eighteen (18) hours following wound creation, the untreated HPMECs exhibited 63% wound closure, while at 24 h closure reached 72%. The rate of wound closure in the HPMECs treated with LPS and the LPS-exposed HIF1A-silenced HPMECs, at 18 h was 39% and 40%, respectively, while at 24 h closure reached 55% and 57%, respectively. Hence, as seen in HIF1A-silenced HPMECs exhibited a slower wound healing rate compared to untreated HPMECs (p < 0.01).The impact of AQP1 was explored in a wound healing assay. Alongside, we also examined whether LPS exposure and HIF1A-silenced HPMECs, no increase in the mRNA and protein expression of AQP1 was observed. In the same manner, at the functional level, silencing of HIF1A once again abolished the effect of LPS on the osmotic cell challenge. Our results could indicate a possible role of HIF1A in the LPS-induced changes observed in the mRNA and protein expression levels and function of AQP1.In the present study, we demonstrated that the AQP1 and HIF1A systems interact under inflammatory conditions. The exposure of HPMECs to LPS resulted in an upregulation of the mRNA and protein expression of AQP1. Moreover, LPS treatment increased the relative cell volume when compared to untreated HPMECs. However, in the LPS-exposed LPS is a component of the outer membrane of Gram-negative bacteria and a known immunostimulatory agent inducing pro-inflammatory responses . Many exInflammatory stimuli such as bacteria and bacterial components, including LPS, are known to upregulate HIF1A. HIFs are transcription factors that under normoxia are inactivated; however, under hypoxic conditions become activated. In particular, hypoxia enables HIF1A stabilization, enabling it to form heterodimers with the HIF1B subunit. In the nucleus, the heterodimeric transcription factor HIF1A/HIF1B binds to promoters containing the hypoxia-response element (HRE), inducing the HIF-dependent transcription of target genes ,25.HIF1A in HPMECs seems to abrogate the LPS-induced AQP1 upregulation. HIF1A-silenced HPMECs exposed to LPS did not exhibit increased mRNA and protein expression of AQP1, indicating a possible interplay between the two systems.Several studies, examining both in vitro and in vivo septic lung inflammation models, revealed that following lung injury HIF1A is activated in endothelial cells; hence, inducing HIF signaling pathways has a protective role, by promoting cell proliferation, vascular repair, and resolution of inflammation. These studies imply a potential treatment strategy based on the therapeutic activation of HIF1A signaling pathways ,27,28. OHIF1A silencing on the hypoxia-induced AQP1 upregulation was studied in an in vitro model of primary cells [AQP1. Silencing of AQP1 caused cell shrinkage, whereas AQP1 overexpression caused cell swelling [2, an AQP1 blocker, promoted cell shrinkage. Moreover, Zhang et al. investigated the involvement of HIF1A in AQP1 regulation. In agreement with our results, they concluded that silencing of HIF1A decreased the hypoxia-induced mRNA and protein AQP1 upregulation. In our study, we also demonstrated that HIF1A silencing abrogated the LPS-induced increase in AQP1 mRNA and protein levels, and the water permeability function of AQP1. The role of AQP1 in water permeability and edema formation, and the effect of ry cells . To examswelling . These rAQP1 or exposed to HgCl2, compared to untreated HPMECs. Moreover, the apoptosis rate remained stable in the HPMECs silenced for HIF1A and treated with LPS. We conclude that in our model, AQP1 does not affect cell apoptosis.Many studies have underlined the importance of AQP1 in cell survival and cell migration. However, the exact role of AQP1 in the abovementioned processes differs based on the model examined. In an in vitro model of septic acute kidney injury, overexpression of AQP1 promoted cell viability and reduced apoptosis , while iHIF1A-silenced HPMECs, wound closure was slower than the closure rate observed in untreated HPMECs. This might suggest that in pulmonary microvascular endothelial cells, overexpression of AQP1 possibly interferes with the processes of endothelial migration and/or proliferation, which could result in the loss of vascular integrity.Endothelial cell migration is important in injury repair and vascular integrity maintenance, while it also occurs during vasculogenesis and angiogenesis . AQP1 exSince the pulmonary endothelium has been recognized as a key modulator of lung disorders, such as ARDS, we performed our experiments on human pulmonary microvascular endothelial cells. It should be noted that widespread vascular endothelial injury is thought to be a major mechanism for multiorgan dysfunction in sepsis, and pulmonary microvascular endothelial injury in ARDS . The cel\u00ae Fast PCR Master Mix, Merck KGaA, Darmstadt, Germany), which was carried out on a BioRad CFX Connect thermocycler . Primers were designed for specific genes using information from the NCBI Sequence database. The primers\u2019 sequences are listed in \u2212\u0394\u0394CT [GAPDH was used to normalize the levels of expression of the target genes.RNA was extracted, using the TRI reagent , reverse transcribed , and the resulting cDNA was used as a template for the real-time PCR analysis  method [Using a hand-held homogenizer, cells were homogenized on ice in phosphate-buffered saline (PBS) with the addition of 1 mM dithiothreitol (DTT) , 1 mM phenylmethylsulfonyl fluoride (PMSF)  in isopropanol, and 1% protease inhibitors . The homogenate was centrifuged at 4 \u00b0C for 5 min at 1000\u00d7 ) method  was used) method  was usedOsmotic swelling was measured as previously reported . HPMECs Apoptosis was measured using the caspase 3 kit from Sigma-Aldrich , following the manufacturer\u2019s instructions. Briefly, cells were incubated in the presence of 1 \u03bcg/mL staurosporine  for 3 h to induce apoptosis, and cell lysates were collected for the measurement of caspase 3 activity.Twenty-eight thousand  HPMECs were seeded in 24-well cell culture plates (60\u201370% confluency the next day). When cells reached 100% confluence, a wound in the shape of a cross was created using a 200 \u03bcL pipette tip. Cells were then washed once with PBS to remove cell debris and were observed on a Zeiss Axiovert 25-phase contrast microscope  when the first image was retrieved (time-point 0 h) using a Canon digital camera . The second image was retrieved after 18 h, while the last image was retrieved after 24 h. Images were analyzed using the ImageJ software. The migration rate was expressed as the percentage of wound closure over time. p-values under 0.05 were considered significant.Data are presented either as means \u00b1 standard deviation (SD) or medians with the interquartile range (IQR), accordingly. Statistical analysis was performed by one-way or 2-way ANOVA followed by Kruskal Wallis, Tukey\u2019s, or Wilcoxon tests, as appropriate. For the statistical analysis, the GraphPad Prism 6 for Windows  statistical program was used. HIF1A in the LPS-induced changes observed in the mRNA and protein expression levels and function of AQP1, in the setting of lung injury. A better understanding of the emerging roles of AQP1 in both physiological and pathological lung processes could mark the beginning of the investigation of new therapeutic strategies, which could eliminate the overwhelming edema formation and inflammatory responses induced by the infectious injury present in the lungs.Our results indicate a possible role of"}
+{"text": "Case studies further demonstrated that formulaic sequences could contribute to oral fluency development by promoting speed and reducing pausing when retrieved holistically, but they sometimes lost processing advantages when retrieved and processed in a word-by-word manner. The inappropriate use of formulaic sequences also neutralized the facilitative effects of formulaic sequences on repair fluency and could mirror speakers\u2019 occasional tendency to sacrifice repair fluency for the improvement of speed and breakdown fluency when using formulaic sequences. Pedagogical implications were provided accordingly to promote sustainable oral fluency development through the use of formulaic sequences.Psycholinguistics has provided numerous theories that explain how a person acquires a language, produces and perceives both spoken and written language, including theories of proceduralization. Learners of English as a foreign language (hereafter referred to as EFL learners) often find it difficult to achieve oral fluency, a key construct closely related to the mental state or even mental health of learners. According to previous research, this problem could be addressed by the mastery of formulaic sequences, since the employment of formulaic sequences could often promote oral fluency in the long run, reflected in the positive relationship between formulaic sequence use and oral fluency. However, there are also findings contradicting the abovementioned ones, without adequate explanations. This study aims to explore the roles of formulaic sequences in oral fluency, taking into account the relationship between formulaic sequence use and oral fluency. This study investigated 120 pieces of spoken narratives by Chinese EFL learners, using both quantitative and qualitative methods, combined with artificial intelligence techniques. Results of canonical correlation analysis showed that the frequency of formulaic sequences was significantly related to speed fluency ( Traditionally speaking, in language learning and teaching, teachers and students have been pursuing three goals, namely fluency, nativeness, and intelligibility . In thisHowever, the pursuit of fluency is never a piece of cake. Instead, it could be a hot potato both for teachers and students. Although many people may have certain knowledge of another language which is not their mother tongue, they are usually much less fluent in their second language than their first language, and there is a \u201cfluency gap\u201d , p. 2. PSome scholars have claimed that a possible solution to the problem of oral fluency is the mastery of formulaic language, retrieved from long-term memory as if they were single words . The ideThe findings of previous research did demonstrate the significant influence of formulaic language on the natural and fluent proficiency of English , or specTo fill these research gaps, the present study was carried out to explore the roles of formulaic sequences in the three dimensions of oral fluency, namely speed, breakdown, and repair fluency, reflected in Chinese EFL learners\u2019 spoken narratives. The positivity of the proposed work consists in the fact that the present study investigated sophomore English majors in China, whose treatment of instructions was similar to each other, so that the proposed work could better showcase the influences of formulaic sequences on oral fluency under similar circumstances. Results of the present study systematically displayed the correlations between indices of formulaic sequences and the three dimensions of oral fluency, and illustrated the conditions on which formulaic sequences could contribute to oral fluency development by promoting speed and reducing pausing, as well as the situations on which they lost processing advantages. Based on the relationship between formulaic sequence use and oral fluency, the present study could help demonstrate the role of psycholinguistic theories related to proceduralization, in language learning. Such findings also helped students improve their oral fluency by learning formulaic sequences.In previous research, the term \u201cformulaic sequence\u201d was often used interchangeably with \u201cformulaic language,\u201d as an umbrella term , which rThe most widely employed definition of formulaic sequence is by a sequence, continuous or discontinuous, of words or other elements, which is, or appears to be, prefabricated: that is, stored and retrieved whole from memory at the time of use, rather than being subject to generation or analysis by the language grammar. (p. 9)However, this definition was later regarded as a stipulative and not operational one , and thaIn her later work, Based on this distinction, The present study adopts the speaker-external approach. The main reason is that the speaker-external approach generally involves the phraseological approach regarding the identification of formulaic sequences, which is essentially meaningful, since apart from frequency there should be other features that make formulaic language formulaic . The speThe present study combines the definition by Different from the broad-narrow division, The present study only focuses on utterance fluency. The reason is that only this sense of oral fluency has a concrete and measurable nature, allowing objective and systematic examination and measurement based on the acoustic characteristics of speech , and it Utterance fluency, which is the focus of the present study, can be further divided into two aspects, namely speed and smoothness. Related to these two aspects of fluency is another triadic framework of fluency constructed by The present study adopts the aforementioned triadic framework of fluency constructed by The relationship between the use of formulaic sequences and oral fluency has psycholinguistic basis. The facilitative effect of formulaic sequences on oral fluency could be illustrated from a cognitive perspective, concentrating on proceduralization. The holistic hypothesis could reflect the issue of proceduralization, which holds that formulaic sequences are stored as single units that are directly addressable and retrievable from the mental lexicon and that they have been lexicalized . Pawley The issue of proceduralization is also reflected in the language production model. According to the language production model , languagMost of the previous studies investigated the relationship between the use of formulaic sequences and oral fluency from a quantitative approach. They usually designed experiments to investigate participants\u2019 processing of formulaic sequences, or calculated the correlations between indices of formulaic sequences and those of oral fluency. Quantitative analysis in previous research employed different measurement of oral fluency when investigating the relationship between the use of formulaic sequences and oral fluency.Some of the previous studies employed subjective judgment to measure fluency, so what they actually examined was the relationship between the use of formulaic language and perceived fluency. As for utterance fluency in contrast to perceived fluency, some other studies employed objective measurement to measure the speed of speech, focusing on the relationship between the use of formulaic language and speed fluency. There are also several studies that involved breakdown fluency (pausing) when exploring the relationship between the use of formulaic language and oral fluency. Compared with speed fluency and breakdown fluency, repair fluency was not often involved regarding the relationship between the use of formulaic language and oral fluency. Only a few studies have taken repair fluency into consideration. As such, most of the previous research has reported a positive correlation between the use of formulaic language and overall oral fluency. Since formulaic sequences fall into the scope of formulaic language, to a large degree, the positive relationship between the use of formulaic language and oral fluency could mirror the positive relationship between the use of formulaic sequences and oral fluency. These results could correspond well to the findings that formulaic sequences play a crucial role in language use .On the other hand, results of previous research on the relationship between the use of formulaic sequences and oral fluency were not always consistent. Some studies found that the correlations between indices of formulaic sequences and oral fluency did not always reach a significant level. For instance, Also, previous research hardly explored when, where, why, and how could formulaic sequences promote different dimensions of fluency so as to help maintain sustainable oral fluency development in different aspects. Traditional models of the relationship between formulaic sequence use and oral fluency usually treated oral fluency as a whole, or only focused on some of the aspects of oral fluency. However, speed, breakdown, and repair fluency are the three key components indispensable to oral fluency, and their different features suggested the possibility that they would make different contributions to the relationship between formulaic sequence use and oral fluency, thereby deserving more research . Thus, tThis study aims to investigate the relationship between formulaic sequence use and oral fluency from both quantitative and qualitative perspectives. Specific research questions were:To what extent is formulaic sequence use related to speed fluency?To what extent is formulaic sequence use related to breakdown fluency?To what extent is formulaic sequence use related to repair fluency?The Test for English Majors-Band 4  is a standardized proficiency test held in China to measure oral proficiency of English majors in the sophomore year. The TEM-4 oral test includes three tasks, often on the similar or related topics. The second task, used to elicit data in the present study, is a spoken narrative task in which the students are required to talk on a given topic. Students are required to prepare for 3\u2009min and then talk for 3\u2009min. This preparation time is regarded as an opportunity to \u201creduce the cognitive load and communicative pressure of the task\u201d by providing students with an opportunity to make a plan about the content of their talk , p. 513.Test-takers are rated on a 100-point scale based on five sub-scales. The overall numeric score is eventually transformed into one of the four ranks considering their positions among all speakers based on numeric scores. The highest is Rank 4, featuring \u201cexcellent\u201d speakers. The second is Rank 3, featuring \u201cgood\u201d speakers. The third is Rank 2, featuring \u201cqualified\u201d speakers. The last is Rank 1, featuring \u201cunqualified\u201d speakers.The present study selected recordings from 2021\u2019s TEM-4 as the data. The present study focused on Task 2, and the topic of Task 2 of 2021\u2019s TEM-4 oral test was to \u201crecall an experience in which you made a mistake and eventually put it right.\u201d Test-takers were required to prepare for 3\u2009min and talk for 3\u2009min as has been mentioned.The present study sampled 120 recordings of the TEM-4 oral test in 2021, together with a detailed list of scores, as well as three separate recordings of performance in three tasks by all the students taking TEM-4 oral test in 2021. Names and other private/confidential information of the test-takers were not shown. While these students\u2019 overall scores and ranks were based on the assessment of their performance in all three tasks, the data used in the present study came from their performance of only Task 2 . The data set contained 120 task performance, 30 from each Rank, totaling 362\u2009min of recordings .Otter,Before the measurement of formulaic sequences, all the recordings were automatically transcribed with the assistance of artificial intelligence techniques, using the online transcription tool The identification of formulaic sequences was completed manually according to a set of standards, including:An FS should be composed of two or more than two words;An FS should be at the phrasal or clausal level;An FS should be contained in the Longman Dictionary of Contemporary English.r\u2009<\u20090.60 in r\u2009=\u20090.92 in All the formulaic sequences were coded with BFSU Qualitative Coder. The whole data set was coded separately by three coders. A major issue with the phraseological approach to measuring formulaic sequences is its involvement of \u201ca fair degree of subjectivity\u201d , p. 516,Then, BFSU Qualitative Explorer was used to calculate the number of formulaic sequences. The frequency, proportion, and variety of each type of formulaic sequences were calculated, since these three indices were shown to be related to oral fluency. Frequency refers to the total output of formulaic sequences in the given time, or in other words, the total number of formulaic sequences in each text transcribed from every test-taker\u2019s recording, reflecting the overall quantity of formulaic sequences employed by speakers; proportion refers to the ratio of formulaic sequences produced in each text, which is the total number of formulaic sequences divided by the total number of words in each text, reflecting speakers\u2019 degree of tendency to use formulaic sequences; and variety concerns how multifarious are the formulaic sequences produced by each test-taker, which is the total types of formulaic sequences divided by the total number of formulaic sequences, and the \u201ctypes\u201d here refer to the total number of formulaic sequences excluding the repeated ones, rather than the so-called types based on structures or functions.Since speed, breakdown, and repair fluency are closely related, there is not a clear boundary between indices measuring speed, breakdown, and repair fluency as they sometimes overlap. Nevertheless, for the sake of convenience and clarity, the present study has been sticking to the tripartite taxonomy, categorizing each index according to the dimension of fluency that is most reflected by it. In the present study, speed fluency was measured by speech rate (SR), articulation rate (AR), mean length of run (MLR), and phonation time ratio (PTR). Breakdown fluency was measured by three indices, namely frequency of silent pauses (FSP), mean length of silent pauses (MLP), and pause time ratio (PAR). Repair fluency was measured by frequency of all repairs per 60\u2009s (FAR), frequency of false starts and reformulations per 60\u2009s (FFR), frequency of partial or complete repetitions per 60\u2009s (FRP), and frequency of self-corrections per 60\u2009s (FSC).Speed fluency and breakdown fluency were measured automatically with artificial intelligence techniques, while repair fluency was measured manually. To measure speed fluency and breakdown fluency, the recordings were analyzed with Praat, using a script developed by X denote the first set of variables, which is m dimensional, and let Y denote the second set of variables, which is n dimensional. Let m\u2009\u2264\u2009n.Let Cov (X), Cov (Y) and Cov  be denoted by Quantitative analysis employed SPSS 25 for the exploration of the relationship between the use of formulaic sequences  and oral fluency . Specifically, the present study employed not only Pearson correlation analysis, but also canonical correlational analysis (CCA), a statistical technique initially developed by is positive definite, where m and n dimensional coefficient vectors. Then, the following equations could be derived:in which The equation of the canonical correlation is as follows:The general procedure of the whole framework is displayed as r\u2009=\u20090.559, p\u2009<\u20090.001), frequency of FSs and articulation rate , as well as frequency of FSs and phonation time ratio , respectively. Thus, total frequency of formulaic sequences is positively related to speed fluency to a large degree, total proportion of formulaic sequences only shows the tendency to be positively related to speed fluency, while total variety of formulaic sequences shows the tendency to be negatively related to speed fluency.To sum up, quantitative analysis indicates a significant positive relationship between frequency of formulaic sequences and speed fluency.According to detailed analysis, the employment of formulaic sequences could promote speed fluency when retrieved and processed as a whole, but failed to promote speed fluency when retrieved and processed in a word-by-word manner, losing their processing advantages.As has been mentioned, there are actually two approaches to formulaic sequences, namely speaker-external and speaker-internal . Based oAs the present study mainly focuses on the speaker-external approach, all the formulaic sequences in the present study are linguistic clusters (LC), but they did not always function as processing units (PU). When a formulaic sequence functioned both as a linguistic cluster (LC) and a processing unit (PU) in the present study, it was stored and retrieved holistically, showcasing proceduralization and processing advantages, promoting speed and breakdown fluency. On the contrary, when a formulaic sequence functioned only as a linguistic cluster (LC) but not as a processing unit (PU), then it was retrieved and processed in a word-by-word manner, similar to that of the non-formulaic language, losing processing advantages, and did not promote speed and breakdown fluency.Formulaic sequences were often uttered at a faster speed. Speakers often uttered formulaic sequences faster than other words. Consider the following example:got around it.[B2-3] But there\u2026 and there were not any snack in my house when I got around,\u201d her speed rose immediately.In example [B2-3], the speaker B2 maintained a slow speed when uttering most of the words in the sentence \u201cand there were not any snack in my house,\u201d but when she began to utter the formulaic sequence \u201cIt should also be noted that the facilitative effects of formulaic sequences were often not influenced by their positions in a sentence. No matter a formulaic sequence occurred at the beginning or end of a sentence, it could promote speed, as long as it was retrieved and processed as a whole. The processing advantages of formulaic sequences would not disappear along with the change of their positions. Consider the following group of examples:at that moment.[C3-1] Everything was fine At that moment, I felt a sense of relief.[C3-4] at that moment\u201d twice in total in his speech, one of which at the beginning of a sentence, and the other at the end of a sentence. In both cases, the formulaic sequence \u201cat that moment\u201d was uttered much faster than other sentences, demonstrating the facilitative effect of formulaic sequences on speed, regardless of their location in a sentence.In examples [C3-1] and [C3-4], the speaker C3 employed the formulaic sequence \u201cThe facilitative effects of formulaic sequences on speed fluency were sometimes due to their easiness of pronunciation in the articulation stage. The speakers sometimes obscured the pronunciation of a component of the word, thereby pronouncing them faster, consistent with previous findings . Consideat home, I felt\u2026 I started to feel guilty about it.[B3-5] But after that, after we have our model planes at home,\u201d the speaker B3 obscured the sound of [t], which made the utterance of \u201cat home\u201d even faster, greatly increasing speed, contributing to speed fluency.In example [B3-5], when uttering the formulaic sequence \u201cSpeakers sometimes chose to link a few formulaic sequences so as to extend their length of run, creating a longer run, consistent with the findings of picked me up as usual\u2026[B6-3] And he picked me up\u201d and \u201cas usual,\u201d hence creating a longer run, namely \u201cpicked me up as usual,\u201d which contained no pauses, posing a contrast to the utterance of \u201cand he,\u201d as he paused for 1\u2009s, respectively, before and after uttering \u201che.\u201dIn example [B6-3], the speaker B6 strung together two formulaic sequences, namely \u201cThe string of two or more formulaic sequences could benefit speakers who often made pausing or repairs in their speech. This would be further illustrated in the next section when discussing the influence of formulaic sequences on breakdown fluency. Consider the following example:I know that a hardworking student do not have to go to the library every day, but have to be hardworking\u2026[C8-1] And after that, have to go to the library every day.\u201d In her speech, this speaker C8 frequently paused and made repairs. But thanks to these formulaic sequences in this entry, she made no pausing or repairs and uttered this sentence faster.In example [C8-1], the speaker used five formulaic sequences in this entry, three of which were strung together to make a longer run, namely \u201cTo sum up, the use of formulaic sequences could often facilitate speed fluency by increasing rate and amount of speech. Formulaic sequences were often uttered at a faster rate or strung up together to create a longer run. The facilitative effects were not influenced by the position of formulaic sequences in a sentence. Formulaic sequences could promote speed fluency for both fast speakers and slow speakers, but the facilitative effects were especially prominent for slow speakers. The facilitative effects were sometimes due to the easiness of pronunciation.Despite the processing advantages of formulaic sequences reported above, it is also possible for formulaic sequences to lose their processing advantages, when they functioned as only linguistic clusters (LCs), failing to promote speed. Actually, in the present study, those speakers who performed not so well in speed fluency, or in other words, whose indices of speed fluency were among the last 50% of all speakers, were more likely to retrieve and process formulaic sequences in a word-by-word manner. Consider the following example:care for\u2026 for everything.[D1-3] And from the things I get a good lesson we should care for\u201d in a word-by-word manner, rather than holistically. She repeated the word \u201cfor\u201d as a filler, instead of repeating the formulaic sequence \u201ccare for\u201d as a filler. This suggests that she did not regard \u201ccare for\u201d as a prefabricated chunk, or as a whole. Consequently, the utterance of \u201ccare for\u201d was slow, even more slowly than other words in this sentence, showing no processing advantage to facilitate speed.In example [D1-3], the speaker D1, whose indices of speed fluency were among the last 20% of all speakers, obviously retrieved the formulaic sequence \u201cIn short, formulaic sequences failed to facilitate speed fluency when they were retrieved and processed in a word-by-word manner. This phenomenon usually occurred in the performance of those slow speakers.r\u2009=\u2009\u22120.298, p\u2009<\u20090.01). Thus, total frequency of formulaic sequences is negatively related to pausing to a certain degree, while proportion of FSs and variety of FSs show a mixed pattern regarding their relationships with pausing.To sum up, quantitative analysis indicates a significant negative relationship between frequency of formulaic sequences and pausing.According to detailed analysis, the employment of formulaic sequences could help reduce pausing when retrieved and processed as a whole, but failed to reduce pausing when retrieved and processed in a word-by-word manner. On the former condition, the formulaic sequences functioned as both linguistic clusters (LCs) and processing units (PUs), while on the latter condition, the formulaic sequences only functioned as linguistic clusters (LCs).The employment of these formulaic sequences could reduce pausing, reflecting their negative relationship with pausing. The effects of formulaic sequences on reduction of pausing are reflected in two aspects. For one thing, formulaic sequences could help reduce the number of pauses. For another, formulaic sequences could help shorten the length of pausing. Consider the following two examples:getting home that night, I was very guilty.[B4-2] \u2026that was my younger sister\u2019s mistake. After getting home,\u201d he uttered it smoothly without any pauses.In example [B4-2], the speaker B4 paused a few times when uttering \u201cthat was my younger sister\u2019s mistake\u201d as well as after uttering \u201cafter.\u201d However, when he retrieved \u201cas usual.[C10-1] And unfortunately, I took her offer as (2\u2009s) \u2026 as usual,\u201d which was uttered with a fast speed and no pausing in between.In example [C10-1], the speaker C10 might originally intend to say something with the meaning of \u201ctake something for granted.\u201d However, he failed to come up with a phrase like that, leading to a pause of 2\u2009s after the utterance of \u201ctook her offer as.\u201d Then, he had to find other expressions to shorten the length of pause, so he finally employed the formulaic sequence \u201cThe two examples above showcased the effects of formulaic sequences on the reduction of the number of pauses (Example [B4-2]) and on the shortening of the length of pausing (Example [C10-1]) respectively.Besides, after the utterance of formulaic sequences, speakers could sometimes uttered the later words smoothly, with few pauses. Consider the following example:in my life that it is uncountable.[B5-3] I made so many (2\u2009s) mistakes in my life,\u201d however, the speech became quite smooth, displaying no pausing. Also, after uttering \u201cin my life,\u201d the speaker also uttered \u201cthat it is uncountable\u201d much more smoothly than \u201cI made so many mistakes,\u201d which might be due to the processing time saved from the holistic retrieval and processing of \u201cin my life.\u201dIn example [B5-3], the speaker paused frequently before uttering the formulaic sequence \u201cin my life.\u201d He even paused for 2\u2009s between the utterances of \u201cmany\u201d and \u201cmistakes.\u201d When uttering the formulaic sequence \u201cTo sum up, formulaic sequences could help facilitate breakdown fluency by reducing the number and length of pauses. The use of formulaic sequences helped reduce pausing not only during their utterance, but also after their utterance. Speakers sometimes borrowed formulaic sequences from task prompts, and these formulaic sequences were usually uttered with no pausing, facilitating breakdown fluency.As has been mentioned in Section \u201cLoss of Facilitative Effects of FSs (LCs) on Speed Fluency\u201d, it is possible for formulaic sequences to lose their processing advantages when they functioned as only linguistic clusters (LCs), failing to reduce pausing. Again, in the present study, those speakers who performed not so well in breakdown fluency, or in other words, whose indices of breakdown fluency were among the last 50% of all speakers, were more likely to retrieve and process formulaic sequences in a word-by-word manner. It should be noted that the indices of breakdown fluency of all speakers were sequenced from the smallest numbers to the largest numbers. Thus, the so-called \u201clast 50%\u201d means that the numbers themselves were larger than the \u201cfirst 50%,\u201d indicating that these speakers were among the last 50% regarding their breakdown fluency performance. Consider the following example:took (1\u2009s) part in a\u2026 in a speaking competition.[C22-2] I took part in\u201d in a word-by-word manner, since she paused for 1\u2009s between the utterance of \u201ctook\u201d and that of \u201cpart,\u201d suggesting that she actually retrieved \u201ctook\u201d and \u201cpark\u201d separately, rather than as a whole.In example [C22-2], the speaker C22, whose indices of breakdown fluency were among the last 20% of all speakers, retrieved the formulaic sequence \u201cIn short, formulaic sequences failed to facilitate breakdown fluency when they were retrieved and processed in a word-by-word manner. This phenomenon usually occurred in the performance of those speakers with more pausing.r\u2009=\u2009\u22120.285, p\u2009<\u20090.01), variety of FSs and frequency of false starts and reformulations , as well as variety of FSs and frequency of repetitions . Thus, total variety of formulaic sequences is negatively related to repairing to a certain degree.To sum up, quantitative analysis indicates a significant negative relationship between variety of formulaic sequences and repairing.Similar to the cases of speed and breakdown fluency, for repair fluency, formulaic sequences also had facilitative effects on the reduction of repairing to a certain degree. However, what distinguished the case of repair fluency from those of speed and breakdown fluency is that formulaic sequences were often entwined with repair phenomena, washing away their facilitative effects on the reduction of repairing. According to detailed analysis, participants often made correction and reformulation of formulaic sequences, repeated the same formulaic sequence as a filler, and reused the same formulaic sequence in self-correction or reformulation. All these led to the decrease of the variety of formulaic sequences along with the increase of repairs, explaining the negative relationship between the variety of formulaic sequences and repairing.The employment of formulaic sequences sometimes helped to reduce the number of repairs, reflecting the facilitative effects on repair fluency. Consider the following example:pocket money at that time.[B20-3] Because I did not have any pocket money\u201d and \u201cat that time\u201d so as to create a longer run of \u201cpocket money at that time.\u201d This speaker B20 often made repairs in her speech. However, the utterance of this run constituting \u201cpocket money\u201d and \u201cat that time\u201d was very smooth, with no repairs.In example [B20-3], the speaker strung together two formulaic sequences, namely \u201cIn this way, the use of formulaic sequences did help to reduce repairing from time to time, promoting repair fluency.When making self-corrections, speakers sometimes did not restart with the targeted information alone, but reused the same formulaic sequences, uttering them again. In other words, they sometimes repeated formulaic sequences together with the targeted information. Consider the following example:quarrel with her\u2026 quarrel with him.[D18-1] I should not quarrel with.\u201d When she made the self-correction, she did not directly utter \u201c(quarrel with her\u2026) him\u201d but uttered \u201c(quarrel with her\u2026) quarrel with him,\u201d reusing the formulaic sequence \u201cquarrel with.\u201dIn example [D18-1], the speaker D18 mistakenly uttered \u201cher\u201d instead of \u201chim,\u201d after uttering the formulaic sequence \u201cSimilarly, when speakers decided to abort their original expressions and restarted their utterances, they sometimes did not restart with the targeted information alone, but reused the same formulaic sequences in their reformulations. Consider the following example:have to for\u2026 have to stop on each floor\u2026[B15-1] The elevator have to for,\u201d but changed his mind and decided to switch to another expression, namely \u201cstop on each floor.\u201d However, he did not directly utter \u201c(have to for\u2026) stop on each floor,\u201d but reused the formulaic sequence \u201chave to,\u201d and uttered \u201c(have to for\u2026) have to stop on each floor.\u201dIn example [B15-1], the speaker B15 originally intended to say something beginning with \u201cTo sum up, speakers often employed formulaic sequences when making self-corrections, restarting utterances, or switching to new information. During these processes, the use of formulaic sequences generally did not have extra facilitative effects on repair fluency, since these formulaic sequences were already being used in self-corrections or reformulations.When speakers failed to come up with words, they sometimes repeated formulaic sequences or those expressions containing formulaic sequences. On these occasions, the formulaic sequences or those expressions containing formulaic sequences actually acted as \u201cfiller words.\u201d This process might help avoid a pause, thereby mirroring the facilitative effects of formulaic sequences on breakdown fluency. However, the frequency of repetitions was counted as an index of repairing, so that the use (repetition) of formulaic sequences here actually had negative effects on repair fluency.The repetition of the same formulaic sequences could sometimes help avoid a pause, which could also help illustrate the facilitative effect of formulaic sequences on reducing the frequency of pauses. Consider the following example:went out\u2026 went out from school.[B6-2] And I went out,\u201d so he repeated the formulaic sequence \u201cwent out\u201d to buy time, avoiding a pause here.In example [B6-2], the speaker B6 did not know what to say after uttering the formulaic sequence \u201cOn some other occasions, the repetition of the same formulaic sequences could not avoid pausing, but could help shorten the length of pausing, which could illustrate the facilitative effect of formulaic sequences on shortening the length of pausing. Consider the following example:went to a supermarket to look for\u2026 look for the best car and I bought it.[C15-2] I have earned about 100 money and I happily went to\u201d with a fast speed and no pausing. But after he uttered the formulaic sequence \u201clook for,\u201d he failed to come up with what he intended to say and paused. Consequently, he repeated the formulaic sequence \u201clook for\u201d as a filler, so as to buy time for later processing shortening the length of the pause here.In example [C15-2], the speaker C15 uttered the formulaic sequence \u201cIn short, speakers in the present study sometimes repeated formulaic sequences. This did lead to the reduction of pausing, but this also led to the increase of repairing.Speakers sometimes made mistakes when using formulaic sequences, and then they had to correct or reformulate their expressions. The frequency of false starts and reformulations as well as the frequency of self-corrections were counted as indices of repairing, so that the use of formulaic sequences here actually had negative effects on repair fluency. Consider the following example:report card\u2026 report card was very bad.[B16-2] So in the first month of the test, my report card\u201d for the first time, he pronounced the word \u201creport\u201d incorrectly, with the wrong stress location. Then he made a self-correction and uttered \u201creport card\u201d again, with a correct pronunciation this time. In this way, the speaker actually used the same formulaic sequence \u201creport card\u201d twice, reducing variety of formulaic sequences while increasing repairs.In example [B16-2], when the speaker B16 uttered \u201cTo sum up, the use of formulaic sequences could bring about self-corrections and reformulations when they contained mistakes. Under these circumstances, they actually had negative effects on repair fluency.Quantitative analysis indicates significant relationships between formulaic sequence use and oral fluency. For speed fluency, quantitative analysis indicates a significant positive relationship between frequency of formulaic sequences and speed fluency, proving the findings of many of the previous studies . For breQualitative analysis suggests the roles and functions of formulaic sequences through proceduralization. The use of formulaic sequences promoted speed fluency and reduced pausing when retrieved and processed holistically, but lost their processing advantages when retrieved and processed in a word-by-word manner. Some formulaic sequences seemed to be retrieved and processed as wholes , in lineMoreover, thanks to the proceduralization, the holistic retrieval and processing of formulaic sequences could help free up the attentional resources for other aspects of language production, reducing the amount of language planning and processing, thereby facilitating oral production . BesidesThe facilitative effects of formulaic sequences on speed or breakdown fluency are shown in different stages of speech production, reflecting proceduralization. As has been mentioned, among the three stages in language production, namely conceptualization, formulation, and articulation , formulaDespite the processing advantages of formulaic sequences reported above, it should be noted that the proceduralization could not be the same for everyone. As a matter of fact, a word sequence processed holistically by one person may not be processed holistically by another, and the matter of holistic processing may be a matter of degree . It is pSpeakers sometimes did not resort to holistic processing, but to analytical processing when retrieving formulaic sequences , 2002, fThese findings could explain why the overall proportion of formulaic sequences was not significantly related to speed or breakdown fluency, while the overall frequency of formulaic sequences was significantly positively related to speed fluency and negatively related to pausing. Those speakers who performed well in speed and breakdown fluency were more likely to resort to the idiom principle  when usiSimply put, as for speed and breakdown fluency, the key problem in the employment of formulaic sequences of those less fluent speakers does not lie in the quantity of formulaic sequences, but lies in the quality of formulaic sequences. It is not that those less fluent speakers were not so prone to use formulaic sequences as fluent speakers, but that they failed to use formulaic sequences properly to give full play to the processing advantages of formulaic sequences.The negative relationship between variety of formulaic sequences and repairing does not mean that more varied usage of formulaic sequences could help reduce repairing. Instead, this negative relationship resulted from speakers\u2019 reuse of the same formulaic sequence in self-correction or reformulation, repetition of the same formulaic sequence as a filler, and correction and reformulation of formulaic sequences. All these could lead to the decrease of variety of formulaic sequences and increase of repairs at the same time, explaining the negative relationship between the variety of formulaic sequences and repairing.The proceduralization of formulaic sequence use did facilitate repair fluency on some occasions, but on other conditions, such as those mentioned in the previous paragraph, the use of formulaic sequences lost their facilitative effects or even had negative effects on repair fluency. In this way, the original facilitative effects on repair fluency was washed away, explaining the absence of significant relationship between formulaic sequence use and repair fluency.First, when making self-corrections and reformulations, speakers repeated the proceduralized information , which could help them compensate for the limited capacity of working memory , freeingSecond, although the holistic retrieval of some formulaic sequences did help free up processing load and contribute to language production through proceduralization, they themselves could also contain mistakes and lead to self-corrections, exerting negative impact on repair fluency.Third, speakers sometimes repeated the proceduralized information  to buy more processing time for the preparation of other processing needs . The natBased on the results of quantitative and qualitative analyses, a new framework of proceduralization of formulaic sequence use could be constructed (see The present study investigated the relationship between Chinese EFL learners\u2019 use of formulaic sequences and oral fluency, reflecting the roles of psycholinguistic theories related to proceduralization in language learning. Total frequency of formulaic sequences was positively related to speed and negatively related to pausing, while total variety of formulaic sequences was negatively related to repairing, and all these relationships reached significant levels. Formulaic sequences could promote speed and reduce pausing when retrieved holistically, but sometimes lose their processing advantages when retrieved and processed in a word-by-word manner. Less fluent speakers are much more likely to retrieve and process formulaic sequences in a word-by-word manner, and their key problem lies in the quality rather than quantity of formulaic sequences. Formulaic sequences are also reused in self-corrections, repeated as fillers, corrected or reformulated, reducing variety while increasing repairs. These also neutralize the facilitative effects of formulaic sequences on repair fluency and could mirror speakers\u2019 occasional tendency to sacrifice repair fluency for the improvement of speed and breakdown fluency when using formulaic sequences. Theoretically, the present study reflects the roles of psycholinguistic theories of proceduralization in teaching and learning by unpacking the relationship between formulaic sequence use and three dimensions of oral fluency. A new framework of the proceduralization of formulaic sequence use was also constructed accordingly (see Despite the advantages and contributions mentioned above, the main disadvantage or limitation of the study is that it does not consider the categories of formulaic sequences or the oral proficiency of speakers when exploring the relationship between formulaic sequence use and oral fluency. Future research could consider these factors that might interplay with the functions of formulaic sequences in the mechanism of proceduralization, helping students know how to further improve their oral fluency, which is closely related to their mental state or mental health.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.The author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "The objective for this systematic review was to examine how the record linkage process was reported and to understand challenges related to accessing, linking, and analysing linked routinely collected data used for multimorbidity research.Twenty studies were included, of which seventeen looked at the relationship between two specified long-term conditions. Fourteen studies received the linked dataset from an external data linkage provider. Hospital Episode Statistics was the most common source of data (n=5). Eight studies reported variables used for the data linkage, while only two studies reported pre-linkage checks. The quality of the linkage was assessed by three studies, of which two reported linkage rate and one reported raw linkage figures. Only one study checked for bias by comparing patient characteristics of linked and non-linked records.The findings from this study will feed into further guidance to understand and minimise bias due to linkage error in medical research.Twenty studies were included, of which seventeen looked at the relationship between two specified long-term conditions. Fourteen studies received the linked dataset from an external data linkage provider. Hospital Episode Statistics was the most common source of data (n=5). Eight studies reported variables used for the data linkage, while only two studies reported pre-linkage checks. The quality of the linkage was assessed by three studies, of which two reported linkage rate and one reported raw linkage figures. Only one study checked for bias by comparing patient characteristics of linked and non-linked records.UK LLC provides a strategic research-ready platform for longitudinal research: a clear step change from pre-pandemic capability. With sustained investment, and through exploring options to extend linkages and generalise to wider purposes, UK LLC is positioned to inform cross-cutting themes such as understanding health and social inequalities, health-social-environmental interactions, and managing the COVID-19 recovery."}
+{"text": "The results obtained indicate variability with respect to a particular irrigation dose and the applicable assessment method. The results were reviewed by one-way ANOVA analysis where observed coefficients and irrigation dose were considered as dependence factors. The results indicate a statistically significant impact of the applied quality coefficient of work and thus the impact of a particular device . When evaluating the effect of the included irrigation dose, we also showed a statistically significant effect in both facilities . By checking the operation of the hose reel irrigation machine, we managed to successfully apply the proposed classifications, which also perform the function of fault prediction. The proposed facilities show that proper plant operation and timely response can help create more efficient and sustainable irrigation services, not only saving water but also reducing costs for the owner.The aim of this paper was to design a device for monitoring the work of irrigation technology . Two devices for monitoring selected irrigation operating parameters for two hose reel irrigation machines were designed. During the monitored period of connection of the equipment to the sprinkler, 15 irrigation doses were carried out for both sprinklers. Irrigation operating characteristics working pressure, hose reel speed and selected weather conditions temperature and humidity were monitored. When evaluating the results, we proved the need to monitor the operation of the sprinkler not only by the coefficient of variation  Given that there is currently a shortage of water, which is a global problem and growing every day, the issue of economic management of this resource needs to be addressed. Approximately 70% of the fresh water used in many parts of the world is used for irrigation, from agricultural fields to gardens . Excess Monitoring hose reel irrigation quality of work and proper deployment, however, is one of the essential features, which we are convinced not only by us but also by other authors ,6,7. IrrAs already mentioned, among the solutions for spray irrigation, irrigation with hose reel irrigation machines have been used in practice, which is most often connected to hydrants with a flexible hose. The systems consist of components for mobile water distribution with a big gun sprinkler (combination with a stativ-travelers on an irrigation boom) or a bracket (located at the end of the hose). The hose is wound on a drum and in most cases, the result is an irrigated rectangle. For their function, they need to have pressurized water from the hydrant (pressure approx. 0.5\u20130.6 MPa). The principle of operation is therefore to slowly wind the hose on a spool using the most used turbine, hydraulics, electric linear motor, or hydrostatics with an independent combustion engine. To ensure even irrigation or programmed distribution of the required dose, the hose retraction rate must be continuously controlled by mechanical or hydraulic devices that often operate automatically ,4,8.Modern models are equipped with many automatic operations, including limiting the pressure drop in the turbine, stopping the winding at the end of irrigation, etc. . To ensuThe literature review shows that there are devices in the category of hose reel irrigation machines that have no electronic equipment (older and simplest devices), have monitoring of some functions  oThe winding system is one of the most complex and most researched mechanical-hydraulic components because it affects the overall operation of the machine. The even distribution of water, the reduction of pressure losses, the setting of the winding speed of the travelers or the irrigation booms with precise control of the irrigation dose height depend on this modern system . Until nThe overview shows that research and development in the field of modernization are also being carried out in hose reel irrigation machines. A key component of the hose reel irrigation machine is the mechanism for extending and retracting the hose guide. It affects the uniformity of water distribution, load loss, precise regulation of hose retraction speed, folding of irrigation arms and the resulting accuracy of irrigation. Technological developments have begun to focus on better hose reel systems to increase efficiency by reducing the power required for operation and increasing the quality of work even at lower operating pressures. The modernization is a turbine with a choke perpendicular to the inlet with a built-in bypass, which achieves a reduction of load losses to a minimum and more precise control of a wide range of speeds .The difference between the designed devices and commercialy available equipment is that commercial systems do not employ GPS coordinate monitoring systems and that parameter monitoring is provided on the basic design of the machine . Our solution is to create new and modern hardware and software equipment that can ensure sustainable irrigation, not only in spray irrigation conditions. The proposed system collects and analyzes the current machinery datasets and on the basis of those data, evaluates and predicts potential faults. This means that the quality of work does not depend on the quality of the GPS signal. The aim of our research was therefore to create a monitoring system that monitors the working activity of the sprinkler  and informs the operator about the specific operating state.The less researched area of irrigation concerning the use of informatization in irrigation technology inspired us for more attention. This is also due to reducing the overall water consumption and increasing the quality of work by monitoring the operating parameters of irrigation equipment. Research in this area has been going on for several years, where the development of the device has been gradually improved and the results of the initial versions have been gradually published in scientific journals. As already mentioned, an older version of the hose reel speed monitoring system was presented in a previous publication . The verTwo hose reel irrigation machines (Z1 and Z2) were used for practical tests, where the designed device E1 , 14 digital input/output pins (of which six can be used as PWM outputs), six analog inputs, USB connection, power connector, ICSP interface and reset button, contains everything needed to operate a microcontroller, and connection to a computer via USB cable is easy .\u00ae single-/dual-core 32-bit LX6 microprocessor). The operating voltage is in the range of 2.7 to 3.6V with a working current of 70 mA [The LilyGO TTGO v1.4 microcontroller  is a more modern development board equipped with additional components . The communication between the data collection device using the SIM800L module and the server will be discussed in the following sections.A standard pressure transducer  uses I2C bus . The I2A combined DHT11 sensor  with a capacity of 4Ah,  and the terminal designed by us  for its connection to the device. The basic output voltage of 20 V is adjusted to 5 V DC using an LM2596 converter  . The batAt the beginning of 2021, another important functionality was added to the data collection system\u2014MessageCenter  with an assigned phone number. In the next step, the operator is assigned a device to manage. One operator usually has more than one device, but it is possible to assign one device to more employees. This is done in the device pairing section, where you can assign, edit, and delete pairings.The software was designed and programmed on the open-source development platform Arduino . It is bThe first lines of the program are devoted to the import of libraries needed for various peripherals, which are planned to connect to development boards of various manufacturers such as Arduino LilyGO TTGO and the like. In Another rule when writing a program is to define global variables and constants that are used throughout the program. The practical solution is to define them immediately after the import of the corresponding libraries, as it was solved in our case  and then only work with it (pinDHT11).After defining the header, the program consists of two main parts, setup, and loop. Setup is used for the initial setup of the program and is performed only for the first time after starting the microchip board. Loop (cycler) is the part of the program that is executed continuously while the microchip board is running. User-defined functions are written after the end of the loop. The programmer can thus define his own functions that need to be used several times in the program . In thisAnother important part of the proposed communication system  between The server has scripts used to authorize devices and process received data. The server also includes an administration interface for device management, display, and export of measured data.The administration interface  is desigInverted metering logic (+ = \u2212);Distance constant (ratio of measured and required distance);Pressure tuning (\u00b1Bar);Temperature tuning (\u00b1\u00b0C);Humidity tuning (\u00b1%).When selecting under windows (device settings) from the administrator main menu, setting options are offered to the user . DiffereWith these tools, the operator can react relatively quickly to device measurement inaccuracies. Communication and sending of data with the device take place at certain time intervals set in the device itself, except for crisis situations such as pressure loss or excessive speed change. Communication takes place on a secure SSL layer. The server authorizes the communication link, verifies its correctness, and processes it. The processed data are then stored in a database so that they can be presented to the operator. For the communication of the device with the server, a so-called REST API  is an interface that can be used to create, read, and edit data using simple http calls (links).Within the created hardware component (MessageCenter), software was also created for better communication and crisis prediction options. The types of output messages are listed in The upgraded version of the device running on the LilyGO TTGO v1.4 motherboard has a subsystem for checking the machine\u2019s crisis situations (programmed to constantly check the database of error messages with the designed device). This means that it can intervene by sending a unique line in the event of a machine failure. This link contains the type of machine fault and the exact GPS coordinates. The machine\u2019s crisis subsystem also has an informative character where it can tell the operator the approaching end of the irrigation cycle. The principle of the functionality of the MessageCenter subsystem consists of three points .Correct functionality of the device was secured by communication based on the GSM network, which is one of the most widespread standards for voice and data communication. The proposed device with the connection of hardware and software via the GSM network (with the possibility of data transmission) sends data to a server, which decodes them, verifies their correctness, and processes them . As alreThe data acquisition device is programmed to send data to the server at a certain time interval, except for fault prediction, which does not follow a time schedule. The data are sent over a Secure Sockets Layer (SSL) and authenticated on the server using a unique key.The practical verification of the designed devices\u2019 correct operation took place in the field conditions during the irrigation of seed maize. The irrigation dose realized by the hose reel irrigation machines was set to an average value depending on the winding speed of the hose on the spool. The irrigation dose represents the amount of water delivered per unit area in one uninterrupted time interval until the effective watering depth is reached. The lands covered by these enterprises belong to a warm, slightly humid climate area, which is characterized by a mild winter with January temperatures above \u22123 \u00b0C. Average annual temperatures range from 9 \u00b0C to 10 \u00b0C, while average temperatures during the growing season range from 15 to 16 \u00b0C. Temperatures below 0 \u00b0C start, on average, from 12.10. and end on 24 April. The multi-annual average atmospheric precipitation in the area ranges from 600 to 650 mm. For the growing season, the average total is 350\u2013400 mm. The area is in a slightly humid area, which significantly affects the soil formation process. The increased amount of precipitation determines the shift of organic minerals in the soil. As a result of this process, a cadastre of brown earth with lying and rusty subsoil, popularly called \u201cJune\u201d, was created on the ridges and flat plateaus. The slopes of the ridges are affected by water erosion, because of which the soils are heavily washed away. Cadastral soils are characterized by the following genetic types: brown soil, alluvial soil, and chernozem. The area is dominated by brown earth floodplain soils and belongs to a slightly humid area.The goal of the agricultural company SLOV-MART, ltd., K\u00e1tlovce is, together with top technicians, ensuring and maintaining the functionality of irrigation equipment, during their maintenance, protection, repairs, and operation . When growing crops and providing the necessary amount of irrigation water, the company envisages improving the economic result and facilitating its restructuring and modernization, especially to increase its market participation and agricultural diversification.In this paper, we focused on the deployment of the proposed equipment on two Irtec hose reel irrigation machines . Selected plots were irrigated with hose reel irrigation machines, which were equipped with refurbished irrigation water distributors . The sprThe technical and operational parameters show that the total length of the Z1 hose reel irrigation machine hose is 380 m with a hose diameter of 90 mm. The range of the long-range sprayer is up to 35 m, i.e., with an overall irrigation width of 70 m. The operating pressure reached 0.59 \u00b1 0.02 MPa. The total length of the hose at the second sprinkler Z2 was 350 m with a hose diameter of 82 mm. The range of the big gun sprinkler was also 35 m, and the working pressure reached 0.59 \u00b1 0.01 MPa. The source was a water pumping station, which takes water from underground sources . Water eThe Z1 and Z2 sprinklers in the basic version consist of a classic or tandem chassis, a hose wound on a spool, a control unit, and travelers with big gun sprinklers. The movement of the coil is ensured by a transmission and a turbine to which the pressurized water is led. These sprinklers are widely used not only in agriculture but also in irrigation of playgrounds and in domestic conditions. During practical tests, the settings of the spray angle stop (more oriented to one side) were changed for some hose reel irrigation machines, depending on the conditions in the field or obstacles contained .A manometer and a flexible connection hose are available as standard. Since the available irrigation devices had different hose diameters, 90 mm for the Z1 sprinkler and 82 mm for the Z2 sprinkler, calibration of the water flow was performed to secure the same irrigation water volumes applied from both irrigation systems. The standard hose length is 400 m, but for specific machines, the hoses are shortened to 380 m (Z1) and 350 m (Z2) for practical reasons of maximum available irrigation distance, which was limited by field boundaries. These changes, however, do not affect the measurements or limit irrigation device operation parameters. The equipment is protected with safety panels as required and the sprayers are supplied with the prescribed nozzles. The upper (hose with a spool) is rotatable relative to the chassis and its components are galvanized. Some types allow hydraulic travelers to lift. In addition, a tachometer or control unit may be included. The turbine is directly connected to a gearbox with a built-in bypass. The speed is changed mechanically (if there is no control unit) using a four-speed transmission. The traveler is galvanized and mounted on four variable wheels. The device also has an output for the tractor Power Take-Off shaft (max. speed 540 rpm).The plot on which the measurement took place is located near the village of \u017dlkovce, after which the \u017dlkovce 1 pumping station is named. Opposite the plot is the Mal\u017eenice steam-water power plant. The proposed equipment was used on hose reel irrigation machines when irrigating seed maize. The quality of work of a hose reel irrigation machine is formed on the one hand by transverse (application of one of the methods and the coefficient of spray uniformity) and longitudinal spray uniformity, and on the other hand by the continuous hose winding speed and the correct value of operating pressure. Both designed devices were tested for deployment in field conditions for 16 calendar days under variable weather conditions. As already mentioned, the device was equipped with pressure (EARU), humidity (DHT11), and temperature (DHT11) sensors and a speed monitoring device . The output will be graphical waveforms of the observed parameters. The average humidity values were for device E1 (58.97%) and for device E2 (57.29%).As it can be seen from the available resources, standards, and methodologies, there are several evaluation options for determining the quality of work, that is, coefficients and degrees of uniformity or non-uniformity of spraying, resp. coefficient of variation. Because the device designed by us is focused on measuring the pressure and winding speed of the hose, we focused our efforts on observing the values of the coefficient of variation. An important factor was also the state of pressure, the results of which could be monitored at any time because the pressure gauges installed by the manufacturers proved to be unreliable. This condition could also be caused by the age of the machine.A . From the achieved values of A, the value of non-uniformity is determined and according to the following formula :To evaluate the quality of the work (in our case variability of hose winding speed and pressure variability), the methodology applied by Oehler  was useda\u2014uneven winding speed, %mN\u2014average value, mm,A\u2014average deviation, mm.vC [mh value %):The second method, to determine the quality of work unevenness, was used to calculate the coefficient of variation Cv , the depANOVA analysis was used to evaluate and compare the results of the coefficient of non-uniformity and variation Formula (3), but also to evaluate the impact of the included irrigation dose Formula (4):Statistically significant differences are assumed from the applied equations for evaluating the quality of work of the designed equipment (E1 and E2) mounted on hose reel irrigation machines (Z1 and Z2). From the obtained results, demonstrable or unprovable changes within the applied designed equipment are evaluated. For this purpose, the statistical apparatus STATISTICA  was usedijy\u2014measured value,\u00b5\u2014overall mean,iC\u2014the effect of the coefficient of non-uniformity and variation,iF\u2014effect of the serial number of the applied irrigation dose,ije\u2014random error with mean 0 and variance \u03c32.Finally, an overall evaluation of the achieved results was performed.www.irrigationbugs.com; accessed on 21 March 2021).As part of our long-term experience in the Department of Machinery and Production Biosystems , we managed to gain a lot of experience and thus design equipment for monitoring selected weather conditions and operating parameters of a sprinkler. In the paper, two devices E1 and E2 were tested on two different hose reel irrigation machines Z1 and Z2. Since we aimed to test the functionality of the proposed monitoring equipment and, on the other hand, the quality of the work of the irrigators, we had an agricultural plot on which seed maize was grown . In the The implementation of the system made it possible to collect enough of the obtained results for the subsequent assessment of the quality of work of the hose reel irrigation machine and the monitored variability of information on weather parameters during irrigation.Graphic . In the event of a fault that occurred during the tests, an SMS message was sent to the mobile device (sending a unique line). This link  contained the type of machine failure and the exact GPS coordinates. The machine\u2019s crisis subsystem also has an informative character when it can tell the operator the approaching end of the irrigation cycle. An example of a message is shown in The program interface allows you to add operator identification data, telephone numbers, pair the device with telephone numbers, and then inform the irons about the specific status of the device. The devices were therefore successfully set up and the data were sent to the two telephone numbers we had chosen.vC of the selected hose reel irrigation machine Z1 at 15 irrigation doses realized in succession. The results show that the results of the coefficient of variation vC vary from 14.26 to 52.20% and the coefficient of non-uniformity and from 11.69 to 42.45%. vC vary from 27.23 to 62.29% and the coefficient of non-uniformity and from 21.80 to 49.74%. From the above, it can be concluded that with the reliability of monitoring the results of work designed by the equipment E1 and E2, the quality of hose reel irrigation machines was different. Based on the achieved measured and graphical results, statistical verifications of the significance of the impact of the selected hose reel irrigation machine and the coefficient of quality of work, on the one hand, and the impact of the included irrigation levy, on the other hand, were also performed. ANOVA one-factor analysis was used for statistical evaluation of the results, where the results showed a statistically significant influence of the applied quality coefficient of work and thus also the influence of a specific device . When evaluating the effect of the included irrigation dose, we also showed a statistically significant effect in both facilities . This means that when all coefficients and methods are introduced, there is a statistically significant difference between the examined coefficient values between at least two evaluation methods. If the null hypothesis is rejected, the alternative hypothesis comes into play. This means that not all mean values are the same .In the tested period, which lasted 16 days, irrigation took place with several Irtec hose reel irrigation machines , of which two irrigators were subjected to quality testing. The speed of winding the hose on the spool was evaluated, and the values were calculated from monitoring the wound distance in 10-min time sequences. The working position of irrigators (both) changed 15 times during the monitoring period. Irrigation time periods depended on the set irrigation dose (related to the speed of winding the hose on the spool), they ranged from 11 h to 16 h. The value of the operating pressure was monitored in the values of 0.5 to 0.6 MPa, the pressure failure occurred only once, and we removed it. The evaluation of the quality of work was based on the determination of the proposed coefficient of non-uniformity and the coefficient of variation for both proposed devices E1 and E2. In addition to operating parameters, the proposed equipment helped to monitor weather conditions (humidity and temperature). In the future, it is planned to expand the equipment with the possibility of a power supply with an energy source connected to the solar panel. The endurance of the energy source will thus enable the monitoring device of the quality of the irrigator\u2019s work during the entire irrigation season. Through the proposed devices, irrigators were notified in good time of a pressure failure via a fault prediction function. They were able to monitor the progress of the irrigators and thus take measures to improve them.The plant was designed over several months to years , with research and calibration taking place during the irrigation season and the growing season of the crop. The value of the set irrigation dose depended on the decade of the growing season and affected the speed of winding the hose . In addition to this investigated property, the measurements also focused on the working pressure and selected weather conditions (humidity and air temperature).There are many publications focused on monitoring the issue of irrigation that deal with irrigation in terms of monitoring evapotranspiration , the amoFor example, in Pakistan, surface irrigation methods are commonly used to grow crops to meet the demand of an ever-growing population . PracticApplication and distribution uniformity (main monitored parameter\u2014operating pressure) was determined by standard evaluation methods. The authors found that the application efficiency and uniformity of distribution of the hose reel irrigation system vary from 66 to 74% with a corresponding base pressure range of 0.38 to 0.46 MPa. These irrigation systems are suitable for all types of land and small plots, are easy to move and operate, and are cost-effective from an economic point of view. Therefore, surface irrigation methods are commonly used to grow crops to meet the demand of an ever-growing population ,33. The In recent years, modern irrigation technologies have come to the forefront of agricultural development for water conservation; these are highly efficient and crucial in the absence of agricultural water resources ,34,35.The essence of today\u2019s modern irrigation system is to reduce water consumption and thus ensure the needs of regional sustainable development and local conditions ,35. The This is a global problem and water-saving measures and scenarios need to be developed to sustain production for a growing number of consumers and water shortages . SustainFrom the research, the achieved results, and their comparison in terms of environmental, economic, and energy performance of irrigated and non-irrigated crops are therefore important. The output should be a sustainability analysis of irrigation practices within different irrigation systems. The sustainability of irrigation systems can be assessed by water-related indicators , crop  or energy and economic indicators . Various conclusions lead from various sources aimed at increasing the quality of irrigators\u2019 work, introducing informatization and monitoring not only the operational characteristics of machinery but also weather conditions ,43,44. DThe paper paid attention to the design of two devices for monitoring the most important operating characteristics of the sprinkler and selected weather conditions. The result of the research was the design of hardware and software equipment that are improving the field operation of selected irrigation technology. Designs were tested on hose reel irrigation machines with possibilities to be applied to other types of irrigation technologies after adaptations. By suitable modification of the selected component (gear) and subsequent calibration, it is possible to use the designed equipment for wide-area irrigators. A highly valued benefit was the fitting of the proposed equipment into practical conditions. The facilities informed the irrigators about the current values of the activities of the hose reel irrigation machines, or about the specific monitored failure. The results of this new study show that the use of the obtained real-time weather data is a great advantage. It was also possible to understand that is only the state of the environment but also the operating parameters increased the efficiency of irrigation.The results show that the hypotheses concerning the significant impact of the used irrigator or the applied coefficient of quality of work have been confirmed to us. In the case of confirmation of the second established hypothesis, concerning the order of the applied irrigation benefit, the results again showed a significant effect. So far, we have not made any publications that focus on the design of equipment for monitoring hose reel irrigation machinery speed and working pressure. The difference in this study in the evaluation of the quality of work of hose reel irrigation machines was also in the application of a new parameter, the irrigation non-uniformity coefficient. Another difference from commercial equipment is that parameter monitoring is provided on the basic design of the machine. Our solution was to create new and modern hardware and software equipment that can ensure sustainable irrigation, not only in spray irrigation conditions. The application of the equipment created by us allows irrigators to respond in a timely manner at a precisely determined place, and thus reduce the excess irrigation dose, respectively there is no state of hose reeling without irrigation.In many agricultural companies, reel hose irrigators without modern electronics are still used, in which case the operator will later find out the current state of the irrigator or the event of a failure. Depending on the operator\u2019s arrival delay to the device and in the event of a failure (assumed in our case) of winding the hose on the spool, the set irrigation dose is still applied to the same spot. A given irrigation dose would clearly increase water consumption, and thus economic costs, which were not quantified in the paper. This issue is a topic of future research, pointing out the overall losses that could occur at different time intervals for the operator\u2019s arrival at the irrigator. If these devices are used, the operator can react immediately and react in time based on fault prediction."}
+{"text": "To compare two approaches for linking multiple datasets: using all \"pairwise linkages\", linking each dataset to every other dataset; versus linking each dataset to a designated \"spine dataset\", by: considering the differences between these approaches, and illustrating using real-world data on patients undergoing emergency bowel cancer surgery.We linked an administrative hospital dataset  capturing patients admitted to hospitals in England, and two clinical datasets comprising patients undergoing emergency bowel surgery  and patients diagnosed with bowel cancer , with study period from 31 October 2013 to 30 April 2018. We compared pairwise linkage to spine linkage, designating HES as the spine dataset, by considering the number of eligible patients linked by each approach, characteristics of linked patients, levels of missing data, and whether analysis results were sensitive to the approach used.The spine linkage approach resulted in an analysis cohort of 15,826 patients, equating to 98.3% of the 16,100 patients identified with the pairwise linkage approach. Of 274 additional patients captured in the pairwise approach, approximately two-thirds were only in the emergency surgery dataset (NELA) and one-third were only in the bowel cancer dataset (NBOCA). There were no systematic differences in patient characteristics between these analysis cohorts. Associations of patient and tumour characteristics with mortality, complications, and length of stay were not sensitive to the linkage approach. When eligibility criteria were applied before linkage, spine linkage included 14,509 patients (90.0% compared to pairwise linkage).Spine linkage can be an efficient alternative to pairwise linkage, if case ascertainment in the spine dataset and data quality of linkage variables are high. These aspects should be systematically evaluated in the nominated spine dataset before spine linkage. Results are sensitive to order of linkage steps."}
+{"text": "Here, antique devices are restored, and their electrophysiological qualities ascertained.Desperation for cure led to 19Determine the comparative capabilities of these devices in delivering electrostimulation and compare with modern standards to understand possible electrophysiological sequelae.Devices known as \u201cmedical batteries\u201d were analyzed. Power delivery utilized a \u201cvoltaic battery\u201d, simple circuit, and a conductor wrapped around an iron core. When the circuit is energized, the core is magnetized by direct current of the battery which induces an alternating current that electrifies probes used on the body. Due to their marked age, a common 9-volt battery was exchanged for the corrosive dry cell paste batteries. Electrical parameters were then measured.Devices for electrotherapeutics ranged from anemic vibrations to dangerous tetany inducing shocks. Measuring the capabilities of these devices shows the robust yields possible if the original higher capacity batteries were utilized. The reality is, cure or not, the devices were surprisingly potent. It is interesting that, albeit unrefined, efficacious doses were available before modern electrification.No significant relationships."}
+{"text": "Progressive collapsing foot deformity (PCFD) is a complex 3-dimensional (3-D) deformity with varying degrees of hindfoot valgus, forefoot abduction, and midfoot varus. The first aim of this study was to perform a 3-D analysis of the talus morphology between symptomatic PCFD patients that underwent operative flatfoot correction and controls. The second aim was to investigate if there is an impact of individual talus morphology on the success of operative flatfoot correction.We reviewed all patients that underwent lateral calcaneal lengthening for correction of PCFD between 2008 and 2018 at our clinic. Radiographic flatfoot parameters on preoperative and postoperative radiographs were assessed. Additionally, 3-D surface models of the tali were generated using computed tomography (CT) data. The talus morphology of 44 flatfeet was compared to 3-D models of 50 controls without foot or ankle pain of any kind.p\u2009=\u20090.02) and medial deviation  of the talar head in relation to the body in PCFD patients compared to controls. Moreover, PCFD were characterized by an increased valgus  alignment of the subtalar joint. Satisfactory correction was achieved in all cases, with an improvement of the talometatarsal-angle and the talonavicular uncoverage angle of 5.6\u00b0\u2009\u00b1\u20099.7 (p\u2009=\u20090.02) and 9.9\u00b0\u2009\u00b1\u200916.3 (p\u2009=\u20090.001), respectively. No statistically significant correlation was found between talus morphology and the correction achieved or loss of correction one year postoperatively.Groups were comparable regarding demographics. Talus morphology differed significantly between PCFD and controls in multiple aspects. There was a 2.6\u00b0 increased plantar flexion (22.3\u00b0 versus 26\u00b0; The different morphological features mentioned above might be contributing or risk factors for progression to PCFD. However, despite the variety of talar morphology, which is different compared to controls, the surgical outcome of calcaneal lengthening osteotomy was not affected.III. The pathophysiology of the development of adult-acquired flatfoot deformity (AAFD), or more recently termed progressive collapsing flatfoot deformity (PCFD), is not yet fully understood . AlthougThe purpose of this study was to perform a detailed and comprehensible 3-D analysis of the talus morphology between symptomatic PCFD patients that underwent operative flatfoot correction and controls. We hypothesized that (1) anatomical/morphological differences of the talus may be detected and (2) that its morphology affects the outcome of surgical PCFD correction.The local ethical committee approved this study  and all patients (controls included) gave their informed consent for the use of their data for research purposes. All methods were performed in accordance with relevant guidelines and regulations.This study had two aims: (1) to compare the talus morphology between symptomatic flatfeet and controls, and (2) to investigate if morphological differences of the talus affects the deformity correction after lateral calcaneal lengthening procedures. For (1), 3-D talus surface models of PCFD patients and controls were generated and compared. For (2), preoperative and postoperative radiographic parameters of symptomatic flatfeet were collected and correlated to the 3-D talus morphology.n\u2009=\u2009121). Only symptomatic stage II flexible AAFD/PCFD treated with lateral calcaneal lengthening were included in the study. Patients with ankle osteoarthritis were excluded [n\u2009=\u200937) or medial sliding calcaneus osteotomy (n\u2009=\u20099)] or the medial column ), lack of preoperative MRI or CT of the hindfoot (n\u2009=\u200910), and lack of pre- and postoperative complete conventional radiographs  (n\u2009=\u20092) with a minimum follow-up of 3 months.Data was collected of patients that were operatively treated at out clinic from January 2008 to July 2018 for PCFD . At the time of surgery, the average age was 43.5\u2009\u00b1\u200911.1 years and the average BMI was 27.6\u2009\u00b1\u20096.2 kg/mThe control group consisted of 50 lower legs of 48 patients , which were assigned out of the department\u2019s database to the study cohort. The controls obtained the CT scan of one or both legs for preoperative planning of surgical procedures around the knee , using patient-specific instruments. Medical records were reviewed and patients with prior osseous surgery, posttraumatic leg deformity, or foot or ankle pain in any kind, were excluded. The exclusion criteria did not necessarily omit patients with asymptomatic pes planus. However, previous research reported morphologic differences between neutrally aligned feet and symptomatic pes planus, but not when compared to asymptomatic pes planus . As a loAccording to Hintermann , lengthePreoperative and postoperative radiographic parameters of the PCFD group were assessed by two senior orthopaedic residents using conventional weight-bearing lateral and dorsoplantar foot radiographs, which included the talometatarsal-angle (Meary\u2019s angle), calcaneal inclination-angle , talocalcaneal-angle (Kite angle), and talonavicular uncoverage-angle , 22. TheCT scans of all PCFD patients and controls were segmented using the global thresholding and region growing functionality of a standard segmentation software  to generate 3D bone models. Afterwards, the models were imported into the preoperative planning software CASPA  . All CT X-axis was pointing anteriorly, the Y-axis laterally and the Z-axis proximally.An oriented bounding box of the talus was generated based on the principal component analysis. Then, a local coordinate system of the talus based on the geometric principal axes was generated Fig. a 24, 2525. The oX-axis of the talus coordinate system was projected onto the sagittal and the transversal plane.The centre of mass of the superior articular surface of the talus dome was determined, which was defined as the area between the transition from convexity and concavity from talus dome to talus neck anteriorly and to the processus posterior tali posteriorly Fig. b 26]. T. T26]. TY-axis. Subtalar joint orientation was assessed as the projected angle between those two axes on the transversal and frontal planes, respectively.The subtalar joint axis was defined as the axis of a cylinder, which best fitted the posterior calcaneal facet of the talus. Subtalar joint axis was decomposed in relation to standard axes of the talus coordinate system as well, using the Interobserver ICC and intraobserver ICC for 3-D measurement of the talus neck torsion was 0.886 and 0.956, respectively. For measurement of the subtalar joint orientation in the frontal plane, interobserver and intraobserver repeatability was 0.955 and 0.979, respectively.All relevant data were entered in a spread-sheet program and statistically analysed with SPSS software version 23.0 . Descriptive and continuous variables were calculated as means\u2009\u00b1\u2009standard deviation (SD), and range, when appropriate. Mean values are given as the average of both raters. Normal distribution was confirmed with the Kolmogorov\u2013Smirnoff test. Group comparison  of preoperative morphologic factors was performed with an unpaired t test. Pearson correlation coefficient was used for continuous variables searching for a correlation between (1) conventional radiographic flatfeet assessment  and talur\u2009=\u20090.332, p\u2009=\u20090.028), inferior alignment of the talus neck and dorso-plantar talocalcaneal angulation , and frontal subtalar joint orientation and talonavicular uncoverage .Preoperative values of radiographic parameters are shown in Table p\u2009=\u20090.02) (Table p\u2009=\u20090.04)   Table ; Fig. 2bNo influence of talus morphology was found on the correction achieved when comparing PCFD preoperatively and three months or one year postoperatively . In addition, there was no correlation (n.s.) of talus morphology and loss of correction (radiographic PCFD parameters three months postoperatively versus one year postoperatively) Table .Table 3CWhen flatfoot is acquired during adulthood, the shape of the foot changes. Accordingly, the condition was recently renamed to progressive collapsing foot deformity (PCFD), a complex 3-D deformity with varying degrees of hindfoot valgus, forefoot abduction, and midfoot varus . EvidencIn the current detailed and comprehensible 3-D analysis, an overall significant different morphology of the talus between symptomatic flatfeet and controls was found. In addition to Louie et al. , the talNevertheless, the question remains unanswered, if mentioned deformities occurred due to musculotendonous imbalances and subsequent mechanical bone adaption, or if the morphology of the talus leads to PCFD. Concerning the last, one might hypothesize that adult acquired flatfeet become symptomatic with progressive deformity, predisposed through their talus morphology. In contrast, innate pes planus with near-normal tali remain mostly stationary (with regard to deformity) and therefore asymptomatic. However, even though the talar morphology differed between symptomatic flatfeet and controls, there was an only weak correlation to radiographic pes planus assessment Table . This miRegarding operative management for symptomatic PCFD, it was hypothesized that differences in talar neck and subtalar articular facet morphologies might affect hindfoot correction, especially since the \u201cChopart joint\u201d and the subtalar joint are anatomically and functionally coupled. By lateral lengthening of the calcaneus, significant radiographic correction of the deformity is achieved, reflected in the improved talometatarsal and talonavicular uncoverage angles . HoweverThis study should be interpreted in light of its potential limitations. First, this study did not explore talonavicular joint coverage or position of the navicular relative to the talus. One reason was, that only supine CT was used. Weightbearing CT of the foot and ankle is an emerging technology . HoweverTalus morphology differs between flatfeet and controls, suggesting morphological features to be contributing or risk factors for progression to PCFD. Despite the variety of talar morphology, the surgical outcome of calcaneal lengthening osteotomy in case of symptomatic PCFD was not affected. In the future, assessment of 3-D weightbearing CT data of the hind- and midfoot pre and postoperative are needed."}
+{"text": "This erratum corrects the following:FASEB BioAdvances. 2022;4:408\u2013434. doi: 10.1096/fba.2021\u201000131Yasom, S., Watcharanurak, P., Bhummaphan, N., Thongsroy, J., Puttipanyalears, C., Settayanon, S., Chalertpet, K., Khumsri, W., Kongkaew, A., Patchsung, M., Siriwattanakankul, C., Pongpanich, M., Pin\u2010on, P., Jindatip, D., Wanotayan, R., Odton, M., Supasai, S., Oo, T. T., Arunsak, B., Pratchayasakul, W., Chattipakorn, N., Chattipakorn, S., Mutirangura, A.  The roles of HMGB1\u2010produced DNA gaps in DNA protection and aging biomarker reversal. The authors report that inadvertent errors were made in assembling two of the figures submitted for publication. In both Figures\u00a0The correct versions of Figures"}
+{"text": "MineralMate is a standalone MATLAB-based program designed to optimize the workflow associated with the magnetic separation of minerals. For nearly every bulk geochemical analysis some amount of mineral separation must occur, and the use of an electromagnetic separator is ubiquitous and considered as standard practice in many fields. Despite the commonality in which magnetic separation is used, there are considerable shortcomings. Electromagnet overheating and composite mineral grains are frequently encountered, as well as poorly constrained mineral behavior. These complications ultimately reduce the quality of downstream geochemical data. MineralMate is designed to alleviate these shortcomings by quickly and efficiently producing a magnetic separation workflow allowing the user to: (1) identify and compare optimal recovery ranges for different minerals from a bulk mineral assemblage, (2) identify the parameters on a conventional magnetic separator required to magnetically separate composite grains, (3) create/update user-specific magnetic susceptibility databases through empirical data collection, and (4) utilize an alternative magnetic separation equation.  Frantz; Geochemistry; Geochronology; MATLAB; Gagnetic separation; Mineral separation. A standardized approach to mineral separation is therefore beneficial for studies that require the efficient extraction of trace mineral phases . Indust22.1m). This parameter is specific to each mineral species, and is the quantifiable degree to which said mineral is magnetized by an external magnetic field. Magnetic susceptibilities in practice are typically positive , but can be negative in impurity-free diamagnetic minerals . Within individual particles, minor elements, trace elements, and crystal\u00a0structure can cause variations in Km  powering the electromagnet, and the gravitational force defined by the side slope of the chute (\u03b1), allows mineral separation over a large range of Km. The equation that has been provided by numerous authors that shows this relationship is of the form:Geology and geochemistry labs employ magnetic separators (a Frantz LB-1 was used in this study and described herein) to concentrate a desired mineral from a bulk mineral assemblage. The magnetic separator consists of a vibrating inclined chute that is placed between two poles of an electromagnet that directs the sample into one of two collection cups . The elem obtained from m of forsterite from \u22125 to 3.59 \u00d7 10\u22126. The Km from \u22125 to 3.82 \u00d7 10\u22126. If the Km from \u22125 to 3.74 \u00d7 10\u22126. The proximity of these Km to the conventional Km values obtained using \u03b2 of 20 and/or Km, however since mineral separation is routinely performed in a controlled laboratory environment at room temperature, then, as Particle grains that are introduced to the magnetic separator typically have an average diameter of \u223c125 \u03bcm . The par33.1m and seeks to constrain and identify the collected particles. MineralMate addresses these goals by allowing the user to create workflows for magnetic separation based on m databases of https://github.com/MineralMate-Program.The primary application of MineralMate  will be 3.1.1m values for over 350 minerals by varying I at \u03b1 = 15\u00b0. However, by fixing \u03b1 the tabulated values are limited in their flexibility. Given that Frantz electromagnets overheat at high currents (>1.7 A) it is beneficial to determine how to manipulate \u03b1 to maintain a lower I at a particular Km. MineralMate allows for users to rapidly identify the equivalent combinations of \u03b1 and I by fixing Km and solving for either \u03b1 or I in m values in the Database are calculated using the currents reported in the full range for each mineral of Previous workers 3.1.2m values of minerals within a bulk sample and provide a workflow that will best isolate the constituent minerals from the bulk assemblage  by first comparing (any) overlapping Km ranges of different minerals  and the maximum current allowable on the Frantz to minimize overheating effects. In this example there are 4 recovery steps required and if followed would produce five separates  he3.1.3m has been determined and compiled. The observed Km for a specific mineral from a bulk whole rock is expected to fall within the total range provided by these workers. To supplement existing databases, MineralMate is designed to calculate, save, and export Km values calculated from user entered I and \u03b1 used during mineral separation. For example, empirical results of a nearly monomineralic forsterite sample are provided below. The experiment is explained for other users wishing to determine the Km value of either a new mineral, or to update a mineral already in the Database (see Table\u00a0S1).1.Maintain a constant \u03b1 throughout the separation process2.Begin at I = 0 A, introduce sample to the top of the chute3.Measure the magnetic mass % and the non-magnetic mass % of the total sample (non-magnetic mass % = 100) using a balance4.Recombine the magnetic and non-magnetic concentrates, mix well5.Increase I to 0.1 A, introduce sample to the top of the chute6.Repeat Steps 3\u20135 until all particles enter the magnetic section of the chute (magnetic mass % = 100)The databases of m < Kforsterite, while the low current tail likely contains a mineral phase with Km > Kforsterite). To avoid interference of these tails in evaluating the Km, MineralMate allows the user to select the domain that best constrains a linear relationship-excluding both extremes. In m. It is assumed that at these extremes of the Km distribution, the purity of the mineral sample likely worsens (the tail Km is attributed to composite grains). The cutoff is flexible and can be made in accordance to observations made regarding sample purity from an optical microscope. This decrease in sample purity  will manifest as Km much greater or much lower than that of the median Km . If, however, as in this example, there are no datapoints existing at exactly 10% and 90%, then MineralMate linearly interpolates between the two nearest bounding points (i.e. 90.7% and 83.5% for the 90% cut-off) to calculate the Km values. The Km values in this example range from 5.96 \u00d7 10\u22125 and 1.18 \u00d7 10\u22124. These values can then be added to the Database for future use.Enter the experimental parameters into a data table and upload to MineralMate (see Table\u00a0S2). In this example, the curve in m Database to the open access spreadsheet at https://github.com/MineralMate-Program. This link contains the converted Km information from By explicitly defining how the \u201cbest recovery range\u201d is determined, MineralMate improves upon existing databases, which provide only a qualitative \u201cbest recovery range\u201d, defined as \u201ccontain[ing] the greatest amount\u201d . We enco3.1.4m of these particles will be a linear combination of its constituents. For a multiphase particle, the magnetic susceptibility will be a weighted average, denoted as KComposite and expressed asA, KB, KC,\u2026,KN are the Km values for the respective phases in the composite particle. The constituent components of a composite grain may be quickly verified using an optical microscope. If the composition of the mineral phases present are representative of the composite grains in the sample, then this information may be entered into MineralMate. For example, a composite grain consisting of four unique mineral phases in equal fractional abundance, then n = 0.25 (or 25 %) for each KN. MineralMate offers users the ability to create and add multiphase particles to their Database by solving for KComposite in composite is adjusted accordingly. The composite grains may then be reprocessed through the magnetic separator at the new side slope and current suitable for extraction. This approach increases the total number of whole or partial desired grains. The concept outlined above could be scaled-up for industrial purposes, as Physical disaggregation of geologic materials commonly results in fractured particles that do not align with precise mineral-specific grain boundaries or cleavage planes. This physical disaggregation therefore results in mineralogically heterogeneous particles . The Km 44.1t operating conditions at lower current. This allows continuous operation by minimizing cooldown times. MineralMate also provides predictability regarding mineral output, therefore limiting redundant steps during mineral separation.MineralMate was designed to have an intuitive and easy to use interface that can increase the efficiency of magnetic separation through idealized workflows and optimized working conditions. The operating conditions suggested by MineralMate will reduce the probability of the electromagnet overheating by offering equivalen4.2One of the primary applications of MineralMate will be providing workflows for geoscientists preparing samples for bulk geochemical methods. For example, sulfide Re\u2013Os geochronology e.g. , as wellCreation of multiphase particles during the crushing of material may be extensive  and Minem values-improving upon the qualitative evaluation used in some publications (e.g. m values is recommended in any study that uses magnetic separation because this documentation allows for independent analysis, scrutiny, and standardization of the magnetic separation procedure.MineralMate streamlines magnetic separation procedures by providing a standardized protocol for the calculation of Kons e.g. . Documen5m databases of over 350 minerals, as well as providing a method to create personal Databases. The operating conditions suggested by MineralMate are optimized to provide the lowest currents in order to minimize the risk of overheating the electromagnet. MineralMate provides an intuitive platform to visualize and calculate the expected behavior of different mineral phases. Therefore, inverse problems can also be considered, where the mineral species of collected separates can be investigated based on the recorded operating conditions. MineralMate along with user-created Databases can be downloaded and shared to the global community for iterative improvement and expansion.The MATLAB-based, but standalone program MineralMate, provides a flexible, and easy to use platform that is designed to enable researchers to maximize their mineral separation efficiency when using a conventional, laboratory-type magnetic separator. MineralMate allows the user to create a high precision workflow that will maximally recover minerals based on existing KBowman, Samuel; Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper. Hnatyshin, Danny: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.Database data is available with submission as Supplementary Data. Also, we plan to add a User Database on the MineralMate Github account.The authors declare no conflict of interest.https://doi.org/10.1016/j.heliyon.2022.e10411.Supplementary content related to this article has been published online at"}
+{"text": "To investigate the effect of a rotary agitation method or ultrasonically activated irrigation on the antibiofilm effect of a mixture of sodium hypochlorite (NaOCl) and etidronate  using a dual-species biofilm model in root canal system.Mature dual-species biofilms of Enterococcus faecalis and Streptococcus gordonii were formed in root canals of mandibular premolars. Teeth were randomly allotted (n\u2009=\u200912) to group 1, XP-endo Finisher (XPF); group 2, ultrasonically activated irrigation (UAI); group 3, syringe-and-needle irrigation (SNI). In all groups, canals were instrumented with a rotary instrument (XP-endo Shaper) prior to irrigant agitation/activation. A mixture containing 2.5% NaOCl and 9% HEBP was used throughout the experiment. Bacterial counts from the canal were determined using qPCR before preparation (S1), after preparation (S2), and after final irrigation agitation/activation (S3). Bacterial viability within the dentinal tubules in the coronal, middle and apical root-thirds was quantified using confocal microscopy after Live/Dead staining. The bacterial counts and viability were compared between groups using one-way ANOVA and post-hoc Tukey\u2019s tests. Paired t-test was used to compare the bacterial counts within groups.Instrumentation alone could significantly reduce the microbial counts in all the groups (P\u2009<\u20090.0001). Subsequent agitation/activation resulted in significant microbial reduction only in XPF and UAI (P\u2009<\u20090.05), both of which reduced significantly more microbial counts than SNI (P\u2009<\u20090.05). Live/Dead staining revealed that XPF and UAI showed significantly greater percentage of dead bacteria within the dentinal tubules than SNI in the coronal third (P\u2009<\u20090.05); UAI resulted in the significantly highest percentage of dead bacteria in the middle third (P\u2009<\u20090.05); while there was no significant difference between the groups in the apical third (P\u2009>\u20090.05).When using the sodium hypochlorite/etidronate mixture for irrigation, final irrigant agitation/activation with XP-endo Finisher or ultrasonic can improve disinfection of the main root canal space and the dentinal tubules in the coronal third, while ultrasonically activated irrigation appears to exhibit better disinfection within dentinal tubules in the middle third.The online version contains supplementary material available at 10.1186/s12903-022-02222-1. The main goal of root canal debridement is to reduce the microbial load to a subcritical level which can facilitate periapical healing . HoweverIrrigants play a central role in disrupting biofilms and killing microbes that colonize in root . Sodium Multiple agitation/activation methods have been proposed to improve the efficacy of irrigants, including rotary instruments, oscillating instruments, sonics, ultrasonics, multi-sonics and lasers. While the evidence\u00a0based on the efficacy of ultrasonically activated irrigation (UAI) is contradictory \u201313, it iTo the best knowledge of the authors, there is only one study  which deThe sample size was calculated based on the data of a previous study  by the PAll chemicals and reagents were of reagent grade and purchased from Sigma Aldrich  unless otherwise indicated. Normal saline (0.9% NaCl), NaOCl and EDTA were prepared with deionized water solvent. A pH of 7.5 was maintained for EDTA. A commercially available 0.9 \u0261 HEDP powder  was freshly mixed with 10\u00a0mL 2.5% NaOCl according to the manufacturer\u2019s instructions.Single-rooted mandibular premolars were obtained by extraction for orthodontic treatment after informed consent of patients and approval of the institutional ethics committee (No. KQEC-2020-63-01). Teeth were stored in 0.9% saline at 4\u00a0\u00b0C until use. Teeth with cracks, fractures or fused roots were excluded. Radiographs were obtained in the buccolingual and mesiodistal directions to exclude teeth with calcification, multiple root canals or internal resorption. The teeth were decoronated with a diamond disc under copious water cooling. And the root length was further standardized to 13\u00a0mm. Teeth with an initial apical file greater than size 15 were excluded. Thirty-six teeth were included in this study. A size 10\u00a0K-file was placed in the canal until its tip was visible at the apex, and the working length (WL) was established 1\u00a0mm short of this measurement.Root canal contamination was performed as described previously . Briefly2, 10%CO2, 10%H2) on separated BHI agar plates for 24\u00a0h, and a single colony was cultured overnight in BHI broth and then adjusted to 1\u2009\u00d7\u2009107 colony-forming units/mL (CFUs/mL). The bacterial suspensions were mixed (1:1) and each canal was filled with 200 \u03bcL of the suspension, placed in 2\u00a0mL tubes containing BHI broth and incubated anaerobically at 37\u00a0\u00b0C for 21\u00a0days. The culture medium was replenished every other day.A dual-species biofilm of Enterococcus faecalis  OG1RF and Streptococcus gordonii (S. gordonii) DL1 was used to infect the root canals. The two species were cultured anaerobically at 37 \u2103 (80%NScanning electron microscopy (SEM) was used to confirm if a biofilm was formed throughout the length of the root canal and into dentinal tubules. Two randomly chosen specimens were vertically split through the root canal into 2 halves and fixed in 2.5% glutaraldehyde for 4\u00a0h, dehydrated with increasing grades of\u00a0ethanol, dried using critical point drying and sputter-coated with iridium. The root canal walls and dentinal tubules were observed under SEM .Thirty-six specimens were randomly distributed into 3 groups n\u2009=\u200912) based on the final irrigant agitation/activation protocol: group 1, XPF; group 2, UAI and group 3, SNI. A closed-ended root canal system  instrument according to the manufacturer\u2019s guidelines. Briefly, canals were irrigated with 2\u00a0mL of NaOCl/HEBP and instrumented to working length with XPS for 10\u00a0s. Then the canals were rinsed with 2\u00a0mL NaOCl/HEBP, and the entire cycle of instrumentation and irrigation was repeated 6 times. The residual NaOCl/HEBP was then absorbed completely by sterile paper points, and canals were rinsed with 1\u00a0mL 5% sodium thiosulfate. Then, the sample after preparation (S2) was obtained as described for S1. Following this, the NaOCl/HEBP was agitated/activated as described below.Group 1 (XPF) The canals were flushed with 1\u00a0mL NaOCl/HEBP and activated by a new XPF instrument that was placed in the canal to 1\u00a0mm short of the working length and powered by the motor at 800\u00a0rpm (1 Ncm) for 30\u00a0s according to the manufacturer\u2019s instructions. Then the canals were rinsed with 1\u00a0mL NaOCl/HEBP for 30\u00a0s, and the cycle was repeated once. Finally, the canals were dried and rinsed with 1\u00a0mL 5% sodium thiosulfate. The sample after final irrigation (S3) was taken from the canal as described for S1 and S2.Group 2 (UAI) A new ultrasonic tip  placed 1\u00a0mm short of the working length was activated by an ultrasonic unit  at a power setting of 5 for 30\u00a0s, in the presence of 1\u00a0mL of NaOCl/HEBP. Then the canals were rinsed for another 30\u00a0s with 1\u00a0mL NaOCl/HEBP, and the cycle was repeated once. Finally, the canals were dried and rinsed with 1\u00a0mL 5% sodium thiosulfate. S3 was taken from the canals as described above.Group 3 (SNI) Root canals were irrigated with 4\u00a0mL of NaOCl/HEBP for 2\u00a0min using a 30-G side-vented needle placed 1\u00a0mm short from the working length. The canals were dried and rinsed with 1\u00a0mL 5% sodium thiosulfate. Finally, S3 was taken from the canals as described.DNA was extracted from the samples using the TIANamp Bacteria DNA Kit  according to the manufacturer\u2019s instructions. The extracted DNA\u00a0was used to quantify the amount of E. faecalis and S. gordonii by 16sRNA gene-based qPCR with SYBR Green Premix Pro Taq HS qPCR Kit  on an ABI QuantStudio 5 real-time PCR instrument . The total reaction volume was 20 \u03bcL. The primers used for E. faecalis and S. gordonii were F: 5\u2032-CGCGAACATTTGATGTGGCT-3\u2032 and R: 5\u2032-GTTGATCCGTCCGCTTGGTA-3\u2032, F: 5\u2032-GCCTTAATAGCACCGCCACT-3\u2032 and R: 5\u2032-CCATCTCTGTTGTTAGGGCGT-3\u2032 respectively [Specimens were sectioned at 3, 7 and 11\u00a0mm from the apical foramen with a diamond disc under water cooling. The root sections were washed gently with sterile saline and stained with 200 \u03bcL of LIVE/DEAD BacLight bacterial viability stain  and incubated in the dark for 15\u00a0min. The specimens were visualized under a confocal laser scanning microscope  at the excitation wavelengths of 480/500\u00a0nm for SYTO 9 and 490/635\u00a0nm for propidium iodide to determine the green and red fluorescence within the dentinal tubules. Three random locations were scanned at each section at a resolution of 1024\u2009\u00d7\u20091024 pixels. Finally, the area ratio of red-to-total fluorescence was calculated to provide the red (apparently dead cells) %, using the Image J program  . Two sam10CFUs/mL and fluorescence ratios between the groups was analyzed by one-way ANOVA and post-hoc Tukey\u2019s tests. To compare the samples within each group, the paired t-test was used. Statistical Package for Social Science  was used to perform the statistical analysis. The alpha-type error was set at the 5% significance level (\u03b1\u2009=\u20090.05).The normality of the data was determined using Shapiro\u2013Wilk test. Comparison of mean LogThe SEM images validated the formation of a dense biofilm, with large bacterial clusters covering the root dentin Fig.\u00a0a\u2013c and p10CFUs/mL) was assessed by qPCR before preparation (S1), after preparation (S2) and after final irrigation (S3) Figure\u00a0The present study aimed at comparing the effect of a rotary instrument-based irrigant agitation strategy (XP-endo Finisher) with a commonly used irrigant activation strategy (ultrasonic) on the removal of dual-species biofilms from root canals and bacterial killing within dentinal tubules, after root canal instrumentation with an anatomically conforming coreless instrument (XP-endo Shaper). It has been shown that mixed-species biofilms display higher resistance to intracanal treatment procedures . It was The irrigation regimen used in this study was a mixture of sodium hypochlorite with etidronate i.e., continuous chelation. It has been shown in vitro that this approach results in superior root canal disinfection compared to the traditional NaOCl/EDTA irrigation , withoutThe XPS is composed of the MaxWire alloy and has been shown to change shape with changes in temperature . It is iThe results of this study showed that instrumentation with XPS using NaOCl/HEBP significantly reduced the bacterial burden from root canals. Notably, it could remove more than 99% of the bacteria from root canals. This corroborates with well-established findings that instrumentation alone can remove a substantial part of the septic content from root canals . Such fiThe main outcome variable in this work was the bacterial counts within the root canal and dead bacterial percentage within the dentinal tubules, when NaOCl/HEDP was subject to XPF agitation, UAI or syringe irrigation. The results showed that there was no significant difference between XPF and UAI in the bacterial reduction from root canals. Such results have also been demonstrated in a clinical trial where UAI and XPF agitation of NaOCl showed no difference between each other in bacterial reduction, but both were significantly superior to syringe irrigation . While tCLSM analysis showed that the percentage of dead bacteria within dentinal tubules ranged from 58 to 76%, indicating that none of the methods used in this work completely eliminated bacteria from the dentinal tubules. As expected, syringe-and-needle irrigation resulted in the least percentage of dead bacteria within the dentinal tubules in the coronal-third. The use of XPF and UAI promotes irrigant exchange and improves the flow dynamics of the irrigant across all regions of the canal , 37. PreIndeed, one may question the rationale behind not including the traditional NaOCl/EDTA protocol in the experimental design. However, the focus of this work was to investigate the methods to enhance disinfection using the NaOCl/HEBP protocol with the instruments that are currently available. Comparing the NaOCl/EDTA and NaOCl/HEBP protocols in the current study design is challenging in terms of standardization of duration and volume as the former is a sequential regimen and the latter is a combined regimen. Furthermore, it has been reported that the NaOCl/HEBP protocol is superior to the NaOCl/EDTA protocol in eliminating biofilms from root canals , 40. PreThe results of study highlight that XP-endo Finisher and ultrasonically activated irrigation are equally effective but significantly more effective than syringe irrigation in disinfecting the main canal space and dentinal tubules in the coronal third when a mixture of NaOCl/HEBP is used for irrigation, while ultrasonically activated irrigation displayed better disinfection within dentinal tubules in the middle third.Additional file 1.\u00a0Bacterial counts of E. faecalis (a) and S.gordonni (b) before preparation (S1), after preparation (S2) and after final irrigation (S3)."}
+{"text": "Precision deuteration has become part of the medicinalchemist\u2019stoolbox, but its usefulness can be undermined by unpredictable metabolicswitch effects. Herein we report the deuteration of doxophylline,a drug used in the treatment of asthma and COPD that undergoes extensiveoxidative metabolism. Labeling of the main metabolic soft spots triggeredan unexpected multidirectional metabolic switch that, while not improvingthe pharmacokinetic parameters, changed the metabolic scenario and,in turn, the pharmacodynamic features in two murine models of lunginjury. In contrast, their use in bronchiectasisis off-label, and their potential in this clinical setting is stillelusive and requires further investigations.Asthma, chronic obstructivepulmonary disease (COPD), and bronchiectasis fall under the umbrellaof chronic respiratory diseases and are three closely linked disorderswhose phenotype and etiology frequently overlap. Despite the factthat novel medications have emerged, methylxanthines are still indicatedas add-on agents for the treatment of both asthma and COPD by GINA1) is the most widely used methylxanthinebecause of its marked bronchodilator activity and anti-inflammatoryproperties  to the inhibition of phosphoinositide3-kinases \u03b4 (PI3K\u03b4) and from adenosine receptor antagonismto the restoration of histone deacetylase (HDAC) activity.5 Moreover, theophylline significantly reducesthe number of neutrophils and eosinophils in the airways. While itsuse is encouraged by its low cost and high oral bioavailability, theophyllinesuffers from side effects such as CNS stimulation, cardiac arrhythmias,and gastrointestinal effects, leading to a narrow therapeutic windowthat warrants strict monitoring of its levels in the blood. It alsointerferes with CYP1A2, CYP2E21, and CYP3A4, resulting in drug\u2013druginteractions with many drugs metabolized by these pathways.6 Because of these drawbacks, theophylline is relegatedto second- or third-line therapy in most treatment guidelines.Theophylline , acebrophylline (3), and doxophylline (4)  1 and hav8 At the pharmacological level, it has been demonstratedto positively interact with \u03b22 adrenoreceptors, withless affinity for \u03b11 and \u03b12 receptors,eliciting relaxation of blood vessel and bronchial smooth muscles.9 Unlike theophylline, it has a low affinity foradenosine receptors, does not inhibit any of the known PDE isoforms(except for PDE2A1), and does not interact with HDACs.10 With regard to its anti-inflammatory activity,doxophylline has been shown to reduce leukocyte count and recruitmentinto the airways.11 A recent report showedthat it reduces the oxidative burst in human monocytes, an effectmediated by the inhibition of protein kinase C (PKC) activity, differentlyfrom theophylline.12 From a toxicologicalperspective, indirect comparisons through meta-analyses suggest thatit might have a favorable risk-to-benefit ratio compared with theophylline,17 pointing to this novophylline as a safer therapeutic option forthe treatment of chronic respiratory diseases.19Doxophylline differsfrom theophylline in that it contains a methylene1,3-dioxolane group at position 7 1, result8In vitro metabolic studies in rat liver microsomes identified theophyllineand the 7-hydroxyethyl ester of 7-theophylline acetic acid  as metabolites of doxophylline.20 Zhao et al. showed that in men, doxophylline undergoesa more extensive oxidative metabolism.21 After both incubations in human liver fractions and intravenousadministration, a metabolic scenario is proposed where different metabolicpathways occur (5), which is further converted toT-COOH 6 or reduced to 7-hydroxyethyltheophylline  (route 1). The second pathway is involved oxidation ofthe tertiary carbon atom on the 1,3-dioxolane ring, with the formationof the 7-hydroxyethyl ester of T-COOH, which can hydrolyze in vivo to afford T-COOH 6 (route 2). In addition,four minor biotransformations are identified (routes 3\u20136),leading to the formation of theophylline, N-demethylation to formdm-doxophylline (9), dehydrogenation of the 1,3-dioxolanering to give dh-doxophylline (10), and oxidation of thexanthine nucleus to give ox-doxophylline (11), respectively,deuterium has beenshown to increase the stability toward the C\u2013D cleavage stepand induce resistance from oxidative metabolism. Many successful exampleshave recently been reported in which deuterium incorporation in placeof protium leads to a beneficial effect in terms of longer half-life,higher exposure, or reduced toxicity, drug\u2013drug interactions,and interpatient variability.d4-doxophylline(16) and d7-doxophylline(20)  or Pseudomonas aeruginosa.With the aim of investigating the effectof deuterium incorporationat the main metabolic soft spots of doxophylline, we synthesized twodeuterated analogues, ine(20) 1,27 and d4-acetaldehyde or/and d4-ethylene glycol. The general procedure, independentof the isotopic composition, consists of three steps -dione in the presence of potassiumcarbonate in dimethylformamide at 115 \u00b0C to yield the final product.The synthesis proceeded smoothly in high yields for both productsand allowed the preparation of the two final compounds on a scaleof 15 g.To this aim, a straightforward \u201cdeuteratedpool strategy\u201dwas undertaken using commercially available deuterated substrates,namely, ee steps 1. Acetald4-doxophylline and d7-doxophylline,respectively; 28 a PK study was also performed in minipigs. However,following both oral and intravenous administration, the two deuteratedanalogues showed very similar pharmacokinetic profiles  triggered considerable alterations inthe relative abundances of the metabolites generated. In more detail,increased formation of T-COOH 6 was evident (about 3-foldhigher level at 0.5 h), concomitant with a decreased level of etophylline 7  retain deuterium atoms (see the Supporting Information for structures). Indeed,the presence of deuterium lowers the signals in the electrospray ionizationsource, and the corresponding deuterated standards of the metaboliteswould have been necessary to perform an accurate quantification. Moreover,no reference standard was available for the quantification of themetabolites dm-doxophylline 9 and dh-doxophylline 10, and their relative abundances were expressed as peak areasof the corresponding accurate-mass ions. Despite these limitations,we could detect diminished levels of all of the above-mentioned metabolitesexcept for dm-doxophylline 9, suggesting that, althoughto a lesser extent than for d4-doxophylline,the metabolic switch occurs also for d7-doxophylline and contributes to the reduction of its bioavailability, and PAR . Finally, TUNEL assays were done to evaluate apoptosisin treated tissues, and again there was an increased apoptotic signaturein lungs exposed to P. aeruginosa thatwas reverted both by doxophylline and d7-doxophylline, whereas d4-doxophyllinedid not have any effect .The trend of an effect of doxophylline and position 7f, immund7-doxophylline, whereas d4-doxophylline has a lesser effect or no effect,according to the model used. Even if there are no sufficient elementsto draw a clear correlation between the metabolic stabilities of d4- and d7-doxophylline,the corresponding plasma levels of metabolites, and the invivo efficacy, it is evident that the different deuterationpatterns lead to distinct metabolite scenarios, which in turn appearsto influence the effect on lung injury. Indeed, it must be taken intoaccount that the pharmacological activity of methylxanthines observed in vivo is the result of the interaction of both the drugitself and its structurally similar metabolites with multiple targetsthat are still not fully elucidated and that on each of these targetsevery single metabolite has a different potency. It must be also recognizedthat besides the metabolic fate, other factors might contribute todetermining such differences in vivo, including aneffect of deuterium incorporation on plasma protein binding.32Overall, the two animal models are concordant in showing that doxophyllinehas an important protective effect in lung injury and that this ismimicked by 34 While this disorder shows an ever-increasingand worrying prevalence,35 no effectivepharmacological treatment is currently approved, and the search fora therapy is complicated by the lack of an understanding of its pathophysiologyand by the complex etiology of the disease.36 Indeed, it can be the result of several underlying causes, includinginfections by P. aeruginosa, Haemophilus influenzae, or other pathogens, dysregulatedimmunity, and impaired mucociliary clearance. Therefore, no animalmodel closely recapitulates the disorder, and different preclinicalsettings must be used to represent the different subpopulations ofpatients.37 In spite of the surge of interestin this disease, only a few disease-modifying agents are being evaluatedin clinical trials ,38 and they mainlytarget neutrophiles, the most abundant population recruited in airways,together with macrophages.39 In this context, doxophyllinemight represent an effective treatment as it both relaxes airway smoothmuscle and has anti-inflammatory properties, with a profound impactnot only on neutrophils but also on monocytes and alveolar macrophages.In February 2014, the U.S. Food and Drug Administration granted anorphan drug designation to doxophylline for treatment of bronchiectasis.40 Moreover, a couple of patents claim a pharmaceuticalcomposition comprising doxophylline and the mucolytic agent erdosteineto be used in treatment of bronchiectasis41 and dosage forms of doxophylline to treat orphan respiratory diseases.42 However, no data have been reported to supportthis hypothesis to date.7 In this Letter,we have shown for the first time that both doxophylline and its d7 analogue are effective in two animal modelsof chronic lung diseases that partly recapitulate bronchiectasis.Contrary to our expectations, no improvement in pharmacokinetics betweendoxophylline and its d4 and d7 analogues was observed, offering an enlightening exampleof a deuterium-promoted multidirectional metabolic switch and confirmingthe challenges associated with precision deuteration in drug R&D.Bronchiectasis is characterized by permanentenlargement of peripheralbronchi accompanied by repeated respiratory infections, disablingproductive cough, and shortness of breath, resulting in loss of lungfunction."}
+{"text": "Drosophila, Polo cooperates with the conserved centrosome proteins Spd\u20102/CEP192 and Cnn/CDK5RAP2 to assemble a PCM scaffold around the mother centriole that then recruits other PCM client proteins. We show here that in Drosophila syncytial blastoderm embryos, centrosomal Polo levels rise and fall during the assembly process\u2014peaking, and then starting to decline, even as levels of the PCM scaffold continue to rise and plateau. Experiments and mathematical modelling indicate that a centriolar pulse of Polo activity, potentially generated by the interaction between Polo and its centriole receptor Ana1 (CEP295 in humans), could explain these unexpected scaffold assembly dynamics. We propose that centrioles generate a local pulse of Polo activity prior to mitotic entry to initiate centrosome maturation, explaining why centrioles and Polo/PLK1 are normally essential for this process.Mitotic centrosomes are formed when centrioles start to recruit large amounts of pericentriolar material (PCM) around themselves in preparation for mitosis. This centrosome \u201cmaturation\u201d requires the centrioles and also Polo/PLK1 protein kinase. The PCM comprises several hundred proteins and, in  In vivo data combined with mathematical modelling reveal the interplay of Polo kinase and conserved centrosome proteins Ana1/CEP295, Spd\u20102/CEP192 and Cnn/CDK5RAP2 during centrosome maturation in Drosophila embryos. We propose that centrioles generate a local pulse of Polo activity that initiates mitotic centrosome assembly in syncytial fly embryos prior to mitotic entry. We speculate that the ability of centrioles to locally activate Polo/PLK1 prior to mitosis may be a conserved feature of mitotic centrosome assembly\u2014explaining why centrioles and Polo/PLK1 are both normally required to initiate this process.Here, we focus on the kinetics of mitotic PCM scaffold assembly in living Drosophila embryos during nuclear cycles 11\u201313  that drives progression through these early nuclear cycles  between S\u2010phase length and the Spd\u20102 and Polo growth period (measured as the time it takes for Spd\u20102 and Polo levels to peak in S\u2010phase)  Fig\u2009. This suet\u2009al, et\u2009al, et\u2009al, et\u2009al, Spd\u20102/CEP192 is thought to be the major protein that recruits Polo into the assembling mitotic PCM in vertebrates  is generated at the surface of mother centrioles, with levels peaking at mid\u2010S\u2010phase (we explore below how this pulse might be generated). We allow centrosomal receptors (RS) to recruit cytoplasmic Spd\u20102 (S) to the centriole to form the complex\u2009R\u00afS. The Spd\u20102 bound to this complex can be phosphorylated by P\u2217 and converted to a form that can form a scaffold (S\u2217) that is released from R\u00afS to flux outwards. This scaffold is unstable and can be rapidly converted back to S by a phosphatase, which we allow to be active in the cytoplasm at a constant level. However, S\u2217 can also bind cytoplasmic Polo (P) and Cnn (C), to form a more stable scaffold (S\u00af) that converts back to S relatively slowly. When bound to S\u00af, Polo is activated so that it can phosphorylate the S\u00af\u2010bound Cnn and convert it into a form (C\u2217) that can form a scaffold and be released from S\u00af to flux further outwards. In this way, the Spd\u20102 scaffold acts to convert catalytically C into the scaffold C\u2217. The C\u2217 scaffold disassembles when it is dephosphorylated by a cytoplasmic phosphatase (PPTase), which we allow to be active in the cytoplasm at a constant level. Note that this PPTase activity drives a low\u2010level of Cnn scaffold disassembly during the assembly process, but it is not intended to mimic the high levels of PPTase activity that are thought to drive the rapid disassembly of the PCM scaffold at the end of mitosis .We turned to mathematical modelling to test whether imposing an underlying pulse of centriolar Polo activity on these proposed molecular interactions could explain the observed kinetics of PCM scaffold assembly. In this model  Theet\u2009al, et\u2009al, et\u2009al, et\u2009al, et\u2009al, et\u2009al, et\u2009al, How might the centrioles generate such a pulse of Polo activity? This pulse of activity is unlikely to simply reflect the general activity of Polo in the embryo, which, like Cdk/Cyclin activity   and new mother centriole (hereafter NM centrosomes) separately, as they behaved differently. In embryos expressing Polo\u2010GFP and WT\u2010Ana1\u2010mCherry, the Polo pulse was similar on OM and NM centrosomes, although NM centrosomes initially organised significantly less Polo than OM centrosomes  and its centriolar Receptor (RP) (in these embryos most likely Ana1) could generate a pulse of Polo activity.Intriguingly, expressing either Ana1\u2010S34T or Spd\u20102\u2010S16T in embryos perturbed not only the amplitude of the Polo pulse, but also its period Fig\u2009. With thModel 2; Fig\u2009RPoff). Mitotic PCM recruitment is initiated when RPoff is phosphorylated on S\u2010S(P)/T(P) motifs by a kinase  to generate RP. These activated receptors can recruit and activate cytoplasmic Polo to form the complex R\u00afP. This pool of centriole\u2010bound active Polo can phosphorylate the Spd\u20102 bound to the centriolar Receptor complex R\u00afS\u2014also potentially Ana1/CEP295  to generate R\u00afPoff, which can no longer recruit Polo. In this way, an activator (RP), activates its own inhibitor (P\u2217) to form a classical delayed negative feedback network . When we used the pulse of Polo activity generated by Model 2 to feed into Model 1 to generate the PCM scaffold, it produced assembly kinetics that were similar to the original Model 1 (where we simply imposed a Polo pulse on the system)  Fig\u2009. Hence, et\u2009al, et\u2009al, As Polo/PLK1 turns over rapidly at centrosomes  at which the relevant protein kinase phosphorylates the centriolar Polo receptor   Fig\u2009. If thiscles Fig\u2009, consistet\u2009al, et\u2009al, In interpreting this experiment, it is important to remember that Spd\u20102 and Cnn do not \u201cturn\u2010over\u201d at centrosomes in the classical sense, as both proteins incorporate into the PCM in the central region around the mother centriole and then flux outwards to leave the PCM from the more peripheral regions (Conduit Spd\u20102 (+/\u2212Spd\u20102 embryos) or ana1 (+/\u2212ana1 embryos)  Figs\u2009 and EV5.os) Figs\u2009 did not os) Figs\u2009 (potentios) Figs\u2009. Perhapshase Fig\u2009. In our pink boxing, Fig\u2009unboxed white areas, Fig\u2009To test the robustness of these predictions, we performed a \u201cparameter sweep\u201d Fig\u2009, individWe conclude that the surprising observation that centrosomal Polo levels are actually higher at the end of S\u2010phase in the Ana1 half\u2010dose embryos is robustly predicted by Model 2. Importantly, this finding could explain our earlier puzzling observation that OM centrosomes recruit more Polo than normal in embryos expressing some Ana1\u2010S34T protein Fig\u2009. If theset\u2009al, Finally, we note that these models are purposefully minimal to reduce the number of parameters and test possible mechanisms rather than to mimic experimental data. This approach explains why the overall shape of the predicted growth curves do not exhibit all of the finer characteristics of the experimental data. For instance, in our models, the Polo and Spd\u20102 pulses consistently have higher amplitudes and earlier peaks compared to experimental data  activity would not only maintain the phosphorylation of the Cnn scaffold (and perhaps that of other mitotic PCM components), but would likely also suppress the activity of protein phosphatases (PPTases) that promote the disassembly of the mitotic PCM Glover, . Such PPCaenorhabditis elegans embryo indicate that centriolar Polo/PLK\u20101 activity is also required to initiate mitotic PCM assembly in this system. Worm embryos build a mitotic PCM scaffold using a PLK\u20101/SPD\u20102/SPD\u20105 system that is analogous to the fly Polo/Spd\u20102/Cnn system /T(P) motifs in Ana1 and Spd\u20102. Although PLK1 binding to these motifs can activate its kinase activity  in 8\u2009cm x 2.5\u2009cm plastic vials or 0.25\u2010pint plastic bottles.The et al, et al, et al, in\u2009vivo, the cleavage sites (sgRNA target sequence) were introduced on either side of the 3\u2009kb sequence in the donor plasmid. In addition, the sgRNA target sequences within the homology arm of the donor plasmid were mutated (without affecting the amino acid sequence) to prevent the Cas9 from cleaving within the repair template and the knock\u2010in construct once it had been inserted into the endogenous locus in vivo. The mixture of both constructs\u2014Guide RNA (sgRNA) and donor plasmid\u2014was injected into Cas9\u2010expressing CFD2 embryos  and donor plasmid for homology\u2010directed repair (HDR) were generated respectively, injected into Cas9\u2010expressing CFD2 embryos and screened as previously described  supplemented with fresh yeast suspension. For imaging experiments, embryos were collected for 1\u2009h at 25\u00b0C, and aged at 25\u00b0C for 45\u201360\u2009min. Embryos were dechorionated by hand, mounted on a strip of glue on a 35\u2010mm glass\u2010bottom Petri dish with 14\u2010mm micro\u2010well (MatTek), and desiccated for 1\u2009min at 25\u00b0C before covering with Voltalef grade H10S oil (Arkema). Embryo collections for western blotting experiments were performed as described previously . A 63\u00d7, 1.4NA oil objective was used for all acquisition. The oil objective was covered with an immersion oil  with a refractive index of 1.518 to minimise spherical aberration. The detector used was a charge\u2010coupled device (CCD) camera , with a gain of 200\u2009V. The system was equipped with 405, 488, 561\u2009nm and 642 solid\u2010state lasers (Oxxius S.A.). All red/green fluorescently tagged samples were acquired using UltraVIEW ERS \u201cEmission Discrimination\u201d setting. The emission filter of these images was set as followed: a green long\u2010pass 520\u2010nm emission filter and a red long\u2010pass 620\u2010nm emission filter. For dual channel imaging, the red channel was imaged before the green channel in every slice in a z\u2010stacks. For Fluorescent Recovery after Photobleaching (FRAP) experiments, circular regions of interests (ROI) of diameter 4\u2009\u03bcm were defined around selected centrosomes of interest . A 488\u2009nm laser at 50% laser power was\u2009used to FRAP each sample in 10 iterations over a period of 2\u2009s. 0.5\u2010\u03bcm z\u2010sections were acquired, with the number of sections, time step, laser power and exposure depending on the experiment.et al, initial intensity of the centrosomes as they first separated in early S\u2010phase and their maximum intensity at the oscillation peak; the time between these points represented the growth period, while the growth rate was calculated as: /growth period. To extract these features for Cnn, several mathematical models were fit to the data from each embryo, and the model that best fit the majority of the embryos was then applied to all embryos: linear increase (Cycle 11); linear increase\u2009+\u2009plateau (Cycle 12); linear increase\u2009+\u2009linear decrease (Cycle 13) , and the box was linked across multiple frames using a custom Python script. In experiments where the centrosomes organised by the old mother centriole and new mother centriole  were tracked independently, two centrosomes with the shortest inter\u2010centrosomal distance at the start of S\u2010phase and within a preset distance threshold were annotated as a pair. The brighter centrosome in a pair was annotated as the OM while the dimmer one was annotated as NM (Conduit The details of statistical tests, sample size and definition of the centre and dispersion are provided in individual Figure legends.https://github.com/SiuShingWong/Wong\u2010et\u2010al\u20102021. A copy is archived at https://github.com/RaffLab/Wong\u2010et\u2010al\u20102021 where it is maintained and updated.Custom Python scripts for data analysis and MATLAB scripts for mathematical modelling are available open\u2010source on Github, For a detailed description of the mathematical modelling, please refer to the Siu\u2010Shing Wong: Conceptualization; Data curation; Software; Formal analysis; Investigation; Visualization; Writing\u2014original draft; Writing\u2014review & editing. Zachary M Wilmott: Conceptualization; Formal analysis; Validation; Investigation; Visualization; Writing\u2014original draft; Writing\u2014review & editing. Saroj Saurya: Resources; Methodology. Ines Alvarez\u2010Rodrigo: Conceptualization; Resources. Felix Y Zhou: Software. Kwai\u2010Yin Chau: Software. Alain Goriely: Conceptualization; Supervision; Investigation; Writing\u2014original draft; Project administration; Writing\u2014review & editing. Jordan W Raff: Conceptualization; Supervision; Funding acquisition; Writing\u2014original draft; Project administration; Writing\u2014review & editing.CRediT author contributions listed above, the contributions in detail are:In addition to the This study was conceptualised by S\u2010SW, ZMW, IA\u2010R, AG and JWR. Investigation was done by S\u2010SW and ZMW. Key reagents were generated by SS. Computational analysis pipelines were developed by S\u2010SW, K\u2010YC and FYZ. Data was analysed by S\u2010SW, ZMW, AG and JWR. The project was supervised and administered by AG and JWR. The manuscript was initially drafted by S\u2010SW, ZMW, AG and JWR. and all authors contributed to the editing of the manuscript.The authors declare that they have no conflict of interest. JR is an EMBO member. This has no bearing on the editorial consideration of this article for publication.AppendixClick here for additional data file.Expanded View Figures PDFClick here for additional data file."}
+{"text": "Our first objective is to investigate how between subject differences in the likelihood of record linkage consent and record linkability determine the composition of and so risks of biases in estimates from linkable business datasets. The utility of datasets linking information from multiple sources is compromised by such non-linkage biases, but both components of the linkage process have rarely been considered. Our second objective is to introduce methods for evaluating non-linkage bias risks in datasets. Such evaluations can inform linkage method choice and assessment of the validity of linked dataset findings. Previous work, often lacking non-linked subject information on non-sample dataset covariates, tends to utilise overall linkage rates as quality measures, but in the related area of survey non-response correlations between analogous response rates and non-response biases are weak.We utilise the UK 2010 Small Business Survey (SBS) dataset. If a survey subject consents to record linkage, an attempt is made to append their Inter-Departmental Business Register (IDBR) identifier (if one exists), enabling linkage to other surveys etc. Given this, we evaluate bias risks arising from variation in subject linkage consent and identifier appendability, as well as its product, overall linkability, utilising representativeness indicators developed to evaluate survey non-response bias risks. These measure risks in terms of sample-subset similarity (representativeness) given an attribute covariate set obtained from the sample dataset, based on variation in subject inclusion propensities estimated by logistic regression, and are decomposable to assess correlates of inclusion propensity variation. Specifically, we use the CV (the standard deviation of inclusion propensities divided their mean), computed given nine attribute covariates describing business demography and perceived performance.We give full details in our presentation. Briefly, overall CVs suggest the linkable dataset exhibits substantial non-representativeness and non-linkage bias risk. Decompositions suggest main impacts on the linkable dataset are under-representation of very small businesses , due to being both less likely to consent and less likely to have an identifier appended, and under-representation of businesses unable / refusing to respond to survey items, due to being less likely to consent.Our analyses provide evidence of non-linkage bias risks in linked SBS datasets caused by under-representation of several sample subgroups. Each is explicable given known IDBR under-coverage or knowledge of business response processes. We also conclude that representativeness indicators are an easily applied method by which such risks can be evaluated."}
+{"text": "However, current Li\u2013O2 batteries are suffering from severe barriers, such as sluggish reaction kinetics and undesired parasitic reactions. Recently, molecular catalysts, i.e. redox mediators (RMs), have been explored to catalyse the oxygen electrochemistry in Li\u2013O2 batteries and are regarded as an advanced solution. To fully unlock the capability of Li\u2013O2 batteries, an in-depth understanding of the catalytic mechanisms of RMs is necessary. In this review, we summarize the working principles of RMs and their selection criteria, highlight the recent significant progress of RMs and discuss the critical scientific and technical challenges on the design of efficient RMs for next-generation Li\u2013O2 batteries.Aprotic lithium\u2013oxygen (Li\u2013O 2 batteries.This review provides the operation and design principles, latest development, and future challenges and perspectives of redox mediators, which demonstrate a promising strategy to solve the issues in Li\u2013O I. I+\u2013Nafiet\u00a0al. electrochemically fabricated a thin conductive polymer film of poly-anthraquinone (PAQ) octane (DABCO), the most efficient quencher used in Li\u2013O2 batteries, was proposed to protect DMPZ from the attack by 1O2 and achieved satisfactory performance . S. S2 battRMs Fig. b 89]. T. T2 batt results ; compareox) Fig. c. It sugs theory . Howeveratteries . This tr2 batteries, including organic, organometallic and inorganic compounds. Moreover, we discuss the main challenges associated with RMs-assisted Li\u2013O2 batteries. Although several pioneering investigations have been performed to understand RMs-assisted Li\u2013O2 batteries, notable advances are still desired to meet the requirements for practical applications. We also outline several possible research directions for advanced RMs and hope that our perspectives would contribute to the future development of RMs-assisted Li\u2013O2 batteries. Concretely, the outlook will be propagated according to the following five aspects: understanding the oxidation kinetics of Li2O2 with RMs, regulating the molecular structure of RMs, optimizing the components of RMs-assisted Li\u2013O2 batteries, analysing the catalytic efficiency of RMs and exploring the guideline for seeking new RMs.In this review, we summarize the operation mechanisms and properties of typical RMs for Li\u2013O2O2 oxidation by RMs that need to be further studied. To date, there is relatively little research on the kinetic feature of RMs-assisted charging. Besides, it is also unclear whether there is a relationship between the kinetics of the chemical decomposition of Li2O2 by RMs and the kinetics of the electrochemical oxidation of RMs. Due to the complexity of Li\u2013O2 batteries, involving gas, liquid and solid phases, traditional kinetic analytic methods are unsuitable. An appropriate electrochemical model is expected to overcome this obstacle and provide a guide for exploring the factors on reaction kinetics. Moreover, most current research focuses on understanding and optimizing the OER RMs. Only a few systematic studies were performed based on a general standard for an ideal ORR RM, which has severely hindered the development of the ORR RMs due to the lack of deep understanding.The most vexing obstacle is the kinetics of Li2O2 by RMs.An ideal RM is supposed to be highly soluble, fully reversible and stable against active oxygen species. It should also yield proper redox potential and high diffusion coefficient. In addition, under the aim of practical applications, all the discussed RMs should have low cost and little toxicity. As discussed above, the physiochemical properties of RMs greatly depend on their molecular structure and operational environment. Rationally modifying the molecular structure of RMs may enable to address some awkward problems, such as the deterioration of RMs, shuttle effect and lower solubility. Furthermore, adjusting the RM diffusion kinetics may provide a new sight on the oxidation kinetics of Li2 and all redox-active species, which can realize a Li\u2013O2 battery with larger capacity, better rate capability and longer cyclability. Besides, an electrolyte with low viscosity is beneficial to the diffusion of RMs. Notably, when RMs oxidize Li2O2, RMs may also oxidize or reduce the solvent. Side products from the decomposition of electrolytes and electrodes would block the O2-evolving interface. Therefore, improving the stability of electrodes and electrolytes should proceed in parallel with the efforts described herein. High concentration electrolytes (HCEs) have led to significant stability improvement in various electrochemical fields. The salts with high concentration in HCEs can coordinate with most solvent molecules and thus increase the stability of electrolytes without or with limited unstable free solvent molecules. As a result, the parasitic products associated with electrolytes are significantly reduced, thereby enhancing the transport current of the cathode and the accessibility of RMs to Li2O2 products, and ameliorating the catalytic efficiency of RMs. Besides, the HCEs can greatly improve the stability of Li-metal anodes because of the construction of an electrochemically stable SEI layer, which is expected to relieve the \u2018redox shuttle\u2019 of RMs.Reasonable match with the battery components is expected to achieve synergy and further improve battery performance. Engineering cathodes with abundant channels can provide efficient transport pathways for O2O2, the overpotential observed from discharge or charge curves only provided partial information about the suitability of RMs in Li\u2013O2 batteries. Some possible undesired reactions, widely observed as the detrimental decomposition of electrodes and electrolytes, might be missing in the unilateral electrochemical assessments. In addition, both the shuttle effect and stability issues for RMs confuse the precise assessment of the effectiveness of RMs. Any claim about the true catalytic effectiveness of RMs in Li\u2013O2 batteries is inadequate without quantitative measurement. Therefore, multiple quantitative analyses are urgently required to investigate the yield of Li2O2, oxygen consumption and evolution. The appropriate measurement techniques could provide clear interpretation of the catalytic efficiency of RMs.Although the RMs did facilitate the formation and decomposition of Liet\u00a0al. suggested using ionization energy (IE) as a key indicator for designing RMs, where specific organic molecules with a certain range of IE values (5.8\u20136.8\u00a0eV) can be utilized as RMs in Li\u2013O2 batteries [Although numerous RMs have been investigated and applied, the general principles of seeking and designing a new type of RMs remain a mystery. Kang atteries . Regretf2 batteries, although it is unlikely that all the problems in Li\u2013O2 batteries can be addressed with RMs at the same time. More advanced experimental, computational and applied investigations are needed to advance the practical development of RMs-assisted Li\u2013O2 batteries. The current status of practical applications of Li\u2013O2 batteries seems extremely challenging. Major drawbacks, such as Li dendrite growth, electrolyte decomposition, unstable electrodes and operation in pure oxygen, prevent the progress. Future work towards practical Li\u2013O2 batteries should primarily focus on the following three aspects. (i) Fundamental mechanisms underpinning Li\u2013O2 electrochemistry. Performing theoretical modeling of the reactions between oxygen species and battery components, and combining electrochemical measurements with spectroscopic methods and online technology can identify possible electrochemical and chemical reactions in Li\u2013O2 batteries. In addition, follow-up research should also provide some additional electrochemical performances, including self-discharge rate, performance at different temperatures and safety issues. (ii) Further optimization of battery components. It is generally accepted that the current electrodes and electrolytes, as well as cell structures, are far from real applications. Cathode materials with more stability, lower cost and higher catalytic activity play an important role in determining the Li\u2013O2 battery performance. Besides, similarly to other Li-metal-based batteries, the safety issue of Li-metal anodes is also unavoidable. The research progress of Li-metal anodes in other batteries is helpful to the development of Li\u2013O2 batteries. Especially, the influence of oxygen species  on Li-metal anodes must be considered in future research. Meanwhile, electrolyte evaporation also needs to be addressed by optimizing the battery structure or employing polymer and solid electrolytes [2 batteries with polymer and solid electrolytes are still needed but extremely difficult. Anchoring RMs at the electrode surface or introducing RMs to the working gas outside the assembled battery might be considered in the future, which can overcome the limitation of dissolution characteristics while maintaining the catalytic function of RMs. (iii) True Li\u2013air batteries. Most reported Li\u2013O2 batteries are operated under a pure-oxygen environment. However, to achieve a true \u2018Li\u2013air\u2019 battery in the future, the battery should eventually be operated in ambient air. Although an appropriate amount of impurity gas can improve the battery performance, the fickle external environment have made it difficult to achieve Li\u2013air batteries until now. Designing an O2 selective membrane is a feasible strategy to ensure that the battery works under a constant O2 atmosphere, thereby indirectly realizing the operation of Li\u2013O2 batteries under ambient conditions. Furthermore, more research should be devoted to understanding the influence of other gases in air on battery performances and then developing high-efficiency multifunctional catalysts to simultaneously catalyse the reversible reactions of other gases, especially CO2 and water, eventually realizing true Li\u2013air batteries.Objectively speaking, employing RMs is the most promising approach to tackle the sluggish reaction kinetics of Li\u2013Otrolytes . Though"}
+{"text": "Mechanochemistryhas become a sustainable and attractive cost-effectivesynthetic technique, largely used within the frame of crystal engineering.Cocrystals, namely, crystalline compounds made of different chemicalentities within the same crystal structure, are typically synthesizedin bulk via mechanochemistry; however, whereas the macroscopic aspectsof grinding are becoming clear, the fundamental principles that underliemechanochemical cocrystallization at the microscopic level remainpoorly understood. Time-resolved in situ (TRIS) monitoring approacheshave opened the door to exceptional detail regarding mechanochemicalreactions. We here report a clear example of cocrystallization betweentwo solid coformers that proceeds through the formation of a metastablelow melting binary eutectic phase. The overall cocrystallization processhas been monitored by time-resolved in situ (TRIS) synchrotron X-raypowder diffraction with a customized ball milling setup, currentlyavailable at \u03bcSpot beamline at BESSY-II, Helmholtz-Zentrum Berlin.The binary system and the low melting eutectic phase were furthercharacterized via DSC, HSM, and VT-XRPD. Mechanochemical cocrystallization between thymol and hexamethylenetetramineoccurs through the formation of a low melting eutectic (LME) phase.The overall process, going to completion in 5 seconds, is monitoredat the subsecond regime by time-resolved in situ (TRIS) synchrotronXRPD with a customized ball milling setup. Binary phase diagram, HSM,and VT-XRPD complete the characterization of the LME. A direct correlation of crystal structure/properties is at thebasis of the cocrystal design and application of molecular materials.37Cocrystalsare crystalline compounds made of different molecularentities taken together by intermolecular forces within the same crystalstructure.41 however, whereas the macroscopic aspects of grinding are becomingclear, the fundamental principles that underlie mechanochemical cocrystallizationat the microscopic level remain poorly understood.42 Despite their evident utility, this lack of comprehension de facto inhibits the outbreak of cocrystals that remainconfined within the boundaries of the pharmaceutical industry,46 with the exception of a few examples.51Cocrystals are typicallysynthesized in bulk via mechanochemistry;53 liquid,55 or amorphous56 phase as a function of the coformers used.Clearly, the mass transport and reagent diffusion representthekey step of the overall cocrystal formation process. Only a few interpretationsreported in the recent literature suggest that the diffusion processcan occur through a gas,60Time-resolvedin situ (TRIS) monitoring approaches have openedthe door to exceptional detail regarding mechanochemical reactions.We here report direct evidence of solid\u2013solid cocrystalformation between thymol  and hexamethylenetetramine (HMT) that proceeds throughthe formation of a metastable binary low-melting eutectic (LME) 1.LME is a binary phase compositionwhose melting point lies belowambient temperature. The formation of a liquid intermediate has akey role in the mass transport of the coformers in solventless cocrystalformation.62 at a subsecond data collection frequency, and the low-melting eutectichas been fully characterized by thermal analyses .The whole mechanochemical process has been monitoredby fast time-resolvedin situ (TRIS) synchrotron radiation X-ray powder diffraction (XRPD)50 andfood preservative alternatives51 with thethymol biologically active against Gram\u2013 and Gram+ pathogens.64The present cocrystal THY:HMT 3:1 has already been proposed elsewherewithin the frame of green pesticides50However, the low water solubility and high volatility of purethymolintrinsically limit its direct application in the agrochemical andfood industry. Cocrystallization has been recently proposed to mitigateits negative performances, thus obtaining a stimuli-responsive materialable to tune the release of the essential oils components as a functionof the environmental conditions.60 X-ray powder diffraction (XRPD) data were collectedat \u03bcSpot  with a low-energyincident beam (17 KeV) of \u00f8 150 \u03bcm and an Eiger 9 M 2Ddetector. Data were collected with an accumulation time of 500 msper frame while the mill was shaking. Sample-to-detector distancewas set at ca. 250 mm. Sequential multiphase Rietveld refinement wasperformed with TOPAS v 665 to extrapolatethe relative amount of the chemical species involved in the mechanochemicalreaction.The ball millgrinding experiments were performed by means of a Fritsch Pulverisette23 shaker mill with a vertical movement. This mill has a fixed amplitudeof 9 mm and adjustable frequency from 15 to 50 Hz with an adjustabletimer. A 2.3 mL jar was custom-designed at BAM and consists of threepieces, two stainless steel or polyvinyl chloride (PVC) end piecesand a transparent Perspex middle segment of 0.75 mm thickness. Theoverall size of the jar is 40 mm with an internal diameter of 12 mm.\u20131. The sample was thencooled at 5 \u00b0C min\u20131 down to 10 \u00b0C andthen heated again at 40 \u00b0C. The whole process was recorded bymeans of an Euromex 18MP camera placed on a trinocular optic microscopeequipped with a 100\u00d7 magnification lens.Cocrystallization of THYand HMT was monitored placing a few crystals (\u03bcm order of magnitude)of the two coformers on a glass slide and brought them into contactwith a spatula. Different firing profiles (heating and cooling) havebeen performed by means of a Linkam LTS420 hot stage. The first heatingprofile was performed by increasing the temperature from 10 to 30\u00b0C at 1 \u00b0C min\u20131, then cooling it to 10 \u00b0C at 5 \u00b0C min\u20131 and heating again to 60 \u00b0C at 5 \u00b0C min\u20131. At the end of the firing profile, the melt samplewas slowly thermalized to ambient conditions. Powder patterns wereextrapolated integrating the resulting 2D images in the range of 163\u00b0< \u03b2 < 197\u00b0 to obtain the powder pattern in the rangeof 5\u201319\u00b0 2\u03b8. Results are reported in Supporting Information Figures 19\u201324.VT-XRPD measurements of theTHY:HMT 3:1 cocrystal were carried outin parallel beam geometry with CuK\u03b1 radiation on a Rigaku SmartlabXE diffractometer equipped with an Anton-Paar TTK600 nonambient chamberwith flat copper sample holder. Data were collected in Bragg\u2013Brentanogeometry with the radiation source fix at \u03c9 = 4\u00b0 and theHypix3000 2D solid-state detector at 2\u03b8 = 13\u00b0. The solid-statedetector was used in 2D mode and still images were collected withan accumulation time of 3 s. Data collection was performed at ambientpressure heating the sample from 20 to 60 \u00b0C at 5 \u00b0C minHMT <0.33 were exposed to a 20 \u00b0C/100 \u00b0C/\u201320 \u00b0C/100\u00b0C heating\u2013cooling\u2013heating firing profile. Forthe mixtures with \u03c7HMT \u2265 0.33, thus with anexcess of HMT with respect to the 3:1 cocrystal, a single heatingramp from 20 to 300 \u00b0C was performed due to the decompositionprocess of HMT. All measurements were performed at 5 \u00b0C min\u20131 at atmospheric pressure under a constant flow ofnitrogen (20 \u03bcL min\u20131). The enthalpy of theendothermic or exothermic events, reported in J g\u20131, were determined by integrating the area underneath the thermalpeaks.Binary mixtures of THY and HMT weremechanochemically prepared by grinding the coformers at differentmolar fractions for 30 min at 500 rpm in a Retsch 100 PM planetaryball mill. A 12 mL steel jar was loaded with ca. 300 mg of each mixtureand two 9 mm steel ball bearings. Differential scanning calorimetry(DSC) analysis was performed with a PerkinElmer Diamond equipped witha ULSP 90 ultracooler. Thermal analyses were carried out in closed10 \u03bcL Al-pans. All mixtures with \u03c766 while the cocrystal liquiduscurve was obtained by fitting the experimental data with a second-orderpolynomial function (see SI for details).Due to the HMT decomposition, the liquidus curve of the HMT couldonly be approximated.THY:HMT binary mixtures at differentmolar ratios were tested to extrapolate the solid/liquid equilibriumcurves for THY:HMT 3:1 cocrystal and the single coformers. The thymolliquidus curve was experimentally calculated according to the Schr\u00f6der\u2013Laarequation,TheTHY:HMT 3:1 cocrystal has been synthesizedby grinding the two coformers together in the stoichiometric ratio. As soon as the two solids were gentlybent together, a low melting eutectic formed that became dominantafter a few minutes of blending. By grinding the so-formed stickypaste for about 30 min, a whitish solid was obtained. The titled compoundwas alternatively synthesized by grinding the two coformers in theappropriate stoichiometry in a Retsch 100 PM planetary ball mill for30 min at 500 rpm. A 12 mL steel jar was loaded with ca. 300 mg andtwo 9 mm steel ball bearings.50 consistsof supramolecular HMT:THY3 aggregates that crystallizein the P1\u0305 space group with a very high molecularmultiplicity . Each independent HMTis hydrogen bonded to three THY molecules in a pseudotrigonal arrangement,thus forming columns of the HMT:THY3 aggregates that runalong the a-axis .To deconvolute the intrinsic amorphous contributionto the massiveextrinsic background due to the Perspex jar, the XRPD pattern of theempty jar was collected in the same experimental conditions and includedin the Rietveld Refinement input . A linear fit passing through the pure HMT melting/decompositionand the cocrystal melting represent a first-order approximation ofthe liquidus curve of HMT. The liquidus curves of THY and HMT intersectat the metastable eutectic compositions \u03b5TH, whichis characterized by a melting point below ambient temperature in thestandard laboratory conditions (T\u03b5TH = 24.38 \u00b0C)  4.HMT < 0.04) showedan indented exothermic peak in the cooling run that can be attributedto the concomitant crystallization of the single coformers. The binaryeutectic phase thus clearly melts at 24.86 \u00b0C in the second heatingrun as reported in As a further proof, the thermal analyses performed on binary mixturewith a large excess of THY  evidenced by hole triangles in SI.The \u20131; thus a massive melting of the LMEwas observed along with a solid residue of the coformers. The samplewas then cooled to 10 \u00b0C, and a clear crystallization processoccurs  were placed on a glass slideat 10 \u00b0C. The temperature was then raised up to 30 \u00b0C at1 \u00b0C minsoccurs 6.HMT = 0.25) surprisingly showed in DSC an endothermic event in the secondheating of the firing profile at a lower temperature with respectto the first heating run. The thermogram reported in The thermal analysis performed on the cocrystal (\u03c7A TRIS-VT-XRPD experiment was then performed to clarify the natureof these thermal events. The THY:HMT 3:1 cocrystal was placed intoa nonambient chamber mounted on a laboratory diffractometer see . 2D dataThis suggests that the thermal profile influences the formationof a metastable phase that can only be isolated by a kinetically controlledcooling ramp.HMT = 0.25, corresponding to the3:1 THY:HMT stoichiometry.The cocrystallizationof thymol and hexamethylenetetramine occursvia solvent-free mechanochemical reaction and proceeds through theformation of a metastable low melting eutectic phase that plays akey role in the mass transport of the coformers. The whole processwas monitored via time-resolved in situ X-ray powder diffraction witha customized ball milling setup, currently available at the \u03bcSpotbeamline at the BESSY-II synchrotron facility. The two coformers reactas soon as they are blended, thus forming a low-melting eutectic phase.In the experimental XRPD patterns collected every 500 ms, the intensitiesof the coformers monotonically decrease, while the background increasesas a symptom of the growth of the liquid phase. From the metastableeutectic binary composition, the cocrystallization occurs in lessthan 5 s. The binary phase diagram suggests that the metastable eutecticphase is indeed characterized by a melting point below ambient temperature,which was further confirmed by hot stage microscopy. A new kineticphase was observed and isolated through VT-XRPD performed on the binarycomposition with \u03c7"}
+{"text": "Sharing aggregated electronic health records (EHRs) for integrated health care and public health studies is increasingly demanded. Patient privacy demands that anonymisation procedures are in place for data sharing.Traditional methods such as k-anonymity and its derivations are often overgeneralising resulting in lower data accuracy. To tackle this issue, we proposed the Semantic Linkage K-Anonymity (SLKA) approach to balance the privacy and utility preservation through detecting risky combinations hidden in the record linkage releases.K-anonymity processing quasi-identifiers of data may lead to \u2018over generalisation\u2019 when dealing with linkage data sets. As most linkage cases do not include all local patients and thus not all modifying data for privacy-preserving purposes needs to be used, we proposed the linkage k-anonymity (LKA) by which only obfuscated individuals in a released linkage set are required to be indistinguishable from at least k-1 other individuals in the local dataset. Considering the inference disclosure issue, we further designed the semantic-based linkage k-anonymity (SLKA) method through extending with a semantic-rule base for automatic detection of (and ruling out) risky associations from previous linked data releases. Specially, associations identified from the \u201cprevious releases\u201d of the linkage dataset can become the input of semantic reasoning for the \u201cnext release\u201d.The approach is evaluated based on a linkage scenario where researchers apply to link data from an Australia-wide national type-1 diabetes platform with survey results from 25,000+ Victorians about their health and wellbeing. In comparing the information loss of three methods, we find that extra cost can be incurred in SLKA for dealing with risky individuals, e.g., 13.7% vs 5.9%  however it performs much better than k-anonymity, which can cause 24% information loss (k=4). Besides, the k values can affect the level of distortion in SLKA, such as 11.5% (k=2) vs 12.9% (k=3).The SLKA framework provides dynamic protection for repeated linkage releases while preserving data utility by avoiding unnecessary generalisation as typified by k-anonymity."}
+{"text": "Do physician group practices participating in bundled payments among Medicare beneficiaries exhibit similar or different changes in episode outcomes compared with participating hospitals?This cohort study with a difference-in-differences analysis found that physician group practices participating in bundled payments had associated savings with surgical but not medical episodes, whereas participating hospitals had savings associated with both episode types.The findings of this cohort study suggest that policy makers should consider the comparative performance of participant type when designing and evaluating future bundled payment models. Hospital participation in bundled payment initiatives has been associated with financial savings and stable quality of care. However, how physician group practices (PGPs) perform in bundled payments compared with hospitals remains unknown.To evaluate the association of PGP participation in the Bundled Payments for Care Improvement (BPCI) initiative with episode outcomes and to compare these with outcomes for participating hospitals.This cohort study with a difference-in-differences analysis used 2011 to 2018 Medicare claims data to compare the association of BPCI participation with episode outcomes for PGPs vs hospitals providing medical and surgical care to Medicare beneficiaries. Data analyses were conducted from January 1, 2020, to May 31, 2022.Hospitalization for any of the 10 highest-volume episodes  included in the BPCI initiative for Medicare patients of participating PGPs and hospitals.The primary outcome was 90-day total episode spending. Secondary outcomes were 90-day readmissions and mortality.The total sample comprised data from 1\u2009288\u2009781 Medicare beneficiaries, of whom 696\u2009710  received care through 379 BPCI-participating hospitals and 1441 propensity-matched non\u2212BPCI-participating hospitals, and 592 071  received care from 6405 physicians in BPCI-participating PGPs and 24 758 propensity-matched physicians in non\u2212BPCI-participating PGPs. For PGPs, BPCI participation was associated with greater reductions in episode spending for surgical  but not for medical episodes . Hospital participation in BPCI was associated with greater reductions in episode spending for both surgical  and medical  episodes.This cohort study and difference-in-differences analysis of PGPs and hospital participation in BPCI found that bundled payments were associated with cost savings for surgical episodes for PGPs, and savings for both surgical and medical episodes for hospitals. Policy makers should consider the comparative performance of participant types when designing and evaluating bundled payment models. This cohort study evaluates the association between bundled payments and episode outcomes per medical and surgical episodes for physician groups compared with hospitals participating in the Medicare bundled payments initiative. Along with hospitals, PGPs participated in model 2 of Medicare\u2019s Bundled Payments for Care Improvement (BPCI) initiative,10 assuming accountability for the quality and costs of medical and surgical episodes spanning hospital admission and up to 90 days of postacute care. Although PGP participation in BPCI was associated with reduced Medicare payments and improved quality for joint replacement episodes,11 data are lacking for the hundreds of groups participating in other medical and surgical episodes.Although hospital participation in bundled-payment programs has been well studied,12 We addressed this knowledge gap by evaluating the association between PGPs and hospitals participating in BPCI model 2 on episode outcomes and comparing PGP vs hospital performance.To coordinate participation in future payment models, policy makers must understand the dynamics of PGP vs hospital performance, particularly given the evidence from other payment models indicating that physician groups may perform differently than hospitals in managing quality and costs.STROBE) reporting guideline.13This study was approved by the University of Pennsylvania Institutional Review Board and informed consent was waived because only historical data with minimal risk of harm were used. This study followed the Strengthening the Reporting of Observational Studies in Epidemiology  and a subsequent intervention period . The study sample included Medicare fee-for-service beneficiaries receiving care through BPCI PGPs and hospitals for 1 of 10 episodes, each defined by a set of Medicare Severity-Diagnosis Related Group codes: the top 5 highest-volume medical episodes  and the top 5 highest-volume surgical episodes . We excluded beneficiaries with end-stage kidney disease or insurance coverage through Medicare Advantage, as well as beneficiaries who had any non-Inpatient Prospective Payment System claims, lacked continuous primary Medicare fee-for-service coverage during or in the 12 months preceding the episode, or died during the index hospital admission.3 We excluded episodes between January 1, 2013, and September 30, 2013, to account for the transitional period during which PGPs and hospitals may have implemented care process changes in anticipation of BPCI.We constructed episodes beginning with hospital admission and spanning 90 days after hospital discharge, capturing episodes beginning on or before September 30, 2017. To avoid bias arising from Medicare precedence rules for overlapping episodes between participating PGPs and hospitals, we followed prior methods and constructed naturally occurring episodes by assigning overlapping ones to the earlier hospitalization.12 and those with any BPCI PGPs or BPCI hospitals were defined as BPCI Markets. We categorized patients based on hospitalization for 1 of 10 episodes of interest through BPCI PGPs, non-BPCI PGPs, BPCI hospitals, or non-BPCI hospitals. Patients receiving care through BPCI PGPs and hospitals were categorized as \u201cBPCI-both.\u201dWe used Medicare claims data (2011-2018) to identify BPCI PGPs and BPCI hospitals. Groups and hospitals that never participated in any of the 48 episodes in BPCI were categorized as non-BPCI PGPs and non-BPCI hospitals. We used propensity scores to match BPCI with non-BPCI PGPs and BPCI with non-BPCI hospitals , and other responses were classified in the \u201cother\u201d category.The study exposures were dichotomous indicators of PGP participation and hospital participation in BPCI. To reflect participant entry into BPCI at different times, participation indicators were time-varying and specific to each PGP or hospital. Groups and hospitals were considered as participants after enrolling in BPCI, regardless of subsequently dropping out. Covariates were chosen based on prior studies and included patient demographic and clinical variables, such as age, sex, and disease severity (defined using Elixhauser comorbidities), as well time-varying market variables, such as Medicare population size and Medicare Advantage penetration.The study\u2019s primary outcome was 90-day total episode spending . Secondary outcomes were 90-day readmissions and 90-day mortality. We also evaluated a number of exploratory spending and utilization outcomes (eMethods 2 in 18 We used a difference-in-differences (DID) method to mimic a 2\u2009\u00d7\u20092 factorial experiment comparing BPCI participation among hospitals and PGPs. Per the factorial design, we classified the treatment groups to reflect patient exposure to BPCI hospitals, BPCI PGPs, both (BPCI-both), and neither (non-BPCI). This approach enabled the comparison of episode performance for BPCI PGPs (vs non-BPCI) and BPCI hospitals (vs non-BPCI), as well as between BPCI PGPs and BPCI hospitals).Characteristics between the propensity-matched BPCI and non-BPCI PGPs and BPCI and non-BPCI hospitals were compared using standardized differences of means and proportions.19 Nonparticipating PGPs and hospitals were assigned the same treatment indicators as their propensity matched organizations. We evaluated medical and surgical episodes separately because they involve different care processes that may have different associations with outcomes.19In adjusted analyses, DID models included PGP- and hospital-specific indicators of BPCI participation as treatment to reflect the time-varying nature of BPCI participation\u2014that is, the fact that PGPs and hospitals could start participating at different times. This approach contrasted with traditional DID models, in which the baseline and treatment periods are fixed regardless of timing of actual contract initiation.We used generalized linear models with identity links and normal distributions for all outcomes. All models included episode (Medicare Severity-Diagnosis Related Group code) and market  fixed effects to generate within-episode type, within-market estimates that addressed time-invariant episode type, and geographic differences. Models also included time  fixed-effects to account for secular trends. Robust standard errors were clustered at the hospital level.20 we assessed the relationship between the primary outcome and potentially time-varying market-level covariates and how those covariates changed over time across the 4 study groups .Final model specifications for the primary outcome included only covariates deemed as potential confounders according to this process  as being in the BPCI PGP group. Finally, we repeated analyses for the mortality outcome by including individuals who died during index hospitalization.In response to an association observed between BPCI participation and differential changes in mortality that, to our knowledge, has not been previously described in the literature, we conducted post hoc analyses to explore robustness. First, we described selection based on observable and unobservable patient characteristics. Second, we tested approaches to mitigate bias from unobserved clinical severity of patients. Third, we repeated analyses using our modeling approach, but using data from that earlier time period, to assess the ability to replicate those mortality results.The total study sample comprised 2011 to 2018 Medicare claims data for 1\u2009288\u2009781 beneficiaries, of whom 696\u2009710 patients  received care through 379 BPCI hospitals and 1441 propensity-matched non-BPCI hospitals; and 592\u2009071 patients  received care from 6405 physicians in BPCI PGPs and 24\u2009758 propensity-matched physicians in non-BPCI PGPs.Compared with non\u2212BPCI-participating hospitals, participating hospitals tended to be larger, nonprofit, teaching hospitals located in urban areas and markets with larger populations and smaller proportions of low-income individuals . In contrast, there were no differential changes in spending between BPCI PGP and non-BPCI groups  or between BPCI-both and non-BPCI groups . The BPCI hospitals had differentially greater reductions in total episode spending compared with BPCI PGPs .In adjusted analysis of medical episodes , but not between BPCI PGP and non-BPCI groups . Compared with the non-BPCI group, there were differential decreases in mortality for BPCI PGPs  and BPCI hospitals . The BPCI hospitals and BPCI PGPs did not exhibit comparatively differential changes in mortality or readmissions. The BPCI hospitals differed from nonparticipants with respect to exploratory outcomes for medical episodes , BPCI PGP group , and BPCI-both  compared with the non-BPCI group. The magnitude of spending changes did not differ between BPCI hospitals and PGPs .In adjusted analysis . In contrast, there were no differential changes in readmissions for the BPCI hospital or BPCI-both groups compared with the non-BPCI group .Compared with patients in the non-BPCI group, those in the BPCI PGP group had differentially greater changes in 90-day readmissions  and BPCI hospitals  compared with patients in the non-BPCI group. The magnitude of these changes was not different for BPCI hospitals vs BPCI PGPs . Compared with nonparticipants, BPCI hospitals and PGPs differed in exploratory surgical episode outcomes (eTable 10 in Mortality changed differentially for patients cared for through BPCI PGPs (difference, \u20130.5 pp; 95% CI, \u20130.8 to \u20130.2 pp; 3 that found no association between BPCI hospital participation and differential mortality changes.Compared with the main study analyses, results of sensitivity analyses were qualitatively similar (eFigures 7-12 in In this cohort study with DID analysis, participation of PGPs and hospitals in BPCI was associated with cost savings for surgical episodes; however, only hospital participation was associated with cost savings for medical episodes. Hospital and PGP participation were associated with different patterns of changes in postacute utilization and mortality. For example, for medical episodes, hospital participation in BPCI was significantly associated with reductions in length of stay at skilled nursing facilities, whereas PGP participation was not. For surgical episodes, PGP participation in BPCI was associated with reductions in home health use, whereas hospital participation was not. These findings pose 3 implications.11 Although it is only one aspect of a payment model\u2019s success, spending reductions are critical because policy makers increasingly judge the viability of bundled payment programs by their cost savings.22First, these findings underscore the benefit of engaging PGPs in episode-based payment models. This analysis adds to prior work by describing the association of PGP participation in BPCI with cost savings for multiple surgical episodes, extending beyond hip and knee replacements.12 demonstrating that PGPs may be more successful than hospitals at reducing spending in population-based payment models, such as acute care organizations. Future work should elucidate drivers underlying this distinction. For example, hospitals may be better positioned to coordinate with postacute care organizations such as skilled nursing facilities given their high volume of shared patients. These strategies may be particularly important for medical conditions where the episode cost savings come from reductions in postacute care facility length of stay rather than reductions in the proportion of individuals discharged.3 Policy makers may consider these facets of performance when considering participant types in future alternative payment models.Second, these study findings affirm the suitability of hospitals to bundled payment models, specifically highlighting their relative advantage over PGPs in achieving cost and potential quality outcomes for medical conditions. These findings contrast with prior researchThird, these study findings emphasize the need for future research on the drivers of cost savings and quality improvements under bundled payments. Our results regarding BPCI-participating PGPs point to the importance of changes in readmissions and postacute care utilization in determining episode savings. Yet as observed from BPCI-participating hospitals, different patterns of utilization changes may drive savings for different episode types. Specifically, in these findings medical episode savings were associated with reductions in length of stay within skilled nursing facilities, whereas surgical episode cost savings came from fewer discharges to skilled nursing facilities.Additional work is also needed to assess the relationship between bundled payments and quality improvements. Although differential mortality reductions were observed by this study, there was also evidence of observable and unobservable favorable patient selection under bundled payments. This makes definitive conclusions regarding changes in health care quality challenging. Clarity on whether apparent quality changes represent true improvements, patient selection, or measures of both is highly relevant to policy and should be the focus of future studies.This study had some limitations worth noting. Findings may have been subject to residual confounding; however, we mitigated concerns by using a DID design that accounted for unobserved heterogeneity and patient and hospital characteristics. We evaluated the highest-volume episodes under a single program; however, BPCI model 2 was the direct basis for ongoing PGP and hospital participation in BPCI Advanced. Also, we did not include more recent data from BPCI Advanced because physician group participation files were not available. Moreover, we were unable to match physicians or identify episodes using tax identification number-level information owing to a lack of data availability. However, our approach using all episodes per National Provider Identification number and any participation in BPCI was conservatively biased toward the null hypothesis. Furthermore, to our knowledge, mortality reductions have not been previously described, and although we tested the robustness of the findings using a range of sensitivity and post hoc analyses, the analyses suggested the presence of unobservable patient selection that precluded definitive conclusions regarding any changes in health care quality. Despite conducting sensitivity analyses using alternative modeling approaches for episode spending as the primary outcome, future work should assess the use of modeling approaches beyond ordinary least-squares for other outcomes.This cohort study with DID analysis found that PGP participation in BPCI was associated with cost savings for surgical episodes but not for medical episodes, whereas hospital participation in BPCI was associated with savings for both episode types. Policy makers should consider the comparative performance of participant type when designing and evaluating future bundled payment models."}
+{"text": "Currently, the optimal adjuvant regional nodal irradiation (RNI) volume for breast cancer (BC) remained controversial. We aimed to define the optimal RNI treatment volume for BC by using a comprehensive network meta-analysis (NMA) of published studies.PubMed, Embase, Medline, and Cochrane Central Register of Controlled Trials were searched from database inception to 30 May 2022. Studies assessing different adjuvant RNI volumes for BC were eligible for inclusion. The primary outcome was overall survival (OS), and secondary outcome was disease-free survival (DFS) and distant-metastasis-free survival (DMFS).p<0.001), DFS with HR of 0.78 , and DMFS with HR of 0.87  when compared to controls. Sub-group analysis indicated that RNI with IMNI significantly improved OS , DFS , and DMFS  when compared to RNI without IMNI. NMA showed that CW/WB  + RNI with IMNI significantly improved DFS  and DMFS , but not for OS  when compared to CW/WB alone. Based on the analysis of the treatment ranking, CW/WB+RNI with IMNI appeared as the best treatment approach for BC patients.A total of 29,640 BC patients from twenty studies were included. The pooled hazard ratio demonstrated that internal mammary node irradiation (IMNI) in BC patients significantly improved OS giving HR (hazard ratio) of 0.87 (95%CI: 0.83\u20130.91, Our pooled results demonstrated that RNI with IMNI yielded a significant survival advantage for BC patients. NMA showed that CW/WB+RNI with IMNI was the optimal radiation volume for BC patients. Studies comparing different regional nodal irradiation volumes were included. The search keywords were breast cancer, breast carcinoma, radiotherapy, regional node radiation, and clinical studies. Clinical studies should meet the following criteria (1): clinical studies involving BC patients (2), clinical studies comparing efficacy of different adjuvant RNI volumes, and (3) available survival data regarding RNI in BC patients. BC patients treated with neoadjuvant therapy were excluded for analysis in the present study.Four independent investigators conducted the data extraction, and any discrepancy between the reviewers was resolved by consensus. The following information was extracted for each study: first author\u2019s name, year of publication, number of enrolled patients, study design, radiation regimen, main inclusion characteristics, radiotherapy dose, and median follow-up time. If the radiation volume of RNI was not specifically defined including RNI+SCN (supraclavicular lymph node) and/or RNI+IMNI and/or RNI+SCN+IMNI, we defined it as mixed RNI group. The primary outcome of interest was OS, and the secondary outcomes were DFS and DMFS.2-based Q statistics . Between-study heterogeneity was estimated using the \u03c7atistics . Heteroger tests , 8. A ster tests . The quaer tests . For stuer tests .An NMA offered methods to visualize and interpret a broader picture of current evidence and assessed the comparative effectiveness among various RNI volumes. Therefore, a network meta-analysis was performed using a frequentist framework . A netwoAccording to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) statement, we conducted the present meta-analysis . Our iniThe main characteristics of included studies are summarized in For the five prospective randomized studies \u201317, 27, p<0.01, I2=26%, p=0.16), and the pooled HR for OS was performed by using fixed-effects model. As for DFS, 16 studies were included for analysis , and the pooled HR for DFS was performed by using random-effects model. A total of 10 studies included DMFS data for analysis , and the pooled HR for DMFS was performed by using random-effects model.A total of 17 studies , 33, 34 analysis , 32, 34.analysis , 26, 31,We then performed sub-group analysis according to different RNI volumes and found that CW/WB+RNI with IMNI significantly improved OS  CW/WB+RNI with IMNI-R (right-side breast cancer), 2) CW/WB+RNI with IMNI, 3) CW/WB+ RNI without IMNI, 4) CW/WB +mixed RNI, 5) SW/WB+SVC, 6) CW/WB alone, and 7) no radiotherapy (RT).An NMA of seven different RNI radiotherapy regimens was conducted with regards to OS in BC patients  received RNI, while 82.2% of patients with four or more positive lymph nodes were treated with RNI. Among patients treated with RNI, 60.9% of the patients RNI targeting only one regional node area, while only 3.9% received RNI targeting the three regional nodal areas . One posNevertheless, there were some limitations that needed to be concerned of. First, both prospective and retrospective studies were included in this meta-analysis. Although the quality of included studies was high, selection bias between groups could not be avoided despite the fact that we performed subgroup analysis according to RNI volumes that successfully reduced the heterogeneity to a low grade. Second, this study was conducted at the base trial level but not at the individual level. Therefore, we could not perform pooled analysis according to patient characteristics, such as nodal stage or tumor location, although it had been established that medial-located tumor is a well-known predictor for IMNI benefit. In addition, both early-stage and locally advanced BC were included in the present meta-analysis, which might be another source of heterogeneity. Finally, the present study included both old or modern systematic therapy, and advances in systematic treatment in the modern era might affect the benefit of RNI, which might be another source of heterogeneity.In conclusion, the present study demonstrated that RNI with IMNI yielded a significant survival advantage for BC patients. Subgroup analysis according to RNI volumes showed that CW/WB+RNI+IMNI significantly improved DFS and DMFS when compared to CW/WB+RNI without IMNI, but not for OS. NMA found that CW/WB+RNI+IMNI was the optimum RNI treatment strategy for BC patients that reduced mortality and disease progression. Additionally, further studies evaluating the impact of RNI volume on late cardiac and lung toxicities are strongly needed.The original contributions presented in the study are included in the article/Conceptualization: JC and W-XQ. Project administration: CX and LC. Methodology: W-XQ, GC, and JC. Data curation: JC and CX. Formal analysis: W-XQ. Manuscript preparation: W-XQ, CX, GC, JC, and LC. Final approval of manuscript: all authors."}
+{"text": "The protocol involves direct Regitz diazo transfer onto readily available 3(2H)-isoquinolones followed by TfOH-promoted hydroarylation by an arene molecule. Screening of the novel 1,2,4-trisubstituted 1,4-DHIQs against cancer cell lines confirmed high cytotoxicity of selected analogs, which validates this new chemotype for further investigations as anticancer cytotoxic agents.A practically convenient and streamlined protocol for the  H)-isoquinolones  undoubtedly represent a privileged scaffold . He. HeH)-isH)-isoquinolones 11 from phenylacetyl chlorides 12 and Schiff bases 13 using conditions described in the literature . Th. Th10a a9a was unavoidably accompanied by a varying amount of byproduct 15a (vide infra for the mechanistic reasoning for its formation) which demonstrated, along with other analogs of 15 which were isolated and characterized, limited chemical stability and deteriorated on prolonged standing as a solution in CDCl3 at ambient temperature. Compound 15a was the exclusive isolable product when benzene was eliminated from the reaction mixture  via theC-arylation of diazo substrates 10 , we proceeded to investigate the scope of this transformation for 4-diazo-3(2H)-isoquinolones 10a\u2013s as well as various arenes (C-arylation products 10 which in some cases was accompanied by a regioisomer formation (with respect to the entering arene moiety) and the formation of 3-isoquinolones 15 . Not unexpectedly, less electron-rich arenes furnished higher amounts of the 3-isoquinolone 15 byproduct compared to their electron-rich counterparts (cf. 9o vs 9p).Having identified the optimum conditions for the s arenes . In all H)-isoquinolone benzene ring increased the tendency of 3-isoquinolones 15 to form (cf. 9(15)f\u2013h). Using more reactive (electron-rich) arenes results in lower diastereoselectivity and regiospecificity of the reaction . The structure and the initially anticipated trans-configuration of the products 9 was unequivocally confirmed by 1H and 13C NMR spectroscopy as well as, in the case of compound 9a, by single-crystal X-ray analysis.Likewise, electron-donating substituents in the 4-diazo-3 by the formamide carbonyl oxygen atom followed by hydrolysis of the iminium moiety  reduced the number of viable cells by >95% at 30 \u03bcM concentration. Dose\u2013response testing of this compound against both NCI-H460 and A549 lung carcinoma cell lines resulted in the determination of IC50 values for compound 9j as 31.4 \u00b1 0.48 \u03bcM and 13.6 \u00b1 3.34 \u03bcM, respectively  scaffold. The protocol relies on hitherto undescribed direct Regitz diazo transfer onto readily available 3(2H)-isoquinolones followed by TfOH-promoted arylation. The generally high-yielding two-step sequence was shown to be applicable to a wide range of substrates. To a varying degree, the arylation step was accompanied by the elimination of the nitrogen molecule and deprotonation to furnish 3-isoquinolone byproducts (formed exclusively in the absence of the arene). The extent of this side reaction was found to be dependent on the electronic character of the 1,4-DHIQs and the carbocation-intercepting arene molecule. Considering the pronounced three-dimensional character of 1,2,4-trisubstituted 1,4-DHIQ adducts synthesized in this work, they were deemed efficient probes for the perturbation of vital cellular targets. Screening of these compounds against lung carcinoma cancer cell lines confirmed high cytotoxicity of selected analogs, which validates this new chemotype for further investigation as anticancer cytotoxic agents.In summary, we have presented a practically convenient and streamlined protocol for the 9a), 2170881 (for 10c) and 2170877 (for 16) contain the supplementary crystallographic data for this paper. These data are provided free of charge by the joint Cambridge Crystallographic Data Centre (http://www.ccdc.cam.ac.uk/structures) and Fachinformationszentrum Karlsruhe Access Structures service.Deposition Numbers 2158046 (for File 1General experimental information, X-ray crystallographic data, synthetic procedures, analytical data and NMR spectra for the reported compounds."}
+{"text": "Irrigation of root canal system is of great significance to the success of endodontic treatment, where sodium hypochlorite (NaOCl) is the most widely used irrigant in chemical preparation. NaOCl functions by eliminating bacterial biofilms and dissolving organic tissue, which may vary according to several factors such as the microbiology of root canal infection and the concentration of the irrigant. It has been proposed that the effectiveness of NaOCl could be enhanced via several methods, including heating the irrigant, applying in conjunction with certain reagents, or activating by agitation techniques. Despite its antibacterial and tissue-dissolving capacities, NaOCl should be used with caution to avoid detrimental effect due to its cytotoxicity and negative effect on dentin properties. In this narrative review, we discussed the factors that affect the properties of NaOCl, the methods to improve its efficacy, and the side effects that might occur in clinical practice. The elimination of microorganisms, necrotic tissues, and accumulated hard tissue debris is pivotal to the success of endodontic treatment . Since mSodium hypochlorite (NaOCl) is the most commonly used irrigant in endodontics . It is rIn an attempt to provide guideline for clinical practice and future studies, this paper is aimed at providing an overview of the published work discussing the parameters affecting the efficacy of NaOCl, clinical strategies that could enhance its effectiveness and side effects caused by NaOCl that could possibly occur during chemical preparation.Verification has been established regarding the etiologic role of intracanal microorganisms in the evolution of pulpal and periapical diseases, the elimination of which is a crucial function of NaOCl irrigant. The features of bacterial biofilm, including the bacterial species, biofilm structure, and maturity, could significantly influence the antibacterial effectiveness of NaOCl irrigant.in vitro. Enterococcus faecalis  is one of the main pathogens of persistent endodontic infection and has been long used to test the efficiency of root canal irrigants [E. faecalis. Darrag [Streptococcus mutans (S. mutans) was significantly more sensitive to 5.25% NaOCl compared to E. faecalis in planktonic condition. Using 0.5% NaOCl for 10\u2009s lowered the colony-forming unit (CFU) below the limit of detection in the case of Actinomyces naeslundii (A. naeslundii) and Candida albicans , while it took 30\u2009min for the same irrigant to reduce the CFU of E. faecalis to zero, suggesting that E. faecalis was more resistant to NaOCl [E. faecalis. Ghivari et al. [E. faecalis and Staphylococcus aureus (S. aureus) were both 100% eliminated after 10\u2009s of contact with 5.25% NaOCl in vitro. Moreover, the NaOCl-resistant capability of E. faecalis is also strain-related as Yang et al. [E. faecalis VP3-181 biofilms were more sensitive than Gel 31 biofilms when treated with 2% NaOCl.Numerous studies have investigated the NaOCl-resistant capability of different bacterial species rrigants . Some ba. Darrag  revealedto NaOCl . In conti et al.  reportedg et al.  reportedE. faecalis survival [E. faecalis with a multispecies biofilm containing E. faecalis, Fusobacterium nucleatum (F. nucleatum), Prevotella intermedia (P. intermedia), and Porphyromonas gingivalis  and found that E. faecalis was significantly less susceptible to NaOCl treatment in the multispecies biofilm, regardless of the NaOCl concentration applied (0.025%-2.5%). In addition, it has been reported that the resistance to NaOCl in a dual-species biofilm containing S. mutans was 30-fold higher than that of the E. faecalis single-species biofilm [E. faecalis biofilm groups after 0.9% NaOCl was applied, suggesting that when E. faecalis is grown as part of a multispecies biofilm, it did not gain any further resistance to NaOCl.The structure of bacterial biofilm plays an important role in its susceptibility against NaOCl irrigant, possibly due to the evolution of the microenvironment within the biofilm . Matrix survival . Bacterisurvival  compared biofilm . However biofilm  revealedThe biofilm architecture, especially its bacterial compactness, is another influential factor that affects the antibacterial efficacy of NaOCl. 2% NaOCl had a significantly weaker effect on the bacterial dense biofilms with lower water and EPS content, resulting in even stiffer biofilms, as evidenced by the significant reduction of the biofilm stress relaxation, compared to biofilms which are richer in water and EPS . An ensuThe maturity of biofilm also contributes to its susceptibility to NaOCl irrigant. It has been widely reported that the relatively mature biofilms  were more resistant to NaOCl compared to young biofilms  , 16, 17.Compared to younger biofilm, more viscous water molecules bounding to the EPS structure, along with more viscous EPS material, were found in mature biofilm, which could be a potential explanation of the age-dependent resistance to NaOCl . The turThe remnant pulp tissue could provide ideal conditions for microorganisms to survive and proliferate , 23; theAll forms of chlorine in NaOCl solution, including hypochlorous acid (HOCl) and hypochlorite ion (OCl-), are collectively referred to as \u201cfree available chlorine\u201d. By direIt is noteworthy that EDTA reduces the tissue-dissolving capability of NaOCl due to the dramatic depletion of free available chlorine content , 32, as Previous studies have reported that the presence of dentin has a detrimental effect on the ability of NaOCl to dissolve pulp tissue , 36. Thiin vitro studies.Tissues from different sources were used to test the tissue dissolution effectiveness of NaOCl due to the availability, including bovine pulp and muscle as well as porcine pulp, muscle, and palatal mucosa , 38\u201342. The tissue-dissolving capability of NaOCl could also be influenced by various factors such as concentration, temperature, agitation, and pH, which will be discussed in the following sections.In addition to the various uses mentioned above, NaOCl also possesses other functions in endodontic treatment. Tooth discoloration caused by the accumulation of hemoglobin or other forms of hematin molecules in dentinal tubules after endodontic treatment is a clinical esthetic problem . BleachiIt is widely agreed that the increase of exposure time would lead to a significantly better antibacterial efficacy, though the testing exposure time varies from 1\u2009min to 30\u2009min due to the difference in experimental design.Wang et al.  found thE. faecalis biofilms was fastest during the first 3 minutes and slowed down greatly after 10 minutes [Petridis et al.  investig minutes .E. faecalis, suggesting that with 0.5% NaOCl as irrigant, an exposure time of 30\u2009min reduced viable count to zero, compared with 10\u2009min for 1%, 5\u2009min for 2.5%, and 2\u2009min for 5.25%. Similarly, Tawakoli et al. [Radcliffe et al.  studied i et al.  reportedThe tissue-dissolving capacity of NaOCl was also reported to be time-dependent . When boNaOCl demonstrates its antibacterial and tissue-dissolving activity through the interaction between free available chlorine (active chlorine) and the organic composition of bacteria biofilm/residual pulp tissue . HoweverRefreshment of NaOCl irrigant is considered a practical method to maintain its efficacy by compensating the loss of free available chlorine during the oxidating process . A numerE. faecalis biofilms. Moreover, some studies suggested that high concentrations of NaOCl are necessary to eliminate bacterial biofilms. Golob et al. [E. faecalis vitality in the biofilm while the 2.5% and 5.25% NaOCl solutions caused complete inhibition of the biofilm bacterial growth [NaOCl irrigant is used at concentrations varying between 0.5% and 6% with no consensus for the optimal concentration . Dumitrib et al.  proposedl growth .Penetration depth of NaOCl is vital to the disinfection within dentinal tubules. It was found that the increase of NaOCl concentration resulted in deeper penetration depth, with the observed depth of 1% NaOCl was approximately 60%-80% of that of 6% NaOCl . Likewisin vitro studies described above, it has been reported that biofilm removal from isthmus and lateral canal during syringe irrigation was independent from NaOCl concentration [Although the application of concentrated NaOCl presented considerable disinfecting capacity in the ntration . In addintration  found thpH value significantly influences the chemical properties of NaOCl irrigant by altering the relative ratio of OCl- and HOCl . The decThe application of NaOCl gel, instead of solution, has been proposed with the objective to reduce the risk of apical extrusion and thereby preventing NaOCl accident . Zand etOne of the drawbacks of NaOCl as root canal irrigant is its high surface tension, which limits its ability to penetrate the root canal irregularities and dentinal tubules . EndeavoSurface active agent resulted in lowered contact angle, while the amount of free available chlorine, cytotoxicity, and antibacterial efficacy of NaOCl solution was unaffected , 88. HowContinuous chelation is an innovative root canal irrigation protocol where NaOCl is mixed with an etidronate powder (HEDP) to create a new endodontic irrigant . HEDP isHeating NaOCl irrigant could enhance the chemical properties of NaOCl by increasing its reaction rate . WarmingIt was reported that greater tissue dissolution, bacteria elimination, and smear layer removal could be achieved by heating NaOCl while maintaining its free available chlorine , 109. IaDue to the presence of organic content such as dentin collagen and microbial biomass, the efficacy of NaOCl irrigant would be compromised, resulting in bacterial persistence . While tTraditionally, NaOCl is delivered into root canal system by syringe, referred to as traditional needle irrigation (CNI), which failed to demonstrate satisfactory irrigating effectiveness . Therefo\u03bcs), Er:YAG laser could induce intracanal cavitation and shockwaves as a result of photoacoustic and photomechanical effects, known as photon-induced photoacoustic streaming (PIPS) [Passive ultrasonic irrigation (PUI) is a type of ultrasonic-driven irrigation that has become one of the most widely used technique to activate NaOCl irrigant in clinical practice . Passiveg (PIPS) . Recentlg (PIPS) . XP-endog (PIPS) . Negativg (PIPS) .In this segment, we compared the differences of the aforementioned agitation techniques in terms of their abilities to enhance the efficacy of NaOCl, including the tissue-dissolving and antibacterial properties, the penetration and distribution in the root canal system, the removal of dentin debris and smear layer, and the clinical outcomes.The removal of pulpal remnant and organic tissue is of great significance to the success of root canal treatment, the achievement of which depends on the application of NaOCl , which iGW has been proven to be a very effective agitation technique in terms of tissue dissolution. Compared to PUI and EA, GW demonstrated a tissue dissolution rate that was more than 8 times faster than the second fasted device using 6% NaOCl as irrigant . In addiThe removal or dissolution of inflamed pulp tissue is essential in the treatment of internal root resorption cases . PUI demin vitro studies found a superior antimicrobial effect compared to syringe irrigation [PUI is an effective irrigation technique that improves the action of NaOCl against root canal bacteria and the endotoxins \u2013133. Howrigation , while arigation .\u03bcm.Compared to PUI, PSI demonstrated similar antibacterial efficacy in both curved and straight root canals . MoreoveNeelakantan et al.  comparedXPF has also demonstrated promising antibacterial effectiveness compared to PUI and CNI . Azim etThe EndoVac apical negative pressure irrigant delivery system was found to be effective compared to CNI in terms of antimicrobial efficacy \u2013151. It As a novel activation device, GW demonstrated promising antibacterial effectiveness. Zhang et al.  revealedIt has been clearly demonstrated by the microbiology of endodontic infection that bacteria can be found in main canal space, lateral canals, and dentinal tubules . The penSeveral agitation techniques have been verified to significantly increase NaOCl penetration into dentinal tubules, including PUI, PSI, LAI, and EndoVac , 155. AkIn addition to the penetration into the dentinal tubules, the distribution of NaOCl irrigant in the root canal system is also essential to the success of chemical preparation. It was reported that XPF was more effective in distributing irrigant throughout the mesial root canal system compared to PUI and CNI . FurtherThe main purpose of root canal preparation is the cleaning and shaping of root canal system, the cleanliness of which is dependent on the proper removal of hard tissue debris and smear layer . Severalin vitro studies have evaluated the effectiveness of activation methods, in vivo studies are required to provide higher-level evidence regarding the performance and benefits of irrigation devices. Liang et al. [Although a great number of g et al.  comparedg et al. , 184. Mog et al. . More rain vivo, the data obtained in vitro studies cannot be directly extrapolated to conditions in clinical practice.Apical extrusion, known as hypochlorite accident, is defined as the incidence which NaOCl irrigant extrudes beyond the apex, with or without dentin debris . Large aAs a strong oxidizing agent, NaOCl is cytotoxic to the periapical tissue and stem cells, especially in case of regenerative endodontic treatment . The cytA major complication of root canal treatment is the occurrence of postoperative pain caused by various factors, one of which is the extrusion of NaOCl irrigant . Due to Theoretically, as mentioned above, the application of NaOCl gel is an alternative that could reduce the risk of apical extrusion and thereby reducing the occurrence of postoperative pain. A study regarding this topic showed that the NaOCl gel group resulted in significantly less postoperative pain compared to its solution counterpart within the first 24\u2009h, while no significance difference was recorded on days 2, 3, and 7 . More raSurprisingly, although it was claimed that the activation of irrigant might lead to a greater amount of apical extrusion , currentThe irrigation protocol using NaOCl irrigant would impose a time- and concentration-dependent adverse effect on mechanical properties of dentin , such asex vivo experimental model, Ioannidis et al. [Due to the reaction of chlorine or hypochlorite with natural organic matter (NOM) such as pulpal tissue and bacterial biofilm, harmful volatile organic compounds (VOCs) and chlorinated disinfection by-products (DBPs) can be generated during chemical preparation with NaOCl. The carcinogenic and mutagenic properties of these VOC and DBP compounds may further impair human health via leaking through the apical foramen, biologically interacting with periapical tissue and even entering blood circulation . By usins et al.  found chIn vitro studies suggested that the characteristics of bacterial biofilms could affect their susceptibility against NaOCl. In addition, the efficacy of NaOCl could be affected by various parameters such as concentration, temperature, exposure time, and pH value of the irrigant. Numerous techniques have been developed to activate NaOCl since the application of NaOCl alone could be ineffective during chemical preparation, though controversy remained regarding their clinical effectiveness. Despite its antibacterial and tissue-dissolving capacities, NaOCl possesses cytotoxicity against periapical tissue whereas the strong oxidant property would impose negative impact on the mechanical properties of dentin. Therefore, NaOCl should be used with caution to avoid its detrimental effect in clinical practice.NaOCl plays an indispensable role in chemical preparation of root canal treatment. We recommend that future studies should focus on the disinfection in apical third and deeper layer of dentin, since these areas are potential harbors of bacterial biofilms. Furthermore, more clinical studies are warranted to provide due credence to the efficacy of agitation techniques."}
+{"text": "The supplementary figures are now available online."}
+{"text": "For the \u03b2\u2013\u03b2\u2032 linkage, the alpha carbons had the lowest BDEs of the ring opening reactions due to excessive electron delocalization around the aromatic rings. The bonds of the 4-O-5 linkage had similar BDEs but were appreciably higher than the BDEs for other ether linkages, such as \u03b2-O-4 and \u03b1-O-4. The higher BDEs of the 4-O-5 bonds is a result of the radical being formed on an aromatic carbon compared to an aliphatic carbon. Our results indicate the ring-opening reactions around the alpha-carbon of the \u03b2\u2013\u03b2\u2032 linkage would be a major reaction point during thermal deconstruction of the chosen oligomers. This work provides valuable information on the thermal deconstruction behavior of two lesser studied interunit linkages that builds on the authors' previous work, on \u03b2-O-4, \u03b1-O-4, and \u03b2-5 linkages, to develop a library of reaction information for various lignin interunit linkages.Model compounds that represent important substructures in lignin have popularly been used to gain a better understanding of the behavior of lignin during thermal deconstruction, such as fast pyrolysis. The \u03b2-O-4 linkage of lignin has previously been the focus of many model compound studies as it is the most prevalent linkage found in native lignin. In this work, two lesser studied linkages, the \u03b2\u2013\u03b2\u2032 and 4-O-5, were investigated with density functional theory (DFT). Bond dissociation enthalpies (BDEs) were calculated for the relevant bonds along each interunit linkage for two model compounds containing these linkages. Conformational analysis of the first model oligomer has a relative enthalpy difference of 1.55 kcal mol \u03b1-containing bonds look to be the primary points of reaction along the \u03b2\u2013\u03b2\u2032 linkage during thermal conversion.Density functional theory (DFT) simulations were performed on two lesser understood linkages in lignin. The C Lignocellulosic biomass is an abundant, renewable resource with the potential to serve as a sustainable alternative to fossil fuel derived products such as liquid transportation fuel and specialty chemicals. Increasing concern over greenhouse gas emission from fossil fuels has sparked interest in understanding how to effectively convert lignocellulosic biomass into value-added materials typically derived from fossil fuels. Fast pyrolysis is a thermochemical conversion process that can create a liquid product, which can potentially be upgraded into transportation fuel and other valuable products.1\u20134 The cellulose found in lignocellulosic biomass is structurally the same regardless of material except for the degree of polymerization. Cellulose has received significant attention in fast pyrolysis reaction studies and its reaction mechanism is consequently more understood relative the other major components.5\u201310 Hemicellulose comprises 20\u201335 wt% of lignocellulosic biomass and is an amorphous heteropolymer of pentose and hexose sugars.11,12 The composition and structure of hemicellulose is dependent on the biomass source; however, it be classified in terms of well-defined polysaccharide types.13 Hemicellulose has not been investigated to the extent of cellulose, but mechanistic information about its fast pyrolysis process has been achieved.11 Lignin, the last major component of lignocellulosic biomass, constitutes up to 35 wt% of lignocellulosic biomass.14,15 Unlike cellulose and hemicellulose, lignin does not have a polysaccharide structure. Instead, lignin is made up of aromatic units and is the most abundant natural source of aromatics in the world.16 Lignin is synthesized via enzymatic dehydrogenation of three monolignols, p-coumaryl alcohol, sinapyl alcohol, and coniferyl alcohol. The resulting phenoxyl radicals are randomly polymerized into a complex and not well-defined three-dimensional structure with a variety of different interunit linkages.15,17 Lignin's fast pyrolysis mechanism is less understood compared to cellulose and hemicellulose, owing to its complex and less well-defined structure. The limited knowledge of lignin's fast pyrolysis mechanism leaves a significant knowledge gap in whole lignocellulosic biomass' fast pyrolysis reaction mechanism. Therefore, it is difficult to implement mechanistic reaction schemes into models of biomass fast pyrolysis and has led to the use of lumped kinetic schemes.19 Consequently, the overall goal of this study is to improve our understanding of lignin fast pyrolysis by examining the pyrolytic breakdown of specific interunit linkages in model compounds through bond dissociation enthalpies (BDE). The determination of BDEs of lignin interunit linkages provides valuable insights on initial reaction sites and can guide the proposal of future lignin pyrolysis reaction mechanisms using a combination of both computational and experimental approaches.Lignocellulosic biomass is primarily comprised of three major components: cellulose, hemicellulose, and lignin. Cellulose makes up 40\u201350 wt% of lignocellulosic biomass and is a linear polymer of glucose monomers joined together through a \u03b2- glycosidic linkage. It is considered the most abundant naturally occurring polymer in the world.20\u201332 The kinetic information gathered from monomer and dimer studies has greatly improved our understanding of the thermal degradation behavior of the smallest lignin building blocks; however, due to the complex, non-linear nature of lignin, it is not advisable to assume the kinetics are directly applicable to the larger native lignin structure. Therefore, the knowledge gap for lignin pyrolysis is how to get from model dimers to a native lignin structure. The authors believe it is possible to bridge that gap by increasing the size of the model compounds and begin to investigate oligomers and other important lignin substructures. Recently, there have been studies that have explored the thermal decomposition behaviors of larger, linear oligomeric species.33 The author's previous work investigated a model tetramer containing \u03b2-O-4, \u03b1-O-4, and \u03b2-5 interunit linkages and found the trends between dimer and oligomer for each linkage hold true. The C\u2013O bonds of both the \u03b2-O-4 and \u03b1-O-4 linkages had lower BDEs than their C\u2013C counterparts while the C\u03b1 of the \u03b2-5 ring exhibited the lowest BDEs for ring-opening. However, the magnitudes of the bond dissociation enthalpies (BDE) differ based on the surrounding substituents.34 Through continuing investigations into the variety of interunit linkages and substructures found in native lignin, it is possible to build a library of potential reactions. Given you have characteristics of the lignin, this library will form a reaction mechanism comparable to the mechanisms proposed for cellulose and hemicellulose.6,7,11Despite lignin's unclear native structure, it is still possible to identify prevalent and important linkages found in lignin. We can gain valuable understanding of the reaction behavior of these linkages and substructures using model compounds. Model compounds are smaller, simpler compounds that either contain a significant interunit linkage found in lignin or represent possible substructures of a larger lignin polymer. Most lignin model compound studies have focused on model monomers, such as guaiacol and syringol, and dimers, such as phenethyl phenyl ether (PPE).28,35 In a DFT study of pinoresinol, a dimer with a \u03b2\u2013\u03b2\u2032 linkage, the C\u03b1\u2013O and C\u03b1\u2013C\u03b2 bonds exhibited the lowest BDEs, which were supported by visualization of significant delocalization of the unpaired electrons from the resulting product.28 Additionally, the 4-O-5 linkage, as part of a large BDE study of multiple linkage types, was found to have high BDEs among the ether linkages even higher than the C\u03b1\u2013C\u03b2 bond of the \u03b2-O-4 linkage.35 The authors hypothesize similar trends will be present in the oligomers where the ring-opening reactions involving the alpha carbon of the \u03b2\u2013\u03b2\u2032 linkage will have the lowest BDEs of the \u03b2\u2013\u03b2\u2032 linkage. This is due to the resulting radicals on the alpha carbon should exhibit higher electron delocalization along the neighboring aromatic ring. Additionally, the authors believe both 4-O-5 bonds will have appreciable higher BDE than the \u03b2\u2013\u03b2\u2032 and other ether linkages as the carbons of this ether linkage are directly part of an aromatic ring. These hypotheses line up with the previously reporting findings for model dimers.28,35 Based on previous oligomer works, the authors believe the general trends found in BDE for each linkage will agree with the trends published for their respective dimers, but the differences will be found in the magnitude of the BDEs.In this study, two model oligomers, containing \u03b2\u2013\u03b2\u2032 and 4-O-5 linkages, are investigated using DFT to determine the BDEs of homolytic cleavage reactions at relevant bonds along the interunit linkages. The \u03b2\u2013\u03b2\u2032 and 4-O-5 have not received the level of attention as other more prevalent linkages, such as \u03b2-O-4; however, there are previous dimer studies for both linkages that serve as good points of comparison.36 The second tetramer, hereafter referred to as model compound 2 (MC2), contains a \u03b2\u2013\u03b2\u2032 linage and two \u03b2-O-4 linkages.37 The structures of MC1 and MC2 are shown in 29,34,38\u201341 this work will focus primarily on the bond dissociation enthalpies of the \u03b2\u2013\u03b2\u2032 and 4-O-5 linkages.Two model oligomers were chosen for this study, both containing at least one \u03b2\u2013\u03b2\u2032 and one containing an additional 4-O-5 linkage. The first tetramer, hereafter referred to as model compound 1 (MC1), contains two \u03b2\u2013\u03b2\u2032 linkages and one 4-O-5 linkage.et al.37 proposed a single absolute configuration of the four chiral centers of the \u03b2\u2013\u03b2\u2032 linkage. Additionally, both \u03b2-O-4 linkages were reported as erythro, which further reduces the number of stereoisomers that should be considered. The reported configurations of each linkage reduce the number of possible stereoisomers from 64 to 4. The naming convention for these configurations is also shown in R or S). The chiral centers are ordered from left to right and the resulting combination of absolute configurations is how each stereoisomer will be differentiated from each other. An example of the naming convention using MC1 is as follows: if the four chiral centers of both \u03b2-O-4 linkages have the R configuration, the resulting stereoisomer will be labeled RRRSSRRR.MC1, also known as hedyotisol, contains eight chiral centers, which produces 64 possible stereoisomers. However, in the initial identification of hedyotisol, Matsuda 36 Similar to MC1, MC2 has eight chiral centers located around both of the \u03b2\u2013\u03b2\u2032 linkages. Assuming the same configuration of the \u03b2\u2013\u03b2\u2032 in MC1, there is only one possible stereoisomer to account for, which is RSSRRSSR.MC2 is a previously identified model lignin oligomer that contains two \u03b2\u2013\u03b2\u2032 linkages and a 4-O-5 linkage.et al.34The computational work in this study was done using Spartan'18 , GaussView 6, and Gaussian 16 . The calculations in this project can be broken down into two categories that are discussed in the following sections: conformational analysis and bond dissociation enthalpy (BDE) calculations. The conformational analysis of each stereoisomer was performed using Spartan 18 on a local desktop using a single processor, while the density functional theory (DFT) calculations were performed using the Gaussian suite of software. The DFT simulations were carried out on the Infrastructure for Scientific Applications and Advanced Computing (ISAAC) high-performance computing resource at the University of Tennessee. Each DFT simulation was performed in parallel using eight processors. Additional discussion on the computational details employed in this work can be found in Houston 18 These oligomers can be present in different three-dimensional configurations due to the rotations of individual bonds throughout the molecule. To ensure our calculations are carried out on the lowest energy structure for each stereoisomer of our model oligomers, a Monte Carlo conformational analysis was performed to identify the lowest energy conformer. The initial Monte Carlo search found the 500 lowest energy conformers using the molecular mechanics force field MMFF94.42 The remaining conformers were further refined down to the 10 lowest energy conformers by performing a geometry optimization using a semi-empirical PM6 level of theory.43 The 10 conformers were then optimized by DFT using the M06-2X a 6-31+G(d) to find the lowest energy conformer, which was further optimized using a more robust 6-311++G basis set.44\u201348 The dispersion interactions were described using the GD3 empirical dispersion correction.49In both model oligomers, the chiral centers are located at the \u03b1 and \u03b2 positions of each respective \u03b2\u2013\u03b2\u2032 and \u03b2-O-4 linkage, due to the free-radical nature of lignin polymerization.via a two-step calculation procedure consisting of geometry optimization and frequency analysis at 773.15 K (500 \u00b0C). This is an established moderate temperature for biomass fast pyrolysis and energetics determined for this temperature will be relevant for comparisons with experimental studies. For each stereoisomer, the starting oligomers as well as the resulting products from each homolytic cleavage were generated from the lowest energy conformer determined by conformational analysis. The products from cleaving the bonds in the \u03b2-O-4 and the 4-O-5 linkages are two, separate radical species, while most of the products from scission of bonds in the \u03b2\u2013\u03b2\u2032 are single, di-radical species. In the case of the di-radical species, special care was taken to ensure they did not immediately re-bond during geometry optimization. The cleavage location for every relevant bond along each interunit linkage is shown in 50The energetics associated with the homolytic cleavage of relevant bonds along each interunit linkage were determined \u22121 and 10.07 kcal mol\u22121, respectively. The lowest energy conformer of each stereoisomer was then optimized using the same Minnesota 06 functional with a 6-311++G basis set to determine the initial structure of each stereoisomer for MC1 and MC2. The relative enthalpy differences of stereoisomers for MC1 are shown in \u22121, which exceeds the accepted \u00b11.00 kcal mol\u22121 threshold for chemical accuracy so we are unable to say that these stereoisomers have the same total enthalpy.51 Therefore, BDEs of relevant linkages will be calculated for all four stereoisomers of MC1. The optimized initial geometries of each stereoisomer for MC1 and MC2 are shown in The thermal enthalpies, the sum of the electronic and thermal enthalpies, of each stereoisomer's ten conformers were calculated at 298.15 K using the M06-2X functional with a 6-31+G(d) basis set for both model compounds. The conformers of stereoisomers for MC1 and MC2 were shown to have an appreciable difference in their enthalpies, ranging from 4.52 to 9.14 kcal mol\u03b1\u2013O and C\u03b3\u2013O bonds, were not considered.34 The BDEs of each homolytic cleavage were determined at 773.15 K (500 \u00b0C) to represent a well-accepted operating temperature for biomass fast pyrolysis. Upon determination of the BDEs for each relevant bond, trends were identified for each interunit linkage. Heat maps of the BDEs for MC1 and MC2 are shown in The bond dissociation enthalpies (BDE) were calculated for each linkage along the \u03b2\u2013\u03b2\u2032 and 4-O-5 linkages. The three-dimensional coordinates for each investigated compound are included in the ESI.\u03b2\u2013O bond has a lower BDE than the other bonds along the \u03b2-O-4 linkage, ranging from 77.39\u201390.06 kcal mol\u22121. The non-aromatic component of the ether bond has previously been shown to be the lowest BDE bond in the \u03b2-O-4 linkage, therefore, our trends are consistent with previous reports.35,41,52,53 The overall trends agree; however, the magnitudes of the BDEs are slightly higher than what has typically been reported for \u03b2-O-4 dimers .35,52,54 Inspection of the three-dimensional geometry shows MC1 is not necessarily linear and can fold in a way that brings the \u03b2-O-4 into closer proximity to other aromatics and linkages, which could serve to stabilize the bond. Further investigation into the cause of the magnitude discrepancy is needed; however, it is outside the scope of this study.For the \u03b2-O-4 linkages of MC1, the C\u22121. The reactions involving each C\u03b1 (excluding bonds to an aromatic ring) exhibited lower BDEs then the other \u03b2\u2013\u03b2\u2032 ring-opening reactions. The C\u03b1\u2013O bond had a range of 70.29\u201380.04 kcal mol\u22121, while the C\u03b1\u2013C\u03b2 had a range of 68.43\u201381.45 kcal mol\u22121. This trend agrees with previous BDEs published for a pinoresinol dimer.28 The magnitudes of the BDEs for these reactions are slightly larger than previously reported dimer values but have much closer agreement than the BDEs for the \u03b2-O-4 linkage. Spin density plots for each di-radical produced from the \u03b2\u2013\u03b2\u2032 ring opening scission of MC1 are shown in \u03b1\u2013O and C\u03b1\u2013C\u03b2 scissions, there is significantly more delocalization of the unpaired electrons, which leads to more stable, di-radical products. The more stable the resulting product, the lower the BDE for the scission of that bond will be. The di-radical species produced from the scission of other bonds along the \u03b2\u2013\u03b2\u2032 linkage do not possess similar levels of electron delocalization. Instead, the spin density is centered around the remaining \u03b2\u2013\u03b2\u2032 ring. This same trend and spin density phenomenon holds true for the linkages of MC2 as well.The ring-opening scissions in the \u03b2\u2013\u03b2\u2032 linkage, for both MC1 and MC2, were shown to have the BDEs in the range of 70.29\u201387.06 kcal mol\u22121) was shown to have a slightly lower BDE than the O-5 bond . The 4-O-5 linkage is a strong ether linkage due to the proximity of the aromatic rings. The cleavage of these bonds is not favored compared to other ether linkages, such as the \u03b2-O-4, due to the fact scission would result in a radical on an aromatic carbon, which would disrupt the aromaticity. Additionally, the spin density is concentrated around the radical making the product unstable. Therefore, we see the 4-O-5 linkage has higher BDEs than other ether linkages.The 4-O-5 linkage found in MC2 has similar BDEs for both the 4-O and the O-5 bond. The 4-O bond  with ranges of 70.29\u201380.04 kcal mol\u22121 and 68.43\u201381.45 kcal mol\u22121, respectively. The 4-O-5 bonds of MC2 had much higher BDEs than other ether linkages such as the \u03b2-O-4 linkage in MC1 and other ether linkages in literature. Spin density plots showed excessive delocalization of radicals around the alpha carbons, which helps explain the lower BDEs of the respective bonds. Even though the trends were the same as previous dimer work, the magnitudes of the BDEs for the model compounds in this work were typically larger than what was reported for their dimer counterparts. Further investigation is needed to determine the exact cause of the discrepancies in the magnitudes of the BDEs. This study builds on a previous model oligomer study and provides another step towards developing a library of interunit linkage behavior that can potentially be used for generalized reaction rules for the pyrolysis of lignin.The use of larger model compounds, such as oligomers, attempts to bridge the knowledge gap between model monomers and dimers and native lignin. In this work, the authors computationally determined, with density functional theory, the bond dissociation enthalpies of two model lignin oligomers containing two underrepresented interunit linkages, \u03b2\u2013\u03b2\u2032 and 4-O-5. Understanding the bond dissociation energies (BDEs) of relevant bonds provides information about the most likely points of reactions during the thermal deconstruction of these oligomers during biomass fast pyrolysis. Conformational analysis of every relevant stereoisomer for both model compounds showed a relative enthalpy difference of 1.55 kcal molThe authors declare no competing financial interest.RA-013-D2RA07787F-s001"}
+{"text": "Mindfulness techniques, which are currently widely used in psychosomatics and psychotherapy, pose challenges when treating people coming from Buddhist groups for several reasons.For their treatment, it is important to take into account decontextualized terms that underlie crucial group dynamics and the effects of damaging neologisms in international Buddhist organizations.In the current research project, this topic is approached in combining quantitative with qualitative data. Whereas the data collection is still ongoing, the replies of twelve people are presented.As commitments to secrecy hinder people to ask for psychotherapy for long, they were asked on their thoughts about secrecy in Buddhist groups. Five of them agreed that acts against them were declared secret, which they then further specified. Six probands agreed having witnessed acts directed toward others being sworn to secrecy, four of which told this was about sexual abuse. Whereas nine agreed having experienced enemy images being built up, three agreed and specified how their own freedom was impaired and six witnessed and specified other group members\u2019 freedom having been constrained. While six persons agreed that it was assumed in their group one or more persons could \u2018purify\u2019 someone else in the sense of a \u2018karma purification\u2019 and specified their replies, two replied this concept was used to rationalize actions towards themselves and how it has affected.As for psychotherapy, it is important to take into account rationalization of violence and abuse through neologisms, pseudotherapies and structural issues in context.This research is funded by the German Federal Ministry of Education and Research (BMBF)."}
+{"text": "The COVID-19 Infection Survey (CIS) allows us to understand many aspects of the pandemic. To enable more in-depth accurate analysis on positivity and vaccine effectiveness we have needed to link the survey at pace to NHS Test and Trace (T&T) data to better inform policy decisions at the highest level.The complex nature of the T&T dataset has been challenging to the development of a robust linkage method capable of producing regular timely updates. The data first required extensive cleaning, editing and standardisation. A combination of deterministic, probabilistic, and associative linkage techniques was used to produce four pairwise linkages: T&T was linked to itself to create person-level test clusters and a unique person ID; CIS-Personal Demographic Service (PDS) and T&T-PDS linkage tables were then produced assigning NHS number to maximise the quality of the linkage between the datasets, before producing a final separate anonymised CIS-T&T linked product fit for analysts.Post-linkage quality assessment to estimate the number of false positives  and false negatives (missed matches) was undertaken and utilised in the context of true positives to estimate precision (accuracy) and recall (coverage) of the linked method, both found to be over 98% for the CIS-T&T linkage. Continuous methodological development has significantly improved the linkage quality and match rate over time. The additional test data provided by the linkage enables analysts to identify participants testing positive between their scheduled monthly visits. This adds key missing information, enabling the level of protection from re-infection afforded by previous infection to be estimated with increased accuracy and reduced bias. Analysts can now access enriched linked datasets with confidence that the linkage method meets the required quality standards.The analysis produced from this linked data has provided crucial evidence to government on re-infection and infection following vaccination, which has enabled more targeted public health policies to help avoid unnecessary economic harm. Widely published statistics have helped inform the public\u2019s understanding of the pandemic and their personal risk."}
+{"text": "Over the past few decades, there has been a noticeable surge in the market of plant-based meat analogs (PBMA). Such popularity stems from their environmentally friendly production procedures as well as their positive health effects. In order to meet the market demand, it is necessary to look for plant protein processing techniques that can help them match the quality of conventional meat protein from the aspects of sensory, quality and functionality. Bean proteins are ideal options for PBMA with their easy accessibility, high nutrient-density and reasonable price. However, the high polyunsaturated lipids content of beans inevitably leads to the unpleasant beany flavor of soy protein products, which severely affects the promotion of soy protein-based PBMA. In order to solve this issue, various methods including bleaching, enzyme and fermentation etc. are developed. Among these, fermentation is widely investigated due to its high efficiency, less harm to the protein matrix, targeted performance and low budget. In addition, proper utilization of microbiome during the fermentation process not only reduces the unpleasant beany flavors, but also enhances the aroma profile of the final product. In this review, we provide a thorough and succinct overview of the mechanism underlying the formation and elimination of beany flavor with associated fermentation process. The pros and cons of typical fermentation technologies for removing beany flavors are discussed in alongside with their application scenarios. Additionally, the variations among different methods are compared in terms of the strains, fermentation condition, target functionality, matrix for application, sensory perception etc. The Food and Agriculture Organization of the United Nations estimates that in 2019, humans consumed about 3.25 million tons of meat; demand for meat is expected to increase by another 12% by 2029; and by 2050, demand for meat will increase by about 70%. If traditional meat production and management patterns remain unchanged, an additional 30%\u201350% of the land will be needed for livestock and meat production by then . MoreoveRecent PBMA research and development has been focusing on utilizing raw materials such as soy and pea proteins to mimic the flavor, smell, appearance and texture of traditional meats. It is beneficial for the whole mankind not only in terms of promoting a sustainable development, but also from a nutrition aspect . Compare2 extraction, new cultivars breeding, genetic engineering, etc.  had the most obvious effect on the removal of beany flavor. Because the selection of compounds involved in the beany flavor included nine aldehydes, one furan, four alcohols, four ketones, three sulfides, and five pyrazines, the compound with the greatest effect on \u201cbeany\u201d are aldehydes . ADH conLactobacillus acidophilus and Streptococcus thermophiles  Oblrogenase . (c) Fac pathway . (d) Alcrevisiae . (e) Oxiboxydans .Previous studies have indicated that the beany flavor in legumes is primarily the result of interactions between lipid oxidation products, proteins, and phytochemicals. Lipid-derived off-flavors are believed to be the main cause of beany flavor. Among them, the precursors of the flavor of legumes are mainly phospholipids (PLs) and free fatty acids (FFAs).Phospholipase can be used to remove such precursors, among which the combined phospholipase A2 (PLA2) and cyclodextrin mixed with soybean meal in a water bath and it is found that the removal rate of phospholipids is more effective .In previous studies, it was found that the principle of removing beany flavor was mainly divided into two steps: hydrolyzing PLA 2 to decompose PL, and then removing the hydrolyzed product by forming an inclusion complex through \u03b2-cyclodextrin (\u03b2-CD). PLA2 selectively cleaves the ester bond of the acyl chain at the sn-2 position of PL and generates Lyso-PL and FFA. The product is a non-polar material. \u03b2-CD is a cyclic non-reducing oligosaccharide. Its special construction makes its inner cavity hydrophobic. \u03b2-CD and other cyclodextrins can form water-soluble inclusion complexes with insoluble non-polar substances. The water-soluble inclusion compound can be dissolved into the supernatant by a polar solvent such as deionized water, so as to achieve the effect of separation from soybean flour.Additional studies have shown that after the application of Alcalase, papain or a combination of enzymes, the acidic subunits of \u03b2-conglycinin and glycinin disappear completely, resulting in the removal of some of the precursors of the beany flavor, thereby reducing the beany flavor .L. rhamose L08 can bring floral and honey-like aromas  Fermentation using some strains may produce products with high acidity, which may not be preferred by consumers. More research is needed to select suitable strains and optimize fermentation process to meet the preferences of consumers . (b) AsiXY and TZ contributed to conception and design of the study. AT, HZ, and JD wrote the first draft of the manuscript. YX, YL, JL, and JH helped review and revise the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the Science and Technology Development Funds, Macau SAR (0024/2022/A and 0004/2021/ITP), the Science and Technology Planning Project of Guangdong Province (2020B1212030008), Innovation Cultivation Project of Zhuhai College of Science and Technology (2019XJCQ006), and the Open Fund of Guangdong-Hong Kong-Macao Joint Laboratory for Contaminants Exposure and Health (GHMJLCEH-05).JL and JH were employed by Macau Uni-Win Biotechnology Co., Ltd.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "In early 2020 there was intense media speculation that ethnicity and Covid-19 deaths were correlated. However, the existing method of adding ethnicity to death records resulted in low linkage rates for very recent deaths. We designed and implemented a bespoke linkage in three days enabling accurate reporting to the nation.We linked the 2011 England and Wales Census to death records using a range of personal identifiers. Due to time pressure, we focused on executing a single linkage method well. Deterministic linkage was chosen, using a variety of matchkeys which were tested via clerical review. To overcome the issue of addresses changing since 2011, we also linked 2020 death record residuals to the 2019 Patient Register (PR) and then made use of the 2011 PR address where it existed. This additionally provided an indication of whether unmatched death records might be attributable to migration into England and Wales post-2011.The prior linking method used NHS Number only. Although the overall linkage rate was approximately 90%, the rate for recent deaths (2nd March 2020 to 10th April 2020 in the first iteration of the linkage) was closer to 30% due to an administrative lag in adding NHS Numbers to death records. Our novel bespoke linkage method linked over 39,000 extra death records. Whilst this had minimal impact on the overall linkage rate, it improved the linkage rate for recent deaths to approximately 90%. This was without an impact on accuracy: clerical review demonstrated that the false positive rate was approximately 0.2%. A report was published using this data showing that the risk of death involving Covid-19 among some ethnic groups was significantly higher than others.Determining whether Covid-19 disproportionally affected certain ethnicities was of crucial importance in the early phase of the pandemic to enable appropriate government strategies to be developed. We delivered a bespoke linkage under an exceptional time-limit without compromising on accuracy, enabling this impactful analysis with nation-wide interest and impact."}
+{"text": "Self/informant convergence was robust for traits of boldness and disinhibition, but weaker for meanness. Informant\u2010rated traits showed incremental predictive validity over self\u2010report traits, both within and across assessment domains. These findings indicate that simple prototype\u2010based informant ratings of the triarchic traits can provide a useful supplement to self\u2010report in assessing psychopathy within forensic\u2010clinical settings.The validity of self\u2010report psychopathy assessment has been questioned, especially in forensic settings where clinical evaluations influence critical decision\u2010making . Informant\u2010based assessment offers a potentially valuable supplement to self\u2010report but is challenging to acquire in under\u2010resourced forensic contexts. The current study evaluated, within an incarcerated sample ( Informant ratings of all three triarchic traits showed higher validity coefficients for informant\u2010assessed criterion variables, and informant report did not contribute incrementally to the prediction of any self\u2010report criteria over/above self\u2010report triarchic scores. In another separate study, Gerbrandij et\u00a0al.\u00a0(rs\u00a0=\u00a00.23\u20130.45) and predicted several criterion variables in both domains, with disinhibition ratings contributing to prediction of self\u2010reported symptoms in areas of anxiety, depression, hostility, and paranoid ideation  convergence between informant ratings and counterpart self\u2010report scores would be significant, but likely lower than those reported by Kelley et\u00a0al.\u00a0 and Gerb22.1n\u00a0=\u00a0362) consisted of 277 incarcerated males and 85 incarcerated females from 10 separate medium\u2010 and high\u2010security prisons in Italy. The National Administration of Prisons  and its local division for the Lombardy region in Northern Italy (PRAP Regione Lombardia) granted approval for data collection at these 10 prisons. Study procedures adhered to the Declaration of Helsinki and were approved by the DAP and by the Institutional Review Board of the University of Firenze.The study sample (Study personnel reviewed prison files to determine eligibility in relation to exclusion criteria as follows: (i) criminal history limited to minor property offenses or illicit substance use/trafficking in small quantities (ii) current serious mental illness , or intellectual disability, (iii) involvement with the institution's Drug Addiction Services over the last six months , (iv) lack of fluency in the Italian language, (v) visual or hearing impairments, and (vi) impending release from the prison. Eligible individuals were invited by case managers at each prison to participate on a completely voluntary basis. No direct incentives were offered for participation in the study, and there were no negative consequences for declining to participate. For those eligible and invited to participate, the enrollment rate was approximately 95%, with no substantial differences across the 10 prison facilities.A total of 49 case managers across the 10 prisons volunteered to serve as informant raters in the study. Each case manager rated between 3 and 16 prisoners  so that each prisoner was rated once. Case managers were given prototypic descriptions of boldness, meanness, and disinhibition , followed by the self\u2010report measures described in the Section p\u00a0<\u00a00.01. This resulted in 24 participants (7% of the sample with full questionnaire data) being excluded, yielding a final analysis sample of 322.Four male and two female participants provided incomplete self\u2010report data and were omitted from analyses. 10 participants for whom informant ratings were not provided by case managers were also omitted. Multivariate outliers were excluded following the procedure described by Leys and colleagues, in which a variant of the Mahalanobis distance based on the Minimum Covariance Determinant was used  of the participants in the final analysis sample were serving life sentences. The remaining 297 were serving an average sentence of 9.7\u00a0years (SD = 8.0\u00a0years).The mean age of the final analysis sample was 45.9\u00a0years , with an average of 9.4\u00a0years of education (SD\u00a0=\u00a03.5\u00a0years). Approximately one\u2010third of the sample was single (34.5%), 37.3% were married or cohabitating, 21% were separated or divorced, and 7% were widowed. Additional demographic data such as race, ethnicity, and gender identity were not collected. Nearly all participants had a history of multiple criminal offenses; however, for a large portion of the sample, data were not available to characterize the nature of prior convictions. Prison personnel reported the offense of the most serious type associated with each participant's current sentence: 21% were convicted of homicide, 20% pedophilia/sexual aggression, 17% armed robbery, 10% fraud and extortion, 10% Mafia affiliation, 7% drug trafficking, 7% aggression and fighting, 6% criminal conspiracy, and 3% bankruptcy. About 8% : \u03b1s for Boldness, Meanness, and Disinhibition\u00a0=\u00a00.70, 0.87, and 0.85, respectively, M item\u2010total rs\u00a0=\u00a00.28, 0.47, and 0.42.The 58\u2010item TriPM Patrick,\u00a0 providesdoes not describe this person at all) to 7 (perfectly describes this person).Informant ratings were based on prototypic descriptions of boldness, meanness, and disinhibition generated by Hall et\u00a0al.\u00a0 and used2.2.2\u03b1) was 0.86 and the mean\u2010corrected item\u2010total correlation (r) was 0.52.Self\u2010report criterion measures for the current study consisted of inventories assessing depressive hopelessness, propensities toward self\u2010harm, and substance problems. The first of these, the Beck Hopelessness Scale . The Italian translation used in the current study has shown good reliability in other research .The self\u2010harm measure was the Deliberate Self\u2010Harm Inventory , one third relate to marijuana , and the final third reference other substances . For the substance use problem scale in the current sample, Cronbach's \u03b1 was 0.90 and the mean\u2010corrected item\u2010total r was 0.55.The Substance Abuse factor scale from the ESI , (ii) social connectedness with relatives or friends outside prison , and (iii) reintegration prognosis . Case managers made these ratings based on information contained in prison files together with observations of or direct experiences with the designated prisoner in various contexts . Archival information in prison files included records of social contacts  and reports concerning daily behavior filed by correctional staff.As mentioned above, case managers rated each prisoner on three aspects of adjustment, using one to five scales for which higher values represented more favorable ratings: (i) behavior in prison  to better approximate normality. In the case of missing score values, expectation\u2010maximization (EM) algorithm\u2010based maximum likelihood estimates were computed and utilized in analyses. Simple regression analyses were used to quantify how informant ratings for Boldness, Meanness, and Disinhibition predicted self\u2010report trait scores and other criterion variables, both individually and alongside the other two traits, when accounting for variation in scores due to differing raters. To account for rater effects, raters were assigned numbers and represented in regression analyses through the use of dummy\u2010coding. Hierarchical regression analyses were performed to evaluate whether informant ratings of traits contributed incrementally to prediction of criterion measures over and above self\u2010report trait scores. In these analyses, dummy\u2010coded variables representing different raters were entered in Step 1, self\u2010reported Boldness, Meanness, and Disinhibition were entered in Step 2, and informant\u2010rated Boldness, Meanness, and Disinhibition were entered in Step 3.3r\u00a0=\u00a00.16, p\u00a0<\u00a00.01) but was not associated with Disinhibition , and Meanness correlated moderately with Disinhibition . Comparatively, inter\u2010correlations among informant\u2010rated traits were stronger: Boldness correlated modestly with both Meanness  and Disinhibition , and Meanness and Disinhibition ratings were strongly correlated .Table\u00a0\u03b2s) from (a) regression analyses including only that trait along with dummy\u2010coded raters as predictors , and (b) regression analyses including that trait together with the other two, along with dummy\u2010coded raters, as predictors . Correlations between self\u2010report triarchic trait scores and criterion variables in this sample were reported previously by Brislin et\u00a0al.\u00a0. As shown in Table\u00a0\u03b2D\u00a0=\u00a00.15, p\u00a0<\u00a00.05), but not in the regression model including all three ratings as predictors . Informant\u2010rated Meanness did not predict self\u2010reported Boldness. Meanness and Disinhibition ratings each significantly predicted self\u2010reported Meanness when evaluated individually , but when included together along with boldness in the joint regression model, the beta coefficient for informant\u2010rated Meanness fell short of significance  and the coefficient for Disinhibition was clearly nonsignificant \u2014indicating that variance shared between the two traits contributed to the independent beta coefficients for each. Significant beta coefficients were also evident for both informant\u2010rated Meanness  and Disinhibition  when evaluated individually as predictors of self\u2010reported Disinhibition, but the joint regression model in this case revealed a unique predictive association for informant\u2010rated Disinhibition (\u03b2T\u00a0=\u00a00.31 p\u00a0<\u00a00.001) but not for Meanness , whereas it was unrelated to informant\u2010rated Meanness or Disinhibition , but not to either Boldness or Meanness. Substance use problems showed positive associations with all three informant\u2010rated traits when evaluated individually , though only Meanness and Disinhibition ratings significantly predicted substance use problems in the joint regression model .Hopelessness was associated with lower informant\u2010rated Boldness in both the individual\u2010trait and joint\u2010trait regression models , but not Boldness. However, when the three informant\u2010rated traits were entered together in a regression model, informant\u2010rated Boldness positively predicted prison behavior ratings , whereas Disinhibition predicted worse behavior ratings  and Meanness was no longer significant. The same pattern was evident for the criterion of informant\u2010rated social connectivity: Meanness and Disinhibition but not Boldness emerged as significant in their individual regression models , but in a joint regression model with all three traits predicting social connectivity, Boldness predicted better ratings , Disinhibition predicted worse ratings , and Meanness was no longer significant. Similarly, for informant\u2010rated reintegration prognosis, the individual regression analyses yielded significant predictive effects for informant\u2010rated Meanness and Disinhibition , but all three traits emerged as significant predictors in the joint regression analysis. Again, Boldness predicted more favorable reintegration prognosis ratings , whereas Meanness  and Disinhibition  were associated with poorer ratings.In individual\u2010trait regression models, ratings for behavior in prison were negatively associated with both Meanness and Disinhibition . Informant\u2010rated Meanness and Disinhibition did not significantly predict years of sentence either on their own, or alongside the other informant\u2010rated traits.Across all regression analyses, years of sentence was significantly predicted only by informant\u2010rated Boldness . To avoid misrepresenting the degree of variance in each criterion measure explained by self\u2010 and informant\u2010report, Table\u00a0R2 values for Step 2  and Step 3 . Based on significant \u0394R2 values, informant\u2010rated triarchic traits contributed incrementally to the prediction of self\u2010reported hopelessness and substance use problems, all informant\u2010rated criterion variables, and years of sentence. For these models in which significant contributions were evident, the mean increase in variance explained by adding the informant\u2010rating scores at Step 3 was 7.2%.Table\u00a03.3.1\u03b2\u00a0=\u00a0\u22120.30, p\u00a0<\u00a00.001) and informant\u2010rated Boldness , with positive predictive relations evident for self\u2010reported but not informant\u2010rated Meanness , and adding informant ratings at Step 3 increased variance explained by 2.7% (p\u00a0<\u00a00.05). Substance use problems were predicted by higher levels of self\u2010reported Disinhibition  and informant\u2010rated Meanness , with the addition of self\u2010report scores at Step 2 and informant ratings at Step 3 accounting for 22.1% and 3.9% additional variance in substance problems, respectively (ps\u00a0<\u00a00.001). For self\u2010harm frequency, only self\u2010reported Disinhibition and Boldness were significant in the Step 3 model . Informant\u2010rated Disinhibition fell just short of significance . While the incremental contribution of self\u2010report trait scores at Step 2 was significant , the addition of informant\u2010reported traits at Step 3 was not . Self\u2010harm versatility was associated with higher levels of both self\u2010 and informant\u2010reported Disinhibition , but again, self\u2010report added significantly to the variance explained  whereas informant report did not .In Step 3 of the model for the hopelessness criterion, significant negative predictive associations were evident for both self\u2010reported Boldness . Self\u2010reported and informant\u2010rated Boldness, on the other hand, predicted behavior ratings in opposing directions at Step 3, with self\u2010reported Boldness predicting lower (less favorable) ratings  and informant\u2010rated Boldness predicting higher (more favorable) ratings . A similar pattern was observed in the model for social\u2010connectivity ratings, which showed a significant increase of 7.8% in variance explained (p\u00a0<\u00a00.001) with the addition of informant trait scores at Step 3: Although higher levels of informant\u2010rated Disinhibition were associated with lower ratings of social connectivity at Step 3 , higher informant\u2010rated Boldness predicted elevated ratings of social connectivity at this step . By contrast, adding self\u2010report scores to the model at Step 2 did not significantly increase variance explained , and none of the self\u2010reported traits were predictive of social connectivity ratings at Step 3. Likewise, for reintegration prognosis, both informant\u2010rated Meanness  and Disinhibition  were predictive of lower ratings of expected reintegration into society, whereas informant\u2010rated Boldness predicted more positive expectations . The addition of self\u2010report scores at Step 2 and informant ratings at Step 3 resulted in significant increases of 8.0% and 14.6%, respectively, in variance explained (ps\u00a0<\u00a00.001). Of note, self\u2010reported Disinhibition was also predictive of lower reintegration ratings at Step 3 .In the model for ratings of behavior in prison, both self\u2010 and informant\u2010rated Disinhibition showed negative predictive relations at Step 3 , but adding informant\u2010rated traits at Step 3 produced a 3.4% increase in variance explained (p\u00a0<\u00a00.01). However, only informant\u2010rated Boldness evidenced significant prediction at this final step ; neither self\u2010reported Boldness, nor Meanness or Disinhibition assessed either by self\u2010report or informant rating, showed a significant predictive association  than in a study of forensic patients by Gerbrandij et\u00a0al.\u00a0(r\u00a0=\u00a00.45). Self/informant convergence for boldness in our study was also lower than that reported by Kelley et\u00a0al.\u00a0(r\u00a0=\u00a00.52). The convergence for the trait of disinhibition in our study (\u03b2D\u00a0=\u00a00.30) was modestly higher than for boldness, and less discrepant from the figures of 0.34 and 0.36 reported by Gerbrandij et\u00a0al.\u00a0 were more equivocal for meanness. As expected, however, lower self/informant convergence was observed for these traits in our study than in prior studies that have obtained informant ratings of psychopathy using detailed item\u2010based measures. Specifically, lower convergence was observed for boldness in the current study  than that reported by Gerbrandij et\u00a0al. , and appreciably lower than that reported by Kelley et\u00a0al. . Moreover, in a regression model that included informant ratings for all three triarchic traits as predictors of self\u2010reported meanness, the beta coefficient for informant\u2010rated meanness fell short of significance\u2014indicating a lack of selective self/informant convergence for this triarchic trait. This was in contrast to informant\u2010rated boldness and disinhibition, both of which evidenced unique predictive associations with their self\u2010report counterparts in corresponding regression models.Although challenging to adequately compare across studies, the convergence between informant\u2010rated and self\u2010report Meanness was somewhat lower (r\u00a0=\u00a00.27), the domain most related to meanness  than for either Boldness (r\u00a0=\u00a00.45) or Disinhibition (r\u00a0=\u00a00.34). These authors postulated that informant ratings were more influenced by observable, behavioral\u2010deviancy features of meanness  whereas self\u2010report scores included greater representation of internal, affective\u2010deficiency features .Of note, other studies have also reported lower cross\u2010modality convergence for assessments of meanness\u2010related attributes as compared to bold or disinhibited characteristics. For example, Carnovale et\u00a0al.\u00a0 obtainedr\u00a0=\u00a00.31 vs. 0.16 for Boldness and Meanness; r\u00a0=\u00a00.26 vs. \u22120.02 for Boldness and Disinhibition; r\u00a0=\u00a00.65 vs. 0.55 for Meanness and Disinhibition), and these augmented associations may be a result of informants' reliance on overt behavior in assigning their ratings, compared to the prisoners who based their self\u2010report on internal experience as well. This discrepancy may be especially relevant to the meanness domain, which has fewer unique interpersonal and behavioral indicators compared to boldness and disinhibition.The same interpretation can be applied to the findings of the current study, particularly given our use of global, prototype\u2010based informant ratings. The Meanness scale of the self\u2010report based TriPM includes a substantial portion of items that assess emotional sensitivity and empathic concern , along with items pertaining to more observable indicators of callousness . Case managers, on the other hand, especially those less familiar with the participant to be rated, would be expected to base their global meanness ratings predominantly on observable behaviors indicative of antagonism and callous\u2010disregard  that overlap more with disinhibition. In fact, informant ratings exhibited stronger inter\u2010correlations compared to self\u2010report scores  may account for its unique predictive relation with substance problems.Notably, all informant\u2010rated criterion variables benefited from the addition of informant ratings at Step 3 of the hierarchical regression model. Interestingly, in predicting ratings of behavior in prison, the beta coefficients observed for self\u2010 and informant\u2010rated boldness were in opposing directions, such that higher informant\u2010perceived boldness was associated with significantly higher (more favorable) behavior ratings, whereas higher self\u2010perceived boldness was associated with lower (less favorable) behavior ratings. Paralleling our interpretation for meanness, this divergence may be a function of differing features entering into informant\u2010rated boldness as compared to self\u2010reported boldness. Like meanness, boldness encompasses features pertaining to internal experience  along with behaviorally observable features . Consistent with the idea of boldness as a \u201cmask of sanity,\u201d the overt features of boldness could be expected to contribute more uniformly to impressions of positive adjustment on the part of observers.\u03b2|\u00a0=\u00a00.13\u20130.37) than for self\u2010report criteria (range of |\u03b2|\u00a0=\u00a00.17\u20130.19). The reverse was true for validity coefficients associated with self\u2010report trait scores, which were generally stronger for self\u2010report criterion variables (range of |\u03b2|\u00a0=\u00a00.12\u20130.49) than for informant\u2010rating criteria (range of |\u03b2|\u00a0=\u00a00.11\u20130.21). Likewise, the incremental contribution of informant\u2010rated traits was larger for predicting informant\u2010rating criteria (\u0394R2s\u00a0=\u00a00.08\u20130.15) than for predicting self\u2010report criteria  or years of sentence (\u0394R2\u00a0=\u00a00.03). Again, the reverse was true for self\u2010report, in that the incremental contribution of self\u2010reported trait scores was larger for predicting self\u2010report criteria (\u0394R2s\u00a0=\u00a00.07\u20130.22) than for predicting informant\u2010rated criteria , and self\u2010report did not significantly add to the variance explained for years of sentence. These differential effects are theoretically coherent given the well\u2010documented impact of method variance on validity coefficients in prior research of this kind , several limitations must be noted as well. First, limited data were available for characterizing the study informants, in terms of their familiarity with each rated participant, their degree of experience serving in a case manager role, and other factors that could have influenced ratings. As a result, we were unable to evaluate these factors as potential moderators of self/informant convergence and the predictive validity of informant ratings. Future studies of this kind should explicitly assess how the extent of contact between prisoners and informant prison staff affects agreement across report modalities. Along similar lines, detailed demographic data were not available for the prisoners, preventing a more comprehensive look at potential moderators of the relationships observed in this study . Of note, a recent study by Sica et\u00a0al.\u00a0 that empFinally, similar to other studies of self and informant\u2010rated psychopathic traits, the current findings are limited to a research setting in which neither the incarcerated individuals nor the informant raters faced consequences for inaccurate reporting. Thus, while incentive to provide false report in the current study was low, it is unclear whether findings would generalize to real\u2010world settings where the incentive to misrepresent is higher .5Notwithstanding these limitations, the current study provides support for the use of a simple global\u2010rating approach to collecting collateral data for boldness, meanness, and disinhibition as described by the triarchic model of psychopathy (Patrick et\u00a0al.,\u00a0The authors declare no conflict of interest.Supplementary MaterialClick here for additional data file."}
+{"text": "Scientific Reportshttps://doi.org/10.1038/s41598-021-04445-8, published online 10 January 2022Addendum to: Following publication, concerns have been raised that annotations in some figures depicting magnetic resonance images mask important features. A supplementary information file containing un-annotated images corresponding to Figures 1-4 is linked to this notice.Supplementary Information."}
+{"text": "Introduction: Linking longitudinal cohort resources with police-recorded records of criminal activity has the potential to inform public health style approaches to policing, and may reduce potential sources of bias from self-reported criminal data collected by cohort studies. A pilot linkage of police records to the Avon Longitudinal Study of Parents and Children (ALSPAC) allows us to consider the acceptability of this linkage, its utility as a data resource, differences in self-reported crime according to consent status for data linkage, and the appropriate governance mechanism to support such a linkage.Methods: We carried out a pilot study linking data from the ALSPAC birth cohort to Ministry of Justice (MoJ) records on criminal cautions and convictions. This pilot was conducted on a fully anonymous basis, meaning we cannot link the identified records to any participant or the wider information within the dataset. Using ALSPAC data, we used summary statistics to investigate differences in socio-economic background and self-reported criminal activity by consent status for crime linkage. We used MoJ records to identify the geographic and temporal concentration of criminality in the ALSPAC cohort.Results: We found that the linkage appears acceptable to participants (4% of the sample opted out), levels of criminal caution and conviction are high enough to support research, and that the majority of crimes occurred in Avon & Somerset . Those who did not respond to consent requests had higher levels of self-reported criminal behaviour compared to participants who provided explicit consent.Conclusions: These findings suggest that data linkage in ALSPAC provides opportunities to study criminal behaviour and that linked individual-level records could provide robust research in the area. Our findings also suggest the potential for bias when only including participants who have explicitly consented to data linkage, highlighting the limitations of opt-in consent strategies. ALSPAC, Avon Longitudinal Study of Parents and ChildrenMoJ, Ministry of JusticePNC, Police National ComputerSEP, socio-economic position. This approach is multi-disciplinary, takes a joint agency approach, and relies on \u2018the skilled use and interpretation of data and the evidence base to ensure that interventions are designed, delivered and tailored to be as effective as possible\u2019. This can now be seen in operation within some UK police forces \u2013 for example, within Thames Valley Police. Epidemiological analysis is an important approach to identify risk and protective factors for criminal and antisocial behaviours. Police records of criminality (e.g. convictions and cautions) do not contain data relating to an individual\u2019s exposure to potential risk factors for perpetrating crime, whereas longitudinal birth cohort studies have a wealth of data on the lives of their participants, and often their families, peers, and wider contexts, across the life course. Therefore, linking police data with cohort studies has the potential to add considerable value to research on criminal behaviour.Policing in the UK increasingly seeks to take a public health approach to tackling crime, where the focus is on proactive prevention, the tackling of upstream risk factors, and on populations rather than individuals.Accurate measures of participants\u2019 criminal behaviours are necessary for any such research to be valid. Some cohorts contain their own measures of criminality \u2013 these may be self-reported by the participants or by related individuals (e.g. teachers or parents). While this is a relatively straightforward way of measuring such behaviours, and has the advantage of capturing crimes irrespective of whether they appear on any official records, measurement error may be introduced through recall error , or social desirability bias (choosing not to disclose certain behaviours). Further, there is a potential for measurement error based on questionnaire design (e.g. study wording or response options) and valuable data may not be recorded . Finally, a known limitation of cohort studies is that attrition is associated with socio-economic, demographic and health status which, in turn, may be associated with criminal behaviour. By relying on self-report measures of criminality, it is likely that cohort studies underestimate rates of criminality compared to the wider population. There is evidence to suggest that violence between people who know each other, less serious violence, and violence that involves alcohol areless likely to be reported to the police, and males are less likely to report violent victimisation than females. Conversely, violence that involves injury or weapons, and violence perpetrated by a stranger aremore likely to come to the police\u2019s attention. Finally, there is some evidence that offences against residents of the most deprived neighbourhoods are less likely to be reported to the police than offences against residents of less deprived areas. The impact of this on accurate estimates would be enhanced where the factors (e.g. ethnicity) associated with policing practice were also predictive of failure to participate in study follow-up. In police records, data quality issues within the records may also lead to error  and this may disproportionately impact some population groups. Linking cohort and official records also enables research questions to be addressed that would not otherwise be possible e.g. investigating self-reported and official records measuring different constructs and analysing discrepancies in these data sources, and comparing outcomes of those who self-report criminality vs. those with officially recorded criminality .Record linkage of cohort data to official police records has the potential to address some of the limitations of self-reported data. As official records are not affected by recall error or social desirability bias, they can potentially provide greater detail and accuracy than would be feasible via self-report. Furthermore, attrition can be addressed using record linkage as criminality outcomes can be followed in individuals even if they miss opportunities to participate in study data collections. However, not all crimes come to the attention of the police or result in a formal record and so to rely solely on police records would under-estimate the prevalence of criminality in a cohort. That cohort study has a criminality focus, they work closely with Scottish criminal justice policy makers and practitioners, and police record linkage was integral to the study\u2019s design from the start. The legal, ethical and practical example set through their successful linkage therefore isn\u2019t a precedent that other UK cohorts with a more general purpose can necessarily follow. Also, Scotland and England have differing legal systems and police records. However, linkages of police records to general cohorts have been achieved in other countries, such as the NSW-CDS cohort study in Australia, and the Swedish National Cohort Study.In sum, a combination of official police records with self-reported criminal behaviours could allow research that uses the strengths of both sources of crime data while addressing some of their respective limitations. However, achieving linkage of a longitudinal cohort to any routine health or administrative data can be a complex and time-consuming process. In Scotland, the Edinburgh Study of Youth Transitions and Crime has successfully linked police records to a longitudinal population-based cohortAs with all data linkage projects in longitudinal studies, there are specific considerations relating to data protection and confidentiality, and wider considerations relating to participant trust and the acceptability of novel forms of data use. In the UK, criminal records were deemed \u2018sensitive\u2019 data in the Data Protection Act 1998 and are now considered \u2018special category\u2019 data in the EU General Data Protection Regulations (GDPR) and the UK\u2019s Data Protection Act 2018 (DPA). Both categories are subject to elevated levels of protection. The DPA 1998 allowed for the use of criminal records where studies gained explicit consent from study participants or where the data were anonymised . In contrast, the new DPA 2018 provides a separate legal basis for using identifiable \u2018special category\u2019 records for scientific research which is in the public interest, subject to utilising sufficient safeguards (GDPR Article 89). Nevertheless, these routes available to meet DPA 2018 requirements do not alter the requirement for research use of individual data to meet the Common Law Duty of Confidentiality, which can be met through consent, anonymisation or meeting a public interest test. However, data linkage based on consent may systematically omit some individuals and population sub-groups and introduce bias into study findings. Therefore, alternative mechanisms to use data for individuals who have not necessarily provided consent are needed to minimise the risk of selection bias. Further to addressing the legal basis for record linkages, it is also necessary to examine the acceptability of data linkage to crime records for cohort participants and \u2013 in order to justify the intrusion to privacy of non-consented approaches - to determine whether the group of participants who do consent to data linkage are, in terms of criminal behaviour, representative of the wider cohort .It is also necessary to consider if any linkage is proportionate \u2013 to be ethical, it has to be useful. In the case of linking cohorts to police records, it is currently unclear whether the levels of criminality are sufficient for a longitudinal population study to be a viable resource for future research projects. Furthermore, gaining a better understanding of the age crimes are committed and in which areas can help to identify key age periods and geographical locations for where data linkage may be the most valuable for research.This paper describes a pilot linkage project of participants from the Avon Longitudinal Study of Parents and Children (ALSPAC) to criminal conviction and official caution records in the UK Police National Computer (PNC) database held by the Ministry of Justice (MoJ). To our knowledge, this pilot project is the first to link criminal records to an English general population longitudinal cohort. The overall aim of this pilot was to test the feasibility of linking ALSPAC to official criminality records, and to determine if full linkage is likely to be worthwhile in terms of creating a useful resource for future research. Our specific research questions were: (1) What can participant responses to the study\u2019s proposed linkage to criminality records suggest about the level of acceptability of this to ALSPAC participants? (2) Are there sufficient levels of recorded criminal caution or conviction for the data resource to be useful in future research? (3) In what geographical area are crimes most commonly committed by ALSPAC participants? (4) Are those we have consent to link to crime data representative of the wider cohort in terms of their self-reported criminal behaviours?The linkage in our pilot was restricted to an anonymous data extract of historic criminal convictions and cautions of ALSPAC study participants. No identifiers are present in the file meaning it cannot be linked to any participant records held within the ALSPAC databank. and a searchable data dictionary can be accessed from the study\u2019s website . In brief, there were 14,541 pregnancies resulting in 13,988 children alive at one year of age (known as the \u2018core sample\u2019). By age 18 years, an additional 718 children, who were eligible under the original study eligibility definition, but whose mothers had not joined the study during pregnancy, had also been recruited. The mothers, their partners, and the study children have been followed ever since through questionnaires and clinic visits.ALSPAC is a birth cohort study that recruited pregnant women who were resident in and around the city of Bristol, with a due date between April 1991 and December 1992. Full details are available in the cohort profiles and other study factors, the participants selected to be in batch 1 and batch 2 were not selected at random and are likely to over represent participants with good histories of study participation.When the ALSPAC children reached legal adulthood (age 18 years), there was a postal campaign that aimed to re-enrol them into the study and to seek permission for linkage to their routine health and administrative records, including education, employment, earnings and benefits, and criminal conviction and caution records . This was part of the Wellcome Trust funded \u2018Project to Enhance ALSPAC through Record Linkage\u2019 (PEARL). Each participant was sent a pack that included an information booklet and consent form, which provided a clear means to opt-out of ALSPAC, or to any of the proposed linkages. Due to factors related to establishing an appropriate ethico-legal basis for record linkage in ALSPAC and the negotiation of access to linked health records (i.e. unrelated to this crime data linkage), the participant information materials were initially issued in two batches. Batch one sought opt-in consent, which stated that linkage would only occur with explicit participant approval, while batch two was structured as an opt-out approach and notified participants that their routine records would be linked to ALSPAC unless they specifically opted-out (i.e. linkage would occur in the event of non-response). Participants that did not respond to batch 1 were a sent a new opt-out pack. Following participant consultation, the opt-in/out materials were structured as a series of specific linkage permission options to allow for individual level decision making. This led to participants returning forms that in effect indicated consent for some linkage categories even when the overall campaign was structured as an opt-out . The following participants were excluded from the pilot criminality linkage: participants who no longer wished to be part of ALSPAC; those who objected to linkage to their criminality records; those where we had evidence the participant had not received their information pack ; and those who lacked capacity to consent. Due to the inclusion of a randomised controlled trial of linkage information materialsFollowing the ALSPAC \u2013 MoJ pilot linkage, the study continued to issue opt-out linkage materials to all participants via postal campaigns and online promotion. Where practicable, consent was sought where participants attended a study clinic visit. This means there is an increasing number of participants who have opted-in to record linkage over time.. Following negotiations between ALSPAC and the MoJ, it was agreed to conduct apilot linkage exercise which would test the feasibility of the linkage mechanism through the production of an anonymous linked extract. For individuals for whom ALSPAC had permission to link to criminality records , the following identifiers were sent to the MoJ: forename, surname, date of birth, current address, last four known addresses. No attribute data about the participants was provided. This linkage was done in March 2013.The Police National Computer (PNC) is a large administrative database that was started in 1974 and contains information about police cautions and court convictions held on individual offenders in England and WalesThe MoJ conducted the linkage to the PNC using a deterministic linkage protocol with manual review (see \u2019Linkage Protocol\u2019 section below). Once linked, the MoJ provided an anonymised data extract detailing all historic criminal convictions and cautions that were linked to study participants. Direct individual identifiers were removed and replaced with two pseudonymised identifiers: 1) \u2018lcr_id\u2019, which uniquely identified individuals in the data set; and, 2) \u2018lcr_caseid\u2019, which identified unique cases and the criminal acts associated with it, which were nested within each individual\u2019s overall record . ALSPAC has no means to reverse these pseudonyms to the participants\u2019 personal identifiers. The extract was securely sent to the ALSPAC data linkage team for analysis within their PEARL Data Safe Haven (at the University of Bristol).The linkage was conducted by MoJ staff. In summary, they received a file of identifiers from ALSPAC and then processed (cleaned) these. They then searched the Home Office Police National Computer (HOPNC) live database. Where matches were found, the individual\u2019s PNC ID was extracted and subsequently used to extract criminality outcomes.The automated HOPNC database search process returns a set of results, indicating varying levels of matching success according to a set of deterministic match rules. Matches are graded from 01 to 24, and in general, the higher the number, the more suspect the match. The process accommodates the tendency for criminal convictions to be assigned to alias identities rather than true identities. Each match level may be sub-divided into A or B levels, where B also uses data contained in Alias and AliasDateOfBirth tables. \u2018Suspect\u2019 matches are manually matched against the HOPNC live database by MoJ staff in order to obtain either an accurate PNCID or a status of no match.ALSPAC was not provided with information on match strength or as to whether suspect matches were manually reconciled, dropped or retained. This was due to the primary aim of the project being to demonstrate the feasibility of subsequent research and to test the workflow process (i.e. the aims did not require the full linkage protocol to be implemented).The cleaning process used aimed to standardise identifiers prior to matching:Adding centuries to the PNCID year portionLimiting Gender / Sex to 1st character of \u2018Male\u2019 / \u2018Female\u2019 / \u2018Unknown\u2019Supplying dummy date of birth where none provided. (29 Feb 2004 suggested)Splitting forenames into 3 columns; First forename, Second forename & Other forenamesRemoval of hyphens, spaces, apostrophes, full stops, commas from name elementsRemoving leading zeros from \u2018Nibnum\u2019 field if provided, which converts it to a CRONumberCorrecting date formatsRemoving rows with insufficient mandatory fieldsThe MoJ linkage operator followed a linkage protocol including manual check rules and rules for dealing with duplicate entries. Where in doubt, the operator was instructed to not establish a link which, theoretically, increases the rate of false negative linkages but reduces the rate of false positive linkages. Where there was a high degree of missing data  then no link would be established. Where duplicates exist, and there is no conclusive evidence from other PNC information that they are a link, then none of the candidate entries are set to a match. The full HOPNC linkage protocol of the time is available from the authors on request.Ethical approval for ALSPAC was obtained from the ALSPAC Ethics and Law Committee (ALEC) and the Local Research Ethics Committees. The PEARL project received approval from ALEC and the Haydock NHS Research Ethics Committee (REF: 10/H1010/70) for the use of NHS records. Approval for the MoJ to link ALSPAC participants to their PNC records was granted by the PNC Information Access Panel (PIAP). When the study children reached legal adulthood (age 18), ALSPAC initiated a postal fair processing campaign to formally re-enrol the children into the study  and to simultaneously establish permissions for ALSPAC to link to their health and administrative records. All participants have been offered the right to opt-out (which is respected). This approach was developed with participant involvement..Data was cleaned, managed and analysed using STATA version 15Police National Computer (PNC) data. The variables provided by the MoJ included: date of offence; offence class ; police force that processed the case; adjudication code ; disposal type .ALSPAC data. A variable was derived to summarise criminality linkage consent status at the time of the pilot linkage: opted-in to criminality linkage; non-responder to batch 2; not sent to MoJ for criminality linkage . Current (September 2019) criminality linkage consent status was also summarised in a similar way. Measures related to family socio-economic position (SEP) were reported by the mother during her pregnancy with the study child: family occupational social class, defined as the higher of maternal and paternal social class and categorised as high  and low ; highest maternal education ; housing tenure ; and financial difficulties  is considered high). Child variables included sex and ethnicity .Antisocial and criminal behaviours were reported by the children at ages 14, 15.5, 17.5 and 18 years. A series of binary variables were derived based on whether they reported doing each of the behaviours in the previous 12 months : theft ; hit, kicked or punched someone on purpose; carried a knife or weapon for protection or use during a fight; deliberately damaged or destroyed property belonging to someone else; deliberately set fire to property or building (or attempted to); rowdy or rude in a public place. At 17.5 years, they also reported a series of measures related to having had involvement with the police and criminal justice system in the past year. A series of binary variables were derived (no/yes): in trouble with police; picked up by police and taken home; picked up by police and taken to station; told off/told to move on by police officer; stopped and told to empty pockets or bag; received official police caution; charged for committing a crime; been on trial in court. Due to the small numbers reporting these outcomes, two further aggregate variables were derived: received any \u2018punishment\u2019 , or having mediation as an offender); and any criminal justice involvement .Statistical analyses. We used descriptive statistics to summarise the number of convictions and cautions, the year the offences were committed (as a proxy for age of the participants), and where they were committed (which policing area). We then used ALSPAC questionnaire date to compare: (1) participants whose identifiers were sent to the MoJ to those whose identifiers were not sent; and (2) within the sent for linkage group, the participants who explicitly opted-in to linkage to those in batch 2 who did not opt-out, in terms of child sex and ethnicity, early life family SEP, and child-reported anti-social and criminal behaviours. Finally, we repeated these comparisons by current criminality linkage consent status. For these descriptive analyses, we excluded triplets and quadruplets (as their ALSPAC data are not released to researchers) and those who have withdrawn consent from ALSPAC participation, giving a sample size of 14,683. Note that due to missing data in the ALSPAC measures, the denominator for each individual comparison varies.At the time of the pilot linkage (March 2013), batch 1  sought opt-in consent, while batch 2  gave participants the option to opt-out of the linkage, which would proceed in the event of non-response . This resulted in permission to link to the criminality records of 7,361 participants  . Note thOf those whose identifiers were sent to the MoJ for linkage , 885 (12%) were successfully linked to a criminality record. These participants had a conviction, caution, reprimand or warning for 4,000 separate offences, comprising 2,635 criminal convictions and 1,365 official cautions, warnings or reprimands. Of those linked, 394 (44.5%) had received at least one conviction and 84 (9.5%) had received 10 or more convictions.The offence class with the greatest number of offences was summary offences excluding motoring, followed by theft and handling of stolen goods, breach offences, drug offences, and violence against the person. Almost a third (31.6%) of offences related to serious crimes (defined as class 1-5).The majority of the offences  were committed in the area covered by the Avon and Somerset constabulary . Neighbowithin the sent for linkage group (i.e. between those who were opt-in and those who were non-responders) than between the sent for linkage group overall compared to the not sent for linkage group. Those who opted-in to criminality data linkage were more likely to be female, of White ethnicity, and from a socio-economically advantaged background, compared to those in batch 2 who did not respond to the opt-out request  had their identifiers sent to the MoJ for linkage (2963 of these had opted-in and 4394 were Batch 2 non-responders) and 7326 did not have their identifiers sent . The overall pattern was of greater differencesrequest . Particirequest . Furtherrequest .The comparisons by current consent status included 4884 opt-in, 7612 non-responders, and 2187 individuals with no permission for linkage. Overall, the patterns observed by current day consent status in SEP , self-reIt is important to note that there are also differences in the proportions of missing data by consent status for each variable: those with opt-in consent have a lower proportion of missing data than those who have not responded to the consent campaign. This is true of both early-life (reported by participant\u2019s mother) variables and those reported by the participant themselves later in adolescence. For example, of those who had opted-in at the time of the pilot, 7% are missing maternal education data, compared to 25% of those who were non-responders and 13% of those who were not sent for linkage . In general, the proportion of missing data increases over time; the differences between the consent groups in terms of missing data also increase. For example, for theft reported at age 18 years, the opt-in group at time of pilot linkage had 37% missing data, compared to 96% of the non-response group and 82% of the not sent for linkage group. The equivalent numbers by current consent status are 48%, 93% and 89% respectively.Finally, we did not find a consistent pattern in self-reported anti-social and criminal behaviours when comparing participants who dissented to criminality linkage but did agree to at least one other linkage, compared to participants who did not dissent to any data linkage, or those who dissented to all data linkage options (but agreed to continue in ALSPAC). Comparison of these groups in terms of self-reported involvement with the criminal justice system was precluded by small numbers.We completed a pilot record linkage in 2013 to determine the feasibility of linking an English population-based cohort study (ALSPAC) to official criminality records, and to inform whether a full linkage would be a worthwhile future endeavour in terms of creating a useful resource for research. The pilot was conditional on the extract being anonymous and not able to be linked to information on individual participants within the ALSPAC databank.We first aimed to determine whether linkage to criminality records was acceptable to study participants, and whether there was sufficient criminality in the sample for research purposes. Criminal behaviour is a potentially sensitive area and so it was a positive finding that almost 900 participants with criminality record(s) enabled the linkage to happen through either explicit consent (in response to the opt-in request) or not objecting (in response to the opt-out fair processing campaign): out of a sample of 7,361 ALSPAC participants, 885 participants were linked to one or more criminality records held in the Police National Computer database. Further, our finding that - to date - only 4% of the sample have explicitly opted-out of linkage to criminality records supports the view that such linkage is acceptable to the majority of study participants. The group of participants who dissented to criminality linkage - but not to all linkage data sources - was small and within this group levels of self-reported criminality were low. With the available data we cannot determine if this sub-group of dissenters had engaged in a greater level of criminality compared to the rest of the sample and considered the research use of their criminality record to be sensitive. However, the proportion of participants who self-reported criminality and who did provide explicit consent could imply that participants trust the study to use these records appropriately for research. Whilst this could benefit from further research , this could inform future study designs and governance frameworks, and the considerations of ethical review boards.In the sample of participants with criminal records, 4,000 convictions and cautions were recorded, many relating to serious crimes. If the linkage were repeated today, we would expect the number of criminal records to be substantially higher because (1) we now have permission to link to a larger sample and (2) there would now be more than 7 years of additional data. Therefore, we believe that there is a sufficient level of criminality in the ALSPAC sample for it to be a useful resource for crime-related research. However, it is unlikely that ALSPAC would have sufficient rates of less common crimes for these to be studied individually. We found criminal records from around the age of 12 years, but the majority of offences in our sample were committed later in adolescence. Therefore ALSPAC may not have sufficient numbers for research using linked criminality records at younger ages. Note that the PNC database is not \u2018weeded\u2019  therefore this is not an explanation for the small amount of records at younger ages in our sample.. This suggests that studies using only an opt-in sample may underestimate rates of criminal behaviour in the full study population. As such, it is necessary to consider the potential for selection bias when using a sample that relies on explicit opt-in consent status when designing linkage methodologies and considering the appropriateness of data sharing requests.While all participants in the pilot were informed about the linkage and had not objected, only a sub-set of these had provided explicit consent. We found evidence suggesting different rates of self-reported criminal activity, and socio-demographic differences, according to consent status. Participants who explicitly consented to data linkage were more likely to be female, have higher socioeconomic status, lower levels of missing data and were less likely to self-report criminal behaviour. This pattern is similar to that found for general ALSPAC participationFinally, in order to inform which sources of crime data could be worthwhile pursuing for future linkage and research, we determined where the crimes committed by our sample took place. Our finding that the majority of offences in the pilot linkage were committed in the Avon and Somerset Police (A&SP) area, which has a similar geographical footprint to the ALSPAC recruitment area, suggests linkage to local police data held by A&SP, which contain more detail than that held in the national PNC, would capture most offences (at least to age 18). Working at a local level provides the opportunity to identify areas of research of local importance. However, at older ages criminal activity may become less geographically clustered, meaning centralised national records may be of increasing value.There are several strengths and limitations to be considered in our pilot study. A strength was the wealth of data available on demographic measures and self-reported crime collected at multiple time-points, which allowed us to examine patterns in these variables by consent status. The ability to disaggregate our \u2018sent for linkage\u2019 group into those who actively opted-in and those who did not respond was a further strength as it enabled us to highlight the many differences between these groups. This is an important finding for other studies who are considering how to structure their consent campaigns, and will help inform the decision making of those reviewing this use of linked data in longitudinal studies. However, our evaluation is complicated by the fact that the sub-sample of participants included in the consent campaign, and those who were included in batch 1 versus batch 2, were not selected at random. This weakness is mitigated by the fact that this pilot study is intended to demonstrate viability rather than provide accurate association or prevalence estimates. Also, given that the sub-sample disproportionately included participants with strong levels of engagement, it can be hypothesised that this has led to an underestimate of recorded criminality within the sample.. For example, individuals may report a false identity to the police. The linkage methodology used relied on deterministic matching that incorporates fuzzy parameters . ALSPAC was not provided with a match quality score , which is counter to expectations that linkage quality estimates are transparent and available to the analyst.The quality of data linkage relies on the accuracy of identifier records in both datasets . While ALSPAC\u2019s administrative database is generally of good quality, it is likely to be out of date for some participants who are lost to follow-up. For the PNC data, the identifier database is known to have accuracy problems and includes pseudonyms, out of date information and duplicatesIt is also important to consider the quality of the data that is being linked from both sources and their potential limitations for answering questions in this research area. For PNC data, this depends on reliable and accurate testimony and record keeping. For ALSPAC records, the use of self-reported measures of criminal behaviours are vulnerable to social desirability bias, although the figures provided here illustrate that many participants are willing to report criminal and anti-social activity. Furthermore, drop out by participants may lead to bias.Finally, as this pilot only produced a fully anonymous file, which cannot be linked to the wider ALSPAC dataset, there were limits to what could be included in this evaluation. For example, we could not examine relationships between official criminal records and the self-reported measures.We found differences in socio-demographic characteristics and rates of criminality according to the consent status of participants (i.e. explicit consent versus non-response to opt-out approaches), which suggest that methods of securing data must be considered carefully in future studies to reduce the risk of bias.This pilot study illustrates that a full linkage of ALSPAC to crime records at an individual level would be a worthwhile future endeavour that would create a valuable resource for crime related research. Both local (Avon and Somerset) and national police records would be suitable for linkage, and linkage to both would be worthwhile. Advances in privacy preserving record linkage and \u2018Trusted Research Environment\u2019 secure research infrastructure and legislative changes  may now enable linkage and the joint analysis of linked study-criminal record data under sufficiently controlled conditions to mitigate potential risks to confidentiality and help ensure that this form of data use is publicly acceptable. Individual-level linkage would enable direct comparisons between police-collected and self-reported criminal data, inform statistical strategies to account for missing data, and allow investigation of research questions related to the causal pathways to criminal behaviours using the wealth of life-course information collected by ALSPAC or other longitudinal studies. Once linked, these studies could provide valuable evidence to inform public health approaches to tackling crime.ALSPAC data access, including linked PNC data, is through a system of managed open access. The steps below highlight how to apply for access to ALSPAC data.Please read the ALSPAC access policy which describes the process of accessing the data in detail, and outlines the costs associated with doing so.research proposals database, which lists all research projects that have been approved since April 2011 including those using linked data.You may also find it useful to browse the fully searchabledata-linkage@alspac.ac.uk.For enquiries regarding linked data, please contact This paper is much improved by the revisions - happy to approve.Is the work clearly and accurately presented and does it cite the current literature?PartlyIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?NoIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Criminology.I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Happy with revisions \u2013 no further comments.Is the work clearly and accurately presented and does it cite the current literature?PartlyIf applicable, is the statistical analysis and its interpretation appropriate?PartlyAre all the source data underlying the results available to ensure full reproducibility?PartlyIs the study design appropriate and is the work technically sound?PartlyAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:NAI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. et al. (2020)).There have been other studies employing a similar approach  that could have been cited ; the authors should consider being clearer about this and explain why direct linkage is not possible; it is certainly managed in other settings  which undertakes the linkage to manage privacy/confidentiality).The most interesting aspect of the study is really the reported differences between those consenting to linkage, those not consenting and those not objecting ; the authors should consider formally testing these differences rather than just presenting descriptive statistics.The submitted manuscript describes a pilot study in which a longitudinal cohort sample is linked to official criminal records. The submission is interesting and presents a useful contribution to the important and growing field of research using data arising from the linkage of cohorts/samples to large administrative datasets. The authors could consider the following points in making any amendments to the paper:Is the work clearly and accurately presented and does it cite the current literature?PartlyIf applicable, is the statistical analysis and its interpretation appropriate?PartlyAre all the source data underlying the results available to ensure full reproducibility?PartlyIs the study design appropriate and is the work technically sound?PartlyAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Forensic Mental Health, Psychiatric Epidemiology, Record linkage research.I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. This is a very helpful paper describing a pilot project linking Avon Longitudinal Study of Parents and Children (ALSPAC) to Ministry of Justice (MoJ) records on criminal cautions and convictions. The paper is well-written, and provides useful information about the pilot linkage. I agree with the authors' conclusions that there are 'enough' people in ALSPAC with official cautions/convictions to make further linkage valuable analytically, and think that the further linkage discussed would be incredibly valuable for the reasons listed below. For such a future linkage MoJ including information on linkage error would be useful as the authors state. There are a few areas where I think the paper could be strengthened with some additional information about the datasets involved, for those not familiar with ALSPAC/PNC, which are listed below.Are all the source data underlying the results available to ensure full reproducibility? I answered no, but this is not applicable given that it is a linkage study.Is the work clearly and accurately presented and does it cite the current literature? There are two reasons I responded 'partly' to this question.1. Summary of criminological literature From a criminologist's perspective, this linkage may be even more important than is emphasised in the paper. This is why I responded 'partly' to 'Is the work clearly and accurately presented and does it cite the current literature?'.\u00a0, of understanding self-reported and official records measuring different constructs and analysing discrepancies in these data sources. There is a lot to be learned about the impacts of contact with the justice system itself from comparing the subsequent official criminal histories of people with similar levels of self-reporting but who do and do not come to the attention of the police. The capacity to make this kind of comparison in a contemporary cohort would be a real strength of linked ALSPAC/MoJ data. First, a cohort study with both self-reported and official offending data is particularly useful. Throughout the paper having both data sources is framed as a way to reduce measurement error in offending. This is a valid conceptual understanding of these two measures, but there is also a tradition in criminology, exemplified by the Edinburgh Study of Youth Transitions and Crime ,, but the number of cohort studies which can be used for such comparisons is limited. As such, linking ALSPAC to MoJ records can provide an incredibly valuable resource to understand this important phenomenon. Second, one of the most important issues in contemporary criminology is understanding the international 'crime drop' over the last 25-30 years, and within this area of study one of the most informative but scarce resources are cohort studies. Comparing both self-reported and official offending records across different birth cohorts is a crucial part of understanding how this overall fall in crime is reflected in individual 'criminal careers'  However, given that the primary audience for this paper might not be criminologists I don't necessarily think these points need to be discussed at length in the paper, but the first point in particular is important context. I leave it to the discretion of the authors as to whether to include the second point.\u00a0 N.B. Both these potential benefits would come from a 'full' linkage of ALSPAC to MoJ records, rather than the pilot project described in this paper.\u00a02. Description of the dataset/linkage For someone who is not familiar with the ALSPAC dataset, it was tricky to reconcile the numbers in tables 3 and 4 with the study N listed on page 4. It would be helpful to add the total relevant N to these tables to make them a bit easier to understand. Similarly, for those not familiar with ALSPAC it would be helpful to provide a flow chart (or similar) which showed: 1. total ALSPAC N; 2. permission gained to link N; 3. successfully linked N etc. From Table 3 in particular, it would be helpful to clarify whether all ALSPAC children are asked the self-report questions at every age/are included in every ALSPAC questionnaire . The terminology in Table 3  does not map directly onto the questionnaire names included in the link at the bottom of the table, which makes finding more information about the questionnaires slightly cumbersome. It would also be useful to know how far back the PNC linkage goes, and whether Home Office Police National Computer (HOPNC) live database is ever 'weeded' . Would we expect future linkage to list all convictions for the ALSPAC cohort ? If PNC is not weeded it would be useful to say this explicitly in the text.Is the work clearly and accurately presented and does it cite the current literature?PartlyIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?NoIs the study design appropriate and is the work technically sound?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Criminology.I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above."}
+{"text": "For 23 years, Charles V \u201cThe Wise\u201d of France suffered from a mysterious fistula on his left arm that continuously drained pus. For all this time, he believed that as soon as the fistula ceased to weep, he would have a mere 15 days before death followed. His death in 1380, at the young age of 42, seemingly proved this assumption correct. This paper explores the possible explanations from arsenic poisoning at the hands of his longtime nemesis Charles II, as many of his contemporaries believed, to an undiagnosed case of hidradenitis suppurativa, to an underlying tuberculosis infection, to the possibility that his condition was entirely self-inflicted. While it is impossible to determine a definitive cause, it is highly unlikely that Charles II \"The Bad\" of Navarre had anything to do with his rival's strange condition. For nearly a quarter of a century, Charles V \u201cThe Wise\u201d of France suffered from a mysterious illness: a fistula on his left arm that continuously drained pus. A foreboding prophecy had warned the king that as soon as the fistula ceased to weep, he would have a mere 15 days before death followed. His death in 1380, at the young age of 42, seemingly proved this assumption correct. Many of his contemporaries suspected the hand of his longtime nemesis, Charles II \u201cThe Bad\u201d of Navarre, in the king\u2019s strange condition. The evidence available to us today does suggest that perhaps Charles II may have poisoned his rival with arsenic around the time that the strange fistula developed. But would this have been enough to cause 23 years of pus drainage? Or was Charles V\u2019s affliction due to another disease process entirely? The key to unraveling this mystery may lay in the decades-long rivalry between the two kings.The bad and the wiseA Distant Mirror: The Calamitous 14th Century, the historian Barbara Tuchman describes Charles II of Navarre as a \u201c\u2026small slight youth with glistening eyes and a voluble flow of words\u2026 he was a plotter, subtle, bold, absolutely without scruple, but so swerving and unfixed of purpose as to undo his own plots. His only constancy was hate\u201d [\u00a0In her book as hate\u201d . The rulas hate\u201d .Charles II first emerged onto the political scene at the age of 21 years, in an episode that encapsulated his lifelong approach to the power struggles of the nobility. King Jean II of France had recently granted the county of Angouleme, a holding that Charles regarded as his birthright, to Charles d\u2019Espagne, a royal favorite and the Constable of France. Although Jean II attempted to repair the insult by giving the hand of his daughter, Jeanne, to Charles II, he withheld the dowry for the princess\u2019s hand, only further inflaming the situation. Furious for revenge against his new father-in-law, Charles II had the Constable of France brutally assassinated. On January 8, 1354, his younger brother Phillip led a group of noblemen that broke into the Constable\u2019s room and hacked him to death; by some accounts inflicting 80 wounds upon the dead man\u2019s body before they left [And yet, thanks in a large part to his eloquence, with which he varnished over his actions his entire life , and by It was in this setting that the two Charles (Charles II and Charles V), one a king and one a prince, would first become friends. Thanks to Charles II\u2019s marriage into the Valois royal family, the two young men doubtless knew of one another, but their first significant interactions appear to have occurred in September 1355, when Charles II spent several days at Prince Charles\u2019  castle in Vaudreuil. Charles II\u2019s magnetic personality quickly won over the Dauphin . PerhapsThis was a miscalculation on the king\u2019s part. With the Dauphin acting as the new Duke of Normandy, Charles II had even easier access to the young prince than before. As whispers of conspiracies between the two Charles again spread, by the spring of 1356 Jean II had had enough . In fullAs luck would have it, barely five months later, Jean II found himself a captive as well. The defeat that the French suffered against the English at the Battle of Poitiers on September 19, 1356, was an utterly crushing one; Jean II and a significant chunk of the French nobility were captured and taken to the Tower of London to serve as hostages until the already-bankrupt kingdom could raise the eye-watering ransom that the English demanded [But the cunning king of Navarre had more tools at his disposal than political lobbying. While the French signed a humiliating truce after the battle of Poitiers, the frenzied bloodshed of the Hundred Year\u2019s War did not stop. The English armies may have withdrawn, but massive Free Companies (bands of mercenaries that offered their services to whichever side gave the most coin) still roamed the countryside, burning and looting as they had before. Even from his imprisonment, many believed that Charles II, acting through his brother Phillip, had a hand in funding many of the worst offenders, likely to apply further pressure for his release . Even thWhatever affection or cooperation the brothers-in-law may have displayed in the past was forever gone upon Charles II\u2019s escape. His objectives shifted, and whether out of cold ambition or blazing hatred for the man who had kept him imprisoned for over a year, a wave of violence was unleashed as he made a bid for the French throne. In the maddened political atmosphere, one of his key allies stormed the royal palace at the head of the angry mob, and in front of the Dauphin\u2019s very own eyes, in the very bedchamber of the Prince Regent, had the Dauphin\u2019s two marshals  murdered. The attack, so similar to the death of Charles d\u2019Espagne just a few years earlier, was widely believed to have been directly orchestrated by Charles II . Soon af\u00a0And yet, within a year, Charles II abandoned the campaign against the Dauphin. He revoked his alliance with the English in August 1359 and performed a ceremony of reconciliation with his brother-in-law, just as he had with his father-in-law [The poisoned prince?It was around this period that the Dauphin\u2019s contemporaries started to comment on his health. According to the chronicler Froissart, he was \u201c\u2026seized with an illness, which very much disheartened all who loved him; for as no remedy could be found for it, they foresaw that in a very short time he must depart this life\u2026 the reports were firmly believed that the King of Navarre, during the time he resided in Normandy, had attempted to poison him\u2026\u201d . The futAfter being treated by a physician named George of Prague, who performed the \u201c\u2026greatest cure known\u2026\u201d by draining out the venom \u201cthrough a small fistula on (the Dauphin\u2019s) arm\u201d the future King\u2019s symptoms were soon completely resolved . HoweverImmediate suspicion fell upon Charles II, at this time an exile in the Kingdom of Navarre. It was widely believed by contemporaries that \u201c\u2026 he had caused poison to be administered (when Charles V was) Dauphin; the effects of which, tho\u2019 retarded, or mitigated by medicine, are nevertheless said to have yet eventually terminated in his premature death\u201d .Charles II was no stranger to direct, immediate violence. In addition to his penchant for orchestrating bloody assassinations, his forces slaughtered an estimated 3000 peasants during the ill-fated Jacquerie Rebellion, a 10th of whom were consumed by the flames of a burning monastery .It was stories such as this and his vassal\u2019s constant betrayals that made the Dauphin, now King Charles V of France, launch a campaign in 1378 that forever tore Normandy out of Charles II\u2019s grasp. For the rest of his life, Charles was forced to live in the cramped confines of his mountain kingdom, all political influence stripped from him . It appeBut the question remains: is it possible that Charles V\u2019s fistula could have been due to poisoning by his lifelong rival?The possibilityAt the time, arsenic was not a difficult substance to obtain and could be found almost ubiquitously in most apothecaries. During the Middle Ages and Renaissance, it was often referred to as the \u201cking of poisons\u201d and the \u201cpoison of kings\u201d due to its odorless and tasteless nature . Even thWhile arsenic does not directly interact with DNA, it is co-mutagenic, enhancing the mutating effects of ultraviolet light, and inhibiting the process of DNA repair. This often manifests as hyperkeratosis, altered skin pigmentation, and dermatological cancers . The mosOf these, if we assume that Charles V\u2019s weeping fistula was due to chronic exposure to arsenic, the most likely explanation appears to be Bowen\u2019s Disease. This is an in-situ squamous cell carcinoma that typically manifests as a solitary lesion, often with a scaled surface. It is both a persistent and progressive pathology, with approximately a 3% chance of progression to invasive carcinoma . The indHowever, Bowen\u2019s Disease, while attributable to arsenic, is not one that satisfactorily matches Froissart\u2019s description of the ever-draining fistula. The King\u2019s discomfort and constant production of pus are far more consistent with hidradenitis suppurativa (HS). HS is a chronic, recurrent auto-inflammatory skin disease, often found in the axillae or groin, that may be associated with the formation of painful abscesses, deep nodules, and sinus tracts . While iRejecting the poison hypothesisHowever, while HS is an autoinflammatory condition, the evidence that we present for it being tied to possible arsenic exposure is relatively scarce . If indeCharles II\u2019s characteristics further support this. While the king of Navarre was certainly capable of intricate plotting, he routinely suffered from a lack of focus . His truEven his poisonings were, so far as we now know, acute and fast-acting ones. The dog that ate the poison intended for the Count of Foix died within hours . The merOther possibilitiesStaphylococcus aureus infection, it may be associated with minor traumas of the bone without an associated fracture [With both Bowen\u2019s Disease and HS deemed unlikely culprits, a new possibility for Charles\u2019 condition arises, one that is far more consistent with the descriptions of his disease that we currently have available. As Froissart writes, the king\u2019s mysterious fistula developed after George of Prague allegedly drained out the poison from Charles\u2019 arm . Brodie\u2019fracture . There ifracture . This raEven if the king did not have Brodie\u2019s abscess, there are a host of other pathologies that may be responsible for his condition. Tuchman theorizes that he may have suffered from tuberculosis . In the However, a cutaneous infection of tuberculosis is not strictly necessary to mimic the king\u2019s symptoms; tuberculosis osteomyelitis may present with swelling, pain, and a bone-to-skin fistula that drains purulent fluid. One case report of an Italian man with right shoulder pain secondary to tuberculosis osteomyelitis  reads reThere is another possibility as well, one grimmer than Brodie\u2019s abscess or some other form of infection. For 23 years, Charles V emphatically believed that he would die when the fistula ceased to drain . Given tAs for Charles the Bad, the \u201cwithered old serpent\u201d, as Tuchman describes him, he lived on for another seven years in exile. After one last plot to poison two of Charles V\u2019s surviving brothers, he finally met his fate in a freak accident that saw him burned alive and left to suffer for two agonizing weeks before he passed in 1387 at the age of 54. It was a fate that his contemporaries felt was well-deserved.It appears possible that Charles II may have successfully poisoned the future Charles V, likely with arsenic, around the year 1360. Such an act was consistent with both his previous and future behavior. However, even if this was the case, the Dauphin soon recovered. The strange fistula that he suffered from for the next quarter-century likely had nothing to do with any attempted poisoning. Possibilities range from the unlikely, such as HS, to the likely, such as an undiagnosed Brodie\u2019s abscess, an underlying osteomyelitis, or a tuberculosis infection. It is also possible that the ever-weeping fistula was maintained by the king himself in an attempt to stave off George of Prague\u2019s dire warning."}
+{"text": "It is possible to generate small amounts of electrical power directly from photosynthetic microorganisms\u2014arguably the greenest of green energy. But will it have useful applications, and what are the hurdles if so? It is now possible to generate small amounts of electrical power directly from photosynthetic microorganisms. Can this electricity be used outside of the lab, and what are the hurdles to be overcome? There has long been interest in using microorganisms to generate electricity directly, in biologically driven electrochemical systems. The first such systems were operated with heterotrophic microorganisms and are known as microbial fuel cells. They rely on some of the electrons generated during metabolism being exported from the cell and collected by an anode. Microbial fuel cells offer the attractive possibility of simultaneously breaking down waste material and producing electricity, and have been used, for example, to produce power to illuminate lavatories from urine harvested there . More reTypical devices \u20134, referThat is all very well in the lab, but will BPVs powered by photosynthetic microorganisms ever have real-world applications, and how soon? Laboratory studies have shown maximum power outputs in the region of 0.5 to 0.8 watts per square meter  and estiWith the power densities presently achieved, BPVs could in principle be used now to run small devices with low power requirements (up to a mW or so) , Stage 1It might become feasible to run small domestic appliances such as laptops, especially in off-grid locations (Stage 3), but this will require improvements in the performance of BPVs. We think it is unlikely to be feasible to run larger domestic or industrial appliances with BPVs (Stage 4), whether the power is generated locally or supplied by a grid, because of the large surface area that would be needed for power generation. For the same reason, it is even less likely to be feasible to incorporate BPVs into transport vehicles to power them. The surface area required and the resulting weight would be too great.\u22122 of cathode, which is equivalent to only a few cents or pence for the microprocessor BPV [How can we improve the performance of BPVs? We need to optimise power output while using sustainable materials. Highly structured anodes containing indium and tin are efficient , but usessor BPV ) and it What other hurdles are there? There is a tendency among researchers to judge BPVs against conventional photovoltaics purely in terms of their power generation when operating. However, comparisons are more complicated than that. One needs to assess the whole life cycle\u2014building, decommissioning and recycling the components of the devices. The reliance of BPVs on self-replicating organisms  rather than energy-intensive photovoltaic materials is a particular attraction. Once we have a better idea of how BPVs will be built for use in the real world , we can carry out the life cycle assessments that will help us judge objectively the benefits of BPVs. It will also be essential to understand the needs of local communities where BPVs are to be installed, especially in LMICs, and to involve the communities closely. A third challenge will be to persuade stakeholders (including research funders) to switch to a novel technology like BPVs. In spite of all these challenges, we believe that successful applications of BPVs in the lab suggest they can contribute to energy provision in the near future, especially for situations requiring small amounts of power without a direct supply."}
+{"text": "Trametes versicolor in association with a cocktail of natural phenol redox mediators efficiently degraded eumelanin from Sepia officinalis, offering an alternative procedure to traditional whitening agents. Redox mediators showed a synergistic effect with respect to their single-mediator counterpart, highlighting the beneficial role of the cocktail system. The pro-oxidant DHICA sub-units of eumelanin were degraded better than the DHI counterpart, as monitored by the formation of pyrrole-2,3,5-tricarboxylic acid (PTCA) and pyrrole-2,3-dicarboxylic acid (PDCA) degradation products. The most effective laccase-mediated cocktail system was successively applied in a two-component prototype of a topical whitening cream, showing high degradative efficacy against eumelanin.The overproduction of eumelanin leads to a panel of unaesthetic hyper-pigmented skin diseases, including melasma and age spots. The treatment of these diseases often requires the use of tyrosinase inhibitors, which act as skin whitening agents by inhibiting the synthesis of eumelanin, with harmful side effects. We report here that laccase from  These compounds covered a wide range of possible oxidative reaction mechanisms [The overproduction of eumelanin by melanocytes causes skin hyperpigmentation disorders, such as melasma as well as age and post-inflammatory spots, which can affect human health and social relationships ,2. Curreritation ,23,24,25ritation . In thisritation . These critation . Exampleetail in ,29,30,31mediator ,33,34. Wchanisms ,36,37. Echanisms ,42,43,44Sepia officinalis by the traditional LMS using a single redox mediator [MAX of eumelanin (540 nm) [Abs0 is the initial value of absorbance, and fAbs is the value of absorbance after 4 h. The treatment of eumelanin in the absence of the redox mediator was performed as a reference [Initially, we studied the degradation of eumelanin from mediator . The expmediator . The selmediator , ionic Imediator , and hydmediator . As a ge(540 nm) , while t(540 nm) : (1)(DE)eference . In thiseference .Lentinus polychrous [The highest value of DE was obtained in the presence of V , entry 3lychrous . MAX of 540 nm [The laccase mediator cocktail system (LMCS) shows the contemporary presence of (at least) two redox mediators, sometimes involving different reaction mechanisms. In nature, LMCS is involved in the degradation of lignin by fungi through the cooperative action of lytic polysaccharide monooxygenase and oligomers deriving from lignin degradation ,48,49. If 540 nm . Irrespen-dodecane (7.8 \u03bcmol) as an internal standard and analyzed by GC-MS. PTCA and PDCA were identified at 19 min and 21 min, respectively. Mass fragmentation analysis confirmed the structure of these compounds [In order to gain information about the degradation pathway of eumelanin by LMCS, we analyzed the sample after its treatment with the five best pairs of redox mediators by gas chromatography associated with mass spectrometry (GC-MS), quantifying the formation of PTCA and PDCA, which are considered the main degradation products of eumelanin. The formation of these compounds is indicative of the degradation of specific melanin sub-units, PTCA being correlated with the degradation of DHICA, whilst PDCA with that of DHI ,51. The ompounds . Note thompounds ,42,43,44ompounds ,54. \u22121, 1513 cm\u22121, and 1139 cm\u22121 were observed and assigned to alkyl groups [Field emission scanning electron microscopy (FE-SEM) of residual eumelanin after LMCS treatment with ABTS/V, TEMPO/V, Syr/V, As/V, and Va/V are reported in l groups .w/w) and sodium hyaluronate (1% w/w) and a variable amount of laccase in citrate buffer , whilst chamber B contained dimethyl isosorbide (5% w/w) and sodium hyaluronate (1% w/w) as well as the V/Va couple in citrate buffer . The DE of these formulations was evaluated by treating eumelanin (800 \u03bcg) dissolved in NaOH  with 1.0 mL of the content of chamber A, followed by the addition of 1.0 mL of the content of chamber B. The DE was calculated as described above and monitored by measuring the change in the absorbance of the sample (\u028eMAX 540 nm) at 37 \u00b0C after 24 h. Irrespective of the experimental conditions, the whitening cream formulations showed appreciable capacity to degrade eumelanin, with the highest DE value being obtained in the case of the 6X sample (55%) , and three concentrations of laccase , taking care to separate laccase (chamber A) from the pair of redox mediators (chamber B) by an airless dispenser A. Chambele (55%) . With thle (55%) . The sta4 [As reported in 4 . It is w4 ,62. In a4 .Sepia officinalis was purchased from Sigma-Aldrich ; laccase from Trametes versicolor, 2,2\u2032-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), 2,2,6,6-Tetramethylpiperidin-1-yl-oxyl (TEMPO), Vanillin, 4-hydroxy-3-methoxybenzyl alcohol , Acetosyringone, Syringaldehyde, and Acetovanillone were purchased from Sigma-Aldrich  and were used without any further purification. All experiments were performed in a citrate buffer pH 6.0 (0.1 M). Spectrophotometric measurements were taken with a Varian Cary 60 UV/Vis spectrophotometer equipped with a single-cell peltier thermostated cell holder. Spectrophotometric data were analyzed with the Cary Win UV software.Melanin from MAX of 540 nm [As a general procedure, different amounts of redox mediators  were added to eumelanin (800 \u03bcg) in an NaOH  and citrate buffer  mixture, followed by the addition of laccase (0.79 U/mg) in buffer citrate  at 37 \u00b0C for 4 h. The degradation of eumelanin was monitored by measuring the absorbance at the maximum melanin \u028ef 540 nm .MAX of 540 nm [As a general procedure, the selected redox mediator couple (1.6 \u03bcmol) was added to eumelanin (800 \u03bcg) in an NaOH  and citrate buffer  mixture, followed by the addition of laccase (0.79 U/mg) in buffer citrate  at 37 \u00b0C for 4 h. The degradation of eumelanin was monitored by measuring the absorbance at the maximum melanin \u028ef 540 nm .n-dodecane (7.8 \u03bcmol) as an internal standard, under vigorous stirring conditions at room temperature for 2 h. The analyses were performed using a VF-5MS column and an isothermal temperature profile of 100 \u00b0C for 2 min, followed by a 10 \u00b0C/min temperature gradient to 280 \u00b0C for 25 min. The injector temperature was 280 \u00b0C. Chromatography-grade helium was used as the carrier gas, with a flow of 2.7 mL/min. The mass spectra were recorded with an electron beam of 70 eV.Mass spectrometry was performed with the use of a 450 GC-320 MS apparatus  in comparison with commercial samples. Regarding the preparation of the sample, the residue was treated with pyridine, bis-trimethyl silyl trifluoro acetamide  in the presence of The field emission scanning electron microscopy (FE-SEM) results of the eumelanin samples were acquired by FE-SEM ZEISS GeminiSEM500 at 5 kV after a 20 mL drop (with deionized water) of the sample dispersion onto the specimen stubs, which were air-dried and coated with gold by sputtering with AGAR (Auto Sputter Coater). Before the observations, the samples received a deposition of chromium thin film (5 nm) by sputter coating with the use of a QUORUM Q 150T ES plus coater. \u22121.Attenuated total reflection infrared (ATR-IR) spectroscopic analyses were performed at room temperature with a Perkin\u2212Elmer Spectrum One spectrometer equipped with an ATR-IR cell. IR spectra were recorded by averaging 32 scans, with a resolution of 4 cmTrametes versicolor, we used the 2,2\u2032-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) assay. Laccase (0.79 U/mg) was added to citrate buffer  containing ABTS (0.58 \u03bcmol). The absorbance of the cationic radical ABTS+ was measured at 420 nm (molar extinction coefficient \u03b5420 = 36 mM\u22121 cm\u22121). The increase of absorbance at 420 nm was measured at room temperature for 10 min. The enzyme activity was calculated, as described in the following equation, from the slope of absorbance versus the time curve: To determine the activity parameters of laccase from One unit of specific activity was defined as 1.0 mg of the enzyme consumed by 1.0 \u00b5mol of the substrate per minute. LMCS was an efficient tool for the degradation of eumelanin. We have demonstrated that the degradation significantly increased in the presence of the Syr/V and Va/V couples. In these cases, the degradation efficiency was 67% and 87%, respectively, after only 24 h of treatment. Worthy of note is the fact that the couples Syr/V and Va/V showed a synergic effect, probably due to their high difference in the value of the redox potential. The quantitative degradation of eumelanin was obtained by repeating the treatment with the selected LMCS in a second run. The prevalence of PTCA with respect to PDCA after the LMCS treatment suggests that the degradation of the DHICA sub-units was more effective than its DHI counterpart, favoring the removal of the more pro-oxidant residues in the starting material. It is also worth noting that LMCS based on the Va/V couple worked with high efficacy even when embedded in a whitening cream formulation that resembled a standard commercial product. The use of the airless dispenser was necessary to avoid the premature activation of laccase. The 6X whitening cream formulation performed similarly to commercially available tyrosinase inhibitors, reaching high stability at 4 \u00b0C . OverallPending patent resulting from this work\u2014Title: Procedure for the decolorization of eumelanin based on laccase cocktail mediators system and cosmetic and cosmeceutical application in the field of whitening cream. Number: 102022000008453; Deposition: 28 April 2022."}
+{"text": "Optimizing the retaining ring structure can improve the quality of Chemical Mechanical Polishing (CMP). This study establishes a two-dimensional Computational Fluid Dynamics-Discrete Element Method (CFD-DEM) model, while the model is validated by experiments. The results graphically demonstrate the influence of the retaining ring groove design on the motion of the slurry abrasive particles. The size of the retaining ring groove appears to have a threshold value, above which the abrasives start to have significant distribution in the wafer region. As the groove size continues to increase, the number of abrasives entering the ring increases abruptly and oscillates at specific nodes. The abrasive transfer rate increases with the number of grooves in the early stage but reaches a limit at a certain number of grooves. Meanwhile, the retaining ring position affects the transfer of the abrasives. This study provides a base for optimizing the design of retaining rings. CMP is the process of achieving global flattening in advanced semiconductor manufacturing ,11,12,13In the CMP process, the wafer is attached to the polishing head and confined by the retaining ring. Then, it is pressed face-down onto the polishing pad. The head carrying the wafer and the pad carrying the slurry rotate around their respective centers. The slurry, which is composed of liquid and abrasive particles, is carried by the rotating pad to the polishing gap between the wafer and the pad ,17. The The retaining ring groove design had significant impacts on improving material removal rate (MRR), flatness, and providing a way to exchange the slurry with the external polishing slurry into the pad-wafer interface ,21,22. FSome studies have shown that the flat surface of the wafer can be obtained by repeatedly sliding, rolling, and squeezing the abrasives in slurry ,24,25,26In this paper, a two-dimensional multi-physics model is established using the Finite Element Method and the Discrete Element Method, aiming to study the effect of the retaining ring design on the abrasive movement during the CMP process. The effects of the retaining ring groove size on the abrasive transfer rate are investigated, and the periodic variation patterns of abrasive transfer through the grooves are observed. On this basis, the number of grooves can be changed to optimize the distribution of abrasives in the wafer region and to improve the flatness of the wafer.In the CMP procedures, there are three contact models that exist depending upon the material and process parameters, namely (i) boundary lubrication, (ii) partial lubrication, and (iii) hydrodynamic lubrication, as depicted in nd wafer . With a nd wafer . The disIn a CMP process, there are two main sources of polishing slurry in the polishing gap: one part already exists on the polishing pad before the polishing head is pressed down, and the other part flows through the grooves of the retaining ring. To simplify the modeling, it is assumed that the abrasives in the polishing gap before the polishing head is pressed down are not affected by the retaining ring and are in a stable state. Therefore, this part of the abrasives can be ignored in the modeling of the abrasive distribution, and the focus of this study was set on the abrasives passing through the grooves of the retaining ring. Before the abrasives entered the polishing gap, they were scraped flat by the conditioner, as shown in 1 is the outer diameter of the retaining ring, and d2 is the inner diameter. The surface slurry is distributed throughout the pad and flows freely with the rotation of the pad, the wafer, and the retaining ring. The nano-sized spherical abrasives are uniformly dispersed in the slurry except when colliding with an object. Collisions between abrasives are not considered in this work.Based on the general polishing process and the parameters of the polishing pad, retaining ring, and slurry, a two-dimensional finite element model is constructed, as shown in Slurry and abrasives are incompressible;The gap between the wafer and the polishing pad is the near-surface layer of the wafer. The velocity of the liquid layer on the wafer surface is approximately that of the wafer ;The total amount of slurry and abrasives is in a stable state.COMSOL Multiphysics 6.0 is used to conduct Finite Element Analysis. The slurry flow in the chemical mechanical polishing device is relatively turbulent, so this paper uses the k-epsilon turbulent model and the particle tracing model in a turbulent flow. The model is based on the following assumptions:The continuity equation and momentum equation describing the flow process of the polishing slurry are as follows:Continuity equation:u is the slurry velocity, p is the slurry pressure, \u03c1 is the slurry density, \u00b5 is the slurry dynamic viscosity, T\u00b5 is the eddy viscosity, and F is the additional force acting on the slurry.Momentum equation:F represents the equivalent replacement force of polishing pad rotation, which is composed of Coriolis force and centrifugal force:\u03c1 is the slurry density, u is the slurry velocity, \u03c9 is the angular velocity of circular motion, and r is the distance between the abrasive and the center of the circle.Among them, \u03b5 is the turbulent dissipation rate, k is the turbulent kinetic energy, k\u03c3 and \u03b5\u03c3 are the respective turbulent Prandtl numbers of \u03c3 and k, T\u00b5 the eddy viscosity coefficient, and kP is the generation term of turbulent kinetic energy caused by the average velocity gradient.The k-epsilon equation is as follows:In order to consider the rotation of both the pad and the polishing head, the model is calculated in two reference frames. One is a moving zone consisting of the retaining ring and the internal flow region, and the other is a stationary zone for the region outside the retaining ring.The computational domain with respect to the moving frame is defined such that an arbitrary point in the CFD domain is located by a position vector from the origin of the moving frame. The fluid velocity can be transformed from the stationary frame to the moving frame based on the following equation:Based on the turbulence model and the multiple moving frames, the flow rate of the slurry can be obtained, and the movement of the abrasives can be analyzed. The abrasives are completely immersed in the slurry. During the rotation process, the turbulent flow produces a drag force on the abrasives, which drives the movement of the abrasives.v and pm represent the speed and the mass of the abrasive, respectively, and DF is the drag force on the abrasives.The movement of the abrasives in the flow field can be expressed as:p\u03c4 is the abrasive speed response time indicating how fast the abrasive is accelerated to the slurry velocity, which is expressed by the formula:p\u03c1 is the abrasive density, pd is the abrasive diameter, and \u00b5 is the hydrodynamic viscosity.The drag force on the abrasives is related to the size, mass, and velocity of the abrasives, as well as the velocity and dynamic viscosity of the slurry, and can be calculated by the following formula:The flow in the slurry is turbulent, so the reaction of the abrasive colliding with the wall surface of the retaining ring is a diffuse reflection.Based on the high rotational speed, the contact mode among the pad, slurry, and wafer can be defined as hydrodynamic lubrication, in which case the pad and the wafer are not in direct contact but separated by the slurry layer. Therefore, the slurry flows freely in the model;u is the fluid velocity, v is the abrasive velocity):Initial value  and a diameter of 356 mm.The grid density has an impact on the computational results, and the grid density reaches a threshold value to keep the influence of the grid at a low level. Therefore, a mesh-independent verification of the model is required to determine the non-correlation between the number of meshes used in the model and the results obtained from the calculations. As the abrasive transmission through the retaining ring is of the main concern in this paper, the average number of the abrasives in the first cycle is selected as the basis for judgment, as shown in In the CMP retaining ring model, The retaining ring groove design in the simulation is shown in 2 abrasive slurry  was used for polishing a 300 mm silicon bare wafer to evaluate the effect of the retaining ring groove. A polyurethane polishing pad  was used in the experiment. The speed of the polishing head/pad was set to 120/120 rpm, and the flow rate of the slurry was 300 mL/min. The retaining ring structure used in the experiments is shown in The polishing data was obtained using a 300 mm wafer polishing machine Universal\u2014300Dual. SiOThe MRR and ESFQR (Edge Site Front Least Squares Range) were measured to verify the simulation model in a real manufacturing process. The MRR and ESFQR of wafers were measured by Bare Wafer Geometry Metrology Systems . MRR is defined as the amount of material removed per minute, while ESFQR is used to characterize the flatness of the wafer surface. As shown in aN is the number of abrasives entering the polishing gap, and N is the total number of abrasives in the polishing pad.The abrasive transfer rate is defined as the ratio of the amount of abrasive moving to the polishing gap to the total global abrasive as follows:In order to study the effect of the groove width of the retaining ring on the ability of the abrasive to enter the polishing gap through the groove, the abrasive transfer rate was calculated with a different groove width of the retaining ring.In order to observe the distribution of the abrasives in the polishing gap, the abrasive movement is tracked and visualized at an interval of 0.01 s. It can be seen from the above that the groove size of the retaining ring has a large effect on the abrasive transfer rate. The abrasive transfer rate in Under the same process parameters, a retaining ring model with a groove width of 7 mm is selected as the reference since it is the size when the abrasives start to be distributed significantly. The number of grooves is varied to study the effect on the abrasive transfer rate.In this section, the abrasives are tracked at a frequency of 0.01 s to observe the abrasive distribution in the polished area. \u22121 for 18 grooves to 1.8\u20132.2 m\u00b7s\u22121 for 48 grooves. Because abrasives have more kinetic energy, the displacement distance of the abrasives is increased. When the number of grooves is increased from 18 to 48, the abrasives are able to move closer to the center of the wafer. This reduces the non-uniformity caused by a high concentration of abrasive at the wafer edge. Therefore, the increase in the number of retaining ring grooves will improve the distribution of abrasives and reduce the unevenness of the wafer surface.As shown above, the number of grooves in the retaining ring affects the abrasive distribution, and the abrasive distribution tends to move closer to the wafer center as the number of grooves increases. The speed of the abrasive entering the retaining ring is an important factor affecting its distribution. The simulation results show that the structure of the retaining ring groove will affect the abrasive transfer rate and the abrasive distribution in the wafer region. In order to verify the simulation results, three retaining ring structures were chosen to conduct CMP polishing experiments. The material removal rate and ESFQR improvement are shown in It can be seen that Ring3 has the highest MRR of 597 nm/min, while Ring2 has an MRR of 557 nm/min, and Ring1 has an MRR of 531 nm/min. Therefore, it is clear that both MRR and ESFQR are improved with the increase of retaining ring groove size, indicating that the flatness of the wafer surface is improved. As analyzed in the above simulations, the abrasive transfer rate of Ring3 is greater than that of Ring2 and Ring1. In addition, Ring3 can also help to distribute the abrasive more evenly in the wafer area, so Ring3 has the highest MRR and flatness. The experiment proves the trend of the influence of the retaining ring structure on the abrasive movement state in the simulation. Meanwhile, the conclusion agrees well with the simulation results of Ring1, Ring2, and Ring3 from the perspective of MRR and uniformity. It can also be obtained that the increase in the number of abrasives leads to an increase in MRR, which explains the highest MRR of Ring3. Furthermore, from Ring1 to Ring3, the abrasives are evenly distributed over the entire wafer area, thus achieving the effect of improving ESFQR.Abrasives can either enter or exit through the grooves at different positions of the retaining ring. Proper adjustment of the size and number of grooves of the retaining ring can result in more abrasives entering than exiting, thus improving the utilization rate of the abrasives. Under the influence of the retaining ring, the abrasive transfer rate varies periodically and becomes stable with time. When the groove size is small, the abrasive transmission through the groove and utilization is poor. Increasing the groove size within a certain range can effectively improve the abrasive transfer rate. The abrasives start to have significant distribution in the wafer region when the groove size reaches 7 mm. When the groove size reaches 13 mm, the abrasive inflow is significantly higher than the outflow, and the transfer rate is at a high level in the later stable stage. However, by continuously increasing the retaining ring groove size, the abrasive transfer rate decreases again. This is due to the fact that at a certain point, the contribution of increasing the groove size to abrasive outflow starts to be greater than that of inflow;The results of the retaining ring model based on 7 mm groove size show that the abrasive transfer rate increases to varying degrees at the beginning as the number of grooves in the retaining ring increases and peaks at the number of grooves of 42. In addition, increasing the number of grooves results in the abrasive entering the retaining ring at a higher speed and greater displacement of the abrasive towards the wafer center after passing through the grooves, which helps optimize the abrasive distribution.In this paper, a fluid-particle multi-physics model is established based on the finite element method and the discrete element method, and the effects of the groove width and the number of grooves of the retaining ring on the abrasive transfer rate and the distribution during the CMP process are studied and validated by experiments, and the following conclusions can be drawn:The above conclusions show that the design of the retaining ring grooves affects the abrasive motion near the retaining ring. Therefore, improving the design of the retaining ring grooves can improve the abrasive transfer rate and optimize the abrasive distribution in slurry, thus improving the polishing removal rate and reducing the wafer surface non-uniformity. It can also enhance the utilization of slurry and save the polishing cost."}
+{"text": "In order to improve the nutritional value and reduce starch the digestibility of black soybean cookies, superfine black soybean flour was modified by heat-moisture treatment (HMT). The physicochemical properties, structure analysis of the flour samples and corresponding dough, and nutritional, physical, and textural properties of the cookies were investigated. After HMT, the water and lactic acid retention capacity and the oil binding capacity of mix powder dramatically increased, being almost twice the value of the untreated sample. HMT increased gelatinization temperature by about 10 \u00b0C but decreased gelatinization enthalpy. HMT had no apparent effect on the morphology and size of granules, but some cracks and pores appeared on the HMT-mix powder granules and corresponding dough. Fourier transform infrared spectroscopy analysis showed that the ordered structure of dough was unaffected during HMT. After HMT, the thickness, density, and baking loss of the cookies increased, and the spread ratio decreased. HMT dramatically increased the chemical score of cookies from 12.35% in mix powder cookies to 19.64% in HMT-mix powder cookies. HMT decreased the rapidly digestible starch content, while the slowly digestible starch increased from 45.97% in mix powder cookies to 49.31% in HMT-mix powder cookies, and RS increased from 21.64% to 26.87%. Overall, HMT did not have a negative effect on the processing properties and microstructure and secondary structure of the dough, or the physical properties and quality of the cookies, but significantly improved the nutritional properties and decreased the starch digestibility of the cookies. Cookies are a popular bakery product for most consumers, including children and the elderly. Its popularity is due to its ready-to-eat nature, convenience, rich shapes and tastes, relatively longer shelf-life, and ability to serve as a vehicle for various nutrients and supplements ,2,3. HowBlack soybean is an important variety of coarse grains that contains rich dietary fibers, proteins, vitamins, micronutrients, and lysine. It has many physiological functions, including antioxidative capacity, risk reduction in obesity, diabetes, hypercholesterolemia, coronary heart diseases, and other diseases ,8. Thus,Heat-moisture treatment (HMT) is a physical modification method used to change the physicochemical properties of starch. It is the process in which the starch is heat-treated at a low moisture content and a relatively high temperature . Many stThus, in this study, HMT was used to process mix powder of wheat flour and superfine black soybean powder. The physicochemical properties of mix powder and the structure of the dough were analyzed, as well as the physical characteristics, texture, in vitro starch digestibility, and nutritional properties of the cookies. This study aims to decrease the starch digestibility of black soybean cookies, while maintaining the texture of the cookies, which is of great significance to consumers of obesity diseases and other chronic diseases. The results can provide theoretical guidance for the deep processing of black soybeans and the development of high-quality and low-digestibility black soybean cookies with HMT-black soybean flour.Wheat flour and black soybeans were purchased from a local market, and the superfine black soybean flour was prepared using a high-energy nano-impact mill . Mix powder was obtained by replacing wheat flour with 35% superfine black soybean flour. The porcine pancreas \u03b1-amylase  and amyloglucosidase (A7095 \u2265 300 U/mL) were obtained from Sigma\u2013Aldrich Co. . All other chemicals were of analytical grade.The HMT of wheat and black soybean mix powder was conducted referring to Wang et al.  with somThe ash, total protein, fat, and total dietary fiber content of wheat flour, mix powder, and HMT-mix powder were determined using standard AACC (2000) methods . The nitX-ray diffraction patterns of flour samples were obtained with the X-ray diffractometer  at 40 kV and 80 mA. The diffraction angle (2\u03b8) scanning region range was 4\u00b0\u201350\u00b0, and the rate was 1\u00b0/min. The crystalline peak area and amorphous area were separated by PeakFit software . The relative crystallinity of the samples was calculated as the ratio of the crystalline peak area to the total diffraction area .w/w), and maize oil were added to determine the WRC, LARC, and OBC, respectively. The mixtures were vigorously shaken for 10 s. After that, the tubes were incubated in a 30 \u00b0C water bath for 30 min, shaken for 5 s every 10 min, and then centrifuged at 3000\u00d7 g for 10 min to remove the supernatant. Centrifuge tubes with precipitate were weighed, and the WRC, LARC, and OBC values were calculated as the weight of the solvent contained in the sample. The formula is as follows:The water retention capacity (WRC), lactic acid retention capacity (LARC), and oil binding capacity (OBC) of wheat flour, mix powder, and HMT-mix powder were determined by the method described by Cappa et al. . Briefly\u22121 using an empty pan. The gelatinization temperature at onset (To), peak (Tp), and end (Tc) and gelatinization enthalpy (\u0394H) were calculated based on the thermogram.The thermal properties of the wheat flour, mix powder, and HMT-mix powder were measured using a differential scanning calorimeter . The method was referred to Yang et al.  with somThe surface morphology of wheat flour, mix powder, HMT-mix powder, and their doughs were observed under cold field-emission scanning electron microscopy . For the dough preparation, wheat flour, mix powder, and HMT-mix powder were separately mixed with water, and the dough was prepared by stirring the flour at 50 rpm for 10 min in a dough mixer . Then, the dough was freeze-dried and cut into small pieces . The floA412 means the absorbance at 412 nm; D means the dilution factor (D1 is 5.02 and D2 is 10); C denotes the sample concentration in mg/mL.The determination of free sulfhydryl groups (-SH) in the gluten was performed according to the spectrophotometric assay method reported by Bressiani et al.  and Cao w/w) and compressed into thin disk-shaped pellets. FTIR spectra were recorded from 4000 to 400 cm\u22121 with a resolution of 4 cm\u22121.The secondary structures of the gluten were studied by an FTIR spectrometer , according to the method described by Xu et al. . After lCookies were separately prepared using wheat flour, mix powder, and HMT-mix powder. The cookies were produced using the method reported by Sulieman et al.  with a sThe amino acid composition of the cookie samples was measured by Amino Acids Automatic Analyzer  following the method described by Zhao, Mu, and Sun  with fewThe physical parameters of the cookies, such as weight, diameter, thickness, spread ratio, density, and baking loss, were determined according to the method reported by Sulieman et al.  and HeraThe cookies were ground into powders to determine the in vitro starch digestibility. The enzymatic hydrolysis of the cookies was determined as described by Yang et al. .G20 represents the glucose released after 20 min; G120 represents glucose released after 120 min; G0 represents free glucose.The fractions of RDS, SDS, and RS were calculated by the following formulas:The textural properties of the cookies were determined by texture profile analysis using a CT3 texture analyzer  with a TA39 probe. The test parameters were set as follows: the pre-test speed, test speed, and post-test speed were 1 mm/s; the degree of compression was 50%; the dwell time between two compressions was 2 s, and cycled twice. The TextureLoader software was used for data collection and processing. The hardness, fracturability, gumminess, and chewiness were recorded.p < 0.05.All analyses were conducted in triplicate using SPSS 17.0 . Significant differences were determined by comparing the means using Duncan\u2019s multiple range test at a significance level of \u22121, respectively, while in wheat flour, they were 1.67, 0.90, and 0.13 g\u00b7kg\u22121, respectively. This result was mainly because of the characteristics of the raw materials. After HMT, some changes in the proximate composition of the HMT-mix powder occurred. In summary, the ash, protein, lipid, and dietary fiber all decreased after HMT. The decrease in these components may be due to degradation during the higher temperature treatment.The proximate composition of wheat flour, mix powder, and HMT-mix powder is presented in XRD assay was conducted to analyze the changes in the crystalline type and relative crystallinity of the mix powder after HMT. The X-ray diffractograms and relative crystallinities of wheat flour, mix powder, and HMT-mix powder are displayed in Although HMT did not change the crystal pattern, it increased the relative crystallinity. Moreover, the relative crystallinity significantly increased from 20.40% in mix powder to 26.02% in HMT-mix powder. This result is consistent with Na et al.  who founThe WRC, LARC, and OBC of powders need to be noticed when developing new products. In this study, these parameters of the wheat flour, mix powder, and HMT-mix powder are shown in DSC allows the analysis of transition temperatures and the transition enthalpies, corresponding to the melting of starch crystallites during heating. The DSC curves are showed in Compared with the dough of wheat flour, the dough prepared from mix powder displayed a more compact gluten network, in which most wheat starch granules were firmly embedded. In wheat flour dough, some starch granules dissociated with the gluten network. It revealed that adding black soybean powder improved the continuity and stability of gluten, which was also verified by the S\u2013S content of the samples. The higher protein content of black soybean powders contributes to the formation of the gluten network. According to Rakhesh, Fellows, and Sissons , the addFree SH and S\u2013S significantly affect the structure and functional characteristics of the dough . S\u2013S bonThe FTIR spectrum of gluten from the mix powders and wheat flour dough is shown in \u22121. This result was consistent with the findings of Zhao et al. [\u22121) was significantly greater than that of wheat flour cookies (2.42 g\u00b7kg\u22121). Thus, the addition of black soybean flour effectively increased the content of limited amino acids in wheat flour cookies and promoted protein complementation. This result is important for improving the amino acid composition of ordinary wheat flour cookies.The amino acid composition of the cookies is presented in o et al.  that theo et al.  that the\u22121 in mix powder cookies to 34.80 g\u00b7kg\u22121 in HMT-mix powder cookies, and leucine increased from 3.20 g\u00b7kg\u22121 to 11.13 g\u00b7kg\u22121, both more than threefold. The tyrosine increased from 2.15 g\u00b7kg\u22121 in mix powder cookies to 6.10 g\u00b7kg\u22121 in HMT-mix powder cookies, and proline increased from 5.58 g\u00b7kg\u22121 to 14.99 g\u00b7kg\u22121, both by more than two times. These results could be explained as follows: phytic acid, condensed tannins, and polyphenols may interact with proteins to form complexes that reduce protein solubility, and thus affect the action of proteases on unstable peptides bonds. Furthermore, the increase in bioavailability of amino acids after HMT may be caused by the reduction or removal of the above antinutritional factors, which increases protein digestibility. Embaby [Based on . Embaby  found thThe weight, diameter, thickness, spread ratio, specific volume, and baking loss of the cookies, as affected by the particle size of black soybean flour, are shown in Compared to the mix powder cookies, the thickness, density, and baking loss of the HMT-mix powder cookies increased, and their spread ratio decreased. The increased baking loss of HMT-mix powder cookies can be explained by the changes in the microstructure of the dough. Based on SEM analysis, the dough of HMT-mix powder presented some pores, and the structure became loose and less dense. These changes may contribute to the water flow and evaporation during baking.The textural properties of the cookies made from wheat flour, mix powder, and HMT-mix powder are presented in In this study, HMT was used to treat the mix-powder of wheat flour and black soybean to improve the nutritional value and reduce the starch digestibility of cookies. The physicochemical properties, structural characteristics of the flour samples and corresponding dough, and physical, nutritional, and textural properties of the cookies were investigated. The results showed that HMT could significantly vary the properties and structure of the dough and cookies. After HMT, the water retention, lactic acid retention capacity, and oil binding capacity of the mix powder dramatically increased. The gelatinization temperature  increased by approximately 10 \u00b0C, while \u0394H decreased. The morphology and size of the granules were not affected by HMT, but some cracks and pores appeared on the HMT-mix powder granules and HMT-mix powder dough, respectively. HMT facilitated the formation of disulfide bonds and improved the stability of the dough. The amino acid analysis showed that HMT significantly increased the chemical score of the cookies. It increased from 12.35% in mix powder-cookies to 19.64% in HMT-mix powder cookies. Notably, HMT dramatically decreased the in vitro digestion characteristics of starch. These results suggest that HMT can be used as an effective \u2018green\u2019 process for increasing the total SDS content, RS content, and chemical score of cookies with superfine black soybean flour. In addition, HMT can effectively improve the nutritional value of cookies without negative effects on the dough\u2019s processing properties and the cookies\u2019 physical properties. Therefore, this study can provide guidance for the applications of HMT-superfine black soybean flour in baked foods. Considering that starch is the main component of all ingredients (wheat flour and mix powders) in this paper, their starch digestibility was emphasized; while protein was another important component, its role in the whole digestion process was unfortunately ignored, which is worthy of further study as one of the research directions."}
+{"text": "For the 2022 Rwanda Census, data will be collected electronically for the first time. This provides both an opportunity and a challenge to use technology for census processing. We are working with National Institute of Statistics Rwanda (NISR) to help overcome linkage problems and produce robust population estimates.Working with NISR we aim to create an achievable Census solution given the constraints on time, computing power and experience. We identified key issues, such as the lack of addresses and full date of birth, and developed a series of recommendations. These combined our experience of census processing and NISR\u2019s knowledge of Rwandan geography, conventions and culture to develop the simplest solutions possible, ensuring that each recommendation gave maximum benefit for the effort required. Next, we plan to use 2021 Rwanda Census Pilot data to begin development of an end-to-end matching exercise including pre-processing, automatic linkage, clerical resolution and quality assurance.We developed recommendations, including small changes to the data collection application and creating a Unique Property Reference Number by concatenating the Enumeration Area code and the Structure Number written on the building during Census collection. Following this, we produced a synthetic Rwandan Census and Post Enumeration Survey (PES) which was used to demonstrate and collaboratively develop matching algorithms. Probabilistic linkage methods were considered but discarded due to lack of sufficient infrastructure. Instead, deterministic methods were used. Match keys are currently being developed by NISR with clerical review undertaken using our open-source Clerical Matching Online Widget (CROW) with the synthetic data. Future plans include using the Census Pilot to estimate overcount and developing a modified version of the Soundex phonetic encoder for use on Rwandan names.The resource available for the Rwandan Census is comparatively small, but even the smallest resource can have a massive impact when used productively. This collaborative project takes the key concepts of well-developed linkage strategy and makes appropriate modifications to enable an achievable and effective Rwandan Census linkage strategy."}
+{"text": "The present work expects to meet the personalized needs of the continuous development of various products and improve the joint operation of the intraenterprise Production and Distribution (P-D) process. Specifically, this paper studies the enterprise's P-D optimization. Firstly, the P-D linkage operation is analyzed under dynamic interference. Secondly, following a literature review on the difficulties and problems existing in the current P-D logistics linkage, the P-D logistics linkage-oriented decision-making information architecture is established based on Digital Twins. Digital Twins technology is mainly used to accurately map the P-D logistics linkage process's real-time data and dynamic virtual simulation. In addition, the information support foundation is constructed for P-D logistics linkage decision-making and collaborative operation. Thirdly, a Digital Twins-enabled P-D logistics linkage-oriented decision-making mechanism is designed and verified under the dynamic interference in the linkage process. Meanwhile, the lightweight deep learning algorithm is used to optimize the proposed P-D logistics linkage-oriented decision-making model, namely, the Collaborative Optimization (CO) method. Finally, the proposed P-D logistics linkage-oriented decision-making model is applied to a domestic Enterprise H. It is simulated by the Matlab platform using sensitivity analysis. The results show that the production, storage, distribution, punishment, and total costs of linkage operation are 24,943\u2009RMB, 3,393\u2009RMB, 2,167\u2009RMB, 0\u2009RMB, and 30,503\u2009RMB, respectively. The results are 3.7% lower than the nonlinkage operation. The results of sensitivity analysis provide a high reference value for the scientific management of enterprises. Production and Distribution (P-D) are the two core links that directly affect the Enterprise (ENT)'s production efficiency and overall operation efficiency. Traditionally, P-D are operated independently by the production and logistics departments within an ENT or by the third-party logistics ENTs , 2. P-D In recent years, P-D-oriented Collaborative Optimization (CO) from a global perspective has attracted the extensive attention of scholars and production ENTs. Scholars study the optimization of P-D parameters under different customer demand and delivery parameters. Loro et al.  used actBased on the above research, in Intelligent Manufacturing (IM), the integrated optimization method is mainly used to optimize P-D. Therefore, combined with the actual production distribution operation problem, this paper expands the application of the Digital Twins from independent objects to multiple related objects' optimization. It makes up for the lack of research on global correlation twinning in complex multiunit production and operation systems in the past. It enriches the application scenario of Digital Twins.The intelligent enabling technologies provide a solid basis for online collaboration and independent DM of P-D. Digital Twins technology features excellent characteristics, such as data-driven process, iterative optimization, real-virtual integration, and real-time interaction. These features also meet the needs of the current research problems. Thus, this paper intends to use Digital Twins intelligent technology to support the linkage Production-Distribution (PD) DM.Digital Twins was first proposed by professor Grieves of the University of Michigan in 2003. At that time, Grieves defined Digital Twins as a Three-Dimensional (3D) model including virtual products, physical products, and their connection. Over time, scholars continue to supplement and improve the connotation of Digital Twins. For example, the National Aeronautics and Space Administration (NASA) claims Digital Twins to be a highly integrated simulation model for aerospace vehicles. Digital Twins can map or reflect the real-time operation state, future evolution trend, and basic functions of entity objects in the computer information world. It usually employs historical data, real-time sensing data, and physical models.Based on the Digital Twins, foreign scholars have designed a Manufacturing Execution System (MES) to integrate data from two different sources: machine tool operation data and production execution data. The MES realizes data-driven production optimization. Noticeably, Digital Twins technology can also combine knowledge learning technology to improve reasoning ability by extracting and reasoning relevant knowledge from production data. As a result, the Digital Twins system can improve the manufacturing process and production management.Arguably, Digital Twins technology provides a feasible technical framework and management mode for the interactive control and digital construction of physical entities. Digital Twins technology is mainly used to optimize and control a single independent object. However, there is still a lack of research on online collaborative decision control and global correlation virtual twins for multiple correlated physical objects.Under the customized production model, the demand uncertainty can lead to a significant increase in the complexity of P-D joint operation. At the same time, all links in the P-D process tend to make independent decisions. The traditional integrated optimization method is challenging to solve the P-D collaborative problem under dynamic interference. Therefore, a distributed DM-oriented CO approach must be devised for ENT P-D.MDO, proposed in 1982, is an effective global optimization methodology for designing and optimizing complex systems by analyzing the synergy and interaction between large-scale complex systems and their multiple subsystems. Over the years, the MDO method has been widely used in the design and optimization of large and complex equipment, such as automobile, aerospace, and machinery, as well as Supply Chain (SC) operation. According to the decomposition levels, complex systems are divided into single-level MDO, two-level MDO, and multilevel MDO. Single-level MDO mainly includes All At Once (AAO) and feasible multidisciplinary methods. By comparison, two-level MDO methods (such as CO method) and multilevel MDO methods include the target cascade method and Lagrange coordination. P-D linkage operation can be decomposed into a two-level coordination optimization problem, so the present work focuses on the research progress of CO methods.The CO method decomposes the complex system optimization into a two-level optimization problem of system-level and multiple-discipline levels. Under the coordination of system-level, discipline level can realize distributed parallel optimization. The CO method has the characteristics of high efficiency, rapidity, simplicity, and organization form of design division in line with the actual project. It has become one of the more studied MDO methods. Like most methods, the CO method includes theoretical and applied research.Theoretically, scholars have studied how to improve the solution performance and practicability of the CO method. The CO method can be optimized either by equality constraint relaxation or the Response Surface Method (RSM). These improvements effectively improve the solution efficiency of the CO method. The application and research of the CO method in large-scale system optimization mainly stay in the static design optimization of collaborative structure and parameters. There is less research on CO method-based dynamic modeling and online DM for the complex multiunit system.CO of P-D refers to comprehensively considering the actual production, inventory, and distribution situation, formulating the optimal P-D cooperation plan, meeting customer needs with the minimum total operation cost, and improving market competitiveness. The P-D-oriented CO includes production scheduling and distribution allocating. Production scheduling is responsible for formulating the order production sequence, determining the start processing time, and allocating production resources. By contrast, distribution allocation determines the distribution start time of Finished Products (FPs) and formulates the distribution sequence for each order. It then administers vehicles to undertake distribution tasks and plans distribution routes. The overall operation cost of SC can be effectively reduced by coordinating production scheduling and distribution allocation.The existing research on P-D-oriented CO is mainly carried out in a stable production and operation environment. There is less research on P-D's dynamic, collaborative operation under the uncertain production and operation environment. In addition, P-D mainly uses the integrated optimization method. The study is still very few on the P-D-oriented multiunit distributed CO method under the dynamic operational environment.Here, the logistics linkage system is optimized based on Digital Twins and optimization algorithms , 10. BasLogistics linkage is a process including P-D links, with each link affecting one another and the overall system stability. Therefore, the collaborative process and high relevance of all logistics links are the basis for ensuring the efficiency of the P-D links , 12. In P-D logistics linkage involves an interrelated but self-sustained set of dynamic production, storage, and distribution link. Remarkably, the production link manufactures customization products (home-deliverable over the distribution link). It involves multiple departments , 15, amoAs in Uncertain production completion time. The complex production and operation process includes various production factors, such as personnel, materials, equipment, and technology. The change of each production factor may affect regular production and operation. Significant uncertainty makes the production operation vulnerable to dynamic interference, thus significantly increasing possible mismatch in production factors and affecting the normal operation. As a result, it prevents the accurate control of the product production process, resulting in large fluctuations in the offline time and offline quantity of products.Long queueing time for order completion. Personalized demand orders often contain multiple types of products. Due to the universal application of professional division of labor in production ENTs, customers' orders are usually re-distributed into different production workshops. They can be delivered until all production workshops complete the products order and the warehouse is ready. However, due to random dynamic interference, the offline time of products in each production workshop fluctuates significantly. Thus, each production workshop's offline time cannot be synchronized, resulting in a long queueing time in the warehouse, increasing the storage cost.The operation efficiency of FP distribution is low. The number of distribution vehicles owned by third-party logistics companies is extremely limited. When the uncertainty of offline order production is more significant, the demand for distribution vehicles will be more unpredictable. Given the random uncertainty, it is difficult for the third-party logistics company to effectively integrate the distribution demand of each order to realize the scale effect. In that case, low vehicle loading rate and long distribution path occur from time to time. At the same time, due to the great uncertainty of products offline, the offline FPs have no vehicles to match sometimes. It is necessary to pay a huge penalty fee for delayed delivery, which will affect customer satisfaction in the long run.Poor execution of production plan and distribution plan. The randomness and dynamics often lead to dynamic changes in P-D's execution and operation environment. Under such circumstances, the production scheduling plan and FP distribution plan are challenging to meet the operation requirements of the actual site. At the same time, due to the lack of real-time and effective coordination and response mechanisms and methods among the DM links of P-D, the implementation effect of the initial plan is poor.The P-D logistics linkage is subject to the P-D departments' decisions. For example, a short supply of raw materials will cause production suspension, thus extending the product output time. The distribution vehicle failure or traffic jams can also hinder products' on-time delivery. Insufficient cooperation of P-D links might cause associated overstock and broken FP inventory, which adds up the overall production and operation costs. The random dynamic interference generated in production execution harms the P-D operation , 21. TheP-D logistics linkage operation is a complex process including multiple independent DM units. The highly efficient operation is inseparable from each object-specified and constrained DM unit , 23. UsuThe production link's main DM goal is to consider the actual personnel, materials, equipment, and other resources in the production workshop. DM can help formulate the production scheduling plan with optimal resource allocation and complete the production requirements of customer orders. In making the production scheduling plan, it is unclear whether the resources and capabilities of the storage link and distribution link can meet the planning requirements.The main DM goal of the storage link is to maximize the utilization rate of storage space and warehouse turnover to complete the storage of FPs. It does not consider whether its operation scheme benefits the production and distribution links.For the distribution link, the main DM goal is to distribute the FPs from the warehouse to each customer with the maximum vehicle loading rate, the minimum distribution cost, or the shortest distribution path according to the existing vehicle resources (model and number of vehicles). Generally, the actual situation of production and warehouse operations is not considered, resulting in the inability to optimize time or cost from the system's perspective.Digital Twins-enabled P-D linkage-oriented DM can accurately describe physical entities and reflect their essential characteristics , 25, as As in The physical object layer uses Intelligent Sensing Technologies (IST), such as Radio Frequency Identification (RFID) tag, RFID reader, Global Positioning System (GPS) device, and Personal Digital Assistant (PDA) handheld device to sense the multidimensional real-time operation data of personnel, equipment, materials, process, and environment generated in the P-D process in real time. It also provides data support for each DM unit's interconnection, cooperative operation, and linkage DM.The virtual object layer supports online linkage DM through an accurate virtual image of physical objects and dynamic virtual simulation of digital objects. The virtual image maps the whole production process based on the geometric and relational models. It is reconstructed using the multidimensional and real-time production operation data to present the actual operation of physical objects in the information world. Virtual simulation makes accurate and comprehensive operation effect judgment on the production system according to the real-time image data and constantly iterates and optimizes the linkage DM strategy to support the online linkage DM.The linkage service layer is driven by virtual simulation data and independently analyzes and discriminates the execution process of P-D and the behavior of resources. Meanwhile, it provides intelligent production scheduling DM service and intelligent vehicle scheduling DM service. Thus, the adaptive DM is realized to control the whole P-D process.The linkage application layer provides a Human-Computer Interaction (HCI) application system with complex production and operation systems and multiunit joint operations. Users interact with the application system through the intelligent mobile terminal or Personal Computer (PC) to make online linkage decisions to reduce the adverse impact of dynamics on the production system.In summary, P-D logistics linkage mainly involves three objects: production workshop, warehouse, and distribution vehicle. As in Production workshop is the core component of P-D operation. The real-time acquisition of its dynamic data directly affects the efficient linkage between P-D. Production workshops can install intelligent sensing devices on production equipment and pastes RFID tags on material and FP pallets. It can equip workshop operators with intelligent wearable devices and install RFID readers at stations and workshop offline points. Besides, it can provide Wireless Sensor Networks (WSNs), such as Fourth Generation (4G)/Fifth Generation (5G) networks and WiFi. Thus, the workshop data can be accurately obtained and transmitted in real time, including personnel, materials, and environment.The FP warehouse is the buffer in the P-D operation process, which determines the continuity and smoothness of P-D. The FP warehouse can place a mobile access control system at the door like the production workshop. Meanwhile, the workshop can paste bar codes on the shelves and equip forklift drivers with onboard tablet computers. It can also configure readers for warehouse staff and cover antennas, 4G/5G, and other wireless transmission networks in the whole warehouse. As such, real-time operation data can be obtained, including the use of cargo spaces, the FP storage information, and the access of forklifts in and out.(1) Distribution Vehicle. The relevant parameter data and dynamic operation data of distribution vehicles are an essential basis for the linkage operation of P-D. Specifically, tablet computers can obtain real-time cargo loading and unloading data and customer location. The dynamic operation data, such as the position, speed, and loading rate of distribution vehicles, are obtained in real-time through GPS technology.Supported by P-D linkage DM architecture, a P-D logistics linkage DM mechanism is proposed, as in \u2009 Initial planning stage. Before production, IST can collect and integrate real-time operation data, such as personnel, equipment, materials, methods, and environment, in the whole P-D process. Driven by real-time fusion data, the virtual object layer in the multiunit linkage DM architecture based on Digital Twins simulates the linkage DM model. The model aims to achieve the minimum total system cost. It plans the optimal collaborative initial work through the linkage DM of multiple independent DM units with heterogeneous DM structures and control objectives.\u2009 Dynamic revision planning stage. In P-D, the virtual object layer in the Digital Twins-enabled linkage DM architecture accurately reflects the production and operation environment. Then, it dynamically simulates, evaluates, and optimizes the whole process based on relevant models. At the same time, the execution deviation is obtained by comparing the actual operation state of the system with the dynamic optimization state. According to the deviation value, four online linkage decisions are triggered: internal rescheduling linkage of the unit, rescheduling linkage of the associated unit, system resource reconfiguration linkage, and customer demand readjustment linkage. These online linkage decisions maintain the performance of the P-D system in the feasible optimal state under dynamic interference.\u2009 Dynamic coordination and control stage. Feeding back the linkage DM results of dynamic revision planning to relevant units in real time realizes the real-time online adjustment of the production system. The whole process of production execution is a timely, adaptive, and feasible optimal state of system control under the dynamic action.The Digital Twin-enabled P-D linkage-oriented DM mechanism is divided into three main linkage stages: initial planning stage, dynamic revision planning stage, and dynamic coordination and control stage. The specific description is as follows:Based on the real-time perception of the dynamics of the production execution environment, the linkage control scheme is continuously revised and optimized to eliminate the adverse interference of the dynamics to the production system and realize the coordinated management and control of the whole KC of the complex production system.The methods of implementing the P-D logistics linkage mathematical model are introduced. The two-level CO is used for systematic coordination in distributed DM and global optimization to avoid the active interference of P-D linkage information.Collaborative Optimization (CO) is a two-level optimization method that can effectively solve the coupling problem of large-scale complex systems. It decomposes the complex optimization design problem into a two-level structure, a multiple parallel discipline level and a system level. It can solve the coupling problem between different disciplines by simplifying their relationship. Due to its high efficiency, rapidity, simplicity, and many other characteristics, CO has been successfully applied in many fields, such as aerospace equipment manufacturing, automobile manufacturing, mechanical equipment design, and supply chain optimization , 27.American scholars proposed GA in the early 1970s, inspired by the theory of evolution. GA has many advantages, such as simple principle, easy operation, strong universality, and robust global optimization ability. It is widely used in solving and optimizing large-scale complex combinatorial optimization problems , 29. TheAs in GA can solve practical problems by simulating biological evolutionary processes in nature. It determines the optimal solution to practical problems through repetitive genetic operations and refers to some biogenetics knowledge and terms. In biogenetics, biological evolution is realized through the continuous crossover and mutation of chromosomes in biological individuals. The chromosomes in GA have two expression forms: genotype and phenotype. Genotype refers to the various gene strings making up individual chromosomes, and phenotype is the form in vitro of organisms made up of chromosomes. Fitness (adaptability) is borrowed from the biological concept of individuals adapting to their living environment. According to Darwin's Survival of the Fittest (SOTF) theory, only biological individuals with stronger adaptability can survive. Then, they inherit and evolve through gene mutation and recombination. When GA is used, feasible solutions to practical problems are called chromosomes, and genes are elements of chromosome coding, which mainly represent the characteristics of individuals. GA cannot directly solve practical problems through genetic operations. Therefore, to use GA to solve practical problems, it is necessary to convert the problem parameters into the chromosomes in the GA. This conversion process is called coding. On the contrary, converting the GA individuals into the actual problem solutions is called decoding.Broader search scope: Traditional optimization algorithms usually calculate a single initial value when solving practical problems. Thus, it has a slow operation and cumbersome solution process. In contrast, GA solves problems in a larger domain through population search. As a result, the problem becomes easier to solve, and GA can calculate quickly with high precision.Probabilistic search over deterministic search: GA uses a certain probability to search the individual population and randomly search for the optimal solution of the problem. Such a mechanism improves optimization efficiency. Conversely, the deterministic search method easily misses individuals with high fitness, thus missing the optimal solution.Fitness-based search: When solving practical problems, GA first constructs the fitness function and determines the search range and direction accordingly to fitness. There is no need to determine the search direction through Objective Function (OF)'s derivation, so the search efficiency is greatly improved.Strong expansibility: GA can combine with other optimization algorithms to complement one another, give full play to their advantages, and improve the ability and efficiency of solving optimization problems.Compared with the traditional optimization algorithm, GA has the following characteristics:(1)Direct conversionFitness function can be directly converted from OF, thus the direct conversion method. Equation  gives thLikewise, equation  gives thThe direct conversion method is relatively simple and convenient to operate, but there are some shortcomings. For example, the direct conversion uses the roulette selection that demands a nonnegative function value. Still, the fitness cannot always be calculated as nonnegative.(2)Boundary constructionThe boundary construction is an improved direct conversion method. It designs the fitness function by adding the corresponding boundary value, divided into two cases.Equation  gives thEquation  gives thCmin\u2014OF minimum estimation; Cmax\u2014OF maximum estimation.In equation , Cmin\u2014OFIn nature, biological individuals follow the natural law of SOTF. Biological individuals with strong adaptability have a high probability of survival against a dynamic natural environment. Therefore, these individuals can inherit excellent genes and pass them down to the next generation. GA is established based on biological evolution theory. Similarly, in GA-based optimization problems, the fitness represents the quality of the problem solution. The greater the fitness is, the better the gene individual is, and the closer it is to the optimal solution. The fitness is calculated through the fitness function. Thus, fitness function design has a certain impact on GA's operation efficiency and solution accuracy. Therefore, it is necessary to design an appropriate fitness function. Generally, there are the following methods to design a fitness function:This section discusses the optimization modeling of P-D links. The optimization of the P-D linkage model is as follows. 1) A low-cost plan is formulated according to customers' requests. 2) Then, the dynamic process is modified in the P-D link, reducing the cost of the whole system. After that, the following hypotheses are put forward, as listed in Tables  A low-co Then, thAs mentioned above, the CO-based P-D logistics linkage system aims to reduce the target difference between the P-D subsystem and minimize the whole system's cost. The cost is reduced by:The OF is counted by equation  to minimk=1mFCdisk is the fixed vehicle allocation cost, \u2211k=1m\u2211a=1c\u2211b=1cyabkdabVCdisk is the variable vehicle allocation cost, \u2211p=1P\u2211i=1nVCwarQiptwar,p is the inventory cost, and [\u2211c=1cmax+\u2211c=1cmax] is the distribution liquidated damages.Equations  and 7)  7) calcuAccording to the above hypotheses, the production subsystem model is optimized, and the subsystem optimization mathematical model is implemented by:Equation  is used Then, the distribution subsystem model is optimized by:Equation  is the oDistribution vehicle capacity constraints are calculated by:Vehicle uniqueness constraints are calculated by:Constraints on vehicles returning to warehouse after delivery are calculated by:Vehicle consistency constraint:Vehicle delivery time constraints:Order delivery constraints:(1)CodingThe coding rule is: if there are three orders to be processed, each order has three processes, each process has 2, 2, and 3 parallel processing machines, and the numbers are 1, 2, 1, 2, 1, 2, and 3, respectively, a real-numbered double-layer matrix is generated:In matrix equation , the fir(2)Initializing populationi=1nQi, and the population size is between 50 and 500, forming the initial population.Determine the chromosome length as \u2211(3)Fitness functionHere, the OF of minimizing the related costs of the distribution subsystem is taken as the fitness function.(4)(a)Cross operationTwo-point crossover is used to cross chromosomes, as shown in Crossover operation is the main way to maintain population diversity, effectively solving premature convergence. The steps are as follows. Based on the set crossover probability, select a crossover mode in line with the characteristics of the problem, and exchange some loci of the paired parent individuals selected by the selection operation. The basic GA usually adopts single-point crossover or multipoint crossover. Neither of these operations can guarantee the legitimacy of the second layer machine code.(b)Mutation operationIn the first layer, two-point reciprocity is used for chromosome mutation, as illuminated in The specific steps of mutation operation are as follows: the first step is to randomly select a crossed chromosome and randomly select a mutant gene on the chromosome machine code. Then, the second step randomly generates a random number, compares the generated random number with the crossover probability, and decides whether to carry out a mutation operation. Suppose the random number is less than the mutation probability. In that case, the mutation is selected. A machine number is randomly selected from the remaining available parallel processing machine set according to the process number corresponding to the gene. Finally, the machine number chosen is replaced with the mutated gene.Genetic evolutionary operation(5)Optimization of constraint rulesThe mathematical model of the distribution subsystem is solved on the maximum iteration, and the optimization results are output.According to the research problem, the production subsystem and distribution subsystem models are simulated and solved by GA.Z\u2217 and transferring it to the distribution and production subsystem; (2) Conducting internal optimization to obtain optimal solutions Z\u2217p and Z\u2217d; (3) Getting optimal solution Z\u2032 of the system according to the optimal solution; and (4) Judging whether optimal solution Z\u2032 conforms to penalty factor \u03b5. If not, the iteration continues until the system consistency constraint is met.As in This section employs the proposed P-D logistics linkage optimization model to a large domestic coating ENT H. Then, a case study is conducted using the Matlab simulation platform and comparative analysis. ENT H is a well-known coating manufacturer in China. Since its establishment, it has been deeply engaged in the Research and Development (R&D), production, and sales of coating products for a long time. Due to the large variety of coating products and its seasonal market demand, the demand for different coatings will fluctuate greatly. Then, to avoid the operation risk from market fluctuation, H ENT adopts the Make-To-Order (MTO) production mode. MTO can reduce the inventory of products while meeting customer needs.Due to the gaps in coating products' physical and chemical properties, coating products have different requirements for production equipment and the environment. According to the characteristics of coating products, coating ENTs choose to arrange various kinds of products in other production workshops. At present, ENT H has three production workshops capable of producing three types of coating products: exterior wall paint, interior wall paint, and wood paint. After accepting the customers' orders, the customer service department will split the orders per product type and release the production requirements of different products to the corresponding production workshops. Then, the FPs will be transported by the forklift to the FP warehouse for temporary storage until all products in the same customer's order are ready. The third-party logistics company will arrange the distribution vehicles to distribute the FPs to the customers.ENT H adopts the MTO production mode. Such a model can respond to customer needs to a certain extent. Yet, as people's life pursuit sublimes and vision broadens, more people choose personalized coating products over traditional mass production products. Generally, personalized demand features small-batch, multiple varieties, high timeliness requirements, and high randomness. Thus, it makes the production and operation of ENT H vulnerable to dynamic interference from orders, resources, and quality. This condition challenges ENT H's operations and unveils its low production efficiency, distribution, operation plan, and high production and operation cost. So far, almost all ENTs face dynamic customer demands during production execution, which dramatically affect the efficient implementation of production operation of ENT H. The dynamic customer demands faced by ENT H mainly include the following three situations:(1) Temporary order increase. Simply put, customers add a new order based on the original demand order. (2) The demand quantity of the original order increases. That is, the order that has been planned or is being produced dynamically increases in quantity. (3) The demand quantity of the original order decreases. In other words, the order quantity reduces for the products being planned or produced. The first case has the most significant impact on the P-D operation in the MTO mode, and the probability of occurrence is greater than the latter two. Therefore, this paper studies the P-D linkage operation of ENT H under the interference of dynamic new demands to prove the effectiveness of the proposed linkage DC method. Basic information of customers is plotted in The basic information of distribution vehicles is manifested in The optimization results of vehicle scheduling initialization are charted in As in As in As in P-D's dynamic modification and optimization results corroborate that the random emergence of dynamic demand changes the scheduling plan made in advance by the two subsystems of P-D. However, due to the timely and effective online collaborative operation and independent linkage DM of P-D, each DM unit dynamically adjusts its operation state according to the dynamic interference. The adverse effects caused by dynamic interference are effectively eliminated. The results are detailed in As in Based on the initial planning, the present work analyzes the impact of linkage DM and nonlinkage DM on P-D operation with different interference degrees. The effectiveness of DM linkage is verified in dealing with active interference. As a result, changes of five indexes are summarized, including production cost, storage cost, penalty cost, distribution cost, and total system cost. The effects of linkage and nonlinkage DM with different periods and requests are compared and analyzed. Sensitivity analysis is conducted under other requests. From Management enlightenment: When the quantity of new demand changes, the cost of linkage operation is lower than that of nonlinkage operation. When the number of new demands is small, the cost-effectiveness of linkage operation over nonlinkage operation is insignificant. At this time, the production ENTs can selectively adopt linkage operation according to their conditions. As the number of new demands transcends a threshold, the cost-effectiveness of linkage operation is significantly better than that of the nonlinkage operation. Then, production ENTs should actively adopt linkage operations to respond to dynamic demands.Furthermore, As in Management enlightenment: when dynamic orders appear earlier, the cost of linkage operation is lower than that of nonlinkage operation due to the ample optimization space of the production system. Therefore, for an early initiation order, production ENTs should actively carry out linkage operations as soon as possible. With the postponement of the start-up time of new orders, the space limitation of linkage optimization will also be enhanced. The linkage operation often increases the resource investment, with an insignificant optimization effect, resulting in the overall rise of the total system cost. Therefore, the production ENTs should adopt the nonlinkage method to address delayed dynamic demands.Based on DT, the dynamic virtual simulation of P-D logistics linkage is carried out, and the framework and mechanism of P-D DM logistics linkage are constructed.The case study shows that the production cost, storage cost, distribution cost, penalty cost, and total system cost of the optimized linkage are 24,943\u2009RMB, 3,393\u2009RMB, 2,167\u2009RMB, 0\u2009RMB, and 30,503\u2009RMB, respectively. The total cost is 3.7% lower than that of a nonlinkage system. Sensitivity analysis shows that ENT managers should adjust the linkage operation state to reduce the manufacturing cost according to different requests.The problems of P-D DM logistics linkage in the dynamic production environment are analyzed through global optimization to minimize the impact of active interference in the P-D links. Consequently, a set of solutions is proposed, including the architecture and mechanism of P-D DM logistics linkage. The contributions and conclusions are as follows:"}
+{"text": "Bacillus cereus sensu lato species complex, also known as the B. cereus group, vary in their ability to cause illness but are frequently isolated from foods, including meat products; however, food safety surveillance efforts that use whole-genome sequencing (WGS) often neglect these potential pathogens. Here, we evaluate the surveillance and source tracking potential of WGS as applied to B. cereus sensu lato by (i) using WGS to characterize B. cereus sensu lato strains isolated during routine surveillance of meat products across South Africa (n\u2009=\u200925) and (ii) comparing the genomes sequenced here to all publicly available, high-quality B. cereus sensu lato genomes . Strains sequenced here were collected from meat products obtained from (i) retail outlets, processing plants, and butcheries across six South African provinces (n\u2009=\u200923) and (ii) imports held at port of entry (n\u2009=\u20092). The 25 strains sequenced here were partitioned into 15 lineages via in silico seven-gene multilocus sequence typing (MLST). While none of the South African B. cereus sensu lato strains sequenced here were identical to publicly available genomes, six MLST lineages contained multiple strains sequenced in this study, which were identical or nearly identical at the whole-genome scale (\u22643 core single nucleotide polymorphisms). Five MLST lineages contained (nearly) identical genomes collected from two or three South African provinces; one MLST lineage contained nearly identical genomes from two countries (South Africa and the Netherlands), indicating that B. cereus sensu lato can spread intra- and internationally via foodstuffs.Members of the IMPORTANCE Nationwide foodborne pathogen surveillance programs that use high-resolution genomic methods have been shown to provide vast public health and economic benefits. However, Bacilluscereus sensu lato is often overlooked during large-scale routine WGS efforts. Thus, to our knowledge, no studies to date have evaluated the potential utility of WGS for B. cereus sensu lato surveillance and source tracking in foodstuffs. In this preliminary proof-of-concept study, we applied WGS to B. cereus sensu lato strains collected via South Africa\u2019s national surveillance program of domestic and imported meat products, and we provide strong evidence that B. cereus sensu lato can be disseminated intra- and internationally via the agro-food supply chain. Our results showcase that WGS has the potential to be used for source tracking of B. cereus sensu lato in foods, although future WGS and metadata collection efforts are needed to ensure that B. cereus sensu lato surveillance initiatives are on par with those of other foodborne pathogens. Bacillus cereus sensu lato, also known as the B. cereus group, is a complex of closely related, Gram-positive, spore-forming species, which are widespread throughout the environment   \u20136, otheranisms) \u2013, 7\u20139. Il illness , 10, 11, illness \u201315, foodillness \u2013, 15, andillness \u2013, 17. As ach year , althougB. cereus sensu lato strains in a wide variety of foodstuffs (\u2013B. cereus sensu lato in (i) retail meats sold at supermarkets in the Pretoria area   . Most reectively .B. cereus sensu lato and may pose a potential food safety risk to South African consumers. However, it is unclear which B. cereus sensu lato lineages are present in South African meat and poultry products on a genomic scale. Here, we used whole-genome sequencing (WGS) to characterize 25 B. cereus sensu lato strains isolated from raw intact, processed, and RTE meat and poultry products collected from processing plants, butcheries, and retail outlets across South Africa as well as imported meat products tested for B. cereus sensu lato at port of entry. By comparing South African strains sequenced here to all publicly available B. cereus sensu lato genomes , we identified multiple B. cereus sensu lato species present among South African meat and poultry products, and we detected multiple potential international and interprovincial B. cereus sensu lato dissemination events. Overall, our proof-of-concept study serves as the first genome-scale study of South African B. cereus sensu lato in foodstuffs and showcases the utility of WGS for B. cereus sensu lato surveillance and source tracking.Ongoing surveillance efforts in South Africa have indicated that meat and poultry products can harbor B. cereus sensu lato strains that had been isolated from meat and poultry products collected across South Africa in 2015 and 2016  to assign B. cereus sensu lato strains to one of seven or more major phylogenetic groups, which have been proposed to conceptually serve as \u201cspecies\u201d  of the strains sequenced here were assigned to panC group IV  were each assigned to separate GTDB genomospecies (Using the Genome Taxonomy Database (GTDB) taxonomy, the 25 ospecies . The 18 ospecies . The panospecies .B. cereus sensu lato in 2020 (panC group IV strains were assigned to the B. cereus sensu stricto genomospecies (B. cereus sensu stricto genomes (using BtToxin_scanner2\u2019s \u201cold\u201d gene detection approach), meaning that these 18 strains were predicted to belong to the Thuringiensis biovar  were assigned to genomospecies B. mosaicus within the 2020 GSB framework  was assigned to PubMLST sequence type 26 (ST26) and possessed cereulide (emetic toxin) synthetase-encoding cesABCD and was thus assigned to the cereus subspecies and biovar Emeticus , and thus this strain was predicted to belong to biovar Thuringiensis  nomenclatural framework proposed for  in 2020   were each assigned to separate pseudo-GFUs, respectively -based pseudo-gene flow unit (GFU) assignment scheme, which attempts to assign ene flow , the 25 in vitro or in vivo. No anthrax toxin- or capsule-encoding genes were identified within the genomes of the isolates sequenced here  encompassed 11 STs, with eight strains (32.0%) assigned to unknown STs , contained two isolates sequenced in this study , which were assigned to GTDB\u2019s \u201cB. thuringiensis_S\u201d genomospecies ; however, this strain was not closely related to the three nearly identical South African ST2668 strains sequenced in this study  was assigned to GTDB\u2019s B. paranthracis genomospecies and possessed cereulide synthetase-encoding genes   , 40, 41. or not) .panC group III B. cereus sensu lato strains sequenced in this study (S59 and S62) were assigned to ST2413 within GTDB\u2019s B. paranthracis species  and was assigned to ST794 within GTDB\u2019s B. wiedmannii species .One  species . Strain  species . ComparepanC group V B. cereus sensu lato strain was sequenced in this study (S72) and was assigned to ST223 within GTDB\u2019s B. toyonensis species (GCF_002552615.1), which had been isolated in 2014 from a tree leaf in North Carolina, United States (NCBI BioSample accession SAMN07598612) (One  species  to 3. S7 species . Strain 7598612) ; the two7598612) .The movement of commodities  through inter- and intranational trade can contribute to the global, regional, and local dissemination of microorganisms, including pathogens \u201348. The \u2013B. cereus sensu lato lineages, which showcased evidence of interregional dissemination   ; howeverB. cereus sensu lato lineages, which showed evidence of interprovincial spread within South Africa: four panC group IV lineages and one panC group III lineage were each composed of (nearly) identical strains, which were isolated from meat products in two or more South African provinces  may preferentially select for or against some B. cereus sensu lato lineages  have long been known to be inconsistent with genome evolution  , consumer products , and soil, indicating that these organisms are not uncommon in environmental and industrial settings , the  Manual) , 54, and anthrax . These nsettings . Thus, a produce , 34. Her disease . These sl assays , 54; how methods . While i storage , the iso storage . Thus, rB. cereus sensu lato strains from beef and poultry products, which were assigned to the \u201cB. paranthracis\u201d genomospecies via GTDB and similar ANI-based methods , which can use genomic, genetic, and/or phenotypic information for taxonomic classification  ; biovar terms can thus be appended to B. cereus sensu lato lineage names . Overall, standardized taxonomic frameworks that can incorporate both genomic/genetic and phenotypic information may improve strain-level risk evaluation of B. cereus sensu lato.Recently, we proposed a standardized nomenclatural framework for fication , 34. Impctively) , 34. Wit strains . While tospecies  and 2 . This can be contrasted with other foodborne pathogens for which source tracking and surveillance have proven to be successful . Thus, future B. cereus sensu lato surveillance and WGS initiatives in clinical, industrial, and environmental settings are needed to improve B. cereus sensu lato source tracking and traceback efforts. Furthermore, it is essential that data and metadata obtained in such future initiatives are made publicly available, as international sharing of WGS data can decrease both the amount of time required to solve foodborne outbreaks and the public health burden caused by foodborne pathogens  genomic data and (ii) corresponding metadata associated with ytogenes , 59. Whicreasing , effortsdatabase , 61 duriccessful , 59, as athogens .B. cereus sensu lato WGS data and metadata, a final limitation of our study is that there are very few existing studies that have used WGS to characterize B. cereus sensu lato isolates known to come from a single source . Consequently, metrics that are used to determine whether two B. cereus sensu lato genomes are \u201cidentical\u201d or derived from a common source  (B. anthracis)  , 64 are thracis) , 38, 65.nsu lato , as moreB. cereus sensu lato surveillance and source tracking, even among closely related lineages, and future studies will benefit from increasingly available publicly available WGS data and metadata.Overall, the proof-of-concept study detailed here highlights the benefits of WGS for B. cereus sensu lato isolates from our previous study  was used to assess the quality of the resulting trimmed paired-end reads.Paired-end reads associated with each of the 34 isolates were supplied as input to Trimmomatic v0.38 , which wN50, number of contigs). Genomes with (i)\u2009<95% completeness (via CheckM), (ii)\u2009>5% contamination (via CheckM), and/or (iii) an N50\u2009of <20 kbp were considered to be of low quality and were excluded (n\u2009=\u20094), yielding a preliminary set of 30 genomes used in subsequent analyses.SKESA v2.4.0  was usedB. cereus sensu lato (in silico seven-gene MLST using the PubMLST B. cereus database (accessed 25 October 2020), and (vi) panC phylogenetic group assignment using an adjusted eight-group (groups I to VIII) framework  ANnsu lato   and were thus excluded, yielding a final set of 25 B. cereus sensu lato genomes used in subsequent analyses  and GTDB-Tk v. 1.3.0  using GTDB-Tk\u2019s \u201cclassify_wf\u201d workflow \u201377. Nota\u2013B. cereus sensu lato genomes sequenced in this study. Protein-coding sequences derived from the type strain genomes of each of the 23 validly published and effective B. cereus sensu lato species (accessed 28 August 2021) were downloaded from NCBI\u2019s RefSeq Assembly database  (B. cereus sensu lato genomes sequenced in this study plus 23 B. cereus sensu lato species type strain genomes) using MAFFT v7.475 (Prokka v1.14.6  was usednumbers) . OrthoFinumbers) , 81 was T v7.475 , 83 for T v7.475  for phylB. manliponensis\u201d    and annosu lato)  in R v4.su lato) .B. cereus sensu lato species  . QUAST and CheckM were used to assess the quality of each assembled genome , and BTyper3 (using default settings) and GTDB-Tk were used for typing and/or taxonomic assignment as described above (see In silico typing and taxonomic characterization). The rentrez package (v1.2.3) was used to download metadata associated with each genome\u2019s BioSample in R v3.6.1 : (i)\u2009>95% completeness (via CheckM), (ii)\u2009<5% contamination (via CheckM), (iii) N50\u2009of >20 kbp (via QUAST), and (iv) composed of\u2009<1,000 contigs (via QUAST).All assembled genomes submitted to the National Center for Biotechnology Information RefSeq database , 61 as odmannii) , 93\u2013105 R v3.6.1 , 107. PuB. thuringiensis in France (In silico typing and taxonomic characterization). Four genomes did not meet the quality thresholds used in this study (see Acquisition of publicly available B. cereus sensu lato genomes) and were thus excluded, yielding 167 genomes from the study that were used in subsequent analyses (Table S3).All sequencing reads associated with isolates from a previous study of outbreaks caused by n France  were down France , 110. GeB. cereus sensu lato strains isolated in conjunction with a 2016 emetic outbreak in New York State (United States) were downloaded, preprocessed, and assembled as described previously (In silico typing and taxonomic characterization). Two genomes did not meet the quality thresholds used in this study (see Acquisition of publicly available B. cereus sensu lato genomes) and were thus excluded, yielding 31 genomes from the study that were used in subsequent analyses (Table S4).The genomes of 33 eviously . The quaB. cereus sensu lato strains sequenced here spanned four major phylogenetic groups based on their panC sequence  B. mosaicus, and (iii) B. toyonensis genomospecies within the 2020 GSB taxonomic framework  pa B. luti , and  files associated with each genome were supplied as input to Panaroo v1.2.8 , which wpanC group IV phylogeny, panC group III B. anthracis strain Ames Ancestor was used as an outgroup (NCBI RefSeq accession number GCF_000008445.1). For the panC groups II/III and panC group V phylogenies, panC group IV B. cereus strain ATCC 14579 was used as an outgroup (NCBI RefSeq accession number GCF_006094295.1). Additionally, only genomes with detailed metadata  were included in this analysis (Tables S1 to S4). The resulting phylogenies were annotated using the bactaxR package in R.For each of the three major lineages, all aforementioned steps were repeated, with the addition of an outgroup genome. For the B. cereus sensu lato genomes sequenced in this study , and all genomes assigned to the panC group of the query genome (panC groups II and III were aggregated); genomes were then grouped into lineages based on STs assigned using seven-gene MLST (see In silico typing and taxonomic characterization). For each of the resulting MLST lineages, FastANI was used to calculate pairwise ANI values between all genomes within the MLST lineage  was used to identify core SNPs among all genomes assigned to the respective MLST lineage using (i) a genome sequenced in this study as a reference genome  pae genome , 108 ande genome  was usede genome  using the genome , 116 in B. cereus sensu lato isolates sequenced in this study have been deposited in NCBI\u2019s SRA database under BioProject accession number PRJNA798224. Metadata and quality information for all genomes queried in this study are available in Table S1 (the 25 isolates sequenced in this study) and Tables S2 to S4 .Paired-end Illumina reads associated with the 25"}
+{"text": "The prepared SPLTOPD can effectively barrier the pass\u2010through of polysulfides, catalyze the reactions of polysulfides into S2\u2212, and increase the ionic conductivity of the Li\u2010S batteries. The SPLTOPD can also prevent the aggregation of insulating sulfur species on the surface of the cathode. The assembled Li\u2010S batteries with the SPLTOPD can cycle 870 cycles at 5 C with the capacity attenuation of 0.066% per cycle. When the sulfur loading is up to 7.6 mg cm\u22122, the specific discharge capacity at 0.2 C can reach 839 mAh g\u22121, and the surface of lithium anode after 100 cycles does not show the existence lithium dendrites or a corrosion layer. This work provides an effective way for the preparation of commercial separators for Li\u2010S batteries.The market demand for energy pushes researchers to pay a lot of attention to Li\u2010S batteries. However, the \u2018shuttle effect\u2019, the corrosion of lithium anodes, and the formation of lithium dendrites make the poor cycling performances  of Li\u2010S batteries, which limit their commercial applications. Here, a separator is prepared and modified with Super P and LTO (abbreviation SPLTOPD) through a simple coating method. The LTO can improve the transport ability of Li A multifunctional separator is prepared by coating the Super P and LTO onto DKJ\u201014 separator for the Li\u2010S battery. The lithium anodes are uncorroded and dendritic free, the \u2018shuttle effect\u2019 is greatly depressed, and the charge transfer resistance is reduced, leading to high\u2010performance Li\u2010S batteries. Li\u2010S batteries have attracted extensive attention due to the high theoretical capacity of S (1675 mAh g\u22121) and energy density (2600 Wh kg\u22121). In addition, the advantages of sulfur, which has low cost, is easily available, and environmentally friendly, have also attracted wide attentions. However, The \u2018shuttle effect\u2019 and lithium anode corrosion have always restricted the development of Li\u2010S batteries. During the charging and discharging process, the soluble lithium polysulfides  are formed and dissolved in the electrolyte, resulting in the loss of active sulfur and corrosion of lithium anodes, which leads to the sharp capacity decline and poor safety performance.With the development of electric vehicles, the market has higher requirements for energy storage devices.1, 2 Besides, inorganic compounds are often used to tailor the cathodes because of their excellent adsorption or catalysis on polysulfides,  which improves the utilization of sulfur and accelerates the redox reaction kinetics. For the anodes, many measures are taken to prevent the corrosion of lithium anodes and the growth of lithium dendrites, among which the method of constructing artificial protective layers on lithium anodes is effective. These mentioned strategies have greatly improved the electrochemical performance of Li\u2010S batteries, but the tailored preparation processes for positive and negative electrode structures often lead to an increase in time and cost, which restricts the further development of Li\u2010S batteries.Many efforts have been made to solve the above problems in Li\u2010S batteries. As for the cathodes, various materials and structures are designed to anchor the active sulfur. Different carbon materials  are used as the hosting framework for sulfur cathodes.17 Bes The idea of separator modification is to add functional modification layers on the separators to prevent the active sulfur from moving to the anode side. The modified material should have the function of accelerating redox reactions, forming a stable modification layer on the separators, and improving the ionic transport ability. In recent years, there are quite some works related to the modification on separators, and outstanding progresses have undoubtedly been achieved. The simple, fast, and low\u2010cost coating method for preparing modified separators can greatly improve the electrochemical performance of Li\u2010S batteries. However, the prepared Li\u2010S batteries are difficult to have good cycling performance under high current density and high sulfur loading, which are not conducive to the commercialization of Li\u2010S batteries. For the separators that can promote the commercialization of Li\u2010S batteries, it is supposed that they should have the advantages of cheap modified materials, simple preparation, and short preparation time. They can also simultaneously suppress the \u2018shuttle effect\u2019 and protect the anodes. In addition, the prepared Li\u2010S batteries have excellent cycling performance under high sulfur loading and high current density.As an important part of the batteries, the separators play an important role in preventing short circuits and maintaining lithium ions. In recent years, it was found that simple modification of the separators can greatly improve the electrochemical performance of the batteries.25 The4Ti5O12 (LTO) and a conductive carbon (Super P) (SPLTOPD) for Li\u2010S batteries, the Super P modified separators (SPD), and the LTO modified separators (LTOPD) were prepared for comparison, too. LTO has the advantages of low cost and good stability as commercial anode material for lithium ion batteries. Furthermore, it can improve the Li+ transport ability. It can also form a stable additional layer on the separators to barrier the transport of polysulfides, which improves the utilization of active sulfur and accelerates the redox kinetics of S electrode.Here, we prepared a separator modified with Liatteries.35 Fur4Ti5O12/graphene coated separator for Li\u2010S batteries. The above work demonstrates the feasibility of using LTO nanospheres as modified materials for separators of Li\u2010S batteries, but the preparation of special structure LTO will increase costs. In our work, we found that commercial LTO together with PVDF in combination with Super P conductive carbon could be used as the modification material for separator of Li\u2010S batteries. It is found the Super P modified layers on the separators can improve the utilization of insulating active substances, buffer the shuttle of polysulfides, and provide a reaction site for polysulfides. Super P as an additional layer on the LTO modified layer can further improve the barrier of the separators to polysulfides and reduce the charge transfer resistance of the batteries. The prepared Li\u2010S batteries show excellent cycling performance under high current density and high sulfur loading.In 2016, Yang et\u00a0al. prepared a Liatteries.36 The24Ti5O12, JCPDS No. 49\u20130207). The LTO powders were pressed into a pellet and assembled in stainless steel//stainless steel (SS//SS) cells for the EIS test, showing an ionic conductivity of 2.53 mS cm\u22121 at 25 \u00b0C  with the same quality. The Li2S6 solution with Super P+LTO\u2010PVDF becomes clear and transparent after 12 h ,  the ionic conductivity of the LTOPD is significantly improved at 25 \u00b0C, indicating that the LTO can promote the migration of Li+. Both Super P and LTO can promote the movement of ions. However, there is a limit for both of them. As a result, from the SPD to SPLTOPD, the increase of ionic conductivity is not much. From the test results of contact angle , and the temperature range of the ionic conductivity test was 25\u201385 \u00b0C. There were short straight lines in the EIS plots  were measured by applying a constant voltage (10 mV) to the Li//Li symmetrical cells with different separators. Based on the calculation of i\u2010t curves and Nyquist plots  was tested for comparison. The Li+ transference number of the separator through LTO modified is significantly increased, indicating that the LTO crystal is favorable for the mobility of Li+ cations.The Li\u22121 , respectively. The anodic peak \u22482.4 V correspond to the oxidation of sulfides. Comparison presents that the peak area of CV curve with the SPLTOPD is the largest, indicating that the SPLTOPD can promote redox kinetics, and improve the utilization of active sulphur. The distance between oxidation peak and reduction one is the narrowest, indicating that the SPLTOPD can reduce polarization in Li\u2010S batteries. From the CV curves of cells with different separators for the first three cycles at 0.1 mV s\u22121  were prepared with different separators and tested by cyclic voltammetry at 1.8\u20132.8 V. From the CV curves of the first cycle of cells with different separators at 0.1 mV s1 Figure\u00a0, it can sulfides.44 Com1 Figure , the calFigure\u22121 at current densities of 0.1, 0.2, 0.5, 1, 2, 3, and 5 C, respectively. In addition, from the charge and discharge curves under different current densities  of cells with the SPLTOPD is 7.7 \u03a9 after 100 cycles, lower than those in the SPD (12.1 \u03a9) and the LTOPD (18.1 \u03a9) (Table  The charge transfer resistances (RCT) of cells with the LTOPD, SPD, and SPLTOPD before cycling are 70.2, 19.3, and 19.1 \u03a9, respectively, and the RCT after 100 cycles are 50.4, 4.1, and 3.2 \u03a9, respectively. The RCT values of the cell with the SPLTOPD are the lowest, indicating that the SPLTOPD can improve the utilization of insulating sulfur species in Li\u2010S batteries. Besides, the RCT values decreased significantly after coating of Super P, indicating that Super P can effectively reduce the charge transfer resistance of Li\u2010S batteries. The above results show that the SPLTOPD can effectively block the polysulfides and enhance the reaction kinetics of Li\u2010S batteries.EIS tests were carried out before and after 100 cycles with different separators  during the cycling. From the test results at the high current density, it can be seen that Li\u2010S batteries with the SPLTOPD show outstanding cycling stability.The long\u2010term cycling stability of Li\u2010S batteries at the high current density is also explored. Herein, the cell with the SPLTOPD was tested for ultra\u2010long cyclability at 5 C Figure\u00a0. After a density.48 Aft\u22122, the maximum specific capacity of the cell with the SPLTOPD after activation could reach 1200 mAh g\u22121 at 0.2 C, and could retain 676 mAh g\u22121 after 210 cycles (coulomb efficiencies > 99%). When the sulfur loading increases to 5.8 mg cm\u22122, the maximum specific capacity after activation could reach 1016 mAh g\u22121 at 0.2 C, and could retain 460 mAh g\u22121 after 210 cycles  was tested for comparison  because of its good electronic conductivity, which accelerate the redox reaction in the Li\u2010S battery. Meanwhile, the SPLTOPD can prevent the aggregation of insulating sulfur species on the surface of the cathode and improve the utilization of active substances in the cathode. As a result, the Li\u2010S battery with the SPLTOPD exhibits good electrochemical performance under high sulfur loading and high current density, and the lithium anode has no lithium dendrites or a corrosion layer after 100 cycles at 1 C.The actions of the SPLTOPD and DKJ\u201014 in the Li\u2010S battery are schematically shown in 34Ti5O12) and Super P for Li\u2010S batteries through a simple doctor coating method. LTO can improve the Li+ cation transport ability of the separators. Super P can reduce the charge transfer resistance of the Li\u2010S batteries and increase the kinetics of S/S2\u2212 couple. The modified layer can effectively barrier the pass\u2010through of polysulfides, catalyze the reactions of polysulfides into S2\u2212, and prevent the aggregation of insulating sulfur species on the surface of the cathode. As a result, the prepared Li\u2010S batteries can cycle 870 cycles at 5 C with small capacity attenuation of 0.066% per cycle. When the sulfur loading is 4.0 mg cm\u22122, the specific discharge capacity after activation at 0.2 C can reach 1200 mAh g\u22121, and when the sulfur loading is high up to 7.6 mg cm\u22122, it can still reach 839 mAh g\u22121. In addition, there are no lithium dendrites or a corrosion layer on the lithium anode of the cell with the SPLTOPD after 100 cycles at 1 C. It is no doubt that this work can greatly promote the commercialization of the functionally modified separators of Li\u2010S batteries.In summary, we prepared a separator modified with commercial LTO  was dissolved in DMF, stirred for 6 h, and uniformly coated on the commercial separators (DKJ\u201014). After drying overnight under vacuum at 60 \u00b0C LTO modified separators (LTOPD) were obtained. After PVDF was added to a certain amount of NMP and stirred until completely dissolved, then a certain amount of super P was added. After stirring for 6 h, it was coated on the LTOPD to obtain SPLTOPD. The loading amount of LTO and Super P on the SPLTOPD was about 2.64 mg cm\u22122. Super P modified separators (SPD) without LTO were made in the same way for comparison. All prepared separators were cut into 19 mm discs before use.LTO  and super P modified separators (SPLTOPD) were prepared by a simple doctor blading coating. First, LTO and PVDF with a mass ratio of 4:1 were put into a ball milling tank and milled overnight at 400 r\u00a0min Briefly, a certain amount of CNTs was acidified in dilute nitric acid, washed and filtered until it was neutral, and dried in an 80 \u00b0C oven overnight. Sulfur and acidified CNTs with a mass ratio of 7:3 was put into the ethanol, stirred until the sulfur was completely dissolved. Then, deionized water was added until the sulfur was completely dissipated. The dispersion was washed, filtered, and vacuum dried overnight at 60 \u00b0C to obtain S composites. S composites, carbon black, and PVDF (mass ratio: 8:1:1) were put into NMP and stirred for 6 h until a uniform slurry was formed. The slurry was coated on aluminum foil, vacuum dried at 60 \u00b0C overnight, and cut into small discs to prepare S cathodes. The sulfur loading is \u22481.0 mg cm\u22122.The preparation method of normal S cathodes was the same as we mentioned earlier.29 Bri\u22122) cathodes were prepared according to the previous report. Typically, CMC (80 mg) was added to deionized water (2.5 mL) and stirred until completely dissolved. Then, Super P (80 mg) was added, stirred until a uniform suspension was formed, and then the above S composite (560 mg) was added to obtain a uniform slurry. The high sulfur\u2010loaded cathodes were obtained by coating (carbon\u2010coated Al foil), vacuum drying, and cutting.High sulfur\u2010loaded .The morphology of the samples was observed by a scanning electron microscope . The crystalline phase of the materials was tested by X\u2010ray diffraction analysis . A contact angle meter (Kino) was used to record contact angles. The Li2<0.1 ppm, H2O<0.1 ppm), the cathodes, separators, electrolyte and anodes were successively encapsulated in the coin shells under the pressure of 500 kg cm\u22122 to prepare the CR2025 type coin cells. The amount of electrolyte in all cells is the same. The cycling and rate performance of the batteries were tested by the battery test system . Cyclic voltammetry  and electrochemical impedance spectra  were tested by electrochemical workstation .In a glove box filled with argon , the ionic conductivity, and activation energy were calculated based on the EIS data of the battery at different temperatures.2S6 symmetric cells were obtained by loading LTO and Super P on the aluminum foil. The electrolyte is 0.2 m Li2S6 solution in the electrolyte (1 m LiTFSI in DOL/DME = 1/1 by volume). The symmetric cells were assembled with the above electrodes as working and counter electrodes with 50 \u00b5L of Li2S6 solution. CV tests were carried upon a voltage window from \u22120.8 to 0.8 V.The electrodes of Li2S6 (7 mL) solution and DME were separately placed on both sides of the H\u2010type battery, and different separators were placed in the middle.LiThe authors declare no conflict of interest.Supporting InformationClick here for additional data file."}
+{"text": "BackgroundAQP1 gene in the tunica vaginalis of patients with adult-onset non-communicating hydrocele testis to elucidate the cause of enhanced AQP1 protein expression.Water channel aquaporin 1 (AQP1) protein expression is enhanced in the tunica vaginalis of patients with adult-onset non-communicating hydrocele testis and may contribute to the development of non-communicating hydrocele testis. We performed genetic and epigenetic analyses of the MethodologyAQP1 gene and SNPs in the 5\u2019-upstream region of the AQP1 gene. Then, by performing association analysis, the applicability of various genetic models was investigated for each SNP. Moreover, the methylation rate of CpG sites was examined for the CpG island related to the AQP1 gene.The genotype was determined for Tag single-nucleotide polymorphisms (SNPs) representing the ResultsThere was no significant association between each SNP and hydrocele testis for any of the genetic models. The average methylation rate of the 17 CpG sites evaluated was not significantly different between controls and hydrocele testis, but the methylation rate was lower in hydrocele testis than in controls at one CpG site.ConclusionsAQP1 gene in the tunica vaginalis of patients with non-communicating hydrocele testis. This may increase AQP1 protein expression and contribute to the formation of hydrocele testis. SNPs related to the AQP1 gene were not associated with hydrocele testis.There was a significant decrease in the methylation rate at one of the CpG sites in the CpG island associated with the  Hydrocele testis involves the accumulation of clear fluid between the tunica vaginalis and testis. Adult-onset, primary, non-communicating hydrocele testis causes progressive swelling and local discomfort on the scrotum, which has been attributed to the enhanced influx and/or impaired absorption of fluid in the space between the tunica vaginalis and testis.Aquaporins (AQPs) are a family of water channels that are conserved from lower organisms to mammals. Thirteen mammalian AQPs AQP0-AQP12) are known to be widely expressed in various epithelia and endothelia in many organs. AQPs form tetramers in membranes, each monomer of which contains six transmembrane \u03b1-helical domains, with the amino and carboxyl termini being located on the cytoplasmic surface of the membrane 2 are kno. Their pWe previously reported that AQP1 protein expression is enhanced in the tunica vaginalis of patients with adult-onset non-communicating hydrocele testis compared with controls  and may AQP1 gene in the tunica vaginalis of patients with adult-onset non-communicating hydrocele testis to elucidate the cause of enhanced AQP1 protein expression.However, the reason why AQP1 protein expression in the tunica vaginalis is enhanced in patients with non-communicating hydrocele testis has remained unclear. Thus, we performed genetic and epigenetic analyses of the AQP1 gene and SNPs in the 5\u2019-upstream region of the AQP1 gene. Then, by performing association analysis, the applicability of various genetic models was investigated for each SNP. Moreover, the methylation rate of 17 CpG sites was examined for the CpG island related to the AQP1 gene.Specifically, the genotype was determined for Tag single-nucleotide polymorphisms (SNPs) representing the An SNP is a variation of a single nucleotide in the genome sequence of an organism compared with the population, where the variation is found at a frequency of 1% or more within the population. The human genome contains approximately 3 billion base pairs, and SNPs occur in approximately one in every 1,000 base pairs. SNPs can be used as DNA markers to examine the genetic background, and SNP-based linkage and association analysis can help identify disease susceptibility genes. If two SNPs are in strong linkage disequilibrium, information about one SNP can provide information about the other. Thus, a representative SNP chosen to characterize a region of the genome is called a Tag SNP. Tag SNPs save cost and effort by eliminating the need to examine all SNPs in a region.A CpG island is a region of the genome where CpG dinucleotides occur more frequently than in other regions of the genome. By definition, a CpG island is at least 200 base pairs long, has a GC content of at least 50%, and has an observed-to-expected CpG ratio >0.6 . The CpGCpG island shores are 2,000 base pair-long regions located on both sides of a CpG island, and gene expression levels are found to be negatively associated with methylation levels at CpG island shores . Thus, tPatients and samplesIn this case-control study, 119 male patients (66 with hydrocele testis and 53 controls) were enrolled. Among them, 105 were from Kanto Rosai Hospital and 14 were from Toshiba Hospital. Their ages were 65.1 \u00b1 14.5 and 66.1 \u00b1 16.2  for hydrocele testis patients and controls, respectively. If yellowish hydrocele fluid was identified at the time of surgery, the diagnosis of hydrocele testis was determined regardless of its volume. The volume of hydrocele fluid measured was less than 10 mL in 20 cases, 10-100 mL in 15 cases, and more than 100 mL in 31 cases.Genomic DNA was extracted from frozen (n = 86) and paraffin-embedded (n = 21) tunica vaginalis samples excised at surgery of hydrocele testis, other intrascrotal lesions, and bilateral orchiectomy for prostate cancer using a DNA Extractor\u00ae\u00a0TIS Kit  and TaKaRa Dexpat\u2122 Easy Kit . In addition, genomes were extracted from whole blood samples of 12 male patients with hydrocele testis using a DNA Extractor\u00ae\u00a0WB-Rapid Kit .Selection of Tag SNPs2\u00a0\u2265\u00a00.8 and MAF \u2265\u00a00.1, six Tag SNP sites for the human\u00a0AQP1\u00a0gene located on chromosome 7 were determined. These Tag SNP sites are listed in Table Using the HapMap site  with a cAnalysis of Tag SNPs by the Invader\u00ae assayFor 88 genomic DNAs , the genotypes of the Tag SNPs were determined by the Invader\u00ae assay ,11 usingAQP1\u00a0geneAnalysis of the 5\u2019-upstream region of the\u00a0AQP1 gene was amplified using a thermal cycler  and volumes of 50 \u03bcL with 0.3 \u03bcM of sense/antisense primers (5\u2019-tcccagagactggaatgctgagcca-3\u2019/5\u2019-gcttcttcttgaactcgctggcca-3\u2019), 1 unit of KOD Plus Ver.2 polymerase , 4% dimethyl sulfoxide, and a buffer supplied with the enzyme as follows: 94\u00b0C for two minutes, 36 cycles  of two-step polymerase chain reaction (PCR). Amplified PCR products (10 \u03bcL) were resolved by electrophoresis in 1% agarose gel. PCR products were directly Sanger sequenced with three sequencing primers, namely, 5\u2019-cagggctcctcagaggaaagg-3\u2019, 5\u2019-gtggggctgccattccttccacc-3\u2019, and 5\u2019-tggcagggggcttggcctgagac-3\u2019, and the genotypes of the SNPs were determined. The analyzed SNPs are listed in Table For 92 genomic DNAs , the 5\u2019-upstream region of the Analysis of genetic modelsAQP1\u00a0gene and its 5\u2019-upstream region, we analyzed the applicability of four different genetic models [For each SNP in the\u00a0c models  by assocAnalysis of CpG island methylationAQP1\u00a0gene was evaluated using genomes extracted from the tunica vaginalis. A 495 bp CpG island was detected in the\u00a0AQP1\u00a0gene and its upstream sequence using Methyl Primer Express v1.0 software. The island was mostly located within the coding region of exon1, downstream of the core promoter. Furthermore, the GC content was 60.6%, and the observed-to-expected CpG ratio was 59.4%. CpG islands were not identified within the promoter region of the\u00a0AQP1\u00a0gene. Sixty-six genomes (32 hydrocele testis and 34 controls) extracted from frozen tunica vaginalis were bisulfited using an EpiSight\u2122 Bisulfite Conversion Kit Ver.2  and amplified with two pairs of bisulfite primers  using a PyroMark\u00ae PCR Kit  at an annealing temperature of 56\u2103, following the manufacturers\u2019 instructions. Pyrosequencing was performed on the amplified products, and the percentage of methylation was measured for 17 CpG sites using PyroMark Q24 . The sequences analyzed after bisulfitization were yggyggtttaggataaygtgaaggtgtyg, tygttaygttggygtagagtgtgggttatattagyggyg, ttatygtttagtgygtgggggttatygtygttatyg, and tygtttggtygtaatgayg.Methylation of the CpG island of the\u00a0Statistical analysisStatistical analysis was performed using R.\u00a0In the genetic model for SNPs, Fisher\u2019s exact test was used. Welch t-test was used for the methylation rate of each CpG site.Genotyping of SNPs and analysis of genetic modelsThere was no significant association between each SNP and hydrocele testis for any of the genetic models Table . SimilarAnalysis of CpG island methylationThe average methylation rate of the 17 CpG sites evaluated was not significantly different between controls and hydrocele testis . Considering the individual CpG sites, the methylation rate was lower in hydrocele testis than in controls at CpG position 16 , but at other CpG sites, there was no significant difference in methylation rates between controls and hydrocele testis Figures -1C.AQP1\u00a0gene.Most of the CpG island detected in this study was located within the coding region of exon1, downstream of the core promoter. Thus, although this was detected as a CpG island, it may be closer to a CpG island shore. CpG islands were not detected in the promoter region of the\u00a0Overall, although there was no difference in the average methylation rate of the CpG sites within the CpG island between hydrocele testis and controls, one CpG site showed a decreased methylation rate in hydrocele testis. This hypomethylation may contribute to enhanced AQP1 protein expression in the tunica vaginalis of patients with hydrocele testis and the development of hydrocele testis. However, the methylation status of the CpG island shore, which extends further on both sides of the CpG island, should be investigated more extensively to clarify its relationship with enhanced AQP1 protein expression.AQP1\u00a0gene and its 5\u2019-upstream region were not associated with the development of hydrocele testis in this study. Eight SNPs were examined herein, of which a few have been reported to be associated with diseases and pathological conditions. A minor allele of rs2075574, located in the 5\u2019-upstream region of the\u00a0AQP1\u00a0gene, was associated with decreased peritoneal ultrafiltration and increased mortality in peritoneal dialysis patients, as well as decreased promoter activity of the\u00a0AQP1\u00a0gene and decreased AQP1 protein expression [AQP1\u00a0gene, was previously associated with sudden infant death syndrome [AQP1\u00a0gene, was reported to be a risk SNP for malignant mesothelioma [SNPs in the\u00a0pression . This susyndrome . The majthelioma , and thethelioma  and runnthelioma .AQP1\u00a0gene and AQP1 protein production was not directly examined, but hydrocele testis was analyzed as a target factor. The development of hydrocele testis involves not only an increase in fluid influx due to an increase in AQP1 protein but also a factor of decreased fluid absorption due to changes in the lymphatic vessels of the tunica vaginalis. Thus, it cannot be said that SNPs associated with the\u00a0AQP1\u00a0gene do not cause changes in AQP1 protein levels. If fluid absorption from the lymphatic vessels is sufficient, even a small increase in AQP1 protein levels does not result in fluid retention as a subtraction. Figure In the present study, the relationship between genetic and epigenetic changes in the\u00a0There are a few limitations of this study. Decreased methylation of one CpG site in the CpG island was suggested as a contributing factor to the increase in AQP1 protein in the tunica vaginalis. Further pursuit will require detailed methylation analysis in the CpG shore, which extends on both sides of the CpG island.AQP1 gene in the tunica vaginalis of patients with non-communicating hydrocele testis. This may increase AQP1 protein expression and contribute to the formation of hydrocele testis. SNPs related to the AQP1 gene were not associated with hydrocele testis.Adult-onset, primary, non-communicating hydrocele testis involves the accumulation of clear fluid between the tunica vaginalis and testis with the enhanced influx of fluid in the space. The enhanced influx can be contributed by the enhanced water channel AQP1 protein in the tunica vaginalis. There was a significant decrease in the methylation rate at one of the CpG sites in the CpG island associated with the"}
+{"text": "Glycine max) is an increasingly relevant crop due to its economic importance and also a model plant for the study of root symbiotic associations with nodule forming rhizobia. Plant polyesters mediate plant-microbe interactions with both pathogenic and beneficial microbes; suberin has been hypothesized to play a key role during the early steps of rhizobia attachment to the root. The downside is that suberin chemistry in soybean root is still scarcely studied. This study addresses this outstanding question by reporting a straightforward workflow for a speedy purification of suberin from soybean root and for its subsequent detailed chemical analysis. To purify suberin, cholinium hexanoate (an ionic liquid) was used as the catalyst. The ensuing suberin is highly esterified as observed by a precise Nuclear Magnetic Resonance quantification of each ester type, discriminating between primary and acylglycerol esters. Moreover, the composing hydrolysable monomers detected through GC-MS revealed that hexadecanoic acid is the most abundant monomer, similar to that reported before by others. Overall, this study highlights the adequacy of the ionic liquid catalyst for the isolation of suberin from soybean roots, where the polymer natural abundance is low, and builds new knowledge on the specificities of its chemistry; essential to better understand the biological roles of suberin in roots.Soybean ( Another unresolved question, is if the ionic liquid catalyst can be applied to purify suberin from plant roots where its abundance is extremely low. The present study builds foundational proof of concept that the ionic liquid catalysts is appropriate to ensure uncomplicated and efficient recovery of suberin present in soybean roots. The polymeric arrangement of the recovered root suberin (NMR), and its monomeric composition (GC-MS), were analyzed, and the data significance discussed.Soybean (Glycine max) is a model plant with high economical relevance used for animal feeding, biodiesel fuel production or bio-composite materials development . MoreoveSodium hydroxide (>98%) was purchased from Jos\u00e9 Manuel Gomes dos Santos; methanol (\u226599.8%), dimethyl sulfoxide , dichloromethane (>99.99%) and glycerol (\u226599%) from Fisher Chemical; cholinium hydrogen carbonate (\u223c80% in water), hexanoic acid (>99.5%), hexadecane (>99%), 2.0\u00a0M (trimethylsilyl)diazomethane (TMSD) in hexane, N,O-bis(trimethylsilyl)trifluoroacetamide (99%) containing 1% (v/v) of trimethylchlorosilane , chromium (III) acetylacetonate (97%), N-hydroxy-5-norbornene-2,3-dicarboxylic acid imide (97%), 2-chloro-4,4,5,5-tetramethyl-1,3,2-dioxaphospholane (95%) and toluene (ACS >99.5%), heptadecanoic acid (\u226598%), hexadecanedioic acid (96%) and pentadecanol (99%) from Sigma-Aldrich; hydrochloric acid (37%) from Riedel-de-Ha\u00ebn and deuterated dimethyl sulfoxide  from Merck. Hoagland\u2019s basal salt mixture was acquired from MP Biomedicals. Cholinium hexanoate was synthesized by dropwise addition of hexanoic acid to aqueous cholinium hydrogen carbonate in equimolar quantities, as previously described .Glycine max) seeds of cv. Williams 82 were kindly provided by Dr. S Subramanian . For surface sterilization seeds were immersed in 2.25% sodium hypochlorite for 3\u00a0min, rinsed with sterile deionized water, and treated with 70% ethanol for 5\u00a0min. Seeds were washed seven times and soaked in sterile deionized water for 20\u00a0min before incubating on R2A agar at 30\u00b0C for 4\u00a0days to ensure sterility. Sterile seeds showing a radicle were potted in square pots (3\u2033 x 3\u2033 x 4\u201d deep) containing a sterile mixture of 3:1 vermiculite: perlite. Pots were saturated with sterilized Hoagland solution (5\u00a0mM Ca(NO3)2.4H2O, 2\u00a0mM MgSO4.7H2O, 5\u00a0mM KNO3, 0.8\u00a0mM KH2PO4, 92\u00a0\u00b5M Na2Fe EDTA, 0.36\u00a0\u00b5M MnCl2, 0.034\u00a0\u00b5M ZnSO4, 1.15\u00a0\u00b5M H3BO3, 0.0125\u00a0\u00b5M CuSO4, and 52\u00a0\u00b5M H2MoO4), and incubated in a growth chamber at 25\u00b0C under lights for 14\u00a0h (day temperature) and 20\u00b0C for 10\u00a0h in the dark (night temperature). Plants were watered every 3\u00a0day and Hoagland solution was provided every seventh day. After 17\u00a0days, plants were harvested and the roots were decapitated, washed, dried at 50\u00b0C for 48\u00a0h, and autoclaved. Afterwards the roots were milled  and used for analysis.Soybean (Quercus suber L.) was obtained from Amorim & Irm\u00e3os SA . Afterwards it was milled ; then, the extractives were removed by sequential Soxhlet extraction with solvents of increasing polarity  as previously described  and subsequently freeze-dried. The ensuing suberin powders were kept at room temperature until further analysis.Suberin was extracted from oak cork (hereafter simply defined as cork) or soybean roots, as previously described . In briein planta suberin profile, 100\u00a0mg of soybean roots were washed with 25\u00a0mL of deionized water, sonicated during 30\u00a0min, mixed and then centrifuged during 1\u00a0min, 2655\u00a0g at 4\u00b0C (Eppendorf centrifuge 5810\u00a0R) to recover the washed precipitate.To analyze the RESTCH Cryomill equipped with 5\u00a0mL grinding jars with two stainless steel grinding balls (4\u00a0mm) was used. Soybean roots (washed as described above) and isolated suberin were cryogenically milled at \u2212196\u00b0C (liquid nitrogen) using 80\u00a0milling cycles as follows: 3\u00a0min of precooling followed by nine milling cycles, each cycle consisting of 3\u00a0min of milling at 30\u00a0Hz followed by 0.5\u00a0min of intermediate cooling at 5\u00a0Hz.1H, 1H\u201313C HSQC, 1H\u201313C HMBC) were acquired in DMSO-d6 using 5\u00a0mm diameter NMR tubes at 60\u00b0C as follows: 15\u00a0mg of either soybean root or purified suberin in 500\u00a0\u03bcL of DMSO-d6 adding 10\u00a0\u00b5L of a solution of Benzene in DMSO-d6 as an internal standard. Quantitative 31P NMR spectra of suberin was also recorded using an Avance III 500  (NMR spectra of soybean roots (subjected to cryogenic milling as mentioned above) and suberin extracted from soybean roots with the ionic liquid were recorded using an Avance III 800 CRYO . All NMR spectra (Germany) . MestReNi.e., composing of hydrolyzable monomers), as well as cork hydrolysates, the samples were submitted to alkaline hydrolysis. In brief, the samples were added to a solution of 0.5\u00a0M NaOH in methanol/water  at 95\u00b0C for 4\u00a0h (3:1\u00a0m/v). The mixture was cooled to room temperature and acidified to pH 3\u20133.5 with HCl 1\u00a0M, spiked with a known concentration of hexadecane , and extracted three times with dichloromethane. Sodium sulphate anhydrous was added to the organic phase to remove water, which was then concentrated in a nitrogen flux. The dried combined organic extracts were immediately processed for GC-MS analyses (see below).To obtain suberin hydrolysates extracted from soybean roots or from cork with the ionic liquid catalyst (To quantify the amount of hydrolyzable monomers composing the purified suberin samples (or cork) an Agilent GC (7820A) equipped with an Agilent (5977B) MS (quadrupole) was used. For the first step of derivatization, 750\u00a0\u00b5L of MeOH:Toluene (2.5:1) and 500\u00a0\u00b5L of TMSD (2\u00a0M in hexane) were used . For the second step of derivatization N,O-bis(trimethylsilyl)trifluoroacetamide containing 1% of trimethylchlorosilane in pyridine (5:1) were used . The ensuing samples were analyzed by GC\u2013MS (HP-5MS column) with the following ramp temperature: 80\u00b0C, 4\u00b0C/min until 310\u00b0C for 15\u00a0min. MS scan mode, with a source at 230\u00b0C and electron impact ionization , was used for all samples. The GC\u2013MS was first calibrated with pure reference compounds  relative to hexadecane . Triplicates, each with technical triplicates were analyzed. Data acquisition was accomplished by MSD ChemStation (Agilent Technologies); compounds were identified based on the equipment spectral library (Wiley-NIST) and references relying on diagnostic ions distinctive of each derivative and its spectrum profile.Levene\u2019s test was used to analyze the variance of the GC-MS data. Statistical differences for each monomer type between samples were analyzed using a two-way Analysis of Variance (ANOVA).i.e., outer bark of Q. suber) due to its high suberin content and extant knowledge on the chemistry of cork suberin purified using a similar process  through Gas Chromatography Mass Spectrometry (GC-MS), upon standard derivatization of free OH groups   . This re1H) . The isolated suberin was analyzed through NMR: unidimensional proton (1H)  and two-1H) .1H\u221213C HSQC, and 1H\u221213C HMBC) and using as reference previous assignments of suberin  and 9-octadecenoic acid (oleic acid), could be unequivocally assigned. Moreover, the signals related to \u03b1(C=O) acids at a13C shift of 33.13\u00a0ppm and 1H shift at 2.18\u00a0ppm, \u03b1(C=O) esters at a13C shift of 32.99\u00a0ppm and 1H shift at 2.27\u00a0ppm, and \u03b2-PAE  at a13C shift of 27.62\u00a0ppm and 1H shift at 1.54\u00a0ppm, were observed. In the glycerol CH-acyl region all five glycerol configurations were assigned: 1,2,3-triacylglycerol (TAG), 1,2-diacylglycerol , 1,3-diacylglycerol , 2-monoacylglycerol (2-MAG) and 1-monoacylglycerol (1-MAG). The relative contribution of each acylglycerol configuration was quantified  of free acid and hydroxyl groups in the extracted suberin   , then waThe diversity of hydrolysable monomers detected in the soybean root suberin that was purified using the ionic liquid catalyst is depicted in i.e., the glycerol content in the purified suberin is time-dependent. Accordingly, to purify suberin close to its native polymeric arrangement, short ionic liquid extraction periods are required. Taken cork as a model, the 2\u00a0h reaction results in the extraction of ca. 50% of the extant suberin but the native levels of glycerol are preserved in the polymer. The hypothesis that the monomeric composition of the extracted suberin is not affected by the usage of sequential reactions was validated here using cork as model. The results showed that the ionic liquid catalysis did not result in the purification of a specific sub-type of suberin when used to extract, consecutively, two times suberin from cork  as well as in OH aliphatic groups (quantitative 31P NMR data). Interesting, the in planta suberin shows high proportion of both TAG and 1,2 DAG (\u223c50% each); this feature is different from that observed in both cork (P. radiata bark (P. radiata bark suberin (7-fold), but higher than that of cork suberin (2.3-fold). Finally, the overall distribution of the acylglycerol configurations is similar to that of P. radiata bark, both of which compared to cork suberin showed higher abundance of 1-MAG  which could not be precisely assigned in the 1H NMR spectra due to the low signal intensity of these spectral region. The low abundance of aromatic features in the soybean root suberin\u2013extracted suberin and in planta suberin\u2013is likely explained by the specificity of the used cultivar. In fact, it has been reported that different cultivars have differences not only in response to drought and pathogens but also in root architecture and traits (Phytophthora sojae respectively) differ greatly. The first is much richer in both the aliphatic and aromatic components of the polymer than the last (p < 0.05) , as well as the presence of the signal assigned to the esterified ferulic acid (trans-FA-\u03b1-ester)  . In addi < 0.05) . Taken a\u03b1-ester) .P. radiata bark noic acids (nearly 60%, in planta. Despite low abundance of suberin in root, the ionic liquid extraction was time efficient and simple to implement. The recovered suberin largely preserves its acylglycerol-esterified backbone. Subsequent analyses through NMR and GC-MS allowed quantification of the suberin key molecular features and monomeric composition, respectively. Further methodological developments are possible to target suberin deposited in specific cell layers within the root. The methodological workflow here presented, which combines a catalyst that specifically mediates acylglycerol ester cleavage and quantitative solution NMR analyses, secures access to essential information on the polymeric arrangement of suberin. It is therefore undeniable that this workflow can help solving outstanding questions on suberin structural chemistry, biosynthesis and function in roots. Importantly, protocols described here to extract suberin will open the door to study the significance of suberin chemistry in the overall root microbiota and potential resistance to root pathogens, as well as the attachment of bacteria such as rhizobia to this polymer, as yet, poorly studied soybean root component.The soybean suberin monomeric composition as shown by GC-MS  revealedata bark . The maj"}
+{"text": "The innate immune system in our bodies responds to pathogenic infections and cellular damage by inducing pyroptosis through the assembly of inflammasome complexes. The apoptosis-associated speck-like protein containing a CARD (ASC) serves as an adapter, recognizing pattern recognition receptors (PRRs) and procaspase-1 based on the homotypic interactions of the pyrin domains (PYD) and the caspase recruitment domains (CARD) within the inflammasome complexes. The structural diversity of ASC and the role of the semi-flexible linker in structural transitions are critical in understanding the biological functions of ASC. This study employs molecular dynamics simulations to explore the structural dynamics of ASC and to analyze the potential relationship between interdomain dynamics and the biological roles of ASC as an adapter. The findings suggest that ASC dynamics partially originate from the movement of the linker and that the type I interaction surface on PYD is generally exposed and inaccessible to the CARD domain. These insights are consistent with experimental results and shed light on the function-related dynamic behaviors of ASC.The canonical ASC domains, PYD and CARD, are interconnected by a lengthy, semi-flexible linker. The molecular basis and purpose of ASC\u2019s highly dynamic feature remain elusive. In this study, all-atom molecular dynamics simulations were utilized to examine the role of the linker and the interdomain dynamics of the ASC monomer. As revealed in the principal component analysis (PCA), the flexible linker enables interdomain dynamics and rotation. The stumbling between domains is partially attributed to the helical portion of N-terminal residues in the linker. Additionally, the linker exhibits a certain structural preference due to the turn-type structural inclination of the N-terminal and the presence of several prolines on the linker. Such structural preferences lead to the unavailability of regions for PYD type I interactions to CARDs, as evidenced by the CARD spatial restraint analysis. In conclusion, the semi-flexible linker introduces functionally relevant interdomain dynamics, potentially enhancing PYD self-assembly and the subsequent assembly of the inflammasome complex. The immune system can rapidly recognize and respond to damage- or pathogen-associated molecular patterns (DAMPs and PAMPs) in order to maintain homeostasis and promote health. These DAMPs and PAMPs can be detected by host pattern recognition receptors (PRRs), such as NOD-like receptors (NLRs) and absent in melanoma 2 (AIM2), via inflammatory pathways ,2,3. PRRHuman ASC is a protein comprised of 195 amino acids . The solAtomic resolution information on structure and dynamics is pivotal for understanding the fundamental physicochemical properties of the multi-talented ASC. Previous NMR experimental studies have shown that the ASC linker governs its interdomain dynamics by partially restricting the accessible conformational space of each domain relative to the other . In this+ or Cl\u2212) to neutralize the whole system at 300 K. Long-range electrostatics were calculated using the particle-mesh Ewald (PME) algorithm [Two systems were simulated: the ASC full-length monomer (195 residues), as shown in lgorithm ,35. Perilgorithm ,37, whiclgorithm , using tlgorithm ,37 was uThe hierarchical cluster analysis was performed using Ward\u2019s minimum variance method . It is bDistance and angle analyses were performed using the GROMACS package . The cenAs observed from the ASC monomer simulations, there is no stable packing structure between PYD and CARD. As shown in To delve deeper into the interdomain dynamics, a thorough analysis of the ASC monomer was conducted, employing time-resolved force distribution analysis (TRFDA) and correlation plots. TRFDA is a useful tool for detecting residues under significant punctual stress, which are integral to maintaining the protein\u2019s structural integrity and contribute to the ASC interdomain dynamics. Here, TRFDA was applied to evaluate the interaction between PYD and CARD using six 1 \u00b5s trajectories. The punctual stress was averaged over the six trajectories. The ASC monomer is highly dynamic, with its PYD and CARD domains arranged differently in terms of spatial positions and orientations. The loop regions in the two domains and the linker demonstrate significantly more fluctuation compared to other regions. One domain interacts with varying surfaces of the other domain across different clusters. TRFDA results suggest some weak interactions between PYD and CARD, with the motion of these two domains showing correlation in certain segments. In order to further understand the dynamics of the ASC monomer, we have isolated the linker from the simulation for a detailed analysis, which we will present in the following section.13C\u03b1 secondary chemical shifts, implying the possible adoption of turn-type conformations. In contrast, residues 95\u2013112 had primarily negative 13C\u03b1 chemical shifts, indicating the formation of sparsely populated extended structures. We utilized the DSSP program [The linker, as observed in the TRFDA results  and corr program ,46 to asThough the linker exhibits some structural preference, its probability is relatively low. Consequently, a more in-depth analysis of the linker\u2019s flexibility is required. As demonstrated in The linker is highly dynamic, but we do observe the low-populated structures with a turn-type structure transiently formed at its N-terminus, especially in the monomer. In combination with the presence of prolines, the linker is not fully flexible but displays a structural preference to some extent.To study the interdomain dynamics, it is essential to conduct principal component analysis (PCA). In Moving on to the ASC monomer, the linker region has a very similar movement to the one described above, albeit with some distortion in shape. It is important to analyze how the movement of the linker is coupled with changes in rotation and orientation of the PYD and CARD domains. As shown in While observing these varied movements, it is important to emphasize that the interfaces formed by H1\u2013H4 and H2\u2013H3 of PYD (Type I interaction) are consistently maintained at a distance from CARD. Previous NMR work  proposedThe interdomain movement was further evaluated by analyzing the torsional angle illustrated in PCA results on the isolated linker and ASC monomer reveal that the interdomain dynamics of ASC originate from the linker. Moreover, interdomain dynamics are accompanied by interdomain rotation. We characterize the interdomain rotation using a torsional angle, as shown in De Alba previously proposed that the ASC interdomain topological organization aids binding by avoiding steric interference between the two interacting regions of both domains and by favoring a specific protein binding orientation . AlthougWe utilized the acquired frames to cluster the structures and calculate the probability of each spatial arrangement of CARD . This shThe spatial distribution of CARD around PYD implies that the ASC dynamics contribute to its function as an adapter in the inflammasome. In general, the Type I interaction on the surface of PYD, which is responsible for ASC self-assembly, is not blocked by CARD. Consequently, the preferred dynamics have evolved over time to serve the function.The loop regions in PYD and CARD and the central linker are the regions with the largest fluctuation. The interdomain bending angle (defined by the C\u03b1 atoms of residues 49\u201369\u2013163) varied from 30\u00b0 to 160\u00b0, and the COM distance distribution between PYD and CARD ranges from 2 to 8 nm, indicating that ASC is relatively flexible. The structure with the greatest COM distance seems to be the inter-conversion state of various compact structures. The representative structures from the cluster analysis of the ASC monomer demonstrate the interdomain flexibility and varied contact interfaces between PYD and CARD.The structural features of the linker were characterized to study the origin of ASC interdomain dynamics. It is worth noting that the N-terminus of the linker prefers the helical structure in the ASC monomer, as shown in Subsequently, we conducted an in-depth study on the potential role of interdomain dynamics in ASC assembly. PCA results show that CARD can rotate relative to PYD. This is important because several adjacent CARDs can rotate their binding interfaces to form homotypic interactions after PYD nucleation and assembly. Furthermore, the torsional angle between PYD and CARD (defined by C\u03b1 of residues 81\u201369\u2013163\u2013177), employed to describe the relative stumbling of PYD and CARD, is widely distributed in the range of \u2212180\u2013180\u00b0. The time evolution of the above-mentioned torsional angle shows that the angle sometimes oscillates frequently between \u2212180\u00b0 and 180\u00b0, indicating the interdomain stumbling of the ASC monomer, which is consistent with the PCA results. The rotation may be related to the transient contacts involving the linker and domains. Some interactions were observed in one conformation, while others were observed in other conformations. In other words, these transient interactions were not strong enough to stabilize ASC in one single structure during the entire simulation, yielding structural transitions. In addition, the interdomain rotation appears to be correlated with the dynamic variation of helical probability of residues 90\u201394 in the linker. Given the flexibility of the linker, CARD can access many regions around PYD. However, the helices involved in the Type I interactions in PYD, namely H1\u2013H4, are inaccessible to CARD. The interdomain spatial restrain is highly related to the properties of the linker, which is flexible but also has structural preferences, such as the turn-type structural preference in the N-terminal region of the linker and geometry limitation of prolines in the linker. Taken together, the structural features of the linker and the weak interdomain interplays lead to the ASC monomer favoring interdomain dynamics that seem to benefit ASC assembly and ultimately the assembly of the inflammasome complex.With the MD simulation on the ASC monomer and the isolated linker, we analyzed the origin of the interdomain dynamics of ASC. According to the PCA results, the interdomain dynamics stem from the semi-flexible linker since the ASC monomer showed similar motions as the linker. Additionally, the residues at the N-terminus of the linker show a weak tendency to form a turn-type structure, one possible factor driving the interdomain rotation. ASC is observed to show evident interdomain dynamics, but the dynamics are not entirely free to adopt diverse organizations for the two domains. Analysis of the spatial restraint of CARD suggested that it is likely to populate in certain positions with the Type I interface of PYD being exposed. Therefore, such positions contribute to the subsequent assembly of inflammasome complexes. These findings can be verified by further assembly simulations of more complex systems. With more information on the dynamic behaviors of ASC, we will be able to reveal the activation mechanism of the inflammatory signaling pathway and target ASC to regulate the immune response in inflammasome-related disorders."}
+{"text": "Science Progress 103(3): 1\u201422. DOI: Figures 7 to11 were misprinted in the article. The correct figures should be read as follows:"}
+{"text": "One hundred and five oval-shaped mandibularpremolars were instrumented, sterilized, and inoculated withEnterococcus faecalis, Candida albicans, andStaphylococcus aureus, divided into: control group -saline; O3 group - ozonated water; O3 PUI group - ozonatedwater with PUI agitation; O3 PUI+EA group - ozonated water withPUI+EA agitation; NaOCl group - NaOCl; NaOCl PUI group - NaOCl with PUIagitation; and NaOCl PUI+EA group - NaOCl with PUI+EA agitation. Microbiologicalsamples were collected before (S1) and after (S2) the disinfection proceduresand the data were statistically analyzed using the Kruskal-Wallis test. In theculture method, there was significant disinfection in the O3 PUI+EA,NaOCl, NaOCl PUI, and NaOCl PUI+EA groups (p\u02c20.05). The combination of NaOClwith PUI+EA reduced microbial counts to zero (p\u02c20.05). In the qPCR method, therewas a significant reduction in the total count of viable microorganisms in theO3 PUI, O3 PUI+EA, NaOCl, NaOCl PUI, and NaOCl PUI+EAgroups (p\u02c20.05). It can be concluded that 2.5% NaOCl with and without agitation,as well as 8 \u00b5g/mL ozonated water with its action enhanced by the agitationtechniques, were effective in root canal disinfection, and their antimicrobialefficacy is related to the microorganisms present in the biofilm.This  Apical periodontitis is an infectious disease caused by microorganisms that colonizethe root canal system. To improve the prognosis of endodontic treatment, bacterialpopulations should ideally be eliminated or reduced to biologically compatiblelevels to permit the periapical tissues to heal. Bacterial persistence afterchemomechanical preparation, supplemented or not with intracanal medicament, posesan increased risk of adverse prognosis in endodontic treatment. Furthermore, thepresence of bacteria in the root canal at the time of filling has been shown to be arisk factor for post-treatment apical periodontitis Enterococcus faecalis is a Gram-positive bacterium thatappears individually and usually occurs in pairs or short chains. This facultativeanaerobic bacterium is commonly isolated from root canals with persistent periapicaldisease. E. faecalis can colonize dentin, invade dentinal tubules,and resist the antimicrobial actions of irrigating solutions and intracanalmedicaments Candida albicans is a eukaryotic fungus found in infectedroot canals, with a prevalence ranging from 0.5% to 5.5%, mainly insecondary/persistent infections, and virulence factors that may play an importantrole in the onset of endodontic pathologies Staphylococcus aureus is a Gram-positive, facultativebacterium that produces enzymes potentially important in microbial pathogenicity,such as coagulase, hyaluronidase, lysozyme, and lipases, as well as several toxins,such as hemolysins, epidermolytic toxin, and some enterotoxins, causing severaldiseases E. faecalis, C. albicans, and S.aureus were selected because they represent species that are resistantto disinfection protocols, often being present in secondary/persistent infectionsApproximately 150 species of microorganisms can colonize the root canal system, andsome of them can induce or maintain periapical lesions 3), a naturally occurring gas and a strong and selective oxidant,for therapeutic purposes 3 rapidly dissociates intowater and releases a reactive form of oxygen that can oxidize cells, thus exhibitingantimicrobial activity without inducing drug resistance In endodontics, irrigation is an integral part of canal preparation, playing acritical role in disinfection and debris removal. Sodium hypochlorite (NaOCl) hasbeen widely used as an irrigant in endodontic treatment due to its ability todissolve tissue and excellent antimicrobial activity. However, a major drawback isits toxicity to periapical tissues when it extrudes through the apex Not only the irrigant but also the irrigation technique plays an important role.Conventional syringe-needle irrigation cannot clean hard-to-reach areas of thecanal, being considered insufficient for complete cleaning of the root canal spacePassive ultrasonic irrigation (PUI) is the most commonly used technique for agitationof the chemical irrigant. PUI promotes a cavitation effect by producing bubbles thatburst close to the dentin walls, in addition to generating microacoustic streamingthat promotes the hydrodynamic agitation of the irrigant, enhancing cleaning, but ithas the limitation of the ultrasonic insert being positioned 2 mm short of theworking length in straight canals and up to the beginning of the curvature in curvedcanals E. faecalis, C. albicans, and S. aureus. Thenull hypotheses tested for each method, culture and molecular (quantitativepolymerase chain reaction [qPCR]), were that In view of the foregoing, the eradication of multispecies biofilms containingresistant microorganisms is crucial for successful endodontic treatment. Therefore,the purpose of this study was to compare the antimicrobial efficacy of 2.5% NaOCland 8 \u00b5g/mL ozonated water agitated by PUI or PUI combined with sonic agitationusing the EndoActivator (EA) system against mature multispecies biofilms containingThe local research ethics committee (protocol number 5.129.512) approved this study.The sample size of 15 specimens per group was based on the results of a pilot testand the study by Moraes et al. The teeth were obtained from the Biorepository Bank of the S\u00e3o Leopoldo Mandic DentalResearch Center, Campinas, SP, Brazil. Mandibular premolars extracted fororthodontic, prosthetic, or periodontal indication were selected after obtainingwritten informed consent from the patient. Inclusion criteria were single-rootedteeth with a fully formed apex and curvature angle of 0\u00ba to 5\u00ba, according toSchneider A total of 105 single roots from extracted mandibular premolars were used. Thesample was prepared by a single experienced endodontist. The crowns weresectioned using Zekrya burs driven by a high-speed motor , underwater cooling. The tooth length was standardized at 15 mm.The root canals were initially prepared with a #10 K-file with oscillatory movements until 2 mm short of the initial tooth length,followed by a #15 K-file  until 5 mm short of the initialtooth length, also with oscillatory movements. The canals were instrumented withthe Protaper Next rotary system  driven by the X-Smart Plusmotor  at a speed of 300 rpm and a torque of 2 Ncm in anin-and-out pecking motion, combined with a brushing motion. Each file was used 4times and then discarded. Pre-flaring was performed with the X1 file (17.04)until 5 mm short of the initial tooth length. A #10 K-file was inserted into theroot canal until its tip was visible at the apical foramen, and the workinglength was visually determined at 1 mm short of the foramen. The anatomicdiameter of the root canal corresponded to a #15 K-file .The canal was prepared with a #15 K-file to the working length and theninstrumented with X1 (17.04), X2 (25.06), and X3 (30.07) files also to theworking length. At each instrument change, a #10 K-file was inserted to a lengthof 1 mm beyond the apical foramen to ensure patency, and the specimens wereirrigated with 3 mL of 2.5% NaOCl ,for a total of 25 mL, using a disposable syringe and 0.55 x 20 mm needle. The flutes of the instrumentwere cleaned with gauze.The smear layer was removed by irrigating the canals with 3 mL of 17% EDTA for 3minutes, followed by aspirtion with a 0.014\u02ba capillary tip . Finally, the canals were irrigated with 3 mL of 2.5%NaOCl, also for 3 minutes, and aspirated with a 0.014\u02ba capillary tip. All canalswere dried with sterile absorbent paper points .The apical foramen of all teeth was sealed with light-cured composite resin to prevent bacteria from enteringthrough the foramen and to create a closed system. Two layers of nail polishwere applied to the outer surface of all roots. Heavy-body silicone putty was used to createniches for the roots, which were inserted into 24-well cell culture plates forpreparation of test specimens and bacterial colonization. The specimens wereautoclaved at 121\u00b0C for 15 minutes and then incubated at 37\u00b0C for 24 hours toevaluate the occurrence of bacterial growth.E. faecalis (ATCC 29212), S.aureus (ATCC 25923), and C. albicans (ATCC 10231)were activated in brain-heart infusion (BHI) broth ,matched to a 10 McFarland standard - suspension containing 3.0 \u00d7 109colony forming units (CFUs) per mL. The colonies were injected into the rootcanals using a sterile insulin syringe with a 30-gauge needle and incubated at37\u00b0C for 21 days. During this period, a 20-\u00b5L aliquot of the suspension wasreplaced daily for each specimen using a sterile pipette in a laminar flow hoodunder a 5% CO2 atmosphere. Two random specimens were checked weeklyfor the viability of microorganisms by inserting a sterile paper point into theroot canal and then incubating it at 37\u00b0C for 24 hours.Standard strains of Ozonated water was prepared following the protocol described by Nogales et al.2 atmosphere, before (S1) and after (S2) thestudy disinfection procedures. The first sample (S1) was collected by insertinga sterile FM paper point (Tanari) to the working length for 1 minute and thenplacing it in an Eppendorf tube containing 900 \u00b5L of saline solution, which wasvortex mixed for 30 seconds. The bacterial suspension was serially diluted at10-1, 10-2, 10-3, and 10-4concentrations, and aliquots were seeded into Petri dishes containing BHI agarand incubated at 37\u00baC under a 5% CO2 atmosphere for 24 hours.Microbial growth was measured by the CFU/mL counts. CFU counting was performedby a single calibrated and trained operator using a digital colony counter.Microbiological samples were collected, under aseptic conditions in a laminarflow hood under a 5% CO2 atmosphere.The roots were randomly divided into 7 groups (n=15 each) according to thetreatment performed, as shown in -Negative control group  - the canals were irrigated with 5 mL of sterilesaline solution for 1 minute, using a 0.55 x 20 mm needle (Embramac) inserted toa length of 10 mm.-NaOCl group - the canals were irrigated with 5 mL of 2.5% NaOCl (F\u00f3rmula &A\u00e7\u00e3o) for 1 minute, using a 0.55 x 20 mm needle (Embramac) inserted to a lengthof 10 mm.-NaOCl PUI group - the canals were irrigated with 1.6 mL of 2.5% NaOCl (F\u00f3rmula& A\u00e7\u00e3o), using a 0.55 x 20 mm needle (Embramac) inserted to a length of 10mm, activated using an Irrisonic tip  coupled to an ultrasonic piezoelectric unit at 30% power, inserted into the rootcanal until 2 mm short of the working length for 20 seconds and aspirated with aneedle and syringe (Embramac). NaOCl was replaced with fresh irrigant andagitation was repeated 3 times, for 20 seconds each. Total irrigation time wasstandardized at 1 minute and total irrigant volume, at 5 mL.-NaOCl PUI+EA group - the canals were irrigated as described for the NaOCl PUIgroup, activated using an Irrisonic tip (Helse Ultrasonic), repeated 2 times for20 seconds each, until 2 mm short of the working length. NaOCl was replaced withfresh irrigant and final agitation was performed with EA , using the medium activator tip (#25/.04) to the working length for20 seconds. Total irrigation time was standardized at 1 minute and totalirrigant volume, at 5 mL.3 group - the canals were irrigated with 5 mL of ozonated water at aconcentration of 8 \u00b5g/mL for 1 minute, using a 0.55 x 20 mm needle (Embramac)inserted to a length of 10 mm.-O3 PUI group - the canals were irrigated with 1.6 mL of ozonatedwater at a concentration of 8 \u00b5g/mL, using a 0.55 x 20 mm needle (Embramac)inserted to a length of 10 mm, activated using an Irrisonic tip (HelseUltrasonic) coupled to an ultrasonic piezoelectric unit (J. Morita) at 30%power, inserted into the root canal until 2 mm short of the working length for20 seconds and aspirated with a needle and syringe (Embramac). Ozonated waterwas replaced with fresh irrigant and agitation was repeated 3 times. Totalirrigation time was standardized at 1 minute and total irrigant volume, at 5mL.-O3 PUI+EA group - the canals were irrigated as described for theO3 PUI group, activated using an Irrisonic tip (HelseUltrasonic), repeated 2 times for 20 seconds each, until 2 mm short of theworking length. Ozonated water was replaced with fresh irrigant and finalagitation was performed with EA , using themedium activator tip (#25/.04) to the working length for 20 seconds. Totalirrigation time was standardized at 1 minute and total irrigant volume, at 5mL.-O2 atmosphere, by inserting a sterile FM paperpoint (Tanari) to the working length for 1 minute and then placing it in anEppendorf tube containing 900 \u00b5L of saline solution, which was vortex mixed for30 seconds. The bacterial suspension was serially diluted at 100,10-1, and 10-2 concentrations, and aliquots wereseeded into Petri dishes containing BHI agar and incubated at 37\u00baC under a 5%CO2 atmosphere for 24 hours. Microbial growth was measured by theCFU/mL counts. CFU counting was performed by a single calibrated and trainedoperator using a digital colony counter .Immediately after the disinfection procedures, the second microbiological sample(S2) was collected as described for S1, under aseptic conditions in a laminarflow hood under a 5% COS1 and S2 samples were kept frozen until DNA extraction for real-time qPCRanalysis using the Maxima SYBR Green/ROX qPCR Master Mix detection system in the 7500 Fast Real-Time PCRSystem .E. faecalis; C. albicans ; and S. aureus. Prior to the real-time PCR assays, concentration-effect and melting curveswere performed to determine the working concentration and specificity of theprimers, respectively. The amplification reaction consisted of cycling at 50\u00baCfor 2 minutes, at 95\u00baC for 10 minutes, followed by 40 cycles at 95\u00baC for 15seconds and 60\u00baC for 1 minute. The results were analyzed based on the thresholdcycle (Ct) value, which is the point corresponding to the number of cycles inwhich the amplification of the samples reached a threshold  that allowed the quantitative analysis ofthe expression of the evaluated gene. The Ct values \u200b\u200bwere used to determine theabsolute number of microorganisms in each experimental group, based on thepreviously established standard curve.The DNA of each previously isolated microorganism was extracted by using QIAampDNA Mini Kit  according to the manufacturer\u2019sinstructions. The DNA concentration (absorbance at 260 nm) was determined with aspectrophotometer . Afterextraction, the sequences of the specific primers for the microorganisms studiedwere designed using the Primer-BLAST software from the National Center forBiotechnology Information and were as follows: To calculate the percentage of microbial reduction per sample, the followingformula was used, where MB is the total number of microorganisms beforedisinfection, obtained by collecting the first microbiological sample (S1), andMA is the total number of microorganisms after disinfection, obtained bycollecting the second microbiological sample (S2):The results were analyzed using Biostat 5.3 and tested for normality using theShapiro-Wilk test. The data were not normally distributed and, therefore,analyzed using the nonparametric Kruskal-Wallis test, followed by Dunn\u2019s test.The significance level was set at 5%.3 PUI+EA, NaOCl, NaOCl PUI, and NaOClPUI+EA (p\u02c20.05). Microbial counts before the various disinfection protocols did notdiffer significantly (p>0.05), indicating standardization of the studymethodology. The most marked reduction in viable CFUs occurred in the NaOCl, NaOClPUI, and NaOCl PUI+EA groups, with a significant difference in relation to the othergroups. The combination of NaOCl with PUI+EA was the most effective in disinfectingthe root canal system, reducing microbial counts to zero after the application ofthe protocol (p\u02c20.05)  . However(p\u02c20.05) .E. faecalis and C. albicans afterusing the following disinfection protocols: O3 PUI, O3 PUI+EA,NaOCl, NaOCl PUI, and NaOCl PUI+EA (p\u02c20.05). Regarding E. faecalisand C. albicans counts after the various disinfection protocols,saline (control) and O3 were the least effective disinfecting agents,with a significant difference in relation to the other groups (p\u02c20.05)  .S. aureus counts after the various disinfection protocols,there was a significant microbial reduction in the O3 PUI, O3PUI+EA, NaOCl, NaOCl PUI, and NaOCl PUI+EA groups (p\u02c20.05). When comparing groupsregarding S. aureus counts, saline (control) was the leasteffective disinfecting agent, with a significant difference in relation to the othergroups (p\u02c20.05) (Regarding (p\u02c20.05) .E. faecalis, S. aureus, andC. albicans in all experimental groups in relation to thecontrol group (p\u02c20.05), with no significant differences between the experimentalgroups (p>0.05)  .According to the results obtained by the culture method, the first null hypothesiswas rejected. The 8 \u00b5g/mL ozonated water and 2.5% NaOCl were not equivalent to eachother, with 2.5% NaOCl being more effective. The second null hypothesis was alsorejected because 8 \u00b5g/mL ozonated water combined with agitation improved theantimicrobial efficacy of this irrigant and 2.5% NaOCl combined with agitationyielded results superior to those of the other disinfection protocols. By the qPCRanalysis, the first null hypothesis was rejected, since 8 \u00b5g/mL ozonated waterwithout agitation performed worse than 2.5% NaOCl in the before-and-aftercomparison. The second null hypothesis was accepted for 2.5% NaOCl but rejected for8 \u00b5g/mL ozonated water, as its effect was enhanced by agitation.E. faecalis, which can invadedentinal tubules, shows strong adhesion to collagen, and is resistant to irrigatingsolutions commonly used during root canal instrumentation E. faecalis have been widely used inresearch settings ,,,,E.faecalis, C. albicans, and S. aureus,microorganisms that are equally resistant and contribute to persistent intracanalinfection, as done in previous studies using multispecies biofilms ,Several microorganisms and their byproducts are involved in the etiology ofendodontic infections, including ,,,In addition, it is important to highlight the biofilm formation time of 21 days, asused in previous studies Pseudomonas aeroginosa, S.aureus, and E. faecalis and concluded that the highestconcentration was effective in eliminating the three bacteria. In a later study, inaddition to investigating the antimicrobial efficacy of ozonated water at the threeconcentrations previously described, Nogales et al. Despite being a potent antimicrobial agent, NaOCl in its different concentrations hasdisadvantages such as cytotoxicity, which encourages the investigation of othersubstances such as ozone, which has been used in the form of ozone gas, ozonatedwater, or ozonated oil as an optimal protocol for its use has yet to be established,,,,Agitation potentiates the effects of the irrigant, which can be agitated by PUI ,,The S1 and S2 samples were collected with sterile paper points, as previouslydescribed E.faecalis. Although the culture method was used in many other studies,,,,The current study used both culture and molecular (qPCR) methods to analyze the levelof disinfection achieved with different irrigants and agitation methods, as was donein the study by Moraes et al. E. faecalis. In the studyby Nunes et al. The results of our culture analysis showed that in the groups irrigated with 8 \u00b5g/mLozonated water, the combination with PUI+EA agitation produced better results in thebefore-and-after disinfection comparisons, but the absence of this methodology inthe literature precluded a direct comparison with the results of previous studies.Moraes et al. 3 and NaOCl obtained the same results ,,In the present study, the results of culture analysis also showed that irrigationwith 2.5% NaOCl had superior antimicrobial efficacy, with emphasis on the group withPUI agitation supplemented with EA, the only treatment regimen able to reducemicrobial counts to zero. This result may have been caused by irrigation with NaOCl,well known as a potent disinfectant, supplemented with a hybrid agitation technique,thus obtaining the best of both techniques: cavitation and microacoustic streaminggenerated by PUI with the Irrisonic tip working until 2 mm short of the workinglength and sonic agitation to the working length. Previous studies comparing theantimicrobial efficacy of OE.faecalis counts in the groups irrigated with 8 \u00b5g/mL ozonated water, inthe groups agitated by PUI and by PUI+EA, when comparing S1 and S2 samples. Similarresults were reported by Hubbezoglu et al. The qPCR results showed a significant difference in the reduction of S. aureus was the least resistant microorganism when comparing thedisinfection protocols vs control . Therefore, 8 \u00b5g/mL ozonated water and2.5% NaOCl, both with and without agitation, significantly reduced S.aureus counts. Comparing different concentrations of ozonated waterwithout agitation, Nogales et al. S.aureus. Nogales et al. S. aureus.According to Estrela et al. E. faecalis is the dominant microorganism.C. albicans was found to be significantly reduced by irrigationwith 8 \u00b5g/mL ozonated water with agitation and by irrigation with 2.5% NaOCl withand without agitation in our before-and-after comparisons of disinfection protocols.However, none of the treatment regimens were able to reduce microbial counts tozero. Huth et al. C. albicans only when5.25% NaOCl was used, followed by a reduction of more than 96% with gaseous ozone,ozonated water, and chlorhexidine. Cardoso et al. C.albicans and E. faecalis and reported a reduction inCFUs for both microorganisms. Both studies, however, used only the culture methodfor analysis.S.aureus was eliminated by irrigation with ozonated water with agitationand by irrigation with NaOCl with and without agitation, but the othermicroorganisms under study were present in all groups, supporting that theusefulness of the culture method is limited.It is important to note that there was a reduction in the total count of viablemicroorganisms in all experimental groups, an extremely satisfactory outcomeaccording to Siqueira and R\u00f4\u00e7as invitro research, such as the difficulty in reproducing the biofilm as itis organized in vivo.Despite the positive aspects, such as studying multispecies biofilm, researching thehybridization of irrigant agitation, in an attempt to overcome the limitations ofsonic and ultrasonic agitation methods, and studying two different methodologies toquantify the disinfection of samples, this study has limitations inherent E.faecalis, C. albicans, and S. aureus.Also, the antimicrobial efficacy of the irrigant is related to the microorganismpresent in the root canal system.It can be concluded that there was a reduction in microorganisms in all experimentalgroups. By the culture method, 2.5% NaOCl agitated in a hybrid way sterilized allspecimens, whereas by the qPCR method, 2.5% NaOCl with and without agitation, aswell as 8 \u00b5g/mL ozonated water with its action enhanced by the agitation techniques,was effective in reducing mature multispecies biofilms containing"}
+{"text": "Background: The ultrathin-strut drug-eluting stent (DES) has shown better clinical results than thin- or thick-strut DES. We investigated if re-endothelialization was different among three types of DES: ultrathin-strut abluminal polymer-coated sirolimus-eluting stent (SES), thin-strut circumferential polymer-coated everolimus-eluting stent (EES), and thick-strut polymer-free biolimus-eluting stent (BES) to gain insight into the effect of stent design on promoting vascular healing.Methods: After implanting three types of DES in the coronary arteries of minipigs, we performed optical coherence tomography (OCT) at weeks 2, 4, and 12 . Afterward, we harvested the coronary arteries and performed immunofluorescence for endothelial cells (ECs), smooth muscle cells (SMCs), and nuclei. We obtained 3D stack images of the vessel wall and reconstructed the en face view of the inner lumen. We compared re-endothelialization and associated factors among the different types of stents at different time points.Results: SES showed significantly faster and denser re-endothelialization than EES and BES at weeks 2 and 12. Especially in week 2, SES elicited the fastest SMC coverage and greater neointimal cross-sectional area (CSA) compared to EES and BES. A strong correlation between re-endothelialization and SMC coverage was observed in week 2. However, the three stents did not show any difference at weeks 4 and 12 in SMC coverage and neointimal CSA. At weeks 2 and 4, SMC layer morphology showed a significant difference between stents. A sparse SMC layer was associated with denser re-endothelialization and was significantly higher in SES. Unlike the sparse SMC layer, the dense SMC layer did not promote re-endothelialization during the study period.Conclusion: Re-endothelialization after stent implantation was related to SMC coverage and SMC layer differentiation, which were faster in SES. Further investigation is needed to characterize the differences among the SMCs and explore methods for increasing the sparse SMC layer in order to improve stent design and enhance safety and efficacy. In the Intracoronary imaging, including intravascular ultrasound (IVUS) and optical coherence tomography (OCT), is used to clinically estimate the re-endothelialization of the strut surface and predict the risk of LST. OCT is the method of choice to characterize fibrous tissue, plaque, thrombus, and neointimal coverage, due to its axial resolution of 5\u201320\u00a0\u03bcm. Recently, it has been reported that the evaluation of strut coverage by OCT can be one of the factors for clinicians to decide the duration of dual antiplatelet therapy . HoweverIn this preclinical study, we investigated the relation between re-endothelialization, SMC pathophysiology, and neointimal coverage after stent implantation using quantitative histological examination and OCT data. Particularly, we compared the process of SMC differentiation and its effect on re-endothelialization after the implantation of EES, BES, and SES in the coronary artery of a porcine model.n = 12; body weight = 25\u201335\u00a0kg) were administered aspirin (300\u00a0mg) and clopidogrel (300\u00a0mg). On the day of the experiment, the pigs were premedicated with atropine sulfate  and subsequently anesthetized with zoletil  and xylazine . Afterward, the pigs were intubated and ventilated with room air and sevoflurane. We inserted a 6 Fr sheath via the right femoral artery using an ultrasound-guided puncture.The animal experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Seoul National University Bundang Hospital (BA 1808-253/067-03) and performed in accordance with the Guide for the Care and Use of Laboratory Animals from the Institute of Laboratory Animals Resources. The day before the experiment, male minipigs , BES , and SES . The thin-strut EES has 81-\u03bcm-thick cobalt\u2013chromium struts and a PVDF-HFP polymer-containing everolimus . The thiThese three stents were implanted in the three coronary arteries of 12 pigs. After stent implantation, the pigs were dosed with 100\u00a0mg aspirin and 75\u00a0mg clopidogrel daily. We assigned each of the four pigs to 2-, 4-, and 12-week post-stent implantation groups and compared the results from OCT and histology .\u00ae image catheter and C7-XR\u2122 OCT system  after an intra-coronary injection of nitroglycerine before autopsy. From the OCT images, the cross-sectional area (CSA) of the coronary artery and stent strut apposition or coverage were evaluated using a dedicated imaging analysis program .Coronary angiography and OCT were performed using a DragonflySamples were fixed in 10% neutral buffered formalin at room temperature. Dehydration treatment was performed using 50%\u2013100% ethanol. Blood vessels were longitudinally cut, opened to expose the endothelium, and dried naturally. All samples were coated with gold to impart conductivity. The samples were imaged after selecting the magnification while appropriately adjusting the electron beam voltage and working distance by field-emission scanning electron microscopy .After the coronary arteries were harvested, endothelialization and neointima growth were evaluated by staining with antibodies against CD31, a glycoprotein expressed on ECs, alpha-smooth muscle actin (SMA-cy3), and nucleic acid . Histological quantification was performed by obtaining three to six random field images from each coronary artery . Confocapost hoc test was performed to find the statistical significance of the difference between one group and another. Statistical analyses were performed using R . A two-sided p-value <0.05 was considered statistically significant.Continuous variables are reported as the mean \u00b1 standard deviation. One-way ANOVA and a statistical model constructed by generalized estimating equations with a variable number of repeated measures on every millimeter of OCT images and random field histologic images of each stent were applied. The main independent variable was the type of stent implanted, whereas the covariates included the major branches of the coronary artery where the stent was implanted. The dependent variables included the CSA of the stent, lumen, and neointima on OCT images, as well as the area of the stent strut, the area of the stent strut covered by ECs, or SMCs on histologic images. If there was significance among the three groups, Bonferroni\u2019s All pigs survived the in-life phase of the study. All stents were evenly expanded at a 1:1 stent-to-vessel ratio. Angiography and OCT were performed immediately after stent implantation and at 2, 4, and 12\u00a0weeks . No signUsing confocal microscopic images, we identified stent strut endothelialization and SMC coverage, as shown in p = 0.100) or EES (62.2% \u00b1 30.7%)  . At week\u00b1 30.7%) . The SMC\u00b1 30.7%) . The are\u00b1 30.7%) .r = 0.899, p < 0.001). However, there was a very weak correlation at week 4  and no statistical significance at week 12   .p = 0.005). The sparse SMC area was significantly different at weeks 2 and 4, higher in the SES group . Interestingly, the re-endothelialization on each SMC morphology layer was significantly different. On the no/rare SMC area, approximately 1% of the area showed re-endothelialization , and dense (spindle) . SMCs inlization ; Table 3lization .This study demonstrates the difference between neointimal coverage and re-endothelialization of stents in the coronary artery and the effect of stent design on endothelialization after different types of DES implantation. Neointimal coverage of the stent struts was almost complete at 4\u00a0weeks after implantation, but re-endothelialization was not completed until week 12. Re-endothelialization seemed to be affected by the underlying SMC layer, particularly in the early phase of vascular healing, as there was a correlation between the appearance of the endothelium and the stent coverage of SMC until 4 weeks. In addition, we found no significant difference in the neointimal coverage of each type of stent tested in this study observed by the OCT. However, there was a significant difference in the endothelial coverage of the struts of each stent. The ultra-thin strut and abluminal biodegradable polymer SES showed the best re-endothelialization in the early and late phases of vascular healing. Re-endothelialization in the late phase of vascular healing seemed to be affected by the underlying SMC morphology as re-endothelialization progressed only on the sparse SMC area.Stent thrombosis is caused by delayed healing of the coronary artery. It is also affected by persistent fibrin deposition, hypersensitivity, bifurcation lesions, and stent-strut malapposition . EspeciaImaging methods, such as IVUS or OCT, are used to clinically evaluate re-endothelialization of the stent strut and assess the risk of LST indirectly as it is impossible to evaluate re-endothelialization of the coronary arteries histologically. OCT offers the highest resolution among all the currently available coronary imaging modalities , which is \u223c10 times greater than that of IVUS . FurtherOne possible indication for re-endothelialization is SMC. There was a correlation between stent coverage of the SMC and re-endothelialization until 4\u00a0weeks after implantation. SMC coverage was much faster than re-endothelialization. Therefore, re-endothelialization seemed to require an underlying SMC layer, particularly in the early phase of vascular healing. In addition, the shape of the SMC on the surface of the stent strut changed from dense and spindle to sparse and stellate over time in the present study. SMC is known to change its shape and respond to damage once vascular damage occurs. Specifically, spindle-shaped SMCs are known to transform into a stellate shape by various factors, including miRNA, to promote the recovery of blood vessels . The conStent design influences re-endothelialization and intimal hyperplasia . Blood fThis study had several limitations. First, the healing processes in humans and pigs are not the same. Neointimal responses are pronounced, and the time course of healing is five-to-six times longer in humans than in animals . This stRe-endothelialization after stent implantation was related to SMC coverage at the early phase and SMC layer differentiation at the late phase. Ultrathin-strut SES showed faster recovery than thin-strut EES or thick-strut BES. The pathophysiology of SMC and its characterization and modification should be further investigated to improve stent design to improve re-endothelialization and its clinical efficacy and safety.OCT is frequently used to clinically estimate re-endothelialization of stent struts, indirectly assessing the risk of stent thrombosis as it is impossible to test re-endothelialization of the coronary arteries histologically. In this study, we found that it is not the neointimal coverage observed on OCT that is critical, but that the SMC layer and its differentiation is essential for the re-endothelialization of the stented vascular wall. Therefore, the pathophysiology of SMC and its modification should be further investigated to improve stent design to improve re-endothelialization and its clinical efficacy and safety.In this study, the DES design affects the rapidity of SMC coverage and morphology of the SMC layer, leading to different re-endothelialization and vascular healing. Therefore, it is necessary to further renovate the coronary stent to enhance the initial proliferation and differentiation of SMC, which can improve re-endothelialization."}
+{"text": "Corylus spp.) is known as one of the four famous tree nuts in the world due to its pleasant taste and nutritional benefits. However, hazelnut promotion worldwide is increasingly challenged by global climate change, limiting its production to a few regions. Focusing on the eurytopic Section Phyllochlamys, we conducted whole-genome resequencing of 125 diverse accessions from five geo-ecological zones in Eurasia to elucidate the genomic basis of adaptation and improvement. Population structure inference outlined five distinct genetic lineages corresponding to climate conditions and breeding background, and highlighted the differentiation between European and Asian lineages. Demographic dynamics and ecological niche modeling revealed that Pleistocene climatic oscillations dominantly shaped the extant genetic patterns, and multiple environmental factors have contributed to the lineage divergence. Whole-genome scans identified 279, 111, and 164 selective sweeps that underlie local adaptation in Corylus heterophylla, Corylus kweichowensis, and Corylus yunnanensis, respectively. Relevant positively selected genes were mainly involved in regulating signaling pathways, growth and development, and stress resistance. The improvement signatures of hybrid hazelnut were concentrated in 312 and 316 selected genes, when compared to C. heterophylla and Corylus avellana, respectively, including those that regulate protein polymerization, photosynthesis, and response to water deprivation. Among these loci, 22 candidate genes were highly associated with the regulation of biological quality. Our study provides insights into evolutionary processes and the molecular basis of how sibling species adapt to contrasting environments, and offers valuable resources for future climate-resilient breeding.Hazelnut ( Corylus L. genus within Betulaceae, includes approximately 17 polymorphic deciduous tree and shrub species that produce healthful nuts and oils [Corylus originated in southwest China during the middle Eocene and has spread to Europe and North America through two long-distance dispersal routes: the Himalayan-Central Asia Corridor and Beringian Land Bridge [Hazelnut, the and oils . The genand oils . HazelnuAcanthochlamys, Siphonochlamys, Phyllochlamys, and Colurnae) have been identified within Corylus [Phyllochlamys has long been the source of hazelnut breeding because of its superior agronomic traits such as open nuts without bracts completely enclosed and easy artificial propagation. Traditionally, the section is widely recognized to contain three intercontinental disjunct taxa: European hazelnut (Corylus avellana), American hazelnut (Corylus americana), and Asian hazelnut (Corylus heterophylla species complex) [Corylus species, is distributed in the Europe and Mediterranean region, while American hazelnut occurs only in eastern North America. Asian hazelnut complex is widely distributed in East Asia and shows high genetic diversity, in which three cryptic species  were further proposed [C. avellana \u00d7 C. heterophylla) [C. avellana \u00d7 C. americana) [Phyllochlamys more complex.To date, four sections  . Europeaproposed , 11. Theproposed , 13. Dueproposed , 14, comproposed , and nucophylla)  and Nortericana) , has madC. avellana, C. americana, and C. heterophylla species complex) of Section Phyllochlamys have formed unique adaptation to different continental environments, making them the main body of hazelnut breeding in the world. This local adaptation triggered by divergent selection [C. avellana have become the most commercially valuable Corylus species around the world; however, it is confined to Mediterranean-like climates  that allow consistent yields. Unsurprisingly, the promotion of C. avellana is restricted in non-native cultivation regions, including in mainland China and eastern North America, mainly due to climate inadaptation and severe pathogens [C. heterophylla species complex spans a broad range of growing zones, climates, and soil types, suggesting that the complex holds diverse sources of stress resistance and climate adaptation [C. heterophylla complex occur successively along the altitudinal and hydrothermal gradients, forming their suitable regions in northern, central and eastern, and southwest China, respectively. However, their morphology deviates somewhat from commercial standards, such as husk enclosed shells, thick shells, and small kernels. Fortunately, interspecific hybridization between C. heterophylla and C. avellana occurs very readily, generating excellent hybrid cultivars that not only inherit the commercial traits of C. avellana, but also expand more suitable regions than C. heterophylla. Therefore, Section Phyllochlamys is an excellent system for illuminating the genetic basis of evolutionary adaptation at the intercontinental scale. However, traditional molecular markers  only produce a few variable loci, and so are generally insufficient to characterize the genetic structure of species with complex demographic history and adaptive pattern [Current hazelnut breeding programs mainly emphasize climate adaptation, and a fundamental biological question that has recently emerged concerns how hazelnut adapt to regional environments. In the long evolutionary process, these closely related taxa , European hazelnut (C. avellana), and hybrids of C. avellana \u00d7 C. heterophylla. Specifically, we aim to address the following questions: (i) characterize the genomic diversity and differentiation level among different genetic lineages; (ii) clarify the evolutionary history of C. heterophylla complex and explore the genetic footprints involved in geographic adaptation; (iii) capture the selection signals in C. avellana and hybrid hazelnut during adaptation and improvement.In order to explore genomic divergence, demographic history, and selection signatures, we here re-sequenced 125 representative accessions of Section Phyllochlamys, including 76 Asian hazelnut , 12 European hazelnut, and 37 hybrids of Asian and European hazelnuts (C. heterophylla reference genome. The mapping rate varied slightly in C. avellana (94.5%), C. heterophylla (95.5%), hybrid hazelnut (95.1%), C. kweichowensis (93.6%), and C. yunnanensis (85.8%), respectively. The visible differences in mapping rates were mainly caused by the reference genome and divergence among sequenced genotypes  was more closely to its paternal species (C. avellana) than to maternal species (C. heterophylla). Principal component analysis (PCA) also illustrated five distinct groups observed in the ML tree, and the first two axes accounted for 15.40% and 9.26% of the total variance, respectively. PC1 clearly distinguished the hybrid combination , and PC2 further separated three geo-ecotypes of Asian C. heterophylla complex  tree indicated that these 125 accessions could be divided into five species-specific clades , which w complex .K\u2009=\u20092, C. heterophylla complex and C. avellana clustered into two distinct groups, while hybrid hazelnut contained a mixture of genetic components of the former two groups , more complex admixture scenarios occurred within C. heterophylla and hybrid hazelnut.ADMIXTURE analysis revealed significant population division and extensive admixture among different groups. In the scenario of o groups . When K\u2009en K\u2009=\u20095 , which cFST to evaluate patterns of genomic diversity and genetic divergence among the five genetic lineages. The heterozygosity ranged from 0.334% to 0.855% across all accessions. As expected, hybrid hazelnut harbored the highest heterozygosity (0.822\u2009\u00b1\u20090.046%), whereas C. yunnanensis exhibited the lowest heterozygosity (0.429\u2009\u00b1\u20090.060%). C. heterophylla (0.508\u2009\u00b1\u20090.075%), C. kweichowensis (0.482\u2009\u00b1\u20090.059%), and C. avellana (0.479\u2009\u00b1\u20090.024%) showed very close and moderate heterozygosity (\u22123), followed by C. heterophylla (\u03c0\u00a0=\u20093.677\u2009\u00d7\u200910\u22123) and C. kweichowensis (\u03c0\u00a0=\u20093.540\u2009\u00d7\u200910\u22123). C. yunnanensis (\u03c0\u00a0=\u20092.691\u2009\u00d7\u200910\u22123) and C. avellana (\u03c0\u00a0=\u20092.646\u2009\u00d7\u200910\u22123) had similar but low nucleotide diversity . Within C. heterophylla complex, C. heterophylla and C. kweichowensis showed the lowest differentiation (FST\u2009=\u20090.099), whereas C. yunnanensis displayed relatively high differentiation with the former two (FST\u2009=\u20090.193 and 0.143), which further supported their evolutionary relationships in the phylogenetic tree. Within the hybrid combination, the differentiation level of hybrid hazelnut with its male parent (C. avellana) (FST\u2009=\u20090.126) was slightly lower than that with its female parent (C. heterophylla) (FST\u2009=\u20090.137) , C. avellana vs hybrid hazelnut (0.289), C. heterophylla vs hybrid hazelnut (0.268) had low and similar thresholds.Pairwise =\u20090.137) . At the r2) in all the five genetic groups (C. avellana showed the highest LD level and the slowest decay rate (19.6\u00a0kb) to half of the maximum r2 (0.326), which is in accord with the fact that the species has experienced long generations of selection and domestication in Europe and Mediterranean regions. By contrast, C. kweichowensis had the lowest LD level and the fastest decay rate (2.9\u00a0kb), which was comparable to that of C. heterophylla (3.8\u00a0kb) at the same r2 threshold (~0.19). The similar LD pattern between the two  is consistent with their low differentiation level and similar nucleotide diversity. The LD level and decay distance (4.9\u00a0kb) of C. yunnanensis were significantly higher than those of C. kweichowensis, although they showed the lowest genetic differentiation. This difference is supposed to be caused by the strong selection pressure exerted by the unique environment in southwest China. As expected, the LD level and decay distance (8.2\u00a0kb) of hybrid hazelnut were between the two parents (C. avellana and C. heterophylla).Linkage disequilibrium (LD) decay of a population can be affected by many factors, including genetic drift, mutation, selection, recombination, population structure, etc. Thus, population attributes, such as generation length, mating system, natural or domesticated population, and selection intensity, are closely related to the state of LD decay. We observed decreased signatures of genome-wide LD (estimated by c groups . C. avelNe) of four evolutionary lineages   during 0.8\u20130.1 Mya, with the divergence peak occurring at about 0.3 Mya. By contrast, the latter three lineages exhibited similar historical trajectories until 0.1 Mya, implying a more recent divergence. Although the PSMC profiles ostensibly displayed significant population changes within the last approximately 3000 generations, it could be implausible because PSMC has difficulties in evaluating very recent demographic dynamics.We performed the pairwise sequentially Markovian coalescent (PSMC) analyses to trace historical trajectory in effective population size (anensis) . The resC. heterophylla complex, and 17 three-population models with different divergence scenes, gene flow, and varying population sizes were tested . This phenomenon was largely due to the geographic isolation in glacial refugia. The divergence between C. kweichowensis and C. heterophylla was accompanied with constant and symmetric gene flow (m\u2009=\u20092.273). Hereafter, significant population expansions were observed in all three species, with the Ne values of C. yunnanensis, C. kweichowensis, and C. heterophylla estimated to be 32\u2009439, 34\u2009186, and 33\u2009023, respectively . Based on the topological trios P3)O), the ABBA-BABA statistics revealed significant introgression signal between C. kweichowensis (P2) and C. yunnanensis (P3)  (C. heterophylla (P1) and C. yunnanensis (P3).We further employed \u2202a\u2202i to simulate recent demographic history and gene flow within ectively . These pC. avellana, C. heterophylla, C. kweichowensis, and C. yunnanensis, respectively. The predicted ecological spaces of the four species were basically consistent with their actual distributions. During the glacial period from last interglacial (LIG) to last glacial maximum (LGM), C. avellana retreated to southern refuges due to extensive ice sheets in northern Europe, and then recolonized northern regions during the warm interglaciation from LGM to middle Holocene (MH). Within the Asian hazelnut complex, both C. heterophylla and C. kweichowensis showed clear southward range shifts during the transition from LIG to LGM, while C. yunnanensis exhibited significant northward expansion. This phenomenon is in line with the characteristics of global cold in northern China and favorable climate in southwest China during glacial periods. Since the MH period, all the four lineages have experienced population recovery or expansion, and basically formed their distribution patterns similar to the current predications  was constructed to predict the current and past distributions for four evolutionary clades. All models displayed high predictive ability with area under the curve (AUC) values greater than 0.95. Jackknife tests of ENMs showed that precipitation of driest month (bio14), temperature seasonality (bio4), temperature seasonality (bio4), and annual precipitation (bio12) had the highest predictive contribution of 42.9%, 31.1%, 26.7%, and 36.9% for D and I significantly lower than null distributions  , whereas slight overlap existed between parapatric species such as C. kweichowensis and C. heterophylla  or C. yunnanensis .PCA analysis of environmental data indicated strong habitat differentiation among the four species . The hieC. heterophylla complex have adapted locally through long-term selection under local environments. To identify genomic loci that favor local adaptation, we applied an integrative approach  to detect signatures of selective sweeps for each species  in C. kweichowensis, respectively  was compared to C. yunnanensis    on Chr11 (position: 1.02\u20131.06\u00a0Mb) for the latter  . We founC. heterophylla/C. avellana) to explore the adaptive signals of C. avellana under the European Mediterranean climate. The three selection tests  together identified 59 overlapped genomic regions and 109 PSGs  to probe into the improvement signatures of hybrid hazelnut relative to its parents. The three methods combined allowed us to identify a total of 161 genomic regions (312 PSGs) in hybrid hazelnut when compared to its female parent C. heterophylla, and 124 genomic regions (316 PSGs) when compared to its male parent C. avellana  and their hybrids  widely distributed and cultivated in three continents. In this study, we focused on the eurytopic C. heterophylla complex in East Asia, the most commercially valuable C. avellana in Europe, and the improved hybrid hazelnut (C. avellana \u00d7 C. heterophylla) in Mainland China, to investigate the genetic basis of evolution, adaptation, and improvement signatures in the intercontinental scale. The results could provide a valuable resource for research of hazelnut evolutionary biology and breeding, especially in terms of enhancing stress resistance.The Section C. heterophylla, C. kweichowensis, and C. yunnanensis) within C. heterophylla complex has long been unsolved, with several controversial treatments proposed: (i) both C. kweichowensis and C. yunnanensis were the botanical varieties of C. heterophylla [C. yunnanensis was treated as a distinct species, while C. kweichowensis was the variety of C. heterophylla [C. heterophylla and C. kweichowensis were supported by phylogenetic inference and low differentiation index. Besides, demographic simulation revealed that C. yunnanensis was the first to split from the species complex, while C. kweichowensis and C. heterophylla diverged from each other at a more recent scale  at that time. However, the combination of Ne dynamics  C.rophylla ; and , with the FST values ordered as C. yunnanensis\u2009>\u2009C. heterophylla\u2009>\u2009C. kweichowensis  of C. heterophylla. In line with this, we detected PSGs associated with improvement in hybrid hazelnut, including those that regulate protein polymerization (e.g. EVM0015097 and EVM0001784) and photosynthesis  (EVM0017023) and response to water deprivation (e.g. EVM0008440 and EVM0013486). These findings are compatible with the improved traits expected to be influenced by selection breeding in hybrid hazelnut.After long generations of selection and domestication, enotypes , 56. Hyb0010405) , which sPhyllochlamys were collected from the hazelnut germplasm repository of Research Institute of Forestry, Chinese Academy of Forestry , including 32 C. heterophylla, 25 C. kweichowensis, 19 C. yunnanensis, 12 C. avellana, and 37 hybrid cultivars. The 76 accessions of the three geo-ecotypes within Asian hazelnut complex basically cover the natural distribution regions in northern China, central and eastern China, and southwest China. The 12 C. avellana accessions represent six domesticated breeds widely cultivated in most European countries, and the 37 hybrid cultivars have been identified to have wider suitable regions than their female parent (C. heterophylla) in northern China  was employed to remove PCR duplicates. The mapping rate, sequencing depth, and coverage rate were calculated with the DepthOfCoverage program in GATK v4.0.5.1 [C. heterophylla reference genome [The raw reads were filtered using Trimmomatic v0.36  to remove genome  using BWe genome  with defe genome  was usedv4.0.5.1 . Single v4.0.5.1  with thev4.0.5.1  based one genome . The finhttps://itol.embl.de/). Principal component analysis (PCA) was performed using GCTA v1.26 [K) from 2 to 8 and each running 10\u2009000 iterations. The optimum number of clusters (K) was determined based on the minimum cross-validation (CV) error value.We produced a maximum likelihood (ML) tree based on the LD-pruned SNP dataset (7177893), as implemented by RaxML v8.2.12 . The ML TA v1.26  and the TA v1.26  based on\u03c0) values and pairwise fixation index (FST) were calculated for each locus using VCFtools v0.1.13 [C. heterophylla genome was assigned for window calculation of \u03c0 and FST. The interaction figure of \u03c0 and FST values of different species was drawn by Cytoscape v3.5.1 [To compare the genetic diversity and genetic differentiation among different species, nucleotide diversity ( v0.1.13 . A slidie v3.5.1 . Besidese v3.5.1 . Heteroz2r) was calculated for pairwise SNPs along 500\u00a0kb windows using the PopLDdecay v3.31 [2r dropped by half.To measure the LD level, the squared correlation coefficient (ay v3.31  in each Ne) for four evolutionary clades  was inferred by the pairwise sequentially Markovian coalescent (PSMC) model [\u03bc\u2009=\u20093.75\u2009\u00d7\u200910\u22128 per base per generation) and average generation time (g\u2009=\u00a015\u00a0years) were applied to convert the population sizes and scaled times into real sizes and times [The trajectory of effective population size (C) model . Becausend times .C. heterophylla complex under different divergence scenarios. Based on a coalescent framework, \u2202a\u2202i predicts the site frequency spectrum (SFS) of genetic variation among populations enabling statistically rigorous assessments on population size, migration rates, and divergence times [https://github.com/isaacovercast/easySFS). Based on threefold randomly perturbed starting parameters, we conducted 50 optimized replicates using the Nelder\u2013Mead method with a maximum of 20 iterations for each model included in the 3D model sets. The global maximum log-likelihood model was selected after correcting for number of estimated parameters using Akaike information criterion. To obtain 95% confidence intervals based on the best fitting parameters, simulation was carried out 100 times. The parameters estimated by \u2202a\u2202i were scaled by Ne which was calculated through the formula Ne\u2009=\u2009\u03b8/(4\u00a0\u03bcL), where \u03b8 represents the effective mutation rate of the ancestral population, \u03bc is the mutation rate (3.75\u2009\u00d7\u200910\u22128 per base per generation), and L is the genome size of C. heterophylla (370.75\u00a0M). The above parameters were then used to estimate the divergence time and population size.In order to explore the intracontinental divergence pattern, we further used \u2202a\u2202i to estimate the demographic histories of ce times . We testC. heterophylla complex, we performed Patterson\u2019s D-statistic (ABBA-BABA statistic) and the related admixture fraction estimates (f4-ratio statistics) using Dsuite software [D-statistic test was used to assess the imbalance between ABBA and BABA allele patterns. Tests were conducted with a four-taxon fixed phylogeny: given an outgroup C. avellana (O), we set three ingroup taxa of C. heterophylla (P1), C. kweichowensis (P2), C. yunnanensis (P3) with the relationship P3)O). Without introgression, conflicting ABBA and BABA patterns should arise with equal frequencies via incomplete lineage sorting, producing a D-statistic equal to 0. Whereas, if introgression between P1 or P2 and P3 has occurred, the ABBA pattern should be excessive relative to BABA, and the D-statistic deviates significantly from zero. First, we used the Dtrios program to calculate f4-ratio for all species based on the phylogeny; then, f-branch statistics were estimated for each phylogenetic branch by the Fbranch program; finally, we adopted the f-branch metric to tease apart potentially correlated f4-ratio statistics and identify introgression events between internal branches.To further explore introgression patterns between taxa of software . D-statiPhyllochlamys, we further conducted ENM to predict potential distribution for four evolutionary taxa at the current time, during the last glacial maximum , the last interglacial , and the middle Holocene . Nineteen bioclimatic variables and one elevation layer used for ENM analysis were retrieved from the WORLDCLIM database (www.worldclim.org). To avoid multicollinearity, environmental variables were subjected to redundancy removal using a threshold for Pearson\u2019s correlation coefficients (r) >0.75 [http://www.gbif.org) and field investigations, including 100 C. avellana, 131 C. heterophylla, 121 C. kweichowensis, and 46 C. yunnanensis , which covered nearly the whole natural ranges of these species. ENM analyses were performed based on the current variables and projected against the other two historical variables using the maximum entropy model in MAXENT v3.3.4 [To explore the patterns of distribution shifts within Section r) >0.75 . FinallyT v3.3.4  with thePhyllochlamys, we performed PCA based on 10 environmental variables and 400 occurrence records. The climate data were retrieved using ArcGIS 10.3 [D and the standardized Hellinger distance (I) in ENMtools v1.4.3 [D and I (0\u20131) indicate the similarity or difference of the niches, which represent no overlap and completely identical, respectively.To better elucidate the habitat differentiation within Section GIS 10.3  for all s v1.4.3 . The valFST), nucleotide diversity ratio , and the cross-population composite likelihood ratio test (XP-CLR). On the basis of potential evolutionary scenario and breeding background, two types of selection signals were investigated. First, we explored the genetic footprints of local adaptation within the Asian C. heterophylla complex. For C. heterophylla and C. kweichowensis, reciprocal comparisons were performed to investigate their adaptive divergence caused by north\u2013south climate differences. To identify positive selection signals involved in high-altitude adaptation of C. yunnanensis, we conducted a one-way comparison between lowland species (C. heterophylla) and highland species (C. yunnanensis) for identification. Second, we scanned the genomic signatures associated with adaptation and improvement within the hybrid combination . Taking Asia C. heterophylla as the control, we conducted a unidirectional comparison with C. avellana so as to explore the adaptive signals of the latter under the European Mediterranean climate. Moreover, two unidirectional comparisons (i.e. C. heterophylla/hybrid and C. avellana/hybrid) were implemented to identify the improvement signals of hybrid hazelnut relative to its parents. The statistics of FST and \u03c0 ratio were calculated with 100-kb sliding windows and 10-kb steps across the genome using vcftools [FST and \u03c0 ratio. The top 5% genomic regions identified by all the three metrics were designated as putative selective sweeps.To detect selection signals during geographic adaptation and improvement for the five species, selective sweep analyses were performed based on three genome-wide metrics, including genetic differentiation index (vcftools . For XP-P\u2009<\u20090.05) in corresponding lineages.Functional analysis for candidate genes was performed against the Gene Ontology (GO) database . FunctioWeb_Material_uhad065Click here for additional data file."}
+{"text": "Ribosomal proteins are fundamental components of the ribosomes in all living cells. The ribosomal protein uS5 (Rps2) is a stable component of the small ribosomal subunit within all three domains of life. In addition to its interactions with proximal ribosomal proteins and rRNA inside the ribosome, uS5 has a surprisingly complex network of evolutionarily conserved non-ribosome-associated proteins. In this review, we focus on a set of four conserved uS5-associated proteins: the protein arginine methyltransferase 3 (PRMT3), the programmed cell death 2 (PDCD2) and its PDCD2-like (PDCD2L) paralog, and the zinc finger protein, ZNF277. We discuss recent work that presents PDCD2 and homologs as a dedicated uS5 chaperone and PDCD2L as a potential adaptor protein for the nuclear export of pre-40S subunits. Although the functional significance of the PRMT3\u2013uS5 and ZNF277\u2013uS5 interactions remain elusive, we reflect on the potential roles of uS5 arginine methylation by PRMT3 and on data indicating that ZNF277 and PRMT3 compete for uS5 binding. Together, these discussions highlight the complex and conserved regulatory network responsible for monitoring the availability and the folding of uS5 for the formation of 40S ribosomal subunits and/or the role of uS5 in potential extra-ribosomal functions. Despite the expanding number of roles played by noncoding RNAs, proteins remain key actors involved in nearly every operation required for cellular life, from proliferation to differentiation, internal organization, intercellular communication, and cell death. In order to set in motion the synthesis of new proteins, the information encoded by genes as messenger RNAs (mRNAs) is decoded into polymers of amino acids by a highly complex cellular machine, the ribosome, in a process known as translation. The ribosome is one of the most important ribonucleoprotein complexes in the cell, as demonstrated by its essential role in protein synthesis, its highly conserved nature, and its dominating abundance in most cell types. In fact, the fundamental structure and function of the ribosome were highly conserved throughout the evolution from bacteria to humans ,2. SinceThe 80S ribosome is a large RNA\u2013protein complex with a molecular mass of 4.3 megadalton in humans -x(8)-H-x-H) and three containing atypical consensus motifs. Similar to PRMT3, the interaction between uS5 and ZNF277 depends on the integrity of its zinc finger domains, especially the two most C-terminal zinc fingers of ZNF277 . FurtherC. elegans shows that the homolog of human ZNF277, ZTF-7, as well as 40S RPs are required for the nucleolar depletion of the RNA exosome after a cold shock [S. cerevisiae does not appear to code for a protein with homology to ZNF277, whereas a ZNF277 homolog is found in the S. pombe genome.To date, the molecular and cellular function of ZNF277 remains unclear. The homolog of human ZNF277 in mice, Zfp277, was shown to function as a transcriptional regulator  and to ild shock . As the ZNF277 overexpression is associated with improved prognosis of human cancers according to the Human Protein Atlas Project [PRMT3 overexpression appears to be associated with poor prognosis, uncovering the process by which ZNF277 and PRMT3, two C2H2-type zinc finger proteins, compete for uS5 binding is likely to have relevance to cancer biology. As the PRMT3\u2013uS5 complex is exclusively cytosolic [Since the initial discovery of the first non-ribosomal uS5-associated protein in fission yeast almost twenty years ago , studies Project , whereasytosolic , why wouytosolic ?Further studies will also be required to clarify the role of uS5 in the nuclear export of pre-40S particles ,11,32 an"}
+{"text": "Then, a study was conducted on the dissipation of prochloraz in strawberries under greenhouse conditions. The dissipation of prochloraz in strawberries followed the first-order kinetic equation, and its half-life was 8.06 days. The health risk associated with prochloraz in strawberries was evaluated using the target hazard quotient (THQ) method and EFSA PRIMo model. The results showed that the THQ values, %ARfD values, and %ADI values were less than 1. These results indicate that no health concerns of prochloraz are associated with the consumption of the studied strawberries. The government can use the results of this study to support the establishment of a maximum residue level for prochloraz in strawberries.Prochloraz and its metabolites in strawberries have not been determined until now. Meanwhile, few reports in the literature have concerned the dissipation behavior and risk assessment of prochloraz and its metabolites in strawberries under greenhouse conditions in Beijing. A method for the determination of prochloraz and its metabolites in strawberries was developed using QuEChERS in combination with ultra-performance liquid chromatography\u2013tandem mass spectrometry (UPLC-MS/MS). Prochloraz and its metabolites recovered from strawberries were present in concentrations of 73.06% to 116.01%, their RSDs ranged from 1.12% to 9.17%, and their limits of detection ranged from 0.1 to 1 \u03bcg kg Prochloraz is a highly effective and broad-spectrum imidazole fungicide used in crop growth to provide preventive protection and curative functions. In addition, it is used for the storage and preservation of fruits and vegetables ,2,3. ProN\u2032-formyl-N-propyl-N-[2-ethyl]urea (BTS44596) and N-propyl-N-[2-ethyl]urea (BTS44595). These two compounds can break the carbon\u2013oxygen bond and degrade into 2,4,6-trichlorophenol (BTS45186), which is a free and conjugated metabolite. There are also traces of 2\u2013\u2013acetic acid [\u22121, respectively [Fragaria and Myrica rubra, and the LOD was \u22643 \u03bcg kg\u22121 [\u22121 [\u22121 [In plants, prochloraz breaks its imidazole ring and produces its main metabolites , includitic acid ,13,14,15tic acid . The strtic acid . An extrnol BTS456, which  \u03bcg kg\u22121 . Tian etkg\u22121 [\u22121 . Radio-U [\u22121 [\u22121 . NeverthUntil now, prochloraz dissipation has been reported in previous research. It is important to note that the dissipation rate of prochloraz is affected by various factors. Its half-life varies with its matrix. A study reported that the dissipation half-life of prochloraz was 8.89\u201318.02 days and 5.79\u201312.38 days in soil and apple, respectively . It was Therefore, this study aims to establish an efficient, sensitive, accurate, and reliable analytical method based on QuEChERS combined with UPLC-MS/MS to determine prochloraz and its metabolites simultaneously. In this study, the dissipation kinetics of prochloraz in strawberries was studied under greenhouse conditions in Beijing, and the dietary risk was assessed. Based on the results of this research, supporting data could be provided for setting a maximum residue limit (MRL) of prochloraz in strawberries in China to ensure food safety.\u22121 single standard solution. After the liquid phase flow rate was set to 0.3 mL min\u22121, the standard was injected into the mass spectrum directly. Then, the tuning function of Masslynx 4.1 software was applied to find parent and daughter ions of the target compounds. The optimal mass spectrum parameters of each ion were fixed by optimizing the cone voltage, collision energy, retention time, etc., as shown in Special consideration was given to the optimization of the MS/MS parameters to enable highly sensitive and reliable quantification for prochloraz and its metabolites. Four target compounds were prepared in a 100 \u03bcg LOptimization of the chromatographic column and mobile phase is an effective means of separating and characterizing prochloraz and its metabolites ,25,26. A\u22121. The study results indicated that the method was linear, and the determination coefficient R2 was higher than 0.9969. LOD was defined as the concentration of analyte detectable at a signal-to-noise ratio of 3:1. The LOD of prochloraz, BTS44595, BTS44596, and BTS45186 in strawberries was 0.1 \u03bcg kg\u22121, 0.1 \u03bcg kg\u22121, 0.1 \u03bcg kg\u22121, and 1 \u03bcg kg\u22121, respectively  of five replicate analyses on the same day, while within-laboratory reproducibility was determined on five consecutive days by three operators at three fortification levels. The RSDs were 1.12\u20139.17%, which indicates that this method could be used to determine prochloraz and its metabolites in strawberries. The MRM images of prochloraz and its metabolites are shown in The linearity, sensitivity, precision, and accuracy of the method were validated according to European Commission SANTE/11312/2021 . A matriectively . The lowIn summary, the validation of this method  demonstr2 of the dissipation equation was 0.995. The half-life of prochloraz in strawberries was 8.06 days according to Formula (2). This result is similar to the half-life of prochloraz obtained in pear peel [Spraying in the field experiment for prochloraz EC was carried out three times over a 35 d period. The first and second sprayings were insufficient to fit the curve. Therefore, the dissipation of prochloraz in strawberries under greenhouse conditions after the third spraying was analyzed. The kinetic equation of prochloraz dissipation in strawberries is shown in ear peel .C0) was 185.3 \u03bcg kg\u22121. On the 35th day, the concentration reduced to 24.3 \u03bcg kg\u22121. The main metabolite of prochloraz is BTS44596. After prochloraz EC spraying, the BTS44596 concentration showed an increasing trend in the first three days; then, it decreased. Its residue was 169.2 \u03bcg kg\u22121 on the 35th day. The concentration of BTS44595 residue was lower than the LOD within the first 5 days after spraying, and the concentration was 1.7 \u03bcg kg\u22121 on the first day after the second spraying. The compound concentration reached the highest value of 5.1 \u03bcg kg\u22121 on the 15th day, and the residual concentration was 2.2 \u03bcg kg\u22121 on the 35th day. The concentration of BTS45186 decreased to 11.9 \u03bcg kg\u22121 on the 35th day. The sum of prochloraz, BTS44595, BTS44596, and BTS45186 residues was 195.7 and 101.6 \u03bcg kg\u22121 in the high- and low-concentration groups, respectively. The residue concentrations were higher than the 30 \u03bcg kg\u22121 specified in the EU standards for residue.In this study, the spraying of 25% prochloraz EC at different concentrations (0.015 kg a.i./ha and 0.01 kg a.i./ha) in strawberries was conducted under greenhouse conditions. After spraying, prochloraz in strawberries dissipated, while BTS44595, BTS44596, and BTS45186 were generated, as shown in The dissipation of prochloraz metabolites in strawberries is a dynamic process that is affected by their formation rate and dissipation rate. In strawberries, residual concentrations of prochloraz metabolites decrease when the generation rate is lower than the decomposition rate, and vice versa. The formation and degradation rates of chemicals in crops are affected by many factors, such as ambient temperature, humidity, and light ,29,30. T\u22124 and 1.5 \u00d7 10\u22124, respectively. In the second approach (EFSA PRIMo), the ARfD and ADI of prochloraz were set at 0.025 and 0.01 mg/kg bw/day, respectively, by EFSA [The prochloraz concentration of the low-concentration spraying group on the 35th day was used for analyzing the dietary risk assessment. The dietary risk of prochloraz and its metabolites in strawberries is related to the consumer\u2019s age, daily intake, and body weight, and pesticide residues, as shown in  by EFSA . The calProchloraz and its metabolites BTS44595, BTS44596, and BTS45186 were purchased from Dr. Ehrenstorfer ; LC-MS-grade methanol, acetonitrile, ammonium formate, and ammonium acetate were purchased from Thermo Fisher Scientific ; and analytical-grade magnesium sulfate and sodium chloride were purchased from Aladdin .The experiment was carried out at the Tongzhou Yujiawu strawberry experimental base of the Institute of Forestry, Beijing Academy of Agriculture and Forestry Sciences, in 2021\u20132022. The trials were conducted according to the \u201cGuidelines for the testing of pesticide residues in crops NY/T 788-2018\u201d . We spraIn accordance with the strawberry sample collection requirements, strawberries of consistent growth maturity were randomly sampled at no less than 12 sampling points throughout the strawberry plots. Each strawberry sample collected in each plot should not be less than 1 kg. The strawberry samples were then homogenized. All the homogenized samples were sealed and stored at \u221220 \u00b0C.4 and 1 g of NaCl were added to the sample, which was vortexed for 1 min. The sample was vibrated mechanically for 30 min, and then, centrifuged for 5 minutes at 5000 rpm. An amount of 1 mL of supernatant was sampled, and d-SPE adsorbent  was added to the samples, which were vortexed for 1 min and centrifuged at 10,000 rpm for 3 min. The supernatant was passed through a 0.22 \u03bcm filter membrane  and transferred to a 2 mL microvial for UPLC-MS/MS analysis.Strawberry samples were pretreated using the QuEChERS method, as in our previous work . A 10 g \u22121, 40 \u2103, and 5 \u03bcL, respectively. The TQS was operated in positive and negative ion modes, with the following settings: ESI+/ESI\u2212 of 3 kV/1 kV; source temperature, 150 \u00b0C; desolvation temperature, 400 \u00b0C; flow of desolvation gas, 800 L h\u22121. Optimized multiple-reaction monitoring (MRM) mode was chosen to detect target compounds. The acquisition parameters are summarized in UPLC-MS/MS analysis was performed using a Waters UPLC system interfaced with a triple quadrupole mass spectrometer (TQS). An ACQUITY UPLC HSS T3 column  was selected for the analysis. Water + 5 mM ammonium acetate (A) and acetonitrile (B) were used as the mobile phase with the following gradient program. Initially, 20% B at 0 min, 100% B at 3 min, 100% B at 5 min, 20% B at 5.5 min, and20% B at 7 min. Flow rate, column temperature, and injection volume were 0.3 mL min\u22121) in triplicate. Accuracy was determined in the recovery experiments using five replicates at three  fortification levels. LOD was defined as the concentration of analyte detectable at a signal-to-noise ratio of 3:1. LOQ was estimated as the lowest fortified concentration yielding suitable recovery and precision. Precision was expressed as the relative standard deviation (RSD) of five replicate analyses on the same day, while within-laboratory reproducibility was determined on five consecutive days by three operators at three fortification levels.The linearity, accuracy, limit of detection (LOD), limit of quantification (LOQ), and precision of the UPLC-MS/MS method were validated according to European Commission SANTE/11312/2021 . LineariThe concentration of prochloraz is equal to the sum of the residues of prochloraz, BTS44596, and BTS44595 according to the definition of the European commission .Strawberry samples were collected at specified sampling times after the spraying of prochloraz. Then, prochloraz residue was determined in the samples. The dissipation behavior of prochloraz was described using the first-order kinetic equation:t (d);\u22121);k is the dissipation rate constant (d\u22121).The dissipation half-life of prochloraz was obtained from:Risk assessment was calculated using two different approaches. In the 1st approach, the THQ method was used to evaluate the health risk of prochloraz according to the following equations.\u22121 d\u22121). EDI was calculated using the following formula:The estimated daily intake (EDI) of pesticide was expressed as milligrams of pesticide intake per kilogram of body weight per day by eating strawberries (mg kgC is the residue concentration in strawberry samples (mg kg\u22121).\u22121 d\u22121). The average daily fruit intake for children and adults was 80.4 and 64 g d\u22121, respectively [ectively .BW is the average body weight (kg). The average weights of children and adults were 32.7 and 55.9 kg, respectively .The target hazard quotient (THQ) was calculated using Equation (4):\u22121, which refers to the regulation on prochloraz in the GB 2763-2021 National Food Safety Standard Maximum Residue Limit of Pesticides in Food [The ADI value is 0.01 mg kg in Food . If the In the 2nd approach, a standard deterministic risk assessment model, EFSA PRIMo revision 3.1, was applied to estimate the health risks for acute/chronic dietary exposure to pesticides . In thisA simple, sensitive, and efficient method that combines the QuEChERS method and UPLC-MS/MS to simultaneously and quantitatively determine the residues of prochloraz and its metabolites in strawberries was established in this study. The proposed method with high recovery and accuracy is suitable for monitoring prochloraz and its metabolites in strawberries from a greenhouse. Prochloraz and its metabolites in strawberries were rapidly degraded following first-order kinetics models, with dissipation half-lives of 8.06 days under greenhouse conditions. The health risk associated with prochloraz in strawberries was evaluated using the THQ method and EFSA PRIMo model. In the risk assessment, the THQ values, %ARfD values, and %ADI values of Chinese children and adults were less than 1, indicating that no obvious health concerns of prochloraz are associated with the consumption of the studied strawberries. However, this study only investigated the dissipation of prochloraz and its metabolites in strawberries. Farmers may use multiple pesticides to control strawberry diseases. Therefore, the dissipation and risk assessment of prochloraz combined with other pesticides used in strawberries should be studied further."}
+{"text": "Adavosertib may alter exposure to substrates of the cytochrome P450 (CYP) family of enzymes. This study assessed its effect on the pharmacokinetics of a cocktail of probe substrates for CYP3A (midazolam), CYP2C19 (omeprazole), and CYP1A2 (caffeine).Period 1: patients with locally advanced or metastatic solid tumors received \u2018cocktail\u2019: caffeine 200\u00a0mg, omeprazole 20\u00a0mg, and midazolam 2\u00a0mg (single dose); period 2: after 7- to 14-day washout, patients received adavosertib 225\u00a0mg twice daily on days 1\u20133 (five doses), with cocktail on day 3. After cocktail alone or in combination with adavosertib administration, 24-h pharmacokinetic sampling occurred for probe substrates and their respective metabolites paraxanthine, 5-hydroxyomeprazole (5-HO), and 1\u2032-hydroxymidazolam (1\u2032-HM). Safety was assessed throughout.0\u201312), respectively; AUC0\u2013t increased by 61%, 98%, and 55%. Maximum plasma drug concentration (Cmax) increased by 4%, 46%, and 39%. Adavosertib co-administration increased 5-HO and 1\u2032-HM exposure by 43% and 54% (AUC0\u201312) and 49% and 58% (AUC0\u2013t), respectively; paraxanthine exposure was unchanged. Adavosertib co-administration decreased Cmax for paraxanthine and 5\u2013HO by 19% and 7%; Cmax increased by 33% for 1\u2032-HM. After receiving adavosertib, 19 (63%) patients had treatment-related adverse events (six [20%] grade\u2009\u2265\u20093).Of 33 patients  receiving cocktail, 30 received adavosertib. Adavosertib co-administration increased caffeine, omeprazole, and midazolam exposure by 49%, 80%, and 55% (AUCAdavosertib (225\u00a0mg bid) is a weak inhibitor of CYP1A2, CYP2C19, and CYP3A.NCT03333824The online version contains supplementary material available at 10.1007/s00280-023-04554-3. Cyclin-dependent kinase 1  drives a cell from the G2 phase of the cell cycle into mitosis. In response to DNA damage, the nuclear tyrosine kinase Wee1 inhibits CDK1 to prevent the cell from dividing until the damaged DNA is repaired (G2 checkpoint arrest) ) and accuracy (percentage bias) for the quality control (QC) samples were\u2009\u2264\u20097.0% and\u2009\u2264\u20097.5% CV and within\u2009\u2212\u20093.1% to\u2009\u2212\u20091.8% and\u2009\u2212\u20094.9% to\u2009\u2212\u20091.8% bias for caffeine and paraxanthine, respectively. The lower limit of quantification (LLOQ) was 25.0\u00a0ng/mL for caffeine and paraxanthine.Plasma concentrations of caffeine and paraxanthine collected with KPlasma concentrations of omeprazole and 5-HO were collected with lithium heparin as anticoagulant and were determined by HPLC\u2013MS/MS detection simultaneously. The standard curves of omeprazole and 5-HO both ranged from 20.0 to 20,000\u00a0nmol/L. Precision (%CV) and accuracy (percentage bias) for the QC samples were\u2009\u2264\u20093.9% and\u2009\u2264\u20094.4% CV and within 0.0\u2009\u2212\u20099.0% and\u2009\u2212\u20091.9% to 2.2% bias for omeprazole and 5-HO, respectively. The LLOQ was 20.0\u00a0nM for omeprazole and 5-HO.2EDTA as anticoagulant and were determined by HPLC\u2013MS/MS detection simultaneously. The standard curves of midazolam and 1\u02b9-HM both ranged from 0.100 to 100\u00a0ng/mL. Precision (%CV) and accuracy (percentage bias) for the QC samples were\u2009\u2264\u20096.4% and\u2009\u2264\u20095.1% CV and within\u2009\u2212\u20090.8% to 1.0% and\u2009\u2212\u20091.0% to 0.0% bias for midazolam and 1\u02b9-HM, respectively. The LLOQ was 0.1\u00a0ng/mL for midazolam and 1\u02b9-HM.Plasma concentrations of midazolam and 1\u02b9-HM were collected with KThe concentration of adavosertib in human plasma was determined by Labcorp Bioanalytical Laboratories using the same validated method described previously . The assAll plasma samples were stored at \u2212\u00a070\u00a0\u00b0C (or below) prior to analysis and analyzed within the validated time frame.Safety was appraised throughout the study by the assessment of clinical and laboratory adverse events , physical examination, and evaluation of vital signs and laboratory data .No formal sample size estimation was conducted. Based on an estimate of within-patient standard deviation of 0.294 and assuming a true interaction effect of 100%, it was estimated that 20 evaluable patients would provide 80% power to show that the 90% confidence interval (CI) for the adavosertib effect lies entirely below 2.67, i.e. it would rule out a 167% increase of exposure in the presence of adavosertib. The number of patients was based on careful clinical consideration to gain adequate information on the primary endpoints while exposing as few patients as possible to study procedures. Enrollment of approximately 30 patients, with a target of 20 evaluable patients, was considered adequate and sufficient to meet the objectives of this study.The safety analysis set included all patients who received at least one dose of study treatment (adavosertib or probe cocktail drugs). Patients were evaluated according to the treatment received. A treatment-emergent AE was defined as an AE with its start date and time on or after the first dose of probe cocktail on day\u00a0\u2212 8 up to and including 30\u00a0days after the last dose date of adavosertib, or, for pre-existing pre-treatment AEs, the date on which they worsened in severity after the first dose of study treatment. The PK analysis set included all dosed patients who had at least one quantifiable plasma concentration for any of the cocktail drugs (or metabolites) or adavosertib collected post-dose without protocol deviations or events that could affect the PK analysis.\u00ae WinNonlin\u00ae version 6.4 . All descriptive and inferential statistical computations were performed using SAS\u00ae version 9.1 . Estimates of the mean difference between treatments  and corresponding 90% CIs were calculated using a linear mixed-effects model, with a fixed effect for treatment and a random effect for patient. The natural-log-transformed PK parameters  of the cocktail parent compounds and metabolites were used in the mixed-effects models as dependent variables. The mean differences and CIs were back transformed to the original scale to give estimates of the ratios and the associated 90% CIs; additionally, back-transformed geometric means, together with 95% CIs, were estimated and are presented by treatment.PK parameters were derived using non-compartmental methods with Phoenix0\u201312 and Cmax obtained following adavosertib administration on period 2\u00a0day 3 were compared with those obtained on period 2\u00a0day 3 following the same adavosertib dosage schedule but without cocktail  to probe the potential effects of the cocktail drugs and to obtain an estimate of intra-patient adavosertib variability.Results for AUCOf 57 patients enrolled , 33 were assigned to treatment , CYP2C19 (omeprazole), and CYP3A (midazolam) and their metabolites  in the presence and absence of adavosertib are shown in Table 0\u201312) to 61%  to 98% (AUC0\u2013t) and increased Cmax by 46%  versus adavosertib alone (part B), and the individual and geometric mean AUC0\u2013t and Cmax of caffeine, omeprazole, and midazolam alone versus each cocktail drug plus adavosertib, are shown in Fig.\u00a0The individual and geometric mean AUCThe geometric mean plasma concentrations of caffeine, omeprazole, and midazolam versus time, with and without adavosertib, are shown in Fig.\u00a00\u201312 and Cmax geometric least-squares (LS) mean ratios were 108% and 97%, respectively. Accumulation over 2.5\u00a0days of bid dosing with adavosertib was approximately 2.3 and 2.1 for AUC0\u201312 and Cmax, respectively. The adavosertib concentration in human plasma ranged from 4 to 2000\u00a0nM. Precision (%CV) was\u2009\u2264\u20094.2% and accuracy (percentage bias) was within 0.7\u20131.5% for the QC samples. Reproducibility was confirmed by re-analysis of 87 study samples (with adavosertib concentrations above the LLOQ) selected at random; 98.9% of the results obtained following the initial and repeat analyses were within 20.0% of the mean of the two values, thus meeting the acceptance criteria [The PK parameters of adavosertib following co-administration of cocktail are shown in Table criteria .Table 3Sn\u2009=\u200926 patients) were diarrhea and nausea (both in four [15.4%] patients) and dizziness (in two [7.7%] patients). The most common treatment-related AEs in patients receiving adavosertib alone (n\u2009=\u200930 patients) were diarrhea (in 10 [33.3%] patients), vomiting (in six [20.0%] patients), and nausea (in three [10.0%] patients).Causally related AEs (stratified by any grade and grade\u2009\u2265\u20093) are shown in Supplementary Table 2 for patients who received only adavosertib and patients who received both adavosertib and the probe cocktail. Four of 33 (12.1%) patients experienced AEs with cocktail alone, compared with 16/26 (61.5%) patients who received probe cocktail plus adavosertib and 16/30 (53.3%) patients after receiving adavosertib alone on days 1 and 2. Treatment-related AEs were reported by 12/26 (46.2%) patients receiving probe cocktail plus adavosertib and 11/30 (36.7%) patients after receiving adavosertib alone on days 1 and 2. The most common treatment-related AEs in patients receiving probe cocktail plus adavosertib (Adverse events of CTCAE grade\u2009\u2265\u20093 were observed in: 4/26 (15.4%) patients receiving probe cocktail plus adavosertib ; 5/30 (16.7%) patients receiving adavosertib alone on days 1 and 2  patients]); and 0/33 patients receiving cocktail alone.One patient receiving adavosertib alone on days 1 and 2 reported two serious AEs (SAEs) considered related to adavosertib  that led to discontinuation; four patients reported five SAEs that were not considered related to treatment .Two patients discontinued treatment because of AEs: one as a result of grade 3 diarrhea considered related to adavosertib, which resolved, and the other for grade 3 small-bowel obstruction, which was not considered related to adavosertib and did not resolve.No AEs with an outcome of death were observed; one patient died as a result of disease progression (pancreatic cancer) following receipt of the probe cocktail but prior to receiving adavosertib.0\u201312 and Cmax), and adavosertib accumulation over 2.5\u00a0days of bid dosing was approximately twofold. The detailed PK data from this study reveal that the point estimates of the AUC0\u201312 and AUC0\u2013t geometric LS mean ratios of all three probe cocktail drugs in the presence of adavosertib (225\u00a0mg bid) compared with probe cocktail drugs administered alone showed a 1.25- to\u2009<\u2009twofold increase [In this study, adavosertib exposure, when given alone or co-administered with the cocktail drugs, was similar to that observed in previous studies when given alone . Therefoincrease . Adavoseincrease .A physiologically based PK (PBPK) model  to assess multifaceted CYP modulation predicted that adavosertib is mainly metabolized by CYP3A with an FMO3 and/or FMO5 component and is a time-dependent (irreversible) inhibitor of CYP3A and a weak reversible (direct) inhibitor of CYP2C8, CYP2C9, and CYP2C19 , 24. ModThe observed safety profile of adavosertib in this study was concordant with that found in previous clinical studies , 15. Themax were especially prevalent for caffeine and paraxanthine, despite the study design including washout periods and restriction of caffeine consumption. A potential explanation for this finding is that caffeine/paraxanthine elimination may be slower in this patient population than in participants in previous DDI studies; also, and perhaps the most likely explanation, patients may not have been strictly compliant with the caffeine restrictions in the protocol. The true exposure increase when caffeine/paraxanthine were co-administered with adavosertib may therefore be lower than reported here. Compared with the recommended phase II dose for monotherapy  and the maximum tolerated dose of monotherapy , both of which were determined after the present study commenced, the adavosertib dose in this study (225\u00a0mg bid) was higher and the dosing duration (2.5\u00a0days) sufficient to reach steady state; therefore, DDIs at the recommended phase II dose for monotherapy are not expected to be worse than observed during this study [Although the study was terminated early because of the difficulty in enrolling patients (i.e. screening/rescreening failures) and evaluable data not being available for all enrolled patients (as a result of protocol deviations/events), this had minimal impact on the interpretation of the PK results. Overall, the data from evaluable patients were adequate and representative of the primary objective of assessing the DDI potential of adavosertib. Instances of pre-dose concentrations\u2009>\u20095% of COverall, the results from this study suggest that adavosertib is a weak inhibitor of CYP1A2, CYP2C19, and CYP3A according to the US FDA guidance for definition of a weak inhibitor . AlthougAdavosertib, when administered at a dose of 225\u00a0mg bid, is a weak inhibitor of CYP1A2, CYP2C19, and CYP3A and therefore has a low risk of causing clinically significant DDIs with drugs metabolized by these enzymes.Supplementary file1 (PDF 304 kb)Below is the link to the electronic supplementary material."}
+{"text": "The percentage of killed bacteria was not correlated with the depths in any group (p\u2009=\u20090.633).The percentage of dead bacteria was higher both in the NaOCl  and in the hClO2 until at least the measured 950\u00a0\u03bcm. However, both were only able to eradicate the intratubular bacteria partially.Our results suggest that the functional penetration depth of NaOCl is at least 2\u20133 times more than published to date. There is no difference in disinfection effectiveness along the dentin tubules between NaOCl and hClO2 could be used as an alternative or final adjuvant irrigant in endodontic treatment.Hyper-pure ClOThe online version contains supplementary material available at 10.1186/s12903-023-03685-6. Root canal treatment of infected teeth aims to completely eradicate pathogenic microorganisms embedding and penetrating the root canal system before the final obturation. Mechanical preparation alone has proven to be inefficient due to the complexity of the root canal system . ConsequEnterococcus faecalis  is an opportunistic pathogen in the oral cavity [E. faecalis, and 24 to 77% of teeth with persistent infection present cultivable, that is viable E. faecalis [l cavity . It is al cavity . It is rl cavity . Four tofaecalis . TherefoE. faecalis had higher resistance to NaOCl than did other microorganisms [The gold-standard disinfectant irrigant solution in endodontics is still sodium hypochlorite (NaOCl). Commercial products of NaOCl are most commonly around pH 12 showing strong base characteristic. At this pH the solution contains 5 to 12% free available chlorine. These strong oxidizing agents that react with aminoacids, peptides, proteins, lipids, and DNA by attacking the C\u2009=\u2009C double bonds . NaOCl hrganisms .2) has been used as a disinfectant in the food industry and water treatment since 2008 [2 (hClO2) [2 desinfectants. This hyper-pure solution does not contain any acidic by-products from manufacturing as ClO2 solutions prepared with other processing technologies. As a true gas solution, it is very volatile [2 is limited. Due to the short contact time and its reactiveness, it could kill a bacterial cells, but not harm human cells, which, on the other hand, also have a natural protection mechanisms such as the antioxidant glutathione [E. faecalis, irrigation with hClO2 showed significantly less rebound of bacterial count in the samples after 2 and 5 days of temporization than NaOCl [Chlorine dioxide   to avoidvolatile \u201323. It rtathione . Studiestathione . In extran NaOCl . On the an NaOCl .2, we hypothesized that it could exert its antimicrobial efficacy at least as deep as NaOCl along the dentin tubules and at least with the same effectiveness.Currently, no method or irrigant solution can achieve total elimination of biofilm from the root canal system. The biocompatibility of the gold-standard NaOCl is also of concern especially if used in regenerative endodontics . Based o2 along the dentin tubules and to compare their antimicrobial effectiveness at various depths in an in vitro root segment model.We aimed to determine the depth of antimicrobial activity of both NaOCl and hClOThe distal root of 27 extracted lower mandibular molar teeth with no prior endodontic treatment were collected. Patient consent was obtained for the in vitro use of the teeth according to conditions set by the Regional Ethical Committee of Scientific Affairs (SE-RKEB 205/2021). The teeth were stored in saline at 4\u00a0\u00b0C until use.\u00ae blue file  1\u00a0mm shorter than the apical foramen. In order to open the dentin tubules and to remove smear layers from both endings, they were immersed in 17% EDTA  for 1\u00a0min. Additionally, it was stimulated by sonic intracanal activation using the EDDY system  for 60\u00a0s. Next, the roots were washed with distilled water, dried, and embedded in Eppendorf tubes . They were subsequently autoclaved at 121\u00a0\u00b0C for one hour in saline . Three roots were randomly chosen to be kept sterile as the first absolute controls in group 1 (n\u2009=\u20093)  centrifugation protocol [The teeth were prepared for inoculation by the modified protocol of Andrade et al. . Brieflylis ATCC ,212 cent2   in the depth of working length minus 1\u00a0mm. The irrigants were manually activated by #15 K-file  for another 30\u00a0s. After eight minutes, the irrigation was repeated. A total of 4 mL irrigant solution was used for each root for an effective time of 10\u00a0min. The disinfectant reaction was stopped by a final rinse with 2 mL saline.After four days of incubation, completed with a centrifugation procedure, the culturing BHI medium  was removed. The remaining medium was washed out with 2 mL saline. The dentin tubules and the apical foramen were sealed from the outside of the root by covering the outer surface of the roots with two layers of nail polish. We waited two minutes between each layer for the drying of the nail polish. The teeth were randomly divided into four groups. The second absolute control group 2 (n\u2009=\u20093) received no disinfection. Group 3 was irrigated with saline (n\u2009=\u20097), group 4 with 5% NaOCl (n\u2009=\u20097), group 5 with 0.12% hyper-pure ClOTM Viability stain  for 20\u00a0min according to the protocol of Andrade et al. [A groove was prepared by a diamond disc along the axis of the root on the mesial side until the root canal. A blade was fit in the groove, then the roots were longitudinally split with a hammer and were mounted on glass slides. The split surface to be stained and examined was cleaned with 17% EDTA for 10\u00a0min to remove the smear layer. The samples were then washed with saline and dried. They were stained with 30 \u00b5L of LIVE/DEAD\u00ae BacLighte et al. . The viae et al. . The scaThe fluorescence of dead and live bacteria was measured at the following distance intervals starting from 50\u00a0\u03bcm from the root canal surface: 50\u2013150\u00a0\u03bcm, 150\u2013250\u00a0\u03bcm, 250\u2013350\u00a0\u03bcm, 350\u2013450\u00a0\u03bcm, 450\u2013550\u00a0\u03bcm, 550\u2013650\u00a0\u03bcm, 650\u2013750\u00a0\u03bcm, 750\u2013850\u00a0\u03bcm, 850\u2013950\u00a0\u03bcm  compared to group 4 . The effect size was estimated from the result of a previous study using the same method [2. The required sample size was estimated by the G*power . The alpha was set to 0.05, and the power was 0.80 (1-\u03b2). The calculations revealed that six samples per group were needed to detect a 50% drop in viable cells.The primary outcome was to detect a significant decrease in the percentage of viable bacteria in group 5 . Values in the text and graph are given as a mean\u2009\u00b1\u200995% confidence interval.2) are shown in Fig.\u00a0In the first absolute control group, no bacteria were found in the dentin tubules Fig.\u00a0. A, B. HBoth disinfectants penetrated dentin tubules at least 950\u00a0\u03bcm, as seen in the bar graph Fig.\u00a0..The linear regression equation for each irrigant was calculated from the model. The interaction between penetration depth and irrigants (p\u2009=\u20090.893) and the main effect of penetration depth (p\u2009=\u20090.633) were insignificant. As seen in Fig.\u00a0NaOCl = 42.7\u2009+\u20090.005x.yClO2 = 42.2\u2009+\u20090.006x.ysaline = 23.2\u20130.001x.y2  was significantly higher than that of saline (23\u2009\u00b1\u20094.5%). No significant difference was found between the overall effectiveness of NaOCl and hClO2  and hClO2 seems to fulfill these criteria.The basic requirements of endodontic irrigant solutions are (1) to be able to penetrate into the site to be disinfected, (2) to suppress the growth of pathogenic microorganisms, (3) to have an effective concentration on pathogenic microorganisms, but at the same time to be non-toxic to human cells and (4) to disinfect without pathogenic microorganisms developing resistance against it . Hyper-pE. faecalis successfully at least 950\u00a0\u03bcm deep. Furthermore, the NaOCl and hClO2 killed bacteria at the measured 850\u2013950\u00a0\u03bcm. Contrarily, a previous study using a dye bleaching technique showed 300\u00a0\u03bcm diffusion of NaOCl along the dentin tubules [In the root segment model, the dentin tubules were inoculated by  tubules . Other s tubules , 35- dem tubules , 31\u201332 ( tubules . Further2 is only 0.12%, which is an order of magnitude less than that of 2.5-5% NaOCl. In an in vitro study, after irrigation and temporization of the roots, it was able to reduce the regrowth of bacteria after both 2 and 5 days following disinfection [2 was much more effective on E. faecalis than NaOCl due to its gas and redissolved phases.The clinically relevant concentration of hClOnfection , showingnfection , 26 indi2 was still similarly effective as NaOCl on E. faecalis in the same depth of dentin tubules, even in a smaller concentration. Both disinfectants were statistically more effective than saline, but unfortunately, they could only partially eradicate the intratubular bacteria. Considering the natural background amount of dead bacteria seen by irrigation with saline, the effective antibacterial effect of both tested irrigants was around 22%. A stronger antimicrobial effect of both irrigants was expected. The inability of hClO2 to interact with other irrigant solutions [The effective antibacterial concentration in endodontics showed no toxic effect on human periodontal ligamental stem cells compared to NaOCl . Nevertholutions , its higolutions , and itsolutions \u201326, 36 sE. faecalis by centrifugation. Previously, the unsatisfactory and heterogeneous inoculation resulted in poor reproducibility; therefore, several modifications have been made since its introduction [2 react with inorganic dental tissues, thereby preventing a decrease in their gradient [The patency of the dentin tubules of the pre-sterilized teeth used in the artificially infected model showed significant heterogeneity in bacterial presence. Both ends of the tubules were opened up to facilitate the inoculation of oduction , 37. Theoduction , the denoduction \u201340, and oduction \u201343. Thesoduction . The limoduction , 45. Scloduction \u201345. A higradient , 46.2 until at least the measured 950\u00a0\u03bcm, but both were unfortunately only able to eradicate the intratubular bacteria partially. Hyper-pure ClO2 was similarly effective as NaOCl, but in ten times less concentration, therefore, due to its extremely low toxicity, it is suggested as a final irrigant after smear layer removal.The functional penetration depth of NaOCl along dentin tubules is at least 2\u20133 times more than what has been published to date. There is no difference in disinfection effectiveness along the dentin tubules between NaOCl and hClOBelow is the link to the electronic supplementary material.Supplementary Material 1"}
+{"text": "Avicennia marina, Kandelia obovata, and Aegiceras corniculatum, respectively. These Snakin/GASA family members were identified and categorized into three subfamilies via phylogenetic analysis. The genes coding for the Snakin/GASA family members were unevenly distributed on chromosomes. Collinearity and conservative motif analyses showed that the Snakin/GASA family members in K. obovata and A. corniculatum underwent multiple gene duplication events. Snakin/GASA family member expression in normal leaves and leaves infected with pathogenic microorganisms of the three mangrove species was verified using real-time quantitative polymerase chain reaction. The expression of KoGASA3 and 4, AcGASA5 and 10, and AmGASA1, 4, 5, 15, 18, and 23 increased after microbial infection. This study provides a research basis for the verification of HDPs from mangrove plants and suggests directions for the development and utilization of marine biological antimicrobial peptides.Host defense peptides (HDPs) are components of plant defensive barriers that resist microbial infection. Members of the Snakin/GASA protein family in plants have functions of regulating plant growth, defense, and bacteriostasis. Most mangrove plants grow in coastal zones. In order to survive in harsh environments, mangrove plants have evolved complex adaptations against microbes. In this study, Snakin/GASA family members were identified and analyzed in the genomes of three mangrove species. Twenty-seven, thirteen, and nine candidate Snakin/GASA family members were found in  Plants have evolved sophisticated defense mechanisms in the natural environment to protect themselves from bacteria, fungi, viruses, and protozoa . For exaSolanum tuberosum and was named Snakin because of some common sequence motifs with snake venom [Arabidopsis thaliana) gene family in A. thaliana is consistent with the structural features of Snakin [Allium cepa L.; fourteen Snakin/GASA family members were found in Vitis vinifera L., and thirty-seven Snakin/GASA family members were found in Glycine max [According to the sequence, cysteine number, and protein structure, plant HDPs can be divided into eight families: defensins, thionins, nonspecific lipid transfer proteins (LTPs), Snakins, hevein-like peptides, knottins, \u03b1-hairpinins, and cyclic peptides . Snakin ke venom ,14. Sequke venom ,14. The ke venom ,14. The f Snakin ,16. Incrf Snakin ,13,17. Fcine max ,19,20. TAtGASA4, 6, 7, 8, and 13 in A. thaliana, PeuGASA5, 6, 12, 17, and 19 in Populus euphratica, and OsGASA1 in Oryza sativa [PeuGASA4, 8, 9, and 15 in P. euphratica [AtGASA2, 3, 5, and 14 in A. thaliana and PeuGASA9, 10, and 14 in P. euphratica but inhibit the expression of AtGASA7 and 9, PeuGASA8, 11, 15, 17, and 18 [AtGASA6 caused early flowering in A. thaliana. The overexpression of GmGASA32 can promote soybean height [Various plant hormones regulate the expression of Snakin/GASA family members. For example, gibberellins (Gas) can induce a sativa ,15,16,21n height ,23.StSN1 isolated from S. tuberosum inhibited the growth of fungal pathogens such as Fusarium solani, Fusarium culmorum, Bipolaris maydis, and Botrytis cinerea, as well as bacterial pathogens such as Clavibacter michiganensis at low concentrations (EC50 < 10 \u03bcM) [PnSN1 found in Panax notoginseng inhibited the mycelial growth of four phytopathogenic fungi  and the spore germination of F. solani [PdSN1 (Peltophorum dubium Snakin peptide) inhibited Streptomyces scabies at 1.8 \u03bcM [GmSN1 enhanced viral resistance in A. thaliana and G. max [Snakin/GASA family members can inhibit the growth of various bacteria and fungi at very low concentrations. < 10 \u03bcM) . PnSN1 f. solani . PdSN1  found few research reports concerning HDPs from mangrove plants. Therefore, we first identified Snakin/GASA family members in K. obovata were obtained from Genome Warehouse (GWH) with the accession code PRJCA002330/GWHACBH00000000 [A. corniculatum genome data were obtained from the China National GeneBank (CNGB), accession number CNA0017738 [A. marina genomic data were obtained from NCBI , Genebank numbers GCA_019155195.1  and PRJNA392013  and Pfam . Finally, conserved domains of all candidate protein sequences were verified by manually removing incomplete domain sequences.The genomic data of 00000000 . The A. A0017738 . A. marinscripts ). The hihttps://web.expasy.org/protparam/, 3 April 2022). The chromosomal locations of Snakin/GASA family members were confirmed in the gene annotation files of K. obovata and A. corniculatum and mapped using MapChart software [A. marina. Then the chromosomal location information of Snakin/GASA family members was obtained in file 1 by blastn and visualized using MapChart software [S. tuberosum.Snakin/GASA family members\u2019 physicochemical properties were predicted using ProtParam  was used to visualize the evolutionary tree. Gene structure was analyzed using GSDS tools . Motifs in sequences were analyzed using the MEME online tool . Prediction of cis-acting elements was performed on the upstream sequence of each gene (1.5 KB) using the PlantCARE online tool , and the final images were created using TBtools software [A phylogenetic tree was constructed using the neighbor-joining method with 1000 bootstrap replicates via MEGAX software with Snakin/GASA sequences of software .https://www.genscript.com/wolf-psort.html, 7 April 2022) and Plant-PLoc . Protein 3D structure prediction was performed with SWISS-MODEL  and illustrated with Cherima1.14. The occurrence and duplication events of the Snakin/GASA family members in the three mangrove species were analyzed and visualized by MCScanX . Non-synonymous (ka) and synonymous (ks) substitution rates of each duplicated WRKY gene were calculated using KaKs_Calculator 2.0.Subcellular localization analysis of GASA was performed using the online sites WoLF PSORT . According to the manufacturer\u2019s instructions, the extracted RNA was reverse transcribed into cDNA using the Hifair III 1st Strand cDNA Synthesis SuperMix for qPCR . NanoDrop nucleic acid concentration tester  was used to determine the concentration and purity of RNA. Only samples with OD 260/OD 280 ratios between 1.8 and 2.0 could proceed to the next step. RNA samples were randomly selected and electrophoresed on 0.8% agarose gels to check the integrity of the RNA. The loading volume was 1 \u03bcg total RNA, and the voltage was 6 V/cm. The specific primers for Snakin/GASA family members from three mangrove species were designed using Premier 6.0. The specificity of primers was determined by 0.8% agarose gel electrophoresis. Real-time PCR was run with the qTower3 instrument  to detect the chemical SYBR Green. The established reaction system was as follows: 5 \u03bcL 2 \u00d7 SYBR Green Pro Taq HS Premix , 0.5 \u03bcL of each forward and reverse primer (10 \u03bcM), 1 \u03bcL of diluted cDNA template, and RNase-free ddHThe infected part was cut out, and the Ezuo Column Fungal Genomic DNA Extraction Kit DNA  instructions were followed to obtain. PCR amplification was performed using the fungus ITS universal primer pair  that amplifies the ITS1-5.8S-ITS2 region, and the bacteria 16S universal primer pair  that amplifies the V1-V5 variable regions. The nucleic acid was recovered from an agarose gel and sent for sequencing. The sequencing results were compared to the NCBI database to identify the pathogenic microbes.p-value < 0.05 was considered statistically significant.GraphPad Prism 7.0  was selected for statistical analysis. Statistical significance was determined via a one-way analysis of variance. All data are presented as mean \u00b1 SE and K. obovata and A. corniculatum were reported in 2020 [A. marina was published in 2021 [A. corniculatum; nine candidate Snakin/GASA family members were found in K. obovate; and twenty-seven candidate Snakin/GASA family members were found in A. marina. The Snakin/GASA family members of the three mangrove species were named according to their locations on the chromosomes (A. corniculatum genome contained 13 Snakin/GASA family members spread across six chromosomes. Eight Snakin/GASA family members of A. corniculatum (AcGASA1\u2013AcGASA8) were located on chromosome 1. Nine Snakin/GASA family members were spread across six chromosomes in the K. obovata genome. On chromosomes 4 and 2, there were three and two Snakin/GASA family members, respectively. AmGASA27 was not placed on a scaffold and could not be located on a chromosome. The remaining 26 Snakin/GASA family members were distributed on 15 chromosomes in the A. marina genome. Furthermore, three Snakin/GASA family members were respectively identified on A. marina\u2019s chromosomes 2, 20, 22, and 25.Chromosome-scale assemblies of the genomes of  in 2020  and 2021 in 2020 . A refer in 2021 . We firsomosomes . As showA. marina, AmGASA22 had 88 amino acids and was the smallest. AmGASA19 had 307 amino acids. Among the Snakin/GASA family members of K. obovata, KoGASA9 was the largest with 186 amino acids. There were 421 amino acids in AcGASA6 of A. corniculatum, the Snakin/GASA family member with the most significant number of amino acids among the three mangrove species. AcGASA7 and AcGASA8 had 346 and 387 amino acids, respectively. KoGASA1, 4, 5, and 8 in K. obovata, AcGASA5, 9, 10, 12, and 13 in A. corniculatum, and AmGASA2, 5, 11, 16, 17, 21, 22, and 23 in A. marina had fewer than 100 amino acids. As shown in A. corniculatum, AcGASA2, 5, 10, and 12 were predicted to be localized in the extracellular matrix, AcGASA6 in the nucleus, and AcGASA3, 4, 8, and 9 in the chloroplast  and synonymous substitution rate (Ks) and their ratio were estimated for Snakin/GASA family members. The gene duplication event of Snakin/GASA family members occurred on chromosome 1  suggested negative or purifying selection pressure during evolution. T = Ks/2r, where r is the expected clock sample rate of synonymous substitution in dicotyledons, and r = 1.5 \u00d7 108 substitutions/synonymous sites/year is the formula used to determine divergence time. The range of divergence times of two gene pairs calculated using Ks values was 32.28 to 33.13 million years ago (MYA)  . AcGASA5K. obovata and A. thaliana. Between A. corniculatum and A. thaliana, two collinear gene pairs of Snakin/GASA family members were found. Between K. obovata and P. trichocarpa and K. obovata and V. vinifera, there were nine and seven collinear gene pairs of Snakin/GASA family members, respectively. Two and three collinear gene pairs of Snakin/GASA family members existed between A. corniculatum and V. vinifera, respectively. Finally, K. obovata and V. vinifera shared three gene pairs with Snakin/GASA family traits. K. obovata consisted of two exons and introns. Other Snakin/GASA family members of K. obovata had multiple exons and introns. Eight Snakin/GASA family members on chromosome 1 of A. corniculatum did not have a UTR and had many introns and exons. AcGASA6 of A. corniculatum had the largest number of introns and exons, with 12 exons and 11 introns. AmGASA21 and AmGASA22 of A. marina had only one intron and one exon, while AmGASA19 had the largest number of exons and introns, with five exons and four introns.As shown in AcGASA3, AcGASA7, and AcGASA8 consisted of motif 1 and motif 4. The GASA domain of most Snakin/GASA family members in G-II and G-III consisted of motif 1 and motif 4, except for AmGASA27 and AcGASA9. The GASA domains of AcGASA9, AmGASA3, AmGASA12, and AmGASA25 had deletions compared to the conserved GASA domain.The GASA domain of most Snakin/GASA family members in G-I consisted of motif 3 and motif 2 or motif 3 and motif 4. The GASA domains of AcGASA2, 3, 5, 7, 8, 9, and 12 had methyl jasmonate (MeJA) elements upstream. AcGASA4, 8 had SA elements upstream. AcGASA3, 4, 5, 8, and 10 had abscisic acid (ABA) elements upstream, and there were four ABA regulatory elements upstream of AcGASA10. AcGASA2, 3, 5, 6, 8, 11, and 13 had regulatory elements that could respond to defense  regulatory elements upstream of KoGASA4. All Snakin/GASA family members of K. obovata were regulated by ABA except for KoGASA8  protein structure prediction. The predicted structures of these Snakin/GASA family members were similar, with all of them having random coils, extended strands, and two long \u03b1-helices .To investigate the role of the GASA family in response to environmental stress, infected leaves were collected from the Dapeng mangrove forest  in Shenzhen, China . Tropicoporus texanus caused marginal and vein scorching in A. corniculatum. K. obovata became ulcerated as a result of Jattaea spp. infection. It should be Berkeleyomyces basicola that caused the infection of A. marina  have been reported [Most members of the Snakin/GASA family are small peptides with multiple functions. They are regulated by various hormones involved in plant development, stress response, and antibacterial activities ,14. The reported ,19,21. A. thaliana has 87 amino acids, while the largest has 275 amino acids [K. obovata, AcGASA5, 9, 10, 12, and 13 in A. corniculatum, and AmGASA2, 5, 11, 16, 17, 21, 22, and 23 in A. marina had fewer than 100 amino acids. Their amino acid numbers met the criteria for HDPs, but their antibacterial functions remain elusive.Peptide lengths vary greatly among Snakin/GASA family members in the same plant species. For instance, the smallest Snakin/GASA protein in no acids . The smano acids . The pepno acids . Howeverno acids . KoGASA1In cotton and potato, longer chromosomes did not necessarily contain more Snakin/GASA family members. This suggests that the number of Snakin/GASA family members on each chromosome of the three mangrove species was unrelated to chromosome length. Additionally, the pI values (from 4.11 to 10.14) also vary widely among different plant species and individual members . The nonA. thaliana is present in the cell wall and extracellular matrix [Hevea brasiliensis are present in the nucleus and the cytoplasm [The GASA domain of Snakins is typically situated at the C-terminal and consists of approximately 60 residues, including 12 conserved cysteines ,39. The r matrix ,44, and ytoplasm . The traytoplasm . Based oytoplasm ,13. As aA. corniculatum may have evolved leading to variation in motifs and introns in some groups. Some members of the Snakin/GASA family can be strongly induced to respond to temperature variation [A. corniculatum. AtGASA4, 6, and 14 in A. thaliana are critical in promoting plant development [AtGASA4 and 6 has been shown to cause delayed flowering [KoGASA6 and AmGASA13 are adjacent to AtGASA4 and 6 ; AmGASA7 and AmGASA26 were adjacent to StGASA2 (Snakin-2). Therefore, KoGASA4, AmGASA7, 11, and 26 may have the same function as Snakin-2 and Snakin-3.The adjacent Snakin/GASA family members in the evolutionary tree had similar sequences and thus may have similar functions. Although many plant Snakin/GASA family members have been identified, few have been functionally validated ,19,44,46ariation . The Oliariation , which melopment ,34,51. Ilowering . KoGASA6A4 and 6 , and thuar genes . TherefoAmGASA1-7, 10, 12, 13, 15, 18, 21, 23, and 25 had MeJA and defense regulatory elements upstream, consistent with their up-regulated expression after pathogenic microorganism infection. Not all Snakin/GASA family members of the three mangrove species with MeJA and defense regulatory elements were up-regulated after infection by pathogenic microorganisms. Salicylic acid (SA) can act as a signal to promote the expression of downstream defense genes and limit the growth of pathogenic microorganisms [KoGASA4, and its expression was up-regulated after pathogenic microorganism infection. KoGASA4 is a secreted protein with a signal peptide and was predicted to localize in the extracellular milieu. Therefore, KoGASA4 has some characteristics of plant HDPs, and its function is worthy of further verification. Although AcGASA6 had two SA upstream regulatory elements and its expression could be induced by pathogenic microorganisms, it did not have a signal peptide and was predicted to localize in the nucleus. Therefore, after pathogenic microorganisms have invaded the plants, the increased level of SA induces the expression of AcGASA6. A recent report has shown that the type one protein phosphatases (TOPP)-SnRK2 module of ABA signaling in A. thaliana was disturbed by the pathogenic effector AvrE, resulting in up-regulation of ABA signaling and stomatal closure that promoted the generation of interstitial waterlogging and pathogenic infection [AcGASA10 may account for its up-regulation after infection by pathogenic microorganisms. The regulatory relationship between the Snakin/GASA family members of the three mangrove species and hormones should be further investigated.Snakin/GASA family members are regulated by gibberellin (GA), abscisic acid (ABA), and other plant hormones ,19. The rganisms ,55. Fournfection . Four ABBased on prediction, the structure of Snakin/GASA family members is highly conserved . The strT. texanus can cause browning of the leaf edges and veins, Jattaea spp. will cause yellowing and ulceration of plant leaves; and Fusarium spp. will cause the plant to wilt [Rhizoctonia solani and Erwinia carotovora in transgenic potato plants [B. cinerea [CaSn gene from the Snakin family induces resistance against root-knot nematode infection in Capsicum annuum [Pseudomonas syringae pv. Tabaci [PnSN1 in P. notoginseng roots was induced 24 h after infection by F. solani [TcGASA12 and TcGASA13 of Theobroma cacao were up-regulated after infection by Phytophthora megakarya [KoGASA3, 4, AcGASA5, 6, 9, 10, AmGASA1-5, 7, 12, 13, 15, 18, 23, and 25 were significantly higher than those of normal leaves. They may be candidates for HDP and deserve further verification. The prediction of HDPs relies on multiple criteria, including sequence features such as the presence of conserved motifs, charge, hydrophobicity, and secondary structure. Additionally, subcellular localization and phylogenetic analysis provide valuable information on the potential function of the protein. Based on these criteria, it is suggested that AcGASA6 may not be an HDP, as it lacks a signal peptide and is predicted to be localized in the nucleus. Regarding AcGASA9, its GASA domain is incomplete, which may affect its functionality as an HDP. AmGASA12 was predicted to be located in chloroplasts, and its GASA domain is incomplete. According to sequence characteristics, amino acid number, and subcellular localization prediction, they may not be HDPs. AmGASA1-5, 7, 13, 15, 18, 23, and 25 can be regulated by ABA or MeJA, while KoGASA3, 4, and AcGASA5, 10 are regulated by ABA. These genes may be induced by pathogenic microorganisms, resulting in increased expression. Thus, they may play a role in the plant\u2019s defense against pathogenic microorganisms. Further verification is needed to confirm their functions. That is very worthy of study; there has been much discussion and in-depth excavation, and it is also one of the directions for future work.Microorganisms often cause diseases in plants. For instance,  to wilt ,58,59. Go plants . Express cinerea . The newm annuum . Snakin-. Tabaci . PnSN1 i. solani . TcGASA1egakarya . Snakin/K. obovata, A. corniculatum, and A. marina had 9, 13, and 27 candidate Snakin/GASA family members, respectively. Sequence alignment showed that the cysteine residues in the GASA domains of Snakin/GASA family members were relatively conserved within the three mangrove species. Additionally, the peptide lengths of the Snakin/GASA family members in the three mangrove species were different. Through evolutionary analysis, prediction of physicochemical properties, and qRT-PCR results, we have identified several potential candidates for HDPs. Taken together, these findings suggest that AmGASA1-5, 7, 13, 15, 18, 23, 25, KoGASA3, 4, AcGASA5, and 10 are most likely involved in plant defense. These candidates warrant further investigation to determine their involvement in plant defenses. This study lays the foundation for further investigation and exploration of the HDPs present in mangrove plants.In this study, the number of Snakin/GASA family members varied among the three mangrove species. Specifically,"}
+{"text": "Developing new energy vehicles (NEVs) is necessary to grow the low-carbon vehicle industry. Many concentrated end-of-life (EoL) power batteries will cause large-scale environmental pollution and safety accidents when the time comes to replace the first generation of batteries if improper recycling and disposal methods are utilized. Significant negative externalities will result for the environment and other economic entities. When recycling EoL power batteries, some countries need to solve problems about lower recycling rates, unclear division of echelon utilization scenarios, and incomplete recycling systems. Therefore, this paper first analyzes representative countries\u2019 power battery recycling policies and finds out the reasons for the low recycling rate in some countries. It is also found that echelon utilization is the critical link to EoL power battery recycling. Secondly, this paper summarizes the existing recycling models and systems to form a complete closed-loop recycling process from the two stages of consumer recycling and corporate disposal of batteries. The policies and recycling technologies are highly concerned with echelon utilization, but few studies focus on analyzing application scenarios of echelon utilization. Therefore, this paper combines cases to delineate the echelon utilization scenarios clearly. Based on this, the 4R EoL power battery recycling system is proposed, which improves the existing recycling system and can recycle EoL power batteries efficiently. Finally, this paper analyzes the existing policy problems and existing technical challenges. Based on the actual situation and future development trends, we propose development suggestions from the government, enterprises, and consumers to achieve the maximum reused of EoL power batteries. Under the dual pressure of global ecological degradation and the energy crisis, the world landscape is undergoing significant and profound changes. The global transportation sector will consume 60% of total oil consumption, contributing 26% of carbon dioxide emissions, and has become the major source of urban air pollution ,2,3,4. AA strategic emerging industry, the NEV industry has received substantial support from government . In 2021In order to reduce the negative externalities of EoL power batteries to the environment and relieve the pressure caused by insufficient raw materials, there has been widespread attention given to the issue of reuse and recycling of EoL power batteries. Existing studies have also confirmed the necessity and feasibility of the economic, environmental, and resource benefits . Neubaue(1)The recycling rate in some countries is relatively low.(2)Countries worldwide are exploring the echelon utilization of EoL power batteries. However, the specific application scenarios and implementation process need to be clearly delineated.(3)To guide remanufacturing enterprises toward a sustainable direction, policies for recycling EoL power batteries should be formulated from a systems perspective in some countries.Therefore, it is highly significant to sort through current policies, recycling methods, and systems to classify different application scenarios and propose reasonable recycling systems for EoL power batteries. These actions will help to address the challenges in EoL power battery recycling operations and improve laws and regulations related to the recycling process.This paper is different from the above-mentioned literature studies as it provides a detailed overview of the power battery recycling policies of typical countries and analyzes the characteristics of countries with high recycling rates. Second, this paper provides a comprehensive summary of the existing recycling models and systems for power batteries to fill the gap in this field. Third, this paper clearly divides the application fields of power battery gradient utilization to clarify the specific classification. Fourth, this paper proposes the 4R power battery recycling system in the context of circular economy to provide a theoretical basis for industrial development.A country\u2019s policies and regulations play a crucial role in developing emerging industries. Some countries have begun to pay attention to recycling EoL power batteries and have introduced many regulations. By analyzing the policies of the EU, U.S., Japan, and China, we summarize the policy characteristics of different regions and propose the development trend of recycling EoL power batteries.In 1991, the EU adopted its first Battery Directive (Council Directive 91/157/EEC), which emphasized the reduction of toxic emissions . The EndEU member states have also developed regulations that are appropriate for the development of their respective industries. Germany has extended its legal framework based on EU directives. The legal system specifies the registration requirements for battery manufacturers and retailers and the recycling responsibilities of battery manufacturers, retailers, and consumers. Importers and manufacturers of batteries must register with the government before production, retailers must work with manufacturers to educate consumers on how to recycle batteries, and consumers must return used batteries to designated recycling institutions . GermanyThe U.S. was the first nation to regulate the recycling of EoL batteries with laws. As a result, the U.S. has a practical legal framework, technical specifications, and a recycling system for managing the recycling of EOL power batteries. The U.S. primarily constructs its system at the federal, state, and local levels . There aThe \u201cResource Conservation and Recovery Act\u201d and \u201cMercury-Containing and Rechargeable Battery Management Act\u201d regulate federal recycling laws for EoL batteries. The acts mandate that battery manufacturers design batteries by keeping disassembly and recycling in mind and controlling EoL batteries in transportation, manufacturing, and recycling . The staThe U.S. Department of Energy recognizes the critical role of EoL power batteries in the clean energy industry. It is gradually focusing on applying EoL power batteries in the field of echelon utilization in light of the increasing popularity of NEVs. The \u201cNational Lithium Development Blueprint 2021\u20132030\u201d, released by the Department of Energy in 2021, proposes to maximize the use of EoL lithium batteries, establish a special fund for battery recycling, recover vital raw materials, and develop a competitive industry chain for lithium battery recycling . The DepGeneral Motors (GM) has recognized the potential advantages of recycling power batteries and has invested in \u2018Lithion Recycling\u2019, which has a recycling rate of over 95%. GM utilizes advanced recycling technology to recycle battery materials and co-design recyclable power cells. GM ensures a stable vehicle production supply chain and reduces production costs by investing .The circular economy (CE) law in Japan is one of the most inclusive in the world. The Japanese legal system consists of three levels, from highest to lowest: basic law, comprehensive law, and special law, which form a product, consumption, reuse, and based circular system. The complete law framework of the CE addresses the negative externalities of batteries and provides specific operational guidance and a legal basis for battery recycling . Japan bJapan has pioneered the commercialization of hybrid vehicles to improve fuel efficiency and reduce greenhouse gas emissions. NiMH batteries are commonly used to power hybrid vehicles, so the initial recycling policy for power batteries centered on NiMH batteries . Since NAs Japan is prone to natural disasters, emergency equipment is in high demand. Nissan, Toyota, and Mitsubishi have implemented power batteries in the step-up and reuse industries. Nissan Motor and Sumitomo Corporation have established the 4R Energy joint venture. According to the remaining capacity of EoL power batteries, 4R Energy has divided the echelon utilization scenario and applied it primarily to energy storage systems and grid energy storage. Using EoL power batteries in home emergency power and energy storage devices is a viable solution pushed by battery manufacturers and vehicle manufacturers.China needs to establish a comprehensive battery recycling model to alleviate the shortage of critical resources, such as lithium, nickel, and cobalt . The polChina has prioritized the recycling of EoL batteries since 2003. Recycling focuses on EoL rechargeable batteries and disposable button batteries. With the advancements in science and technology and the rise in popularity of electronic devices, the following policies emphasized lead\u2013acid recycling batteries. In 2006, the National Development and Reform Commission (NDRC) proposed the first \u201cTechnical policy on recycling and utilization of automobile products\u201d based on the system of EPR, offering the recycling of automotive product parts and components. Since 2014, the government has begun to increase its support policies for the NEV industry, resulting in its rapid development. As a result, the Chinese demand for NEV power batteries has proliferated, and the country will soon deal with an abundance of EoL batteries. However, the government should have paid more attention to recycling EoL batteries then, so no separate document existed. The policy document for the creation of NEVs merely calls for the recycling of EoL batteries.In 2016, NDRC issued the \u201cTechnical Policy on Electric Vehicle Power Battery Recycling (2015 Vision)\u201d. The policy implemented the EPR, directed enterprises to recycle and reuse EoL power batteries, and established upstream and downstream enterprises to form a closed-loop recycling system. Since then, China has gradually unveiled its recycling policy for power batteries. Chinese approach focuses more on recycling lithium batteries and encourages manufacturers to establish product recycling channel facilities, as lithium batteries are the most common type of battery used in power batteries ,49. In 2Based on the national industrial development policies, local governments should consider factors such as urban construction planning, regional economic development, talent pools, and echelon utilization technology to formulate targeted local policies. The policies and local governments should complement each other to form \u201cone local, one policy\u201d to better implement the echelon utilization of EoL power batteries , such asBy analyzing the regulations of different countries, we can observe that each country has distinct characteristics. However, all of them recognize the significant role of EPR in recycling power batteries . We anal(1)Some countries face low recycling rates, with China having a lower recycling rate for end-of-life (EoL) power batteries compared to other countries. High recycling rates are typically observed in nations that implement specific characteristics, i.e., (1) the establishment of regulations that govern enterprise behavior toward recycling EoL batteries; (2) the imposition of hefty fines for any violations; (3) the regulation of recycling processing fees, taxes, and deposit payments, which serves to oversee recycling enterprises and enforce consumer recycling obligations. However, in China, the recycling of rechargeable batteries is encouraged by charging consumers a fee for recycling treatment and providing subsidies. As a result, China has an imperfect and ineffective recycling system in comparison to several other countries, and most EoL power batteries have not been recycled through formal methods . Countri(2)Currently, the policies of several countries prioritize the recycling and echelon utilization of power batteries, which are important for implementing sustainable development. The field of power battery echelon utilization is still in its early stages, and the policies introduced by various countries primarily focus on funding the R&D of enterprises in this field. The application scenarios for echelon utilization are rather vague, and there has yet to be a comprehensive and specific summary of them.EoL power batteries are associated with strong negative externalities . Two maiThe battery manufacturer recycling model involves battery manufacturers collecting EoL batteries from consumers through methods such as NEV manufacturers, NEV retailers, and EoL NEVs dismantling companies, establishing a recycling method led by battery manufacturers and based on reverse logistics . In recyJapan has established a recycling method mainly led by battery enterprises, with the idea of \u201creverse logistics\u201d. Battery manufacturers use service networks such as retailers, sellers, and gas stations to recover EoL batteries from consumers free of charge and then hand them over to professional battery recycling companies for disposal. Toyota Motor Corporation offers customers cash subsidies or purchase discounts for returning EoL batteries to dealers or retailers. The Nissan Leaf once offered a battery replacement business for customers .Under the NEV enterprises recycling model, NEV enterprises will be responsible for recycling (EoL) vehicles. NEV enterprises can use their existing sales distribution network to establish recycling service stations, such as automobile sales, 4S stores, and after-sales service stations, or they can establish specialized recycling service stations. NEV enterprises recycle power batteries and transfer the recovered batteries to regular dismantling enterprises. The batteries undergo echelon utilization and resource recovery. The recycling process is shown in BYD is the first company in China to lay out the NEV market. BYD\u2019s recycling method for EoL power batteries is mainly through authorized retailers or dealers. When a customer needs to replace the EoL power battery, the retailer or dealer will transport the EoL power battery to the BYD factory for testing. Trumpchi, FAW-Volkswagen, and Geely are establishing special recycling service stations for recycling. The three companies have established the largest number of recycling service stations in China, accounting for 26.9%. In addition, many NEV manufacturers actively sign strategic cooperation agreements with battery recycling and dismantling enterprises to jointly establish power battery recycling networks. According to data from the MIIT, as of November 2022, China has established 15,251 power battery recycling service stations nationwide.The industry alliance recycling model refers to an incentive alliance between manufacturers, sellers, recycling, and traceability technology enterprises. In the recycling process, the sales and service networks among the members combine to build a closed-loop recycling network to centralize the recycling of EoL power batteries. The EoL power batteries are transported to the professional recycling centers of the alliance members for recycling and processing. Compared with individual manufacturers or retailers, industry alliances can divide and collaborate according to their strengths, ensuring the integrity of the entire recycling industry chain. The specialized division of labor within the industry alliance can improve recycling efficiency, allowing the entire recycling system to have a more professional recycling network and processing equipment at a lower cost . The recThe industry alliance recycling model is popular in the U.S. The \u2018Portables\u2019 team in the U.S. is an incentive consortium that was formed by Everledger, HP, Fairphone, and Call2Recycle. Everledger provides technical support for the battery traceability feature, which creates a battery passport for manufactured batteries. HP and Fairphone are the manufacturers of lithium batteries, and Call2Recycle is the company responsible for recycling lithium batteries, including EoL power batteries . Call2ReUnder the third-party recycling model, manufacturers pay a service fee to third-party companies so that third-party companies can assist companies in completing the EPR. The service fee is calculated based on the quantity and type of EoL power batteries returned by the manufacturers. This model can transfer the responsibility and risk of recycling to the third-party company, so it is more suitable for small manufacturers . Third-pIn China, GEM is a third-party recycling company approved by China. As a leading company in recycling EoL power batteries, GEM built a full life cycle value chain from recycling, remanufacturing, and echelon utilization to ensure maximum reuse of EoL power batteries. Moreover, MAE built an \u201cInternet+recycling\u201d platform for EoL lithium batteries by cooperating with battery manufacturers, NEV manufacturers, EoL battery recycling and dismantling companies, automobile retailers, and internet e-commerce platforms. This innovative recycling model has built a nationwide recycling service network and realized the efficient recycling of metal resources refer to .A low-carbon, resource-efficient, and inclusive future requires CE systems that emphasize reducing, reusing, and recycling . CE systReducing means that society uses as few non-renewable and natural resources as possible in the process of production and consumption, reduces the amount of energy used, and improves the utilization of resources; it is also a prerequisite and basis for recycling and resourcefulness . ReducinAfter a battery is repaired (after its first use), it can be reused in the same vehicle model. This method reduces the need for new batteries to be produced for use in vehicles . The batEssentially, remanufacturing is the process of repairing, replacing, or restoring EoL products to \u2018like-new\u2019 conditions . This reRecycling companies have become the most popular method for recovering rare metals and valuable materials from power batteries ,74. By aIn conclusion, each system places much emphasis on reusing. However, the existing studies do not specify the process of reusing. In the EoL power battery recycling process, we specify that the process of echelon utilization involves reusing. From the policy analysis in the previous section, it is clear that echelon utilization is already a key area for future research and development.Echelon utilization can be defined as reusing the EoL power batteries. Echelon utilization can relieve the pressure of recovery, reduce environmental pollution, improve economic efficiency, and help the development of renewable energy. Echelon utilization aims to reduce the battery capacity to less than 80%, which is a slight scrap, and is not suitable for power batteries used in NEVs . EoL powAs the reserve energy of EoL batteries can be applied to some scenarios, it is extremely critical to pay more attention to the echelon utilization. There are three categories of application scenarios for echelon utilization: generation-side energy storage, user-side energy storage, and mobile power supply. The classification of echelon utilization scenarios presented in this paper is based on the classification of energy storage proposed by  as well In general, the division of power generation-side and user-side energy storage systems is based on the size of energy storage facilities and the standard of centralized physical distribution of energy storage systems. The scale of the generation-side energy storage is usually large and the level of battery consistency and balance management is high. ThU.S., it often needs the support of a large power grid , such asThere are two main application scenarios of energy storage cited in this paper  to ensure the stability of the power grid on the generation side.As an energy storage facility, batteries are primarily used for storing sustainable renewable energy . ApplicaUsing batteries as energy storage systems on the generation side can also cut and fill peaks and valleys. Due to the greater relative cost of energy consumption during peak periods than during low peak periods, the battery can be used as an energy storage device at a low cost to store excess energy during peak periods and then output the stored energy during low peak periods to the power grid. Generally, the application scenarios for energy storage on the power generation side have high demands on battery specifications, packaging forms, and quantity. In addition, they also have high demands on the level of selection and balance of management of batteries. For example, GM and ABB are further exploring the use of used battery packs to provide backup power for small commercial buildings when power is lost as a means of compensating for the intermittent power generation limitations of renewable energy sources, such as solar and wind power, as well as storing and using electricity during preferred periods in a way known as \u201cpeak-load shifting\u201d.The main characteristics of the application scenarios of user-side energy storage of EoL batteries involve distributed energy storage, such as small charging stations, commercial power supply, remote areas without power grids, and scattered villages with high construction costs for power grids.In economics, the EoL battery industry is still in its infancy, and there is less risk associated with small applications. The current application of user-side energy storage has greater economic potential than power generation-side energy storage. In this area, some companies have already begun implementing plans. For example, Japanese automaker Nissan has pre-ordered a residential energy storage unit called \u201cxstorage\u201d, which uses 12 retired EV batteries and is connected to the grid to provide backup energy . To demoMobile power supply mainly refers to the echelon utilization of used batteries in low-speed vehicles and electric motorcycles that have less battery performance requirements than NEVs. Applications include power batteries for vehicles with low-performance requirements, backup power supplies for short trips in NEVs, and emergency start energy. Jin  demonstrEchelon utilization was first studied in Europe, the U.S., and Japan. Currently, chemical batteries, fuel batteries, and solar batteries make up the majority of power batteries on the market. Chemical batteries include lead\u2013acid batteries and lithium-ion batteries. Lead\u2013acid batteries are the most widely used in the automotive industry due to their low price and mature technology . There hRapid echelon utilization development has led to the significant strengthening of cooperation among leading enterprises within the foreseen scope, with the strong support of national policies . The govEoL power batteries refer to three types of situations. First, EoL batteries are created during the production process by battery manufacturers. Second, NEVs cannot guarantee regular operation due to the remaining capacity or the charge/discharge performance. Thirdly, EoL power batteries have been reused after echelon utilization. The ability to recycle power batteries effectively will directly impact national energy savings and emission reduction strategies. Integrating the battery recycling production network into the industrial ecological chain will optimize the allocation of system resources and minimize external diseconomies. However, we found that there currently needs to be clearer guidance for recycling EoL power batteries. Based on the analysis in In general, recycling EoL power batteries can be divided into two parts: recycling methods and recycling systems. Countries around the world can determine a reasonable recycling paths according to the country\u2019s development and combine it with the four recycling methods proposed in In the early stages of developing NEV batteries, the open-loop production mode of \u201ccradle to grave\u201d was mainly utilized. The industrial ecological chain ends at the product\u2019s EoL point. This paper presents a 4R recycling system for EoL batteries. In this paper, we aimed to design a 4R system for recycling EoL power batteries, addressing the industry\u2019s original crude open-loop production structure and achieving a \u201ccradle-to-recycle\u201d production model ,92.In After 5\u20138 years of consumer use, the power battery will be retired. Consumers take their EoL power batteries to a standard recycling service station and receive a subsidy offer. The recycling service station hands the EoL power battery to the NEV manufacturer. NEV manufacturers fulfill the EPR by returning EoL power batteries to the upstream battery manufacturer. Prior to battery echelon utilization, the battery manufacturer calculates the SOH of the battery through BMS. The enterprise can assess the economy, safety, and availability of power batteries by reasonably evaluating the SOH data of vehicles and assessing the application field of the secondary reuse of power batteries ,94,95. WThe 4R recycling system for EoL power batteries improves the components\u2019 utilization rate and reduces the industry\u2019s ecological disorder. Power batteries can be extended in life, and a \u201cproduce-use-recycle-reuse\u201d production model can be used and enterprises will be able to implement a \u201ccradle-to-cradle\u201d production model for power batteries. Meanwhile, it creates several market opportunities in the recycling system, reducing waste and negative externalities on the environment. If the government and enterprises actively implement the 4R recycling system in the future, it will be of great importance to improve the country\u2019s economic, environmental, and social well-being.Previous studies have shown that recycling power batteries is effective, i.e., economically, environmentally, technically, and resourcefully ,25,74. HGovernment policy plays a critical role in the emerging industry, but five issues remain regarding policies. (1) The NEV battery recycling industry needs a transparent system and specific recycling and dismantling standards. NEV manufacturers consider factors such as safety, space optimization, and applicability during the design process. However, there are differences in the battery packs, modules and battery packs produced according to the requirements of different vehicle models . It willRecycling EoL power batteries may have some negative externalities on the environment . We analDue to the rising costs of raw materials for power batteries and graphite, recycling (EoL) power batteries has gained worldwide attention. In this paper, we analyze the recycling policies of representative nations and find that nations worldwide are gradually focusing on the scenario of hierarchical utilization of EoL power batteries. Based on the current recycling model and system, we propose that the 4R recycling system can effectively manage EoL power batteries in the future. Regarding the six presented development obstacles, we propose the development of recycling EoL power batteries from the perspectives of government, enterprises, and consumers.(1)According to the 4R EoL power battery recycling system, governments consider echelon utilization and clarify the responsibilities of recycling enterprises. To achieve the goals of environmental protection, resource conservation, and safe use, the government must also improve its policies and regulations, technical guidelines, evaluation standards, and industrial chain for echelon utilization.(2)The government should bolster oversight of EPR implementation by battery manufacturers and NEV manufacturers and improve the regulatory framework. In addition, the government should increase the penalties for companies that violate recycling laws.(3)The government establishes technology subsidies, focusing on subsidizing research and technological breakthroughs in critical echelon utilization applications. The government has increased support for recycling companies to promote the growth of the recycling power battery industry.(4)The government increased subsidies to consumers to incentivize consumer participation in recycling. The government and competitors should educate consumers about environmental awareness and formal recycling procedures.(5)The government rationalizes the planning of recycling infrastructures for power batteries. It may be convenient for consumers to return their power batteries at these locations and for enterprises to reduce transportation distances and reduce costs by replacing them at these locations.(1)Battery manufacturers, NEV manufacturers, NEV retailers, and hierarchical utilization enterprises must engage in collaborative design, management, and data sharing, and cooperate to address the issues of constructing battery recycling methods, building a recycling evaluation system for the entire life cycle, and establishing a service system. According to the four existing recycling methods, technology integration among enterprises will be strengthened, thus reducing investment risks and achieving win-win cooperation.(2)Battery manufacturers should increase their investments in recycling equipment, research, development, and technical personnel. They should establish a traceable power battery recycling regulatory system using blockchain technology to ensure real-time monitoring of companies\u2019 implementation of full life cycle tracking. Enterprises should also enhance their awareness of recycling and improve their sense of corporate social responsibility.(3)NEV retailers should use advertising and education to increase consumer participation in battery recycling or promote \u201ctrade-in\u201d activities to attract more consumers to participate in recycling. When the recycling volume reaches a particular scale, it can also increase the profit of the recycler. In addition, recycling companies should set reasonable prices to balance the interests of enterprises and consumers.As essential participants in recycling EoL power batteries, consumers should recognize the critical role of environmental protection and resource conservation. Consumers should consciously comply with their recycling obligations and take the initiative to send EoL batteries to designated recycling service stations. By consciously recycling, consumers can gain considerable benefits and effectively reduce the costs of government regulation, thus achieving the goal of maximizing social welfare.(1)This paper examines the battery recycling regulations of the EU, the U.S., Japan, and China. The government regulates countries with high recycling rates through legislation, deposit systems, high fines, and subsidies. Upon analyzing the policies, it was found that echelon utilization of EoL power batteries has become a significant concern in all nations.(2)This paper summarizes the four existing recycling \u201cmodels\u201d: battery manufacturers recycling model, NEV enterprises recycling model, the industry alliance recycling model, and the third-party recycling model. This paper summarizes the recycling systems in EoL power batteries, including reducing, reusing, remanufacturing, and recycling, by combining circular economy theory and closed-loop supply chain systems.(3)Combined with the development policy and recycling system, EoL power battery echelon utilization will be the focus of future development. However, several studies clearly delineate the application scenarios of echelon utilization. This paper presents the application scenarios of echelon utilization in terms of generation-side energy storage, user-side energy storage, and mobile power supply; we use actual cases for analysis.(4)The government must establish clear and effective recycling methods and normative recycling systems. Therefore, this paper proposes a 4R EoL power battery recycling system that accounts for echelon utilization and suggests the use of state of health (SOH) to assess the states to determine the recycling steps.(5)This paper analyzes the existing policy problems and technological challenges and proposes three aspects of development for the government, businesses, and consumers based on the above analysis.With the rapid popularity of NEVs, large-scale EoL power batteries will face retirement. There are substantial potential development opportunities and broad market prospects for recycling EoL power batteries. In order to reduce environmental damage, save resources, and protect health, it is important for the government, enterprises, and consumers to remanufacture, utilize them hierarchically, and recover resources from EoL power batteries through cooperation under a circular economy. This paper compares the current industrial development status and analyzes the problems of the existing recycling process in terms of policy and technology from a practical problem perspective. The main conclusions of this paper are summarized as follows:This paper has a limitation in that it mainly summarizes the literature studies and actual situations without using bibliometric analysis, which is commonly used by most scholars. Therefore, future studies can use this approach to analyze the literature in this field from multiple perspectives."}
+{"text": "This perspective discusses the fundamental benefits and drawbacks of aqueous batteries and the challenges of the development of such battery technology from laboratory scale to industrial applications. As estimated up to 2022, global installed capacity of lithium-ion batteries (LIBs) has increased dramatically from 148 to 580 GWh in the past five years. Their wide range of applications is mainly attributed to their high energy density, excellent cycle life, and low self-discharge rate . Gen. Gen4]. 5O8 as an example, bare Mn5O8 exhibits a stable potential window of 2.5\u00a0V, but surface hydroxylated Mn5O8 shows a higher operating potential range of 3.0\u00a0V in the same electrolyte [At present, a series of optimization measures have been proposed for electrode materials to enhance their energy density. For example, pre-intercalation chemistry methods can be applied to enlarge the diffusion path of charge carriers and create more active sites inside electrode materials, which could thus enhance the specific capacity for insertion-type hosts . Surfacectrolyte . MateriaThe nature of the electrolyte is another crucial factor. In aqueous batteries, electrolyte is formed by dissolving inorganic salt in water. Taking water as a solvent, aqueous electrolyte not only has the advantages of low cost, non-flammability, and non-toxicity, but also offers higher ionic conductivity beneficial to ion migration, which is two orders of magnitude higher in the case of aqueous electrolyte compared to that of organic electrolyte . AlthougTo widen the ESW of aqueous batteries, various solutions have been proposed Fig.\u00a0. First, oC. Finally, to promote its application in flexible electronics, aqueous flexible batteries are expected to be developed in the future.In addition to the rational design of electrode materials and electrolytes, the following aspects should also be considered in the practical applications of aqueous batteries. First, compared with laboratory studies, the usage amount of electrolyte in practical aqueous batteries will be greatly reduced. It is therefore crucial to maximize the electrochemical performance of electrode materials with limited electrolyte usage. Second, aqueous batteries should make optimal use of its advantages of high ionic conductivity to achieve fast energy storage (fast charging capability). At the same time, the possible increase in OER and HER cannot be ignored at high rates. Third, aqueous batteries must have the ability to work in extreme operating environments, such as high ambient temperatures and severe deformations. So far, through the regulation of electrolytes, aqueous batteries can operate in a wide temperature range from \u221290 to 80In short, the energy density and cycling performance of current aqueous batteries have met the demand of energy storage systems to a certain extent Fig.\u00a0. There i"}
+{"text": "Aiming at the shortcomings of the traditional sparrow search algorithm (SSA) in path planning, such as its high time-consumption, long path length, it being easy to collide with static obstacles and its inability to avoid dynamic obstacles, this paper proposes a new improved SSA based on multi-strategies. Firstly, Cauchy reverse learning was used to initialize the sparrow population to avoid a premature convergence of the algorithm. Secondly, the sine\u2013cosine algorithm was used to update the producers\u2019 position of the sparrow population and balance the global search and local exploration capabilities of the algorithm. Then, a L\u00e9vy flight strategy was used to update the scroungers\u2019 position to avoid the algorithm falling into the local optimum. Finally, the improved SSA and dynamic window approach (DWA) were combined to enhance the local obstacle avoidance ability of the algorithm. The proposed novel algorithm is named ISSA-DWA. Compared with the traditional SSA, the path length, path turning times and execution time planned by the ISSA-DWA are reduced by 13.42%, 63.02% and 51.35%, respectively, and the path smoothness is improved by 62.29%. The experimental results show that the ISSA-DWA proposed in this paper can not only solve the shortcomings of the SSA but can also plan a highly smooth path safely and efficiently in the complex dynamic obstacle environment. Path planning is one of the key technologies for the autonomous navigation of mobile robot, which has been widely used in the fields of intelligent warehousing and logistics, autonomous driving and aerospace. The definition of path planning is that the mobile robot designs the route from the starting point to the end point according to the specified performance index to avoid collision with obstacles in a challenging and uncertain environment . DependiScholars\u2019 interest in some heuristic algorithms has recently increased. There are many classical heuristic algorithms used for the solution of the path planning, such as the particle swarm optimization algorithm (PSO) , ant colWith the intensive research of scholars, some new intelligence heuristic optimization algorithms have emerged. The sparrow search algorithm (SSA), as a brand-new meta-heuristic algorithm first put forward by Xue  in 2020,In line with the above research, this paper focused on the innovative improvement and application of the SSA. When the literature is examined, studies combining the improved SSA with the DWA to solve the optimal path problem of the mobile robot have not been found. Aiming at the shortcomings of the SSA, this paper proposes an improved SSA based on Cauchy reverse learning, the sine-cosine algorithm, the L\u00e9vy flight strategy and the DWA algorithm, which is called the ISSA-DWA algorithm. Firstly, the Cauchy reverse learning technique was used to initialize the sparrow population, which enriches the diversity of sparrow population, avoids the premature convergence of the algorithm due to an uneven population distribution in the later iteration and improves the efficiency of path planning. Secondly, the sine\u2013cosine algorithm was used to update the producers\u2019 position of the sparrow population, which balances the exploration and exploitation ability of the algorithm. Then, the L\u00e9vy flight strategy was adopted to update the position of scroungers so as to optimally improve the ability of the algorithm to jump out of the local optimum. Finally, the improved DWA was used for the local path planning and dynamic obstacle avoidance of mobile robot. By improving the azimuth evaluation function of the DWA, fusing the optimal path point information given by the ISSA and setting temporary sub-target points, the mobile robot can not only move smoothly along the global optimal path planned by the ISSA but can also avoid the dynamic obstacle in real time.1.A novel swarm-based algorithm for solving the optimal path planning problem of the mobile robot, named as ISSA-DWA, is proposed by combining several advanced strategies in this paper.2.In the proposed ISSA-DWA method, the Cauchy reverse learning theory is used to enrich the diversity of sparrow population to avoid the algorithm falling into premature maturity.3.In order to solve the problem of the weak search ability in the later iterations of the SSA, the sine\u2013cosine algorithm is used to update the location of the producers so as to balance the exploration and exploitation capability of the algorithm.4.Since the SSA is prone to fall into local optimal solutions, the L\u00e9vy flight strategy is used to update the scroungers\u2019 position in the ISSA-DWA, which could increase the probability of jumping out of the local optimum.5.An improved azimuth evaluation function is proposed in the ISSA-DWA. Combined with the optimal path point information given by the ISSA, the proposed ISSA-DWA makes the mobile robot move smoothly to the optimal path.6.The experimental results and analysis show that the proposed ISSA-DWA is more competitive in solving the optimal path planning problem of mobile robot.The main contributions of this paper are as follows:The rest of this paper is organized as follows: the standard SSA introduction is given in The standard SSA is a brand-new swarm intelligence optimization technique that mimics the predation and anti-predation of sparrows. According to Ref. , it has n is the number of sparrows, i =  and d is the sparrows\u2019 dimension.It is assumed that the sparrow set matrix is:According to Formula (1), the sparrow\u2019s fitness function can be expressed as Equation (2):i-th sparrow is represented by i)f, i-th sparrow in the j-th dimension, itermax indicates the max iterations and B represents the In the standard SSA, the whole population will move to the food source, and the sparrows with priority access to food and good fitness will become producers. The producers\u2019 location is updated as follows:Z is the i-th scroungers did not obtain food due to its poor fitness value. To increase its fitness value, the scroungers need to go to various locations to hunt for food.Except for the producers, most of the remaining sparrows are scroungers. The scroungers\u2019 position is updated as follows:k represents the direction of the sparrows\u2019 movement.Approximately 10\u201320% of the sparrow population will be randomly chosen as scouters to remain watchful of their surroundings throughout the foraging phase. All sparrows, producers and scroungers alike, must abandon their present food source and migrate to a new area once the sparrows identify its natural opponents. The scouters\u2019 location is updated as follows:The flow chart for the standard SSA is shown in 1.The initial population of the standard SSA is generated at random, and there are several issues existing, including an uneven distribution of the population and poor diversity. In the subsequent iterations, this will lead to an insufficient search scope, low quality of the initial solution and slow convergence speed of the algorithm.2.The update method of the producers\u2019 location of the standard SSA is poor, where it is unable to balance both the exploration and exploitation capability. The dimensions of producers will decrease slowly in the later iteration, which will lead to a decline in its search ability and slow convergence speed.3.Since the standard SSA algorithm iterates for a specific amount of times, if the fitness value of the producers stayed constant, the producers will become the scroungers and the algorithm will fall into the local optimum easily.In summary, the standard SSA has the following disadvantages:1.Pointing at the disadvantages of the standard SSA, such as an insufficient population distribution and population diversity, Cauchy reverse learning was used to initialize the sparrow population, which enriches the diversity of the sparrow population and improves the quality of the initial solution of the algorithm.2.Aiming at the problem of the poor update method of the producers\u2019 location, the sine\u2013cosine algorithm and dynamic learning factor were used to balance both the exploration and exploitation capability.3.To solve the problem of it being easy for the standard SSA to fall into the local optimum, the L\u00e9vy flight strategy was used to update the position of the scroungers and increase the probability of the algorithm jumping out of the local optimal solution.In view of the shortcomings of the standard SSA, this paper proposes the following methods to improve its performance:Through the reverse learning mechanism, the Cauchy reverse learning algorithm can generate the reverse population of the initial population. Compared with the initial population, the better individuals will be chosen as the next generation population in the reverse population. The selected individuals are currently approaching the ideal solution than those in the reverse population and the initial population, which significantly accelerates the convergence of the algorithm. In addition, compared with the random initialization and chaotic mapping initialization, the initialization of the Cauchy reverse learning algorithm has several advantages, such as a large effective searching area, rich population variety and excellent global search ability. Therefore, this paper used the Cauchy reverse learning algorithm to establish the sparrow initialization population to address the issues of the uneven population distribution and poor population diversity of the standard SSA.d-dimensional space, where P\u2019s i-th dimensional space are P is:P is:i = 1, 2, \u2026, d. The Cauchy reversal point is a randomly produced point that is between the middle point and the typical reversal point, as shown by Formula (7). As a result, in accordance with the definition of the Cauchy reverse point, the Cauchy reverse population of the initial population is created by using the Cauchy reverse learning algorithm. Then, the fitness values of the Cauchy reverse population and the initial population are calculated and compared. Finally, the individuals with good fitness values are added to the final initialization population of the sparrows.Assume that the point se point , it can The sine\u2013cosine algorithm (SCA), a dynamic adjustment algorithm, can reduce algorithmic unpredictability in the later iterations of the algorithm . When coBy combining the SCA algorithm with the learning factor, the improved producers\u2019 location is updated as follows:d) stands for the moving distance of d-dimensional sparrows using the L\u00e9vy flight strategy. Other variables have been clarified in Formula (4), so they will not be described here again.The L\u00e9vy flight strategy, which combines continuous small steps with sporadic big steps, prevents the algorithm from exploring the same area twice, broadens the algorithm\u2019s search space and enhances its search ability . BecauseThe formula for the L\u00e9vy flight strategy is expressed as follows:Here, For the convenience of modeling, we regard the mobile robot as a particle. In Ref. , a lineaIn As can be seen from the figure, the path length was changed significantly by adding the ILPS strategy and INSS strategy to the proposed ISSA algorithm. In The standard dynamic window approach has the advantages of real-time obstacle avoidance and a smooth route. In the velocity vector space composed of multiple velocities and accelerations, the DWA method samples multiple groups of velocities of the mobile robot model according to its own finite velocity and acceleration constraints, and deduces the motion trajectories of these velocities in a certain time interval. At this time, these trajectories can be scored according to certain evaluation indexes, and the trajectory with the highest score can be selected as the final trajectory of the mobile robot. The core of the DWA is to transform the local path planning problem into a constrained optimal solution problem in the velocity vector space by controlling and searching the velocity of the mobile robot .t of the mobile robot, respectively. Similarly, t+1 of the mobile robot, respectively. t of the mobile robot, respectively.To implement the DWA method, the mobile robot\u2019s motion model must first be constructed. Assuming that the mobile robot cannot move in all directions, its motion trajectory is composed of each small arc, and the motion state of the small arc can be described by a two-dimensional velocity space essed as :(13){xt+1.Restricted by the maximum and minimum velocity of the robot\u2019s own model:After the mobile robot model is established, it needs to be sampled in multiple groups to calculate the range of the sampling velocity of the mobile robot\u2019s motion trajectory. The gamut of the sampling velocity is typically governed by the following three factors:2.Limited by the safe distance between the robot and the obstruction:3.Limited by the performance of the robot\u2019s motors:After velocity sampling, several feasible simulation trajectories can be calculated. In order to ensure that the mobile robot can reach the target position safely and effectively, it is necessary to evaluate these simulated trajectories in multiple dimensions and select the simulated trajectory with the highest score as the final trajectory of the mobile robot. There are three criteria for evaluating the simulated trajectory by the standard DWA: azimuth, obstacle distance and velocity. The overall evaluation function of the standard DWA can be expressed as:Since the azimuth evaluation function Algorithm 1: ISSA-DWA pseudo codeInput:2.RWarning value: ST.Safety value: n.The number of sparrow populations: itermax.The maximum number of iterations: PDNumber.The initial number of producers: SDNumber.The initial number of sparrows in charge of vigilance: The parameters of the DWA: Output: The smooth optimal trajectory.1.Initialize the population of sparrows by applying Cauchy reverse learning to Formula (7);2.Choose the sparrow in the initial population with the lowest fitness value as the present best option;3.While (t < itermax)4.Use the ILPS strategy to reduce the path node;5.fori = 1: PDNumber6.Update the location of the producers with the improved Formula (9);7.end for8.for\u00a0i = (PDNumber + 1): n9.Update the location of the scroungers with the improved Formula (10);10.end for11.for\u00a0i = 1: SDNumber12.Update the location of the sparrows with Formula (5);13.end for14.Obtain the current new location;15.Use the INSS strategy to reduce the fitness of the optimal individuals;16.Update the optimal location of the current individual;17.t = t + 1;18.end While19.ISSA;Acquire global optimal path point information of the 20.Fuse the point information and put it into local planner DWA;21.Control the robot to move according to the global optimal path;22.return the smooth optimal trajectory.After improving the DWA, it can then be combined with the ISSA to be a novel dynamic path planning fusion algorithm, which is called the ISSA-DWA algorithm. The ISSA-DWA algorithm proposed in this paper can not only solve the shortcomings of the standard SSA and standard DWA, but also the local dynamic obstacle avoidance ability and path smoothing ability of the algorithm are further improved. The pseudo code of the ISSA-DWA is as follows Algorithm 1:n), the dimensions of the path planning problem (d), the number of iterations (itermax) and the function assessment\u2019s cost (c). In specific terms, the overall computational complexity of the proposed ISSA-DWA can be given as below:The computational complexity of an algorithm can be described by the Big-O notation. When solving the optimal path planning problem of the mobile robot, the complexity relies on the number of sparrows  Core(TM) i7-5500 @ 2.40 GHz, memory 8 GB and Windows 7 64-bit system with MATLAB R2017b software.It was assumed that the mobile robot can be regarded as a point and that it can only move in the two-dimensional grid area. In the static obstacle avoidance simulation experiment, this paper set up three groups of comparative experiments for three different map environment models. Since the proposed ISSA-DWA algorithm increases its path smoothing ability and dynamic obstacle avoidance ability on the basis of the proposed ISSA algorithm, the global path planning ability is not affected. The static obstacle avoidance simulation experiment actually tests the static characteristics of the path planning algorithm. When only the path length, path turning times, path smoothness and execution time of the algorithm are considered as the measurement criteria, the static obstacle avoidance effect of the proposed ISSA-DWA algorithm can be regarded as equivalent to that of the proposed ISSA algorithm. Therefore, in the static obstacle avoidance simulation experiment, we only needed to compare the proposed ISSA algorithm with four basic heuristic algorithms, such as the ACO, MRFO, WOA and SSA algorithm. To obtain a fair and valid comparison of the results, all experiments were conducted on the same computer. The population size for both the proposed ISSA and the other four basic heuristic algorithms was set to 50, and the maximum number of iterations was set to 200. In environmental model 1, the ACO, MRFO, WOA, SSA and ISSA algorithms were applied to the static path planning of the mobile robot, and the ISSA-DWA algorithm was applied to the dynamic path planning of the mobile robot. The experimental simulation results of the first group are shown in As can be seen from the convergence curve plot, with an increase in iteration times, the advantages of the proposed ISSA algorithm are more obvious. The convergence speed of the proposed ISSA algorithm is faster than the other four basic heuristic algorithms from the beginning, and the final convergence accuracy of the proposed ISSA algorithm is higher than that of the other four basic heuristic algorithms.In order to further verify the performance of the proposed ISSA algorithm, the second group of static obstacle avoidance simulation experiments was carried out by selecting environment model 2, which has a more complex obstacle distribution, instead of environment model 1. In environment model 2, the proposed ISSA algorithm and the other four heuristic algorithms, such as the ACO, MRFO, WOA and SSA, were applied to the static path planning of mobile robot, and the proposed ISSA-DWA algorithm was applied to the dynamic path planning of the mobile robot. The simulation results of the second group of experiments are shown in As can be seen from the curve chart, in the complex environment model 2, with an increase in iteration times, the proposed ISSA algorithm has an excellent performance. Regarding the convergence speed, the MRFO algorithm is the slowest and the proposed ISSA algorithm is the fastest. Regarding the convergence accuracy, the ACO algorithm is the worst and the proposed ISSA algorithm is the best. Obviously, considering the convergence speed and convergence accuracy, the convergence speed of the ISSA algorithm is faster than that of the other four basic heuristic algorithms, and the final convergence accuracy is also higher than that of the other four basic heuristic algorithms.To verify the generalization of the proposed ISSA algorithm, the third group of static obstacle avoidance simulation experiments was carried out by selecting environment model 3, which has a more complicated obstacle distribution than environment model 1 and environment model 2. In environmental model 3, the proposed ISSA algorithm and four other heuristic algorithms, such as the ACO, MRFO, WOA and SSA, were applied to the static path planning of the mobile robot, and the proposed ISSA-DWA algorithm was applied to the dynamic path planning of the mobile robot. The simulation results of the third group of experiments are shown in As can be seen from the figure, when increasing the complexity of the obstacle distribution, the excellent performance of the proposed ISSA algorithm is more apparent. In environmental model 3, when the convergence speed and convergence accuracy are considered as the measurement criteria, with an increase in iteration times, the convergence speed of the algorithms can be ranked from fast to slow as ISSA, SSA, WOA, MRFO and ACO. The convergence accuracy of the algorithms can be ranked from high to low as the proposed ISSA, MRFO, SSA, WOA and ACO. Therefore, through a series of experiments, such as environment model 1, environment model 2 and environment model 3, the convergence performance of the proposed ISSA algorithm is the best, which also proves that the ISSA algorithm proposed in this paper is not only effective in improving the standard SSA algorithm but also superior to the other four basic heuristic algorithms.s = 1, 2, \u2026, m) within the path P are denoted as smt is defined and expressed by the following formula:m is the number of path points of the set P, and smt, the smoother the path.After completing the three groups of static obstacle comparison experiments, the performance of the ACO, MRFO, WOA, SSA and ISSA algorithm should be summarized in digital form. It is assumed that the path planned by the algorithms can be described as a set of the path points, and then the continuous turning angles between the path points In summary, the results of the three groups of static obstacle avoidance show that the proposed ISSA-DWA algorithm can plan a smooth global optimal path in environmental model 1, environmental model 2 and environmental model 3 safely and efficiently, no matter how the obstacle distribution changes. Since the proposed ISSA-DWA algorithm adds its dynamic characteristics on the basis of the proposed ISSA algorithm, when only the static characteristics of path planning are considered, it can be regarded that the effect of the proposed ISSA-DWA is equivalent to that of the proposed ISSA. Therefore, when summarizing the results of the three groups of static obstacle avoidance comparative experiments in digital form, it is only necessary to compare the performance of the proposed ISSA with the other algorithms, such as the ACO, MRFO, WOA and SSA. The algorithm performance indexes of the three groups of static simulation experiments are shown in 2 and 80 rad/s2, respectively. The linear velocity resolution was 0.01 m/s. The angular velocity resolution was 1 rad/s. The four weighting factors In the improved DWA section, the maximum linear velocity and maximum angular velocity were set to 3.6 m/s and 45 rad/s, respectively. The maximum linear acceleration and the maximum angular acceleration were set to 0.35 m/sTo further verify the dynamic obstacle avoidance ability of the proposed ISSA-DWA, the second group of dynamic obstacle avoidance simulation experiments was carried out in environmental model 2, and the experimental results are shown in In order to verify the excellence dynamic obstacle avoidance ability and generalization of the proposed ISSA-DWA algorithm, the third group of dynamic obstacle avoidance simulation experiments was carried out in environmental model 3, and the experimental results are shown in In summary, the results of the three groups of dynamic obstacle avoidance for the mobile robot show that the proposed ISSA-DWA algorithm can not only smooth the path but can also avoid dynamic obstacles in real time. In addition, this paper innovatively proposed three dynamic obstacle avoidance performance indexes, such as obstacle avoidance distance, obstacle avoidance time-consumption and obstacle avoidance angle of the mobile robot. The performance index results of the three groups of dynamic obstacle avoidance simulation experiments are summarized in In summary, the results of three groups of static and dynamic obstacle avoidance comparative experiments show that the proposed ISSA-DWA fusion algorithm is better than the proposed ISSA algorithm and the other four heuristic algorithms, such as the ACO, MRFO, WOA and SSA, when both static and dynamic characteristics of the algorithm are considered. It not only retains the global optimal path planning ability of the proposed ISSA algorithm but also has the ability to smooth the path and avoid dynamic obstacles in real time.In this study, a novel ISSA-DWA was proposed to solve the optimal path planning problem of mobile robot. Firstly, in order to enrich the diversity of the sparrow population and avoid a premature convergence of the algorithm, the Cauchy reverse learning strategy was used to initialize the sparrow population. Secondly, the sine\u2013cosine algorithm was used to update the producers\u2019 position to balance the global search and local exploration capabilities of the algorithm. Furthermore, the L\u00e9vy flight strategy was used to update the scroungers\u2019 position, which not only improves the ability of the algorithm to jump out of the local optimum, but also accelerates the convergence. Finally, the proposed ISSA algorithm and the improved DWA method were combined as a novel fusion algorithm, named ISSA-DWA algorithm. The effectiveness of the proposed ISSA-DWA was verified in the three groups of static and dynamic avoidance comparison experiments. When both static and dynamic characteristics of the algorithm are considered, the proposed ISSA-DWA\u2019s performance on the optimal path planning problem outperforms the proposed ISSA and the other four heuristic algorithms, such as the ACO, MRFO, WOA and SSA. The experimental results demonstrate that the proposed ISSA-DWA can not only solve the shortcomings of the standard SSA but can also plan the global optimal path, which has the ability to smooth the path and avoid dynamic obstacles in real time. The proposed ISSA-DWA was only verified on the mobile robot\u2019s simulation of the optimal path problem. In order to apply it to the practical engineering problem, future research should focus on extending it to the real mobile robot to verify the effectiveness of the proposed methods."}
+{"text": "NHP2, encoding for one component of the telomerase cofactor H/ACA RNA binding complex together with Dyskerin, NOP10 and GAR1, have been previously reported in rare cases of TBDs. Here, we report two novel NHP2 variants (NHP2-A39T and NHP2-T44M) identified in a compound heterozygous patient affected by premature aging, bone marrow failure/myelodysplastic syndrome and gastric cancer. Although still able to support cell viability, both variants reduce the levels of hTR, the telomerase RNA component, and telomerase activity, expanding the panel of NHP2 pathological variants. Furthermore, both variants fail to be incorporated in the H/ACA RNA binding complex when in competition with wild-type endogenous NHP2, and the lack of incorporation causes their drastic proteasomal degradation. By RoseTTAFold prediction followed by molecular dynamics simulations, we reveal a dramatic distortion of residues 33\u201341, which normally position on top of the NHP2 core, as the main defect of NHP2-A39T, and high flexibility and the misplacement of the N-terminal region (residues 1\u201324) in NHP2-T44M and, to a lower degree, in NHP2-A39T. Because deletion of amino acids 2\u201324 causes a reduction in NHP2 levels only in the presence of wild-type NHP2, while deletion of amino acids 2\u201338 completely disrupts NHP2 stability, we propose that the two variants are mis-incorporated into the H/ACA binding complex due to the altered dynamics of the first 23 amino acids and/or the distortion of the residues 25\u201341 loop.Telomere biology disorders (TBDs) are characterized by short telomeres, premature aging, bone marrow failure and cancer predisposition. Germline mutations in  Telomeres comprise thousands of TTAGGG duplexes bound by the six-protein shelterin complex and folded back in the t-loop structure (reviewed in Germline mutations affecting telomere stability are associated with telomere biology disorders (TBDs) and are characterized by short telomeres and a spectrum of conditions, including premature aging, oral leukoplakia, skin pigmentation, nail dystrophy and bone marrow failure in dyskeratosis congenita (DC), intrauterine growth retardation and microcephaly in H\u00f8yeraal\u2013Hreidarsson syndrome (HHS), and aplastic anemia (AA), idiopathic pulmonary fibrosis (IPF) and liver cirrhosis  variants have been identified in unrelated TBDs patients  identified in an adult patient diagnosed with DC clinical features, bone marrow failure/myelodysplastic syndrome (MDS) and gastric cancer, and we validate both of them as pathological for hTR expression and telomerase activity in two cancer cell lines. By molecular modeling and dynamics simulation, we predict that the first 41 amino acids of NHP2 are either too flexible or misplaced in both variants, potentially affecting their binding to hTR or NOP10. We then show that protein expression of both NHP2-A39T and NHP2-T44M is severely compromised in the presence of endogenous NHP2 due to reduced binding to NOP10 and Dyskerin and high proteasomal degradation, but it is restored when endogenous NHP2 is deleted. A similar phenotype is observed for an NHP2 mutant lacking the first 23 amino acids, whereas the deletion of all the first 37 amino acids completely compromises NHP2 protein levels. Therefore, we propose that all the NHP2 not incorporated in the H/ACA RNA binding complex is rapidly degraded by the proteasome and that the correct positioning of the first 24 amino acids of NHP2 plays a key role in the kinetics of such incorporation.In this study, we present two novel The index patient with no family history of MDS was diagnosed at age 38 with gastric cancer and low-risk MDS, with normal karyotype . Cancer was successfully removed by surgery (Billroth II). After 7 years, his cytopenia (mainly anemia) aggravated. Further clinical investigation revealed nail dystrophies, gray hair, tooth loss and oral leukoplakia with a bone marrow cellularity between 30 and 40%, which decreased to 10\u201330% in the following 8 months, with normal karyotype but erythro- and megakaryopoiesis dysplasia, suggesting TBDs. Consistently, PCR showed short telomeres compared with same-age controls . Family\u2019NHP2 at c.115G\u2009>\u2009A (chr5:177580704) and c.131C\u2009>\u2009T (chr5:177580688) leading to p.Ala39Thr (hereafter indicated as NHP2-A39T) and p. Thr44Met (NHP2-T44M), respectively (\u22124 (Subsequent whole-genome sequencing performed on DNA extracted from the patient\u2019s fibroblasts identified compound heterozygote missense variants in ectively . The firvely (\u22124 . HoweverNHP2 mutation (R41H) (in silico to be highly deleterious for NHP2 protein function (SIFT tolerance score of 0.00 and PolyPhen confidence score of 0.999 for both).These two mutations are located in the N-terminal region of NHP2, in proximity to only one other previously identified n R41H)  29). Ta. TaNHP2 n (R41H) . Althoug1H  29). n (R41H) , both suNHP2-A39T and NHP2-T44M variants on telomerase activity in human cell lines. Because NHP2 is essential, MYC-tagged wild-type NHP2 (NHP2-WT) or mutant NHP2 (NHP2-A39T or NHP2-T44M) were expressed in telomerase-positive HeLa cancer cell line first, and then the bulk endogenous NHP2 was targeted with CRISPR/Cas9. The Myc-tag allowed to differentiate between exogenous Myc-NHP2 (NHP2*) and endogenous NHP2 (endo-NHP2) on the immunoblot . Al. Alin sieTTAFold . HowevereTTAFold \u2013S5. CluseTTAFold , accounteTTAFold . From reeTTAFold , whereaseTTAFold  and stabeTTAFold .In parallel, the five initial models predicted by RoseTTAFold for either NHP2-A39T or NHP2-T44M  against Myc in HT1080 cells expressing either NHP2-WT, NHP2-A39T or NHP2-T44M after deletion of endo-survival  and the survival , demonstNHP2 with lentiviral CRISPR Cas9/sgRNA caused a significant increase in the levels of NHP2-A39T and NHP2-T44M, about 50- and 4-fold, respectively, whereas only slightly affecting NHP2-WT levels , we, wein si2 levels . On the 2 levels , demonstImportantly, similar competition between endo-NHP2 and exogenous NHP2 and the instability of NHP2-A39T and NHP2-T44M was also observed in U-2OS, HeLa cancer cells and in normal BJ fibroblasts S6A, B. NSupplementary Material, Figs S3D and Because both variants were predicted to affect the positioning/folding of the NHP2 N-terminal, we investigated the role of the N-terminal in the formation of the H/ACA RNA binding complex by expressing untagged NHP2 mutants deleted of amino acids 2\u201324 (NHP2-\u039424) or amino acids 2\u201338 (NHP2-\u039438) before or after deletion of endo-NHP2 . NHP2-\u03942Importantly, either NHP2-A39T or NHP2-T44M variants were stabilized by the deletion of amino acids 2\u201324  , suggestGermline predisposition for bone marrow failure and hematological disorders, including malignancies, is a major clinical challenge, and numerous novel mutations have been identified during the last decade, including many associated with telomere biology . IdentifNHP2 gene (OMIM #613987), NHP2-A39T and NHP2-T44M, identified in a patient diagnosed with pancytopenia and signs of early aging. Both variants affect hTR stability and telomerase activity, validating them as pathogenic for TBDs. However, and consistent with the late onset of TBD, neither of the variants compromises the essential functions of NHP2. A specific impairment in binding hTR versus other H/ACA RNAs could explain the different effect on telomerase activity versus cell survival for both mutations. Another possibility is that lower NHP2 levels, as observed for both mutants even in the absence of wild-type NHP2, are sufficient to perform NHP2\u2019s primary functions but are not enough for the secondary functions such as telomerase formation. Because NHP2-A39T is highly unstable in the presence of wild-type NHP2 while NHP2-T44M is still, at least partially, incorporated in the complex in the presence of wild-type NHP2, the NHP2-A39T could be reasonably considered recessive, whereas NHP2-T44M could be co-dominant. Although further analysis on individuals carrying NHP2-T44M in heterozygosity will be needed to confirm this hypothesis, this proposed analysis would be in agreement with the previous identification of monoallelic NHP2 variants in adult patients diagnosed with AA and/or IPF  of NHP2 in promoting H/ACA RNA binding complex formation and NHP2 stability. Residues 2\u201324 are the most divergent between species and even absent in the archaea paralogue RL7. Furthermore, they have never been resolved by X-ray crystallography, cryo-EM  due to tAs for the first 23 amino acids, it is possible that they actively promote transient interactions with NOP10 and/or Dyskerin. Indeed, also Dyskerin has several unresolved regions that are predicted to be highly flexible and could mediate such interaction. Another non-mutually exclusive possibility is that the correct binding of this flexible N-terminal tail to the external surface of NHP2 stabilizes the folding of the 25\u201338 loop, therefore promoting the formation of the interface with NOP10. Because the addition of a Myc-tag before the N-terminal does not compromise NHP2\u2019s stability and/or functions, we do favor the second hypothesis, but further studies will be required to characterize the dynamic mechanism underlying the interaction of NHP2 with NOP10/Dyskerin.in silico and genetic analysis, we would like to propose that the main effect of the T44M mutation is to increase the flexibility of the N-terminal aberrantly. This increased flexibility would then compromise the correct positioning of the N-terminal and its function in the formation of the H/ACA RNA binding complex. Furthermore, mispositioning of the N-terminal could also directly reduce the ability of NHP2 to bind hTR. The effect of A39T substitution seems instead to be more deleterious since the torsion of the P33-R41 loops could severely affect the interface between NHP2 and NOP10, unless the correct positioning of the first 23 amino acids compensates for it. This would explain why the removal of the N-terminal tail in the NHP2-A39T variant exacerbate NHP2 instability.In this context, the telomerase-deficient phenotype observed in these new NHP2 variants could be explained by three non-mutually exclusive hypotheses: defective binding to hTR (i), low levels of active H/ACA RNA binding complex due to reduced intrinsic stability of NHP2 (ii) and/or defective incorporation of NHP2 into the complex (iii). In fact, both mutants are predicted to have reduced interaction with hTR but are also less expressed than wild-type NHP2. Furthermore, as discussed before, it is not possible to discriminate between intrinsic instability due to misfolding and proteasome-dependent degradation of all the NHP2 not incorporated into the H/ACA RNA binding complex. However, based on our The lack of incorporation of NHP2 in the H/ACA RNA binding complex promotes dramatic NHP2 degradation. Tight regulation of NHP2 levels has been previously shown to be associated with ALT  cells. HRelative telomere length (RTL) was determined by quantitative PCR on 17.5\u00a0ng DNA extracted from peripheral blood, as described in , using sSequencing libraries were prepared from 50\u00a0ng of genomic DNA using TruSight Myeloid Sequencing Panel  according to the manufacturer\u2019s instructions. Ingenuity Variant Analysis (Qiagen) was used to detect SNVs and small indels with detection thresholds of 500\u00d7 coverage, variant allele frequency of 5% and minimum 50 reads for variant. No variants met the conditions to be included in downstream analysis.https://github.com/lh3/bwa). The alignments were deduplicated (PicardMarkDuplicates), recalibrated and indel realigned using Genome Analysis Toolkit, GATK v3.3.0 (https://github.com/broadinstitute/gatk-docs). Small nucleotide variants and indels were called using the GATK Haplotypecaller and annotated using snpEff v4.1 (http://pcingola.github.io/SnpEff/). Variant files and patient HPO term descriptions were uploaded to Moon  for variant prioritization. Highlighted variants were inspected using Integrative Genomics Viewer  (Sequencing libraries were prepared from 1\u00a0\u03bcg DNA using the TruSeq PCRfree DNA sample preparation kit (Illumina) targeting an insert size of 350\u00a0bp. The library was sequenced with paired-end 150\u00a0bp reads on NovaSeq S4 flowcell, with v1 sequencing chemistry (Illumina). Raw sequence reads were aligned to the human reference GRCh37.75 using bwa 0.7.12  .https://www.uniprot.org) and aligned in SnapGene (GSL Biotech) using Clustal Omega or MUSCLE multiple sequence comparison. Alignments were colored with ClustalX coloring (properties\u2009+\u2009conservation) or by identity using Homo sapiens NHP2 as reference.Protein sequences were obtained from BLAST-Uniprot (https://sift.bii.a-star.edu.sg) (http://genetics.bwh.harvard.edu/pph2/dokuwiki/start)  , where bi/start) , where tl-glutamine (Gibco), non-essential amino acids (Gibco) and penicillin\u2013streptomycin (Gibco) at 37\u00b0C and 5% CO2. Bortezomib  was dissolved in DMSO (Sigma) and added to the cells  for 6\u00a0h before harvest.HeLa S3, HT1080, U-2OS, BJ, 293T and Phoenix AMPHO  were cultured in Dulbecco\u2019s modified Eagle medium (DMEM) (Corning) supplemented with 10% fetal bovine serum (FBS) (Gibco), NHP2-WT was cloned in pWZL-MYC hygromycin-resistant retroviral vector (GenScript). The mutations G115A (NHP2-A39T) or C131T (NHP2-T44M) were inserted by site-directed mutagenesis. The gRNA against endogenous NHP2 (5\u2032-CAAATGCATCAAGAAAGGTG-3\u2032) was cloned in pLentiCRISPR v2 puromycin-resistant lentiviral vector. Depletion of Dyskerin was obtained using shRNA cloned in pLKO.1 vector . Phoenix AMPHO and 293T were transfected by CaPO4 precipitation with 20\u00a0\u03bcg of plasmid DNA for Retroviral and Lentiviral transduction, respectively. Viral supernatant, supplemented with 4\u00a0\u03bcg/ml polybrene (Merck Millipore), was filtered through a 0.45-\u03bcm filter and used for transduction of target cells two/three times per day over 2 days. Target cells were selected in 300\u00a0\u03bcg/ml Hygromycin B  or 300\u00a0nm Puromycin  for 3\u20136\u00a0days.Deletions of amino acids 2\u201324 or 1\u201337 were performed by PCR with the following primers:NHP2\u2014Rev: 5\u2032-CATGGTGGCTGATCCGGCCGGC-3\u2032.NHP2-\u039424\u2014For: 5\u2032-CAGGAGCTGCTGGTCAACCAG-3\u2032.NHP2-\u039438\u2014For: 5\u2032-GCTTCTCGCCGCCTCACGCGG-3\u2032.m Tris\u2013HCl pH\u00a06.8, 200\u00a0\u03bcm DTT, 3% SDS, 20% glycerol, 0.05% bromophenol blue) at 5\u2009\u00d7\u2009103 cells/\u03bcl. The lysate was denatured for 10\u00a0min at 96\u00b0C, and genomic DNA was sheared with 29-gauge needle. Proteins were separated by SDS/PAGE and transferred to a nitrocellulose membrane. The membrane was blocked in 5% milk dissolved in PBS with 0.1% Tween-20 and incubated with primary antibodies for 2\u00a0h at RT. Secondary anti-mouse or anti-rabbit IgG HRP antibodies  were incubated for 1\u00a0h at RT. The signal was detected by ChemiDoc  after adding Chemilimunescence western blotting reagents  and quantified using ImageJ  and PhosSTOP phosphatase inhibitor mix (Roche)] ] . After s6 cells using RNeasy Mini Kit (Qiagen). RNA (500\u00a0ng) was reverse transcribed using First Strand cDNA Synthesis Kit (Thermo Scientific). SYBR Green PCR Master Mix (Applied Biosystems) was used for qPCRs. BioRad software was used to automatically calculate thresholds. Primers used in this study were as follows:RNA was extracted from 10GAPDH-F: 5\u2032-GTCTCCTCTGACTTCAACAGCG-3\u2032 (OriGene).GAPDH-R: 5\u2032-ACCACCCTGTTGCTGTAGCCAA-3\u2032 (OriGene).hTR-F: 5\u2032-AACCCTAACTGAGAAGGGCG-3\u2032.hTR-R: 5\u2032-AGAATGAACGGTGGAAGGCG-3\u2032.NHP2-F: 5\u2032-AGACACACTGCCCATTGAGG-3\u2032.NHP2-R: 5\u2032-TCCAGGCACTCATCGTAAGC-3\u2032.hTR or NHP2 values were normalized on GAPDH mRNA.m ACX primer and 0.3\u00a0\u03bcm TS primer , neocarzinostatin  , cisplatin   or their respective controls . For \u03b3-irradiation, cells were exposed to 1\u20133\u00a0Gy/min. Cells were incubated for 10\u201314\u00a0days with one change of medium at day 6. Plates were stained with 50% methanol/2% methylene blue and colonies were scored manually.HeLa and U-2OS cells were seeded in 6-well plates in triplicate or duplicate at 100 or 250 cells per well, respectively. After 24\u00a0h, cells were incubated with aphidicolin  (50\u2013300\u00a0n2P2 disorder predictor database (http://d2p2.pro/) (The predicted disordered agreement for NHP2 was obtained by entering hNHP2 sequence into the Dp2.pro/) .https://robetta.bakerlab.org/), with no template provided and standard parameters. MD simulations were performed using GROMACS 2020.3 software package (www.gromacs.org) and CHARMM36m force field  within NPT thermodynamic ensemble at 300\u00a0K and 1\u00a0atm using a time step of 2\u00a0fs. Cluster analysis was then performed on a skipped trajectory of 5000 frames for each system. The RMSD matrices were computed on backbone atoms and then processed using the Gromos (Five structures of full-length NHP2-WT, NHP2-A39T and NHP2-T44M were modeled using RoseTTAFold  at the Rce field . Each moe Gromos  algorithGraphPad Prism was used to perform statistical analysis on at least three separate experiments.P-value using unpaired t-test, ordinary one-way ANOVA for multiple comparisons, two-way ANOVA for multiple comparisons or ratio paired t-test. P-Values \u22640.05 were considered statistically significant. ns, not significant, *P\u2009\u2264\u20090.05, **P\u2009\u2264\u20090.01, ***P\u2009\u2264\u20090.001, ****P\u2009\u2264\u20090.0001.Significance was assessed by calculating the HMG-2023-CE-00181Malinski_Supplementary_Data_proof_ddad114Click here for additional data file.Supplemental_Material_Table_S1_ddad114Click here for additional data file.Supplemental_Material_Table_S2_ddad114Click here for additional data file.Supplemental_Material_Table_S3_ddad114Click here for additional data file.Supplemental_Material_Table_S4_ddad114Click here for additional data file.Supplemental_Material_Table_S5_ddad114Click here for additional data file.Movie_S1_ddad114Click here for additional data file.Movie_S2_ddad114Click here for additional data file.Movie_S3_ddad114Click here for additional data file.Movie_S4_ddad114Click here for additional data file.Movie_S5_ddad114Click here for additional data file.Movie_S6_ddad114Click here for additional data file.Movie_S7_ddad114Click here for additional data file.Movie_S8_ddad114Click here for additional data file.Movie_S9_ddad114Click here for additional data file.Movie_S10_ddad114Click here for additional data file.Movie_S11_ddad114Click here for additional data file.Movie_S12_ddad114Click here for additional data file.Movie_S13_ddad114Click here for additional data file.Movie_S14_ddad114Click here for additional data file.Movie_S15_ddad114Click here for additional data file."}
+{"text": "The retaining wall is a passive engineering measure to prevent and control unsafe factors caused by rock collapse in the valleys. Existing studies have mainly focused on its functional robustness and safety features, with few exploring its visual quality in the landscape. A multiple regression analysis was applied to evaluate the Scenic Beauty Estimation (SBE) of the giant retaining wall in Jiuzhaigou\u2019s  Heye Village, then the factors affecting SBE were analyzed. It is found that enhancing the sense of perspective and spatial hierarchy of retaining-wall murals in narrow roads contributes to the extension of observers\u2019 sight, which is the key to improving SBE. Furthermore, the showcase of folk culture in murals can realize the beautification function of the giant retaining walls. In addition, the SBE of giant retaining walls is also linked to coordination, where the walls embellished with the natural landscape and folk culture murals have better SBE performance than those with local stones. This study provides a reference for constructing scenic beauty after fulfilling the safety function of retaining wall engineering. Located in the southeastern margin of the Qinghai-Tibet Plateau, the western part of Sichuan province is a typical mountainous area covered by plateau mountains and hills . In the The giant retaining wall  is a cruAdministrators, engineers, and designers have attempted to dissipate the negative visual impact of these structures on the environment. Although some studies point out that retaining walls can achieve positive visual effects in urban streets and improve the scenic appearance , its effExisting landscape assessment methods include \u201cLandtype Hypothesis\u201d , \u201cLandcoThe landscape visual quality of retaining walls is assessed through the sensitivity, universality, and applicability of SBE . This isTherefore, this paper adopts the SBE method to examine the giant retaining walls in Jiuzhaigou\u2019s Heye Village, Sichuan Province. Based on the evaluation indexes of regional characteristics, the giant retaining walls with natural scenery murals, folk culture murals, and local stones are evaluated. This paper attempts to answer the following questions: 1) What are the main factors affecting the aesthetic value of giant retaining walls in valleys? 2) What effects do different beautification approaches have on the aesthetic value of giant retaining walls?The research object of this study is the giant retaining wall of Heye Village, a typical mountain village in Jiuzhaigou Scenic Area, Aba Prefecture, Sichuan Province, China.  . In 1992A 7.0-magnitude earthquake struck Jiuzhaigou on August 8, 2017. The earthquake not only caused severe infrastructure damage and casualties but also triggered a large number of landslides . FurtherIn reality, there are several ways to decorate the giant retaining wall, such as planting greeneries, painting murals, and embedding materials. Constrained by budget, Heye village settled on murals and local stones. Jiuzhaigou\u2019s unique landscape and regional culture  were painted on the wall. Local stones were embedded in certain parts of the wall. The purpose was to reduce the adverse effects of the giant retaining wall by shifting visual attention.The index construction of the SBE for retaining walls is multi-dimensional. For such unique engineering structures as giant retaining walls, no clear evaluation metrics have been proposed in the existing evaluation methods. Therefore, new evaluation indexes must be developed based on available visualization resources. Furthermore, the giant retaining walls in valleys are ubiquitous in the mountainous areas of western Sichuan and have a unique regional cultural background. Accordingly, this study innovates the evaluation indexes with \u201cregionalization\u201d under the mature SBE method system.First, the retaining-wall SBE needs to follow the basic principles of landscape aesthetics, in which the evaluator\u2019s visual perception is the most significant . Second,Regarding the previous research results on influencing factors of SBE value [31-33], five perceptual indicators are identified .Previous studies showed no significant difference between photos-based landscape quality evaluation and on-site evaluation . HoweverEach evaluator rated the five perceptual indicators (independent variables) for each photo; then, the mean score of each indicator of each photo was taken as the quantitative value. The dependent variable is the SBE value calculated by the standardized formula after the evaluator scores the five perceptual indicators of the retaining-wall photos , 22. To Previous studies also showed no significant differences in landscape evaluations between students and the general public , 37. In The evaluation value of each sample photo is standardized  to elimiij represents the standardized value of image i by evaluator j. Rij is the evaluation value of evaluator j for image i, while j. Sj is the standard deviation of the evaluation value of all images by evaluator j(1).Zi stands for the standardized value of image i by all evaluators. The average of all standardized scores for each image is the standardized value of SBE for each image. Zij is the standardized value of evaluator j for image i, and Nj is the number of total evaluators for image i(2).SBEX1 (color richness), X2 , X3 , X4 (vegetation coverage), and X5 , and SBE value of the giant retaining wall in Heye Village. Multivariate linear regression analysis was adopted, and the retaining-wall SBE model was established. The validity of the model was verified through the multicollinearity test, degree of fitting (R2) test, t-test, and F-test. The Data processing adopted EXCEL and IBM SPSS 23.0 , including standardization of SBE values, analysis of variance, and regression analysis.This paper assumes that there is a linear relationship between the five perceptual evaluation indicators of The results indicated that the SBE values of the 20 sample photos of the giant retaining wall in Jiuzhaigou\u2019s Heye Village range from 0.82 to -0.575 , of whicP value) (P<0.05), the P values of the sample images were all greater than 0.05. This indicated that the professional background of elevators had no significant impact on the SBE of the giant retaining wall of Heye Village.The standardized value of each sample photo was analyzed by ANOVA to obtain the significance test value  . The resBy comparing the evaluation results of the giant retaining wall of the Heye Village between the professional and the non-professional groups , it was X1 color richness and X4 vegetation coverage. The factors X2 visual effect (sig = 0.000<0.05), X3 cultural connotation (sig = 0.000<0.05), and X5 overall coordination (sig = 0.000<0.05), which contributed more to the SBE value, were retained. A regression equation for predicting the SBE of the giant retaining wall of Heye Village was formulated: y = -3.297+0.904X2+0.508X3+0.746X5. The absolute value of the standardized regression coefficient reflects the weight of the indicators\u2019 contribution to SBE, where X2 is the most important, followed by X5 and then X3. The positive and negative signs reflect the direction of the contribution to SBE.The stepwise method in linear regression excluded the less important factors, R2 reflected the effect of linear regression. The closer the value of R2 to 1, the better the fitting degree [R2 of the regression model was 0.984 , indicating that the dependent variable y had a significant linear regression effect on the independent variables. Therefore, the three perceptual evaluation indicators selected were significantly correlated with the SBE value of the giant retaining wall. This model can be applied to predict the SBE value of the giant retaining wall of Heye Village.First, the regression model was tested for multicollinearity through the variance inflation factor (VIF). According to the results, the VIF ranged within 1.064\u223c8.282 (VIF<10), and the tolerance was above 0.1, indicating no collinearity in the independent variables of the regression equation. The results of partial correlation coefficients were all significant by t-test . The coeg degree . The R2 as 0.984 , and theson test  value wa test [F , and F wThe SBE results of the giant retaining wall agreed with previous studies, indicating that groups with different backgrounds are roughly consistent in natural landscape aesthetics , 46. TheX1) can significantly improve the visual environment quality of landscapes [X1) and vegetation coverage (X4) of the giant retaining wall had no significant effect on the SBE value of the giant retaining wall, which is inconsistent with the findings of previous research. The possible reason is that designers are reserved about the color properties  for murals on the giant retaining wall. The excessive color expression may strengthen the difference between murals and the surrounding environment and thus affect visual perception. The giant retaining wall intersections with the mountain in the valley, forming a steep and undulating narrow slope (average slope above 50\u00b0) at the roadside. In addition, the soil and water loss is severe, and the soil layer of the slope is thin, which makes it difficult to form a coordinated, green space. So the impact of natural vegetation coverage on the overall visual aesthetics is weak.The purpose of psychophysical SBE is to study the consistency and difference of landscape in people\u2019s eyes and to analyze the main factors affecting landscape evaluation . In prevndscapes , 26. In ndscapes , and divndscapes , 50. HowX2) of the retaining-wall mural is the most significant factor affecting the SBE of the giant retaining wall. The giant retaining wall at the edge of Heye Village forms a narrow space with the road, with the narrowest being less than 5 meters  also significantly impacted the SBE value of the retaining wall. Previous research has shown that the mural consists not only of an artistic aspect but also depicts the culture and way of life at the time it was created [According to the results, 9 out of the top 10 sample photos with the highest SBE values exhibited folk culture scenes. The results of multiple regression analysis also showed that the evaluators\u2019 awareness of Jiuzhaigou folk culture , and contribute to SBE improvement. Nevertheless, A16 (-0.575), A9 (-0.324), and A1 (-0.226) gain the lowest SBE values, and these samples all use the local stones to embellish some parts of the murals. In other words, murals with local stones are less effective than natural scenery murals and folk culture murals in beautifying the giant retaining wall. The reason may be that the local stones only cover a smaller area compared to the mural area, which is less likely to attract the viewer in their visual perception. Furthermore, if large numbers of local stones are used to upgrade the texture of the vertical wall, it will increase the load of the giant retaining wall and thus affect its safety and stability. Another point of concern is that, from the perspective of engineering cost, the giant retaining walls in mountain streams and valleys are often built at the steep slopes at the foot of the mountain. Therefore, the transportation and construction of local stones invariably consume plenty of resources and funds. Nevertheless, the enhancement of the SBE is less effective. This reminds the administrators and designers to make wise selections of materials for retaining wall beautification.As an engineering protection measure made of reinforced concrete, the giant retaining wall is poorly coordinated with the surrounding environment, reflecting the inhomogeneity and complexity of the spatial distribution of landscape elements and their properties, that is, the heterogeneity of landscapes. The murals with different contents  and local stones alleviate the heterogeneity between the giant retaining wall and the surrounding environment, promote the visual coordination of the landscape Click here for additional data file."}
+{"text": "Aquaporins  are water channels expressed in many and diverse cell types, participating in various functions of cells, tissues, and systems, including the central nervous system (CNS). AQP dysfunction and autoimmunity to AQPs are implicated in several diseases. The best-known example of autoimmunity against AQPs concerns the antibodies to AQP4 which are involved in the pathogenesis of neuromyelitis optica spectrum disorder (NMOSD), an autoimmune astrocytopathy, causing also CNS demyelination. The present review focuses on the discovery and the potential role of antibodies against AQP1 in the CNS, and their potential involvement in the pathophysiology of NMOSD. We describe (a) the several techniques developed for the detection of the AQP1-antibodies, with emphasis on methods that specifically identify antibodies targeting the extracellular domain of AQP1, i.e., those of potential pathogenic role, and (b) the available evidence supporting the pathogenic relevance of AQP1-antibodies in the NMOSD phenotype.  Aquaporins (AQPs) represent an important group of transmembrane proteins, having a key role in the transport of water across biological membranes acting as a water channel ,2. AQPs In mammals, 13 distinct types of AQPs have been identified (AQP0 to AQP12), which are classified into three families based on their permeability status: (a) the AQPs , (b) the aquaglyceroporins  which are additionally permeable to urea and glycerol, and (c) the \u2018\u2018unorthodox\u2019\u2019 AQPs (AQP11 and AQP12) [AQPs assemble as tetramers in the cell membrane which are present in various tissues such as the kidneys, lungs, and brain ,3,5. In Defects and/or mutations in aquaporin-related genes can lead to various diseases. For example, mutations in the AQP2 gene can cause nephrogenic diabetes insipidus (NDI) , in whicFurthermore, there is ongoing research for the development of small molecule inhibitors targeting AQPs to affect their function; an example of this category of drugs is the tolvaptan, a selective vasopressin V2 receptor antagonist that targets AQP2 in the kidneys. By blocking the interaction between vasopressin and AQP2, tolvaptan reduces water reabsorption in the collecting ducts, thereby decreasing urine concentration and increasing urine volume. It has been used for the treatment of conditions including hyponatremia and polycystic kidney disease . Other AAntibodies against AQP channels are involved in the pathogenesis of autoimmune disorders of the central nervous system (CNS) , blood  and kidnAQP4 is the most abundant aquaporin in the CNS, found in the optic nerve , brain ,22, and NMO, also known as Devic\u2019s disease, is a relapsing-inflammatory autoimmune astrocytopathy, more commonly associated with longitudinally extensive transverse myelitis (LETM) and optic neuritis . The cliSeveral reports have shown that, in addition to AQP4, human CNS astrocytes also abundantly express AQP1 on their surface ,32. SpecAQP1 is the first identified member of the aquaporin superfamily. The structure of AQP1 has been extensively studied using X-ray crystallography and electron microscopy . The AQPAQP1 is expressed in numerous tissues of the body. In the kidney, AQP1 is abundant in the proximal tubules and is particularly important for water reabsorption from the urine back into the bloodstream, regulating fluid balance, retaining necessary fluids, and preventing excessive water loss. ,39. In rAQP1 antibodies have been detected in autoimmune hemolytic anemia (A\u0399HA) and in S\u00f6gren\u2019s syndrome (SS).AQP1 contains the Colton blood group antigen expressed on erythrocytes . AntibodSS is a chronic autoimmune disease that primarily targets the lacrimal and salivary glands causing dryness in the mouth and eyes. AQPs seem to play a role in salivary gland secretions/function. Alam et al.  reportedFurthermore, Tzartos et al. screened 34 sera from SS patients for binding to the extracellular domains of various AQPs . Thirteeneg cases antibodies against myelin oligodendrocyte glycoprotein (MOG) can be detected as well. It must be pointed out that the diagnosis of AQP4-Abneg/MOG-Abneg (seronegative) NMOSD phenotype remains extremely challenging.Human CNS astrocytes express both AQP4 and AQP1 on their surface . It is wIn 2013, Tzartos et al.  used var125-labelled intact AQP1, Western-blot with SDS-denatured AQP1 polypeptide, ELISA with intact AQP1, ELISA with synthetic peptides corresponding to the extracellular and cytoplasmic loops of AQP1 channel, and importantly, an indirect live cell-based assay (indirect CBA) with similar reliability with the classical CBA, which is used for the detection of antibodies to several membrane antigens. Yet, probably due to the reasons explained below, a reliable classical (live) CBA has not been established until now.Although a gold-standard method for detecting AQP1-Abs is not yet available, several different methodologies have been proven successful in detecting AQP1-Abs, which confirm one another. These include radioimmunoprecipitation assay (RIPA) with IRadioimmunoprecipitation assay (RIPA)125-streptavidin. This test identified 58/348 (17%) patients with suspected NMOSD phenotype as positive for AQP1-Abs (AQP1-Abpos sera) (pos patients was somewhat higher than that of the AQP4-Abpos patients (12%) in the studied group. No AQP1-Abs were detected in the 242 control sera [pos/AQP4-Abpos) was also detected in some patients with definite NMOSD phenotype [Initially, a sensitive RIPA for AQP1-Abs was developed, using biotinylated human AQP1 indirectly labeled with Ios sera) . The antrol sera  and 4% .The use of AQP1-ELISA for the detection of AQP1-Abs in patient sera was also attempted by at least another two groups. S\u00e1nchez Gomar et al. , in addiJasiak-Zato\u0144ska et al.  using a d.ELISA with AQP1 synthetic peptides (extracellular vs. intracellular location of the epitopes)pos sera were bound to some AQP1 synthetic peptides. Interestingly, most sera were bound selectively only to one peptide group, either the extracellular or cytoplasmic peptides. In competition experiments, in which positive sera were preincubated with a mixture of AQP1 peptides before testing the available free antibodies with RIPA or ELISA using intact AQP1, the binding to intact AQP1 was reduced by more than 50%. This confirmed that most AQP1-Abs bind to the synthetic peptides and therefore the peptide binding findings are representative of the major fraction of the AQP1-Abs repertoire. Using the peptide-ELISA it was found that out of 16 patients with NMOSD phenotype or myelitis and AQP1-Abs, 13 had predominantly antibodies to extracellular loops (mostly to Loop-A) and only three patients had predominantly antibodies to the cytoplasmic peptides. In contrast, all three AQP1-Abpos patients with classical MS profile  had only antibodies to the cytoplasmic AQP1 epitopes . These are analyzed in more detail in To differentiate between sera with potentially pathogenic antibodies  and those with antibodies to in vivo unavailable epitopes, i.e., to intracellular sites, an ELISA with synthetic peptides corresponding to all extracellular parts of the AQP1 molecule was employed . These iDirect CBALive-cell CBA  is currently considered the gold standard method for detecting antibodies to membrane antigens of the nervous system. This approach detects only the antibodies that can bind to the cell-exposed part of the antigen, in its native form, which are obviously more likely to be disease-relevant . The proSubsequently, the following three different groups also attempted to detect AQP1-Abs in patients with NMOSD phenotype and other CNS demyelinating diseases using different CBA approaches ,57,58. Opos and 151 with AQP4-Abneg sera. AQP1-Abpos were found in 74.5% AQP4-Abpos sera  and 32.5% of the AQP4-Abneg sera. Triton-X permeabilization of the cells allows access of the antibodies to the abundant AQP1 molecules inside the cells but also allows access to the cytoplasmic side of the AQP1 molecule. However, as also explained above, antibodies targeting the cytoplasmic side of the molecule are not expected to be pathogenic [Long et al.  developethogenic ,62 thus pos sera were detected with the intact fixed cells. Yet, contrary to Long et al. [Gomar et al.  developeg et al.  in cellspos. In contrast, similar to the commercial anti-AQP1 mAb, no serum was found positive for AQP1-Abs [neg NMOSD sera (103-81 AQP4-Abpos). Nevertheless, the authors concluded that there are no AQP1-Abs in NMOSD [Finally, Schanda et al.  developeAQP1-Abs . Yet, itin NMOSD . Tzartosin NMOSD  in a repin NMOSD , suggestAlthough all the above four studies acknowledge the difficulty in the development of a live-cell CBA capable of detecting AQP1-Abs in NMOSD sera, overall, they neither confirm nor exclude the presence of antibodies capable of binding to the cell-exposed AQP1.b.Indirect CBASince major difficulties were met during the efforts to establish a reliable and sensitive direct live-cell CBA, an indirect live-cell CBA, which, like the classical live CBA detects only the antibodies capable of binding to the extracellular side of the cell-embedded AQP1, was then developed . This assay involves preincubation of the test serum (which may contain AQP1-Abs), with intact live cells transfected with human AQP1 (or AQP4 or control plasmid). This allows only serum AQP1-Abs capable of binding to the extracellular side of the AQP1 molecule in its natural environment, embedded within the plasma membrane of the live cells, to bind to the AQP1-transfected cells. The treated (possibly antibody-depleted) serum, in parallel with the control-treated serum, is then tested by RIPA or ELISA with purified AQP1 to measure the unbound antibodies. The potential reduction of antibodies in the sample pretreated with AQP1-expressing cells  represents the fraction of the serum\u2019s antibodies capable of binding to the surface-exposed side of the cell-embedded AQP1  live-CBA, but it has the advantage over direct CBA, that it is quantitative while it detects exactly the same antibodies with the live CBA. Therefore, it is at least as reliable as the direct CBA. Unfortunately, we are not aware of other groups\u2019 efforts to reproduce this \u201cindirect CBA\u201d approach. pos). During the same period, we identified, by CBA, 119 new AQP4-Abpos patients, meaning that the frequency of the AQP1-Abpos patients was approximately half of that of the AQP4-Abpos patients. This frequency of AQP1-Abpos patients is very considerable, which could be invaluable for NMOSD diagnosis. Lastly, no serum was found double positive for extracellular AQP1 and AQP4-Abs. However, we did identify two cases of AQP4-Abpos sera containing additional antibodies against the cytoplasmic side of AQP1 (unpublished data). Since a combination of ELISAs with intact AQP1 and with the two groups of synthetic peptides (corresponding to the extracellular and cytoplasmic side of AQP1) corelated well with the results of the more laborious indirect CBA, we routinely screened sera from patients suspected for NMOSD phenotype only with the combined ELISAs. In selected ELISA-positive cases, we confirmed the result with the indirect CBA. Between 2016 and 2022, we conducted screening for the potential detection of AQP1-Abs in all patient samples that were referred to Tzartos NeuroDiagnostics for testing against NMOSD antigens. Our analysis revealed 55/121 (45%) new patients with AQP1 antibodies had antibodies against the extracellular peptide-loops of AQP1, i.e., potentially pathogenic antibodies. All sera with antibodies to the AQP1 synthetic peptides were also bound to the intact AQP1, and all sera bindings to the intact AQP1, were additionally bound to the peptide group. On the other hand, no serum showed binding to both extracellular and cytoplasmic peptides. Only sera positives for the extracellular peptide group were considered positive  with five of them also presenting with optic neuritis. Another five patients had MS phenotype, although two of them had also predominant spinal cord lesions. The clinical and laboratory data of this cohort are summarized in In the initial study , both mapos patients with NMOSD phenotype of neg patients, with a possible pathogenic role. Therefore, we conclude that the AQP1-Ab indirect CBA test is quite competent in \u201cdetecting\u201d patients with NMOSD clinical characteristics.It should be stressed that the 17 AQP1-AbInterestingly, the AQP1-Abs in the three patients with typical MS phenotype  and a clinical relapse with activity on brain MRI while a high AQP1-Ab titer was measured [As described in measured . Subsequmeasured . FurtherThe high AQP1-Ab titer, in vivo antibody-binding to RBCs during hemolytic anemia and demyelinating relapse, and the drop of AQP1-Ab-titer during the recovery of both clinical syndromes suggest a pathogenic role of these AQP1-Abs. Interestingly, the patient\u2019s antibodies were bound to AQP1 loop-A which contains the Colton group antigens , which have been previously linked to hemolytic reactions . AIHA haDisease transfer models would be highly valuable for demonstration of the pathogenicity of AQP1 antibodies although such evidence is currently not present. There are a few AQP4-antibody mouse models recapitulating major pathological features of NMO  enablingThe present review focuses on the reliable detection of AQP1-Abs by a panel of different methods and on the possible relevance of these antibodies with CNS astrocytopathy and associated demyelination. We also discuss the presumed implication of AQP1-Abs in the development of autoimmune disorders, especially in the occurrence of CNS demyelination. Despite controversies in the detection of AQP1-Abs, it seems that the RIPA, the ELISA, and the Western blot methods with purified AQP1 have led to the reliable identification and quantification of these AQP1-Abs, while ELISA with AQP1 synthetic peptides and especially the indirect CBA suggest that some of these antibodies bind to the extracellular side of AQP1.The detection of AQP1-Abs in some patients with NMOSD phenotype, as well as the binding of AQP1-Abs to the extracellular loops of the AQP1 molecule, are indicative of the possible pathogenicity of these AQP1-Abs. The fact that the majority of the AQP1 antibodies are of the IgG1 subtype  further The two published case reports that demonstrated the pathogenic impact of AQP1-Abs in the CNS and RBCs were also analyzed; specifically, these included (a) the selective AQP1 (but not AQP4) loss in a tumefactive demyelinated lesion in WM of a patient with NMOSD phenotype in conjunction with the antibody deposition in the astrocytes of the WM, and (b) the concurrence of hemolytic anemia and demyelinating relapse with high AQP1 titer, followed by the simultaneous drop of antibody titer and remission of both diseases after \u201cantibody diminishing\u201d therapies  ,49.Future studies are needed to further confirm the role of the AQP1-Abs as biomarkers and pathogens in NMOSD, to characterize in depth the clinical phenotype of the AQP1-NMOSD subtype, and to determine the effect of the known therapies for AQP4-NMOSD cases, versus those for MS, in AQP1-NMOSD cases. Finally, despite the variety of currently developed assays for AQP1-Abs , the development of a reliable and easy direct live-cell CBA  would further enhance the use of these antibodies in NMOSD diagnosis and treatment. neg patients with NMOSD phenotype, along with their likely pathogenic role. In this context, AQP1-Abs could play a pivotal role, providing a promising diagnostic biomarker for patients with NMOSD phenotype; in the future, these antibodies could also be incorporated into the therapeutic targets of NMOSD, thus leading to novel treatment interventions.In conclusion, we presented and discussed the presence of antibodies specific for the cell-surface exposed AQP1 domain, in some AQP4-Ab"}
+{"text": "In this research, muffin-type bakery products were developed based on wheat flour (WF) and mesquite flour (MF) in the following proportions: WFMF 90:10, WFMF 75:25, and WFMF 50:50. The products were characterized based on various properties in which it was possible to observe that the water activity (aw) did not show a significant change with the increase in the concentration of MF. In addition, the increase in the concentration of MF modified the sensory properties , further decreasing the luminosity and increasing the values of the a* and b* coordinates. Moreover, in the texture profile analysis, it was found that the increase in the MF concentration increased hardness, fracturability, and gumminess and decreased adhesiveness and cohesiveness. All the previously mentioned changes were more evident in the WFMF50:50 and, to a lesser degree, in WFMF75:25. In general, in most evaluations realized, the WFMF90:10 treatment was the most similar to the control (without MF). However, WFMMF75:25 provided a higher protein and fiber content and a lower fat content. Finally, it is possible to use the flour obtained from the mesquite fruit to make bakery products since it is an important source of food due to the wide distribution of mesquite in the country. Food is an essential factor for the well-being of the human being. Likewise, the lifestyle of today\u2019s society has presented changes in recent years since the quantity and quality of food, as well as feeding habits, are factors that directly influence health. Therefore, the consumer is not only looking for sensory-acceptable foods but also healthy products that contain quality nutritional components that provide a beneficial effect on health ,2,3.Bakery products are among the most consumed foods worldwide . Bread, Moreover, the most commonly used cereal for making bread is wheat flour , but this type of product is restricted for people with conditions, such as celiac disease (gluten intolerance), D\u00fchring\u2019s disease (dermatitis herpetiformis), gluten ataxia, gluten sensitivity (non-celiac), wheat allergy, autism spectrum disorders, among others, so they can only consume gluten-free bakery products . RecentlProsopis spp.) are species of the legume family found mainly in semi-arid and arid areas of Mexico. Most of the species are native to the Americas (40), some to Asia (3), and Africa (1) 1/2, where Lc, ac, and bc represent the color measurements of control samples, and L, a, and b are the color measurements of the different treatments [Color determinations  were performed in a Brookfield texture analyzer  using the TA11/1000 probe, with an activation load of 0.07 N at a test speed of 0.50 mm/s. The parameters evaluated were fracturability (N), hardness (N), cohesiveness (dimensionless), adhesiveness (J), springiness (dimensionless), gumminess (N), and chewiness (J).The TPA measurements of the products  were selected for a bromatological analysis. The determinations made were moisture  , ash conp \u2264 0.05), using the SPSS software .The results of the sensory, texture profile, and physicochemical analyses were subjected to an analysis of variance (ANOVA) and Tukey\u2019s method with a confidence level of 95% (p < 0.05) were found among the different treatments. The highest scores were for the control (4.50 \u00b1 0.69), followed by WFWM 90:10 (4.35 \u00b1 0.75), WFWM 75:25 (4.30 \u00b1 0.92), and WFWM 50:50 (3.40 \u00b1 1.05); this effect can be attributed to a color change in the products as the mesquite flour content in the formulations increases , WFMF 90:10 (4.65 \u00b1 0.67), and WFMF 75:25 (4.70 \u00b1 0.47) were significantly similar. The WFMF 50:50 product presented the lowest scores (3.15 \u00b1 0.69) but within the acceptance limit \u22652.5; b. Accord9, WFMF 9p > 0.05) among the scores of WFMF 75:25 (4.20 \u00b1 0.95), WFMF 90:10 (4.30 \u00b1 0.73), and control (4.50 \u00b1 0.89), whose scores fluctuated between 4.2 and 4.5. Nonetheless, the lowest values (p < 0.05) were for WFMF 50:50 (3.15 \u00b1 1.09) since the high concentration of mesquite flour modified the characteristic flavor of the muffin , with scores that fluctuated between 4.45 and 4.65. Furthermore, the lowest scores (p < 0.05) were for WFMF 50:50 , followed by WFMF 90:10 (4.30 \u00b1 0.66), WFMF 75:25 (4.20 \u00b1 0.89) and WFMF 50:50 (3.25 \u00b1 1.02) e, indicaProsopis pallida) flour (0.972\u20130.978); both studies with substitution levels of up to 15% flour. This effect could be related to higher protein content in the mesquite flour used in our research; it has been reported that some globular proteins can absorb large amounts of water, thus decreasing the water activity of breadcrumbs. Additionally, the presence of carbohydrates, such as galactomannan, retains moisture, forming a gel-like structure that directly influences the aw of the product.Regarding the aw determinations in bakery products, the values fluctuated between 0.89 and 0.91, below the values found in two studies carried out by Gonzales-Barron et al. ,13 in whp < 0.05) the values of the L, a*, and b* coordinates in crumb and crust. In the case of luminosity, the values were higher for the control (76.15 and 63.66), followed by the WFWM 90:10 (60.26 and 51.45), and WFWM 75:25 (46.83 and 40.56) treatments; the lowest value was for WFWM 50:50 (41.22 and 32.29)  a. This rp < 0.05) was observed in this parameter as the concentration of mesquite flour in the formulation increased  was found in the values of the b* coordinate as the amount of mesquite flour in the formulation increased  with the increase in the mesquite gum concentration, particularly in the WFMF 50:50 treatment, whose value was 10.47 \u00b1 1.20 N  was found when increasing the concentration of mesquite flour; the highest values were for the control (3.30 \u00d7 10\u22124) and WFMF 90:10 (3.00 \u00d7 10\u22124), while the lowest values were for WFMF 75:25 (7.00 \u00d7 10\u22125) and WFMF 50:50 (3.00 \u00d7 10\u22125)  b. Comparp < 0.05) was observed as the concentration of mesquite gum in the formulation increased, the highest values being for WFMF 50:50 (10.47 \u00b1 1.20 N), followed by WFMF 75:25 (5.37 \u00b1 0.10 N), WFMF 90:10 (1.39 \u00b1 0.78 N), and control (0.32 \u00b1 0.16 N)  c.Fracturability or brittleness refers to the hardness with which the food crumbles or breaks ,25. Therp > 0.05), which fluctuated between 0.67 and 0.71, while the lowest value was for the WFMF 50:50 treatment   d. Accordp < 0.05) was found between the different treatments, whose values fluctuated between 3.96 and 4.29  in gumminess was found as the concentration of mesquite flour increased; the control presented the lowest value (1.88 \u00b1 0.06 N), followed by WFMF 90:10 (2.08 \u00b1 0.00 N) and WFMF 75:25 (3.60 \u00b1 0.10 N). The highest value was found in the WFMF 50:50 treatment (6.52 \u00b1 0.71 N)  f. These p < 0.05) was observed as the concentration of mesquite gum increased. Control and WFMF presented similar values , followed by WFMF 75:25 (1.49 \u00d7 10\u22122) and WFMF 50:50 (2.59 \u00d7 10\u22122) , a s \u00d7 10\u22122) g. This p \u00d7 10\u22122) a,g. LikeConcerning the moisture content and dry matter of the bakery products, the values found in the bakery products  was observed in the treatments when adding mesquite flour in the formulations , in general, WFMF 75:25 provided a higher protein, minerals, and crude fiber content, as well as lower fat and carbohydrate content (nitrogen-free extract), which could be associated with beneficial health effects. Finally, mesquite is a plant species whose fruits can be used to make flour and incorporated into various foods such as bakery products. Moreover, at the same time, mesquite represents a source of healthy food for rural communities in much of Mexico due to the wide distribution of this species in the country."}
+{"text": "Dear Editor,The high diagnostic potential of urinary extracellular vesicles (uEVs) for urogenital disease has been recognized for more than a decade. This is emphasized by the identification of different molecular biomarkers  in uEV preparations that may assist the clinical management of prostate, bladder, and renal cancer  published a position paper summarizing the current state of the art and listing detailed recommendations for improved rigor, reproducibility and inter\u2010operability in uEV research  position paper (Thery et\u00a0al.,  positionSTORAGE of urinary EVs (Figures\u00a0To support the implementation of the published recommendations, and enhance their application in daily research practices, here we provide a Quick Reference Card on  Figures\u00a0. The Qui Figures\u00a0. The Car Figures\u00a0 and acco Figures\u00a0. The CarTo conclude, we present a novel format of communication for EV study guidelines and recommendations that can also be applied to other topics within, but importantly also outside the field of urinary EVs. Ultimately, by using this format, we endeavor to enhance adherence to pre\u2010analytical best practice guidelines in order to promote reproducibility and, above all, the translational potential of uEV studies.The authors report no conflict of interest.Conceptualization: M.v.R., C.S., C.G., J.W., T.T., M.D., A.B., B.G., A.L., C.B., D.B., U.E., E.M.U. Writing, original draft preparation: M.v.R., E.M.U.; Writing, review and editing: M.v.R., C.S., C.G., J.W., T.T., M.D., A.B., B.G., A.L., C.B., D.B., U.E., E.M.U.All authors have read and agreed to the published version of the manuscript.Supplementary File 1. Quick Reference Card: Storage of Urinary EVsClick here for additional data file."}
+{"text": "In recent years, sodium hypochlorite and chlorhexidine digluconate have been the gold standard of irrigation solutions utilized within the disinfection protocol during root canal treatments. Nowadays, it is known that, during chemical disinfection of the root canal, consecutive application of sodium hypochlorite and chlorhexidine digluconate leads to the formation of an orange-brown precipitate. This precipitate is described as being chemically similar to para-chloroaniline, which is suspected to have cytotoxic and carcinogenic effects. Concerns also exist regarding its influence on the leakage of root canal fillings, coronal restorations, and tooth discoloration. The purpose of this article is to review the literature on the interaction of sodium hypochlorite and chlorhexidine digluconate on the tooth and its surrounding tissues, and to discuss the effect of the precipitate formed during root canal treatment. We further address options to avoid the formation of the precipitate and describe alternative irrigation solutions that should not interact with sodium hypochlorite or chlorhexidine digluconate. Howevcid (PA) , as wellGermany) . For thiGermany) ,10.Unfortunately, endodontic irrigation solutions may interact chemically with each other during an alternating irrigation technique, potentially forming unwanted by-products, which may be toxic or cause allergic reactions . Sodium Sodium hypochlorite  is the mSodium hypochlorite (NaOCl) is the most common irrigant used in root canal treatments. NaOCl is an effective tissue solvent and antimicrobial agent. It is usually used in a concentration range from 0.5 to 8.25% ,19,20. IChlorhexidine digluconate (CHX) is the gluconate salt form of chlorhexidine, a biguanide compound used as an antiseptic agent with topical antibacterial activity . ChlorheChlorhexidine digluconate (CHX) can be used as a complement to increase the antibacterial action of NaOCl solutions during root canal preparation. CHX shows similar antimicrobial effects to sodium hypochlorite ,24 in viChlorhexidine digluconate is a broad-spectrum antibacterial agent with substantivity to tooth structures, i.e., it binds to the hydroxyapatite of the enamel and dentin or to anionic groups of glycoproteins, is slowly released and, due to the moderate concentration decrease, its antibacterial effects are prolonged for an extended period of time .+, O2\u2212, and Cl\u2212). The chloride group then reacts with the chlorhexidine molecule in the guanine group (NH). This leads to the formation of chlorhexidine chloride (N+ and Cl\u2212). In this reaction, the formation of an orange-brown precipitate is described. This precipitate contaminates the dentin and adheres to the canal walls [When NaOCl and CHX are mixed, NaOCl dissociates into different ions  was carried out through the website of the National Center for Biotechnology Information (NCBI), utilizing the combination of the Medical Subject Headings (MeSH terms) \u201csodium hypochlorite\u201d (NaOCl) AND \u201cchlorhexidine\u201d (CHX) and yielded 955 results from the years 1974\u20132022. Specifying the search term to \u201cchlorhexidine AND sodium hypochlorite AND interaction\u201d, 64 publications remained from the original result. By individually reviewing the references and abstracts of these 64 publications the keywords \u201cprecipitate\u201d and \u201cpara-chloroaniline\u201d were regularly found in the keywords of the relevant articles \u201d, which resulted in a selection of 88 articles that included the manually determined references. The abstracts of all articles of the final online search result were evaluated and 25 articles that showed no relevance to the question were sorted out .The 63 papers included in this review are listed in 58 publications  were relevant to the topic; another 5 were excluded after reading the full texts. Sources to which the research publications referred were included if they were relevant to the topic, even if the date of their publication was before 1994.The PubMed search found 63 publications, 58 of which were relevant. After full text analysis, 49 were studies that have been published since 2006 in the medical and especially in the dental-endodontic field, which have dealt with the interaction of NaOCl and CHX. Eight reviews with different focuses giving an overview of the state of knowledge at the date of publication were also selected. Furthermore, one article made recommendations on how to avoid formation of the precipitate, and thus was included.The 1998 study by Kuruvilla and Kamath  indicateRegarding the toxicity of the precipitate, Cintra et al.  found a Marchesan et al.  evaluateIn order to prevent the formation of precipitates when using NaOCl and CHX, an irrigation protocol that includes intermediate rinses has frequently been recommended. For example, Zehnder  recommen\u00ae, Dentsply Sirona, Konstanz, Germany) significantly, it was subsequently shown that the adhesion of Resilon\u00ae-Epiphany SE obturation system  was not affected by the precipitate. In addition, Magro et al. [In addition, Mortenson et al.  found tho et al. ,62 foundo et al. ,62.\u00ae, VDW, Munich, Germany) or ultrasound devices, is superior to syringe rinsing [Even by activating the rinsing solutions, the removal of the precipitate is only possible to a limited extent. However, it was found that activation of the chelating agents EDTA and citric acid, in particular using sonic  and QMix\u00ae  are products that combine a biguanide and a chelator. After their application, the penetration depths of the sealer into the dentin were greater than those after sequential rinsing with 17% EDTA, saline solution, and CHX. While QMix\u00ae is used after saline or distilled water (2-phase), SmearOFFTM combines the intermediate rinse and the final rinse, which simplifies and shortens the rinsing protocol. According to the manufacturer, the use of SmearOFFTM after NaOCl in the root canal does not lead to the formation of precipitates; this is also indicated by the sealer penetration depths. Since the manufacturers have not disclosed the formulation of the preparations, further studies on effectiveness and interactions are required [Furthermore, CHX alternatives were also considered and examined. The substitution of CHX by the herbal antimicrobial substances neem, tulsi, aloe vera, and garlic was not successful, as the amount of precipitate resulting from these substances in combination with NaOCl was a factor of 4\u20137.5 higher than that with CHX . ALX, a a et al.  saw poterequired .2) can be utilized as an alternative means of root canal irrigation instead of NaOCl, due to its antimicrobial activity, biocompatibility, and ability to dissolve organic tissue [The possibility of exchanging NaOCl in the combination of NaOCl and CHX was only considered possible by Buyukozer et al. . Chlorinc tissue ,88,89.In 2023, the best possible cleaning and disinfection of the root canal system by means of chemomechanical preparation is still an indispensable prerequisite for the success of endodontic treatment ,91.-Dissolution of necrotic and vital tissue;-Effectiveness against bacteria;-Effectiveness against fungi;-Neutralization of endotoxins;-Opening of the dentinal tubules;-Removal of iatrogenic impurities;-Economic efficiency;-Practicality.The desirable properties of the various irrigation solutions are:-Irritation of neighboring tissues;-Cytotoxicity;-Mutagenicity;-Changes in the color of dentin or tooth enamel;-Occlusion of the dentinal tubules;-Undesirable interactions with other endodontic irrigating solutions and materials.Unwanted properties are:Since no irrigation solution is known that combines all the necessary properties and can be solely applied clinically, different solutions are used consecutively . CertainAlthough it can be considered unlikely that the precipitate of NaOCl and CHX contains free para-chloroaniline, a substance that is suspected of being mutagenic, it is important to avoid precipitation in the root canal. After formation, the complete removal of the precipitate from the root canal system is difficult or impossible, even with advanced methods of activating irrigation solutions with sound, ultrasound, or laser pulses ,57. The In earlier studies, the combination of CHX and NaOCl was determined to have a better effect against Enterococcus faecalis and gram-positive germs compared to NaOCl alone ,93,94,95A strict avoidance of the possible interaction between NaOCl and CHX in all its variants  is desired. According to the literature evaluated, this works best when an intermediate rinse with citric acid is utilized, as this removes the smear layer of the mechanical treatment without causing a precipitate with NaOCl or CHX and thereby triggering other complications.In rinsing protocols that use NaOCl as the sole antimicrobial rinse, based on current knowledge, the final rinse to remove the smear layer should be carried out with citric acid or EDTA before the final use of NaOCl in an activated manner .Since 2006, there has been a sharp increase in publications addressing the interactions between NaOCl and CHX. A total of 88 publications from the PubMed database were identified and evaluated. Of those, 58 publications were relevant to the topic. The results of the studies examined are often controversial, but certain aspects show a tendency over time.-The chemo-mechanical preparation of the root canal system is currently the gold standard;-NaOCl should be used as the sole agent during mechanical reprocessing, due to its tissue-dissolving and antimicrobial properties;-The smear layer can be removed with CA or EDTA after the mechanical preparation. NaOCl should not be mixed with CA or EDTA, since chelators neutralize the tissue-dissolving effect of NaOCl;-The consecutive use of NaOCl and CHX is obsolete due to the precipitate that forms;-If NaOCl and CHX (or CHX derivatives) are used in the same tooth, intermediate rinsing is required. Since CHX also forms a precipitate with EDTA, CA is recommended for this.The following findings relate to the endodontic irrigation protocol:These recommendations are useful in clinical practice to effectively avoid the formation of the undesirable precipitate."}
+{"text": "Following a witnessed lethal lightning strike of an adult male who was standing outside in a storm, numerous Lichtenberg figures were identified upon external examination of the body. Sectioning across multiple areas of linear erythema in the figures showed no subcutaneous hemorrhage. This was later confirmed on histology which showed only subtle dermal capillary dilatation with no interstitial hemorrhage or inflammation in these areas. The only areas of interstitial hemorrhage were present in adjacent scattered punctate burns from arcing. The documented resolution of Lichtenberg figures within hours would be more in keeping with temporary functional capillary dilatation, shown in this case, rather than with tissue alteration by interstitial hemorrhage or inflammation. Two men who were standing outside watching an approaching thunderstorm were struck by lightning. One was rendered unconscious and the other immediately killed. The decedent was referred for medicolegal assessment which consisted of an external examination with skin sampling, CT examination, and toxicological evaluation of blood, all performed 3 days after death. There was no history of significant medical illness.At autopsy, the body was that of an adult male in his twenties. He had only been wearing a pair of underpants. The only signs of injury consisted of multiple Lichtenberg figures on the inner upper left thigh, the right cubital fossa Fig.\u00a0, the uppHistological examination of the Lichtenberg figures showed subtle dilatation of superficial dermal capillaries with no evidence of any associated interstitial hemorrhage or inflammation Fig.\u00a0. The onlLichtenberg figures were first described by a German physicist in the late eighteenth century during experiments with static electricity . TransieAs there have been surprisingly few reports of the histological characteristics of these lesions and as there also appears to be some confusion in the literature regarding the microscopic morphology of these figures, the current case was reported.Lightning occurs when a very rapid (0.0001\u20130.001\u00a0s) discharge of static electricity occurs during a thunderstorm. The current can be as high as 200,000\u00a0A with voltages between 20\u00a0million and 1\u00a0billion volts. It may occur in the absence of thunder. Skin lesions caused by a strike range from singeing of hair to extensive deep charring , althougThe etiology of Lichtenberg figures remains unclear with no theory adequately explaining their occurrence. While it has been suggested that they follow lines of skin moisture, their absence in the axillae and groin creases in favor of drier more exposed areas would not fit with this concept. Similarly the idea that they follow superficial vessels has been discounted . AlthougThere has also been some disagreement as to the precise morphology of these figures, with some authors attributing their appearance to interstitial hemorrhage  or to inHowever, in the reported case, cutting across the lines of erythema showed no macroscopic evidence of hemorrhage, and in the histologic sections, the most characteristic finding was simple dermal vessel dilatation. If this is the underlying pathology, then the mystery of the rapid resolution of the figures in survivors could be readily explained, as dilated capillaries would certainly have the capacity to more rapidly revert to normal compared to extravascular aggregates of red cells, which require erythrocyte breakdown and removal by macrophages. For example, histologic examination of the linear areas of erythema in Fig.\u00a0In conclusion, Lichtenberg figures are a striking skin finding in a minority of cases of lightning strike that may be present in both the living and the dead. Their etiology is poorly understood; however, their rapid resolution in survivors would be more in keeping with temporary capillary dilatation rather than with interstitial hemorrhage or inflammation, as has been previously suggested."}
+{"text": "Morchella galilaea, the only species of the genus Morchella known to fruit in the autumn. Based on the observation of five samples, we first discovered significant variation in the shape and size of ascospores in Morchella. One to sixteen ascospores were found in the asci. Ascospore size correlated negatively with ascospore number, but positively with ascus size, and ascus size was positively correlated with ascospore number. We noted that ascospores, both from fresh collections and dried specimens, germinated terminally or laterally either by extended germ tubes, or via the production of conidia that were formed directly from ascospores at one, two or multiple sites. The direct formation of conidia from ascospores takes place within asci or after ascospores are discharged. Using laser confocal microscopy, we recorded the number of nuclei in ascospores and in conidia produced from ascospores. In most ascospores of M. galilaea, several nuclei were observed, as is typical of species of Morchella. However, nuclear number varied from zero to around 20 in this species, and larger ascospores harbored more nuclei. One to six nuclei were present in the conidia. Nuclear migration from ascospores to conidia was observed. Conidia forming directly from ascospores has been observed in few species of Pezizomycetes; this is the first report of the phenomenon in Morchella species. Morphological and molecular data show that conidial formation from ascospores is not found in all the specimens of this species and, hence, is not an informative taxonomic character in M. galilaea. Our data suggest that conidia produced from ascospores and successive mitosis within the ascus may contribute to asci with more than eight spores. The absence of mitosis and/or nuclear degeneration, as well as cytokinesis defect, likely results in asci with fewer than eight ascospores. This study provides new insights into the poorly understood life cycle of Morchella species and more broadly improves knowledge of conidia formation and reproductive strategies in Pezizomycetes.Spores are important as dispersal and survival propagules in fungi. In this study we investigated the variation in number, shape, size and germination mode of ascospores in  Ascospores are produced after meiosis and conidia are produced by mitosis . In addiThe conidium is the main type of mitospores produced by ascomycetes and serves as a propagule for rapid dissemination or as a \u201csafe house\u201d for the fungal genomes under adverse environmental conditions . TypicalThe phenomenon of formation of conidia directly from ascospores, without mycelial growth, can be termed microcyclic conidiation . HoweverPurpureodiscus subisabellinus (Morchella spp.) belonging to Morchellaceae are well-known as one of the most popular edible fungi in the world, due to their highly desirable flavor  (RPB1) (RPB2)  ; RPB1Y-F) (RPB1) ; and, RP) (RPB2) . The amp. Mes-26  were retEF1a-RPB1-RPB2) by using RAxML v.8.2.4  was used to rehydrate dried specimens prior to morphological analysis. Specimens were stained with 1% aqueous Congo red solution when necessary. Microscopic features were observed using an Optec BK-FL light microscope  at magnifications of 40\u00d7, 100\u00d7, 400\u00d7, and 1,000\u00d7, and were drawn by hand. Images were captured with an Optec CCD TP510 digital camera (Optec). Measurements of ascospores are presented with 95% confidence intervals. Ascospore sizes are presented using a range notation in the form (a-) b-c (\u2212d), where the range b-c contains a minimum of 90% of the measured values and extreme values  are shown in parentheses.Nuclei were stained with 4\u2032,6-diamidino-2-phenilindole  for 10\u2009min directly on microscope slides. Excess stain was removed with filter paper. Nuclei were visualized with a Fluorview 1,000 laser confocal fluorescent microscope . Images were analyzed using FV10-ASW 4.0 Viewer software (Olympus). The descriptions and abbreviations follow those of 3.3.1.M. galilaea. The alignments of the twenty sequences of four markers that were newly generated in this study , and the ML phylogeny is presented in M. galilaea  or fewer than six nuclei were commonly found. In ascomycetes, the fusion nucleus in the ascus regularly divides in succession by meiosis and post-meiotic mitosis to produce eight nuclei before spore delimitation for typical eight-spored asci  values indicated that 21.6% of the variability in ascospore length may be explained by ascospore number which also was responsible for 8.5% of the observed variability in ascospore width (2) values indicated that only 0.02% of the variability in ascus length was explained by ascospore number, which was responsible for 13.9% of the observed variability in ascus width  ; this agre width . In contre width . Coefficus width .2) values indicated that 29.4% of the variability in ascus length may be explained by ascospore length, and 5.8% may be explained by ascospore width. Weak positive linear correlations were shown for ascus width and ascospore size  values indicated that 0.09% of the variability in ascus length was due to ascospore length, and 2% was related to ascospore width.Apparent positive linear correlations were shown for ascus length and ascospore size  and ascospores . The production of fewer than eight spores in asci increases maximum potential spore size. Large, compartmentalized spores have clear advantages in a harsh environment and also increase impaction efficiency and the amount of mycelium which could be produced . The hyaline conidia in  2\u201310\u2009\u03bcm . The ont 2\u201310\u2009\u03bcm . More moThe nuclear status of ascospores at germination either by long germ-tubes  or by prFigure 11Morchella, conidia are produced on upright, aerial conidiophores after a period of vegetative growth during their hyphal development (this state was previously identified as Costantinella). These conidia are spherical and 2.5\u20138\u2009\u03bcm in diameter, and harbor one to three nuclei   . The cursolation  but cautP. subisabellina  can be found in the article/X-HD: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing \u2013 original draft, Writing \u2013 review & editing. S-YW: Methodology, Writing \u2013 review & editing. MR: Writing \u2013 review & editing. Y-JG: Resources, Writing \u2013 review & editing. J-YW: Resources, Writing \u2013 review & editing. DP: Supervision, Writing \u2013 review & editing, Conceptualization. HJ: Supervision, Writing \u2013 review & editing, Conceptualization."}
+{"text": "Sternocera aequisignata, wing cases, we found that iridescence provides initial protection against avian predation by significantly reducing the willingness to attack. Importantly, we found that the main factor explaining this aversion is iridescence, not multiple colours per se, with surface gloss also having an independent effect. Our results are important because they demonstrate that even when prey are presented up close and against a mismatching background, iridescence may confer a survival benefit by inducing hesitation or even, as sometimes observed, an aversion response in attacking birds. Furthermore, this means that even postdetection, prey do not necessarily need to have secondary defences such as sharp spines or toxins for iridescence to have a protective effect. Taken together, our results suggest that reduced avian predation could facilitate the initial evolution of iridescence in many species of insects and that it is the defining feature of iridescence, its colour changeability, that is important for this effect.It has recently been found that iridescence, a taxonomically widespread form of animal coloration defined by a change in hue with viewing angle, can act as a highly effective form of camouflage. However, little is known about whether iridescence can confer a survival benefit to prey postdetection and, if so, which optical properties of iridescent prey are important for this putative protective function. Here, we tested the effects of both iridescence and surface gloss (i.e. specular reflection) on the attack behaviour of prey-na\u00efve avian predators. Using real and artificial jewel beetle,  \u2022Iridescence and gloss are usually associated with display, but can they also defend?\u2022We tested na\u00efve birds' willingness to attack glossy and iridescent prey.\u2022Iridescence reduced attack probability, with gloss also having an independent effect.\u2022Iridescence and gloss could provide a survival benefit to prey, even postdetection. Iridescence is a striking form of structural coloration in which hue and intensity vary with the angle of view or illumination . Our undOreina cacaliae, Another putative antipredator function of iridescence is aposematism: signalling unprofitability e.g. . HoweverGallus gallus domesticus, presented with real and artificial jewel beetle, Sternocera aequisignata, wing cases, all of which were conspicuous on the background against which they were displayed. We used both iridescent and noniridescent prey with the same overall range of colours, as well as manipulating their level of specular reflection (gloss). We predicted that if iridescence (where hue and intensity vary with angle) is an important factor in predator aversion, the birds would be less willing to attack the iridescent than the noniridescent prey. Alternatively, if gloss (where just intensity varies with angle) is more influential, the birds would be less willing to attack the prey items with higher specularity.One unexplored effect of iridescence, relevant to both the rate of avoidance learning and evolutionary considerations of initial viability, is whether it induces an aversion response in na\u00efve predators. To investigate this, and to isolate the effects of gloss and iridescence, we measured the attack willingness of na\u00efve chicks of the domestic fowl, Tenebrio molitor, covered by a real or artificial jewel beetle elytron. Iridescent targets were natural wing cases, where iridescence is structurally produced by a chitin multilayer stack with a refractive index contrast phase, probably melanin (N\u00a0=\u00a0100) inside the experimental room and photographed them in plan view using a Nikon D90 DSLR camera . Photographs were taken at 1:1 reproduction, and the printed size matched that of the real beetles. Photographs contained an X-Rite ColorChecker Passport , which was used to calibrate the images for bird vision , demonstrating the desired difference between the glossy and matte targets , and the other half with matte spray . We quantified the differences in specular reflection between treatments with a ZGM1120 Glossmeter . The Glossmeter provides the ratio of specular to diffuse reflectance (\u2018gloss units\u2019). We recorded all specular measurements at 60\u00b0, because that is the recommended measurement angle for small surfaces . The Glo targets . The hypNote that the colour spectra illustrated in www.gewiss.com; twin 26 W LED). Water and chick starter crumbs  were provided ad libitum, except during the experimental phase when chicks had a 30 min pretrial deprivation to increase their foraging motivation. Chicks also received mealworms in their home pens, as these were used as rewards in the experiment.We used 51 domestic chicks from Hy-Line Millennium Hatchery . The chicks arrived within 24 h of hatching. Once they were settled in their home pens and were eating well, each chick was marked with Porcimark spray  for identification. Of the 51 chicks, 32 were marked as experimental chicks and 19 as \u2018buddies\u2019 . Utmost care was taken to minimize stress, including the use of \u2018buddy chicks\u2019 to prevent experimental chicks feeling lonely in the arena, pre-exposure to the foraging board and appropriate ambient lighting. At the end of the experiment and with veterinary approval, all chicks were successfully rehomed to local small-holders.Chicks were initially trained to eat chick starter crumbs from a foraging plate made from A4-sized cardboard, placed in their home pen, to establish an association between the plate and food. The experiment took place in an identical room opposite their home pens, where there were two identical arenas measuring 120\u00a0\u00d7\u00a050 cm and 50 cm high. Both arenas contained a section measuring 20\u00a0\u00d7\u00a050 cm and 50 cm high, partitioned with wire mesh to create a separate \u2018buddy area\u2019.The experiment started on the second day after the arrival of the chicks. Just before the experiment, two chicks were placed in the buddy arena to reduce any potential distress from isolation. These buddy chicks were changed every three trials .Each of the 32 experimental chicks underwent four experimental trials, one per day. In each trial, chicks were presented with all four prey items randomly distributed on the foraging plate. From approaching the foraging plate within pecking distance from the first target, each experimental chick had a maximum of 4\u00a0min to attack all four items  from the foraging plates see . Any tarOne chick failed to attack any prey in trials 1 and 2; these were treated as missing values. Four chicks attacked some, but not all, targets in trial 1; these prey were assigned a tied rank of 4, equivalent to \u2018last to be eaten\u2019.We analysed attack order using mixed model ordinal logistic regression . These two-way interactions were only evident in the first prey attacked .The order of attack varied significantly with respect to the two-way trial\u2217gloss and trial\u2217iridescence interactions , but noniridescent prey were 3.27 times (CI 1.39\u20138.26) more likely to be attacked than iridescent prey . In trial 2, there was no significant effect of gloss  or iridescence . In trial 3, matte targets were 3.43 times more likely to be attacked first than glossy targets , but there was no effect of iridescence . In trial 4, noniridescent prey were 5.27 times (CI 2.16\u201314.36) more likely to be attacked than iridescent prey  but there was no effect of gloss . Finally, direct observations of the chicks' avoidance behaviour obtained from video recordings in trial 4 revealed that 9/32 chicks walked off the board or actively avoided the prey item before the trial ended . These displays of avoidance behaviour occurred significantly more often when there was only iridescent prey left (eight of nine cases), compared to when there was only static spectrum prey left .To investigate the trial-to-trial differences in the main effects of iridescence and gloss, we fitted separate GLMMs to each trial's data. In trial 1, the probability of attacking the first prey did not vary with gloss (matte:gloss odds ratio 0.63, 95% confidence interval, CI 0.26\u20131.46; \u03c7This experiment demonstrates a possible antipredator function of iridescence, and potentially gloss, against avian predators. Considering that all birds in our experiment were na\u00efve, any behavioural response they showed in their first prey encounter can be considered unlearnt. Interestingly, the birds initially hesitated to attack the iridescent, but not the static spectrum prey: having multiple colours displayed at the same time is not enough to elicit aversion. Rather, it is the key feature of iridescence, its colour changeability, that is important for this protective effect . Our stuThe variation in attack willingness across trials could be due to opposing forces: the aversive properties of the coloration and the positive value of the mealworm reward, as with aposematic prey . We suggAs our prey did not have any secondary defences and yet still induced an aversion response, it is possible that iridescence could facilitate the evolution of aposematism. Future studies could establish the frequency with which the combination of iridescence and secondary defences occurs. As the intensity of the iridescent effect is also dependent on the illuminant, future experiments could investigate the protective function of iridescence and gloss relative to the specularity of the background and the level of, and variation in, ambient light, both of which affect detectability . NeverthK.K., A.L., I.C.C., H.M.W. and N.E.S.-S. conceived the experiment. K.K., A.L., R.M., L.C. and J.R.H. collected the data. K.K. and R.M. conducted spectral measurements of the targets. K.K. and I.C.C. performed the statistical analyses. K.K. wrote the first draft, with contributions by all authors.https://doi.org/10.5523/bris.32jo661m7sdkw2751vtphaq7vl.The data are deposited in the University of Bristol data repository: The authors declare no conflict of interest."}
+{"text": "Pseudomonas aeruginosa is attributed to the production of many virulence factors and its resistance to several antimicrobials. Among them, sodium hypochlorite (NaOCl) is a widely used disinfectant due to its strong antimicrobial effect. However, bacteria develop many mechanisms to survive the damage caused by this agent. Therefore, this study aimed to identify novel mechanisms employed by P. aeruginosa to resist oxidative stress induced by the strong oxidizing agent NaOCl. We analyzed the growth of the P. aeruginosa mutants \u0394katA, \u0394katE, \u0394ahpC, \u0394ahpF, \u0394msrA at 1\u2009\u03bcg/mL NaOCl, and showed that these known H2O2 resistance mechanisms are also important for the survival of P. aeruginosa under NaOCl stress. We then conducted a screening of the P. aeruginosa PA14 transposon insertion mutant library and identified 48 mutants with increased susceptibility toward NaOCl. Among them were 10 mutants with a disrupted nrdJa, bvlR, hcnA, orn, sucC, cysZ, nuoJ, PA4166, opmQ, or thiC gene, which also exhibited a significant growth defect in the presence of NaOCl. We focussed our follow-up experiments  on mutants with defect in the synthesis of the secondary metabolite hydrogen cyanide (HCN). We showed that HCN produced by P. aeruginosa contributes to its resistance toward NaOCl as it acts as a scavenger molecule, quenching the toxic effects of NaOCl.The high pathogenicity of  For eashcnB) mutant from the PAO1 transposon mutant library , according to the manufacturer\u2019s instructions. BM2 minimal medium was used to mitigate side reactions between NaOCl and growth medium components. Additionally, to confirm that the addition of NaOCl to BM2 did not reduce the amount of overall RCS, RCS concentration was measured as previously described . In this2.3.P. aeruginosa stress resistance to NaOCl, we initially screened the P. aeruginosa PA14 transposon insertion mutant library , washed twice with phosphate-buffered saline (PBS), and resuspended in BM2 medium. The optical density at 600\u2009nm (OD600nm) was adjusted to 0.2 (2\u2009\u00d7\u2009108\u2009CFU/mL), and 50\u2009\u03bcL was mixed in 96-well plates with 50\u2009\u03bcL of serial dilutions of NaOCl (0.125\u2013128\u2009\u03bcg/mL) or H2O2  prepared in BM2 . The plates were incubated for 24\u2009h at 37\u00b0C, and the MIC was considered the lowest concentration of oxidizing agent that inhibits the visual growth of bacteria.The MIC for NaOCl and Heviously . Briefly2.5.Pseudomonas aeruginosa overnight cultures grown at 37\u00b0C and 220\u2009rpm in LB were washed twice with PBS, resuspended in BM2, and the OD600nm was adjusted to 0.2 (2\u2009\u00d7\u2009108\u2009CFU/mL). Then, 50\u2009\u03bcL of bacterial suspension and 50\u2009\u03bcL of oxidizing agent were mixed in flat-bottom polystyrene 96-well microtiter plates, leading to a final concentration of NaOCl of 1\u2009\u03bcg/mL and H2O2 of 400\u2009\u03bcg/mL and the final cell concentration of 1\u2009\u00d7\u2009108\u2009CFU/mL. The OD600nm was read every hour for 20\u2009h at 37\u00b0C using the Epoch plate reader . Growth curves were statistically analyzed by measuring the area under the curve (AUC) using GraphPad Prism version 9.5.1 .2.6.hcnB, and \u0394hcnB-phcnBC grown in LB were collected by centrifugation and washed twice with PBS. The cells were resuspended in 2\u2009mL of LB and transferred to a small Petri dish (35\u2009cm diameter) placed in the middle of a 100-mm diameter Petri dish. The small petri dish was covered with chromatography paper soaked in HCN detection reagent: 100\u2009mg of copper (II) ethyl acetoacetate and 100\u2009mg of 4,4\u2032-methylenebis- solubilized in 20\u2009mL chloroform . The Shapiro\u2013Wilk test was used to confirm the normality of the data. Parametric data were analyzed by One Way ANOVA, followed by Tukey or Dunnett\u2019s post-test for multiple comparisons or Student\u2019s t-test for comparison between two groups. Non-parametric data were analyzed by the t-test and Mann\u2013Whitney test for comparison between two groups. All experiments were performed in at least three independent experiments, and results were considered statistically significant when 3.3.1.2O2 has identified several detoxifying enzymes and oxidative stress repair systems in P. aeruginosa. To evaluate if these previously described genes involved in H2O2 adaptation also play a role in the adaptation of P. aeruginosa to NaOCl, we examined growth of the P. aeruginosa PAO1 and PA14 mutants \u0394katA and \u0394katE , \u0394ahpC and \u0394ahpF , \u0394msrA (methionine sulfoxide reductase), and \u0394ohrR (organic hydroperoxide resistance protein) exposed to NaOCl at 1\u2009\u03bcg/mL for 20\u2009h at 37\u00b0C. This sub-lethal concentration was chosen based on the MIC of the WT strains (2\u2009\u03bcg/mL). In accordance with Previous work on HPseudomonas aeruginosa PAO1 and PA14 WT treated with 1\u2009\u03bcg/mL NaOCl took approximately 5\u20136\u2009h to reach an OD600nm of 0.2 . Overall, all mutants presented reduced growth at 1\u2009\u03bcg/mL NaOCl compared to the WT strains. Furthermore, the AUCs were statistically significant for both the PA14 and PAO1 mutant strains compared to the WT strains treated with 1\u2009\u03bcg/mL NaOCl. An OhrR mutant, a transcriptional repressor involved in oxidative stress response in P. aeruginosa, was used as a control and presented growth compared to the untreated controls and WT strains , except for OhrR, which presented a MIC of 4\u2009\u03bcg/mL . These r3.2.hcnA, cyanide production), PA0846 (sulfate uptake protein), PA4110 , PA5446 , PA0040 (hemolysin activation/secretion protein), PA1046 , PA4973 , and PA1315 ].To identify novel genes involved in NaOCl resistance, we screened the comprehensive Harvard PA14 transposon insertion mutant library  for muta3.3.600nm of 0.2  after 11\u2009h of incubation, while it took approximately 5\u20136\u2009h for the WT strains to get to this OD. Among them, \u0394bvlR, \u0394hcnA, and \u0394thiC presented overall reduced growth at 1\u2009\u03bcg/mL NaOCl, reaching the maximum OD600nm of 0.364, 0.382, and 0.405, respectively, after 20\u2009h compared to the PA14 WT (OD600nm of 0.6 after 20\u2009h).To further characterize the susceptibility phenotype of the mutants identified in the MIC screening in more detail, we analyzed the growth of these 48 mutants in the presence of 1\u2009\u03bcg/mL NaOCl for 20\u2009h at 37\u00b0C in microtiter plates. Overall, 10 PA14 mutants identified in the library screening also presented a significant delay in growth in the presence of NaOCl compared to the WT-treated strains . nrdJa, \u0394cysZ, \u0394opmQ, and \u0394thiC presented delayed growth  and statistically different AUCs compared to the NaOCl-treated WT strains for both PA14 and PAO1 mutants, suggesting that the phenotype found is not strain specific. PAO1 homologs for the mutants \u0394hcnA and \u0394sucC were unavailable for testing.To evaluate if our findings are strain-specific, we also assessed the growth of the PAO1 mutant homologs from the PAO1 two-allele transposon mutant library from the University of Washington Genome Center  under th2O2. This concentration was chosen based on the growth curve of PA14 and PAO1 WT strains previously conducted  by the transfer of the phcnBC plasmid, which expresses the genes hcnBC. Since both PA14 and PAO1 mutants presented increased susceptibility to NaOCl, we focused our analyzes on the PAO1 \u0394hcnB mutant since this mutant has been characterized in a recent study . The PA4134 gene forms a gene cluster together with PA4133, with PA4133 being located upstream of PA4134;In order to provide further insight into the underlying mechanism of the HCN phenotype found in this study, we formulated two hypotheses: (i) HCN-related NaOCl resistance is mediated by cellular effects caused by HCN, or (ii) HCN acts as an extracellular metabolite, directly reacting with NaOCl and quenching its antimicrobial effect. In a previous study, th NaOCl ; Table 6hcnB and PAO1 WT strains by evaluating the kill kinetics in response to NaOCl. Cells grown overnight were washed twice, resuspended in PBS to remove the HCN from the medium, and NaOCl was added to a final concentration of 2\u2009\u03bcg/mL. The absence of HCN in the medium did not provoke a difference in the NaOCl susceptibility of PAO1 WT and \u0394hcnB  and potassium cyanide (KCN)]. For this, we resuspended overnight \u0394hcnB cells in the supernatants of \u0394hcnB, PAO1 WT, and \u0394hcnB-phcnBC grown in BM2 minimal medium. Then, the bacterial suspensions were treated with NaOCl at 4\u2009\u03bcg/mL for 60\u2009min. hcnB-phcnBC supernatants to cells of \u0394hcnB complemented the \u0394hcnB phenotype, and \u0394hcnB cells showed increased resistance to NaOCl  compared to the \u0394hcnB cells resuspended in \u0394hcnB supernatant . These results suggest that HCN reacts with NaOCl, quenching its lethal effect.Next, we aimed to complement the susceptibility phenotype of \u03944.Bacteria have developed several mechanisms to mitigate the harmful and often irreversible damage caused by oxidizing agents. Most of these resistance mechanisms are not specific but rather provide a general defense against a broad range of oxidizing agents. In this context, the knowledge of HOCl-specific responses is still limited . Among tP. aeruginosa PAO1 and PA14 to NaOCl compared to the WT strains, showing that these H2O2 responses are employed by this bacterium as a general response against oxidizing agents. We then identified 48 mutants with increased susceptibility to NaOCl and characterized 10 mutants in more detail. Among them, we found that HCN acts as a scavenger molecule and increases the survival of P. aeruginosa in the presence of NaOCl.In this study, we demonstrated that the loss of KatA, KatE, AhpC, AhpF, and MsrA increases the susceptibility of 2O2 and other ROS, while their roles in RCS and NaOCl resistance remain mostly unknown. Therefore, in the first part of this study, we showed by growth kinetics analyzes that well-known H2O2 responses are also involved in the survival of P. aeruginosa to NaOCl. Catalases and peroxidases are specialized enzymes that convert H2O2 into less toxic species  and four alkyl hydroperoxide reductases , in which KatA is considered the main catalase and its expression is controlled by various systems, such as OxyR, quorum sensing, and ANR  in more detail  has a mutation in a sulfate uptake protein. Proteins, mainly the sulfur-containing ones, are the main target of HOCl in the cells  and one implicated in thiamine biosynthesis (thiC). Thiamine, for example, has been explored as a target in the development of antibiotics . BvlR is a transcriptional repressor that belongs to the LysR-type transcriptional regulator (LTTR) family. It is upregulated during exposure to epithelial cells , cupA-associated fimbrial-based surface attachment, and toxin A. Furthermore, it was shown to promote tight microcolony formation, which is associated with the formation of biofilms in the lung of cystic fibrosis (CF) patients (katA mRNA (In our screening, we also identified genes involved in the pathogenicity of al cells  and contpatients . The olipatients  and contpatients  and aminatA mRNA .hcnA gene. The absence of HCN in our P. aeruginosa mutant strains increased their susceptibility to NaOCl, while the complementation of \u0394hcnB with an HCN-producing plasmid recovered the phenotype in our growth kinetic analyzes. HCN is a toxic volatile secondary metabolite as it has no apparent function in primary metabolism and is produced at later stages during the exponential phase and offers an advantage for the producing strain, which is tolerant to it (Alcaligenes, Aeromonas, Bacillus, Pseudomonas, and Rhizobium (P. aeruginosa, HCN is synthesized by the HCN synthase, encoded by the hcnABC operon, and regulated by quorum sensing and the ANR regulator (P. aeruginosa cultures produce up to 300\u2009\u03bcM of HCN by the decarboxylation of glycine, and the hcnABC operon is induced by low oxygen (P. aeruginosa has two systems to avoid HCN intoxication. One involves a cyanide-insensitive terminal oxidase, CioAB, which allows aerobic respiration in the presence of cyanide (Another finding in our screening was a mutant with a disrupted nt to it . It is shizobium . In P. aegulator . P. aeruw oxygen  and highw oxygen . The toxw oxygen . P. aeru cyanide  and the  cyanide .CF patients (P. aeruginosa could also employ this metabolite as a virulence factor to increase its pathogenicity. Due to its high toxicity, HCN also exerts a toxic effect on non-producing strains (P. aeruginosa has been shown to control the growth of S. aureus, contributing to P. aeruginosa competition (P. aeruginosa to the strong antioxidant NaOCl. Recently, studies have shown the effect of signaling molecules on the resistance profile of bacterial species (P. aeruginosa, we first tested the hypothesis that the production of HCN induces cellular mechanisms that, in turn, activate resistance mechanisms. For instance, indole produced by bacterial species induces bacterial resistance endogenously and exogenously by many mechanisms, including efflux pump regulation, biofilm formation, and induction of the persister state (P. aeruginosa genes that were up or down-regulated under endogenously produced HCN (HCN has been detected in the breath  and the patients , suggest strains . In thispetition . Here we species . Therefoer state . We thenuced HCN . Except 2, N2, and HCl. However, due to its toxicity, we could not evaluate if adding HCN to P. aeruginosa would rescue the NaOCl-susceptibility phenotype found for the \u0394hcnB mutant, and we used different supernatants from WT and HCN-deficient strains as an alternative approach.We then assessed if the NaOCl susceptibility found was due to the reaction of HCN with NaOCl and found evidence supporting the scavenger effect of HCN in the presence of NaOCl, quenching the toxic effect of this oxidizing agent. Due to its high reactivity, HOCl is known to react rapidly with sulfur- and nitrogen-containing compounds, producing chlorinated derivatives with impaired functions . In thisP. aeruginosa infection, such as wound infection in which the environment presents low oxygen levels, favoring anaerobic microorganisms (P. aeruginosa and fight infections and the spread of this bacterium.It is believed that the production of HCN by bacteria serves various purposes, including defense mechanisms, helping the bacteria compete with other microorganisms for resources, and antimicrobial effect, inhibiting the growth of other microorganisms. Here, we have identified a new role for HCN produced by pathogenic bacteria as a NaOCl scavenger molecule, contributing to bacterial resistance under NaOCl stress conditions. We hypothesize that HCN is produced in the context of rganisms , and helrganisms  that prorganisms . The seaOf note, due to the high reactivity of NaOCl and its active ingredient, HOCl, the chemistry behind the formation and consumption of RCS in media is complex . For ins5.2O2, and the knowledge on adaption to RCS, including NaOCl, is still in its infancy. Our PA14 mutant library screening identified 48 genes and showed that P. aeruginosa relies on diverse mechanisms to survive the potent and often irreversible stress caused by NaOCl. Among them, we identified the hcnA gene and showed that HCN contributes to the resistance of P. aeruginosa by quenching the toxic effect of NaOCl. To our knowledge, this is the first study reporting the roles of HCN in NaOCl resistance.Although much effort has been made to uncover the mechanisms employed by bacteria to resist oxidative stress, most of the studies have focused on HThe original contributions presented in the study are included in the article/WSdCN: Conceptualization, Formal analysis, Investigation, Methodology, Project administration, Visualization, Writing \u2013 original draft, Writing \u2013 review & editing. MEA: Formal analysis, Investigation, Methodology, Writing \u2013 original draft. VI: Formal analysis, Investigation, Methodology, Writing \u2013 original draft. CB: Methodology, Writing \u2013 original draft, Investigation. JO: Methodology, Writing \u2013 original draft, Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Writing \u2013 review & editing."}
+{"text": "Phosphonates are compounds containing a direct carbon\u2013phosphorus (C\u2013P) bond, which is particularly resistant to chemical and enzymatic degradation. They are environmentally ubiquitous: some of them are produced by microorganisms and invertebrates, whereas others derive from anthropogenic activities. Because of their chemical stability and potential toxicity, man-made phosphonates pose pollution problems, and many studies have tried to identify biocompatible systems for their elimination. On the other hand, phosphonates are a resource for microorganisms living in environments where the availability of phosphate is limited; thus, bacteria in particular have evolved systems to uptake and catabolize phosphonates. Such systems can be either selective for a narrow subset of compounds or show a broader specificity. The role, distribution, and evolution of microbial genes and enzymes dedicated to phosphonate degradation, as well as their regulation, have been the subjects of substantial studies. At least three enzyme systems have been identified so far, schematically distinguished based on the mechanism by which the C\u2013P bond is ultimately cleaved\u2014i.e., through either a hydrolytic, radical, or oxidative reaction. This review summarizes our current understanding of the molecular systems and pathways that serve to catabolize phosphonates, as well as the regulatory mechanisms that govern their activity. Organophosphonates (henceforth termed phosphonates for simplicity) are compounds containing a direct C\u2013P bond instead of the more usual C\u2013O\u2013P ester linkage . DespiteThe most common natural phosphonates are shown in Anthropogenic phosphonates in the environment derive primarily from large-scale human activities, such as agricultural treatments  and industrial processes . BecauseWhile phosphorus is an essential nutrient for living organisms, its simplest bioavailable form (inorganic phosphate) is found in low concentrations in various environments, often limiting microbial growth. To cope with the scarcity of inorganic phosphate, many microorganisms have developed peculiar strategies, one of which is scavenging the phosphorus from environmentally available phosphonates ,15,16. SSo far, all the metabolic systems identified as degrading the C\u2013P bond can be subdivided based on the mechanism by which the bond is ultimately cleaved. Accordingly, we schematically distinguish the hydrolytic, oxidative, and radical pathways. The hydrolytic and oxidative pathways are generally specific for one or a few substrates, whereas the radical mechanism of cleavage is typical of the C\u2013P lyase complex, which has a broad substrate scope.This paper provides an updated overview on the biochemical and regulatory strategies that microbes adopt to degrade phosphonates, as well as the abundance and distribution of phosphonate degradative pathways in natural environments. Our focus is primarily on the catabolism of naturally occurring molecules, but we also provide information on the degradation of anthropogenic compounds. While some phosphonate catabolic pathways have been individually reviewed in recent years ,22,23, tAn early step in the microbial process of scavenging the phosphorus from phosphonates is the uptake of the latter from the environment, and since biological membranes are generally impermeable to charged substances, the import of phosphonates implies the involvement of transporters. Indeed, the bacterial gene clusters dedicated to phosphonate degradation very often include genes encoding transporter proteins. While direct experimental data on these transporters are quite limited, it is evident that different types of bacterial phosphonate transporters exist, arguably distinct on the basis of structure, specificity, and regulation.Most of the transporters seem to belong to two main classes: ABC transporters and symporters/antiporters.ATP binding cassette (ABC) transporters are multiprotein complexes that encompass one (or more) membrane-associated ATPase subunits and exploit ATP hydrolysis to drive the translocation of substrates across membranes , potentially against their concentration gradient. A typical ABC transporter consists of four components: a substrate-binding protein (SBP), two transmembrane domains, and an ATPase domain . The SBPEscherichia coli are encompassed in the phn operon, whose first three genes are phnC, phnD, and phnE. They encode, respectively, an ATP-binding subunit . Ho. HoStappAntiporters (which move two substrates across a membrane in opposite directions) and symporters (which transport two substrates in the same direction) do not require ATP hydrolysis but, in general, utilize some ion gradient as a secondary energy source.Pseudomonas putida BIRD-1 on AEP. The expression of the aepP gene occurred both in the presence and in the absence of phosphate, and it significantly increased during growth on AEP as a sole N source (phosphate-insensitive) )(R)-HAEP , yieldin(R)-HAEP a.phnWX and phnWAY types and in some cases to just phnX . A. ABurkhoe  ,66. PalAor PnPyr .2+-dependent hydrolases and is likely to use a Thr residue as the nucleophile to attack PnAc ..E. coli 4) and \u03b1-D-ribose-1,2-cyclic-phosphate-5-phosphate (PRcP) . Ho. Ho98]. E. coli ,87. Whilstoc sp. , as well strains . Bacteri strains .The dominant pathway for glyphosate usage is the AMPA pathway , where fAMPA is also the main microbial degradation product of NTMP, EDTMP, and DTPMP in soil . Its envO. anthropi GPK 3 )Finally, while there is much left to discover about phosphonate cycling in prokaryotes, phosphonates remain even more cryptic in eukaryotes, even though these molecules were originally found in eukaryotic cells. The recent progress that has been made in phosphonate biochemistry has significantly improved our understanding of how these molecules can be catabolized, but much remains to be learned about their synthesis and function in cells and environments."}
+{"text": "Flexibility has become a certain trend in the development of secondary batteries to meet the requirements of wide portability and applicability. On account of their intrinsic high energy density, flexible alkali metal\u2010chalcogen batteries are attracting increasing interest. Although great advances have been made in promoting the electrochemical performance of metal\u2010S or metal\u2010Se batteries, explorations on flexible chalcogen\u2010based batteries are still limited. Extensive and rational use of soft materials for electrodes is the main bottleneck. The re\u2010emergence of safe liquid metals (LMs), which provide an ideal combination of metallic and fluidic properties at room temperature, offers a fascinating paradigm for constructing flexible chalcogen batteries. They may provide dendrite\u2010free anodes and restrain the dissolution of polysulfides and polyselenides for cathodes. From this perspective, we elaborate on the appealing features of LMs for the construction of flexible metal\u2010chalcogen batteries. Recent advances on LM\u2010based battery are discussed, covering novel liquid alkali metals as anodes and LM\u2010sulfur hybrids as cathodes, with the focus placed on durable high\u2010energy\u2010density output and self\u2010healing flexible capability. At last, perspectives are proposed on the future development of LM\u2010based chalcogen batteries, and the viable strategies to meet the current challenges that are obstructing more practical flexible chalcogen batteries. Liquid metal (LM) offers a fascinating paradigm for constructing flexible chalcogen batteries. This perspective elaborated the appealing features of LMs and discussed recent advances on LM based battery. The viable strategies and future development of LM\u2010based chalcogen batteries are highlighted. Recently, much effort has been put into the development of flexible batteries; however, improving the energy density while keeping the flexibility and lightweight of the devices is still challenging due to the lack of flexible materials as electrodes with high electrochemical capacity and energy\u2010density.Rigid\u2010type lithium\u2010ion batteries (LIBs) dominate the current battery technology for portable electronics due to their high energy density and long cycle\u2010life.1 On t Therefore, flexible alkali metal\u2010chalcogen batteries, such as lithium\u2010sulfur (Li\u2010S) batteries, sodium\u2010sulfur (Na\u2010S) batteries, and lithium\u2010selenium (Li\u2010Se) batteries, are attractive due to the low\u2010cost and high theoretical capacity. Recently, there has been impressive progress on flexible batteries involving alkali metal\u2010chalcogen analogs, especially for flexible Li\u2010S batteries and flexible Na\u2010S batteries. Many advanced materials were developed for fabricating flexible alkali metal\u2010chalcogen batteries, such as the flexible cathodes consisting of sulfur hybridized with carbon nanotubes, graphene materials, carbon fibers, and metal\u2010organic framework\u2010based materials, as well as soft anodes based on lithium metal and elastic quasi\u2010solid state electrolytes. Nevertheless, these materials are still not flexible enough to afford different types of deformations covering from bending, twisting, or even stretching. The insufficient performance is mainly displayed in the instability or degradation of the performance output during repeated deformation of the whole battery system. Compared to conventional batteries, the challenges toward alkali metal\u2010chalcogen batteries are complicated: (1) the mechanical properties of each component in the cell should be flexible, and maybe more importantly, these components should be mechanically matched and steadily connected during dynamic deformation; (2) the electrochemical activity of electrode materials should maintain a high and stable level during deforming operation, and smooth electron and ion transport through the interface are also essential; (3) the safety standards should be much stricter due to the massive mechanical strains.Taking the electrochemical potential and capacity into consideration, the electrochemistry between alkali metals and chalcogen elements (excluding oxygen) offers alkali metal\u2010chalcogen batteries a balanced performance toward compelling batteries.5 Ther These metals or alloys are characterized by owing low melting points which make them keep liquid state for applications. As an intrinsically flexible conductor and pool of metal ions, applying LMs in the fabrication of batteries can not only enhance the flexibility of batteries but also upgrade the operational mode of metal\u2010ion based secondary batteries. It is believed that the change transfer kinetic and the mass (ions) transport in the liquid\u2013liquid electrode\u2010electrolyte interfaces would be fast for these LM\u2010based batteries, which results in low ohmic losses. Especially, the low melting point of chalcogen elements offers an intrinsic advantage to build an all\u2010liquid electrochemical cell. From this perspective, we focus on overviewing the current progress and then discussing the feasibility of more possibilities , which provide an ideal combination of metallic properties and fluidity, presents a tremendous potential for flexible electronics.14 The2 Other than rigid\u2010type batteries, flexible batteries usually require more stable electrochemical activities, because they would be repeatedly bent, folded, or stretched under working conditions. Consequently, all the components of the cell, covering the current collector, anode, electrolyte, separator, and cathode, should be flexible enough to afford the mechanical deformation. Obviously, a rigid lithium metal tablet (melting point of Li \u223c180\u00b0C) is not a good choice for achieving a flexible anode. This is because the dendritic growth on the surface of the anode during Li stripping\u2010deposition process could seriously affect the cycling performance and result in safety issues, which are certain to be even worse if bending or folding action is applied. Applying a metal that is liquid as anode is expected to significantly enhance the flexibility and self\u2010healing capability of the battery due to the infinite ductility originating from the fluidity. Furthermore, the intrinsic dendrite problem or volume expansion of alkali metals could be also eliminated by the adoption of LMs, while maintaining a durable high specific capacity.The Li\u2010S battery is the most common example in the family of alkali metal\u2010chalcogen batteries, which has been widely investigated.17 Oth In the early exploration of the LM batteries, they usually operated at high temperatures >400\u00b0C, due to the fact that these LM electrodes are high\u2010melting\u2010point materials. The attractive performance of LM\u2010S batteries has been already demonstrated, in which the working anodes are melted alkali metals, such as Na (melting point \u223c97.8\u00b0C), and K (melting point \u223c63.7\u00b0C). In 1967, Kummer et\u00a0al. (Ford Motor Company) invented the first high temperature (300\u2013350\u00b0C) sodium\u2010sulfur (Na\u2010S) batteries, integrating a molten sulfur cathode, a Na+\u2010conducting \u03b2\u2032\u2032\u2010Al2O3 solid electrolyte, and a molten Na anode, as shown in Figure\u00a0\u22121), and long cycle life. Nevertheless, the high operation temperature will result in safety concerns, such as corrosion of the electrode, the fire risk of solid electrolyte, etc. The transport of the Na+ through the electrolyte between the S cathode and Na metal anode is displayed in Figure\u00a0+ will go through the electrolyte to arrive at the other side and react with the sulfur cathode forming various sodium polysulfide intermediates. In the charging process, reversible reactions will take place, where Na+ ions will be transported back to the Na metal anode. The commercial high\u2010temperature Na\u2010S batteries are predominantly used for stationary energy storage. Later, Lu et\u00a0al. proposed a new potassium\u2010sulfur (K\u2010S) battery consisting of a liquid K anode, a sulfur cathode, and a K+\u2010conducting \u03b2\u2032\u2032\u2010Al2O3 solid electrolyte 6 was employed as the cathode, these LM cells operated at 25\u00b0C and delivered a high capacity of 629\u00a0mA h g\u22121 based on the Na metal as anode and 579\u00a0mA h g\u22121 for the K metal as anode , and their intrinsically insulating property, which is also true of their discharge products , and the shuttle effect of polysulfides. The poor conductivity of sulfur and the products after discharge will result in inefficient use of active materials and irreversibility in cycling, which are the major reason for their low accessible capacity. Additionally, the shuttle effect would also result in the loss of polysulfides into the electrolyte, and thus lead to fast capacity fading during cycling. These practical challenges limit the practical application of alkali metal\u2010chalcogen batteries.The ultrahigh theoretical energy density of alkali metal\u2010chalcogen batteries originates from the chalcogen materials as cathodes. For example, Li\u2010S batteries possess a high theoretical capacity of 1672\u00a0mA h gn sulfur,24 and polymer composite materials, and inorganic composite materials. Although these cathode hosts usually possess many merits, including high conductivity, high surface areas, and excellent flexibility, they still cannot be directly employed as single constituents of a flexible electrode.For flexible alkali metal\u2010chalcogen batteries, such issues become even more serious, due to the lack of alternatives for electrode materials, especially the cathode materials. In comparison to rigid ones, the flexible chalcogen batteries have to maintain durable electrochemical performance during repeated bending or even stretching. Therefore, this requires all the electrode materials to be flexible enough to endure the possible mechanical deformation. The conventional chalcogen cathodes, such as sulfur, selenium, and tellurium, consists of active materials coated on a conductive metal foil collector. These cathodes can hardly meet the requirements of the flexible electrodes due to the possible cracking and delamination that can occur during the electrode deformation. Besides the mechanical instability, the problem of low electrical conductivity and the migration of soluble polysulfides would be intensified. Recently, a tremendous amount of work has been carried out in attempting to solve the above issues by novel chalcogen cathode hosts, including carbonaceous materials,30 pol Even at the solid state, sulfur may also react with LM to form a buffer layer to enhance the wettability. For example, Zhu et\u00a0al. proposed uniform S@Ga core\u2010shell structures as the cathode for the Li\u2010S batteries  skeleton to the cathode operation. In addition, the surface of a LM is usually electron\u2010rich, which can enhance the conversion of discharge product intermediates, such as polysulfides and polyselenides. For constructing a hybrid of LM and chalcogen such as sulfur, the interfacial wettability has to be considered. It is reported that, after the liquification and polymerization of sulfur, the resulted polysulfide loops/thiol groups could strongly interact with LM, which increases the interfacial strength.33 Eves Figure\u00a0.[34] The electrons tend to be transferred to the LM metal Ga and form an electron\u2010rich surface, which can promote the electrocatalytic conversion from lithium polysulfides to Li2S. Therefore, this S/Ga composite presented a specific capacity of 1044\u00a0mA h g\u22121 after 1000 cycles at the density current of 1\u00a0mA cm\u22122 with a Coulombic efficiency of 98.7%.LM is not only a good conductor, but also can be regarded as a good liquid catalyst. For example, micro/nanometer scale liquid Ga materials have been considered as good catalysts for liquid\u2010based reactions owing to the dynamic distribution of active atoms at the surface. It is worth noting that, in comparison to solid nano\u2010catalyst supports, the LM\u2010based catalysts exhibit extraordinary longevity. In addition, liquid Ga is also regarded to favor the electrocatalytic reactions due to the high conductivity and electron\u2010rich surface. The electron\u2010rich surface of LM offers sufficient active sites to catalyze the redox reactions of lithium polysulfides, and then improve the final performance. Li et\u00a0al. reported 3D porous S/Ga hybrids as cathode materials for Li\u2010S batteries Figure\u00a0.35]35[34 Recently, Liu et\u00a0al. demonstrated a deformable battery based on LM materials and an elastic polyacrylic acid\u2010based gel electrolyte. With flexible electrode materials and gel electrolyte, such battery cells can be stretched from 12 to 24\u00a0cm, and show excellent shape recoverability with 98.87% performance retention during discharging. Even with these possible SSEs, the problems of interfacial contact and low ionic conductivity still need to be solved for achieving durable deformation repeatability and high charge\u2010discharge cycling capacity. The design concept of an integrated anode/SSE/cathode architecture is ideal for addressing these issues for the LM\u2010based flexible metal\u2013chalcogen batteries, but the problems are still very challenging. Nevertheless, the development of LM\u2010based flexible metal\u2013chalcogen batteries is still in its infancy, and inspiration from other more developed solid\u2010state batteries, for example, LIBs, will be very valuable.As we reviewed, impressive improvement and innovation have been achieved in both liquid alkali metal anodes and LM\u2010based chalcogen cathodes. Nevertheless, the assembly of LM\u2010based anodes and cathodes toward integrated flexible metal\u2013chalcogen batteries has been not realized yet. To achieve an actual flexible battery, each cell component has to be assembled into a compact and pliable device. The current fabrication mode for LM\u2010based batteries is still mainly based on the standard battery components with liquid electrolyte, which is not suitable for repeatable bending or twisting. In addition, these liquid organic electrolytes may raise the possibility of electrolyte leakage and fire, raising safety risks. Using solid\u2010state electrolytes (SSEs) to support the electrode materials that are in the liquid state would be an ideal choice to realize the final flexibility of devices. The SSEs usually can be classified into polymer electrolytes, including solid polymer and gel polymer electrolytes, as well as inorganic electrolytes. Among these candidates, polymer electrolytes are especially preferred for flexible metal\u2013chalcogen batteries because the inorganic electrolytes are too rigid to bear the bending or twisting.36 RecTaking advantage of their fluidity, although LM anodes possess excellent mechanical pliability, challenges remain to balance the energy density, safety, and weight in practical application. Such issues would become even tougher in view of the requirements on the melting point and electrochemical reduction potential. Owning to the intrinsic strong metallic bonds, the liquid alloys that can work at room temperature are limited. Alkali alloys with low melting points, such as Na\u2010K alloy, are promising choices of anode materials for the direct stripping\u2010deposition. In view of the quite high reactivity of Na\u2010K alloy, even though the problem of dendrites has been overcome, the risk of the battery safety in air is still high especially in the case of packaging broken. Another thing that should be noted, the alkali alloys that are liquid at room temperature are still limited, which astricts the performance optimization and wide application. Solid\u2010liquid mixtures or hybrids that contain active metal and room temperature LMs may also be a possible strategy to enrich the materials variations and construct soft anodes for flexible batteries. For example, the Ga\u2010based liquid alloys usually have low melting temperatures and possess self\u2010healing capability during the electrochemical processes, but their reduction potentials are obviously higher than that of the alkali metal. Constructing Ga\u2010based alloys with Li or Na, or forming hybrids containing metal species and metal droplets are all optional plan. Nevertheless, the intermetallic phases formed between post\u2010transition metals and alkali metals can affect the final kinetic and charge transport. Establishment of electrochemical mechanism on the multi\u2010cation systems of these elements is highly demanded. They would be meaningful to guide the designs of LM alloy anodes and help in understanding the interfacial charge and mass transport phenomena.To pursue high energy density, chalcogen elements should be the main active part of the cathodes. The introduction of LM could offer a flexible and conductive binder for sulfur and other chalcogens including selenium and tellurium, forming a hybrid cathode. However, the balance of the content's ratio between chalcogens\u2010based active materials and LMs requires careful consideration. The high content of LMs may reduce the portability and gravimetric energy density of the whole electrode. It is expected to obtain satisfied bending or self\u2010healing capability with the least amount of LM additives in the cathode materials. The atomic metal species in LM offer a possibility to catalytic regulate the redox kinetics of chalcogens; nevertheless, in\u2010depth investigations in the form of both theoretically and experimentally are highly demanded.In conclusion, with further explorations on the design of LM favored electrolyte, LM anode, and LM\u2010based cathode, such novel LM\u2010based chalcogens battery system will promise an attractive future of applications for next\u2010generation flexible energy storage. It is believed that the introduction of LMs into chalcogen batteries can not only offer a design principle for high\u2010performance flexible batteries but also inspire extensive and in\u2010depth application of LMs.The authors declare no conflict of interest."}
+{"text": "Glucocorticoid usage and alcohol abuse are the most widely accepted risk factors for nontraumatic osteonecrosis of femoral head (ONFH). Despite distinct etiologies between glucocorticoid-associated ONFH (GONFH) and alcohol-associated ONFH (AONFH), little is known about the differences of the microarchitectural and histomorphologic characteristics between these subtypes of ONFH.To investigate bone microarchitecture, bone remodeling activity and histomorphology characteristics of different regions in femoral heads between GONFH and AONFH.From September 2015 to October 2020, 85 patients diagnosed with GONFH and AONFH were recruited. Femoral heads were obtained after total hip replacement. Femoral head specimens were obtained from 42 patients (50 hips) with GONFH and 43 patients (50 hips) with AONFH. Micro-CT was utilized to assess the microstructure of 9 regions of interest (ROIs) in the femoral head. Along the supero-inferior orientation, the femoral head was divided into necrotic region, reactive interface, and normal region; along the medio-lateral orientation, the femoral head was divided into medial region, central region and lateral region. Decalcified and undecalcified bone histology was subsequently performed to evaluate histopathological alterations and bone remodeling levels.In the necrotic region, most of the microarchitectural parameters did not differ significantly between GONFH and AONFH, whereas both the reactive interface and normal region revealed a less sclerotic microarchitecture but a higher bone remodeling level in GONFH than AONFH. Despite similar necrotic pathological manifestations, subchondral trabecular microfracture in the necrotic region was more severe and vasculature of the reactive interface was more abundant in GONFH.GONFH and AONFH shared similar microarchitecture and histopathological features in the necrotic region, while GONFH exhibited a less sclerotic microarchitecture and a more active bone metabolic status in both the reactive interface and normal region. These differences between GONFH and AONFH in bone microarchitectural and histopathological characteristics might contribute to the development of disease-modifying prevention strategies and treatments for ONFH, taking into etiologies. Osteonecrosis of femoral head (ONFH) is known as avascular necrosis or aseptic necrosis of the femoral head, associated with an interruption of the blood supply . One of via direct or indirect harmful effects, with the potential mechanism being vascular damage, increase in intraosseous pressure, intraosseous coagulation, disruption of osteogenic differentiation, and mechanical stresses : GONFH or (2) AONFH. Patients were recruited to the GONFH group if they met the classification criteria of GONFH included the following: (1) Patients should have a history of glucocorticoid use more than 2\u00a0g of prednisolone or its equivalent within 3 months;(2) ONFH was diagnosed within 2 years after glucocorticoid administration;(3) There were no other risk factors except glucocorticoids . A totalRegarding AONFH, we collected information about age at starting and cessation of alcohol consumption, usual frequency of drinking, and the usual volume of alcohol intake by beverage type. This volume was consequently converted to grams of ethanol and values for each beverage type were added. The ethanol content for calculation was as follows: 4.5% for beer, 12% for wine, 43% for whisky, 15.5% for rice wine and 50% for baijiu  . We descGONFH and AONFH subjects were well matched on demographic factors and medical conditions Table\u00a01,Exclusion criteria for ONFH patients were as follows: (1) Other recognized metabolic or bone conditions, such as thyroid or parathyroid illness, and malignancy, that may impact bone metabolism; (2) undergoing additional medications that influence bone metabolism such as anti-resorptive drugs, calcitonin, thyroid or parathyroid hormone therapy, or hormonal replacement therapy; or (3) history of hip fracture and osteotomy \u201331. InfoFollowing THA surgery, specimens of the femoral head were collected and preserved in a -80\u00b0C freezer. Then, they were scanned using the high resolution micro-CT system , with the following scan settings: X-ray tube voltage, 70 kVp; tube current, 200 \u03bcAs; exposure time, 300 ms; projection number, 1000; and voxel size, 73.6 \u03bcm. The scan length was approximately 49\u00a0mm, resulting in 1200 slices and a 45-minute scan time.Orientation adjustment of the femoral head, segmentation of the regions of interest (ROIs), and measurement of bone microstructural parameters in each region were performed by the built-in software. Orientations of all femoral heads were adjusted based on anatomical landmarks using the fovea capitis femoris and the three\u2212pillar structure theory , 35. TheFollowing scanning and reconstruction, the pictures were converted to binary images using a fixed threshold. To distinguish between soft tissue and calcified tissue, a consistent global threshold range (90 to 255) based on grayscale histogram analysis and empirical observations was applied , 37. ColThe following microarchitectural parameters were measured: bone volume fraction (BV/TV) (%), bone surface/volume ratio (BS/BV) (1/mm), trabecular thickness (Tb.Th) (\u03bcm), trabecular separation (Tb.Sp) (\u03bcm), trabecular number (Tb.N) (1/mm), structure model index (SMI), degree of anisotropy (DA), connectivity density (Conn.D) (1/mm3) .After micro-CT scanning, all femoral heads were cut in half with a diamond saw, coronally separating the anterior hemisphere from the posterior hemisphere  in the medial, central, and lateral reactive interfaces, respectively. The evaluation was performed by two independent researchers who were blind to the identity of group.2/mm3), percentage eroded surface (ES/BS) (%), specific eroded surface (ES/BV) (mm2/mm3) and eroded surface in bone tissue volume (ES/TV) (mm2/mm3) . The following bone formation and resorption parameters were measured in each ROI: thickness of osteoid , percentage osteoid volume (OV/BV) (%), percentage osteoid surface (OS/BS) (%), specific osteoid surface (OS/BV) (mmmm2/mm3) .We randomly selected 10 samples and assessed the inter-observer and intra-observer agreements using Root Mean Square Error Estimation (RMSE). Two experienced observers determined the ROIs of selected samples independently. We reported the RMSE of the difference between two observers for each region stratified by subtype of ONFH. The results showed good intra and inter-observer reproducibility in different regions . Continuous data are presented as mean \u00b1 standard deviation (SD). Gender, smoker, diabetes, hypertension, side of ONFH, ARCO stage  distribution and no significant departures were identified. The unpaired Student\u2019s t test was used to compare the microarchitecture parameters, vessel number, and bone remodeling parameters between groups. A conservative two-tailed P < 0.05 was chosen CO stage , JIC staIn Sup-Med region, none of the microarchitecture parameters differed significantly between GONFH and AONFH, with the exception of DA. There were significantly lower values of DA in GONFH, compared to AONFH. Similarly, in Sup-Cen region, none of the microarchitecture parameters differed significantly between GONFH and AONFH, with the exception of SMI Table\u00a02.In Cen-Med region Table\u00a02,In Cen-Cen region, all the microarchitecture parameters were significantly different between GONFH and AONFH, with the exception of DA Table\u00a02.In Cen-Lat region, Tb.Th, Conn.D, SMI and DA were not significantly different between GONFH and AONFH Table\u00a02.In Inf-Med region, there were higher values of SMI in GONFH than in AONFH, while the other parameters showed no significant difference. In Inf-Cen region, BV/TV, BS/BV, Tb.Th, Tb.N, Tb.Sp, Conn.D and DA were not significantly different between GONFH and AONFH. However, there were significantly higher values of SMI in GONFH, compared to AONFH. In Inf-Lat region, none of the microarchitecture parameters differed significantly between GONFH and AONFH Table\u00a02.In the necrotic region of the femoral head, bone marrow necrosis and osteocytic death were observed in both groups. Hematopoietic cells disappeared and adipose nucleus staining was absent in the bone marrow, indicating the necrosis of bone marrow Figure\u00a05In the necrotic-normal junction region, reparative process, such as vascular-rich granulation tissue, was observed. Histiocytes and giant cells aggregated in the reactive fibrous interface, especially in the lateral aspect. Although vessels penetrated into the fibrotic capsule, the angiogenesis was blocked at the boundary of the sequestrum and reparative fibrovascular tissue could not penetrate into the bone marrow space of the necrotic region. Constantly, the reparative fibrovascular tissue would convert to a sclerotic rim in the late stages of the disease, which was attributed to the bone formation by osteoblasts.In the medial reactive interface, the vessel number was 8.76 \u00b1 3.47 in the GONFH group and 4.90 \u00b1 2.03 in the AONFH group. In the central reactive interface, the vessel number was 9.74 \u00b1 3.33 in the GONFH group and 5.72 \u00b1 1.72 in the AONFH group. In the lateral reactive interface, the vessel number was 10.72 \u00b1 3.94 in the GONFH group and 6.82 \u00b1 2.27 in the AONFH group. There were significant statistical differences in the vessel number between the two groups. Accordingly, the angiogenesis was more abundant in the reactive interface in GONFH, bringing about a more active bone remodeling status, despite the failure of thorough reparative substitution of the sequestrum in both groups  for the publication of any potentially identifiable images or data included in this article.All authors listed have read and approved all versions of the manuscript. YC had full access to all data in the study and took responsibility for the integrity of the data and the accuracy. Critical revision of the article for important intellectual content, YC, GL, KL, YM, BZ, FX, JY, JZ, CZ, and YF. All authors contributed to the article and approved the submitted version."}
+{"text": "Background and Objectives: The aim of this study was to compare the accuracy of three types of electronic apex locators (EALs) when two different concentrations of NaOCl irrigation solutions are used by two operators. Materials and Methods: After creating the access cavities for 20 single rooted extracted teeth, the actual canal length (ACL) of each canal was determined visually using a #10 file and magnification. The teeth were subsequently inserted in plastic molds filled with alginate. The electronic measurement of root canal length (EWL) was performed using three different electronic apex locators: Root ZX II, Apex ID, and Dual Pex. Two independent operators, an endodontic specialist with 20 years practice and an undergraduate student in the final year of study, performed the irrigation procedures with two different concentrations of NaOCl (2% and 5.25%), and then measured the EWL using each of the EALs. The accuracy of all EALs, was determined in each case by subtracting the EWL from the ACL. Statistical analyses were performed using one-way ANOVA test. Results: In the presence of 2% NaOCl solution, for a margin error of \u00b10.5 mm, Root ZX II, Apex ID, and Dual Pex presented an accuracy of 90%, 80%, and 85% respectively. The increase in the concentration of the irrigation solution affected the accuracy of Root ZX II and Apex ID for both operators, diminishing it to 75% for the same margin error, but improved Dual Pex\u2019s accuracy to 100%. Conclusions: The best accuracy in working length determination was obtained by Root ZX II for 2% NaOCl solution and by Dual Pex for 5.25% NaOCl solution with no significant statistical difference when compared. Among the factors that significantly influence the success of an endodontic treatment are the correct instrumentation and obturation of root canal to a precise working length (WL). Therefore, an accurate determination of the apical limit during an endodontic treatment is of extreme importance . DiffereMethods of determining the working length (WL) include radiological and electronical means. Conventional periapical radiograph was the most used method for determining the length of the root canal. Using this method, the position of the apical AC was chosen according to the radiographic apex. Several microscopic studies have shown that the vast majority of ACs were found between 0.5 and 1 mm from the radiographic apex . AlthougEven though the idea of using electricity to locate the canal terminus appeared in the beginning of the twentieth century , the production of these electronic apex locators (EALs) began only after Sunada (1962) built the first device that electronically determined the WL. The accuracy of early first and second generation EALs was strongly influenced by the presence of strong electrolytes, excessive hemorrhage, or remnant pulp tissue in the root canal . The thiSince irrigation is of essential importance in root canal treatment for the removal of dentine, remnant tissues, and individual bacteria or microbial communities ,21, the Therefore, the aim of this in vitro study was to compare the accuracy of three types of EALs when two different concentrations of NaOCl irrigation solutions are used by operators with different experience in using these devices. The null hypothesis tested was that there are no differences in accuracy among the three EALs tested, regardless of the concentration of the irrigation solution used and operator\u2019s experience.This study was performed after ethical approval was obtained from the Ethics Committee of the \u201cIuliu Ha\u021bieganu\u201d University of Medicine and Pharmacy, Cluj-Napoca, Romania .A number of 20 single rooted teeth (incisors and premolars), extracted due to periodontal or orthodontic reasons, were used in the present study. After extraction, all teeth were immersed in 2.5% NaOCl solution for 4 h  in order to disinfect them and allow the disintegration of remained organic tissues. Then they were cleaned from calculus with hand or ultrasonic scalers and stored in saline solution until their use. Before their preparation, all teeth were X-rayed from a buco-oral and mesio-distal incidence, to ensure the presence of a single root canal with a mature apex, without internal or external root resorption and to observe root canal\u2019s degree of curvature. Only teeth with moderate degree of curvature (less than 20\u00b0) according to Schneider\u2019s method of evaluation were chosen . All incThe teeth were numbered from 1 to 20 and cut 4 mm from the cemento-enamel junction in coronal direction with a diamond disc 345 22 MM 0.25  fitted to a dental hand piece to provide a flat surface and a reliable occlusal reference for all working length (WL) measurements . After cThe ACL of each canal was determined by the insertion of an MMC file #10  into the root canal, until its tip could be visualized tangential to the AF under 8\u00d7 magnification microscope . A rubber stop was carefully placed at the reference point, represented by the flat edge of the cut, and fixed to the file with polymerized fluid resin. The file was then removed from the canal and the distance between the rubber stop and the tip was measured with an endodontic ruler under endodontic microscope obtaining the actual canal length (ACL). This process was repeated twice by the same operator for each tooth and the measurements recorded in a table. If the AF was located sideways on the surface of the root, the coronal edge of the foramen was taken as a reference for positioning the tip of the file.The teeth were subsequently inserted separately in plastic molds filled with freshly prepared alginate  up to the cemento-enamel junction ,27,30. T(a)Root ZX II :(b)Apex ID :(c)Dual Pex .The electronic measurements of root canals length were performed using 3 different electronic apex locators:For the irrigation protocol, two solutions of 2% NaOCl (S1) and 5.25% NaOCl (S2) were used at room temperature . The bottles were opened just before use. Two independent operators, an operator with 20 years of experience in endodontics (Op1) and an undergraduate student (Op2) in the last year of study performed the irrigation procedures and the electronic measurements of the WL with each of the 3 devices. The undergraduate student knew the working principle of EALs and, before starting the research, was trained on the working method of each EAL used in the present study and measured the WL of ten root canals with each of those devices.In order to ensure an adequate alginate humidity, all measurements were carried out within a 2-h interval ,31. PrioRoot canals were initially irrigated with S1 and then with S2 solution, following the same protocol. Every root canal was first irrigated with 1 mL of S1 solution using a 5 mL syringe and 30-gauge needle . The excess irrigation solution from the pulp chamber was removed by air drying, keeping the root canal moist. The external tooth surface was wiped with a cotton pellet. For Root ZX II, the 10 MMC file was connected to the holder and advanced into the root canal until the last green bar appeared on the device display, and \u201cApex\u201d began to flash. For Apex ID and Dual Pex, the 10 MMC file was advanced into the root canal until the flashing bar \u201c00\u201d was reached. If the instrument remained in this position for at least 5 s, the measurement was considered appropriate. A rubber stop was carefully placed at the reference point of each sample and fixed on the file with light-curing fluid resin. Then, the distance between the rubber stop and the tip of the instrument was measured with an endodontic ruler under dental loupe with 2.5\u00d7 magnification. After recording the EWL, the irrigation solution (S1) was sucked out and the canal was dried with paper points, adapted to root canal, to avoid the modification of NaOCl concentration .The same protocol was repeated for S2 solution by both operators. Two measurements were carried out for every tooth with each solution (S1 and S2), for all three devices and by both operators. The EALs were used according to the indications given by the manufacturer.After performing the measurements according to the steps described above, all data were recorded in Excel tables. These EWL measurements were compared with ACL measurements, obtained before the irrigation procedures under the endodontic microscope. The accuracy of EAL was determined in each case by subtracting the EWL from the ACL. Positive or negative values were recorded when the length measured with EALs was lower  or higher (the tip exceeded the AF) than the one measured by visual detection of the tip under the microscope. When the EALs measurement coincided with the actual length, the value was reported as \u201c0\u201d. All measurement errors were calculated as the absolute difference, in millimeters.Prior to statistical analyses, a sample size of 20 per group was calculated for one-way ANOVA using G3*Power , considering alpha-error = 0.05 and power = 0.8.p < 0.05). Pairwise comparisons between groups were assessed using post hoc standard and Dunn tests with Bonferroni corrections. \u03b1 = 0.05 was chosen as level of statistical significance. All graphics were prepared and analyses performed using JASP .The normality of statistical distributions was visually assessed using Q-Q plots. One-way ANOVA was applied to determine the accuracy of each EAL in relation with the AF and to assess the influence of operator experience and NaOCl concentration on each EAL separately. Levene\u2019s test was used to establish the homogeneity of variance. With one exception (Op1 vs. Op2 for NaOCl = 5.25%), the equality of variance assumption was not rejected . In the presence of 2% NaOCl solution, Root ZX II obtained the closest EWL mean value to that of ACL (0.29 mm), when compared to the values obtained by the two other EALs. Yet, no statistically significant difference among the means of the three EALs was found. When irrigation of root canals was performed with 2% NaOCl solution, Root ZX II, Apex ID, and Dual Pex managed to locate in 90%, 80%, and 85%, respectively, the major foramen with a margin of error of \u00b10.5 mm for Op1. For Op2 the accuracy of the three devices was 85% (Root ZX II), 80% (Apex ID), and 90% , respectively (When the measurements obtained with the endodontic microscope (ACL) were compared with those obtained by the three EALs, no statistically significant difference was found among them (ectively .The increase in concentration of the irrigation solution (from 2% to 5.25%) affected the accuracy of Root ZX II and Apex ID for both operators, diminishing it to 75% for the same margin error of \u00b10.5 mm. On the other hand, for Dual Pex, the increase in concentration of the irrigation solution improved its accuracy, achieving the closest value to ACL (0.225 for Op1). As for Op2, an increase in concentration of NaOCl solution did not influence the accuracy of Dual Pex .p < 0.05) was observed for Op1. However, when the three devices were compared two by two by applying the Dunn test with Bonferroni corrections, no significant statistical difference was found among them for Op1  was 22.5% for Root ZX II, 12.5% for Apex ID, and 32.5% for Dual Pex. When 5.25% NaOCl solution was employed, the ratio of the measurements beyond the foramen was 57% for Root ZX II, 60% for Apex ID, and 47% for Dual Pex.When 2% NaOCl solution was used for root canal irrigation, the proportion of EWL measurements that were beyond the major foramen for Op1 and Op2 together (p > 0.05) showed that the experience of the two operators did not influence the accuracy of the three EALs, regardless of the concentration of the irrigation solution used  in the accuracy of the three EALs tested were noticed within a tolerance of \u00b10.5 mm, except when Op1 used 5.25% NaOCl solution.The null hypothesis tested in the present study was partially accepted because, regardless of the concentration of the irrigation solution used and operator\u2019s experience in irrigating root canals, no significant differences , while Dual Pex used by Op1 and Root ZX II used by Op2 showed an accuracy of 85%. The smallest accuracy in the location of the AF, for this solution, was shown by Apex ID, for both operators (80%). The results obtained by Root ZX II in the present study, when root canals were irrigated with 2% NaOCl solution, are comparable with those of other studies obtained by the same device but with 2.5% NaOCl solution. Therefore, accuracy of 97.44%  and 93% Regarding Apex ID accuracy in localizing the AF, the present results do not corroborate with those of Oliviera et al. , where tp < 0.05) between the results of Dual Pex used by Op1 and the results obtained with the other two EALs by the same operator. Op2 also obtained better results with Dual Pex (80% for a tolerance range of \u00b10.5 mm) compared to the two other EALs for the same NaOCl solution but without any statistical significance. These findings are in agreement with those of Mahmoud et al. [Since NaOCl solution has electrical conductivity properties, the question arises whether its concentration influences the efficiency of EALs. The present findings revealed that for 5.25% NaOCl irrigation solutions, the accuracy of Root ZX II and Apex ID decreased from 90% to 75% for Root ZX II and from 80% to 75% for Apex ID (within a tolerance range of \u00b10.5 mm), irrespective of the device or operator, while Dual Pex used by Op1 showed an accuracy of 100% for the same tolerance limit. Therefore, in this case, there was a statistically significant difference (d et al.  who compd et al.  using a d et al. . They coPrevious studies have shown that a possible inaccurate measurement of the EWL could be caused by the bending of the measuring file . In ordeBy determining the AF with EALs, the WL can be overestimated ,38,39,40In this regard, Ding et al.  showed tThe measurements of this investigation consistently located the AF within \u00b10.5 mm tolerance for all EALs, except three or four cases when the AF was recorded beyond the major apical foramen with 0.5\u20131 mm. This indicates that, using these devices, after locating the AF and taking off 0.5 mm from the root canal working length obtained, the canal will not be prepared and filled beyond the AF. Nekoofar et al.  claim thSome discussion might also be related to the material used for embedding the teeth when determining the EWL. There are a few methods of EWL determination described in literature where gelatin agar or alginate is used to recreate the clinical conditions needed for this procedure . In the Concerning operator experience in the measurement of EWL, no significant differences in the location of the AF between the two operators were observed, regardless of the device or concentration of the irrigation solution used. The results of this study are consistent with those obtained in several studies ,30 whereUnder the limitations of these experimental conditions, including the usage of alginate models for embedding the studied teeth, the three EALs tested in this study located the position of the AF with a similar accuracy. The best results were obtained with Root ZX II for 2% NaOCl and Dual Pex for 5.25% NaOCl, but without statistically significant difference. The concentration of NaOCl solution did not significantly affect the performance of EALs. Electronic root canal measurements represent an objective technique in determining the WL regardless of operator experience. However, the use of these EALs does not entirely prevent the risk of overestimating the WL. Future investigations are needed to determine if other factors, e.g., heated NaOCl solution, the presence of blood or other types of secretions in the root canal, or the rotary motion of endomotors, could influence the accuracy of EALs in determining the WL."}
+{"text": "Poly(ADP-ribose) polymerase 2 (PARP2) alongside PARP1 are responsible for the bulk of cellular PARP activity, and they were first described as DNA repair factors. However, research in past decades implicated PARPs in biological functions as diverse as the regulation of cellular energetics, lipid homeostasis, cell death, and inflammation. PARP activation was described in Th2-mediated inflammatory processes, but studies focused on the role of PARP1, while we have little information on PARP2 in inflammatory regulation. In this study, we assessed the role of PARP2 in a Th17-mediated inflammatory skin condition, psoriasis. We found that PARP2 mRNA expression is increased in human psoriatic lesions. Therefore, we studied the functional consequence of decreased PARP2 expression in murine and cellular human models of psoriasis. We observed that the deletion of PARP2 attenuated the imiquimod-induced psoriasis-like dermatitis in mice. Silencing of PARP2 in human keratinocytes prevented their hyperproliferation, maintained their terminal differentiation, and reduced their production of inflammatory mediators after treatment with psoriasis-mimicking cytokines IL17A and TNF\u03b1. Underlying these observations, we found that aromatase was induced in the epidermis of PARP2 knock-out mice and in PARP2-deficient human keratinocytes, and the resulting higher estradiol production suppressed NF-\u03baB activation, and hence, inflammation in keratinocytes. Steroidogenic alterations have previously been described in psoriasis, and we extend these observations by showing that aromatase expression is reduced in psoriatic lesions. Collectively, our data identify PARP2 as a modulator of estrogen biosynthesis by epidermal keratinocytes that may be relevant in Th17 type inflammation.PARP2 mRNA expression is increased in lesional skin of psoriasis patients.PARP2 deletion in mice attenuated IMQ-induced psoriasis-like dermatitis.NF-\u03baB activation is suppressed in PARP2-deficient human keratinocytes.Higher estradiol in PARP2-deficient keratinocytes conveys anti-inflammatory effect.The online version contains supplementary material available at 10.1007/s00109-023-02338-z. Psoriasis is a chronic, immune-mediated, inflammatory skin disorder affecting approximately 2% of worldwide population. The etiology of psoriasis is multifactorial, with genetic and environmental triggers being the most prominent contributors to disease development . To datePsoriasis is considered primarily a T cell-mediated disease, where the T helper type (Th)1 and Th17 subsets have central role . The intPARP enzymes constitute a superfamily of 17 members in humans. Majority of cellular PARP activity is covered by PARP1 (85\u201390%) and PARP2 (5\u201315%), which are ubiquitously expressed in mammalian tissues . PARP1 aPARPs are associated with inflammatory mechanisms in several tissues . The rolAn extended description of materials and methods is given in Chromogenic in situ hybridization (CISH) was performed on paraffin embedded skin specimens with double-digoxigenin-labeled PARP2 specific  or mRNA scramble negative control  locked nucleic acid detection probes according to the manufacturer\u2019s instructions. For visualization alkaline phosphatase conjugated anti-DIG antibody  and 4-nitro-blue tetrazolium/5-bromo-4-chloro-indolylphosphate AP chromogen substrate was applied. Slides were counterstained with liquid-stable nuclear fast red.\u2212/\u2212 and littermate PARP2+/+ male mice on a C57BL/6 J background from heterozygous crossings were used (age 8\u201312 weeks). The IMQ-induced psoriasis model was performed on mice as in [Homozygous PARP2ce as in . For aroImmunohistochemistry analyses were performed on 4-\u03bcm-thick sections cut from formalin fixed, paraffin embedded mouse or human skin samples, similar, as in . EpidermSections of mouse skin stained with hematoxylin and eosin were examined. The thickness of the epidermal layer was measured using the Fiji ImageJ software. Five points were selected at random on each sections and measured from stratum basale to stratum corneum on vertical sections.2+ for 3 days.HPV-Ker cell line was provided by Creative Laboratory Ltd, Szeged, Hungary. HPV-Ker keratinocytes were maintained as in . Post-coPARP2 knocked-down HPV-Ker keratinocytes (shPARP2) were generated by stable transfection with a predesigned shRNA targeting PARP2  that was cloned into a pLKO.1 vector and packed in lentivirus carrier . Negative control HPV-Kers (sc) were created by transfection with a non-target shRNA-containing pLKO.1 vector carrying transduction particles . After transfection, puromycin-resistant cells were selected with 2.5 \u03bcg/ml puromycin and subsequently maintained in culture media supplemented with puromycin.Total RNA from cells was routinely isolated using TRIzol reagent  according to the manufacturer\u2019s instructions. For RNA-sequencing, samples were isolated with the RNeasy Kit from Qiagen . RNA samples were treated with Ambion DNase I ; afterwards, samples were reverse transcribed using a High-Capacity cDNA Reverse Transcription Kit . The RT-qPCR reactions were performed in a Light-Cycler 480 Detection System  using TaqMan assays  terms. Two-sided hypergeometric test with Bonferroni step down correction was performed using the list of differentially expressed genes and GO Biological process database.High throughput mRNA sequencing analysis was performed on Illumina sequencing platform. RNA sample quality was checked on Agilent BioAnalyzer using Eukaryotic Total RNA Nano Kit according to manufacturer\u2019s protocol. RNA-Seq libraries were prepared from total RNA using Ultra II RNA Sample Prep kit (New England BioLabs) according to the manufacturer\u2019s protocol. Moderated In silico analysis was performed on transcriptomics data from healthy skin and lesional skin of psoriasis patients already published by other groups (PubMed Unique Identifier (PMID): 24,441,097 ) and ourWhole-cell or nuclear protein samples were isolated and further processed for SDS-PAGE/Western blotting as detailed in . AntibodCells were fixed with ice-cold 70% ethanol. Cells were washed with staining buffer; then, 100 \u00b5g/ml propidium iodide stock solution  was added to the samples (1 \u00b5l/sample) and incubated for 20 min in dark. Cell cycle analysis was carried out by NovoCyte Flow Cytometer .Mouse skin samples were flash frozen in liquid nitrogen after excise. Tissues were homogenized in a TissueLyser II device . Supernatants were used for the cytokine array according to the manufacturer\u2019s protocol . The immunoblot images were analyzed using the CellProfiler for non-cell images analysis software  as in .Supernatants were collected from HPV-Kers, and mouse skin protein samples were analyzed for cytokines and estradiol using commercially available ELISA kits according to the manufacturers\u2019 protocols. The ELISAs used in this study are listed in Confocal microscopy was performed as in . AntibodProtein was extracted from the cells using Complete Lysis Buffer AM2 . NF-\u03baB p65 subunit activation was determined in 4 \u00b5g of whole-cell protein extracts from each sample using the TransAM\u2122 NF\u03baB p65 Chemi Kits  according to the manufacturer\u2019s instructions. Luminescence was measured using a Spark 10 M microplate reader .t-test (two-tailed), as the Shapiro\u2013Wilk test showed normal distribution. If the distribution was not normal, Mann\u2013Whitney test was used. When we compared more than two groups and if the distribution was normal, we used ANOVA followed by Tukey\u2019s post hoc test. In case the data did not show a normal distribution, Kruskal\u2013Wallis test were applied complemented by Dunn\u2019s post hoc test. Intensity of IHC reactions was scored on a scale 0\u20133, and subsequently, \u03c72-test was applied for statistical analysis between the groups. The significance level was set at 0.05.The distribution of data was analyzed by Shapiro\u2013Wilk test. If two groups were compared, we used independent \u2212/\u2212 and littermate PARP2+/+ male mice. Macroscopically, the developed lesions were less severe in the IMQ-treated PARP2\u2212/\u2212 (PP) mice than in IMQ-treated wild-type (WP) mice  mice RNA-containing lentiviral vector that resulted in approximately 70% decrease in PARP2 protein expression in shP2 HPV-Ker cells compared to the negative control sequence-bearing sc cells  that were induced in shP2 HPV-Kers compared to their sc counterparts Fig. a, which First, we confirmed that silencing of PARP2 induced the expression of aromatase in keratinocytes Fig. b. In accWhen HPV-Kers were kept in estradiol containing media prior to stimulation with IL17A and TNF\u03b1, lower IL6 mRNA expression Fig. e and secWe detected IL17A\u2009+\u2009TNF\u03b1-induced degradation of inhibitor of nuclear factor kappa B alpha (I\u03baB\u03b1) that retains NF-\u03baB in cytoplasm in sc HPV-Kers that was less pronounced in shP2 cells Fig. g. HoweveTo functionally link estrogen action to the antipsoriatic phenotype of PARP2 silencing, estradiol synthesis was inhibited in HPV-Kers by the application of the aromatase inhibitor exemestane. The activation of p65 in response to IL17A and TNF\u03b1 increased in shP2 cells to the level measured in sc cells upon exemestane treatment Fig. h. In agr+/+ and PARP2\u2212/\u2212 mice that were treated with vehicle and IMQ , and PARP2+/+ and PARP2\u2212/\u2212 mice that were treated with topical exemestane solution prior to treatment with IMQ .We studied the effect of cutaneous aromatase inhibition on the IMQ-induced psoriasis in mice. We performed the IMQ-induced dermatitis as follows: PARP2+/+ and PARP2\u2212/\u2212 mice , an enzyme involved in androsterone biosynthesis, and 5\u03b1-reductases  that catalyze the conversion of testosterone to dihydrotestosterone (DHT) [\u2212/\u2212 mice, without changing systemic levels of DHT [PARP2 has been associated with several aspects of steroid synthesis and steroid mechanism of actions mostly in metabolic tissues . In skelne (DHT) . As a cos of DHT . In addis of DHT . In thisThe therapeutic potential of estrogens in psoriasis has been suggested , 37, but\u221210 M range, which approximately corresponds to the concentration we detected in the supernatants of sc and shP2 HPV-Kers. It is therefore possible that subtle increases in estradiol production may suffice to turn on the anti-inflammatory machinery in keratinocytes, such as in the case of PARP2-silenced cells. The estradiol level in the skin may show strong intrapersonal and interpersonal variability that requires further investigations to determine the potentially therapeutic concentration of estrogens in psoriasis.In fact, the role of estrogens in immune modulation is not straightforward. Although psoriasis tends to improve during pregnancy and exacerbate in menopause, a minority of patients experienced worsening of symptoms during pregnancy , 39. The\u2212/\u2212 mice [\u2212/\u2212 and PARP2\u2212/\u2212 mice displayed opposing phenotypes. In experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis, PARP1 deletion increased [PARP2 may also have a complex role in inflammatory regulation. PARP2 deletion does not seem to influence Th2-mediated inflammatory processes, whereas the deletion or inhibition of PARP1 was anti-inflammatory in mouse models of contact hypersensitivity reaction \u201317, asth\u2212/\u2212 mice , while tncreased , while Pncreased . Interesncreased \u201350 that \u2212/\u2212 mice; however, the mechanism we demonstrate in keratinocytes might be an important piece in the pathomechanism of psoriasis.A limitation of our study is the utilization of whole-body knock-out mice instead of knock-out mice with conditional deletion of PARP2 in keratinocytes or immune cells. Indeed, we cannot exclude the contribution of distinct skin cells or distant organs to the observed skin phenotype of PARP2Currently, the most successful therapies against psoriasis are biologic agents targeting IL17A or TNF\u03b1, but there are several safety concerns about the usage of such therapies, and there is a constant search for better-tolerated solutions. Several PARP inhibitors are in clinical use for systemic application in tumor therapy, involving FDA-approved veliparib (Abbvie), rucaparib (Pfizer/Clovis), olaparib (KuDOS/AstraZeneca), niraparib (Merck/Tesaro), talazoparib (Lead/Biomarin/Medivation/Pfizer), and fluzoparib and pamibarib approved by the Chinese NMPA. These are pan-PARP inhibitors that equally target PARP1, PARP2, and even PARP3. Hence, selective targeting of PARP2 cannot be done by these inhibitors, and their effects may be affected by the concurrent inhibition of PARP1 and PARP3. However, here we show that talazoparib could recapitulate the effect of depletion or specific inhibition of PARP2 with UPF1069 on estradiol production. Given that the protective effect of genetic deletion or depletion of PARP2 in psoriasiform inflammation may probably be boiled down to increased estradiol synthesis of keratinocytes, our data might raise the potential of repurposing PARP inhibitors in the treatment of psoriasis. Still, the applicability of PARP inhibitors will have to be assessed in subsequent studies.In summary, we highlighted a yet unknown mechanism by which PARP2 may be involved in inflammatory regulation and identified a potential targetable player in psoriasis. Our study may promote the development of PARP2 specific inhibitors and encourage that more studies be conducted on the elucidation of the role of PARP2 in inflammation and immune regulation.Supplementary file1 (DOCX 22 KB)Below is the link to the electronic supplementary material."}
+{"text": "Epidemiological research may require linkage of information from multiple organizations. This can bring two problems: (1) the information governance desirability of linkage without sharing direct identifiers, and (2) a requirement to link databases without a common person-unique identifier.We develop a Bayesian matching technique to solve both. We provide an open-source software implementation capable of de-identified probabilistic matching despite discrepancies, via fuzzy representations and complete mismatches, plus de-identified deterministic matching if required. We validate the technique by testing linkage between multiple medical records systems in a UK National Health Service Trust, examining the effects of decision thresholds on linkage accuracy. We report demographic factors associated with correct linkage.\u03b8 and a leader advantage threshold \u03b4. Defaults were chosen to penalize misidentification 20-fold versus linkage failure. By default, complete DOB mismatches were disallowed for computational efficiency. At these settings, for non-self database comparisons, the mean probability of a proband being correctly declared to be in the sample was 0.965 (range 0.931\u20130.994), and the misidentification rate was 0.00249 (range 0.00123\u20130.00429). Correct linkage was positively associated with male gender, Black or mixed ethnicity, and the presence of diagnostic codes for severe mental illnesses or other mental disorders, and negatively associated with birth year, unknown ethnicity, residential area deprivation, and presence of a pseudopostcode (e.g. indicating homelessness). Accuracy rates would be improved further if person-unique identifiers were also used, as supported by the software. Our two largest databases were linked in 44\u00a0min via an interpreted programming language.The system supports dates of birth (DOBs), forenames, surnames, three-state gender, and UK postcodes. Fuzzy representations are supported for all except gender, and there is support for additional transformations, such as accent misrepresentation, variation for multi-part surnames, and name re-ordering. Calculated log odds predicted a proband\u2019s presence in the sample database with an area under the receiver operating curve of 0.997\u20130.999 for non-self database comparisons. Log odds were converted to a decision via a consideration threshold Fully de-identified matching with high accuracy is feasible without a person-unique identifier and appropriate software is freely available.The online version contains supplementary material available at 10.1186/s12911-023-02176-6. A common problem in epidemiological research involving health data is the linkage of information about the same person from multiple sources. This brings two significant problems: information governance (IG) constraints and the potential lack of a common unique identifier. Medical records contain health information  , plus idWhen two organizations A and B communicate to link health data, with the ultimate purpose of creating de-identified data for research, further IG challenges arise. The simplest technique would be for A to send its identifiable data wholesale to B, so that B can identify patients common to A and B, and prepare a de-identified data set. However, this provides B with a great deal of information about A\u2019s patients, which is undesirable. Such work is likely to require special approvals, e.g. in the UK under Section\u00a0251 of the National Health Service (NHS) Act . NeverthA more desirable process would be to exchange irreversibly encrypted or hashed identity information for linkage, removing the need for identifiable data to leave any organization Fig.\u00a0B,D,F. SuLinkage is substantially harder between organizations that do not share a common identifier. If the only shared identifiers are non-unique ones such as names, DOBs, or addresses, then linkage must deal with the possibility of non-unique matches. If errors are present, the process becomes harder.Conceptually, probabilistic linkage systems  address Probability-based techniques centre around Bayes\u2019 theorem . For a gFor practical use, probability or a related quantity must be converted to a categorical decision about linkage. In the Fellegi\u2013Sunter method , pairs oFor identifiable linkage, exact identifier comparison is trivial, and a range of approaches have been used for fuzzy comparison. These include fuzzy string comparison, such as via the Levenshtein edit distance , 26 or vq-grams of a string), permitting string similarity measures to be obtained from pairs of hashed values themselves, and thus inexact comparison in the de-identified domain , whether the candidate is  or  does not matter: both would be treated as giving A\u2013A and B\u2013B matches.P(D | \u00acH) may require correction in some circumstances. Corrections are not required if multiple comparisons are not in fact made. If the proband has a single identifier [A] and the candidate also has a single identifier, then we will declare a match if the candidate is A, an event whose probability for a random candidate is P(D | \u00acH)\u2009=\u2009q\u2009=\u20091/26, and this situation requires no further correction.However, n\u2009=\u20091, m\u2009=\u20092), a match will be declared when the candidate is  or . We would therefore declare a match with a random candidate  with probability 1/26\u2009+\u20091/26\u2009\u2212\u20091/262\u2009=\u20092q\u2009\u2212\u2009q2. The subtracted component is for a candidate , who would otherwise be counted twice. Generalizing, for n\u2009=\u20091, the probability of a random candidate matching is 1\u2009\u2212\u2009(1\u2009\u2212\u2009q)m, because the non-match probability for each candidate identifier is (1\u2009\u2212\u2009q) and it takes m individual non-matches to achieve an overall failure to match. By the Bonferroni approximation, this is approximately mq, and never more, so mq is a slightly conservative correction for multiple comparisons. We used the Bonferroni correction rather than the accurate value because this is substantially easier to implement in a cumulative log odds system with varying q. Since an uncorrected comparison would give P(D | \u00acH)\u2009=\u2009q, the correction can be implemented by adding\u2009\u2212\u2009ln(m) to the cumulative log odds.If the proband is [A] and our candidate has two identifiers q2, and so on. We implement this by adding\u2009\u2212\u2009ln(m\u22c5[m\u2009\u2212\u20091]\u22c5\u2026) to the cumulative log odds.Generalizing further to multi-named probands, we can work sequentially: the first proband name is matched by the candidate with approximate probability No correction is required for \u201cnon-match\u201d comparisons, however, since no \u201cfishing\u201d for the correct order is then needed. This process is summarized in Table If the order of the comparisons is significant (e.g. forenames), we modify the process slightly. If only one comparison could be made, no correction was necessary. Otherwise we proceed as follows.P(D | H), we define po as the probability of an ordered match (given H) if there is more than one possible comparison ordering, and its converse pu\u2009=\u20091\u2009\u2212\u2009po, the probability of an unordered match in these circumstances, which can be thought of as the probability of a recording error that alters the sequencing of these identifiers. We ranked potential pairwise matches and picked the most likely as before. If that best set of matches reflected strict ordering, such that the proband\u2019s and candidate\u2019s identifier indices were identical for all pairs (e.g. P1\u2013C1 and P2\u2013C2), we weighted P(D | H) by po, by adding ln(po) to the cumulative log odds. There was only one way in which an ordered match could be achieved. Otherwise, if any matches occurred (defined as LLR\u2009>\u20090 per comparison), we weighted P(D | H) by pu.For P(D | \u00acH), we did not apply a correction if the matches were strictly ordered, since there is only one way in which such a combination can be achieved. If there was a set of matches that were not strictly ordered, we applied the same correction as for the unordered process (as above), subtracting one (for the strictly ordered option). This process is summarized in Table For We applied this system to the following types of identifier.pfDOB\u2009=\u2009P(same DOB | different person)\u2009=\u20091/b365.25\u22c5 where b is a configurable parameter related to the birth year range\u2014the age range but including deceased people\u2014of the population of interest. The parameter b reflects the intuitive concept that if everyone in the population of interest is aged 42, the chance of an exact DOB (year\u2013month\u2013day) match between two random people is about 1/365, whereas in a population with an evenly distributed age range of 1\u2013100, it is about 1/36500. In populations with an uneven DOB distribution, b may be lower than the full range . In principle, a birthday of 29 February is\u2009~4 times less common than all others, so it may merit special treatment, but for simplicity, and to reduce re-identification risks from marking it as different in de-identified data, we treated it just like all other days. We calculated ppnfDOB and pnDOB accordingly, using the birth year range, the number of months per year, and the mean number of days per month (ppnfDOB\u2009=\u20091/365.25\u2009+\u20091/b30.4375\u22c5\u2009+\u20091/b12\u22c5\u2009\u2212\u20093/b365.25\u22c5\u2009=\u2009b + 631)(16\u22c5/b5844\u22c5). We took error rates pepDOB and penDOB as configurable parameters.DOB is a core personal identifier that always exists (in principle) and should never change. We used a three-state comparison Table . A full pengender\u2009=\u2009P(different gender | same person), and pfgender\u2009=\u2009P(same gender | different person), which depends on the gender in question, as configurable parameters. Not all systems may allow three-state recording and the UK Census did not ask about gender in addition to sex prior to 2021. If gender was absent from the proband or candidate, we did not infer evidence.We used standard genders of F , M , X (other) . Given tA forename is any name that precedes a surname . When comparing an individual name between proband and candidate, we used a four-state comparison Table . Exact mGender and forename are not statistically independent. Forenames vary from those that are very strongly predictive of gender  to those with relatively weak gender associations (e.g. Rowan); the gender association also varies with country of birth (e.g. Andrea). Therefore, we made all forename-related frequencies conditional upon the proband\u2019s gender: for example, for a female proband, the population frequency was that among females.We calculated population frequencies for names, metaphones, and F2C from public US historical baby name frequencies . For a ppoforename and puforename.If applicable, we applied a correction for ordered multi-identifier comparisons Table E, using In this system, if either person had no forename recorded, no evidence was inferred. Optionally, we allowed start/end dates to be associated with any forename. If there was an explicit temporal non-overlap between any two names, they were not compared.We used broadly the same approach for surnames  as for forenames, with two modifications.First, surnames may contain multiple components , which may be recorded variably. To handle this problem, we split each surname into\u2009\u2265\u20091 fragments, by splitting whitespace- or punctuation-separated parts of double-barrelled surnames and standardizing the whole component by removing whitespace/punctuation , and by creating versions with accents removed and transliterated . We maintained a user-configurable list of nonspecific name components to ignore, such as nobiliary particles . Individual fragments were compared using the four-state method described as above. For a given name comparison, when choosing which fragments to compare, we preferred full match\u2009>\u2009metaphone match\u2009>\u2009F2C match\u2009>\u2009no match, and within each category preferred the most informative match . Population frequencies were taken from the fragment that did in fact match; thus, for example, for proband \u201cMozart-Smith\u201d versus candidate \u201cSmith\u201d, the population frequency was taken to be that of the fragment SMITH. We used a large-scale US dataset for surname frequencies , 62, desSecond, we regarded multiple recorded surnames (if present) as representing alternatives, not sequenced names, so we used an unordered multiple-comparisons method Table D. We didpenpostcode. We did not use information about the duration of the period for which a postcode is shared by the proband and sample except to exclude matches when the overlap was zero, as above.This part of the system is specific to the UK, but adaptable in principle to similar systems internationally. UK addresses all have a postcode, which is a 5\u20137-character alphanumeric string. Postcodes have four nested components: area , district, sector, and unit . Postcodes are a relatively weak identifier because people move home, so a postcode mismatch between two databases is not uncommon, but sharing a postcode at a given point in time provides some evidence towards an identity match. We allowed postcodes to be associated with a start date and an end date, though these can be null, e.g. for an unknown start date or an absent end date representing a current postcode. We used a three-state comparison Table , in whicffpostcode\u2009=\u2009postcode population\u2009\u00f7\u2009total UK population, and fppostcode\u2009=\u2009sector population\u2009\u00f7\u2009total UK population. Postcode and sector populations were estimated from national data. Postcodes contribute to larger units called Output Areas (OA) )\u2009\u00f7\u2009(84.3%\u2009\u00d7\u200927.8\u2009\u00d7\u2009106\u2009\u00d7\u20092.4)\u2009=\u20090.0203%. However, empirically, rates were an order of magnitude higher in our healthcare context. In the SystmOne database, for people with valid NHS numbers and postcodes , 2336 (0.382%) had a pseudopostcode (defined as starting ZZ99), 2250 (0.368%) had a ZZ993 sector, and 1232 (0.201%) had a NFA postcode. We used 0.201% as the estimate of ppseudopostcode_unit, and 2250/1232\u2009=\u20091.83 as the estimate of kpseudopostcode.For pseudopostcodes, we estimated frequencies based on national data for homelessness and then measured rates empirically. Pseudopostcodes cover overseas addresses and \u201cunknown\u201d plus \u201cno fixed abode\u201d (NFA) status . ZZ99 3VWe report, using anonymous Structured Query Language (SQL) queries, some empirical estimates of error rates for the linkage between the two largest databases, RiO and SystmOne. Original records were linked by NHS number and counted by discrepancy type . We did not perform NHS number checksum validity checks for these queries, but eliminated test NHS numbers. We also report metaphone match probabilities. Name comparisons in SQL removed spaces and were case-insensitive; name/metaphone comparisons for this purpose via Python also removed punctuation and, for metaphones, accents (due to limitations of the metaphone package), giving rise to very slight discrepancies with SQL-based totals.The error rate priors derived from these analyses represent an example of limited information derived from known match status , whose NHS number was invalid , or without a known DOB . NHS numbers are not a perfect standard for personal identity\u2014for example, overseas nationals requiring emergency healthcare may not yet have one, and people changing gender in the UK may apply for a fresh NHS number \u2014but theyThe following information was extracted from each database separately, for each patient with an NHS number recorded. (1)\u00a0For matching we extracted forename(s), surname, gender, DOB, and all known postcodes with associated start/end dates if available  . Each daThe settings for the de-identification and linkage system are shown in Table We linked databases in pairs, using only de-identified data. For example, we used CDL patients as probands against the RiO data set, and vice versa. We used an additional validation option in our software that included the (encrypted) ID of the leading candidate even if there was no clear winner, to examine the effects of varying match thresholds. We also linked databases to themselves, to estimate best-case matching performance.P(match declared | proband in sample); true negative rate \u2009=\u2009P(no match declared | proband not in sample); false positive rate \u2009=\u2009P(match declared | proband not in sample); and false negative rate \u2009=\u2009P(no match declared | proband in sample). Additionally, we calculated the misidentification rate (MID): P(person misidentified | match declared). Note that this two-stage method differs slightly from some others\u2019 approaches; see Appendix A given proband from the first database is either present or absent in the sample from the second database. The matching system involves both detecting the existence or non-existence of the proband in the sample, and identifying the correct candidate. We examined the first aspect of performance using standard signal detection theory (SDT) methods . We defi\u03b8 and \u03b4 (range 0\u201315 in steps of 1). Sensible defaults are required as end users are likely to require de-identified linkage without the possibility of gold-standard verification. We established values of \u03b8 and \u03b4 that minimized a weighted performance metric WPM\u2009=\u2009FNR\u2009+\u2009wMID\u22c5MID for non-self database comparisons, setting wMID\u2009=\u200920 to reflect a preference for accuracy over comprehensive linkage; we provide these values of \u03b8 and \u03b4 and used them as defaults.We examined the effects on these measures of varying the thresholds We measured the effects of bias according to factors known about the proband, namely birth year; sex/gender ; ethnicity; deprivation centile; diagnostic groupings . We did not use age at first contact with MH services as a predictor, as it was strongly anticorrelated with birth year; compare .For this purpose, we used the RiO database as probands, as it was the best characterized in terms of mental health diagnostic coding and the second best for ethnicity coding (see Results), and the SystmOne database as the sample, since this was the largest and broadest (see Results). We restricted to probands known to be in the sample, and with demographic information categorizable as above (i.e. excluding those with no recorded postcode and thus no deprivation centile known). We used a binary dependent variable of correct linkage (declaring a match and to the correct person), calculated using default thresholds, and predicted it via logistic regression.We established categories of linkage failure reason for the RiO\u2009\u2192\u2009SystmOne pair, as this gives an indication of where our system could most usefully be improved. Using only de-identified data, we examined probands who should have been matched but were not matched at the software\u2019s default thresholds (including misses and misidentifications), and compared each to their corresponding true match (themself) in the sample. This established the proportion of failed linkages in which forenames differed, DOBs differed, and so forth.Random matches on the first two characters of a name (F2C) were commoner than random matches on metaphone. From the US forename/surname frequency data, we found that the probability of two randomly selected people sharing a metaphone was 0.0124 (surname) or 0.00503 (forename); the probability of sharing a metaphone but not a name was 0.000842 (surname) or 0.00259 (forename); and the probability of sharing a metaphone but not a name or F2C was 0.000633 (surname) or 0.00147 (forename). Similarly, the probability of sharing a F2C was 0.0221 (surname) or 0.0185 (forename); the probability of sharing a F2C but not a name was 0.0105 (surname) or 0.01602 (forename); and the probability of sharing a F2C but not a name or metaphone was 0.0103 (surname) or 0.0149 (forename).  These data contributed to the decision to use metaphone as the more specific first partial match for names, and F2C as the less specific second partial match.pfDOB\u2009=\u20099.03\u2009\u00d7\u200910\u22125, equivalent to b\u2009=\u200930.3; we therefore used b\u2009=\u200930 for validation , there was a birth year range from 1906\u20131996, but on Table . The empOther population frequencies used for validation are summarized in Table Subject counts and demographics for all databases are shown in Table n\u2009=\u2009126,904), no DOBs were absent. DOBs, which cannot change veridically, did not match in 0.492% of people. Of DOB mismatches, 93.3% were \u201csingle component\u201d mismatches . Of DOB mismatches, 30.9% were out-by-one errors  and 14.7% were out-by-two errors. Digit transposition errors  accounted for 2.7% of DOB mismatches, and day/month transposition errors (e.g. United States versus UK format errors) accounted for 0.48% of DOB mismatches. Where DOBs did not match, the mean absolute difference in date was 772\u00a0days. Thus, DOB errors were fairly rare and most were accounted for by single-component errors, but we elicited no further dominant category beyond that. We took 0.00459 (93.3%\u2009\u00d7\u20090.492%) as pepdob , while surname mismatches occurred in 6.78% of females  and 2.22% of males , a highly significant difference . We did not analyse genders other than M/F as numbers were very small and there was no consistent matching . We therefore used separate error rates by gender.Other identifiers may change veridically as well as through error. For the same linkage, the primary forename  differed for 2992/126,884 people (2.36%), and surname differed for 6166/126,904 people (4.86%). Both differed for 560/126,884 (0.44%), and forename/surname were different and transposed for 98/126,884 (0.077%). Thus, overall, name discrepancies were relatively common. When restricted to people of M/F gender and whose gender was identically recorded in both databases, forename mismatches occurred in 2.37% of females  and 2.16% of males , a small but significant difference , M 334/53,474 (0.625%), taken as penforename.For forenames, the probability of a metaphone match but not a name match  was F 652/72,948 (0.894%), M 449/53,474 (0.840%), taken as pep1surname as F 402/72,958 (0.551%), M 252/53,484 (0.471%); pep2np1surname as F 276/72,958 (0.378%), M 132/53,484 (0.247%); penforename as F 4140/72,958 (5.67%), M 719/53,484 (1.34%).For surnames, similarly, we took \u03c721\u2009=\u20093.24, p\u2009=\u20090.0719), for those with matching gender. We took the probability across all matched people, 184/96,569 (0.191%), as puforename  and at least one forename in a notional proband (RiO) database. Of these, the proportion deviating from strict name ordering , was F 90/54,480; M 91/41,736 , to reduce the possibility that differences represented a home move, or occasionally a change in the postcode system itself, rather than an error, though this possibility is far from eliminated.  Postcodes differed for 31.0% of these people. Postcode units differed but the sectors matched for 0.97% of people  corner were all optimized at r\u2009=\u2009\u2009+0.0275 to r\u2009=\u2009\u2009+0.0875 across databases.) Males were more likely to be matched correctly. People in the Black or mixed ethnicity groups were more likely to be matched correctly, but people with unknown ethnicity were less likely to be matched correctly, and there was no effect of Asian or \u201cother\u201d ethnicity. People living in more deprived areas were slightly less likely to be matched correctly. The presence of a pseudopostcode had a small simple effect to reduce the likelihood of linkage (by Z test of coefficient), but this effect did not persist over and above all others (by F test with type III sums of squares). The presence of MH ICD-10 codes was associated with a greater likelihood of linkage.Linkage success was associated with several demographic factors Table . Youngern\u2009=\u20093058 comprising misses and misidentifications), DOB matched fully in 94.5%, exhibited a partial match only in 4.09%, and was mismatched in 1.37%. Gender was correct in 97.3%, mismatched in 2.65%, and missing (on at least one side) in 0.0327%. The first name matched in 73.1%, exhibited a metaphone partial match (but no better) in 4.84%, exhibited a F2C partial match (but no better) in 7.13%, was mismatched completely in 14.8%, and was missing in 0.131%. Surnames exhibited a full match in 31.3%, a metaphone partial match in 2.22%, an F2C partial match in 1.77%, a complete mismatch in 64.7%, and there were no missing values. Postcodes exhibited at least one full match in 10.3%, at least one postcode partial match (but no better) in 14.0%, and complete mismatch in 75.0%, with missing values in 0.621%.Among people in the RiO\u2009\u2192\u2009SystmOne pair who were not correctly linked at default thresholds  taking 44\u00a0min.We developed a Bayesian method for linking records relating to people based on personal identifiers  that can deal with a variety of error types and operate either with direct identifiers or de-identified (hashed) versions. We validated this by linking multiple independent but overlapping electronic health records databases in an NHS Trust. Calculated log odds predicted probands\u2019 presence in the sample database well, with AUROC 0.997\u20130.999 for comparisons between different databases. Decision threshold defaults were chosen to penalize misidentification over linkage failure 20-fold. By default, complete DOB mismatches were disallowed for computational efficiency. At these defaults, for comparisons between different databases, the mean probability of a proband being correctly declared to be in the sample was 0.965 (range 0.931\u20130.994), and the misidentification rate was 0.00249 (range 0.00123\u20130.00429). Performance was satisfactory despite the use of an interpreted programming language, with a linkage of 217\u00a0k people to 619\u00a0k people (1.34\u2009\u00d7\u200910The de-identified linkage files contain irreversibly hashed identifiers (e.g. DOB) and irreversibly hashed \u201cfuzzy\u201d versions of identifiers (e.g. year and month of DOB), with accompanying frequencies. Frequency representations are rounded and a floor applied, to avoid a 1:1 mapping between identifier and frequency. If start/end dates are provided (e.g. for postcodes), they are reproduced as plaintext in the output, but these are not direct identifiers. Gender is trivial to attack because gender is a three-state space whose frequencies are public knowledge, but gender by itself is not identifying, even for gender X. Assuming the user does not actively elect to add identifiable information via the \u201cother information\u201d feature , the resulting linkage files contain nothing that would allow a human to re-identify a data subject by inspection.Two re-identification attack methods should be considered. The first is that if the organizations leak their shared secret hash key, an attacker with access to a linkage file could re-identify some aspects by exhaustively hashing the entire space of possible identifiers\u2014such as by hashing all DOBs in a 100-year period, or all names from a public name dictionary. This is a known problem with all such similar schemes, and the standard mitigation is of careful attention to the security of both keys and linkage files.only identity information; even a plaintext version would not reveal, for example, medical information about a person.The second is that weak information is of course provided by the frequencies themselves (e.g. a high-frequency surname is more likely to be SMITH than MOZART) and the combination of multiple such data points might support a sophisticated jigsaw attack. The incorporation of frequency information aims to improve Bayesian accuracy but any frequency information increases vulnerability to cryptographic frequency attack (see Introduction); however, this may often be unimportant when the objective of de-identified linkage is simply to remove direct identifiers rather than to pass strong anonymity tests, such as for linking data in trusted research environments where the data itself is person-level and thus potentially vulnerable to jigsaw attack even without linkage data \u201351. The Linkage was affected by demographic factors in ways that were generally as expected Table . Males wYounger people (those with greater birth year) were slightly less likely to be matched correctly. This might reflect a number of potential causes, such as forename frequencies not matching our reference public dataset, or an association between age and accuracy of recording. It did not appear to be due to an \u201caccumulation\u201d of postcodes (and thus points of identification) with age, as the number of postcodes was positively, not negatively, correlated with birth year. Previous work with children  found vaPeople in the Black or mixed ethnicity groups were more likely to be matched correctly than those of white ethnicity, but people with unknown ethnicity were less likely to be matched correctly, and there was no effect of Asian or \u201cother\u201d ethnicity. These effects are quite similar to those seen by Downs et al.  prior toThe association of worse linkage with pseudopostcodes is expected, as pseudopostcodes may indicate homelessness or visitor status. Both might be associated with fewer identifier verification opportunities, and the single national NFA pseudopostcode is a much less informative identifier than a true postcode (being shared by many more people). People living in more deprived areas were less likely to be matched correctly, over and above any pseudopostcode effect, which does not match some previous work finding better linkage in the most deprived quartile , though The association of MH diagnostic codes with better linkage is plausible, not least because more contact with MH services increases both the chance of diagnostic coding and the chance of accurate and complete recording of other details. Since SMI is associated with homelessness \u201394, it iOur work was motivated by the need to link health data to external data sources, and an excellent demonstration of this is by Downs et al. , who linThere have been many linkage approaches in the non-Bayesian domain, including deterministic (rule-based) approaches , systemsp\u2009=\u20090.5, equivalent to our \u03b8\u2009=\u2009\u03b4\u2009=\u20090), our system achieved a mean TPR of 98.0% (range 95.9%\u201399.6%) and a mean error rate of 0.965% (range 0.259%\u20132.18%). In contrast, performance at our default thresholds was numerically similar . Notably, recent Bayesian linkage work for the large-scale SAIL Databank has used an SQL-based algorithm called MACRAL (Matching Algorithm for Consistent Results in Anonymised Linkage) to achieve sensitivity (TPR)\u2009>\u200994.6% and error rate\u2009<\u20090.2% ; this syThe primary strengths are that we provide a new technique for fully de-identified (or identifiable) linkage based only on common, non-unique personal identifier types, in portable cross-platform open-source software, and show that it has high accuracy. This may permit fully automatic linkage in situations where manual assistance is currently used, and permit fully de-identified linkages between organizations that do not share a common person-unique identifier , improving IG, and requiring slightly less stringent regulatory burdens by eliminating identifiable data from the linkage pipeline. We also examine in detail the predictors of, and reasons for, non-linkage.\u03b8, \u03b4) influence error rates, which users can configure to their own preference regarding the relative costs of false negatives and misidentification, and the system yields an estimate of absolute linkage probability based on locally provided baseline priors. We also provide a full framework with which users can validate performance against their own data where gold-standard comparators are available, as we did in the present study.The primary weakness of fully de-identified linkage is that it is not verifiable intrinsically, only extrinsically. The proportion of probands left unlinked is readily apparent from the results, but how many are false negatives will be unknown; similarly, where people are linked, the proportion that are misidentified will remain unknown. We provide data to gauge how the software\u2019s thresholds , which might have conferred an advantage on that pairing if those discrepancies were not mirrored elsewhere; however, that pairing was not even the one with the best linkage Table . Other pOur system provides user-configurable minimum values for name frequencies and user-configurable rounding for all frequencies. This is a trade-off provided to ensure de-identified representations are less likely to contain unusual or unique values, and to reduce spurious matches for extremely rare names, but both could reduce the accuracy of probability calculations. We report empirical validated accuracy at default settings. Security considerations around frequency information are discussed above.n-grams [Not all categories of error were explicitly represented. We attempted to model, and thus improve linkage despite, a number of \u201cincomplete\u201d error types, including name alterations , DOB component alterations, and incomplete postcode errors, as well as complete mismatches in all identifiers. However, there were types of error we did not model (e.g. forename/surname transposition), and fuzzy representations that we did not use. Phonetic approximations, like the metaphones used here, are known to be worse for approximate matching than n-grams , but havn-grams \u201336. HoweWe prohibited \u201ccomplete\u201d DOB mismatches during validation, which prevented some linkages being made\u20140.033% of people in the RiO/SystmOne pair had a DOB mismatch that would have been eliminated by this pre-filtering , so this is one cap on current performance. To link large databases whilst permitting complete DOB mismatches is certainly possible using the current system, but re-implementation in a compiled language might be desirable for better performance .Postcodes are of course a UK-specific address abstraction, but the principles of the system are readily adaptable to similar coding systems internationally, and we make our code freely available for use and adaptation.Fully de-identified matching with high accuracy is feasible and practical, even without a person-unique identifier and despite real-world errors and variations in data recording. We hope this method will facilitate fully automated linkage, subject to ethical and regulatory approvals and appropriate security measures, between organizations that do not share a common person-unique identifier, and do so in a way that is safer and more acceptable from an IG perspective than transient sharing of direct identifiers. A hybrid approach incorporating deterministic matching using person-unique identifiers, where available, would be expected to increase accuracy still further, and this is supported by our system. The software we developed to perform these tasks is freely available.Additional file 1."}
+{"text": "The early effects of progressive collapsing foot deformity (PCFD) on the ankle and syndesmotic joints have not been three-dimensionally quantified. This case-control study focused on using weight bearing CT (WBCT) distance (DM) and coverage maps (CM) and volumetric measurements as 3D radiological markers to objectively characterize early effects of PCFD on the ankle and syndesmotic joints. Seventeen consecutive patients with symptomatic stage I flexible PCFD and 20 matched controls that underwent foot/ankle WBCT were included. Three-dimensional DM and CM of the ankle and syndesmotic joints, as well volumetric assessment of the distal tibiofibular syndesmosis was performed as possible WBCT markers of early PCFD. Measurements were compared between PCFD and controls. Significant overall reductions in syndesmotic incisura distances were observed in PCFD patients when compared to controls, with no difference in the overall syndesmotic incisura volume at 1, 3, 5 and 10\u00a0cm proximally to the ankle joint. CMs showed significantly decreased articular coverage of the anterior regions of the tibiotalar joint as well as medial/lateral ankle joint gutters in PCFD patients. This study showed syndesmotic narrowing and decreased articular coverage of the anterior aspect of the ankle gutters and talar dome in stage I PCFD patients when compared to controls. These findings are consistent with early plantarflexion of the talus within the ankle Mortise, and absence of true syndesmotic overload in early PCFD, and support DM and CM as early 3D PCFD radiological markers. Subluxation of the foot underneath the talus, or Peritalar Subluxation (PTS)8, describes the hindfoot component of the deformity with the foot progressively dorsiflexing, rotating externally, and abducting under the talus. It is thought that changes resulting from PTS in PCFD can cause stresses on lateral ligamentous supports and lead to ankle involvement and arthritis11. Described markers of PTS severity in PCFD patients are the presence of sinus tarsi impingement , subtalar joint subluxation, and subfibular impingement 20. These markers were initially assessed on isolated single coronal plane WBCT images21.The assessment of the overall three-dimensional (3D) deformity in patients with Progressive Collapsing Foot Deformity (PCFD)11. They concluded that increased stresses in the lateral aspect of the ankle/hindfoot resulting from PTS may lead to this widening, especially in the more chronic and severe deformities. However, their study again relied on isolated single axial plane WBCT images neglecting true out-of-plane 3D assessment21.Examining the impact of PTS directly on the syndesmosis, Auch and colleagues recently called the attention to significant syndesmotic widening and possible syndesmotic instability in PCFD patients secondary to chronic and progressive PTS23. Dibbern and colleagues implemented DM to evaluate intra- and extra-articular interactions between the talus and the calcaneus across the entire peritalar surface in a case-control study of PCFD patients and healthy controls24. In that study, they introduced the concept of coverage maps (CM), where utilizing the calculated DM between talus and calcaneus, colored maps were generated demonstrating the amount of normal interaction and coverage of the articular facets of the subtalar joint as well as abnormally increased coverage and impingement of extra-articular peritalar regions, more specifically in the sinus tarsi and subfibular areas. Later, Behrens et al. applied the same technique to the Chopart joints, demonstrating a plantarmedial coverage decrease in PCFD patients25.More recently, the calculation of 3D distance maps (DM) assessing the relative position between two opposing articular surfaces (joint interaction) was validated in the foot and ankle11.To the authors knowledge, a similar 3D investigation of joint interaction utilizing DM to analyze the condition of the ankle and syndesmotic joints in PCFD has not been performed. Therefore, we sought to use 3D DM and CM, as well as volumetric measurements to more precisely and objectively characterize the effects of PCFD on the tibiotalar, distal tibiofibular, and ankle joint medial and lateral gutters under normal standing weightbearing conditions in early stage I (flexible) PCFD deformities. We hypothesized that compared to controls, PCFD patients would have significantly reduced anterior talar dome articular coverage due to plantarflexion of the talus, as well as increased syndesmotic volumes and distances due to chronic syndesmotic overloadThis case-control study obtained institutional review board approval (IRB #201904825), complied with the Health Insurance Portability and Accountability Act (HIPAA) and the Declaration of Helsinki. The patients signed the informed consent.1. Patients with class E deformity  were not included. Control patients were selected from our WBCT imaging dataset including healthy volunteers and patients that underwent WBCT imaging for non-related acute and chronic foot and ankle pathologies, with Foot and Ankle Offset (FAO) values of normality (\u2212\u20090.6 to 5.2%)26, and with no history of PCFD, midfoot arthritis or hallux valgus deformity  and the center of the talus, the percentage of the talar deviation from the tripod was given as the FAO27.A retrospective review of patient data collected between 2014 and 2020 was conducted to identify patients with clinical and radiographic diagnosis of PCFD who underwent a standard of care WBCT examination. Inclusion criteria for PCFD patients were age of 18\u00a0years or older, no history of prior surgical treatment in the assessed foot, complete foot, and ankle WBCT scan, flexible (PCFD Stage I) deformity,  and no valgus tilting of the ankle joint1 were selected for the study. Stage I flexible PCFD patients were selected to represent an earlier subset of deformity, allowing assessment of earlier and potentially progressive changes associated with PCFD. Twenty control patients with bilateral WBCT foot and ankle imaging were matched to have similar distributions of age, sex, and BMI . Participants were instructed to bear weight in a natural, upright standing position with the feet approximately at shoulder width and to distribute body weight evenly between the two lower limbs.24.Volumetric measurements of the distal tibiofibular syndesmosis at the first 1\u00a0cm, 3\u00a0cm, 5\u00a0cm, and 10\u00a0cm from the tibiotalar joint  were performed, as well as the volume of the medial and lateral ankle joint gutters . Custom Matlab  code was used to automatically obtain volumes for these regions. This code first identified interfacing surfaces using the normal projections to identify opposed regions of the tibia and fibula, tibia and talus, and fibula and talus to measure volume in the syndesmosis and medial and lateral gutter, respectively. Then faces within a 30\u00b0 tolerance of each other were selected to remove outliers. This defined opposed sets of points and faces on the surfaces to be enclosed to measure volume. To enclose the region between the surfaces with concave alpha shapes, points needed be filled between them at an interval less than the alpha radius. Therefore, the averaged surface normals between the bones were used to project a series of points at these intervals between the opposed surfaces. Averaged normals were used to ensure smooth boundary edges that did not include projections outside of the incisura. Points were then wrapped by a watertight concave alpha-shape using Matlab\u2019s built in alphaShape function and an initial alpha radius of 3\u00a0mm. Bone models were compared to the alpha-shapes to ensure there were no residual convexities enclosing points inside the bone. If convexities enclosing bone existed, triangular edge subdivision was used to increase mesh density and the alpha radius and point projection were iteratively halved before being rewrapped to remove them. The 1, 3, 5, and 10\u00a0cm subdivisions of the syndesmosis were achieved by identifying the most distal point on the tibial syndesmotic interface and selecting the subset of interfacing points superiorly at those intervals using the scanners coordinate system. The resulting watertight volumes for the medial and lateral gutter and syndesmosis were then measured . Distance measurements were made based on a previously published protocol28.DMs were colored to highlight regions of interest Fig.\u00a0. Red/Ora24, aiming to better depict and highlight areas of adequate joint interaction, joint subluxation, and extra-articular impingement25. Pink was chosen to highlight uncoverage of articular regions that were either completely uncovered or had distances greater than 5\u00a0mm  were found to be normal, between 0.5 (no impingement) and 5\u00a0mm. The 5\u00a0mm upper threshold was chosen based on prior literature that demonstrated cartilage thickness and bone to bone distances rarely exceed 3\u00a0mm in the tibiotalar joint28. Finally, gray was used to indicate a shadow from the tibia and fibula in non-articular regions of the talus, where DMs were greater than 5\u00a0mm.Colored CMs were also created using the measured DMs and following previously published methodologyP values of 0.05 or lower were considered significant.Descriptive statistics of means, standard deviation, and range were reported for the DM, CM and volumetric data. Raw continuous data was initially checked for normality using the Shapiro\u2013Wilk test. Two-tailed independent samples Student t-tests or Wilcoxon Tests were used to assess differences between control and PCFD groups, depending on the normality of their distributions. Each region in the talar dome, ankle gutters and syndesmotic space was considered independent and compared in isolation between PCFD and controls. Statistical analysis was performed by an independent observer using a dedicated software . 11. The sample of 17 and 20 patients for each group was found to provide a 98.8% power based on mean syndesmosis distances at 1\u00a0cm and a 93.9% power based on anterior talar uncoverage.A post-hoc analyses power was executed using G*Power . The calculation was based on WBCT syndesmosis widening values from a prior studyUniversity Ethics Committee approved this research under the number 201904825 in accordance with the Declaration of Helsinki.The author affirms that this manuscript is an accurate, honest, and transparent account of the study reported; that no important aspects of the study were omitted; and that any discrepancies from the study as planned  were carefully explained.P\u2009=\u20090.87), and BMI (P\u2009=\u20090.98) distributions, though there were more males in the control group (Table\u00a0There were no significant differences in patient characteristics between the PCFD and control groups with respect to age (up Table\u00a0. A summap\u2009=\u20090.012), by an average of 30.3%, with no difference observed in the lateral gutter . However, significantly smaller medial gutter volumes were detected in PCFD patients when compared to controls p\u2009=\u20090.01, by an ap values 0.009 and 0.017, respectively) and 10\u00a0cm proximally to the ankle joint . These findings can be visualized on Fig.\u00a0When assessing syndesmotic distances Table\u00a0, we obsep\u2009=\u20090.021), and no significant differences in the medial gutter.When evaluating the mean distances in each region of the tibiotalar joint, there were no significant differences in any of the nine regions assessed Table\u00a0, Fig.\u00a05.p\u2009=\u20090.01) and 25.2% (p\u2009=\u20090.021) in PCFD when compared to controls. Similarly, the anterior regions of both the medial and lateral gutters in the talus showed reduction in articular coverage in PCFD patients, with mean reductions of respectively 33% (p\u2009=\u20090.006) and 40.1% (p\u2009=\u20090.013). No differences were observed in the posterior aspect of the gutters. Conversely, there were significant increases in the articular coverage of all three posterior tibiotalar joint zones  as well as the central medial tibiotalar zone . Comparison of CMs for all PCFD and control patients are visualized in Fig.\u00a0When calculating the CMs however Figs.\u00a0, Table\u00a0530. This talar malpositioning is a known phenomenon in the pathogenesis of PCFD, a true manifestation of PTS31. As the foot moves in external rotation under and distal to the talus, the talar bone assumes a internal rotation and plantarflexed position33. The talar head becomes uncovered and some of the collapse is established25. Reduced outward forces from the talus on the syndesmosis in this position can also be expected potentially explaining the decreased syndesmotic distances that contrasted with our hypothesis and previous findings of widening. These changes were observed despite a notable absence of any decreases in tibiotalar joint distance measures across all regions in the articulation.To the author\u2019s knowledge, this is the first study to investigate the use of three-dimensional distance maps, coverage maps, and volumetric measurements of the ankle and distal tibiofibular syndesmotic joints as WBCT-based markers for PCFD. In accordance with our hypothesis, joint coverage analysis revealed significant and prominent respective decreases and increases in joint coverage of the anterior and posterior aspects of the talar dome articular surface in PCFD patients consistent with plantarflexion of the talus inside the mortise. Plantarflexion of the talus can also explain the larger averaged distances observed in the posterior aspect of the lateral gutter and decreased joint coverage in the anterior aspect of the medial and lateral gutters of the ankle joint as talar plantarflexion places the narrower posterior talus in articulation with the distal fibula and medial malleolus35. Syndesmotic widening has also been recently demonstrated in PCFD patients in a case-control study with 62 symptomatic PCFD and 29 controls where the authors measured the distal tibiofibular syndesmotic incisura area in a single axial WBCT image, 1\u00a0cm proximally to the ankle joint11. An explanation for syndesmotic widening and possible syndesmotic instability in PCFD patients would be related to persistent and progressively increased stresses in the lateral aspect of the tibiotalar, talofibular and subtalar joints secondary to PTS, sinus tarsi and subfibular impingements, as well as possible valgus tilting of the talus under the mortise39. The same authors demonstrated that although there was syndesmotic widening in PCFD patients, the correlation between deformity severity, measured by the Foot and Ankle Offset (FAO), and syndesmotic widening was weak11. They also demonstrated that the widening was more pronounced when FAO values were between 7 and 9.3%, a range that can be considered as moderate to severe collapse and hindfoot valgus in PCFD patients, since values of FAO in healthy non-PCFD patients being reported as normal when up to 5.2%26. The assessments performed in our study should be considered as a more complete and 3D evaluation when compared to Auch et al.11 since this study measured distance and volumes throughout the entire imaged extension of the distal tibiofibular syndesmosis, up to 10\u00a0cm proximal to the ankle joint, rather than a 2D area assessment performed in a single axial plane image. In our assessment, we did not confirm the initially expected syndesmotic widening observed by previous authors. Conversely, we identified that overall syndesmotic distances were smaller in PCFD patients when compared to controls. A possible explanation for this could be that as the talus plantarflexes inside the mortise, likely secondarily to a loss of bony and ligamentous support related to the PTS, the thinner posterior part of the talus moves to interface with the mortise, explaining the increased averaged distances in the posterior aspect of the medial and lateral gutters of the ankle joint. This same talar plantarflexion motion would also allow the distal fibula to rotate internally and displace medially42, decreasing width of the mortise and potentially explaining the overall smaller averaged syndesmotic incisura distances observed in PCFD patients when compared to controls. Another possible explanation for decreased syndesmotic distances in our cohort of PCFD patients would be that our cohort, composed of patients with early and less pronounced deformity, was not severe enough to be consistent with chronic lateral impingement and increased syndesmotic overload. In that hypothetical scenario, the resultant effects associated with talar plantarflexion could occur earlier, bringing the narrower part of the talar dome into the mortise, unloading the syndesmosis, allowing distal fibular internal rotation and subsequent decrease in the overall distances between the distal tibia and fibula. The anterior talofibular ligament could exaggerate this movement pulling the fibula along with the talus into internal rotation. Taken together, our results suggest that syndesmotic widening may be a symptom of more severe later stage PCFD, occurring secondary to chronic lateral stresses associated with sinus tarsi and primarily subfibular impingement18. It also suggests that subfibular narrowing/impingement may provide an early marker for subsequent progression to increased syndesmotic overload, widening and instability. However, additional research is needed to confirm these possibilities.Volumetric syndesmotic incisura measurements have been shown to be accurate in diagnosing traumatic syndesmotic instability44. However, the 3D CMs assessment of relative positioning between talus and distal tibia revealed significantly increased joint uncoverage in the anterior aspect of the talus and increased coverage of the posterior talar dome. These findings can be explained by increased plantarflexion and possible slight anterior translation of the talus relative to the tibia in patients with PCFD46. Our hope and understanding is that 3D DM assessment in PCFD can serve as a better WBCT-based radiologic marker for initial and subtle deformities in PCFD when compared to conventional angular assessments of severity of longitudinal medial arch collapse and talus plantarflexion, such as of talar position relative to the distal tibia, calcaneus, and first metatarsal. PCFD measurements that include assessment of the longitudinal axis of the talus have been shown to not to have high reliability and reproducibility due to challenges and inconsistency in reproducing accurate measurements47; that could hinder gauging and recognition of mild, initial, and earlier deformities. Thus, the 3D DMs could represent a more complete, multiplanar and accurate assessment that may provide early identification of previously underappreciated tibiotalar joint changes in early stages of PCFD. Additional longitudinal and prospective studies with different severities of PCFD would help to confirm these possibilities. It is also our hope that through objective quantification using 3D DM and CM we will be able to identify earlier the influences of PCFD in the ankle and syndesmotic joints, potentially preventing late complications such as deltoid ligament insufficiency, syndesmotic instability as well as ankle arthritis. Our findings of significantly decreased articular coverage in the anterior aspects of both the medial and lateral gutters of PCFD patients also support the relatively plantarflexed and possibly anteriorly translated position of the talus in relation to the distal tibia, highlighting the accuracy and potential clinical applicability of 3D CMs as radiologic markers of early changes in PCFD.As previously mentioned, the distance maps of all nine talar dome zones did not show any differences between PCFD and control patients. This indicates that our PCFD cohort had not yet developed any important arthritic changes (narrowing) or valgus talar tilting (widening) in the tibiotalar joint. It is well accepted that these are usually late findings in more severe PCFD49. However, the correlations seen were relatively weak (r\u2009=\u2009\u2009\u2212\u20090.29) and based on a single 2D angular measure of a complex 3D correction. Thus, the regional 3D measures developed herein may provide a foundation for more objective and direct detection of early and subtle malpositioning of the ankle and syndesmotic joints in PCFD patients. They have the potential to extract meaningful information beyond simple plantarflexion performing localized assessments of subregional impingement and widening that may be used to identify more direct correlates of pain generation. Such detailed assessments could enhance precision and enable novel objective analyses of deformity correction following conservative or surgical treatment.The possible clinical applicability of 3D DMs and CMs as earlier WBCT-based radiologic markers of PCFD is supported by considering the results of a recent study. Conti et al. demonstrated that improvements in talar plantarflexion as measured by Meary\u2019s angle  following PCFD reconstructive surgery were associated with significant improvements in patient reported outcomes indicating that some aspects of plantarflexion are associated with pain generation50. Future studies should optimally seek to normalize these measures to leg length or subject height to account for variability in size. Third, only stage I flexible PCFD patients were evaluated. Future work should seek to study the full range of PCFD including stages I and II, as well as to consider the possible specific findings in the ankle and syndesmotic joints for the different classes of the deformity . The lack of formal assessment of deformity severity measurements such as FAO, PTS markers such as subtalar joint subluxation, and presence of sinus and/or subfibular impingement also limit the interpretation of our results. Finally, although a formal power analysis was performed demonstrating sufficient strength, our sample could be underpowered to demonstrate population normality values. However, given the significant findings observed mainly on coverage maps for the tibiotalar joint and ankle gutters, we believe the size of our cohort was robust enough to achieve our objective of examining the utility of CMs as radiologic markers of PCFD.There are several limitations to this study. First, controls were primarily selected from a group of patients with contralateral foot and ankle injuries and deformities. Therefore, subtle asymmetries resulting from antalgic stance may confound our results. We acknowledge that a direct matching process with healthy normal volunteer controls would improve the quality of data collected. However, we believe it would be unlikely to have a substantial impact on DM, CM, or volumetric measurement findings. Second, the volumetric measurements may be impacted by subject size. Our study wanted to enable comparison to syndesmotic injury literature and therefore measured at 1, 3, 5, and 10\u00a0cm from the from the tibiotalar joint as reported previouslyIn conclusion, in this study with PCFD patients and controls, we utilized 3D WBCT distance and coverage maps as well as volumetric measurements to assess joint interaction within the ankle and distal tibiofibular syndesmotic joints. We observed significant decreases in joint coverage of the anterior aspect of the tibiotalar joint, medial and lateral ankle gutters, consistent with early plantarflexion of the talus within the ankle mortise in PCFD patients. Contrary to our hypothesis, syndesmotic volumes were not different when comparing PCFD patients and controls, and the averaged syndesmotic incisura distances were actually significantly smaller in PCFD patients than in controls. These findings, consistent with an absence of syndesmotic widening in PCFD patients, could be explained by talar plantarflexion and resultant internal rotation and medial translation of the fibula. In the presence of peritalar subluxation and syndesmotic stressors like sinus tarsi and particularly subfibular impingement, they may provide important contextual clues about staging and potentially imminent progression. It is our hope that the novel 3D WBCT distance and coverage mapping assessment can enable this kind of early and accurate quantification of the initial and progressive changes in tibiotalar and tibiofibular positioning as well as assisting with treatment decision-making. Further studies are needed to confirm these findings in a prospective and more complete cohort, representing the myriad of different deformities and stages for PCFD, as well as assessing the correlation of these WBCT deformity markers with patient outcomes."}
+{"text": "Research with administrative records involves the challenge of limited information in any single data source to answer policy-related questions. Record linkage provides researchers with a tool to supplement administrative datasets with other information about the same people when identified in separate sources as matched pairs. Several solutions are available for undertaking record linkage, producing linkage keys for merging data sources for positively matched pairs of records. In the current manuscript, we demonstrate a new application of the Python RecordLinkage package to family-based record linkages with machine learning algorithms for probability scoring, which we call probabilistic record linkage for families (PRLF). First, a simulation of administrative records identifies PRLF accuracy with variations in match and data degradation percentages. Accuracy is largely influenced by degradation  compared to the percentage of simulated matches. Second, an application of data linkage is presented to compare regression model estimate performance across three record linkage solutions . Our findings indicate that all three solutions, when optimized, provide similar results for researchers. Strengths of our process, such as the use of ensemble methods, to improve match accuracy are discussed. We then identify caveats of record linkage in the context of administrative data. Record linkage, also known as entity resolution, is the process of identifying records across unique datasets that capture information for the same entity. In the context of research, record linkage has been applied to merge administrative data to address research questions related to social sciences and public policy \u20133. TheseRecord linkage can be applied using deterministic  or probabilistic  methods. Given the nature of administrative data, which may include missing, incomplete, or inaccurate values for fields , there is an advantage to implementing probabilistic linkage methods, and studies have shown data simulated to mimic real linkage parameters are more accurately linked with probabilistic methods than deterministic methods . AdditioThe key steps in probabilistic record linkage are: (1) standardizing and cleaning data; (2) blocking; (3) generating a comparison feature matrix; and (4) assigning match probability. The standardization and cleaning step ensures that all columns are comparable between the two datasets in the linkage by performing transformations such as removing special characters from fields, capitalizing letters in name and address fields, and setting invalid data to blanks . The blocking step reduces all possible record pairs between the two datasets in the linkage to only the most likely pairs, which can be implemented via hard rules , fuzzy matching , or both. The next step performs pairwise comparisons between the blocked pairs, exporting the results in the form of a Boolean matrix, where a row represents a pair to be compared and a column represents the comparison . Based on the results of this matrix, a match probability is assigned to each row via human review or machine algorithm and a final decision is made on whether the pair is a match. Further reading on probabilistic record linkage can be found in Christen , Sayers Link Plus, developed by the Centers for Disease Control and Prevention , is a maChoiceMaker  is a fulOther solutions such as Link King , Dedupe https://recordlinkage.readthedocs.io/). PRLF performs data cleaning, blocking, feature extraction, model training, and match probability assignment while also generating optimized models for scoring based on various algorithms of interest and training data supplied. All components of PRLF are accessible via a single install, and although it has default settings that allow users to perform linkages \u201cout of the box,\u201d users easily can control parameters at each step of the linkage process to fit their needs with minimal editing of Python code. Further, PRLF is agnostic to system software and hardware and can be run on Windows, Mac OS, and Linux machines of varying hardware capabilities. A detailed process flow and available user customizations can be found in PRLF was developed to allow end users more control over the linkage task while automating as many steps as possible. The solution is an automated, Python-based, end-to-end linkage pipeline built on the open-source RecordLinkage package  to estimate match probabilities, which allows for the creation of ensembles of probability scores for further scrutiny to identify matched pairs. Thus, PRLF builds on the combined strengths of current linkage solutions while addressing their gaps. A comparison of PRLF with other linkage solutions is displayed in In the current study, we compared several known probabilistic record linkage solutions and PRLF, a novel record linkage solution designed to provide researchers with greater control over characteristics dictating linkage performance. We conducted comparisons using two methods: simulation and applied analysis.First, we ran PRLF to link sets of simulated data with known parameters to provide insight into the performance of the linkage solution. Our simulation analysis was guided by several questions: (1) How well does blocking correctly document true matches? (2) Are blocking accuracy and block size associated with dataset size, percentage of overlap, and percentage of rows with typos introduced? (3) What is the proportion of matches identified in the blocked pairs and how does it compare to the total match pairs? (4) What is the proportion of false positives?Next, we compared the linkage results of PRLF to other linkage solutions using real-world administrative data. For our applied analysis, we sought to address the following questions: (1) Does PRLF, with trained algorithms, perform similarly to other linkage solutions? (2) Does varying the PRLF algorithm threshold for identifying a match pair affect analyses based on linkages? (3) Is it possible to establish recommended thresholds that researchers can use as a starting point and modify based on desired accuracy?https://faker.readthedocs.io/en/master/index.html). The birthdate field was created using a random number generator, ensuring all birthdays were in 2016. The state field was set to \u201cCalifornia\u201d for all individuals, and the ZIP code field was populated using random selection (with replacement) from all valid California ZIP codes (performed once per row). The dataset sizes included 10,000, 50,000, and 100,000 rows. The row percentage overlap parameter values were 1%, 3%, 5%, 10%, 20%, 40%, and 50%. The percentage error parameter values were 0%, 2%, 5%, 10%, 15%, 20%, 30%, and 40%. In total, 168 pairs of datasets were created, one pair for each parameter.The data created through simulation were designed to mimic data observed in records from California  . SimulatAll simulated pairs underwent the \u201cout of the box\u201d PRLF processes  for dataComparison of model performance was done with two main simulation parameters, overlap between dataset pairs and percentage of rows with administrative errors. Several metrics for exploring the resulting linkages from simulated data were presented from a typical confusion matrix. That is, pairs identified as matches (positives) and nonmatches (negatives) through model scoring were identified as true or false based on the data simulation procedure. We calculated the sensitivity, specificity, precision, and F1 score for each condition in the simulation grid. We additionally calculated metrics of blocking performance .N = 487,853). These data were linked to child protection service (CPS) records from the California Department of Social Services from 2016 . Identifiers for both datasets included child names ; date of birth; parent or guardian names  and dates of birth; and complete addresses . These identifiers were used in three linkage solutions: Link Plus, ChoiceMaker, and PRLF. Link Plus and ChoiceMaker have been used for previous analyses with these data [Data from the California Department of Public Health Vital Statistics was obtained for the birth cohort year 2016 . For PRLF, we used the combination of these approaches with birth year, birth month, first name, middle name, and last name meeting a similarity threshold based on TF-IDF to establish a set of blocked record pairs for which to generate comparison features. Pairs were placed into a block if their first names or last names were in the top 3 matches as determined by TF-IDF, with a match probability cutoff of 80% using n-grams of length 4 (excluding leading and trailing spaces). Of these pairs, only those that matched on exact birth month and birth year continued to feature extraction. The extracted features of these blocked pairs were then used to determine a match probability using the XGBoost algorithm. The parameterization for this model is based on a grid search outlined in the simulation data linkage solution. We compared results of the PRLF linkage with varied thresholds for establishing a positive match between data sources to those of the established solutions.Referral for abuse or neglect to CPS (the first stage of contact with CPS) was identified by a positive linkage between the birth cohort and a CPS maltreatment record with a referral within 3 years of the birthdate of birth (yes or no). A 3-year window of observation was used based on data available at the time of data linkage .Birth records offered covariates as a measure of model accuracy using various linkage solutions. Child sex , low birthweight (yes or no) and variables related to the mother: age , race and ethnicity , non-U.S.-born (yes or no), and education . Variables related to the household were also coded: public insurance (yes or no), first trimester prenatal care (yes or no), established paternity (yes or no), and WIC . Later sections show the distribution of these variables in the subset of children for each linkage solution.An initial bivariate comparison of linkage tools tested the equality of group distributions on the outcome  and model covariates from the birth records using chi-square tests. Then, a generalized linear model with a Poisson distribution with a log link function and robust standard errors was used to show the multivariable relationship of the covariates with CPS referral likelihood . To testSimulations were run for the grid of 168 linkages specified in the methodology. Results were replicated with 10,000 and 100,000 rows per dataset pair and provided comparable results, as presented in Training data consisted of 5,771 record pairs of both known matches and nonmatches. These pairs included various prototypes of matches that would provide notably discriminable data for identifying high probability of a positive match . Parameters for each algorithm were selected through a grid search process, and parameters that maximized accuracy were selected. Hyperparameters are specific to the algorithm selected. PRLF includes the capability to train additional classifiers available in SciKit Learn with model-specific grid search parameters and selects the hyperparameters producing the best fit. Examples of other trainable models include logistic regression, random forests, XGBoost, and artificial neural networks. The XGBoost algorithm with match probability threshold set at 80% produced a solution that identified 93.8% of true matches, with 1.4% of nonmatches mislabeled a match. This is similar in performance to ChoiceMaker  and neural networks . Logistic regression  and random forests  exhibited inflated levels of false positives compared to the other algorithms. As a result of these comparisons, the applied data example compared the performance of PRLF with XGBoost to Link Plus and ChoiceMaker.n = 58,377) to CPS before age 3 years, and ChoiceMaker provided a 12.2%  linkage rate with a 80% threshold for match probability as suggested by ChoiceMaker and in line with prior research using current linkage solutions. These two methods overlapped with 97.18% of Link Plus pairs and 95.05% of ChoiceMaker pairs. PRLF using XGBoost identified 12.1%  birth records as a match to a CPS record. When compared to the other solutions, 95.6% of PRLF pairs matched with Link Plus record pairs and 97.2% PRLF pairs matched with ChoiceMaker record pairs. As shown in Records from birth records and CPS records were linked using the three linkage tools: Link Plus, ChoiceMaker, and PRLF with XGBoost. The manual review of Link Plus record pairs yielded a linkage rate of 12.0% of the total birth record population . The PRLF solution allows researchers to adjust these settings and compare results across various linkage assumptions. The applied analysis identified a typical research use case of PRLF\u2014the linkage of a population-based vital records dataset with an administrative dataset and analysis of program interaction for a birth cohort.The simulation analysis identified the various metrics affected by row errors and dataset overlap. Many performance metrics improved as the theoretical overlap between the dataset pairs increased, in line with expectations for well-defined linkage solutions. Accuracy and precision showed the largest improvements, with other metrics stable for a specified proportion of row errors. That is, as the proportion of overlapping records increased between two datasets, the proportion of false positives fell while holding the match threshold constant.Proportion Blocked = 1\u2212row errormean. Two data sources with relatively clean records  would produce a blocked set with nearly all true matched record pairs.Alternatively, the introduction of row errors reduced the ability of blocking to successfully retain true matches between datasets. This is shown in the proportion of matches identified in the blocked data , Panel FResearchers may adjust the threshold for an acceptable match pair between datasets . The applied analysis presented here shows how threshold selection, examined over a large range (40%\u201395%), can be tested in the context of PRLF if other linkage tools have been used. Similarly, the development of PRLF allows for multiple algorithms to be deployed simultaneously. Machine learning encompasses a wide variety of methods for classification problems, and these methods may be suitable across varying types of classification problems and combined to create an overall voting system for detecting classification accuracy \u201327. ThisThere are several limitations to note in our current work. First, the variation in unique name values available in our simulation was lower than the variation encountered in real-world data . This seems to be a product of the data generation functions used, and future work should test how increasing this source of variance affects results. Second, our simulation linkage used person and address information, whereas our applied analysis included guardian information in addition to the fields used in the simulation analysis. The introduction of more fields for comparing record pairs allows for a more nuanced and accurate scoring model based on increasing the number of points of comparison and thus, points where records with more than one match pair can differ.The PRLF record linkage solution encapsulates all aspects of data cleaning, blocking, match probability modeling and scoring, and final linkage production. Our linkage solution, PRLF, is adaptable and allows researchers to adjust each step to best fit their needs. That is, if accuracy is of highest import , then a high threshold for selecting a match should be prioritized. These requirements will vary by use case, and PRLF has many ways to handle a wide range of applications for research making use of administrative data.S1 FigThe inner two columns show the two workflows of PRLF: 1. Assigning match probabilities to potential shared pairs between two datasets, and 2. Generating models based on user-provided training data which can be used as score functions for Workflow 1.(TIF)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.S2 Text(DOCX)Click here for additional data file.S3 Text(DOCX)Click here for additional data file.S4 Text(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file."}
+{"text": "The lithium\u2013sulfur (Li-S) battery is considered to be one of the attractive candidates for breaking the limit of specific energy of lithium-ion batteries and has the potential to conquer the related energy storage market due to its advantages of low-cost, high-energy density, high theoretical specific energy, and environmental friendliness issues. However, the substantial decrease in the performance of Li-S batteries at low temperatures has presented a major barrier to extensive application. To this end, we have introduced the underlying mechanism of Li-S batteries in detail, and further concentrated on the challenges and progress of Li-S batteries working at low temperatures in this review. Additionally, the strategies to improve the low-temperature performance of Li-S batteries have also been summarized from the four perspectives, such as electrolyte, cathode, anode, and diaphragm. This review will provide a critical insight into enhancing the feasibility of Li-S batteries in low-temperature environments and facilitating their commercialization. In the context of the green and environmental protection society, the demand for rechargeable batteries is ever increasing, and the application range is also gradually becoming wider. Meanwhile, it is a significant challenge to break the theoretical bottleneck for commercial lithium-ion batteries (LIBs), consisting of a low-capacity anode and cathode.Moreover, with the in-depth research of Goodenough et al. on lithium\u2013sulfur (Li-S) batteries, such as the invention and development of key cathode materials for lithium-ion batteries, the potential development of lithium-ion batteries is being limited ,2. Li-S 2Sx, 2 < x \u2264 8), the insulation of S and insoluble sulfide (Li2S2 and Li2S) ..2S film The high desolvation energy barrier of the Li ions at low temperature was determined to be the key factor leading to the failure of the battery. When the Li ions are bonded with the solvent molecule, the energy barrier for desolvation substantially increases at the electrochemical interface and further weakens local charge transfer capability . To thisThe uncontrolled nucleation and deposition of lithium have an extremely negative effect on cycle performance. As the temperature decreases, the large-size deposited Li will evolve into dead lithium with an extensive surface, significantly reducing the Coulomb efficiency . Accordi2S4 clusters . T. T\u22121 at e (GRAL) d. Moreovectively .2S cathode and Si anode. The rationally designed CoN@MCNF/Li2S cathode shows good temperature adaptability at around \u221220 \u00b0C . Si. Si81]. 2 Wh L\u22121 .Recently, much endeavor has been paid into the advanced hollow structure of sulfur ,85. A un\u22121 (0.5 \u00b0C) over 100 cycles at \u221220 \u00b0C . L. L4 has f \u221240 \u00b0C . With ref \u221240 \u00b0C . It is rf \u221210 \u00b0C . All in 3 is a popular additive due to its excellent character of dissolving the lithium deposit. Xiao et al. employed LiNO3 as additive for Li-S batteries, showing high Coulombic efficiency over 95% [3 can reduce the nucleation overpotential and achieve excellent cycle stability over 300 h [3 easily dissolve in the electrolyte and efficiently preventing the Li nucleation. Due to its exceptional role, the Li/LiFePO4 with TPPO still performed a stable performance when tested for stability at the low temperature of \u221215 \u00b0C  the uncontrolled lithium nucleation and deposition and low Li+ diffusion rate; (4) the cathode passivation caused by aggregation of LiPSs and Li2S; and (5) Li plating on the surface of the anode, contributing to the short circuits.In short, the advances for low-temperature Li-S batteries have been reviewed, and the challenges have also been proposed: (1) the wettability and ionic conductivity decrease in the electrolyte; (2) the sluggish interfacial reaction kinetics, attributed to the high Li+ diffusion; (3) suppressing Li seeding and dendrites; and (4) other strategies, such as self-heating and/or optimization of battery configuration.To overcome these challenges, a few implementable strategies are proposed: (1) rational tailoring of solvents, lithium salts, and additives to boost low-temperature ionic conductivities, reduce desolvation energy, and form thin, inorganic-rich SEI layers; (2) doping, surface coating, as well as structural design for electrode, etc., to stimulate LiPay more attention to the performance of the low-temperature Li-S batteries with high loading. In the current research, a considerable electrochemical performance has been achieved under a relatively low mass loading. However, it is inevitably necessary to enhance the mass loading when the Li-S batteries are for commercial application.Construct an artificial SEI layer towards cryogenic applications. As ideal SEI layers are difficult to achieve at low temperatures, it may be an effective and reasonable strategy to pretreat the lithium surface at room temperature, constructing artificial SEIs and, thus, improving low-temperature performance prior to low-temperature operation.Further investigate the mechanism for low-temperature Li-S batteries. In contrast to the transformation process of LiPSs at room temperature, aggregation and slow ion transportation at low temperatures can affect the kinetics of sulfur species. It is, therefore, suggested that experimental investigation and theoretical simulation should be combined for future studies. It is of significant importance to explore the critical rate-limiting steps in the transformation of LiPSs at low temperatures.Evaluate the pouch-cell performance. Li-S batteries are gradually being developed from basic research to practical applications. To bridge the gap between laboratory experiments and industrial production, there is a growing body of work to verify the feasibility of the proposed strategies in practical applications. Consequently, in future research and development of low-temperature Li-S batteries, pouch cells should also be fully utilized to assess battery performance.Currently, research on low-temperature Li-S batteries has made significant progress and laid the groundwork for future research. There is, however, still a considerable gap in practical application. Additionally, the advanced prospects for the development of low-temperature Li-S batteries have been proposed:\u22121. Their batteries have shown excellent results in unmanned aerial vehicle applications [As the requirements for flexible batteries, such as the foldable smartphones from Huawei and Samsung, in the current market continue to increase, there is a growing interest in wearable electronic devices . Li-S baications . These r"}
+{"text": "Summary: At Disease Models & Mechanisms, we are prioritising and investing in high-quality scientific figures to ensure that the communication of disease biology is accessible and engaging to all. Images are a cornerstone of scientific communication and are crucial in conveying complex biological concepts and functions. The technical art of biological and medical illustration is not a new concept and has been supporting the dissemination of science for centuries, initially through anatomical diagrams; then, as we explored biology at a molecular level, illustrations displayed more granular biological processes. Whilst modern research articles fully rely on figures to communicate results, authors have much more choice when crafting figures for their review-type articles. Whether this freedom represents an exciting or daunting challenge, it is important to remember that, not only does this imagery act as a visual aid for our understanding of biology, but figures are important to engage readers with varying levels of expertise on the topic. Whether perusing a primary research article or a review, it is likely that one of the first things a reader will do is scan through the figures, debating whether it is worth their time to delve deeper into the text. Well-designed figures can capture their attention and deliver complex scientific information with a clarity that plain text alone cannot achieve. If you're already investing your time in writing, it will be worth investing some more in the figures.1. Structure graphics logically. Each figure tells a story; it might explain a molecular process in a cell, a key disease mechanism or the features of a model system. Keep this story aspect in mind when designing your figure. Use left-to-right/top-to-bottom arrangement so that the information flows logically. The vast majority of readers will look top left first, so this is the recommended starting point of a diagram. Avoid arranging your panels in a circle, unless you are presenting a \u2018spider\u2019 diagram with a clear central focus, or you are showing a cycle.2. Clear hierarchy of the text helps readers perceive the structure of the graphic. Headings and sub-headings should be made clear with larger and/or bold font and this should be consistent throughout the figure. When emphasising any text, bold letters are preferable to underlined, which can be harder to read for people with dyslexia.3. The format of the font, in general, is very important. It should be large enough to read easily and with good contrast, although black on white can be jarring and difficult to read for people with dyslexia so non-white block backgrounds are preferrable.4. Avoid overcrowding your image. Remove unnecessary detail to allow the essential elements to stand out. Do not repeat information that you explained in the text.5. Minimise text \u2013 it's a figure for a reason. Instead of including long passages of text, you can move this text to the figure legend or use concise bullet points. Elements should be labelled clearly in the figure but avoid detail overload. You can include a Key if appropriate but keep in mind that this can create extra work for the reader under certain circumstances. If preparing a Key, use concise labels and adequate sizing of the graphic elements, so they are clearly distinguishable and easy to recognise in the figure.6. Include recognisable \u2018landmarks\u2019. For example, if you are showing a signalling pathway, the addition of the cell and nuclear membranes in the background will help orient the readers without overwhelming the imagery.7. Use colours mindfully. Not every element needs to have its own colour. You can use the same colour to group objects or showcase their relationship, then use different shapes to distinguish between individual elements. Be mindful of colour combinations that may not be accessible to colour-blind readers, such as red-green and blue-yellow.8. Be consistent within each figure and between figures. Elements that are in aesthetic agreement are easier to be perceived as also being in narrative agreement. This will help readers identify parallels between the figures.In review-type articles, which synthesise vast bodies of knowledge, custom schematics aim to translate complex concepts into accessible at-a-glance visual resources. These figures are an integral part of the article and should be planned early on, such that they seamlessly fit into the article's structure. This will ensure the figures complement, not repeat, concepts already explained in the text. It is important to think about whether you should \u2018show\u2019 or \u2018tell\u2019 information and what format would be easiest for the reader to digest. At Disease Models & Mechanisms (DMM), we provide guidelines  to help Despite figures being a pillar of scientific communication, they are often an afterthought when preparing manuscripts for publication. This can often be the case for research articles, although most of the principles outlined for review-type articles also apply to figures in research articles. An analysis of 580 articles, published in the top 15 journals across three fields of biology, showed that as low as 2\u201316% of articles met \u2018good practice criteria\u2019 in all of their figures . Some ofOur goal is to ensure that the crucial scientific information presented in the figures within research- and review-type articles can be appreciated by the broadest possible readership. As a fully Open Access journal, we strive to communicate high-quality basic and translational research in disease biology to a broad audience of scientists, clinicians and patients . The twi"}
+{"text": "Ruminococcus taxa. This study provides valuable insights into the health benefits of HA-HMT rice and supports its potential as a functional food ingredient in the management of obesity and cholesterol-related disorders.The rising prevalence of lifestyle diseases, such as type 2 diabetes, cardiovascular diseases, and metabolic syndrome, has increased the need for effective dietary interventions. This study aimed to evaluate the effects of heat-moisture-treated high-amylose rice (HA-HMT) on body weight, lipid metabolism, and gut microbiome composition in a rat model of obesity. Starch digestibility\u2014specifically, resistant starch\u2014has been shown to provide various health benefits, including improved metabolic health and gut microbiome composition. We employed a sequential approach: firstly, utilizing diet-induced obesity rat models fed with HMT-processed and HMT-non-processed low- or high-amylose rice to investigate the potential of amylose content or HMT to alter phenotypic characteristics and lipid metabolism; and secondly, using the optimal rice flour identified in the previous step to explore the underlying mechanisms. Our findings indicate that heat-moisture treatment, rather than the level of the amylose content of the rice, contributes to the observed anti-obesity and cholesterol-lowering effects. We identified candidate genes contributing to the cholesterol-regulating potential and demonstrated that HMT rice flour could influence the gut microbiome, particularly the  The global prevalence of lifestyle diseases, including type 2 diabetes (T2D), cardiovascular diseases (CVD), and metabolic syndrome, is on the rise and has become the leading cause of death and disability . ImproviStarch is a major component of plant-based foods and the primary carbohydrate in human diets . AbsorptOryza sativa) is a staple food and a significant source of carbohydrates in various cultures. During processing, the micronutrient-rich outer layers are often removed to improve taste. Rice grains that retain the bran and germ layers typically have a brown color and are, therefore, commonly referred to as brown rice (BR), distinct from the outer layer-ground, refined, white-colored rice. Whole grains, such as brown rice, may contribute to better glycemic control and reduced risk of type 2 diabetes when they replace refined grains and simple sugars in the diet, given that the latter are associated with increased risk of these conditions [Rice  is a processing technique that involves the application of heat and moisture to starch-based foods, resulting in alterations to their physicochemical properties and, consequently, their potential health benefits . HMT hasPrevious studies have investigated the impact of HMT on the functional properties of HA rice ; howeverThe present study employed two approaches: the first utilized HMT-processed and HMT-non-processed low- or high-amylose rice and diet-induced obesity rat models to investigate the potential of amylose content or HMT to alter phenotypic characteristics and lipid metabolism; the second used the optimal rice flour identified previously to study the underlying mechanisms. Our findings will not only expand the current understanding of the nutritional and health benefits of HMT but also provide valuable insights for the development of effective dietary strategies to combat obesity and associated metabolic disorders.The rice flour used for the studies was obtained from the National Institute of Technology. The fodder was made using different kinds of rice flour and other components. The nutritional information of the fodder is shown in 3) in a room maintained at 22 \u00b1 2 \u00b0C with a relative humidity of 40\u201360% and a 12 h light (8:00\u201320:00) and 12 h dark (20:00\u20138:00) cycle. The animals were allowed free access to water and food throughout the experiment. After a 1-week adaptation, they were then randomly divided into four groups with the same average weight and four kinds of high-fat diets (HFDs): two diets whose carbohydrate source was rice flour obtained from low-amylose Koshihikari rice species, with or without HMT ; and two diets whose carbohydrate source was rice flour obtained from high-amylose Koshinokaori rice species, with or without HMT . LA-nHMT, LA-HMT, HA-nHMT, and HA-HMT rice flour were obtained from the National Institute of Technology (Kosen), Nagaoka College . The nutritional information of the diet is shown in Twenty-nine male Wistar rats (5-week-old) were purchased from Charles River Japan  and kept to cages  after a 16 h fasting. After euthanization, blood samples were collected from the carotid arteries. The liver, muscle, and abdominal fat pads were harvested and weighed. Plasma was separated from the blood, while the organs and tissues were snap-frozen in liquid nitrogen and stored at \u221280 \u00b0C until use.n = 10); HFD, whose carbohydrate source was rice flour obtained from low-amylose Koshihikari rice species, without HMT ; and HFD, whose carbohydrate source was rice flour obtained from high-amylose Koshinokaori rice species, with HMT . For the HFD and HMT groups, extra cholesterol was added. Group 2 (HFD + 0.25% CHO) was used as a negative control of non-HMT processed diet, while CHO was supplemented. The nutritional information of the diet is shown in Study 2 was conducted to examine the triglyceride- and cholesterol-lowing effects we observed in study 1. Thirty male Wistar rats (5-week-old) were obtained and kept as described in Blood and tissue biochemical parameters were measured according to the manufacturer\u2019s protocol. Briefly, plasma biochemical parameters, including glucose, triglyceride (TG), total cholesterol (T-Cho), and high-density lipoprotein cholesterol (HDL-Cho) concentrations, were determined using commercial kits .v/v) chloroform/methanol. The extracts were incubated overnight at 4 \u00b0C. Further, 0.4 mL of 0.8% KCI solution were added to each sample. After a series of vacuum evaporations, the organic phase was recovered using isopropanol. The recovered samples were tested for the presence of biochemicals. Hepatic TG and T-Cho levels were determined using commercial kits .For hepatic TG and T-Cho, the lipid content was extracted from frozen livers using the modified Folch method . BrieflyFor fecal determination of bile acids, 50 mg of each fecal sample was dissolved in ethanol (0.5 mL) and kept for 2 h at 70 \u00b0C. After centrifugation for 5 min at 10,000 rpm, the supernatants were collected for subsequent analyses. Fecal bile acid was measured using a commercial kit .Total RNA was isolated from homogenized liver tissue using NucleoSpinRNA , according to the manufacturer\u2019s instructions. The total RNA concentration was measured using a NanoDrop spectrophotometer . RNA quality was determined by assessing the A260/280 and A260/230 ratios and performing agarose gel electrophoresis.Twelve rats from Study 2 (four from each treatment group), whose body weights were similar to the average weight of the respective group, were selected. DNA microarray analysis was performed using the Genechip Rat Genome 230 2.0 Array . Data were normalized by using the Robust Multi-array Average (RMA) method in R software, and differentially expressed genes between the groups were identified using the rank products 2.0 method. The probesets were filtered under the condition of a false discovery rate < 0.05. Bioinformatics analysis was performed using Ingenuity Pathway Analysis  to examine the underlying molecular mechanisms. IPA Core Analysis was used to identify the most significant biological canonical pathways in which the molecules in the dataset were involved.Rps18) was used as an internal reference for the normalization of target gene mRNA expression.cDNA was synthesized from the extracted total RNA using PrimeScript RT Master Mix (Takara Bio), according to the manufacturer\u2019s instructions. RT-qPCR was performed using a Thermal Cycler Dice Real Time System TP800 . The expression level of ribosomal protein s18 (The plasma samples of nine rats from Study 2 (three from each treatment group), whose body weights were similar to the average weight of the respective groups, were selected for metabolome analyses. Briefly, samples were filtrated through 5 kDa cut-off filters  to remove macromolecules, and then resuspended in 50 \u03bcL of ultra-pure water using CE-TOFMS immediately before the metabolome analysis. The detected peaks were annotated based on the m/z value and normalized migration time. The relative area value of each peak was calculated and subsequently used for the intergroup comparison.http://www.qiime.org accessed on 20 May 2023) was used to analyze the data against the Greengenes alignment (v gg_13_8).QIAamp Stool Mini Kit  was used to extract DNA from cecal contents, following the manufacturer\u2019s instructions. The variable regions 3 and 4 of the 16S rRNA were amplified using the primers, 5\u2032-CCTACGGGNGGCWGCAG-3\u2032 and 5\u2032-GACTACHVGGGTATCTAATCC-3\u2032, followed by a modification to include the Illumina adapters and barcode sequences for the next sequencing. Library size and quantification analysis were conducted using the Agilent 2100 Bioanalyzer . All libraries were pooled in a single Illumina MiSeq run . The software of Quantitative Insights Into Microbial Ecology (QIIME v1.8.3) . Multiple-group comparisons were performed using two-way (Study 1) and one-way (Study 2) analysis of variance (ANOVA), followed by Tukey\u2019s test using the GraphPad Prism software . Statistical significance was set at a After euthanization, the final body weight, organ, or tissue mass was recorded, and the ratio of organ or tissue mass/final body weight was calculated. As shown in p = 0.05), compared to CON. However, despite the fact that the ratio of liver mass/body weight in the HMT group remained significantly higher than that in the CON group, HMT ameliorated the abnormal increase in total adipose tissue mass and the ratio of adipose tissue mass/body weight caused by HFD. Furthermore, as shown by the weight change curve presented in After euthanization, the final body weight and organ and tissue masses were recorded, and the ratio of organ or tissue mass/final body weight was calculated. As shown in p < 0.001) reduced the plasma concentration of HDL-Cho . As campesterol can compete with cholesterol for absorption and lathosterol is a cholesterol precursor, the above results indicate that less absorption and precursor might partially explain why rats in the HMT group had low circulating and hepatic cholesterol levels.We also conducted metabolome analyses using plasma samples . The camp-value (y-axis) using volcano plots. Employing QIAGEN IPA software, we focused on the comparison between HFD and HMT. The top 10 altered functional categories included several lipid metabolic pathways: triacylglycerol biosynthesis, acetyl-coA biosynthesis III (from citrate), palmitate biosynthesis I , and fatty acid biosynthesis initiation II. Thus, we focused on the top altered genes in these categories, which are listed in Cyp7a1, a gene that encodes cholesterol 7 \u03b1-hydroxylase (CYP7A1), a key enzyme in cholesterol-to-bile acid metabolism, was significantly upregulated in the HMT group compared to in the CON or HFD groups  to 31.3 \u00b1 7.2% (HFD) and 25.0 \u00b1 5.6% (HMT) for Bacteroides, and a significant elevation from 11.9 \u00b1 4.4% (CON) to 21.8 \u00b1 4.6% (HFD) and 21.3 \u00b1 4.1% (HMT) for Parabacteroides. Another noteworthy genus is Blautia, which was significantly downregulated from 14.7 \u00b1 5.5% (CON) to 6.7 \u00b1 2.2% (HFD) and 5.7 \u00b1 1.8% (HMT), respectively. Ruminococcus was 11.4 \u00b1 2.2% in the CON group and decreased to 5.9 \u00b1 3.1% in the HFD group. However, its levels were elevated in the HMT group (17.5 \u00b1 6.4%). Moreover, the HMT group showed area intersection with the HFD group in the principal component analysis (PCA) plot (CA) plot C, indicaAlpha diversity of the bacterial communities was calculated using Shannon index. We found that the Shannon index was significantly higher in the HMT group than that in the CON group. Treatment with HFD decreased alpha diversity indices to some extent compared to the CON group, although the difference was not statistically significant D. No sigStudies in recent years have frequently reported the health-promoting effects of HA rice flour and HMT starches . The mecAcat1, Cyp8b1, and Hmgcoa-R, while decreasing the hepatic expression of Ldlr and Serbps in ovariectomized (OVX) rats [Cyp7a1 and Scd1 were candidate genes. CYP7A1 is a rate-limiting enzyme in cholesterol metabolism and is a therapeutic target for cholesterol-lowering [Cyp7a1 induces a hypercholesterolemic phenotype [Cyp7a1 as a novel gene that prevents hyperlipidemia in OVX rats [Cyp7a1 could be regulated through HMT. Scd1 is downstream of Serbp and is reported to be regulated through HACs [Scd1 deficiency or disruption protects against hypertriglyceridemia [Fasn, Gpt3, Lipin, and Fabp5 might also be involved in the mechanism through which HMT contributes to cholesterol-lowering. However, further studies are warranted.As predicted, HMT rice flour reversed the weight increase caused by the high-fat component, improving circulating and hepatic lipid metabolism. Liu et al. reported that heat-moisture-treated high-amylose starch (HACs) increased the hepatic expression of VX) rats . Our PCRlowering . In humahenotype . In factOVX rats . Howeverugh HACs ; studieseridemia . In addiFasn), glycerol-3-phosphate O-acyltransferase 3 (Gpat3), and lipin 1 (Lpin1), may also contribute to the lipid-regulating effects. Hence, future studies should focus on investigating these genes.Campesterol is a plant sterol that is structurally similar to cholesterol and is found in many foods, especially vegetable oils, nuts, and seeds . It is aFirmicutes and Bacteroidetes, respectively, and showed similar regulating properties as inulin, a well-studied prebiotic [Thiotrichaceae, Bifidobacteriaceae, Streptococcaceae, Enterococcaceae, Thermoanaerobacteraceae, Deferribacteraceae, Porphyromonadaceae, Muribaculaceae, Barnesiellaceae, Lactobacillaceae, and Mogibacteriaceae [Bacteroides, Ruminococcus, and Bifidobacterium [Ruminococcus was enhanced through HMT. Previous reports have suggested that Ruminococcus plays a key role in the degradation of dietary RS into small molecular fragments for further digestion [Ruminococcus taxa in the HMT group might be a consequence of the RS component. The differentially regulated microbial profiles in our study might be attributable to the amylose content and animal species; therefore, further studies are required to reveal how HMT rice flour affects the human gut microbiome.Several studies have reported that a high amylose or RS content regulates gut microbial communities and microbial metabolites ,29. In Hrebiotic . High-ameriaceae . In HFD-acterium . In the igestion . BecauseCyp7a1 and Scd1 as candidate genes involved in the cholesterol-regulating potential of HMT rice flour, and highlighted the roles of Fasn, Gpt3, Lipin, and Fabp5 in this process. Furthermore, our study has shown that HMT rice flour can influence the gut microbiome, particularly the Ruminococcus taxa, which is likely attributable to the RS component of the rice flour. However, further research is needed to elucidate the precise mechanisms underlying these effects and to establish their relevance to human gut microbiome and health.In conclusion, our findings indicate that the HMT, rather than the inheritance of HA rice, is responsible for the observed anti-obesity and cholesterol-lowering effects. We identified"}
+{"text": "Irrigation solutions might affect dentin surface characteristics and, consequently, endodontic sealers adhesion.  This study analyzed the effect of different final irrigation protocols on push-out bond strength (BS) of AH Plus to dentin seven days and 20 months after obturation. Scanning electron micrographs were obtained from the dentin surface of one sample/group after final irrigation. Canals of bovine incisors were instrumented and received final irrigation with (n=21): G1 \u2013 2.5% sodium hypochlorite (NaOCl) + distilled water; G2 \u2013 2.5% NaOCl + 17% EDTA; G3 \u2013 2.5% NaOCl + 17% EDTA + 2.5% NaOCl; G4 \u2013 2.5% NaOCl + 17% EDTA + 2% chlorhexidine (CHX); G5 \u2013 mixture 5% NaOCl + 18% etidronate (HEDP); and G6 \u2013 mixture 5% NaOCl + 10% tetrasodium EDTA (Na4EDTA). After irrigation, one root/group was split and images were obtained by scanning electron microscopy (SEM). The other 20 roots/group were filled with only AH Plus sealer. Three slices/root were used for push-out assessment seven days and 20 months after obturation. One-way analysis of variance and Tukey (\u03b1<0.05) were used to compare the results among experimental groups, and unpaired t-test (\u03b1<0.05) was used to compare the results of the same group over time. The photomicrographs showed that, excepting G1, all groups completely removed the smear layer from the samples. In G2 and G4, the opening of the dentin tubules enlarged. In G3, erosion was observed in the peritubular and intertubular dentin. Values of the BS in the seven days were G2=G3=G4=G5>G6=G1 and in the 20 months were G3=G5>G6=G4>G1=G2. G3, G5, and G6 presented values of BS in 20 months similar to the values of seven days (P>0.05). The final irrigation protocols tested produced dentin surfaces with different characteristics. Only G3 and G5 presented high BS values that were stable over time. It can be obtained by physical, chemical, and/or mechanical bonding between materials, being the latter the most effective in creating strong joints.1 Studies have shown that irrigation solutions applied in the process of shaping and cleaning of the root canal system might alter the characteristics of dentin substrate7 and consequently interfere with the adhesion of the endodontic sealers.11 Sodium hypochlorite (NaOCl), the main irrigant in Endodontics , denatures the collagen of dentin7 and its oxidizing effect can compromise the polymerization of the resin sealers.12 The EDTA solutions used to remove the smear layer increase the roughness of the surface13 and produces a large demineralized dentin zone with great amounts of collagen fibrils exposed.7 These fibrils can contribute to the adhesion of endodontic sealers, such as AH Plus.14 However, an incomplete infiltration of the dental materials into the full extension of the exposed collagen matrix results in a weak bond interface that is degraded over time.17 This degradation is caused by the action of host-derived proteolytic enzymes called metalloproteinases on the denuded collagen fibrils.17The properties of the dentin surface are crucial to reach good interaction with the dental materials and, among the relevant features of this substrate are the cleanliness, roughness, and wettability.18 Another option is to use weaker chelating agents, such as etidronate (HEDP) or tetrasodium EDTA (Na4EDTA), since they promote moderate demineralizing effects that supposedly allow for an adequate infiltration of the filling material throughout the depth of the exposed collagen matrix.8 Removing the exposed collagen fibrils with a deproteinizing agent, such as NaOCl, after the chelating agents is a third alternative.19In theory, some irrigation protocols could help to overcome the problem of the adhesive interface degradation by providing stronger, favorable, and stable bonding of endodontic sealers over time. One of the protocols proposes the application of chlorhexidine (CHX) after the use of the decalcifying agent to inhibit the matrix metalloproteinases.20 When the sealer does not perform its function, a lack of adequate seal is implicated as a source of failure due to the movement of tissue fluids, bacteria, and their toxins into or out the canal space, by permeable spaces and voids in the filled canal.22 AH Plus is an epoxy resin-based sealer widely used and considered the gold standard for experiments assessing root canal obturation.23 The reasons for its use are the physical properties including adequate dimensional stability,24 low solubility and disintegration, and excellent adhesion to dentin, higher than other sealers.25Also, the selection of an endodontic sealer is mandatory for the long-term success of root canal treatment and the sealing ability is one of the most important properties to be considered.Due to a lack of studies evaluating long-term effects of different irrigation protocols on the dentin bond strength (BS) of endodontic sealers, this article presents the impact of different irrigation protocols in the BS of AH Plus to dentin in seven days and 20 months after the obturation. Moreover, scanning electron micrographs were obtained from one sample per group to verify the dentin surface. This study was designed to test if different irrigation protocols have a similar impact on the immediate and on the long-term BS of AH Plus sealer to dentin.This study was submitted and registered at the Animal Ethics Committee of the institution where the study was conducted under the number 016/2017.Bovine incisors with completely formed roots were sectioned transversally at the cementoenamel junction and apically at the root end to obtain a root segment with approximately 14 mm of length. The cuts were performed using a diamond disc , under water cooling. The apex portion of the canals was sealed with wax to keep the irrigants inside the root canal. Then, the canals were instrumented using the ProTaper Universal system . The cervical third was prepared with the SX instrument and the other thirds with the instruments S1, S2, F1, F2, F3, F4, and F5.The root canals were irrigated with 2 mL 2.5% NaOCl before the use of the SX file and at each instrument change. Irrigation was performed with a 10 mL disposable plastic syringe attached to a polypropylene capillary tip . After the final irrigation, the root canals were completely dried with absorbent paper points .4EDTA (5 min). For the irrigation procedure, 1 mL of irrigant was used per min of the time set per solution. After using each irrigant, the solutions were aspirated, and the canals were rinsed with 2 mL of distilled water for 1 min to remove the remnants of the irrigators. At the end, the canals were dried with absorbent paper points.The roots were then distributed, with the aid of the Bioestat 5.0 program , into six groups (n=21) according to the final irrigation protocols: G1 \u2013 2.5% NaOCl (5 min) + distilled water (1 min); G2 \u2013 2.5% NaOCl (5 min) + 17% EDTA (1 min); G3 \u2013 2.5% NaOCl (5 min) + 17% EDTA (1 min) + 2.5% NaOCl (1 min); G4 \u2013 2.5% NaOCl (5 min) + 17% EDTA (1 min) + 2% CHX (2 min); G5 \u2013 Mixture 5% NaOCl + 18% HEBP (5 min); and G6 \u2013 Mixture 5% NaOCl + 10% Na26 The root canals of 20 samples/group were filled using only the sealer to have exclusively the results of the BS between the sealer and the dentin and avoid confounding factors.27 AH Plus was inserted into canals with the aid of a lentulo spiral #40 (Dentsply/Maillefer) until the entire canal was filled. The roots were digitally radiographed in two angulations: mesiodistal and buccolingual directions. Those with voids or bubbles were discarded and replaced with new samples. The roots of all groups were stored at 37\u00b0C and 100% humidity, and on the day after the obturation, the wax on the apical portion of the roots was removed and the coronal and apical extremities of the canals were sealed with glass ionomer cement.For the obturation of the canals, the AH Plus sealer  was prepared according to the manufacturer\u2019s instructions. To obtain a similar composition of the sealer in all groups, only the middle portion of the tubes was used.After one week, 10 roots of each group were sectioned in a precision cutting machine  under constant water cooling. To remove the glass ionomer cement from the extremities of the canal, a 1 mm slice was cut at the cervical and another at the apical portion of the roots and discarded. Then, cuts were performed with 2 mm of distance between each other, so six slices were obtained from each root (two per root third). The most cervical slice of each third was selected for the push-out test and the other one was discarded. The other 10 roots/group were stored for 20 months and then were cut in slices, in the same way.Each slice was labelled. Thickness was confirmed with a digital caliper with 0.02 mm accuracy , and the apical and cervical diameters of the obturated area were measured with the stereomicroscope Stemi 2000 C .For the push-out assessment, the slices were placed on a base with a central hole. The tests were performed by applying a compressive load to the apical-coronal direction by using different cylindrical plunger tips. The selection of the plunger for each slice was based on the measures previously obtained by the stereomicroscopy. The biggest plugger, compatible with the obturation area in the apical surface of the slice, was used. The plunger tip was centred on the filling material without touching the surrounding dentin surface. Loading was performed on a universal testing machine  at a 0.5 mm/min speed until bond failure occurred.2 ), using the equation \u03c3=F/A. The bonding surface area of each sample was estimated by the equation of a conical frustum: A=\u03c0(R2+R1)\u00d7[h2+(R2-R1)2 ]0, where R1=base radius, R2=top radius, and h=height of the frustum.The megapascals (MPa) value of push-out BS of each specimen was estimated by dividing the force required to dislodge the filling material (F in kN) by the bonding surface area of the root canal filling (mmOne root of each group was not obturated and was used to obtain images of the dentin surface by scanning electron microscopy (SEM). The roots were split longitudinally and mounted on SEM stubs using a double-faced carbon tape, then they were sputter coated with platinum and palladium, and evaluated under the FEI Nova NanoSEM 230 ultra-high resolution scanning electron microscope . Photomicrographs were taken from the middle portion of the root fragments at 25000\u00d7 magnification.t -test with 80% power and a 5% bilateral a, to detect a 20% difference between the same group over time, the required sample size was 27 specimens per group, which means one slice/root third of nine teeth. Considering that samples could be lost during the experiment, 10 teeth were prepared per group for each period of analysis.The sample size was determined based on a pilot study in which the push-out BS mean was 15.9 \u00b1 4.2 (\u00b1 standard deviation). Thus, using the unpaired post-hoc tests were used to compare the results among the different irrigation protocols at the same experimental time. Unpaired t -test was used to compare the results of the same irrigation protocol seven days and 20 months after the obturation. The confidence level of all tests was set at 95%.The data of push-out BS obtained showed a normal distribution in the preliminary normality test (P>0.05); thus, the one-way analysis of variance (ANOVA) and Tukey The effect of root canal thirds was assessed. However, no significant differences were found among all groups in both time periods (P>0.05).t -test showed that only G3, G5, and G6 presented results of push-out BS in 20 months similar to the values obtained in the seven days (P>0.05).The comparison of the values of the same group over time by the unpaired The SEM images taken from all groups showed that, except for G1, all irrigation protocols completely removed the smear layer produced by the root canal preparation   . In G2 2 bacterial re/contamination,3 and displacement of the obturation during a subsequent handling.5 This study results showed that the different irrigation protocols tested have a significant impact on the immediate and on the long-term dentin BS of AH Plus rejecting the null hypothesis. Although many studies evaluated the long-term stability of fibre-post bonding to dentin, the authors failed to find studies analyzing the effect of different irrigation protocols on the BS of endodontic sealers over time, except for two studies that presented the results after three and six months of the obturation.29It is important to identify materials and techniques to be used during the endodontic treatment, to obtain stable BS values of the endodontic sealers to the root canal in a long term, since a good adhesion of the sealers to the dentin walls can reduce fluids percolation,30 No differences were found between the bovine collagen and demineralized human dentin and from mineral matrices of human and bovine dentine.32 A study showed that the bovine dentin substrate did not influence the push-out BS of sealers compared to human dentin when using an intratooth model.33 Although bovine teeth can have dentin tubules larger than human teeth, the results of the groups are not skew since the push-out test can only rank the bonding of the root canal materials.34 Also, the samples from all groups can be more standardized than when human teeth are used, as they can be acquired from animals with similar age and stored for the same amount of time.In this study, bovine teeth were used instead of human teeth since the dentin of the bovine incisor has a similar structure and number of tubuli to the human molar dentin.34 In this study, the root canals were filled only with the AH Plus sealer to have values of the BS truly between the dentin and the sealer. The complementary analysis of the failure patterns was not performed, since once the canals were filled only with the sealer, all failures had undoubtedly adhesive nature.34When root canals are filled with a core material, such as gutta-percha, and a sealer at least two interfaces can be dislodged from the canal space, this can create a systematic source of error.35 which favours the micromechanical interlocking of the endodontic sealers.1 The use of CHX after the decalcifying agent in G4 (NaOCl + EDTA + CHX) did not improve the initial BS when compared with G2 (NaOCl + EDTA), as observed in previous studies related to adhesion.36 In G2 (NaOCl + EDTA) and G4 (NaOCl + EDTA + CHX), the exposed collagen on the dentin surfaces probably contributed to the adhesion of AH Plus, since it was reported that the amino groups of the collagen fibers can bond chemically with this sealer.14 However, this contribution was not significant to produce statistical differences between these groups and G3 (NaOCl + EDTA + NaOCl), which had the collagen matrix deproteinated by the final irrigation with NaOCl.7 Another research with different sealers did not find differences in the BS with the use of NaOCl after the EDTA when compared with the group that applied EDTA as the last irrigant as well.14 These results reinforce the concept that the strong joints are most effectively created by the mechanical bonding.1The best results of initial BS were obtained in G2 (NaOCl + EDTA), G3 (NaOCl + EDTA + NaOCl), and G4 (NaOCl + EDTA + CHX). These groups were similar to each other and presented a clean surface with high roughness due to EDTA use,14 A paper on FTIR analysis showed that after using the mixture of NaOCl and HEDP, more mineral than collagen can be found on the surface of the substrate.7 Consequently, the high push-out BS obtained in this group might have resulted from the cleanliness of the surface and reasons other than the interaction between collagen amino groups and AH Plus.10 Once the roughness of the dentin surface is not increased with the use of HEDP as much as when EDTA is used,13 an important factor to be considered is that HEDP is a bisphosphonate that strongly adsorb to hydroxyapatite surface and increase the surface-free energy.37 In the root dentin, this increase in surface-free energy results in high wettability of the canal walls,13 providing the necessary proximity between materials, facilitating molecular attraction. It results in a major penetration of the sealer into the irregularities of the surface, observed by the low gap distance between the filling material and the root dentin,38 and in an enhancement of the BS values due to the increase in the mechanical interlocking of endodontic sealers to dentin when HEDP is used to irrigate the root canals.10The specimens treated with the mixture of NaOCl and HEDP (G5) had initial BS similar to the groups with EDTA as the decalcifying agent . However, previous studies obtained higher values of BS for the mixture of NaOCl and HEDP compared to the conventional irrigation protocol of NaOCl followed by EDTA.10 The possible reason for this outcome was the sealer adhesion to the inorganic components of the smear layer reducing the sealer resistance to the dislodgement forces.8 Since the mechanical bonding is the most effective mean to create a strong joint1 and NaOCl does not significantly increase the roughness of the dentin surface,13 this irrigation protocol does not favour a mechanical interlocking of the sealer to the dentin. Another reason that could be suggested for the low BS values is the oxidizing effect of the NaOCl that remains inside the root canal and negatively affects the BS of the resin-based sealers.12 However, this effect might have been insignificant in this study due to the final rinse with distilled water.8 Besides, in G3 (NaOCl + EDTA + NaOCl), the last irrigant used was also NaOCl and the push-out BS values of this group were significantly higher than the values of G1 (NaOCl).Amongst the six irrigating protocols tested, G1 (NaOCl) was one of the groups that presented the lowest initial push-out strength value. This result agrees with previous studies that obtained lower values of BS for the group that used only NaOCl as the irrigant compared to groups that employed NaOCl associated with EDTA and/or HEDP.4EDTA). In this group, the smear layer is completely removed after the 5 min of use of the mixture, and the chemical composition of the surface is similar to a surface not treat with irrigants.7 However, no results were published regarding the effects of the mixture of NaOCl + Na4EDTA on the dentin roughness and, since this is the first study that tested the effects of this irrigation protocol on the BS of sealers to dentin, comparisons of these results with the literature were not possible.The other group that presented the lowest initial push-out BS values was G6  and G5 (Mixture of NaOCl + HEDP) was the high occurrence of fractures of the dentin slices during the push-out tests.7 In G1 (NaOCl), the values of BS reduced after 20 months of the obturation when compared with the initial values, suggesting that the presence of smear layer on the surface can negatively affect the adhesion over time. Although G2 (NaOCl + EDTA) and G4 (NaOCl + EDTA + CHX) presented initial high BS values, a reduction occurred after 20 months of the obturation. The likely main reason for this find was a suboptimal infiltration of the sealer in the demineralized collagen matrix, followed by the degradation of the denuded collagen fibers by the action of metalloproteinases.15 G4 (NaOCl + EDTA + CHX) had a significantly better preservation of the BS than G2 (NaOCl + EDTA) after aging, agreeing with previous studies that reported some long-term stability of BS with the use of the CHX.39 Probably, in G4 (NaOCl + EDTA + CHX), the action of the metalloproteinases was partially suppressed by the protease inhibitor effect of CHX.18 However, the protective effects of CHX do not remain for long aging periods.39When analysing the results of the same group over time, those with BS preserved  were smear layer free and did not present the collagen matrix exposed on the dentin.7 the smear layer was not completely removed by this protocol and it is possible to observe inorganic matter covering the surface of the dentin on the photomicrography. The use of NaOCl associated with chelating solutions G2 (NaOCl + EDTA), G3 (NaOCl + EDTA + NaOCl), G4 (NaOCl + EDTA + CHX), G5 (mixture of NaOCl + HEDP), and G6 (mixture of NaOCl + Na4EDTA) promoted a complete removal of the smear layer in the samples of these groups. However, probably due to the higher demineralization capacity of 17% EDTA compared to Na4EDTA and HEDP40 , it is possible to observe an enlargement of the entrance of dentin tubules in G2 (NaOCl + EDTA), G3 (NaOCl + EDTA + NaOCl), and G4 (NaOCl + EDTA + CHX). In G3 (NaOCl + EDTA + NaOCl), the intercalated use of NaOCl and EDTA resulted in dentin erosion and irregular and rough tubules orifices, as previously described.41 In the SEM images, the microtopography appears to be rougher and irregular in G1 (NaOCl), G3 (NaOCl + EDTA + NaOCl), and G4 (mixture of NaOCl + HEDP). This feature might be the result of the higher amounts of mineral on the surface of these groups due to the collagen deproteination by the NaOCl,7 since the exposition of the collagen has been associated with smooth and plane images.41 However, this appearance is not related to major values of surface roughness.13This study did not have the intention to quantify the smear layer removal by the irrigation protocols tested since this is already well established in the literature, thus only one sample of each group was used to obtain the SEM images from the dentin surface. The photomicrographs obtained showed different characteristics on the dentin surface according to the irrigation protocols tested   . Since 1 However, more studies should be performed to understand the adhesive interface and the changes that occur in this region over time when different irrigation protocols and different types of endodontic sealers are used.Within the limitations of this study, it was possible to rank the irrigation protocols tested that promote strong and stable dentin BS of AH Plus in a long term. The final irrigation protocols of NaOCl + EDTA + NaOCl and the mixture of NaOCl + HEDP resulted in high BS values for AH Plus to the dentin that were stable over time. These results seemed to be influenced by the alterations on the dentin surface characteristics promoted by the irrigation solutions. It reinforces the importance achieving clean surfaces that present high roughness and wettability to obtain good interfacial interaction between the adherend and the adhesive.Only the final irrigation protocols of NaOCl + EDTA + NaOCl and the mixture of NaOCl + HEDP resulted in high BS values for AH Plus to the dentin that were stable over time. These results seemed to be influenced by the alterations on the dentin surface characteristics promoted by the irrigation solutions."}
+{"text": "The crk10-A397T mutant is a dwarf that displays collapsed xylem vessels in the root and hypocotyl, whereas the vasculature of the inflorescence develops normally. In situ phosphorylation assays with His-tagged wild type and crk10-A397T versions of the CRK10 kinase domain revealed that both alleles are active kinases capable of autophosphorylation, with the newly introduced threonine acting as an additional phosphorylation site in crk10-A397T. Transcriptomic analysis of wild type and crk10-A397T mutant hypocotyls revealed that biotic and abiotic stress-responsive genes are constitutively up-regulated in the mutant, and a root-infection assay with the vascular pathogen Fusarium oxysporum demonstrated that the mutant has enhanced resistance to this pathogen compared with wild type plants. Taken together our results suggest that crk10-A397T is a gain-of-function allele of CRK10, the first such mutant to have been identified for a CRK in Arabidopsis.Cysteine-rich receptor-like kinases (CRKs) are a large family of plasma membrane-bound receptors ubiquitous in higher plants. However, despite their prominence, their biological roles have remained largely elusive so far. In this study we report the characterization of an Arabidopsis mutant named  crk10-A397T) causes defects in the vasculature and activates defence pathways in hypocotyls and roots of Arabidopsis.A gain-of-function allele of the cysteine-rich receptor-like kinase CRK10 ( Plant growth and development are modulated by a multitude of intrinsic growth regulators and environmental cues. Factors regulating development as well as environmental and pathogenic signals are mostly recognized by receptor-like kinases (RLKs), membrane-localized receptors that perceive and transduce these signals to the intracellular environment. Similar to animal receptor tyrosine kinases (RTKs), these receptors consist of an extracellular domain that perceives specific ligands, a single-pass transmembrane domain, and a cytoplasmic kinase domain that transduces the signal via phosphorylation of downstream target proteins in the cytoplasm in order to tailor a cellular response . The welGingko biloba  is one of the largest with over 40 members in Arabidopsis. The signature motif for CRKs is the presence of, in most cases, two repeats of the DOMAIN OF UNKNOWN FUNCTION 26 (DUF26) in their extracellular domain, which contains three cysteine residues in the conserved configuration C-X8-C-X2-C . AlthougDespite the large number of CRKs among the RLK superfamily, very little is known about their specific biological roles and the regulation of downstream signalling events. Efforts to assign functions to members of this family have involved a comprehensive analysis of a collection of T-DNA knockout lines for 41 CRKs of Arabidopsis, which suggested a role for several members in the fine-tuning of stress adaptation and plant development  of Arabidopsis. This mutation leads to the replacement of alanine 397 with threonine in the \u03b1C helix of the kinase domain of the protein, with the newly introduced Thr397 acting as an additional phosphorylation site in situ. We show that the crk10-A397T allele causes a dwarf phenotype in Arabidopsis, which is associated with the collapse of xylem vessels in roots and hypocotyls. Analysis of the transcriptome shows the occurrence of extensive transcriptional re-programming of immune- and cell wall-related genes in the hypocotyls of the mutant plants. Biochemical analyses reveal that these transcriptional changes are accompanied by alterations in cell wall composition. A pathogen assay indicates enhanced resistance to the soil-borne vascular pathogen Fusarium oxysporum. Taken together our results suggest that crk10-A397T is a gain-of-function allele of CRK10 that leads to xylem vessel collapse and the activation of defence responses to pathogens.Establishing ligand\u2013receptor pairs is not a trivial task and exploring regulation and function of RLKs without the knowledge of their activating ligands poses a challenge. In these circumstances, mutants harbouring gain-of-function alleles of RLKs, where kinase activity occurs in the absence of the ligand, can be a useful resource to study receptor regulation and function. In this study we describe the characterization of one such gain-of-function allele, \u22122 s\u22121 light intensity. For in vitro experiments, surface-sterilized seeds were cultivated on \u00bd Murashige and Skoog (MS) plates (Duchefa Biochemie). The T-DNA lines SAIL_427_E09 and SALK_116653 were obtained from the Nottingham Arabidopsis Stock Centre. Micrografting was performed according to the procedure described by Arabidopsis ecotype Col-0 plants were grown in Grobanks cabinets  in Levington F2 + Sand compost in long day conditions (16 h/8 h), 23/18 \u00baC day/night temperature, and 200 \u00b5mol mTotal RNA was extracted from hypocotyls of Arabidopsis plants using the RNeasy Mini Kit (Qiagen). Four biological replicates were isolated per genotype per time point with each biological replicate consisting of a pool of 50\u201360 hypocotyls. Samples were treated with DNase Turbo DNA-free kit (Thermo Fisher Scientific). Prior to library preparation RNA quality was assessed on a 2100 Bioanalyzer (Agilent Technologies). Library preparation and paired-end sequencing was performed by Exeter Sequencing Service  using the Illumina HiSeq 125 PE sequencing platform.https://usegalaxy.org/) bioinformatics web pipeline, the quality of the reads was assessed using MultiQC (https://multiqc.info/), reads were trimmed using Trimmomatic (https://daehwankimlab.github.io/hisat2/). The table of counts was acquired using the featureCounts functions in the Subread (https://bioconductor.org/packages/release/bioc/html/Rsubread.html), on the R Bioconductor platform (https://bioconductor.org/). Genes that did not meet a threshold of 5 counts in at least three out of four biological replicates were discarded. Differential expression analysis was performed  in the R (v3.6.1) Bioconductor package DESeq2 (https://bioconductor.org/packages/release/bioc/html/DESeq2.html). Gene Ontology (GO) enrichment analysis  and AtUBC21 For/Rev  were used as internal controls (see RNA was isolated using TRI Reagent (Merck) and treated with DNase I, Amplification Grade (Thermo Fisher Scientific) prior to cDNA synthesis with SuperScript III Reverse Transcriptase (Thermo Fisher Scientific). All qPCR reactions were performed in a LightCycler 96 Real-Time PCR System (Roche Diagnostics) using the FastStart Essential DNA Green Master (Roche Diagnostics). Primers AtCRK10 forward (For)/reverse (Rev) were used for quantification of rols see . The 2\u2212\u0394t method  was usedhttps://ibmcp.upv.es/services/plant-hormone-quantification/).Three replicates, each containing between 75 and 100 mg (fresh weight) of hypocotyls isolated from 3-week-old wild type (WT) and mutant plants were prepared. Hormone analysis was performed using the platform provided by The Plant Hormone Quantification Service from Universitat Polit\u00e8cnica de Val\u00e8ncia according to their protocol . Vector pJD330 was kindly provided by Dr D. R. Gallie, and vector RS 3GSeedDSRed MCS by Edgar B. Cahoon, University of Nebraska-Lincoln. A list of primers used in this study can be found in CRK10-NOSt construct was generated by amplification of the full-length cDNA of CRK10 from clone U60398 with primers CRK10 SalI For and CRK10 SacI Rev. The CRK10 cDNA sequence was subcloned into pJD330 between the 35S promoter and NOS terminator after removal of the \u03b2-glucuronidase (GUS)-containing fragment by restriction digestion (pJD330 35S:CRK10-NOSt). The 35S:CRK10-NOSt fragment was then amplified with 35S AscI For and NOSt AscI Rev primers and inserted into the binary vector RS 3GSeedDSRed MCS. The CRK10Pro:crk10-A397T-NOSt construct was generated by replacing the 35S promoter in pJD330 35S:CRK10-NOSt with 1 kb of the native promoter of CRK10 (CRK10Pro) obtained by amplification of genomic DNA with primers CRK10 Pro SphI For and CRK10 Pro SalI Rev. In vitro mutagenesis  with primers CRK10 A397T For and CRK10 A397T Rev was used to introduce the G>A mutation responsible for the replacement of A397 by T. CRK10 Pro AscI For and NOSt AscI Rev primers were used to amplify CRK10Pro:crk10-A397T-NOSt for cloning into the RS 3GSeedDSRed MCS binary vector. The CRK10Pro:GUS-NOSt construct was generated by replacing 35S in pJD330 with the CRK10Pro sequence obtained by amplification with primers CRK10 Pro SphI For and CRK10 Pro NcoI Rev through restriction digestion. The CRK10Pro:GUS:NOSt fusion was amplified with primers CRK10 Pro AscI For and NOSt AscI Rev for transfer into RS 3GSeedDSRed MCS. The translational fusion of CRK10 with mCherry was obtained by replacing the stop codon of CRK10 in pJD330 35S:CRK10-NOSt with a SacI restriction site (primers CRK10 wsc SacI For and CRK10 wsc SacI Rev) by in vitro mutagenesis. This restriction site was used to insert in frame the mCherry sequence that had been amplified with compatible primers (mCherry SacI For and mCherry SacI Rev). The CRK10-mCherry-NOSt fragment was then amplified with primers CRK10 SalI For and NOSt NotI Rev and cloned into the pENTR1A Dual Selection Vector (Thermo Fisher Scientific). Gateway cloning (Thermo Fisher Scientific) into destination vector pB2GW7 (CRK10-mCherry-NOSt.The cDNA clone (U60398) containing full length Nicotiana benthamiana leaves according to Agrobacterium tumefaciens GV3101 containing the respective constructs.Detection of transient expression of fluorescent fusions was performed by infiltration of Thin sections were prepared by fixing plant tissue in 4% paraformaldehyde\u20132.5% glutaraldehyde followed by gradual dehydration with ethanol and infiltration with LRWhite resin (Agar Scientific). Sections were prepared using a Reichert ultramicrotome (section thickness: 1\u20132 \u00b5m) and stained with 0.5% potassium permanganate. Thin sections were observed with a Zeiss Axiophot microscope equipped with a Q-Imaging Retiga EXi CCD camera .N. benthamiana leaves or stably transformed Arabidopsis hypocotyls was detected with a laser excitation wavelength of 561 nm and collection of emission at 578\u2013639 nm. Plasmolysis was performed using a 0.8 M mannitol solution for 40 min.Confocal microscopy was performed using the Zeiss 780 LSM system. For detection of the autofluorescence of lignin, non-stained resin-embedded thin sections were imaged with an excitation wavelength of 405 nm and emission was collected at 451\u2013480 nm and 560\u2013612 nm. mCherry fluorescence from transiently transformed Samples were prepared by fixing plant material by high-pressure freezing using a Leica HPM100, followed by freeze substitution with 100% ethanol (Leica EM Auto Freeze substitution) and infiltration with LRWhite resin (Agar Scientific). Ultra-thin sections were prepared with a Leica EM UCT ultramicrotome (section thickness: 90 nm) and were collected on pioloform/carbon-coated nickel grids (Agar Scientific) and stained with 2.5% uranyl acetate and Reynolds lead citrate . Ultrathcrk10-A397T mutant plants for Fourier-transform infrared spectroscopy (FTIR) analysis were prepared using a cryostat  with three replicates per sample. Cross sections were washed with 70% ethanol and air-dried on barium fluoride (BaF2) discs prior to analysis using a Nicolet iN10MX infrared microscope (Thermo Scientific) equipped with a \u00d715 infrared (IR) objective. FTIR maps were obtained by transmission aperture mapping with a mercury\u2013cadmium\u2013telluride detector and an x\u2013y step size of 10 \u03bcm (20 \u00d7 20 \u03bcm2 aperture). A total of 128 scans were averaged at 8 cm\u22121 for each image pixel. An empty spot on the BaF2 disk was used as background. The maps were exported in ENVI format and processed in MATLAB . The spectra were truncated to 1800\u2013700 cm\u22121 and the density map was calculated by averaging the IR absorption from 1800 to 800 cm\u22121. Characteristic band intensities for the following components were mapped to visualize their distribution: 1018 cm\u22121 for pectin, 1033 cm\u22121 and 1050 cm\u22121 for hemicelluloses and cellulose, 1511 cm\u22121 for lignin, 1650 cm\u22121 for protein, and 1735 cm\u22121 for ester groups.Transverse cross sections of hypocotyls of 3-week-old WT and crk10-A397T mutant plants were collected per replicate with three replicates per sample. Alcohol insoluble residue (AIR) preparation was performed according to Thirty hypocotyls of 3-week-old WT and Plant tissue was incubated overnight in X-gluc (Melford) solution at 37 \u00b0C. Chlorophyll was removed with 80% ethanol prior to imaging using a Leica M205 FA stereomicroscope (Leica Microsystems).CRK10 was amplified with primers CRK10 KD SalI For and CRK10 KD NotI Rev and cloned into pENTR1A Dual Selection Vector (Thermo Fisher Scientific). In vitro mutagenesis was used to generate the gain-of-function mutation of CRK10 (primers CRK10 A397T For and CRK10 A397T Rev) and the dead kinase variant (primers CRK10 D473N For and CRK10 D473N Rev) prior to Gateway cloning with pDEST17 (Thermo Fisher Scientific). The 6\u00d7 His tagged proteins were expressed in BL21 AI One-Shot Escherichia coli cells (Thermo Fisher Scientific). Bacterial cultures were grown to an OD600 of 0.4\u20130.5 after which protein expression was induced by adding l-arabinose to a final concentration of 0.2%. After 3 h bacterial cells were lysed by sonication, His-tagged proteins were purified with the HIS-Select Nickel Affinity Gel (Sigma-Aldrich) and protein concentration determined by the Bradford method . For phosphatase treatment, 5 \u00b5g protein extract was treated with Lambda Protein Phosphatase  for 1 h 30 min at 30 \u00baC. Samples were resolved by SDS-PAGE  and gels were stained with Quick Coomassie Stain (Generon). For western blotting, proteins were transferred to a polyvinylidene difluoride membrane  and hybridized with a His-probe (H-3) horseradish peroxidase monoclonal antibody (Santa Cruz Biotechnology). The membrane was washed and incubated with Amersham ECL Western Blotting Detection Reagent  according to manufacturer\u2019s instructions.Primers used to generate the 6\u00d7 His-tagged CRK10 kinase domain (KD) constructs are listed in WT and His-CRK10kdA397T were excised from an acrylamide gel and sent to the Cambridge Centre for Proteomics, (https://proteomics.bio.cam.ac.uk/core-facility) for analysis by LC-MS/MS. Data were submitted to the Mascot search algorithm  against a custom database consisting of the CRK10WT and CRK10A397T sequences and the UniProt Arabidopsis database . A significance threshold value of P<0.05 and a peptide cut-off score of 20 were applied.Protein bands corresponding to His-CRK10kdA structural model of the kinase domain of CRK10 was generated by homology modelling using PyMOD 3.0 with default parameters (Fusarium oxysporum f. sp. conglutinans 699 (kindly provided by Prof. Antonio Di Pietro) was assessed by a root infection assay according to in vitro were inoculated by immersing their roots for 20 min in a suspension of 1 \u00d7 106 microconidia ml\u22121 of F. oxysporum f. sp. conglutinans 699. Subsequently seedlings were transferred to soil and cultivated in a growth chamber under long day conditions and a temperature set at 28 \u00baC during the day and 25 \u00baC at night. For each repetition of the experiment, 80 plants per genotype were arranged in a randomized blocked tray design, and mortality was assessed daily between 7 and 20 d post-inoculation. The experiment was performed twice. To determine fungal burden, seedlings were inoculated as described above and sampled at 2 and 7 d post-inoculation. At these time points, total DNA was extracted (protocol adapted from ACTIN1 For/Rev (F. oxysporum) and normalized to the Arabidopsis ACTIN2 gene (primers AtACT2 For/Rev) (see Susceptibility to infection by Rev) see . The expt-test was used to assess statistical differences between two variants. To assess whether the pattern of segregation of the dwarf phenotype followed the expected 1:2:1 ratio, the Oi is the observed count for group i and Ei is the expected count for group i. Under the null hypothesis of 1:2:1 segregation, this test statistic should follow a chi-square distribution with 2 degrees of freedom. The probability of survival of each genotype in the bioassay with F. oxysporum was assessed with a generalized linear model ; statistical significance of the genotypic effect was tested after removing variation associated with plant position within rows of different trays and quantified through a chi-square statistic of the difference in deviance. Statistical significance of the differences in fungal burden between genotypes was tested by ANOVA .Statistical tests were performed using Genstat software . Student\u2019s 2 progeny of the sixth backcross revealed the semi-dominant nature of the mutation, as WT, intermediate and dwarf phenotypes segregated according to a 1:2:1 ratio with heterozygous plants being clearly discernible . To determine the underlying mutation responsible for the dwarf phenotype, whole genome sequencing was performed on bulk segregants derived from the sixth backcross. This returned a list of 15 candidate genes containing point mutations in coding regions. We noticed that a point mutation (G>A) in the fourth exon of CYSTEINE-RICH RECEPTOR-LIKE KINASE 10  causes the replacement of alanine 397 by a threonine in the kinase domain of the protein  background  under the control of the 1 kb genomic region containing the putative native promoter of CRK10. A total of 25% of the recovered transformants were dwarfs, establishing a direct link between the dwarf phenotype and the mutant allele . GUS expression was detected in the vasculature of the roots, cotyledons, petioles, leaves, hypocotyls, and inflorescence stem . Both transient expression of the construct in N. benthamiana leaves and stable expression in transgenic Arabidopsis plants indicated that the fusion protein localized to the plasma membrane  and vice versa . Compared with WT, qPCR performed on 4-week-old leaves detected a CRK10 transcript increase of 15 and 6 times for CRK10 OE-1 and OE-2, respectively  and crk10-4 (SALK_116653). Quantification of CRK10 transcript levels from leaves of 4-week-old plants by qPCR confirmed that crk10-2 and crk10-4 are a knockout and knockdown line of CRK10, respectively  from E. coli cells, as well as its \u2018dead\u2019 kinase counterpart that harboured the substitution of the essential aspartic acid 473 with an asparagine residue (His-CRK10kdWT-D473N). Following separation of the recombinant proteins by SDS-PAGE and detection by anti-His immunoblotting, the dead kinase version His-CRK10kdWT-D473N migrated at the predicted molecular mass of 40 kDa, while the WT kinase version His-CRK10kdWT showed an electrophoretic mobility shift to a larger molecular mass, known to occur for phosphorylated proteins (in vitro mutagenesis (His-CRK10kdA397T and His-CRK10kdA397T-D473N). Although the dead kinase version His-CRK10kdA397T-D473N migrated at the same molecular mass as His-CRK10kdWT-D473N on SDS-PAGE gels, the mobility shift of His-CRK10kdA397T was increased when compared with the one observed for His-CRK10kdWT  in the CRK10 kinase domain could have introduced a potential additional phosphorylation site. We therefore wanted to determine whether WT and mutant CRK10 are enzymatically active kinases and if differences in their autophosphorylation pattern could be detected. We addressed this question by investigating the autophosphorylation activity of the cytoplasmic kinase domain of CRK10 proteins ; Fig. 5Cblotting . HoweverWT and His-CRK10kdA397T to analysis by LC-MS/MS. The Mascot probability-based algorithm was used to confirm the peptides match to the CRK10 kinase domain sequence. Individual MS/MS spectra were inspected for confirmation of phosphorylation sites, which led to the unambiguous identification of Thr340, Tyr363, Thr507, Ser508, Tyr514, Thr625, Ser662, and Thr664 as phosphosites in both His-CRK10kdWT and His-CRK10kdA397T proteins , we obtained 523 (2 weeks), 1836 (3 weeks), and 913 (5 weeks) DEGs, of which 274 were common to all time points. These DEGs were selected as the core set and taken forward for analysis  and exposed to abiotic stresses (treatment with fenclorim and sulfometuron methyl) (P=2.30 \u00d7 10\u221226), \u2018Response to stimulus\u2019  and \u2018Response to stress\u2019  are significantly over-represented  . Equallyresented . In accoT mutant . Transcresponses . The anaesponses . Also atesponses . In summirregular xylem (irx) mutants. As our transcriptomic dataset revealed the reprogramming of numerous cell wall-related genes, we proceeded with the characterization of the cell wall composition of the intact and collapsed xylem vessels in the hypocotyls of 3-week-old WT and crk10-A397T mutant plants by using FTIR spectroscopy, a powerful and rapid technique for analysing cell wall components and putative cross-links. The difference spectrum between the cell wall of an intact and collapsed xylem vessel revealed that complex changes had occurred in the collapsed vessel cell walls, with changes in hemicellulose composition and a reduced amount of ester cross-links among the most noticeable  confirmed the differences in the probability of survival between genotypes, with the crk10-A397T mutant having the highest chance of survival of 81.25%, followed by 52.5% for the CRK10 OE-1 plants, and just over 30% for both crk10-2 and WT  lies in subdomain III of the kinase domain, at the C-terminus of the \u03b1C helix and at the start of the short \u03b1C\u2013\u03b24 loop that links the \u03b1C helix to the \u03b2 strand 4 , where it provides a firm docking site for the C-terminal residues of the kinase that keeps it in an inactive conformation  and genes involved in the synthesis of the tryptophan-derived antimicrobial compounds  are significantly up-regulated in the mutant, as are numerous transcription factors usually associated with co-ordinating stress responses  plot of RNA sequencing samples and number of differentially expressed genes identified in crk10-A397T mutant hypocotyls shows marked differences from the spectrum of intact vessels in the WT.Fig. S11. The FTIR spectrum profile of collapsed xylem vessels in the crk10-A397T mutant plants shows marked differences from that of WT plants.Fig. S12. The total monosaccharide content of hypocotyls of Fig. S13. The ultrastructure of the secondary cell wall of collapsed xylem vessels resembles that of intact vessels in the WT.F. oxysporum.Fig. S14. Fungal burden quantification at 2 and 7 d post-inoculation with Fig. S15. Alignment of the \u03b1C helix segment of the kinase domain of the CRK family from Arabidopsis shows members of the family that contain alanine, threonine, or serine residues on position equivalent to Ala397 in CRK10.Fig. S16. Phylogenetic tree of the CRK family from Arabidopsis and their respective residue on position equivalent to Ala397 in CRK10.Table S1. Table of primers.crk10-A397T mutant hypocotyls.Tables S2\u2013S5. Differentially expressed genes in the Tables S6, S7. Gene Ontology analysis.Tables S8\u2013S11. Tables of differentially expressed genes per functional category.crk10-A397T mutant plants.Table S12. Quantification of hormones in the hypocotyl of WT and erad080_supp_Supplementary_Figs_S1-S16_and_Tables_S1_S8-S12Click here for additional data file.erad080_suppl_Supplementary_Tables_S2-S7Click here for additional data file."}
+{"text": "However, some transcripts from active young SINEs showed high tissue-specificity, as confirmed by analyzing 3570 RNA-seq samples. We also detected 211,067 dimorphic SINEs in 374 individuals, including 340 population-specific ones associated with local adaptation. Mapping these dimorphic SINEs to genome-wide associations of 97 complex traits in pigs, we found 54 candidate genes  that might be mediated by TEs. Our findings highlight the important roles of young SINEs and provide a supplement for genotype-to-phenotype associations and modern breeding in pigs.Transposable elements (TEs) are a major source of genetic polymorphisms and play a role in chromatin architecture, gene regulatory networks, and genomic evolution. However, their functional role in pigs and contributions to complex traits are largely unknown. We created a catalog of TEs ( A catalog of over 3 million transposable elements in pigs reveals that evolutionarily young SINEs are associated with increased epigenomic silencing, and may be related to expression of nearby genes. The movement of TEs is often accompanied by an increase in their abundance, comprising a large fraction of genomic sequences9. According to the mechanism of transposition, TEs can be generally classified into (1) RNA-mediated class I elements (retrotransposons), including long terminal repeats (LTRs), long interspersed nuclear elements (LINEs), and short interspersed nuclear elements (SINEs); and (2) RNA-independent class II elements (DNA transposons)10. TE classes could be further divided into distinct families or subfamilies based on their age (active period) and DNA sequence characteristics.TEs and common repeats are ubiquitous sequences that can copy and insert themselves throughout the eukaryotic and prokaryotic genomes11. Thanks to the availability of whole-genome sequence of various species and the ongoing development of bioinformatics tools15, our knowledge of TEs has progressed at a fast pace. TEs are known to play an essential role in shaping genomic sequences and contributing to the diversity in genome size and chromosome structure17. Most TEs, in fact, are fixed, inactive, and not randomly distributed in the genome19. However, several TE families are still actively transposing and serving as a major source of genetic polymorphisms between individuals, such as the Alu, L1, and SVA TE families in the human genome20.At the predominant view of the 1960s\u20131990s, TEs were described as selfish or junk DNA24. For example, the polymorphic TEs detected in the 1000 Genomes Project, consisting of 16,192 loci in 2504 individuals across 26 human populations, successfully recapitulated human evolution and captured the signal for positive selection on recent human TE insertions26.It is evident in many species  that the impacts of active TEs on genome evolution are wide-ranging, including admixture, adaptation, footprints of selection, and population structure30. TEs can disrupt the existing cis-regulatory elements, such as promoters, enhancers, and insulators, or provide novel ones34. They can also serve as a rich source of non-coding RNAs, including lncRNAs, circRNA, small RNAs, and microRNA targets38. Moreover, the silencing of TEs has a close connection with epigenetic regulatory mechanisms, such as DNA methylation, piRNA, histone modifications, and RNA interference42. Importantly, it has been reported that the complex interactions between TEs and epigenetic elements could allow for rapid phenotypic adaptation to environmental changes43.In addition to their direct influence on DNA sequence, there is also emerging evidence that TEs have important functional contributions to gene regulatory networks and epigenome variation. For instance, TEs can directly affect gene transcriptional structure by provoking various forms of alternative splicing, including exonization, exon skipping, and intron retention (3\u2032 and 5\u2032), to generate novel protein-coding sequences or premature endsSus scrofa), one of the earliest domesticated animals, is estimated to have been domesticated approximately 10,000 years ago in Asia and Europe independently44. It serves as an indispensable source of animal protein and an important biomedical model for humans46. Currently, a total of 22 pig assemblies are publicly available on NCBI51, accompanied by the availability of massive high-throughput whole-genome sequences. These provide researchers with ideal materials to advance the current development of genomic research in pigs. However, the study of TEs in the pig genome is still in its infancy. A few previous studies have mainly focused on their diversity and distribution51, yet the functional and evolutionary importance of TEs in pigs has largely been overlooked. In our recent study52, we identified novel introgressions in Eurasian boars from Asian and European pig populations using SINE (PRE-1 subfamily) polymorphisms, suggesting that a portion of TEs are still active in the current pig genome. However, these studies are far from sufficient to comprehensively understand the important roles of TEs in pigs.Pig , and DNA methylation. We estimated the contribution of active SINEs to tissue-specific gene expression by cross-examining 3570 published RNA-seq samples from 52 tissues and 27 cell types. Furthermore, we created an atlas of SINEs using 374 whole-genome sequence data to study the roles of young SINEs in pig population admixture and local adaptation. The TE-mediated adaptation has been found in functional regions, such as the almost fixed dimorphic SINEs observed in laboratory-inbred Bama Xiang pigs at the upstream region of the Sus scrofa 11.1). This pipeline used a combination of similarity-, structure-, and de novo-based methods. We also classified all potential pig TEs into classes/superfamilies and families and derived their consensus sequences based on existing TE repositories (RepBase update and Dfam 2.0 databases).To thoroughly detect TEs, we developed the Pig TE Detection and Classification (PigTEDC) pipeline Fig.\u00a0 and appl51, LTR (9.25%), LINE (27.57%), and SINE (54.95%) were the most common retrotransposons, whereas DNA transposons only accounted for 8.12% of TEs  of the pig genome. Two-thirds of TE copies (insert in the genome) were assigned to a specific family, with retrotransposons being the most common type (~90%). Similar to previous studiesTEs Fig.\u00a0. SINE beTEs Fig.\u00a0.53. Obviously, most TE families amplified around 70\u201350 Mya (divergence at 30\u2009\u00b1\u20095%). This was during the Paleocene Epoch (65\u201354 Mya) which created new ecological niches for surviving mammals, birds, reptiles, and marine animals54. The most recent burst of TEs was mainly related to the SINE/tRNA, LINE/L1, and LTR/ERV1 families. Among these, SINE/tRNA remains the most active in the modern pig genome57. Further exploring the ages of highly homologous subfamilies in SINE classes  make up 84.6% of all classified TEs Fig.\u00a0. These iWe next analyzed young SINE families  to classify them into subfamilies with high resolution. We examined all the full-length young SINEs from 14 publicly available pig genomes, identified 978,506 non-redundant young SINEs, and created their consensus sequence through multiple sequence alignment  structure variations (SVs) in 14 assemblies to the pig reference, we found that most polymorphic SINEs belonged to the PRE1-SS, PRE1a, and PRE0-SS families, accounting for an average of 90.75% of the medium-length SVs Fig.\u00a0. Especia61. To test this, we examined how SINE subfamilies affect genome features such as 3D chromatin architecture62, chromatin accessibility, histone modifications, TFBS, and DNA methylation after only mapping unique reads , while there was a depletion in the B compartments (inactive)  class II promoter binding proteins that functioned as trans-activators of the hepatitis B virus enhancer73.TEs carrying TFBS may contribute to the regulation of genesons Fig.\u00a0. In tota74, we analyzed the epigenetic states of SINE families by examining DNA methylation (MeDIP), density of CG (CpG) sequence contexts, and AT:GC content (CpG islands)  Fig.\u00a0. The resds) Fig.\u00a0.75. We further distinguished small non-coding RNAs into three classes to investigate the relationship between piRNA density and SINE families  were TE-related transcripts (the transcripts containing TE sequences)83. Notably, 68.81%  of the TE-derived transcripts were recognized as SINE-associated transcripts inserted by nearly full-length SINE (average coverage of 87.76%). This suggests that TE-derived transcripts, particularly SINEs, are abundant in the pig transcriptome. We next classified 337,746 young (younger and youngest) SINE-associated transcripts into four categories based on their genomic location compared to known transcripts in the pig genome annotations81 85. For example, we found a full-length PRE0-SS was inserted in the 3\u2032-UTR of the pig PDK1 gene, which is consistent with a previous report that Alu and B1 regulate both human and mouse orthologs of PDK1 by SMD86. Besides, we found that young SINEs that produced transcripts (Young-T) had lower average CG methylation levels than all young SINEs in most tissues . Normalized gene expression by DESeq288 allowed us to create t-SNE plots that is mostly consistent with tissue types , with most modules showing high tissue specificity and playing key roles in particular organ systems in pigs , brain development (GO:0007420), and neuron projection morphogenesis (GO:0048812)  using a uniform sequencing depth of 10\u00d7  were rare, with minor allele frequencies of less than 5% in the entire pig population  showed genetic separation between Asian and Western breeds  analysis of dimorphic SINE genotypes distinguished four species of the Suidae Fig.\u00a0. PC1 sepeds Fig.\u00a0. Korean eds Fig.\u00a0, consistFsti values and alpha coefficients (using Bayescan)97 to measure their divergence in allele frequencies at specific loci. Loci with higher Fst values and positive alpha values indicate positive selection. We found 337 dimorphic SINEs with high Fsti and positive alpha coefficient in the gene functional regions, including exon, splice, UTR5, UTR3, and upstream regions, of 330 genes (Supplementary Data\u00a0n\u2009=\u2009223) and ISEA (n\u2009=\u200942), while the remaining 75 were linked to breed-specific traits of domestic pigs  and SIRT1 (UTR3) genes having the dimorphic SINEs with a perfectly fixed frequency.A fixed dimorphic SINE was found in the first exon of the igs Fig.\u00a0. RUNX3 g104  data associated with phenotypic traits104 Fig.\u00a0. Four ofr2\u2009>\u20090.3) with T-SNPs were identified using 296 domestic pigs (109 Asian and 187 European). Specially, it was found that these dimorphic SINEs were more prevalent in the TxFlnkWk (Weak transcribed at gene), indicating their potential for gene regulation  from 79 published GWAS studies of 97 complex traits in pigs, including reproduction, production, meat and carcass, health, and exterior traits , such as the nervous system , reproductive system , and muscle satellite cells  linked to intramuscular fat composition, containing six dimorphic SINEs and eight genes. Two genes, C14H10orf76  and GBF1 (r2\u2009=\u20090.86), are essential for Golgi maintenance and secretion106. The ELOVL3 gene is a strong candidate gene for fatty acid composition108. A low-frequency dimorphic SINE was found in its intron region, while multiple T-dimorphic SINEs were found within its upstream region of 15 to 50\u2009kb. The dimorphic SINE  near 27\u2009kb upstream was found at high frequency in Chinese domestic pigs, especially Southern Chinese domestic pigs  was observed between the dimorphic SINE  located in the ANK2 intron and the T-SNP linked to C14:0, C16:0, and C16:1n7 fatty acid content in backfat109. Ankyrin-B (AnkB), an alternatively spliced variant of ANK2, is linked to obesity susceptibility in humans110. We found that the insertion of the T-dimorphic SINE was almost fixed in Western domestic pig populations  between a T-SNP  and a T-dimorphic SINE  in the first intron of the VRTN gene\u2014the gene suggested to be associated with teat number and the most promising candidate gene to increase the number of thoracic vertebrae (ribs) in pigs113. We observed a clear difference in frequency of the dimorphic SINE between Chinese indigenous breeds and commercial breeds  of the pig genome is made up of TEs, mainly non-LTR retrotransposons (SINE and LINE). SINE is shorter and more complete than LINE. Similar to our previous findingsGene regulatory network is influenced by genomic components, chromatin accessibility, histone modifications, DNA methylation, and cis-regulatory elements such as TFBS, promoters, and enhancers. TEs linked to specific chromosome features can impact gene regulatory networks in multiple ways as listed above. To our knowledge, this is the first time that large-scale multi-omics data were used to fully explore the relationships between TEs and chromosome features in the pig genome. Our findings showed that SINEs were highly enriched in the A compartment, and that the enrichment of SINEs in chromatin was associated with their ages. For instance, young SINE families were frequently enriched in close chromatin-like nucleosomes but highly depleted from open chromatin.As expected, SINEs were highly depleted from all active chromatin tags, and more signals of constitutive heterochromatin tags (H3K9me3 peaks) were observed on SINEs. The exception was H3K27me3, which was associated with facultative suppressor genes and cannot permanently silence SINEs. Most histone modifications in SINE decreased as the TE\u2019s age increased, which was in line with the distribution of DNA methylation on SINE and its contribution to TFBS. However, young SINEs, especially the youngest SINE family, were highly enriched in weakly active enhancer regions of hypothalamus tissue (Fold >1.5). We speculate that the relationship between SINE and its host genome is a combination of both arms race and co-evolution, depending on how the symbiosis turned out.In the former case of parasitism, the young SINEs were more likely to be treated as new invaders that were constitutively silenced by histone modifications and DNA methylation of the host genome , while the old SINEs mutated and gained new regulatory potential, and thus were tolerated or even co-opted by the pig genome. In the latter case of mutualism, there might be rare cases where the SINEs were positively selected by nature, thereby helping the host genome better adapt to the local environment in the long run.The use of long-read isoform sequencing provided us a more complete characterization of full-length transcripts, which made it possible to identify the young SINE-associated transcripts. Meanwhile, the Iso-seq reads we used here were collected from ~40 pig tissues, which ensured the investigation of the abundance and tissue specificity of young SINE-associated transcripts.116, suggesting that SINE insertions may be a crucial component of genes and regulate tissue-specific expression of their target genes.Our findings showed that the vast majority of young SINE-associated transcripts were non-coding RNAs that covered exons or fell within introns. A total of 3112 genes were found to be associated with young SINE-associated transcripts, and nearly 88% of them were enriched in co-expressed modules with high tissue specificity. The transcripts derived from young SINEs exhibited lower levels of CG methylation and were more enriched in open chromatin and histone modifications than whole young SINEs. Specifically, some young SINEs exhibited strong and consistent tissue specificity in both transcript expression and epigenetic regulation. This is consistent with previous findings in other speciesRef+ and Ref- detection  had robust performance in both The contribution of TEs was underestimated in pig genomic research, despite the active role of SINEs under selective pressure. However, our understanding of SINEs in pig population genetics is limited without a comprehensive map of dimorphic SINEs based on large-scale re-sequencing data.Fst value can help us understand local adaptation in domestic pigs and identify candidate genes for economically important traits. Our findings confirm previous studies  and identify new candidate genes.We genotyped and analyzed 211,067 dimorphic SINE loci in 374 individuals from 25 pig populations. These loci showed high variability in allele frequencies among populations. Based on these SINEs, we identified ten major clusters that corresponded to geographic differentiation. These SINEs with high pairwise VRTN gene. Future research is needed to validate how these dimorphic SINEs regulate target genes in specific tissues  and affect complex traits.GWAS studies have discovered thousands of QTLs for important pig traits based on SNPs, but most of these loci remain functionally uncharacterized. One possible reason is that phenotypic changes may be affected by SVs (TEs) in linkage disequilibrium with SNPs. In this study, 127 dimorphic SINEs were found to be in linkage disequilibrium with significant GWAS SNPs of complex traits, and nearly a third of them showed high tissue specificity in expression. Some of these dimorphic SINEs can generate novel transcripts (in H3K4me1 and H3K27me3), as exon-covered transcripts were found upstream of the The PigTEDC pipeline was composed of three TE detection approaches using different algorithms to achieve its overall efficiency:http://www.repeatmasker.org), which search sequence using the TE consensus sequence from RepBase Update (https://www.girinst.org/repbase/) and Dfam 2.0 databases117 against homologous regions of the pig genome. The method was used to detect all known TEs, including DNA transposons, LTR, SINE, and LINE.The similarity-based method was represented by the widely known RepeatMasker (V4.0.6) 119 and LTR-Finder (V1.07)120 were used to enrich the LTR results, and SINE-Finder (V1.1)121 was used to increase SINE detection.The structure-based method was used to capture the particular TE families based on their known sequence structure and motifs, which further enhanced the results of the similarity-based method to enhance the power of detecting known TEs. In our pipeline, HelitronScanner (V1.1)http://www.repeatmasker.org/RepeatModeler/). Red122 and P-Clouds123 were used to capture all potential repetitive sequences that included TEs and simple repeats in the pig genome using machine learning and oligonucleotide clustering methods, respectively.The de novo-based method was used to identify the missing pig TEs from the known TE database, which employed the clusters of the repetitive sequences in the genome based on various methods. There were a total of three tools in this category. RepeatModeler (V1.0.11) was used to automate the runs of RECON  and RepeatScout (Consensus seed clustering) for the pig genome . The parameters of cross_match was set as \u201c-gap_init \u221225 -gap_ext \u22125 -minscore 10 -minmatch 6 -alignments -bandwidth 50 -word_raw\u201d. Parts of TEs failed to pass the paired match with their family consensus sequences, thus were regarded as overly fragmented TEs. The passed TEs consisting of full-length and fragmented TEs, had clear and specific family classifications.Match of known TEs with their family consensus sequences. Considering the different TEs have different coverage towards their family consensus sequences, we located the coverage area for each TE family using cross_match software  and picked the representative one of each cluster as the consensus sequence for each unknown TE family. Finally, to obtain the genomic locations of unknown TEs, we performed the genome-wide identification for each unknown TE family using RepeatMasker with the custom-build library.124. The divergence levels were corrected for CpG content by DCpG = D/(1\u2009+\u20099FCpG)125. Histograms with a bin size of 0.01 were plotted to show the distribution of divergence levels. Activity periods were estimated assuming a substitution rate of 5\u2009\u00d7\u200910\u22129 substitutions/site per year127. Distribution histograms for sequence divergence of TE were plotted using a 0.01 bin size.Divergences of TEs from consensus sequences were obtained using the Kimura two-parameter model from RepeatMaskerAnnotation of young SINEs on 14 genome assemblies. For each pig genome assembly, we employed RepeatMasker to search for young SINEs in our customized repeat library . And then \u201cparseRM_GetNesting.pl\u201d was used to remove the nesting and nested young SINEs, followed by a length filtering to remove elements with length <200\u2009bp and >300\u2009bp using a shell script.128 with the parameter that \u201c-T 0 -c 0.9 -M 0 -n 5 -p 0\u201d. we finally retained 52 of 17,294 SINE clusters (n\u2009>\u2009500) involving 1,157,133 young SINEs.Clustering and filtering of young SINEs. To ensure that the young SINEs used for subfamily classification were highly homologous, we utilized a clustering-based approach to keep all non-nested young SINEs from 14 genomes with a sequence identity of >90%, implemented in CD-HIT-ESThttps://github.com/shenwei356/seqkit).Removing the duplicated sequences of young SINEs. Due to the existence of shared SINEs from different pig genomes, we removed the completely duplicated sequences from our young SINE datasets using seqkit (http://web.mit.edu/meme_v4.11.4/share/doc/motif-consensus.html), we scanned each column in a letter\u2019s  frequency matrix of MSA using the \u201c50% rule\u201d that any letters with frequency less 50% of the maximum were discarded. Finally, a consensus sequence with a length of 261\u2009bp was created for the subfamily classification.Construction of consensus sequence. Because the running time and memory usage for multiple sequence alignment (MSA) of large-scale genomic sequences can be enormous, we randomly extracted 100,000 young SINEs to conduct the MSA. Using MEME suite programs (129 (V7.407) at the default setting. Then, IQ-TREE130 was used to create maximum likelihood (ML) trees for SINE (with 100 fast bootstrap replicates). Finally, EVOLVIEW131 was used to visualize the phylogenetic tree for SINE families.26 SINE families were used to construct the phylogenetic tree. Multiple sequence alignments of their consensus sequences were performed with maffthttp://www.repeatmasker.org/COSEGDownload.html). First, sequence homology analysis was done using cross_match software (https://www.phrap.com/) with the parameters . Then, \u201cpreprocessAlignments.pl\u201d was used to determine the consensus range and create input files for COSEG programs with the following parameters: the minimum distance between sites (-maxEdgeGap) was 10; the consensus sequence ranged from 1 to 251\u2009bp  was used132; 2\u2009bp (-t) co-segregating mutations were used when developing subfamilies.We conducted subfamily classification for young SINEs using the COSEG pipeline  for each genome assembly using Minimap2 with the following parameters: \u201c-c -x asm20 \u2013cs\u201d. Then, we used paftools.js (a JavaScript script within Minimap2) to identify the confident/callable regions and call the variants from the asm-to-ref alignment with the following parameters: \u201ccall -L1000\u201d.Detection of SVs among the 14 genome assemblies. We employed Minimap2 (V2.17)Identification of dimorphic SINEs from SVs. We first retained the SVs that had a similar size (>200\u2009bp and <300\u2009bp) with overlapping SINE using a shell script. Then, the inserted sequences of the resulting SVs were then aligned against the consensus sequences of SINE families and young SINE subfamilies using BLASTn version 2.2.31+ with default parameters. Finally, the SINE families or subfamilies of these SVs were defined by their best high-quality match between consensus sequences of SINE families or subfamilies.62. MNase-seq reads were filtered with Trim_galore tools to obtain and retain high-quality reads with the following parameters: \u201c-q 20 --phred 33 --stringency 3 -e 0.1\u201d. The filtered reads were then aligned to the Duroc reference genome (Sus scrofa 11.1) using the BWA134. DANPOS2135 (V2.26) was then used to call nucleosome binding peaks and to generate nucleosome occupancy profiles with the following parameters: \u201c--span 1 --smooth_with 20 --wideth 40\u201d, which were further normalized with the mean score of the whole genome.The 3D chromatin architecture and ATAC-seq used in this study, including A/B compartments, Hi-C, and TADs, were downloaded from the FRAGENCODE project136 (V2.1.1) with the following parameters: \u201c-q 0.05\u201d. The computeMatrix module from deepTools137 (V.3.5.0) was used to transform and compute the corresponding data matrix from the modification signal over a set of SINE regions. The plotProfile module was used to turn the compressed matrix into summary plots.The histone modifications used in this study include four active epigenetic marks  and two repressive marks (H3K9me3 and H3K27me3). Histone modification data were quality-filtered using Trim_galore tools with the following parameters: \u201c-q 20 --phred33 --stringency 3 -e 0.1\u201d. ChIP-seq data were aligned to the Duroc reference genome using BWA. Peaks were called using MACS266, we defined 15 distinct chromatin states across 14 tissues and grouped them into the following seven categories: (1) promoters included TssA (Strongly active promoters/transcripts), TssAHet (Flanking active TSS without ATAC), and TssBiv (Transcribed at gene); (2) TSS-proximal transcribed regions included TxFlnk (Transcribed at gene), TxFlnkWk (Weak transcribed at gene), and TxFlnkHet (Transcribed region without ATAC); (3) enhancers included EnhA (Strong active enhancer), EnhAMe (Medium enhancer with ATAC), EnhAWk (Weak active enhancer), EnhAHet (Active enhancer no ATAC), and EnhPois (Poised enhancer); (4) repressed regions included Repr (Repressed polycomb) and ReprWk (Weak repressed polycomb); (5) quiescent regions; (6) ATAC_Is (ATAC island); (7) TssBiv .As described in a previous study138 from the MEME Suite139 to look for their occurrences in the Duroc reference genome for the 746 Transcription Factors (TFs) cataloged in the Vertebrate 2020 JASPAR database140. We set the parameters of fimo software as \u201cp-value\u2009=\u20091e-4\u201d, \u201c--max-stored-scores 100000000\u201d and \u201c--alpha=1\u201d. Among the predicted TFBS, those obtained from the 31 known TF motif were used to profile the resulting density of different SINE families.To identify TFBS genome-wide, we used the FIMO software141 with the following parameters: \u201c-q 20 -u 30 -l 30 -w 16\u201d. MeDIP-Seq data were aligned to the Duroc reference genome using Hisat2142. Duplicate reads were removed from the bam files using Sambamba tools143. The filtered bam files were used to identify the MeDIP-enriched regions based on a clustering approach using SICER2144.MeDIP-Seq data were quality-filtered using Fastp tools145 (V0.23.0) with the parameters that \u201c--ambig-bam\u201d. The statistical analysis and visualization of DNA methylation levels on TEs and the whole genome were performed using the methPlot script from BatMeth2146, and the R language utilizing the \u201cdata.table\u201d and ggplot2 packages.The WGBS data were aligned to the Duroc reference genome using Bismark147. (2) We used the \u201ccollapse_reads_md.pl\u201d script of Mirdeep2148 (V0.1.2) to remove repetitive sequences. (3) We downloaded known pig piRNAs from piRBase149 (V3.0) (http://bigdata.ibp.ac.cn/piRBase/) and used Bowtie to map the collapsed reads to them, saving the unaligned reads. (4) We used Mirdeep2\u2019s mapper and miRDeep2 to identify candidate microRNAs and their genomic locations based on these unaligned reads. (5) We mapped the remaining reads that did not align to piRNAs or miRNAs to the Duroc reference genome using Mirdeep2\u2019s mapper, filtering for a length of 21 nt to identify candidate siRNAs. We excluded any small non-coding RNAs that had multiple hits in the reference genome.To study non-coding RNAs in young SINE families, we did the following steps: (1) We merged all raw data and removed adapters with sRNAseqAdapterRemover from TBtools82 software with parameters \u201c-k 21 -s 3\u201d, we processed the raw data by their corresponding RNA-seq data. After merging the error-corrected Iso-seq reads, we aligned the consensus sequence of young SINE families to it using BLAST software with parameters \u201c-evalue 1e\u22125 -max_target_seqs 1\u201d. We identified 2,267,973 candidate TE-derived transcripts containing young TE insertions (transcripts contain more than 80% of the sequence of SINE). As previously studied81, we categorized young SINE-associated transcripts into four groups by comparing their genomic location to known transcripts in the currently available pig genome annotations. These four groups, as shown in Supplementary Fig.\u00a0Using LoRDEC87 to measure the abundance (RNA-seq counts) of genes and SINE-associated transcripts. The RNA-seq counts were transformed to log2-counts per million .To study the cis-functionality of young SINE-associated transcripts, we used Salmon tools88 and built a co-expression network with WGCNA R package89. The network was constructed with the following parameters: \u201ccorType = pearson, maxBlockSize = 20,000, power = 4, minModuleSize = 30, mergeCutHeight = 0.3\u201d. The power was set to 4 , TranSurVeyor154 (https://github.com/Mesh89/TranSurVeyor), MELT155 (https://melt.igs.umaryland.edu/), and RetroSeq156 (https://github.com/tk2/RetroSeq). Their performance was evaluated using Sniffles157 (https://github.com/fritzsedlazeck/Sniffles) with 20\u00d7 PacBio sequencing from the same individual. We compared the counts and verification rates (the proportion of dimorphic SINEs supported by Sniffles) of identified dimorphic SINEs across a range of sequencing depths  to assess their detection powers . We found that the number of dimorphic SINEs increased with the sequencing depth, especially from 5x to 10x that increased the average number of dimorphic SINEs by nearly one-fold , we retained 374 individuals whose sequencing depth was greater than 10x for dimorphic SINEs identification (average mapped bases: 27.17 GB and average mapping rates: 99.43%).We benchmarked four types of dimorphic SINEs detection tools that have been reported to have superior performance in the previous projectshttps://github.com/OpenGene/fastp) with the following parameters: The quality value that a base was qualified (-q) was 20; The percent of bases were allowed to be unqualified (-u) was 30; The required length of kept reads (-l) was 50; Step 2: Read alignment. Clean reads from all individuals were aligned to the Duroc reference genome (Sus scrofa 11.1) using BWA134 (https://github.com/lh3/bwa) with the BWA-MEM algorithm; Step 3: Processing the alignment files. The mapped reads were subsequently processed for format conversion (view), position sorting (sort), merging (merge), and statistics (stats) using Samtools152 (http://samtools.sourceforge.net/). Then, the MarkDuplicates method from the Picard package (https://sourceforge.net/projects/picard/) was used to remove the PCR duplicates that were introduced during library construction.Preprocessing of next-generation sequencing. The 374 individuals were selected to identify the SNP and dimorphic SINEs, after their sequencing depth was standardized around 10x. We first performed the preprocessing as following steps: Step 1: Quality control. Quality control was conducted for each raw re-sequencing data using the fastp (https://github.com/Sentieon/sentieon-dnaseq) was next used to identify genome-wide SNP in the following steps: Step 4: Indel realignment and Base quality score recalibration (BQSR). The processed alignment bam files were subjected to realign and recalibration with the parameters of \u201cRealigner\u201d and \u201cQualCal\u201d; Step 5: SNP genotype calling. Genotype calling of SNP for each individual was performed using the \u201cHaplotyper\u201d algorithm. Step 6: Merge of VCF files. All 374 g-vcf files were joined together using the \u201cGVCFtyper\u201d algorithm. Then, the newly merged VCF were filtered to retain high-quality SNPs using the vcftools with the following parameters: \u201c--max-missing 0.8 --maf 0.05 --min-alleles 2 --max-alleles 2 --recode\u201d.Genome-wide SNP detection. After preprocessing of the alignments, the DNAseq mode of Sentieon (https://melt.igs.umaryland.edu/) was used to discover the dimorphic SINEs (Ref+ and Ref-) in the following steps: Step 7: Discovery of deletion. With the processed bam files from Step 3, we used the Deletion-Genotype module of MELT to identify the \u201cRef-\u201d dimorphic SINEs based on our previously identified TE sets ; Step 8: Building retrotransposon. Referring to the results of Step 7, we build three customized reference files for PRE1-SS, PRE0-SS, and PRE1a families using the \u201cBuildTransposonZIP\u201d module; Step 9: Discovery of insertion. For each bam file, we carried out the identification of \u201cRef+\u201d dimorphic SINEs using four modules of MELT step by step, including \u201cIndivAnalysis\u201d, \u201cGroupAnalysis\u201d, \u201cGenotype\u201d, and \u201cMakeVCF\u201d.Genome-wide dimorphic SINEs detection. The MELT tool (158. The pig annotation files were downloaded from the NCBI database for the Duroc reference genome (Sus scrofa 11.1). Dimorphic SINEs and SNPs were classified into eight categories based on their genome locations, including exonic regions, splicing sites, intronic regions, 5\u2032 and 3\u2032 untranslated regions (UTRs), upstream and downstream regions, and intergenic regions.All detected dimorphic SINEs and SNPs were processed using gene-based annotations in ANNOVARRef+ and Ref-) from 374 pigs using PLINK159 v1.9 with following parameters: \u201c--maf 0.01 --mind 0.8 --geno 0.8\u201d. Next, we performed principal component analysis on the filtered dimorphic SINEs using PLINK v1.9 with the parameters: \u201c--pca\u201d. We presented the eigenvectors and eigenvalues for each individual in the PCA biplot using R packages.We filtered identified dimorphic SINEs (Ref+ and Ref-) into a pseudo-SNPs dataset (A/T/C/G) by randomly replacing data under the condition that individual polymorphism was not changed. The transformed dataset was reduced to 44,192 dimorphic SINEs using PLINK v1.9 with the following parameters: \u201c--maf 0.1 --indep --pairwise 50 10 0.2\u201d, based on a linkage disequilibrium threshold of 0.2 and a minor allele frequency threshold of 0.1. The phylogenetic tree was constructed with 1000 bootstrap replicates using the maximum-likelihood approach implemented in SNPhylo160 (V1.10.2).To establish evolutionary relationships between individuals, we transformed the dimorphic SINEs dataset (161 (V1.3.0) to analyze population structure in our study. This estimated genetic admixture among different pig breeds using all identified dimorphic SINEs. We tested 14 cases (ranging from K\u2009=\u20092 to 15) to identify genetic clusters for 374 pigs, using default parameters. Results were visualized using StructureSelector162.We used the program ADMIXTURE163, G08: Korean domestic pigs (JEJ and PEN) plus European domestic pigs, G09: European wild boars plus IBE and Yucat\u00e1n miniature pigs, G10: Landrace/Yorkshire crossbreeds (YL)164, and G11: Duroc pigs.As shown in Fig.\u00a0Fsti value and estimated alpha coefficient97 of dimorphic SINEs between cluster i and the remaining clusters to measure their locus-specific divergence in allele frequencies. Pairwise Fsti values were calculated for each dimorphic SINEs using the vcftools165 (V0.1.16) with the default parameters. BayeScan program97 (v2.1) was used to identify putative adaptive dimorphic SINEs. based on different allele frequencies among populations, and we performed it using default settings .We calculated the pairwise ANK2 gene  was performed using Haploview software166. LDBlockShow167 (V1.40) tool was used for the linkage disequilibrium analysis of large regions like 320\u2009kb dimorphic SINEs hotspots .We collected a total of 4072 trait-associated SNPs (T-SNPs) from 79 published GWAS studies of 97 complex traits. These included 18 reproduction, 22 production, 36 meat and carcass, six health, and two exterior traits. We combined the dimorphic SINEs and 4072 T-SNPs into a new variant dataset from 296 domestic pigs (109 Asian and 187 European domestic pigs). We analyzed the linkage disequilibrium between T-SNPs and dimorphic SINEs using PLINK v1.9 with the parameter \u201c--ld\u201d. We only considered dimorphic SINEs with a relative T-SNP distance less than 10\u2009kb and R2 greater than 0.3 as candidate dimorphic SINEs. Linkage disequilibrium analysis for 168, with the number of permutations set to 1000. For the association analysis of young SINE-associated transcripts and genes, we used R code and generalized linear models (GLM) to model transcript expression levels of each gene. We considered the expression level of young SINE-associated transcripts (within their gene body) and the type of tissues or cells as explanatory variables. We used a Bonferroni significance threshold of 1.42\u2009\u00d7\u200910\u22126  as the standard threshold.The Z-score of each chromatin state for each TE group was calculated using permutation tests in regioneRFurther information on research design is available in the\u00a0Peer Review FileSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1-13Reporting Summary"}
+{"text": "Pseudostellaria heterophylla (Miq.) Pax is a popular clinical herb and nutritious health food. However, leaf spot disease caused by fungal pathogens frequently occurs and seriously influences the growth, quality, and yield of P. heterophylla. In this work, the field control roles of difenoconazole, chitosan, and their combination in the leaf spot disease in P. heterophylla and their effects on the disease resistance, photosynthetic capacity, medicinal quality, and root yield of P. heterophylla are investigated. The results manifest that 37% difenoconazole water-dispersible granule (WDG) with 5000-time + chitosan 500-time dilution liquid had a superior control capacity on leaf spot disease with the control effects of 91.17%~88.19% at 15~30 days after the last spraying, which significantly (p < 0.05) exceeded that of 37% difenoconazole WDG 3000-time dilution liquid and was significantly (p < 0.01) higher than that of 37% difenoconazole WDG 5000-time dilution liquid, chitosan 500-time dilution liquid, or chitosan 1000-time dilution liquid. Simultaneously, this combination could more effectively enhance the disease resistance, photosynthetic capacity, medicinal quality, and tuberous root yield of P. heterophylla compared to when these elements were applied alone, as well as effectively reduce difenoconazole application. This study emphasizes that chitosan combined with a low dosage of difenoconazole can be proposed as a green, efficient, and alternative formula for controlling leaf spot disease in P. heterophylla and enhancing its resistance, photosynthesis, quality, and yield. Pseudostellaria heterophylla (Miq.) Pax, a popular clinical herb and nutritious health food, is widely distributed in China, Japan, the Russian Far East, South Korea, and North Korea [2 [Alteraria tenuissima, Phyllosticta commonsii, Septoria sp., Arcopilus versabilis, and Phoma sp. frequently occurs and seriously influences the growth, quality, and yield of P. heterophylla, as well as constantly causing over 50% of economic losses [Bacillus subtilis [P. heterophylla.th Korea . Its drith Korea ,3,4. As th Korea ,6,7. Mosth Korea . CurrentKorea [2 . Despitec losses ,11,12,13subtilis . ConsideP. heterophylla, He et al. [Alteraria tenuissima with an EC50 value of 27.31 \u03bcg mL\u22121. Li et al. [Arcopilus versabilis mycelium was higher than 90%. Li et al. [Phyllostatita commonsii. Although difenoconazole has excellent control potential in leaf spot disease in P. heterophylla, it is substantially and permanently applied to P. heterophylla fields, which might bring several hidden troubles to human health, animals, hydrobios, and the ecosystem [Rose roxburghii, to improve its resistance, photosynthesis, yield, quality, and amino acid content, as well as to reduce pyraclostrobin application [P. heterophylla, a decline in difenoconazole application and its hidden troubles, and delayed pathogen resistance are worthy of further attention.Difenoconazole, a systemic triazole fungicide, is one of the most long-term and mainstream applied fungicides in the world ,16. Due e et al.  found thi et al.  reportedi et al.  showed tcosystem ,24,25,26lication . In thisPlatycodon grandifloras treated with chitosan soaking were effectively improved, and they found that chitosan also promoted the drought resistance of Sctellaria baicalensis seedlings [R. roxburghii against powdery mildew, and enhanced its photosynthesis and quality [Pinellia ternata [Rosa roxburghii powdery mildew, and reduce their application dosage [P. heterophylla and whether their co-application can more effectively enhance the growth, quality, and yield of P. heterophylla is also worth further study.Chitosan, a natural non-toxic biomolecule from chitin deacetylation, is widely used as a growth promoter, resistance inducer, biological fungicide, or fertilizer in agricultural production ,31,32,33eedlings , induced quality . Our pre ternata . Meanwhin dosage ,41,42,43P. heterophylla. Subsequently, the influences of difenoconazole, chitosan, and their combination on the disease resistance and photosynthetic characteristics of P. heterophylla leaves were investigated, as were the influences of difenoconazole, chitosan, and their combination on the medicinal quality and yield properties of P. heterophylla roots. This work aimed to provide an alternative natural biomolecule-assisted fungicide technology to control leaf spot disease in P. heterophylla and reduce chemical pesticide application.This study evaluated the field control effects of difenoconazole, chitosan, and their combination on leaf spot disease in P. heterophylla are shown in p < 0.01) diminished the disease index of leaf spot disease in P. heterophylla 15 days and 30 days after the last spraying. D 3000 and D 5000 had good control capacities on leaf spot disease in P. heterophylla, with control effects of 89.30~80.13% and 75.44~69.92% at 15 days and 30 days after the last spraying, respectively. Although chitosan exhibited a relatively inferior control potential compared with difenoconazole, the control effects of C 500 and C 1000 on leaf spot disease in P. heterophylla reached 62.96~57.85% and 56.70~53.07% at 15 days and 30 days after the last spraying, respectively. D 5000 + C 500 displayed an optimal control capacity on leaf spot disease in P. heterophylla, with control effects of 91.17% and 88.19% at 15 days and 30 days after the last spraying, respectively, which significantly (p < 0.05) exceeded those of D 3000 and were significantly (p < 0.01) higher than those of D 5000, C 500, and C 1000. Simultaneously, the difenoconazole application dosage of D 5000 + C 500 effectively declined compared with that of D 3000. These results indicate that chitosan had a favorable induced control effect on leaf spot disease in P. heterophylla, and combined with low-dosage difenoconazole, it was more effective at controlling leaf spot disease in P. heterophylla than high-dosage difenoconazole, as well as effectively reducing the application dosage of difenoconazole.The field control effects of difenoconazole and chitosan on leaf spot disease in P. heterophylla leaves are displayed in p < 0.05) increased the soluble protein, total phenols, and flavonoid contents in P. heterophylla leaves; D 5000 and C 1000 slightly increased the soluble protein, total phenols, and flavonoid contents in P. heterophylla leaves; and D 5000 + C 500, D 3000, D 5000, C 500, and C 1000 significantly (p < 0.05) decreased their MDA contents. Moreover, the soluble protein content in P. heterophylla leaves treated with D 5000 + C 500 was significantly (p < 0.05) higher than that in leaves treated with D 3000, D 5000, C 500, or C 1000; their MDA content was significantly (p < 0.05) lower than that in leaves treated with D 3000, D 5000, C 500, and C 1000; their total phenol content was significantly (p < 0.05) higher than that in leaves treated with D 5000; and their flavonoid content was significantly (p < 0.05) higher than that in leaves treated with D 5000 and C 1000. However, there were no significant (p < 0.05) differences in the soluble protein, total phenols, and flavonoid contents in P. heterophylla leaves between D 3000 and D 5000, or C 500 and C 1000, but those for leaves treated with D 3000 and C 500 were slightly higher than those for leaves treated with D 5000 and C 1000, respectively. These results show that chitosan combined with difenoconazole can effectively promote the soluble protein, total phenols, and flavonoid contents in P. heterophylla leaves and decline their MDA content, thereby enhancing their disease resistance to leaf spot disease.The influences of difenoconazole and chitosan on the soluble protein MDA, total phenols, and flavonoids in P. heterophylla leaves are shown in p < 0.05) enhanced the SOD, PPO, PAL, and POD activities in P. heterophylla leaves; C 500 significantly (p < 0.05) promoted their SOD activity; D 5000 significantly (p < 0.05) enhanced their PAL activity; and D 3000, D 5000, C 500, and C 1000 significantly (p < 0.05) improved their POD activity. Furthermore, the SOD activity in P. heterophylla leaves treated with D 5000 + C 500 was significantly (p < 0.05) higher than that in leaves treated with D 3000, D 5000, C 500, and C 1000; their PPO activity was slightly higher than that in leaves treated with D 3000, D 5000, C 500, and C 1000; their PAL activity was significantly (p < 0.05) higher than that in leaves treated with D 5000, C 500, and C 1000; and their POD activity was significantly (p < 0.05) higher than that in leaves treated with D 3000, D 5000, and C 1000. Moreover, the POD activity in P. heterophylla leaves treated with D 3000 and C 500 was significantly (p < 0.05) higher than that in leaves treated with D 5000 and C 1000, respectively. Meanwhile, the SOD, PPO, and PAL activities in P. heterophylla leaves were not significantly (p < 0.05) different between those treated with D 3000 and D 5000, or C 500 and C 1000, but those treated with D 3000 and C 500 showed slightly higher activities than those treated with D 5000 and C 1000, respectively. These results show that difenoconazole in combination with chitosan can effectively improve the enhancing effects of their treatment alone on the SOD, PPO, PAL, and POD activities in P. heterophylla leaves, reliably promoting the disease resistance of P. heterophylla.The influences of difenoconazole and chitosan on the SOD, PPO, PAL, and POD activities in P. heterophylla leaves are shown in p < 0.05) enhanced the chlorophyll content in P. heterophylla leaves; D 5000 + C 500, D 3000, D 5000, C 500, and C 1000 significantly (p < 0.05) enhanced their Pn and Tr contents; D 5000 + C 500, D 3000, D 5000, and C 500 significantly (p < 0.05) improved their Gs content; and D 5000 + C 500 significantly (p < 0.05) increased their Ci content. Moreover, the chlorophyll, Pn, Tr, Gs, and Ci contents in P. heterophylla leaves treated with D 5000 + C 500 were 6.04 mg g\u22121, 4.03 \u03bcmol m\u22122 s\u22121, 2.15 mmol m\u22122 s\u22121, 5.03 \u00d7 10\u22122 mol m\u22122 s\u22121, and 3.18 \u00d7 102 \u03bcmol mol\u22121, which were increased by 1.01, 1.04, 1.01, and 1.03 fold; 1.06, 1.10, 1.03, and 1.07 fold; 1.06, 1.16, 1.11, and 1.14 fold; 1.05, 1.08, 1.03, and 1.07 fold; and 1.03, 1.08, 1.04, and 1.08 fold compared with D 3000, D 5000, C 500, and C 1000, respectively. Additionally, the chlorophyll, Pn, Tr, and Gs contents in P. heterophylla leaves treated with D 3000 were significantly (p < 0.05) higher than in those treated with D 5000, and the Pn and Gs contents in P. heterophylla leaves treated with C 500 were significantly (p < 0.05) higher than in those treated with C 1000. Nevertheless, the WUE content in P. heterophylla leaves was not significantly (p < 0.05) different between all treatments. The results demonstrate that chitosan used in combination with low-dosage difenoconazole can effectively promote the chlorophyll, Pn, Tr, Gs, and Ci contents in P. heterophylla leaves compared with treatments using difenoconazole or chitosan alone, favoring growth.The influences of difenoconazole and chitosan on the chlorophyll, Pn, Tr, Gs, Ci, and WUE contents in P. heterophylla roots are shown in p < 0.05) improved the ash, extractum, and polysaccharide contents in P. heterophylla roots, and D 5000 + C 500, D 3000, D 5000, C 500, and C 1000 significantly (p < 0.05) enhanced their total saponin contents. Moreover, the ash and extractum contents in P. heterophylla roots treated with D 5000 + C 500 were significantly (p < 0.05) higher than in those treated with D 5000 and C 1000, and their polysaccharide and total saponin contents were significantly (p < 0.05) higher than in those treated with D 3000, D 5000, C 500, and C 1000. Simultaneously, the ash, extractum, and total saponin contents in P. heterophylla roots treated with D 3000 and C 500 were significantly (p < 0.05) higher than in those treated with D 5000 and C 1000, respectively, and their polysaccharide contents were not significantly (p < 0.05) different from those in roots treated with D 3000, D 5000, C 500, and C 1000. These findings show that the co-application of chitosan and difenoconazole can more effectively improve the medicinal qualities of P. heterophylla roots.The influences of difenoconazole and chitosan on the ash, extractum, polysaccharide, and total saponin contents in P. heterophylla roots are shown in P. heterophylla roots. D 5000 + C 500 increased the yield capability of P. heterophylla with a root length, root diameter, fresh weight, and dry weight of 5.65 cm, 0.40 cm, 228.54 g m\u22122, and 44.16 g m\u22122, which were increased by 1.04, 1.08, 1.05, 1.07, and 1.09 fold; 1.05, 1.14, 1.05, 1.18, and 1.21 fold; 1.03, 1.09, 1.10, 1.01, and 1.17 fold; 1.03, 1.09, 1.08, 1.12, and 1.19 fold; and 1.03, 1.08, 1.04, and 1.08 fold compared with D 3000, D 5000, C 500, C 1000, and the control, respectively. The results show that using chitosan combined with low-dosage difenoconazole can more effectively enhance P. heterophylla\u2019s root growth and yield increase.The influences of difenoconazole and chitosan on the root length, root diameter, fresh weight, and dry weight of Difenoconazole WDG of 37% was obtained from Jiangxi Heyi Chemical Co., Ltd. . Chitosan with more than 90% deacetylation was purchased from Mingrui Bioengineering Co., Ltd. . The other chemicals were of chromatographic or analytical grade.P. heterophylla was carried out in a P. heterophylla herbal garden in Wujiatang village, Niudachang Town, Shibing country, Guizhou Province, China . P. heterophylla of the \u2018Shitai 1\u2019 cultivar was cultivated via ridging, where each plot area was 3.0 m2  and the application dosage of seed roots was 300 kg per 667 m2. The previous crop in the herbal garden was also P. heterophylla. The annual sunshine, annual rainfall, average temperature, average altitude, and frostless season in the P. heterophylla herbal garden were 1197 h, 1060 mm, 15.0 \u00b0C, 943 m, and 274.5 d, respectively. Additionally, P. heterophylla herbal garden.The field control experiment on leaf spot disease in P. heterophylla: (1) a 37% difenoconazole WDG 5000-times + chitosan 500-times dilution liquid (D 5000 + C 500), (2) a 37% difenoconazole WDG 3000-times dilution liquid (D 3000), (3) a 37% difenoconazole WDG 5000-times dilution liquid (D 5000), (4) a chitosan 500-times dilution liquid (C 500), (5) a chitosan 1000-times dilution liquid (C 1000), and (6) clear water (control). Each treatment had 3 replicates with a total of 18 plots. Fungicide or chitosan dilution liquid was sprayed on the above-ground parts of P. heterophylla plants using an electrostatic atomizer  on 26 March, 2 April, and 9 April, respectively. The application amount of dilution liquid was 60 L per 667 m2.A completely randomized method and the foliar spray method were applied to delineate the experimental plots and spray the fungicide or chitosan dilution liquid, respectively. Six treatments were projected to control leaf spot disease in P. heterophylla plants were randomly selected from each area in the east, west, south, north, and central areas in each plot, and a total of thirty plants were used to investigate the total leaf number and diseased leaf number. The incidence grades were classified as follows: grade 0\u2014no disease spots; grade 1\u2014the disease spots are small and few, and their area accounts for less than 5% of the whole leaf area; grade 3\u2014the disease spots are small and numerous, or large and few, and their area accounts for 6~10% of the whole leaf area; grade 5\u2014the disease spots are large and numerous, and their area accounts for 11~20% of the whole leaf area; grade 7\u2014the disease spots are large and numerous, or multiple disease spots are connected to form large disease spots, and their area accounts for 21~50% of the whole leaf area; and grade 9\u2014the disease spot area accounts for more than 51% of the whole leaf area. Furthermore, Equations (1) and (2) were used to calculate the disease index and control effect of leaf spot disease in P. heterophylla, respectively.The disease index of leaf spot disease in P. heterophylla was investigated on 24 April (15 days after the last spraying) and 9 May (30 days after the last spraying) as described by Wang et al. , with slP. heterophylla on 9 May, as described by Wang et al. [P. heterophylla leaves was ground and homogenized with 10 mL of 80% methanol, extracted in a refrigerator at 4 \u00b0C for 24 h, and then centrifuged at 5600\u00d7 g for 10 min. The supernatant was kept at \u221220 \u00b0C to determine the total phenols and flavonoid contents. An amount of 5.0 mL of water, 1 mL of Folin\u2019s reagent, and 1.5 mL of a 20% sodium carbonate solution were successively added to 1.0 mL of the supernatant, shaken well, and then sheltered from light at 35 \u00b0C for 0.5 h. The light absorption value was determined at 765 nm, corresponding to the gallic acid standard curve to determine the total phenol content in P. heterophylla leaves. An amount of 7 mL of methanol was added to 1 mL of the supernatant in a centrifuge tube, and 6 mL of methanol and 1 mL of a 2% zirconium dioxide methanol solution were added to 1 mL of the supernatant in another centrifuge tube, after which they were mixed and bathed in water at 30 \u00b0C for 60 min. The light absorption values of the two tubes were measured at 420 nm, taking their difference as corresponding to the rutin standard curve to measure the flavonoid content in P. heterophylla leaves. Simultaneously, the superoxide dismutase (SOD), polyphenoloxidase (PPO), phenylalaninammonialyase (PAL), and peroxidase (POD) activities in P. heterophylla leaves were measured using the nitrogen blue tetrazole, catechol, trans-cinnamic acid, and guaiacol methods, respectively.Fresh leaves of the same orientation were collected and taken to the laboratory to determine the disease resistance parameters of g et al.  and Zhang et al. ,45. The \u22122 s\u22121 at 8:00~10:00 a.m. Meanwhile, the chlorophyll a and chlorophyll b contents of P. heterophylla leaves were determined with acetone\u2013ethanol  extraction using a UV-5800PC spectrophotometer at 663 nm and 645 nm, respectively. The chlorophyll content in P. heterophylla leaves was the sum of the chlorophyll a and chlorophyll b contents.Healthy leaves of the same orientation were selected on 9 May to measure their photosynthesis parameters according to Chen et al.  and ZhanP. heterophylla in each plot were collected and washed on 30 June. The average length and diameter of 100 roots in each plot were measured using a ruler and vernier scale, and the fresh weight and dry weight of the P. heterophylla roots in each plot were also determined using the gravimetric method. Moreover, the ash, extractum, polysaccharide, and total saponin contents in the P. heterophylla root were checked according to the general principles of four parts of the Chinese Pharmacopoeia 2020 [The underground roots of eia 2020 .All data are displayed as mean values \u00b1 standard deviation (SD). The significant differences in the data were analyzed using Duncan\u2019s test with one-way analysis of variance (ANOVA) with SPSS 18.0 software . Origin 10.0 software  was applied to draw the figures.P. heterophylla caused by Alteraria tenuissima, Arcopilus versabilis, and Phyllostatita commonsii [R. roxburghii [Alternaria tenuissima and Lasiodiplodia theobromae with a control effect of 56.61% [P. heterophylla, with control effects of 91.17% and 88.19% at 15 days and 30 days after the last spraying, respectively, which significantly (p < 0.05) exceeded those of the 37% difenoconazole WDG 3000-times dilution liquid and was significantly (p < 0.01) higher than those of the 37% difenoconazole WDG 5000-timex dilution liquid, chitosan 500-times dilution liquid, and chitosan 1000-times dilution liquid. These results indicate that the control effect of difenoconazole on leaf spot disease in P. heterophylla was significantly enhanced with chitosan, and their co-application effectively reduced the application dosage of difenoconazole. The mechanism of this synergistic effect may derive from difenoconazole\u2019s and chitosan\u2019s abilities to prevent and kill fungal pathogens and chitosan\u2019s ability to induce plants\u2019 disease resistance. Difenoconazole is a systemic, broad-spectrum, and mainstream triazole fungicide that can inhibit the ergosterol biosynthesis in the fungal pathogenic cell membranes of various plant diseases such as anthracnose, scab, false smut, powdery mildew, etc. ,18,19,20ommonsii ,13,22. Mommonsii ,48,49,50xburghii  and induf 56.61% . In this2O2 [P. heterophylla leaves and reduce their MDA content compared with the application of difenoconazole or chitosan alone, thereby more reliably improving the disease resistance of P. heterophylla against leaf spot disease. These results emphasize that the induced disease resistance performance of chitosan had a positive role in the efficient control of leaf spot disease via the combination of difenoconazole and chitosan.Proteins are the life activity basis of plants, and the disease-course-related proteins plants contain are considered an important indicator of induced resistance. MDA also reflects the lipid peroxidation degree of cell membranes ,41,51. P2O2 ,41,51. A2O2 ,48,49,502O2 . In thisR. roxburghii, P. Grandiflorus, S. baicalensis, P. ternata, Actinidia chinensis, etc. [P. heterophylla leaves compared with treatment using high-dosage difenoconazole or chitosan alone, which was related to the protection of P. heterophylla from leaf spot diseases via the combination of difenoconazole and chitosan and their growth promotion effects. The unique medicinal and nutritional functions of P. heterophylla are determined by the various active ingredients in their tuberous roots, including polysaccharides, saponins, and minerals [P. heterophylla roots and their root length, root diameter, fresh weight, and dry weight. These results emphasize that chitosan is an effective natural adjuvant of low-dosage difenoconazole in enhancing the medicinal quality, root growth, and yield increase of P. heterophylla.Notably, triazole fungicides have a good growth-regulating effect on plants, which can effectively enhance plants\u2019 photosynthesis and increase carbohydrate accumulation and crop yields ,54. For is, etc. ,38,39,40is, etc.  indicateminerals . In thisP. heterophylla and enhanced its disease resistance, photosynthesis, quality, and yield, as well as effectively reducing difenoconazole application. The possible mechanism of these positive synergistic effects is as follows: Difenoconazole can prevent and kill fungal pathogens and can be applied as a growth enhancer to promote the photosynthesis, carbohydrate accumulation, and root yield of P. heterophylla. Chitosan can induce the disease resistance of P. heterophylla and enhance its growth, photosynthesis, quality, and yield. Furthermore, the difenoconazole dosage in combination with chitosan is very low (5000-times dilution liquid), chitosan is natural and non-toxic, and the safe interval time of 80 days is very long. Hence, the potential food risk caused by their combination is almost nonexistent. This work proposes the 37% difenoconazole WDG 5000-times + chitosan 500-times dilution liquid as a feasible candidate formula for controlling leaf spot disease in P. heterophylla and reducing chemical pesticide application.In recent years, the reduced use of chemical pesticides and supplemental or alternative methods for plant disease management have attracted increasing attention ,41,56. SP. heterophylla was effectively improved using chitosan. Moreover, chitosan combined with low-dosage difenoconazole could more effectively enhance the disease resistant substance contents and resistant enzyme activities of P. heterophylla leaves compared to their alone application. Meanwhile, the co-application of chitosan and difenoconazole effectively improved the enhancing effects of their treatment alone on the photosynthetic capacity, medicinal quality, and tuberous root yield of P. heterophylla. This work highlights that chitosan combined with low-dosage difenoconazole can be used as an efficient and green candidate formula for controlling leaf spot disease in P. heterophylla and reducing chemical pesticide application.In conclusion, the control effect of low-dosage difenoconazole on leaf spot disease in"}
+{"text": "These types of plants are undervalued, because there is a lack of knowledge about their bioactive composition and nutritional/functional potential. (2) Scope and Approach: The main aim of this review is to fully identify the potential uses and importance of WEPs in certain regions based on (i) their sustainability, because they grow with their own resources, (ii) their content of bioactive compounds and consequently nutritional and functional value, (iii) their socio-economic relevance, and (iv) their ability to be useful in the agri-food industry in the short term. (3) Results: This review found evidence that a consumption of between 100 and 200 g of some of these WEPs can cover up to 50% of the recommended daily intake of proteins and fiber, being also a natural source of macro- and micro-minerals. Regarding their bioactive composition, most of these plants contain phenolic compounds and flavonoids, which determine their antioxidant capacity. (4) Conclusions: These reported results clearly demonstrate the high potential of the WEPs from a nutritional, economic and social point of view; although further studies are needed to gather deeper scientific information about their potential role in the socio-economic sustainability of specific groups of farmers worldwide. Rumex sp. and Calendula sp. The conclusion of this ethnobotanical study was that these plants were used as food and/or as flavoring additives [\u201cWild Edibles\u201d is a term used to describe both plants and animals consumed by humans. In 1999, the Food and Agriculture Organization of the United Nations (FAO) described the term wild plants as \u201cthose that grow spontaneously in self-maintaining populations in natural or semi-natural ecosystems and can exist independently of direct human action\u201d ; this fidditives . The impdditives , China [dditives , Ethiopidditives , Guatemadditives , Icelanddditives , India [dditives , and Tundditives . In Eurodditives  wrote a dditives ,15,16,17These plants may play an important role in environmental sustainability as they grow wildly and can be used as a functional ingredient to develop new food products. This sustainable character is persuading more and more consumers, chefs and nutritionists to introduce WEPs in their dishes and the diets they prescribe. The relevance of this review is supported by the growing global demand for a change in eating habits, where key new trends are essential and include: (i) reduction in gasses\u2019 emissions, (ii) growth of sustainable crops, and (iii) greater environmental awareness. An example of this change and this new trend is the guide that FAO published in 2017 on wild food plants, or current campaigns for the consumption of edible insects as a new and sustainable source of protein . The futTo identify interesting scientific publications dealing with the composition and relevance of WEPs, this review was based on the 2020 update of the PRISMA approach . The litThis review collects information on 115 WEPs  located Proximate analysis is used in foods to estimate the values of energy, moisture, protein, lipids, water, ash, and carbohydrate in the samples under study. Proteins, lipids and carbohydrates contribute to the total energy content in an organism, while ash and water also contribute to the organism mass . TherefoThe different parameters of the proximate characterization were compiled and summarized for 47 WEPs. The families with the highest representation in the proximate characterization were Asteraceae (9) > Lamiaceae (5) > Polygonaceae (4) .moisture is one of the most important parameters in a plant. For a plant to produce 1 kg of organic matter, it needs to absorb 500 kg of water, which is subsequently eliminated by different processes  [2 through photosynthesis, and even in the cooling of the leaves during hot hours [Silybum marianum (Asteraceae family) when it reached 93.4 g per 100 g [Thymus pulegioides (Lamiaceae family) with 47.6 g per 100 g [The water content or oration) . In herbot hours . The aveer 100 g ,26; on ter 100 g . In WEPsBlumea lacera (Asteraceae family) and Hygrophila schulli (Amaranthaceae family) with values of 24.05% and 23.36%, respectively [Rumex acetosella, Rumex induratus, Rumex papillaris, and Rumex pulcher) with values of 1.2, 1.0, 1.0 and 1.9 g per 100 g, respectively [Studies dealing with 45 out of the 115 studied WEPs (from 24 families), provided results on ash contents. The two WEPs that showed the highest ash contents were: ectively . On the ectively ,30.Portulaca oleracea with 27.8 g per 100 g [Blumea lacera and Asparagus acutifolius with 22.52 and 22.40 g per 100 g, respectively [Silybum marianum (Asteraceae family), with a protein content as low as 0.6 g per 100 g [With respect to protein, three WEPs stood out for their high protein content, each belonging to a different family : er 100 g , followeectively ,32. On ter 100 g ,26.Bryonia dioica (Cucurbitaceae family) showed the highest content at 15.1 per 100 g dry weight . The family with the highest number of plants (Asteraceae) had, in general, low contents, reaching its maximum with Silybum marianum and Enhydra fluctuans (1.1 g per 100 g), and its minimum with Scolymus hispanicus (0.09 g per 100 g) [Fat content was only reported in 47 of the available 115 WEPs. r 100 g) ,26,31.Thymus pulegioides and Mentha pulegium had the highest content, reaching 89.35 and 84.74 per 100 g, respectively [Thymus pulegioides having the highest value (89.35 g per 100 g), while Glechoma hederacea had the lowest with 21 g per 100 g [The scientific literature only provides the carbohydrates content for 46 WEPs out of the 115 reviewed plants. In this regard, ectively ; both pler 100 g .Blumea lacera (20.68 g per 100 g dw) and Berberis aristata (18.10 g per 100 g dw), belonging to the Asteraceae and Berberaceae families, respectively [The content of dietary fiber was only found for 36 WEPs. The WEPs that stood out for their high fiber content were ectively . In geneectively ,37,39,42Energy is calculated from the determination of food macro-nutrients including protein, fiber, carbohydrates, fat and alcohol [Bryonia dioica with 440 kcal per 100 g dw [Rumex acetosella and Rumex induratus with 368 and 376 kcal per 100 g dw, respectively [ alcohol . Nowaday alcohol ,49. Howe alcohol . This di100 g dw . Regardiectively .The role of the sugars, which are generated through the photosynthesis process, in plants is fundamental; they are the main source of carbon and energy for the plants, and participate in the plant metabolism control, for example participating in multiple biological processes, from embryogenesis to plant senescence ,52. SugaSugar contents of 22 WEPs (from 16 families) are summarized in Malva sylvestris  and Tamus communis (Dioscoraceae family), with 8.72 and 3.83 g/100 g dw, respectively [Malva sylvestris also showed the highest glucose content at 7.35 g/100 g dw [Mentha pulegium, which had 4.62 g/100 g dw, and Asparagus acutifolius with a sucrose concentration of 4.27 g/100 g dw [The most commonly studied plant family regarding sugars was Lamiaceae with five plants. Regarding fructose, the WEPs that had the highest content were ectively ,36. Malv100 g dw ,37.At a general level, organic acids are weak acids, which can be classified mainly by four criteria: (i) nature of the carbon chain ; (ii) saturation or unsaturation properties; (iii) substituted or unsubstituted characteristics; and (iv) number of functional groups. Acids play an essential role in the physiology of plants, participating in processes such as pH regulation, balancing the redox potential cells, the Krebs cycle, or even in organoleptic properties such as color, taste and aroma of both fruits and vegetables ,59,60.Three organic acids  were mainly found in WEPs, and especially in four families: Asteraceae, Brassicaceae, Crassulaceae, and Portulacaceae .Raphanus raphanistrum (Brassicaceae family); the second cluster was based on the association of oxalic acid with Sonchus oleraceus (Asteraceae family); while in the third group both oxalic acid and shikimic acid were associated with Hymenonema graecum (Asteraceae family).One of the most representative organic acids in the Asteraceae family is oxalic acid ,26, whicRaphanus raphanistrum and Sonchus oleraceus  [Hymenonema graecum > Sonchus oleraceus > Raphanus raphanistrum, with concentrations between 972 and 706 mg/100 g [Hymenonema graecum and Sonchus oleraceus, with 244 and 166 mg/100 g, respectively; the other WEPs where this acid was found had values of below 100 mg/100 g [In this way, the highest contents of malic acid were reported in ctively) ,61. The mg/100 g ,61. RegaThe Mediterranean diet is considered by many experts as one of the best in the world at a nutritional level, and one of its main strengths is the contribution of minerals and vitamins . In bodyPrincipal Component Analysis (PCA)  was carrRegarding Foeniculum vulgare was that with the highest content of this mineral, followed by Chondrilla juncea  [Calcium was not associated with any specific WEPs, although ctively) ,35.Chondrilla juncea and Malva sylvestris. Regarding Cu and Mn, the WEPs that had the highest simultaneous contents, were Chondrilla juncea  followed by Malva sylvestris ; although the WEP that had the highest Mn content was Montia fontana with 1.08 mg/100 g [Malva sylvestris had the highest contents  compared to Chondrilla juncea  [Scolymus hispanicus, the content of which was much higher than in the rest of the WEPs (1040 mg/100 g) [Beta maritima, containing the highest concentration of this mineral, 171 mg/100 g) [The first group was mainly associated with two plants: mg/100 g . Regardictively) . In the g/100 g) ,26. In tg/100 g) .Chondrilla juncea, which can provide almost 50% of the recommended daily intake of Cu after consumption of 200 g of the plant. Montia fontana, with its Mn content, could also cover approximately 50% of the recommended daily amount after consumption of 200 g  had FA profiles published; the most commonly studied families regarding FAs were: Asteraceae (8) > Lamiaceae (5) > Polygonaceae (4) > Brassicaceae (3). As can be seen in Bryonia dioica Jacq. (70.3%) > Malva sylvestris (67.8%) > Papaver rhoeas (65.0%) > Anchusa azurea (64.7%) > Rumex pulcher (63.0%) > Origanum vulgare (62.3%) > Umbilicus rupestris (62.0%) > Cichorium intybus (62.0%) [Linolenic acid was by far the FA with the highest percentage in most plants, showing in eight of these 42 plants a percentage above 60%; the list of plants in decreasing order of abundance was as follows:  (62.0%) ,46,56,70Allium ampeloprasum (53.5%) > Asparagus acutifolius (44.5%) > Tamus communis (42.0%) [Linoleic acid was the second most abundant FA, highlighting its importance in WEPs, especially in  (42.0%) ,67,72; tViola x wittrockiana (Violaceae family) with 36.41 g/100 g dw [Silybum marianum (Asteraceae), Allium ampeloprasum (Amaryllidaceae) and Portulaca oleracea (Portulaceae) had values of 28.7%, 26.4% and 24.7%, respectively [Another of the most representative FAs, palmitic acid, was present in all the WEPs analyzed. The WEP that obtained the highest value in palmitic acid was ectively ,67,70,72Glechoma hederacea with 35.1% [Portulaca oleracea with 12% [Umbilicus rupestris (0.6%) [Regarding oleic acid, the highest content was found in th 35.1% , followewith 12% ; whilst,s (0.6%) .In general, the main FAs identified in WEPs were linolenic and linoleic acids. On the other hand, it should be noted that palmitic acid was identified in all plants, although at lower concentrations.Phenolic compounds are the most abundant secondary metabolites in plants, helping in various functions of great importance, such as pigmentation, growth, and/or resistance to pathogens, also playing a fundamental role in maintaining redox homeostasis of cells ,74,75. AIn the references evaluated in this review, instudying up to 115 WEPs (from 47 families), the total phenolic contents (TPC) were provided for 100 plants; for 51 plants, the total flavonoids content (TFC) was also provided  5,30,32,72,80,81Tamus communis reaching 404 mg GAE/g, this plant being the only from the Diocoreaceae family [Youngia japonica) to 184 mg GAE/g (Helichrysum stoechas) [Rumex pulcher) and 142 mg GAE/g (Rumex acetosella) [The WEP with the highest TPC was e family . Regarditoechas) ,82. It itosella) ,72.Origanum vulgare (Laminaceae family) and Malva sylvestris  having the highest values at 224 and 211 mg CE/g, respectively [Sophora viciifolia (Fabaceae family) with values of 237 mg rutin/g dry extract [Sesbania sesban (Fabaceae family) > Hygrophila schulli (Acanthaceae family) > Berberis aristata (Berberidaceae family)  [Diplotaxis erucoides (Brassicaceae family) and Asparagus acutifolius (Asparagaceae family) showed the highest content of flavonoids expressed in mg QE/kg WW, at 2877 and 2263, respectively [Data regarding TFC (mg EC/g) were found for 31 WEPs, with ectively ,80. Rega extract . On the ctively) . Diplotaectively .These data show the great importance of the WEPs due to their bioactive composition, and also their great diversity, with so many families represented.Nowadays, a fundamental aspect in society is the economy and its linkage with sustainability is essential. Therefore, the economic value of WEPs must be highlighted and studied, in which edible flowers can play a key role; they can be prepared for sale in different formats, such as fresh, dried, or even candied. This variability together with the color of these flowers makes them highly attractive and a good business proposition within the flowers market. Globalization and online marketing have created an emerging market for this type of product worldwide. The sale of these flowers is usually in punnets of between 6 and 15 flowers, depending on the type of flower and the season. The price of these punnets also depends on the type of flower, but is usually between 8 and 17 euros, some of them reaching approximately 40 euros ,86,87,88Apart from this direct way of selling edible flowers, there are also lollipops with edible flowers inside, or crystallized. The price range is between 21 and 60 euros .Other ways in which these plants contribute their economic value is through disclosure through books. In 2004, the book \u201cClorofilia\u201d by Andoni Luis Aduriz was released, where information was collected on 50 wild plants, their flowering calendar, taxonomic information and recipes . AnotherSo far, we have studied the entire market for WEPs, either directly or through other sources; however, for many families these plants are their livelihood. In the study by Mokria et al. , the neeNot only are these plants important in ethnic communities, but the study carried out by Matsuura  showed tWith all these data, the economic importance of WEPs is evident, being essential for many families around the world to survive.The topic of this review is a step into the future, because developing countries are facing a serious problem, the fast growth of their population and the consequent increase food needs/availability. Furthermore, in many rural communities it is a necessity, because WEPs are not only a food but a way of earning a living. WEPs are a natural source of minerals, vitamins, fiber, and antioxidants, and at the same time they are inexpensive, as they are not cultivated. Therefore, they are a real alternative in trying to reduce this gap between food production and demand, especially to produce natural and sustainable food additives, such as flavorings and aromas. In addition, the food industry could take advantage of the properties of WEPs to develop nutritional, organic, and sustainable foods. Nevertheless, more research is needed to go beyond just their composition information (which is the most commonly found in the scientific databases for this type of plant); information on their technological and functional properties is needed to include them in industrial processes. Toxicological studies are also needed to determine their activities . Thus, a deep investigation into WEPs is needed to start developing commercial products based on these inexpensive and sustainable plants/ingredients. Sustainability, nutrition, and the agri-food industry converge around these plants."}
+{"text": "Although there is a wide use of wild edible plants (WEPs) in Ethiopia, very little work has so far been done, particularly, in the Tigray Region, northern Ethiopia, to properly document the associated knowledge. The purpose of this study was, therefore, to document knowledge and analyze data related to the use of wild edible and nutraceutical plants in Raya-Azebo District of Tigray Region. The district was prioritized for the study to avoid the further loss of local knowledge and discontinuation of the associated practices because of the depletion of wild edible plants in the area mainly due to agricultural expansion and largely by private investors.A cross-sectional ethnobotanical study was carried out in the study District to collect data through individual interviews held with purposively selected informants, observation, market surveys, and ranking exercises. Descriptive and inferential statistical methods were employed to analyze and summarize the data using Statistical Package for Social Sciences (SPSS) version 16.Ziziphus spina-christi L. Desf., Balanites aegyptiaca (L.) Del. and Opuntia ficus-indica (L.) Miller were the most preferred WEPs. Both interviews and local market surveys revealed the marketability of Opuntia ficus-indica, Ziziphus spina-christi, Ficus vasta Forssk., Ficus sur Forssk., and Balanites aegyptiaca. Of the total WEPs, 21 were reported to have medicinal  values, of which Balanites aegyptiaca and Acacia etbaica scored the highest rank order priority (ROP) values for their uses to treat anthrax and skin infections, respectively.The study documented 59 WEPs, the majority of which (57.63%) were sought for their fruits. Most of the WEPs (49 species) were consumed in the autumn, locally called qewei, which includes the months of September, October, and November. The current investigation demonstrated the wide use of WEPs in the district. In future nutritional composition analysis studies, priority should be given to the most popular WEPs, and nutraceutical plants with the highest ROP values. Wild edible plants (WEPs) play an important role in the livelihood of many rural communities across the world, particularly, in providing reliable alternatives when the production of cultivated crops decreases or fails \u20135. Wild There is a wide use of WEPs in Ethiopia as supplement foods as revealed by different ethnobotanical studies \u201324. Furto 15\u2019and 13o 41\u2019 North and longitudes between 38o 59\u2019and 39o 54\u2019 East [Raya-Azebo District belongs to the Southern zone of the Tigray Region in northern Ethiopia and is located at latitudes between 1254\u2019 East . Raya-Az54\u2019 East . The dis54\u2019 East , and has54\u2019 East . Ninety 54\u2019 East . The disFor the study, nine tabiyas that were relatively considered to have better vegetation cover and availability of knowledgeable individuals concerning use of WEPs were purposively sampled out of the total 18 rural tabiyas of the district with the help of experts at Raya-Azebo District Agriculture and Natural Resources Conservation Office. The selected tabiyas included Ebo, Erba, Genete, Hade Alga, Hadis Kigni, Hawelti, Mechare, Tsigea and Ulaga , field observation and market surveys following the methods stated in Martin . Attemptp-value\u2009\u2264\u20090.05 were considered statistically significant. Mean values are presented as mean plus or minus standard error of the mean (mean\u2009\u00b1\u2009SEM). Preference ranking exercises were performed on WEPs of the highest informant consensus by involving individuals randomly sampled from the list of informants who participated in interviews following the method of Martin [Microsoft Excel version 2016 was employed to enter and organize the data. Descriptive statistical methods were used to analyze and summarize the data using Statistical Package for Social Sciences (SPSS) version 16. Comparison of mean differences between informant groups was made using one-way analysis of variance (ANOVA) and differences in means with f Martin . Preferef Martin . Howeverf Martin . RPL valGrewia contributed four species, the genera Acacia and Ficus contributed three species each, and the genera Rhus, Cordia, Brassica, Dovyalis and Rumex contributed two species each, while the remaining 31 genera were represented by one species each. Of the plants that were determined, at least, to a genus level, 18(35%) were shrubs, 18 (35%) were herbs and 15 (29%) were trees.The study documented a total of 59 WEPs, of which 51 (belonging to 33 families and 40 genera) were, at least, identified to a genus level (44 to a species level and seven to genus level). The remaining eight species were only known by their Tigrigna names, as informants were not willing to travel to far distances to collect their specimens for identification purpose (Table The majority (57.63%) of the WEPs in the study area were sought for their fruits, and few were harvested for their leaves (13.60%) and roots (8.5%) Fig.\u00a0. The ediEragrostis tef (Zucc.) Trotter).Most fruits were consumed raw by peeling off their skin (exocarp) and then chewing and swallowing with occasional spitting of seeds or stones Table . On the Amaranthus hybridus L., Capsella bursa-pastoris (L.) Medic., Cleome gynandra L., Commiphora Africana (A.Rich.) Engl., Cynanchum abyssinicum Decne., Echidnopsis sp., Huernia macrocarpa (A.Rich.) Sprenger, Eragrostis sp., Dobera glabra (Forssk.) Pair., Pentarrbinum insipidum E.Mey and Rumex nervosus Vahl) were only consumed at times of famine as reported by informants. Fruits were predominantly consumed by children, especially when herding animals in places that were far away from homesteads. On the other hand, leafy vegetables were usually harvested by women and prepared at home for household consumption.The great majority of the wild edible plants in the study area were frequently harvested and consumed as supplementary/complementary foods at time of plenty or seasonal shortage of staple food. However, some  cif. and Rhus natalensis Krauss, Smilax aspera L., and a plant locally known as katoita were reported to be available for harvest throughout the year.Analysis of data shows that the highest number of WEPs (49 species) in the study district were available for harvest in the autumn season , followed by those (37 species) that were harvested in the summer season . The autumn season, which includes the months of September, October and November, comes after the long rainy summer season that includes the months of June, July and August. Twenty-six WEPs were consumed in the winter season , and 25 plants were consumed in the spring season which includes the months of March, April and May  M.C. Johnston, Grewia villosa, Balanites aegyptiaca, Ficus vasta, Dovyalis abyssinica (A.Rich.) Warb., Ximenia americana, Opuntia ficus-indica, Ziziphus spina-christi, Ficus sur and Diospyros mespiliformis Hochst. ex. A.DC. were sold at local markets for their food values. Whereas, market surveys witnessed the marketability of only four of the aforementioned plants that included Opuntia ficus-indica, Ziziphus spina-christi, Ficus vasta, Ficus sur and Balanites aegyptiaca.Interviews data showed that Ficus sur, Rhus natalensis, Ximenia americana and Ziziphus spina-christi were reported to have rare occurrences in the study area.Most of the WEPs consumed in the study area were harvested from farmlands and other disturbed habitats, roadsides, and woodlands. Very few were harvested from forested area. Nearly half of the reported WEPs were reported to have scarce occurrence in the area with the population of each plant continuing to decline from time to time. However, as interview reports indicated, very little effort has so far been made in the area to spare them from further devastation. The frequently mentioned threats of WEPs in the study area included agricultural expansion, recurrent drought and cutting of trees . Ranking exercise conducted by informants revealed agricultural expansion and cutting of trees for firewood making as the main factors responsible for the depletion of WEPs in the district Table . Of the p\u2009<\u20090.05) in the mean number of WEPs reported by literate and illiterate informants; the mean number WEPs reported by literate and illiterate informants were 6.69\u2009\u00b1\u20090.37and 5.45\u2009\u00b1\u20090.22, respectively. However, there was no significant difference in the number of WEPs reported by male (6.08\u2009\u00b1\u20090.23) and female (4.90\u2009\u00b1\u20090.43) informants, and those reported by informants above the age of 40\u00a0years and above (5.94\u2009\u00b1\u20090.23) and those who were below the age of 40\u00a0years (5.94\u2009\u00b1\u20090.52).Analysis of data collected revealed that there was a significant difference  values. Balanites aegyptiaca scored RPO value of 58.6 for its use to treat anthrax, and Acacia etbaica scored an RPO value of 43.8 for its use to manage skin infections  uses Table . Of thesSmilax aspera, Cynanchum abyssinicum and Pentarrbinum insipidum), were also found to be consumed elsewhere in the country, which may be related to their better preference and/or wide occurrence in different agro-ecological zones of the country.Results of the current study demonstrates that there is a wide use of wild edible as supplementary/complementary foods and nutraceuticals in Raya-Azebo District of the Tigray Region as revealed by the high diversity of the reported plant species. Relatively higher number of WEPs (59 species) was recorded from the study District as compared with those reported from other districts of the same region by Girmay et al. in Asgede Tsimbla, Tahtay Koraro and Medebay Zana districts 41 spp.) , Adhena  spp. , AThe fact that the families Asclepiadaceae and Fabaceae and Tiliaceae contributed a relatively higher number of wild edible species could be due to a combination of factors that, among others, may include their species diversity in Ethiopia and/or better nutritional value. Fabaceae is one of the few dominant dicotyledonous families in Ethiopia contributing 486 species . This faMost of the WEPs in the district were sought for their fruits, which could be due to rich nutritional content and good taste of fruits as also claimed by informants involved in the study. Many other studies conducted elsewhere in the country also witnessed the dominance of wild edible fruits , 58\u201372.The fact that there was little practice of harvesting and storing WEPs in the study district for later consumption may be attributed to the perishable nature of the consumed parts, especially the fruits and leaves, which were reported to be popular. Studies conducted elsewhere in Ethiopia also reported the perishability of wild fruits and leaves , 71, indAcacia abyssinica, Acacia seyal, Balanites aegyptiaca, Carissa spinarum, Cordia monoica, Cynanchum abyssinicum, Grewia sp., Grewia villosa, Huernia macrocarpa, Olea europaea subsp. cuspidata and Rhus natalensis, Smilax aspera, and a plant locally known as katoita were revealed to be harvested and consumed year-round because of the availability of their edible parts, although the yield each plant may differ from season to season.The majority of the WEPs in the district were harvested and consumed during the summer and autumn seasons including June, July, August, September, October and November, and that may attributed to the fact that their edible parts (mostly fruits) abundantly ripen at that time of the year. Several studies conducted in different parts of the country also reported better harvest and consumption of WEPs in the aforementioned seasons , 68, 73 Ziziphus spina-christi, Balanites aegyptiaca and Opuntia ficus-indica were revealed as the most popular and preferred plants in the district, which may be attributed to their good harvest, taste and nutritional value. The fact that the three plants served as a good source of financial income, as also noted during interviews and market surveys, could have also contributed to their popularity. These plants were also found popular elsewhere in the northern part of the country [Ziziphus spina-christi in fiber, carbohydrate and different minerals [Balanites aegyptiaca in protein, fiber and different minerals [Opuntia ficus-indica in carbohydrate, fiber and vitamin C [ country , 56, 64.minerals , 75, Balminerals \u201376, and itamin C , 78. Preitamin C .Analysis of data revealed that literate people (those who read and write) had better knowledge of the use of WEPs plants as compared to illiterate ones (those who do not read and write), which was in contrast to results of some studies conducted elsewhere in the country where illiterate people are more knowledgeable than literate ones , 58\u201370. Balanites aegyptiaca and Acacia etbaica scored the highest rank order priority (ROP) values, Balanites aegyptiaca for its use to treat anthrax and Acacia etbaica for its use to manage skin infections. Investigations conducted elsewhere in the country also revealed the use of Acacia etbaica against skin infection [Balanites aegyptiaca against anthrax [Acacia etbaica [Balanites aegyptiaca [Of the WEPs reported to have medicinal  values in the study district, nfection \u201383, and  anthrax , 85. Fur etbaica , 87 and gyptiaca \u201390, whicZiziphus spina-christi, Balanites aegyptiaca and Opuntia ficus-indica were found to be the most preferred WEPs. Agricultural expansion and cutting of trees for firewood purpose were found to be the main conservation threats for WEPs. Of the total WEPs, 21 were reported to also have medicinal  values. Balanites aegyptiaca and Acacia etbaica scored the highest rank order priority (ROP) values, the former for its use to treat anthrax and the later for its use to manage skin infections. In future evaluation of the nutritional value of the documented WEPs, priority should be given to those that were found popular in the study district. Likewise, priority should be given to nutraceutical plants that scored the highest ROP values in the investigation of pharmacological properties and phytochemical profiles. Furthermore, immediate attention should be given by concerned individuals and institutions in the country to manage (in situ and ex situ) wild edible and nutraceutical plants that were reported to have rare occurrences in the study District by involving the local community.The current investigation demonstrated a wide use of WEPs in Raya-Azebo district as revealed by the high diversity of recorded plants (59 species), the majority of which were sought for their fruits. Most of the plants were consumed, as supplementary foods, and often by children. The highest number of WEPs was consumed in the autumn season, which includes the months of September, October and November from which September took the lead. The plants"}
+{"text": "Fundulus notatus and F. olivaceus, are reciprocally monophyletic, and co\u2010occur in drainages throughout much of the central and southern United States. Hybridization rates vary substantially among populations in isolated drainage systems. We employed genome\u2010wide sampling to investigate geographic variation in hybridization, and to assess the possible importance of chromosome fusions to reproductive isolation among nine separate contact zones. The species differ by chromosomal rearrangements resulting from Robertsonian (Rb) fusions, so we hypothesized that Rb fusion chromosomes would serve as reproductive barriers, exhibiting steeper genomic clines than the rest of the genome. We observed variation in hybridization dynamics among drainages that ranged from nearly random mating to complete absence of hybridization. Contrary to predictions, our use of genomic cline analyses on mapped species\u2010diagnostic SNP markers did not indicate consistent patterns of variable introgression across linkage groups, or an association between Rb fusions and genomic clines that would be indicative of reproductive isolation. We did observe a relationship between hybridization rates and population phylogeography, with the lowest rates of hybridization tending to be found in populations inferred to have had the longest histories of drainage sympatry. Our results, combined with previous studies of contact zones between the species, support population history as an important factor in explaining variation in hybridization rates.Pairs of species that exhibit broadly overlapping distributions, and multiple geographically isolated contact zones, provide opportunities to investigate the mechanisms of reproductive isolation. Such naturally replicated systems have demonstrated that hybridization rates can vary substantially among populations, raising important questions about the genetic basis of reproductive isolation. The topminnows,  Fundulus notatus and F. olivaceus, co\u2010occur in drainages throughout much of the central and southern United States. We compared hybridization rates and genomic clines among nine contact zones. We found substantial variation among contact zones in hybridization rates, but no consistent patterns of variation in genomic clines across linkage groups. We did observe a relationship between hybridization rates and population phylogeography.The topminnows,  We genotyped F2 progeny using genotype\u2010by\u2010sequencing (GBS) following Elshire et al.\u00a0 and Ouachita Highland (Glover) populations. Although these parents were members of different F. notatus clades, both populations exhibit n\u2009=\u200920 chromosomes with four Rb fusions. F2 progeny was genotyped following the same approach as F. olivaceus. We aligned short reads from the F. notatus F2 family to the F. olivaceus draft genome contigs (because an F. notatus draft genome was not available) and then assembled those contigs into 20 F. notatus linkage groups. The synteny of linkage groups between the two species was then established by aligning the F. olivaceus and F. notatus assemblies to each other using MUMmer 4.0  was selected to represent the Western Gulf Slope clade. The Glover (Glv) and Cossatot (Cos) rivers in the southwestern Ouachita Highlands were selected to represent the Red River basin clade. The Tombigbee (Tom) and Noxubee (Nox) rivers represent the Mobile River basin clade. Finally, the Mississippi River basin clade was represented by contact zones in the Spring River (Spr), Horse Creek (Hrs), Saline River , and Pascagoula River (Pas). In each sampling region, the distribution of parental species and the center of the contact zone were known from earlier surveys \u2009>\u20090.95 for one species or the other from our Entropy analysis (see next section). We selected a set of phylogeographically informative loci from our Stacks SNP library by specifying a minimum allele frequency of 0.05, and minimum interlocus distance of 50,000\u2009bp. A small subset of around 6% of loci exhibited substantial excess observed heterozygosity that may be caused by paralog alignment  and interspecific heterozygosity (Q12) for individuals in each contact zone using the hierarchical Bayesian model implemented in Entropy  with 50,000 iterations sampling every 10th iteration after discarding an initial burn\u2010in of 2000 iterations. Population statistics were averaged over three replicate runs after convergence among runs was confirmed visually.We estimated the proportion of ancestry (3.5\u03b1 and \u03b2) describing introgression of each locus. The cline parameter \u03b1 indicates a bias in the directionality of introgression relative to the genome average. Specifically, \u03b1 indicates the magnitude and directionality of introgression at a single locus relative to the genome\u2010wide average. The cline parameter \u03b2 specifies the rate of transition from one parental to the other. Negative values of \u03b2 correspond to loci that introgress more readily (wider cline) than the genome\u2010wide average, and positive values correspond to loci that resist introgression (steeper cline). We used the BGC model to test the prediction that SNP markers mapped to Rb fusions in F. notatus would exhibit more positive \u03b2 values than SNPs mapped to unfused linkage groups.We used the Bayesian genomic cline (BGC) model , making comparisons of BGC runs among contact zones problematic. Consequently, to simplify the analysis, we chose to use a set of \u201cspecies\u2010diagnostic fixed loci\u201d (between F. olivaceus and F. notatus) that could be used to estimate BGC parameters at the same set of loci across all contact zones.The BGC model requires specification of reference population samples to define population gene pools. Our first efforts to employ the BGC model utilized the same set of phylogeographically informative SNP loci as the TreeMix analysis, while using either individuals from our allopatric samples from neighboring drainages, or individuals from within contacts zones exhibiting Entropy F. notatus and F. olivaceus reference individuals in equal proportions  of the published F. olivaceus draft genome. Chromosome fusions in the F. notatus genome were identified with a separate linkage map constructed from an F2 family of 53 F. notatus offspring. The F. notatus map included 5860 markers in 20 linkage groups totaling 916\u2009Mb (75.1%) of the published draft genome. Alignment of the assembled genomes identified the four paired linkage groups that have undergone fusion in an F. notatus ancestor. These four paired linkage groups were numbered , , , and  in our F. olivaceus linkage map.Contigs in the published unmapped draft genome assembly for 4.2F. notatus and 5 F. olivaceus). After quality filtering of SNPs, the median read coverage was 26x, and the median proportion of loci with GQ\u2009>\u200930 was 82%.After quality filtering, there were a total of 453,154,662 reads from 1269 individuals distributed over nine contact zones and 12 reference populations  was possible in some of these active hybrid zone individuals. The fifth migration edge identified an intraspecific F. notatus migration event connecting the Red River drainage clade to the Horse/Spring clade with an admixture value of 10%. The most substantial migration edge inferred in the analysis connected the root of the F. notatus Mobile drainage clade to the root of the F. olivaceus clade with an admixture value estimated at 36%.A maximum likelihood phylogeny constructed with TreeMix using 3314 phylogeographically informative loci confirmed the relationships among s Figure\u00a0. The phy4.3F. olivaceus was 0.36 in the Cossatot and 0.34 in the Pascagoula; it ranged between 0.45 and 0.60 in all other contact zones. We observed substantial variation in levels of hybridization among contact zones. There was also variation in distribution of ancestry proportion (q) and interspecific heterozygosity (Q12) among contact zones  of which mapped to the 24 \u03b1 and \u03b2 were highly variable in some contact zones, and invariant in others  ranged from none in the Pascagoula and Saline contact zones to as high as 14% and 16% of loci in the Tombigbee and Noxubee contact zones, respectively  supporting the validity of the cross\u2010study comparisons using different sets of SNP markers. Altogether, estimates of FIS were available for 14 contact zones distributed over most of the co\u2010occurrence of the two species  were all restricted to contact zones in the Mississippi River basin and adjacent coastal drainages. These included the Pascagoula and Pearl coastal drainages, as well as the Saline, Elk, Black, and St. Francis River drainages in the Mississippi River basin. This study indicated that hybridization was entirely absent in the Pascagoula River and nearly so in the Saline River. Previous studies of other contact zones utilizing a small number of species\u2010diagnostic PCR\u2010RFLP loci detected an absence of heterozygous genotypes among mixed\u2010species samples collected in the Pearl, Black, and St. Francis Rivers, and only minimal hybridization in the Elk River .Sites where hybridization rates were very low or absent , and low interspecific FIS consistent with close to random mating and at least partial hybrid viability. In other contact zones, both species were observed co\u2010occurring within the same habitats in equal proportions, with no F1 or early generation backcross individuals, and correspondingly, FIS nearly 1. Our results, based on a genome\u2010wide distribution of SNPs on all linkage groups, confirmed previous reports of similarly wide ranging hybridization rates, which were based on a small number of loci  matched high and low hybridization rates, respectively, observed in natural contact zones in this study.Contrasting patterns of hybridization in this study suggest that reproductive isolation is highly variable among populations across drainages. The genetic basis of reproductive isolation is supported by previous work. A study of mate selection (probability of spawning) and backcross hybrid offspring viability (hatching success) reported evidence of both prezygotic (conspecific mate preference) and postzygotic (low hatching success) barriers  from providing informative genomic cline data.F. notatus genome would exhibit steeper genomic clines than the 16 unfused linkage groups. Our results did not uncover any consistent patterns among linkage groups for genomic clines based on either \u03b1 or \u03b2 parameters or support a specific role of Rb fusions in promoting reproductive isolation. We detected no positive \u03b2 outliers that would be indicative of reproductive barriers in any linkage groups, and there were no consistent differences in inferred chromosome\u2010level mean \u03b2 across independent contact zones. We found no evidence of consistent differences in genomic clines between fused and unfused linkage groups, respectively. This study fits with some other studies and systems in which Rb fusions have appeared to not limit gene flow per se or disproportionately contribute to reproductive isolation between species that differ in karyotype  were generally distributed uniformly across linkage groups, and in similar proportions . In the absence of any consistent patterns of \u03b1 outliers among linkage groups, we interpret these results as uninformative overdispersion of the \u03b1 parameter.We found that substantial numbers of s Figure\u00a0. There wIt remains unclear the nature and extent of reproductive isolation in topminnows. It is possible that Rb fusions were not found to be disproportionately associated with steeper genomic clines if, perhaps, postzygotic isolation is distributed more widely across chromosomes, and not inherently associated with specific linkage groups or Rb fusions. These topminnow species are estimated to be of Pliocene origin, having diverged over several million years  are distributed ; data curation ; formal analysis (lead); funding acquisition ; investigation ; methodology ; project administration ; resources ; supervision (lead); writing \u2013 original draft (lead); writing \u2013 review and editing (lead). Naznin S. Remex: Formal analysis (supporting); investigation (supporting); project administration (supporting). Jeffrey T. Miller: Formal analysis (supporting); methodology (supporting); software (supporting). Jacob F. Schaefer: Conceptualization ; data curation ; formal analysis ; funding acquisition ; investigation ; methodology ; project administration ; resources ; supervision ; writing \u2013 original draft (supporting); writing \u2013 review and editing (supporting).Benefits Generated: Benefits from this research accrue from the sharing of our data and results on public databases as described above.Data S1Click here for additional data file.Table S1Click here for additional data file."}
+{"text": "Greenhouse gases absorb the Earth\u2019s thermal radiation and partially return it to the Earth\u2019s surface. When accumulated in the atmosphere, greenhouse gases lead to an increase in the average global air temperature and, as a result, climate change. In this paper, an approach to measuring CO There are many point sources that produce anthropogenic greenhouse gas emissions, including coal mines, landfills, sewage treatment facilities and animal husbandry. Although current ground-based detection methods can detect gases at low thresholds, their application time and coverage area are constrained. Rapid and precise identification of greenhouse gas emissions can be achieved through satellite monitoring. Deploying a constellation of satellites can remove restrictions on the observation time.Hydrometeorological spacecraft, such as Meteor-M , MeteoroSpectrometers placed on board spacecraft are a marker of the spacecraft\u2019s capabilities for the remote measurement of greenhouse gases. The OCO-2  instrumeNSO-FTS)  onboard  10.5 km . GHTSat coverage . The IKFcoverage ; it allout 50 kg . The Gaout 50 kg . GaoFen-ut 50 kg . GMI-II The devices for measuring greenhouse gases discussed above have properties that make them useful for resolving a variety of issues in remote sensing of the Earth\u2019s properties. However, the creation of instruments for spacecraft in the CubeSat design is currently a critical endeavor. Due to its size and weight, most sensing equipment cannot be fitted in a CubeSat spacecraft. Even a low-resolution FTIR spectrometer such as the Bruker EM27/SUN  cannot bMeznSat-A is a 3U CubeSat for monitoring greenhouse gases using short-wave infrared (SWIR) spectroscopy. The spacecraft is equipped with an Argus 2000 spectrometer. The spectral range of the grating spectrometer is 1.0 to 1.65 nels. In , a groundvantage  can prodToday, many COtroscopy ,29. Devitroscopy . FTIR sptroscopy  simultantroscopy .This paper is devoted to the analysis of using infrared spectroscopy for remote measurements of greenhouse gas concentrations and the development of a compact, low-resolution FTIR spectrometer for the CubeSat spacecraft. We focus on CubeSat satellites because they are the cheapest to launch into space as an additional payload. Monitoring should be carried out by CubeSat spacecraft to solve the task of remote sensing in the field of greenhouse gas emissions.The external and internal layout of the developed small spacecraft follows the logic of the CubeSat standard ; the spaAn FTIR spectroradiometer unit is integrated into the design of the small spacecraft developed for greenhouse gas remote sensing. Namely, to determine the oxygen and carbon dioxide concentrations in the air along the line of sight of the device, it measures the reflected sun radiation which passes the air column twice . Ground-Carbon dioxide molecules have fundamental absorption bands around 1.61 mosphere . The use.765 \u03bcm)  creates .765 \u03bcm) .The device is based on a Michelson interferometer . SatelliThe operating principle of the FTIR spectrometer is as follows: Solar radiation reflected from the Earth\u2019s surface passes through the atmosphere and is collected by the telescopic system  and inexpensive production conditions.An FTIR spectrometer mockup b for groExperimental measurements were taken in September 2022 near the Baumanskaya district . Solar radiation passes through the air masses of the atmosphere and is reflected off both inhomogeneities of the atmosphere and off topographic objects. The mockup registered reflected solar radiation. The line of sight direction did not change during the experiment. The overall time of measurements was 5 h. The ambient temperature was 12 The OCO retrieval algorithm  uses theThe spectral transmittance of the atmosphere obtained with an FTIR spectrometer is shown in database , see FigGraphs of COMeasurements of greenhouse gases were obtained from the transmittance spectra in stations  and CO2  data in . The vartrometer ,58.The presented results experimentally demonstrate the possibility of measuring greenhouse gas concentrations by reflected solar radiation using an IR Fourier spectrometer. The developed compact, low-resolution FTIR spectrometer is designed to be placed in CubeSat spacecraft for remote measurement of greenhouse gas concentrations.The functional parts of the CubeSat FTIR spectrometer are the same as for the ground-based mockup. The differences are in the mechanical design, for example, the use of special materials with an extended temperature range. To limit the radiation flux, a motorized diaphragm can be installed in the optical system. A diaphragm allows us to change the size of the radiation flux in the range from 1 mm to 27 mm. To ensure functioning in outer space, special heat sinks from electronic circuit boards can be used, special vacuum lubrication of moving parts can be provided and a radiation-resistant element base can be used. The CubeSat FTIR spectrometer will be tested in conditions close to those in outer space.For the CubeSat FTIR spectrometer, we assume a detection limit of about 1% of the nominal values of COo HITRAN  and GEISo HITRAN .An FTIR spectrometer mockup for recording IR absorption spectra in the wavelength range from 1.0 to 1.7 We developed an optical scheme  and desiThe FTIR spectrometer allows for simultaneous concentration measurements of several greenhouse gases and is designed for CubeSat spacecraft."}
+{"text": "In water polo, the team\u2019s technical and tactical performance is determined by the sum of the players\u2019 activities. This study aimed to investigate the playing offensive performance of an Italian First League team performed during all matches (n = 19) of the 2021/22 championship using the Team Sport Assessment Procedure (TSAP). For all subjects (n = 15), gaining possession of the ball  and conquered balls (CB)) and disposing of the ball ; lost balls (LB); offensive ball (OB) and successful Shots (SS)) parameters, as well as volume of play (VP), efficiency index (EI) and performance score (PS) indexes, were analyzed in relation to the playing positions, season phase, match location and final score difference. Multiple linear regression showed a significant association between the playing position and VP and PS. Perimetral players showed the highest VP (65%) and PS (66%) values, and center defenders showed the highest values of CB (30%), while center forwards gained the highest amount of exclusion when handling the ball (48%). Although they were not significant, the other contextual factors showed that season phase and match location could affect the TSAP indexes. For water polo coaches, the TSAP represents an effective tool to assess how players interpret the match. Water polo is an intermittent, high-intensity and body-contact aquatic team sport  played bThe last decade has seen an increase in interest for research lines related to time motion and notational analyses , even thMen\u2019s national and international matches were compared in a study by Lupo et al. , showingThe 2019 revision of the water polo rules of play  states that each team may retain ball possession for up to 30 s during a regular action or for up to 20 s in case of events as exclusion, corner throw or rebound to the attacking team after a shot . Players\u2019 individual skills, such as ball technique, accuracy when passing the ball while under pressure from the opponent and shooting accuracy, are crucial to successfully completing a ball possession phase [However, despite the fact that the analysis of the number and duration of ball possession phases  combinedThe Team Sport Assessment Procedure (TSAP) was first developed by Grehaigne et al.  in the cTherefore, the purpose of this study was to use the TSAP tool to provide coaches with objective data on individual and team offensive performances in relation to the phase of the competitive season, match location, final score difference and playing positions for male elite water polo players. The study\u2019s hypotheses were that over the course of a competitive water polo season, there might be noticeable variations in the TSAP performance indexes between the regular phase and play-out matches, balanced and unbalanced matches, home and away matches, as well as based on the players\u2019 playing position.ADrive.com\u2019 database (https://www.adrive.com (accessed on 30 September 2021)) , which is a platform with free access to clubs and where the Italian First League water polo clubs were obliged by the Italian Swimming Federation to upload a professional video  of all their matches after their public streaming on YouTube or other public platforms. For this reason, since the current study used footage with free public access, no informed consent was required according to the ethical standards outlined by the local research ethics committees.All matches\u2019 footage were downloaded after each weekly match (first access on 30 September 2021) from the \u2018All matches were classified according to the following contextual factors: (a) season phase, based on the Italian water polo First League, which divided the 2021/22 championship schedule into regular season and play-out and play-off phases; (b) match location (home or away); (c) final score difference, which is defined as the difference of goals scored between the two teams in a match  ; and (d)Fifteen male elite water polo players belonging to the Italian elite First League team \u2018S.S. Lazio Nuoto\u2019 were observed during all 19 official matches of the 2021/22 Italian men\u2019s First League championship (Serie A1) and classified according to their principal playing position as: perimetral  players (n = 8); center defenders (n = 3); center forwards (n = 2); and goalkeepers (n = 2) .Following them being downloaded, all the matches were reproduced using QuickTime for Mac OS X , which allowed the researchers to pause the video and use replay. Then, a specific water polo dashboard was created using LongoMatch Pro software , which allowed the researchers to collect the TSAP parameters throughout all individual and team offensive actions. In line with previous notational analysis studies on water polo ,16,21, tNotational analysis data are provided by means of the TSAP observational instrument , including received balls (RB) and conquered balls (CB) counted as variables for gaining the ball possession, while neutral balls (NB), lost balls (LB), offensive balls (OB) and successful shots (SS) were counted as variables for disposing of the ball . As perfStatistical analyses were performed using Excel version 2016 (Microsoft Office) and SPSS version 26.0 . For each TSAP variable, descriptive data are expressed as means \u00b1 standard deviations (SD).p-value.Two multiple linear regressions were generated, with VP and PS as dependent variables, while in both analyses, the playing position, season phase, match location and final score difference were the constant predictors. Then, the analysis of covariance (ANCOVA) with playing time as a covariate was applied to assess differences in the VP and PS in relation to the playing positions. ANCOVA analysis was shown to be robust to violations of either the conditional normality or homoscedasticity assumption in previous studies . For thip < 0.05.Finally, the k-means cluster analysis was applied to identify 3 groups of players   in relatFollowing the regular phase (thirteen matches), the observed team participated in the play-out phase (six matches) reserved for the last seven teams of the final league table at the end of the regular phase. Of these nineteen official matches, ten were played at home, and nine matches were played away. Regarding the final score difference factor, ten matches registered a balanced result , while nine matches ended with an unbalanced result .Multiple linear regression analyses, except those for the playing position, showed no significance for the season phase, match location and final score difference in terms of the VP and PS indexes .p < 0.001; mean difference \u00b1 SE = \u221216.3 \u00b1 1.9), goalkeepers and center defenders , perimetral players and center defenders , perimetral players and center forwards  and between center defenders and center forwards . According to the players\u2019 playing position, the highest VP mean values (mean \u00b1 SE) were registered for perimetral players (22.9 \u00b1 0.8), followed by center defenders (18.8 \u00b1 1.1), center forwards (9.8 \u00b1 1.4) and goalkeepers (6.5 \u00b1 1.7). ANCOVA analysis (with Bonferroni pairwise comparison) showed significant differences for the VP index between the goalkeepers and perimetral players , goalkeepers and center defenders , goalkeepers and center forwards , perimetral players and center defenders , perimetral players and center forwards  and between the center defenders and center forwards . For PS, significant differences were found between the goalkeepers and perimetral players . The first cluster included eight players , and the second cluster included five players , while the third cluster included only two players . This study aimed to use the TSAP tool to provide water polo coaches objective data on individual and team offensive performances in relation to the phase of the 2021/22 competitive season (regular and play-out phases), match location (home or away), final score difference  and playing positions among male elite  water polo players.The main findings of this study were that the TSAP parameters registered different values for the four playing positions  and that cluster analysis provided three groups of players for the VP and PS indexes.The analysis of technical-tactical components of the TSAP parameters showed, as the main results, that 51% of the LB was determined by the shots, 53% of the OB was determined by the assist and offensive passages, while 68% of the SS was determined by scoring a goal. In a water polo match, the LB, OB and SS parameters should be considered as intertwined since their interactions could provide a positive or a negative outcome for the team. The high number of missed shots registered as LB could be explained by the players\u2019 technical and decision-making skills. Indeed, a missed shot could lead a team to concede a counterattack or a goal to their opponents, as previously revealed in a study by Perazzetti et al.  on eliteBased on our study\u2019s hypotheses, that there might be a noticeable variation in the TSAP indexes between the regular phase and play-out matches, balanced and unbalanced matches, and home and away matches; analysis of these three contextual factors and the players\u2019 playing positions was conducted. Multiple linear regression analysis showed a not significant relationship between the season phase, match location and final score difference and the VP and PS indexes, while only the players\u2019 playing positions showed a significant adherence.Although they were not statistically significant, our study found that the VP and PS indexes were higher during the play-out phase, which is chronologically played after the regular one. Even though this is the first study investigating the season phase factor in water polo, the results are consistent with earlier research on other elite invasion team sports like football and Australian football, which showed that the technical and tactical performances improved toward the last part of the competitive season ,45. An aConsidering the pooled data for the entire season (regular and play-out phases), our results showed higher mean values for the balanced matches compared to those of the unbalanced matches for the PS . This result could be explained by the higher percentage of goals scored the during power play situation in balanced matches than that in unbalanced ones . In fact, Lupo et al.  suggesteAdditionally, in our study, the analysis of the match location factor revealed higher mean values in the TSAP parameters and indexes  for homeduel\u2019 actions before performing the offensive phase. As demonstrated in the study by \u00d6zkol et al. [Regarding the playing position variable, ANCOVA analysis showed significant differences between the different positional roles, with perimetral players registering the highest mean values for both the VP and PS indexes. Although the goalkeeper position is typically excluded from water polo studies , we madel et al. , the higl et al. , as showl et al.  on eliteThe k-means cluster analysis provided in this study on the pooled data identified three groups of players based on their values of the VP and PS. According to the classification suggested by Nadeau et al. , in our This kind of analysis, using such a classification, could be helpful for water polo head coaches as a source of valuable information to organize players\u2019 substitutions during matches, plan specific trainings drills, as well as to be used at the beginning of the season for the roster construction, since many head coaches still base their choices on the players\u2019 defensive skills rather than their offensive ones .Based on the observation of all official matches played during the 2021/22 Italian First League water polo championship by a male elite team, through technical and tactical analyses of the offensive actions, this study has provided sufficient information on elite players\u2019 offensive skills, as well as their ability to dispose of the ball. However, over an entire season, this research included only one elite men senior team, so future research might include a larger number of teams, as well as different competitive levels (elite and non-elite), age categories (senior and different youth categories) and different gender competitions (men and women).The Team Sport Assessment Procedure was initially designed by Grehaigne et al.  for bothThe use of TSAP as an observational instrument has confirmed that it is a valid tool to assess players\u2019 playing abilities, which is why the coaching staffs could regularly adopt it to monitor training sessions  and competitions, as an important and useful tool for monitoring players.However, even if it is restricted to the analysis of only offensive games, this research can be regarded as an original and beneficial contribution to a better comprehension of water polo performances. Furthermore, considering that the TSAP observational tool was designed to assess only players\u2019 offensive behavior, to provide a more complete technical and tactical analysis, this tool should also include their defensive behavior. For this reason, it could be interesting to elaborate and validate specific water polo defense indexes to correlate them with the TSAP ones."}
+{"text": "Wild edible plants (WEPs) grow naturally in self-maintaining ecosystems. WEPs are harvested for consumption, sale, and medicinal uses. We hypothesize that WEPs play a major role in supplying food and generating income for the rural people in a world that is increasingly recognising its emerging conservation issues. We tested this hypothesis by identifying the reasons for harvest, consumption, and conservation of WEPs using focus group discussion, field observations and questionnaire surveys in south eastern Bhutan in late 2019.Focused group discussions were held with the local people to identify reasons for harvest and consumption of WEPs. Data on the identified reasons for harvest, consumption, and conserving WEPs were determined using a questionnaire survey with ranking scales for a set of 76 randomly selected households. Representative field-observations and questionnaire surveys were carried out in villages close to forests. Parts of the plant used, how these were consumed, harvest season, and plant (life form) were recorded. The data was subjected to a Kruskal-Wallis rank sum test and weighted averages calculated.A total of 120 WEPs belonging to 63 families (including Agaricaceae) were reported. Most of the WEPs recorded were trees (45.0%) then herbs (25.8%), vines (13.3%) and shrubs (10.8%). The commonly consumed plant parts were the fruit (43.3%), shoots (28.3%) and leaves (20.8%).The purposes for harvesting and consumption, conservation of WEPs were significantly (P<0.001) different, while the motivations for collecting WEPs were not. The motivation for collecting WEPs were family consumption > sale > medicinal uses > preservation for future use > insufficient food from cultivated source\u2019s. The two most important strategies for conservation were to domesticate the WEPs and cultivate in forests. The findings reveal valuable lessons and insights about the reasons for harvesting, collection, consumption, and conservation of WEPs. Forests represent a crucial resource stock for local people . They suHumans have been using forests intensively since the dawn of civilization and this has often resulted in the wise management of forest resources . These pApproximately two billion people globally experience hunger due to lack of nutrition  and WEPsMany indigenous populations have relied on wild products for aeons in order to eat a balanced diet particularly during times of critical food shortages ,27,28. IDioscorea species are harvested and consumed in special preparations during Losar (New Year), Lochoe  and Prechula  in the Kheng region of central Bhutan [Traditional knowledge of WEPs is associated with indigenous food systems . Culinarl Bhutan . Valuabll Bhutan .The decline in the availability of WEPs also diminishes the valuable traditional knowledge pertaining to diverse use of WEPs . A studyThere is also a vast and valuable traditional knowledge that Bhutanese farmers have not documented around traditional health care systems in Bhutan , on forePsilotrichum axilliflorum is listed as endangered, and Mitragyna stipulosa and Vitellaria paradoxa are listed as vulnerable [The potential loss of some WEPs is of global conservation concern. Species has been for example lnerable . Unregullnerable . Declinelnerable ,62.There is a widespread ignorance about the loss of WEPs diversity and decline in abundance at policy level e.g. ,64. A redungkhag) of Samdrup Jongkhar district and lies between the coordinates of 26.7848 and 27.2473\u00b0 N and 90.9981 and 92.1187\u00b0 E in south eastern Bhutan. Samdrup Choeling dungkhag is further divided into four administrative areas viz. Samrang, Pemathang, Phuntshothang and Martshala. Samdrup Choeling dungkhag is located 68 km away from the district headquarters of Samdrup Jongkhar. A modern motorable road connects it. Similarly, modern bitumen roads connect each administrative area; and farm roads  connect the villages within. There are also government enterprises such as coal mines, fisheries, poultry farming and dairy farming on small and medium scales, and public institutions like schools , health care and Renewable Natural Resources (RNR) centres. Apart from the local inhabitants, there are also public servants; private employees and business people living in the dungkhag. Most importantly, there is a designated area for weekend\u2019s market at Tshangchutham village (Phuntshothang). The present study was conducted in the nearby forests surrounding the villages  of Samrang, Pemathang, Phuntshothang and Martshala areas. Samdrup Choeling is surrounded by evergreen tropical forest covering the foothills, plateaus, and plain lands. The sub-district has subtropical climatic conditions with annual rainfall of 3582 mm, average monthly minimum temperature of 16.72\u00b0C and average monthly maximum temperature of 25.94\u00b0C [Bhutan lies between the coordinates 26.7263 and 28.2466\u00b0 N, and 88.7495 and 92.1236\u00b0 E in the eastern Himalayas. It is geographically located between India in the East, West and South, and China in the North. Bhutan is divided into twenty districts. Samdrup Choeling is one of the sub-districts (Tshogpa) and consisted of those who were aware of the use and knowledgeable of WEPs. Eight to 10 village elderlies of both the genders represented the group in a one-day consultation meeting in the participants\u2019 mother tongue to discuss the availability and uses of WEPs in each village. The identified WEPs along with the characteristics of each WEP were recorded. Additionally, the focus group discussions helped formulate the questions for the follow-up questionnaire survey with particular emphasis on identifying the reasons for (a) harvest, (b) collection, (c) consumption, and (d) conservation of WEPs. Five representative attributes or ranks for each of these four major reasons were selected though group discussions and consensus. At the end of each focus group discussion, a key informant was identified to assist in constructing a sampling frame or system for the field survey.A village nearby a forest was purposively selected in each of the four areas (Geogs) of Samrang, Pemathang, Phuntshothang and Martshala under Samdrup Choeling sub-district where focus group discussions were held in November and December 2019. Members of the focused groups were selected in consultation with heads of villages  assigned to each of the data sets. The generated Kruskal-Wallis test by ranks were plotted along with the calculated weighted averages so that comparisons could be made.Since the data generated in this study were non-parametric in nature (and not fulfilling the assumptions of an ANOVA), a Kruskal-Wallis test by ranks  were used for statistical data analyses using the IBM SPSS Statistics (Version 22) predictive analytics software. This methodology is more appropriate to analyse non-parametric or ordinal data ,80. The A total of 120 species from 63 families (including one Agaricaceae) of wild edible plants were reported in the study sites . Most ofThere were 76 farmers (>20 years of age) who took part in the voluntary questionnaire survey. In terms of gender representation, 55.3% were male and 44.7% female farmers. Seventy-five per cent of the interviewees were between 20 and 50 years of age while the remainder were > 50 years old. The plurality of the respondents was 36.8% illiterate; few had a diploma/degree or higher education; while the remaining fell into other categories .The median, spread and skewedness of the four data sets are graphically demonstrated  in boxplnd rank)  . HoweverThe results of Kruskal-Wallis rank sum test showed that most of the purposes of harvesting WEPs were highly and significantly different (P<0.001) to each other . The higThe weighted averages values show the degree of importance and closely follow the mean ranks. The mean ranks and weighted averages for purposes of collecting WEPs were: family consumption > for sale > medicinal uses > preserve for future use > insufficient food from cultivated sources .Data for good taste, salability and time-efficient are symmetrically distributed (rank 3 as median), while that of availability is slightly skewed towards the upper extreme value even though the median is rank 3 . HoweverThe data for consume differently, easily available and palatability are fairly symmetrically distributed with a median rank of 3. However, for health benefits there is a strong skewedness towards the upper extreme value (median = rank 5) and 64% of the respondents ranked it as 5. The mean ranks for most of the reasons for consuming WEPs were statistically different (P<0.001) to each other  with heaThe data for decrease harvest and implement regulations are symmetrically distributed (median = rank 3), while those for cultivate in forest and domesticate are skewed towards rank 5 (median = rank 4) . HoweverThe least important strategy to conserve WEPs was to stop harvesting (mean rank = 135) and this had a significantly lower mean rank than the other four strategies . StrategBhutan is one of the 10 global hotspots for biodiversity . The larOf 120 species consumed in our study, most were consumed cooked, raw, pickled, chewed or in other forms (Tables The most commonly consumed plant parts in the study were fruits 43.3%), shoots 28.3%) and leaves (20.8%) Tables  and 2. T8.3% and .3%, shooThe WEPs reported in the study were also classified into plant types  with mosThe purposes for harvesting WEPs were significantly different (P<0.001) to each other  with theDioscorea species due to the insufficient rice and maize yields from cultivation in Nangkor in the Zhemgang district in central Bhutan in a separate study [The median of for sale and medicinal uses was rank 3  and theyte study . Furtherte study  reportedThe relative importance of the purposes for collecting WEPs were family consumption > for sale > medicinal uses > preserve for future use > insufficient food from cultivated sources indicating varying degree of role played by the WEPs in supporting the rural livelihood in general. Moreover, the data of Kruskal-Wallis rank sum test closely match that of the weighted averages across all the analyses confirming the findings.The common motivating attributes of local people collecting WEPs were assessed by ranking the five questions on salability in the market, time efficiency for collection, availability in the vicinity of the locality, they tasted good, and ability to generate high price. The median, which is a measure of central tendency for a non-parametric analysis, which is same for all five attributes (rank 3)  except fCordyceps  as the motivation to harvest it from localites in the Bhutanese highlands.In contrast to this study, WEPs collected and sold in Nepal were reported to fetch high prices ,23: the The five most important reasons for consuming WEPs were palatability, nutritious, consumed in different ways, easily available and health benefits or of medicinal value. The mean ranks for the reasons for consuming WEPs were statistically different  with heaArtocarpus heterophyllus Lamk., Bauhinia purpurea L., Bauhinia retusa Roxb., Colocasia esculenta (L.) Schott, Emblica officinalis Gaertn., were reported to be domesticated in home gardens in Odisha, in eastern India [An understanding of local people\u2019s priority for WEPs conservation strategies were projected from their ranking responses because WEPs are under various constant natural and anthropological threats ,88. The rn India . People rn India . Hence cDioscorea in Nangkor Geog under Zhemgang district in south central Bhutan [Gnetum africanum and G. buchholzianum) has been pushed towards vulnerable status due to over exploitation [Local people consider decrease frequency of harvest and implement of regulation as other prioritised methods (median = rank 3) of conserving WEPs because the high prices in the local markets could stimulate over-harvesting behaviour leading to conservation concerns such as those reported in Nepal . Likewisl Bhutan  is clearoitation . Besidesoitation . The leaoitation . In respoitation ,93, whicoitation , even thLantana camara L. is an example of invasive WEP reported in Samdrup Choeling [Tropaeolum majus L., Boerhavia diffusa L., Aconogonon molle (D. Don) H. Hara, Bidens pilosa L., Girardinia diversifolia (Link) Friis, Urtica dioica L., Nasturtium officinale R.Br., Oenanthe javanica (Blume) DC., Adhatoda vasica L., Houttuynia cordata Thunb., Solanum nigrum L., Amaranthus spinosus L., Pteridium aquilinum (L.) Kuhn, Remusatia vivipara (Roxb.) Schott., Elatostema lineolatum J.R.Forst. & G.Forst., Chenopodium album L. and Boehmeria sp Jacq. [Most studies have not considered the status on WEPs as native or invasive. In the current study, 25% of 120 species reported were invasive , which pChoeling ,87, and Choeling . Consumisp Jacq. ,78,98. Msp Jacq. .The presence of 120 WEPs species belonging to 63 families (including one Agaricaceae) were reported. Most of the WEPs recorded were trees followed by herbs, vines and shrubs, while the commonly consumed plant parts were fruit, shoots and leaves. The relative importance of the purposes of collecting WEPs were family consumption > for sale > medicinal uses > preserve for future use > insufficient food from cultivated sources. There are important reasons why WEPs should be and are consumed by the local people, including the overlapping reasons of food and medicinal value from WEPs. The data revealed that the palatability, nutritious and consumed in different ways categories of WEPs were of similar priority, whereas the health benefits associated with consumption was of the highest priority to the respondents. Two of the most important conservation strategies were to domesticate and cultivate in forests. The data of Kruskal-Wallis rank sum test closely match with that of weighted averages across all the analyses reinforcing the findings. The results reveal a range of valuable understandings and insights into the purposes of harvesting, motivations for collection, reasons for consumption and strategies on conservation of WEPs.S1 File(XLSX)Click here for additional data file."}
+{"text": "The figures were published out of order. See here the correct figures and their captions."}
+{"text": "We tested the hypothesis that the use of outward displacement of the soft tissue between the apex and the chest wall as seen in TTE, is a sign of apical displacement and would allow for more accurate diagnosis of apical dyskinesis. This is a retrospective study of 123 patients who underwent TTE and cardiac magnetic resonance imaging (MRI) within a time frame of 6\u00a0months between 2008 and 2019. 110 subjects were deemed to have good quality studies and included in the final analysis. An observer blinded to the study objectives evaluated the echocardiograms and recorded the presence or absence of apical dyskinesis. Two independent observers evaluated the echocardiograms based on the presence or absence of outward displacement of the overlying tissue at the LV apex. Cardiac MRI was used to validate the presence of apical dyskinesis. The proportion of studies which were identified as having apical dyskinesis with conventional criteria defined as outward movement of the left ventricular apex during systole were compared to those deemed to have dyskinesis based on tissue displacement. By cardiac MRI, 90 patients had apical dyskinesis. Using conventional criteria on TTE interpretation, 21 were diagnosed with apical dyskinesis (23.3%). However, when soft tissue displacement was used as the diagnostic marker of dyskinesis, 78 patients (86.7%) were diagnosed with dyskinesis, p\u2009<\u20090.01. Detection of displacement of soft tissue overlying the LV apex facilitates better recognition of LV apical dyskinesis.The online version contains supplementary material available at 10.1007/s10554-023-02856-4. The evaluation of wall motion of left ventricular (LV) segments is an integral component of cardiac imaging. During systole, the myocardium contracts and thickens inwards toward the ventricle cavity. In normal functioning myocardium, the LV motion is synchronous. American Society of Echocardiography (ASE) guidelines recommend visually assessing the LV regional function on transthoracic echocardiography (TTE) using a 17-segment model and grading abnormal wall motion qualitatively using a range of severity from normal to hypokinetic (reduced thickening), akinetic (absent thickening), dyskinetic (systolic thinning or stretching), and aneurysm  [The left ventricular apex is usually affected in disease states such as ischemic heart disease with LV apical infarction, hypertrophic cardiomyopathy, and Takotsubo cardiomyopathy. On physical exam, LV apical dyskinesis may be detected as systolic apical displacement during palpation. Although wall thickening of most LV segments can be assessed by transthoracic echocardiography, complete visualization of the LV apex is often difficult and confounded by foreshortening of the LV apex. This is due to nearfield artifact from the proximity of the transducer to the LV apex. These factors may result in underestimation of apical akinesis and dyskinesis by two-dimensional TTE. The outward movement of soft tissue overlying the LV apex seen in patients with apical dyskinesis in the apical 2- and 4-chamber views is associated with infarctions due to left anterior descending (LAD) artery occlusion due to involvement of the blood supply affecting the apex and its proximity to surrounding structures, including the pericardial fat pad, which can transmit dyskinetic segments [Dyskinesis is associated with an infarcted myocardial segment and is more commonly seen at the LV apex. The prevalence and characteristics of apical dyskinesis following myocardial ischemia and necrosis, however, is not well described. Our objective was to test the hypothesis that the use of soft tissue displacement can be identified by TTE  in the apical views, improving the accuracy of detecting apical dyskinesis in patients suspected to have apical wall motion abnormalities.We performed a computerized search on all cardiac MRI and TTE studies performed at Cedars-Sinai Medical Center (CSMC) in Los Angeles, CA, USA, which contained the diagnosis of \u201capical dyskinesis\u201d in the report. All clinical and imaging data were collected from the electronic medical records. The study met ethical requirements set forth by the Institutional Review Board and approved at Cedars Sinai Medical Center.Patients who underwent TTE and cardiac MRI within 6\u00a0months at CSMC from 2008 to 2019 were included in the study. The time delay between the two studies was\u2009<\u20096\u00a0months with average delay being 21\u00a0days (range 1\u2013154\u00a0days). 103 patients with apical dyskinesis on MRI were initially reviewed; however, 90 were selected as having decent visualization of the LV apex. An additional 20 patients with normal LV ejection fraction, no history of ischemic or nonischemic cardiomyopathy and who underwent cardiac MRI for evaluation of the etiology of premature ventricular beats were selected as controls . Wall motion was assessed with a standard 17-segment model using apical 4-, 3-, and 2- chamber view. Scoring was based on visual assessment of motion of LV segments and apical cap. As the study exams took place before longitudinal strain and contrast echo were used as standard methods to better evaluate the LV apex, it could not be included as part of our evaluation. Apical wall motion was graded by standard echocardiographic analysis on echo reports. The same studies were reviewed by the authors for presence of outward motion of the soft tissue overlying the LV apex . Additional three-dimensional (3D) TTE images were obtained in one example to better illustrate the presence of soft tissue displacement overlying the apex in diastole versus systole , with electrocardiogram gating and phased-array surface coil , and subjects in the supine position. Steady state free precession images were obtained with axial, vertical long axis and three chamber views. Short axis stack of SSFP cine images was obtained with post-processing LV apical dyskinesis was assessed by a level 3 certified CMRI reader. LV ejection fraction was measured using Circle CVI42 software.Descriptive statistics were summarized as mean or number with percentage. Ejection fraction measurements were further evaluated using mean, median, and interquartile range. Analysis was obtained by Cohen\u2019s kappa statistic using a table comparing initial echocardiographic evidence of dyskinesis to reclassification of dyskinesis on echo read. This statistic is used to define inter-rater reliability for qualitative items. Negative numbers indicate no agreement and the closer the number to 1, the more congruent the observers. Additional evaluation with Fisher\u2019s exact test using a contingency table with p-values were subsequently obtained with significance defined as p\u2009<\u20090.05.2, with an LV ejection fraction of 40.7 \u00b1 16.5%  had dyskinesis on the initial formal echo read. However, after reclassification with interpretation of soft tissue displacement, 78 (86.7%) were found to have dyskinesis  . AdditioIn the control arm with MRI indicating no LV dyskinesis, 4 of the 20 subjects had displacement of the LV apex on TTE. Of note, 3 of those had moderate aortic regurgitation and ascending aortic root dilation, which may have contributed to finding of soft tissue displacement. The remaining patient had evidence of apical-variant hypertrophic cardiomyopathy.This is a single-center retrospective study of patients who underwent CMRI and TTE for clinical indications, which were different for each patient. Furthermore, the TTE and CMRI were not performed specifically for identifying apical dyskinesis which could affect windows and views. As this is an observational study the authors do not know the exact mechanism behind why the apical dyskinesis is present and further studies using our findings should be undertaken to better understand the significance.As noted above, there was evidence of soft tissue displacement in the echocardiograms of 4 patients without dyskinesis on MRI. It is not clear to the authors why this may be the case but it is important to note that while this novel finding may increase the sensitivity of determining dyskinesis, it may negatively affect specificity.In this study we report a novel transthoracic echocardiographic finding  that facilitates the detection of left ventricular apical dyskinesis.Video 1: Video demonstrating soft tissue displacement of a 2-chamber view transthoracic echocardiogram (TTE). Supplementary file1 (MP4 632 kb)Video 2: Video demonstrating soft tissue displacement of a 4-chamber view transthoracic echocardiogram (TTE). Supplementary file2 (MP4 746 kb)Below is the link to the electronic supplementary material."}
+{"text": "The retraction has been agreed due to the identification of duplications in Figures 2A and 3J and the lack of compelling raw data underlying these figure panels. As a result, the key conclusions of the paper are considered unreliable. The results of an institutional investigation support this decision.The above article, published online on 2 June 2020 in Wiley Online Library ("}
+{"text": "The presence of thyroglobulin (Tg) in needle washouts of fine needle aspiration biopsy (Tg-FNAB) in neck lymph nodes (LNs) suspected of metastasis has become a cornerstone in the follow-up of patients with papillary thyroid carcinoma (PTC). However, there are limited data regarding the measurement of anti-Tg antibodies in these washouts (TgAb-FNAB), and it is not clear whether these antibodies interfere with the assessment of Tg-FNAB or whether there are other factors that would more consistently justify the finding of low Tg-FNAB in metastatic LNs. We investigated 232 FNAB samples obtained from suspicious neck LNs of 144 PTC patients. These samples were divided according to the patient\u2019s serum TgAb status: sTgAb- (n = 203 samples) and sTgAb+ (n = 29). The TgAb-FNAB levels were measured using two different assays. Tg-FNAB was also measured using two assays when low levels (< 10 ng/mL) were identified in the first assay of the metastatic LNs from the sTgAb+ samples. The TgAb-FNAB results were negative in both assays in all samples. Low levels of Tg-FNAB were identified in 11/16 of the metastatic LNs of the sTgAb+ patients and 16/63 of the sTgAb- patients (p < 0.05) using assay 1. The measurement of the Tg-FNAB levels using assay 2 indicated additional metastases in 5 LNs of the sTgAb+ patients. Factors other than the presence of TgAb-FNAB may contribute to the higher number of metastatic LNs with undetectable Tg-FNAB in the sTgAb+ group. In addition, the measurement of Tg-FNAB using different assays was useful to enhance the diagnosis of metastatic LNs, particularly when cytological and Tg-FNAB results are discordant. Papillary thyroid carcinoma (PTC) is the most common endocrine malignancy, with a propensity for cervical lymphatic spread, which occurs in 20% to 50% of patients based on reports of surgical pathological specimens using standard methods of description; however, the prevalence may reach 90% of patients when the surgical sample is scrutinized in detail for micrometastases ,2.The confirmation of malignancy in lymph nodes (LNs) with a suspicious ultrasonographic (US) appearance is achieved by US-guided fine needle aspiration biopsy (FNAB) for cytological study (cyto-FNAB) and the measurement of thyroglobulin levels (Tg) in needle washouts (Tg-FNAB) -8.Considering only the data from cyto-FNAB, approximately 6% to 8% of metastases are misdiagnosed as a result of false-negative results ,10. ThesAnother aspect for consideration during the follow-up of patients with PTC is the interpretation of serum Tg (sTg) values in the presence of serum anti-Tg antibodies (sTgAb), which occurs in approximately 25% of patients with PTC; these antibodies may interfere with the measurement of sTg, which compromises the use of this tumor marker in the follow-up of patients with PTC . It remaTherefore, the objectives of the present study were to: 1) assess the presence of TgAb in a substantial amount of washout fluid samples (TgAb-FNAB) of cervical LNs suspicious for PTC metastases; 2) analyze whether the presence of serum sTgAb+ interfered with the assessment of Tg-FNAB for the management of neck lymphadenopathy in PTC patients; and 3) compare the TgAb values of the serum and FNAB washout.Escola Paulista de Medicina, Universidade Federal de Sao Paulo and the Instituto Israelita de Ensino e Pesquisa Albert Einstein, both in Sao Paulo, Brazil.We assessed 144 patients who presented suspicious LNs via US assessment during follow-up for PTC. These patients were followed by a single team of physicians at associated Thyroid Disease Centers at the Division of Endocrinology, Department of Medicine, Suspicious LNs were submitted to FNAB guided by neck US assessment. The minimum follow-up period was 4 years after the last FNAB. Some patients had more than one FNAB performed when multiple suspicious LNs appeared during the follow-up or the FNAB had to be repeated because of inadequate or non-diagnostic cyto-FNAB results with undetectable Tg-FNAB values. Two hundred eighty-six FNABs were performed, and 232 FNAB samples were analyzed. Fifty-four FNAB samples were excluded because the patients were lost during follow-up (n = 32) or the diagnosis of lymphadenopathy could not be confirmed (n = 22), as cytopathology indicated that these samples were obtained from other cervical lesions rather than LNs.131I uptake after radioiodine treatment. In two particular situations, LNs that initially fulfilled suspicious criteria via US assessment were considered reactive over time: 1) LNs that spontaneously disappeared at the subsequent US assessment performed during follow-up; and 2) LNs that initially had inadequate or non-diagnostic cytology results on the first FNAB attempt and presented a reactive cytology with Tg-FNAB < 10 ng/mL after a second FNAB.The confirmation of LN metastases was obtained via histopathological examination after surgery or the All diagnostic procedures were performed in accordance with the regulations of the local ethics committee. Written informed consent was obtained from each patient.\u00ae, PerkinElmer, Turku, Finland), with a functional sensitivity of 1.0 ng/mL (assay 1). A second immunofluorometric Tg assay (assay 2), using mono and polyclonal antibodies, was performed on the washout fluids of sTgAb+ patients with a Tg-FNAB value < 10 ng/mL in assay 1 to determine whether low Tg-FNAB persisted using a different method. Assay 2 has a functional sensitivity of 0.3 ng/mL  were immediately fixed and stained with panoptic dye. All cytological examinations were performed by the same cytopathologist.e.g., abnormal nuclear shape, nuclear enlargement and nuclear polymorphisms, presence of papillae, and/or characteristic nuclear changes, such as grooves and pseudo-inclusions).The cyto-FNAB results were classified into 3 distinct diagnostic categories: 1) reactive: presence of lymphocytes and occasional plasma cells without malignant epithelial cells; 2) inadequate or non-diagnostic: presence of blood cells and absence of inflammatory and epithelial cells; and 3) positive for PTC metastases: presence of epithelial cells with malignant cytological characteristics . All analyses were performed using the Statistical Package for Social Science professional software version 15.0 .The number of metastatic LNs in Group 1 (sTgAb-) and Group 2 (sTgAb+), as well as their Tg-FNAB levels (assay 1) and cyto-FNAB results were compared using Fisher\u2019s Exact Tests and \u03c7A general overview of the present study that indicates the results of 232 FNABs in 144 patients is presented in We identified metastases in 63 (31%) LNs in Group 1 (sTgAb-) and 16 (55%) LNs in Group 2 (sTgAb+). This difference was statistically significant  . All met2 test)  had a higher number of metastatic LNs with Tg-FNAB values < 10 ng/dL than Group 1  . ConsistThe cutoff of Tg-FNAB of 10 ng/mL (assay 1) for the diagnosis of metastatic PTC in cervical LNs in Group 1 (sTgAb-) exhibited a sensitivity of 74.6%, with a specificity of 100%, a positive predictive value of 100% and a negative predictive value of 89.7%; Group 2 (sTgAb+) exhibited a sensitivity of 31.2%, with a specificity of 100%, a positive predictive value of 100% and a negative predictive value of 54.2%. Notably, it was not possible to perform the same analysis for Tg-FNAB using assay 2 because it was only tested in the washout fluids of the sTgAb+ patients with Tg-FNAB levels < 10 ng/mL in assay 1.131I treatment, and the subsequent scintigraphy indicated substantial 131I uptake in the area of the corresponding LN. Consequently, in this patient, the measurement of Tg-FNAB using assay 2 was important for the diagnosis of metastasis.To evaluate the potential differences in the performance of Tg assay 1 in the sTgAb+ patients (Group 2), we performed a different Tg-FNAB assay  in all washout fluid samples of metastatic LNs with Tg-FNAB levels < 10 ng/mL using assay 1 . We obtained different results between both assays in 4 patients whose cyto-FNABs were positive for PTC metastases and the levels of Tg-FNAB were low (< 10 ng/mL) according to assay 1 and high (\u2265 10 ng/mL) according to assay 2 . In thesHowever, the direct measurement of TgAb-FNAB based on immunofluorometric assays was negative in all 232 samples of both groups, including the FNAB washouts of the sTgAb+ patients. Using a second assay , we identified 7 samples with positive TgAb-FNAB values, all of which were obtained from Group 1 (sTgAb-) patients, with high levels of Tg-FNAB, which likely lead to false elevations of TgAb-FNAB. Moreover, this artifact represents an established competitive effect that occurs when the Tg levels are > 2000 ng/mL, and these values must be considered false-positive results, as described by the manufacturer.The evaluation of cervical LNs is difficult during the follow-up of PTC patients because inflammatory LNs are frequently present. The use of Tg-FNAB is well recognized as a valuable instrument in the examination of suspicious LN metastases ,4,11-23.The presence of TgAb in FNAB washout fluids may reflect the active LN synthesis of TgAb  or the cOther studies have presented similar results. Following the inclusion of a limited number of TgAb-FNAB measurements of LN washout in sTgAb+ patients, the authors did not detect TgAb via direct measurement in the washout fluids of LNs, which suggests that sTgAb do not interfere with the detection of Tg-FNAB levels ,18,20,26Boi and cols. evaluated washout fluid samples from 8 sTgAb+ patients and suggested that the presence of TgAb-FNAB may interfere with the interpretation of Tg-FNAB results because lower levels of Tg-FNAB were detected in 2 metastatic LNs with positive TgAb-FNAB levels compared with metastatic LNs with negative TgAb-FNAB levels . These aTgAb-FNAB was negative in all samples examined in the present study; thus, further comparisons between the TgAb values in the serum and FNAB washout would not be plausible. Instead, we considered other potential reasons why low levels of Tg-FNAB were present in the metastatic LNs of patients with PTC, including difficulties in sampling a representative area of neoplasia during the FNAB procedure, heterogenic Tg production by metastatic cells and differences in Tg assay performance.Regarding the difficulties in sampling a representative area of neoplasia during FNAB, we identified metastatic LNs with Tg-FNAB < 10 ng/mL in both Group 1 (n = 16) and Group 2 (n = 11). We proposed that this Tg-FNAB value represented a true negative result in most cases based on the finding that the majority of these LNs also exhibited inadequate/non-diagnostic cyto-FNAB results (Group 1: n = 13/16 and Group 2: n = 5/11) . HoweverIn the present study, we did not identify undifferentiated variants of PTC carcinoma in metastatic LNs with low Tg-FNAB levels (< 10 ng/mL). In both Groups 1 and 2, all metastatic LNs with low Tg-FNAB values presented classical and follicular PTC histological variants. However, variable levels of Tg-FNAB have been described, even in samples with the same variant, which reflects heterogenic Tg production by differentiated metastatic cells .Differences in Tg assays were also identified in the present study. The finding of negative TgAb-FNAB in all samples and a higher percentage of metastatic LNs with Tg-FNAB < 10 ng/dL in the sTgAb+ patients than the sTgAb- patients  prompted us to perform a second Tg assay in the washout fluids of all proven metastatic LNs of the sTgAb+ patients with Tg-FNAB < 10 ng/dL . In someSimilar to the results obtained in the present study, Jeon and cols.  and Jo aIn conclusion, we propose that other factors, rather than the presence of TgAb, may contribute to the higher number of metastatic LNs with undetectable Tg-FNAB identified in the sTgAb+ group because TgAb-FNAB was negative in all samples according to 2 different assays. Furthermore, for the analysis of Tg-FNAB in the sTgAb+ patients, assay 2  had a better performance in the detection of metastatic LNs than the monoclonal assay; however, it exhibited limitations in the identification of some LN metastases. Nevertheless, this analysis was useful in some cases in which there was no concordance between the cyto-FNAB and Tg-FNAB results or when the cyto-FNAB was inadequate/non-diagnostic and the Tg-FNAB levels were low based on the monoclonal assay. Therefore, we proposed that the measurement of Tg using another assay would be helpful in LNs with a positive cytology for PTC and low levels of Tg-FNAB using the first assay and in cases of inadequate/non-diagnostic cytology and low Tg-FNAB levels. However, in this latter situation, we emphasize that the low value of Tg-FNAB may persist in every additional assay assessed, as a matter of material scarcity. When employed alone, cyto-FNAB or Tg-FNAB (independent of the tested assay) did not provide a guarantee of establishing the diagnosis of cervical lymphadenopathy in patients.In addition, false-positive and false-negative results may occur, which reinforce the recommendations that these LNs require monitoring and physicians must interpret clinical, laboratory, cytology, and ultrasound data together to achieve successful management in the follow-up of patients with PTC."}
+{"text": "Mycobacterium kansasii infections predominantly manifest in immunocompromised people and are primarily responsible for lung disease and systemic disseminated infection. Osteopathy is a rare consequence of M.\u00a0kansasii infection. Here, we present imaging data from a 44-year-old immunocompetent Chinese woman diagnosed with multiple bone destruction, particularly of the spine, secondary to M. kansasii pulmonary disease, which is easily misdiagnosed. The patient underwent an emergency operation after experiencing unexpected incomplete paraplegia during hospitalization, indicating an aggravation of bone destruction. Preoperative sputum testing and next-generation sequencing of DNA and RNA of intraoperative samples confirmed the diagnosis of M.\u00a0kansasii infection. Treatment with anti-tuberculosis therapy and the subsequent patient response supported our diagnosis. Given the rarity of osteopathy secondary to M.\u00a0kansasii infection in immunocompetent individuals, our case offers some insight into this diagnosis. Mycobacterium kansasii is one of the most prevalent species worldwide [M. kansasii is primarily responsible for lung disease and systemic disseminated infection in immunocompromised individuals and is rare in immunocompetent people. Osteopathy is a rare sequela of such M. kansasii infections that commonly manifest as osteomyelitis. Only seven such cases involving immunocompetent individuals have been documented in the literature, of which four involving the spine are identical to our case [M. kansasii infection with spinal involvement and show her imaging performance before  remains unknown. Patients without HIV infections may be affected by anomalies in the IFN\u2013interleukin-12 (IL-12) axis and T cell + lymphopenia [M. kansasii infection, as in this case. This patient was previously suspected and misdiagnosed as having lung cancer with bony metastases in the long term because of the resembling imaging performances of the lung and multiple bone lesions and a family history of lymphoma contributing to her father\u2019s death. This misdiagnosis is clinically common if species identification results are not obtained [Although the pathogen development resembles that of tuberculous bacteria, the mechanism of non-tuberculous phopenia ,14. In tobtained ,16. Ther"}
+{"text": "Cepaea nemoralis for which classical genetic studies have shown that around nine loci, several physically linked and inherited together as a \u2018supergene\u2019, control the shell colour and banding polymorphism. As a first step towards identifying the genes involved, we used whole-genome resequencing of individuals from a laboratory cross to construct a high-density linkage map, and then trait mapping to identify 95% confidence intervals for the chromosomal region that contains the supergene, specifically the colour locus (C), and the unlinked mid-banded locus (U). The linkage map is made up of 215,593 markers, ordered into 22 linkage groups, with one large group making up ~27% of the genome. The C locus was mapped to a ~1.3\u2009cM region on linkage group 11, and the U locus was mapped to a ~0.7\u2009cM region on linkage group 15. The linkage map will serve as an important resource for further evolutionary and population genomic studies of C. nemoralis and related species, as well as the identification of candidate genes within the supergene and for the mid-banding phenotype.Molluscs are a highly speciose phylum that exhibits an astonishing array of colours and patterns, yet relatively little progress has been made in identifying the underlying genes that determine phenotypic variation. One prominent example is the land snail  In C. nemoralis into pseudochromosomes. We then use trait mapping to infer the map position of the shell ground colour (C) and mid-banded (U) loci, which together represent a major part of the phenotypic variation of the shell.In general, high-density genetic linkage maps are useful to order and orient even fragmented assemblies  locus within the supergene, as well as the mid-banded locus (U). Two grandparents were also included, making 79 snails in total , aiming for ~10\u00d7 fold haploid coverage. A single library was prepared from DNA of each individual and run over two lanes. Sequence data were binned by barcode and trimmed for known adapters.Linkage mapping was performed using Lep-MAP3 Rastas  because CPCY) and mid-banding (U3U-) loci. Additionally, SNPs were excluded where all individuals were heterozygous. The resulting list of SNPs was extracted from the VCF file with vcftools v. 0.1.17 , with SNPs that failed the filtering criteria set for removal using the removeMarkers option. The order of markers was then evaluated for an additional six iterations.SNPs were removed from the map if the contigs they were on were assigned to multiple linkage groups, under the conditions that contigs with 10 or less mapped SNPs were removed from the map completely. The exception was in cases where contigs had >10 SNPs in the map, for which markers were removed only from a single linkage group if the number of SNPs assigned to that LG constituted less than 10% of markers from that contig in the map. Maps were then reconstructed for each linkage group using C. nemoralis are hermaphrodites  was calculated as the sum of the total length of all linkage groups divided by the number of markers minus the number of linkage groups. The expected genome length was calculated by adding 2s to each linkage group, to account for terminal chromosome regions, and summing all linkage groups  using logistic regression analysis for binary traits (module scan1 with model = binary). This produced an LOD score, where the null hypothesis was that there were no loci anywhere in the genome associated with the binary trait, and the alternate hypothesis was that there was a binary trait locus near a specific position. The LOD scores were plotted across the linkage groups to identify peaks associated with a putative binary trait locus.To begin, the module scan1perm, model = binary, n_perm = 1000)  were considered significant. Utilising a Bayesian credible interval, these thresholds were then used to identify LOD peaks on the genomic map associated with each of the phenotypes, either yellow or mid-banded.The threshold of the significance level of the LOD score was determined by a permutation test for each binary trait with the recommended number of permutations (module find_peaks (threshold = (P), peakdrop = 1.8), where LOD peaks above the significance level threshold were considered independent only if the LOD score dropped by at least 1.8 between the lower limits of the two adjacent peaks. The effects of the different genotypes on the phenotypic traits, so-called allele effects, were estimated for individual linkage groups ; this indicates the likely position of the binary trait locus, and ascertains which genotypes are associated with the presence/absence of the phenotype in question. Finally, putative locations for the colour/mid-banding locus on the linkage map were examined for each phenotype against the raw, phased genotypes.Multiple peaks across a chromosome were identified using the module LinkageMapView package implemented in R  than the contigs not placed in the map   or absence (0) of a yellow shell, the trait described by shell ground colour locus ome Fig. . The anaome Fig. .Fig. 2ReThe phenotype of each individual was compared against the raw, phased genotypes along linkage group 11 Fig. . As expeIn the first interval (Y1), three map locations  is on the low side compared with others in the superfamily Helicoidea, which range from n\u2009=\u200922 to n\u2009=\u200930 , and ~21% for the power to be less than 95%.A further potential limitation of the study is the relatively small sample size of 75 F2 individuals. In a similar manner to Bu et al. , we usedCepaea. The map regions identified are sequence-incomplete, both because of the draft nature of the assembly and because of the rigorous filtering during processing. A further consideration is that there are likely to be assembly and other errors in the existing contigs, such as false SNPs caused by repetitive paralogous sequences, which did not segregate properly in the map. For example, it is otherwise difficult to explain why SNPs from a contig such as tig00045252 (Supplementary Table Despite these considerations, an improved assembly and/or linkage map is a necessity in any future trait mapping in Cepaea nemoralis and mapped two important shell phenotype loci. The shell ground colour locus C identifies a limited region of linkage group 11 within which the colour and pattern supergene must reside. For the first time we show that the locus U controlling for the mid-banded phenotype is on an unlinked region in linkage group 15. We look forward to the further integration of the linkage map with improved C. nemoralis genome assemblies to fully define the supergene region and this accelerated discovery and understanding of the causative loci. It will be particularly fruitful to compare genes expressed in the mantle with candidates identified by linkage mapping, and to better define the gene regulatory networks controlling trait expression. The linkage map will also be useful in studies of synteny across related species and as a resource in population genomic studies of C. nemoralis.Here we have presented the first high-density, complete linkage map for Cepaea as possibly \u2018a problem with too many solutions\u2019. We hope that the whole-genome linkage map presented here, as well as the mapping of shell-polymorphism loci can serve as a further impetus to understand the genetics underlying the polymorphism in Cepaea, as well as the much wider group of colour polymorphic snails.In their famous paper, Jones et al.  questionSupplementary Tables S4 and S5 and Supplementary Figures S2\u2013S5Figure S1Table S1Table S3Table S2"}
+{"text": "Here, we utilized a cellulose matrix as an absorptive probe for bELF sampling, subsequently testing the design of a device and sampling technique CF-12)] was first tested through tissue-contact experiments on porcine airway tissue. The absorption and elution capacity of the matrix, as well as the laboratory processing and analysis method, was validated with a range of Interleukin-8 (CXCL8) and C-Reactive protein (CRP) stock solutions. Subsequently, the device\u2019s design was optimized for universal in-house production and both, safe and efficient sampling. The airway sampling method was then tested in a group of 10 patients with Chronic Obstructive Pulmonary Disease (COPD). For each patient, a bELF sample was obtained using the newly developed bELF probe, as well as a reference 20\u2009mL saline bronchial wash sample. Supernatants were assessed, using an immunoassay, for levels of the pro-inflammatory markers CXCL8, Myeloperoxidase (MPO), and CRP. The bELF samples were compared to bronchial wash.The absorptive matrix [Whatman\u00ae qualitative filter paper (Grade CF-12) bELF probes adhered to porcine airway tissue, softening slightly upon wetting. The material maintained architectural integrity following the removal of the probes, leaving no residual fibers on the porcine airway mucosa. The bELF probe design was optimized for bronchoscopic delivery and in-house production. On average, a fully saturated bELF probe carried 32\u2009\u03bcL of protein-rich fluid. The mean return of CXCL8 and CRP from samples collected from a serial dilution series  was 69% (range 48%\u201387%). The bELF probe detected, on average, 7 (MPO), 14 (CRP), and 59 (CXCL8) times higher equivalent inflammatory protein concentrations in the collected bELF probe samples compared to the bronchial wash.The Whatman\u00ae qualitative filter paper (Grade The bELF probe is an effective absorptive technology for high-precision bELF sampling without dilution. With a simple in-house production procedure and bronchoscopic sampling technique, this method can be introduced in any bronchoscopic center for a consistent sampling of bELF. With thAn effective alternative to saline lavage is the application of a sampling probe, developed from a synthetic or cellulose-based absorptive matrix , 10, ontOlympus first developed the bronchoscopic microsampling (BMS) method and commercialized a synthetic hydroxylated polyester (FHPE) and a cellulose-based absorptive tip into catheter systems for bELF sampling . RecentlIn this study, we set out to develop and investigate a novel bELF sampling probe with a high capacity of bELF absorption and reliable output while being simple and inexpensive to produce.2.2.1.CF-12 qualitative filter paper .Three absorptive matrices were tested, the Whatman\u00ae Grade GF/C Glass microfiber filters (WHA1822090), Whatman\u00ae Grade 3 qualitative filter paper (WHA1003185), and Whatman\u00ae Grade 2.2.CF-12) A4 sheet, Whatman\u00ae Glass microfiber filters (Grade GF/C) circles (\u2300 23\u2009mm), Whatman\u00ae qualitative filter paper (Grade 3) circles (\u2300 23\u2009mm), using an ethanol-sterilized hole puncher , producing near-identical oval probes with dimensions of 15x3mm  were applied onto porcine airway tissue. Probes from each matrix were gently pressed against the surface of the freshly harvested wet porcine airway for 30\u2009s. After the removal of the probe, the contact surface of porcine airway tissue was imaged with a camera and evaluated for the deposition of any residual material.All three absorptive technologies were tested with airway tissue contact and evaluated on the adhesion to the surface and material strength. Freshly harvested porcine lungs were purchased from a commercial vendor . Probes cut from Whatman\u00ae Glass microfiber filters (Grade GF/C), Whatman\u00ae qualitative filter paper (Grade 3), and Whatman\u00ae qualitative filter paper (Grade 2.4.CF-12) matrix. Two serial dilution series  were prepared from a recombinant standard of human interleukin-8 (CXCL8) and human C-reactive protein . A sample of each solution from both series was collected to measure the original analyte concentration. For the experimental setup, 40 bELF probes stored in pre-weighed assigned 0.5\u2009mL tubes were used. Two sets of four 1\u2009mL aliquots containing 20, 10, 5, and 1\u2009ng/mL of CXCL8 and CRP were prepared. Five bELF probes were designated for each of the eight solutions. One at a time, a bELF probe was removed from its assigned tube using a clean stainless steel tweezer and submerged for 10\u2009s in its designated solution. Five bELF probe samples were collected from each of the eight serial dilution solutions.Testing for protein analyte collection and elution capacity was only carried out on the Whatman\u00ae qualitative filter paper  for further processing within 2\u2009h.2.7.The collected bELF probes were processed and stored according to the primary processing method detailed above, and a dilution factor was calculated. The bronchial wash was centrifuged at 500\u2009g at 4\u00b0C for 10\u2009min, and supernatants were aliquoted. The collected supernatants were stored at \u221280\u00b0C for further analysis.2.8.Bronchial epithelial lining fluid was collected from patient group 2 using the bronchosorption catheter system . One sample was collected from each patient. The sampling and primary processing method was carried out according to the protocol described by Thwaitse et al., and aliquoted supernatants were stored at \u221280\u00b0C for further analysis .2.9.Levels of CXCL8 (DY20), CRP (DY1707), or MPO (DY3174) were measured in duplicate using R&D systems human enzyme-linked immunosorbent assays  according to the manufacturer\u2019s instructions. Absorbance was measured using the EL808 multi-well microplate reader, and the data was analyzed using Gen5\u2122 data analysis software .2.10.U-test was also performed. The points of sample measurements, above or below the limit of detection, were not included in the statistical analysis but were manually plotted for visual representation. All data were assessed and statistically analyzed using GraphPad Prism version 9.5.1 . A value of p \u2264 0.05 was considered significant.Validation data and patient data were characterized descriptively. Descriptive statistics, including mean, median, and 95% confidence intervals, were used to represent the variables of bELF, bronchial wash, and bronchosorption cytokine concentrations. A Mann\u2013Whitney 3.3.1.CF-12 were evaluated through contact with airway tissue. All three filter papers adhered to the porcine airway tissue upon wetting. From observation, grade GF/C filter paper adhered the strongest and grade 3 the weakest. The Whatman\u00ae Glass microfiber filters (Grade GF/C) softened the most upon wetting, and residual fibers were consistently observed on the airway tissue following the removal of the probe, shown in CF-12 softened slightly when wet but remained structurally intact, with no evidence of residual cellulose fibers remaining on the airway tissues  replicates measurements was 0.13 .3.3.p <\u20090.0001) in bELF samples compared to bronchial wash and for CRP 14 times higher (p <\u20090.0001). The median concentration of MPO was 7 times higher, but there was no difference between the two groups . The median concentration of CRP was approximately 4 times higher (p =\u20090.0040). The median concentration of MPO was 2 times higher, but there was no difference between the two groups (p =\u20090.4535).The concentrations of CXCL8, CRP, and MPO were measured in the supernatants of the bronchosorption samples collected from 6 current and ex-smokers \u2013C. The l4.CF-12 was selected as the optimal absorptive matrix for the bELF sampling probe due to its maintenance of structural integrity during sampling and absorption and elution efficiency. The bELF probe production, sampling, processing, and correction method were developed to be universally applicable in any bronchoscopy center with basic laboratory equipment. The bELF probe was well-tolerated by patients and returned a higher yield for inflammatory biomarkers in bELF compared to the current standard bronchial wash.This study explored a novel cellulose-based absorptive matrix as a candidate for a respiratory epithelial lining fluid sampling device. The Whatman qualitative filter paper grade CF-12 filter papers were found to be the most optimal candidate sampling matrix. The CF-12 filter paper technology was developed and optimized for protein analysis with Whatman\u2122 903 cards, a bio-sample  collection, and transport product. These filter papers are 100% pure cotton linter with no-wet strength additives. This material composition equipped the bELF probes with high absorption capacity and rapid wicking  from the serial dilution series of CXCL8 and CRP. The low variability between the replicate measurements highlights the high precision of the measurements. To improve the returned yield, non-specific binding to the inner surfaces of the sample tubes could be reduced by using specific low-protein binding tubes or pre-coating the tubes with a protein matrix, such as albumin. Additionally, to improve protein extraction, parameters of the elution buffer, such as pH and salt content, could be adjusted. Likewise, the time, temperature, and speed of shaking with the thermomixer could be optimized. Lastly, an electrochemiluminescence-based immunoassay could have been employed to improve the sensitivity of the analysis.The dilution factor calculation was critical to maximize the accuracy of quantified values. Under our controlled experimental setup dipping 40 bELF probes until complete saturation, a relatively small range of dilution factors (13 to 21) was calculated. Dilution factors calculated from bELF samples collected from the 10 COPD patients ranged from 8 to 78. The small range of dilution factors during the optimization experiments is likely due to a consistent dipping method till full saturation and collection of a uniform PBS-based protein stock solution. The viscosity of bELF remains a challenge for all respiratory sampling methods and was also encountered with the bELF probe when sampling severe COPD respiratory tracts layered intermittently with highly viscous bELF. When samples collected from the same airway segment vary in collected bELF viscosity, the concentrations of suspended analytes may also vary, leading to variability in the quantification of analytes and greater variation across samples from the same segment.The bELF probe significantly improved spatial informativity and sensitivity in measuring inflammatory biomarkers compared to bronchial wash. The bELF probe samples are obtained from the specifics section of the airway with which the device is in contact. This level of spatial informativity is most relevant for biomarker profiling in heterogenous airway disease, where lavage methods are limited in resolution. For instance, the bELF probe could be used to profile tissue reactions at the implant sites of airway devices . FurtherQuantified levels of CXCL8, CRP, and MPO were, on average, higher in the bELF samples measured with the bELF probe than those measured with the commercially-available bronchosorption system. First, it is important to highlight this comparison\u2019s limitations, which include the comparison of inflammatory biomarker concentrations of two different patient groups and the falling of several measurements from the limited sample outside of the ELISA dynamic range. Nevertheless, the data obtained shows that the bELF probe is a comparable absorptive matrix for measuring inflammatory biomarkers. Furthermore, the difference in concentrations may be attributed to the lack of dilution correction described in the bronchosorption method . Our datGuiding the bELF probe requires a degree of bronchoscopy handling competency. Transforming the bELF probe into a single-use catheter-based system, similar to the BMS and bronchosorption devices , 10, wouThe bELF probe is currently an effective alternative to bronchial wash for biomarker analysis, but future studies are necessary. Optimizing elution yields will require further investigation into the impact of viscosity, the type of biomarkers, and elution buffer constituents. Exploration of new applications, such as microbiological and proteomics analysis, will require validation. Additionally, testing for batch variation and implementation in multiple centers is needed to evaluate the replicability of this method.Overall, the bELF probe is a simple, safe, and effective bronchoscopy-guided technique to generate samples for analyzing biomarkers in bELF from the lower respiratory tract. The bELF probe equips physicians with a reliable diagnostic, prognostic, and monitoring tool for various airway diseases by improving the precision and accuracy of obtained bELF biomarker measurements.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Medical Ethics Review Board University Medical Center Groningen. The patients/participants provided their written informed consent to participate in this study.AG, JB, D-JS, and SP were involved in the conception and design of the study, were involved in interpretation of the generated data, and were involved in revising the manuscript. AG performed the experimental work, analyzed the data, generated figures, and was involved in drafting the manuscript. D-JS contributed by performing the bronchoscopic sampling. All authors contributed to the article and approved the submitted version.This project was co-financed by the Ministry of Economic Affairs and Climate Policy by means of the PPP-allowance made available by the Top Sector Life Sciences and Health to stimulate public-private partnerships, with project code PPP2019-043.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "The sparrow search algorithm (SSA) is a novel swarm intelligence optimization algorithm. It has a fast convergence speed and strong global search ability. However, SSA also has many shortcomings, such as the unstable quality of the initial population, easy to fall into the local optimal solution, and the diversity of the population decreases with the iterative process. In order to solve these problems, this paper proposes an improved sparrow search algorithm (ISSA). ISSA uses Chebyshev chaotic map and elite opposition-based learning strategy to initialize the population and improve the quality of the initial population. In the process of producer location update, dynamic weight factor and Levy flight strategy are introduced to avoid falling into a local optimal solution. The mutation strategy is applied to the scrounger location update process, and the mutation operation is performed on individuals to increase the diversity of the population. In order to verify the feasibility and effectiveness of ISSA, it is tested on 23 benchmark functions. The results show that compared with other seven algorithms, ISSA has higher convergence accuracy, faster convergence speed, and stronger stability. Finally, ISSA is used to optimize the sound field of high-intensity focused ultrasound (HIFU). The results show that ISSA can effectively improve the focusing performance and reduce the influence of sound field sidelobe, which is of great benefit for HIFU treatment. Optimization methods are widely used in many fields, such as signal processing , image pThe sparrow search algorithm (SSA) is a swarm intelligence optimization algorithm proposed by Xue and Shen in 2020 and inspired by foraging and antipredation behaviors of sparrows . It has Many scholars have made improvements to SSA. Lyu et al.  use the In order to overcome the above shortcomings of SSA, an improved sparrow search algorithm (ISSA) is proposed in this paper. In the initial population stage, ISSA uses Chebyshev chaotic map to improve the diversity of the population and uses an elite opposition-based learning strategy to produce a high-quality population. When updating the producer's location, the dynamic weight factor is introduced to balance the producer's ability of global exploration and local exploitation, and the Levy flight strategy is used to expand the search space to avoid falling into the local optimal solution. The mutation strategy is used to update the scrounger's position, guide individuals to approach the optimal solution, and improve population diversity and global search ability.High-intensity focused ultrasound (HIFU) is a high technology for the treatment of tumors. HIFU has been initially applied to the clinical treatment of soft tissue tumors such as breast cancer and uterine leiomyoma by virtue of its advantages of minimally invasive and noninvasive, less complications, and repeatable treatment . The priSSA is improved from the perspective of elite individuals, initial population, and search spaceISSA is verified by Wilcoxon's rank-sum test and time complexity analysisISSA is used to optimize the HIFU sound field and suppress the acoustic sidelobeStudies by many scholars have shown that by optimizing the sound field of focused ultrasound and suppressing the acoustic sidelobe, the focusing performance and the therapeutic effect of focused ultrasound can be improved. Wang et al.  proposedThe rest of this paper is organized as follows. t represents the current number of iterations, Xi,j represents the position of the ith sparrow on the jth dimension , \u03b1 \u2208 \u22121 represents the MP inverse of A.The scroungers always follow the producers to obtain high-quality food and increase their energy reserves. Some scroungers monitor the producers and compete with them for food. When the energy reserve of the scroungers is low, they will fly away from the population and look for food by themselves to survive. The location update equation of the scroungers is as follows:Xbestt is the current global optimal location; \u03b2, as the step size control factor, is a random number that obeys the normal distribution with mean value of 0 and variance of 1; K \u2208  is a random number; fi is the fitness value of the current individual ; fg and fw represent the current global optimal and worst fitness values, respectively; \u03b5 is a very small number so as to avoid denominator being 0.In the sparrow population, some individuals play the role of the scouter. These individuals can detect the threat posed by predators and send out alerts to other individuals to avoid. In the simulation experiment, it is assumed that such individuals account for 10% to 20% of the total population, and their initial positions are randomly assigned. The location update equation of the scouters is as follows:x1 \u2208  is a random number. After obtaining the Chebyshev chaotic sequence, the initial population is generated by the following equation:lbj and ubj represent the lower and upper boundary of the jth dimension of the search space, respectively.In swarm intelligence optimization algorithm, the quality of initial population directly affects the convergence performance of the algorithm. In SSA, the initial population is generated randomly, which makes the distribution of the initial population uneven and the quality unstable, and reduces the convergence accuracy and convergence speed. Chaotic mapping has the characteristics of randomness, ergodicity, and regularity. In recent years, it has been used in swarm intelligence algorithm to improve the quality of the initial population. Commonly used chaotic maps include Tent chaotic map , Kent chThe elite opposition-based learning strategy (EOLS) is used to improve the quality of the initial population . In the \u03bc \u2208  is a random number, and n individuals with small fitness values are selected to form the real initial population.Generally speaking, the elite individuals are individuals with small fitness value in the population. After obtaining the initial population, the individuals are sorted according to the fitness value, and several individuals with small fitness value are selected to form the elite group. For each elite individual in the elite group, its elite opposition can be calculated by the following equation:Xi,j\u02dc=\u03bclb\u03b4 \u2208  is a random number, which is used to avoid the dynamic weight factor \u03c9 being too small in the later stage of iteration. It can be seen from , v ~ N, \u03c3u and \u03c3v is defined as\u03b2 \u2208  is a random number.If the producers have fallen into the local optimal solution in the early stage of iteration, exploitation can only be performed near the local optimal solution in the later stage of iteration. In order to avoid such a situation, this paper adopts the Levy flight strategy to help the producers still have the opportunity to jump out of the local optimal solution in the later stage of iteration. Levy flight is a non-Gaussian random process, and its step size obeys Levy distribution. It is very difficult to calculate the step size of Levy flight, so the Mantegna algorithm  is oftens of Levy flight, the position of the producers is updated according to the following equation:Xit is the location of the producers calculated by  to the projection point of the array element center  in the xy plane. For the calculation of other parameters, please refer to reference [In the simulation experiment, 256 rectangular array elements  of each focal point, but does not specify the phase, so each focal point is formed at the same time. In fact, by adjusting the phase of each focal point, not only focusing can be achieved, but the optimal focusing effect can also be obtained. Rewrite the sound pressure vector P asThe above method only specifies the sound pressure value Optimal focusing effect is achieved by maximizing the sound pressure gain functionAll simulation experiments are performed on an Intel Core i7-11800H CPU @2.30GHz. All codes are implemented on MATLAB R2020b.In the simulation experiments, the performances of the ISSA, GA, PSO, GWO, WOA, HHO, SSA, and SNS on 23 benchmark functions are compared to verify the feasibility and effectiveness of ISSA. These 23 benchmark functions are divided into three categories: unimodal functions, multimodal functions (both of which have a dimension of 30), and fixed-dimension functions. The details are shown in Tables 1n is set to 100, and the maximum number of iterations Tmax is set to 500. In ISSA and SSA, the proportion of the producers and the scroungers is set to 20% and 80%, respectively, the proportion of the scouters is set to 20%, and the safety threshold ST is set to 0.8. In GA, the crossover probability pc and mutation probability pm adaptively change. In PSO, learning factor c1=c2=1.5, inertia weight wmax=0.8, wmin=0.4, speed vmax=1, vmin=\u22121.When comparing the performance of the eight algorithms, in order to ensure the fairness and objectivity of the results, the same values are set for the common parameters: the population size 30 experiments are conducted independently on each benchmark function, and the convergence curve of each algorithm is drawn. The results are shown in For unimodal functions, i.e., F1 to F7, ISSA is superior to SSA in all indicators. For F1 to F4, ISSA can accurately find the optimal value of zero, and the average and standard deviation are also zero, indicating that the convergence accuracy and stability of ISSA are excellent. For F2 to F4, although SSA can also find the optimal value of zero, this does not mean that SSA can find the optimal value every time, because the average is not zero. The other six algorithms fail to find the optimal value of zero. For F5 to F7, ISSA and SSA do not converge to the global optimal solution, but ISSA converges faster. From the data in For multimodal functions, i.e., F8 to F13, ISSA is superior to SSA in most indicators. On F8, ISSA is not as stable as SSA, but the convergence accuracy is one order of magnitude higher than SSA, and the convergence speed is also much faster than SSA. For F9 to F11, the performance of ISSA is similar to that of SSA. On both F12 and F13, ISSA is two to three orders of magnitude higher than SSA, both in terms of convergence accuracy and stability. For the other six algorithms, except that the GWO, WOA, HHO, and SNS perform slightly worse than ISSA, both the GA and PSO are far inferior to ISSA.For fixed dimension functions, i.e., F14 to F23, due to the low dimension, the indicators of ISSA and SSA are relatively close. On F15 to F20, except GA, the other seven algorithms can find or approach the optimal value, but ISSA is always the most stable. On F14, SNS has the best convergence accuracy and stability, while SSA has the worst convergence accuracy and stability. On F21, SNS has the best convergence accuracy and stability. On F22 and F23, ISSA has excellent convergence accuracy and stability.Based on the three strategies proposed in As shown in It can be seen from SSA01 adopts the Chebyshev chaotic map to improve the diversity of the population and uses elite opposition-based learning strategy to generate high-quality populations. SSA02 introduces a dynamic weight factor to balance the ability of global exploration and local exploitation and uses the Levy flight strategy to expand the search space, avoid falling into the local optimal solution, and improve the convergence accuracy. SSA03 uses a mutation strategy to perform mutation operations on individuals to increase the diversity of the population and improve the ability to jump out of local optimal solutions. This further explains the feasibility of three strategies adopted in this paper.p is used to compare the differences between the two algorithms. When p < 0.05, H0 is rejected, indicating that there is a significant difference in performance between the two algorithms. When p > 0.05, H0 is accepted; that is, the two algorithms have the same global optimization performance.Derrac et al.  suggest R is the significance evaluation result: \u201c+,\u201d \u201c\u2212,\u201d and \u201c\u2009=\u2009,\u201d respectively, represent the performance of ISSA is superior, inferior, and equivalent to the algorithms under comparison. NAN means that it cannot be compared; that is, the two algorithms under comparison both find the global optimal solution and cannot make a significant difference judgment.p values of ISSA and SSA on F3 are slightly greater than 0.05, and the other p values are much less than 0.05. This indicates that the performance of ISSA and SSA on F3 is similar, while on other benchmark functions, the performance of ISSA is significantly different from the other seven algorithms. The p values of ISSA and SSA on F1, F9 to F11, F17, and F19 are NAN because both algorithms find the global optimal solution. The results of the Wilcoxon rank-sum test further illustrate that the performance of ISSA is indeed significantly improved compared with other algorithms.It can be seen from N, the dimension of solution space is D, and the maximum number of iterations is Tmax. Suppose the time required for initializing population parameters is t0, the time required for generating random numbers in each dimension is t1, the time for solving fitness function is f(D), and the time for sorting sparrows by fitness value is t2, and then, the time complexity of SSA in initializing population stage isSuppose the number of individuals in the sparrow population is PD, the time required to update the position of each dimension according to . The time complexity of this stage isWhen updating the location of producers, suppose the number of producers is rding to  is t3, tN \u2212 PD), the time required to update the position of each dimension according to . The time complexity of this stage isWhen updating the position of scroungers, the number of scroungers is . The time complexity of this stage isWhen updating the position of scouters, suppose the number of scouters is rding to  is t6, tTo sum up, the time complexity of SSA is\u03b70, the time required to initialize the position of each dimension according to . Suppose the proportion of elite individuals is r, then the number of elite individuals is rN. Suppose the time required to generate the elite individual position of each dimension according to , and the time to sort and generate the real initial population is \u03b73. Then, the time complexity of this stage isNow, the time complexity of ISSA is analyzed. Suppose the time required for initializing population parameters is rding to  and 5)  \u03b70, the rding to  is \u03b72, t\u03b74, the time required to update the position of each dimension according to . Then, the time complexity of this stage isWhen updating the position of producers, suppose the time required to generate the dynamic weight factor according to  is \u03b74, trding to  is \u03b75, arding to  is \u03b77, tequation  is \u03b78, a\u03b79, the time to generate the random number Q is still \u03b76, and the time to solve the fitness function is f(D). Then, the time complexity of this stage isWhen updating the position of scroungers, the time required to update the position of each dimension according to  or 12)  12) is \u03b7T3\u2032 of updating the position of scouters is the same as equation mm, mm, mm, and mm, respectively. The distribution of unoptimized sound field and the ISSA-optimized sound field on the z=100mm plane is shown in x=\u221210mm, the variation curve of sound pressure P with y is shown in In the case of symmetric focal point, we set four focal points, whose coordinates in the E\u2009\u2212\u200905\u2009Pa, and the energy of the acoustic wave is more concentrated to the focal points. It can be seen from the calculation that the percentage of sound pressure improvement is nearly 100%. This improvement can be more clearly observed in y=0mm, and the blue curve is always above the red curve, which means that the acoustic sidelobes are very obvious in the unoptimized sound field, while after ISSA optimization, the acoustic sidelobes in the sound field have been suppressed. Here, the focal point sound pressure is set to 600\u2009Pa, and the acoustic sidelobe sound pressure has reached 94.5\u2009Pa, accounting for 15.75% of the focal point. It can be seen that there is a lot of waste of sound energy. It is very necessary to suppress the acoustic sidelobe and improve the focal point energy.As shown in z direction are 200mm, and their coordinates in the xy plane are mm, mm, mm, and mm, respectively. The distribution of the unoptimized sound field and the ISSA-optimized sound field on the z=200mm plane is shown in x=\u221220mm, the variation curve of sound pressure P with y is shown in In the case of the asymmetric focal point, we also set four focal points. Their coordinates in the y=0mm before optimization is 168.11\u2009Pa, accounting for 28.02% of the focal point. After ISSA optimization, the acoustic sidelobe is weakened to 116.05\u2009Pa, accounting for 19.34% of the focal point. The improvement is about 10%. The energy of the sound field is more concentrated after ISSA optimization, which is very beneficial for HIFU treatment.As shown in Improve the sparrow search algorithm, enhance the quality of the initial population, and the ability to jump out of the local optimal solutionEstablish a 256-element concave spherical phased array transducer model, use ISSA to optimize the HIFU sound field, effectively suppress the acoustic sidelobe, improve the focusing performance, and provide a new idea for the research of HIFU technologyThis paper presents an improved sparrow search algorithm, which overcomes some shortcomings of SSA and improves the convergence performance and stability of SSA . ISSA usIn future work, we will further optimize ISSA and use it to solve other engineering problems. At the same time, we will also pay attention to other advanced optimization algorithms and make further research."}
+{"text": "Mitotic count in breast cancer is an important prognostic marker. Unfortunately, substantial inter\u2010 and intraobserver variation exists when pathologists manually count mitotic figures. To alleviate this problem, we developed a new technique incorporating both haematoxylin and eosin (H&E) and phosphorylated histone H3 (PHH3), a marker highly specific to mitotic figures, and compared it to visual scoring of mitotic figures using H&E only.Two full\u2010face sections from 97 cases were cut, one stained with H&E only, and the other was stained with PHH3 and counterstained with H&E (PHH3\u2013H&E). Counting mitoses using PHH3\u2013H&E was compared to traditional mitoses scoring using H&E in terms of reproducibility, scoring time, and the ability to detect mitosis hotspots. We assessed the agreement between manual and image analysis\u2010assisted scoring of mitotic figures using H&E and PHH3\u2013H&E\u2010stained cells. The diagnostic performance of PHH3 in detecting mitotic figures in terms of sensitivity and specificity was measured. Finally, PHH3 replaced the mitosis score in a multivariate analysis to assess its significance.P\u2009<\u20090.001. The concordance between pathologists in identifying mitotic figures was highest when using the dual PHH3\u2013H&E technique; in addition, it highlighted mitotic figures at low power, allowing better agreement on choosing the hotspot area (k\u00a0=\u00a00.842) in comparison with standard H&E (k\u00a0=\u00a00.625). A better agreement between image analysis\u2010assisted software and the human eye was observed for PHH3\u2010stained mitotic figures. When the mitosis score was replaced with PHH3 in a Cox regression model with other grade components, PHH3 was an independent predictor of survival , and even showed a more significant association with breast cancer\u2010specific survival (BCSS) than mitosis  and Ki67 (P\u00a0=\u00a00.27).Pathologists detected significantly higher mitotic figures using the PHH3\u2013H&E  compared with H&E alone , Using PHH3\u2013H&E\u2010stained slides can reliably be used in routine scoring of mitotic figures and integrating both techniques will compensate for each other's limitations and improve diagnostic accuracy, quality, and precision. Table\u00a01. Correlation between PHH3 expression with clinicopathological variables; Table\u00a02. Correlation between MAI, with PHH3 and Ki67; Table\u00a03. Multivariate cox regression analysis results for (A) adding PHH3 to predictors of survival (grade and stage), (B) adding PHH3 to mitosis and Ki67 score (C) components of grade (D) components of grade with the replacement of mitosis score with PHH3 score (E) components of grade with the replacement of mitosis score with Ki67 score. Mitotic score is a key component of breast cancer (BC) grading and is a strong predictor of survival,Histone H3 is one of the five histone proteins that together form the major protein constituents of chromatin in eukaryotic cells.Although staining results of both H&E and PHH3 can be viewed using a conventional bright\u2010field microscope, H&E alone cannot reflect the presence and distribution of underlying specific antigens, just as PHH3 protein expression alone cannot be evaluated adequately without the context of tissue morphology. The dual\u2010staining technique proposed in this work enables visualization of morphology and molecular profiling over the same tissue section and can thus improve the overall accuracy, quality, and diagnostic precision. Another advantage of this approach is that computational stain separation can be performed on a dual\u2010stained image to obtain an H&E and an immunohistochemistry (IHC)\u2010stained whole\u2010slide image from the same tissue section thus eliminating the need for image registration from serial sections. Consequently, the proposed scheme can be used for the development of computational pathology\u2010based biomarker prediction algorithms directly from dual\u2010stained histopathological images without the need for image registration or correspondence analysis.Combining both H&E and IHC techniques might achieve an optimum method for mitosis detection and counting in BC, and that dual staining of BC tissue sections with PHH3 and H&E will improve the concordance of mitosis counting, and hence the overall grade.This study was conducted on a cohort of primary invasive BC where pseudonymised patient tissue samples were used. Two full\u2010face tumour sections 4 \u03bcm thick from 97 cases were cut; one was stained with H&E only, and the other was stained with PHH3 counterstained with H&E. The cases were selected to represent different grades of BC.Clinical information and tumour characteristics including patient's age at diagnosis, histological tumour type, grade, tumour size, lymph node status, Nottingham Prognostic Index (NPI), and lymphovascular invasion (LVI) were available. Outcome data were calculated and these included BC\u2010specific survival (BCSS), defined as the time (in months) from 6\u2009months after the date of primary surgical treatment to the time of death due to BC, and distant metastasis\u2010free survival (DMFS) defined as the time (in months) from 6\u2009months after surgery until the first event of distant metastasis. Data for oestrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), and Ki67 were available as previously published.in\u00a0situ hybridisation (CISH), using the HER2 CISH pharmDx kit , as previously described.ER and PR positivity were defined as positive nuclear staining in\u2009\u22651% of the invasive tumour cells.Representative paraffin\u2010embedded tissue blocks of BC tissue were retrieved and processed using a protocol for the dual H&E and IHC staining; 4\u2010\u03bcm tissue sections were cut onto charged slides, and then placed on a 60\u00b0C hotplate for 20\u00a0min. After rehydration, slides were submerged in citrate buffer at pH\u00a06.0. Water bath heat\u2010assisted retrieval for 30\u00a0min at 96\u00b0C was applied with citrate buffer.Rabbit polyclonal anti PHH3  was diluted at 1:100 in Leica antibody diluent  and incubated with the sections for 60\u00a0min at room temperature. The DAB  working solution was applied. Haematoxylin nuclear stain was applied for a longer period (8\u2009min), to remove nonspecific background staining and to improve contrast, weak acid alcohol was used, and then eosin counterstain was applied (2\u2009min); Figure\u00a0Stained slides were scanned at 40\u00d7 magnification using a high\u2010throughput slide scanner , and the slides were then viewed with case viewer software program .We assessed the utility of adding PHH3 to routine H&E in scoring mitosis and grading BC by comparing counting mitosis using this technique with traditional mitoses scoring using H&E only.2 rectangle was drawn, in the exact region in each of the two slides, and mitotic figures within each rectangle were counted: Figure\u00a0For assessment of the reproducibility of each staining technique, two sections from each case were utilised, one stained with H&E only and the other was stained with PHH3 and counterstained with H&E. A 3\u2009mmMitotic counts using H&E and dual PHH3\u2013H&E immunostaining techniques were independently scored by two certified pathologists to measure the agreement between them.The technique that achieved the highest level of agreement was considered the most reliable one. For each staining technique, the average time required to count mitoses was recorded.2 circle in the area with the highest number of mitotic figures using the circle annotation tool in the toolbar. Agreement was reached when these circles overlapped or intersected.To determine the most effective method for revealing the greatest number of mitotic figures (hotspots), we evaluated the agreement of two pathologists in detecting mitotic hotspots in 20 whole\u2010slide images (WSIs) by having each of them draw a 5\u2010mmWe assessed the degree of agreement between manual and digital image analysis (DIA) tools  and specificity (shown on the X\u2010axis) of a test as the threshold for defining test positivity, which varies over the entire range of possible test outcomes. Sensitivity and specificity were calculated as follows:true\u2010positive mitotic figures.Brown\u2010stained nuclei with loss of nuclear membrane or the presence of chromosome condensation arranged along a plane or separated were considered false\u2010negative mitotic figures.Unstained or missed mitotic figures showing the above criteria were considered false\u2010positive mitotic figures, or PHH3\u2010stained G2 phase cellsWhile intact brown\u2010stained nuclei or nuclei with smooth membrane and the absence of chromosome condensation were considered Using this method, we were able to assess PHH3's diagnostic performance and accuracy in detecting true mitotic figures. The relative ability of PHH3 to distinguish mitotic figures from other cells in the cell cycle was determined by performing the receiver operating characteristic (ROC) curve. ROC curves demonstrate the coordinate variation in sensitivity  and low (<20 mitoses /3\u2009mm2). Differences between the two independent groups were compared by the Mann\u2013Whitney U\u2010test. The degree of interobserver agreement was assessed using the intraclass correlation coefficient (ICC) for continuous data. The Kappa statistic was used to assess the concordance between observers for categorical variables. Outcome analysis was assessed using Kaplan\u2013Meier curves and the log\u2010rank test. The Cox regression model was used for the univariate and multivariate analysis. For all tests, P\u2009<\u20090.05 (two\u2010tailed) was considered statistically significant.All statistical analyses were performed using SPSS v. 26 . The correlations between categorical variables were analysed by the Chi\u2010square test. The total number of PHH3\u2010stained mitotic figures was dichotomised based on BCSS using X\u2010tile bioinformatics software version 3.6.1 When using H&E stain only, the number of mitoses was significantly underestimated as compared to those identified using the PHH3\u2013H&E staining technique; Figure\u00a0P\u2009<\u20090.001.Pathologists detected significantly higher mitotic figures using the PHH3\u2013H&E  compared with the H&E method , High agreement between pathologists was observed when using PHH3\u2013H&E (ICC\u00a0=\u00a00.820) in comparison with standard H&E (ICC\u00a0=\u00a00.514). The concordance between pathologists in identifying mitotic figures was highest when using the dual PHH3\u2013H&E technique and was lowest using H&E\u2010stained slides only.2 for HE only and median\u2009\u00b1\u2009SD, 120\u2009\u00b1\u200970\u2009sec/3\u2009mm2 for PHH3\u2013H&E; P\u2009<\u20090.001); Figure\u00a0For both pathologists, the time taken to score mitotic figures stained with H&E only was significantly longer than the scoring time for those mitotic figures stained with PHH3\u2013H&E  was better in comparison with H&E (k\u00a0=\u00a00.605).PHH3\u2010labelled\u2010mitotic figures were easily seen and permitted quick identification of hotspots; it highlighted mitotic figures at low power at ease without strain. Agreement between pathologists when using PHH3\u2013H&E , between ImageJ and pathologist eye (ICC\u00a0=\u00a00.337), and between pathologist and QuPath (ICC\u00a0=\u00a00.405) was observed.For PHH3\u2010stained mitotic figures, a good agreement between QuPath and ImageJ was observed (ICC\u00a0=\u00a00.692), between ImageJ and pathologist eye\u00a0(ICC\u00a0=\u00a00.706), and between pathologist and QuPath (ICC\u00a0=\u00a00.824); Figure\u00a0Regarding the distinction between PHH3\u2010stained mitotic cells and G2 cells, a good agreement was observed between QuPath and ImageJ (ICC\u00a0=\u00a00.643), ImageJ and pathologist eye (ICC\u00a0=\u00a00.791), and between pathologist and QuPath (ICC\u00a0=\u00a00.834), in detecting PHH3\u2010stained G2 cells only.PHH3 diagnostic performance using diagnostic testing metrics such as the sensitivity, specificity, and area under the ROC curve (AUC), revealed that AUC was equal to 0.84, suggesting that PHH3 can be used as a good accurate test in detecting mitotic figures; Figure\u00a0Using PHH3\u2013H&E, 9 cases of grade 1 were upgraded to grade 2 and 15 cases of grade 2 were upgraded to grade 3 . None of the cases were downgraded.The associations between the PHH3 expression level and clinicopathological features of the tumours are summarised in Table\u00a0P\u00a0=\u00a00.01), tumour size \u22652\u00a0cm, high grade, nuclear pleomorphism, few tubule formations, and poor NPI (P\u2009<\u20090.001).PHH3\u2010positivity was significantly associated with aggressive characteristics, including higher tumour stage , while a weak positive correlation was observed for the Ki67 score ; Table\u00a0r\u00a0=\u00a00.177, P\u00a0=\u00a00.016).A strong positive significant correlation was found between mitotic count per 3\u2009mm2, had a significantly shorter BCSS and DMFS  and  respectively; Figure\u00a0Univariate survival analysis revealed that patients with a high number of PHH3\u2010stained mitotic figures with a cutoff of PHH3 mitotic figures >20 per 3\u2009mmP\u2009<\u20090.001), and occurrence of distant metastasis (P\u2009<\u20090.001). However, such an association was not observed in patients who received chemotherapy.In the nonchemotherapy\u2010treated cohort, a high number of PHH3\u2010stained mitotic cells were predictive of a higher risk of death from BC (P\u2009<\u20090.001), and occurrence of distant metastasis (P\u2009<\u20090.001). However, such an association was not observed in patients who received hormonal therapy; Figure\u00a0Similarly, in the nonhormonal therapy\u2010treated cohort, high PHH3 was predictive of a higher risk of death from BC (P\u00a0=\u00a00.039) and worse DMFS . When PHH3 was added to mitosis and the Ki 67 score, it was an independent predictor of BCSS , and DMFS , and when mitosis was replaced with PHH3 in a Cox regression model with other grade components, PHH3 was an independent predictor of survival , and even showed more significant association with BCSS than mitosis  and Ki67 (P\u00a0=\u00a00.27) in our sample; Table\u00a0In the multivariate Cox regression model including other prognostic covariates , PHH3 was an independent predictor of shorter BCSS  potential of tumours, including assessing the overall proliferation index (average mitotic score), and mitotic evaluation in randomly selected areas and the highly mitotic areas of the tumour (hotspots). In a previous study carried out by our group, we found that there is a tendency to underestimate mitotic count in randomly selected areas or the whole tumour slide compared to hotspots.Accurate histologic grading is required for effective clinical staging and treatment decisions; however, distinguishing mitotic figures in H&E\u2010stained slides from similar chromatin changes is a subjective process that can be subjected to intra\u2010 and interobserver variation.Our study evaluated this subjectivity by assessing the interobserver reproducibility of mitotic count using this new technique of counterstaining PHH3 with H&E among pathologists, we have found that the agreement among pathologists in recognizing mitotic figures was highest when employing the dual PHH3\u2013H&E staining approach. In accordance with other studies,Moreover, PHH3 staining within a given tumour was heterogenous and allowed for easy identification\u00a0of mitotic hotspots; lastly, it was significantly less\u00a0time\u2010consuming than counting mitoses on conventional H&E preparations, sparing precious diagnostic time, and efficiently increasing the number of cases diagnosed daily, while improving the quality of diagnosis.The added value of using PHH3\u2013H&E immunostaining is that it allows pathologists to assess the morphologic features of mitosis at the same time, with the tumour histological features increasing the specificity of quantification.We also consider that using H&E in staining along with PHH3, or other diagnostic antibodies, can spare important diagnostic areas that could be lost during sequential sectioning, sparing the valuable tissue biopsies as serial sectioning may cut through the area of interest and may result in the loss of regions necessary for critical diagnosis. This is particularly an issue with smaller core needle biopsies that are of limited size and number. And if we considered the removal of the H&E stains, it does not always leave the target epitopes intact for potential reuse of the slide for selective biomarkers in current existing protocols.Another advantage of using dual\u2010stained slides is that the rapidly expanding use of WSI and artificial intelligence allowed the use of more objective measurements, including DIA for more accurate and objective grade reporting.Thus, mitotic count based on PHH3 staining appears a robust, easy, and reliable method and could potentially decrease interobserver variability, especially with less experienced pathologists.We also demonstrated that using ImageJ analysis\u2010assisted techniques was comparable to the human eye in terms of the detection of mitotic figures, and the agreement even improved when these mitotic figures were labelled with PHH3, and the distinction between PHH3\u2010labelled mitotic figures and G2 phase\u2010stained cells are possible with good agreement.Using this technique, we were able to test the accuracy of mitosis detection by PHH3, and it showed high accuracy reflected by the sensitivity, specificity, and ROC curve. Despite missing a few mitotic cells, this may be due to IHC\u2010related technical issues with tissue fixation and antigenic retrieval.We examined the clinical outcome of the patients, and based on our findings we found that PHH3 has the\u00a0capability for a further contribution to BC grading and classification, and could be especially beneficial for\u00a0pathologists, and training machine\u2010learning algorithms.2, whereas the Ki67 score showed only a mild positive correlation. Although Ki67 is a widely used and well\u2010known proliferation marker in BC, it is not specific for mitosis, but is expressed in all phases of the cell cycle. Many cells that are not committed to cell division (not in the mitosis phase of the cell cycle) will be positive for Ki67. In contrast, PHH3 specifically identifies cells undergoing mitosis; therefore, it would provide a better representation of proliferation activity in BC and can be used in the clinical setting to identify mitoses.The mitotic count showed a significant positive association with PHH3 score, per 3\u2009mmPHH3 was an independent predictor of survival when it was added to grade and nodal stage, and it even showed a more significant association with survival than mitosis score, and Ki67 in the multivariate analysis; therefore, the PHH3 score could be more predictive of outcome than mitosis and Ki67. This agrees with other studies, where it has been proposed as a replacement for the Ki67 index in several cancers.A challenge that might face the implementation of PHH3 staining in routine practice is the cost burden on the pathology service, especially in places where healthcare is not extensively subsidised. It would be a trade\u2010off between precision and expense. Healthcare providers in general and pathologists specifically should supply the best possible service to the patients whenever possible and they should be responsible for the decision and diagnoses made. Another point to mention is that PHH3 staining has the same cost as other routinely assessed IHC markers in BC such as ER and Her2, and can provide prognostic value at a lower cost than existing multigene assays and will refine BC grading when using WSIs, which are associated with lower mitoses detection abilityMoreover, utilizing PHH3 to refine mitosis counting, requires readjusting the range and the cutoffs used to define mitosis scores in BC, as it was shown that the number of mitotic figures detected using PHH3 is higher than that detected using H&E. This refining would need multicentric validation on a large number of cases and with long follow \u2010p data.Histopathological diagnoses of tumours depend mainly on H&E and IHC staining. These are the gold standards in clinical care. We are developing a new technique that combines both and can be tissue\u2010 and timesaving, while improving the diagnostic quality. It provides a more sensitive approach for training artificial intelligence IHC prediction models while using the exact same slide. Our results demonstrated a tendency to undergrade BCs based on H&E compared with PHH3, which alters the stage, risk of disease progression, and treatment recommendations. We, therefore, show for the first time the potential of using PHH3 counterstained with H&E for precise routine mitotic scoring in clinical practice.This work was approved by the Nottingham Research Ethics Committee 2 under the title Development of molecular genetic classification of breast cancer, and obtained ethics approval by the Northwest\u2014Greater Manchester Central Research Ethics Committee under the title; Nottingham Health Science Biobank, reference number 15/NW/0685.AI stained and scored all the cases, took the lead in writing the article, data analysis and interpretation, SM helped with double scoring, ER: conceived and planned the study, contributed to data interpretation, made critical revisions, and approved the final version. All authors contributed to writing the article and approved the final version.The authors declare no conflicts of interest."}
+{"text": "The permanence of microorganisms in the root canal system represents the main cause of endodontic failure. Considering the impossibility of effective action of the endodontic files in ramifications of the main canal and mainly inside the dentinal tubules, a better understanding of the irrigation dynamics to enhance endodontic prognosis is essential. Objective: To evaluate the depth of intratubular penetration values of sodium hypochlorite (NaOCl) (dependent variable) by comparing different concentrations, methods of irrigation, and root canal thirds (independent variables) and to investigate the existence of interactions among them, capable of influencing the dependent variable. p< 0.05). 40 roots from extracted human maxillary central incisors were stained and instrumented according to four irrigation protocols (n. 10): conventional irrigation (CI) at each use or change of instrument, and final irrigation with 5ml of 2.5% or 5.25% NaOCl, with or without passive ultrasonic irrigation (PUI), respectivelly. Measurements based on stereomicroscopic images were obtained, and the data were subjected to statistical analysis (The highest depth values of intratubular penetration of NaOCl were observed in the cervical third, at 5.25%, and by PUI. When only two independent variables were analyzed in association, the highest penetration depth values of NaOCl were obtained at 5.25%, regardless of irrigation method, at 5,25%, in the cervical third, and; in the cervical third, despite of irrigation method. Considering the three independent variables simultaneously, the highest depth values of intratubular penetration of NaOCl were observed in the cervical third, at 5.25%, no matter the irrigation method. The interaction between the independent variables on the penetration depth values of NaOCl was only confirmed considering the irrigation method and root canal third. Intratubular penetration of NaOCl was influenced by the three independent variables individually and when the irrigation method and root canal third were considered simultaneously. Key words:Dentinal tubules, Depth, Disinfection, Irrigation, Root canal system, Sodium hypochlorite. The main goal of Endodontics is to keep natural teeth by preserving or restoring the health of periapical tissues. In necrotic teeth, the pulp cells are compromised, thus favoring root canal system (RCS) infection . Even ifThe biomechanical preparation is the main factor responsible for endodontic infection control . It is pAntimicrobial strategies during endodontic interventions are much more effective in the main root canal. However, the maintenance of remnants of microorganisms, including in the dentinal tubules, has previously been evidenced by several studies ,7, beingPassive ultrasonic irrigation (PUI) is one of the most studied methods to optimize the disinfection of the RCS. The energy released by inserts during PUI produces cavitation and acoustic streaming, resulting in microbubbles and hydrodynamic waves that promote agitation of the liquid against the root canal walls, favoring their cleaning and the penetration of the irrigating solution into the dentinal tubules .et al.  to evaluate the depth of intratubular penetration values of NaOCl (dependent variable), comparing two concentrations (2.5 and 5.25%) and two irrigation methods (CI and PUI) in the three root canal thirds  (independent variables), and; 2) to investigate whether there were interactions between these variables, capable of influencing the depth of penetration of the irrigant, by using a comprehensive statistical analysis performed with data obtained by the histochemical method proposed by P\u00e9cora, et al. . Bearing-Approval by the Research Ethics Committee, selection, and preparation of specimensFor the development of this study, after authorization issued by the Research Ethics Committee of the Ribeir\u00e3o Preto School of Dentistry \u2013 FORP/USP (CAAE: 14962013.4.0000.5419), 40 extracted human maxillary central incisors were selected. The teeth were supplied by the Tooth Bank of the same Institution and had a single canal, complete rhizogenesis, without abrupt curvatures, resorption processes, or previous endodontic treatments. These characteristics were confirmed visually and on radiographs taken in the mesiodistal and buccolingual directions. The specimens selected were stored in 0.1% thymol solution at 48\u00b0C and washed in running water for 24 hours before use . After tAfter the canal was irrigated with 5 ml of distilled and deionized water using a Navitip needle and a 5 ml plastic syringe , it was initially explored through an n. 20 K-FlexoFile . Then, the specimens were placed in a vacuum desiccator  for 120 minutes to allow complete dehydration. After this, they were immersed in a copper sulfate solution and placed in a desiccator coupled to a vacuum pump  for 15 minutes to remove air from the main canal to enable more extensive penetration of the solution mentioned above. Subsequently, the specimens remained immersed in this solution for another 75 minutes at ambient atmospheric pressure, and the root surface and the canal were then dried with paper towels and n. 20 absorbent paper cones (Dentsply-Maillefer), in that order. Finally, all specimens were immersed in a rubeanic acid solution, transferred to and stored in the desiccator coupled to a vacuum pump, and exposed to ambient atmospheric pressure for 15 and 225 minutes, respectively.-Biomechanical preparation Before performing biomechanical preparation, each root was wrapped with a thin layer of hydrophilic vinyl imprint vinyl polysiloxane  to simulate the periodontal ligament space and embedded in acrylic resin blocks   to avoid\u2022 CI with 5ml of 2.5% NaOCl at each use or change of instrument, and final irrigation with 5ml of 2.5% NaOCl for 30 seconds*;\u2022 CI with 5ml of 2.5% NaOCl at each use or change of instrument*, and PUI** with 5ml of 2.5% NaOCl for 30 seconds*;\u2022 CI with 5ml of 5.25% NaOCl at each use or change of instrument, and final irrigation with 5ml of 5.25% NaOCl for 30 seconds*;\u2022 CI with 5ml of 5.25% NaOCl at each use or change of instrument*, and PUI** with 5ml of 5.25% NaOCl;*Procedure performed with a Navitip irrigation tip (Ultradent) inserted at -2mm from the WL.**Procedure performed with an Irrisonic ultrasonic insert (Helse), inserted at -2mm from the WL, at a power of 20kHz.Subsequently, the root canal was dried with absorbent paper cones (Dentsply-Maillefer) with a diameter corresponding to the final instrument used during the biomechanical preparation.-Analysis of intratubular penetration values of NaOClEach specimen was sectioned horizontally at 2, 8, and 14mm from the apex using a low-speed steel cutting disc . Three slices per root were obtained, resulting in 120 slices. A standard polishing procedure using SiC paper  followed by three \u03bcm diamond paste was employed on the cervical surface of each slice to produce a high-reflection surface, and each slice was observed in a high-resolution stereomicroscope to acquire images at 1048x1048 pixels, covering the entire root surface. For each image, the areas corresponding to discolored dentin and the perimeter of the root canal were measured using the AxioVision Software 4.11 . By subtracting the area corresponding to the perimeter of the root canal from the region corresponding to the discolored dentin, intratubular depth penetration of the irrigant was determined in mm2.-Statistical analysisp > 0.05). Therefore, the 3-way parametric analysis of variance (ANOVA) test  was applied to identify statistical differences concerning the mean values of the dependent variable studied (intratubular penetration depth of NaOCl), according to independent variables: concentration (2.5% and 5.25%); irrigation method (CI and PUI) and root canal third , as well as to analyze the paired and associated interaction between them. Since there were statistically significant differences, the Tukey HSD and Games-Howell parametric tests for multiple comparisons were applied for homogeneous and heterogeneous variances. The Levene test was used to identify the homogeneity of variances in the depth of intratubular penetration values of NaOCl among the irrigation protocols. The significance level was set at 0.05, and all analyses were performed using the IBM SPSS Statistic 25.0 software   .-Data availabilityThe complete statistical analysis of this research can be ac-cessed at: https://1drv.ms/f/s!AnuvHAhxDtNGg75jJv8e-jlJeQcGcqg?e=qNqX9X.-Depth of intratubular penetration values of NaOCl, considering the irrigation method, concentration, and root canal third, individuallyp < 0.01, with a test power of 99.99% determined based on the sample size. Thus, the existence of statistically significant differences was proved by comparing concentrations of the irrigant (2.5% and 5.25%), irrigation methods (CI and PUI), and root canal thirds evaluated . The highest depth values of intratubular penetration of NaOCl were observed in the cervical root canal thirds (0.72 \u00b1 0.28 mm2), using the product at the concentration of 5.25% (0.68 \u00b1 0.31 mm2) and in an activated manner (PUI) (0.63 \u00b1 0.30mm2) (p < 0.01). The lowest values occurred in the apical root canal thirds (0.38 \u00b1 0.32mm2), with no statistically significant differences in comparison to the middle root canal thirds (0.48 \u00b1 0.20mm2) (p \u02c3 0.05)  test . This test showed F values of 68.88, 29.41, and 28.46, respectively, and -Depth of intratubular penetration values of NaOCl, considering concentration/irrigation method, concentration/root canal third, and irrigation method/root canal thirdp < 0.01, with a test power of 99.99% determined based on the sample size. The highest penetration depth values of NaOCl were obtained by the following combinations, without significant statistical differences between them (p \u02c3 0.05): 5.25%/PUI (0.78 \u00b1 0.31mm2) and 5.25%/CI (0.58 \u00b1 0.28mm2); 5.25%/cervical root canal third (0.90 \u00b1 0.26mm2), and; CI/cervical root canal third (0.64 \u00b1 0.30mm2) and PUI/cervical root canal third (0.79 \u00b1 0.26mm2)  test , showed F values of 21.33, 19.71 and 12.01, respectively, and -Depth of intratubular penetration of NaOCl considering the irrigation method, concentration, and root canal third, simultaneouslyp < 0.01, with test power considering the sample size of 99.99%, indicating that there were statistically significant differences. The highest depth values of intratubular penetration of the irrigant were observed in the cervical root canal thirds, using it at the concentration of 5.25%, without statistically significant differences between irrigation methods ; PUI/2.5%: 0.64 \u00b1 0.10mm2; CI/2.5%: 0.43\u00b1 0.13mm2). (p < 0.05). In the middle root canal third, there was no statistically significant di-fference when irrigation methods and concentrations of irrigant were compared  (p < 0.05). In the apical root canal third, the highest penetration depth values of NaO-Cl were obtained when it was used in higher concentration (5.25%), regardless of the irrigation method (PUI or CI) (p < 0.01)  , Fig. 3.-Interaction between the irrigation method, concentration, and root canal third on the intratubular penetration depth of NaOClp > 0.05)   , Fig. 4Cp > 0.05)  , Fig. 4DIn most cases, endodontic failure is caused by remaining microorganisms inside the RCS , which can even reach up to half the distance between the root canal walls and the cementum-dentin junction . Consideet al. ; therefore, the first null hypothesis established was rejected. Virdee, et al.  reported . However, the trend towards achieving higher penetration depth values of NaOCl rose significantly because of its activation . Furthermore, the proven interaction between the irrigation method and root canal third demonstrated that PUI promoted relevant increases in the depth values of intratubular penetration of the irrigant in all root canal thirds, which were considerably higher in the apical third (0.35 mm2); therefore, leading to rejection of the second null hypothesis established.Considering concentration/irrigation method, concentration/root canal third, and irrigation method/root canal third, the highest penetration depth values of NaOCl were obtained by the following combinations, without statistically significant differences between them: 5.25%/PUI (0.78 \u00b1 0.31mm2) and 5.25%/CI 0.58 \u00b1 0.28mm2); 5.25%/cervical root canal third (0.90 \u00b1 0.26mm2), and; CI/cervical root canal third (0.64 \u00b1 0.30mm2) and PUI/cervical root canal third (0.79 \u00b1 0.26mm2). When assessing the impact of the three independent variables together on the depth of intratubular penetration of the irrigant, the highest values were observed in the cervical root canal third of the specimens irrigated with 5.25% NaOCl, with no relevant statistical differences between PUI (0.94 \u00b1 0.28mm2) and CI (0.86 \u00b1 0.25mm2). Because of the higher volume and larger diameter of the dentinal tubules present in the cervical root canal third, the use of 5.25% NaOCl only, with the consequent release of a larger quantity of chlorine ions .\u2022 Regarding concentration/irrigation method, concentration/root canal third, and irrigation method/root canal third, the highest penetration depth values of NaOCl were obtained by: 5.25% NaOCl, regardless of the irrigation method (PUI or CI), 5.25% NaOCl in the cervical root canal third, and cervical root canal third, despite of the irrigation method.\u2022 Considering the three independent variables simultaneously, the irrigant\u2019s highest depth values of intratubular penetration were observed in the cervical root canal thirds, using it at the concentration of 5.25%, without statistically significant differences between irrigation methods. In the middle root canal third, there was no statistically significant difference when irrigation methods and concentrations of irrigant were compared. In the apical root canal third, the highest penetration depth values of NaOCl were obtained by PUI, using it at the concentration of 5.25% with significant statistical differences compared to the other combinations. In CI, the highest penetration depth values of the irrigant were also observed using it in the higher concentration, with significant statistical differences compared to the other combinations.\u2022 Analysis of the interaction between the concentration of NaOCl and irrigation method and NaOCl and root canal third on the intratubular penetration depth of the irrigant, indicating their absence. An interaction was identified by considering the irrigation method and root canal third on penetration depth values of the irrigant. Investigating the interaction among all independent variables simultaneously on the depth of intratubular penetration values of NaOCl, no interaction was proven."}
+{"text": "This review aims to highlight the capabilities and advantages of neutron imaging in characterizing lithium batteries, as well as its current state of application in this field. Additionally, we discuss the potential of neutron imaging to contribute to the ongoing development of advanced batteries through its ability to visualize internal evolution.Lithium batteries have been essential technologies and become an integral part of our daily lives, powering a range of devices from phones to electric vehicles. To fully understand and optimize the performance of lithium batteries, it is necessary to investigate their internal states and processes through various characterization methods. Neutron imaging has been an indispensable complementary characterization technique to X-ray imaging or electron microscopy because of the unique interaction principle between neutrons and matter. It provides particular insights into the various states of matter inside lithium batteries, including the Li Neutron imaging is an indispensable technique for understanding batteries by providing unique insights into their internal states and processes, including lithium ion concentration, Li-metal anode behavior, electrolyte distribution, and gas generation. Batteries have been extensively used in intelligent electronics, electric vehicles and smart grids, etc., due to their characteristics of high open-circuit voltage, large discharge capacity and long cycle life . However\u22121) in lithium-ion batteries (LIBs) ..30\u201332].  cameras\u00a0. Typical cameras\u00a0.In this review, we focus on the application of NI in observing different states of matter\u2014classified by solid, liquid and gaseous phases\u2014inside lithium batteries. Also, the current development of the NI technique and possible future directions for its application in advanced batteries are discussed.in situ by nondestructive NI\u00a0[In solid electrode materials, the lithium concentration typically fluctuates significantly during charge and discharge\u00a0. Lithiumctive NI\u00a0, which cctive NI\u00a0.et\u00a0al.\u00a0[in situ that the attenuation coefficient of the whole graphite electrode gradually increased during discharge  can be related to the formation of SEI  anode to the LiCoO2 (LCO) cathode was highly dependent on the discharge rates, which was the visual evidence of the correlation between discharge capacity and current density. This provided visual evidence of the correlation between discharge capacity and current density  and an ultralow electrochemical potential (\u22123.04\u00a0V versus the standard hydrogen electrode), thereby Li metal has been referred to as the \u2018holy grail\u2019 anode of lithium batteries to fulfill the need for energy density\u00a0[in situ observe the dynamic evolution process of Li metal in batteries. Compared with X-ray imaging, neutron imaging exhibits its superiorities in observing Li metal. As Fig.\u00a02 electrode, but it is difficult to distinguish the morphology of Li metal. By contrast, a neutron is favorable to detect the consumption of the Li-metal anode during discharge because of its remarkable attenuation coefficient to lithium  a.\u00a0et\u00a0al.\u00a0 performeing Fig.\u00a0. Accordiing Fig.\u00a0. Also, tity Fig.\u00a0, which m6Li and 7Li isotopes. For instance, when exposed to thermal neutrons with an energy of 25\u00a0meV, 6Li demonstrates a considerable absorption cross section\u00a0of 940\u00a0b. In contrast, 7Li behaves nearly transparently with an absorption cross section\u00a0of 0.0454\u00a0b\u00a0[6Li and can easily pass through a 7Li atom, which allows for the visualization of the diffusion profile of lithium ions in electrode/electrolyte materials and the analysis of the tracer diffusion coefficient. Takai et\u00a0al.\u00a0[1.33Ti1.67O4. As presented in Fig.\u00a07Li, and annealing was carried out for diffusion couples of Li1.33Ti1.67O4 at 900\u2009\u25cbC. By comparing it with the calibration curve based on the digitized gray level of the neutron image  in comparison to LiFePO4 (LFP). As presented in Fig.\u00a0Also, the gas generation behavior in LIBs depends on the electrochemical and thermodynamic stability of the cathode materials\u00a0. Starke et\u00a0al.\u00a0, employiIn situ monitoring of the gas distribution within the battery using NI, coupled with gas chromatography-mass spectrometry analysis to determine the gas composition, can provide valuable insights into the underlying chemical and electrochemical processes involved in gas generation.Typically, the gas production process of batteries is intricate, influenced by various factors including electrolyte consumption, battery abuse, impurity introduction, inadequate packaging, etc. The development of neutrons as a tool for research has been driven by advances in instrumentation and experimental techniques. In recent years, neutron imaging has emerged as a powerful alternative to X-ray imaging for studying the internal structure and morphology features of materials. New neutron imaging modalities, such as phase contrast imaging\u00a0, Bragg eNeutron source. Research reactors are the main source of neutron imaging, but they are not portable and are only available at a few facilities\u00a0[(1) cilities\u00a0. The higcilities\u00a0. As a recilities\u00a0. Supplemcilities\u00a0.Advanced neutron imaging. In traditional neutron imaging techniques, the intensity of the transmitted beam is modulated by inhomogeneous neutron attenuation, known as absorption contrast. However, this approach does not work with materials of high transmittance or when one is interested in probing the magnetic or electric field distribution inside a bulk. The concept of wave-particle duality of neutrons can be used to overcome these limitations and treat neutron-matter interaction on par with wave-matter interaction. This allows the concept of \u2018phase of wave\u2019 to be implemented in phase-based neutron imaging to improve existing techniques\u00a0[(2) chniques\u00a0, such aschniques\u00a0. Besideschniques\u00a0.The neutron and X-ray tomography system. Neutron tomography and X-ray tomography are both nondestructive, penetrating methods used to examine the internal structure and composition of materials. However, each method has its strengths and limitations, and using them separately can lead to incomplete analyses. In the past, researchers have tried to overcome this issue by using separate experimental setups for neutron and X-ray imaging\u00a0[(3)  imaging\u00a0,58,59, w imaging\u00a0,120\u2013123. imaging\u00a0. For exaThe limitations of neutron imaging and strategies to enhance its functionality. Currently, whether for a reactor or spallation-based neutron source, the flux, monochromaticity and divergence of the generated neutron beam are inferior to those of X-rays. Furthermore, the spatial resolution of detectors used for NI typically ranges in the order of tens of micrometers, which presents a notable limitation compared to the submicron-level imaging resolution achievable with X-rays. Therefore, the relatively lower measurement accuracy and imaging quality of NI hinder the advancement of high-resolution imaging techniques. Here are several strategies that can enhance the functionality of NI techniques in lithium batteries. Firstly, implementing Bragg-edge NI based on time-of-flight methods can be advantageous for characterizing lithium batteries. This approach allows for the acquisition of both morphology and structure information of the materials inside the batteries (e.g. the phase transition of cathode). Secondly, coupling the NI facility with X-ray imaging can provide complementary benefits. By combining these two imaging techniques, researchers can obtain a more comprehensive understanding of the evolution inside the batteries. Furthermore, the design of an in situ imaging device, specifically tailored for lithium batteries and located at the sample position of the NI facilities, can serve a highly functional purpose by enabling real-time observation of the internal evolution and dynamic processes within the batteries.(4) in situ analysis of the lithiation/delithiation process. In addition, the dynamic evolution process of lithium dendrites can be visualized in the neutron images. Generally, the displacement of liquid electrolytes inside the batteries is coupled with gas generation. NI can be applied not only to observe the soaking process and consumption of electrolytes, but also to track the evolution of gas production in the battery.To summarize, we have reviewed the research progress of NI in observing lithium batteries based on the particularity of the interaction between neutron beams and matter. In terms of the solid phase, NI allows for the quantitative calibration of the lithium concentration distribution within the electrode, enabling 2 batteries, remains rare currently. Therefore, it is necessary to extend the powerful capabilities of NI to the characterization of advanced batteries. In light of this, we would like to propose several prospects for the future application of NI, specifically in observing the internal evolution of advanced batteries , to compensate for the shortage of in situ electron microscopy that is mainly applicable to smaller size cells. In addition, taking advantage of NI in liquid electrolytes and gas, the consumption of electrolytes and generation of gas caused by SEI fracture can be analyzed in situ , as a supplement to the measurement of ionic conductivity by electrochemical impedance spectroscopy. Secondly, the density or porosity of the solid-state electrolytes, as well as the evolution of the interface morphology during the cycle of the solid-state batteries, can be visualized by neutron tomography. Thirdly, the gradient distribution of the lithium-ion concentration at various interfaces inside the solid-state batteries can be calibrated by NI, which can provide important information for the ion diffusion mechanism at the interfaces. Fourthly, NI can be applied to observe the dynamic evolution of lithium dendrite growth along the grain boundary of solid electrolytes in situ. Besides, the molds utilized during the assembly of solid-state batteries to ensure tight electrode-electrolyte contact can introduce interference with the transmitted neutron beam, potentially compromising image quality. To mitigate the adverse effects on the neutron beam and signals, molds for solid-state battery assembly can be designed and fabricated using materials with low neutron absorption .benefits . The mecLi-S batteries. Li-S batteries possess the merits of high energy density and low cost, making them promising candidates for rechargeable batteries in the future\u00a0[2S) will cause an 80% volume change, which is associated with the lithiation of the cathode in Li-S batteries. Therefore, the evolution of cathode morphology can be observed in situ from both the volume change and lithium concentration through NI. On the other hand, the high-order polysulfide  can be observed in neutron images. Nonetheless, the data acquisition process of NI is often slower compared to X-ray imaging, resulting in limited temporal resolution. Consequently, this may lead to the loss of valuable information concerning complex thermodynamic and dynamic processes . To obtain more comprehensive chemical/electrochemical information, it is advisable to complement NI with in situ spectroscopic characterization techniques, allowing for a deeper understanding of the intricate processes occurring within Li-O2 batteries.00\u00a0Wh/kg\u00a0. The Li-ion Fig.\u00a0. In addiOverall, lithium batteries heretofore have experienced remarkable leaps in the past decades with commercialization. Meanwhile, researchers are unremittingly developing advanced batteries aiming at expanding the boundaries of energy density, safety and cycle life. Still, these advanced batteries involve complex internal components, including solid, liquid and gaseous phases, as well as light elements  widely existing in active materials, electrolytes and lithium metal. Therefore, we believe that NI can give full scope to its unique functions and advantages in the flourishing progress of lithium batteries in the future.nwad238_Supplemental_FileClick here for additional data file."}
+{"text": "Damage to the primary visual cortex following an occipital stroke causes loss of conscious vision in the contralateral hemifield. Yet, some patients retain the ability to detect moving visual stimuli within their blind field. The present study asked whether such individual differences in blind field perception following loss of primary visual cortex could be explained by the concentration of neurotransmitters \u03b3-aminobutyric acid (GABA) and glutamate or activity of the visual motion processing, human middle temporal complex (hMT+).We used magnetic resonance imaging in 19 patients with chronic occipital stroke to measure the concentration of neurotransmitters GABA and glutamate (proton magnetic resonance spectroscopy) and functional activity in hMT+ . We also tested each participant on a 2-interval forced choice detection task using high-contrast, moving Gabor patches. We then measured and assessed the strength of relationships between participants\u2019 residual vision in their blind field and in vivo neurotransmitter concentrations, as well as visually evoked functional magnetic resonance imaging activity in their hMT+. Levels of GABA and glutamate were also measured in a sensorimotor region, which served as a control.Magnetic resonance spectroscopy-derived GABA and glutamate concentrations in hMT+ (but not sensorimotor cortex) strongly predicted blind-field visual detection abilities. Performance was inversely related to levels of both inhibitory and excitatory neurotransmitters in hMT+ but, surprisingly, did not correlate with visually evoked blood oxygenation level\u2013dependent signal change in this motion-sensitive region.Levels of GABA and glutamate in hMT+ appear to provide superior information about motion detection capabilities inside perimetrically defined blind fields compared to blood oxygenation level\u2013dependent signal changes\u2014in essence, serving as biomarkers for the quality of residual visual processing in the blind-field. Whether they also reflect a potential for successful rehabilitation of visual function remains to be determined. Strokes affecting the visual system occur in \u224820% to 57% of stroke survivors and can significantly affect daily living and quality of life.10 Although this type of relationship has not been explored in stroke survivors with visual loss, the presence of blood oxygenation level\u2013dependent (BOLD) activity in hMT+ has been related to blindsight performance,5 and baseline BOLD signal change in both V111 and hMT+ appears to be predictive of visual rehabilitation success.12Over recent years it has become possible to measure the neurochemistry of specific brain regions in vivo in addition to structure and function. Indeed, with strokes that affect the motor system, a reduction in the inhibitory neurotransmitter \u03b3-aminobutyric acid+macromolecules (GABA+) has been related to increased plasticity and rehabilitation success.In the current study, we asked if the levels of activity and concentrations of key neurochemicals in hMT+ may underlie residual vision in the blind field of those with vision loss from V1 damage. We assessed how GABA+, glutamate (Glx), or BOLD signal changes in the ipsilesional hMT+ relate to individual differences in residual motion detection in the blind field of 19 chronic, unilateral, occipital stroke survivors. In additional to behavioral performance data, we collected structural magnetic resonance imaging (MRI), functional MRI (fMRI), and proton magnetic resonance spectroscopy (MRS) to determine how major inhibitory (GABA+) and excitatory (Glx) neurotransmitter levels, and neural activity in hMT+ related to residual vision. MRS data were collected in hMT+ of the damaged hemisphere and in sensorimotor cortex (M1) of the same hemisphere to use as a control region.We hypothesized that in stroke survivors with V1 damage (1) lower GABA+, (2) higher Glx, and (3) increased BOLD signal change in hMT+ might underlie superior residual motion processing in the blind field.Anonymous MRI and behavioral data will be made available on request.Figure S1 for average lesion location) resulting in contralesional visual field deficits . All participants had sustained damage in adulthood, at least 6 months before the study . There was no history of diagnosed cognitive or psychiatric disorders (including executive deficits), motor impairments (including those caused by the stroke), previous eye disease, or impairment other than visual loss after stroke .Nineteen, otherwise-healthy, stroke survivors with damage to V1 .14 but involved indicating the time interval in which a drifting, achromatic, Gabor stimulus  of varying contrasts  appeared . Participants were instructed to fixate on the central cross. Eye position was monitored using an Eyelink 1000 eye tracker .A psychophysical task (contrast detection task) was used to measure residual vision in the perimetrically defined blind and sighted fields. This task has been described in detail in prior publications,16 was used to acquire fMRI data for the motion localizer and contrast fMRI tasks .MRI scans were acquired on a 3 Tesla MRI scanner Siemens Prisma  using a 64-channel head/neck coil. A multiband gradient echo sequenceSupplemental Material for task details), and (2) determine BOLD signal change in hMT+ (contrast fMRI task). Stimuli were generated using MATLAB (2015b or 2018b) and Psychtoolbox (version 3.0.11) and presented on a screen at the back of the MRI scanner bore (viewing distance=127.5 cm). The contrast fMRI task has been described previously,12 and involved passively viewing a drifting achromatic Gabor patch  presented at the same location in the blind field and the equivalent location in the sighted field in a block design. In each 20 s block, a single contrast  or a rest block was presented 8\u00d7 . Each participant completed 3 runs. Participants fixated on a static cross and had to press a button when the cross changed from black to red. Color changes were random and lasted for 300 ms. In 4 cases, the visual field loss was in the far periphery. The fixation cross was then positioned eccentrically to enable the Gabor stimulus to be presented in the blind region.Two fMRI tasks were used to (1) localize hMT+  hemisphere . In the case of bilateral damage, the voxel was located in the hemisphere with the worse visual function (left hemisphere).MRS data were acquired in 2 separate scans from 25\u00d725\u00d720 mmSupplemental Material).GABA+ and Glx were measured from spectra acquired using a locally developed MEGA-PRESS (Mescher-Garwood point resolved spectroscopy) sequence, derived from the Center for Magnetic Resonance Research spectroscopy package . During MRS acquisition participants watched a nature documentary to reduce boredom and sleepiness. Participants were instructed to keep their eyes open throughout, and this was monitored using the eye tracker.In 12 participants, the lesion-affected zone (defined as damaged tissue and fluid-filled space resulting from the stroke) overlapped with the hMT+ voxel, occupying on average 5.68%\u00b19.86% (mean\u00b1SD) of the voxel volume. In 7 out of 12 the overlap was >2.5%, with the largest overlap at 36.8%  Gannet failed to fit the data, (2) the neurochemical concentration value exceeded 3SD from the mean group, and (3) the voxel was incorrectly positioned. All data were included in the hMT+ MRS analysis (N=19). One participant was excluded for each of these reasons for the M1 MRS analysis.http://www.fmrib.ox.ac.uk/fsl; see the Supplemental Material).Preprocessing and statistical analysis of the contrast task fMRI were carried out using Oxford Center for Functional MRI of the Brain software library . The FSL Featquery tool was used to calculate BOLD percentage signal change within the mask.Ipsilesional hMT+ masks were created for each participant using the intersection of the motion localizer activity, J\u00fclich histological atlas hMT+ mask, and MRS voxel volume and thresholded to produce a mask guided by the volume of hMT+ in the human literaturehttp://www.gabamrs.com/; see the Supplemental Material). Metabolite concentrations are given as a ratio to total creatine .MRS data from both voxels were analyzed using Gannet 3.1, an open-source toolbox for analysis of edited MRS data  were interpreted using standard convention.17Multiple linear regression and correlation analyses were carried out in R studio (version 4.1.2). Partial correlation analyses were carried out using the P<0.05 and a cumulative binomial distribution.14 Percentage correct on this contrast detection task was used as the measure of residual vision for all subsequent analyses.Residual blind-field visual performance was measured in each participant using a 2-alternative, forced-choice, psychophysical, contrast detection task  and Bayesian statistics (BF10=3.53). Comparable, strong evidence for a negative relationship was also present between Glx:tCr and behavioral performance . There was no strong evidence for a relationship between contrast detection performance and either GABA+ =1.51; P=0.896; \u03b2=0.04; BF10=0.50) or Glx concentrations in the M1 voxel .There was substantial evidence for a negative relationship between GABA+:tCr measured within the hMT+ voxel and behavioral contrast detection performance using both conventional , but not in the M1 voxel . Patient age, months since lesion, and proportion of gray matter volume within the voxel were controlled in all analyses. When a ratio of Glx/GABA+ was calculated to estimate excitatory-inhibitory balance, there was no correlation with behavior .We also found a significant, positive correlation between GABA+ and Glx concentrations in hMT+ .Percentage BOLD signal change in hMT+, measured while participants viewed a moving, achromatic, Gabor patch of high contrast in their perimetry-defined blind fields, was compared with rest periods Figure . There wThe present study shows, for the first time, that MRS-derived measures of neurochemistry in hMT+ are directly related to individual differences in the ability to detect drifting, high-contrast, Gabor patches inside perimetrically defined blind fields in patients with chronic stroke with V1 damage. Specifically, participants with lower levels of both GABA+ and Glx in hMT+ were better at performing this contrast detection task in their blind field than those with higher levels of both neurotransmitters. In contrast, there was no relationship between BOLD signal change in hMT+ when exposed to the same stimulus, and blind-field ability to detect it during psychophysical testing.9 Here, we posited that, as in the motor system,18 decreased inhibitory activity in the residual visual system long after occipital stroke\u2014especially in intact extrastriate visual areas\u2014could play a significant role in supporting neuroplastic mechanisms. In the healthy visual system, Frangou et al19 found that reduced GABA in the occipital lobe was related to the ability to detect targets in clutter, while GABA was increased when discriminating fine feature differences. The psychophysical task used to measure residual perceptual abilities in the blind field required detection of a moving Gabor patch rather than fine feature discrimination. Given that the V1-damaged visual system appears to suffer from increased levels of internal processing noise,20 a plausible hypothesis emerging from the present results is that GABAergic disinhibition in the motion processing visual cortical area hMT+ may work to enhance target detection by increasing signal-to-noise. The current findings are also consistent with Lunghi et al21 who showed that decreased GABA following short-term monocular deprivation correlated with greater plasticity of binocular vision in the healthy system.In chronic motor stroke survivors, GABA+ and Glx decrease in M1 relative to controls, and greater decreases in GABA+ correlate with improvement due to rehabilitation.22 This mechanism is necessary to prevent the system from becoming overactive  or overly suppressed . Previous MRS studies in humans indicate that the balance between excitation and inhibition depends on the anatomic region being measured. Studies in humans have found a positive relationship between GABA+ and Glx in the medial parietal cortex,23 but not early visual cortex.24 However, a subsequent study by the same authors using a large dataset found a positive relationship between GABA+ and Glx in the early visual cortex but not in the prefrontal cortex.25 Moreover, evidence from the same anatomic location as the current study, extrastriate hMT+,26 indicates a positive relationship between GABA+ and Glx. We originally hypothesized that increased plasticity after stroke, and superior residual vision, would be associated with lower GABA+. If this were the case, an equivalent reduction in Glx would be required to maintain the inhibitory-excitatory balance. In the current study, GABA+ and Glx were highly correlated in hMT+, and when the variance between GABA+ and behavior was controlled, the relationship with Glx disappeared. However, our control analyses indicated that the relationship was unlikely to be driven by data quality . These results further suggest that the 2 neurochemicals are not functionally independent and that changes in both neurochemicals affect behavior. Although it is not possible to determine whether the differences observed are driven by GABA+ or Glx, we theorize that the higher levels of Glx and GABA+ in those who have the least residual vision may reflect reduced plastic potential of hMT+, driven by higher GABA+. However, future studies are needed to explore how these neurochemicals relate to training-induced recovery.Of note, we were surprised to find that reduced Glx levels in hMT+ were related to superior motion detection in the blind field, despite initially hypothesizing the opposite effect. Research from the animal literature indicates that excitatory and inhibitory activity must remain in balance in the brain, with individual neurons receiving roughly equal levels of excitation and inhibition.29 they did not relate the extent of activity to performance of a detection task with respect to these stimuli, as done here. Nonetheless, they provided evidence that the pathway between the dorsal lateral geniculate nucleus and hMT+ likely carried relevant visual information.31 This motivated our hypothesis that V1-independent residual vision for moving Gabor patches might be linked to increased BOLD signal in hMT+. However, no such relationship was found in the present study.Although prior studies investigating residual vision or blindsight reported activity in hMT+ when moving stimuli were passively presented in the blind field,32 Furthermore, multimodal imaging studies in motor stroke survivors have shown the presence of neuronal responses measured with magnetoencephalography33 and transcranial magnetic stimulation34 even in the absence of a BOLD response. The lack of relationship between motion detection and BOLD signal evoked by passive exposure of a motion stimulus while performing an attentionally demanding task at fixation in the current study may, therefore, not reflect a lack of neuronal activity in hMT+ in stroke survivors with V1 damage.BOLD signal changes are thought to indirectly reflect changes in neural activity through neurovascular coupling; however, the relationship between neural activity and the hemodynamic response depends on a combination of cerebral blood flow, blood oxygenation levels, and blood volume, some of which may be abnormal in stroke survivors. In strokes affecting the motor system, the BOLD signal is delayed and of lower amplitude in the affected and unaffected sensorimotor cortex.12 and perilesional V1,11 and BOLD resting-state connectivity between the anterior precuneus and the occipital pole network35 correlate with the extent of improvement following rehabilitation. This suggests that the relationship between the BOLD signal and residual vision is likely to be complex.Although there was no relationship between performance and BOLD activity in this and previous studies, recent work found that baseline BOLD activity in hMT+36 meaning it would not be a pure control structure.The extensive time required to setup and acquire MRS data from each voxel (\u224820 minutes) meant that we were unable to collect neurotransmitter concentration data from >2 voxels. Although it would have been beneficial to also collect data from hMT+ in the intact hemisphere, there is a known interhemispheric interaction between hMT+ after damage to V1The data were collected during the baseline session of a rehabilitation study, so there is no control group with normal visual function. Thus, while we can correlate neurochemical concentrations with residual visual detection in the blind field, we cannot determine whether there is a change in concentration as a result of the occipital stroke itself. This is compounded by the lack of any normative data for neurochemical concentrations in hMT+ and the variability in concentrations across different brain regions, individuals, or time.37 Much of the research investigating residual vision after damage to V1 has focused on individual case studies.39 When larger sample sizes are used, patients with a variety of causes, including stroke, trauma, or tumor resections,14 are often included. In the current study, as in several more recent publications,40 we aimed to minimize the variability by limiting our inclusion criteria to stroke damage in adulthood. However, to achieve the sample size for the study, it was not possible to control for the type of stroke . The large range of age (24\u201374 years) and time since stroke (6\u2013297 months) is a further challenge. Although controlled for in all analyses, the limited sample size means it is not possible to fully account for any effects on neurochemistry. It is, therefore, possible that the cause of stroke, age, or time since lesion may also impact on residual vision and neurochemistry, and future studies with an increased (or homogeneous) sample will be needed to investigate this further.A major challenge in all stroke-related research is the heterogeneity among stroke survivors. Damage caused by stroke is highly variable, even when it is confined to a single area, such as the occipital cortex, and is dependent on factors such as age and type of stroke.3) averaged across the duration of the scan (\u22488 minutes). At these spatial and temporal scales, it is not possible to obtain a direct measure of synaptic activity, nor to determine where within the voxel the neurochemical concentration may vary. Moreover, using GABA-editing MEGA-PRESS sequences in a 3 Tesla MRI scanner it is not possible to separate the resonances of glutamate and glutamine (resulting in the combined measure of Glx) and the GABA signal is contaminated by macromolecules41 and homocarnosine.42 Thus, MRS-derived quantifications of GABA and glutamate may be driven by these alternative signals. This could account, at least in part, for the lack of relationship between the ratio of Glx/GABA+ and behavior in the current study. It is possible that acquiring at higher field strengths to separate glutamate and glutamine, or using macromolecular suppression to reduce contamination of GABA, could produce different results. At ultra-high field strength (7 Tesla MRI scanner), where glutamate and glutamine can be separated, a recent study found evidence for a relationship between GABA+ and glutamate in both prefrontal and occipital cortex.25 However, they only found a relationship with Glx in the occipital lobe and not the prefrontal cortex, pointing towards regional differences between measures of glutamate and Glx.Although MRS is a valuable technique that allows us to quantify measurements of neurochemicals in vivo, there are several limitations when interpreting the relationship between neurochemicals and behavior. First, MRS measures total concentration of each neurochemical within a defined area of cortex  or endogenous reference such as water. The specific reference used varies, with creatine, N-acetylaspartate, or an unsuppressed water signal commonly used. Here we referenced GABA and glutamate to creatine, a commonly used method in MRS studies.In conclusion, this is the first study to explore neurochemistry in the visual system of chronic stroke survivors with visual loss. Lower levels of GABA+ and Glx in the motion-selective extrastriate region hMT+ are linked to better residual visual detection performance in the blind field. Despite the lack of normative data, the presence of this correlation suggests that the neurochemistry of hMT+ is altered to different degrees in different patients with V1 damage. Importantly, neurochemistry in hMT+ may provide useful information about neural functioning not captured by BOLD signal change. It may thus serve as a biomarker for enhanced visual processing abilities inside the blind field which, in turn, may reflect increased capacity for neurorehabilitation.The authors thank our participants for generously giving up their time to participate. Additionally, the radiographers David Parker, Michael Sanders, Nicola Aikin, and Juliet Semple, optometrist Patsy Terry, and optometric technician Charlene Hennesey for assistance with training and data collection. Also, Ione Fine for her comments on the article.The project was funded by European Research Council grant to Dr Tamietto (prot. 772953) and a British Medical Association Foundation John Moulson Grant to H. Bridge. H.E. Willis was supported by the Medical Research Council and a Waverley Scholarship from The Queen\u2019s College, Oxford. I.B. Ip was supported by a Royal Society Dorothy Hodgkin Research Fellowship (DHF\\R1\\20114112). The Wellcome Center for Integrative Neuroimaging is supported by core funding from the Wellcome Trust (203139/Z/16/Z). For the purpose of Open Access, the author has applied a CC BY public copyright license to any author-accepted article version arising from this submission.None.Supplemental MethodsSTROBE ChecklistMinimum Reporting Standards for MRS Checklist"}
+{"text": "Gel polymer electrolytes (GPEs) hold tremendous potential for advancing high-energy-density and safe rechargeable solid-state batteries, making them a transformative technology for advancing electric vehicles. GPEs offer high ionic conductivity and mechanical stability, enabling their use in quasi-solid-state batteries that combine solid-state interfaces with liquid-like behavior. Various GPEs based on different materials, including flame-retardant GPEs, dendrite-free polymer gel electrolytes, hybrid solid-state batteries, and 3D printable GPEs, have been developed. Significant efforts have also been directed toward improving the interface between GPEs and electrodes. The integration of gel-based electrolytes into solid-state electrochemical devices has the potential to revolutionize energy storage solutions by offering improved efficiency and reliability. These advancements find applications across diverse industries, particularly in electric vehicles and renewable energy. This review comprehensively discusses the potential of GPEs as solid-state electrolytes for diverse battery systems, such as lithium-ion batteries (LiBs), lithium metal batteries (LMBs), lithium\u2013oxygen batteries, lithium\u2013sulfur batteries, zinc-based batteries, sodium\u2013ion batteries, and dual-ion batteries. This review highlights the materials being explored for GPE development, including polymers, inorganic compounds, and ionic liquids. Furthermore, it underscores the transformative impact of GPEs on solid-state batteries and their role in enhancing the performance and safety of energy storage devices. It enables the reversible transport of ions between these terminals, enabling the conversion of stored chemical energy into electrical energy ,2,3. Theformance ,17. Addiformance ,19.\u22124 to 10\u22123 S/cm, which is comparable to commercial liquid electrolytes. This high ionic conductivity, combined with the operational safety, mechanical flexibility, and non-combustible nature of GPEs, makes them suitable for use in various battery types, including lithium-ion batteries, sodium-ion batteries, zinc\u2013air batteries, and aluminum\u2013air batteries [In terms of establishing a stronger interface with electrodes, gel polymer electrolytes (GPEs) offer advantages over solid electrolytes. Unlike liquid or solid electrolytes, GPEs possess a unique combination of properties, exhibiting both the diffusive characteristics of liquids and the cohesive properties of solids ,21. GPEsatteries ,24,25. Tatteries ,27. Thisatteries . 4, LiSnOS, and sodium bis(fluorosulfonyl)imide [The polymers used as matrixes for gel polymer electrolytes (GPEs) need to meet specific requirements. The liquid electrolytes are formed by dissolving salts in solvents. Common salt electrolytes include bis-(trifluoromethane)sulfonimide lithium (LiTFSI), LiClOyl)imide . To incoyl)imide . Additioyl)imide . These cyl)imide ,33.2CH2O repeating unit in the PEO backbone exhibits a strong interaction with Li ions, enabling better charge transport [Commonly used polymer matrixes for gel polymer electrolytes (GPEs) include poly(vinylidene fluoride) (PVDF), poly(vinylidene fluoride-hexafluoropropylene) (PVDF-HFP), poly(methyl methacrylate) (PMMA), polyacrylonitrile (PAN), and polyethylene oxide (PEO) ,31. PVDFransport ,38. HoweIn order to meet the practical application requirements of batteries, gel polymer electrolytes (GPEs) with excellent affinity to electrodes, high ionic conductivity, and cyclic stability have been developed. However, there is often a trade-off between mechanical strength and ionic stability when attempting to improve these properties. Therefore, various combinations of GPEs have been prepared with the objective of achieving the following merits: good mechanical and thermal stabilities, a high level of ionic conductivity comparable to that of liquid electrolytes at an ambient temperature, a high transference number, and a favorable electrolyte and electrode interface. Significant progress has been made in developing GPEs with improved properties, leading to their widespread application in various battery systems. This comprehensive review aims to explore the advancements in GPE technology which have greatly transformed energy storage solutions, particularly with respect to batteries with high energy densities and safety. Within this review, we extensively discuss different types of GPEs, including GPEs with flame-retardant properties, GPEs that prevent dendrite formation, hybrid solid-state batteries, and GPEs that are suitable for 3D printing. Moreover, we investigate the methods used to prepare GPEs, such as solution casting, phase inversion, in situ polymerization, and electrospinning. We provide detailed coverage of a wide range of battery systems, including lithium-ion batteries (LiBs), lithium metal batteries (LMBs), lithium\u2013oxygen batteries, lithium\u2013sulfur batteries, zinc-based batteries, sodium-ion batteries, and dual-ion batteries. Additionally, we highlight the materials currently being studied for GPE development, including polymers, inorganic compounds, and ionic liquids. By presenting the latest advancements and their impacts on solid-state batteries, this review aims to provide a comprehensive understanding of the potential of GPEs in improving the performance and safety of energy storage devices. The insights presented in this review are highly valuable to researchers, industry professionals, and enthusiasts involved in advanced battery technologies.In a GPE, the polymer chains form a network with interconnected pores that facilitate the absorption of the liquid electrolyte and enable charge transfer. GPEs can be prepared using two different methods, depending on the nature of the preparation process. One method involves using a porous polymer matrix that allows the liquid electrolyte to be absorbed through swelling. Porous structures in the polymer matrix can be achieved through the controlled removal of a solvent from a polymer solution or by directly forming fibrous structures with inherent porosity. The other method involves storing the liquid electrolyte between polymer chains generated via in situ polymerization. The common methods used for preparing GPEs include solution casting, phase inversion, and in situ polymerization. These methods are considered energy-efficient, cost-effective, and environmentally friendly compared to other available methods for GPE preparation ,40,41. ASolution casting is considered advantageous compared to other methods due to its simplicity and the absence of specialized equipment requirements. In this technique, the matrix polymer and salt electrolytes are dissolved in a solvent, forming a homogeneous solution. The solution is then spread onto a suitable substrate and allowed to dry at a low temperature, typically below 60 \u00b0C. Solvents with low boiling points are usually selected to facilitate the easy removal of the solvent at low temperatures. The low processing temperature restricts the movement of polymer chains, resulting in a less crystalline polymer matrix. Solution-cast GPEs offer several benefits. They are easy to process, flexible, compact, and allow for the tuning of their weight and thickness. They exhibit resistance to pressure and are non-volatile. However, due to their low crystallinity and the presence of salt, solution-cast GPEs may have lower levels of mechanical strength. To address this, crosslinking agents can be added to connect the matrix polymer chains, thereby enhancing the mechanical properties. It is important to note that electrolytes, especially lithium salts, are sensitive to moisture. Therefore, strict measures must be taken to avoid any interference from moisture or water during the solution casting process.N,N\u2032-dimethylformamide, N,N\u2032-dimethylacetamide, and N-methylpyrrolidone is unavoidable. While these solvents can be blended with up to 30% acetone to aid in dissolving these polymers, temperatures above 50 \u00b0C are required to effectively remove these solvents and obtain GPEs with sufficient mechanical strength [In the case of PVDF and PVDF-HFP, the use of high-boiling-point solvents such as strength ,39,42. TThe phase inversion method is a relatively simple and cost-effective technique that holds great potential for scalability. In this method, a polymer solution or a polymer\u2013liquid composite is solidified through the introduction of a non-solvent. The process involves coating the polymer solution onto a substrate, such as glass or between two glass slides, and immersing this system in a non-solvent. During the phase inversion process, the non-solvent replaces the solvent in the polymer solution, leading to the precipitation and solidification of the polymer. The addition of oligomers as additives can enhance the porosity of the resulting GPE by being extracted out during phase inversion. By carefully tuning the experimental conditions and the composition of the polymer solution, GPEs with honeycomb-like and network morphologies can be achieved. The formation of a network-like morphology with a narrow pore distribution helps to prevent the leakage of liquid electrolytes and enables the GPE to exhibit high ionic conductivity. The phase inversion method offers advantages such as the production of continuous and defect-free GPEs ,44,45. HIn the in situ polymerization method, polymerization or the growth of polymer chains occurs in the presence of a liquid electrolyte. The monomer or prepolymer, initiators, and the liquid electrolyte to be loaded in the gel polymer electrolyte (GPE) are mixed together in a matrix and subjected to polymerization under light or heat. This process enables the formation of GPEs with uninterrupted ion transport networks ,41. WhenN,N\u2032-dimethylformamide (boiling point = 153 \u00b0C) and N,N\u2032-dimethylacetamide (boiling point = 165 \u00b0C), are non-volatile. To facilitate solidification, up to 30% of acetone (boiling point = 56 \u00b0C) is added. It is important to note that concentrated polymer solutions are more prone to drying and should not be stored for extended periods [In the electrospinning method, the polymer matrix is transformed into a porous layer of nanofibers. A sufficient voltage is applied to a polymer solution with the required viscosity, which is then drawn into fibers through a conducting nozzle. These fibers are collected on a conducting substrate which serves as the bottom electrode. The thickness of the fibrous layer can be controlled by adjusting the volume of the polymer solution. Parameters such as the concentration of the polymer solution, the applied voltage, the inner diameter of the nozzle, and the boiling point of the solvent are optimized to control the thickness of the fibers. The electrospun polymeric layer is utilized as a matrix for the gel polymer electrolyte (GPE). The electrolyte is loaded by impregnating a liquid electrolyte solution with the fibrous matrix. The interconnected pores within the fibrous structures provide a larger surface area and efficient ion-conducting channels ,48. PVDF periods ,49,50.Lithium-ion batteries (LIBs) utilize the reversible reduction of lithium ions for energy storage. They have emerged as a promising type of energy storage device due to their high energy density, long lifespan, and low self-discharge properties. LIBs have gained a dominant position in the market and are considered ideal choices for powering portable electronic devices, electric vehicles, and implantable medical devices. However, the use of liquid electrolytes in LIBs raises intrinsic safety concerns, such as the potential for overheating, which can result in bursting and catastrophic thermal accidents. In 1975, PEO-based gel polymer electrolytes (GPEs) for LIBs were developed by Wright . Since t2S, LiSnOS was used as a precursor to avoid the hygroscopic nature. LiSnOS was prepared via the sulfurization of LiNO3 with SnOS. By adjusting the composition of LiSnOS, the highest Li ion conductivity achieved was 1.92 \u00d7 10\u22124 S cm\u22121. A GPE containing 30 wt% LiSnOS exhibited the highest discharge capacity of 134.6 mA h/g at 0.2 C and remained stable over 30 cycles. A GPE with 30% LiSnOS facilitated fast ion transport and proved to be beneficial for maintaining flexibility and flame retardancy in lithium-ion batteries (LiBs) [Li superionic conductor (LISICON)-type solid electrolytes are known to be promising, but they suffer from a hygroscopic nature and limited chemical stability. In an effort to address these challenges, Kuo et al. developed a LISICON-based gel polymer electrolyte (GPE) using a PVDF-HFP matrix with varying compositions of LiSnOS. Instead of the moisture-sensitive Lis (LiBs) .\u22123 S/cm at 25 \u00b0C. The ionic conductivity of five layers of 3D printed GPE (240 \u00d7 175 \u03bcm) was 2 \u00d7 10\u22123 S/cm. Good adhesion was observed in the 3D printed GPE, and there was no contact resistance. The number of interfaces in the 3D printed layers was considered to be larger than that in molded GPE samples [Three-dimensional printing is a valuable technique for fabricating layered and complex structures. Gambe et al. developed a UV-curable gel polymer electrolyte (GPE) using 3D printing. This method allows for the direct printing of the GPE onto various substrates at room temperature, making it suitable for processing thermally unstable materials. The electrolyte ink was formulated using a UV-curable monomer, ionic gels, an ionic liquid (IL) capable of conducting lithium, and silica nanoparticles. In this study, lithium(trifluoromethanesulfonyl)imide (Li-TFSI) was dissolved in 1-ethyl-3-methyl imidazolium bis(trifluoromethanesulfonyl) imide (EMI-TFSI) and used as an Li-conducting IL. The structural stability of the GPE was optimized by varying the concentration of the silica nanoparticles. A mixture of bis(trifluoromethanesulfonyl) imide powder, 1-ethyl-3-methyl imidazolium bis(trifluoromethanesulfonyl)imide, 2-hydroxyethyl methacrylate (a monomer of poly(hydroxyethylmethacrylate)\u2014P(HEMA)), silica nanoparticles, ethylene glycol dimethacrylate (a crosslinker), and 2,2-dimethoxy-2-phenylacetophenone as a photo initiator was prepared at an appropriate ratio to serve as a 3D printable ink. The printed patterns were then exposed to UV light to form the GPE. Quasi solid-state lithium-ion batteries (LiBs) were assembled by printing the GPE ink onto cathodes coated with a slurry of lithium cobalt oxide and a lithium foil anode. Fully 3D printed LiBs were obtained by using a 3D printed lithium cobalt oxide cathode and a lithium titanium oxide anode. This 3D printing approach enabled the simple fabrication of flexible LiBs. The initial capacity of the batteries was approximately 100 mA h/g (at 0.1 C). The ionic conductivity of the GPE, regardless of the filler content (ranging from 3 to 7 wt.%), was measured to be 2.9 \u00d7 10 samples . 7La3Zr2O12 (LLZO) materials with tantalum (Ta) doping (referred to as LLZTO) have emerged as potential candidates for solid-state electrolytes, despite the challenge of large interface resistances arising from inactive cathodes and the inherently difficult garnet\u2013electrode interfaces. In a study by Meng Liu et al., a cathode material was prepared using a mass ratio of 5:5:90 of carbon black, poly(vinylidene fluoride) (PVDF), and LiCoO2. The cathode was fabricated by mixing the components into a slurry, coating it onto aluminum foil, and subsequently drying it. The anode material consisted of lithium metal. For the solid-state electrolyte, a precursor mixture for the garnet electrolyte was prepared using ethylene glycol diacrylate and coated onto an LLZO pellet. Thermally induced in situ polymerization was employed to form a poly(ethylene glycol) diacrylate (PEGDA) layer at 60 \u00b0C for 1 h. The incorporation of this layer led to a decrease in the interface resistance from 6880 to 473 \u03a9. Furthermore, a Li symmetric cell demonstrated a stable galvanostatic charge/discharge profile for 400 h without the formation of lithium dendrites. Additionally, a solid-state Li|GPE@LLZO|LiCoO2 battery exhibited a retention of the initial discharge capacity (104.1 mA h/g) of 82.6% after 100 cycles (at 0.5 C) at room temperature. These findings highlight the potential of garnet LLZO-based electrolytes to reduce interface barriers and improve compatibility in solid-state lithium-ion batteries [Due to their electrical stability, high ionic conductivity, and mechanical strength, garnet Liatteries .4, 10 wt% PEO/TPU/LiTFSI composite, and 10 wt% carbon black, with N-methyl pyrrolidone (NMP) serving as the solvent medium on an aluminum foil substrate. A polyethylene oxide (PEO)/thermoplastic polyurethane (TPU)/Li7La3Zr2O12 (LLZO) nano-network composite electrolyte was prepared using the solution casting method. LLZO nanonetworks were synthesized via electrospinning, followed by thermal treatment. A mixture of LiNO3, La(NO3)3\u22c56H2O, and ZrO(NO3)2\u22c56H2O in stoichiometric proportions was prepared. To compensate for lithium loss during calcination, more than 15 wt% of LiNO3 was added. Polyvinylpyrrolidone (PVP) was dissolved in N,N-dimethylformamide (DMF) at a concentration of 8 wt% and added to a solution containing glacial acetic acid mixed with deionized water. The PVP and salt solution were mixed at a weight ratio of 1:1.2 and stirred at 60 \u00b0C for 8 h. The resulting mixture was electrospun at a high voltage, with a distance of 18 cm between the needle and the nanowire collector, and a constant voltage of 26 kV was maintained. The electrospun precursor was pre-oxidized at 280 \u00b0C for 2 h, followed by calcination at 750 \u00b0C for 2 h in an air atmosphere with a heating rate of 1.0 \u00b0C/min to obtain the LLZO nanonetwork. The preparation process is illustrated in \u22123 S cm\u22121 at 60 \u00b0C and a high electrochemical stability window of 5.6 V. To enhance the electrochemical properties and mechanical stability, thermoplastic polyurethane (TPU) was widely employed. The discharge capacity was observed to be 170 mA h g\u22121 at 0.1 C after 100 cycles, demonstrating improved cycle durability. At 0.5 C and 60 \u00b0C, the initial capacity retention was 96.1%. Solid polymer composite electrolytes offer several advantages, including a high degree of safety and excellent electrochemical performance. As a result, solid-state cells hold great promise as future candidates for lithium batteries [To overcome the disadvantages associated with liquid electrolytes, such as electrolyte leakage and the risk of explosions due to lithium dendrite formation, solid-state electrolytes (SSEs) have gained significant attention in recent years. In a study by Haoshan Xu et al., cathode materials were prepared using 80 wt% LiFePOatteries .2-x@Li anodes and a highly conductive solid-state electrolyte (GPE) by Jiao et al. not only addresses the challenges associated with Li-metal anodes but also presents a promising solution for enhancing the performance and commercial viability of lithium-ion batteries (LIBs). Their initial findings demonstrate the effectiveness of utilizing nanofibrous TiO2-x@Li anodes and a highly conductive solid-state electrolyte (GPE) to enhance the stability of the Li-metal anode and improve its compatibility with electrolytes. The GPE was prepared by dissolving LiTFSI and LiPF6 in DMF with 1,3-dioxolane (DOL) and allowing it to form poly(dioxolane) via a 6 h room temperature reaction. The nanofibrous TiO2-x@Li anodes promote the uniform deposition of Li by facilitating the conduction of Li ions along the GPE and their deposition onto the electronically conductive TiO2-x nanofibers. When combined with high-loading cathodes such as LiFePO4 or LiNi1/3Co1/3Mn1/3O2, the quasi-solid-state Li batteries exhibited excellent long-term cycling stability (>500 cycles), high-rate capability (>1 C), and a stable charge/discharge capacity with minimal overpotential. The ionic conductivity of the GPE was approximately 0.6 \u00d7 10\u22122 S/cm at room temperature. 2\u2212x@Li anodes and integrated quasi-solid-state Li-batteries (QSSLBs). The incorporation of the in situ polymerization technique into commercial lamination manufacturing enables the production of 63-Ah pouch cells using LiFePO4-graphite. These pouch cells exhibit a high initial Coulombic efficiency, a notable energy density at 0.3 C, an excellent rate capability ranging from 0.25 to 2 C, and long-term cycling performance at 0.3 and 1 C. These results indicate promising prospects for commercial applications and address interfacial issues in lithium-ion batteries, thereby presenting a novel approach for the development of commercially viable Li-metal anodes. [The integration of nanofibrous TiO anodes. .\u22123 S/cm, a Li transference number of 0.43, excellent thermal stability up to 372 \u00b0C, and a wide electrochemical window of 5.3 V. Furthermore, the researchers also prepared anodes using HMSFs/Fe-N-doped carbon composite nanofibers. The lithium-ion battery (LIB) assembled using the HMSF-based GPE and anode demonstrated a high specific capacity of 1290 mA h/g at 0.3 C, and it exhibited excellent stability over 500 cycles. The presence of HMSFs in both the electrolyte and electrode facilitated effective interphase bridging and the easy transport of Li ions, leading to the enhanced electrochemical performance of the assembled LIB [A study by Zhu et al. introduced an innovative approach by incorporating helical mesoporous silica nanofibers (HMSFs) into a PVDF-HFP matrix, resulting in the development of an inorganic polymer gel electrolyte (GPE). This GPE exhibited remarkable characteristics, including a high ionic conductivity of 1.2 \u00d7 10bled LIB . 7La3Zr2O12 (LLZO) materials with tantalum (Ta) doping have demonstrated promise in reducing interface barriers and improving compatibility in solid-state LiBs. Solid-state electrolytes based on polymeric composites have also emerged as a viable option, offering a high degree of safety and excellent electrochemical performance. Finally, the integration of nanofibrous anodes and highly conductive GPEs has shown potential in enhancing the stability of Li-metal anodes and improving LiB performance. These advancements in GPE technology hold great promise for enabling the development of high-performance and safe LiBs for future applications. The development of gel polymer electrolytes (GPEs) has shown significant potential for advancing the performance and safety of lithium-ion batteries (LiBs). Various strategies have been explored to overcome the limitations of traditional solid electrolytes, such as their hygroscopic nature, limited chemical stability, and interface resistances. Researchers have successfully utilized alternative precursors, such as LiSnOS, to improve the hygroscopic properties of Li superionic conductor (LISICON)-type solid electrolytes. Additionally, 3D printing techniques have enabled the fabrication of GPEs with precise structures, offering opportunities for the direct printing of electrolytes onto various substrates and the simplification of LiB manufacturing. Furthermore, garnet LiLithium metal batteries (LMBs) are highly regarded as potential energy storage devices because of their high theoretical capacity (3860 mA h/g) and low electrochemical potential (\u22123.04 V). These batteries, with lithium metal as the anode, have found widespread use in various applications such as implantable medical devices, digital watches, and calculators. However, the practical application of LMBs has been hindered by the instability of the lithium metal\u2013liquid electrolyte interface, which is primarily caused by the uncontrolled growth of lithium dendrites. This issue raises serious safety concerns and limits the practical viability of LMBs . Fortuna\u22124 S/cm at 25 \u00b0C, confirming its suitability for ambient temperature battery operation. The LIB utilizing the DPN-GE as an electrolyte showed an initial discharge capacity of 159 mA h/g at 0.1 C rate. At a higher rate of 0.5 C, the capacity remained at approximately 140 mA h/g, and after 100 cycles, 79% of the initial capacity was retained. In contrast, the LIB employing the liquid electrolyte LiTFSI exhibited a rapid capacity decay after only 50 cycles [The stability of the lithium (Li) anode and gel electrolyte interface plays a crucial role in improving the coulombic efficiency and cycling performance of lithium-ion batteries (LIBs). In a study by Zuo et al., a gel electrolyte based on a double-polymer network (DPN-GE) was prepared using a single-step photopolymerization process. The researchers employed a solid electrolyte interphase (SEI) strategy to stabilize the interface between the Li anode and the electrolyte. The DPN-GE was formed by creating an interpenetrating network of poly(ether-acrylate) containing silica microspheres as a scaffold and introducing lithium nitrate for the generation of an SEI. The gel electrolyte was composed of poly(ethylene oxide) (PEO), pol(acrylate) (PA), tetraethylene glycol dimethyl ether (TEGDME), and LiTFSI. The DPN-GE exhibited excellent thermal and mechanical stabilities, high room-temperature ionic conductivity, and a stable Li-electrolyte interface. When integrated into quasi-solid-state LIBs, the stable interface provided by the DPN-GE resulted in a dendrite-free morphology and impressive stripping and plating efficiencies. The ionic conductivity of the DPN-GE was measured to be 6.4 \u00d7 104 cathodes compared to organic liquid electrolytes [Organic electrolytes with good wetting characteristics have been preferred for use in lithium-ion batteries (LiBs) due to their ability to form a favorable interface and improve the overall performance. The interfacial chemistry is particularly sensitive to the wetting properties of liquid electrolytes. Ionic liquid-based gel polymer electrolytes (ILGPEs) have shown promise in maintaining suitable viscosity and interface properties compared to common organic liquid electrolytes. In a study by Pan et al., a composite gel polymer electrolyte (ILGPE) based on poly(vinylidene fluoride-co-hexafluoropropylene) (PVDF-HFP) and an ionic liquid was prepared and utilized in LiBs. The ionic liquid used was N-Methyl-N-propylpiperidinium bis(trifluoromethanesulfonyl) imide (PP13TFSI), which was mixed with LiTFSI and dissolved in N-methyl-2-pyrrolidone along with PVDF-HFP. The resulting solution was cast into a film to form the gel polymer electrolyte. When the ILGPE was used, the bulk resistance and ionic conductivity remained unaffected even after 50 cycles of testing. However, when an organic liquid electrolyte was used, significant increases of 50- and 825-fold was observed in the transfer and interfacial resistances, respectively. Wetting the electrode with liquid electrolytes resulted in a maximum discharge capacity of 151.1 mA h/g, while the cells wetted with the ILGPE exhibited a slightly lower discharge capacity of 128.7 mA h/g. However, during cyclic testing, the discharge capacity of the liquid electrolyte-wetted gel polymer electrolyte exhibited a sharp decay, and it eventually short-circuited after 400 cycles. In contrast, the discharge capacities of the ILGPE-wetted cells remained stable even after 400 cycles. The superior performance of the ILGPE can be attributed to the compatibility and stability of the electrode-electrolyte interphase. Furthermore, the ILGPE demonstrated better stability and compatibility with LiFePOtrolytes .13TFSI. PVDF-HFP was incorporated into the matrix to provide flexibility, while poly(ether-acrylate) (PEA) was selected to form a rigid network through photopolymerization. LiNO3 was utilized to create a stable solid electrolyte interphase. The DPGPE facilitated the uniform deposition of Li ions at the interface, enabling a highly reversible plating/stripping process. The process of creating a double-polymer network gel electrolyte (DPNGE) is illustrated schematically in \u22124 S cm\u22121 at room temperature, along with an electrochemical window of 5.0 V. When assembled into an LIB, the DPGPE-based battery demonstrated a discharge capacity of 153.7 mA h/g at a 0.5 C rate, with about 92.7% of the initial capacity retained after 500 cycles [The capability of ILs to control the growth of Li dendrites and address interfacial issues is useful for preparing GPEs. ILs offer advantages such as high levels of thermal stability, low viscosities, high levels of room-temperature ionic conductivity, large electrochemical windows, and improved anode stability. Thus, the use of ILs enhances the performance of GPEs and ensures the safety of LIBs. To harness the benefits of ILs, Wu et al. formulated a double-polymer network gel electrolyte (DPNGE) using PVDF-HFP and branched acrylate with the IL Py0 cycles . \u22125 S/cm at room temperature [Solid-state batteries are subject to several disadvantages, including: (i) the breakage of the Li and electrolyte interface due to volume changes during Li anode stripping/plating, (ii) the creation of gaps at the electrolyte\u2013electrode interface, leading to increased electrochemical impedance, and (iii) limited ionic conductivity, resulting in a lower current density than required. To address these issues, Zhu et al. proposed a simple solution using a coiling GPE membrane. They prepared a composite GPE by combining PVDF-HFP and LiTFSI. The composite anode was formed by coiling and cutting stacked GPE films along with Li foil. This vertical alternating array design offers a large electrolyte-Li interface area and enables the continuous transmission of Li ions across the composite anode. The structure also reduces the local current density and resistance at the interface, resulting in more uniform Li stripping/plating. The initial discharge capacity of the cell was 87.5 mA h/g, and after 830 cycles, it decreased to 81.6 mA h/g. The stability of the battery was measured at 93.26%. The GPE demonstrated an ionic conductivity of 3.11 \u00d7 10perature .2S molecular cathode and a fireproof gel electrolyte based on solid-state redox chemistry. In a study by Xiangyu Meng et al., a cathode composed of MXene with polyacrylonitrile (PAN)-modified Li2S was employed, while silicon (Si) was used as the anode material. The advantages of a quasi-solid-state lithium battery based on the LiPS-free solid-state redox chemistry of a molecular-Li2S cathode in a fireproof, mixed ether\u2013carbonate gel electrolyte with MXene as a fire-retardant are illustrated in 2S molecular cathode demonstrated outstanding performance, with a remarkable lifetime of 2000 cycles, an excellent capacity of 830 mA h g/L, 100% coulombic efficiency, and an ultralow capacity loss of 0.005\u20130.01% per cycle. The battery also exhibited high-rate capability, with achievable rates of up to 10 C. To further enhance the lifetime, cell energy, and compatibility, high-capacity, carbonate-friendly anodes were employed. Looking ahead, the utilization of fire-retardant gel electrolytes has promise for mitigating the risks of short circuits and overheating, and ensuring high levels of safety [High-energy, safe, and cost-effective batteries have garnered significant attention in recent years. One approach to achieving high-energy lithium batteries involves utilizing a Lif safety .To develop high-energy-density lithium metal batteries (LMBs), it is crucial to utilize low-cost and environmentally friendly gel polymer electrolytes (GPEs) that possess wide electrochemical windows, optimal compatibilities, and structural stability. The electrospinning method was employed by Simin Chai et al. to fabricate a three-dimensional biodegradable composite (PAL) composed of Poly-L-lactic acid (PLLA) and poly(acrylonitrile) (PAN) nanofibers. + ions along the polymer chain. The incorporation of the biodegradable nanofiber membrane led to improvements in the corrosion resistance, ionic conductivity, electrochemical window, and dielectric properties of the gel polymer electrolyte. The cathode material consisted of a LiFePO4/PAL composite, while Li metal was used as the anode. The optimized PAN-based gel polymer electrolytes exhibited a high ionic conductivity of 5.17, along with excellent cycling stability of 1000 h at a current density of 0.15 mA/cm2 or a charge density of 0.15 mA h/cm2. After 140 cycles at a 1 C rate, the LiFePO4-based whole cell demonstrated a capacity retention of 97.63%. The utilization of this nanofiber membrane holds significant implications for the design and fabrication of gel polymer electrolytes for lithium metal batteries. Moreover, it contributes to the pursuit of sustainable and environmentally friendly development in energy storage systems [The PLLA/PAN nanofiber matrix was then immersed in a mixture of 1,3-dioxolane (DOL) and lithium bis(trifluoromethanesulfonyl)imide (LiTFSI) and subjected to in situ polymerization, resulting in the formation of a gel polymer electrolyte (PDOL-LiTFSI). The formation and strengthening of hydrogen bonds within the PAL composite membrane plays a role in facilitating the migration of Li systems .2O3 composite was utilized by Shaolun Cui et al. To fabricate the nanofibrous matrix, a matrix solution was prepared by dissolving PVDF-HFP, cellulose acetate (CA), and succinonitrile (SN) in a mixture of acetone and N,N\u2032-dimethylacetamide. The solution was then electrospun to form the nanofibrous matrix. Subsequently, a dispersion of Z1F8 (zeolite imidazolate framework) nanoparticles and poly(ethylene oxide) (PEO) in anhydrous acetonitrile was solution-casted onto the nanofibrous matrix. Finally, an ultrathin layer of Al2O3 was deposited using the atomic layer deposition method. Metal organic frameworks (MOFs) were chosen due to their stable skeletons, high specific surface areas, well-defined pore structures, and abundant Lewis acid sites. These properties make MOFs ideal for hosting lithium ions and serving as electrolyte fillers, thereby enhancing ion transport and charge transfer processes while inhibiting the growth of lithium dendrites. As for the anode material, a cathode composed of Mn-based layered oxide with a Li-rich and lithium metal configuration was employed. To ensure uniform lithium-ion interaction and stabilize the interfacial lithium anode, an ultrathin layer of Al2O3 and the MOF was coated on the same side of the polymer matrix. The incorporation of this composite structure resulted in significant improvements. The transference number of Li+ ions increased to 0.74, leading to enhanced cycling stability of over 1000 h. Moreover, the battery retained 84.6% of its initial discharge capacity (257.5 mA h/g at 0.2 C) after 500 cycles. The mechanical strength and interfacial stability were further enhanced by introducing heterostructured ZIF-8 with an ultrathin layer of Al2O3. 2O3 films and the EDS mapping results [Gel polymer electrolytes (GPEs) are highly promising and practical options for portable lithium metal batteries (LMBs). The unique composite design of GPEs allows for the regulation of uncontrolled lithium dendrite growth in liquid electrolytes, as well as the improvement of interface interaction in solid-state electrolytes. A hierarchal layered GPE with a nanofibrous polymer matrix\u2013metal organic framework (MOF)-Al results .3. The in situ polymerization reaction led to the formation of the GPE, which exhibited both autogenous stability and facial stability. The anode material employed in this study was Li metal, while LiFePO4 was used as the cathode material. By employing this method and utilizing LiFePO4 cathodes, a strategy involving the regulation of the -CF3 functional group was implemented to achieve a high capacity after 1000 cycles. This strategy resulted in a more stable cycling process, with 90% retention of the initial capacity (170 mA h/g at 0.5 C) after 1000 cycles. Among the trifluoroethyl methacrylate molecules, the reactivity of the -CF functional group (in the order of -CF > -CF2 > -CF3) provided high stability to the anode\u2013electrolyte interface. This contributed to the improved performance and longevity of the battery. For the development of next-generation power supplies, there is a high demand for quasi-solid-state lithium-metal batteries (LMBs) due to their superior safety and high energy density. To establish these quasi-solid-state batteries, it is essential to introduce fluorine-containing polymer skeletons through in situ gelation. A hybrid framework consisting of poly(ethylene glycol) dimethacrylate (PEGDMA), trifluoroethyl methacrylate (TFMA), and lithium fluoride (LiF) was utilized by Qiyu Wang et al. to prepare a gel polymer electrolyte (GPE) via thermally induced in situ polymerization at 60 \u00b0C. The addition of a LiF-enriched solid\u2013electrolyte interphase (SEI) facilitated the growth of dendrite-free Li crystalline grains. The hybrid framework formation resulted in the creation of functional groups, namely -C=C- and -CF4 as a cathode material, and a gel polymer electrolyte (GPE) was prepared via thermally induced polymerization. The GPE was prepared using the following procedure: a solution of P(VDF-HPF), EMITFSI, PEGDGE, and D-400 (a polyetheramine curing agent) in dimethylformamide (DMF) was mixed. The resulting mixture was solution-casted and subjected to a two-step heating process, with temperatures of 80 \u00b0C for 12 h and 120 \u00b0C for 24 h. Subsequently, the GPE was dried at 100 \u00b0C for 24 h. This GPE exhibited high ionic stability, compatibility, and flexibility. The ionic conductivity of the GPE ranged from 0.05 \u00d7 10\u22124 to 1.69 \u00d7 10\u22123 S/cm. After 50 cycles at 60 \u00b0C, the GPE-based cells showed sustained discharge capabilities of 157 mA h/g. At 0.1 \u00b0C and 60 \u00b0C, the initial discharge capacity was 162 mA h/g [Ionic liquids are considered promising due to their advantageous properties such as negligible evaporation, high thermal stability, large potential window, high ionic conductivity, and safety. Zhennan Wang et al. studied the use of LiFePO2 mA h/g .\u22124 S/cm at 60 \u00b0C. These findings highlight the reliable performance and safety of using this GPE in LIBs [During the process of in situ polymerization, monomers are mixed with the liquid electrolyte, and gelation occurs as polymerization progresses. The low viscosity of the monomers allows for the good wettability of the GPE by the liquid electrolyte, facilitating well-connected charge transport pathways. In a study by Ma et al., a polyethylene (PE)-based GPE was prepared using in situ polymerization.  in LIBs .\u22124 S cm\u22121 at 25 \u00b0C and a wide electrochemical window of up to 4.82 V. The outstanding interfacial stability of Te in Li-Te batteries contributed to good rate capability and high electrical conductivity. This resulted in superior electrochemical performance compared to S/C and Se/C cathodes [Solid-state Li-Te batteries have drawn significant interest because of their excellent performance and security. In order to develop quasi-solid-state Li-Te batteries, Yue Zhang and colleagues used a gel polymer electrolyte (GPE), Li metal as the anode, and Te/C as the cathode. A comparison was also made with S/C and Se/C cathodes in which the Te/C exhibited superior electrochemical performance. The gel polymer electrolyte (GPE) was fabricated via a solution-casting technique by blending poly(vinylidene fluoride-co-hexafluoropropylene) (PVDF-HFP) and N-methyl-2-pyrrolidone (NMP) in a 1:4 ratio. The cathode material was prepared using a mixture of Te/C composite in a 2:1 ratio. S/C and Se/C cathodes were prepared using a mixture in a 1:1 ratio. The PVDF-HFP polymer in the GPE provided thermal and electrochemical stability, a suitable dielectric constant, and mechanical strength. The GPE exhibited a high ionic conductivity of 8.0 \u00d7 10cathodes .\u22123 S/cm at 25 \u00b0C. Moreover, it displayed a large potential window of 4.5 V, stability for over 1200 h, and a high specific capacity of 135.4 mA h/g. By combining a lithium metal anode with a LiFePO4 cathode and the DES-based GPE, high-energy-density and fire-resistant LiBs were achieved. These findings highlight the use of DES-based electrolytes in the design of advanced lithium-ion batteries [Deep eutectic solvents (DES) are being developed as potential electrolytes for lithium-ion batteries (LiBs) due to their low cost, low vapor pressure, high electrochemical stability, superior flame retardance, and properties similar to ionic liquids (IL). However, modifications of DES-based gel polymer electrolytes (GPE) are necessary to attain high a specific capacity and stability. A DES-based GPE with a rapid self-healing capability was prepared by Qiqi Li et al. via the in situ copolymerization of pentaerythritol tetraacrylate (PETEA), cystine, and 2-isocyanoethyl methacrylate (ICEM). This GPE demonstrated the ability to self-smooth cracks within 30 min. The resulting GPE exhibited a high ionic conductivity of 1.1 \u00d7 10atteries .GPEs have the potential to significantly enhance the performance, stability, and safety of LMBs. The utilization of double-polymer network (DPN-GE) gel electrolytes, interpenetrating networks, and SEI strategies has resulted in stable Li-electrolyte interfaces, dendrite-free morphologies, and enhanced stripping/plating efficiencies. Ionic liquid-based gel polymer electrolytes (ILGPEs) have demonstrated superior wetting properties, compatibility, and stability, leading to improvements in the interfacial chemistry and cycling performance. Incorporating organic electrolytes with high thermal stabilities and low viscosities, as well as employing coiling GPE membranes and fireproof gel electrolytes, has addressed interfacial issues, enhanced anode stability, and facilitated continuous Li ion transmission. Additionally, the utilization of biodegradable nanofiber membranes has improved corrosion resistance, ionic conductivity, and cycling stability in GPEs. 2O3 composite, has allowed for effective regulation of lithium dendrite growth and enhancement of interface interaction. By incorporating stable MOFs with high surface areas and Lewis acid sites, ion transport and charge transfer processes are improved while inhibiting dendrite formation. Additionally, the introduction of fluorine-containing polymer skeletons through in situ gelation has enabled the development of quasi-solid-state LMBs with enhanced safety and capacity retention. The utilization of ionic liquids as next-generation electrolytes has shown advantageous characteristics, including wide electrochemical windows, high ionic conductivity, and high thermal stability. In situ polymerization techniques have been employed to create gel polymer electrolytes (GPEs) with excellent ionic stability, compatibility, and flexibility. Moreover, the combination of gel polymer electrolytes with specific anode and cathode materials, such as Li metal and LiFePO4, has resulted in significant improvements in cycling stability and capacity retention. These advancements in GPE technology contribute to the development of high-energy-density, safe, and environmentally friendly lithium batteries and hold promise for future applications in the field of energy storage.The utilization of composite designs in GPEs, such as the nanofibrous polymer matrix-metal organic framework (MOF)-AlSimilar to lithium-ion batteries (LIBs), lithium metal is used as an anode in lithium\u2013oxygen batteries (LOBs). In LOBs, a conducting porous matrix is employed as the cathode. The anode undergoes Li metal dissolution, while oxygen reduction/oxygen evolution takes place at the cathode. LOBs offer high operational voltages (\u223c2.96 V) and formal specific energies (\u223c3500 Wh/kg). To address the performance and safety concerns common to LIBs, gel polymer electrolytes (GPEs) with high levels of ionic conductivity and interconnected polymer networks have been utilized. GPEs play a vital role in enhancing the performance of LOBs by preventing Li metal corrosion and electrolyte evaporation. Recently, GPEs incorporating inorganic fillers and polymer matrixes with high thermal and mechanical stabilities, as well as biodegradable polymers, have been developed for LOBs. These advancements aim to improve the overall efficiency, safety, and lifespan of LOBs ,73. Tabl2-graphene oxide composites onto nickel foam as the cathode, and a monomer mixture capable of forming a gel via in situ polymerization was applied by brushing. The monomer mixture consisted of tetraethylene glycol dimethyl ether, ethoxylated trimethylolpropane triacrylate (ETPTA), and 2-hydroxy-2-methyl-1-phenyl-1-propanone. This hybrid solid\u2013gel electrolyte served as both the separator and the electrolyte in the LiB. After 140 cycles at a current density of 0.4 mA/cm2, the flexible LiB demonstrated a terminal voltage of 2.2 V and a capacity of 1000 mA h/g. The solid\u2013gel electrolyte exhibited a lower activation energy and higher ionic conductivity compared to typical liquid electrolytes. Furthermore, this solid\u2013gel electrolyte effectively protected the lithium metal anode during long-term cycling processes [To safeguard the lithium anode from electrolyte evaporation, a hybrid solid\u2013gel electrolyte was investigated by Wen-Bin Luo et al. for use in lithium batteries (LiBs). The GPE was prepared by coating RuOrocesses .4), lithium bis(trifluoromethanesulfonyl)imide (LiTFSI), and lithium hexafluorophosphate (LiPF6) were examined. The results showed that LiPF6 exhibited the highest ionic conductivity (8 \u00d7 10\u22125 S/cm) compared to LiClO4 (3.7 \u00d7 10\u22125 S/cm) and LiTFSI (3 \u00d7 10\u22125 S/cm). However, LiTFSI demonstrated the highest Li ion transference number (0.77) and the widest electrochemical window (4.9 V). Additionally, the cyclic stability of LiTFSI was four times higher than that of the other lithium salts studied. The highest overall ionic conductivity was observed in the PVDF-HFP/LiPF6 system, reaching 9.44 \u00d7 10\u22125 S/cm. This enhanced conductivity can be attributed to the smaller size of the LiPF6 anions [GPEs have found applications in lithium\u2013oxygen batteries, which are well-known for their high energy densities. Among the various options, PVDF-HFP-based GPEs filled with different lithium salts have been preferred over liquid electrolytes. In a study conducted by Celik et al., the effects of different lithium salts on GPE performance, including ionic conductivity, cyclic stability, electrochemical stability, and interfacial stability, were investigated. Specifically, lithium perchlorate  dimethyl ether, to obtain the final PI GPE. \u22124 S/cm) and a lithium transference number of 0.596. Upon its integration into a lithium\u2013oxygen battery, the PI GPE demonstrated remarkable cyclic stability, enduring up to 366 cycles at a current density of 0.1 mA. The discharge capabilities of a lithium\u2013air pouch cell with a default end-of-discharge voltage of 2 V are shown in Polyimide (PI) is a highly durable polymer known for its exceptional thermal and structural stability. In the context of gel polymer electrolytes (GPEs), PI-based cross-linked formulations have demonstrated impressive thermal stability, withstanding temperatures of up to 334 \u00b0C. Moreover, these GPEs exhibit a remarkable organic liquid electrolyte uptake of 178%. Leveraging these properties, Xu et al. developed a PI-based GPE to boost lithium\u2013oxygen battery cycle performance. Despite facing challenges such as electrolyte evaporation, lithium\u2013oxygen batteries have garnered significant attention due to their exceptionally high theoretical power density, making them a promising candidate among secondary batteries. The anode material consists of lithium foil, while foamed nickel serves as the cathode current collector. In the pursuit of developing a gel polymer electrolyte (GPE) for these batteries, a combination of cellulose acetate (CA) and poly(vinylpyrrolidone) was dissolved in NMP. The resulting solution was cast onto a glass plate and subjected to phase inversion by immersing it in water. Subsequently, the CA film was immersed in a solution of lithium bis(trifluoromethanesulfonyl)imide (LiTFSI) to obtain the final CA-based GPE. 2-graphene oxide composites and a gel-forming monomer mixture serves as both the separator and electrolyte, effectively protecting the lithium metal anode and demonstrating excellent cyclic stability. Similarly, GPEs based on poly(vinylidene fluoride)-based matrixes, super-high ionic conductive gel polymers (SHGPs), and polyimide (PI) have shown significant improvements in ionic conductivity, cyclic stability, and interfacial stability. These GPEs enable enhancements to the performance and longevity of lithium\u2013oxygen batteries, offering higher specific capacities, improving voltage retention, and extending the cycling life. The choice of lithium salts in GPEs also influences their performance, with lithium bis(trifluoromethanesulfonyl)imide (LiTFSI) demonstrating superior characteristics, including a wide electrochemical window and high transference number. The development of hybrid solid-gel electrolytes and gel polymer electrolytes (GPEs) has made significant progress in developing the high performance and stable lithium\u2013oxygen batteries. The hybrid solid\u2013gel electrolyte composed of RuOMoreover, the incorporation of renewable and biodegradable materials, such as cellulose acetate (CA), adds sustainability and cost-effectiveness to the GPE formulations. Collectively, these advancements in GPE technology contribute to the advancement of lithium battery systems and enable the creation of effective and dependable energy storage systems.In lithium\u2013sulfur batteries (LIS), the power generation process involves the gradual transition of sulfur through charge and discharge cycles. The use of elemental sulfur, a byproduct of the petrochemical industry, as the cathode material in LIS significantly reduces the cost compared to lithium-ion batteries (LIBs). Theoretically, LIS has a high energy density of 2600 Wh/kg and a specific capacity of 1675 mA h/g. However, there are several challenges associated with LIS that must be addressed to ensure their safe and efficient operation. One of the main issues in LIS is the volume expansion of sulfur during cycling, which can lead to mechanical stress and electrode degradation. Furthermore, the development of lithium dendrites on the anode surface raises safety issues since they may result in short circuits and even thermal runaway. These challenges can be mitigated by incorporating a uniform charge distribution and utilizing the flexibility of gel polymer electrolytes (GPEs). GPEs provide a stable and conductive environment for the movement of lithium ions, reducing the likelihood of dendrite formation and enabling safer battery operation. A further issue that is addressed by the usage of GPEs in LIS is the capacity fading issue brought on by repeated charge and discharge cycles. GPEs can effectively encapsulate the active sulfur material and provide a stable interface between the cathode and electrolyte, preventing the loss of active material and maintaining consistent electrochemical performance over time . A compa\u22124 S/cm at room temperature, which is higher than those of the GPEs made using Celgard (3.03 \u00d7 10\u22124 S/cm) and PVDF (4.51 \u00d7 10\u22124 S/cm). Therefore, the PDA modification of PVDF was found to be effective in improving the performance of Li-S batteries, particularly in terms of cyclic stability and ionic conductivity [In order to enhance the interface between lithium (Li) and gel polymer electrolytes (GPEs), lithiophilicity has been introduced to improve the stability of the Li anode during cycling in lithium\u2013sulfur (Li-S) batteries. Han et al. employed poly(dopamine) (PDA) modification on a polyvinylidene fluoride (PVDF)-based GPE to impart lithiophilicity. The lithiophilic nature of PDA, which contains pyrrolic nitrogen, facilitates Lewis acid\u2013base interactions. Solution casting was first used to prepare PVDF film, followed by coating both sides of the PVDF film with dopamine, which underwent oxidative polymerization to form PDA. The resulting PVDF-PDA film was then infiltrated with a mixture of liquid electrolyte containing lithium bis-(trifluoromethane)sulfonimide (LiTFSI), 1,3-dioxolane, and 1,2-dimethoxyethane (DME). The strong confinement of polysulfide species by the polydopamine layer provided improved the interfacial stability of the Li anode and increased the cyclic stability of the Li-S battery. The quasi-solid-state cell with the polydopamine-modified GPE exhibited an extended cycling life. The specific capacity demonstrated by the PVDF-PDA GPE was 1215.4 mA h/g at a current rate of 0.1 C. After 200 cycles, the initial specific capacity decreased to 868.8 mA h/g, corresponding to a decay of approximately 0.14% per cycle. The stability after 200 cycles was 71.48%. In comparison, the specific capacities exhibited by cells with PVDF and Celgard films were 1164.58 mA h/g and 1186.8 mA h/g, respectively. After 200 cycles, the respective initial specific capacities of the PVDF and Celgard films decreased to 733.6 mA h/g and 509.7 mA h/g. The PVDF-PDA GPE demonstrated an ionic conductivity of 5.71 \u00d7 10uctivity .2S deposition and lithium dendrite formation caused by repeated stripping/plating and the shuttle effect resulting from soluble polysulfides in lithium\u2013sulfur (Li-S) batteries, a super-high ionic conductive gel polymer (SHGP) electrolyte was developed by Zhou et al. 2S8 (lithium octasulfide) and facilitates the efficient deposition of Li2S. This enhanced performance is attributed to the presence of abundant nucleation sites within the SHGP electrolyte, which are derived from its enriched polar functional groups. The SHGP electrolyte demonstrated an ionic conductivity of 2.2 \u00d7 10\u22123 S cm\u22121 at 60 \u00b0C and 0.75 \u00d7 10\u22123 S cm\u22121 at 30 \u00b0C. The Li-S battery with the SHGP electrolyte had a specific capacity of 950 mA h/g at 0.2 C, and it maintained 98% of its original capacity after 100 cycles. Theoretical simulations verified that the polar functional groups of SHGP interacted with lithium polysulfides, preventing their migration and hindering their detrimental effects. When tested in quasi-solid-state Li-S batteries, the SHGP electrolyte exhibited improved performance, maintaining its stability over 400 cycles [To address the challenges of Li0 cycles .2S deposition, lithium dendrite formation, and the shuttle effect. The SHGP electrolyte demonstrates excellent Li ion conductivity and exhibits a high specific capacity, retaining its stability over a significant number of cycles. These advancements in GPE technology contribute to the overall performance enhancement and viability of Li-S batteries, offering a promising solution for next-generation energy storage systems.The development of GPEs has proven effective in addressing key challenges in lithium\u2013sulfur (Li-S) batteries. Enhancing the interfaces between lithium (Li) and GPEs has been achieved by introducing lithiophilicity, as demonstrated via the poly(dopamine) (PDA) modification of a polyvinylidene fluoride (PVDF)-based GPE. The lithiophilic nature of PDA enhances the interfacial stability and cyclic stability of Li-S batteries, resulting in an extended cycling life and improved specific capacity. The quasi-solid-state cell with a polydopamine-modified GPE exhibited a high specific capacity and stability after 200 cycles. Furthermore, the development of a super-high ionic conductive gel polymer (SHGP) electrolyte addresses the challenges of LiZinc-based batteries are emerging as next-generation energy storage technologies with significant potential, finding applications in wireless devices, hearing aids, and signal lamps. Unlike lithium-based batteries, zinc batteries do not require stringent dry room conditions for their operation. Moreover, zinc is abundantly available in the Earth\u2019s crust and can be easily recycled, resulting in lower manufacturing costs. The The required changes have been adopted. of gel polymer electrolytes (GPEs) in zinc batteries has shown great potential, particularly in wearable electronics in which the ability to withstand mechanical deformation is crucial. GPEs play a significant role in influencing the power output and cyclic stability of zinc batteries. Therefore, the rational design of GPEs becomes essential in achieving superior mechanical stability and accelerated ion transport. Recent years have seen significant efforts made in developing GPEs tailored for various types of zinc batteries. The primary objective is to enhance mechanical stability, ionic stability, mechanical stability, and cyclic stability, thereby optimizing the overall performance of zinc batteries ,81. Tabl\u22122, which is advantageous for enhancing battery performance. By incorporating the PAM-AGE, the power density of the ZAB was significantly improved, reaching up to 105.0 mW cm\u22122. This enhancement in power density is a noteworthy achievement in the development of flexible ZABs [Zinc\u2013air batteries (ZABs) have gained significant interest as potential substitutes for LIBs. However, their development has been hindered by challenges related to their low energy conversion efficiency and output power density, especially in flexible ZABs. To address these limitations, a polyacrylamide (PAM)-based alkaline gel electrode (PAM-AGE) was utilized in this study. The PAM-AGE demonstrated a high hydroxide ion conductivity, reaching 0.2156 S cmble ZABs .\u22123. The discharge capacity reached 406.4 mA h/g at a rate of 0.2 C, and the battery showed remarkable stability, with a capacity retention of 96% after 300 cycles. Notably, this battery exhibited heat resistance and excellent mechanical stability. The PVA/G electrolyte demonstrated environmental adaptability and suitability for practical applications [The safety and affordability of aqueous zinc-ion batteries (AZIBs) is well known. However, their industrial applications are limited due to the high freezing point of the electrolyte. To address this issue, Zhou et al. developed a polyvinyl alcohol\u2013glycerol (PVA/G) gel polymer electrolyte (GPE) for AZIBs. The PVA/G GPE was prepared by mixing an aqueous solution of polyvinyl alcohol and glycerol, followed by solution casting. The PVA/G GPE was sandwiched over a zinc foil and a titanium foil, with d-MgVO serving as the active material, to create the AZIB. Remarkably, the assembled AZIB exhibited excellent performance over a wide range of temperatures (from \u221230 to 60 \u00b0C). The ionic conductivity of the PVA/G GPE was measured at different temperatures, with values of 22.4, 18.2, 14.3, and 10.7 mS/cm at 60, 25, 0, and \u221230 \u00b0C, respectively. The AZIB also demonstrated a volumetric energy density of 79.35 mW h/cmications . Methanesulfonic acid (MSA) is a commonly used protonating agent for poly(aniline) (PANI) due to its favorable properties, such as its high ionic conductivity, low toxicity, and solubility in water. It plays a crucial role in achieving mechanically and thermally stable PANI with improved conductivity. MSA effectively localizes the charge carriers within the PANI chains, thereby enhancing its overall performance. The interaction between the MSA and the amine/imine groups in protonated PANI forms hydrogen bonds, contributing to enhanced cyclic stability. In the context of fiber-shaped Zn-PANI batteries (Fs-ZPBs), there are certain limitations to be addressed, including low levels of electrolyte conductivity, soluble quinone formation, and capacity fading. To overcome these challenges, Shim et al. utilized MSA to enhance the ionic conductivity of polyvinyl alcohol (PVA)-based gel polymer electrolytes (GPEs). The incorporation of MSA into the PVA matrix resulted in remarkable stability of the GPE, with a capacity retention of 88.1% after 2000 cycles and 92.7% after 500 bending cycles (radius = 2.5 mm). The initial capacity of the battery was measured at 100.3 mA h/g with a 2C rate. The intermolecular hydrogen bonds formed between the PVA and MSA played a crucial role in connecting the PVA molecular chains and PANI chains on the surface of the PVA. These linkages significantly improved the charge transfer properties and ionic conductivity within the battery system. Furthermore, the coordination of large sulfonate ions with PANI effectively prevented the binding of water molecules to the PANI surface, which is a major cause of PANI degradation. This protective effect of MSA on the PANI surface further contributes to the enhanced stability and overall performance of the battery. 2, and a specific capacity of 724 mA h/g at a current density of 3 mA/cm2. Additionally, the cyclic stability of the batteries was maintained for over 56 h. The PVAA-cellulose displayed remarkable mechanical strength, with values of 87 MPa and 818% elongation, which could be due to the formation of an intertwined network structure [The advancement of high-performance, safe, and flexible GPEs is crucial for zinc\u2013air batteries (ZABs). However, poly (PVA)-based GPEs suffer from limitations such as poor mechanical strength, low ionic conductivity, and short cyclic stability. To overcome these challenges, Li et al. devised a novel approach by preparing a multinetwork cross-linked composite GPE. The composite GPE, named PVAA-cellulose, was created by incorporating poly(acrylic acid) (PAA) and ultrafine cellulose into the PVA gel matrix. tructure .42\u2212. As a consequence, the QSZAB\u2019s performance greatly increased, and a long cyclic lifespan of 127 h was achieved. This research provides valuable insights into enhancing the performance of QSZABs by regulating alkali release at the GPE-anode interface [In an effort to enhance the efficiency of quasi-solid-state zinc\u2013air batteries (QSZABs), Li et al. explored the use of hollow Sn microspheres modified with 2-hydroxypropyl-\u03b2-cyclodextrin (hollow Sn-inner HP\u03b2CD) to regulate alkali release within the GPE. The incorporation of hollow Sn-inner HP\u03b2CD proved to be highly effective in preventing alkali leakage, facilitating alkali diffusion back to the GPE during the charging process, and reducing the loss of soluble Zn(OH)nterface .The development of GPEs has emerged as a hopeful avenue for advancing the performance of zinc-based batteries. These batteries offer several advantages such as lower manufacturing costs, the abundant availability of zinc, and the ability to operate without stringent dry room conditions. By incorporating tailored GPEs, notable improvements have been achieved in various types of zinc batteries, including zinc\u2013air batteries (ZABs), aqueous zinc-ion batteries (AZIBs), and fiber-shaped Zn-PANI batteries (Fs-ZPBs). GPEs have demonstrated the capability to enhance the mechanical stability, ionic conductivity, and cyclic stability of batteries, ultimately optimizing the overall performance of zinc batteries. Noteworthy achievements include the utilization of polyacrylamide (PAM)-based alkaline gel electrodes to improve power density in flexible ZABs, the development of polyvinyl alcohol\u2013glycerol (PVA/G) GPEs for AZIBs with wide temperature stability, and the use of methanesulfonic acid (MSA) to improve the stability and ionic conductivity of PVA-based GPEs in Fs-ZPBs. Additionally, the introduction of multinetwork cross-linked composite GPEs and the incorporation of hollow Sn-inner HP\u03b2CD have demonstrated promising results in enhancing the performance and cyclic lifespan of zinc\u2013air batteries. These advancements pave the way for the future development of efficient, safe, and flexible zinc-based energy storage systems.Sodium-ion batteries (SIBs) are a viable replacement for lithium-ion batteries (LIBs) due to the widespread availability of sodium and cheaper manufacturing costs. Gel polymer electrolytes (GPEs) with superior ionic conductivities at ambient temperatures, however, have been investigated due to safety concerns over the combustibility of organic liquid electrolytes that are comparable to LIBs. To advance the widespread commercialization of SIBs, significant efforts have been dedicated to developing GPE-based SIBs that offer high levels of ionic conductivity, mechanical stability, and cyclic stability . In TablN,N\u2032-dimethylformamide and acetone (3:7 ratio), and the GPE was formed via solution casting. This blend was obtained via solution blending. The obtained blend showed an ionic conductivity of 1.086 \u00d7 10\u22123 S cm\u22121. Carbon-coated sodium and sodium metal were employed as the cathode and anode, respectively. The fabricated SIB cell demonstrated a relatively high specific capacity of 109 mA h/g at 1C, attributed to its improved interfacial stability, excellent ionic stability, and low electrolyte leakage. The cyclic stability after 500 cycles was 71.7%. Furthermore, this blend GPE can serve as both a separator and electrolyte in SIBs [To enhance mechanical stability and mitigate dendrite formation, a hydroxyapatite (HA)-incorporated polyvinylidene fluoride-co-hexafluoropropylene (PVDF-HFP) gel polymer electrolyte (GPE) was employed in sodium-ion batteries (SIBs). HA, an environmentally friendly biomaterial known for its mechanical stability and functional groups, offers favorable characteristics for composite structure formation, thereby improving the longevity and safety of SIBs. Specifically, a blend of HA-incorporated PVDF-HFP and poly(butylmethacrylate) (PBMA) was dissolved in a mixture of  in SIBs .\u22124 S/cm. In earlier methods, the polymer matrix was plasticized using carbonate solvents such as ionic liquids, propylene carbonate, ethylene carbonate, and dimethyl carbonate. However, these solvents were flammable and not suitable for large-scale applications. To address this, organic phosphate-based solvents have emerged as a promising alternative due to their nonflammable nature, high solvating capability, wide electrochemical window, low cost, and viscosity. In a study by Chen et al., triethyl phosphate (TEP) was utilized as a flame-resistant solvent for the preparation of a poly(ethylene glycol) methyl ether methacrylate (PEGMA) based GPE for SIBs. Gel polymer electrolytes (GPEs) have proven to be highly effective in enhancing the ionic conductivity of sodium-ion batteries (SIBs), surpassing values of 10\u22124 S/cm at 27 \u00b0C, which closely approaches the ionic conductivity values of liquid electrolytes (ranging from 10\u22123 to 10\u22122 S/cm) [In the fabrication of the GPE, PEGMA was mixed with sodium bis(fluorosulfonyl)imide (NaFSI), TEP, a film-forming additive (fluoroethylene carbonate), and 2,2-azoisobutyronitrile. The mixture was then subjected to thermal-induced polymerization at 80 \u00b0C for at least 3 h. \u22122 S/cm) .0.7CoO2 serving as the cathode and Na metal foil as the anode. The GPE exhibited an electrochemical stability window of approximately 5.2 V, a high ionic conductivity of around 1.12 \u00d7 10\u22123 S/cm at room temperature, a large Na transference number of approximately 0.58, and thermal stability up to approximately 100 \u00b0C. The initial discharge capacity of the NIB cell was measured to be 91.76 mA h/g at a rate of 0.05 C. Over 40 cycles at 0.05 C, the Na metal anode displayed reversible discharge, with a capacity of around 60 mA h/g. Furthermore, extraction or deposition tests indicated excellent stability, with a polarization value limit of \u00b119 mV. The extraction of diglyme\u2019s maximum occupied molecular orbital (HOMO) electrons by the Na salt was predicted to increase the oxidation stability of the metal\u2013diglyme complexes. This suggests that the insertion of NaTFSI into the diglyme contributed to the improved oxidation stability observed in the system [Due to their high degree of safety, favorable transport characteristics, excellent electrochemical stability, and low volatility, sodium-ion batteries (NIBs) have gained significant attention in recent years. Shadma Parveen et al. produced a gel polymer electrolyte (GPE), using solution casting and a mixture of the sodium salt of TFSI (NaTFSI), diglyme (G2), and poly(vinylidene fluoride-co-hexafluoropropylene) (P(VDF-HFP)) dissolved in acetone. The performance of this sodium-ion conducting GPE film was evaluated in NIB cells, with commercial Nae system .The use of gel polymer electrolytes (GPEs) in sodium-ion batteries (SIBs) has demonstrated remarkable advancements in improving their mechanical stability, ionic conductivity, and cycling performance. The incorporation of hydroxyapatite (HA) in a polyvinylidene fluoride-co-hexafluoropropylene (PVDF-HFP) GPE demonstrated enhanced interfacial stability and reduced electrolyte leakage in SIBs. The resulting composite structure, formed through a solution casting process, exhibited a favorable ionic conductivity and facilitated the use of the blend GPE as both a separator and electrolyte. Similarly, the utilization of organic phosphate-based solvents, such as triethyl phosphate (TEP), in a poly(ethylene glycol) methyl ether methacrylate (PEGMA) showcased a nonflammable alternative with a high solvating capability and wide electrochemical window. The optimized composition demonstrated impressive ionic conductivity and enabled the SIB cells to maintain good retention even after repeated cycles. Furthermore, the development of a sodium-ion-conducting GPE using poly(vinylidene fluoride-co-hexafluoropropylene) (PVDF-HFP) and the sodium salt of TFSI (NaTFSI) exhibited a high ionic conductivity, excellent electrochemical stability, and thermal stability. The NIB cells incorporating this GPE demonstrated promising initial discharge capacity and reversible discharge with remarkable stability. These results demonstrate the potential of GPEs in advancing the performance and safety of sodium-ion batteries, bringing us closer to practical and efficient energy storage solutions for various applications. Further research and optimization of GPE formulations and design are crucial for realizing the full potential of sodium-ion batteries and expanding their practical applications in the future.GPEs (gel polymer electrolytes) have been extensively explored, not only in lithium-ion batteries but also in other emerging battery technologies such as dual-ion batteries, solar batteries, and all-organic batteries. A dual-ion battery, also known as a double-ion battery or symmetric battery, is a type of energy storage system in which both cations and anions are involved in charge transfer during battery operation. Unlike conventional batteries, in which only one type of charge carrier (either cations or anions) is responsible for the electrochemical reactions, dual-ion batteries utilize both types of charge carriers to enhance their overall efficiency and energy density. In a dual-ion battery, the electrolyte plays a crucial role in providing the necessary charge carriers for the electrochemical reactions. During the charging and discharging processes, the quantities of both anions and cations in the electrolyte might change. Therefore, the development of suitable GPEs becomes essential to facilitate the efficient transport of both cations and anions in these systems. GPEs offer several advantages in dual-ion batteries. Firstly, they can provide a stable and homogeneous environment for ion transport, allowing for improved ionic conductivity and reduced interfacial resistance. Secondly, GPEs can help mitigate the diffusion mismatch between different ions, ensuring balanced ion transport and preventing ion depletion at the electrode\u2013electrolyte interfaces. Thirdly, GPEs can contribute to enhanced cycling stability and improved safety by inhibiting the development of dendrites, which results in battery failure and short circuits. Moreover, GPEs have been explored in solar batteries and all-organic batteries, which are promising alternatives to traditional inorganic-based energy storage systems. In solar batteries, GPEs can serve as solid-state electrolytes that enable efficient charge separation and transport within the device. In all-organic batteries, GPEs based on organic polymers offer advantages such as high flexibility, tunable properties, and compatibility with organic electrode materials . Table 72) particles and a pore-forming agent . The resulting solution was cast onto an aluminum (Al) foil substrate and subjected to phase inversion via the introduction of methanol. The resulting film was then immersed in a liquid electrolyte containing lithium hexafluorophosphate (LiPF6). The microporous GPE exhibited high levels of ionic conductivity and mechanical strength, providing sufficient ions for efficient charge transport and enabling better contact with the Al foil anodes. The GPE also played a crucial role in reducing gas generation and protecting the Al foil from corrosion. Remarkably, the dual-ion battery using the microporous GPE demonstrated excellent cycling stability, with 1000 cycles retaining 82% of the original capacity (80 mA h/g). Additionally, the dual-ion battery showed an elevated working voltage of 4.2 V, expanding its potential applications. The maximum ionic conductivity observed for the PVDF-HFP-based GPE with a SiO2 content of 15% at room temperature was measured to be 2.40 mS/cm with LiPF6 as the electrolyte. These remarkable results emphasize the potentiality of the dual-ion cell with a microporous GPE for future eco-friendly energy storage applications [In dual-ion batteries (DIBs), the intercalation of anions has been found to be detrimental to the structure of the graphite electrode. This phenomenon leads to early capacity decay, short cyclic stability, and volume expansion issues. To address the challenge of volume expansion, Wang et al. developed a microporous gel polymer electrolyte (GPE) based on polyvinylidene fluoride-hexafluoropropylene (PVDF-HFP) for solid-state DIBs. In their study, a microporous PVDF-HFP film was synthesized using the phase inversion method. The PVDF-HFP solution in N-methyl-2-pyrrolidinone was mixed with silicon dioxide  (PEO) gel polymer electrolyte (GPE). This type of battery is capable of converting solar energy into chemical energy when exposed to light. The GPE was prepared by dissolving PEO and LiClOremained .In pursuit of developing printable gel polymer electrolytes (GPE) for all-organic batteries, Muench et al. successfully formulated a versatile solution. Addressing challenges related to solid electrolyte interface (SEI) formation, electrolyte stability, Li dendrite development, and Li salt requirements, their work introduced a promising approach. By harnessing the unique characteristics of ionic liquids (ILs) as both solvents and salts, the complexity of the electrode system could be simplified. Furthermore, ILs offered additional benefits, including high levels of ionic conductivity and thermal stability, and negligible vapor pressure, making them well-suited for all-organic electrolytes. To achieve their goal, the researchers developed a printable GPE ink formulation comprising nanofillers, methacrylate monomers, and a cross-linking agent. Specifically, benzyl methacrylate, poly(ethylene glycol) methylether methacrylate, triethylene glycol dimethacrylate, and 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide (BMImTFSI) were blended and placed between two siliconized PET foils. By adjusting the spacer for the desired film thickness, the system underwent photopolymerization under UV light for a period of 10 to 60 min. This allowed the formation of a mechanically stable, flexible GPE with an ionic conductivity of around 0.74 S/cm. Remarkably, the printed GPE served a dual purpose as both the electrolyte and the separator in all-organic batteries. It exhibited a maximum specific discharge capacity of 24 mA h/g (at 0.1C), with 77% of the initial capacity retained after 1000 cycles. The mechanical stability of the GPE, as demonstrated through an indentation analysis, enabled its reliable function as a separator, even during the polymerization process in the presence of oxygen. Overall, this innovative printable GPE formulation presents a promising pathway for the advancement of all-organic batteries .The development of gel polymer electrolytes (GPEs) has shown great potential in advancing the performance and endurance of diverse types of batteries. In the context of dual-ion batteries (DIBs), the utilization of a microporous GPE based on polyvinylidene fluoride-hexafluoropropylene (PVDF-HFP) has addressed challenges relating to volume expansion and electrode structure deterioration. The microporous GPE exhibited high levels of ionic conductivity and mechanical strength with improved contact with the anode, which it protected from corrosion, resulting in an excellent cycling stability and a high working voltage. Similarly, in quasi-solid-state dye-sensitized solar batteries, the incorporation of a poly(ethylene oxide) (PEO) GPE enhanced the durability and ionic conductivity of the system, leading to significantly enhanced capacity retention and cycling performance compared to liquid electrolytes. Furthermore, in the pursuit of printable GPEs for all-organic batteries, researchers successfully formulated a versatile solution using ionic liquids (ILs) as solvents and salts. The resultant printed GPE exhibited impressive mechanical stability, ionic conductivity, and specific discharge capacity, serving as both the electrolyte and separator in the all-organic batteries. These advancements in GPE technology offer promising opportunities for the advancement of eco-friendly energy storage technologies, providing improved performance and reliability.The development of gel polymer electrolytes (GPEs) has demonstrated significant potential in enhancing the performance, stability, and safety of various types of batteries. The advancements in GPE technology have addressed key challenges in lithium-ion batteries (LiBs), lithium metal batteries (LMBs), lithium\u2013oxygen batteries, lithium\u2013sulfur batteries, zinc-based batteries, sodium-ion batteries, and dual-ion batteries. Through the utilization of innovative strategies such as alternative precursors, 3D printing techniques, doping, composite designs, the incorporation of ionic liquids, and the introduction of renewable and biodegradable materials, researchers have achieved notable improvements in multiple aspects of battery performance. In LiBs and LMBs, GPEs have resulted in stable Li-electrolyte interfaces, dendrite-free morphologies, and enhanced stripping/plating efficiencies and improved the interfacial chemistry and cycling performance. Additionally, the incorporation of organic electrolytes, coiling GPE membranes, fireproof gel electrolytes, and biodegradable nanofiber membranes has addressed interfacial issues, enhanced anode stability, and facilitated continuous Li ion transmission.For lithium\u2013oxygen batteries, the development of hybrid solid\u2013gel electrolytes and GPEs has shown promise in improving cyclic stability, interfacial stability, and specific capacity. The use of lithiophilic modifications and super-high ionic conductive gel polymers (SHGPs) has addressed challenges relating to lithium dendrite growth, lithium\u2013sulfur batteries, and interface interactions. In zinc-based batteries, GPEs have enhanced mechanical stability, ionic conductivity, and cyclic stability. Notable achievements include improvements in zinc\u2013air batteries, aqueous zinc-ion batteries, fiber-shaped Zn-PANI batteries, and the utilization of multinetwork cross-linked composite GPEs. In sodium-ion batteries, GPEs have shown advancements in mechanical stability, ionic conductivity, and cycling performance. The incorporation of hydroxyapatite, organic phosphate-based solvents, and the sodium salt of TFSI has improved interfacial stability, solvating capability, and thermal stability. Furthermore, in dual-ion batteries, microporous GPEs and poly(ethylene oxide) GPEs have addressed challenges relating to volume expansion, electrode structure deterioration, and stability in quasi-solid-state dye-sensitized solar batteries. The utilization of ionic liquids as solvents and salts has enabled the development of printable GPEs for all-organic batteries demonstrating impressive mechanical stability, ionic conductivity, and specific discharge capacity. These advancements collectively contribute to the development of high-performance, safe, and eco-friendly energy storage systems. Looking ahead, the future scope of gel polymer electrolytes lies in the continued exploration and optimization of new materials, fabrication techniques, and architectures to further enhance the electrochemical performance, mechanical stability, and safety of GPEs. The development of advanced characterization techniques will enable a better understanding of the ion transport mechanisms, interfacial interactions, and degradation mechanisms within GPE-based battery systems. The incorporation of nanofillers, such as graphene, carbon nanotubes, and metal oxides, can improve the ionic conductivity, mechanical strength, and stability of GPEs. Additionally, the utilization of advanced additives, such as functionalized polymers, ionic liquids, and nanoscale solid electrolyte interphases (SEIs), can further optimize the performance and safety of batteries utilizing GPEs. Scalable and cost-effective manufacturing processes, such as roll-to-roll processing and inkjet printing, are crucial for the practical adoption of GPEs in large-scale energy storage systems. These advancements will contribute to the commercial viability of GPE-based batteries and accelerate their integration into electric vehicles, portable electronics, and grid energy storage. With ongoing research and development, GPEs have prospects for enabling the next generation of high-energy-density, long-lasting, and safe energy storage systems. The future of GPEs lies in continued material optimization, advanced manufacturing techniques, and their integration into various applications, driving the transition towards a more sustainable and electrified future."}
+{"text": "Drosophila intestine, an organ dynamically maintained by the intestinal stem cells (ISCs). The results indicated that the polo depletion caused a reduction in the gut size due to a gradual decrease in the number of functional ISCs. Interestingly, the polo-deficient ISCs showed an extended G2/M phase and aneuploidy and were subsequently eliminated by premature differentiation into enterocytes (ECs). In contrast, the constitutively active Polo (T182Dpolo) suppressed ISC proliferation, induced abnormal accumulation of \u03b2-tubulin in cells, and drove ISC loss via apoptosis. Therefore, Polo activity should be properly maintained for optimal stem cell function. Further analysis suggested that polo was a direct target gene of Sox21a, a Sox transcription factor that critically regulates stem cell activity. Together, this study provided a novel perspective on the correlation between the progression of mitosis and the ISC function in Drosophila.Maintaining a definite and stable pool of dividing stem cells plays an important role in organ development. This process requires an appropriate progression of mitosis for proper spindle orientation and polarity to ensure the ability of stem cells to proliferate and differentiate correctly. Polo-like kinases (Plks)/Polo are the highly conserved serine/threonine kinases involved in the initiation of mitosis as well as in the progression of the cell cycle. Although numerous studies have investigated the mitotic defects upon loss of Plks/Polo in cells, little is known about the in vivo consequences of stem cells with abnormal Polo activity in the context of tissue and organism development. The current study aimed to investigate this question using the  Drosophila melanogaster  kinase family, were first found in nogaster , which cnogaster . Five Ponogaster . Drosophectively . Polo kiectively . Plk1 anectively . Thr182 ila Polo , and T21ila Polo . Polo kiila Polo .polo gene can cause a series of problems in mitosis, such as prolonging mitotic duration, affecting spindle orientation, and inducing chromosome misalignment  as compared with the other cell types , enterocytes (ECs), and enteroendocrine cells (EEs) (Dl (ISC), Su(H) and klu (EB), Pdm1 (EC), and Pros (EE) (Drosophila midgut was divided into 5 main regions that were named R1\u2013R5 (The intestine of ls (EEs) . It is aros (EE)   . The Droed R1\u2013R5 . The cured R1\u2013R5 .Drosophila midgut to explore the proliferation and differentiation of ISCs caused by mitotic defects using loss of function and gain of function of Polo. The results showed that the polo-deficient ISCs were correlated with an extended G2/M phase and aneuploidy and were subsequently lost by premature differentiation into ECs. In contrast, the constitutively active Polo (T182Dpolo) suppressed ISC proliferation, induced abnormal accumulation of \u03b2-tubulin in cells, and drove ISC loss via apoptosis. Thus, the Polo activity should be properly maintained for optimal stem cell function in the Drosophila midgut.The current study utilized the The following fly lines were used:tsesg: esg-Gal4, UAS-GFP, tstub-Gal80 (\u2161) (80ts (\u2161) ;tsSu(H)GBE: Su(H)GBE-Gal4, UAS-GFP, ts/Cyotub-Gal80 -Gal80, ts/TM3 sbtub-Ga80 GBE-LacZ; FRT80B.RFP-CycB ; UAS-p35A full list of genotypes used in each experiment is shown in tsesg, tsDl, tsSu(H)GBE, tsISC, and ReDDMesg) were crossed with 1118w or the UAS-linked transgene for polo knockdown or polo overexpression.Female flies were used in all experiments. All flies were fed on a standard medium  at room temperature, unless otherwise mentioned. In most cases, the drive lines , washed in PBST [PBS containing 3% bovine serum albumin (BSA) and 0.1% Triton X-100] 3 times, and then incubated in primary antibodies (at 4\u00b0C overnight) and secondary antibody (at 25\u00b0C for 4 h or 4\u00b0C overnight) prepared in PBST. All antibodies were listed in Erwinia carotovora carotovora 15 (Ecc15) was grown in LB medium at 29\u00b0C with shaking overnight. On next day, Ecc15 cells were harvested after centrifuging at 5,000 rmp at 4\u00b0C for 5 min. The pellet was then washed using 0.85% NaCl and 5% sucrose respectively at the same condition. Finally, Ecc15 cells were suspended with 5% sucrose to OD600 = 200 for oral infection. The control flies were fed with 5% sucrose. Colchicine was prepared as reported previously  was suspended with 5% sucrose for feeding flies of different genotypes for 24 h. The control flies were fed with 5% sucrose. All flies were dry-starved in the empty tubes for 2 h before feeding.eviously . Approxiesg-gal4, Su(H)GBE-gal4, and Dl-gal4) to conditionally express UAS-liked transgenes  technique was used for clonal analysis . Transge\u00b5l TUNEL reaction mixture  as described in the manufacturer's protocol.The dissected midguts were fixed as above and permeabilized using 2 mg/ml Proteinase K for 7 min, followed by washing and incubation in the dark for 1 h at 37\u00b0C in 100-The described fluorescence in situ hybridization (FISH) protocol was followed with some modification . Brieflypolo promoter fragments  was cloned and inserted into the expression vector pET-28b (Novagen) with a His-tag at the C-terminals. The recombinant Sox21aHMG-BD was expressed and purified as described (The sequence of high mobility group box domain (HMG-BD) of Sox21a (named as Sox21aescribed . The proHMG-BD was purified as described above. The Cy5-labeled probe (F2-4) was amplified using 2 rounds of PCR. The Cy5-labeled F2-4-3 probe containing the wild-type sequence and the probe containing mutated sequence were synthesized by Biosune. The Cy5-labeled probe and protein were coincubated in EMSA\u2013binding buffer  at 25\u00b0C for 30 min in dark. After incubation, the reaction mixture was electrophoresed on a 6% native polyacrylamide gel, and then labeled DNA was detected using Typhoon FLA scanners . The sequences of probes were listed in The described electrophoretic mobility shift assay (EMSA) protocol was followed with some modification . Briefly\u00b5g) was applied for reverse transcription with oligo dT, and the first strand cDNA was diluted with water for further use in real-time PCR as described . cDNA was synthesized using the Prime Script RT reagent kit (Toyobo). Total RNA    . Polo-GFolo-GFP) . Since I of ISCs . ISCs we (Delta) . Moreove (Delta) .Drosophila (Su(H)GBE-LacZ) (nubbin (Pdm1), a POU-domain transcription factor expressed specifically in mature ECs  . During osophila . In addiBE-LacZ)  retainedture ECs  and EE mture ECs . These rDrosophila, the clones homozygous for Polo using the null allele 1polo that cannot be phosphorylated   cells in both the anterior and posterior midgut (Dl-LacZ) also decreased significantly (polo were significantly reduced at different time points upon suppressing polo with the tsesg driver (In order to investigate the function of Polo in the midgut of adult orylated  were genorylated . A reducorylated . Furthers esgts) . The flir midgut . Moreover midgut . In addificantly . Consists driver .polo for 10 d, consistent with the loss of +esg > GFP progenitors, the number of PH3-positive (PH3+) cells indeed decreased significantly. However, surprisingly, an obvious increase was detected in the PH3+ cells on days 1 and 5 after initiating the knockdown experiment GBE-Gal4 UAS-GFP tub-Gal80 (ts)GBE -Gal80 tub-Gal80 (tsISC)    and in I (ISCts) . The num for 1 d . polo9 aetaphase , a stagephase   was usedd midgut . These dpolo-depleted G2/M-arrested ISCs were lost. Polo plays a crucial role in regulating the SAC pathway to maintain genomic stability during mitosis  driver to detect apoptotic progenitor cells. The number of progenitor cells being positive for caspase-3 in the midgut was always very low as dying cells were cleared off rapidly from the intestinal epithelium, which hampered quantification  by inducing the differentiation of ISCs were further validated using the 3 methods as follows. First, polo was suppressed by RNAi using the tsesg driver at 29\u00b0C for only 3 d. The ratio of newly formed EC-like cells (GFP+Pdm1+) to all GFP+ cells in R4 region of polo RNAi midguts was significantly increased through staining with EC marker Pdm1, suggesting that these progenitor cells were undergoing rapid differentiation  tracing technique  in esg-Gal4 progenitors, which propagated into the daughter cells, allowing the tracing of newly differentiated epithelial cells. The results showed that the differentiation index  increased after suppressing the polo gene for 5 and 10 d   . MARCM tl clones . The esgP+ cells  and II. eostatic  and infec Ecc15; , III, suosophila . In addiem cells . In contdivision . These rdivision . In summT182Dpolo could drive the loss of stem cells in the same manner as that of polo depletion, the localization of active Polo was first analyzed. The phosphorylation of the conserved Thr210 can activate Polo ortholog Plk1       . Further in ISCs . In addif tumors . The EB section) .polo , was purified (HMG-BD and Cy5-labeled probe (F2-4) were tested using EMSA (HMG-BD with the labeled DNA (https://jaspar.genereg.net), TTTGTTGTTCA was established as a candidate  suppressed ISC proliferation and drove the ISC loss via apoptosis.The current study reported a specific function of Polo-mediated mitosis in regulating the stem cells in function . The resSox21a mutation was rescued by polo depletion  tumorigenesis , p53, Medea (dSmad4), and Pten (Phosphatase and tensin homolog)   will be T182Dpolo led to the abnormal accumulation of \u03b2-tubulin and induced the apoptosis of stem cells in Drosophila midgut. A previous study reported that cohesin controlled ISC identity, in which T182Dpolo showed differentiation phenotype (T182Dpolo with ReDDMesg, some RFP+GFP\u2212 polyploid ECs were also detected. Since the overexpression of T182Dpolo did not affect EB (ReDDMesg flies, which was consistent with the previous studies. The enforced expression of active T182Dpolo has also been proposed to be toxic in other tissues, inhibiting the development of a majority of embryos before hatching or inducing smaller and rougher eyes (This study demonstrated that the constitutively active henotype . Moreoveffect EB , it was her eyes . Altogetjkad084_Supplementary_DataClick here for additional data file."}
+{"text": "Understanding how local communities perceive threats and management options of wild edible plants (WEPs) is essential in developing their conservation strategies and action plans. Due to their multiple use values, including nutrition, medicinal, construction, and cultural as well as biotic and abiotic pressures, WEPs are exposed to overexploitation, especially within arid and semiarid lands, and hence the need to manage and conserve them. We demonstrate how an understanding of indigenous communities\u2019 perceptions could be achieved through an integrated participatory approach involving focus group discussions (FGDs) and field plot surveys.We conducted three FGDs between October 2020 and April 2021 within three community units in northwestern Kenya with different socioeconomic and environmental characteristics. We subsequently surveyed 240 field plots of size 1\u00a0ha each to assess threats facing WEPs within a 5\u00a0km buffer radius in every study community. We compared ranks of threats and management options across community units.Rankings of threats and management options differed across the three study communities. We obtained strong positive linear relationships between field and FGD rankings of threats facing WEPs. Climate change, overstocking, overharvesting, and invasive species were the highest-ranked threats. Mitigation of climate change, local knowledge preservation, selection, propagation, processing, and marketing of WEPs ranked high among possible management options irrespective of the socioeconomic and environmental characteristics of the community unit.Our approach emphasizes the relevance of leveraging indigenous communities\u2019 perceptions and conducting field plot surveys to assess threats and management options for WEPs. Evaluating the effectiveness and cost\u2013benefit implications of implementing the highly ranked management options could help determine potentially suitable habitats of the WEPs for conservation and management purposes, especially for priority WEPs.The online version contains supplementary material available at 10.1186/s13002-023-00584-6. Wild edible plants (WEPs) are \u2018safety nets\u2019 for many communities during lean seasons \u20133 and inWithin Africa, threats to WEPs pose challenges to about 80% of the rural populations that derive food from the wild . The thrAloe vera [Turkana County in northwestern Kenya is one of the affected regions in Africa. It is inhabited by the Turkana people, among others, whose traditional livelihood strategy is nomadic pastoralism , 25. Accloe vera , 29, honloe vera , artisanloe vera , poultryloe vera , basket loe vera \u201335, hideloe vera , local bloe vera , fishingloe vera , 39, andloe vera .Of the 47 counties in Kenya, Turkana County has the highest poverty and malnutrition rates . Only 3.Turkana people have relied on locally constituted management methods like seasonal grazing (via migration with livestock) and clear designation grazing fields . These, We sought to understand the threats and management options that could aid the sustainable use of WEPs in northwestern Kenya. To achieve this, we used an integrated participatory approach to combine FGDs results with field plot surveys guided by three research questions: (i) Which threats do WEPs\u00a0face in Turkana County, and how do they vary across different socioeconomic and environmental settings? (ii) How do indigenous communities\u2019 perceptions of these threats compare with field survey results? (iii) What are possible effective management options and how do they differ across socioeconomic and environmental settings?We conducted the study in three community health units , reflecting the socioeconomic and environmental differences in Turkana County Fig.\u00a0. A commuAtala Kamusio community is situated in the Turkana Border Pastoral livelihood zone , about 1We obtained threats and management options data on WEPs from the literature and discussed these with each of the three community units during FGDs. We also conducted field observations of threats. The research activities were carried out from October 2020 to April 2021.n\u2009=\u200923) that featured either threat or management reports. The list of threats and management options with their corresponding reviewed sources are given in Additional file We extracted threats and management options for WEPs from published literature using a snow-ball\u00a0sampling approach . We wentWe held FGDs with 14 adults (age\u2009>\u2009=\u200918\u00a0years) participants in each of the three study community units . With thVillage elders, for example, oversee matters concerning the use and conservation of community resources, including WEPs. Local administrators maintain peace and ensure adherence to rules, such as settling disputes whenever they arise, including those concerning WEPs. They are also the main entry points to the communities for government and non-government programs. Teachers instill knowledge in the young generation in school settings, including nutrition skills that could involve the use of WEPs. Health workers, such as health extension officers, nutritionists, public health officers, and community health volunteers at the community level, support the improvement of the health and well-being of local people, including advocating for the use of WEPs in their diets. Lastly, other members of the FGDs were drawn from residents who participated in harvesting and use of WEPs for food and medicine, among others. We thus considered all the participants very resourceful in discussing threats and management options for WEPs.Salvadora persica, Ziziphus mauritiana, and Balanites rotundifolia, considered priority [We selected three priority\u00a0woody WEPs, i.e., priority  due to tWe commenced every FGD by allowing participants to free list and discuss threats facing the three priority woody WEPs. We then consensually co-grouped the listed threats into the nine pre-defined cf. 2.2.1) categories with the participants. We added a tenth category for all mentioned threats that were not in our nine pre-selected categories . Treating this as the central point of the community unit, we created a virtual buffer zone of a five-km radius Fig.\u00a0 as buffene sites \u201360.Using nine of our ten threat categories , we scored observable threats to WEPs in each survey plot. Each threat category could receive a score between 1 (least threat) and 9 (highest threat). Scoring was based on the consensus of the threat categories by three observers (two trained research assistants and the corresponding author). Observed indicators of threats included fire scars to denote fire threat, over-browsed seedlings/lower branches of priority woody WEPs to denote overstocking/overgrazing, and plowed land to characterize agriculture expansion threat, among others that were obtained from FGDs  within Turkana County, Kenya. Their representation is summarized in Table n\u2009=\u200913), were ordinary community members, followed by health workers, village elders, and a public health officer. 60% (n\u2009=\u200925) of the participants were female, 40% (n\u2009=\u200917) were male. The participants were considered diverse and knowledgeable enough to give detailed discussions on the WEPs needed for the study.Table Climate change, invasive species, and overstocking/overgrazing ranked highest among the threats facing priority woody WEPs according to scores by FGD participants Table . We obseAt least one community unit was significantly different (\u03b1\u2009<\u20095%) from the other(s) in the ranking of each threat category except for invasive species, pests and diseases, and others Fig.\u00a0.Fig. 2CoWe observed no significant differences in threat scores between riverine and off-riverine field survey plots; hence, we formed a composite of the two datasets resulting in 80 survey plots per community unit. Overstocking/overgrazing, invasive species, and selective harvesting/overharvesting were the top three threats we observed in the field at Nasiger Table . At AtalOur rankings of threat categories facing woody WEPs from the field plots surveys varied significantly among the study community units Fig.\u00a0. We, howTo spatially visualize variations in scores among threats facing priority WEPs in all the 240 surveyed plots and community units, we developed a graduated gray scale map , controlling harvesting for food and fodder, cultivation of WEPs, regulation of charcoal burning, and preservation of local knowledge about WEPs could fit within local community action plans . On the other hand, the assessment of nutritional value and toxicity, the establishment of protected areas, selection, propagation, processing, and marketing require external intervention but with local collaboration. Some measures, such as raising public awareness about the benefits of WEPs, mitigation of climate change, and monitoring and inventorying WEPs, can only be achieved by closely engaging with local communities, policymakers, and any actors attempting to influence the management of WEPs. Involving local communities in implementing any management option is imperative.We understand that cost implications always play a big role in implementing any management options for threats facing biodiversity . HoweverEven though these findings agreed well with most studies on threats to biodiversity across the region, it is important to note that the relative significance varied with environmental and socioeconomic gradients at local scales. Local differences in threats and management options are therefore worth considering in developing sustainable management solutions for WEPs to bring them back into dietary diversification programs sustainably , 88.Climate change, invasive species, and overstocking/overgrazing threaten the sustainable use of WEPs in Turkana County, Kenya. How threats are perceived to affect WEPs depends on socioeconomic and environmental gradients across communities. Our integrated participatory approach, combining local community perceptions and field plot assessments, revealed close links, but some threats were ranked strikingly differently across the three study community units.Across all the study communities, the most plausible management options for the WEPs were mitigation of climate change, preservation of local knowledge, and selection, propagation, processing, and marketing. We propose a detailed cost\u2013benefit analysis of the assessed management options, bringing on-board all stakeholders in the WEP value chain, which should be a prerequisite before conservation plans are implemented. It is also important to establish the extent of the suitable habitats of the WEPs. Such an overview could improve the success of conservation and management interventions.Additional file 1: Supplementary Table 1. Threats facing wild edible plants or biodiversity in general mentioned in 23 literature sources and used to guide development of 10 threat categories for both focus group discussions and field plot surveys in Turkana County, Kenya. Supplementary Table 2. Indicators derived from the focus group discussions on threats facing priority woody wild edible plants in Turkana County, Kenya. The same indicators were used in field plot surveys except for climate change. Supplementary Table 3. Management options for wild edible plants mentioned in 9 literature sources and used to guide development of management categories for focus group discussions in selected community units within Turkana County, Kenya."
\ No newline at end of file